13-Membered Macrolide Compounds for Treating Diseases Mediated by Abnormal Protein Translation

Background

[0001] Macrolides are macrocyclic lactones that contain 12 to 16 ring atoms. The macrolide class of compounds was first discovered in 1952, when scientists isolated erythromycin from soil.

Since then, macrolides have triggered the attention of medicinal chemists and pharmaceutical scientists alike, as targets for synthesis as well as for use as treatments for bacterial infections in humans and animals. In the last 70+ years, synthetic derivatives of erythromycin as well as other natural and man-made macrolides have been a mainstay of the antibacterial pharmacopeia.

Today, macrolides continue to capture the interest of pharmaceutical research and development scientists because of their antibacterial activity, as well as their other potentially beneficial attributes. A small number of macrolides have been found to exert a surprising and an “almost bewildering number of biological effects on a variety of mammalian cells,” ranging from the modulation of inflammatory/immunomodulatory responses, the inhibition of mucus secretion, apparent abilities to block or retard aging as well as enhancing barrier integrity of epithelial cells (Knicker et. al, Pharmacological Reviews October 2021, 73 (4) 1404-1433). This activity has been observed in select 14-, 15-, and 16 membered macrolides including erythromicin, azithromicin, and clarithromicin.

[0002] What is not known more generally is the effect of macrolides on abnormalities in protein translation such as observed in proliferative diseases including cancer as well as in rare genetic diseases (especially those rare diseases caused by nonsense mutations). As noted recently by Song and coworkers, (Song, P., Yang, F., Jin, H. el al The regulation of protein translation and its implications for cancer. Sig Transduct Target Ther 6, 68 (2021). https://doi.org/10.1038/s41392- 020-00444-9). Cancer has been recognized as a disease resulting from the accumulation for multiple genetic and epigenetic changes. Genetic mutations mainly alter the functions of corresponding proteins, while epigenetic changes will change the expression of potential oncogenes and tumor suppressor genes. Although the regulation and function of these alterations have been substantially explored, the relevance of protein translation or the production of nascent proteins from mRNAs to the initiation and progression of human cancers has remained largely overlooked. In the cancer context, protein translation could be affected by various factors including translation initiation factors, RNA-binding proteins, and noncoding RNAs. [0003] Similarly, some diseases such as cystic fibrosis (CF), muscular dystrophy (Duchenne (DMD), Becker (BMD), congenital), spinal muscular atrophy (SMA), ataxia-telangiectasia, mucopolysaccharidosis type 1 (MPS1) (Hurler syndrome), hemophilia (A & B), Usher syndrome (Retinitis pigmentosa, X-linked retinitis pigmentosa), Tay-Sachs, factor VII deficiency, familial atrial fibrillation, Hailey-Hailey disease, McArdle disease, mucopolysaccharidosis, nephropathic cystinosis, polycystic kidney disease, Rett syndrome, cystinosis, severe epidermolysis bullosa, dravet syndrome, X-linked nephrogenic diabetes insipidus (XNDI), dancer, beta-thalassemia, and obesity have been found to be caused by nonsense mutations in genes, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Treatment of these diseases will require suppression of premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon.

[0004] Thus, there is continuing interest and a need to identify macrolides with diverse beneficial properties for treating diseases in humans and animals, particularly for diseases characterized by abnormalities in protein translation such as proliferative diseases including cancer as well as rare genetic diseases (especially those rare diseases caused by nonsense mutations).

Summary

[0005] These and other needs are met by the present invention which is directed to 13-membered macrolide compounds that can be used to treat proliferative diseases and disorders such as cancer as well as rare genetic diseases, including genetic diseases associated with a premature termination codon mutation or other nonsense and/or frameshift mutations. The compounds have an antiproliferative effect by selective inhibition of protein translation and can also induce and/or promote readthrough of the premature termination codon.

[0006] In one aspect, what is provided is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R.2a is selected from the group consisting of H, halo, optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl, wherein C1-10 alkyl, C1-10 alkoxy, and C1-10 alkenyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl; and

R.2b is selected from the group consisting of halo, optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl, wherein C1-10 alkyl, C1-10 alkoxy, and C1-10 alkenyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

R.4a and R4b are each independently selected from the group consisting of -H, and optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Rs is selected from the group consisting of H, an oxygen protecting group, and , ” indicates the point of attachment; Re _a is optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Reb is -H, optionally substituted C1-10 alkyl, optionally substituted C1-10 hydroxyalkyl, and optionally substituted allyl, wherein C1-10 alkyl, C1-10 hydroxyalkyl, and allyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Rsa and R> are each independently selected from the group consisting of -H and optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

R9a is selected from the group consisting of -H, -C(=N)-NH2, -C(=O)-C1-6 alkyl , -C(=O)- C1-6 alkylene-NR9a’R9a”, C1-10 alkyl and C1-10 alkenyl, wherein C1-10 alkyl and C1-10 alkenyl are optionally substituted with 1, 2, or 3 groups independently selected from the group consisting of amino, heterocycloalkyl, halo, hydroxy, alkoxy, aryl, heteroaryl, and heteroaryl ene-heteroaryl, wherein aryl, heteroaryl, and heteroaryl ene-heteroaryl, are optionally substituted with halo, alkoxy, and -NH2; wherein R9a’ and R9a” are selected from the group consisting of H, C1-6 alkyl, cycloalkyl, C1.4 alkylene-cycloalkyl, or R9a’ and R9a” together with the atom to which there are attached from a 3-, 4-, 5-, or 6-membered ring optionally containing an additional heteroatom selected from 0, S, SO, SO2, NH, and N-C1.4 alkyl;

Rioa and Riob are taken together with the carbon atom to which they are attached to form (Rq)o-2 ^— (TV

^K , wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein “ -w ” indicates points of attachment and Rn _a and Rub are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl; or

Riia and Rub are taken together with the carbon atom to which they are attached to form , wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein “ ” indicates points of attachment and Rioa and Riob are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl; Rq at each occurrence is independently selected from the group consisting of H, halo, - NRx’Rx”, C1-6 alkyl, C1-6 haloalkyl, OH, C1-6 alkoxy, -C(=O)-C1-6 alkyl, -C(=O)O-C1-6 alkylene- aryl, -C(=O)-C1-6 alkylene-C1-6 alkyl, -C(=O)-C1-6 alkyl ene-OC1-6 alkyl, -C(=O)-C1-6 alkylene- aryl, -C(=O)O-C1-6 alkylene-heteroaryl, -C(=O)-C1-6 alkylene-heteroaryl, -C1-6 alkylene- cycloalkyl, -C1-6 alkylene-aryl, -C1-6 alkylene-heterocycloalkyl, -C1-6 alkylene-heteroaryl, -C1-6 alkylene-C(=O)-NR _x ’Rx”, -C(=O)-C1-6 alkylene-cycloalkyl, -C(=O)-C1-6 alkylene- heterocycloalkyl, -C(=O)-C1-6 alkylene-NRx’Rx”, -C(=0)-CHCH20H-alkylene-NR _x Rx”, -SO2-C1.6 alkyl, -SO2-C1.6 alkylene-NRx’Rx”, -NR _Z C(=0)-C1-6 alkyl, -NR _Z C(=0)0-C1-6 alkylene-aryl, - NR _Z C(=O)-C1-6 alkylene-aryl, -NR _Z C(=0)0-C1-6 alkylene-heteroaryl, -NR _Z C(=0)-C1-6 alkylene- heteroaryl, -C1-6 alkylene-cycloalkyl, -C1-6 alkylene-heterocycloalkyl, -C1-6 alkylene-aryl, -C1-6 alkylene-heteroaryl, -NRX””C(=0)-C1-6 alkylene-cycloalkyl, -NR _X ” C(=O)-C1-6 alkylene- heterocycloalkyl, -NR _Z C(=0)-C1-6 alkylene-NRx’Rx”, -NR _z C(=0)-CHCH20H-alkylene-NRx’Rx”, - NRx ” ’ SO2-C1.6 alkyl, -NR _x ””SO2-C1-6 alkylene-NRx’Rx”, -C(=O)-heterocycloalkyl, -C(=O)-C1-6 alkylene-heterocycloalkyl, -C1-6 alkyl ene-NR _z C(=0)-C1-6 alkyl, -C(=O)-C1-6 alkylene- NR _x ””cycloalkyl, -C(=O)-C1-6 alkylene-NR _x ””-Ci.6 alkylene-cycloalkyl, -NR _X ””-C(=0)-C1-6 alkylene-heterocycloalkyl, -C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene-cycloalkyl, -C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene- C1-6 alkoxy, -C1-6 alkylene-NR _X ””-C(=O)-C1-6 alkylene-NRx’Rx”, - NRx””-C(=0)-C1-6alkylene -NR _X ” ”-C1-6 alkylene-NRx’Rx”, -C(=O)-cycloalkyl, -C(=O)-aryl, - C(=O)-heterocycloalkyl, C(=O)-heteroaryl, -C1-6 alkylene-arylene-aryl, -C(=O)-cycloalkyl, - C(=O)-heterocycloalkyl, -C(=O)-NR _z aryl, -C(=0)NR _Z -C1-6 alkylene-aryl, -SO2-C1.6 alkyl, -SO2- aryl, -SO2-heteroaryl, -SO2-C1.6 alkylene-NRx’Rx”, and -SO2- C1-6 alkylene-aryl; or or two R _q together with the atom to which they are attached form oxo, or an oxo protecting group, or a 3, 4, 5, or 6-membered cycloalkyl or heterocycle optionally substituted with one or more substituents selected from the group consisting of OH, halo, C1-6 alkyl, Ci/> haloalkyl, C1-6 alkylene-NH2, C1-6 alkyl ene-NH(C1-6 alkyl), C1-6 alkyl ene-N(C1-6 alkyl)2, C1-6 alkylene-NH- C(=O)-C1-6 alkyl, Ci- ₆ alkylene-N(Ci- ₆ alkyl)-C(=O)-Ci- ₆ alkyl, Ci- ₆ alkylene-NH-C(=O)-Ci- ₆ alkylene-NH2, C1-6 alkylene-NH-C(=O)-C1-6 alkylene-NH(C1-6 alkyl), -C(=O)-C1-6 alkyl, -C(=O)- C1-6 alkylene-N(C1-6 alkyl)2, and C1-6 alkylene-NH-C(=O)-C1-6 alkylene-N(C1-6 alkyl)z;

R _X ’ and R _X ” are each independently selected from the group consisting of H, C1-6 alkyl, cycloalkyl, C1-6 alkylene-cycloalkyl, C1-6 alkylene-heterocycloalkyl, C1-6 alkylene-aryl, C1-6 alkylene-heteroaryl, C1-6 alkylene-N-R _z R _X ’” C1-6 alkylene-OH, C1-6 alkylene-O-C1-6 alkyl, cycloalkyl, heterocycloalkyl, or R _x ’ and R _x ” are joined together with the atom to which they are attached to form a 3-, 4-, 5-, 6-, or 7-membered ring optionally containing an additional heteroatom selected from 0, S, SO, SO2, N-R _x ’, wherein said ring is further optionally substituted with halo, OH, C1-6 alkyl, -O-C1-6 alkyl; wherein R _X ”’ is H, C1-6 alkyl, -C(=0)-C1-6 alkyl, -C(=0)0-C1-6 alkylene-aryl, -C(=0)-C1-6 alkyl ene-aryl, -C(=0)0-C1-6 alkylene-heteroaryl, -C(=0)-C1-6 alkylene-heteroaryl, -C1-6 alkylene- aryl, -C1-6 alkylene-heteroaryl, -C(=0)-C1-6 alkylene-cycloalkyl, -C(=0)-C1-6 alkylene- heterocycloalkyl; each R _x ’ and R _z is independently H or C1-6 alkyl; or are joined together with the atom to which they are attached form a 3, 4, 5, or 6-membered cycloalkyl or heterocycle optionally substituted with one or more substituents selected from the group consisting of OH, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkylene-NH2, C1-6 alkylene-NH(C1-6 alkyl), C1-6 alkylene-N(C1-6 alkyl)2, C1-6 alkyl ene-NH-C(=0)-C i-6 alkyl, C1-6 alkylene-N(C1-6 alkyl)-C(=0)-C1-6 alkyl, C1-6 alkyl ene-NH-C(=0)-C i-6 alkylene-NH2, C1-6 alkylene-NH-C(=0)-C1-6 alkyl ene-NH(C1-6 alkyl), - C(=0)-C1-6 alkyl, -C(=0)-C1-6 alkylene-N(C1-6 alkyl)2, and C1-6 alkylene-NH-C(=0)-C1-6 alkyl ene-N(C i-6 alkyl^; and wherein unless otherwise specified, each occurrence of alkylene, alkyl, alkenyl, alkoxy, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl is independently and optionally substituted with halo, OH, C1-6 alkyl, alkoxy, or oxo.

[0007] Pharmaceutical compositions comprising the compounds are also described herein, as are of using the compounds of formula I to treat genetic diseases and also in the manufacture of a medicaments to treat such genetic diseases. Also provided are processes for making compounds of formula I.

Brief Description of the Figures

[0008] FIG.l provides a chart showing that compounds selective for cancerous cells were present in the library.

[0009] FIG.2 indicates that aryl and lipophilic compounds were found to be more potent. [0010] FIGS. 3A and 3B provide an SAR examination of some of the compounds in the series. [0011] FIGS. 4A and 4B indicate that Compound 9 (as provided in the figure) affected protein translation in the CRC cell lines that were tested. [0012] FIGS. 5A and 5B shows that Compound 9 selectively inhibited CRC organoids and was efficacious in vivo.

[0013] FIGS. 6.1A-D and S1A and SIB shows that a representative RMA compound of the invention as disclosed herein is selectively active in a sensitive CRC cell line.

[0014] FIGS. 6.2A-D and S2A-S2D show that the representative RMA compound of the invention inhibits protein translation in a sensitive CRC cell line.

[0015] FIGS. 6.3A-B and S3A and S3B show that the representative compound of the invention alters cell cycle in a sensitive CRC cell line.

[0016] FIGS. 6.4A-D show that CMS2 cell lines display high sensitivity to the representative compound of the invention.

[0017] FIGS. 6.5A-E show that the representative compound of the invention is synergistic with DNA intercalating agents in selected CRC cell lines.

[0018] FIG.7.1 summarizes the approach we employed in identifying RMAs.

[0019] FIGS. 7.2A and B summarize clinical subtypes in solid and hematological cancers that are highly sensitive to the second representative RMA of the invention.

[0020] FIG.7.3 indicates that forty five percent of small cell lung cancer cell lines are highly sensitive to the second representative RMA compound of the invention.

[0021] FIGS. 7.4A-C, indicate that the sensitivity small cell lung cancer cell lines is associated with high expression of ribogenesis, protein translation, and MYC pathway genes.

[0022] FIGS. 7.5A-C indicate that the anti-proliferative effect of RMAs is driven by selective inhibition of new protein synthesis

[0023] FIGS. 7.6A-B show that proteins with higher positively charged regions are more sensitive to translation inhibition by RMAs.

[0024] FIGS. 7.7 indicates that decreased proteins show a large impact on ribogenesis and protein translation machinery.

[0025] FIG.7.8 indicates that the second representative compound of the invention induces apoptosis in sensitive cells.

[0026] FIG.7.9 shows that the second representative compound of the invention selectively inhibits SCLC -patient derived organoids (PDOs).

[0027] FIG.7.10 shows that the second representative compound of the invention demonstrates combination synergy with DNA targeting chemotherapeutics known to inhibit tRNA synthesis. [0028] FIG.7.11 summarizes the multiple levels of selectivity of allosteric inhibition by the RMAs of the invention.

Detailed Description

Definitions

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, including U.S. Pat. Publ. No. 2013/0090326, In case of conflict, the present specification, including these definitions, will control.

[0030] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Thus, the terms “a,” “an,” and “the” as used herein not only include aspects with one member, but also include aspects with more than one member.

[0031] The term “about” as used herein means “approximately” and is used to modify a numerical value indicates a defined range around that value. If “X” were the value, “about X” would generally indicate a value from 0.95X to 1.05X. Any reference to “about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” When the quantity “X” only includes whole-integer values (e.g., “X carbons”), “about X” indicates from (X-l) to (X+l). In this case, “about X” as used herein specifically indicates at least the values X, X-l, and X+l.

[0032] When “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 5 to 20%” is equivalent to “from about 5% to about 20% ” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11% .”

[0033] The following abbreviations and terms have the indicated meanings throughout:

[0034] The symbol means a single bond, “=” means a double bond, “=” means a triple bond, “ means a single or double bond. The symbol “*/vw” refers to a group on a double-bond as occupying either position on the terminus of a double bond to which the symbol is attached; that is, the geometry, E- or Z-, of the double bond is ambiguous. When a group is depicted removed from its parent Formula, the “ ~ ” symbol will be used at the end of the bond which was theoretically cleaved in order to separate the group from its parent structural Formula.

[0035] When chemical structures are depicted or described, unless explicitly stated otherwise, all carbons are assumed to have hydrogen substitution to conform to a valence of four. For example, in the structure on the left-hand side of the schematic below there are nine hydrogens implied. The nine hydrogens are depicted in the right-hand structure. Sometimes a particular atom in a structure is described in textual Formula as having a hydrogen or hydrogens as substitution (expressly defined hydrogen), for example, -CH2CH2-. It is understood by one of ordinary skill in the art that the aforementioned descriptive techniques are common in the chemical arts to provide brevity and simplicity to description of otherwise complex structures.

[0036] If a group “R” is depicted as “floating” on a ring system, as for example in the following Formula. then, unless otherwise defined, a substituent “R” may reside on any atom of the ring system, assuming replacement of a depicted, implied, or expressly defined hydrogen from one of the Ring atoms, so long as a stable structure is formed.

[0037] If a group “R” is depicted as floating on a fused or bridged ring system, as for example in the following Formulas: then, unless otherwise defined, a substituent “R” may reside on any atom of the fused or bridged ring system, assuming replacement of a depicted hydrogen (for example the -NH- in the Formula above), implied hydrogen (for example as in the Formula above, where the hydrogens are not shown but understood to be present), or expressly defined hydrogen (for example where in the Formula above, “Z” equals =CH-) from one of the Ring atoms, so long as a stable structure is formed. In the example depicted, the “R” group may reside on either the 5-membered or the 6-membered ring of the fused or bridged ring system.

[0038] When a group “R” is depicted as existing on a ring system containing saturated carbons, as for example in the following Formula where, in this example, “y” can be more than one, assuming each replaces a currently depicted, implied, or expressly defined hydrogen on the ring; then, unless otherwise defined, where the resulting structure is stable, two “R’s” may reside on the same carbon. In one example, two R’s on the same carbon, including that carbon, may form a ring, thus creating a spirocyclic ring structure with the depicted Ring Ds for example in the following Formula

[0039] The term “acyl” as used herein includes an alkanoyl, aroyl, heterocycloyl, or heteroaroyl group as defined herein. Examples of acyl groups include, but are not limited to, acetyl, benzoyl, and ni cotinoyl. [0040] The term “alkanoyl” as used herein includes an alkyl-C(O)- group wherein the alkyl group is as defined herein. Examples of alkanoyl groups include, but are not limited to, acetyl and propanoyl.

[0041] The term “agent” as used herein includes a compound or mixture of compounds that, when added to a composition, tend to produce a particular effect on the composition’s properties. For example, a composition comprising a thickening agent is likely to be more viscous than an otherwise identical comparative composition that lacks the thickening agent.

[0042] The term “alkenyl” as used herein includes a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. The chain may contain an indicated number of carbon atoms. For example, “C1-C12 alkenyl” indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least one carbon-carbon double bond. When the indicated number of carbon atoms is 1, then the Ci alkenyl is double bonded to a carbon (i.e., a carbon equivalent to an oxo group). In certain aspects, the chain includes 1 to 12, about 2 to 15, about 2 to 12, about 2 to 8, or about 2 to 6 carbon atoms. An alkenyl group can be preferably one stereoisomer (i.e., cis- or, alternatively, trans-). Examples of an alkenyl group may include, but are not limited to, ethenyl (i.e., vinyl), allyl, propenyl, butenyl, crotyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, cyclopentenyl, cyclohexenyl, 2-isopentenyl, allenyl, butadienyl, pentadienyl, 3-(l,4-pentadienyl), and hexadienyl.

[0043] An amino group can be a primary amino group (NH2), or a secondary or tertiary amine, wherein the alkyl groups in the secondary ofr tertiary amine can be the same or different, and can be optionally substituted.

[0044] An alkenyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkenyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, with the proviso that no hydrogen atom substituent on the carbon-carbon double bond is replaced by a hydroxy, amino, or thio group. In some aspects, the alkenyl group is unsubstituted or not optionally substituted. [0045] “Alkenylene” as used herein includes an alkenyl group that is substituted at two points. An example is but-2-enylene (-CH2CH=CHCH2-) and the like.

[0046] The term “alkyl” as used herein includes an aliphatic hydrocarbon chain that may be straight chain or branched. The chain may contain an indicated number of carbon atoms: For example, C1-C10 indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. If not otherwise indicated, an alkyl group contains from 1 to about 20 carbon atoms. In some aspects, alkyl groups have 1 to about 10 carbon atoms. In some aspects, alkyl groups (“lower alkyl”) have 1 to 8, 1 to 6, or 1 to 3 carbon atoms in the chain. Examples may include, but are not limited to, methyl, ethyl, propyl, isopropyl (iPr), 1 -butyl, 2 -butyl, isobutyl (iBu), Ze/7-butyl, pentyl, 2-m ethylbutyl, 1,1 -dimethylpropyl, hexyl, heptyl, octyl, nonyl, decyl, docecyl, cyclopentyl, or cyclohexyl.

[0047] An alkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the alkyl group is unsubstituted or not optionally substituted.

[0048] “Alkylene” as used herein includes an alkyl group that is substituted at two points. An example is methylene (-CH2-), propylene (-CH2CH2CH2-), and the like.

[0049] The term “alkoxy” as used herein includes a straight or branched chain saturated or unsaturated hydrocarbon containing at least one oxygen atom in an ether group (e.g., EtO-). The chain may contain an indicated number of carbon atoms. For example, “C1-C12 alkoxy” indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least one oxygen atom. Examples of a C1-C12 alkoxy group include, but are not limited to, methoxy, ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy, and hexoxy.

[0050] An alkoxy group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkoxy group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, with the proviso that no hydrogen atom alpha to the ether oxygen is replaced by a hydroxy, amino, or thio group. In some aspects, the alkoxy group is unsubstituted or not optionally substituted.

[0051] The term “alkynyl” as used herein includes a straight, branched, or cyclic hydrocarbon containing at least one carbon-carbon triple bond. Examples may include, but are not limited to, ethynyl, propargyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, or decynyl.

[0052] “Alkynylene” as used herein includes an alkynyl group that is substituted at two points. An example is 2-butynylene (-CH2CCCH2-) and the like.

[0053] An alkynyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkynyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, with the proviso that no sp- hybridized hydrogen atom substituent is replaced by a hydroxy, amino, or thio group. In some aspects, the alkynyl group is unsubstituted or not optionally substituted.

[0054] The term “aryl” as used herein includes cyclic aromatic carbon ring systems containing from 6 to 18 carbons. Examples of an aryl group include, but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl, biphenyl and phenanthrenyl.

[0055] An aryl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the aryl group (e.g., from 1 to 5, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of alkyl, cyano, acyl, halo, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the alkoxy group is unsubstituted or not optionally substituted.

[0056] The term “arylalkyl” or “aralkyl” as used herein includes an alkyl group as defined herein where at least one hydrogen substituent has been replaced with an aryl group as defined herein. Examples include, but are not limited to, benzyl, 1 -phenyl ethyl, 4-methylbenzyl, and 1,1,- dimethyl- 1 -phenylmethyl .

[0057] An arylalkyl or aralkyl group can be unsubstituted or optionally substituted as per its component groups. For example, but without limitation, the aryl group of an arylalkyl group can be substituted, such as in 4-methylbenzyl. In some aspects, the group is unsubstituted or not optionally substituted, especially if including a defined substituent, such as a hydroxyalkyl or alkylaminoalkoxy group.

[0058] The term “cycloalkyl” as used herein includes non-aromatic saturated monocyclic or multi cyclic ring system that may contain an indicated number of carbon atoms. For example, C3- C12 indicates that the group may have from 3 to 12 (inclusive) carbon atoms in it. If not otherwise indicated, a cycloalkyl group includes about 3 to about 20 carbon atoms. In some aspects, cyclo alkyl groups have 3 to about 12 carbon atoms in the group. In some aspects, cycloalkyl groups have 3 to about 7 carbon atoms in the group. Examples may include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4,4-dimethylcyclohexyl, and cycloheptyl. [0059] A cycloalkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the cycloalkyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, a substituted cycloalkyl group can incorporate an exo- or endocyclic alkene (e.g., cyclohex-2-en-l- yl). In some aspects, a cycloalkyl group is unsubstituted or not optionally substituted.

[0060] As used herein, “fluoroalkyl” includes an alkyl group wherein the alkyl group includes one or more fluoro- substituents. Examples include, but are not limited to, trifluoromethyl.

[0061] As used herein, “geminal” substitution includes two or more substituents that are directly attached to the same atom. An example is 3,3-dimethyl substitution on a cyclohexyl or spirocyclohexyl ring.

[0062] As used herein, “halo” or “halogen” includes fluoro, chloro, bromo, and iodo.

[0063] The term “heteroaryl” or “heterocycloaryl” includes mono and bicyclic aromatic groups of about 4 to about 14 Ring atoms (e.g., 4 to 10 or 5 to 10 atoms) containing at least one heteroatom. Heteroatom as used in the term heteroaryl refers to oxygen, sulfur and nitrogen. A nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N-oxide. Examples include, but are not limited to, pyrazinyl, furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[l,2-a]pyridine, imidazo[2,l-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thi enopyridyl, quinazolinyl, thienopyrimidyl, pyrrol opyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, and benzothiazolyl.

[0064] The term “heteroarylene” or “heterocycloarylene” as used herein includes a heteroaryl group that is substituted at two points.

[0065] An heteroaryl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the heteroaryl group (e.g., from 1 to 5, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of alkyl, cyano, acyl, halo, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the heteroaryl group is unsubstituted or not optionally substituted.

[0066] The term “heteroaroyl” as used herein includes a heteroaryl -C(O)- group wherein heteroaryl is as defined herein. Heteroaroyl groups include, but are not limited to, thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, and pyridinoyl.

[0067] The term “heterocycloalkyl” may be used interchangeably herein, and as used herein includes a heterocyclyl-C(O)- group wherein heterocyclyl is as defined herein. Examples include, but are not limited to, N-m ethyl prolinoyl and tetrahydrofuranoyl.

[0068] As used herein, “heterocyclyl” (heterocyclo; heterocyclic; heterocycloalkyl) includes a non-aromatic saturated ring of about 3 to about 8 Ring Dtoms (e.g., 5 to about 10 Ring Dtoms, or 3 to about 6 Ring atoms), in which one or more of the atoms in the ring system is an element or elements other than carbon, e.g., nitrogen, oxygen or sulfur. A heterocyclyl group optionally comprises at least one sp ² -hybridized atom (e.g., a ring incorporating an carbonyl, endocyclic olefin, or exocyclic olefin). In some embodiments, a nitrogen or sulfur atom of the heterocyclyl is optionally oxidized to the corresponding N-oxide, S-oxide or S, S-di oxide. The monocyclic heterocycle means a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six- membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of 0, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of 0, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3- dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyridazin-3(2H)-onyl, pyridin-2(lH)-onyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.

[0069] The term “heterocycloalkylene” as used herein includes a heterocyclyl (heterocyclo; heterocyclic) group that is substituted at two points.

[0070] The term “heterocyclyl” also includes multicyclic rings such as a bicyclic heterocycle, or a tricyclic heterocycle which may be in a fused, bridged, or spiro orientation. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the Ring Dre linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, 3- azabicyclo[3.1.0]hexane, 3-azabicyclo[4.1.0]heptane, 3-azabicyclo[3.2.0]heptane, (3aR,6aS)- hexahydro-lH-2Z,2-cyclopenta[c]pyrrole, (3aR,7aS)-octahydro-2X2-isoindole.

[0071] Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic Ring Dre linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms.

[0072] A heterocycyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, a substituted heterocycyl group can incorporate an exo- or endocyclic alkene (e.g., cyclohex-2-en-l-yl). In some aspects, the heterocycyl group is unsubstituted or not optionally substituted.

[0073] The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings, and can be unsubstituted or substituted.

[0074] As used herein, the term “hydrophilic moiety” or “hydrophilic group” includes a moiety or a functional group that has a strong affinity to water. Examples may include, but are not limited to, a charged moiety, such as a cationic moiety or an anionic moiety, or a polar uncharged moiety, such as an alkoxy group or an amine group.

[0075] As used herein, the term “hydroxyalkyl” includes an alkyl group where at least one hydrogen substituent has been replaced with an alcohol (-OH) group. In certain aspects, the hydroxyalkyl group has one alcohol group. In certain aspects, the hydroxyalkyl group has one or two alcohol groups, each on a different carbon atom. In certain aspects, the hydroxyalkyl group has 1, 2, 3, 4, 5, or 6 alcohol groups. Examples may include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 1 -hydroxy ethyl.

[0076] When any two substituent groups or any two instances of the same substituent group are “independently selected” from a list of alternatives, the groups may be the same or different. For example, if R ^a and R ^b are independently selected from the group consisting of alkyl, fluoro, amino, and hydroxyalkyl, then a molecule with two R ^a groups and two R ^b groups could have all groups be an alkyl group (e.g., four different alkyl groups). Alternatively, the first R ^a could be alkyl, the second R ^a could be fluoro, the first R ^b could be hydroxyalkyl, and the second R ^b could be amino (or any other substituents taken from the group). Alternatively, both R ^a and the first R ^b could be fluoro, while the second R ^b could be alkyl (i.e., some pairs of substituent groups may be the same, while other pairs may be different).

[0077] “Oxo” means -C(=O)-

[0078] “Protecting Group” means “amino protecting group,” which is a protecting group that is suitable for preventing undesired reactions at an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, for example alkanoyl groups, such as acetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl (Bn), trityl (Tr), and l,l-di-(4'-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS); and the like. Representative oxygen protecting groups include, but are not limited to silyl groups, such as TMS and TMDMS; esters, such as formate, acetate, and benzoate (Bz); ethers such as methoxymethyl (MOM), tetrahydropyranyl (THP), and benzyl (Bn).

[0079] “ Oxo Protecting Group” means a protecting group for an aldehyde or ketone. Non- limiting examples of “oxo Protecting Groups” include acetals, 1-3 -di oxalanes, mixed ketals and thioketals.

[0080] “Yield” for each of the reactions described herein is expressed as a percentage of the theoretical yield.

[0081] “Subject and “patient” are used interchangeably. A “subject” or “patient” for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a specific embodiment the patient is a mammal, and in a more specific embodiment the patient is human.

[0082] A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington ’s Pharmaceutical Sciences, 17 ^th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference or S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977;66: 1-19 both of which are incorporated herein by reference.

[0083] Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4, 4’-methylenebis-(3-hydroxy-2-ene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p- toluenesulfonic acid, and salicylic acid and the like.

[0084] Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, A-methylglucamine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. “Platin(s),” and “platin-containing agent(s)” include, for example, cisplatin, carboplatin, and oxaliplatin.

[0085] A “therapeutic effect,” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0086] “Therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease. The amount of a compound of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.

[0087] The term "co-administration," "administered in combination with," and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal, including humans, so that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0088] The phrase “genetic disease”, as used herein, means a genetic disorder, genetic disease, genetic condition or genetic syndrome.

[0089] “Preventing” or “prevention” of a disease, disorder, or syndrome includes inhibiting the disease from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome.

[0090] “Treating” or “treatment” of a disease, disorder, or syndrome, as used herein, includes (i) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (ii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.

[0091] As used herein, “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0092] As used herein, “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF).

[0093] The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be "benign" or "malignant," depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A "benign neoplasm" is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain "benign" tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as "pre-malignant neoplasms." An exemplary pre-malignant neoplasm is a teratoma. In contrast, a "malignant neoplasm" is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term "metastasis," "metastatic," or "metastasize" refers to the spread or migration of cancerous cells from a primary or original tumor to one organ or tissue and is typically identifiable by the presence of a "secondary tumor" or "secondary cell mass" of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[0094] As used herein, the term “cancer” refers to a malignant neoplasm (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T- lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

[0095] Rare diseases caused by nonsense mutations are mutations where a stop codon (UAA, UAG or UGA) replaces an amino acid-coding codon, leading to premature termination of translation and eventually to truncated inactive proteins. The Human Gene Mutation Database reports the occurrence of thousands of disease-causing mutations, approximately 12% of which are single point (nonsense) mutations that result in a premature termination codon. (Krawczak M, et al., Hum Mutat. 2000, 15, 45-51.; Mort, et al., M. Hum. Mutat. 2008, 29, 1037-47). Nonsense mutations that result in truncated proteins have been demonstrated to account for many forms of genetic disease including cancer, hemophilia, Tay-Sachs, lysosomal storage disorders or mucopolysaccharidoses such as Hurler Syndrome, Duchenne muscular dystrophy, ataxia telangiectasia, Rett syndrome, various inherited retinopathies, and cystic fibrosis.

[0096] CFTR modulator types as used herein are drugs or compounds that target the underlying defect in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Three types of modulators are potentiators, correctors, and amplifiers, [accessed on May 23, 2019, Cystic fibrosis F oundati on https ://www. cff org/Research/Developing-New-Treatments/CFTR-Modulator- Types/]. Potentiators, such as the drug ivacaftor (Kalydeco®), work on residual function and splice mutations where an insufficient amount of normal CFTR protein is present by holding the gate open so chloride can flow through. Correctors help the CFTR protein with two copies of the F508del mutation to form the right 3-D shape so that it is able to move - or traffic - to the cell surface. Amplifiers increase the amount of CFTR protein that the cell makes. Many CFTR mutations produce insufficient CFTR protein. If the cell made more CFTR protein, potentiators and correctors would be able to allow even more chloride to flow across the cell membrane.

Selected Embodiments

[0097] In one aspect, what is provided is a compound of formula I:

I or a pharmaceutically acceptable salt thereof, wherein:

R.2a is selected from the group consisting of H, halo, optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl, wherein C1-10 alkyl, C1-10 alkoxy, and C1-10 alkenyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl; and

R.2b is selected from the group consisting of halo, optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl, wherein C1-10 alkyl, C1-10 alkoxy, and C1-10 alkenyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

R.4a and R4b are each independently selected from the group consisting of -H, and optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Rs is selected from the group consisting of H, an oxygen protecting group, and , ” indicates the point of attachment;

Rea is optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Reb is -H, optionally substituted C1-10 alkyl, optionally substituted C1-10 hydroxyalkyl, and optionally substituted allyl, wherein C1-10 alkyl, C1-10 hydroxyalkyl, and allyl are optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

Rsa and Rsb are each independently selected from the group consisting of -H and optionally substituted C1-10 alkyl, wherein C1-10 alkyl, is optionally substituted with one or more groups selected from the group consisting of halo, aryl, amino, alkyl, heteroalkyl, heteroalkenyl, heterocycloalkyl, and heteroaryl;

R9a is selected from the group consisting of -H, -C(=N)-NH2, -C(=O)-C1-6 alkyl , -C(=O)- C1-6 alkylene-NR9a’R9a”, C1-10 alkyl and C1-10 alkenyl, wherein C1-10 alkyl and C1-10 alkenyl are optionally substituted with 1, 2, or 3 groups independently selected from the group consisting of amino, heterocycloalkyl, halo, hydroxy, alkoxy, aryl, heteroaryl, and heteroaryl ene-heteroaryl, wherein aryl, heteroaryl, and heteroaryl ene-heteroaryl, are optionally substituted with halo, alkoxy, and -NHz; wherein R.9a' and R9a” are selected from the group consisting of H, C1-6 alkyl, cycloalkyl, Ci-4 alkylene-cycloalkyl, or R9a’ and R9a” together with the atom to which there are attached from a 3-, 4-, 5-, or 6-membered ring optionally containing an additional heteroatom selected from 0, S, SO, SO2, NH, and N-C1.4 alkyl;

Rioa and Riob are taken together with the carbon atom to which they are attached to form , wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein each • ⁿⁿⁿ ' ” indicates a point of attachment and Rn _a and Rub are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl; or

Riia and Rm> are taken together with the carbon atom to which they are attached to form , wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein each “ ” indicates a point of attachment and Rw _a and Rwb are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl;

Rq at each occurrence is independently selected from the group consisting of H, halo, - NRx’Rx”, C1-6 alkyl, C1-6 haloalkyl, OH, C1-6 alkoxy, -C(=O)-C1-6 alkyl, -C(=O)O-C1-6 alkylene- aryl, -C(=O)-C1-6 alkylene-C1-6 alkyl, -C(=O)-C1-6 alkyl ene-OC1-6 alkyl, -C(=O)-C1-6 alkylene- aryl, -C(=O)O-C1-6 alkylene-heteroaryl, -C(=O)-C1-6 alkylene-heteroaryl, -C1-6 alkylene- cycloalkyl, -C1-6 alkylene-aryl, -C1-6 alkylene-heterocycloalkyl, -C1-6 alkylene-heteroaryl, -C1-6 alkylene-C(=O)-NR _x ’Rx”, -C(=O)-C1-6 alkylene-cycloalkyl, -C(=O)-C1-6 alkylene- heterocycloalkyl, -C(=O)-C1-6 alkylene-NRx’Rx”, -C(=0)-CHCH20H-alkylene-NR _X ’Rx”, -SO2-C1.6 alkyl, -SO2-C1.6 alkylene-NRx’Rx”, -NR _Z C(=0)-C1-6 alkyl, -NR _Z C(=0)0-C1-6 alkylene-aryl, - NRZC(=0)-C1-6 alkylene-aryl, -NR _Z C(=0)0-C1-6 alkylene-heteroaryl, -NR _Z C(=0)-C1-6 alkylene- heteroaryl, -C1-6 alkylene-cycloalkyl, -C1-6 alkylene-heterocycloalkyl, -C1-6 alkylene-aryl, -C1-6 alkylene-heteroaryl, -NR _X ” ”C(=O)-C1-6 alkylene-cycloalkyl, -NR _X ” ”C(=O)-C1-6 alkylene- heterocycloalkyl, -NR _Z C(=0)-C1-6 alkylene-NRx’Rx”, -NR _z C(=0)-CHCH20H-alkylene-NRx’Rx”, - NRx ” ’ SO2-C1.6 alkyl, -NR _X ””SO2-C1-6 alkylene-NRx’Rx”, -C(=O)-heterocycloalkyl, -C(=O)-C1-6 alkylene-heterocycloalkyl, -C1-6 alkylene-NR _z C(=O)-C1-6 alkyl, -C(=O)-C1-6 alkylene- NRx””Cycloalkyl, -C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene-cycloalkyl, -NR _X ””-C(=0)-C1-6 alkylene-heterocycloalkyl, -C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene-cycloalkyl, -C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene- C1-6 alkoxy, -C1-6 alkylene-NRx””-C(=O)-C1-6 alkylene-NRx’Rx”, - NRx””-C(=0)-C1-6alkylene -NR _X ’ ”-C1-6 alkylene-NRx’Rx”, -C(=O)-cycloalkyl, -C(=O)-aryl, - C(=O)-heterocycloalkyl, C(=O)-heteroaryl, -C1-6 alkylene-arylene-aryl, -C(=O)-cycloalkyl, - C(=O)-heterocycloalkyl, -C(=O)-NR _z aryl, -C(=0)NR _Z -C1-6 alkylene-aryl, -SO2-C1.6 alkyl, -SO2- aryl, -SO2-heteroaryl, -SO2-C1.6 alkylene-NRx’Rx”, and -SO2- C1-6 alkylene-aryl; or or two R _q together with the atom to which they are attached form oxo, or an oxo protecting group, or a 3, 4, 5, or 6-membered cycloalkyl or heterocycle optionally substituted with one or more substituents selected from the group consisting of OH, halo, C1-6 alkyl, Ci/> haloalkyl, C1-6 alkylene-NEb, C1-6 alkyl ene-NH(C1-6 alkyl), C1-6 alkyl ene-N(C1-6 alkyl)2, C1-6 alkylene-NH- C(=0)-C1-6 alkyl, Ci- ₆ alkylene-N(Ci- ₆ alkyl)-C(=O)-Ci- ₆ alkyl, Ci- ₆ alkylene-NH-C(=O)-Ci- ₆ alkylene-NHz, C1-6 alkylene-NH-C(=0)-C1-6 alkylene-NH(C1-6 alkyl), -C(=0)-C1-6 alkyl, -C(=0)- C1-6 alkylene-N(C1-6 alkyl)2, and C1-6 alkylene-NH-C(=0)-C1-6 alkylene-N(C1-6 alkyl)z;

R _X ’ and R _x are each independently selected from the group consisting of H, C1-6 alkyl, cycloalkyl, C1-6 alkylene-cycloalkyl, C1-6 alkylene-heterocycloalkyl, C1-6 alkylene-aryl, C1-6 alkyl ene-heteroaryl, C1-6 alkylene-N-R _z R _x ’” C1-6 alkylene-OH, C1-6 alkylene-O-C1-6 alkyl, cycloalkyl, heterocycloalkyl, or R _x ’ and R _x ” are joined together with the atom to which they are attached to form a 3-, 4-, 5-, 6-, or 7-membered ring optionally containing an additional heteroatom selected from 0, S, SO, SO2, N-R _x ”, wherein said ring is further optionally substituted with halo, OH, C1-6 alkyl, -O-C1-6 alkyl; wherein Rx’” is H, C1-6 alkyl, -C(=0)-C1-6 alkyl, -C(=0)0-C1-6 alkylene-aryl, -C(=0)-C1-6 alkylene-aryl, -C(=0)0-C1-6 alkylene-heteroaryl, -C(=0)-C1-6 alkylene-heteroaryl, -C1-6 alkylene- aryl, -C1-6 alkylene-heteroaryl, -C(=0)-C1-6 alkylene-cycloalkyl, -C(=0)-C1-6 alkylene- heterocycloalkyl; each Rx”” and R _z is independently H or C1-6 alkyl; or are joined together with the atom to which they are attached form a 3, 4, 5, or 6-membered cycloalkyl or heterocycle optionally substituted with one or more substituents selected from the group consisting of OH, halo, Ci-e alkyl, C1-6 haloalkyl, C1-6 alkylene-NH2, C1-6 alkylene-NH(C1-6 alkyl), C1-6 alkylene-N(C1-6 alkyl)2, C1-6 alkyl ene-NH-C(=0)-C i-6 alkyl, C1-6 alkylene-N(C1-6 alkyl)-C(=0)-C1-6 alkyl, C1-6 alkyl ene-NH-C(=0)-C i-6 alkylene-Mh, C1-6 alkylene-NH-C(=0)-C1-6 alkyl ene-NH(C 1.6 alkyl), - C(=0)-C1-6 alkyl, -C(=0)-C1-6 alkylene-N(C1-6 alkyl)2, and C1-6 alkylene-NH-C(=0)-C1-6 alkyl ene-N(C i-6 alkyl)2; and wherein unless otherwise specified, each occurrence of alkylene, alkyl, alkenyl, alkoxy, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl is independently and optionally substituted with halo, OH, C1-6 alkyl, alkoxy, or oxo.

[0098] In one embodiment, the compound of formula I is a compound of formula IA:

IA.

[0099] In one embodiment, the compound of formula I is a compound of formula IB:

[0100] In one embodiment, the compound of formula I is a compound of formula IC:

[0101] In one embodiment, the compound of formula I is a compound of formula ID:

[0102] In one embodiment, Reb is selected from the group consisting of -H, optionally substituted Ci-C io alkyl, optionally substituted Ci-Cio hydroxyalkyl, and optionally substituted allyl.

[0103] In one embodiment, Reb is selected from the group consisting of methyl, hydroxymethyl hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, -CH2CHOHCH2OH, and allyl.

[0104] In one embodiment, the compound of formula I is a compound of formula IE:

[0105] In one embodiment, the compound of formula I is a compound of formula IF:

[0106] In one embodiment, the compound of formula I is a compound of formula IG:

IG.

[0107] In one embodiment, one of Rza and IGb is optionally substituted C1-10 alkyl.

[0108] In one embodiment, one of R.2a and R.2b is methyl and the other of R.2a and R.2b is H, or both of R _2a and R2b are methyl.

[0109] In one embodiment, one of R2a and R2b is methyl and the other is halo.

[0110] In one embodiment, one of R2a and R2b is methyl and the other is optionally substituted Ci- io alkyl.

[0111] In one embodiment, one of R2a and R2b is methyl and the other of R2a and R2b is selected from the group consisting of optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl, wherein optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, and optionally substituted C1-10 alkenyl are optionally substituted with one or more selected from the group consisting of halo, aryl, and heteroaryl.

[0112] In one embodiment of the compound of formula I, Ry _a is selected from the group consisting of -H and optionally substituted C1-10 alkyl; and

Rioa and Riob are taken together to form wherein Q is a 3-, 4-, 5-, or 6- membered cycloalkyl or heterocyclic ring, wherein each “ ” indicates a point of attachment and Rua and Rub are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl; or Riia and Rm> are taken together to form wherein Q is a 3-, 4-, 5-, or 6- membered cycloalkyl or heterocyclic ring, wherein each ” indicates a point of attachment and Rioa and Rwb are each independently selected from the group consisting of H, halo, and optionally substituted C1-10 alkyl

[0113] In one embodiment, R _q at each occurrence is independently selected from the group consisting of H, halo, -NR _x Rx”, C1-6 alkyl, C1-6 haloalkyl, OH, C1-6 alkoxy, -C(=O)-C1-6 alkyl, - C(=O)O-C1-6 alkylene-phenyl, -C1-6 alkyl ene-phenyl, -C1-6 alkylene-(5-10 membered heteroaryl), -C1-6 alkylene-C(=O)-NR _X ’Rx’’, -C(=O)-C1-6 alkylene-(3-6 membered heterocycloalkyl), -C(=O)- C1-6 alkylene-NRx’Rx”, -SO2-C1.6 alkyl, -SO2-C1.6 alkylene-NRx’Rx”, -NR _X ’ ’ C(=O)-C1-6 alkylene- (3-6 membered heterocycloalkyl), -NR _Z C(=O)-CI.6 alkylene-NRx’Rx”, -C(=O)-(3-6 membered heterocycloalkyl), -C1-6 alkyl ene-NR _z C(=O)-C1-6 alkyl, -C1-6 alkylene-NR _X ””-C(=O)-C1-6 alkylene-NRx’Rx”, -NR _X ” ”-C(=O)-C1-6 alkylene-NRx””-C1-6 alkylene-NRx’Rx”, -C(=O)-phenyl, - C(=O)-(5-10 membered heteroaryl), -C1-6 alkylene-biphenyl, -C(=O)-(3-6 membered cycloalkyl), -C(=O)-NR _z -phenyl, -C(=O)NR _Z -C1-6 alkylene-phenyl, -SO2-phenyl, -SO2-(5-10 membered heteroaryl), and -SO2-C1.6 alkylene-phenyl, wherein phenyl, biphenyl, 3-6 membered cycloalkyl; 3-6 membered heterocycloalkyl, and 5-10 membered heteroaryl are optionally substituted with wherein R _x ’ and R _x ” are each independently selected from the group consisting of H, C1-6 alkyl, C1-6 alkylene-(3-6 membered cycloalkyl), C1-6 alkylene-(3-6 membered heterocycloalkyl), C1-6 alkylene-O-C1-6 alkyl, 3-6 membered cycloalkyl, and 3-6 membered heterocycloalkyl; each Rx ”” and R _z is independently H or C1-6 alkyl; or two R _q together with the atom to which they are attached form oxo, or an oxo protecting group, or a 5 or 6-membered heterocycle optionally substituted with one or more substituents selected from the group consisting of OH, halo, C1-6 alkyl, -C(=O)-C1-6 alkyl, and -C(=O)-C1-6 alkyl ene-N(C i-6 alkyty; wherein each occurrence of alkylene, alkyl, phenyl, heteroaryl, cycloalkyl, and heterocycloalkyl is independently and optionally substituted with halo, OH, C1-6 alkoxy, or C1-6 alkyl.

[0114] In one embodiment, R _q at each occurrence is independently selected from the group consisting of H, -NR _x Rx”, C1-6 alkyl, C1-6 haloalkyl, -C(=O)-C1-6 alkyl, -C(=O)O-C1-6 alkylene- phenyl, -C1-6 alkylene-phenyl, -C1-6 alkylene-(5-10 membered heteroaryl), -C1-6 alkylene-C(=O)- NRx’Rx”, -C(=0)-C1-6 alkylene-(3-6 membered heterocycloalkyl), -C(=O)-C1-6 alkylene-NRx’Rx”, - SO2-C1.6 alkyl, -SO2-C1.6 alkylene-NRx’Rx”, -NRX””C(=0)-C1-6 alkylene-(3-6 membered heterocycloalkyl), -NR _Z C(=0)-C1-6 alkyl ene-NR _x ’R _x ”, -C(=O)-(3-6 membered heterocycloalkyl), - C1-6 alkylene-NR _z C(=O)-C1-6 alkyl, -NR _x -C(=O)-Ci.r, alkylene-NR _X ””-C1-6 alkylene-NRx’Rx”, - C(=O)-phenyl, -C(=O)-(5-10 membered heteroaryl), -C1-6 alkyl ene-biphenyl, -C(=O)-(3-6 membered cycloalkyl), -C(=O)-NR _z -phenyl, -C(=0)NR _Z -C1-6 alkyl ene-phenyl, -SCb-phenyl, - SC>2-(5- 10 membered heteroaryl), and -SO2-C1.6 alkyl ene-phenyl; wherein R _x ’ and R _x ” are each independently selected from the group consisting of H, C1-6 alkyl, C1-6 alkylene-(3-6 membered cycloalkyl), C1-6 alkyl ene-O-C1-6 alkyl, and 3-6 membered cycloalkyl; each Rx”” and R _z is independently H or C1-6 alkyl; or two R _q together with the atom to which they are attached form oxo, or a 5 or 6- membered heterocycle optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, -C(=O)-C1-6 alkyl, and -C(=O)-C1-6 alkylene-N(C1-6 alkyl)2; wherein each occurrence of alkylene, alkyl, phenyl, heteroaryl, cycloalkyl, and heterocycloalkyl is independently and optionally substituted with OH, C1-6 alkoxy, or C1-6 alkyl.

[0115] In one embodiment, R _q is H.

[0116] In one embodiment, R _q is -C1-6 alkylene-aryl. In one embodiment, R _q is — C1-3 alkylene- aryl. In one embodiment, R _q is -C1.2 alkylene-aryl. In one embodiment, Rq is -CTL-aryl. In one embodiment, R _q is -C1-6 alkyl ene-heteroaryl. In one embodiment, R _q is -CHz-heteroaryl. In one embodiment, R _q is -CH2-phenyl. In one embodiment, Rq is -CH2-phenyl wherein phenyl is optionally substituted. In one embodiment, optionally substituted phenyl is alkoxyphenyl or halophenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4-chlorophenyl. In one embodiment, optionally substituted phenyl is biphenyl. In one embodiment, R _q is CH2 -heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, R _q is -CH2-pyridyl, wherein pyridyl can be 2-, 3-, or 4-pyridyl. In one embodiment, Rq is -CH2-imidazolyl. In one embodiment, Rq is -CTb-quinolinyl, or -CH2-isoquinolinyl.

[0117] In one embodiment, R _q is -CTLCTb-aryl. In one embodiment, R _q is -CHzCHi-phenyl wherein phenyl is optionally substituted. In one embodiment, optionally substituted phenyl is alkoxyphenyl or halophenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4- methoxy phenyl or 2-, 3-, or 4-chlorophenyl. In one embodiment, R _q is -CH2CH2-heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, Rq is -CHzCHz-pyridyl. In one embodiment, R _q is -CELCEL-imidazolyl. In one embodiment, Rq is -CHiCHi-quinolinyl, or - CHzCHz-soquinolinyl.

[0118] In one embodiment, R _q is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl. [0119] In one embodiment, R _q is -C(=O)-CHi-6-alkylene-aryl. In one embodiment, R _q is - C(=O)-CHi-3-alkylene-aryl. In one embodiment, R _q is -C(=O)-CHi-2-alkylene-aryl. In one embodiment, R _q is -C(=O)-CH2-aryl, wherein aryl is substituted or unsubstituted. In one embodiment, R _q is -C(=O)-CH2-phenyl. In one embodiment, Rq is -C(=O)-CH2-heteroaryl wherein aryl is substituted or unsubstituted. In one embodiment, Rq is -C(=O)-CH2-imidazolyl. [0120] In one embodiment, R _q is -C(=O)-C1-6 alkyl. In one embodiment, R _q is -C(=O)-Ci-3 alkyl. In one embodiment, R _q is -C(=O)-Ci-2 alkyl. In one embodiment, R _q is -C(=O)-CH3.

[0121] In one embodiment, Rq is -C(=O)-aryl, wherein aryl is optionally substituted. In one embodiment, R _q is -C(=O)-phenyl, wherein phenyl is alkoxypheny or chlorophenyl. In one embodiment, optionally substituted phenyl is chlorophenyl or methoxyphenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4- chlorophenyl.

[0122] In one embodiment, Rq is -C(=O)-cycloalkyl, wherein cycloalkyl is substituted or unsubstituted.

[0123] In one embodiment, R _q is -C(=O)-heterocycloalkyl, wherein heterocycloalkyl is substituted or unsubstituted. In one embodiment, Rq is -C(=O)-pyrrolidinyl. In one embodiment, Rq is -C(=O)-piperidinyl.

[0124] In one embodiment, Rq is -C(=O)-N(H)C1-6 alkyl. In one embodiment, Rq is -C(=O)- N(H)CI-3 alkyl. In one embodiment, R _q is -C(=O)-N(H)CI-2 alkyl. In one embodiment, R _q is - C(=O)-N(H)methyl .

[0125] In one embodiment, R _q is -C(=O)-N(C1-6 alkyl)(C1-6 alkyl), wherein C1-6 alkyl can be the same or different at each occurrence. In one embodiment, Rq is -C(=O)-N(CI-3 alkyl)(C 1.3 alkyl), wherein C1-6 alkyl can be the same or different at each occurrence. In one embodiment, Rq is - C(=O)-N(CI-2 alkyl)(Ci-2 alkyl), wherein C1-6 alkyl can be the same or different at each occurrence.

[0126] In one embodiment, R _q is -C(=O)-N(H)-aryl or -C(=O)-N(Me)-aryl, wherein aryl is optionally substituted. In one embodiment, R _q is -C(=O)-phenyl, wherein phenyl is optionally substituted. In one embodiment, optionally substituted phenyl is chlorophenyl or methoxyphenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4- chlorophenyl.

[0127] In one embodiment, R _q is -C(=O)-heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, R _q is -C(=O)-pyridyl, wherein pyridyl is optionally substituted. In one embodiment, heteroaryl is quinolinyl or isoquinolinyl, either of which is optionally substituted. . [0128] In one embodiment, R _q is -C(=O)-C1-6 alkylene-NRx’Rx”. In one embodiment, R _q is - C(=O)-Ci-3 alkylene-NRxRx”. In one embodiment, R _q is -C(=O)-Ci-2 alkylene-NR _x Rx”. In one embodiment, R _q is -C(=O)-CH2-NR _X ’R _X ”. In one embodiment, R _q is -C(=O)-CH(Me)-NR _x R _x -. In one embodiment, R _q is -C(=O)-C(Me)2-NR _x ’R _x ”. In one embodiment, R _q is -C(=O)CH2-NR _X R _X ”. In one embodiment, R _q is -C(=O)-C(Me)(CH ₂ OH)NRx R _x ” In one embodiment, R _q is - C(=O)CH ₂ CH ₂ -NRX’RX”. In one embodiment, NRx’Rx” is NH ₂ , N(H)Me, NMe ₂ , N(Me)Et, N(Me)isopropyl, NEt ₂ , azetidinyl, azetidinyl-OH, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, N(H)-isopropyl, N(Me)isopropyl, N(H)cyclopropyl, N(Me)cyclopropyl, N(H)cyclobutyl, N(Me)cyclobutyl, N(H)CH ₂ cyclopropyl, N(Me)CH ₂ cyclopropyl, N(Me)isopropyl, N(Me)-tbutyl, N(H)CH ₂ CH ₂ -alkoxy,

[0129] In one embodiment, 2 R _q taken together with the atom to which they are attached form z ^N \X

, or ^Me (as a salt)(e.g., the formate salt), wherein “ ” indicates a point of attachment.

[0130] In one embodiment, R _q is -SO ₂ -C1-6 alkyl. In one embodiment, R _q is -SO2-C1-3 alkyl. In one embodiment, R _q is -SO ₂ -Ci- ₂ alkyl. In one embodiment, R _q is -SO ₂ -Me.

[0131] In one embodiment, R _q is -SO2-C1.6 alkylene-NR _x ’R _x ”. In one embodiment, R _q is -SO2-C1.

3 alkylene-NRxRx”. In one embodiment, R<i is -SO ₂ -Ci- ₂ alkylene-NRx’Rx”. In one embodiment, Rq is -SO ₂ -CH ₂ NRX RX”. In one embodiment, R _q is -SO ₂ -CH ₂ CH ₂ NRxR _x ”. In one embodiment, NRx’Rx” is NH ₂ , N(H)Me, NMe ₂ , N(Me)Et, NEt ₂ , N(H)isopropyl, N(Me)isopropyl, N(H)cyclopropyl, N(Me)cyclopropyl, N(H)cyclobutyl, N(Me)cyclobutyl, N(H)CH ₂ cyclopropyl, N(Me)isopropyl, orN(Me)t-butyl.

[0132] In one embodiment, R _q is -SO ₂ -aryl. In one embodiment, Rq is -SO ₂ -phenyl, wherein phenyl is optionally substituted. In one embodiment, R _q is -SCh-chlorophenyl or -SO2- methoxyphenyl. In one embodiment, Rq is -SO2-(5-10 membered heteroaryl). In one embodiment, R _q is -SO ₂ -pyridyl. In one embodiment, Rq is -SO2-C1.6 alkylene-phenyl. In one embodiment, R _q is -SO ₂ -CH ₂ -phenyl.

[0133] In one embodiment, Rq is NH-C(=O)C1-6 alkylene-NR _X ’R _x or N(Me)-C(=O)C1-6 alkylene- NR _X ’R _X In one embodiment, R _q is NH-C(=O)CI-3 alkylene-NR _x ’R _x or N(Me)-C(=O)Ci-3 alkyl ene-NR _X ’R _x In one embodiment, R _q is NH-C(=O)CI-2 alkyl ene-NR _X ’R _x or N(Me)-C(=O)Ci-2 alkylene-NR _x ’Rx In one embodiment, R _q is N(Me)-C(=O)C1-6 alkylene-NRx’Rx. In one embodiment, R _q is NH-C(=O)CH ₂ -NR _X ’Rx. In one embodiment, R _q is N(Me)-C(=O)CH ₂ -NRxRx. In one embodiment, NR _x ’Rx” is NR _x ’Rx”is NH2, N(H)Me, NMe2, N(Me)Et, NEt ₂ , N(H)isopropyl, N(Me)isopropyl, N(H)cyclopropyl, N(Me)cyclopropyl, N(H)cyclobutyl, N(Me)cyclobutyl, N(H)CH2cyclopropyl, N(Me)isopropyl, N(Me)t-butyl, or N(H)CH2CH2NMe2. In one embodiment, NR _x Rx”is azetidinyl, azetidinyl-OH, pyrrolidinyl, piperidinyl, morpholinyl, or piperizinyl. In one embodiment, NR _X ’R _X ” is NH-C1-6 alkylene-NR _X ””R _z . In one embodiment, NRx Rx” is N(H)CH ₂ CH ₂ -NRx ” ”Rz In one embodiment, NRx’Rx” is N(H)CH ₂ CH ₂ -NMe2.

[0134] In one embodiment, R _q is NH-C(=O)C1-6 alkylene-heterocycloalkyl. In one embodiment, Rq is NH-C(=O)CI-3 alkylene-heterocycloalkyl. In one embodiment, Rq is NH-C(=O)CI- ₂ alkylene-heterocycloalkyl. In one embodiment, R _q is NH-C(=O)CH2-heterocycloalkyl. In one embodiment, R _q is NH-C(=O)CH2-pyrrolidinyl, NH-C(=O)CH2-piperidinyl, NH-C(=O)CH ₂ - piperizinyl, or NH-C(=O)CH2-morpholinyl.

[0135] In one embodiment, R _q is N(Me)-C(=O)C1-6 alkylene-heterocycloalkyl. In one embodiment, R _q is N(Me)-C(=O)Ci-3 alkylene-heterocycloalkyl. In one embodiment, R _q is N(Me)-C(=O)Ci-2 alkylene-heterocycloalkyl. In one embodiment, R _q is NH-C(=O)CH ₂ - heterocycloalkyl. In one embodiment, R _q is NH-C(=O)CH2-morpholinyl.

[0136] In one embodiment, R _q is C1-6 alkylene-C(=O)-NR _x ’Rx. In one embodiment, R _q is C1.3 alkylene-C(=O)-NRx’R _x . In one embodiment, R _q is C1.2 alkyl ene-C(=O)-NR _X ’R _x . In one embodiment, R _q is CH ₂ -C(=O)-NR _X R _X . In one embodiment, NR _X R _X ’’ is NR _X R _X ’’ is NH ₂ , NHMe, NMe ₂ , NMe-Et, NEt ₂ , NH-isopropyl, NMe-isopropyl, NH-cyclopropyl, NMe-cyclopropyl, NH- cyclobutyl, NMe-cyclobutyl, NHCH ₂ cyclopropyl, NMe-isopropyl, or NMe-t-butyl. In one embodiment, NR _x R _x ”is azetidinyl, azetidinyl-OH, pyrrolidinyl, piperidinyl, morpholinyl, or piperizinyl. In one embodiment, NR _X ’R _X ” is NH-C1-6 alkylene-NR _X ””R _z . In one embodiment, NRx Rx” is NH-CH2CH2-NR.X ” ”Rz. In one embodiment, NR _x Rx” is NH-CH2CH2-NMe2.

[0137] In one embodiment, each R _q is independently selected from the group consisting of H, - CH2CI, carboxybenzyl, acetyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertbutyl, benzyl,

attachment.

[0138] In one embodiment, of the compound of formula I, IA, IB, IC, ID, IE, IF, and IG, selected from the group consisting of H, -CH2CI, carboxybenzyl, acetyl, methyl, ethyl, propyl, wherein “ ” indicates a point of attachment.

[0139] In one embodiment, of the compound of formula I, IA, IB, IC, ID, IE, IF, and IG, group consisting of H, -CH2CI, carboxybenzyl, acetyl, methyl, ethyl, propyl, isopropyl, butyl,

[0140] In one embodiment, the compound of formula I, IA, IB, IC, ID, IE, IF, and IG is a compound of formula C-l, or a pharmaceutically acceptable salt thereof: wherein R.2a, Rib, R-t, R5, Rsa, Reb, Rsa, Rsb, and Rq have the definitions as provided for a compound of formula I, IA, IB, IC, ID, IE, IF, IG, and IH; and

R9a is selected from the group consisting of -H and optionally substituted C1-10 alkyl;

A is wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein each “ ” indicates a point of attachment and B is CR’R”, wherein

R’ and R” are each independently optionally substituted alkyl; or

B is wherein Q is a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclic ring, wherein each “ «~w ” indicates a point of attachment and A is CR’R”, wherein R’ and R” are each independently optionally substituted alkyl.

[0141] In one embodiment, Rq _a is -H or C1.4 alkyl. In one embodiment, Rq _a is -H or methyl.

[0142] In one embodiment, A is and Q is a 3-, 4-, 5-, or 6-membered optionally substituted cycloalkyl or a 3-, 4-, 5-, or 6-membered optionally substituted heterocycloalkyl and B is CR’R”, wherein R’ and R” are H, wherein each “ ” indicates a point of attachment.

[0143] In one embodiment, B is and Q is a 3-, 4-, 5-, or 6-membered optionally substituted cycloalkyl or is a 3-, 4-, 5-, or 6-membered optionally substituted heterocycloalkyl and A is CR’R”, wherein R’ and R” are H, wherein each ” indicates a point of attachment.

[0144] In one embodiment, either A or B is , wherein Q is a 3-, 4-, 5-, or 6- membered cycloalkyl or 3-, 4-, 5-, or 6-membered heterocyclic ring, and the other of A and B is - CH2-, and Rq is as defined for formula I compounds.

[0145] In one embodiment, the compound of C-l, or a pharmaceutically acceptable salt thereof, is a compound of formula C-2 or C-3, or a pharmaceutically acceptable salt thereof:

[0146] In one embodiment, the compound, or a pharmaceutically acceptable salt thereof, is a compound of formula C-2a or C-3a, or a pharmaceutically acceptable salt thereof:

C-3a

[0147] In one embodiment, the compound, or a pharmaceutically acceptable salt thereof, is a compound of formula C-2b or C-3b, or a pharmaceutically acceptable salt thereof:

[0148] In one embodiment, the compound of formula C-l, C-2, C-3, C-2a, C-3a, C-2b, and C-3b, or a pharmaceutically acceptable salt thereof, is a compound of formula C-4, C-5, C-6, or C-7, C- 8, C-9, C-10, or C-l 1, or a pharmaceutically acceptable salt thereof:

C-9

[0149] In one embodiment, the compound of formula C-l, or a pharmaceutically acceptable salt thereof, is a compound of formula C-12, C-l 3, C-14, C15, C-16, C-17, C-18, or C-19, or a pharmaceutically acceptable salt thereof:

C-19 wherein Rn _a and Rub are as defined for R’ and R”; each Rt is independently selected from the group consisting of H, halo, -NR _x Rx”, C1-6 alkyl, C1-6 haloalkyl, OH, C1-6 alkoxy, -C(=O)-C1-6 alkyl, -C(=O)O-C1-6 alkylene-aryl, -C(=O)-Ci- 6 alkylene-aryl, -C(=O)O-C1-6 alkylene-heteroaryl, -C(=O)-C1-6 alkylene-heteroaryl, -C1-6 alkylene-aryl, -C1-6 alkylene-heteroaryl, -C1-6 alkylene-C(=O)-NR _x ’R _x ”, -C(=O)-C1-6 alkylene- cycloalkyl, -C(=O)-C1-6 alkyl ene-heterocycloalkyl, -C(=O)-C1-6 alkylene-NR _X ’R _X ”, -C(=O)- CHCH ₂ OH-alkylene-NR _X ’R _X ”, -SO2-C1.6 alkyl, -SO2-C1.6 alkylene-NRx’Rx”, -NR _Z C(=O)-CI. ₆ alkyl, -NR _Z C(=0)0-C1-6 alkylene-aryl, -NRZC(=0)-C1-6 alkylene-aryl, -NR _Z C(=0)0-C1-6 alkylene-heteroaryl, -NR _Z C(=0)-C1-6 alkylene-heteroaryl, -C1-6 alkylene-aryl, -C1-6 alkylene- heteroaryl, -NR _X ””C(=0)-C1-6 alkylene-cycloalkyl, -NR _X ””C(=0)-C1-6 alkylene-heterocycloalkyl, -NRZC(=O)-CI- ₆ alkylene-NRx’Rx”, -NRzC(=O)-CHCH ₂ OH-alkylene-NRx Rx”, -NRX””SO ₂ -C1-6 alkyl, -NR _X ””SO2-C1-6 alkylene-NR _x ’R _x ”, -C(=O)-heterocycloalkyl, -C(=O)-C1-6 alkylene- heterocycloalkyl, -C1-6 alkyl ene-NR _z C(=O)-C 1-6 alkyl, -C(=O)-C1-6 alkylene-NR _x ””cycloalkyl, - C(=O)-C1-6 alkylene-NR _X ””-C1-6 alkylene-cycloalkyl, -NRX””-C(=0)-C1-6 alkylene- heterocycloalkyl, -C(=O)-C1-6 alkylene-NRx” ”-C1-6 alkylene-cycloalkyl, -C(=O)-C1-6 alkylene- NR _X ””-C1-6 alkylene- C1-6 alkoxy, -C1-6 alkylene-NR _X ” ”-C(=O)-C1-6 alkylene-NRx’Rx”, -NR _X ” ”- C(=O)-C1-6 alkylene -NR _X ””-C1-6 alkylene-NRx’Rx”, -C(=O)-aryl, -C(=O)-heteroaryl, -C1-6 alkylene-arylene-aryl, -C(=O)-cycloalkyl, -C(=O)-NR _z aryl, -C(=0)NR _z -Ci.6 alkylene-aryl, -SO ₂ - aryl, -SO2-heteroaryl, and -SO2- C1-6 alkylene-aryl; wherein R _x > and R _x ” are each independently selected from the group consisting of H, C1-6 alkyl, C1-6 alkylene-cycloalkyl, C1-6 alkylene-heterocycloalkyl, C1-6 alkyl ene-O-C1-6 alkyl, cycloalkyl, heterocycloalkyl, or R _x ’ and R _x ” are joined together with the atom to which they are attached to form a 3-, 4-, 5-, 6-, or 7-membered optionally substituted ring optionally containing an additional heteroatom selected from 0, S, SO, SO2, N-R _x ”, wherein R _x ” is H, C1-6 alkyl, - C(=0)-C1-6 alkyl, -C(=0)0-C1-6 alkylene-aryl, -C(=O)-Ci-t> alkylene-aryl, -C(=O)O-Ci.t, alkylene- heteroaryl, -C(=0)-C1-6 alkylene-heteroaryl, -C1-6 alkylene-aryl, -C1-6 alkylene-heteroaryl, - C(=0)-C1-6 alkylene-cycloalkyl, -C(=0)-C1-6 alkylene-heterocycloalkyl; and each R _X ”” and R _z is independently H or C1-6 alkyl, wherein each occurrence of alkylene, alkyl, phenyl, heteroaryl, cycloalkyl, and heterocycloalkyl is independently and optionally substituted with halo, OH, C1-6 alkoxy, or C1-6 alkyl.

[0150] In one embodiment, Rt is H.

[0151] In one embodiment, Rtis -C1-6 alkylene-aryl. In one embodiment, Rtis -C1.3 alkylene- aryl. In one embodiment, Rt is -C1-2 alkylene-aryl. In one embodiment, R _t is -Cfb-aryl. In one embodiment, Rt is -C1-6 alkylene-heteroaryl. In one embodiment, Rt is -CH2-heteroaryl. In one embodiment, Rt is -CTL-phenyl. In one embodiment, Rt is -CH2-phenyl wherein phenyl is optionally substituted, hi one embodiment, optionally substituted phenyl is alkoxyphenyl or halophenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4-chlorophenyl. In one embodiment, optionally substituted phenyl is biphenyl. In one embodiment, Rt is CH2-heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, Rt is -CH2-pyridyl, wherein pyridyl can be 2-, 3-, or 4-pyridyl. In one embodiment, Rt is -CH2-imidazolyl. In one embodiment, Rt is -CTL-quinolinyl, or -CH2-isoquinolinyl.

[0152] In one embodiment, Rt is -CfbCHz-aryl. In one embodiment, Rt is -CfbCHt-phenyl wherein phenyl is optionally substituted. In one embodiment, optionally substituted phenyl is alkoxyphenyl or halophenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4- methoxy phenyl or 2-, 3-, or 4-chlorophenyl. In one embodiment, Rt is -CHiCHi-heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, Rt is -CfbCfb-pyridyl. In one embodiment, Rt is -CTbCTh-imidazolyl. In one embodiment, Rt is -CHzCfh-quinolinyl, or - CHzCTH-soquinolinyl.

[0153] In one embodiment, Rt is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl. [0154] In one embodiment, Rt is -C(=O)-CH2-aryl, wherein aryl is substituted or unsubstituted. In one embodiment, Rt is -C(=O)-CH2-phenyl. In one embodiment, Rt is -C(=O)-CH2-heteroaryl wherein aryl is substituted or unsubstituted. In one embodiment, Rt is -C(=O)-CH2-imidazolyl.

[0155] In one embodiment, Rt is -C(=O)-C1-6 alkyl. In one embodiment, Rt is -C(=O)-C1-6 methyl.

[0156] In one embodiment, Rt is -C(=O)-aryl, wherein aryl is optionally substituted. In one embodiment, Rt is -C(=O)-phenyl, wherein phenyl is alkoxypheny or chlorophenyl. In one embodiment, optionally substituted phenyl is chlorophenyl or methoxyphenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4- chlorophenyl.

[0157] In one embodiment, Rt is -C(=O)-cycloalkyl, wherein cycloalkyl is substituted or unsubstituted.

[0158] In one embodiment, Rt is -C(=O)-heterocycloalkyl, wherein heterocycloalkyl is substituted or unsubstituted. In one embodiment, Rt is -C(=O)-pyrrolidinyl. In one embodiment, Rt is -C(=O)-piperidinyl.

[0159] In one embodiment, R _t is -C(=O)-N(H)C1-6 alkyl. In one embodiment, R _t is -C(=O)- N(H)CI-3 alkyl. In one embodiment, Rt is -C(=O)-N(H)CI-2 alkyl. In one embodiment, Rt is - C(=O)-NHCH ₃ .

[0160] In one embodiment, Rt is -C(=O)-N(C1-6 alkyl)(C1-6 alkyl), wherein C1-6 alkyl at each occurrence can be the same or different. In one embodiment, Rt is -C(=O)-N(Me)Ci- ₃ alkyl. [0161] In one embodiment, Rt is -C(=O)-N(H)-aryl or -C(=O)-N(Me)-aryl, wherein aryl is optionally substituted. In one embodiment, Rt is -C(=O)-phenyl, wherein phenyl is optionally substituted. In one embodiment, optionally substituted phenyl is chlorophenyl or methoxyphenyl. In one embodiment, optionally substituted phenyl is 2-, 3-, or 4-methoxy phenyl or 2-, 3-, or 4- chlorophenyl.

[0162] In one embodiment, Rt is -C(=O)-heteroaryl, wherein heteroaryl is optionally substituted. In one embodiment, Rt is -C(=O)-pyridyl, wherein pyridyl is optionally substituted. In one embodiment, heteroaryl is quinolinyl or isoquinolinyl, either of which is optionally substituted. . [0163] In one embodiment, Rt is -C(=O)-C1-6 alkylene-NR _x ’R _x ”. In one embodiment, Rt is - C(=O)-Ci- ₃ alkylene-NRx’Rx”. In one embodiment, Rt is -C(=O)-Ci-2 alkylene-NRx’Rx”. In one embodiment, Rt is -C(=O)-CH2-NR _x Rx”. In one embodiment, Rt is -C(=O)-CH(Me)-NR _X ’Rx’. In one embodiment, Rt is -C(=O)-C(Me)2-NR _x ’Rx’’ In one embodiment, Rt is -C(=O)CH2-NR _x Rx”. In one embodiment, Rt is -C(=O)-C(Me)(CH2OH)NRx Rx’. In one embodiment, Rt is - C(=O)CH ₂ CH2-NRX’RX”. In one embodiment, NR _X R _X ” is NH ₂ , N(H)Me, NMe ₂ , N(Me)Et, N(Me)isopropyl, NEt ₂ , azetidinyl, azetidinyl-OH, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, N(H)-isopropyl, N(Me)isopropyl, N(H)cyclopropyl, N(Me)cyclopropyl, N(H)cyclobutyl, N(Me)cyclobutyl, N(H)CH ₂ cyclopropyl, N(Me)CH ₂ cyclopropyl, N(Me)isopropyl, N(Me)-tbutyl, N(H)CH ₂ CH ₂ -alkoxy,

[0164] In one embodiment, Rt is -SO ₂ -C1-6 alkyl. In one embodiment, Rt is -SO ₂ -Ci-3 alkyl. In one embodiment, Rt is -SO ₂ -Ci- ₂ alkyl. In one embodiment, Rt is -SO ₂ -Me.

[0165] In one embodiment, Rt is -SO ₂ -C1-6 alkylene-NRx’Rx”. In one embodiment, Rt is -SO ₂ -Ci-3 alkyl ene-NRx’Rx”. In one embodiment, Rt is -SO ₂ -Ci- ₂ alkyl ene-NR _x Rx”. In one embodiment, Rt is -SO ₂ -CH ₂ NRx’Rx”. In one embodiment, Rt is -SO ₂ -CH ₂ CH ₂ NR _x Rx”. In one embodiment, NRx’Rx” is NH ₂ , N(H)Me, NMe ₂ , N(Me)Et, NEt ₂ , N(H)isopropyl, N(Me)isopropyl, N(H)cyclopropyl, N(Me)cyclopropyl, N(H)cyclobutyl, N(Me)cyclobutyl, N(H)CH ₂ cyclopropyl, N(Me)isopropyl, orN(Me)t-butyl.

[0166] In one embodiment, Rt is -SO ₂ -aryl. In one embodiment, Rt is -SO ₂ -phenyl, wherein phenyl is optionally substituted. In one embodiment, Rt is -SO ₂ -chlorophenyl or -SO ₂ - methoxyphenyl. In one embodiment, Rt is -SO ₂ -(5-10 membered heteroaryl). In one embodiment, Rt is -SO ₂ -pyridyl. In one embodiment, Rt is -SO ₂ -C1-6 alkyl ene-phenyl. In one embodiment, Rt is -SO ₂ -CH ₂ -phenyl.

[0167] In one embodiment, Rt is Ci-e alkylene-C(=O)-NR _X ’R _x . In one embodiment, Rt is C1.3 alkylene-C(=O)-NR _x Rx. In one embodiment, Rt is Ci- ₂ alkylene-C(=O)-NR _x ’Rx. In one embodiment, Rt is CH ₂ -C(=O)-NR _x Rx. In one embodiment, NR _x Rx” is NRx’Rx” is NH ₂ , NHMe, NMe ₂ , NMe-Et, NEt ₂ , NH-isopropyl, NMe-isopropyl, NH-cyclopropyl, NMe-cyclopropyl, NH- cyclobutyl, NMe-cyclobutyl, NHCH ₂ cyclopropyl, NMe-isopropyl, or NMe-t-butyl. In one embodiment, NR _x R _x ”is azetidinyl, azetidinyl-OH, pyrrolidinyl, piperidinyl, morpholinyl, or piperizinyl. In one embodiment, NRx’Rx” is NH-C1-6 alkylene-NR _x ””Rz. In one embodiment, NRx Rx” is NH-CH ₂ CH ₂ -NR _X ” ”R _Z . In one embodiment, NR _x Rx” is NH-CH ₂ CH ₂ -NMe ₂ .

[0168] In one embodiment, Rt is selected from the group consisting of H, carboxybenzyl, acetyl,

— N ^/ V ^V methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertbutyl, benzyl, -C(=O)-Me , ,

[0169] In one embodiment, the compound of the invention or a pharmaceutically acceptable salt thereof is selected from Table A.

Table A

[0170] In one embodiment, the compound of the invention or a pharmaceutically acceptable salt thereof is selected from Table B.

Table B

[0171] In one aspect, the invention includes a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. [0172] In one aspect, the invention includes a kit comprising a compound described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and instructions for administering to a subject in need thereof.

[0173] In one embodiment, the compound is selected from Table A, or a pharmaceutically acceptable salt thereof.

Processes for Preparing Compounds

[0174] Compounds disclosed herein can be prepared as described in the following paragraphs.

[0175] Compounds are prepared via two intermediates. The eastern half intermediate is a compound of formula P-1:

P l, or salt thereof. In the compound of formula P-1, R ³ , R ^4a , R ^4b , R ⁵ , R ^6a , R ^6b , R ^8a , and R ^8b are as defined herein; and

G ⁴ is of the formula: each instance of R ¹⁵ is independently silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R ¹⁵ groups are joined to form an optionally substituted heterocyclyl or heteroaryl ring; and each instance of R ^16a is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0176] The uncyclized eastern half intermediate is a compound of formula P-2: or salt thereof, wherein:

PG is a protecting group;

R.4a, Rrb, Rs, Rsa, Reb, Rsa, and Rsb are as defined herein;

G ⁴ is of formula: each instance of R ¹⁵ is independently silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R ¹⁵ groups are joined to form an optionally substituted heterocyclyl or heteroaryl ring; and each instance of R ^16a is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0177] In some embodiments, -OPG is -OBz.

[0178] What is also disclosed is a compound of Formula P-3:

or salt thereof, wherein the variables are as defined herein.

Coupling and Macrolactonization

[0179] In certain embodiments, compounds of the present disclosure are prepared by coupling a compound of Formula Pl (the eastern half) wherein R _s is a sugar residue , wherein PG is a protecting group and “■<««” indicates a point of attachment, and a compound of Formula P-4 (the western half) to provide an uncyclized compound precursor of Formula P-5 as depicted in the following Scheme.

[0180] Formula P-5 is cyclized to give, after deprotection of the sugar residue a compound of Formula I as depicted in the following scheme.

[0181] Alternatively, the compound precursor of Formula P-5 wherein Rga is hydrogen is cyclized to provide a compound of Formula I, which can undergo reductive amination to provide a compound of Formula I wherein R.9 _a is other than H, as shown in the following Scheme.

[0182] Late-stage installment of the R.2b group can be achieved via treatment of a compound of Formula P-6 prepared as provide above with a base and a suitable electrophile group (e.g., halogenating agent or R2-LG, wherein LG is a leaving group) as depicted in the following Scheme. In this process, the sugar residue in P-6 is protected and Rg _a is H or alkyl.

OR ₅ = H

[0183] For all intermediates, the variables are as defined herein for a compound of Formula I.

[0184] Other variables depicted for intermediates and precursors are defined as follows: LG is a leaving group; , ” indicates a point of attachment; each instance of R ¹⁵ is independently silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R ¹⁵ groups are joined to form an optionally substituted heterocyclyl or heteroaryl ring; and each instance of R ^16a is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0185] As noted above, R _s is the sugar moiety . The sugar moiety is typically attached to the compound framework during synthesis of the eastern half, but may also be attached at other stages of the preparation. The sugar moiety may be attached by a chemical or enzymatic glycosylation reaction between the hydroxyl group at the C5 position and a glycosyl donor. In certain embodiments, the sugar moiety is attached to the compound framework as a thioglycoside. In certain embodiments, substituents of the sugar moiety are modified after the glycosylation of the compound or compound precursor (c.g., eastern half).

Methods

[0186] In one aspect, what is provided herein is a method for inhibiting protein translation in a cell, comprising treating the cell with a therapeutically effective amount of a compound of Formula I. In one embodiment, the cell is a cancer cell.

[0187] In one aspect, what is provided herein is a method for treating a disease caused by abnormal protein translation in a patient in need of such treatment, comprising treating the patient with a therapeutically effective amount of a compound of Formula I. In these and other aspects and embodiments, the patient is a human or animal. In one embodiment, the disease is cancer. In one embodiment, the cancer is selected from the cancers provided herein.

[0188] In one embodiment, the disease is caused by a premature codon termination. In one embodiment, the disease caused by a premature codon termination is selected from the diseases disclosed herein.

[0189] In one aspect, what is provided herein is a method for inhibiting protein translation in a patient who has cancer, comprising treating the patient with a therapeutically effective amount of a compound of Formula I.

[0190] In one aspect, what is provided herein is a method for treating cancer, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of Formula I.

[0191] In one aspect, what is provided herein is a method for treating a genetic disorder, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of Formula I. What is also provided is a method of treating a genetic disorder characterized by a premature termination codon mutation, comprising administering to a subject in need thereof a therapeutically effective amount of to a subject in need of such treatment a therapeutically effective amount of a compound of Formula I.

[0192] A genetic disorder characterized by a premature termination codon mutation can be treated by inducing and/or promoting readthrough of the mutation in the complete but otherwise defective transcript (mRNA). That is, a genetic disorder characterized by a premature termination codon mutation can be treated by inducing and/or promoting suppression of the nonsense mutation (the premature termination codon mutation). Thus, as disclosed herein, a genetic disorder that can be treated according to the method disclosed and claimed herein is one that is responds to readthrough-inducing and/or promoting compounds.

[0193] Methods for identifying genetic disorders characterized by a premature termination codon mutation are available to the skilled practitioner, and may involve full or partial genome elucidation, genetic biomarker detection, phenotype classification and hereditary information analysis. These methods often result in pairs of mutant/wild type (WT) sequences. Pairs of WT sequences can be employed to identify whether the genetic disease is characterized by a premature termination codon mutation. Similarly, the ability to determine the ability of a compound or composition to induce or promote readthrough can also is also known in the art. [0194] To that end, a plasmid comprising two reporter genes interrupted by a sequence of the mutated gene (the genetic disease-causing gene) is transected into a protein expression platform, either in full cells or in a cell-free systems, and the ratio between the expression level of the two genes in the presence of a tested compound is measured, typically in series of concentrations and duplications, and compared to the gene expression level ratio of the wild-type and/or to the expression level ratio measured in a control sample not containing the tested compound.

[0195] It is noted that the experimental model for readthrough activity (namely the nucleotide sequence of gene containing the premature stop-codon mutation) is a byproduct of the process of identifying a genetic disorder as associated with a premature stop-codon mutation and/or a protein truncation phenotype, and further noted that with the great advances in genomic data acquisition, this process is now well within the capability of the skilled practitioner.

[0196] Methodologies for testing readthrough of a premature termination codon mutation are known. Experimental methods are provided herein that are designed to gauge the ability of compounds to induce or promote read through.

[0197] A number of in vitro methodologies can be used by the skilled practitioner to test the readthrough-inducing ability of compounds provided herein, as well as their safety as potential drugs.

[0198] Non-limiting examples of genetic diseases that are associated with the presence of at least one premature termination codon mutation or other nonsense mutations include cystic fibrosis (CF), muscular dystrophy (Duchenne (DMD), Becker (BMD), congenital), spinal muscular atrophy (SMA), ataxia-telangiectasia, mucopolysaccharidosis type 1 (MPS1) (Hurler syndrome), hemophilia (A & B), Usher syndrome (Retinitis pigmentosa, X-linked retinitis pigmentosa), Tay- Sachs, factor VII deficiency, familial atrial fibrillation, Hailey-Hailey disease, McArdle disease, mucopolysaccharidosis, nephropathic cystinosis, polycystic kidney disease, Rett syndrome, cystinosis, severe epidermolysis bullosa, dravet syndrome, X-linked nephrogenic diabetes insipidus (XNDI), dancer, beta-thalassemia, and obesity.

[0199] Additional genetic diseases that associated with the presence of at least one premature termination codon or other nonsense mutations are disclosed in, for example, “Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases,” Kim M. Keeling, K. M Bedwell, D.M., Wiley Interdisciplinary Reviews: RNA, 2011, 2(6), p. 837-852; “Cancer syndromes and therapy by stop-codon readthrough,” Bordeira-Carrico, R. et al., Trends in Molecular Medicine, 2012, 18(11), p. 667-678, and references cited therein.

[0200] In one aspect what is provided is a compound or composition as disclosed herein for use in the treatment of a genetic disease associated with a premature termination codon mutation.

[0201] In one aspect, embodiment, what is provided is a use of a compound or composition as disclosed herein in in the manufacture of a medicament for treating a genetic disease associated with a premature termination codon mutation. The genetic disease in this and other aspects and embodiments is selected from the group consisting of cystic fibrosis (CF), muscular dystrophy (Duchenne (DMD), Becker (BMD), congenital), spinal muscular atrophy (SMA), hemophilia (A & B), Usher syndrome (Retinitis pigmentosa, X-linked retinitis pigmentosa), mucopolysaccharidosis, nephropathic cystinosis, Rett syndrome, cancer, Beta-thalassemia, and obesity.

[0202] In one aspect, what is provided is a method of increasing the expression level of a gene having a premature termination codon mutation, the method comprising translating the gene into a protein in the presence of a compound or composition as disclosed herein in any of the respective embodiments and any combination thereof. In one embodiment what is provided is a compound or composition as disclosed herein for use in increasing the expression level of a gene having a premature termination codon mutation. In a further embodiment, what is a provided is the use of a compound or composition as disclosed in the manufacture of a medicament for increasing the expression level of a gene having a premature stop-codon mutation. According to these and other aspects and embodiments, the premature termination codon mutation has an RNA code selected from the group consisting of UGA, UAG and UAA. According to these and other aspects and embodiments, the protein is translated in a cytoplasmic translation system. According to these and other aspects and embodiments, the compound or composition disclosed herein is used in a mutation suppression amount. According to these and other aspects and embodiments, an inhibition of translation IC50 of the compound or composition in a eukaryotic cytoplasmic translation system is greater that an inhibition of translation IC50 of the compound in a ribosomal translation system. According to these and other aspects and embodiments, an inhibition of translation IC50 of the compound in a eukaryotic cytoplasmic translation system is greater that an inhibition of translation IC50 of the compound in a prokaryotic translation system.

Pharmaceutical Compositions and Administration

[0203] The present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable excipient. In certain embodiments, the compound of the present invention is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

[0204] Pharmaceutically acceptable excipients include any and all solvents, diluents, or other liquid vehicles, dispersions, suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and/or manufacture of pharmaceutical compositions agents can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0205] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound of the present invention (the “active ingredient”) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0206] Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0207] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0208] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0209] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0210] Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0211] Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cethyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myij 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Pol oxamer 188, cetrimonium bromide, cethylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

[0212] Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

[0213] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0214] Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0215] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof: malic acid and salts and hydrates thereof: phosphoric acid and salts and hydrates thereof: and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cethylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0216] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0217] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0218] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0219] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

[0220] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0221] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0222] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0223] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0224] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0225] A sterile injectable composition, e.g., a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di glycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0226] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0227] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0228] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cethyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents. [0229] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0230] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the compounds presented herein may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0231] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active aminoglycoside compounds doses.

[0232] The active ingredient can be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner Examples of embedding compositions which can be used include polymeric substances and waxes.

[0233] Dosage forms for topical and/or transdermal administration of a compound of this invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any needed preservatives and/or buffers as can be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively, or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

[0234] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499;

5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851;

5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively, or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. [0235] A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0236] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

[0237] Pharmaceutical compositions of the invention formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface-active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

[0238] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

[0239] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

[0240] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

[0241] Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0242] To practice the method of this invention, the above-described compound or its pharmaceutical composition can be administered intravenously, intravitreally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseosly, periprosthetically, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference). In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

[0243] In certain embodiments, the pharmaceutical composition and/or additional agent is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsules, they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0244] In further embodiments, a composition described herein may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally

[0245] According to some embodiments, the administration is effected orally. For oral administration, the compounds presented herein can be formulated readily by combining the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds presented herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0246] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0247] For administration by inhalation, the compounds presented herein are conveniently delivered in the form of an aerosol spray presentation (which typically includes powdered, liquefied and/or gaseous carriers) from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, tri chlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compounds presented herein and a suitable powder base such as, but not limited to, lactose or starch.

[0248] For administration by injection, the compounds presented herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.

[0249] Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder. Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture. Transdermal administration of the compositions may also comprise the use of a "patch." For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.

[0250] In certain embodiments, the compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in their entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts and could be employed to deliver the compositions described herein. Likewise, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety), and could be employed to deliver the compositions described herein.

[0251] It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.

[0252] For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.

[0253] The compounds presented herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0254] Alternatively, the compounds presented herein may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0255] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[0256] In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.

[0257] In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

[0258] It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. [0259] It will be also appreciated that a compound or composition, as described herein, can be administered in combination with one or more additional therapeutically active agents. The compounds or compositions can be administered in combination with additional therapeutically active agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.

[0260] The compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0261] Also encompassed by the invention are kits (e.g., pharmaceutical packs). The kits provided may comprise an inventive pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of an inventive pharmaceutical composition or compound. In some embodiments, the inventive pharmaceutical composition or compound provided in the container and the second container are combined to form one-unit dosage form.

Combinations

[0262] Combination therapy can be effective with drugs that work by different mechanisms, thereby decreasing the likelihood that resistant cancer cells will develop. When drugs with different effects are combined, each drug can be used at its optimal dose, without intolerable side effects. A compound of Formula I as disclosed herein and a second medicament can be combined for therapeutic benefit, for instance, for treating a proliferative disease or disorder such as cancer. The second medicament can have a different mechanism. Alternatively, it can use the same mechanism as the compound as disclosed herein for therapeutic benefit. The combination can act via a synergistic effect. Combination therapies can also include additional (e.g. a third, fourth, fifth, etc.) medicaments.

[0263] Thus, other embodiments of the invention provide methods for combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes are used in combination with a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, solvate, tautomer, hydrate or derivative thereof. In one aspect, such therapy includes but is not limited to the combination of one or more compounds of the invention with chemotherapeutic agents, therapeutic antibodies, and radiation treatment, to provide a synergistic or additive therapeutic effect.

[0264] Many chemotherapeutics are presently known in the art and can be used in combination with the compounds of the invention. In some embodiments, the chemotherapeutic is selected from the group consisting of DNA intercalating agents, topoisomerase inhibitors, microtubule inhibitors, multi-kinase inhibitors, PARP inhibitor, transcription and translation inhibitors, as well as mitotic inhibitors, alkylating agents, anti-metabolites, growth factor inhibitors, cell cycle inhibitors, enzymes, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens. Therapeutic agents that can be combined with a compound of the invention are found in Goodman and Gilman's "The Pharmacological Basis of Therapeutics" Tenth Edition edited by Hardman, Limbird and Gilman or the Physician's Desk Reference, both of which are incorporated herein by reference in their entirety.

[0265] Non-limiting examples of intercalating agents include, methotrexate, actinomycin, mitoxantrone, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin. [0266] Non-limiting examples of topoisomerase inhibitors include irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), teniposide (VUMON), ellipticine, TAS-103, mitonafide, amonafide, chartreusin, elsamicin, IST-622, ditercalinimum, elinafide, and echinomycin.

[0267] Non-limiting examples of microtubule-disruptor agents include ecteinascidins. Non- limiting examples of microtubule-stabilizing agents include pacitaxel, docetaxel, and epothilones A-F; 4-(3-(l-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluor obenzyl)phtha- lazin-l(2H)- one (olaparib, AZD2281, Ku-0059436), 2-[(2R)-2-methylpyrrolidin-2-yl]-lH-benzimidazole-4- carboxamide (Veliparib, ABT-888), (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4- triazol-5-yl)-8,- 9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one (talazoparib, BMN 673), 4- iodo-3 -nitrobenzamide (iniparib, BSI-201), 8-fluoro-5-(4-((methylamino)methyl)phenyl)-3,4- dihydro-2H-azepino[5,4,3-c- d]indol-l(6H)-onephosphoric acid (Rucaparib, AG-014699, PF- 01367338), 2-[4-[(dimethylamino)methyl]phenyl]-5,6-dihydroimidazo[4,5,l -jk][l,4]benz- odiazepin-7(4H)-one (AG14361), 3 -aminobenzamide (INO-1001), 2-(2-fluoro-4-((S)-pyrrolidin- 2-yl)phenyl)-3H-benzo[d]imidazole-4-carboxa- mide (A-966492), N-(5,6-dihydro-6-oxo-2- phenanthridinyl)-2-acetamide hydrochloride (PJ34, PJ34 HC1), MK-4827, 3,4-dihydro-4-oxo-3,4- dihydro-4-oxo-N-[(lS)-l-phenylethyl]-2-quinazolinep- ropanamide (ME0328), 5-(2-oxo-2- phenylethoxy)-l(2H)-isoquinolinone (UPF-1069), 4-[[4-fluoro-3-[(4-methoxy-l- piperidinyl)carbonyl]phenyl]methyl]-l(2H)-ph- thalazinone (AZD 2461),

[0268] Non-limiting examples of PARP inhibitors include olaparib, niraparib, rucaparib, talazoparib, veliparib, CEP 9722, E7016, and BGB-290.

[0269] Non-limiting examples of transcription and translation inhibitors include alpha-amatinin, CX-5461, TAS-106, BMH-21, metatrestin, JQ-1, birabresib, mivebresib, triptolide, 8-Cl-Ado, 8- NEh-Ado, fludarabine, alvocidib, abemaciclib, palbociclib, ribociclib, rapamycin, everolimus, Torinl, PP242, MLN0128, Rapalink-lsilvestrol, omacetaxine, RG-7388, HDM-201, INCB057643, SY-1365, and BMS-986158.

[0270] In one embodiment, a compound of formula I can be combined with one or more compounds selected from the following compounds to treat cancer: Gleevec® (Imatinib Mesylate), Velcade®. (bortezomib), Casodex (bicalutamide), Iressa®. (gefitinib), and Adriamycin as well as a host of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex®, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norieucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL®., Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®., Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, (Nolvadex®), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin- 11 (CPT-11); topoisomeRASe inhibitor RFS 2000; difluoromethylomithine (DMFO). [0271] Where desired, the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex.RTM., Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17- demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur- uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126 or Zosuquidar; In other embodiments, agents useful in methods for combination therapy with one or more compounds of structure (I) include, but are not limited to: Erlotinib, Afatinib, Iressa, GDC0941, MLN1117, BYL719 (Alpelisib), BKM120 (Buparlisib), CYT387, GLPG0634, Baricitinib, Lestaurtinib, momelotinib, Pacritinib, Ruxolitinib, TG101348, Crizotinib, tivantinib, AMG337, cabozantinib, foretinib, onartuzumab, NVP- AEW541, Dasatinib, Ponatinib, saracatinib, bosutinib, trametinib, selumetinib, cobimetinib, PD0325901, RO5126766, Axitinib, Bevacizumab, Bostutinib, Cetuximab, Crizotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Ibrutinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Trastuzumab, Tofacitinib, Vandetanib, Vemurafenib, Irinotecan, Taxol, Docetaxel, Rapamycin or MLN0128. [0272] The compounds of Formula I can be used in combination with the agents disclosed herein or other suitable agents, depending on the condition being treated. Hence, in some embodiments the one or more compounds of the invention will be co-administered with other agents as described above. When used in combination therapy, the compounds described herein are administered with the second agent simultaneously or separately. This administration in combination can include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any of the agents described above can be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the invention and any of the agents described above can be simultaneously administered, wherein both the agents are present in separate formulations. In one alternative, a compound of the present invention can be administered just followed by and any of the agents described above, or vice versa. In some embodiments of the separate administration protocol, a compound of the invention and any of the agents described above are administered a few minutes apart, or a few hours apart, or a few days apart.

Examples

[0273] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the cwompounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

[0274] The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and methods of preparing such compounds. It is to be understood that the scope of the present invention is not limited in any way by the scope of the following examples and preparations. In the following examples, and throughout the specification and claims, molecules with a single stereocenter, unless otherwise noted, exist as a racemic mixture. Those molecules with two or more stereocenters, unless otherwise noted, exist as a racemic mixture of diastereomers. Single enantiomers/diastereomers may be obtained by methods known to those skilled in the art.

[0275] Intermediate Scheme Cl depicts the process of preparing IS1-1. Intermediate Scheme Cl.

IS1-1

[0276] ter/-Butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-hydroxypyrrolidine- l- carboxylate (IS1-1). A solution of tert-butyl 3 -(aminomethyl)-3 -hydroxypyrrolidine- 1- carboxylate (0.74 g, 3.4 mmol) in DCM (11.3 mL) was cooled to 0°C. Triethylamine (0.95 mL, 6.8 mmol) followed by N-(benzyl oxycarbonyl oxy)succinimide (0.93 g, 3.74 mmol) were added and the reaction mixture was stirred at 0 °C for 30 minutes (mins). The reaction mixture was diluted with ethyl acetate and washed with saturated (sat.) NH4CI. The washed solution was dried over NaiSCU, filtered and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (12 g, 0-60% EtOAc/hexane) to give IS1-1 (1.2 g, 3.4 mmol, 100%). MS (ESI+) mlz-. 373.17 [M + Na] ⁺ ; 'H NMR (400 MHz, Chloroform -J) 8 7.40 - 7.31 (m, 5H), 5.34 (d, 1H), 5.11 (s, 2H), 3.48 (dd, 2H), 3.33 (d, 4H), 3.07 (d, 1H), 1.88 (s, 2H), 1.44 (s, 9H).

IS1-2

[0277] tert- Butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-hydroxy-2-oxopyrrol idine-l- carboxylate (IS1-2). To a solution of IS1-1 (1.2 g, 3.4 mmol) in ethyl acetate (15 mL) was added a solution of sodium periodate (3.27 g, 15.3 mmol) in water (15 mL). Ruthenium chloride (70.9 mg, 0.34 mmol) was added in one portion and the reaction mixture was stirred at room temperature for two hours. The reaction mixture was poured into a separating funnel, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 times). The combined extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (24 g, 0-60% EtOAc/Hexane) to give IS1-2 (140 mg, 0.38 mmol, 11.2%). MS (ESI+) mix. 387.12 [M + Na] ⁺ ;

'H NMR (400 MHz, Chloroform-^/) 8 7.42 - 7.26 (m, 5H), 5.53 (dd, 1H), 5.09 (d, 2H), 3.75 (ddd, 1H), 3.66 - 3.48 (m, 2H), 3.29 (dd, 1H), 2.14 (dddd, 1H), 2.00 (dd, 1H), 1.51 (s, 9H).

19

[0278] 3-( lm/7/f>m(7//)7)-3-hydroxypyrrolidin-2-one (19). A solution of IS1-2 (140 mg, 0.38 mmol) in acetonitrile (1.5 mL) was added with hydrochloric acid (0.29 mL, 4M in 1,4-dioxane, 1.15 mmol). The reaction mixture was stirred at room temperature for 30 mins, at which point ultra performance liquid chromatography (UPLC) showed complete conversion to Boc- deprotected product. The reaction mixture was diluted with dichloromethane (DCM) and washed with sat. aqueous (aq.) NaHCCh. The washed solution was dried over NazSCU, filtered and concentrated in vacuo. The crude material was dissolved in methanol and Pd/C (40.8 mg, 5wt%, 5 mol%) was added. The reaction mixture was bubbled with hydrogen for 15 mins and stirred under an atmosphere of hydrogen for 1 hour (hr). Upon completion, the mixture was filtered through Celite® with ethyl acetate and the filtrate was concentrated in vacuo to give amino alcohol 19 (40 mg, 0.31 mmol, 80.1%) as a clear oil. The crude material was used in the next step without further purification. MS (ESI+) m/z'. 131.03 [M + H] ⁺ ,153.00 [M + Na] ⁺ .

[0279] Tables 1C provides a list of commercially available aminoalcohol intermediates that were used to prepare various compounds.

Table 1C. Intermediate aminoalcohols and their sources.

Scheme SI.

[0280] Benzyl 4-(((2R,4R,5R,6R)-5-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimeth ylamino)-6- methyltetrahydro-2H-pyran-2-yl)oxy)-4-methoxy-2,4-dimethyl-6 -(2,2,5-trimethyl-4-oxo-4H- l,3-dioxin-6-yl)heptyl)amino)-4-(hydroxymethyl)piperidine-l- carboxylate (S1-2-I1). Benzyl

4-amino-4-(hydroxymethyl)piperidine-l-carboxylate (205 mg, 0.775 mmol) and Sl-1 (548 mg, 0.930 mmol) were dissolved in MeOH (3 mL) and Ti(0Et)4 (0.322 mL, 1.54 mmol) was added. After 30 min, a small aliquot was removed from the reaction mixture and was added to a suspension of a small amount of NaBFL in MeOH. LC/MS analysis showed complete conversion. The reaction mixture was cooled in an ice bath and NaBHi (43.8 mg, 1.16 mmol) was added. When gas evolution ceased, 30% aqueous NH4OH (6 mL) was added, and the mixture was filtered through a pad of Celite®, washing with EtOAc. The filtrate was washed with brine, was dried over Na2SO4, was filtered and was concentrated. The residue was purified on a 24 g SiO2 column, eluting with 2-10% MeOH/DCM/0.5% NH ₄ OH, to give SI-2-11 (0.426 g, 66%). MS (ESI+) mlz-. 838.33 [M + H] ⁺ ; 'H NMR (400 MHz, Chloroform-d) 5 8.12 - 8.00 (m, 2H), 7.61 - 7.52 (m, 1H), 7.44 (t, 2H), 7.40 - 7.27 (m, 5H), 5.13 (s, 2H), 5.18 - 5.04 (m, 1H), 4.69 (d, 1H), 3.90 (d, 1H), 3.68 (s, 2H), 3.56 (dd, 1H), 3.30 (s, 4H), 3.23 (s, 1H), 3.06 (s, 3H), 2.95 - 2.83 (m, 1H), 2.42 (s, 1H), 2.39 - 2.28 (m, 1H), 2.32 (s, 6H), 1.86 - 1.65 (m, 10H), 1.64-1.46 (m, 4 H), 1.45 (d, J2H), 1.41 - 1.22 (m, 8H), 0.98 (d, 3H), 0.83 (d, 3H).

[0281] BenzyZ (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimeth ylamino)-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-9, 11,13, 15-tetramethyl-14, 16-dioxo-17- oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (SI-4-11). SI-2-11 (0.426 g, 0.508 mmol) was concentrated twice from toluene. The material was dissolved in chlorobenzene (130 mL), and a stream of nitrogen was bubbled through the solution for 10 min. The mixture was heated at a bath temperature of 150 °C (approximately 130-135 °C internal temperature) overnight. The reaction was allowed to cool to room temperature (rt) and was concentrated. The residue was purified on 24 g of silica gel (elution with 0-10% MeOH-DCM gradient) to give SI-4-11 (243 mg, 61%). MS (ESI+) mlz-. 780.31 [M + H] ⁺ .

[0282] Benzyl (9R,1 lR,12R,13R)-12-(((2S,3R,4S,6R)-4-(J^/ij;Za»Hno)-3-hydroxy-6 - methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-9, 11,13, 15-tetramethyl-14, 16-dioxo-17- oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (Sl-6-Il-l)(Compound 1). SI-4-11 (41 mg, 0.043 mmol) was dissolved in methanol (2 mL), and the mixture was heated at 60 °C (external temperature) overnight. The reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give S1-6-I1- 1 (9.9 mg, 28%). MS (ESI+) m/z: 676.35 [M + H]“, Formate salt, mixture of C2 epimers,

NMR (400 MHz, Methanol-d4) 8 7.33 - 7.15 (m, 5H), 5.01 (s, 2H), 4.31 (dd, 1H), 4.28 - 4.21 (m, 1H), 3.97 (d, 0.7H), 3.88 (d, 0.3H), 3.72 - 3.61 (m, 1H), 3.55-3.35 (m, 6H), 3.26 - 3.11 (m, 6H), 2.77 (s, 0.9H), 2.71 (s, 2.1H), 2.68-2.58 (m, 1H), 2.45-2.38 (m, 0.7H), 2.35-2.22 (m, 6.3H), 2.18- 2.08 (m, 1H), 1.68 (d, 1H), 1.56 - 1.41 (m, 4H), 1.26 - 1.07 (m, 12H), 0.95-0.80 (m, 3H).

[0283] Benzyl (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimeth ylamino)-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15- pentamethyl-14,16-dioxo-17- oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (SI-5-11-1). SI-4-11 (0 243 g, 0 311 mmol) was dissolved in DCM (1.55 mL) and Na(OAc)3BH (131 mg; 0.622 mmol) was added.

Formaldehyde (37 wt% solution in water, 0.175 mL, 1.55 mmol) was added. After 10 min., the reaction mixture was quenched by the addition of NaHCOs (sat., aq. solution). The layers were separated, and the aqueous layer was extracted with DCM (1 time). The combined DCM extracts were dried over ISfeSCL, were filtered, and were concentrated to give crude SI-5-11-1 (244 mg, 99%) which was used without further purification. MS (ESI+) m/z: 794.27 [M + H] ⁺ .

[0284] Benzyl (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15-p entamethyl-14,16-dioxo-17- oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (Sl-6-Il-2)(Compound 2). SI-5-11-1 (17 6 mg, 0.221 mmol) was dissolved in methanol (1 mL), and the mixture was heated at 60 °C (external temperature) for 7 hours (h). The reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give SI-6-11-2 (6.87 mg). MS (ESI+) mlz\ 690.36 [M + H] ⁺ , Formate salt, mixture of C2 epimers, 'H NMR (400 MHz, Methanol-d4) 5 8.55 (s, 2H), 7.45 - 7.29 (m, 5H), 5.14 (d, 2H), 4.51 - 4.37 (m, 1H), 4.30-4.15 (m, 1H), 4.00-3.60 (m, 4H), 3.44 - 3.33 (m, 3H), 3.30 - 3.10 (m, 3H), 3.10-2.95 (m, 2H), 2.80-2.40 (m, 12H), 2.10-1.92 (m, 3H), 1.92-1.60 (m, 6H), 1.52-1.44 (m, 1H), 1.40 - 1.17 (m, 12H), 1.10 - 0.90 (m, 3H).

[0285] (10R,llR,12/?,14R)-ll-(((25,3/?,45,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2//-pyran-2-yl)oxy)-12-methoxy-8,70,12,14,1 6-pentamethyl-6-oxa-2,16- diazaspiro[4.12]heptadecane-l, 7, 9-trione (Sl-6-I9-2)(Compound 112). Prepared according to the methods of SI-6-11-2 from 19. Formate salt. (8.96 mg). MS (ESI+) mlz'. 278.79 [M + 2H] ²⁺ , 556.29 (400 MHz, Chloroform-J) 5 8.53 (s, 2H), 4.43 (dd, 1H), 4.30 (d, 1H), 3.81 - 3.62 (m, 2H), 3.57 - 3.35 (m, 4H), 3.28 - 3.21 (m, 1H), 3.19 (s, 3H), 2.99 (s, 2H), 2.87 (s, 1H), 2.77 (d, 2H), 2.73 (s, 4H), 2.64 - 2.44 (m, 2H), 2.34 - 2.13 (m, 3H), 2.05 - 1.87 (m, 2H), 1.83 (s, 1H), 1.55 - 1.45 (m, 1H), 1.42 (s, 2H), 1.36 (d, 3H), 1.32 (dd, 6H), 1.25 (d, 1H), 1.13 (d, 1H), 1.07 (d, 2H), 0.95 (d, 1H).

[0286] (llR,13R,14R,15R)-14-(((2S,3R,4S,6R)-4-(Drme//ij;/ammo)-3-hy droxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-ll,13,15,17-t etramethyl-l,4,19-trioxa-9- azadispiro[4.2.128.25]docosane-16, 18-dione (Sl-6-I10-l)(Compound 74). Prepared according to the methods of SI-6-11-1 from 110. Formate salt. MS (ESI+) mlz'. 599.28 [M + H] ⁺ ; ^J H NMR (400 MHz, Methanol-d4) 84.35 (dt, 1H), 4.12 - 4.01 (m, 1H), 4.01 - 3.84 (m, 4H), 3.84 - 3.68 (m, 1H), 3.64 (q, 1H), 3.56 - 3.34 (m, 3H), 3.19 (dt, 1H), 3.12 - 2.95 (m, 1H), 2.90 (dd, 1H), 2.84 (s, 1H), 2.69 - 2.35 (m, 3H), 2.27 (d, 6H), 2.18 (t, 1H), 2.06 - 1.35 (m, 13H), 1.35 - 1.08 (m, 12H), 1.09 - 0.83 (m, 4H).

[0287] (HR,13R,14R,15R)-14-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,ll,13,15,17 -pentamethyl-l,4,19-trioxa-9- azadispiro[4.2.128.25]docosane-16, 18-dione (Sl-6-I10-2)(Compound 75). Prepared according to the methods of SI-6-11-2 from 110 and formaldehyde. Formate salt. MS (ESI+) mlz'. 613.32 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-d4) 84.48 - 4.15 (m, 2H), 4.05 - 3.85 (m, 5H), 3.72 (d, 1H), 3.63 - 3.49 (m, 2H), 3.48 - 3.34 (m, 1H), 3.30 - 3.16 (m, 1H), 3.04 - 2.89 (m, 3H), 2.72 - 2.49 (m, 2H), 2.43 (s, 1H), 2.35 (d, 9H), 2.21 - 2.03 (m, 1H), 2.03 - 1.42 (m, 11H), 1.42 - 1.13 (m, 14H), 1.05 - 0.94 (m, 2H), 0.89 (d, 1H).

Scheme S2.

[0288] Benzyl (9R,1 lR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimethylami no)-6- methyltetrahydro-2H-pyran-2-yl)oxy)- 11 -methoxy-7, 9, 11, 13, 15, 15 -hexamethyl- 14, 16-dioxo- 17- oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (S2-1-I1-1). SI-5-11-1 (382 mg, 0.481 mmol) was dissolved in 1,2-dimethoxy ethane (4.8 mL), and the reaction mixture was cooled to -78 °C in a dry ice/acetone bath. Potassium bis(trimethylsilyl)amide (1.0 M solution in THF; 0.625 mL, 0.625 mmol) was added. After 25 min, dimethyl sulfate (0.091 mL, 0.962 mmol) was added. The dry ice was removed from the acetone bath, and the reaction mixture was allowed to slowly warm to 5 °C over 50 min. The reaction was quenched by the addition of NH4C1 (sat., aq. solution) and was diluted with EtOAc. The EtOAc layer was washed with water (2 times) and brine (1 time), was dried over Na2SO4, was filtered, and was concentrated. The residue was purified on 40 g of silica gel (elution with 4-15% MeOH-DCM-0.5% NH4OH gradient) to give S2-1-I1-1 (260 mg, 67%) . MS (ESI+) m/z: 808.35 [M + H]+. 1H NMR (400 MHz, Chloroform-d) 6 8.01 - 7.91 (m, 2H), 7.49 (t, 1H), 7.37 (t, 2H), 7.35 - 7.17 (m, 5H), 5.15-5.00 (m, 3H), 4.58 (br d, 1H), 4.10-90 (m, 2H), 3.80-3.50 (m, 2H), 3.20-2.90 (m, 5H), 2.86 (s, 3H), 2.50-2.00 (m, 11H), 2.00-1.80 (m, 1H), 1.80-1.55 (m, 2 H), 1.49 - 1.32 (m, 1H), 1.31 (s, 3H), 1.29 - 1.05 (m, 12H), 1.03 - 0.860 (m, 5H), 0.85-0.66 (m, 4H).

Scheme S3.

[0289] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((9R,llR,12R,13R)-ll-meth oxy-7,9,77,13,15- pentamethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecan -12-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S3-1-I1-1-1). SI-5-11-1 (224 mg, 0282 mmol) was dissolved in methanol (5.6 mL) and 1 N aqueous HC1 (0.846 mL, 0.846 mmol), and palladium on carbon (5 wt%, 60 mg, 0.028 mmol) was added. An atmosphere of H2 was introduced. After 4 h, the reaction mixture was evacuated and back-filled with N2 (3 times). The reaction mixture was filtered through Celite®, washing with MeOH, and the filtrate was concentrated. This gave the HC1 salt of S3-1-I1-1-1 (226 mg, 100%), which was used without further purification. MS (ESI+) mlz'. 660.29 [M + H] ⁺ .

[0290] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((9R,llR,12R,13R)-ll-meth oxy-7,9,ll,13,15,15- hexamethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecan- 12-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S3-1-I1-1-2). Prepared according to the methods of

S3-1-I1-1-1, substituting S2-2-I1-1. This gave the HC1 salt of S3-1-I1-1-2 , which was used without further purification. MS (ESI+) mlz.' 808.33 [M + H] ⁺ .

[0291] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15-p entamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-2-Il-l-l-l)(Compound 7). S3-1-I1-1-1 (14.7 mg, 0.0191 mmol) was dissolved in methanol (1 mL), and the reaction mixture was heated to 60 °C external temperature. After 3 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCO2H) to give S3-2-I1-1-1-1 (3.61 mg). MS (ESI+) mlz\ 556.32 [M + H] ⁺ . Formate salt, mixture of C2 epimers, 'H NMR (400 MHz, Methanol-d4) 8 8.38 (br s, 3H), 4.46 - 4.21 (m, 2H), 4.19 - 3.99 (m, 2H), 3.96 - 3.77 (m, 1H), 3.67 - 3.50 (m, 2H), 3.40 - 3.20 (m, 2H), 3.18 - 3.08 (m, 2H), 3.08 - 2.97 (m, 2H), 2.94 (s, 1H), 2.89 (s, 2H), 2.80-2.63 (m, 7H), 2.63 - 2.38 (m, 3H), 2.34 - 2.15 (m, 2H), 2.05 -1.80 (m, 3H), 1.79 - 1.52 (m, 3H), 1.49 - 1.30 (m, 3H), 1.29 - 1.09 (m, 12H), 0.95 - 0.77 (m, 3H).

[0292] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-2-Il-l-2-l)(Compound 44). Prepared according to the methods of S3-2-I1-1-1-1 from S3-1-I1-1-2 to provide the title compound (1.52 mg). Formate salt. MS (ESI+) m/z.' 570.33 [M + H] ⁺ . ’H NMR (400 MHz, Methanol-d4) 8 8.42 (br s, 3H), 4.69 - 4.30 (m, 5H), 4.11 - 3.99 (m, 1H), 3.67 - 3.55 (m, 1H), 3.39 - 3.29 (m, 1H), 3.18 - 3.01 (m, 4H), 2.99 - 2.80 (m, 5H), 2.65 (s, 6H), 2.53 - 2.23 (m, 3H), 2.11 - 1.95 (m, 3H), 1.94-1.84 (m, 1H), 1.82 - 1.53 (m, 3H), 1.44 (s, 3H), 1.41 -1.32 (m, 1H), 1.28 (s, 3H), 1.26 - 1.10 (m, 12H), 0.98 - 0.72 (m, 4H).

[0293] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3,7,9,ll,13,1 5-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-2-Il-l-l-2)(Compound 4). S3-1-I1-1-1 (30 mg, 0.039 mmol) was dissolved in DCM (1 mL) and triethylamine (0.016 mL, 0.116 mmol), and Na(OAc)3BH (24.5 mg, 0.116 mmol) was added. Formaldehyde (37 wt% aqueous solution, 0.0157 mL, 0.194 mmol) was added. After 15 min, the reaction mixture was quenched with NaHCCh (sat., aq. solution) and was extracted with DCM (3 times). The combined extracts were concentrated. The crude material was dissolved in methanol (1 mL), and the reaction mixture was heated to 60 °C external temperature. After 3 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give S3-2-I1-1-1-2 (3.36 mg). MS (ESI+) m/z: 570.33 [M + H] ⁺ . Formate salt, mixture of C2 epimers, ¹ H NMR (400 MHz, Methanol-d4) 8 8.34 (br s, 2H), 4.45 - 4.31 (m, 2H), 4.19 - 4.01 (m, 1H), 3.89 - 3.77 (m, 0.5H), 3.70 - 3.54 (m, 1.5H), 3.41 - 3.27 (m, 4H), 3.14 - 3.06 (m, 1H), 3.04 - 2.83 (m, 6H), 2.80 - 2.57 (m, 11H), 2.53 - 2.23 (m, 4H), 2.20 - 1.80 (m, 3H), 1.79 - 1.62 (m, 2H), 1.49 - 1.33 (m, 2H), 1.32 - 1.08 (m, 13H), 1.01 - 0.80 (m, 3H).

[0294] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Z)z7nefZty/znMz7io)-3 -hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15- pentamethyl-3-((l-methyl- lH-imidazol-4-yl)methyl)-17-oxa-3,7-diazaspiro[5.12]octadeca ne-14, 16-dione (S3-2-I1-1-1- 3)(Compound 5). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1-1-1 and 1- methyl-lH-imidazole-4-carboxaldehyde (2.33 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 650.37 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 8 8.44 (s, 1H), 7.46 (s, 1H), 6.93 (s, 1H), 4.60 - 4.42 (m, 0.7H), 4.42 - 4.20 (m, 1.3H), 4.20 - 4.00 (m, 1H), 3.99 - 3.77 (m, 1H), 3.61 (s, 3H), 3.55 - 3.36 (m, 3H), 3.20 - 3.05 (m, 2H), 2.99 - 2.82 (m, 4H), 2.81 - 2.55 (m, 4H), 2.54 - 2.08 (m, 12H), 2.03 - 1.90 (m, 1H), 1.85 - 1.44 (m, 5H), 1.44 - 1.05 (m, 14H), 1.04 - 0.85 (m, 2H), 0.84 - 0.66 (m, 2H).

[0295] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Z)i>M^jZaffM>io )-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-isopropyl-ll-methoxy-7 ,9,ll,13,15-pentamethyl-17- oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-l-4)(Compound 6). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1-1-1 and acetone to provide the title compound (3.87 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlr. 598.37 [M + H] ⁺ . ^r H NMR (400 MHz, Methanol-d4) 8 8.45 (s, 1H), 4.60 - 4.43 (m, 0.5H), 4.42 - 4.20 (m, 1.5H), 4.18

- 4.00 (m, 1H), 3.98 - 3.74 (m, 1H), 3.62 - 3.44 (m, 1H), 3.36 - 3.20 (m, 2H), 3.07 - 2.60 (m, 10H), 2.59 - 2.09 (m, 11H), 2.10 - 1.84 (m, 2H), 1.84 - 1.45 (m, 5H), 1.43 - 1.06 (m, 18H), 1.05

- 0.68 (m, 5H).

[0296] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3,7,9,ll ₉ 13,15,15-heptamethyl-17-oxa- 3, 7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-2)(Compound 78). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1-1-2 and formaldehyde to provide the title compound (5.73 mg). Formate salt. MS (ESI+) mlz\ 584.38 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 8 8.44 (s, 2H), 5.06 - 4.92 (m, 1H), 4.63 - 4.51 (m, 1H), 4.48 (d, 1H), 4.21 (d, 1H), 3.74 (dt, 3.4 Hz, 1H), 3.51 - 3.34 (m, 3H), 3.27 - 3.16 (m, 4H), 3.05 (s, 3H), 2.98 - 2.86 (m, 3H), 2.82 (s, 6H), 2.78 - 2.62 (m, 4H), 2.63 - 2.54 (m, 3H), 2.42 - 2.19 (m, 2H), 2.09 - 1.99 (m, 2H), 1.87 (d, 1H), 1.82 - 1,72 (m, 1H), 1.59 - 1.45 (m, 4H), 1.43 - 1.30 (m, 12H), 1.07 (br d, 3H).

[0297] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Zh>neZZryZawHno)-3 -hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-isobutyl-ll-methoxy-7, 9,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-3)(Compound 79). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1-1-2 and isobutyraldehyde to provide the title compound (7.73 mg). Formate salt. MS (ESI+) mlz\ 626.48 [M + H] ⁺ . ^X H NMR (400 MHz, Methanol-d4) 5 8.40 (s, 1.5H), 5.06 - 4.92 (m, 1H), 4.58 (d, 1H), 4.48 (d, 1H), 4.21 (d, 1H), 3.79 - 3.70 (m, 1H), 3.51 - 3.38 (m, 2H), 3.37 - 3.33 (m, 1H), 3.25 - 3.10 (m, 2H), 3.06 (s, 3H), 3001 - 2.91 (m, 3H), 2.90 - 2.78 (m, 7H), 2.75 - 2.63 (m, 4H), 2.61 - 2.46 (m, 3H), 2.38 - 2.23 (m, 2H), 2.12 - 1.96 (m, 3H), 1.91 - 1.78 (m, 2H), 1.59 - 1.45 (m, 4H), 1.44 - 1.34 (m, 9H), 1.33 (d, 3H), 1.08 (d, 3H), 1.02 (dd, 6H).

[0298] (9R,llR,12R,13R)-3-Benzyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexaniethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-2-Il-l-2-4)(Compound 80). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1-1-2 and benzaldehyde to provide the title compound (7.46 mg). Formate salt. MS (ESI+) mlz\ 660.45 [M + H] ⁺ . ^X H NMR (400 MHz, Methanol-d4) 5 8.48 (s, 3H), 7.39 - 7.25 (m, 5H), 5.01 - 4.88 (m, 1H), 4.56 (d, 1H), 4.47 (d, 1H), 4.21 (d, 1H), 3.79 - 3.67 (m, 1H), 3.60 (br s, 2H), 3.50 - 3.33 (m, 3H), 3.23 - 3.09 (m, 2H), 3.04 (s, 3H), 3.02 - 2.93 (m, 2H), 2.89 (br s, 3H), 2.84 - 2.73 (m, 8H), 2.39 - 2.19 (m, 4H), 2.07 - 1.98 (m, 1H), 1.94 (d, 1H), 1.80 (t, 2H), 1.58 - 1.44 (m, 4H), 1.43 - 1.25 (m, 12 H), 1.07 (d, 3H).

[0299] (9R,llR,12R,13R)-3-([l,l'-biphenyl]-3-ylmethyl)-12-(((2S,3R, 4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-2-I1-1-2- 5)(Compound 116). Prepared according to the methods of S3-2-I1 - 1 - 1-2 from S3- 1 -Il - 1 -2 and 3- phenylbenzaldehyde. 18.1 mg Formate salt. MS (ESI+) mlz'. 736.5 [M + H] ⁺ ; 'H NMR. (400 MHz, MeOD) 5 = 7.64 - 7.58 (m, 3H), 7.56 - 7.51 (m, 1H), 7.46 - 7.38 (m, 3H), 7.37 - 7.29 (m, 2H),

4.40 (d, 1H), 4.25 - 4.15 (m, 1H), 4.12 - 3.98 (m, 2H), 3.80 - 3.74 (m, 1H), 3.70 - 3.49 (m, 3H), 3.47 - 3.35 (m, 1H), 3.28 - 3.21 (m, 1H), 3.11 (s, 1H), 3.05 - 2.85 (m, 4H), 2.80 - 2.57 (m, 3H),

2.40 - 2.21 (m, 13H), 2.05 - 1.78 (m, 4H), 1.78 - 1.64 (m, 3H), 1.54 (br s, 1H), 1.49 (s, 3H), 1.46 - 1.31 (m, 4H), 1.31 - 1.17 (m, 12H), 1.16 - 1.05 (m, 1H), 1.05 - 0.77 (m, 4H).

[0300] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3-(4-methoxyb enzyl)-7,9,ll,13,15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-6)(Compound

120). Prepared according to the methods of S3-2-I1 - 1 -1 -2 from S3-1-I1-1-2 and 4- methoxybenzaldehyde. 24.0 mg Formate salt. MS (ESI+) mlz'. 690.5 [M + H] ⁺ ; ^J H NMR (400 MHz, ACETONITRILES) 8 = 8.33 (br s, 2H), 7.26 - 7.19 (m, 2H), 6.92 - 6.86 (m, 2H), 4.55 - 4.44 (m, 1H), 4.43 - 4.35 (m, 2H), 4.08 (d, 1H), 3.77 (s, 3H), 3.58 (br dd, 1H), 3.50 (s, 2H), 3.25 - 3.17 (m, 2H), 2.96 (s, 3H), 2.86 - 2.67 (m, 7H), 2.62 (br s, 6H), 2.38 (s, 9H), 2.29 - 2.17 (m, 5H), 2.15 - 1.97 (m, 5H), 1.79 (br d, 2H), 1.63 (br d, 1H), 1.53 (br s, 2H), 1.45 (s, 3H), 1.34 - 1.28 (m, 9H), 1.28 - 1.17 (m, 5H), 0.97 (br d, 3H).

[0301] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(isoquinolin-7-ylmethy l)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-2-I1-1-2- 7)(Compound 121). Prepared according to the methods of S3-2-I1 - 1 - 1-2 from S3- 1 -Il - 1 -2 and isoquinoline-7-carboxaldehyde. 12.6 mg Formate salt. MS (ESI+) mlz'. 711.5 [M + H] ⁺ ; 'H NMR (400 MHz, CD ₃ OD) δ = 9.21 (s, 1H), 8.41 (d, 1H), 8.03 (s, 1H), 7.95 - 7.91 (m, 1H), 7.86 - 7.80 (m, 2H), 4.40 (d, 1H), 4.21 (d, 1H), 4.15 - 3.95 (m, 2H), 3.76 (s, 2H), 3.67 - 3.47 (m, 2H), 3.40

(quin, 1H), 3.29 - 3.24 (m, 1H), 3.12 - 2.98 (m, 1H), 2.95 (s, 2H), 2.94 - 2.83 (m, 1H), 2.83 - 2.68 (m, 2H), 2.68 - 2.43 (m, 2H), 2.43 - 2.33 (m, 5H), 2.33 (s, 6H), 2.30 - 2.25 (m, 2H), 2.06 - 1.86

(m, 3H), 1.84 (br d, 1H), 1.77 - 1.66 (m, 3H), 1.55 (br s, 1H), 1.49 (s, 3H), 1.40 (br s, 1H), 1.37 (s,

3H), 1.35 - 1.15 (m, 15H), 1.03 - 0.81 (m, 6H).

[0302] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15,1 5-hexamethyl-3-phenethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-H-l-2-8)(Compound 124). Prepared according to the methods of S3-2-I1 - 1-1 -2 from S3-1-I1-1-2 and phenyl acetaldehyde. 19.2 mg Formate salt. MS (ESI+) mfr. 674.5 [M + H] ⁺ ; ^L H NMR (400 MHz, MeOD) 8 = 7.30 - 7.23 (m, 2H), 7.23 - 7.14 (m, 3H), 4.43 - 4.37 (m, 1H), 4.20 (br d, 1H), 4.14 - 3.98 (m, 2H), 3.78 (s, 1H), 3.56 (ddd, 1H), 3.42 (quin, 1H), 3.27 (dd, 1H), 3.17 - 3.01 (m, 1H), 3.01 - 2.87 (m, 3H), 2.87 - 2.67 (m, 4H), 2.67 - 2.56 (m, 3H), 2.39 - 2.25 (m, 11H), 2.18 - 1.80 (m, 4H), 1.80 - 1.62 (m, 3H), 1.55 (br d, 1H), 1.52 - 1.35 (m, 5H), 1.33 - 1.13 (m, 11H), 0.96 - 0.79 (m, 3H).

[0303] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(pyridin-4- ylmethyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-9)(Compound 126). Prepared according to the methods of S3-2-I1 - 1 -1 -2 from S3-1-I1-1-2 and pyridine-4- carboxaldehyde. 23.1 mg Formate salt. MS (ESI+) m/z'. 661.5 [M + H] ⁺ ; ¹ H NMR (400 MHz, ACETONITRILE-ds) 5 = 8.51 (br d, 2H), 8.27 (br s, 2H), 7.31 (br d, 2H), 4.68 - 4.56 (m, 1H), 4.51 - 4.34 (m, 2H), 4.16 - 4.09 (m, 1H), 3.63 - 3.59 (m, 1H), 3.58 (s, 2H), 3.37 - 3.12 (m, 4H), 3.11 - 3.02 (m, 2H), 2.98 (s, 4H), 2.95 - 2.74 (m, 20H), 2.73 - 2.52 (m, 11H), 2.49 (s, 6H), 2.29 (br t, 3H), 2.20 - 2.06 (m, 3H), 2.05 - 1.97 (m, 2H), 1.89 - 1.79 (m, 2H), 1.66 (br d, 2H), 1.48 - 1.15 (m, 16H), 1.02 (d, 2H).

[0304] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(pyridin-3- ylmethyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-10)(Compound 128). Prepared according to the methods of S3-2-I1 - 1 -1 -2 from S3-1-I1-1-2 and pyridine-3- carboxaldehyde. 16.0 mg Formate salt. MS (ESI+) mlz\ 661.5 [M + H] ⁺ ; 'H NMR (400 MHz, ACETONITRILE-ds) 8 = 8.51 (d, 1H), 8.47 (dd, 1H), 8.29 (s, 2H), 7.72 - 7.66 (m, 1H), 7.31 (dd, 1H), 4.60 - 4.49 (m, 1H), 4.46 - 4.36 (m, 2H), 4.12 - 4.08 (m, 1H), 3.65 - 3.61 (m, 1H), 3.61 - 3.58 (m, 1H), 3.57 (s, 2H), 3.28 - 3.16 (m, 2H), 3.16 (s, 1H), 3.02 (s, 1H), 2.96 (s, 3H), 2.88 (br s, 1H), 2.84 - 2.70 (m, 6H), 2.66 (br s, 4H), 2.52 (br s, 45H), 2.47 - 2.37 (m, 18H), 2.27 (br t, 4H), 2.14 - 2.02 (m, 3H), 1.98 (br d, 1H), 1.92 - 1.72 (m, 4H), 1.70 - 1.54 (m, 3H), 1.51 (br d, 1H), 1.45 (s, 3H), 1.39 (d, 1H), 1.34 - 1.28 (m, 9H), 1.25 - 1.15 (m, 5H), 1.06 - 0.91 (m, 4H).

[0305] (9R,llR,12R,13R)-3-(4-chlorobenzyl)-12-(((2S,3R,4S,6R)-4-(di methylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-ll)(Compound 129).

Prepared according to the methods of S3 -2-11-1 -1 -2 from S3 - 1 -II- 1 -2 and 4-chlorobenzaldehyde. 13.5 mg Formate salt. MS (ESI+) m!z-. 694.4 [M + H] ⁺ ; ^J H NMR (400 MHz, CD ₃ OD) 6 = 7.32 (s, 4H), 4.40 (d, 1H), 4.18 (d, 1H), 3.98 - 3.98 (m, 1H), 3.60 - 3.46 (m, 3H), 3.40 (quin, 1H), 3.27 (dd, 1H), 3.11 (s, 1H), 2.95 (s, 3H), 2.71 - 2.57 (m, 3H), 2.33 (s, 9H), 2.30 - 2.16 (m, 4H), 1.99 - 1.79 (m, 3H), 1.79 - 1.63 (m, 3H), 1.54 - 1.44 (m, 4H), 1.37 (s, 3H), 1.34 - 1.16 (m, 10H), 0.94 - 0.81 (m, 3H).

[0306] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(isoquinolin-6-ylmethy l)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-2-I1-1-2- 12D)(Compound 130). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I1 -1-2 and isoquinoline-6-carboxaldehyde. 21.0 mg Formate salt. MS (ESI+) mfr. 711.5 [M + H] ⁺ ; 'll NMR (400 MHz, CD3OD) δ = 9.21 (s, 1H), 8.42 (d, 1H), 8.08 (d, 1H), 7.88 (s, 1H), 7.81 (d, 1H), 7.74 (dd, 1H), 4.40 (d, 1H), 4.21 (d, 1H), 4.13 - 3.96 (m, 2H), 3.76 (s, 2H), 3.55 (dqd, 1H), 3.40 (quin, 1H), 3.26 (dd, 1H), 2.95 (s, 3H), 2.72 (br dd, 2H), 2.61 (ddd, 1H), 2.40 - 2.34 (m, 4H), 2.32 (s, 6H), 2.32 - 2.23 (m, 3H), 2.03 - 1.90 (m, 2H), 1.84 (br d, 1H), 1.79 - 1.64 (m, 3H), 1.57 - 1.46 (m, 4H), 1.41 (br s, 1H), 1.37 (s, 3H), 1.31 - 1.14 (m, 11H), 0.84 (d, 3H).

[0307] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(2- (pyridin-3-yl)ethyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-1 4, 16-dione (S3-2-I1-1-2- 13)(Compound 134). Prepared according to the methods of S3-2-I1 -1 -1 -2 from S3- 1 -Il -1 -2 and 3 -pyridineacetaldehyde. 22.8 mg Formate salt. MS (ESI+) mfr. 675.5 [M + H] ⁺ ; 'H NMR (400 MHz, CDCh) 5 = 8.43 (d, 1H), 8.38 (dd, 1H), 7.74 (td, 1H), 7.37 (ddd, 1H), 4.44 - 4.38 (m, 1H), 4.21 (d, 1H), 4.12 - 3.98 (m, 2H), 3.78 (s, 1H), 3.70 (s, 1H), 3.60 - 3.52 (m, 1H), 3.42 (quin, 1H), 3.27 (dd, 1H), 3.15 - 3.02 (m, 1H), 3.02 - 2.91 (m, 3H), 2.90 - 2.76 (m, 4H), 2.67 - 2.58 (m, 3H), 2.41 - 2.23 (m, 10H), 2.02 - 1.81 (m, 4H), 1.80 - 1.67 (m, 2H), 1.49 (s, 4H), 1.38 (s, 3H), 1.32 - 1.17 (m, 11H), 0.96 - 0.80 (m, 3H).

[0308] (9R,llR,12R,13R)-3-(3-chlorobenzyl)-12-(((2S,3R,4S,6R)-4-(di methylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-Il-l-2-14)(Compound 135).

Prepared according to the methods of S3 -2-11-1 - 1 -2 from S3 - 1 -II- 1 -2 and 3 -chlorobenzaldehyde 17.3 mg Formate salt. MS (ESI+) mfr. 694.5 [M + H] ⁺ ; ^ NMR (400 MHz, CD ₃ OD) δ = 7.41 - 7.22 (m, 4H), 4.40 (d, 1H), 4.19 (d, 1H), 4.10 - 3.98 (m, 1H), 3.80 - 3.73 (m, 1H), 3.56 (td, 1H), 3.52 (s, 2H), 3.46 - 3.33 (m, 1H), 3.29 - 3.23 (m, 1H), 3.12 - 3.01 (m, 1H), 2.96 (s, 2H), 2.93 - 2.71 (m, 1H), 2.71 - 2.57 (m, 2H), 2.34 (s, 3H), 2.33 (s, 5H), 2.30 - 2.23 (m, 3H), 2.00 - 1.81 (m, 3H), 1.80 - 1.62 (m, 3H), 1.53 - 1.45 (m, 3H), 1.37 (s, 3H), 1.33 - 1.15 (m, 12H), 1.04 - 0.91 (m, 1H), 0.91 - 0.82 (m, 3H).

[0309] (9R,llR,12R,13R)-3-([l,l'-biphenyl]-4-ylmethyl)-12-(((2S,3R, 4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-2-I1-1-2- 5)(Compound 139). Prepared according to the methods of S3-2-I1 - 1 - 1-2 from S3- 1 -Il - 1 -2 and 4- phenylbenzaldehyde. 25.4 mg Formate salt. MS (ESI+) mlz'. 736.5 [M + H] ⁺ ; 'H NMR (400 MHz, MeOD) 8 = 7.65 - 7.57 (m, 4H), 7.47 - 7.38 (m, 4H), 7.37 - 7.30 (m, 1H), 4.83 (s, 11H), 4.40 (br d, J = 7.3 Hz, 1H), 4.20 (br d, J = 11.7 Hz, 1H), 4.12 - 4.01 (m, 2H), 3.61 - 3.48 (m, 3H), 3.45 - 3.35 (m, 1H), 3.35 - 3.24 (m, 5H), 2.95 (s, 3H), 2.80 - 2.68 (m, 2H), 2.68 - 2.55 (m, 1H), 2.34 (br s, 4H), 2.32 (s, 6H), 2.28 (br d, J = 8.6 Hz, 2H), 1.99 - 1.80 (m, 3H), 1.74 (br d, J = 11.1 Hz, 2H), 1.68 - 1.55 (m, 1H), 1.38 (s, 3H), 1.31 - 1.19 (m, 11H), 0.85 (br d, J = 6.5 Hz, 3H).

[0310] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(DiWZ/zj;Z«mino)-3-hy droxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3- (methylsulfonyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-3-Il-l-2-l)( (Compound 82). S3-1-I1-1-2 (21 mg, 0.027 mmol) was dissolved in DCM (1 mL). EtsN (14.8 pL, 0.107 mmol) and MsCl (4.2 pL, 0.054 mmol) were added at rt. The reaction mixture was allowed to stir at rt for 1 h. The reaction was quenched by adding saturated NaHCOs (2 mL) and the aqueous layer was extracted with DCM three times (2 mL). The combined organic layers were dried over MgSCU, filtered and concentrated. The residue was used in the next step without further purification. MS (ESI+) mlz'. 376.7 [M + 2H] ²⁺ , 752.4 [M + H] ⁺ . The material was dissolved in MeOH (0.5 mL) and heated at 60 °C for 16 h. The reaction mixture was filtered through a syringe filter with the aid of methanol and was concentrated. The residue was purified by HPLC (MeCN- water- 0.1% HCO2H) to yield the title compound. Formate salt (2.90 mg). MS (ESI+) mlz-. 324.7 [M + 2H] ²⁺ , 648.4 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 5 8.56 (s, 2H), 4.60 (s, 1H), 4.48 (d, 1H), 4.19 (s, 1H), 3.76 - 3.66 (m, 1H), 3.40 (d, 1H), 3.33 (p, 15H), 3.08 (d, 4H), 2.90 (s, 3H), 2.72 (d, 1H), 2.68 (s, 4H), 2.10 (d, 2H), 1.96 (d, 2H), 1.49 - 1.29 (m, 13H), 0.98 (d, 5H).

[0311] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15,1 5-hexamethyl-3-(pyridin-3- ylsidfbnyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-3-Il-l-2-2)(Compound 127). Prepared according to the methods of S3-3-I1-1-2-1 from S3-1-I1-1-2 and pyridine-3- sulfonyl chloride. 21.8 mg Formate salt. MS (ESI+) mlz'. 711.4 [M + H] ⁺ ; 'H NMR (400 MHz, MeOD) 5 = 8.93 (dd, 1H), 8.82 (dd, 1H), 8.21 (ddd, 1H), 7.67 (ddd, 1H), 4.42 (d, 1H), 4.09 - 4.01 (m, 2H), 3.88 (br d, 1H), 3.60 - 3.51 (m, 1H), 3.37 (quin, 1H), 3.28 (dd, 1H), 3.17 - 3.05 (m, 4H), 2.98 (s, 3H), 2.64 (ddd, 1H), 2.34 (s, 6H), 2.25 - 2.12 (m, 6H), 1.96 - 1.88 (m, 1H), 1.74 (ddd, 2H), 1.67 - 1.54 (m, 3H), 1.53 (s, 3H), 1.35 (s, 3H), 1.33 - 1.17 (m, 13H), 0.62 (br d, 3H).

[0312] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3- (phenylsulfonyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-3-I1-1-2- 3)(Compound 131). Prepared according to the methods of S3-3-H-1-2-1 from S3- 1 -Il - 1 -2 and benzenesulfonyl chloride. 18.7 mg Formate salt. MS (ESI+) mlz'. 710.5 [M + H] ⁺ ; 'H NMR (400 MHz, CD3OD) δ = 7.79 (d, 2H), 7.70 - 7.65 (m, 1H), 7.64 - 7.59 (m, 2H), 4.41 (d, 1H), 4.05 (d, 1H), 4.00 (d, 1H), 3.86 (d, 1H), 3.59 - 3.51 (m, 1H), 3.41 - 3.32 (m, 2H), 3.27 (dd, 1H), 3.14 - 2.99 (m, 4H), 2.97 (s, 3H), 2.62 (ddd, 1H), 2.33 (s, 6H), 2.24 - 2.15 (m, 5H), 2.12 (br dd, 1H), 1.93 - 1.86 (m, 1H), 1.73 (ddd, 2H), 1.66 - 1.56 (m, 2H), 1.55 - 1.48 (m, 4H), 1.39 - 1.17 (m, 15H), 0.63 (d, 3H).

[0313] (9R,llR,12R,13R)-3-((4-chlorophenyl)sulfonyl)-12-(((2S,3R,4S ,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,ll,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octade cane-14, 16-dione (S3-3-I1-1-2- 4)(Compound 132). Prepared according to the methods of S3-3-I1-1-2-1 from S3- 1 -Il - 1 -2 and 3- chlorophenylsulfonyl chloride. 26.5 mg Formate salt. MS (ESI+) mlz'. 744.4 [M + H] ⁺ ; ^r H NMR (400 MHz, CD ₃ OD) 6 = 7.79 - 7.75 (m, 2H), 7.65 - 7.60 (m, 2H), 4.42 (d, 1H), 4.06 (d, 1H), 4.03 (d, 1H), 3.87 (d, 1H), 3.60 - 3.51 (m, 1H), 3.37 (br quin, 1H), 3.28 (dd, 1H), 3.14 - 3.01 (m, 4H),

2.98 (s, 3H), 2,62 (ddd, 1H), 2.33 (s, 6H), 2,20 (s, 4H), 1.94 - 1.86 (m, 1H), 1.74 (ddd, 2H), 1.61 (br d, 2H), 1.56 - 1.49 (m, 4H), 1.35 (s, 3H), 1.31 - 1.19 (m, 12H), 0.63 (d, 3H).

[0314] (9R,llR,12R,13R)-3-(benzylsulfonyl)-12-(((2S,3R,4S,6R)-4-(di methylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-3-H-l-2-5)(Compound 133). Prepared according to the methods of S3-3-I1-1-2-1 from S3-1-I1-1-2 and phenylmethyl sulfonyl chloride.

29.1 mg Formate salt. MS (ESI+) mlz-. 724.5 [M + H] ⁺ ; ^NMR (400 MHz, ACETONITRILE- d ₃ ) 5 = 8.33 (s, 2H), 7.45 - 7.35 (m, 5H), 4.42 (d, 1H), 4.26 (s, 2H), 4.25 - 4.17 (m, 2H), 4.05 -

3.99 (m, 1H), 3.91 - 3.72 (m, 1H), 3.71 - 3.34 (m, 16H), 3.34 - 3.23 (m, 3H), 3.16 - 2.96 (m, 3H), 2.94 (s, 3H), 2.92 - 2.87 (m, 1H), 2.61 - 2.51 (m, 1H), 2.49 (s, 6H), 2.44 (s, 3H), 2.41 - 2.18 (m, 2H), 1.92 - 1.72 (m, 4H), 1.71 - 1.61 (m, 2H), 1.54 (br d, 1H), 1.49 (s, 3H), 1.42 - 1.16 (m, 14H), 1.00 - 0.94 (m, 1H), 0.91 (d, 2H).

[0315] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3-((4-methoxy phenyl)sulfonyl)- 7,9,ll,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octade cane-14, 16-dione (S3-3-I1-1-2- 6)(Compound 136). Prepared according to the methods of S3-3-I1-1-2-1 from S3- 1 -Il - 1 -2 and 4- methoxyphenyl sulfonyl chloride. 26.4 mg Formate salt. MS (ESI+) mlz'. 740.5 [M + H] ⁺ ;

NMR (400 MHz, CD ₃ OD) δ = 7.74 - 7.69 (m, 2H), 7.13 - 7.09 (m, 2H), 4.42 (d, 1H), 4.05 (d, 1H), 4.01 (d, 1H), 3.89 (s, 4H), 3.59 - 3.51 (m, 1H), 3.41 - 3.33 (m, 1H), 3.30 - 3.25 (m, 1H), 3.13 - 3.04 (m, 2H), 3.03 - 2.91 (m, 5H), 2.62 (ddd, 1H), 2.33 (s, 6H), 2.25 - 2.14 (m, 5H), 2.14 - 2.07 (m, 1H), 1.93 - 1.86 (m, 1H), 1.77 - 1.70 (m, 2H), 1.67 - 1.56 (m, 2H), 1.56 - 1.47 (m, 4H), 1.35 (s, 3H), 1.30 - 1.20 (m, 11H), 0.65 (d, 3H).

[0316] (9R,llR,12R,13R)-3-Acetyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15- pentamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-4-I1-1-1-1) (Compound 3). S3-1-I1-1-1 (30 mg, 0.039 mmol) was dissolved in DCM (1 mL) and triethylamine (0.016 mL, 0.116 mmol), and acetic anhydride (0.011 mL, 0.116 mmol) was added. After 45 min, the reaction mixture was quenched with NaHCOs (sat., aq. solution) and was extracted with DCM (2 times). The combined extracts were concentrated. The crude material was dissolved in methanol (1 mL), and the reaction mixture was heated to 60 °C external temperature. After 3 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCO ₂ H) to give S3-4-I1-1-1-1 (9.81 mg). MS (ESI+) m/z: 598.35 [M + H] ⁺ . Formate salt, mixture of C2 epimers, 'H NMR (400 MHz, Methanol-d4) 6 8.44 (br s, 1H), 4.40 - 4.30 (m, 1H), 4.28 - 4.16 (m, 1H), 4.16 - 4.05 (m, 1H), 4.03 - 3.80 (m, 2H), 3.67 - 3.42 (m, 2H), 3.42 - 3.27 (m, 2H), 3.02 - 2.74 (m, 5H), 2.56 - 2.18 (m, 10H), 2.18 - 2.06 (m, 1H), 2.05 - 1.88 (m, 5H), 1.87 - 1.53 (m, 4H), 1.36 - 1.00 (m, 16H), 0.88 - 0.68 (m, 4H).

[0317] (9R,llR,12R,13R)-3-(L-Prolyl)-12-(((2S,3R,4S,6R)-4-(dimethyl amino)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-4-Il-l-2-l)(Compound 56). S3-1-I1-1-2 (63 5 mg, 0.081 mmol) was dissolved in DCM (0.4 mL) and A,A-diisopropylethylamine (0.0563 mL, 0.324 mmol), and 1 -hydroxybenzotriazole (14.1 mg, 0.105 mmol), Cbz-L-Proline (24.2 mg, 0.0971 mmol), and XA-di isopropyl carbodi imide (0.0149 mL, 0.0971 mmol) were added. After 26 h, the reaction mixture was concentrated under reduced pressure, and the residue was purified on 12 g of silica gel (elution with 2-10% MeOH-DCM-0.5% NH4OH gradient) to give the amide intermediate (79.4 mg, more than 100%) as a thick oil contaminated with some minor impurities. MS (ESI+) mlz\ 905.43 [M + H]“. The material was dissolved in methanol (1 mL) and 4 M HC1 in 1,4-dioxane (0.060 mL, 0.240 mmol), and palladium on carbon (5 wt%, 0.86 mg, 0.008 mmol) was added. An atmosphere of H2 was introduced. After 4 h, the reaction mixture was evacuated and back-filled with N2 (3 times). The reaction mixture was filtered through Celite®, washing with MeOH, and the filtrate was concentrated to give the crude amine intermediate (77.0 mg) . MS (ESI+) mlz'. 771.34 [M + H]“. The amine intermediate (31.1 mg, 0.040 mmol) was dissolved in methanol (1 mL) and triethylamine (0.017 mL, 0.12 mmol), and the reaction mixture was heated to 65 °C external temperature. After 4 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give S3-4-I1-1-2-1 (15.1 mg). MS (ESI+) mlz-. 667.39 [M + H] ⁺ . Formate salt. 'H NMR (400 MHz, Methanol-d4) 8 8.51 (s, 2H), 5.11 - 4.95 (m, 1H), 4.81 - 4.71 (m, 1H), 4.72 - 4.52 (m, 2H), 4.48 (d, 1H), 4.22 (d, 1H), 4.06 - 3.87 (m, 1H), 3.80 - 3.67 (m, 1H), 3.52 - 3.33 (m, 6H), 3.07 (s, 3H), 3.03 - 2.94 (m, 1H), 2.89 (br s, 3H), 2.85 - 2.73 (m, 8H), 2.60 - 2.40 (m, 1H), 2.38 - 2.19 (m, 2H), 2.20 - 1.97 (m, 6H), 1.97 - 1.72 (m, 3H), 1.59 - 1.44 (m, 5H), 1.44 - 1.27 (m, 12H), 1.08 (d, 3H).

[0318] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(methyl-L- prolyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-I1-1-2-2) (Compound 108). The amine intermediate from S3-4-I1-1-2-1 above (31.1 mg, 0.040 mmol) was dissolved in DCM (2 mL) and triethylamine (0.017 mL, 0.12 mmol). Na(OAc)sBH (25.4 mg, 0.120 mmol) followed by formaldehyde (37% aqueous solution, 0.0327 mL, 0.403 mmol) were added. After 15 min, the reaction mixture was quenched with NaHCO (sat, aq) and was extracted with DCM (2 times). The combined extracts were concentrated under reduced pressure. The crude material was dissolved in methanol (1 mL) and the reaction mixture was heated to 65 °C external temperature. After 4 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO ₂ H) to give S3-4-I1-1-2-2 (12.9 mg). MS (ESI+) mlz-. 681.41 [M + H] ⁺ . Formate salt. ^X HNMR (400 MHz, Methanol-d4) 5 8.51 (s, 4H), 4.68 - 4.51 (m, 1H), 4.48 (d, 1H), 4.32 - 4.03 (m, 2H), 4.00 - 3.82 (m, 1H), 3.78 - 3.66 (m, 1H), 3.62 - 3.49 (m, 1H), 3.49 - 3.32 (m, 4H), 3.26 - 3.09 (m, 1H), 3.06 (s, 3H), 3.02 - 2.90 (m, 2H), 2.89 - 2.65 (m, 13H), 2.62 - 2.46 (m, 1H), 2.36 - 2.07 (m, 3H), 2.07 - 1.59 (m, 7H), 1.58 - 1.44 (m, 5H), 1.44 - 1.24 (m, 14H), 1.06 (br s, 3H).

[0319] (9R,llR,12R,13R)-3-(D-Prolyl)-12-(((2S,3R,4S,6R)-4-(dimethyl amino)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-4-Il-l-2-3)( (Compound 59). Prepared according to the methods of S3-4-I1-1-2-1 from Cbz-D-proline to provide the title compound (17.7 mg).

Formate salt. MS (ESI+) m/z: 667.43 [M + H] ⁺ . ^X HNMR (400 MHz, Methanol-d4) 1H NMR (400 MHz, Methanol-d4) 5 8.51 (s, 2H), 4.78 - 4.69 (m, 1H), 4.68 - 4.57 (m, 1H), 4.56 - 4.37 (m, 2H), 4.21 (d, 1H), 4.05 - 3.83 (m, 1H), 3.80 - 3.68 (m, 1H), 3.51 - 3.32 (m, 6H), 3.27 - 3.12 (m, 1H), 3.11 - 2.98 (m, 4H), 2.88 (br s, 3H), 2.84 - 2.69 (m, 8H), 2.59 - 2.41 (m, 1H), 2.38 - 1.96 (m, 7H), 1.96 - 1.69 (m, 3H), 1.59 - 1.43 (m, 5H), 1.43 - 1.26 (m, 13H), 1.08 (dd, 3.1 Hz, 3H).

[0320] (9R.HR.12R.13R)-12-(((2S.3R.4S.6R)-4-(/9/7m7//r/u/n/7n>)- 3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(methyl-D- prolyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-Il-5)( (Compound 60).

Prepared according to the methods of S3-4-I1-1-2-3 from Cbz-D-proline to provide the title compound (13.3 mg). Formate salt. MS (ESI+) mlz'. 681.41 [M + H] ⁺ . ^J H NMR (400 MHz, Methanol-d4) 5 8.53 (s, 3H), 5.07 - 4.94 (m, 1H), 4.68 - 4.55 (m, 1H), 4.54 - 4.32 (m, 2H), 4.21 (d, 1H), 3.98 - 3.80 (m, 1H), 3.78 - 3.68 (m, 1H), 3.66 - 3.54 (m, 1H), 3.50 - 3.32 (m, 4H), 3.22 - 2.94 (m, 6H), 2.93 - 2.65 (m, 13H), 2.64 - 2.49 (m, 1H), 2.33 - 1.66 (m, 10H), 1.60 - 1.43 (m, 5H), 1.43 - 1.25 (m, 13H), 1.07 (br s, 3H).

[0321] (9R,llR,12R,13R)-3-(Dimethyl-L-alanyl)-12-(((2S,3R,4S,6R)-4- (dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.72]octadecane-14, 16-dione (S3-4-I1-1-2-5) (Compound 76). S3-1-I1-

1-2 (48 mg, 0.061 mmol) was dissolved in DCM (0.3 mL) and A,A-diisopropylethylamine (0.042 mL, 0.24 mmol), and 1 -hydroxybenzotri azole (10.7 mg, 0.080 mmol), Boc-L-alanine (13.8 mg, 0.0734 mmol), and A,A-diisopropylcarbodiimide (0.0113 mL, 0.0734 mmol) were added. After 21 h, the reaction mixture was concentrated under reduced pressure, and the residue was purified on 12 g of silica gel (elution with 2-10% MeOH-DCM-O.5% NH4OH gradient) to give the amide intermediate (27 mg, 52%) as a thick oil. The material was dissolved in 20% trifluoroacetic acid/DCM (1 mL). After 2 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (2 mL) and Na(OAc)sBH (26.9 mg, 0.0318 mmol) followed by formaldehyde (37% aqueous solution, 0.0513 mL, 0.636 mmol) were added. After 45 min, the reaction mixture was quenched with NaHCCh (sat, aq) and was extracted with DCM (3 times). The combined extracts were dried over Na2SC>4, were filtered, and were concentrated under reduced pressure. The crude material was dissolved in methanol (2 mL), and the reaction mixture was heated to 50 °C external temperature overnight. The reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water- 0.1% HCO2H) to give S3-4-I1-1-2-5 (11.42 mg). MS (ESI+) m/z: 669.48 [M + H] ⁺ . Formate salt. 'H NMR (400 MHz, Methanol-d4) 8 8.52 (s, 3H), 5.09 - 4.92 (m, 1H), 4.70 - 4.51 (m, 1H), 4.48 (d, , 1H), 4.21 (d, 1H), 4.17 - 3.93 (m, 2H), 3.80 - 3.66 (m, 1H), 3.52 - 3.33 (m, 4H), 3.26 - 3.10 (m, 1H), 3.10 - 3.01 (m, 3H), 3.00 - 2.63 (m, 11H), 2.59 (s, 3H), 2.55 (s, 3H), 2.36 - 2.16 (m, 2H), 2.15 - 1.94 (m, 4H), 1.94 - 1.72 (m, 2H), 1.60 - 1.45 (m, 5H), 1.44 - 1.24 (m, 15H), 1.07 (d, 3H).

[0322] (9R,llR,12R,13R)-3-(Dimethyl-D-alanyl)-12-(((2S,3R,4S,6R)-4- (dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-I1-7) (Compound 77). Prepared according to the methods of S3-4-I1-1-2-5 from Boc-D-alanine to provide the title compound (11.5 mg). Formate salt. MS (ESI+) m/z: 669.48 [M + H] ⁺ . ^X H NMR (400 MHz, Methanol-d4) 8 8.51 (s, 3H), 5.07 - 4.92 (m, 1H), 4.62 (br d, 1H), 4.55 - 4.34 (m, 2H), 4.22 (d, 1H), 4.18 - 3.95 (m, 2H), 3.81 - 3.67 (m, 1H), 3.54 - 3.33 (m, 4H), 3.25 - 3.12 (m, 1H), 3.11 - 2.94 (m, 4H), 2.88 (br s, 3H), 2.84 - 2.74 (m, 7H), 2.63 (s, 3H), 2.56 (s, 3H), 2.35 - 1.95 (m, 5H), 1.94 - 1.73 (m, 2H), 1.59 - 1.44 (m, 5H), 1.44 - 1.26 (m, 15H), 1.08 (d, 3H).

[0323] (9R,llR,12R,13R)-3-(2-Amino-2-meZZzj;Z/jropanoj;Z)-12-(((2S, 3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-4-I1-1-2- 7)( (Compound 88). Prepared according to the methods of S3-4-I1-1-2-1 from a-(Boc- amino)isobutyric acid with Boc-deprotection according to the methods of S3-4-I1-1-2-5 to provide the title compound (3.49 mg). Formate salt. MS (ESI+) mlr. 655.42 [M + H] ⁺ . ^J H NMR (400 MHz, Methanol-d4) 8 8.47 (s, 2H), 5.11 - 4.91 (br s, 1H), 4.70 - 4.54 (br m, 1H), 4.49 (d, 1H), 4.45 - 4.24 (br m, 1H), 4.21 (d, 1H), 3.74 (dq, 1H), 3.51 - 3.33 (m, 4H), 3.29 - 3.11 (br m, 2H), 3.06 (s, 3H), 2.95 - 2.84 (br m, 3H), 2.80 (s, 6H), 2.79 - 2.71 (br m, 1 H), 2.37 - 2.19 (br m, 1H), 2.18 - 1.97 (m, 4H), 1.95 - 1.71 (m, 3H), 1.68 (d, 6H), 1.58 - 1.45 (m, 5H), 1.40 (s, 6H), 1.37 (d, 3H), 1.33 (d, 3H), 1.07 (br d, 3H).

[0324] (9R,llR,12R,13R)-3-(2-(Dimethylamino)-2-methylpropanoyl)-12- (((2S,3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-zzzeZZryZteZraZryzZro-2H-pyran-2 -yl)oxy)-l 1-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-4-I1-2-8) (Compound 89). Prepared according to the methods of S3-4-I1-1-2-5 from a-(Boc- amino)isobutyric acid to provide the title compound (6.94 mg). Formate salt. MS (ESI+) mlz\ 683.44 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 8 8.42 (s, 2H), 5.04 (d, 1H), 4.65 (d, 1H), 4.48 (d, 1H), 4.22 (d, 1H), 3.74 (tdd, 1H), 3.51 - 3.32 (m, 4H), 3.28 - 3.14 (m, 2H), 3.06 (s, 3H), 2.89 (br s, 4H), 2.86 - 2.76 (m, 7H), 2.38 - 2.21 (m, 7H), 2.18 - 1.94 (m, 5H), 1.84 (br d, 2H), 1.60 - 1.44 (m, 5H), 1.41 (d, 6H), 1.38 (dd, 3H), 1.33 (d, 3H), 1.28 (s, 6H), 1.09 (d, 3H).

[0325] (9R,llR,12R,13R)-3-(D-Seryl)-12-(((2S,3R,4S,6R)-4-(rfiwietAy /am/no)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-4-I1-1-2-9) (Compound 91). Prepared according to the methods of S3-4-I1-1-2-1 from Boc-D-Ser-OH with Boc-deprotection according to the methods of S3-4-I1-1-2-5 to provide the title compound (5.19 mg). Formate salt. MS (ESI+) mlr. 657.43 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 5 8.48 (s, 1H), 5.05 (br s, 1H), 4.72 - 4.51 (m, 2H), 4.48 (d, 1H), 4.46 - 4.30 (m, 1H), 4.21 (d, 1H), 4.13 - 3.95 (m, 1H), 3.92 - 3.78 (m, 2H), 3.77 - 3.65 (m, 2H), 3.52 - 3.33 (m, 4H), 3.28 - 3.12 (m, 1H), 3.06 (s, 3H), 23.00 - 2.84 (m, 3H), 2.83 - 2.70 (m, 7H), 2.40 - 1.95 (m, 5H), 1.95 - 1.71 (m, 2H), 1.59 - 1.49 (m, 5H), 1.43 - 1.24 (m, 12H), 1.07 (d, 3H).

[0326] (9R,llR,12R,13R)-3-(Dimethyl-D-seryl)-12-(((2S,3R,4S,6R)-4-( dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-I1-1-2-10) (Compound 90).

Prepared according to the methods of S3-4-I1-1-2-5 from Boc-D-Ser-OH to provide the title compound (7.28 mg). Formate salt. MS (ESI+) mlz\ 685.41 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.48 (s, 3H), 5.01 (br s, 1H), 4.69 - 4.51 (m, 2H), 4.48 (d, 1H), 4.22 (d, 1H), 4.19 - 4.07 (m, 1H), 4.04 - 3.84 (m, 3H), 3.73 (dddd, 1H), 3.52 - 3.33 (m, 4H), 3.27 - 3.13 (m, 1H), 3.06 (s, 3H), 3.04 - 2.95 (m, 1H), 2.88 (s, 3H), 2.84 - 2.75 (m, 7H), 2.69 (s, 3H), 2.58 (s, 3H), 2.36 - 2.20 (m, 1H), 2.19 - 2.09 (m, 1H), 2.09 - 1.97 (m, 3H), 1.92 - 1.75 (m, 2H), 1,59 - 1.44 (m, 5H), 1.43 - 1.28 (m, 12H), 1.08 (d, 3H).

[0327] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3-(4-methoxyb enzoyl)-7,9,ll,13,15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-Il-l-2-ll)(Compound

114). Prepared according to the methods of S3-4-I1 - 1 -2-5 from 4-methoxybenzoic acid. 25.0 mg Formate salt. MS (ESI+) m/z: 704.5 [M + H] ⁺ ; ^L H NMR (400 MHz, CD ₃ OD) 8 = 7.44 - 7.33 (m, 2H), 7.03 - 6.96 (m, 2H), 4.42 (d, 1H), 4.24 (d, 1H), 4.17 - 4.00 (m, 2H), 3.98 - 3.84 (m, 1H), 3.84 (s, 3H), 3.70 - 3.39 (m, 5H), 3.28 (dd, 1H), 2.96 (s, 3H), 2.68 - 2.60 (m, 1H), 2.39 (s, 3H), 2.34 (s, 7H), 2.03 - 1.71 (m, 5H), 1.70 - 1.58 (m, 1H), 1.52 (s, 3H), 1.48 - 1.33 (m, 4H), 1.33 - 1.15 (m, 11H), 0.99 - 0.80 (m, 1H).

[0328] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-isonicotinoyl-ll-metho xy-7,9,ll,13,15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-Il-l-2-12)(Compound 115). Prepared according to the methods of S3-4-I1 - 1 -2-5 from isonicotinic acid. 19.2 mg Formate salt. MS (ESI+) m/z: 675.4 [M + H] ⁺ ; 'H NMR (400 MHz, Acetonitrile-d ₃ ) 6 = 8.63 (d, 2H), 7.29 (d, 2H), 4.37 (d, 1H), 4.19 (d, 1H), 4.04 (br d, 1H), 3.94 (d, 2H), 3.57 - 3.30 (m, 5H), 3.25 - 3.14 (m, 1H), 3.14 - 2.99 (m, 1H), 2.89 (br d, 3H), 2.48 (ddd, 1H), 2.37 (s, 3H), 2.34 - 2.20 (m, 9H), 1.92 - 1.66 (m, 6H), 1.65 - 1.49 (m, 1H), 1.48 - 1.44 (m, 3H), 1.42 - 1.36 (m, 1H), 1.33 (br d, 3H), 1.26 (br d, 1H), 1.23 (br s, 2H), 1.22 (s, 4H), 1.20 - 1.15 (m, 4H), 0.83 (br d, 3H).

[0329] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(2- phenylacetyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-I1-1-2- 13)(Compound 117). Prepared according to the methods of S3-4-I1 -1 -2-5 from phenylacetic acid. 16.6 mg Formate salt. MS (ESI+) miz-. 688.5 [M + H] ⁺ ; 'H NMR (400 MHz, MeOD) 5 = 7.36 - 7.20 (m, 5H), 4.40 (d, 1H), 4.21 - 4.09 (m, 1H), 4.09 - 3.94 (m, 2H), 3.91 - 3.83 (m, 1H), 3.83 - 3.72 (m, 2H), 3.72 - 3.44 (m, 3H), 3.44 - 3.32 (m, 3H), 3.30 - 3.23 (m, 2H), 2.95 (d, 3H), 2.66 - 2.58 (m, 1H), 2.33 (s, 6H), 2.28 - 2.24 (m, 3H), 2.20 (br d, 1H), 1.89 - 1.67 (m, 4H), 1.67 - 1.52 (m, 2H), 1.49 (d, 3H), 1.46 - 1.33 (m, 5H), 1.32 - 1.14 (m, 16H), 1.01 - 0.90 (m, 2H), 0.90 - 0.80 (m, 3H).

[0330] (9R,llR,12R,13R)-3-(4-chlorobenzoyl)-12-(((2S,3R,4S,6R)-4-(d imethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-Il-l-2-14)(Compound 118).

Prepared according to the methods of S3 -4-11-1 -2-5 from 4-chlorobenzoic acid. 34.0 mg Formate salt. MS (ESI+) mfr. 708.5 [M + H] ⁺ ; ^L H NMR (400 MHz, CD ₃ OD) 8 = 7.49 - 7.46 (m, 2H), 7.44

- 7.39 (m, 2H), 4.42 (d, 1H), 4.23 (d, 1H), 4.09 (d, 1H), 4.05 (d, 1H), 3.91 (br s, 1H), 3.75 - 3.38 (m, 5H), 3.38 - 3.32 (m, 1H), 3.28 (dd, 1H), 2.96 (s, 3H), 2.66 - 2.59 (m, 1H), 2.39 (s, 3H), 2.34 (s, 8H), 2.09 (br s, 1H), 1.94 (br s, 1H), 1.88 - 1.70 (m, 4H), 1.60 (br s, 1H), 1.52 (br s, 3H), 1.49 - 1.40 (m, 1H), 1.40 - 1.33 (m, 3H), 1.31 - 1.20 (m, 10H), 0.88 (br d, 3H).

[0331] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-N-(4-methoxyp henyl)-7,9,ll,13,15,15- hexamethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecane -3-carboxamide (S3-4-I1-1-2-

15)(Compound 119). Prepared according to the methods of S3-4-I1 -1 - 1 - 1 from S3 -4-11 -1-2- land 4-methoxyphenyl isocyanate. 19.4 mg Formate salt. MS (ESI+) mlz'. 719.5 [M + H] ⁺ ; ^r H NMR (400 MHz, CD ₃ OD) 6 = 7.25 - 7.18 (m, 2H), 6.87 - 6.81 (m, 2H), 4.42 (d, 1H), 4.17 (dd, 2H), 4.05 (d, 1H), 3.80 - 3.68 (m, 5H), 3.61 - 3.53 (m, 1H), 3.44 (quin, 1H), 3.38 - 3.33 (m, 1H), 3.28 (dd, 1H), 3.17 - 3.02 (m, 1H), 2.97 (s, 3H), 2.63 (ddd, 1H), 2.44 - 2.36 (m, 3H), 2.34 (s, 7H), 2.02 - 1.67 (m, 6H), 1.52 (s, 4H), 1.40 (s, 3H), 1.31 - 1.21 (m, 11H), 0.89 (d, 3H).

[0332] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3-(3-methoxyb enzyl)-7,9,ll,13,15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-2-I1-1-2-16) (Compound

122). Prepared according to the methods of S3-2-I1 - 1 -1 -2 from 3 -methoxybenzaldehyde. 16.4 mg, formate salt. MS (ESI+) mlr. 690.4 [M + H]+; 1H NMR (400 MHz, Acetonitrile-d3) 6 8.35 (br s, 2H), 7.25 (t, 1H), 6.94 - 6.86 (m, 2H), 6.83 (dd, 1H), 4.55 - 4.48 (m, 1H), 4.44 - 4.36 (m, 2H), 4.28 - 4.16 (m, 1H), 4.09 (br d, 2H), 4.04 - 3.79 (m, 6H), 3.78 (s, 3H), 3.70 (br s, 2H), 3.66 - 3.56 (m, 3H), 3.54 (s, 2H), 3.43 (br s, 1H), 3.32 - 3.11 (m, 3H), 2.96 (s, 3H), 2.92 - 2.71 (m, 5H), 2.65 (br s, 3H), 2.60 - 2.43 (m, 3H), 2.43 - 2.36 (m, 6H), 2.26 (br t, 3H), 2.06 (br d, 3H), 1.81 (br d, 3H), 1.70 - 1.50 (m, 4H), 1.49 - 1.40 (m, 4H), 1.38 - 1.15 (m, 15H), 0.99 (br d, 3H).

[0333] (9R,llR,12R,13R)-N-benzyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-3,7-diazaspiro[5.12]octadecane-3-carboxamide (S3-4-Il-l-2-17)(Compound 123).

Prepared according to the methods of S3 -4-11-1 - 1 -1 from S3 -4-11- 1 -2- 1 and benzyl isocyanate. 12.5 mg Formate salt. MS (ESI+) m!z-. 703.5 [M + H] ⁺ ; ^J H NMR (400 MHz, CD ₃ OD) 6 = 7.22 - 7.16 (m, 4H), 7.14 - 7.07 (m, 1H), 4.31 (d, 1H), 4.26 (s, 2H), 4.10 (d, 1H), 4.05 - 3.97 (m, 1H), 3.94 (d, 1H), 3.88 - 3.63 (m, 1H), 3.60 - 3.49 (m, 2H), 3.47 (ddd, 1H), 3.33 (quin, 1H), 3.20 - 3.09 (m, 3H), 3.07 - 2.94 (m, 1H), 2.87 (s, 3H), 2.53 (ddd, 1H), 2.27 - 2.14 (m, 11H), 1.94 - 1.60 (m, 6H), 1.55 (br d, 1H), 1.41 (s, 3H), 1.39 - 1.31 (m, 1H), 1.29 (s, 3H), 1.21 - 1.08 (m, 12H), 0.80 - 0.74 (m, 3H).

[0334] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-nicotinoyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-4-Il-l-2-18)(Compound 125).

Prepared according to the methods of S3 -4-11-1 -2-5 from nicotinic acid. Formate salt. MS (ESI+) mfr. 675.5 [M + H] ⁺ ; ^L H NMR (400 MHz, Acetonitrile d ₃ ) 5 = 8.61 (dd, 1H), 8.59 (dd, 1H), 7.75 (td, 1H), 7.39 (ddd, 1H), 4.37 (d, 1H), 4.20 (br d, 1H), 4.04 (br d, 1H), 3.94 (br d, 2H), 3.60 - 3.16 (m, 6H), 3.09 (dd, 1H), 2.90 (s, 3H), 2.49 (ddd, 1H), 2.42 (br s, 1H), 2.37 (br s, 3H), 2.30 (br s, 3H), 2.25 (s, 6H), 1.86 (br d, , 2H), 1.77 (br td, 2H), 1.69 (ddd, 1H), 1.60 - 1.51 (m, 1H), 1.46 (s, 3H), 1.40 - 1.26 (m, 5H), 1.26 - 1.15 (m, 10H), 1.13 (dd, 1H), 0.99 - 0.87 (m, 1H), 0.84 (br d, 3H).

[0335] (9R,llR,12R,13R)-3-benzoyl-12-(((2S,3R,4S,6R)-4-(dimethylami no)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-3,7- diazaspiro [5.12] octadecane-14, 16-dione (S3-4-Il-l-2-19)(Compound 137). Prepared according to the methods of S3 -4-11-1 -2-5 from benzoic acid. 26.0 mg Formate salt. MS (ESI+) mlz\ 674.5 [M + H] ⁺ ; 'H NMR (400 MHz, Acetonitrile d ₃ ) 5 = 8.34 (br s, 2H), 7.53 - 7.34 (m, 5H), 4.54 - 4.10 (m, 4H), 4.06 (br d, 1H), 3.60 (dtd, 2H), 3.44 - 3.10 (m, 20H), 3.06 (br s, 1H), 3.02 - 2.83 (m, 5H), 2.75 - 2.46 (m, 11H), 1.99 - 1.96 (m, 1H), 1.91 - 1.83 (m, 2H), 1.61 (br dd, 2H), 1.47 (s, 3H), 1.45 - 1.24 (m, 11H), 1.22 (d, 3H), 0.95 (br d, 3H).

[0336] (9R,llR,12R,13R)-N-(4-chlorophenyl)-12-(((2S,3R,4S,6R)-4-(di methylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecane-3-carboxam ide (S3-4-I1-1-2-

20)(Compound 138). Prepared according to the methods of S3-4-I1 -1 - 1 - 1 from S3-4-I1 -1 -2- 1 and 3 -chlorophenyl isocyanate. 25.9 mg Formate salt. MS (ESI+) mlz'. 723.4 [M + H] ⁺ ; 'H NMR (400 MHz, CD ₃ OD) 8 = 7.38 - 7.33 (m, 2H), 7.27 - 7.21 (m, 2H), 4.83 (s, 11H), 4.42 (d, 1H), 4.28 - 4.08 (m, 2H), 4.05 (d, 1H), 3.80 - 3.68 (m, 2H), 3.62 - 3.51 (m, 1H), 3.49 - 3.33 (m, 3H), 3.29 - 3.16 (m, 1H), 3.12 (s, 1H), 2.97 (s, 3H), 2.63 (ddd, 1H), 2.38 (s, 3H), 2.36 - 2.27 (m, 8H), 2.03 - 1.65 (m, 6H), 1.52 (s, 3H), 1.46 - 1.33 (m, 3H), 1.31 - 1.20 (m, 11H), 0.89 (d, 3H).

[0337] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- N-phenyl-17-oxa-3,7-diazaspiro[5.12]octadecane-3-carboxamide (S3-4-I1-1-2-

21)(Compound 140). Prepared according to the methods of S3-4-I1 -1 - 1 - 1 from S3-4-I1 -1 -2- 1 and phenyl isocyanate. 26.4 mg Formate salt. MS (ESI+) mfr. 689.5 [M + H] ⁺ ; 'H NMR (400 MHz, MeOD) 8 = 7.36 - 7.32 (m, 2H), 7.28 - 7.23 (m, 2H), 7.01 (t, 1H), 4.42 (d, 1H), 4.23 (br d, 1H), 4.16 - 4.01 (m, 2H), 3.80 - 3.70 (m, 2H), 3.57 (ddd, 1H), 3.49 - 3.32 (m, 3H), 3.29 - 3.23 (m, 1H), 3.12 (s, 1H), 2.98 (s, 3H), 2.63 (ddd, 1H), 2.41 - 2.29 (m, 11H), 2.05 - 1.65 (m, 7H), 1.52 (s, 4H), 1.40 (s, 3H), 1.35 - 1.19 (m, 13H), 1.10 - 0.92 (m, 1H), 0.89 (br d, 3H).

[0338] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(JwietAy/g/ycj7)-ll-me thoxy-7,9,ll,13,15- pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-l-l)(Compound

8). S3-1-I1-1-1 (30 mg, 0.039 mmol) was dissolved in DCM (1 mL) and triethylamine (0.016 mL, 0.116 mmol), and chloroacetyl chloride (0.0046 mL, 0.058 mmol) was added. After 15 min, dimethylamine (2 M solution in tetrahydrofuran, 0.194 mL, 389 mmol) was added. After 15 min, the reaction mixture was heated to 70 °C. After 4 h, the reaction mixture was allowed to cool to rt and stir over the weekend. The reaction mixture was diluted with DCM, was washed with water (1 time), and was concentrated. The crude material was dissolved in methanol (1 mL), and the reaction mixture was heated to 60 °C external temperature. After 3 h, the reaction was allowed to cool to rt and was concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCOzH) to give S3-5-I1-1-1-1 (7.30 mg). MS (ESI+) mfr. 641.42 [M + H] ⁺ . Formate salt, mixture of C2 epimers, 'H NMR (400 MHz, Methanol-d4) 6 8.45 (br s, 1H), 4.81 - 4.68 (m, 1H), 4.66 - 4.40 (m, 2H), 4.36 - 4.22 (m, 2H), 4.21 - 4.15 (m, 0.5H), 4.15 - 4.04 (m, 1H), 4.02 - 3.92 (m, 0.5H), 3.90 - 3.68 (m, 2H), 3.59 - 3.35 (m, 5H), 3.16 - 3.00 (m, 4H), 2.96 - 2.76 (m, 16H), 2.40 - 2.20 (m, 2H), 2.18 - 1.99 (m, 2H), 1.96 - 1.69 (m, 3H), 1.66 - 1.47 (m, 2H), 1.43 (br s, 3 H), 1.40 - 1.25 (m, 10H), 1.16 - 1.02 (m, 3H).

[0339] (9R,llR,12R,13R)-3-(2-(Azetidin-l-yl)acetyl)-12-(((2S,3R,4S, 6R)-4-(dimethylamino)- 3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7 ,9,ll,13,15-pentamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1-2) (Compound 9). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and azetidine to provide the title compound (3.77 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz.- 653.40 [M + H] ⁺ . NMR (400 MHz, Methanol-d4) 6 8.49 (s, 2H), 4.49 (d, 1H), 4.43 (t, 1H), 4.34 - 4,23 (m, 2H), 4.23 - 4.02 (m, 4H), 4.02 - 3.87 (m, 1H), 3.78 - 3.60 (m, 3H), 3.50 - 3.33 (m, 4H), 3.1- 2.94 (m, 4H), 2.88 - 2.59 (m, 10H), 2.49 (d, 2H), 2.25 - 2.05 (m, 3H), 2.06 - 1.98 (m, 2H), 1.93 - 1.58 (m, 5H), 1.57 - 1.43 (m, 2H), 1.43 - 1.19 (m, 12H), 1.12 - 0.95 (m, 3H).

[0340] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15- pentamethyl-3-(2- (pyrrolidin-l-yl)acetyl)-17-oxa-3,7-diazaspiro[5.12]octadeca ne-14, 16-dione (S3-5-I1-1-1-3) (Compound 10). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and pyrrolidine to provide the title compound (3.24 mg). Formate salt, mixture of C2 epimers. MS

(ESI+) mlz-. 667.34 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 8 8.53 (s, 2H), 4.49 (d, 1H), 4.41 (dd, 1H), 4.25 (d, 1H), 4.11 - 3.86 (m, 3H), 3.78 - 3.61 (m, 3H), 3.51 - 3.34 (m, 4H), 3.28 - 2.87 (m, 10H), 2.83 - 2.60 (m, 7H), 2.2.59 - 2.33 (m, 2H), 2.19 - 2.09 (m, 1H), 2.10 - 1.90 (m, 6H), 1.89 - 1.54 (m, 4H), 1.54 - 1.41 (m, 2H), 1.40 - 1.18 (m, 13H), 1.10 - 0.84 (m, 3H).

[0341] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15- pentamethyl-3-(2- (piperidin-l-yl)acetyl)-17-oxa-3,7-diazaspiro[5.12]octadecan e-14, 16-dione (S3-5-I1-1-1- 4)(Compound 11). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and piperidine to provide the title compound (5.25 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 681.37 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 5 8.53 (s, 2H), 4.49 (d, 1H), 4.42 (t, 1H), 4.26 (d, 1H), 3.76 - 3.51 (m, 4H), 3.51 - 3.34 (m, 4H), 3.09 - 2.95 (m, 4H), 2.90 - 2.41 (m, 15H), 2.19 - 1.93 (m, 4H), 1.92 - 1.63 (m, 8H), 1.63 - 1.42 (m, 5H), 1.42 - 1.19 (m, 13H), 1.09 - 0.87 (m, 3H).

[0342] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15-p entamethyl-3-(2- morpholinoacetyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1-5) (Compound 12). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and morpholine to provide the title compound (7.08 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 683.35 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 5 8.54 (s, 1H), 4.48 (d, 1H), 4.40 (d, 0.5H), 4.26 (d, 1H), 4.17 (d, 0.5H), 4.02 - 3.92 (m, 0.5H), 3.84 - 3.79 (m, 0.5H), 3.77 - 3.60 (m, 6H), 3.49 - 3.35 (m, 4H), 3.29 - 3.13 (m, 3H), 3.12 - 2.95 (m, 4H), 2.83 - 2.59 (m, 9H), 2.57 - 2.41 (m, 5H), 2.17 - 2.05 (m, 1H), 2.06 - 1.90 (m, 3H), 1.89 - 1.53 (m, 4H), 1.52 - 1.40 (m, 3H), 1.40 - 1.18 (m, 12H), 1.10 - 0.85 (m, 3H).

[0343] (9R,llR,12R,13R)-3-(Cyclobutylglycyl)-12-(((2S,3R,4S,6R)-4-( dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15-pentamethyl-17- oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1-6) (Compound 17). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and cyclobutylamine to provide the title compound (4.17 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz'. 667.40 [M + H] ⁺ 'H NMR (400 MHz, Methanol-d4) 5 8.51 (s, 2H), 4.70 - 4.52 (br m, 1H), 4.51 - 4.38 (m, 1.5H), 4.26 (d, 1H), 4.17 (d, 0.5H), 4.03 - 3.75 (m, 3H), 3.75 - 3.59 (m, 3H), 3.50 - 3.33 (m, 4H), 3.06 (s, 1H), 3.03 (s, 2H), 2.88 - 2.59 (m, 10H), 2.38 - 2.23 (m, 2H), 2.22 - 2.08 (m, 4H), 2.08 - 1.96 (m, 4H), 1.95 - 1.57 (m, 6H), 1.57 - 1.43 (m, 2H), 1.43 - 1.20 (m, 12H), 1.02 (s, 3H).

[0344] (9R,llR,12R,13R)-3-((Cyclopropylmethyl)glycyl)-12-(((2S,3R,4 S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy-

7,9,11, 13, 15-pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- 7)(Compound 13). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and cyclopropylmethylamine to provide the title compound (11.1 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 667.39 [M + H] ⁺ . ¹ H NMR (400 MHz, Methanol-d4) 6 8.45 (s, 1 ,5H), 4.78 - 4.67 (m, 0.8H), 4.66 - 4.56 (m, 0.2H), 4.56 - 4.40 (m, 2H), 4.27 (d, 0.8H), 4.17 (d, 0.2H), 4.15 - 4.00 (m, 1H), 3.80 - 3.67 (m, 2H), 3.58 - 3.33 (m, 6H), 3.26 - 3.09 (m, 1H), 3.06 (s, 3H), 2.96 - 2.74 (m, 10H), 2,42 (s, 3H), 2.37 - 2.19 (m, 1H), 2.19 - 1.97 (m, 3H), 1.97 - 1.68 (m, 4H), 1.63 - 1.45 (m, 3H), 1.42 (s, 3H), 1.39 - 1.23 (m, 9H), 1.14 - 1.00 (m, 3H), 0.59 - 0.39 (m, 4H).

[0345] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(isopropylglycyl)-ll-m ethoxy-7,9,ll,13,15- pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-l-8)(Compound 14). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and isopropylamine to provide the title compound (8.91 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-.

655.41 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 5 8.52 (s, 1H), 4.49 (d, 1H), 4.44 (d, 0.5H), 4.26 (d, 1H), 4.21 - 4.06 (m, 1H), 4.05 - 3.92 (m, 1.5H), 3.78 - 3.67 (m, 2H), 3.50 - 3.33 (m, 5H), 3.28 - 3.15 (m, 2H), 3.10 - 2.96 (m, 4H), 2.90 - 2.67 (m, 10H), 2.31 - 2.12 (m, 2H), 2.12 - 1.91 (m, 3H), 1.91 - 1.61 (m, 3H), 1.61 - 1.43 (m, 3H), 1.43 - 1.17 (m, 18H), 1.11 - 0.96 (m, 3H).

[0346] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isopropyl-N-methylg lycyl)-ll-methoxy-

7,9,11, 13, 15-pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- 9)(Compound 15). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and N- methylpropan-2-amine to provide the title compound (9.77 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 669.40 [M + H] ⁺ . ¹ H NMR (400 MHz, Methanol-d4) 6 8.53 (s, 2H), 4.49 (d, 1H), 4.26 (d, 1H), 4.13 - 3.85 (m, 3H), 3.78 - 3.65 (m, 2H), 3.53 - 3.33 (m, 5H), 3.10 - 2.94 (m, 3H), 2.89 - 2.56 (m, 14H), 2.30 - 2.14 (m, 1H), 2.14 - 1.89 (m, 3H), 1.88 - 1.57 (m, 3H), 1.57 - 1.43 (m, 2H), 1.43 - 1.18 (m, 22H), 1.11 - 0.95 (m, 3H).

[0347] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-3-((2-methoxy ethyl)glycyl)-7,9,ll,13,15- pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-l-10)(Compound 16). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and 2-methoxyethan- 1-amine to provide the title compound (4.97 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 671.42 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.54 (s, 1H), 4.53 - 4.35 (m, 1H), 4.30 - 4.11 (m, 1H), 3.86 - 3.60 (m, 4H), 3.59 - 3.53 (m, 2H), 3.46 - 3.33 (m, 7H), 3.24 - 3.08 (m, 2H), 3.08 - 2.87 (m, 6H), 2.76 - 2.55 (m, 8H), 2.54 - 2.32 (m, 2H), 2.19 - 1.97 (m, 2H), 1.97 - 1.89 (m, 2H), 1.88 - 1.53 (m, 4H), 1.50 - 1.38 (m, 2H), 1.38 - 1.15 (m, 13H), 1.06 - 0.82 (m, 3H).

[0348] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Z)iW^j;ZawMno)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15-p entamethyl-3- (methylglycyl)-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- ll)(Compound 18). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and methylamine to provide the title compound (3.69 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz-. 627.38 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.52 (s, 2H), 4.72 - 4.54 (br s, 1H), 4.53 - 4.38 (m, 1H), 4.30 - 4.14 (m, 1H), 4.14 - 3.89 (m, 2H), 3.84 - 3.55 (m, 2H), 3.52 - 3.33 (m, 6H), 3.06 (s, 1H), 3.03 (s, 2H), 2.89 - 2.47 (m, 14H), 2.28 - 2.13 (m, 1H), 2.12 - 1.89 (m, 4H), 1.88 - 1.56 (m, 4H), 1.57 - 1.43 (m, 2H), 1.43 - 1.19 (m, 13H), 1.12 - 0.88 (m, 3H).

[0349] (9R,llR,12R,13R)-3-(Diethylglycyl)-12-(((2S,3R,4S,6R)-4-(dim ethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15-pentamethyl-17- oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-l-12)(Compound 28). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and diethylamine to provide the title compound (7.89 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz'. 669.46 [M + H] ⁺ . ^r H NMR (400 MHz, Methanol-d4) 8 8.49 (s, 1H), 4.52 - 4.40 (m, 1H), 4.33 - 4.21 (m, 2H), 4.20 - 4.03 (m, 2H), 3.79 - 3.62 (m, 2H), 3.51 - 3.32 (m, 4H), 3.28 - 3.11 (m, 6H), 3.11 - 2.94 (m, 5H), 2.94 - 2.66 (m, 10H), 2.37 - 2.17 (m, 2H), 2.16 - 1.92 (m, 3H), 1.92 - 1.67 (m, 3H), 1.58 - 1.44 (m, 2H), 1.44 - 1.18 (m, 18H), 1.13 - 0.97 (m, 3H).

[0350] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-ethyl-N-methylglycy l)-ll-methoxy-7,9,ll,13,15- pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-H-l-l-13)(Compound 36). Prepared according to the methods of S3-5-I1 - 1-1- 1 from S3-1-I1-1-1 and N- methylethylamine to provide the title compound (4.79 mg). Formate salt, mixture of C2 epimers. MS (ESI+) m/z: 655.41 [M + H]“. 'H NMR (400 MHz, Methanol-d4) 8 8.54 (s, 2H), 4.52 - 4.42 (m, 1H), 4.25 (d, 1H), 4.03 - 3.92 (m, 1H), 3.71 - 3.58 (m, 2H), 3.57 - 3.37 (m, 4H), 3.23 - 3.10 (m, 1H), 3.08 - 2.91 (m, 4H), 2.79 - 2.50 (m, 10H), 2.48 - 2.29 (m, 6H), 2.22 - 2.08 (m, 2H), 2.07 - 1.97 (m, 2H), 1.96 - 1.84 (m, 2H), 1.83 - 1.52 (m, 2H), 1.51 - 1.20 (m, 16H), 1.19 - 1.07 (m, 4H), 0.96 - 0.80 (m, 3H).

[0351] (9R,llR,12R,13R)-3-(N-(Cyclopropylmethyl)-N-methylglycyl)-12 -(((2S,3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy-

7,9,11, 13, 15-pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- 14)(Compound 41). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and (cyclopropylmethyl)methylamine to provide the title compound (5.0 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz'. 681.39 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.52 (s, 2H), 4.70 - 4.52 (br m, 1H), 4.51 - 4.38 (m, 1H), 4.31 - 4.12 (m, 1H), 4.06 - 3.77 (m, 3H), 3.77 - 3.62 (m, 2H), 3.52 - 3.24 (m, 5H), 3.16 - 2.91 (m, 5H), 2.91 - 2.45 (m, 15H), 2.26 - 1.88 (m, 4H), 1.88 - 1.58 (m, 4H), 1.57 - 1.44 (m, 2H), 1.43 - 1.22 (m, 12H), 1.15 - 0.88 (m, 4H), 0.78 - 0.57 (m, 2H), 0.42 - 0.28 (m, 2H).

[0352] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isobutyl-N-methylgl ycyl)-ll-methoxy-

7,9,11, 13, 15-pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- 15)(Compound 42). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-1 and A-methyl isobutyl amine to provide the title compound (5.03 mg). Formate salt, mixture of C2 epimers. MS (ESI+) mlz'. 683.43 [M + H] ⁺ . ¹ H NMR (400 MHz, Methanol-d4) 6 8.55 (s, 1H), 4.47 (d, 1H), 4.42 - 4.29 (m, 1H), 4.25 (d, 1H), 3.74 - 3.58 (m, 2H), 3.54 - 3.27 (m, 5H), 3.26 - 3.08 (m, 3H), 3.08 - 2.94 (m, 4H), 2.77 - 2.56 (m, 8H), 2.56 - 2.29 (m, 3H), 2.26 (s, 3H), 2.20 (d, 2H), 2.15 - 2.04 (m, 1H), 2.02 - 1.87 (m, 2H), 1.88 - 1.75 (m, 2H), 1.76 - 1.50 (m, 4H), 1.49 - 1.38 (m, 2H), 1.37 - 1.18 (m, 13H), 1.05 - 0.80 (m, 8H).

[0353] (9R,llR,12R,13R)-3-(N-(tert-Butyl)-N-methylglycyl)-12-(((2S, 3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy-

7,9,11, 13, 15-pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-I1-1-1- 16)(Compound 43). Prepared according to the methods of S3-5-I1-1-1- 1 from S3- 1-11-1- 1 and N- methyl t-butylamine to provide the title compound (3.71 mg). Formate salt, mixture of C2 epimers. MS (ESI+) m/z: 683.42 [M + H] ⁺ . ¹ H NMR (400 MHz, Methanol-d4) 6 8.52 (s, 3H), 4.49 (d, 1H), 4.25 (d, 1H), 4.10 - 3.83 (br m, 3H), 3.78 - 3.63 (m, 2H), 3.51 - 3.34 (m, 5H), 3.10 - 2.95 (m, 4H), 2.85 - 2.45 (m, 14H), 2.25 - 2.12 (m, 1H), 2.11 - 1.93 (m, 3H), 1.92 - 1.55 (m, 5H), 1.53 - 1.43 (m, 2H), 1.42 - 1.21 (m, 21H), 1.08 - 0.88 (br m, 3H).

[0354] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(dimethylglycyl)-ll-me thoxy-7,9,ll,13,15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-2-l)(Compound 61). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and dimethylamine to provide the title compound (5.44 mg). Formate salt. MS (ESI+) m/z: 655.40 [M + H] ⁺ . ¹ H NMR (400 MHz, Methanol-d4) 8 8.48 (s, 3H), 5.08 - 4.94 (m, 1H), 4.61 (br d, 1H), 4.53 - 4.38 (m, 2H), 4.22 (d, 1H), 3.99 - 3.83 (m, 2H), 3.84 - 3.67 (m, 2H), 3.52 - 3.33 (m, 4H), 3.06 (s, 3H), 2.99 (d, 1H), 2.92 - 2.84 (m, 3H), 2.83 - 2.74 (m, 7H), 2.69 (br d, 6H), 2.36 - 2.19 (m, 2H), 2.18 - 2.07 (m, 1H), 2.06 - 1.96 (m, 2H), 1.91 - 1.73 (m, 2H), 1.58 - 1.44 (m, 5H), 1.44 - 1.27 (m, 13H), 1.08 (br d, 3H).

[0355] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,H,13,15,1 5-hexamethyl-3-(2- (piperidin-l-yl)acetyl)-17-oxa-3,7-diazaspiro[5.12]octadecan e-14, 16-dione (S3-5-I1-1-2- 2)(Compound 45). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and piperidine to provide the title compound (12.0 mg). Formate salt. MS (ESI+) mlz'. 695.43 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 5 8.50 (s, 3H), 5.05 - 4.93 (br s, 1H), 4.61 (d, 1H), 4.54 - 4.38 (m, 2H), 4.22 (d, 1H), 4.01 - 3.87 (m, 2H), 3.86 - 3.78 (m, 1H), 3.79 - 3.67 (m, 1H), 3.60 - 3.33 (m, 4H), 3.25 - 3.14 (br m, 1H), 3.09 - 2.92 (m, 7H), 2.87 (br s, 3H), 2.84 - 2.72 (m, 7H), 2.37 - 2.19 (m, 1H), 2.19 - 2.07 (m, 1H), 2.07 - 1.95 (m, 2H), 1.92 - 1.74 (m, 6H), 1.74 - 1.54 (m, 4H), 1.54 - 1.43 (m, 5H), 1.40 (d, 6H), 1.37 (d, 3H), 1.33 (d, 3H), 1.13 - 0.99 (m, 3H).

[0356] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isopropyl-N-methylg lycyl)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-5-I1-1-2- 3)(Compound 46). Prepared according to the methods of S3-5-I1- 1-1-1 from S3-1-I1-1-2 and N- methyl-isobutylamine to provide the title compound (13.6 mg). Formate salt. MS (ESI+) ml?.

683.41 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 5 8.50 (s, 2H), 5.01 (br s, 1H), 4.62 (d, 1H), 4.58 - 4.41 (m, 2H), 4.27 - 4.13 (m, 2H), 4.07 (d, 1H), 3.98 - 3.82 (m, 1H), 3.80 - 3.68 (m, 1H), 3.62 - 3.50 (m, 1H), 3.50 - 3.33 (m, 4H), 3.28 - 3.15 (m, 1H), 3.12 - 2.94 (m, 4H), 2.90 (br s, 3H), 2.85 - 2.72 (m, 10H), 2.40 - 2.08 (m, 3H), 2.09 - 1.95 (m, 2H), 1,85 (dd, 2H), 1.59 - 1.43 (m, 5H), 1.43 - 1.27 (m, 18H), 1.08 (dd, 3H).

[0357] (9R,llR,12R,13R)-3-(2-(Azetidin-l-yl)acetyl)-12-(((2S,3R,4S, 6R)-4-(dimethylamino)- 3-hydroxy-6-methyltetr ahydro-2H-pyran-2-yl)oxy)- 1 l-methoxy-7,9, 11,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-2-4)(Compound 47). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and azetidine to provide the title compound (3.49 mg). Formate salt. MS (ESI+) mlz'. 667.41 [M + H] ⁺ . ^J H NMR (400 MHz, Methanol-d4) 5 8.50 (s, 3H), 5.11 - 4.91 (br m, 1H), 4.66 - 4.53 (m, 1H), 4.50 (d, 1H), 4.47 - 4.34 (m, 1H), 4.32 - 4.17 (m, 3H), 4.17 - 4.03 (m, 4H), 3.84 - 3.61 (m, 2H), 3.52 - 3.33 (m, 5H), 3.12 - 2.94 (m, 4H), 2.93 - 2.71 (m, 9H), 2.55 - 2.42 (m, 2H), 2.39 - 2.10 (m, 3H), 2.08 - 1.92 (m, 3H), 1.92 - 1.61 (m, 3H), 1.61 - 1.45 (m, 5H), 1.45 - 1.25 (m, 11H), 1.14 - 1.00 (m, 3H).

[0358] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-3-(2- (pyrrolidin-l-yl)acetyl)-17-oxa-3,7-diazaspiro[5.12]octadeca ne-14, 16-dione (S3-5-I1-1-2- 5)(Compound 48). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and pyrrolidine to provide the title compound (5.04 mg). Formate salt. MS (ESI+) m!z\ 681.38 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.50 (s, 3H), 5.09 - 4.90 (br m, 1H), 4.59 (br d, 1H), 4.52 - 4.35 (m, 2H), 4.32 - 4.04 (m, 4H), 3.90 - 3.64 (m, 3H), 3.51 - 3.33 (m, 5H), 3.19 - 2.93 (m, 4H), 2.92 - 2.70 (m, 10H), 2.34 - 2.14 (m, 2H), 2.15 - 1.93 (m, 9H), 1.93 - 1.66 (m, 3H), 1.60 - 1.43 (m, 5H), 1.43 - 1.26 (m, 12H), 1.13 - 0.97 (m, 3H).

[0359] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-ethyl-N-methylglycy l)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-5-I1-1-2- 6)(Compound 49). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and methylethylamine to provide the title compound (9.04 mg). Formate salt. MS (ESI+) mlz-. 669.42 [M + H] ⁺ . Ti NMR (400 MHz, Methanol-d4) 5 8.47 (s, 2H), 5.03 (br d, 1H), 4.63 (d, 1H), 4.56 -

4.43 (m, 2H), 4.31 - 4.14 (m, 2H), 4.08 (dd, 1H), 3.88 (br d, 1H), 3.80 - 3.68 (m, 1H), 3.52 - 3.33 (m, 4H), 3.27 - 3.09 (m, 3H), 3.07 (s, 3H), 3.03 - 2.95 (m, 1H), 2.95 - 2.87 (m, 3H), 2.86 - 2.74 (m, 10H), 2.38 - 2.15 (m, 2H), 2.08 - 1.97 (m, 3H), 1.93 - 1.77 (m, 2H), 1.59 - 1.44 (m, 5H),

1.44 - 1.28 (m, 15H), 1.13 - 1.05 (m, 3H).

[0360] (9R,llR,12R,13R)-3-(Diethylglycyl)-12-(((2S,3R,4S,6R)-4-(dim ethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-H-l-2-7)(Compound 50). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and diethylamine to provide the title compound (5.78 mg). Formate salt. MS (ESI+) mlz-. 683.35 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.50 (s, 3H), 5.10 - 4.91 (br m, 1H), 4.59 (br d, 1H), 4.52 - 4.39 (m, 2H), 4.26 - 4.16 (m, 1H), 4.16 - 4.03 (m, 1H), 4.03 - 3.83 (m, 2H), 3.80 - 3.68 (m, 1H), 3.50 - 3.33 (m, 5H), 3.21 - 2.97 (m, 9H), 2.93 - 2.69 (m, 10H), 2.35 - 2.08 (m, 2H), 2.07 - 1.94 (m, 3H), 1.93 - 1.64 (m, 2H), 1.58 - 1.43 (m, 5H), 1.43 - 1.21 (m, 17H), 1.07 (br d, 3H).

[0361] (9R,llR,12R,13R)-3-(Cyclobutylglycyl)-12-(((2S,3R,4S,6R)-4-( dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-2-8)(Compound 51). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and cyclobutylamine to provide the title compound (5.90 mg). Formate salt. MS (ESI+) mlz'. 681.34 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.48 (s, 3H), 5.09 - 4.90 (br m, 1H), 4.69 - 4.53 (m, 1H), 4.52 - 4.40 (m, 2H), 4.21 (d, 1H), 4.03 - 3.60 (m, 5H), 3.51 - 3.34 (m, 4H), 3.19 - 2.96 (m, 5H), 2.94 - 2.65 (m, 9H), 2.40 - 2.24 (m, 4H), 2.24 - 2.07 (m, 4H), 2.07 - 1.94 (m, 3H), 1.94 - 1.59 (m, 6H), 1.59 - 1.44 (m, 4H), 1.44 - 1.26 (m, 11H), 1.07 (br d, 3H).

[0362] (9R,llR,12R,13R)-3-(Cyclopropylglycyl)-12-(((2S,3R,4S,6R)-4- (dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl- 17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S3-5-Il-l-2-9)(Compound 52). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and cyclopropylamine to provide the title compound (4.92 mg). Formate salt. MS (ESI+) mlz'. 667.37 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 8 8.45 (s, 3H), 5.10 - 4.90 (m, 1H), 4.63 (br d, 1H), 4.57 - 4.42 (m, 2H), 4.22 (d, 1H), 3.99 - 3.79 (m, 2H), 3.79 - 3.68 (m, 2H), 3.49 - 3.33 (m, 4H), 3.26 - 3.14 (m, 1H), 3.07 (s, 3H), 3.03 - 2.93 (m, 1H), 2.89 (br s, 3H), 2.84 - 2.74 (m, 7H), 2.50 - 2.38 (m, 1H), 2.38 - 2.19 (m, 2H), 2.19 - 1.93 (m, 4H), 1.93 - 1.72 (m, 3H), 1.58 - 1.45 (m, 5H), 1.44 - 1.26 (m, 11H), 1.08 (d, 3H), 0.66 - 0,53 (m, 3H), 0.53 - 0.39 (m, 1H).

[0363] (9R,llR,12R,13R)-3-((Cyclopropylmethyl)glycyl)-12-(((2S,3R,4 S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-5-I1-1-2- 10)(Compound 53). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and cyclopropylmethylamine to provide the title compound (5.22 mg). Formate salt. MS (ESI+) mlz'. 681.35 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 5 8.51 (s, 3H), 5.09 - 4.91 (m, 1H), 4.69 - 4.51 (m, 1H), 4.52 - 4.37 (m, 2H), 4.26 - 4.15 (m, 1H), 4.15 - 3.92 (m, 2H), 3.91 - 3.66 (m, 3H), 3.45 (dd, 1H), 3.41 - 3.32 (m, 3H), 3.11 (d, 1H), 3.06 (s, 3H), 2.93 - 2.83 (m, 4H), 2.82 - 2.68 (m, 7H), 2.37 - 2.06 (m, 2H), 2.07 - 1.91 (m, 3H), 1.91 - 1.62 (m, 2H), 1.57 - 1.43 (m, 5H), 1.43 - 1.25 (m, 11H), 1.18 - 0.91 (m, 5H), 0.75 - 0.65 (m, 2H), 0.56 - 0.47 (m, 1H), 0.44 - 0.34 (m, 2H), 0.23 (dt, 1H).

[0364] (9R,llR,12R,13R)-3-(N-Cyclopropyl-N-methylglycyl)-12-(((2S,3 R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-5-I1-1-2- ll)(Compound 54). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I1-1-2 and N-methyl cyclopropylamine to provide the title compound (3.16 mg). Formate salt. MS (ESI+) mlz\ 681.34 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 8 8.44 (s, 3H), 5.09 - 4.96 (m, 1H), 4.61 (br d, 1H), 4.56 - 4.38 (m, 2H), 4.22 (d, 1H), 4.17 - 4.03 (m, 1H), 3.80 - 3.67 (m, 1H), 3.51 - 3.32 (m, 7H), 3.27 - 3.14 (m, 1H), 3.06 (s, 3H), 3.02 - 2.73 (m, 11H), 2.40 (s, 3H), 2.35 - 2.23 (m, 1H), 2.12 - 1.93 (m, 4H), 1.93 - 1.75 (m, 3H), 1.59 - 1.44 (m, 5H), 1.44 - 1.27 (m, 11H), 1.08 (d, 3H), 0.50 (d, 2H), 0.44 (d, 2H).

[0365] (9R,llR,12R,13R)-3-(N-(Cyclopropylmethyl)-N-methylglycyl)-12 -(((2S,3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-/we^y/te/ra/tyJra-2H-pyran-2-yl) oxy)-l 1-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S3-5-I1-1-2- 12)(Compound 55). Prepared according to the methods of S3-5-I1- 1-1-1 from S3-1-I1 -1-2 and N- methylcyclopropylmethylamine to provide the title compound (4.60 mg). Formate salt. MS (ESI+) m/z: 695.37 [M + H] ⁺ . WMR ^OO MHz, Methanol -d4) 5 8.51 (s, 3H), 5.16 - 4.92 (m, 1H), 4.69 - 4.55 (m, 1H), 4.56 - 4.37 (m, 2H), 4.21 (d, 1H), 3.95 - 3.79 (m, 1H), 3.79 - 3.64 (m, 2H), 3.50 - 3.32 (m, 5H), 3.10 (d, 2H), 3.06 (s, 3H), 2.94 - 2.74 (m, 9H), 2.74 - 2.54 (m, 6H), 2.36 - 2.18 (m, 1H), 2.14 - 1.92 (m, 3H), 1.91 - 1.71 (m, 2H), 1.58 - 1.44 (m, 4H), 1.44 - 1.27 (m, 11H), 1.14 - 0.90 (m, 5H), 0.74 - 0.57 (m, 2H), 0.55 - 0.46 (m, 1H), 0.31 (s, 1H), 0.28 - 0.18 (m, 2H).

[0366] The following Examples were prepared according to the methods of Scheme 3, substituting the appropriate aminoalcohol (I) from Table 1.

[0367] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((llR,12R,13R,15R)-13-met hoxy-9,ll,I3,15,17- pentamethyl-8,10-dioxo-7-oxa-3,17-diazaspiro[5.12]octadecan- 12-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S3-1-I6-1-1) In a 20 mL vial was a solution of Boc piperidine SI-5-16-1 (105 mg, 0.138 mmol) in 20% trifluoro acetic acid (TFA)-DCM (1 mL) to give a yellow solution which was stirred at rt for 1 h. The reaction mixture was diluted with DCM and quenched with saturated aqueous sodium bicarbonate, the aqueous phase was extracted 3 times w/ DCM, dried over sodium sulfate, filtered and concentrated to give S3-1-I6-1-1 which was used without further purification (94 mg). MS (ESI+) mlz: 330.8 [M + 2H] ²⁺ , 660.4 [M + H] ⁺ .

[0368] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,ll,13,15,17 -pentamethyl-7-oxa-3,17- diazaspiro[5.72]octadecane-8, 10-dione (S3-2-I6-l-l-l)(Compound 29). Prepared according to the methods of S3-2-I1-1-1-1 from S3- 1 - 16- 1 - 1 to provide the title compound (9.5 mg). Formate salt, mixture of epimers. MS (ESI+) mfr. 278.9 [M + 2H] ²⁺ , 556.4 [M + H] ⁺ ’H NMR (400 MHz, Methanol-^) 5 8.51 (s, 2H), 4.59 (d, 0.55H), 4.54 - 4.31 (m, 2.9H), 4.22 (d, 0.55H), 3.86 - 3.52 (m, 3H), 3.52 - 3.38 (m, 2H), 3.20 - 3.02 (m, 3H), 3.00 (s, 3H), 2.90 - 2.73 (m, 7H), 2.73 - 2.32 (m, 3H), 2.23 - 1.90 (m, 4H), 1.90 - 1.62 (m, 3H), 1.62 - 1.46 (m, 2H), 1.46 - 1.12 (m, 13H), 1.02 (t, 3H), 0.94 - 0.77 (m, 1H).

[0369] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3,9,77,13,15, 17-hexamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (S3-2-I6-l-l-2)(Compound 111). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I6-1-1 and formaldehyde to provide the title compound (1.0 mg). Formate salt. MS (ESI+) mfr. 285.8 [M + 2H] ²⁺ , 570.3 [M + H] ^{+ J} H NMR (400 MHz, Methanol-^) 5 8.36 (s, 8H), 4.49 (d, 1H), 4.24 (d, , 1H), 4.13 (d, 1H), 3.83 - 3.52 (m, 4H), 3.52 - 3.39 (m, 3H), 3.16 - 2.92 (m, 8H), 2.84 (s, 6H), 2.69 - 2.45 (m, 5H), 2.45 - 2.26 (m, 2H), 2.17 (s, 1H), 2.11 - 1.82 (m, 4H), 1.76 (s, 2H), 1.54 (d, 2H), 1.49 - 1.17 (m, 12H), 1.05 (d, 3H).

[0370] (HR,12R,13R,15R)-3-Benzyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,ll,13,15,17 -pentamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (S3-2-I6-l-l-3)(Compound 31). Prepared according to the methods of S3-2-I6-1-1-2 and benzaldehyde to provide the title compound (8.4 mg). Formate salt. MS (ESI+) mlz'. 323.8 [M + 2H] ²⁺ , 646.4 [M + H] ⁺ ’H NMR (400 MHz, Methanol-^) 6 8.56 (s, 1H), 7.49 - 7.31 (m, 4H), 7.31 - 7.26 (m, 1H), 4.44 (dd, 1H), 4.17 (dd, 1H), 4.03 - 3.87 (m, 1H), 3.69 (d, 3H), 3.58 (q, 1H), 3.48 - 3.35 (m, 1H), 3.30 - 3.19 (m, 2H), 3.09 - 3.00 (m, 3H), 2.99 (s, 3H), 2.88 - 2.78 (m, 3H), 2.78 - 2.72 (m, 6H), 2.69 (s, 1H), 2.59 (t, 1H), 2.39 (t, 2H), 2.35 - 2.15 (m, 2H), 2.05 - 1.96 (m, 1H), 1.88 - 1.64 (m, 3H), 1.55 - 1.41 (m, 3H), 1.40 - 1.14 (m, 12H), 1.07 - 0.96 (m, 3H), 0.96 - 0.82 (m, 1H).

[0371] (HR,12R,13R,15R)-3-Acetyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,ll,13,15,17 -pentamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (S3-4-WFAl-l)(Compound 30) Prepared according to the methods of S3-4-I1-1-1-1 from S3-1-I6-1-1 and acetic anhydride to provide the title compound (3.1 mg). Formate salt. MS (ESI+) mlz-. 299.7 [M + 2H] ²⁺ , 598.4 [M + H] ⁺ (400 MHz, Methanol^) 8 8.56 (s, 2H), 4.61 - 4.32 (m, 2H), 4.32 - 4.10 (m, 1H), 3.99 - 3.50 (m, 4H), 3.46 - 3.34 (m, 1H), 3.25 (d, 1H), 3.15 - 2.89 (m, 1H), 3.02 (s, 6H), 2.84 - 2.50 (m, 6H), 2.50 - 2.19 (m, 4H), 2.17 - 2.07 (m, 3H), 2.05 - 1.52 (m, 4H), 1.52 - 1.14 (m, 16H), 1.14 - 0.77 (m, 5H).

[0372] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(dimethylglycyl)-13-me thoxy-9, 11,13, 15,17- pentamethyl-7-oxa-3,l 7-diazaspiro[5.12}octadecane-%, 10-dione (S3-5-I6-l-l-l)(Compound 34). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I6-1-1 and dimethylamine to provide the title compound (9.8 mg). Formate salt. MS (ESI+) mlz\ 214.6 321.3 [M + 2H] ²⁺ , 641.4 [M + H] ⁺ 'H NMR (400 MHz, Methanol-^) 8 8.52 (s, 3H), 4.53 - 4.39 (m, 2H), 4.24 (d, 1H), 4.09 - 3.88 (m, 2H), 3.76 (dd, 2H), 3.71 - 3.65 (m, 1H), 3.60 (q, 1H), 3.53 - 3.43 (m, 1H), 3.43 - 3.34 (m, 3H), 3.24 (s, 1H), 3.11 - 2.85 (m, 8H), 2.83 (s, 6H), 2.78 (s, 6H), 2.37 (d, 1H), 2.29 (d, 1H), 2.09 - 2.01 (m, 1H), 1.94 - 1.63 (m, 4H), 1.54 (q, 2H), 1.42 (s, 3H), 1.37 - 1.24 (m, 10H), 1.05 (d, 3H).

[0373] (HR,12R,13R,15R)-3-((Cyclopropylmethyl)glycyl)-12-(((2S,3R,4 S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o A:v)-13-methoxy- 9,ll,13,15,17-pentamethyl-7-oxa-3,17-diazaspiro[5.12]octadec ane-8, 10-dione (S3-5-I6-1-1- 2)(Compound 33). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I6-1-1 and cyclopropylmethylamine. Formate salt. MS (ESI+) mlz\ 223.3 [M + 3H] ³⁺ , 334.4 [M + 2H] ²⁺ , 667.4 [M + H] ⁺ ^NMR (400 MHz, Methanol-^) 8 8.65 (d, 2H), 4.49 (d, 1H), 4.44 (d, 1H), 4.23 (d, 1H), 4.02 (q, 2H), 3.85 - 3.51 (m, 4H), 3.51 - 3.36 (m, 2H), 3.19 - 2.91 (m, 3H), 3.04 (s, 3H), 2.87 (d, 3H), 2.76 (s, 6H), 2.37 (d, 3H), 2.06 - 1.94 (m, 1H), 1.94 - 1.59 (m, 4H), 1.58 - 1.18 (m, 16H), 1.18 - 0.80 (m, 5H), 0.75 - 0.63 (m, 2H), 0.46 - 0.29 (m, 2H).

[0374] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9, 11, 13,15, 17-pentamethyl-3-(2- (piperidin-l-yl)acetyl)-7-oxa-3,17-diazaspiro[5.12]octadecan e-8, 10-dione (S3-5-I6-1-1- 3)(Compound 35). Prepared according to the methods of S3-5-I1-1-1-1 from S3-1-I6-1-1 and piperidine. Formate salt. MS (ESI+) mlz\ 227.9 [M + 3H] ³⁺ , 341.4 [M + 2H] ²⁺ , 681.4 [M + H] ⁺ 'H NMR (400 MHz, Methanol-^) 5 8.54 (s, 3H), 4.49 (d, 1H), 4.45 (d, 1H), 4.24 (d, 1H), 4.08 - 3.52 (m, 7H), 3.52 - 3.34 (m, 3H), 3.21 - 2.89 (m, 11H), 2.82 (s, 6H), 2.37 (d, 1H), 2.30 (d, , 1H), 2.11 - 1.99 (m, 1H), 1.97 - 1.47 (m, 12H), 1.47 - 1.20 (m, 12H), 1.06 (d, 3H).

[0375] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isopropyl-N-wet/yVg /ycy/)-13-methoxy- 9,ll,13,15,17-pentamethyl-7-oxa-3,17-diazaspiro[5.12]octadec ane-8, 10-dione (S3-5-I6-1-1- 4)(Compound 32). Prepared according to the methods of S3-5-I1- 1-1-1 from S3- 1-16- 1-1 and N- methylisopropylamine. Formate salt. MS (ESI+) mlz'. 223.9 [M + 3H] ³⁺ , 335.4 [M + 2H] ²⁺ , 669.4 [M + H] ⁺ 'H NMR (400 MHz, Methanol-^) 5 8.48 (s, 3H), 4.52 - 4.32 (m, 2H), 4.28 - 3.98 (m, 3H), 3.81 - 3.49 (m, 5H), 3.49 - 3.31 (m, 3H), 3.25 - 3.10 (m, 1H), 3.10 - 2.88 (m, 7H), 2.81 (s, 6H), 2.78 (s, 3H), 2.44 - 2.09 (m, 3H), 2.09 - 1.97 (m, 1H), 1.97 - 1.61 (m, 4H), 1.59 - 1.44 (m, 2H), 1.44 - 1.12 (m, 19H), 1.04 (d, 3H), 0.93 - 0.80 (m, 1H). Compound 142

[0376] (HR,12R,13R,15R)-3-benzyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (Compound 142). Prepared according to Scheme S3 from 16 and benzaldehyde to provide the title compound (20 mg). Formate salt. MS (ESI+) mlz'.

660.4 [M + H] ⁺ Compound 143

[0377] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-(pyridin-3- ylmethyl)-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 143). Prepared according to Scheme S3 from 16 and pyridine-3-carboxaldehyde to provide the title compound (23 mg). Formate salt. MS (ESI+) mlz'. 661.5 [M + H] ⁺ Compound 144

[0378] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(3-methoxyb enzyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 144). Prepared according to Scheme S3 from 16 and 3 -methoxybenzaldehyde to provide the title compound (11 mg). Formate salt. MS (ESI+) mlz'. 690.5 [M + H] ⁺ Compound 145

[0379] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(4-chlorobe nzyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 145). Prepared according to Scheme S3 from 16 and 4-chlorobenzaldehyde to provide the title compound (22 mg). Formate salt. MS (ESI+) mlz'. 694.5 [M + H] ⁺ Compound 146

[0380] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(3-chlorobe nzyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 146). Prepared according to Scheme S3 from 16 and 3-chlorobenzaldehyde to provide the title compound (21 mg). Formate salt. MS (ESI+) mlz'. 694.5 [M + H] ⁺ Compound 147

[0381] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(isoquinolin-6-ylmethy l)-13-methoxy-

9,9,ll,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]oct adecane-8, 10-dione (Compound

147). Prepared according to Scheme S3 from 16 and isoquinoline-6-carbaldehyde to provide the title compound (20 mg). Formate salt. MS (ESI+) mlz\ 711.5 [M + H] ⁺ Compound 148

[0382] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-(2-

(pyridin-3-yl)ethyl)-7-oxa-3,17-diazaspiro[5.12]octadecan e-8, 10-dione (Compound 148).

Prepared according to Scheme S3 from 16 and 2-(pyri din-3 -yl)acetaldehyde to provide the title compound (11 mg). Formate salt. MS (ESI+) mlz\ 675.5 [M + H] ⁺ Compound 149

[0383] (HR,12R,13R,15R)-3-([l,l'-biphenyl]-3-ylmethyl)-12-(((2S,3R, 4S,6R)-4-

(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-y l)oxy)-13-methoxy-

9,9,H,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octa decane-8, 10-dione (Compound

149). Prepared according to Scheme S3 from 16 and [l,l'-biphenyl]-3-carbaldehyde to provide the title compound (11 mg). Formate salt. MS (ESI+) mlz\ 736.5 [M + H] ⁺ Compound 150 [0384] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3,9,9,H,13,15 ,17-heptamethyl-7-oxa-

3, 17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 150). Prepared according to Scheme

S3 from 16 and formaldehyde to provide the title compound as the formate salt. MS (ESI+) m/z:

584.4 [M + H] ⁺ Compound 151

[0385] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(isoquinolin-7-ylmethy l)-13-methoxy- 9,9,H,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadec ane-8, 10-dione (Compound

151). Prepared according to Scheme S3 from 16 and isoquinoline-7-carbaldehyde to provide the title compound (29 mg). Formate salt. MS (ESI+) m/z: 711.5 [M + H] ⁺ Compound 152

[0386] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(4-methoxyb enzyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 152). Prepared according to Scheme S3 from 16 and 4-methoxybenzaldehyde to provide the title compound (21 mg). Formate salt. MS (ESI+) zw/z: 690.4 [M + H] ⁺ Compound 153 [0387] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-phenethyl-

7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 153). Prepared according to

Scheme S3 from 16 and 2-phenylacetaldehyde to provide the title compound (17.1 mg). Formate salt. MS (ESI+) m!z-. 674.5 [M + H] ⁺ Compound 154

[0388] (HR,12R,13R,15R)-3-([l,l'-biphenyl]-4-ylmethyl)-12-(((2S,3R, 4S,6R)-4-

(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-y l)oxy)-13-methoxy-

9,9,H,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octa decane-8, 10-dione (Compound

154). Prepared according to Scheme S3 from 16 and [l,l'-biphenyl]-4-carbaldehyde to provide the title compound (21 mg). Formate salt. MS (ESI+) mlz'. 736.5 [M + H] ⁺ Compound 155

[0389] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,H,13,15,1 7-hexamethyl-3-(2-

(pyridin-4-yl)ethyl)-7-oxa-3,17-diazaspiro[5.12]octadecan e-8, 10-dione (Compound 155).

Prepared according to Scheme S3 from 16 and 2-(pyridin-4-yl)acetaldehyde to provide the title compound (11 mg). Formate salt. MS (ESI+) mlz\ 675.5 [M + H] ⁺ Compound 156 [0390] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-(pyridin-4- ylmethyl)-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 156). Prepared according to Scheme S3 from 16 and pyridine-4-carboxaldehyde to provide the title compound (20 mg). Formate salt. MS (ESI+) mlz'. 661.5 [M + H] ⁺ Compound 157

[0391] (HR,12R,13R,15R)-3-acetyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (Compound 157). Prepared according to Scheme S3 from 16 and acetyl chloride to provide the title compound as the formate salt. MS (ESI+) m/z:

612.4 [M + H] ⁺ Compound 158

[0392] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-(2- phenylacetyl)-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 158). Prepared according to Scheme S3 from 16 and 2-phenylacetyl chloride to provide the title compound (21 mg). Formate salt. MS (ESI+) mlz'. 688.5 [M + H] ⁺ Compound 159 [0393] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3-nicotinoyl- 7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 159). Prepared according to Scheme S3 from 16 and nicotinoyl chloride to provide the title compound (25 mg). Formate salt. MS (ESI+) mlz-. 675.5 [M + H] ⁺ Compound 160

[0394] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(4-methoxyb enzoyl)-9,9,H,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 160). Prepared according to Scheme S3 from 16 and 4-methoxybenzoyl chloride to provide the title compound (23 mg). Formate salt. MS (ESI+) mlz-. 704.5 [M + H] ⁺ Compound 161

[0395] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,H,13,15,1 7-hexamethyl-3-(2- (pyridin-3-yl)acetyl)-7-oxa-3,17-diazaspiro[5.12]octadecane- 8, 10-dione (Compound 161).

Prepared according to Scheme S3 from 16 and 2-(pyri din-3 -yl)acetyl chloride to provide the title compound (7 mg). Formate salt. MS (ESI+) mlz\ 689.5 [M + H] ⁺ Compound 162 [0396] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(3-methoxyb enzoyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 162). Prepared according to Scheme S3 from 16 and 3 -methoxybenzoyl chloride to provide the title compound (12 mg). Formate salt. MS (ESI+) mlz'. 704.5 [M + H] ⁺

Compound 163

[0397] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-3-(4-chlorobe nzoyl)-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 163). Prepared according to Scheme S3 from 16 and 4-chlorobenzoyl chloride to provide the title compound (22 mg). Formate salt. MS (ESI+) mlz'. 708.5 [M + H] ⁺

Compound 164

[0398] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-isonicotinoyl-13-metho xy-9,9,ll,13,15,17- hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 164). Prepared according to Scheme S3 from 16 and isonicotinoyl chloride to provide the title compound (25 mg). Formate salt. MS (ESI+) mlz'. 675.5 [M + H] ⁺

Compound 165

[0399] (HR,12R,13R,15R)-3-benzoyl-12-(((2S,3R,4S,6R)-4-(dimethylami no)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-7-oxa-3,17- diazaspiro [5.12] octadecane-8, 10-dione (Compound 165). Prepared according to Scheme S3 from 16 and benzoyl chloride to provide the title compound (25 mg). Formate salt. MS (ESI+) mlz'. 674.5 [M + H] ⁺

Compound 166

[0400] (HR,12R,13R,15R)-N-benzyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-8,10-dioxo-7- oxa-3,17-diazaspiro[5.12]octadecane-3-carboxamide (Compound 166). Prepared according to Scheme S3 from 16 and benzyl isocyanate to provide the title compound (14 mg). Formate salt. MS (ESI+) mlz.' 703.5 [M + H] ⁺ [0401] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-N-(4-methoxyp henyl)-9,9,ll,13,15,17- hexamethyl-8,10-dioxo-7-oxa-3,17-diazaspiro[5.12]octadecane- 3-carboxamide (Compound

167). Prepared according to Scheme S3 from 16 and 4-methoxyphenyl isocyanate to provide the title compound (30 mg). Formate salt. MS (ESI+) mlz'. 719.5 [M + H] ⁺

Compound 168

[0402] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-8,10-dioxo-

N-phenyl-7-oxa-3,17-diazaspiro[5.12]octadecane-3-carboxam ide (Compound 168). Prepared according to Scheme S3 from 16 and phenyl isocyanate to provide the title compound (19 mg). Formate salt. MS (ESI+) mlz'. 689.5 [M + H] ⁺

Compound 169

[0403] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-N-(4-chloroph enyl)-9, 9,11,13, 15,17- hexamethyl-8,10-dioxo-7-oxa-3,17-diazaspiro[5.12]octadecane- 3-carboxamide (Compound

169). Prepared according to Scheme S3 from 16 and 4-chlorophenyl isocyanate to provide the title compound (23 mg). Formate salt. MS (ESI+) mlz'. 723.4 [M + H] ⁺ Compound 170

[0404] (HR,12R,13R,15R)-3-((4-chlorophenyl)sulfonyl)-12-(((2S,3R,4S ,6R)-4-

(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-y l)oxy)-13-methoxy-

9,9,H,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octa decane-8, 10-dione (Compound

170). Prepared according to Scheme S3 from 16 and 4-chlorobenzenesulfonyl chloride to provide the title compound (12 mg). Formate salt. MS (ESI+) m/z: 744.4 [M + H] ⁺ Compound 171

[0405] (HR,12R,13R,15R)-3-((4-methoxyphenyl)sulfonyl)-12-(((2S,3R,4 S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-13-methoxy-

9,9,H,13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octa decane-8, 10-dione (Compound 171). Prepared according to Scheme S3 from 16 and 4-methoxybenzenesulfonyl chloride to provide the title compound (12 mg). Formate salt. MS (ESI+) m/z: 740.4 [M + H] ⁺ Compound 172

[0406] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,ll,13,15, 17-hexamethyl-3- (phenylsulfonyl)-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 172).

Prepared according to Scheme S3 from 16 and benzenesulfonyl chloride to provide the title compound (21 mg). Formate salt. MS (ESI+) mlz\ 710.4 [M + H] ⁺ Compound 173

[0407] (HR,12R,13R,15R)-3-(benzylsulfonyl)-12-(((2S,3R,4S,6R)-4-(di methylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9 ,ll,13,15,17-hexamethyl-7- oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 173). Prepared according to Scheme S3 from 16 and benzyl sulfonyl chloride to provide the title compound (24 mg). Formate salt. MS (ESI+) m/z: 724.5 [M + H] ⁺ Compound 174

[0408] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-13-methoxy-9,9,H43,15,17 -hexamethyl-3-(pyridin-3- ylsulfonyl)-7-oxa-3,17-diazaspiro[5.12]octadecane-8, 10-dione (Compound 174). Prepared according to Scheme S3 from 16 and pyridine-3-ylsulfonyl chloride to provide the title compound (22.8 mg). Formate salt. MS (ESI+) m/z: 711.5 [M + H] ⁺ Compound 175

[0409] (HR,12R,13R,15R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isopropyl-N-methylg lycyl)-13-methoxy- 9,9,ll _i 13,15,17-hexamethyl-7-oxa-3,17-diazaspiro[5.12]octadec ane-8, 10-dione (Compound

175). Prepared according to Scheme S3 from 16 and 7V-m ethylisopropylamine to provide the title compound as the formate salt. MS (ESI+) m/z: 683.5 [M + H] ^+y

[0410] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((9R,HR,12R,13R)-ll-metho xy-7,9,ll,1^15,15- hexamethyl-14,16-dioxo-17-oxa-2,7-diazadispiro[3.1.126.14]no nadecan-12-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S3-1-I5-1-2) and (2S,3R,4S,6R)-2- (((7R,9R,10R,llR)-2-(aminomethyl)-2-(chloromethyl)-9-methoxy -5,7,9,ll,13,13- hexamethyl-12,14-dioxo-15-oxa-5-azaspiro[3.12]hexadecan-10-y l)oxy)-4-(dimethylamino)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S3-l-I5b-l-2). S2-2-I5-1 (87 mg, O i l mmol) was dissolved in MeCN (3 mL) and HC1 (4M in dioxane, 0.27 mL, 1.1 mmol) was added at rt and the reaction mixture was allowed to stirred at rt for 10 min. The reaction mixture was concentrated and the crude mixture of S3-1-I5-1-2 and S3-l-I5b-l-2 (83 mg, 95% yield) was used in the next step without further purification. S3-1-I5-1-2: MS (ESI+) mlz'. 229.5 [M + 3H] ³⁺ , 343.7 [M + 2H] ²⁺ , 686.4 [M + H] ⁺ . S3-l-I5b-l-2: MS (ESI+) mlz\ 241.5 [M + 3H] ³⁺ , 361.7 [M + 2H] ²⁺ , 722.4 [M + H] ⁺ .

[0411] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,11513,15, 15-hexamethyl-17-oxa-2,7- diazadispiro[3.1.126.14]nonadecane-14, 16-dione (S3-2-I5-l-2-l)(Compound 96). Prepared according to the methods of S3-2-I1-1-1-1 from S3-1-I5-1-2. Formate salt (6.00 mg, 51% yield). MS (ESI+) mlz\ 194.8 [M + 3H] ³⁺ , 297.1 [M + 2H] ²⁺ , 582.4 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 8.54 (s, 3H), 4.45 (dd, 1H), 4.34 - 3.88 (m, 4H), 3.70 (s, 1H), 3.43 (q, 1H), 3.31 (p, 3H), 3.15 (d, 2H), 3.03 (s, 2H), 2.90 - 2.59 (m, 7H), 2.27 (d, 3H), 2.00 (d, 1H), 1.59 - 1.05 (m, 15H), 0.97 (d, 2H).

[0412] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-2,7,9,H ₉ 13,15,15-heptamethyl-17-oxa- 2, 7-diazadispiro[3.1.126.14]nonadecane-14, 16-dione (S3-2-I5-l-2-2)(Compound 101) and (9R,llR,12R,13R)-12-(((2S,3R?4S,6R)-4-(dimethylamino)-3-hydr oxy-6-methyltetrahydro- 2H-pyran-2-yl)oxy)-ll-methoxy-2,2,7,9,H,13,15,15-octamethyl- 14,16-dioxo-17-oxa-2,7- (ZiazatZispira[3.1.126.14]nonadecan-2-ium (S3-2-I5-l-2-3)(Compound 107). Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I5-1-2 and formaldehyde.

[0413] Data for Compound 101 (S3-2-I5-1-2-2): Formate salt (2.83 mg). MS (ESI+) mlz'. 199.5 [M + 3H] ³⁺ , 298.7 [M + 2H] ²⁺ , 596.4 [M + H] ⁺ ; ³ HNMR (400 MHz, Methanol-^) 5 8.53 (s, 3H), 4.44 (dd, 1H), 4.20 (d, 1H), 4.02 (d, 1H), 3.92 (s, 1H), 3.86 (d, 1H), 3.70 (ddd, 1H), 3.43 (dd, 1H), 3.31 (p, 4H), 3.15 - 3.05 (m, 1H), 3.03 (s, 2H), 2.83 - 2.71 (m, 7H), 2.69 (d, 2H), 2.66 (s, 1H), 2.57 (d, 1H), 2.36 (s, 1H), 2.29 (d, 2H), 1.99 (ddd, 1H), 1.72 (s, 1H), 1.53 (s, 1H), 1.50 (s, 3H), 1.47 (d, 1H), 1.42 (d, 4H), 1.31 (p, 7H), 1.19 (d, 1H), 0.99 (s, 2H).

[0414] Data for Compound 107 (S3-2-I5-1-2-2): Formed during the reaction from S3-l-I5b-l-2. Formate salt (2.13 mg). MS (ESI+) mlz-. 204.1 [M + 3H] ³⁺ , 305.7 [M + 2H] ²⁺ , 610.4 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 6 8.54 (s, 3H), 4.59 (s, 1H), 4.40 (d, 1H), 4.30 - 4.15 (m, 2H), 4.11 (d, 1H), 3.90 - 3.77 (m, 1H), 3.67 (d, 2H), 3.59 - 3.39 (m, 2H), 3.39 - 3.26 (m, 7H), 3.09 (d, 4H), 2.81 (d, 3H), 2.66 (d, 9H), 2.29 (d, 9H), 2.13 (d, 1H), 1.95 (d, 1H), 1.77 - 1.58 (m, 4H), 1.58 - 1.36 (m, 9H), 1.29 (t, 9H), 1.19 (d, 3H).

[0415] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-isopropyl-ll-methoxy-7 ,9,H,13,15,15-hexamethyl- 17-oxa-2,7-diazadispiro[3.1.126.14]nonadecane-14, 16-dione (S8-7a-2)(Compound 102).

Prepared according to the methods of S3-2-I1-1-1-2 from S3-1-I5-1-2 and acetone. Formate salt (4.70 mg). MS (ESI+) mlz\ 208.3 [M + 3H] ³⁺ , 312.7 [M + 2H] ²⁺ , 624.5 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 8 'H NMR (400 MHz, Chloroform-;/) 8 8.52 (s, 3H), 4.46 (d, 1H), 4.21 (d, 1H), 4.06 (d, 1H), 3.96 (s, 1H), 3.88 (s, 1H), 3.78 - 3.65 (m, 1H), 3.44 (dd, 1H), 3.41 - 3.28 (m, 3H), 3.03 (s, 3H), 2.78 (s, 6H), 2.71 (s, 1H), 2.42 (d, 1H), 2.28 (d, 1H), 2.01 (ddd, 1H), 1.57 - 1.46 (m, 4H), 1.48 - 1.25 (m, 12H), 1.13 (d, 5H), 1.00 (d, 2H).

[0416] (9R,llR,12R,13R)-2-Acetyl-12-(((2S,3R,4S,6R)-4-(dimethylamin o)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-17-oxa-2,7- diazadispiro[3.1.126.14]nonadecane-14, 16-dione (S3-4-I5-l-2-l)(Compound 105) and N- (((7R,9R,10R,llR)-2-(chloromethyl)-10-(((2S,3R,4S,6R)-4-(dim ethylamino)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-9-methoxy-5,7,9,ll,13,13 -hexamethyl-12,14-dioxo-15- oxa-5-azaspiro[3.12]hexadecan-2-yl)methyl)acetamide (S3-4-I5b-l-2-l)(Compound 106). Prepared according to the methods of S3-4-I1-1-1-1 from a mixture of S3-1-I5-1-2 and S3-1-I5b- 1-2 with acetic anhydride.

[0417] Data for Compound 105 (S3-4-I5-1-2-1): Formate salt (5.88 mg). MS (ESH) mlz'. 312.7 [M + 2H] ²⁺ , 624.4 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 8.53 (s, 3H), 4.45 (d, 1H), 4.31 (d, 1H), 4.22 (d, 2H), 4.09 - 3.97 (m, 3H), 3.70 (dddt, 1H), 3.42 (dd, 1H), 3.34 - 3.25 (m, 3H), 3.04 (s, 3H), 2.74 (s, 7H), 2.04 - 1.90 (m, 2H), 1.85 (d, 3H), 1.45 - 1.38 (m, 4H), 1.39 - 1.27 (m, 9H), 1.02 (d, 2H).

[0418] Data for Compound 106 (S3-4-I5b-l-2-l): Formate salt (3.05 mg). MS (ESH) mlz'. 330.7 [M + 2H] ²⁺ , 660.4 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 8.55 (s, 3H), 4.56 (d, 1H), 4.20 (s, 2H), 3.83 - 3.57 (m, 2H), 3.51 - 3.22 (m, 4H), 3.07 (d, 3H), 2.56 (d, 5H), 2.22 (d, 3H), 2.04 - 1.79 (m, 5H), 1.38 (s, 7H), 1.33 - 1.17 (m, 6H), 0.94 (s, 2H).

[0419] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Z)i>M^jZawiMo)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(dimethylglycyl)-ll-me thoxy-7,9,ll,13,15,15- hexamethyl-17-oxa-2,7-diazadispiro[3.1.126.14]nonadecane-14, 16-dione (S3-5-I5-1-2- l)(Compound 103) and N-(((7R,9R,10R,HR)-2-(chloromethyl)-10-(((2S,3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)o xy)-9-methoxy- 5,7,9,H,13,13-hexamethyl-12,14-dioxo-15-oxa-5-azaspiro[3.12] hexadecan-2-yl)methyl)-2- (dimethylamino)acetamide (S3-5-I5b-l-2-l)(Compound 104). Prepared according to the methods of S3-5-I1-1-1-1 from a mixture of S3- 1 - 15- 1 -2 and S3- 1 - 15b- 1 -2 with dimethylamine. [0420] Data for Compound 103 (S3-5-I5-1-2-1): Formate salt (5.32 mg). MS (ESI+) mfr. 223.2 [M + 3H] ³⁺ , 334.3 [M + 2H] ²⁺ , 667.5 [M + H] ⁺ ; ³ HNMR (400 MHz, Methanol-^) 8.52 (s, 3H), 4.45 (d, 1H), 4.35 (d, 1H), 4.30 - 4.17 (m, 2H), 4.17 - 3.97 (m, 3H), 3.83 - 3.62 (m, 1H), 3.54 - 3.39 (m, 1H), 3.39 - 3.21 (m, 5H), 3.04 (s, 3H), 2.78 (s, 11H), 2.61 - 2.35 (m, 7H), 2.30 (d, 1H), 2.23 - 2.11 (m, 1H), 2.01 (ddd, 1H), 1.59 - 1.45 (m, 4H), 1.40 (d, 5H), 1.33 (dd, 6H), 1.03 (d, 2H).

[0421] Data for Compound 104 (S3-5-I5b-l-2-l): Formate salt (2.82 mg, 18% yield). MS (ESI+) mfr. 235.1 [M + 3H] ³ ’, 352.2 [M + 2H] ²⁺ , 703.4 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 8.48 (s, 3H), 4.48 (d, 1H), 4.25 (d, 1H), 3.75 (d, 3H), 3.64 - 3.36 (m, 4H), 3.36 - 3.25 (m, 5H), 3.20 (s, 2H), 3,06 (s, 4H), 2.96 (dt, 2H), 2.80 (s, 7H), 2.43 (s, 6H), 2.14 - 1.97 (m, 2H), 1.62 - 1.44 (m, 4H), 1.44 - 1.21 (m, 11H), 1.04 (d, 2H).

[0422] (7R,9R,10R,HR)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydrox y-6- methyltetrahydro-2H-pyran-2-yl)oxy)-9-methoxy-7,9,ll ₉ 13-tetramethyl-15-oxa-2,5- diazaspiro[3.12]hexadecane-12, 14-dione (S3-2-I4-l-l-l)(Compound 20). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3-2-I1-1-1-1. Mixture of epimers. Formate salt. MS (ESI+) mlz-. 514.33 [M + H] ⁺ ; ^NMR (400 MHz, Methanol-^) 5 4.43 (s, 2H), 4.15 - 3.93 (m, 3H), 3.95 - 3.81 (m, 2H), 3.77 - 3.53 (m, 1H), 3.50 - 3.32 (m, 4H), 2.84 (d, 9H), 2.77 (s, 1H), 2.63 (dd, 1H), 2.21 (t, 1H), 2.13 - 1.98 (m, 1H), 1.79 (d, 1H), 1.62 - 1.38 (m, 5H), 1.39 - 1.14 (m, 12H), 1.05 - 0.87 (m, 4H).

[0423] (7R,9R,10R,HR)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydrox y-6- methyltetrahydro-2H-pyran-2-yl)oxy)-9-methoxy-2,5,7,9,ll,13- hexamethyl-15-oxa-2,5- diazaspiro[3.12]hexadecane-12, 14-dione (S3-2-I4-2-l-l)(Compound 25). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3-2-I1-1-1-2 with formaldehyde. Mixture of epimers. Formate salt. MS (ESI+) mlz'. 542.26 [M + H]“; 'H NMR (400 MHz, Methanol-^) 8 4.49 (d, 1H), 4.30-4.40 (m,lH), 4.26 (d, 1H), 3.98 (d, 1H), 3.87 (d, 2H), 3.81 - 3.60 (m, 3H), 3.56 - 3.32 (m, 3H), 2.99 (s, 3H), 2.81 (d, 7H), 2.74 (d, 3H), 2.37 (d, 5H), 2.08 - 1.95 (m, 1H), 1.73 (dd, 1H), 1.61 (s, 1H), 1.50 (q, 1H), 1.42-1.35 (m, lH), 1.34 - 1.24 (m, 13H), 0.97 (d, 3H).

[0424] (7R,9R,10R,llR)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydro xy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(dimethylglycyl)-9-met hoxy-7,9,ll,13-tetramethyl- 15-oxa-2,5-diazaspiro[3.12]hexadecane-12, 14-dione (S3-5-I4-l-l-l)(Compound 24). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3-5-I1-1-1-1 from S3-1-I4-1-1 and dimethylamine. Mixture of epimers. Formate salt. MS (ESI+) mlz\ 599.32 [M + H] ⁺ ; ^} H NMR (400 MHz, Methanol-^) 84.31 (d, 3H), 4.12-3.82 (m,4H), 3.74 (dd, 3H), 3.67 - 3.61 (m, 1H), 3.58 - 3.33 (m, 6H), 2.79 (d, 7H), 2.70 (d, 1H), 2.61 (s, 5H), 2.50 (s, 4H), 2.39 (s, 1H), 2.01 (d, 1H), 1.80 (d, 2H), 1.62 - 1.35 (m, 3H), 1.35 - 1.16 (m, 10H), 0.97 (t, 3H).

[0425] (7R,9R,10R,llR)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydro xy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(N-zso/?ropvZ-N-methyl glycyl)-9-niethoxy- 5,7,9,H,13-pentamethyl-15-oxa-2,5-diazaspiro[3.12]hexadecane -12,14-dione (S3-5-I4-2-1- l)(Compound 40). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3- 5-11-1-1-1 from S3-1-I4-2-1 and N-methylisopropylamine. Mixture of epimers. Formate salt. MS (ESI+) mlz\ 641.42 [M + H] ⁺ ; NMR (400 MHz, Chloroform-J) 8 4.55 - 4.44 (m, 1H), 4.35 - 4.13 (m, 2H), 4.03 (t, 1H), 3.93 (d, 1H), 3.87 - 3.72 (m, 2H), 3.64 - 3.33 (m, 2H), 3.33 - 3.18 (m, 1H), 3.17 - 3.04 (m, 2H), 3.03 - 2.91 (m, 3H), 2.86 (dp, 2H), 2.54 (s, 4H), 2.33 (d, 4H), 2.23 (d, 3H), 2.19 - 1.97 (m, 1H), 1.79 (d, 11H), 1.37 - 1.19 (m, 11H), 1.01 (t, 6H), 0.91 (dd, 3H).

[0426] (5S,8R,10R,l 1R,12R)-1 l-(((2S,3R,4S,6R)-4-(Z)zzMet/zy/azMzno)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-10-methoxy-6,8,10,12,14- pentamethyl-16-oxa-2,6- diazaspiro [4.12] heptadecane-13, 15-dione (S3-2-I3-l-l-l)(Compound 58). Prepared according to the methods of S3-2-I1-1-1-1 from S3-1-I3-1-1. Separated from diastereomer via prep HPLC to provide the title compound (2.91 mg). MS (ESI+) mlz'. 542.2 [M + H]“. [0427] (5R,8R,10R,llR,12R)-ll-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-10-methoxy-6,8,10,12,14- pentamethyl-16-oxa-2,6- (Zi'azasp/ro[4.12]heptadecane-13,15-dione (S3-2-I3-l-l-2)(Compound 57). Prepared according to the methods of S3-2-I1-1-1-1 from S3-1-I3-1-1. Separated from diastereomer via prep HPLC to provide the title compound (2.39 mg). MS (ESI+) mlz'. 542.2 [M + H]“.

[0428] (8R,10R,llR,12R,14R)-ll-(((2S,3R,4S,6R)-4-(Dimethylamino)-3- hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-10-methoxy-2,6,8,10,12,1 4-hexamethyl-16-oxa-2,6- </iazaspira[4.12]heptadecane-13, 15-dione (S3-2-I3-l-l-l)(Compound 72). Prepared according to the methods of S3-2-I1-1-1-1 from S3-1-I3-1-1. Mixture of diastereomers. MS (ESI+) m/z: 279.4 [M + 2H]2+, 556.1 [M + H]+. MS (ESI+) m/z: 271.7 [M + 2H]2+, 542.2 [M + H]+. ’H NMR (400 MHz, Chloroform-d ) 5 4.49 - 4.42 (m, 1H), 4.22 (dd, 1H), 3.98 (q, 1H), 3.73 - 3.65 (m, 1H), 3.51 - 3.32 (m, 1H), 3.08 (d, 3H), 3.03 (s, 3H) 2.73 (s, 6H), 2.48 - 2.22 (m, 6H), 1.97 (d, 1H), 1.53 - 1.06 (m, 15H), 1.11 - 0.98 (m, 2H), 0.96 - 0.82, (m, 4H).

[0429] (8R,10R,llR,12R,14R)-2-Acetyl-ll-(((2S,3R,4S,6R)-4-(dimethyl amino)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-10-methoxy-6,8,10,12,14- pentamethyl-16-oxa-2,6- diazaspiro [4.12] heptadecane-13, 15-dione (S3-4-I3-l-l-l)(Compound 63). Prepared according to the methods of S3-4-I1-1-1-1 from S3-1-I3-1-1. Mixture of diastereomers. MS (ESI+) m/z: 292.8 [M + 2H] ²⁺ , 584.3 [M + H] ⁺ . *H NMR (400 MHz, Methanol-d4) 54.58 - 4.38 (m, 3H), 4.29 (dd„ 1H), 4.04 - 3.92 (m, 1H), 3.73 - 3.51 (m, 3H), 3.47 (q, J 1H), 3.45 - 3.32 (m, 2H), 3.01 (d, 3H), 2.81 (s, 6H), 2.66 (s, 2H), 2.53 - 2.09 (m, 4H), 2.04 (d, 3H), 2.00 (d, 1H), 1.51 (q, 2H), 1.36 - 1.18 (m, 12H), 0.93 - 0.84 (m, 3H).

[0430] (8R,10R,llR,12R,14R)-ll-(((2S,3R,4S,6R)-4-(Dimethylamino)-3- hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(dimethylglycyl)-l 0-methoxy-6,8, 10,12, 14- pentamethyl-16-oxa-2,6-diazaspiro[4.12]heptadecane-13, 15-dione (S3-5-I3-1-1- l)(Compound 62). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3- 5-11-1-1-1 from S3-1-I3-1-1 and dimethylamine. Mixture of diastereomers. MS (ESI+) m/z: 314.3 [M + 2H] ²⁺ , 627.5 [M + H] ⁺ .

[0431] (9R,10R,llR,13R)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(2-(3-hydroxyazetidin- l-yl)acetyl)-ll-methoxy- 7,9,11, 13, 15-pentamethyl-5-oxa-2,15-diazaspiro[3.12]hexadecane-6, 8-dione (S3-5-I8-2-1- l)(Compound 38). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3- 5-11-1-1-1 from S3-1-I8-2-1 and 3-hydroxyazetidine. Mixture of epimers. Formate salt. MS (ESI+) mlz-. 641.38 [M + H] ⁺ ; NMR (400 MHz, Methanol-^) 54.50-4.38 (m, 4H), 4.19 (m, 4H), 3.74 (dd, 4H), 3.66 - 3.33 (m, 6H), 3.25 - 3.12 (m, 1H), 2.90 (d, 13H), 2.04 (d, 2H), 1.96 - 1.61 (m, 2H), 1.62 - 1.11 (m, 16H), 1.01 (dq, 4H).

[0432] (9R,10R,llR,13R)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(N-isopropjZ-N-methylg lycyl)-ll-methoxy- 7,9,11, 13, 15-pentamethyl-5-oxa-2,15-diazaspiro[3.12]hexadecane-6, 8-dione (S3-5-I8-2-1-

2)(Compound 39). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3- 5-11-1-1-1 from S3-1-I8-2-1 and N-methylisopropylamine. Mixture of epimers. Formate salt. MS (ESI+) mlz-. 641.41 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 5 4.50-4.38 (m, 2H), 4.19 (s, 2H), 3.99 (s, 1H), 3.87 - 3.53 (m, 4H), 3.55 - 3.31 (m, 6H), 3.06 - 2.51 (m, 15H), 2.03 (q, 2H), 1.71 - 1.41 (m, 2H), 1.40 - 1.07 (m, 23H), 1.06 - 0.76 (m, 3H).

Scheme S4.

[0433] 3-Benzyl 7-(tert-butyl) (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4- (dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-met hoxy-9, 11,13, 15- tetramethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecan e-3,7-dicarboxylate (S4-1-I1).

SI-4-11 (1.126 g, 1.43 mmol) was dissolved in A. A-di methyl form am ide (7.15 mL), and di-tert- butyl dicarbonate (0.491 mL, 2.14 mmol) and 4-dimethylaminopyridine (17.4 mg, 0.143 mmol) were added. After 1 h, additional di-tert-butyl dicarbonate (0.491 mL, 2.14 mmol) and 4- dimethylaminopyridine (17.4 mg, 0.143 mmol) were added. After an additional 1 h, the reaction mixture was diluted with EtOAc and was washed with NaHCCh (sat, aq, 1 time), water (3 times), and brine (1 time). The EtOAc layer was dried over Na2SO4, was filtered, and was concentrated under reduced pressure. The residue was purified on 80 g of silica gel (elution with 0-10% MeOH-DCM gradient) to give S4-1-I1 (980 mg, 78%) . MS (ESI+) mlz\ 880.33 [M + H] ⁺ .

[0434] tert-Butyl (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimeth ylamino)- 6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-9,ll,13,15- tetramethyl-14,16-dioxo-17- oxa-3,7-diazaspiro[5.12]octadecane-7-carboxylate (S4-2-I1). S4-1-I1 (820 mg, 0 931 mmol) was dissolved in methanol (1.86 mL) and 1 N aqueous HC1 (0.931 mL, 0.931 mmol), and palladium on carbon (5 wt%, 198 mg, 0.093 mmol) was added. An atmosphere of EL was introduced. After 7 h, the reaction mixture was evacuated and back-filled with N2 (3 times). The reaction mixture was filtered through Celite®, washing with MeOH, and the filtrate was concentrated. This gave the HC1 salt of S4-2-I1 which was used without further purification. MS (ESI+) m/z: 746.23 [M + H] ⁺ .

[0435] tert-Butyl (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(rfiwietAy/g/jcj7)-ll- methoxy-9,ll,13,15- tetramethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecan e-7-carboxylate (S4-3-I1-1).

Prepared according to the methods of S3-5-I1-1-1-1 from S4-2-I1 and dimethylamine to give S4- 3-11-1 , which was used without further purification. MS (ESI+) mlr. 727.34 [M + H] ⁺ .

[0436] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(dimethylglycyl)-ll-me thoxy-9, 11, 13,15- tetramethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S4-4-Il-l)(Compound 21). S4-3-I1-1 (26.3 mg, 0.0361 mmol) was dissolved in 4M HC1 in 1,4-dioxane (0.4 mL). After 1 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCO2H) to give S4-4-I1-1 (8.55 mg). MS (ESI+) mlz-. 627.37 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 8 8.08 (br s, 1H), 5.04 - 4.90 (m, 1H), 4.54 - 4.21 (m, 4H), 4.21 - 4.10 (m, 1H), 4.03 - 3.93 (m, 1H), 3.86 - 3.68 (m, 2H), 3.68 - 3.54 (m, 1H), 3.51 - 3.35 (m, 3H), 3.28 - 3.17 (m, 1H), 3.18 - 3.04 (m, 2H), 3.04 - 2.92 (m, 9H), 2.91 - 2.72 (m, 7H), 2.34 - 2.17 (m, 1H), 2.16 - 1.85 (m, 5H), 1.85 - 1.60 (m, 2H), 1.60 - 1.43 (m, 2H), 1.43 - 1.27 (m, 10H), 1.20 - 1.03 (m, 4H).

[0437] (9R,llR,12R,13R)-3-(Cydopropylglycyl)-12-(((2S,3R,4S,6R)-4-( dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxp)-ll-methoxy-9, 11,13, 15-tetramethyl-17- oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S4-4-Il-2)(Compound 22). Prepared according to the methods of S4-4-I1-1 from S4-1-I1 and cyclopropylamine to provide the title compound (10.2 mg). Formate salt. MS (ESI+) mlz-. 639.35 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 8 8.43 (s, 3H), 4.84 - 4.65 (m, 1H), 4.49 - 4.28 (m, 1H), 4.24 - 3.97 (m, 2H), 3.97 - 3.61 (m, 7H), 3.61 - 3.30 (m, 5H), 3.08 - 2.87 (m, 4H), 2.86 - 2.71 (m, 4H), 2.72 - 2.58 (m, 2H), 2.58 - 2.38 (m, 2H), 2.11 - 1.90 (m, 3H), 1.90 - 1.63 (m, 6H), 1.63 - 1.40 (m, 3H), 1.39 - 1.22 (m, 7H), 1.22 - 1.11 (m, 2H), 1.10 - 0.96 (m, 3H), 0.76 - 0.51 (m, 4H).

[0438] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Diwie/Ay/awzino)-3-hy droxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(N-isopropyl-N-methylg lycyl)-ll-methoxy-

9,11, 13, 15-tetramethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S4-4-I1- 3)(Compound 23). Prepared according to the methods of S4-4-I1-1 from S4-1-I1 and N- methylisopropylamineto provide the title compound (9.88 mg). Formate salt. MS (ESI+) m!z\ 655.33 [M + H] ⁺ . ^L H NMR (400 MHz, Methanol-d4) 5 8.49 (s, 2H), 4.78 (d, 1H), 4.46 (d, 1H), 4.23 - 3.95 (m, 5H), 3.83 - 3.65 (m, 2H), 3.64 - 3.51 (m, 2H), 3.50 - 3.33 (m, 4H), 3.01 - 2.85 (m, 4H), 2.82 (s, 6H), 2.79 (s, 3H), 2.62 (q, 1H), 2.10 - 1.93 (m, 3H), 1.92 - 1.76 (m, 3H), 1.75 - 1.65 (m, 1H), 1.65 - 1.44 (m, 3H), 1.40 - 1.22 (m, 18H), 1.05 (d, 3H).

Scheme S5.

[0439] Butyl 3-((((2/?.4/?.5/?.6/?)-5-(((2.S'.3/?.4.S.6/?)-3-(Benzoyloxy) -4-(dimethylamino)-6- methyltetrahydro-2//-pyran-2-yl)oxy)-4-methoxy-2,4-diinethyl -6-(2,2,5-trimethyl-4-oxo-4//- l,3-dioxin-6-yl)heptyl)((benzyloxy)carbonyl)amino)methyl)-3- hydroxypyrrolidine-l- carboxylate (S5-1-I7). Ze/V-Butyl 3 -(aminomethyl)-3 -hydroxypyrrolidine- 1 -carboxylate (17, 124 mg, 0.577 mmol) and Sl-1 (310 mg, 0.525 mmol) were dissolved in MeOH (3 mL) and Ti(OEt)4 (0.217 mL, 1.04 mmol) was added. After 30 min, a small aliquot was removed from the reaction mixture and was added to a suspension of a small amount of NaBLL in MeOH. LC/MS analysis showed complete conversion. The reaction mixture was cooled in an ice bath and NaBHi (29.7 mg, 0.787 mmol) was added. When gas evolution ceased, 30% aqueous NH4OH (2mL) was added, and the mixture was filtered through a pad of Celite® with EtOAc. The filtrate was washed with brine, was dried over Na2SO4, was filtered and was concentrated. The residue was dissolved in DCM (3.5 mL), and triethylamine (0.144 mL, 1.04 mmol) followed by Cbz-OSu (130 mg, 0.525 mmol) were added. After 30 mins, solvent was removed and the crude residue was purified on a 12 g SiCh column, eluting with 2-14% MeOH/DCM/0.5% NH4OH, to give the title compound (0.4 g, 0.432 mmol, 83%). MS (ESI+) mfr. 412.92 [M + 2H] ²⁺ , 924.38 [M + H] ⁺ , 946.42 [M + Na] ⁺ ; ^NMR (400 MHz, Chloroform-;/) 5 8.04 - 7.94 (m, 2H), 7.54 (t, 1H), 7.41 (t, 2H), 7.33 (q, 5H), 5.19 - 5.00 (m, 3H), 4.65 (d, 1H), 3.81 (d, 1H), 3.64 - 3.27 (m, 8H), 3.27 - 3.04 (m, 2H), 3.04 - 2.75 (m, 5H), 2.28 (s, 5H), 2.04 - 1.84 (m, 2H), 1.84 - 1.73 (m, 6H), 1.69 (s, 3H), 1.65 (d, 3H), 1.43 (s, 11H), 1.29 (dd, 5H), 1.10 (s, 3H), 0.86 (t, 3H), 0.76 (td, 3H). [0440] Benzyl (101?,lll?,121?,141?)-ll-(((25,31?,45',61?)-3-(Benzoyloxy)-4 -(diniethylaniino)-6- methyltetrahydro-2//-pyran-2-yl)oxy)-12-methoxy-8, 10,12, 14-tetramethyl-7, 9-dioxo-6-oxa- 2,16-diazaspiro[4.12]heptadecane-16-carboxylate (S5-3-I7). S5-1-I7 (0.4 g, 0 432 mmol) was concentrated twice from toluene. The material was dissolved in chlorobenzene (120 mL), and a stream of nitrogen was bubbled through the solution for 10 min. The mixture was heated at a bath temperature of 150 °C (approximately 130-135 °C internal temperature) overnight. The reaction was allowed to cool to rt and was concentrated. The residue was purified on 12 g of silica gel (elution with 0-10% MeOH-DCM gradient) to give the title compound (253 mg, 67%). MS (ESI+) m/z: 866.36 [M + H] ⁺ , 888.35 [M + Na] ⁺ ; 'H NMR (400 MHz, Chloroform-^/) 5 8.12 -

7.84 (m, 2H), 7.62 - 7.47 (m, 1H), 7.47 - 7.28 (m, 7H), 5.21 - 4.93 (m, 3H), 4.55 (dt, 1H), 4.09 - 3.94 (m, 1H), 3.93 - 3.67 (m, 2H), 3.67 - 3.39 (m, 5H), 3.39 - 3.02 (m, 3H), 2.98 (t, 2H), 2.94 -

2.84 (m, 1H), 2.83 - 2.71 (m, 1.5H), 2.70 - 2.45 (m, 0.5H), 2.32 - 2.23 (m, 6H), 2.09 - 1.98 (m, 1H), 1.98 - 1.71 (m, 3H), 1.53 - 1.35 (m, 11H), 1.33 - 1.06 (m, 11H), 1.06 - 0.81 (m, 6H). A solution of this material (253 mg, 0.29 mmol) in acetonitrile was added with hydrochloric acid (4M in dioxane, 0.22 mL, 0.88 mmol). The reaction mixture was stirred at room temperature for 1 h and concentrated. The residue was suspended in ethyl acetate and washed with sat. aq. NaHCCh (2 times), the washed solution was dried over sodium sulfate, filtered and concentrated to give the title compound as a white foam (210 mg, 0.27 mmol, 94%). MS (ESI+) m/z: 383.86 [M + 2H] ²⁺ , 766.32 [M + H] ⁺ , 788.30 [M + Na] ⁺ .

[0441] (10/?,ll^,121?,141?)-ll-(((25,31?,45,61?)-4-(Diniethylaniino )-3-hydroxy-6- methyltetrahydro-2/Z-pyran-2-yl)oxy)-12-methoxy-8,10,12,14-t etramethyl-6-oxa-2,16- diazaspiro [4.12] heptadecane-7, 9-dione (S5-5-I7)(Compound 65). S5-2-I7 (50 mg, 0 0652 mmol) was dissolved in methanol (1.5 mL), and the reaction mixture was heated to 60 °C external temperature. After 5 h, the reaction was allowed to cool to rt, hydrochloric acid (0.005 mL) and Pd/C (5 wt%, 13.8 mg, 0.0065 mmol) was added. A balloon of hydrogen was bubbling through the reaction mixture. After 30 min, it was filtered through Celite® with ethyl acetate, and the filtrate was concentrated in vacuo. The residue was purified by HPLC (Atlantis T3 column, 5- 50% MeCN-water-0.1% HCO2H) to give the title compound as a formate salt (18.2 mg). MS (ESI+) m!z: 264.76 [M + 2H] ²⁺ , 528.26 [M + H] ⁺ .

[0442] (10^,ll^,12/?,141f)-ll-(((25,3/f,45,6/f)-4-(Dimethylainino)- 3-hydroxy-6- methyltetrahydro-2//-pyran-2-yl)oxy)-12-methoxy-2,8,10,12,14 -pentamethyl-6-oxa-2,16- diazaspiro [4.12] heptadecane-7, 9-dione (S5-4-I7-l)(Compound 64). To a solution of S5-2-I7 (25 mg, 0.0326 mmol) in DCM (1 mL) was added Na(OAc)3BH (10.3 mg, 0.049 mmol) followed by formaldehyde (37 wt% aqueous solution, 0.0261 mL, 0.326 mmol) was added. After 15 min, the reaction mixture was quenched with sat., aq. NaHCOs and extracted with DCM (3 times). The combined extracts were concentrated in vacuo. The residue was dissolved in methanol (1.5 mL), and the reaction mixture was heated to 60 °C external temperature. After 5 h, the reaction was allowed to cool to rt, hydrochloric acid (0.005 mL) and Pd/C (5 wt%, 6.79 mg, 0.0032 mmol) was added. A balloon of hydrogen was bubbling through the reaction mixture. After 30 min, it was filtered through Celite® with ethyl acetate, and the filtrate was concentrated in vacuo. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give the title compound as a diformate salt (9.78 mg). MS (ESI+) m/z'. 271.74 [M + 2H] ²⁺ , 542.28 [M + H] ⁺ ; ^J H NMR (400 MHz, Methanol-^) 5 8.39 (s, 2H), 4.45 (dd, 1H), 4.20 (dd, 0.5H), 4.04 - 3.85 (m, 0.5 H), 3.83 - 3.58 (m, 3H), 3.54 (tdd, 2H), 3.48 - 3.34 (m, 3H), 3.28 - 3.08 (m, 3H), 3.05 - 2.90 (m, 5H), 2.84 (s, 7H), 2.73 - 2.63 (m, 1H), 2.63 - 2.53 (m, 2H), 2.53 - 2.20 (m, 2H), 2.11 - 1.91 (m, 2H), 1.81 - 1.67 (m, 1H), 1.66 - 1.44 (m, 3H), 1.38 (d, 3H), 1.35 - 1.20 (m, 9H), 1.11 - 0.98 (m, 3H). [0443] (101?,117?,121?,141?)-ll-(((25,31?,45,61?)-4-(Diniethylamino )-3-hydroxy-6- methyltetrahydro-2//-pyran-2-yl)oxy)-12-methoxy-2,8,10,12,14 ,16-hexamethyl-6-oxa-2,16- diazaspiro [4.12] heptadecane-7, 9-dione (S5-6-I7-l)(Compound 66). To a solution of S5-5-I7 (17 mg, 0.032 mmol) in DCM (1 mL) was added Na(OAc)3BH (10.2 mg, 0.048 mmol) followed by formaldehyde (37 wt% aqueous solution, 0.0261 mL, 0.322 mmol) was added. After 15 min, the reaction mixture was quenched with sat., aq. NaHCOs and extracted with DCM (3 times). The combined extracts were concentrated in vacuo and the residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to give the title compound (2.2 mg). MS (ESI+) mlz: 338.76 [M + 2H] ²⁺ , 556.23 [M + H]“.

[0444] (10/?J l/?J2/?.14/?)-l l-(((2.S.3/?.4.S.6/?)-4-(l)iinethylaniino)-3-hydroxy-6- methyltetrahydro-2Z/-pyran-2-yl)oxy)-2-(dimethylglycyl)-12-m ethoxy-8, 10, 12,14- tetramethyl-6-oxa-2,16-rfiazas/nro[4.12]heptadecane-7, 9-dione (S5-3-I7-l)(Compound 67).

S5-2-I7 (110 mg, 0.143 mmol) was dissolved in DCM (1 mL) and chloroacetyl chloride (0.0017 mL, 0.021 mmol) was added. After 1 hr, the reaction mixture was diluted with DCM and washed with sat. NaHCCh. The wash solution was dried over sodium sulfate, fdtered and concentrated. The acyl product (29 mg, 0.034 mmol) was dissolved in tetrahydrofuran (0.5 mL) and dimethylamine (2 M solution in tetrahydrofuran, 0.172 mL, 0.344 mmol) was added. After 15 min, the reaction mixture was heated to 70 °C. After 4 h, the reaction mixture was allowed to cool to rt and excess solvent and reagent removed under vacuum. The crude material was dissolved in methanol (1 mL), and the reaction mixture was heated to 60 °C external temperature. After 5 h, the reaction was allowed to cool to rt, hydrochloric acid (0.005 mL) and Pd/C (5 wt%, 7.22 mg, 0.0034 mmol) was added. A balloon of hydrogen was bubbling through the reaction mixture. After 30 min, it was fdtered through Celite® with ethyl acetate, and the fdtrate was concentrated in vacuo. The residue was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCO2H) to give the title compound as a formate salt (3.69 mg). MS (ESI+) m/z: 205.22 [M + 3H] ³⁺ , 307.19 [M + 2H] ²⁺ , 613.19 [M + H] ⁺ ; ^NMR (400 MHz, Methanol-^) 8 8.47 (s, 3H), 4.45 (d, 1H), 4.19 (dd, 1H), 4.14 - 3.99 (m, 1H), 3.83 - 3.49 (m, 8H), 3.49 - 3.34 (m, 4H), 3.26 (p, 1H), 3.08 (dd, 1H), 3.01 - 2.97 (m, 1H), 2.97 - 2.90 (m, 2H), 2.86 (s, 1H), 2.81 (s, 6H), 2.78 - 2.70 (m, 2H), 2.67 (d, 4H), 2.55 - 2.40 (m, 1H), 2.05 - 1.87 (m, 2H), 1.77 - 1.61 (m, 1H), 1.60 - 1.40 (m, 3H), 1.40 - 1.24 (m, 9H), 1.24 - 1.09 (m, 3H), 1.09 - 0.95 (m, 3H).

[0445] (10/?J l/?.12/?.14/?)-l l-(((2.S.3/?.4.S.6/?)-4-(l)iinethylaniino)-3-hydroxy-6- niethyltetrahydro-2//-pyran-2-yl)oxy)-2-( \-isopropyl- \-niethylglycyl)-12-methoxy- 8,10,12,14-tetramethyl-6-oxa-2,l 6-diazaspiro[4.12] heptadecane-7, 9-dione (S5-3-I7- 2)(Compound 68). Prepared according to the methods of S5-3-I7-1, substituting N- isopropylmethylamine. Formate salt (3.47 mg). MS (ESI+) mlz\ 214.55 [M + 3H] ³⁺ , 321.27 [M + 2H] ²⁺ , 641.42 [M + H] ⁺ ; ³ HNMR (400 MHz, Methanol-^) 5 8.47 (s, 2.5H), 4.49 - 4.35 (m, 1H), 4.14 - 3.95 (m, 0.5H), 4.13 - 3.94 (td, 2H), 3.95 - 3.80 (m, 1.5H), 3.73 (dq, 3H), 3.57 (dq, 2H), 3.51 - 3.36 (m, 3H), 3.35 (s, 1H), 3.21 - 3.07 (m, 1H), 3.03 - 2.97 (m, 1H), 2.97 - 2.94 (m, 1H), 2.93 (s, 0.5H), 2.87 (s, 0.5H), 2.82 (s, 6H), 2.77 (q, 2H), 2.64 - 2.42 (m, 1H), 2.42 - 2.18 (m, 1H), 2.07 - 1.88 (m, 2H), 1.73 (dt, 1H), 1.65 - 1.41 (m, 2H), 1.41 - 1.24 (m, 14H), 1.24 - 1.12 (m, 3H), 1.10 - 0.88 (m, 3H).

[0446] (10/?,ll^,12/?,147?)-ll-(((25,3/?,45,6/?)-4-(Dimethylamino)- 3-hydroxy-6- methyltetrahydro-2//-pyran-2-yl)oxy)-2-(isopropylglycyl)-12- methoxy-8, 10, 12,14- tetramethyl-6-oxa-2,16-diazaspiro[4.12]heptadecane-7, 9-dione (S5-3-I7-3)(Compound 69). Prepared according to the methods of S5-3-I7-1, substituting isopropyl amine. Formate salt (5.83 mg). MS (ESI+) m/z: 209.89 [M + 3H] ³ ’, 314.30 [M + 2H] ²⁺ , 627.25 [M + H]“; ^L H NMR (400 MHz, Methanol-^) 5 8.46 (s, 2H), 4.45 (d, 1H), 4.18 (dd, 0.5H), 4.14 - 3.98 (m, 0.5H), 3.99 - 3.89 (m, 2H), 3.88 - 3.50 (m, 6H), 3.50 - 3.34 (m, 5H), 3.29 - 3.16 (m, 1H), 3.15 - 3.01 (m, 1H), 3.01 - 2.92 (m, 2H), 2.91 (s, 0.5H), 2.87 (s, 0.5H), 2.78 - 2.66 (m, 1H), 2.51 - 2.31 (m, 1H), 2.06 - 1.90 (m, 2H), 1.76 - 1.65 (m, 1H), 1.60 - 1.45 (m, 2H), 1.39 - 1.31 (m, 12H), 1.29 (d, 3H), 1.25 - 1.15 (m, 3H), 1.03 (d, 2H).

[0447] (101?,lll?,121?,141?)-2-(Cyclopropylglycyl)-ll-(((25',31?,45 ,61?)-4-(dimethylaniino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12-methoxy-8,1 0,12,14-tetramethyl-6-oxa- 2, 16-diazaspiro[4.12]heptadecane-7, 9-dione (S5-3-I7-4)(Compound 70). Prepared according to the methods of S5-3-I7-1, substituting cyclopropylamine. Formate salt (7.57 mg). MS (ESI+) mlz'.

209.24 [M + 3H] ³⁺ , 313.24 [M + 2H] ²⁺ , 625.36 [M + H] ⁺ ; ’H NMR (400 MHz, Methanol-^) 8 8.47 (s, 3H), 4.45 (d, 1H), 4.22 - 4.04 (m, 1H), 3.98 - 3.81 (m, 1H), 3.80 - 3.64 (m, 5H), 3.63 - 3.35 (m, 6H), 3.17 - 3.09 (m, 1H), 2.99 - 2.94 (m, 1H), 2.92 (s, 1H), 2.87 (s, 1H), 2.79 - 2.71 (m, 0.5H), 2.71 - 2.49 (m, 1.5H), 2.49 - 2.39 (m, 0.5H), 2.39 - 2.28 (m, 0.5H), 2.28 - 2.15 (m, 1H), 2.15 - 1.90 (m, 3H), 1.86 - 1.68 (m, 1H), 1.68 - 1.41 (m, 3H), 1.37 (t, 2H), 1.36 - 1.24 (m, 7H),

1.24 - 1.11 (m, 3H), 1.03 (t, 3H), 0.94 - 0.77 (m, 1H), 0.70 (p, 1H), 0.67 - 0.50 (m, 3H), 0.47 (dt, 1H).

[0448] The following Examples were prepared according to the methods of Scheme 5, substituting SI -2-18.

[0449] (9R,10R,llR,13R)-10-(((2S,3R,4S,6R)-4-(Z)M«ef/iy/awM7io)-3- hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-2-(dimethylglycyl)-ll-me thoxy-7,9,ll,13-tetramethyl-

5-oxa-2,15-diazaspiro[3.12]hexadecane-6, 8-dione. (S5-3-I8-l)(Compound 19). Prepared according to the methods of S5-3-I7-1 from SI-2-18 with dimethylamine. Mixture of epimers. Formate salt. MS (ESI+) w/z: 599.18 [M + H] ⁺ ; ’H NMR (400 MHz, Methanol-^) 5 4.43 (d, 2H), 4.18 (d, 3H), 3.73 (dq, 3H), 3.45 (td, 4H), 3.15 - 2.42 (m, 21H), 2.22 - 1.88 (m, 2H), 1.74 - 1.43 (m, 4H), 1.32 (m, 12H), 1.04 (s, 3H).

[0450] (9R,10R,llR,13R)-2-(cyclopropylglycyl)-10-(((2S,3R,4S,6R)-4- (Dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13-tetramethyl-5-oxa- 2, 15-diazaspiro[3.12]hexadecane-6, 8-dione (S5-3-I8-2)(Compound 26). Prepared according to the methods of S5-3-I7-1 from SI-2-18 with cyclopropylamine. Mixture of epimers, formate salt. MS (ESI+) m/z: 611.02 [M + H]“; ¹ H NMR (400 MHz, Methanol-^) 54.42 (d, 2H), 4.16 (d, 2H), 3.90 - 3.66 (m, 2H), 3.58-3.5 (m,2H), 3.49 - 3.36 (m, 4H), 3.09 - 2.84 (m, 4H), 2.80 (s, 4H), 2.63 (tt, 1H), 2.53 (dt, 1H), 2.45 - 2.26 (m, 1H), 2.27 - 2.14 (m, 2H), 2.07 - 1.90 (m, 2H), 1.89 (s, 1H), 1.79 (dq, 2H), 1.68 - 1.41 (m, 4H), 1.30 (td, 9H), 1.08 - 0.95 (m, 3H), 0.94 - 0.76 (m, 2H), 0.75 - 0.63 (m, 2H), 0.52 (dd, 1H), 0.47 (dq, 3H).

[0451] (9R,10R,llR,13R)-10-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13-tet ramethyl-2-(2-(piperazin-l- yl)acetyl)-5-oxa-2,15-diazaspiro[3.12]hexadecane-6, 8-dione (S5-3-I8-3)(Compound 27).

Prepared according to the methods of S5-3-I7-1 from SI-2-18 with piperazine. Mixture of epimers, formate salt. MS (ESI+) m/z: 640.32 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-6/4) 5 4.49 - 4.38 (m, 3H), 4.22 - 4.01 (m, 2H), 3.83 - 3.63 (m, 1H), 3.62 - 3.34 (m, 5H), 3.23 (q, 7H), 3.04 - 2.89 (m, 2H), 2.88 - 2.70 (m, 14H), 2.69 - 2.45 (m, 1H), 2.16 - 1.86 (m, 2H), 1.72 - 1.42 (m, 3H), 1.40 - 1.20 (m, 13H), 1.02 (dd, 4H). Scheme S6.

S6-1-I2-1

[0452] tert-Butyl (9R,llR,12R,13R,15S)-15-allyl-12-(((2S,3R,4S,6R)-3-(Benzoylo xy)-4- (dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-met hoxy-7,9,ll,13,15- pentamethyl-14,16-dioxo-17-oxa-3,7-diazaspiro[5.12]octadecan e-3-carboxylate (S6-1-I2-1).

SI-5-12-1 (58 mg, 0.076 mmol) was dissolved in THF (2 mL). Allyl tert-butyl carbonate (18 pL. 0.11 mmol) and Pd(PPh3)4 (4.4 mg, 0.0038 mmol) were added at rt. The reaction mixture was degassed with N2 and heated at 60 °C for 16h. LC/MS indicated complete consumption of starting material. The reaction mixture was cooled and concentrated. The residue was purified on 4 g of silica gel (elution with 0-10% MeOH-DCM + 0.5% of 30% aq NH4OH) to give the title compound (47 mg, 79%). MS (ESI+) m/z: 400.8 [M + 2H] ²⁺ , 800.5 [M + H] ⁺ .

[0453] (9R,llR,12R,13R,15S)-15-Allyl-12-(((2S,3R,4S,6R)-4-(dimethyl amino)-3-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-(dimethylglycyl)-ll-me thoxy-7,9,H,13,15- pentamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S14-3)(Compound 73). Prepared according to the methods of S3-1-I6-1-1 (Boc deprotection) and S3-5-I1-1-1-1. Formate salt (8.75 mg). MS (ESI+) m/z: 227.8 [M + 3H] ³⁺ , 341.3 [M + 2H] ²⁺ , 681.5 [M + H] ⁺ ; ³ HNMR (400 MHz, Methanol^) 8 8.52 (s, 3H), 5.62 (ddt, 1H), 5.23 - 5.10 (m, 2H), 4.52 (dd, 2H), 4.18 (d, 1H), 3.91 (s, 1H), 3.84 - 3.68 (m, 3H), 3.50 - 3.32 (m, 4H), 3.04 (s, 4H), 2.94 (dt, 2H), 2.84 (s, 2H), 2.79 (s, 7H), 2.74 (d, 1H), 2.63 (d, 6H), 2.44 (td, 1H), 2.14 - 1.97 (m, 3H), 1.83 (d, 1H), 1.57 - 1.44 (m, 3H), 1.44 - 1.26 (m, 13H), 1.06 (d, 3H).

Scheme S7

[0454] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((9R,llR,12R,13R)-ll-meth oxy-7,9,ll,13,15,15- hexamethyl-14,16-dioxo-3-(vinylsulfonyl)-17-oxa-3,7-diazaspi ro[5.12]octadecan-12-yl)oxy)- 6-methyltetrahydro-2H-pyran-3-yl benzoate (S7-1-I1). S3-1-I1-2 (62 mg, 0 080 mmol) was dissolved in DCM (1 mL). EtsN (44.2 pL, 0.32 mmol) and ethenesulfonyl chloride (20 mg, 0.16 mmol) were added at rt. The reaction mixture was allowed to stir at it for 1 h. The reaction was quenched by adding saturated NaHCCh (2 mL), and the aqueous layer was extracted with methylene chloride three times (2 mL). The combined organic layers were dried over MgSCh, filtered and concentrated. The residue was used in the next step without further purification. MSI (ESI+) mlz'. 382.7 [M + 2H] ²⁺ , 764.4 [M + H] ⁺ .

[0455] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Z)i>M^jZawiMo)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-((2-(dimethylamino)eth yl)sulfonyl)-ll-methoxy- 7,9,H,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadec ane-14, 16-dione (S7-2-I1- l)(Compound 81). The crude S7-1-I1 (8 mg, 0.010 mmol) was dissolved in THF (1 mL) and dimethylamine (2M in THF, 0.016 mL, 0.031 mmol) was added at rt. The reaction mixture was allowed to stirred rt for 2 h. LC/MS shows full conversion. The reaction was quenched by adding saturated NaHCCh (2 mL) and the aqueous layer was extracted with methylene chloride three times (2 mL). The combined organic layers were dried over MgSCL, filtered and concentrated. The residue was used in the next step without further purification. MS (ESI+) m!z\ 270.5 [M + 3H] ³⁺ , 405.3 [M + 2H] ²⁺ , 809.5 [M + H] ⁺ . The material was dissolved in MeOH (0.5 mL) and heated at 60 °C until LC/MS indicated complete consumption of starting material (16 hours). The reaction mixture was filtered through a syringe filter with the aid of methanol and concentrated. The residue was purified by HPLC (MeCN-water-0.1% HCChH) to yield the title compound as a formate salt (4.82 mg, 52% yield in three steps). MS (ESI+) mlz\ 235.8 [M + 3H] ³⁺ , 353.2 [M + 2H] ²⁺ , 705.4 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 5 8.52 (s, 3H), 4.62 (s, 1H), 4.48 (d, 1H), 4.21 (d, 1H), 3.82 (s, 2H), 3.74 (dtd, 1H), 3.44 (dd, 1H), 3.40 - 3.24 (m, 15H), 3.19 - 3.08 (m, 2H), 3.06 (s, 3H), 2.83 (dd, 3H), 2.77 (s, 6H), 2.35 (s, 6H), 2.13 (td, 1H), 2.09 - 1.97 (m, 2H), 1.39 (q, 8H), 1.33 (d, 5H), 1.07 (d, 2H). [0456] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-((2-(ethyl(methyl)amin o)ethyl)sulfonyl)-ll- methoxy-7,9,ll,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.1 2]octadecane-14,16-dione (S7- 2-Il-2)(Compound 83). Prepared according to the methods of S7-2-I1-1, substituting N- methylethanamine. Formate salt (2.42 mg). MS (ESI+) m/z: 240.5 [M + 3H] ³⁺ , 360.3 [M + 2H] ²⁺ , 719.5 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 8 8.55 (s, 3H), 4.48 (d, 1H), 4.20 (s, 1H), 4.01 - 3.64 (m, 2H), 3.47 - 3.36 (m, 1H), 3.29 - 3.15 (m, 5H), 3.06 (s, 3H), 2.95 - 2.83 (m, 3H), 2.71 (s, 6H), 2.56 (q, 2H), 2.32 (s, 3H), 2.12 (s, 1H), 1.98 (d, 1H), 1.47 (d, 4H), 1.43 - 1.25 (m, 12H), 1.18 - 0.98 (m, 6H).

[0457] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-((2-(ethyl(methyl)amin o)ethyl)sulfonyl)-ll- methoxy-7, 9, 11, 13,15, 15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S7- 2-Il-3)(Compound 84). Prepared according to the methods of S7-2-I1-1, substituting N- methylisopropylamine. Formate salt (1.31 mg). MS (ESI+) m/z: 245.2 [M + 3H] ³⁺ , 367.3 [M + 2H] ²⁺ , 733.5 [M + H] ⁺ ; 'H NMR (400 MHz, Methanol-^) 8 8.53 (s, 3H), 4.49 (d, 1H), 4.21 (d, 1H), 3.92 - 3.66 (m, 3H), 3.53 - 3.38 (m, 2H), 3.23 - 3.09 (m, 2H), 3.09 - 2.90 (m, 6H), 2.75 (s, 7H), 2.34 (s, 4H), 2.25 - 1.92 (m, 4H), 1.52 (s, 5H), 1.47 - 1.23 (m, 13H), 1.11 (d, 8H).

[0458] (9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydr oxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-3-((2-(isopropylamino)et hyl)sulfonyl)-ll-methoxy- 7,9,ll,13,15,15-hexamethyl-17-oxa-3,7-diazaspiro[5.12]octade cane-14, 16-dione (S7-2-I1- 4)(Compound 85). Prepared according to the methods of S7-2-I1-1, substituting isopropyl amine. Formate salt (1.85 mg). MS (ESI+) m/z: 240.5 [M + 3H] ³⁺ , 360.3 [M + 2H] ²⁺ , 719.5 [M + H] ⁺ ; ³ H NMR (400 MHz, Methanol-^) 5 8.52 (s, 3H), 4.60 (s, 1H), 4.49 (d, 1H), 4.20 (d, 1H), 3.84 (s, 1H), 3.73 (dd, 2H), 3.53 - 3.36 (m, 2H), 3.20 (q, 5H), 3.06 (s, 4H), 2.75 (s, 9H), 2.07 (dd, 5H), 1.58 - 1.45 (m, 4H), 1.45 - 1.25 (m, 14H), 1.21 (d, 6H), 1.06 (s, 4H).

Scheme S8.

[0459] (2S,3R,4S,6R)-4-(dimethylamino)-2-(((9R,llR,12R,13R)-ll-Meth oxy-7,9,ll,13,15,15- hexamethyl-3,14,16-trioxo-17-oxa-7-azaspiro[5.12]octadecan-1 2-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S8-1-I10-1-2). S2-2-I10-1 (320 mg) was stirred with 50% TFA/DCM (3mL) for 2 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM. This was washed with NaHCCF (saturated, aqueous), and the DCM layer was dried over TsfeSCh, was concentrated, and was dried under vacuum to give the title compound (296 mg, 97%). MS (ESI+) m/z'. 687.41 [M + H] ⁺ .

[0460] (9R,llR,12R,13R)-3-(Dimethylamino)-12-(((2S,3R,4S,6R)-4-(dim ethylaniino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7, 9,11, 13,15, 15-hexamethyl- 17-oxa-7-azaspiro[5.12]octadecane-14, 16-dione (S8-3-I10-l-2-l)(Compound 93). Prepared according to the methods of S3-2-I1-1-1-2 from S8-1-I10-1-2 and dimethylamine to provide the title compound (7.05 mg). MS (ESI+) mlr. 612.4 [M + H]“, Formate salt. 'H NMR (400 MHz, Chloroform- 54.62 - 4.36 (m, 1H), 4.28 - 4.11 (m, 1H), 3.84 - 3.60 (m, 2H), 3.52 (s, 1H), 3.49 - 3.27 (m, 4H), 3.26 - 3.10 (m, 2H), 3.05 (d, 3H), 2.95 - 2.71 (m, 12H), 2.69 (s, 2H), 2.53 (s, 2H), 2.34 - 1.92 (m, 7H), 1.90 - 1.60 (m, 5H), 1.60 - 1.44 (m, 5H), 1.44 - 1.16 (m, 11H), 1.16 - 0.87 (m, 3H).

[0461] (9R,llR,12R,13R)-3-(Dimethylamino)-12-(((2S,3R,4S,6R)-4-(dim ethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15-pentamethyl-17- oxa-7-azaspiro[5.12]octadecane-14, 16-dione (S8-3-I10-l-l-l)(Compound 87). Prepared according to the methods of S3-2-I1-1-1-2 from S8-1-I10-1-1 and dimethylamine. MS (ESI+) mlz: 598.4 [M + H] ⁺ , Formate salt. ^NMR (400 MHz, Chloroform-^/) 84.67 - 4.32 (m, 2H), 4.33 - 4.05 (m, 2H), 3.89 - 3.48 (m, 2H), 3.46 - 3.24 (m, 3H), 3.18 - 2.87 (m, 5H), 2.83 - 2.20 (m, 18H), 2.19 - 1.60 (m, 8H), 1.60 - 1.15 (m, 16H), 1.01 (m, 3H).

[0462] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((9R,llR,12R,13R)-ll-meth oxy-7,9,ll,13,15,15- hexamethyl-3-(methylamino)-14,16-dioxo-17-oxa-7-azaspiro[5.1 2]octadecan-12-yl)oxy)-6- methyltetrahydro-2H-pyran-3-yl benzoate (S8-2-I10-1-2-2). S8-1-I10-1-2 (130 mg, 0.189 mmol) in DCM (4mL) and methylamine in THF (2M, 0.47mL, 0.944mmol, 5eq) were combined and stirred for 15min. NaBH(OAc)3 (80 mg, 0.378 mmol) was added, and the mixture was stirred for 30 min. The mixture was dilute with DCM and washed with water (2 times). The DCM layer was separated, was dried over NazSCU, and was concentrated under reduced pressure. The residue was dried under vacuum to give the crude product (125mg, 95%). MS (ESI+) mlz'. 702.43 [M + H] ⁺ .

[0463] 2-(Dimethylamino)-N-((9R,HR,12R,13R)-12-(((2S,3R,4S,6R)-4-(d imethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9 ,ll,13,15,15-hexamethyl-

14,16-dioxo-17-oxa-7-azaspiro[5.12]octadecan-3-yl)-N-meth ylacetamide (S8-4-I10-1-2-2- l)(Compound 92). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2-I10-1-2-2 and dimethylamine to provide the title compound. MS (ESI+) mlz: 683.48 [M + H] ⁺ , Formate salt. 'H

NMR (400 MHz, Chloroform-t/) 84.64 - 4.28 (m, 2H), 4.27 - 3.85 (m, 4H), 3.72 (d, 2H), 3.43 (d,

4H), 3.26 (d, 1H), 3.07 (d, 3H), 2.99 - 2.55 (m, 18H), 2.36 (d, 3H), 2.16 - 1.66 (m, 9H), 1.64 - 1.18 (m, 17H), 1.08 (d, 3H).

[0464] N-((9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-7-azaspiro[5.12]octadecan-3-yl)-2-(isopropyl(methyl)a mino)-N-methylacetamide (S8- 4-I10-l-2-2-2)(Compound 109). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2- 110-1-2-2 and N-methylisopropylamine to provide the title compound. MS (ESI+) mtz'. 711.43 [M + H] ⁺ , Formate salt. ^NMR (400 MHz, Chloroform^/) 84.60 (d, 1H), 4.47 (d, 1H), 4.36 (s, 1H), 4.22 (d, 1H), 3.99 (dd, 3H), 3.78 - 3.64 (m, 2H), 3.55 - 3.40 (m, 3H), 3.25 (s, 1H), 3.07 (d, 3H), 2.90 (d, 4H), 2.82 - 2.66 (m, 9H), 2.37 (d, 3H), 2.09 - 1.83 (m, 7H), 1.67 (s, 2H), 1.54 - 1.26 (m, 25H), 1.10 (t, 2H), 0.98 (s, 2H).

[0465] 2-(Cyclopropyl(methyl)amino)-N-((9R,HR,12R,13R)-12-(((2S,3R, 4S,6R)-4-

(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-y l)oxy)-ll-methoxy-

7,9,ll _i 13,15,15-hexamethyl-14,16-dioxo-17-oxa-7-azaspiro[5.12 ]octadecan-3-yl)-N- methylacetamide (S8-4-I10-l-2-2-3)(Compound 94). Prepared according to the methods of S3- 5-11-1-1-1 from S8-2-I10-1-2-2 and N-methylcyclopropylamine to provide the title compound.

MS (ESI+) mlz-. 695.47 [M + H]“, Formate salt. ^X H NMR (400 MHz, Chloroform-;/) 8 4.67 - 4.43

(m, 1H), 4.40 - 4.15 (m, 2H), 4.09 (d, 1H), 3.86 - 3.64 (m, 4H), 3.41 (ddd, 3H), 3.21 (t, 1H), 3.16

- 3.03 (m, 4H), 2.97 - 2.76 (m, 11H), 2.39 (dt, 4H), 2.18 - 1.89 (m, 5H), 1.90 - 1.75 (m, 3H),

1.67 (s, 2H), 1.60 - 1.24 (m, 18H), 1.09 (dd, 3H), 0.72 - 0.46 (m, 4H).

[0466] N-((9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-7-azaspiro[5.12]octadecan-3-yl)-2-(3-hydroxyazetidin- l-yl)-N-methylacetamide (S8- 4-I10-l-2-2-4)(Compound 98). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2- 110-1-2-2 and 4-hydroxyazetidine to provide the title compound. MS (ESI+) mlz\ 711.42 [M + H] ⁺ , Formate salt. Ti NMR (400 MHz, Chloroform-;/) 54.69 (dt, 2H), 4.60 - 4.46 (m, 3H), 4.40 - 4.14 (m, 6H), 4.03 - 3.85 (m, 3H), 3.77 (d, 2H), 3.55 - 3.41 (m, 3H), 3.28 - 3.19 (m, 2H), 3.09 (d, 4H), 3.00 - 2.85 (m, 8H), 2.80 (s, 3H), 2.34 (s, 3H), 2.10 - 1.80 (m, 8H), 1.56 - 1.32 (m, 16H), 1.11 (d, 3H).

[0467] N-((9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-7-azaspiro[5.12]octadecan-3-yl)-N-methyl-2-(4-methylp iperazin-l-yl)acetaimde (S8- 4-I10-l-2-2-5)(Compound 99). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2- 110-1-2-2 and 1 -methylpiperazine to provide the title compound. MS (ESI+) mlz\ 738.4 [M + H] ⁺ , Formate salt. ^X HNMR (400 MHz, Methanol-d4) 8 8.52 (s, 2H), 4.58 (d, 1H), 4.47 (d, 1H), 4.22 (d, 1H), 4.08 (d, 1H), 4.01 - 3.81 (m, 1H), 3.77 - 3.61 (m, 2H), 3.57 - 3.37 (m, 3H), 3.35 (s, 1H), 3.17 - 2.89 (m, 7H), 2.89 - 2.50 (m, 16H), 2.50 - 2.20 (m, 7H), 2.20 - 1.17 (m, 28H), 1.17 - 0.88 (m, 3H).

[0468] N-((9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-7-azaspiro[5.12]octadecan-3-yl)-N-methyl-2-morpholino acetamide (S8-4-110- 1-2-2- 6)(Compound 95). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2-I10-1-2-2 and morpholine to provide the title compound. MS (ESI+) mlz\ 725.4 [M + H] ⁺ , Formate salt.

[0469]

[0470] N-((9R,llR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-7-azaspiro[5.12]octadecan-3-yl)-2-((2-(dimethylamino) ethyl)amino)-N- methylacetamide (S8-4-I10-l-2-2-7)(Compound 100). Prepared according to the methods of S3-5-I1-1-1-1 from S8-2-I10-1-2-2 and 2-(dimethylamino)ethylamine to provide the title compound. MS (ESI+) mlz\ 726.5 [M + H] ⁺ , Formate salt.

[0471] 2-(cyclopropylamino)-N-((9R,l lR,12R,13R)-12-(((2S,3R,4S,6R)-4-(dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)- 11 -m ethoxy-7, 9, 11,13,15,15-hexam ethyl- 14, 16- dioxo-17-oxa-7-azaspiro[5.12]octadecan-3-yl)-N-methylacetami de (S8-4-I10-l-2-2-8)(Compound 141) Prepared according to the methods of S3-5-I1 - 1 -1 -1 from S8-2-I10-1-2-2 and cyclopropylamine. 3.4 mg Formate salt. MS (ESI+) mlz'. 695.5 [M + H] ⁺ , ^L H NMR (400 MHz, Methanol </) 8 8.47 (d, 2H), 4.67 - 4.44 (m, 1H), 4.22 (d, 1H), 4.09 (d, 1H), 3.90 - 3.57 (m, 4H), 3.57 - 3.41 (m, 2H), 3.41 - 3.35 (m, 1H), 3.27 - 3.15 (m, 1H), 3.15 - 3.02 (m, 3H), 3.02 - 2.85 (m, 5H), 2.80 (d, 6H), 2.52 - 2.24 (m, 4H), 2.18 - 1.72 (m, 8H), 1.72 - 1.60 (m, 2H), 1.52 (s, 4H), 1.46 - 1.36 (m, 6H), 1.33 (d, 6H), 1.18 - 0.88 (m, 3H), 0.72 - 0.50 (m, 4H). Scheme S9

[0472] 2-((9R,HR,12R,13R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(Dime thylamino)-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,ll,13,15, 15-hexamethyl-14,16-dioxo- 17-oxa-3,7-diazaspiro[5.12]octadecan-3-yl)acetic acid (S9-1-I1-1-2). S3-1-I1-1-2 (56 2 mg, 71.7 mmol) was dissolved in DCM (358 mL) and Et;N (0.0298 mL, 0.215 mmol), and glyoxylic acid monohydrate (19.7 mg, 0.215 mmol) and Na(OAc)3BH (30.3 mg, 0.143 mmol) were added. After stirring overnight, the reaction mixture was quenched with NH4CI (sat, aq) and was extracted with DCM (3 times). The combined extracts were dried over Na2SO4, were filtered, and were concentrated under reduced pressure. LC/MS analysis indicated only trace product. The aqueous layer was adjusted to pH 7-8 with NaHCCh (sat, aq) and was extracted with DCM (4x). The combined extracts were dried over NazSCh, were filtered, and were concentrated under reduced pressure to give the crude title compound (85.4 mg). The material was used without further purification. MS (ESI+) mlz'. 732.35 [M + H] ⁺ .

[0473] 2-((9R,HR,12R,13R)-12-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hy droxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-ll-methoxy-7,9,1143,15,1 5-hexamethyl-14,16-dioxo- 17-oxa-3,7-diazaspiro[5.12]octadecan-3-yl)-N,N-dimethylaceta mide (S9-2-I1-1-2- l)(Compound 71). S9-1-I1-1-2 (52.4 mg, 0.0715 mmol) was dissolved in DCM (0.357 mL) and A. A-di isopropyl ethylamine (0.0132 mL, 0.0858 mmol) and 1 -hydroxybenzotriazole (12.5 mg, 0.0929 mmol), dimethylamine(l M in THF, 0.143 mL, 0.143 mmol), and A,A- diispropylcarbodiimide (0.0132 mL, 0.0858 mmol) were added sequentially. After stirring overnight, the reaction mixture was diluted with EtOAc and was washed with NaHCCL (sat, aq, 2 times) and brine (1 time). The organic layer was dried over NazSCh, was filtered, and was concentrated under reduced pressure. The material was purified by ISCO (24 g column, 4-20% MeOH/DCM/0.5% NH4OH gradient), yielding the benzoate intermediate (21.2 mg, 39%, 2 steps). MS (ESI+) mlz\ 759.37 [M + H] ⁺ . The material was dissolved in MeOH (1 mL), and the reaction mixture was heated to 65 °C. After 3 h, the reaction mixture was concentrated under reduced pressure and the material was purified by HPLC (MeCN-water-0.1% HCO2H) to yield the title compound (14.2 mg, 78%). Formate salt. MS (ESI+) mlz\ 655.41 [M + H] ⁺ . 'H NMR (400 MHz, Methanol-d4) 5 8.38 (s, 2H), 4.59 (d, 1H), 4.48 (d, 1H), 4.22 (d, 1H), 3.74 (ddt, 1H), 3.50 - 3.40 (m, 2H), 3.40 - 3.33 (m, 3H), 3.21 (t, 1H), 3.09 - 2.96 (m, 9H), 2.94 (s, 3H), 2.91 (s, 3H), 2.86 - 2.77 (m, 7H), 2.49 (q, 2H), 2.39 - 2.24 (m, 2H), 2.15 (td, 1H), 2.09 - 2.01 (m, 1H), 1.99 - 1.89 (m, 1H), 1.89 - 1.73 (m, 2H), 1.59 - 1.44 (m, 5H), 1.44 - 1.30 (m, 13H), 1.08 (d, 3H).

Scheme S10.

[0474] (9R, HR, 12R,13R)-3-(Dimethylglycyl)-12-hydroxy-ll-methoxy-7, 9, 11,13, 15,15- hexamethyl-17-oxa-3,7-diazaspiro[5.12]octadecane-14, 16-dione (S10-l-Il-l-2-l)(Compound 97). S3-5-I1-1-2-1 was dissolved in 1 N aqueous HC1, and the solution was freeze-dried. HPLC indicated significant hydrolysis of the desosamine sugar. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCO2H) to yield the title compound as a formate salt (29.7 mg). MS (ESI+) mlz\ 498.18 [M + H] ⁺ . ^NMR (400 MHz, Methanol-d4) 6 8.46 (s, 1H), 5.10 (d, 1H), 4.66 - 4.45 (m, 2H), 4.29 (dd, 1H), 4.16 - 4.04 (m, 1H), 3.93 (d, 1H), 3.84 (d, 1H), 3.47 - 3.14 (m, 2H), 3.13 - 2.99 (m, 4H), 2.99 - 2.75 (m, 11H), 2.41 - 2.18 (m, 2H), 2.10 - 1.98 (m, 2H), 1.94 - 1.77 (m, 2H), 1.50 (s, 3H), 1.37 (d, 6H), 1.29 (dd, 1H), 1.22 (d, 3H), 1.15 - 1.03 (m, 3H).

Compound 113 Compound 113

[0475] Compound 113 was prepared as provided in Scheme SI 1.

Scheme Sil

Compound A

[0476] Compound A was prepared according to the methods of SI-2-11 from II and replacing Sl-

1 with the 06 allyl starting material. See Seiple, et. al. Nature, 533, 338-345 (2016). Compound B

[0477] Benzyl (9R,1 lR,12R,13R,15R)-12-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimethy lamino)- 6-methyltetrahydro-2H-pyran-2-yl)oxy)- 11 -(2-hydroxyethoxy)-7,9, 11,13,15 -pentamethyl- 14,16- dioxo-17-oxa-3,7-diazaspiro[5.12]octadecane-3-carboxylate (Compound B). Compound A (461 mg, 0.562 mmol) was dissolved in DCM (20 mL) and TFA (0.214 mL, 2.81 mmol) was added. The reaction mixture was cooled to -78 °C. A stream of ozone (7 PSI, 2 LPM) was bubbled through the reaction mixture until a blue color persisted (approximately 1 min). The ozone stream was removed, and nitrogen was then bubbled through the solution for 5 min (blue color disappeared). Ph3P (737 mg, 2.81 mmol) was added, and the reaction mixture was removed from the bath and allowed to warm to it After 45 min, the reaction mixture was diluted with NaHCOs (sat, aq) and was extracted with DCM (3x). The combined extracts were dried over NazSCh, were filtered, and were concentrated under reduced pressure. The crude material was dissolved in MeOH (3 mL) and was cooled to 0 °C. NaBEL (42.3 mg, 1.12 mmol) was added. After 15 min, the reaction mixture was quenched with NaHCCh (sat, aq) and was extracted with DCM (3x). The combined extracts were dried over NazSCU, were filtered, and were concentrated under reduced pressure. The material was purified by column chromatography (ISCO 40 g column, 2 to 15% MeOH/DCM/O.5% NH4OH gradient), yielding 174.5 mg (38%) of Compound B. MS (ESI) m/z 824.38 (M+H). Compound C

[0478] Benzyl (lR,8R,10R,15R,16R)-16-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dim ethylamino)-6- methyltetrahydro-2H-pyran-2-yl)oxy)- 1,6,8,10,15 -pentamethyl -2, 14-dioxo-3 , 11 -dioxa-6- azaspiro[bicyclo[8.3.3]hexadecane-5,4'-piperidine]-l'-carbox ylate (Compound C). p- toluenesulfonyl chloride (28.9 mg, 0.152 mmol) was added to a solution of Compound B (115.7 mg, 0.139 mmol) and triethylamine (0.0578 mL, 0.416 mmol) in toluene (0.694 mL) at rt. After 1.5 h, N, A-di methyl ami nopyridine (5 mg) and additional p-toluenesulfonyl chloride (15 mg, 0.0787 mmol) were added. After an additional 1.5 h, additional A,A-dimethylaminopyridine (15 mg) was added, resulting in formation of a precipitate. Acetonitrile was added. After 3 h, additional p-toluenesulfonyl chloride (15 mg, 0.0787 mmol) was added and the mixture was stirred overnight. The reaction mixture was diluted with EtOAc and was washed with NaHCCh (sat, aq, 2 x), water (3x), and brine (lx). The EtOAc layer was dried over NazSCU, was filtered, and was concentrated under reduced pressure. The crude material was dissolved in acetone (1 mL) and sodium iodide (208 mg, 1.39 mmol) was added. The reaction mixture was heated to 60 °C. After 4 h, the reaction mixture was cooled to rt, was diluted with EtOAc, and was washed with water (2x). The EtOAc layer was dried over Na2SO4, was filtered, and was concentrated under reduced pressure. The crude product was dissolved in DMF (1.38 mL) and CS2CO3 (224 mg, 0.690 mmol) was added. After stirring at rt overnight, the reaction mixture was diluted with EtOAc and was washed with water (3x) and brine (lx). The EtOAc layer was dried over Na2SO4, was filtered, and was concentrated under reduced pressure. The material was purified by column chromatography (ISCO 24 g column, 2 to 10% MeOH/DCM/0.5% NH4OH gradient), yielding 31.6 mg (28%, 3 steps) of Compound C. MS (ESI) m/z 806.32 (M+H). Compound D

[0479] Benzyl (lR,8R,10R,15R,16R)-16-(((2S,3R,4S,6R)-4-(dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)- 1,6,8,10,15 -pentamethyl -2, 14-dioxo-3 , 11 -dioxa-6- azaspiro[bicyclo[8.3.3]hexadecane-5,4'-piperidine]-l'-carbox ylate (Compound D). Compound C (4.7 mg, 0.0058 mmol) was dissolved in MeOH (1 mL) and was heated to 65 °C. After 3 h 20 min, the reaction mixture was cooled to rt and was concentrated under reduced pressure. The material was purified by HPLC (Atlantis T3 column, 5 to 50 to 90% gradient of Acetonitrile/0.1% formic acid and Water/0.1% formic acid). Fractions with the desired mass were collected and freeze-dried, yielding 3.00 mg (73%) of the title compound. Formate salt. ^J H NMR (400 MHz, Methanol-^) 6 8.55 (s, 1H), 7.42 - 7.23 (m, 6H), 5.20 - 5.01 (m, 3H), 4.38 (d, 1H), 4.21 - 4.00 (m, 2H), 4.00 - 3.79 (m, 2H), 3.79 - 3.67 (m, 2H), 3.67 - 3.53 (m, 2H), 3.21 - 2.80 (m, 5H), 2.79 - 2.62 (m, 2H), 2.62 - 2.44 (m, 7H), 2.37 (s, 3H), 2.16 (d, 1H), 2.05 - 1.91 (m, 1H), 1.91 - 1.76 (m, 3H), 1.49 - 1.30 (m, 5H), 1.30 - 1.02 (m, 11H), 0.89 - 0.69 (m, 3H); MS (ESI) m/z 702.32 (M+H). Compound E

[0480] (2S,3R,4S,6R)-4-(dimethylamino)-6-methyl-2-(((lR,8R,10R,15R, 16R)-l,6,8,10,15- pentamethyl-2,14-dioxo-3,l l-dioxa-6-azaspiro[bicyclo[8.3.3]hexadecane-5,4'-piperidin]- 16- yl)oxy)tetrahydro-2H-pyran-3-yl benzoate (Compound E). Compound C (26.8 mg, 0.0332 mmol) was dissolved in MeOH (1 mL) and IN aqueous HC1 (0.0996 mL). 5% Pd on carbon (14 mg) was added and an atmosphere of hydrogen was introduced (balloon). After 1.5 h, the reaction mixture was evacuated and back-filled with N2 (3x), and the solution was filtered through a 0.2 pM syringe filter. The filtrate was concentrated under reduced pressure and was used without further purification. MS (ESI) m/z 6T1A (M+H). Compound 113

[0481] (2S,3R,4S,6R)-4-(dimethylamino)-2-(((lR,8R,10R,15R,16R)-T-(d imethylglycyl)- l,6,8,10,15-pentamethyl-2,14-dioxo-3,l l-dioxa-6-azaspiro[bicyclo[8.3.3]hexadecane-5,4'- piperidin]-16-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (Compound 113).

Chloroacetyl chloride (0.0032 mL, 0.040 mmol) was added to a solution of Compound E (25.9 mg, 0.0331 mmol) and trimethylamine (0.0137 mL, 0.0993 mmol) in dichloromethane (2 mL). After Ih and 10 min, the reaction mixture was concentrated under reduced pressure. The crude material was dissolved in dimethylamine solution (2.0 M in THF, 1.23 mL, 2.47 mmol), and the mixture was heated to 65 °C. After 40 min, the reaction mixture was concentrated under reduced pressure. The crude material was dissolved in MeOH (2 mL) and was heated to 50 °C overnight. The reaction mixture was cooled to rt and was concentrated under reduced pressure. The material was purified by HPLC (Atlantis T3 column, 5 to 50 to 90% gradient of Acetonitrile/0.1% formic acid and Water/0.1% formic acid). Fractions with the desired mass were collected and freeze- dried, yielding 6.23 mg (29%) of Compound F. Formate salt. 'H NMR (400 MHz, Methanol-c/v) 5 8.50 (s, 2H, formic acid), 5.19 (d, 1H), 4.50 (dd, 2H), 4.31 (bs, 1H), 4.05 - 3.60 (m, 7H), 3.50 - 3.30 (m, 4H), 2.84 - 2.56 (m, 18H), 2.19 (d, 1H), 2.15 - 1.70 (m, 8H), 1.51 (q, 2H), 1.44 (s, 3H), 1.37 (d, 3H), 1.28 (dd, 7H), 0.98 (s, 3H).; MS (ESI) m/z 653.38 (M+H). Compound 113

[0482] (lR,8R,10R,15R,16R)-16-(((2S,3R,4S,6R)-4-(dimethylamino)-3-h ydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)- 1,6,8,10,15 -pentamethyl -3,11 -dioxa-6- azaspiro[bicyclo[8.3.3]hexadecane-5,4'-piperidine]-2, 14-dione (Compound G). Material was isolated during purification of Compound F, having been formed from unreacted Compound E. 2.29 mg (12%, 4 steps). Formate salt. ^X H NMR (400 MHz, Methanol-^) 8 8.44 (s, 3H), 5.21 (d, 1H), 4.45 (d, 1H), 4.22 - 3.52 (m, 5H), 3.50 - 3.32 (m, 4H), 3.25 - 2.97 (m, 3H), 2.92 - 2.62 (m, 9H), 2.64 - 2.42 (m, 2H), 2.35 (d, 1H), 2.16 - 1.75 (m, 6H), 1.58 - 1.44 (m, 2H), 1.39 (s, 3H), 1.35 - 1.05 (m, 9H), 0.98 - 0.75 (m, 2H); MS (ESI) m/z 568.35 (M+H).

Biological Activity

A novel class of Ribosome Modulating Agents (RMAs) targets ribosome heterogeneity in a subset of colorectal cancers

[0483] Background: Aberrant protein translation is a key driver in cancer downstream of pro- oncogenic stimuli. The aberrant protein translation is frequently carried out by ribosomes that have cancer specific alterations providing an opportunity to selectively target “oncoribosomes”. Our unique synthetic chemistry generates novel macrolides that are Ribosome Modulating Agents (RMAs) capable of selectively targeting oncoribosomes. Wnt-pathway activated colorectal cancers (CRC) tend to be highly dependent on elevated protein translation capacity. These types of cancers maybe vulnerable to RMAs that selectively inhibit oncoribosomes dependent protein translation.

[0484] Material and methods: Cell proliferation was assessed using Cell Titer Gio assay. New protein synthesis was evaluated by metabolic labelling using L-Azidohomoalanine (AHA) followed by biotinylation and detection by Western Blot as provided below. Quantitative Mass spectrometric analysis of proteins was conducted using pulsed Stable Isotope Labeling with Amino acids in Cell culture (pSILAC) proteomics method. Cell cycle arrest and apoptosis were assessed using flow cytometry.

[0485] Results: We evaluated a representative RMA, across a panel of 34 CRC cell lines. The representative compound inhibited of proliferation of 24% of cell lines. Genomic analysis showed that all cell lines sensitive to the compound belonged to the Consensus Molecular Subgroup 2 (CMS2) sub-type. The anti-proliferative effect of the representative compound was confirmed in a panel of CMS2 patient derived organoids while sparing normal colon organoids. Metabolic labelling showed that the representative compound inhibited protein translation in SW 1417 (CMS2) cells but had no impact on protein translation in COLO-320DM (CMS4). Consistent with this, treatment of SW 1417 with the representative compound triggered nucleolar stress response resulting in cell cycle arrest and apoptosis. pSILAC proteomics showed that the compound selectively inhibited translation of a subset of proteins. The impacted proteins were enriched for proteins containing positively charged regions, particularly those involved in ribogenesis and protein translation. Finally, we show that the representative compound causes synthetic lethality with DNA intercalating agents, a class of chemotherapeutics that are known to inhibit ribogenesis. [0486] Conclusions: We have identified a new class of molecules that selectively inhibit protein translation in CMS2 subtype of CRC which is characterized as MicroSatellite Stable (MSS) and tends to harbor high activity of Wnt/Myc pathway. The selectivity of protein translation inhibition is further characterized by preferential effect on positively charged proteins including those involved in ribogenesis and protein translation. This novel mechanism shows synergy with traditional cytotoxic therapy to generate potential new cancer therapeutics.

Proliferation Assay Protocol:

[0487] The anti-proliferative effect of compounds was tested on colorectal (CRC)-derived SW1417 (ATCC, catalog number: CCL-238) and small cell lung cancer (SCLC)-derived NCIH446 (ATCC, catalog number: HTB-171) cell lines using an opaque- walled, clear-bottom, 384-well plate format. Cells were counted with a Vi-cell XR cell counter and subsequently seeded in their corresponding complete medium: DMEM medium (Invitrogen, catalog number: 11995- 040) supplemented with 10% fetal bovine serum (FBS) for SW1417 cells; RPMI-1640 (Invitrogen, catalog number: 11875-093) supplemented with 10% FBS for NCIH446 cells at a density of 600 (SW1417) and 1,200 (NCIH446) cells/well in a final volume of 40ul. Seeded plates were incubated overnight at 37 °C and 5% CO2.

[0488] Plates were prepared from 60mM DMSO compound stocks with intermediate doses in medium using an automated liquid handler, to achieve the following conditions: 60uM as top concentration, 4 doses, 3-fold dilutions all at 0.1% final volume/volume of DMSO/well. 2ul of diluted compounds, from plates where doses were prepared, were added to 40ul of medium with cells already attached. Staurosporine was used as a reference compound at a starting concentration of 2uM, 7 doses, and 3 -fold dilutions for every plate with test compounds.

[0489] Plates seeded with NCIH446 and SW1417 cells were treated for 96 and 144 hours, respectively. The experiment included a time zero (tO) plate for each cell line. All plates were incubated at 37 °C and 5% CO2 for the duration of their corresponding treatments.

[0490] CellTiterGlo Luminescence kit (Coming, catalog number: G7573) was used to measure ATP levels as a readout for cell viability. CellTiterGlo reagents were equilibrated at room temperature prior to use. Plates seeded with cells were also equilibrated at room temperature for approximately 30 minutes. 25ul of CellTiterGlo reagent/well were added to cells. Plates were gently mixed for 10 minutes at room temperature and protected from light. Luminescence was recorded with an EnVision plate reader and measurements were performed with 0.1s iterations. [0491] Data analysis was performed to calculate Minimum Response Percentage values with XLFit curve fitting software using (IF) logical test: =IF(T greater than T0,100*(T-T0)/(C- T0),100*(T-T0)/T0), where T = Luminescence of compound treatment; TO = Luminescence of compound treatment at time zero; C = Luminescence of vehicle treatment.

Results

[0492] Data for individual compounds is presented in the following Table as the Minimum Response Percentage for each cell line tested. ++++ = less than -50% (cytotoxic); +++ = less than 0 to -50% (moderately cytotoxic); ++ = less than 50 to 0% (growth inhibition); + = less than 100 to 50% (moderate growth inhibition); - = greater than 100% (no growth inhibition).

Proliferation Assay Table Nanoluc Assay

[0493] FRT cells expressing CFTR P-globin fusion proteins (W134X) are cultured in Coon’s F- 12 Medium supplemented with 5% fetal bovine serum and 100 units/mL penicillin-streptomycin. Cells are kept in a humidified, 5% CO2 atmosphere at 37°C. Cells are transfected with 0.5 pg/mL of the described construct, and 48 hours later, the levels of luciferase are measured with the luciferase assay lit (Promega, USA), according to the manufacturer’s instructions.

[0494] A NanoLuc luciferase reporter plasmid was developed to target the identification of both readthrough and NMD modulators by using the addition of a nonsense mutation (W134X) in the Nanoluc region to test for readthrough as well as upstream of a P-globin intron to test for NMD attenuation. The G418 used in experiments was commercial G418-sulfate (Gibco, # 10131-027). Following normalization, the differences in luciferase activities reflect the frequency of stop codon readthrough.

[0495] Panel 1: Readthrough was observed when cells were treated with various amounts of Compound 40 alone or in combination with 100 uM G418.

[0496] Panel 2: Readthrough was observed when cells were treated with various amounts Compound 40 alone or in combination with 100 uM G418 alone or in combination with 50 uM G418.

Full method

Cell Culture

[0497] FRT cells expressing CFTR P-globin fusion proteins (W134X) are cultured at 37°C and 5% CO2 in BioChrom Coon’s F-12 Medium (Cedar Lane Labs, #F0855) with 5% fetal bovine serum (Gibco, #26140-079), 1% penicillin-streptomycin (Gibco, #15140-122), L-glutamine (Gibco, #25030) and 200 pg/mL Zeocin (Life Technology, #R25005).

HTS

[0498] Cells are removed from -80 °C freezers, thawed, and pelleted at 1000 rpm for 5 min at room temperature. The cells are resuspended in FRT growth media. A multidrop reagent dispenser (Thermo Fisher Scientific, #22-387-053) is used to plate cells (25,000 cells/well, 50 pL/well) into 384-well assay plates (Coming, #3570BC). The plates are centrifuged at 500 rpm for 2 min at room temperature and incubated for 48 h at 37°C and 5% CO2. before compound administration of 5 pL/well, to yield a final assay volume of 55 pL/well. The cells are incubated for 48 h at 37°C and 5% CO2.

[0499] For the W134X screening, the medium is aspirated and replaced with 16 pL of Opti-MEM medium (Life Technology, #11058), without FBS, buffered with HEPES (Gibco, #15630-080). 4 pL of Nano-Gio Live Cell Reagent (Promega, #N2013) is added and luminescence was read using an integration time of 0.1-2 seconds.

RDEB Assay Rational, Methodology and Results Example 1. Collagen Type VII Immunoblotting Assay [0500] Rationale

[0501] To assess the ability of compounds in inducing the expression of full-length Collagen VII protein in two RDEB patient Fibroblasts (R578X/R578X or R613X/R1683X) or keratinocytes (R2814X/R2814X, R2610X/R2610X or Q251X/Q251X) after 48 hours of treatment.

[0502] Methodology

[0503] This method has been described in detail by Cogan and colleagues (Cogan et al., Molecular therapy, vol. 22,10 (2014): 1741-52). Cells were plated at 70-80% confluency on 6- well plates. On the following day, media were replaced with fresh media and cells were untreated or treated with test compounds (tested range: 10-60 pM, see table 3 for details) or gentamicin (positive control; 200 pg/ml or 400 pg/ml). At 24 hours, the media was replaced with fresh media and compound. After 48 hours of treatment, cells were lysed and subjected to immunoblot analysis using a polyclonal antibody to type VII collagen and a monoclonal antibody to P- Actin (loading control). ImageJ was used to quantify the intensity of the specific bands and all samples were normalized to 0-Actin. Resultant densitometry values were expressed relative to 0-Actin and then expressed as a percentage relative to non-treated cells.

[0504] The ability of the test compounds in inducing readthrough of Collagen VII protein was assessed in 4 different RDEB derived patient cells using two different cell types (fibroblasts and keratinocytes). The average readthrough activity is expressed as a percentage of activity relative to gentamicin: + = 0-33% of gentamicin, ++ = 34-66% of gentamicin, and +++ = greater than 66% of gentamicin.

Example 2. Fibroblast Migration Assay

Rationale

[0505] RDEB patient fibroblasts have a hypermotility phenotype relative to fibroblasts derived from normal subjects. This cellular phenotype is thought to be linked to the inability of RDEB fibroblasts to attach to the growth substrate due to a lack of Collagen VII (Chen et al., Nature genetics vol. 32,4 (2002): 670-5; Cogan et al., 2014). We therefore test the effect of test compounds in reducing/ rescuing the hypermotility phenotype by induction of Collagen VII nonsense mutation readthrough.

Methodology

[0506] This method has been described in detail by Cogan and colleagues (Woodley et al., Journal of cellular physiology vol. 136,1 (1988): 140-6; Chen et al., 2002; and Cogan et al., 2014). Briefly, R578X/R578X, R613X/R1683X and normal human dermal fibroblasts were plated at a density of 300,000 cells per well of a 6-well plates. On the following day, cells were untreated or treated with test compounds for 24 hours (tested range: 10-60 pM, see table 4 for details). Media and compound were then replaced with fresh media containing indicated doses of compounds. After 48 hours of treatment, cells were sub-cultured onto coverslips and subjected to a well-established fibroblast migration assay as follows: Colloidal gold salts were immobilized on coverslips and covered with type I collagen (15 mg/ml). Fibroblast cultures were suspended, plated on the coverslips, and allowed to migrate for 16-20 hours. The cells were fixed in 0.1% formaldehyde in phosphate-buffered saline and examined under dark field optics. 15-20 non- overlapping fields in each experimental condition were analyzed with NIH Image 1.6 and the percentage area of each field consumed by cell migration tracks was determined (termed Migration Index, MI). In this assay migration indexes of treated and untreated RDEB cells were compared to normal human fibroblast (NHF) cells as well as RDEB cells treated with gentamicin (previously shown to rescue hypermotility phenotype by Cogan et al., 2014; positive control).

[0507] The ability of the test compounds in inducing the expression of full-length Collagen VII protein in two RDEB patient fibroblast cell lines with R578X/R578X and R613X/R1683X mutations was assessed after 48 hours of treatment. Compounds were ranked by considering the MI of compound treated fibroblasts relative to untreated and NHF cells. In this assay untreated cells are most motile, (with little/ no readthrough; marked as +) while NHFs are least motile. Compounds that induce a high level of readthrough have a similar motility level (and therefore MI) to NHFs (marked as +++).

Example: CF Assay Rational, Methodology and Results

Automated Equivalent Current (IEQ) Assay

Rationale

[0508] The efficacy of compounds in increasing chloride ion transport by inducing readthrough of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) nonsense mutation was assessed in primary CF human Bronchial Epithelial (hBE) cultures after 96 hours of treatment using a TECC24 assay setup.

Methodology

[0509] This method has previously been described in detail (Vu et al., Journal of medicinal chemistry vol. 60,1 (2017): 458-473). Cell culture: primary CF hBE cells from a donor with the G542X/F508del-CFTR mutation (patient code KK34J) were cultured as monolayers on Transwell 24-well filter plates at an Air Liquid Interface (ALI) for 4-6 weeks until fully differentiated. Treatment procedure: cultures were basolaterally treated with media containing test compounds (diluted in DMSO) at a final concentration of 20 pM and 60 pM. After 2 days of incubation, basolateral media was replaced with fresh media containing test compounds and the apical mucus was washed with media for at least 30 minutes and incubated for a further 2 days (total of 96 hours). 125 pg/mL ELX-02 was used as a positive control and a CFTR activity standard for readthrough ranking. IEQ measurements: custom software was used to measure transepithelial voltage (VT) and conductance (GT) with a 24-channel current clamp circuit and electrode manifold (TECC24; EP Design BVBA, Bertem, Belgium) coupled to a cartesian robot.

Measurements were made every approximately 5 minutes. Electrodes were washed between each plate read cycle. Baseline reads were measured for approximately 20 minutes. After this period for each epithelium, 25 pL of 100 pM benzamil (10 pM final concentration) was added apically to block ENaC currents, and readings were taken for an additional 20 minutes. After this period, forskolin (10 pM final concentration) and the potentiator VX-770 (100 nM final concentration) were added apically (27.8 pL of combined 100 pM forskolin, 1 pM VX-770 stock). After an additional approximately 20 minutes, 30.9 pL of 200 pM CFTRinh-172 (20 pM final concentration) was added to the apical solution to terminate the run by inhibiting the CFTR- dependent current. Data analysis: The Ohm’s law equation IEQ= VTGT was used to calculate current using Microsoft Excel software. IEQ responses to the above experimental reagents were calculated at each step. The change in current from the baseline that is resultant from the simultaneous addition of forskolin and VX-770 is termed activation current and was used to determine the level of CFTR activity (and hence readthrough) in these studies.

[0510] Activity data is expressed as the percent activation relative to vehicle treated (0%) and ELX-02 (100%) treated cells: + = less than or equal to 10%, ++ = 11-50%, +++ = 51-100%, ++++ = greater than 100%.

CF Assay Results Table

In vivo APCmin Study

[0511] ApcMin mice (C57BL/6J-ApcMin/J)JAX, Jackson laboratory, Bar Harbor ME) harboring the L850X mutation are randomized and dosed with either test agent or vehicle control starting at 10 weeks of age. The mice are dosed daily for a period of 8 weeks at which time the mice are sacrificed, and small intestine, large intestine and spleen are harvested. Spleen weights are measured and recorded. Photographs of the whole small intestine and partial large intestine (approximately 300-400mm) are captured. The middle section of each small intestine is fixed in 10% neutral buffered formalin (NBF, RT) for 24-36h and transferred to ethanol. Subsequently, all samples are processed to FFPE blocks for histology analysis.

[0512] During the dosing phase of the study animal body weights are measured and recorded daily. During the dosing phase, any mice showing anemia, greater than 20% body weight loss or clinical signs of pain are euthanized, and small intestine, large intestine and spleen are harvested. Efficacy of test agent is assessed by comparing Polyp counts, spleen weights and histopathology assessments between the vehicle treat mice and the test agent treated mice.

Identification, validation and initial SAR exploration of oncoribosome targeting macrolides [0513] The transformation of normal cells into cancerous cells is associated with changes in the structure and function of ribosomes. This includes an increase in biogenesis, development of mutations, and alterations in post-translational modifications of ribosomal proteins. Sulima, S. 0.; Hofman I. J. F.; De Keersmaecker, K.; Dinman, J. D. “How Ribosomes Translate Cancer.” Cancer Discov. 2017, 7, 1069. These “oncoribosomes” show shifts in the pattern of mRNA translation compared to normal ribosome, which can lead to the genesis or sustainment of tumors. Kampen, K. R.; Sulima, S. O.; De Keersmaecker, K. “Rise of the specialized onco- ribosomes.” Oncotarget. 2018, 9, 35205. Genuth, N. R.; Barna, M. “The discovery of ribosome heterogeneity and its implications for gene regulation and organismal life.” Mol. Cell, 2018, 71, 364. Therefore, we believe that selectively targeting oncogenic ribosome forms is a potential anti-tumor therapy. To achieve this, we are leveraging our knowledge on the synthesis of ribosomal modulating agents (RMAs) and their unique interactions. Clark, R. B.; Myers, A. G. “Discovery of Macrolide Antibiotics Effective against Multi-Drug Resistant Gram-Negative Pathogens.” Acc. Chem. Res. 2021, 54, 1635. ⁴ Van Rechem, C.; Black, J. C.; Boukhali, M.; Aryee, M. J.; Graslund, S.; Haas, W.; et al. “Lysine demethylase KDM4A associates with translation machinery and regulates protein synthesis.” Cancer Discov. 2015, 5, 255.

[0514] We prepared a library of more than 1000 RMAs and screened this library against several cancerous cell lines as well as non-cancerous controls. We found that the phenotypic screen of our library of more than 1000 RMAs identified hits selective for cancer cell lines over non- cancerous cell lines, and demonstrated responsive SAR. The lead compound identified by this screen showed selective inhibition of total protein synthesis in colorectal cancer cell lines and induced increased expression of p21 , a cell cycle regulator that is indicative of ribosomal stress. The compound selectively inhibited the growth of CRC organoids in vitro, and 50 mpk dosing resulted in an 40% inhibition of xenograft tumor growth in mice. As a result of this data, this series of RMAs deserves further exploration and development as a potential anti -tumor therapy.

Assay Methods

[0515] Cell Culture: HEK293 (human embryonic kidney) cells used as counter-screen. SW1417 and SW948 are colorectal cancer derived cell lines. All cell lines grown as recommended by ATCC.

[0516] Screen: HEK293 and SW1417 cell lines were treated with compounds for 72 and 144 hours, respectively. Cell viability was quantitated by assessing ATP levels at the end of the assay. [0517] Protein translation detection: SW1417 cells were seeded for 48 hours and subsequently treated with compounds 9 and 11 for 48 hours for both assays.

[0518] 1. Nascent protein translation was assessed using a metabolic labeling assay that incorporates a methionine analog, which was fluorescently labeled on a 96-well plate and quantitated. ⁴

[0519] 2. p21 protein expression was assessed via Western Blot assay. Protein lysates from treated cells were loaded on 4-12% acrylamide gels. Anti-p21 antibody was used to detect protein on gel. ⁴ Image studio 5.2 software was used to acquire images and quantitate intensities from both protein translation detection assays. GraphPad Prism 9 software was used to calculate tumor volume, IC50 (uM), and relative protein expression. Spotfire (TIBCO) 12.0.0.223 was used to plot bar graph for organoid response.

[0520] FIG.l provides a chart showing that compounds selective for cancerous cells were present in the library.

[0521] FIG.2 indicates that aryl and lipophilic compounds were found to be more potent..

[0522] In-cell Metabolic labeling (AHA) Assay: Cells were seeded at a density of 30xl0 ⁴ cells in a 96-well black clear-bottom plates (Sigma) in triplicate for 48 hours. Cells were treated with DMSO and specified concentrations of test compounds for 24 hours, and 50ug/mL of CHX (Sigma) for 2 hours prior to incubation in methionine free (-Met) media. Cells were washed once with warm -Met media and incubated in -Met media with treatments for 30 minutes. 50uM of AHA (L- Azidohomoalanine) reagent was added to the -Met media for each condition and incubated for 1 hour. Cells were washed with PBS and fixed using 3.7% formaldehyde in PBS followed by permeabilization using 0.5% Triton X-100. The click reaction and detection were performed using the Click-iT™ AHA Alexa Fluor™ 488 Protein Synthesis HCS Assay kit (Invitrogen). Plates were read on Biotek Cytation 5 plate-reader (Agilent) and data was analyzed using GraphPad Prism software.

[0523] FIGS. 3A and 3B provide an SAR examination of the series.

[0524] Western Blotting Assay: Cells were plated at a density of 0.75xl0 ⁶ cells/60mm dish. After 48 hours, cells were treated with either DMSO or test compounds at specified concentrations for 48 hours. Following treatment, cells were lysed with 250mL of RIP A buffer (RIPA lysis and Extraction Buffer, Thermo Scientific) supplemented with IX protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail (I00X), Thermo Scientific) and nuclease (1 :1000, Universal Nuclease, Thermo Scientific). Proteins were separated on 4-12% gradient gels (NuPAGE Bis-Tris protein gels, Invitrogen) using SDS running buffer (NuPAGE MES SDS Running Buffer, Invitrogen). Primary antibodies: p21 Wafl/Cipl (2947, Cell Signaling Technology, 1: 1000) and PSMD14 (4197, Cell Signaling Technology, 1:5000). [0525] FIGS. 4A and 4B indicate that Compound 9 inhibited protein translation in the CRC cell lines that were tested.

[0526] Organoid Culture: Fourteen colorectal cancer-derived (CMS2) organoids and 1 normal organoid from the Hubrecht Organoid Technology (HUB) were thawed, expanded, and screened following HUB’s guidelines and using established formulated media. For cell viability screening assay, organoids were passaged and seeded at high density. After one day of expansion, organoids were harvested by adding Dispase at a final concentration of 1 mg/mL to the culture medium and incubating at 37°C to digest Matrigel (MG)/Basement Membrane Extract (BME). Then, organoids were collected and size-selected between 40 and 100pm using cell strainers. 250 organoids were dispensed/well in a total volume of 40pL of organoid culture medium (with 5% extracellular matrix) in triplicates for all test conditions in ultra-low attachment (ULA) 384-well plates. Two plates were simultaneously seeded from the same organoid suspension, one for CellTiter-Glo measurement directly after plating (“Day 0”) and one to be measured 7 days after incubation with 9 doses in 3 -fold dilutions of compound 9 with 60uM as top concentration with normalization to 0.3% vehicle in all test wells. Immediately after plating, all compounds were dispensed on the “Day 7” assay plate using the Tecan D300. Staurosporine was used at 2pM as a positive control for cell death in all “Day 7” assay plates. After 7 days of exposure to compound 9, luminescence was measured using Cell Titer-Gio 3D on a Tecan Spark 10M plate reader. IC50 values were calculated using the GRmetrics package. [0527] FIGS. 5A and 5B show that Compound 9 (as provided in the figure) selectively inhibited CRC organoids and causes tumor growth inhibition.

Example

A novel class of Ribosome Modulating Agents (RMAs) exploits cancer ribosome heterogeneity to selectively target CMS2 subtype of colorectal cancer

Abstract

[0528] Ribosomes in cancer cells accumulate numerous patient-specific structural and functional modifications with disease progression. These changes facilitate tumor progression by modifying protein translation. However, prior ribosome inhibitors have had limited success in the clinic because they target universally conserved catalytic steps and lack selectivity for cancer cells. To selectively target cancer subtypes, we have taken an orthogonal approach by synthetic chemistry to generate novel macrolides, Ribosome Modulating Agents (RMAs), that act distal to catalytic sites and exploit cancer ribosome heterogeneity. By allosterically targeting ribosomes, an RMA compound of the invention as disclosed herein shows for the first time two levels of selectivity: 1) selective translation inhibition of a subset of proteins enriched for components of the ribosome and protein translation machinery that are upregulated by MYC; and 2) selective inhibition of proliferation of a subset of colorectal cancer (CRC) cell lines. Mechanistically, the selective ribosome targeting in sensitive cells triggered a nucleolar stress response resulting in cell cycle arrest and apoptosis; Consequently, in CRC, sensitivity to the RMA compound of the invention in cell lines and patient derived organoids was restricted to the Consensus Molecular Subtype 2 (CMS2) subtype that is distinguished by high MYC and WNT pathway activity. Not only did the RMA compound of the invention show potential for efficacy as single agent, the potency and efficacy of the RMA compound of the invention synergizes with clinically approved chemotherapeutics known to inhibit ribogenesis. In conclusion, the RMA compound of the invention represents a new class of allosteric ribosome inhibitors that display cancer selectivity through specific ribosome inhibition in the CMS2 subtype of CRC potentially targeting MYC- driven addiction to high protein translation. Moreover, this study demonstrates that ribosome heterogeneity in cancer can be exploited to develop selective ribogenesis inhibitors. The CRC CMS2 subtype, with a high unmet need for therapeutics, shows vulnerability to our novel selective ribosome mdulator. The mechanism suggests that other cancer subtypes with high MYC activation could also be targeted. Introduction

[0529] Over the last 20 years, a growing body of evidence indicates that ribosome abundance, composition, stoichiometry, and function can vary significantly between developmental states, tissues, cell types and even at different subcellular locations in the same cell (1,2). Tissue- or disease-specific expression of Ribosomal Protein Genes (RPGs), rRNA variants, as well as differential chemical modification of rRNA bases contribute to ribosome heterogeneity. This ribosome heterogeneity can alter the structure, processivity, and transcript selectivity of ribosomes and provides one mechanism for selectively regulating protein expression in response to various stimuli or cell states (1,2).

[0530] In cancer, mutations and copy number alterations in RPGs, dysregulated expression of snoRNAs that control chemical modification of specific rRNA residues, and dysregulation of various steps involved in ribogenesis has been shown to facilitate oncogenesis (3-6). These cancer-specific alterations in ribosomes leading to addiction of cancer cells to altered protein translation patterns provide an opportunity to selectively target cancer ribosomes while potentially sparing other cell types.

[0531] Due to the critical function played by ribosomes in oncogenesis, targeting the cancer ribosome has been a focus of earlier drug development efforts (7). Many molecules have been identified that target the different catalytic steps occurring in the ribosome during protein synthesis (8). However, these steps are not just required for oncogenesis, but also required for normal protein synthesis leading to poor selectivity of these agents and resultant narrow therapeutic window.

[0532] Traditional macrolide antibiotics target the bacterial ribosome at the nascent peptide exit tunnel (NPET) to selectively inhibit translation of proteins enriched for positively charged amino acids resulting in a bacteriostatic effect (9). While the macrolide binding site between prokaryotes and eukaryotes is generally conserved, macrolide antibiotics do not bind or modulate eukaryotic ribosome function nor are used in cancer therapy. This is because of a small difference, the adenosine at position 2058 of the 23 S rRNA (Escherichia coli numbering), which in eukaryotes is replaced with guanine (9,10) and consequently precludes binding of macrolide antibiotics to eukaryotic ribosomes. Since small changes in rRNA sequence can drive large differences in selectivity of macrolides for the eukaryotic ribosome, we have synthesized novel macrolides called Ribosome Modulating Agents (RMAs) using our unique chemistry (11) that can explore an alternative chemical space with potential to target the eukaryotic ribosome. These new RMAs can potentially leverage cancer ribosomal heterogeneity and selectively inhibit activity of ribosomes in some cancer subtypes but not others.

[0533] We present data demonstrating that the RMA compound of the invention, a representative RMA, selectively inhibits protein translation in a subset of cancer cell lines. Consequently, treatment of sensitive cells lines with the RMA compound of the invention triggered nucleolar stress, resulting in cell cycle arrest and apoptosis. We identified that the proteins impacted by the RMA compound of the invention in sensitive cancer cells are enriched for proteins containing high density of positively charged regions. This pattern of protein translation inhibition by protein charge characteristics also enriched for MYC regulated components of the ribosome and protein translation machinery. Further evaluation of the RMA compound of the invention across a panel of CRC-derived cell lines and patient derived organoids identified that the Consensus Molecular Subtype 2 (CMS2) (12,13) of CRC characterized by high MYC and Wnt pathway activation is particularly sensitive to the RMA compound of the invention. Finally, we show that the potency and efficacy of the RMA compound of the invention synergizes with chemotherapeutics that are known to inhibit ribogenesis (14). In conclusion, we have invented a new class of molecules that target a critical pathway in cancer to selectively induce cell cycle arrest and apoptosis in cancer cells. This novel mechanism can be used as monotherapy or combined with approved chemotherapeutics in a rational manner to generate potential new cancer therapeutics.

Materials and Methods

Cell Culture

[0534] Cell lines were acquired from ATCC: SW1417, CCL-238, RRID: CVCL 1717; COLO320DM, CCL-220, RRID: CVCL 0219; SW403, CCL-320, RRID:CVCL_0545; and SW948, CCL-237, RRID:CVCL_0632. COLO320DM were cultured in RPMI Media (ATCC 30- 2001). SW1417, SW403 and SW948 were cultured in DMEM (GIBCO 10569010). Cell lines were maintained in humidified 37°C incubators with 5% CO2. All media were formulated using 10% FBS and 1% penicillin/streptomycin. Assay-specific media formulations are described in their corresponding sections. All cell lines were tested for Mycoplasma contamination using the Myco Alert Assay Control Set (Lonza LT07-518) following the manufacturer’s instructions and used only if results were negative. SW1417, COLO320DM, and SW403 cell lines were authenticated by LabcorpBurlington, NC, USA). For all experiments in this study, all cell lines were grown and expanded for a maximum of 15 passages from their thawing.

Proliferation Assay

[0535] Cells were resuspended in 40ul of medium, seeded in 384-well flat transparent bottom tissue culture treated plates (Greiner 781098) at the following densities: COLO320DM (400/well), SW1417, SW403 (600/well) and 1000/well (SW948) and incubated for 24 hours. All treatments were performed using the D300e digital liquid dispenser (TEC AN). A compound of the invention was dosed as a single agent starting at 60uM in both an 8-point three-fold serial dilution, or a 10-point two-fold serial dilution (combinatorial treatments). Mitoxantrone (Selleckchem S2485), Doxorubicin (Selleckchem S1208) or Actinomycin-D (Sigma Aldrich SBR00013) were dosed in a 10-point three-fold dilution series. DMSO’s limit for all treatments was 0.2%. Treatment timepoints for COLO320DM (48 hours) and for SW1417, SW403 and SW948 (144 hours) were selected to span at least two cell doublings. ATP levels were measured as readout for cell viability using CellTiter-Glo 2.0 reagent (Promega G9243) for all timepoints, including day 0 (untreated cells). Luminescence reading was performed using Cytation5 Multi- Mode plate reader (Agilent). Minimum Response Percentage values were calculated using (IF) logical test: =IF(Tgreater than T0,100*(T-T0)/(C-T0),100*(T-T0)/T0). Key: T: Drug Treatment; TO: Drug treatment at time zero; C: Vehicle treatment. To ensure that GI50 in each cell line is measured at the same number of cell doublings (in this case 2 cell doublings), the GI50 for all cell lines were evaluated at timepoints based on their specific doubling time. This is the recommended protocol from the National Cancer Institute (NCI) (dtp.cancer.gov/discovery_development/nci- 60/methodology.htm).

[0536] Drug synergy was analyzed using the Bliss Independence (BI) Principle. BI scores were calculated from averaged values (2 replicates/plate/tested dose as single agent or in combination) using the following formula: Observed - Expected = BI Score. BI scores were defined as follows: antagonistic less than -0.14; -0.14 less than additive less than 0.14; and synergistic greater than 0.14. The first column (Compound of the invention) and bottom row (Actinomycin D, Doxorubicin, or Mitoxantrone) of each matrix were used for single agent treatments.

Metabolic labeling

[0537] Cells were seeded at a density of 7xl0 ⁵ (SW1417) and 5xl0 ⁵ (COLO320DM) in a 6-well plate for 48 and 24 hours, respectively. Cells were treated with DMSO and 40uM of the compound of the invention for 24 hours and 50ug/mL of CHX (Sigma) for 2 hours prior to incubation in methionine free (-Met) media. L- Azidohomoalanine (AHA) incorporation and Click-iT reaction were performed as in (15). Specific modifications are detailed in the supplementary section. pSILAC

[0538] DMEM for SILAC (Gibco) was supplemented with dialyzed Fetal Bovine Serum (FBS) (Gibco). The light media was supplemented with L-Lysine-2HC1 for SILAC (Thermo) and L- Arginine-HCl for SILAC (Gibco). The heavy media was supplemented with L-Lysine-2HC1, ¹³ C6, ¹⁵ N2 for SILAC (Thermo) and L-Arginine-HCl, ¹³ C6, ¹⁵ N4 for SILAC (Thermo). SW1417 cells were seeded at a density of 2xl0 ⁶ cells per 60mm dish in light media (+Light Arg/Lys) for 24 hours. Cells were treated with DMSO and the compound of the invention (20uM) for 1 hour in DMEM for SILAC with dialyzed FBS (-Arg/Lys), followed by a wash with warm heavy media (+Heavy Arg/Lys). Cells in both conditions were incubated in +Heavy Arg/Lys media for a total of 6 and 24 hours. Subsequently, cells were scrapped off, collected in a 1.5mL tube, and centrifuged at 500g for 5 minutes. Cell pellets were washed twice with PBS, centrifuged at 500g for 5 minutes, and stored at - 20C until ready to be processed for Mass Spectrometry.

Mass Spectrometry

[0539] Cell pellets were lysed in 5% SDS, 50 mM tri ethylammonium bicarbonate (TEAB) pH 8.5, and protein concentration was determined using the BCA protein assay. Samples were reduced in 10 mM dithiothreitol for 30 min at 37°C followed by alkylation in 20 mM iodoacetamide for 20 min at room temperature. Samples were acidified and digested on an S- Trap™ spin column (Profiti) by addition of trypsin at 1 :20 for 3 h at 47°C. Peptides were eluted in 50 mM TEAB pH 8.0, 0.2% formic acid and 50% acetonitrile, respectively.

2ug of protein per sample were used for LC-MS/MS analysis. The raw mass spectrometric data were processed in MaxQuant (version 2.0.3.0) for peptide and protein identification. More detailed protocol in the supplementary section.

Immunoblotting

[0540] Assay performed as in (15). Specific modifications are detailed in the supplementary section. Cell cycle analysis

[0541] SW1417 cells were plated at a density of O. lxlO ⁶ cells/well in a 12-well plate, and COLO320DM cells were plated at a density of IxlO ⁶ cells/well in a 6-well plate. After 24 hours, cells were treated with DMSO or the compound of the invention at various concentrations for 48 hours. Following treatment, cell pellets were washed twice with IX PBS, and then fixed with Fixation buffer (BioLegend) for 15 minutes at room temperature. The pellets were then washed with Staining buffer (BioLegend) and then permeabilized with IX Click-iTTM permeabilization and wash reagent (ThermoFisher) for 15 minutes at room temperature. The cells were then stained with DNA-specific dye FxCycleTM Violet Stain (Invitrogen) for 30 minutes and analyzed using the BD FACSCelestaTM Cell Analyzer (flow cytometer).

Organoid Culture

[0542] CRC-derived (CMS2) organoids (14) and a normal organoid from the Hubrecht Organoid Technology (HUB) were thawed, expanded, and screened following HUB’s guidelines and using formulated media internally established. CRC-derived organoid cultures and the normal organoid culture were propagated and screened in colon tumor medium (CTM) and colon surrogate medium (CSM), respectively. CTM medium is routinely used for the culture and screening of colon cancer organoids. CSM contains NGS-Wnt and is routinely used for the culture and screening of colon organoids derived from healthy tissue. Organoid culture composition table, organoid handling, and treatment conditions are detailed in the supplementary section.

[0543] Characterization data for Compound of the invention Citric Acid Salt. 'l l NMR (400 MHz, CD3OD) <57.24 (t, J= 7.8 Hz, 1H) 6.88 - 6.96 (m, 2H) 6.85 (dd, J= 8.2, 2.0 Hz, 1H) 4.56 (br d, 13.6 Hz, 1H) 4.46 (d, J= 7.2 Hz, 1H) 4.2O (d, J = 10.0 Hz, 1H) 3.79 (s, 3H) 3.67 - 3.75 (m, lH) 3.62 (br s, 2H) 3.43 (dd, J= 10.4, 7.2 Hz, 1 H) 3.33 (br s, 1H) 3.23 - 3.29 (m, 1H) 3.11 - 3.20 (m, 1H) 2.94 - 3.07 (m, 5H) 2.87 (br s, 3H) 2.69 - 2.82 (m, 11H) 2.19 - 2.47 (m, 4H) 2.13 (br dd, J= 12.8, 8.0 Hz, 1H) 2.01 (dt, J= 10.6, 2.0 Hz, 1H) 1.90 - 1.97 (m, 1H) 1.80 (br d, J= 13.2 Hz, 2H) 1.28 - 1.56 (m, 17 H) 1.06 (br d, J= 6.6 Hz, 3H). MS (ESI) m/z 690.5 (M+H). 99.7% purity by HPLC (UV @ 220 nM). Yellow solid.

[0544] Quality control HNMR, LCMS, and HPLC data for the Compound of the Invention are provided as a supplementary document.

Software Analysis

[0545] GraphPad Prism 9 (RRID:SCR_002798) was used to plot percent response dose curves and bar graphs, calculate GI50 and LD50 values, and plot bar graphs for protein intensities. Image Studio 5.2 (LI-COR, RRID:SCR_015795) was used to acquire images and quantitate intensities from Smear and Western blot membranes. Spotfire TIBCO 12.0.0.223 (RRID:SCR_008858) was used to plot volcano plots, bar graphs, pie chart, and scatter plot. Fiji 2.6.0 (RRID:SCR_002285) image processing package software was used to analyze in-cell metabolic labeling (AHA) assay.

Data Availability Statement

[0546] The data generated in this study are available within the article and its Supplementary Data files.

Results

[0547] The RMA compound of the invention selectively inhibits protein translation in a sensitive but not insensitive CRC cell line

[0548] We evaluated the impact of the RMA compound of the invention on proliferation of two CRC cell lines (SW 1417 and COLO320DM). Treatment with the RMA compound of the invention shows a robust growth inhibitory effect on SW1417 cells while displaying a minimal anti-proliferative activity in COLO320DM cells (FIG. 6.1A). To determine if the anti- proliferative activity of the RMA compound of the invention is due to impact on protein translation, we conducted a metabolic labeling study using L- Azidohomoalanine (AHA), which is incorporated into newly synthesized proteins in place of methionine (Met) (FIG. 6.1B). L- AHA incorporated into newly synthesized proteins was biotinylated and detected by Western blot. Treatment of SW 1417 with the RMA compound of the invention for 24 hours resulted in a 27% decrease in AHA incorporation into newly synthesized proteins. In contrast, the RMA compound of the invention treatment had no significant impact on AHA incorporation in COLO320DM cells (FIG. 6.1C). These results contrast with the global effect observed after cycloheximide (CHX) treatment, which completely inhibited new protein synthesis in both cell lines. This decrease in new protein synthesis was confirmed in a plate reader assay that uses fluorescence to quantitate AHA incorporation into newly synthesized proteins. In SW1417 cells, the RMA compound of the invention inhibited new protein synthesis in a dose-dependent manner (Supplementary FIG. 6.1S1A and B). Taken together, these data indicate that the RMA compound of the invention selectively inhibited protein translation in SW 1417 cells and that this partial inhibition of protein translation is associated with growth inhibition. [0549] The RMA compound of the invention selectively inhibits translation of a subset of proteins

[0550] To further characterize the impact of the RMA compound of the invention on protein translation, we conducted a pSILAC proteomics study in SW1417 cells. We treated cells with either the RMA compound of the invention (20uM) or DMSO and concomitantly exposed them to heavy isotope-labelled forms of arginine and lysine (heavy medium) (FIG. 6.2 and Supplementary FIG. 6.2S2A). Mass Spectrometric quantitation of heavy and light isotope labelled peptides was used to assess the impact of the RMA compound of the invention on translation rate and abundance of newly synthesized proteins. At the 6-hour time-point, no significant change in global abundance of proteins was observed, but the significant decrease in the heavy-to-light isotope ratio (H/L ratio) of 129 out of 1188 proteins evaluated (Supplementary FIG. 6.2S2B) was indicative of a decrease in protein translation rate. Gene Ontology (GO) analysis (15) indicated that a third of these 129 proteins were nucleic acid binding proteins, 60% of which are known to be involved in protein translation including ribosomal proteins, rRNA maturation factors, and translation factors (Supplementary FIG. 6.2S2C). At the 24-hour time- point, we observed a significant decrease in the abundance of newly synthesized molecules for a large subset (1482 out of 2867) of the detected proteins (FIG. 6.2A). Since macrolide antibiotics selectively inhibit protein translation at peptide motifs enriched for positively charged amino acids (9), analysis of the positive charge density of all detected proteins performed. We observed that, as the density of positively charged regions increased, a larger fraction of proteins was sensitive to translation inhibition (FIG. 6.2B). Gene overrepresentation analysis (16) indicated that proteins sensitive to the RMA compound of the invention mediated translation inhibition were highly enriched for MYC target genes (FIG. 6.2C). A deeper dive into overrepresentation analysis showed that the top gene sets were highly overlapping with MYC targets, with 16 of the top 20 significant gene sets containing protein translation related proteins including ribosomal proteins, translation initiation and elongation factors as the key representative genes (FIG. 6.2C). Since ribosomal proteins have been reported to be highly enriched for positively charged regions (17), it was striking that 68 out of 73 of the detected ribosomal proteins showed a significant decrease in protein synthesis in response to the RMA compound of the invention treatment (FIG. 6.2D) This impact of the RMA compound of the invention on ribosomal protein synthesis was confirmed by Western Blot analysis of a subset of ribosomal proteins (Supplementary FIG. 6.2S2D). These data demonstrate that the RMA compound of the invention selectively inhibited the synthesis of a subset of proteins that are components of the cellular protein translation machinery and map to the MYC pathway. Specifically, the decrease in translation of almost all ribosomal proteins indicated that the RMA compound of the invention selectively inhibited ribogenesis in these sensitive cancer cells.

[0551] The RMA compound inhibits ribogenesis

[0552] Perturbations that disrupt ribogenesis have been reported to trigger nucleolar stress leading to cell cycle arrest or apoptosis (18,19). To characterize the cellular response to the RMA compound of the invention treatment, we assessed the impact of the RMA compound of the invention on the cell cycle. As seen in FIG. 6.3A, a 48-hour treatment with the RMA compound of the invention leads to an increase in p21 protein levels. FACS analysis assessing DNA content showed that the RMA compound of the invention treatment triggered a strong G1 arrest in SW1417 cells. A significant and dose-dependent increase in subGO content, indicative of apoptotic cells, was also observed (FIG. 6.3B). These responses were not observed in the refractory cell line COLO320DM (Supplementary FIG. 6.3S3A and B). These data demonstrate that the RMA compound of the invention inhibits ribogenesis, leading to induction of p21 , and resulting in cell cycle arrest and apoptosis.

[0553] The CMS2 subtype of CRC is highly sensitive to the RMA compound of the invention

[0554] To further characterize the response to the RMA compound of the invention we profiled its activity across a panel of 34 CRC cell lines. We observed that 8 cell lines were highly sensitive to the RMA compound of the invention while 12 cell lines were completely refractory (FIG. 6.4A). The remaining lines showed an intermediate level of sensitivity to the RMA compound of the invention. These CRC lines have been well characterized in the literature and genomic data associated with these lines are publicly available. We leveraged these data sources to characterize the clinical and molecular features associated with sensitivity to the RMA compound of the invention. Our analysis identified that all 8 cell lines sensitive to the RMA compound of the invention belong to the CMS2 subtype of CRC which is characterized by high MYC and WNT pathway activity, whereas the insensitive and intermediate cell lines distributed across all 4 CMS subtypes. Another molecular feature shared by all CMS2 cell lines sensitive to the RMA compound of the invention is the Microsatellite-stable status (MSS), which is known to harbor significantly more copy number variations (CNV) than those of the Microsatellite- instable status (MSI). Interestingly, our analysis also identified copy number gains of Chr20ql 1 as a novel genomic feature present in all CMS2 cell lines sensitive to the RMA compound of the invention (FIG. 6.4B and C). To further validate the sensitivity associated with the CMS2 subtype, we evaluated the RMA compound of the invention across a panel of CMS2, patient- derived CRC organoids, the RMA compound of the invention inhibited proliferation of most of the CMS2, patient-derived organoids without affecting growth of a model organoid derived from normal colon (FIG. 6.4D). These data demonstrate that the RMA compound of the invention selectively targets colon cancer cells of the CMS2 subtype characterized by high MYC and WNT pathway activity.

[0555] DNA intercalating agents demonstrate robust combination synergy with the RMA compound of the invention

[0556] Development of novel cancer therapies not only requires single agent activity, but also requires combinations with existing agents to achieve maximal clinical efficacy. While additivity between two cancer agents can be sufficient, synergy is highly desired to maximize the anti- cancer effect. To identify the therapeutics that could synergize with the RMA compound of the invention, we screened a panel of 14 approved drugs that are Standard-of-Care chemotherapeutics or targeted therapies used to treat CRC. The potential for combination synergy was evaluated in SW403 (CMS2 line) using the Bliss Independence Model (20). A majority of the drugs tested were classified as having an additive effect in combination with the RMA compound of the invention (FIG. 6.5A), but interestingly, the three DNA intercalating agents consistently showed synergistic effects in combination with the RMA compound of the invention in two CMS2 CRC cell lines (FIG. 6.5A-C). In the two CMS2 lines, cytostatic concentrations of single agents, when combined, generated a robust cytotoxic response, demonstrating the combination synergy of DNA intercalating agents with the RMA compound of the invention. (FIG. 6.5D and E)

[0557] Discussion

[0558] Ribosome production in a rapidly dividing cell requires immense cellular resources (21), and thus targeting ribosomes in cancer represents an attractive approach to treatment. As with targeting universal cellular processes to treat cancer through chemotherapy or anti-metabolite therapy where the therapeutic window becomes limiting, targeting catalytic ribosome steps suffers from the same drawback. We have taken an orthogonal approach by leveraging rapid advances in chemistry coupled with detailed understanding of ribosome biology. First, we have used our advanced chemistry platform to create a library of novel macrolides (11) which occupy a new chemical space with increased potential to target the human ribosome due to the high degree of conservation of the macrolide binding site across species (10). Macrolides bind to the nascent peptide exit tunnel, and thus act as allosteric modulators of the ribosome, overcoming issues with inhibiting the highly conserved catalytic steps involved in protein synthesis. Second, we have identified a subset of molecules, including the RMA compound of the invention, which exploit cancer ribosome heterogeneity to selectively target a subset of cancer cell lines.

[0559] The RMA compound of the invention demonstrates two levels of selectivity. First, protein translation inhibition only occurs in some cell types and not others, exemplified by SW1417 vs. COLO320DM. While previously theorized (22), this selectivity ribosome inhibition in one cell line versus one is the first demonstration of differential ribosome targeting. A second level is the propensity of the RMA compound of the invention to selectively inhibit translation of proteins with a high density of positively charged regions. This activity was less surprising given that the mechanism of protein translation inhibition by macrolide antibiotics is well described, and the process of ribosome mediated protein synthesis is conserved across prokaryotes and eukaryotes. It is hypothesized that the positively charged amino acids interact with the negatively charged rRNA lining the NPET which slows down the passage of the nascent peptide through the NPET (17). We observed that as the density of positively charged regions increased, a larger fraction of proteins was sensitive to translation inhibition by the RMA compound of the invention (FIG. 6.2B). The slow translation of sequences enriched for positively charged amino acids is likely further exacerbated by the presence of the RMA compound of the invention in the NPET, resulting in ribosome stalling and translation inhibition. Published reports indicated that nucleic acid binding proteins and ribosomal proteins in particular are highly enriched for positively charged regions (17). Additional classes of proteins enriched for positively charged regions include chromosomal and nucleolar proteins as well as proteins localized to the mitochondria. Consistent with these reports, all these classes of proteins are enriched among the proteins impacted by the RMA compound of the invention. This finding is also a first - all known human ribosome inhibitors target catalytic steps and do not show selectivity for specific protein sequences. [0560] The RMA compound of the invention also sheds new light on mechanisms used by cancer to co-opt normal cellular processes for disease progression. It was unexpected that the RMA compound of the invention-induced protein translation inhibition significantly impacted proteins that map to the MYC pathway. Hence it was not surprising that cell line profiling across 34 CRC lines identified 8 that were sensitive to the RMA compound of the invention belong to the CMS2 subtype of CRC (12). The CMS2 subtype accounts for 37% of CRC and is characterized by activated WNT pathway and high MYC activation (12). Sensitivity of CMS2 subtype was confirmed in a panel of CMS2 patient derived organoids, highlighting the potential for clinical translation of this novel mechanism (7). The spectrum of MYC targets whose protein synthesis was affected were predominantly those that contain high density of positively charged regions and are involved in protein translation either directly, i.e., eukaryotic initiation factors, or indirectly, i.e., ribosomal proteins that are components of ribosomes.

[0561] The nucleolus, the site of ribogenesis, plays a critical role in regulating cell cycle progression, senescence, and apoptosis (19). Perturbations that interfere with ribogenesis trigger a nucleolar stress response that leads to cell cycle arrest and apoptosis via p53-dependent and p53 -independent mechanisms (23). The CRC cell line profiling data indicate that sensitivity to the RMA compound of the invention does not require wild type p53 (data not shown). Consistent with this, inhibition of ribogenesis by the RMA compound of the invention led to a robust G1 arrest and induction of apoptosis.

[0562] Cancer therapy is rarely based on single agents. However, most combinations used in cancer therapy show additivity. We observed that combining the RMA compound of the invention with DNA intercalating agents, that are known to inhibit rRNA expression (14), resulted in a robust synergistic, anti-proliferative effect. Among the various therapeutics tested, only DNA intercalating agents demonstrated synergy with the RMA compound of the invention. [0563] Combining cytostatic concentrations of the RMA compound of the invention and DNA intercalating agents generated a robust cytotoxic response in two CMS2 lines. These data highlight the potential to design mechanism-based combinations that target vulnerability of CMS2 subtype of CRC to ribogenesis inhibition and thereby provide opportunities to optimize therapy with the RMA compound of the invention.

[0564] Many small molecule anticancer agents, especially kinase inhibitors, demonstrate the ability to inhibit targets at a cellular level at sub-micromolar concentrations. However, the RMA compound of the invention requires tens of micromolar concentrations to show effect on protein synthesis. An assessment of target protein abundance showed that while kinases are present at approiximately 10 ³ copy numbers of protein per cell, ribosomes have 10 ⁶ -l 0 ⁷ copy numbers per cell (24), and it is likely higher in cancer cells. Based on average cytoplasmic volume of 1 picoliter (25), the estimated ribosomal concentration ranges from 1 mM to 10 mM, which is aprroximately 1000-fold greater than the concentration of typical kinase drug targets. The potency of the RMA compound of the invention in sensitive lines is consistent with this high concentration of ribosomes in cells.

[0565] Fortuitously, RMAs retain the excellent drug-like properties and high tissue exposure that is inherent to the macrolide class of antibiotics (26). In a single-dose mouse PK study, the RMA compound of the invention dosed PO at 50 mg/kg achieved a terminal skin tissue concentration of 7uM (data not shown). The long half-life and high tissue exposure of macrolides enables coverage of high abundance of ribosomes.

[0566] The activity and selectivity of the RMA compound of the invention exploits two factors: 1) addiction of CMS2 subtype to high protein translation capacity; and 2) ribosome heterogeneity, that enables selective targeting of ribosomes in this subtype of cancers. Mutations and copy number alterations in RPGs, dysregulated expression of snoRNAs that regulate chemical modification of specific rRNA residues, and dysregulation of various steps involved in ribogenesis prevalent in sensitive CMS2 subtype likely enables the RMA compound of the invention to selectively target ribosomes in this subtype of cancer. Additional studies will be needed to further characterize the specific binding interactions resulting from ribosome alterations associated with sensitivity to the RMA compound of the invention. In conclusion, our initial studies demonstrating the novel mechanism of action of the RMA compound of the invention provide evidence that ribosome heterogeneity in cancer can be exploited by this novel class of allosteric ribosome inhibitors to develop selective ribogenesis inhibitors for CMS2 subtype and potentially other cancer subtypes that show high MYC activation and thus potential addiction to high protein translation capacity.

[0567] FIGURES

[0568] FIG. 6.1.

[0569] The RMA compound of the invention is selectively active in a sensitive CRC cell line. A, % Response curves of the RMA compound of the invention treatment in COLO320DM and SW1417 cells. Representative of 3 independent experiments. Error bars are standard deviations of two technical replicates. Table shows GI50 and LD50 values B, Metabolic labeling assay’s workflow. C, Representative Western blot membranes of 2 independent experiments: -AHA (no AHA reagent added to lysate); Cycloheximide (CHX) (positive control for translation inhibition); DMSO (negative control); and the RMA compound of the invention treatment (40uM). Equal protein amount for all samples used for click chemistry reaction. Samples resuspended in equal volume were loaded on gel. Intensity from densitometry of the RMA compound of the invention-treated samples were normalized to those of DMSO samples to calculate relative protein translation. Bar graph shows normalized values from two independent experiments. Error bars are standard deviations.

[0570] FIG. 6.1 SI A

[0571] Panels showing representative immunofluorescence images of SW1417 cells stained for DNA (Hoechst, blue) and AHA incorporated into newly synthesized proteins (Alexa 488, green) after 24hr treatment. -AHA (no AHA reagent added to medium); Cycloheximide (CHX) (positive control for translation inhibition); DMSO (negative control); and Compound of the invention treatment (60uM). Sale bar = lOOum.

[0572] FIG. 6.1 SIB

[0573] Bar graph from representative experiment plotting relative Alexa 488 signal from SW1417 cells treated with DMSO and 5 doses of Compound of the invention for 24 hr. Error bars are standard deviations. Two independent experiments were conducted.

[0574] FIG. 6.2

[0575] The RMA compound of the invention inhibits nascent protein translation in a sensitive CRC cell line. A, Volcano plot showing heavy-isotope intensities (newly synthesized protein abundance) for 2867 proteins detected at 24-hour time point. Protein fraction classification: Decreased (log2FC less than -0.4, adjusted p-value less than 0.05, red); Downward Trend (log2FC less than -0.4, adjusted p-value greater than 0.05, pink); Increased (log2FC greater than 0.4, adjusted p-value less than 0.05, dark green); Upward Trend (log2FC greater than 0.4, adjusted p-value greater than 0.05, light green); Unchanged (-0.4 less than log2FC less than 0.4, adjusted p-value greater than 0.05, dark gray). Dotted lines indicate -0.5 and 0.5 log2FC values. B, Bar graph plotting percent protein fraction of heavy-isotope intensity (categorized as in A) at 24-hour time point versus positively charged regions. Positive charge windows were generated by assigning K and R residues a positive 1 charge and all other amino acid residues a charge of 0. Average charge values were then calculated along a sliding window 10 amino acids long for each protein amino acid sequence (starting at the first residue and ending at the last complete set of 10 residues) C, Overrepresentation analysis and pathway enrichment in significantly downregulated proteins from A. Count refers to the number of genes found in each pathway while Gene ratio refers to the count divided by all genes in the pathway. D, Bar graph showing nascent protein abundance for 68 Ribosomal proteins from heavy-isotope intensity analysis at 24-hour time point (categorized as in A). Dotted line indicates -0.4 log2FC value.

[0576] FIG. 6.2S2A

[0577] pSILAC assay’s workflow.

[0578] FIG. 6.2S2B

[0579] Volcano plot showing heavy-to-light (H/L) ratios for 1188 proteins detected at 6-hour time point. Proteins displaying significantly decreased (adjusted p-value less than 0.05) H/L ratios (129) are depicted in red circles. Detected proteins not significantly affected by the compound of the invention are depicted in black circles.

[0580] FIG. 6.2S2C

[0581] Pie chart of 129 significantly decreased detected proteins grouped in classes curated from Gene Ontology (GO) classification. Bar graph shows 5 subclasses within the Nucleic acid binding class.

[0582] FIG. 6.2S2D, Immunoblots for RPL11, RPS11, RPS29, and RPS21 showing samples from SW 1417 cells treated with DMSO or the compound of the invention (20uM) for 72 hours. Bar graph showing quantitated intensities normalized to PSMD14 (loading control).

[0583] FIG. 6.3.

[0584] The RMA compound of the invention alters cell cycle in a sensitive CRC cell line. A, Immunoblots for p21 following 24-hour treatments with DMSO or the RMA compound of the invention (40uM). Bar graphs show quantitated intensities normalized to B2M (loading control). B, Bar graph plotting percentage of SW1417 cells from cell cycle phases.

[0585] FIG. 6.3S3A and B

[0586] FIG. 6.S3A, Bar graph plotting percentage of COLO320DM cells from cell cycle phases: [0587] FIG. 6.S3B, DNA content analysis by flow cytometry for control (OuM) and 48-hour the compound of the invention treatment (20, 40, and 60uM) conditions from SW147 and COLO320DM cell lines.

[0588] FIG. 6.4.

[0589] CMS2 cell lines display high sensitivity to the RMA compound of the invention and distinct molecular signatures. A, Scatter plot displays drug sensitivity metrics for the RMA compound of the invention across 37 CRC cell lines with colors indicating sensitive, intermediate and insensitive cell lines. B, Heat map of gene expression data for CMS subtype genes across CRC cell lines shows an enrichment of sensitive cell lines in the CMS2 subtype. Row annotations show drug sensitivity, MSI/MSS status, CMS subtypes and average CNV ratios for Chr20ql 1-13. C, Bar graph plotting Chr20 CNV ratios for CRC cell lines, split by CMS2 subtype shows a further enrichment of sensitive cell lines in the CMS2 subtype with Chr20ql 1- 13 CNV gain. D, Bar graph plotting IC50 values for the RMA compound of the invention across CRC-derived CMS2 (light blue bars) and normal (black bar) organoids. Dotted line indicates IC50 30uM.

[0590] FIG. 6.5.

[0591] The RMA compound of the invention is synergistic with DNA intercalating agents on selected CRC cell lines. A, List of 14 inhibitors combined with the RMA compound of the invention. Color coded according to results from BLISS Independence (BI) analysis blue (synergism) or orange (antagonism). B, Matrices with BI scores for combinations of the RMA compound of the invention with Actinomycin D and Doxorubicin on SW948 cells. C, Matrices with BI scores for combinations of the RMA compound of the invention with Mitoxantrone on 2 cell lines: SW948 and SW403. Single agent concentrations in all first columns and bottom rows for all matrices. D, % Response bar graphs showing the RMA compound of the invention’s efficacy enhanced in combination with Actinomycin D (Act-D) and Doxorubicin (Doxo) in SW948 cells. DMSO (black), the RMA compound of the invention (30uM, red), Act-D (lOnM, blue), Doxo (330nM, blue), the RMA compound of the invention+Act-D and the RMA compound of the invention+Doxo (purple). E, % Response bar graphs showing the RMA compound of the invention’s efficacy enhanced in combination with Mitoxantrone (Mitox) in SW948 and SW403 cells. DMSO (black), the RMA compound of the invention (30uM, red), Mitox (6nM, blue) (SW948), Mitox (5.56nM, blue) (SW403), and the RMA compound of the invention+Mitox (purple).

Materials and Methods Cell Culture

[0592] All cell lines (SW1417, COLO320DM, SW403 and SW948) acquired from ATCC. COLO320DM were cultured in RPMI Media (ATCC 30-2001). SW1417, SW403 and SW948 were cultured in DMEM (GIBCO 10569-010). All cell lines were maintained in a humidified 37C incubator with 5% CO2. All media were formulated using 10% FBS and 1% penicillin/streptomycin. Assay-specific media formulations are described in their corresponding sections.

Proliferation Assay

[0593] Cells were resuspended in 40ul of medium, seeded in 384-well flat transparent bottom tissue culture treated plates (Greiner 781098) at the following densities: COLO320DM (400/well), SW1417, SW403 (600/well) and 1000/well (SW948) and incubated for 24 hours. All treatments were performed using the D300e digital liquid dispenser (TECAN). the RMA compound of the invention was dosed as a single agent starting at 60uM in both an 8-point three- fold serial dilution, or a 10-point two-fold serial dilution (combinatorial treatments).

Mitoxantrone (Selleckchem S2485), Doxorubicin (Selleckchem S1208) or Actinomycin-D (Sigma Aldrich SBR00013) were dosed in a 10-point three-fold dilution series. DMSO’s limit for all treatments was 0.2%. Treatment timepoints for COLO320DM (48 hours) and for SW1417, SW403 and SW948 (144 hours) were selected to span at least two cell doublings. ATP levels were measured as readout for cell viability using CellTiter-Glo 2.0 reagent (Promega G9243) for all timepoints, including day 0 (untreated cells). Luminescence reading was performed using Cytation 5 Multi-Mode plate reader (Agilent). Minimum Response Percentage values were calculated using (IF) logical test: =IF(T greater than T0,100*(T-T0)/(C-T0),100*(T- T0)/T0). Key: T: Drug Treatment; TO: Drug treatment at time zero; C: Vehicle treatment.

[0594] Drug synergy was analyzed using the Bliss Independence (BI) Principle. BI scores were calculated from averaged values (2 replicates/plate/tested dose as single agent or in combination) using the following formula: Observed - Expected = BI Score. BI scores were defined as follows: antagonistic: less than -0.14; -0.14 less than additive is less than 0.14; and synergistic: greater than 0.14. First column (the RMA compound of the invention) and bottom row (Actinomycin D, Doxorubicin, or Mitoxantrone) of each matrix were used for single agent treatments.

Metabolic labeling [0595] Cells were seeded at a density of 7x10 ⁵ (SW1417) and 5xl0 ⁵ (COLO320DM) in a 6-well plate for 48 and 24 hours, respectively. Cells were treated with DMSO and 40uM of the RMA compound of the invention for 24 hours and 50ug/mL of CHX (Sigma) for 2 hours prior to incubation in methionine free (-Met) media. L- Azidohomoalanine (AHA) incorporation and Click-iT reaction were performed as in (27).

Metabolic labeling - Supplementary

[0596] Cells were washed once with warm -Met media and incubated in -Met media with DMSO, 40uM the RMA compound of the invention, or 50ug/mL CHX for 30 minutes. 50uM of AHA (L- Azidohomoalanine) reagent was added to the -Met media for each condition and incubated for 1 hour. Cells for each condition were washed once with complete media (culturing media) and incubated for 1 hour in complete media. Cells were washed with PBS and 200uL lysis buffer containing 50mM Tris HC1 at pH 8.0 (Thermo), 1%SDS (Fisher Chemicals), 1: 100 of 100X protease and phosphatase inhibitor (Pierce Biotech) and 1 : 1000 of 20X Universal Nuclease for cell lysis (Invitrogen) were added per well to the plate for SW1417. For COLO320DM, the cells were collected in a 15mL tube and centrifuged at 1200rpm for 5 minutes, washed with PBS once and centrifuged again; 200uL of lysis buffer were added to each tube. For SW 1417, the cells were scraped and transferred to 1 ,5mL tube. The samples were kept on ice for 15 minutes, followed by vortexing for 5 minutes and spinning at 15000g for 10 minutes at 4C. The supernatant was transferred into a new 1.5mL tube. Protein concentration was determined using BCA assay (Pierce Biotech). 200ug of protein from all cell lysates were prepared in 50uL and then brought up to final volume (60uL) with deionized water. For click chemistry, the Click-iT Protein Reaction Buffer Kit (Invitrogen) was used. All reagents in the kit were reconstituted and click reaction was completed following manufacturer's instructions. The dried samples were reconstituted in 25uL of IX LDS sample loading buffer (Invitrogen) and resolved by SDS-PAGE. The Gel was transferred and blocked for Ih in 1 :20000 of IRDye® 800CW Streptavidin (LiCor). The membrane was washed 3 times with IX TBST (Thermo) for 5 minutes.

In-cell Metabolic labeling - Supplementary

[0597] Cells were seeded at a density of 30xl0 ⁴ cells in a 96-well black clear-bottom plates (Sigma) in triplicate for 48 hours. Cells were treated with DMSO or the RMA compound of the invention at various concentrations for 24 hours, and 50ug/mL of CHX (Sigma) for 2 hours prior to incubation in methionine free (-Met) media. Cells were washed once with warm -Met media and incubated in -Met media with treatments for 30 minutes. 50uM of AHA (L- Azidohomoalanine) reagent was added to the -Met media for each condition and incubated for 3 hour. Cells were washed with PBS and fixed using 3.7% formaldehyde in PBS followed by permeabilization using 0.5% Triton X-100. The click reaction and detection were performed using the Click-iT™ AHA Alexa Fluor™ 488 Protein Synthesis HCS Assay kit (Invitrogen). Plates were read on Biotek Cytation 5 plate-reader (Agilent) and data was analyzed using GraphPad Prism software. Images were acquired on the EVOS™ M5000 Imaging System (Invitrogen) with 20X objective lenses. pSILAC

[0598] DMEM for SILAC (Gibco) was supplemented with dialyzed Fetal Bovine Serum (FBS) (Gibco). The light media was supplemented with L-Lysine-2HC1 for SILAC (Thermo) and L- Arginine-HCl for SILAC (Gibco). The heavy media was supplemented with L-Lysine-2HC1, for SILAC (Thermo) and L-Arginine-HCl, ¹³ C6, ¹⁵ N4 for SILAC (Thermo). SW1417 cells were seeded at a density of 2xl0 ⁶ cells per 60mm dish in light media (+Light Arg/Lys) for 24 hours. Cells were treated with DMSO and the RMA compound of the invention (20uM) for 1 hour in DMEM for SILAC with dialyzed FBS (-Arg/Lys), followed by a wash with warm heavy media (+Heavy Arg/Lys). Cells in both conditions were incubated in +Heavy Arg/Lys media for a total of 6 and 24 hours. Subsequently, cells were scrapped off, collected in a 1.5mL tube, and centrifuged at 500g for 5 minutes. Cell pellets were washed twice with PBS, centrifuged at 500g for 5 minutes, and stored at -20C until ready to be processed for Mass Spectrometry.

[0599] Mass Spectrometry

[0600] Cell pellets were lysed in 5% SDS, 50 mM tri ethyl ammonium bicarbonate (TEAB) pH 8.5, and protein concentration was determined using the BCA protein assay. Samples were reduced in 10 mM dithiothreitol for 30 min at 37°C followed by alkylation in 20 mM iodoacetamide for 20 min at room temperature. Samples were acidified and digested on an S- Trap™ spin column (Profiti) by addition of trypsin at 1 :20 for 3 h at 47°C. Peptides were eluted in 50 mM TEAB pH 8.0, 0.2% formic acid and 50% acetonitrile, respectively.

2ug of protein per sample were used for LC-MS/MS analysis. The raw mass spectrometric data were processed in MaxQuant (version 2.0.3.0) for peptide and protein identification. More detailed protocol in the supplementary section. [0601] Mass Spectrometry - Supplementary

[0602] LC-MS/MS Analysis: Dried peptides were resuspended in 0.1% formic acid and then analysed by online nanoflow LC-MS/MS using an Orbitrap Exploris 240 mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLCnano (Thermo Scientific). Peptides (equivalent to 2pg protein) were injected on an Acclaim PepMap 100 C18 LC trap column (100 pm ID x20 mm, 5 pm, 100 A) followed by separation on an EASY-Spray nanoLC C18 column (75 pm ID ><500 mm, 2 pm, 100 A) at a flow rate of 250nl/min. Buffer A contained 0.1% formic acid in water and Buffer B contained 0.1% formic acid in acetonitrile. Peptides were separated with a linear gradient of 2-37% Buffer B over 213 min followed by a step from 37 to 80% Buffer B in 1 min; then, the column was washed with 80% Buffer B and re-equilibrated to 2% Buffer B to complete the 240 min run. The Orbitrap Exploris 240 was operated in positive-ion data-dependent mode. Precursor ion (MSI) scans were performed in the Orbitrap mass analyzer in the range of 350-1,400 m/z, with a resolution of 120,000 (at 200 m/z). Precursor ions were isolated using a quadrupole mass filter with an isolation width of 1.6 m/z and fragmented using higher energy collision dissociation (HCD) with a collision energy of 30%. MS/MS fragment ions were analysed in the Orbitrap mass analyser with a resolution of 15,000 (at 200 m/z). The database search was performed using the Homo sapiens reference proteome from UniProtKB. Oxidation of methionine and acetylation of protein N-terminus were allowed as variable modifications, while carbamidomethylation of cysteine was allowed as a fixed modification. The estimated false discovery rate was set to 1% at the peptide and protein levels. A maximum of two missed cleavages were allowed. Reverse hits, contaminants, and proteins only identified by site were removed before further analysis. Protein groups were further filtered to contain at least two unique peptides. Hypothesis testing was performed in R (version 4.1.0) using the Limma package available in the R/Bioconductor repository and p-values were corrected for multiple hypothesis testing using the Benjamini -Hochberg method. Proteins with a corrected p-value less than 0.05 were considered significantly differential.

Immunoblotting

[0603] Assay performed as in (27). Specific modifications are detailed in the supplementary section.

Immunoblotting - Supplementary

[0604] Cells were plated at a density of 0.75xl0 ⁶ cells/60mm dish. After 48 hours, cells were treated with either DMSO or the RMA compound of the invention at specified concentrations for specified time periods. Following treatment, cells were lysed with 250mL of RIP A buffer (RIPA lysis and Extraction Buffer, Thermo Scientific) supplemented with IX protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), Thermo Scientific) and nuclease (1 : 1000, Universal Nuclease, Thermo Scientific). Proteins were separated on 4-12% gradient gels (NuPAGE Bis-Tris protein gels, Invitrogen) using SDS running buffer (NuPAGE MES SDS Running Buffer, Invitrogen). Primary antibodies: RPL11 (18163, Cell Signaling Technology, 1: 1000), RPS11 (ab 175213, Abeam, 1 :1000), RPS21 (PA5-51914, Invitrogen, 1: 1000), RPS29 (PA5-41744, Invitrogen, 1: 1000), Fibrillarin (2639, Cell Signaling Technology, 1: 1000), p21 Wafl/Cipl (2947, Cell Signaling Technology, 1: 1000), p2-microglobulin (12851, Cell Signaling Technology, 1 :5000), and PSMD14 (4197, Cell Signaling Technology, 1 :5000).

Cell cycle analysis

[0605] SW1417 cells were plated at a density of O. lxlO ⁶ cells/well in a 12-well plate, and COLO320DM cells were plated at a density of IxlO ⁶ cells/well in a 6-well plate. After 24 hours, cells were treated with DMSO or the RMA compound of the invention at various concentrations for 48 hours. Following treatment, cell pellets were washed twice with IX PBS, and then fixed with Fixation buffer (BioLegend) for 15 minutes at room temperature. The pellets were then washed with Staining buffer (BioLegend) and then permeabilized with IX Click-iT™ permeabilization and wash reagent (ThermoFisher) for 15 minutes at room temperature. The cells were then stained with DNA-specific dye FxCycle™ Violet Stain (Invitrogen) for 30 minutes and analyzed using the BD FACSCelesta™ Cell Analyzer (flow cytometer).

Organoid Culture

[0606] 14 CRC-derived (CMS2) organoids and 1 normal organoid from the Hubrecht Organoid Technology (HUB) were thawed, expanded, and screened following HUB’s guidelines and using established formulated media. For cell viability screening assay, organoids were passaged and seeded at high density. Cell handling and treatment conditions are detailed in the supplementary section.

Organoid Culture - Supplementary

[0607] After one day of expansion, organoids were harvested by adding Dispase at a final concentration of 1 mg/mL to the culture medium and incubating at 37°C to digest Matrigel (MG)/Basement Membrane Extract (BME). Then, organoids were collected and size-selected between 40 and 100pm using cell strainers. 250 organoids were dispensed/well in a total volume of 40pL of organoid culture medium (with 5% extracellular matrix) in triplicates for all test conditions in ultra-low attachment (ULA) 384-well plates. Two plates were simultaneously seeded from the same organoid suspension, one for CellTiter-Glo measurement directly after plating (“Day 0”) and one to be measured 7 days after incubation with 9 doses in 3-fold dilutions of the RMA compound of the invention with 60uM as top concentration with normalization to 0.3% vehicle in all test wells. Immediately after plating, all compounds were dispensed on the “Day 7” assay plate using the Tecan D300. Staurosporine was used at 2pM as a positive control for cell death in all “Day 7” assay plates. After 7 days of exposure to the RMA compound of the invention, luminescence was measured using Cell Titer-Gio 3D on a Tecan Spark 10M plate reader. IC50 values were calculated using the GRmetrics package.

Bioinformatic analysis - Supplementary

[0608] Amino Acid Residue Charge Analysis: Protein amino acid sequences were downloaded from Uniprot (Homo sapiens & reviewed_yes) and were cross referenced with mass spectrometry data using the uniport protein ID. Proteins from differential expression analysis with adjusted p value less than 0.05 were analyzed for amino acid sequence charge distributions and gene set enrichment. To investigate consecutive positively charged amino acid sequences effect on ribosomal translation, we focused on positively charged amino acids only, assigning lysine (K) and arginine (R) a positive 1 charge and all other amino acid residues a charge of 0. Average charge values were then calculated along a sliding window 10 amino acids long for each protein amino acid sequence (starting at the first residue and ending at the last complete set of 10 residues). The distribution of average charge windows for all proteins were binned into groups of X<=1, 1<X<=2, 2<X<=3, and 3<X. Geneset enrichment analysis was conducted using the over representation analysis method and an unranked list of significantly downregulated genes from the mass spectrometry experiment. Genesets were obtained from MsigDB using the clusterProfiler package in R. For each geneset and subcategory (Hallmark, CP, GO, CGP, C6, and Cl), over representation analysis was run and results pooled for visualization.

[0609] The ClusterProfiler that was used can be found in: ClusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters. Guangchuang Yu, Li-Gen Wang, Yanyan Han, and Qing-Yu He. OMICS: A Journal of Integrative Biology 2012 16:5, 284-287. (dx.doi.org/10.1089/omi.2011.0118 (last visited 6/14/2023). [0610] ORA: GO::TermFinder — open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Elizabeth I. Boyle, Shuai Weng, Jeremy Gollub, Heng Jin, David Botstein, J. Michael Cherry, Gavin Sherlock. Bioinformatics, Volume 20, Issue 18, December 2004, Pages 3710— 3715. (doi.org/10.1093/bioinformatics/bth456).

[0611] Drug sensitivity cutoffs: Cell lines were grouped into sensitive, intermediate, and resistant groups using empirically derived cutoffs on GI50 and Amax drug response metrics. Expression and CNV data for CRC cell lines were downloaded from the CCLE (DepMap 22Q1; https://depmap.org/). Consensus molecular subtypes (CMS) for colorectal cancer cell lines were computed using the genes associated with each of the subtypes from Guinney et al (Reference). Gene signature scores were computed for each subtype by taking the average z-scaled gene expression for the genes associated with those subtypes. Subtypes were assigned to cell lines based on the highest gene signature score. Chr20ql 1-13 CNV ratios were computed by taking the average of the CNV ratios for genes in this chromosomal segment. Genes were mapped to their chromosomal segments using the latest stable ensemble genome release containing chromosomal band annotations (GRCh37). Chromosomal segment scores were generated for each cell line by taking the average CNV ratios across genes mapping to chromosomal segments Chr20ql l, Chr20ql2, and Chr20ql3. Cell lines having high chromosomal segment scores are indicative of amplified segments while low scores represent segment deletions. Visualizations and statistical analyses were performed using R 4.1.2 version. Packages used: tidyverse 1.3.1, data.table 1.14.2, ComplexHeatmap 2.10.0, dittoSeq 1.6.0.

Software Analysis

[0612] GraphPad Prism 9 was used to plot percent response dose curves and bar graphs, calculate GI50 and LD50 values, and plot bar graphs for protein intensities.

[0613] Image Studio 5.2 was used to acquire images and quantitate intensities for from Smear and Western blot membranes.

[0614] Spotfire (TIBCO) 12.0.0.223 was used to plot volcano plots, bar graphs, pie chart, and scatter plot.

[0615] Fiji 2.6.0 image processing package software was used to analyze in-cell metabolic labeling (AHA) assay.

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Example

A novel class of Ribosome Modulating Agents (RMAs) targets ribosome heterogeneity in a subset of Small Cell Lung Cancers

[0616] Small cell lung cancer (SCLC) has a very poor prognosis and limited treatment options. Genomic studies in SCLC show that MYC isoforms are key oncogenic drivers in most of SCLC tumors. As a result, these tumors tend to have high proliferation and protein translation rates. We synthesized ribosome modulating agents (RMAs), a novel class of macrolides, capable of selectively inhibiting translation of a subset of molecules enriched for ribogenesis and translation-related proteins in sensitive SCLC cell lines. A second RMA representative compound of the invention which is disclosed herein selectively inhibits growth and induces apoptosis in a subset of SCLC cell lines characterized by high MYC activity and high ribogenesis rates. Furthermore, the second representative RMA compound is synergistic with current SCLC standard of care DNA intercalating drugs. The dependency of MYC-driven cancers on abnormally high protein translation rates can therefore be exploited by our RMAs presenting a novel therapeutic opportunity for this recalcitrant disease with high unmet needs. [0617] FIG.7.1 summarizes the approach we employed in identifying RMAs.

[0618] FIGS. 7.2A and B summarize clinical subtypes in solid and hematological cancers that are highly sensitive to the second representative RMA of the invention.

[0619] According to FIGS. 7.3, forty five percent of small cell lung cancer cell lines are highly sensitive to the second representative RMA compound of the invention.

[0620] According to FIGS. 7.4A-C, this sensitivity is associated with high expression of ribogenesis, protein translation, and MYC pathway genes.

[0621] FIGS. 7.5A-C indicate that the anti -proliferative effect of RMAs is driven by selective inhibition of new protein synthesis

[0622] FIGS. 7.6A-B show that proteins with higher positively charged regions are more sensitive to translation inhibition by RMAs.

[0623] FIGS. 7.7 indicates that decreased proteins show a large impact on ribogenesis and protein translation machinery.

[0624] FIG.7.8 indicates that the second representative compound of the invention induces apoptosis in sensitive cells.

[0625] FIG.7.9 shows that the second representative compound of the invention selectively inhibits SCLC-patient derived organoids (PDOs).

[0626] FIG.7.10 shows that the second representative compound of the invention demonstrates combination synergy with DNA targeting chemotherapeutics known to inhibit tRNA synthesis.

[0627] FIG.7.11 summarizes the multiple levels of selectivity of allosteric inhibition by the RMAs of the invention.

Conclusion.

[0628] Several lung cancer subtypes are sensitive to novel allosteric ribosome modulators.

Sensitivity in small cell lung cancer (SCLC) is associated with high expression of MYC target genes involved in Ribogenesis and protein translation. The anti-proliferative effect is driven by selective inhibition of ribogenesis and new protein synthesis. We have found that RMAs show additive and synergistic effects with major chemotherapy backbone agents.

Equivalents and Scope

[0629] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into one claim. For example, any claim that is dependent on one claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g, in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set in verbatim herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0630] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0631] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.