ANTIBACTERIAL AMINOGLYCOSIDE ANALOGS

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Contract No. HHSN272200800043C, awarded by the National Institutes of Health, an agency of the United States Department of Health and Human Services. The government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.

Provisional Patent Application No. 61/250,104 filed October 9, 2009. The foregoing application is incorporated herein by reference in its entirety.

BACKGROUND

Field

The present invention is directed to novel aminoglycoside compounds, and methods for their preparation and use as therapeutic or prophylactic agents. Description of the Related Art

A particular interest in modern drug discovery is the development of novel low molecular weight drugs that work by binding to RNA. RNA, which serves as a messenger between DNA and proteins, was thought to be an entirely flexible molecule without significant structural complexity. Recent studies have revealed a surprising intricacy in RNA structure. RNA has a structural complexity rivaling proteins, rather than simple motifs like DNA. Genome sequencing reveals both the sequences of the proteins and the mRNAs that encode them. Since proteins are synthesized using an RNA template, such proteins can be inhibited by preventing their production in the first place by interfering with the translation of the mRNA. Since both proteins and the RNAs are potential drug targeting sites, the number of targets revealed from genome sequencing efforts is effectively doubled. These observations unlock a new world of opportunities for the pharmaceutical industry to target RNA with small molecules.

Classical drug discovery has focused on proteins as targets for intervention. Proteins can be extremely difficult to isolate and purify in the appropriate form for use in assays for drug screening. Many proteins require post-translational modifications that occur only in specific cell types under specific conditions. Proteins fold into globular domains with hydrophobic cores and hydrophilic and charged groups on the surface. Multiple subunits frequently form complexes, which may be required for a valid drug screen. Membrane proteins usually need to be embedded in a membrane to retain their proper shape. The smallest practical unit of a protein that can be used in drug screening is a globular domain. The notion of removing a single alpha helix or turn of a beta sheet and using it in a drug screen is not practical, since only the intact protein may have the appropriate 3-dimensional shape for drug binding. Preparation of biologically active proteins for screening is a major limitation in classical high throughput screening. Quite often the limiting reagent in high throughput screening efforts is a biologically active form of a protein which can also be quite expensive.

For screening to discover compounds that bind RNA targets, the classic approaches used for proteins can be superceded with new approaches. All RNAs are essentially equivalent in their solubility, ease of synthesis or use in assays. The physical properties of RNAs are independent of the protein they encode. They may be readily prepared in large quantity through either chemical or enzymatic synthesis and are not extensively modified in vivo. With RNA, the smallest practical unit for drug binding is the functional subdomain. A functional subdomain in RNA is a fragment that, when removed from the larger RNA and studied in isolation, retains its biologically relevant shape and protein or RNA-binding properties. The size and composition of RNA functional subdomains make them accessible by enzymatic or chemical synthesis. The structural biology community has developed significant experience in identification of functional RNA subdomains in order to facilitate structural studies by techniques such as NMR spectroscopy. For example, small analogs of the decoding region of 16S rR A (the A-site) have been identified as containing only the essential region, and have been shown to bind antibiotics in the same fashion as the intact ribosome.

The binding sites on RNA are hydrophilic and relatively open as compared to proteins. The potential for small molecule recognition based on shape is enhanced by the deformability of RNA. The binding of molecules to specific RNA targets can be determined by global conformation and the distribution of charged, aromatic, and hydrogen bonding groups off of a relatively rigid scaffold. Properly placed positive charges are believed to be important, since long-range electrostatic interactions can be used to steer molecules into a binding pocket with the proper orientation. In structures where nucleobases are exposed, stacking interactions with aromatic functional groups may contribute to the binding interaction. The major groove of RNA provides many sites for specific hydrogen bonding with a ligand. These include the aromatic N7 nitrogen atoms of adenosine and guanosine, the 04 and 06 oxygen atoms of uridine and guanosine, and the amines of adenosine and cytidine. The rich structural and sequence diversity of RNA suggests to us that ligands can be created with high affinity and specificity for their target.

Although our understanding of RNA structure and folding, as well as the modes in which RNA is recognized by other ligands, is far from being comprehensive, significant progress has been made in the last decade {see, e.g., Chow, C.S.; Bogdan, F.M., Chem. Rev., 1997, 97, 1489 and Wallis, M.G.; Schroeder, R., Prog. Biophys. Molec. Biol. 1997, 67, 141). Despite the central role RNA plays in the replication of bacteria, drugs that target these pivotal RNA sites of these pathogens are scarce. The increasing problem of bacterial resistance to antibiotics makes the search for novel RNA binders of crucial importance.

Certain small molecules can bind and block essential functions of RNA. Examples of such molecules include the aminoglycoside antibiotics and drugs such as erythromycin which binds to bacterial rRNA and releases peptidyl-tRNA and mRNA. Aminoglycoside antibiotics have long been known to bind RNA. They exert their antibacterial effects by binding to specific target sites in the bacterial ribosome. For the structurally related antibiotics neamine, ribostamycin, neomycin B, and paromomycin, the binding site has been localized to the A-site of the prokaryotic 16S ribosomal decoding region RNA (see Moazed, D.; Noller, H.F., Nature, 1987, 327, 389). Binding of aminoglycosides to this RNA target interferes with the fidelity of mRNA translation and results in miscoding and truncation, leading ultimately to bacterial cell death (see Alper, P.B.; Hendrix, M.; Sears, P.; Wong, C, J. Am. Chem. Soc, 1998, 120, 1965).

There is a need in the art for new chemical entities that work against bacteria with broad-spectrum activity. Perhaps the biggest challenge in discovering RNA-binding antibacterial drugs is identifying vital structures common to bacteria that can be disabled by small molecule drug binding. A challenge in targeting RNA with small molecules is to develop a chemical strategy which recognizes specific shapes of RNA. There are three sets of data that provide hints on how to do this: natural protein interactions with RNA, natural product antibiotics that bind RNA, and man-made R As (aptamers) that bind proteins and other molecules. Each data set, however, provides different insights to the problem.

Several classes of drugs obtained from natural sources have been shown to work by binding to RNA or RNA/protein complexes. These include three different structural classes of antibiotics: thiostreptone, the aminoglycoside family and the macrolide family of antibiotics. These examples provide powerful clues to how small molecules and targets might be selected. Nature has selected RNA targets in the ribosome, one of the most ancient and conserved targets in bacteria. Since antibacterial drugs are desired to be potent and have broad-spectrum activity, these ancient processes, fundamental to all bacterial life, represent attractive targets. The closer we get to ancient conserved functions the more likely we are to find broadly conserved RNA shapes. It is important to also consider the shape of the equivalent structure in humans, since bacteria were unlikely to have considered the therapeutic index of their RNAs while evolving them.

A large number of natural antibiotics exist, these include the aminoglycosides, such as, kirromycin, neomycin, paromomycin, thiostrepton, and many others. They are very potent, bactericidal compounds that bind RNA of the small ribosomal subunit. The bactericidal action is mediated by binding to the bacterial RNA in a fashion that leads to misreading of the genetic code. Misreading of the code during translation of integral membrane proteins is thought to produce abnormal proteins that compromise the barrier properties of the bacterial membrane.

Antibiotics are chemical substances produced by various species of microorganisms (bacteria, fungi, actinomycetes) that suppress the growth of other microorganisms and may eventually destroy them. However, common usage often extends the term antibiotics to include synthetic antibacterial agents, such as the sulfonamides, and quinolines, that are not products of microbes. The number of antibiotics that have been identified now extends into the hundreds, and many of these have been developed to the stage where they are of value in the therapy of infectious diseases. Antibiotics differ markedly in physical, chemical, and pharmacological properties, antibacterial spectra, and mechanisms of action. In recent years, knowledge of molecular mechanisms of bacterial, fungal, and viral replication has greatly facilitated rational development of compounds that can interfere with the life cycles of these microorganisms.

At least 30% of all hospitalized patients now receive one or more courses of therapy with antibiotics, and millions of potentially fatal infections have been cured. At the same time, these pharmaceutical agents have become among the most misused of those available to the practicing physician. One result of widespread use of antimicrobial agents has been the emergence of antibiotic-resistant pathogens, which in turn has created an ever-increasing need for new drugs. Many of these agents have also contributed significantly to the rising costs of medical care.

When the antimicrobial activity of a new agent is first tested, a pattern of sensitivity and resistance is usually defined. Unfortunately, this spectrum of activity can subsequently change to a remarkable degree, because microorganisms have evolved the array of ingenious alterations discussed above that allow them to survive in the presence of antibiotics. The mechanism of drug resistance varies from microorganism to microorganism and from drug to drug. The development of resistance to antibiotics usually involves a stable genetic change, inheritable from generation to generation. Any of the mechanisms that result in alteration of bacterial genetic composition can operate. While mutation is frequently the cause, resistance to antimicrobial agents may be acquired through transfer of genetic material from one bacterium to another by transduction, transformation or conjugation.

For the foregoing reasons, while progress has been made in this field, there is a need for new chemical entities that possess antibacterial activity. Further, in order to accelerate the drug discovery process, new methods for synthesizing aminoglycoside antibiotics are needed to provide an array of compounds that are potentially new drugs for the treatment of bacterial infections. The present invention fulfills these needs and provides further related advantages.

BRIEF SUMMARY

In brief, the present invention is directed to novel aminoglycoside compounds, having antibacterial activity, including stereoisomers, pharmaceutically acceptable salts and prodrugs thereof, and the use of such compounds in the treatment of bacterial infections.

In one embodiment, compounds having the following structure (I) are provided:



8

;

each Ri and R2 is, independently, hydrogen or an amino protecting group;

each R3 is, independently, hydrogen or a hydroxyl protecting group; each R4, R ₅ , R ₇ and Rg is, independently, hydrogen or Ci-Ce alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each ¾ is, independently, hydrogen, halogen, hydroxyl, amino or Ci-Ce alkyl;

or R4 and R ₅ together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R ₅ and one Ri together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms, or R4 and one R¾ together with the atoms to which they are attached can form a carbocyclic ring having from 3 to 6 ring atoms, or R ₇ and Rs together with the atom to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms; each R is, independently, hydrogen, hydroxyl, amino or C1-C6 alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each Rio is, independently, hydrogen, halogen, hydroxyl, amino or C1-C alkyl;

or R ₉ and one io together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each R11 and R _[2 is, independently, C1-C6 alkyl or substituted CpCe alkyl;

each n is, independently, an integer from 0 to 4; and

Zi is hydrogen, halogen or -OR3.

In another embodiment, compounds having the following structure (II) are provided:

01)

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

Qi is -NR1R2, -NR1R11, -NRnRu or -OR ₃ ;

Q2 is optionally substituted alkyl,

each Rj and R2 is, independently, hydrogen or an amino protecting group;

each R ₃ is, independently, hydrogen or a hydroxyl protecting group; each R4, R ₅ , R ₇ and Rg is, independently, hydrogen or Ci-Ce alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each Re is, independently, hydrogen, halogen, hydroxyl, amino or Ci-Ce alkyl; or 4 and R5 together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R _s and one Re together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms, or 4 and one R« together with the atoms to which they are attached can form a carbocyclic ring having from 3 to 6 ring atoms, or R7 and Rs together with the atom to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each R is, independently, hydrogen, hydroxyl, amino or C1-C6 alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each Rio is, independently, hydrogen, halogen, hydroxyl, amino or Ci-Ce alkyl;

or R ₉ and one Rio together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each R11 and R12 is, independently, Ci-Q alkyl or substituted C1-C6 alkyl;

each n is, independently, an integer from 0 to 4; and

Z] is hydrogen, halogen or -OR3.

In another embodiment, a pharmaceutical composition is provided comprising a compound having structure (I) or (II), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, a method of using a compound having structure (I) or (II) in therapy is provided. In particular, the present invention provides a method of treating a bacterial infection in a mammal comprising administering to a mammal in need thereof an effective amount of a compound having structure (I) or (II), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof. In addition, the present invention provides a method of treating a bacterial infection in a mammal comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition comprising a compound having structure (I) or (II), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient. These and other aspects of the invention will be apparent upon reference to the following detailed description.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention may be practiced without these details.

Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is as "including, but not limited to".

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

"Amino" refers to the -N¾ radical.

"Cyano" refers to the -CN radical.

"Hydroxy" or "hydroxyl" refers to the -OH radical.

"Imino" refers to the =NH substituent.

"Nitro" refers to the - O2 radical.

"Oxo" refers to the =0 substituent.

"Thioxo" refers to the =S substituent.

"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from one to twelve carbon atoms (C1-C12 alkyl), preferably one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-C ₆ alkyl), and which is attached to the rest of the molecule by a single bond, e.g. , methyl, ethyl, «-propyl, 1-methylethyl (wo-propyl), n-butyl, ra-pentyl, 1 ,1-dimethylethyl (i-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta-l ,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted.

"Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e. , contains one or more double and/or triple bonds), and having from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, w-butenylene, propynylene, w-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

"Alkoxy" refers to a radical of the formula -ORa where Ra is an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.

"Alkylamino" refers to a radical of the formula -NHRa or -NRaR _a where each R _a is, independently, an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkylamino group may be optionally substituted.

"Thioalkyl" refers to a radical of the formula -SR where Ra is an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, a thioalkyl group may be optionally substituted. "Aryl" refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of this invention, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the term "aryl" or the prefix "ar-" (such as in "aralkyl") is meant to include aryl radicals that are optionally substituted.

"Aralkyl" refers to a radical of the formula -Rb-Rc where ¾ is an alkylene chain as defined above and Ro is one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically in the specification, an aralkyl group may be optionally substituted.

"Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted.

"Cycloalkylalkyl" refers to a radical of the formula -Rb d where ¾ is an alkylene chain as defined above and R _g is a cycloalkyl radical as defined above. Unless stated otherwise specifically in the specification, a cycloalkylalkyl group may be optionally substituted.

"Fused" refers to any ring structure described herein which is fused to an existing ring structure in the compounds of the invention. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.

"Halo" or "halogen" refers to bromo, chloro, fluoro or iodo. "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group may be optionally substituted.

"Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to

18-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, j octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclyl group may be optionally substituted.

'W-heterocyclyl" refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. Unless stated otherwise specifically in the specification, a ./V-heterocyclyl group may be optionally substituted. "Heterocyclylalkyl" refers to a radical of the formula -R _b R« where ¾ is an alkylene chain as defined above and ¾ is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom. Unless stated otherwise specifically in the specification, a heterocyclylalkyl group may be optionally substituted.

"Heteroaryl" refers to a 5- to 14-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[6][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, irnidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1- oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, l-phenyl-l//-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

"jV-heteroaryl" refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical. Unless stated otherwise specifically in the specification, an iV-heteroaryl group may be optionally substituted.

"Heteroarylalkyl" refers to a radical of the formula -R _b R _f where ¾ is an alkylene chain as defined above and R _f is a heteroaryl radical as defined above. Unless stated otherwise specifically in the specification, a heteroarylalkyl group may be optionally substituted.

The term "substituted" used herein means any of the above groups (i.e., alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, iV-heteroaryl and/or heteroarylalkyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, CI, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triatylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced with -NR _g R _h , -NR _g C(=0)Ri„ -NR _g C(=0)NRgRh, -NR _g C(=0)OR _h , -NRgC(= Rg)NRgR _h , -NR _g S0 ₂ R _h , -OC(=0)NR _g R _h , -OR _g , -SRg, -SOR _g , -S0 ₂ R _g , -OS0 ₂ R _g , -S0 ₂ OR _g , =NS0 ₂ R _g , and -S0 ₂ NRgR _h . "Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with -C(=0)R _g , -C(=0)OR _g , -C(=0)NR ₆ R _h , -CH ₂ S0 ₂ R _g , -CH ₂ S0 ₂ NR _g R _h . In the foregoing, Rg and R _h are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, Af-heterocyclyl, heterocyclylalkyl, heteroaryl, ΛΓ-heteroaryl and/or heteroarylalkyl. "Substituted" further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, 5 haloalkyl, heterocyclyl, iV-heterocyclyl, heterocyclylalkyl, heteroaryl, iV-heteroaryl and/or heteroarylalkyl group. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the above substituents.

The term "protecting group," as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation,

10 hydroxyl and amino groups, against undesired reactions during synthetic procedures.

Hydroxyl and amino groups which protected with a protecting group are referred to herein as "protected hydroxyl groups" and "protected amino groups", respectively. Protecting groups are typically used selectively and or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected

15 group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Groups can be selectively incorporated into aminoglycosides of the invention as precursors. For example an amino group can be placed into a compound of the invention as an azido group that can be chemically

20 converted to the amino group at a desired point in the synthesis. Generally, groups are protected or present as a precursor that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal, et al., Protocols for Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994; Vol.

25 26 pp. 1-72. Examples of "hydroxyl protecting groups" include, but are not limited to, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, l-(2- chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl (TBDPS), triphenylsilyl,

30 benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate. Examples of "amino protecting groups" include, but are not limited to, carbamate- protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), l-methyl-l-(4- biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl.

"Prodrug" is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term "prodrug" refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention. Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass all 5 pharmaceutically acceptable compounds of structure (I) or (II) being isotopically- labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as ² H, ¾ ^U C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ^l7 0, ¹⁸ 0, ³¹ P,

10 ³² P, ³ S, ¹⁸ F, ³⁶ C1, ¹²³ I, and ¹²⁵ I, respectively. These radiolabelled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds of structure (I) or (II), for example, those incorporating a radioactive isotope, are useful in

15 drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.

3H, and carbon- 14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example,

20 increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ⁿ C, ¹⁸ F, ^ls O and I ³ N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of structures (I) and (II)

25 can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

The invention disclosed herein is also meant to encompass the in vivo

30 metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabelled compound of the invention in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.

"Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

"Mammal" includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

"Optional" or "optionally" means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution.

"Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

"Pharmaceutically acceptable salt" includes both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene- 1, 5 -disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

"Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic 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, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol,

2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benetharaine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, iV-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Often crystallizations produce a solvate of the compound of the invention. As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the invention may be true solvates, while in other cases, the compound of the invention may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

A "pharmaceutical composition" refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

"Effective amount" or "therapeutically effective amount" refers to that amount of a compound of the invention which, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, of a bacterial infection in the mammal, preferably a human. The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. "Treating" or "treatment" as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

(ii) inhibiting the disease or condition, i. e. , arresting its development;

(iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centres of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.

A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds.

As noted above, in one embodiment of the present invention, compounds having antibacterial activity are provided, the compounds having the following structure (I):

(I)

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

Qi is -NR1R2, -NR1R11, -NR11R12 or -OR ₃ ;

Q ₂ is optionally substituted alkyl,



each Ri and R2 is, independently, hydrogen or an amino protecting group;

each R3 is, independently, hydrogen or a hydroxyl protecting group; each R4, R ₅ , R ₇ and R¾ is, independently, hydrogen or C1-C6 alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each Re is, independently, hydrogen, halogen, hydroxyl, amino or Q-C6 alkyl; or R4 and 5 together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R5 and one R together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms, or R and one Re together with the atoms to which they are attached can form a carbocyclic ring having from 3 to 6 ring atoms, or R7 and Re together with the atom to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each n is, independently, an integer from 0 to 4; and

Zi is hydrogen, halogen or -OR3.

In further embodiments, each Ri, R ₂ and R3 are H.

In further embodiments, Qi is -NH ₂ .

In further embodiments, Qi is -NHRn. In further embodiments, Rn is

C1-C alkyl, such as, for example, methyl or ethyl. In other further embodiments, Rn is substituted Ci-C ₆ alkyl, such as, for example, -(CH ₂ ) _m OH, wherein m is an integer from 1 to 6 (e.g., -(CH ₂ ) ₃ OH or -(CH ₂ ) ₂ OH).

In other further embodiments, Qi is -NRnRi ₂ .

In other further embodiments, Qi is -OH.

In further embodiments, Q ₂ is:

wherein: R ₄ is hydrogen; R5 is hydrogen; and n is an integer from 1 to 4. In further embodiments, each R6 is hydrogen. For example, in more specific embodiments of the foregoing, Q ₂ is:

In other further embodiments, at least one R^ is haJogen. For example, in more specific embodiments of the foregoing, Q2 is:

wherein each R _b is halogen (such as, for example, fluoro). in other further embodiments, at least one <s is hydroxyl. For example, in more specific embodiments of the foregoing, (¾ is:

In other further embodiments, Q ₂ is:

wherein: R ₄ is hydrogen; R _s and one ¾ together with the atoms to which they are attached form a heterocyclic ring having from 3 to 6 ring atoms; and n is an integer from 1 to 4. For example, in more specific embodiments of the foregoing, Qi is:

In other further embodiments, at least one ¾ is halog

In other further embodiments, <¾ is:

wherein: R4 and R ₅ together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms; and n is an integer from 1 to 4. In further embodiments, each ¾ is hydrogen. For example, in more specific embodiments of the foregoing, <¾ is:

In other further embodiments, at least one ¾ is halogen.

In other further embodiments, (¾ is:

wherein: R ₅ is hydrogen; 4 and one ¾ together with the atoms to which they are attached form a carbocyclic ring having from 3 to 6 ring atoms; and n is an integer from 1 to 4. For example, in more specific embodiments of the foregoing, Q2 is:

wherein: R4 is hydrogen; R ₇ is hydrogen; ¾ is hydrogen; and n is an integer from 1 to 4. In further embodiments, each ¾ is hydrogen. For example, in more specific embodiments of the foregoing, <¾ is:

wherein: R4 and one R _ft together with the atoms to which they are attached form a carbocyclic ring having from 3 Ιυ 6 ring atoms; R ₇ is hydrogen; Rs is hydrogen; and n is an integer from 1 to 4, For example, in more specific embodiments of the foregoing, Q? is:

In other further embodiments, at least one Re is halogen.

In other further embodiments, Q2 is:

wherein: R ₇ is hydrogen; and Rg is hydrogen. In further embodiments, each Re is hydrogen. For example, in more specific embodiments of the foregoing, <¾ is:

In other further embodiments, at least one ¾ is halogen.

In other further embodiments, (¾ is:

wherein R ₅ is hydrogen. In further embodiments, each R$ is hydrogen. In other further embodiments, at least one R$ is halogen.

In other further embodiments, Q2 is:

wherein: R ₇ is hydrogen; and R¾ is hydrogen. In further embodiments, each Re is hydrogen. In other further embodiments, at least one R$ is halogen.

In other further embodiments, Q2 is:

wherein R ₅ is hydrogen. In further embodiments, each R¾ is hydrogen. In other further embodiments, at least one « is halogen.

In other further embodiments, Q ₂ is:

wherein R ₉ is hydrogen. In further embodiments, each Rio is hydrogen. In other further embodiments, at least one Rio is halogen.

In other further embodiments, Q ₂ is:

wherein: R7 is hydrogen; and Re is hydrogen. In further embodiments, each Rio hydrogen. In other further embodiments, at least one Rio is halogen.

In other further embodiments, (¾ is:

wherein is hydrogen. In further embodiments, each R is hydrogen. In other further embodiments, at least one Re is halogen. In other further embodiments, Q2 is -C(=0)H.

In other further embodiments, (¼ is optionally substituted alkyl. For example, in more specific embodiments of the foregoing, Q2 is unsubstituted or Q ₂ is substituted with one or more halogen, hydroxyl or amino.

In further embodiments, Zi is H.

In other further embodiments, Zi is -OH.

In other further embodiments, Zi is halogen.

In further embodiments, the foregoing compounds of structure (I) have the following configuration:

In further embodiments, the foregoing compounds of structure (I) have the following configuration:

In other further embodiments, the foregoing compounds of structure (I) have the following

In other further embodiments, the foregoing compounds of structure (I) have the following configuration:

As also noted above, in another embodiment of the present invention, compounds having antibacterial activity are provided, the compounds having the following structure (II):

(Π)

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

Qi is -NR1R2, -NR ₁ R ₁₁ , -NRi ₁ R ₁₂ or -OR ₃ ; Q ₂ is optionally substituted alkyl,

each i and R ₂ is, independently, hydrogen or an amino protecting each R ₃ is, independently, hydrogen or a hydroxyl protecting group; each R4, R ₅ , R ₇ and Rg is, independently, hydrogen or C1-C6 alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each R6 is, independently, hydrogen, halogen, hydroxyl, amino or Ci-Ce alkyl;

or ) and R5 together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R ₅ and one R6 together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms, or R4 and one R¾ together with the atoms to which they are attached can form a carbocyclic ring having from 3 to 6 ring atoms, or R7 and Re together with the atom to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each R is, independently, hydrogen, hydroxyl, amino or C\-Ce alkyl optionally substituted with one or more halogen, hydroxyl or amino;

each Rio is, independently, hydrogen, halogen, hydroxyl, amino or Cj-Ce alkyl;

or R9 and one Rio together with the atoms to which they are attached can form a heterocyclic ring having from 3 to 6 ring atoms;

each R11 and R12 is, independently, C1-C6 alkyl or substituted C1-C6 alkyl;

each n is, independently, an integer from 0 to 4; and

Zi is hydrogen, halogen or -OR3.

In further embodiments, each R _r , R ₂ and R3 are H.

In further embodiments, Qi is -NH2.

In further embodiments, Qi is -NHRn. In further embodiments, Rn is C1-C6 alkyl, such as, for example, methyl or ethyl. In other further embodiments, Rn is substituted C1-C6 alkyl, such as, for example, -(CH2) _m OH, wherein m is an integer from 1 to 6 (e.g., -(CH ₂ ) ₃ OH or -(CH ₂ ) ₂ OH).

In other further embodiments, Qi is -N n .

In other further embodiments, Qi is -OH.

In further embodiments, Q ₂ is:

wherein: R4 is hydrogen; Rs is hydrogen; and n is an integer from 1 to 4. In further embodiments, each R _o is hydrogen. For example, in more specific embodiments of the foregoing, Q ₂ is:

Tn other further embodiments, at least one R is halogen. For example, in more specific embodiments of the foregoing, Q ₂ is:

wherein each R ₆ is halogen (such as, for example, fluoro). In other further embodiments, at least one is hydroxyl. For example, in more specific embodiments of the foregoing, is:

In other further embodiments, Q ₂ is:

l U wherein: K is hydrogen; R ₅ and one R ₆ together with the atoms 10 which they arc attached form a heterocyclic ring having from 3 to 6 ring atoms; and n is an integer from 1 to 4. For example, in more specific embodiments of the foregoing, Q ₂ is:

In other further embodiments, at least one ¾ is halogen.

In other further embodiments, (¾ is:

wherein: R4 and R ₅ together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms; and n is an integer from 1 to 4. In further embodiments, each ¾ is hydrogen. For example, in more specific embodiments of the foregoing, (¾ is:

In other further embodiments, at least one ¾ is halogen.

In other further embodiments, <¾ is:

wherein: Rs is hydrogen; R4 and one R _¾ together with the atoms to which they are attached form a carbocyclic ring having from 3 to 6 ring atoms; and n is an integer from 1 to 4. For example, in more specific embodiments of the foregoing, Q ₂ is:

wherein: R4 is hydrogen; R7 is hydrogen; Rg is hydrogen; and n is an integer from 1 to 4. In further embodiments, each ¾ is hydrogen. For example, in more specific embodiments of the foregoing, ⁽ ¾ is:

wherein: R ₄ and one ¾ together with the atoms to which they are attached form carbocyclic ring having from 3 to 6 ring atoms; R ₇ is hydrogen; Rg is hydrogen; and n i an integer from 1 to 4. For example, in more specific embodiments of the foregoing, Q is:

In other further eniborii merits, at least one R<, is halogen.

In other further embodiments, Q2 is: o r jH

wherein: R ₇ is hydrogen; and R _H is hydrogen. Tn further embodiments, each Rg is hydrogen. For example, in more specific embodiments of the foregoing, Q ₂ is:

In other further embodiments, at least one R is halogen.

In other further embodiments, <¾ is:

wherein 5 is hydrogen. In further embodiments, each 6 is hydrogen. In other further embodiments, at least one is halogen.

In other further embodiments, Q2 is:

wherein: R ₇ is hydrogen; and Rg is hydrogen. In further embodiments, each e is hydrogen. In other further embodiments, at least one R is halogen.

In other further embodiments, (¾ is:

wherein R ₅ is hydrogen. In further embodiments, each R¾ is hydrogen. In other further embodiments, at least one R^ is halogen.

In other further embodiments, Q2 is:

wherein R9 is hydrogen. In further embodiments, each io is hydrogen. In other further embodiments, at least one Rio is halogen.

In other further embodiments, (¾ is:

wherein: R ₇ is hydrogen; and Rg is hydrogen. In further embodiments, each Rio hydrogen. In other further embodiments, at least one Rio is halogen.

In other further embodiments, <¾ is:

wherein R4 is hydrogen. In further embodiments, each R« is hydrogen. In other further embodiments, at least one R« is halogen. In other further embodiments, Q2 is -C(=0)H.

In other further embodiments, (¾ is optionally substituted alkyl. For example, in more specific embodiments of the foregoing, (¾ is unsubstituted or Q2 is substituted with one or more halogen, hydroxyl or amino.

In further embodiments, Zi is H.

In other further embodiments, Zi is -OH.

In other further embodiments, Z) is halogen.

In further embodiments, the foregoing compounds of structure (II) have the following configuration:

In other further embodiments, the foregoing compounds of structure (II) have the following

In further embodiments, the foregoing compounds of structure (II) have the following configuration:

In other further embodiments, the foregoing compounds of structure (II) following

In other further embodiments, the foregoing compounds of structure (II) have the following configuration:

It is understood that any embodiment of the compounds of structures (I) and (II), as set forth above, and any specific substituent set forth herein for a Qi, (¾, Ri, R2, R3, R4, R5, R6, R7, Rs, ¾ Rio, R11, R12 and Zi group in the compounds of structures (I) and (II), as set forth above, may be independently combined with other embodiments and/or substituents of compounds of structures (I) and (II) to form embodiments of the inventions not specifically set forth above. In addition, in the event that a list of substitutents is listed for any particular Qi, Q ₂ , Ri, R2, R3, Rt, Rs, Re, R7, Rs, R9, Rio, Ru, R12 and Z[ in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the particular embodment and/or claim and that the remaining list of substituents will be considered to be within the scope of the invention.

For the purposes of administration, the compounds of the present invention may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention comprise a compound of structure (I) or (II) and a pharmaceutically acceptable carrier, diluent or excipient. The compound of structure (I) or (II) is present in the composition in an amount which is effective to treat a particular disease or condition of interest - that is, in an amount sufficient to treat a bacterial infection, and preferably with acceptable toxicity to the patient. The antibacterial activity of compounds of structures (I) and (II) can be determined by one skilled in the art, for example, as described in the Examples below. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

Compounds of the present invention possess antibacterial activity against a wide spectrum of gram positive and gram negative bacteria, as well as enterobacteria and anaerobes. Representative susceptible organisms generally include those gram positive and gram negative, aerobic and anaerobic organisms whose growth can be inhibited by the compounds of the invention such as Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter, Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus, Brucella, Francisella, Anthracis, Yersinia, Corynebacterium, Moraxella, Enterococcus, and other organisms.

Admimstration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention can be prepared by combining a compound of the invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this invention.

A pharmaceutical composition of the invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.

When intended for oral administration, pharmaceutical compositions of the present invention typically are either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical compositions may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. Pharmaceutical compositions of the invention may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, pharmaceutical compositions of the invention typically contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

Liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage will be obtained.

Pharmaceutical compositions of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may mclude a transdermal patch or iontophoresis device.

Pharmaceutical compositions of the invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

Pharmaceutical compositions of the invention may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.

Alternatively, the active ingredients may be encased in a gelatin capsule.

Pharmaceutical compositions of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome.

Pharmaceutical compositions of the invention may be prepared in dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.

One skilled in the art, without undue experimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a compound of the invention with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

The compounds of the invention, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.

Compounds of the invention, or pharmaceutically acceptable derivatives thereof, may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of the compound of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, a compound of the invention and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Where separate dosage formulations are used, the compounds of the invention and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in the synthetic processes described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. As described above, suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, i-butyldimethylsilyl, t- butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like, and suitable protecting groups for amino, amidino and guanidino include f-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although a protected derivative of compounds of this invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". All prodrugs of compounds of this invention are included within the scope of the invention.

Furthermore, compounds of the invention which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the invention can be converted to their free base or acid form by standard techniques.

The following Examples illustrate various methods of making compounds of this invention, i.e., compound of structures (I) and (II):

(II)

wherein Q _b Q ₂ , Ri, R2, R3 and Zi are as defined above. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of structures (I) and (II) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described herein.

As illustrated in the following Examples, compounds of the invention may be made according to methods using an intermediate compound having the following structure (INT-I):

(INT-1)

wherein:

each Ri is, independently, an amino protecting group; each R ₃ is, independently, a hydroxyl protecting group; and each A is, independently, phenyl, optionally substituted with one or more halogen, hydroxyl, amino or C _! -C ₆ alkyl optionally substituted with one or more halogen, hydroxyl or amino.

In more specific embodiments of the foregoing, the intermediate compound is, for example:

It has been found that intermediate compounds of structure (ΓΝΤ-Ι) are useful for the selective modification of neomycin derivatives at the 3 '-position.

The following examples are provided for purposes of illustration, not

EXAMPLES

GENERAL SYNTHETIC SCHEMES

Scheme 1

N-1 Substituted. 3'-Epi Neomycin Analoes

Scheme 2

N-l Substituted. 3'. 4"' Bis-Epi Neomycin Analogs

Scheme 3

N-1 Substituted. 4'-Deoxy. 3'-Epi Neomycin Analogs

Scheme 4

N-1 Substituted. 3', 4' Bis-Epi Neomycin Analogs

Scheme 5

Ν-6'. N-1 Bis-Substituted. 3'-Epi. 4'-Deoxy Neomycin Analogs

Scheme 6

N-6\ N-1 Bis-Substituted, 3'-Epi Neomycin Analogs

5

Scheme 7

N-1 Substituted. 3'-Deoxy. 4'-Epi Neomycin Analogs

Scheme 8

N-6'. N-l Bis-Substituted, 3'-Deoxy, 4'-Epi Neomycin Analogs

Example A N-1 Acylation

Example B N-1 Epoxide Opening

Example C N-1 Sulfonylation

10

Example D N-1 Reductive Amination

Example E N-6' Reductive Amination

Example F N-6' Epoxide Opening

Example G N-6' Amination

REPRESENTATIVE COUPLING REAGENTS

Representative N-1 Coupling Reagents

Representative N-6' Coupling Reagents

GENERAL SYNTHETIC PROCEDURES

Procedure 1 : Reductive Animation

Method A: To a stirring solution of the aminoglycoside derivative (0.06 mmol) in MeOH (2 mL) was added the aldehyde (0.068 mmol), silica supported cyanoborohydride (0.1 g, 1.0 mmol/g), and the reaction mixture was heated by microwave irradiation to 100°C (100 watts power) for 15 minutes. The reaction was checked by MS for completeness, and once complete all solvent was removed by rotary evaporation. The resulting residue was dissolved in EtOAc (20 ml), and washed with 5% NaHCC (2 5 mL), followed by brine (5 mL). The organic phase was then dried over Na ₂ S0 , filtered and the solvent was removed by rotary evaporation.

Method B: To a solution of aminoglycoside derivative (0.078 mmol) in DMF (1 ml) were added 3A molecular sieves (15-20), followed by the aldehyde (0.15 mmol) and the reaction was shaken for 2.5 hours. The reaction was checked by MS for completeness and, if needed, more aldehyde (0.5 eq) was added. The reaction mixture was then added dropwise to a stirring solution of NaB¾ (0.78 mmol) in MeOH (2 mL) at 0°C, and the reaction was stirred for 1 hour. The reaction was diluted with H ₂ 0 (2 mL) and EtOAc (2 ml). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 3 mL). The combined organic layers were dried over Na ₂ S04, filtered and concentrated to dryness.

Procedure 2: Boc deprotection (ferf-butyl dimethyl silyl protecting group is removed under these conditions)

Important: Before Boc deprotection a sample must be dried well by pumping at high vacuum for 3 h.

Method A: To a stirring solution of the Boc protected aminoglycoside (0.054 mmol) in DCM or MeOH (1 mL) were added 3 A molecular sieves (4-6), and trifluoroacetic acid (0.6 mL). The reaction was stirred at room temperature for 1 h, and checked for completeness by MS. Upon completion the reaction mixture was diluted with ether (15 mL) to induce precipitation. The vial was centrifuged and the supernatant was decanted. The precipitate was washed with ether (2 x 15 ml), decanted and dried under vacuum. Procedure 3: PvBOP coupling

To a stirring solution of aminoglycoside derivative (0.078 mmol) in DMF (1 mL) at -40°C was added the acid (0.16 mmol), followed by PyBOP (0.16 mmol) and DIPEA (0.31 mmol) and the reaction was stirred. The reaction mixture was diluted with EtOAc (3 mL) and H ₂ 0 (3 mL), and the aqueous layer was separated and extracted with EtOAc (3 x 3 mL). The combined organic layers were dried over Na2SC> , filtered and concentrated to dryness.

Procedure 4: Epoxide Opening

To a stirring solution of the aminoglycoside derivative (0.06 mmol) in

MeOH (2 mL) was added the epoxide (0.07 mmol), LiC10 ₄ (0.15 mmol), and the reaction mixture was heated by microwave irradiation to 100°C for 90 minutes. The reaction progress was monitored by MS. Upon completion, the solvent was removed by rotary evaporation. The resulting residue was dissolved in EtOAc (20 mL), washed with ¾0 (2 5 mL) and brine (5 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to dryness.

Procedure 5: Phthalimido deprotection

To a stirring solution of the phthalimido protected aminoglycoside (0.064 mmol) in EtOH (3 mL) was added hydrazine (0.32 mmol), and the reaction mixture was heated to reflux for 2 h. The reaction progress was monitored by MS. Upon cooling to room temperature, the cyclic by-product precipitated and was removed by filtration. The filtrate was concentrated to dryness to yield a residue, which was dissolved in EtOAc (20 mL), washed with 5% NaHC0 ₃ (2 x 5 mL) and brine (5 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to dryness.

Procedure 6: Sulfonylation

To a stirring solution of the aminoglycoside (0.067 mmol) in DCM (3 mL) was added DIPEA (0.128 mol) and the sulfonyl chloride (0.07 mmol). The reaction mixture was stirred at room temperature and its progress was monitored by MS. Once complete, the solvent was removed by rotary evaporation and the residue was dissolved in ethyl acetate (20 mL), washed with 5% aHC03 (2 5 mL) and brine (5 mL), dried overNa ₂ S04, filtered and concentrated to dryness. Procedure 7: N-Boc Protection To a stirring solution of the amine (4.64 mmol) in THF (10 mL) was added IN NaOH (10 mL), followed by Boc-anhydride (5.57 mmol) and the reaction progress was checked by MS. Once complete, the THF was removed by rotary evaporation and water (40 mL) was added. The aqueous phase was separated and extracted with Εί ₂ 0 (2 x 30 ml). The aqueous phase was acidified to pH 3 by the addition of dilute H3PO4 and was then extracted with EtOAc (2 x 60 ml). The combined organic layers were washed with H ₂ 0 (2 x 30 mL) and brine (30 mL), dried over NaiSO^ filtered and concentrated to dryness. Procedure 8: Syntheses of Epoxides

To a stirring solution of the alkene (5.16 mmol) in chloroform (20 mL) at 0°C was added m-chloroperbenzoic acid (8.0 mmol) and the reaction mixture was stirred for 30 minutes at 0°C and was then allowed to warm to room temperature. The reaction progress was monitored by MS and TLC, and additional portions of m-CPBA were added as needed. Upon completion, the reaction mixture was diluted with chloroform (50 mL) and washed with 10% aq. Na ₂ S0 ₃ (2 x 30 mL), 10% aq. NaHC0 ₃ (2 x 50 mL) and brine (50 mL). The organic layer was dried over Na ₂ S(¾, filtered and concentrated to yield a crude product, which was purified by flash choromatography (silica gel/hexanes: ethyl acetate 0-25%).

Procedure 9: General Procedure for Synthesis of q-hydroxy carboxylic acids

Step # 1. O-(Trimethylsilyl cvanohvdrines: A 50-mL flask equipped with a magnetic stirring bar and drying tube was charged with the ketone or aldehyde (0.010 mmol), followed by THF (50 mL), trimethylsilyl cyanide (1.39 g, 14 mmol), and zinc iodide (0.090 g, 0.28 mmol), and the reaction mixture was stirred at room temperature for 24 hr. Solvent evaporation gave a residue, which was dissolved in EtOAc (60 mL), washed with 5% aq. NaHC0 ₃ (2 x 30 mL), H ₂ 0 (30 mL), and brine (30 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to dryness to yield a crude, which was carried through to the next step without further purification. Step # 2. Acid hydrolysis to a-hvdroxy carboxylic acid: AcOH (25 ml) and cone. HC1 (25 ml) were added to the unpurified material from step #1 and the reaction mixture was refluxed for 2-3 hr. The reaction mixture was then concentrated to dryness to give a white solid, which was carried through to the next step without further purification.

Step # 3. Boc protection: To a stirring solution of solid from step #2 in 2 M NaOH (20 mL) and i-PrOH (20 mL) at 0°C was added Boc ₂ 0 (6.6 g, 3 mmol) in small portions, and the reaction mixture was allowed to warm to room temperature over 4 h. i-PrOH was then evaporated, and ¾0 (50 mL) was added, and the aqueous phase was separated and extracted with Et ₂ 0 (2 x 30 ml). The aqueous layer was acidified to pH 3 by addition of dilute H ₃ P0 ₄ and was extracted with EtOAc (2 x 60 ml). The combined organic layers were washed with ¾0 (2 x 30 mL) and brine (30 mL), dried over Na ₂ S04, filtered and concentrated to yield the desired N-Boc-a-hydroxy carboxylic acids in 56-72% yield.

Procedure 10: Protection of Amine bv Fmoc Group

To a stirring solution of the amine (0.049 mol) in DCM (100 mL), was added DIPEA (16 mL, 0.099 mol) and the reaction mixture was cooled to 0°C. Fmoc- Cl (12.8 g, 0.049 mol) was then added portion-wise over several minutes, and the reaction was allowed to warm to room temperature for 2 hr. The organic layer was washed with water (2 x 50 mL) and brine (50 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to dryness to yield the Fmoc protected amine (90-95% yield).

Procedure 11 : Synthesis of Aldehydes via TEMPO/Bleach Oxidation

To a vigorously stirring solution of the alcohol (1.54 mmol) in DCM (4 mL) was added TEMPO (0.007 g, 0.045 mmol, 0.03 mol %) and a 2M aqueous KBr solution (75 mL, 0.15 mmol, 10 mol %) and the reaction mixture was cooled to -10°C. In a separate flask aHC03 (0.5 g, 9.5 mmol) was dissolved in bleach (25 mL, Chlorox 6.0% NaOCl) to yield a 0.78 M buffered NaOCl solution. This freshly prepared 0.78 M NaOCl solution (2.3 mL, 1.8 mmol, 117 mol %) was added to the reaction mixture over 5 min and the reaction was stirred for an additional 30 min at 0°C. The organic phase was separated and the aqueous layer was extracted with dichloromethane (2 4 mL). The combined organic layers were washed with 10% aq. Na2S2C>3 (4 mL), sat. aq. NaHC ⁽ ¾ (2 4 mL), brine (5 mL), dried over Na ₂ S0 ₄ and concentrated to dryness.

Procedure 12: Synthesis of alcohols via Borane Reduction

To a stirring solution of the acid (1.5 mmol) in THF (5 mL) at -10°C was slowly added 1.0 M BH ₃ -THF (2.98 mL, 2.98 mmol). The reaction mixture was stirred vigorously for an additional 3 min at -10°C, and was then allowed to warm to room temperature overnight. The reaction was quenched by the dropwise addition of a solution of HOAC/H2O (1 :1 v/v, 2.0 mL). The THF was removed by rotary evaporation and sat. aq. NaHC(¾ (15 mL) was added. The aqueous layer was extracted with DCM (3 x 5 mL) and the combined organic layers were washed with sat. aq. NaHCC>3 (2 x 5 mL), brine (10 mL), dried over Na2SC>4, filtered and concentrated to dryness.

Procedure 13: Ozonolvsis and Pinnick oxidation

The substrate olefin (0.5 to 0.75 mmol) was dissolved in DCM (30 mL) and the reaction was cooled to -78°C. Ozone was bubbled through until a blue color persisted (3 to 5 min), and the reaction was stirred for 1 hr. Argon was then bubbled through to remove excess ozone for 10 minutes. The reaction was further quenched by the addition of dimethyl sulfide (10 equiv.), and was stirred for 30 min with warming to rt. The solvent was reduced under vacuum to yield the crude aldehyde, which was dried under high- vacuum for 10 min, and used without further purification. To a stirring solution of the aldehyde in THF, ffluOH and H ₂ 0 (3:3:2, 10 mL), was added NaH ₂ P0 ₄ (4 equiv.) followed by 2-methyl-2-butene (10 equiv.) and sodium chlorite (2 equiv.), and the reaction was stirred for 4 hr. The reaction mixture was then added to sat. aq. NaCl (10 mL) and extracted with DCM (3x). The combined organic layers were dried over Na ₂ S0 ₄ , filtered and reduced under vacuum to yield a crude, which was purified by flash chromatography (silica gel, 0→ 0.5 or 1% MeOH/DCM). Procedure 14: PvBOP coupling

To a stirring solution of aminoglycoside derivative (0.137 mmol) in DMF (2 mL) at 0°C was added the acid (0.151 mmol, 1.1 eq), followed by PyBOP (0.164 mmol, 1.2 eq) and DIPEA (0.411 mmol, 3 eq) and the reaction was stirred (1-3 h) with warming to room temp until complete (by LC-MS). The reaction mixture was diluted with AcOH (0.2 mL) and was loaded directly onto an HPLC column (Method #3). Fractions were collected, neutralized with 1 M NH ₄ OH and concentrated. The residue was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na ₂ S0 ₄ , filtered and reduced under vacuum to yield the desired product.

Procedure 15: DCC coupling

To a stirring solution of the acid (0.15 mmol) and N-hydroxysuccinimide (0.15 mmol) in EtOAc (1.5 mL) was added Λ '-dicyclohexylcarbodiimide (0.15 mmol) and the reaction mixture was stirred for 1 hr. The resulting white suspension was filtered through cotton, washed with EtOAc (3 x 5 mL), and evaporated to dryness under vacuum to yield the activated ester. To a stirring solution of the activated ester in THF (1.5 mL) was added NaHC0 ₃ (1 mmol) followed by the aminoglycoside (0.138 mmol), and the reaction was stirred for 24 hr. The reaction mixture was quenched with sat. aq. NaHC0 ₃ and extracted with DCM (3 x 30 mL). The combined organic layers were dried over Na ₂ S0 ₄ , filtered and reduced under vacuum to yield a crude product, which was purified by column chromatography (silica gel, 0-100% Hexanes/ethyl acetate over 25 min at 18 mL/min); fractions containing the desired compound were combined and concentrated in vacuo to yield the desired product. Procedure 16: Hvdrogenolvsis with MeOH/TFA

To a stirring solution of aminoglycoside (0.08 mmol) in MeOH (10 mL) was added 20 % PdiOHyC (140 mg) followed by TFA (38 μΐ, 0.49 mmol) and the reaction was stirred under a hydrogen atmosphere until complete (by LC-MS). The reaction was filtered through a 0.45 μπι PVDF filter, concentrated (to 1 -2 mL) and then dripped into Et ₂ 0 (40 mL). The resulting precipitate was collected by centrifugation and dried under vacuum to yield the product as its TFA-salt.

Procedure 17: Hydro genolvsis in THF

To a stirring solution of aminoglycoside (0.15 mmol) in THF (4 mL), was added AcOH (108 L, 1.8 mmol), followed by 20 % Pd(OH) ₂ /C (140 mg) and the reaction was stirred under a hydrogen atmosphere for lh. Then H ₂ 0 (2 mL) was added and the reaction mixture was stirred for 1 h. Additional water (2 x 2 mL) was added and the reaction was stirred under a hydrogen atmosphere overnight. The reaction was filtered through a 0.45 μιη PVDF filter, was diluted with water (50 mL) and lyophilized to yield the product as its acetate salt.

Procedure 18: Hvdroeenolvsis in AcOH/H^O (4:11

To a stirring solution of aminoglycoside (0.2 mmol) in AcOH: H ₂ 0 (5 mL, 4:1 v/v) was added 20 % Pd(OH)2/C (400 mg) and the reaction was stirred under a hydrogen atmosphere overnight. The reaction was filtered through a 0.45 μπι PVDF filter, was diluted with water (50 mL) and lyophilized to yield the product as its acetate salt. Procedure 19: Sulfate salt swap

To a solution of the aminoglycoside salt (0.074 mmol) in H ₂ 0 (1 mL) was added 1 M NH4OH (~ 400 μί) to adjust the pH to 7-8, followed by ( H ₄ ) ₂ S0 ₄ (0.22 mmol, 3 eq.). The resulting solution was filtered through a 0.45 μπι PVDF filter, and the filtrate was dripped into vigorously stirring MeOH (40 mL). After 20 min the precipitate was collected by centrifugation and dried for 1 h under vacuum. The solid was dissolved in ¾0 (1 mL) and precipitated with MeOH (40 mL) a second time. The resulting precipitate was collected by centrifugation, dissolved in H ₂ 0 (3 mL) and lyophilized to yield the product as its sulfate salt. GENERAL PURIFICATION PROCEDURES

Method #1 : Purification by Basic Condition

Mobile Phases:

A - Water with 10 mM N¾OH

B - Acetonitrile with 10 mM NH4OH

Columns:

A: Waters-XBridge Prep Shield RP 18 Column

19x250 mm, 5μιη

Gradient: 20 min at 0%, then 0-20% in 200 min at a flow of 20 ml/min

B: Waters-XBridge Prep Shield RP18 Column

50 xlOO mm, 5μηι

Gradient: 20 min at 0%, then 0-20% in 200 min at a flow of 20 ml/min Method #2: Purification by Acidic Condition

Mobile Phases:

A - Water with 0.1 %TFA

B - Acetonitrile with 0.1% TFA

Columns:

A: Phenomenex Luna C18

21.4 x 250 mm, ΙΟμπι

Gradient: 0-100%, flow 25 ml/min

B: Phenomenex Luna C 18

50 x 250 mm, ΙΟμπι

Gradient: 0-100%, flow 45 ml min

Method #3: Purification by Acidic Condition

Mobile Phases:

A - Water with 0.1 %TFA

B - Acetonitrile with 0.1% TFA Columns: Varian Dynamax 250 x 41.4 mm,

Microsorb 100-8 CI 8

Gradient: 30-100% B over 70 min, flow 50 ml/min

UV detector 215 nm

Method #4: Purification by Basic Condition

Mobile Phases:

A - Water with 0.25 M NH ₄ OH

B - Acetonitrile with 0.25 M N¾OH

Column: Phenomemex Gemini-NX 150 x 21.2 mm,

10μηι Ο18 110A

Gradient: 0% B over 20 min, 0-10% B over 70 min, flow 15 ml/min UV detector 215 nm

Fractions containing the desired compound were combined and lyophilized. To a stirring solution of the aminoglycoside (0.02 -0.05 mmol) in ¾0 (0.5-1 mL) was added 1 M H2SO4 dropwise until pH = 1-2. The solution was filtered through a 0.45 μπι PVDF filter and the filtrate was dripped into vigorously stirring MeOH (25-30 mL). (Et ₂ 0 (10-15 mL) was added if needed to improve the quality of thr precipitate). After 20 min the solids were collected by centrifugation and washed with MeOH - Et ₂ 0 (1:1 v/v, 10 mL), followed by Et ₂ 0 (10 mL). The resulting precipitate was collected by centrifugation to yield the product as its sulfate salt.

REPRESENTATIVE INTERMEDIATES

AyV'-bis-Cbz-2(5)-hydroxy-4-guanidino-butyric acid

DIPEA, DMF, 80° C To a stirring solution of 2(5)-hydroxy-4-amino-butyric acid (0.059 g, 0.50 mmol) in DMF (2 ml) was added iV,W-bis(benzyloxycarbonyl)-lH-pyrazole-l- carboxamidine (0.26g, 0.70 mmol) followed by DIPEA (0.87 mL, 4.99 mmol) and the reaction was heated to 80°C and stirred overnight. The crude mixture was purified on a 2-inch reverse-phase HPLC column (Method 2) to yield JV,A^-bis-Cbz-2(S)-hydroxy-4- guanidino-butyric acid: MS: m/z (M+H) ⁺ calcd. 430.15, found 430.1.

Benzyl-2-(benzoyloxyamino)ethyI carbamate NHCbz

To a solution of benzyl-iV-(2-aminoethyl)carbamate chloride salt (1, 540 mg, 2.34 mmol) in sat. aq. NaHC0 ₃ (45 mL) was added 1 M NaOH (15 mL) and the reaction was stirred vigorously. DCM (30 mL) was added, followed by benzoylperoxide (1.13 g, 4.68 mmol) and the reaction was stirred overnight. The organic layer was separated and washed with brine, dried over MgS0 ₄ , filtered and concentrated to a crude, which was purified on a 1-inch reverse-phase HPLC column (Method 2) to yield benzyl-2-(benzoyloxyamino)ethyl carbamate (2, 252 mg, 0.80 mmol, 34.2%): MS: m/z (M+H) ⁺ calc. 315.13, obs. 315.0. Succinimidyl benzoyloxy(2-Cbz-aminoethyl)carbamate

3

To a stirring solution of disuccinimidyl carbonate (525 mg, 2.05 mmol) in CH ₃ CN (16 mL) was added benzyl-2-(benzoyloxyamino)ethyl carbamate (2, 252 mg, 0.80 mmol) as a solution in CH ₃ CN (12 mL) over 4 hours, and the reaction was stirred overnight. Additional disuccinimidyl carbonate (251 mg, 0.98 mmol) was added and the reaction was heated at 60°C overnight. Solvent removal gave a crude, which was purified on a 2-inch reverse-phase HPLC column (Method 2) to yield succinimidyl benzoyloxy(2-Cbz-aminoethyl)carbamate (3, 81 mg, 0.18 mmol, 22.5% yield).

N-Boc-3-amino-2(5)-hydroxy-propionic acid

To a stirring solution of S-isoserine (4.0 g, 0.038 mol) in dioxane: ¾0 (100 mL, 1:1 v/v) at 0° C was added N-methylmorpholine (4.77 mL, 0.043 mol), followed by B0C2O (11.28 mL, 0.049 mol) and the reaction was stirred overnight with gradual warming to room temperature. Glycine (1.0 g, 0.013 mol) was then added and the reaction was stirred for 20 min. The reaction was cooled to 0°C and sat aq. NaHC<¾ (75 mL) was added. The aqueous layer was washed with ethyl acetate (2 x 60 mL) and then acidified to pH 1 with NaHS0 ₄ . This solution was then extracted with ethyl acetate (3 x 70 mL) and these combined organic layers were dried over Na ₂ SC filtered and concentrated to dryness to give the desired N-Boc-3-amino-2(5)-hydroxy- propanoic acid (6.30 g, 0.031 mmol, 81.5 % yield): ^[ H NM (400 MHz, CDC13) δ 7.45 (bs, 1 H), 5.28 (bs, 1 H), 4.26 (m, 1 H), 3.40-3.62 (m, 2 H), 2.09 (s, 1 H), 1.42 (s, 9 H); ¹³ C NMR (100 MHz, CDC13) δ 174.72, 158.17, 82, 71.85, 44.28, 28.45.

N-Boc-4-amino-2(5)-hydroxy-butyric acid

To a stirring solution of S-4-armno-2-hydroxy-butyric acid (51.98 g, 0.44 mol) in dioxane: H ₂ 0 (2 L, 1:1 v/v) was added K ₂ C0 ₃ (106 g, 0.91 mol) followed by a solution of Boc-anhydride (100 g, 0.46 mol) in dioxane (100 niL), and the reaction was stirred overnight. The reaction was washed with DCM (2 x 300 mL), and the aqueous layer was acidified to pH 2 with H3PO4. The aqueous layer was extracted with DCM (2 x 300 mL), and the combined organic layers were dried over MgS0 ₄ , filtered and concentrated to dryness to yield the desired N-Boc-4-amino-2(5 -hydroxybutyric acid (48.2 g, 50% yield).

N-Boc-3-amirio-propanal

To a stirring solution of 3-(Boc-amino)-l-propanol (25 mL, 0.144 mol) in water saturated DCM (1.0 L) was added Dess-Martin reagent (99.2 g, 233.9 mmol) and the reaction mixture was stirred for 1 hour. The reaction was then diluted with ether (1.0 L), followed by a solution of Na ₂ S ₂ 0 ₃ (250 g) in 80% NaHC0 ₃ (450 g in 1.0 L H ₂ 0). The reaction was stirred vigorously for 30 minutes until two layers formed, the top layer was clear. The reaction was filtered to remove the precipitated solids and the aqueous layer was extracted with ether (1.0 L). The organic layer was washed with sat. NaHC0 ₃ (1.0 L), H ₂ 0 (1.0L), and brine (1L), dried over Na ₂ S0 ₄ and concentrated to a clear oil. The crude oil was dissolved in EtOAc: hexanes (1 :1 v/v, 1.0 L) and filtered through a short silica gel column to yield the desired N-Boc-3-amino-propanal (21.7 g, 0.125 mol, 85.6% yield): ¾ NMR (400 MHz, CDC1 ₃ ) δ 9.77 (s, 1 H, CHO), 4.85 (bs, 1 H, NH), 3.36-3.42 (m, 2 H, CH ₂ ), 2.67 (t, 2 H, CH ₂ ), 1.39 (s, 9 H, (CH ₃ ) ₃ ).

N-Boc-l-oxa-6-azaspiro[2.5]octane

N-Boc-4-Methylene-piperidine (0.222 g, 1.12 mmol) was submitted to Procedure 8 to form the desired N-Boc-l-oxa-6-azaspiro[2.5]octane (0.215 g, 1.01 mmol, 90.2% yield): Ή NMR (250 MHz, DMSO-d ₆ ) δ 3.29-3.61 (m, 6 H), 1.56-1.70 (m, 2 H), 1.30-1.54 (m, 1 1 H).

2-(Pent-4-enyl)-isoin

To a stirring solution of 5-bromo-pentene (6.0 g, 0.040 mol) in DMF (30 mL) was added K ₂ C0 ₃ (4.7 g, 0.034 mol) and potassium phthalimide (6.21 g, 0.033 mmol) and the reaction mixture was heated at 100°C for 1 hr. The reaction mixture was cooled to room temperature, and water (50 mL) was added. The aqueous layer was then extracted with ethyl acetate (2 x 50 mL), and the combined organic layers were washed with 5% aq. NaHC0 ₃ (2 x 20 mL), brine (30 mL) and dried over Na ₂ S0 ₄ . Filtration and solvent evaporation gave an oil, which was purified by flash chromatography (silica gel/ hexanes: ethyl acetate 0-35%) to yield the desired 2-(pent- 4-enyl)-isoindoline- 1,3 -dione as a solid (6.36 g, 0.029 mmol, 72.5 % yield): MS m/e [M+H] ⁺ calcd 216.1, found 216.1; NMR (250 MHz, DMSO-d«) δ 7.79-7.95 (m, 4 H), 5.70-5.91 (m, 1 H), 4.90-5.11 (m, 2 H), 3.58 (t, 2 H), 1.98-2.10 (m, 2 H), 1.59-1.78 (m, 2 H).

2-(3-(Oxiran-2-yl)-propyl)-isoindoline-l,3-dione

2-(Pent-4-enyl)-isoindoline-l,3-dione (6.36 g, 0.029 mmol) was submitted to Procedure 8 for epoxide formation to yield 2-(3-(oxiran-2-yl)-propyl- isoindoline-l,3-dione (5.8 g, 0.025 mmol, 86.2% yield): MS mJe [M+H] ⁺ calcd 232.1, found 232.1; ^! H NMR (250 MHz, DMSO-d ₆ ) δ 7.75-7.90 (m, 4 H, Ar), 3.52 (t, 2 H, CH ₂ ), 2.87-2.96 (m, 1 H, CH), 2.70 (t, 1 H), 2.30-2.45 (m, 1 H), 1.36-1.80 (m, 4 H).

N-Boc-3-hydroxypyrrolidine-3-carboxylic acid

N-Boc-3-pyrrolidone (0.010 mmol) was submitted to Procedure 9 to yield the desired N-Boc-3 -hydroxy-pyrrolidine-3 -carboxylic acid.

N-Boc-l-amino-but-3-ene

3-Buten-l -amine (4.93 g, 0.069 mol) was submitted to Procedure 7 for Boc protection to yield a crude, which was purified by flash chromatography (silica gel hexanes: ethyl acetate 0-30%) to yield N-Boc-l-amino-but-3-ene (6.47 g, 0.038 mol, 55.1 % yield). N-Boc-2-(oxiran-2-yl)-ethyl carbamate

N-Boc-l-amino-but-3-ene (6.47 g, 0.038 mol) was submitted to

Procedure 8 for epoxide formation to yield a crude, which was purified by flash chromatography (silica gel hexanes: ethyl acetate 0-45%) to yield N-Boc-2-(oxiran-2- yl)-ethyl carbamate (6.0 g, 0.032 mol, 84.2 % yield): Ή NMR (250 MHz, DMSO-d ₆ ) δ 2.98-3.09 (m, 2 H), 2.83-2.92 (m, 1 H), 2.65 (t, 1 H), 2.42 (dd, 1 H), 1.44-1.66 (m, 2 H), 1.36 (s, H, (CH ₃ ) ₃ ).

N-Boc-3-hydroxy-azetidin-3-carboxylic acid

N-Boc-3-azetidinone (21.9 g, 0.128 mol) was submitted to Procedure 9 to yield the desired N-Boc-3-hydroxy-azetidin-3-carboxylic acid (18.7 g, 0.086 mol, 67.0% yield): MS m/e [M+H] ⁺ calcd 218.1, found 218.2.

3-Methylene-l-methylamino-cyclobutane To a stirring solution of 3-methylene-l-cyano-cyclobutane (2.5 g, 0.026 mol) in THF (35 ml) at 0°C was slowly added 2M LiAlH ₄ (22 mL, 0.044 mmol) and the reaction was allowed to warm to room temperature. The reaction was then quenched by the addition of sat. aq. NH ₄ CI (10 mL), and THF (10 mL). The organic layer was separated and concentrated to dryness to yield a residue, which was dissolved in ethyl acetate (100 mL). The organic layer was washed with 5% NaHC<¾ (2 x 20 mL), brine (20 mL), dried over NaiSOi _t , filtered and concentrated to yield the desired 3-methylene- 1-memylamino-cyclobutane as an oil, which was carried through to the next step without further purification.

3-MethyIene-l-N-Boc-methylamino-cyclobutane

To a stirring solution of 3 -methylene- 1-methylamino-cyclobutane (2.52 g, 0.026 mol) in IN NaOH (15 ml) and THF (15 mL), was added Boc ₂ 0 (6.7 g, 0.030 mol) and the reaction mixture was stirred overnight. THF was evaporated and the aqueous layer was extracted with ethyl acetate (2 x 40 mL). The combined organic layers were washed with 5% NaHC<¾ (2 x 20 mL) brine (20 mL), dried over Na2SC>4, filtered and concentrated to dryness to yield a crude, which was purified by flash chromatography (silica gel/ hexanes: ethyl acetate 0%-60%) to yield the desired 3- methylene-l-N-Boc-methylamino-cyclobutane (1.9 g, 0.0096 mol, 36.9 % yield): ¾ NMR (250 MHz, DMSO-d ₆ ) δ 6.88 (bs, 1 H), 4.72 (s, 2 H), 2.95-3.05 (m, 2 H), 2.56- 2.71 (m, 2 H), 2.21-2.40 (m, 3 H), 1.20 (s, 9 H).

N-Boc-l-oxaspiro[2.3]hexan-5-yl-methanamine

3-Methylene-l-N-Boc-methylamino-cyclobutane (1.9 g, 0.0096 mol) was submitted to Procedure 8 for epoxide formation to yield N-Boc-1- oxaspiro[2.3]hexan-5-yl-methanamine (1.34 g, 6.27 mol, 65.3 % yield): ¾ NMR (250 MHz, DMSO- ) δ 2.99-3.10 (m, 2 H), 2.60-2.66 (m, 2 H), 1.99-2.47 (m, 5 H), 1.40 (s, 9 H). N-Fmoc-4-amino-butyraldehyde diethyl acetal

4-Amino-butyraldehyde diethyl acetal (8.0 g, 0.050 mol) was Fmoc protected following Procedure 10 to give the desired N-Fmoc-4-amino-butyraldehyde diethyl acetal (22.08 g, MS m/e [M+Na] ⁺ calcd 406.2, found 406.1), which was carried through to the next step without further purification.

N-Fmoc-4-amino-butyraldehyde

To a stirring solution of N-Fmoc-4-arriino-butyraldehyde diethyl acetal (0.050 mmol) in 1,4-dioxane (100 mL) was added aq. HCl (100 ml, 1:1 v/v, H ₂ 0 : cone. HCl) and the reaction progress was monitored by MS. Upon completion, the organic solvent was removed by rotary evaporation, and the aqueous layer was extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with 5% NaHC ⁽ ¾ (2 x 75 mL), brine (75 mL), dried over Na ₂ SC filtered and concentrated to dryness to yield the desired N-Fmoc-4-amino-butyraldehyde (15.35 g, 0.049 mol, 90.0 % yield), which was carried through to the next step without further purification: MS m/e [M+Na] ⁺ calcd 332.1, found 332.0.

3-Methylene-cyclobutane carboxylic acid

To a stirring solution of KOH (70.0 g, 1.25 mol) in EtOH/H ₂ 0 (500 mL, 1:1 v/v) was added 3-methylenecyclobutane carbonitrile (25.0 g, 0.26 mol) and the reaction mixture was refluxed for 6 h. The reaction progress was monitored by TLC and, upon completion, the mixture was cooled and acidified to pH 3-4 with HCl. The ethanol was evaporated, and the remaining aqueous layer was extracted with Et20 (200 mL). The organic layer was washed with water (2 x 20 mL), brine (30 ml), dried over Na2SC filtered and concentrated to dryness to yield 3-methylene-cyclobutane carboxylic acid, which was carried through to the next step without further purification: ¾ NMR (250 MHz, CDC1 ₃ ) δ 10.75 (bs, 1 H), 4.80 (s, 2 H), 2.85-3.26 (m, 5 H).

N-Boc-3-Methylene-cyclobutanamine

To a stirring solution of 3-methylene-cyclobutane carboxylic acid (1.0 g, 8.9 mmol) in THF (90 mL) was added NaN ₃ (2.0 g, 31.1 mmol), followed by tetrabutyl ammonium bromide (0.48 g, 1.5 mmol) and Zn(OTf)2 (0.1 g, 0.3 mmol), and the reaction mixture was heated to 40°C. Boc ₂ 0 (2.1 g, 9.8 mmol) was then added at once, and the reaction was heated at 45°C overnight. The reaction was then cooled to 0°C and was quenched with 10% aq. NaN<¾ (180 mL). The THF was evaporated and the aqueous layer was extracted with EtOAc (180 mL). The organic layer was washed with 5 % aq. NaHC0 ₃ (2 x 20 mL), brine (30 ml), dried over Na ₂ S0 ₄ , filtered and concentrated to dryness to yield a crude, which was purified by flash chromatography (silica gel/hexanes: ethyl acetate: 0-90%) to yield the desired N-Boc-3-methylene- cyclobutanamine (0.57 g, 3.1 mmol, 34.9% yield): *H NMR (250 MHz, CDC13) δ 4.83 (s, 2 H), 4.79 (bs, 1 H), 4.05-4.23 (m, 1 H), 2.92-3.11 (m, 2 H), 2.50-2.65 (m, 2 H), 1.44 (s, 9 H).

N-Boc-l-oxaspiro[2.3]hexan-5-amine

N-Boc-3-methylene-cyclobutanamine (1.65 g, 9.0 mmol) was submitted to Procedure 8 for epoxide formation to yield N-Boc-l-oxaspiro[2.3]hexan-5-amine (1.46 g, 7.33 mmol, 81.5 % yield): Ή NMR (250 MHz, CDC1 ₃ ) δ 4.79 (bs, 1 H), 4.13- 4.31 (m, 1 H), 2.66-2.83 (m, 4 H), 2.31-2.47 (m, 2 H), 1.45 (s, 9 H).

N-Boc-2,2-dimethyl-3-amiiio-propionaldehyde

N-Boc-3-amino-2,2-dimethyl propanol (0.415 g, 2.04 mmol) was submitted to Procedure 11 to yield N-Boc-2,2-dimethyl-3-amino-propionaldehyde (0.39 g, 1.94 mmol, 95.1 % yield): ^] H NMR (250 MHz, CDC13) δ 9.42 (s, 1 H), 4.80 (bs, 1 H), 3.11 (d, 2 H), 1.39 (s, 9 H), 1.06 (s, 6 H).

N-Boc-3-amino-3-cyclopropyl propionaldehyde

N-Boc-3-amino-3-cyclopropyl-propanol (0.130 g, 0.60 mmol) was submitted to Procedure 11 for oxidation to the corresponding N-Boc-3-amino-3- cyclopropyl propionaldehyde, which was carried through to the next step without further purification.

4(5)-tert-Butylditnethylsilyloxy-N-Boc-pyrrolidin-2(J?)-c arboxaldehyde

4(5 -?ert-Butyldimethylsilyloxy-N-Boc-pyrrolidin-2(^)-methanol (0.50 g, 1.50 mmol) was submitted to Procedure 11 for oxidation to the corresponding 4(S)- iert-butyldimethylsilyloxy-N-Boc-pyrrolidin-2( ?)-carboxaldehyde, which was carried through to the next step without further purification. 3-ferf-Butyldimethylsilyloxy-propaiial

3-iert-Butyldimethylsilyloxy-propanol (0.50 g, 2.62 mmol) was submitted to Procedure 11 for oxidation to the corresponding 3-tert- butyldimethylsilyloxy-propanal, which was carried through to the next step without further purification.

2-Methyl-N-Boc-2-amino-propanal

2-Methyl-N-Boc-2-amino-propanol (0.83 g, 4.38 mmol) was submitted to Procedure 11 for oxidation to the corresponding 2-methyl-N-Boc-2-amino-propanal (0.706 g, 3.77 mmol, 86.1 % yield): Ή NMR (250 MHz, CDC1 ₃ ) δ 9.40 (s, 1 H), 1.57 (s, 1 H), 1.41 (s, 9 H), 1.30 (s, 6 H).

N-Boc-l-amino-cyclobutane carboxylic acid

1-Amino-cyclobutane carboxylic acid ethyl ester (1.0 g, 6.28 mmol) was dissolved in IN HCl (10 mL) and the reaction was heated to a reflux for 2 hours. The reaction mixture was then concentrated to dryness to yield a crude which was submitted to Procedure 7 for Boc protection to yield the desired N-Boc- 1-Amino-cyclobutane carboxylic acid.

N-Boc-1 -amino-cy clobu y 1-methanol

N-Boc-l-amino-cyclobutane carboxylic acid (6.28 mmol) was submitted to Procedure 12 for reduction to the corresponding N-Boc-l-Amino-cyclobutyl- methanol. N-Boc-l-amino-cyclobutane carboxaldehyde

N-Boc-l-amino-cyclobutyl-methanol (0.25 g, 1.24 mmol) was submitted to Procedure 11 to yield the corresponding N-Boc-l-amino-cyclobutane carboxaldehyde (0.24 g, 1.20 mmol, 96.8 % yield): Ή NMR (250 MHz, CDC13) δ 9.0 (s, 1 H), 4.91 (bs, 1 H), 3.74 (bs, 2 H), 1.71-2.20 (m, 4 H), 1.42 (s, 9 H).

N-Boc-3-amino-cyclobutanone

To a vigorously stirring solution of N-Boc-3-methylene- cyclobutanamine (9.8 g, 53.5 mmol) in DCM (160 mL) and H ₂ 0 (160 mL) was added K2CO3 (3 g, 21.7 mmol), followed by NaIC>4 (35 g, 163.5 mmol), tetrabutylammonium chloride (0.2 g, 0.72 mmol) and RuCl ₃ (0.6 g, 7.6 mmol). During the course of the reaction, the organic solution turned dark brown, the catalyst turned black, while the upper aqueous layer turned white. The reaction was monitored by TLC, and upon completion, the reaction mixture was filtered through a pad of celite. The filtrates were transferred to a separatory funnel, and the aqueous layer was extracted with DCM (2 x 50 mL). The combined organic layers were washed with 5% NaHC03 (2 x 30 mL), brine (30 mL), dried over Na ₂ S0 ₄ , filtered and evaporated to dryness to yield a crude, which was purified by flash chromatography (silica gel hexanes: ethyl acetate 0-60%) to yield the desired N-Boc-3-amino-cyclobutanone (7.13 g, 38.53 mmol, 72% yield): MR (250 MHz, CDC1 ₃ ) δ 4.88 (bs, 1 H), 4.13-4.29 (m, 1 H), 3.23-3.41 (m, 2 H), 2.9- 3.05 (m, 2 H), 1.39 (s, 9 H).

N-Boc-l-hydroxy-3-amino-cyclobutyl-carboxylic acid

N-Boc-3-amino-cyclobutanone (7.13 g, 38.53 mmol) was submitted to Procedure 9 to yield the desired N-Boc-l-hydroxy-3-amino-cyclobutyl-carboxylic acid (MS m/e [M+H] ⁺ calcd 232.1, found 232.2.

N, N-diBoc-4(5)-amino-2(5)-methanol-pyrrolidine

N, N-diBoc-4(5)-amino-pyrrolidine-2(5)-carboxylic acid (1.03 g, 3.12 mmol) was submitted to Procedure 12 to yield the corresponding N, N-diBoc-4(S)- amino-2(S)-methanol pyrrolidine (0.605 g, 1.91 mmol, 61.2 % yield), which was carried through to the next step without further purification. , N-diBoc-4(5)-amino-pyrrolidine-2(5)-carbaldehyde

N, N-diBoc-4(¾-arnino-2(S)-methanol pyrrolidine (0.486 g, 1.53 mmol) was submitted to Procedure 11 for oxidation to the corresponding N, N-diBoc-4(5)- amino-pyrrolidine-2(5 -carbaldehyde, which was carried through to the next step without further purification. N-Boc-l-aminomethyl-cyclopropyl-methanol

N-Boc-l-aminomethyl-cyclopropane carboxylic acid (1.0 g, 4.64 mmol) was submitted to Procedure 12 to yield the corresponding N-Boc-l-aminomethyl- cyclopropyl-methanol (0.99 g, MS m/e [M+H] ⁺ calcd 202.1, found 202.1), which was carried through to the next step without further purification.

N-Boc-l-aminomethyl-cyclopropane carboxaldehyde N-Boc-l-aminomethyl-cyclopropyl-methanol (0.87 g, 4.32 mmol) was submitted to Procedure 11 for oxidation to the corresponding N-Boc-l-aminomethyl- cyclopropane carboxaldehyde, which was carried through to the next step without further purification.

N-Boc-l-amino-cyclopropyl-methanol N-Boc-l-amino-cyclopropane carboxylic acid (0.25 g, 1.24 mmol) was submitted to Procedure 12 to yield the corresponding N-Boc-l-amino-cyclopropyl- methanol (0.051 g, 0.27 mmol, 21.8 % yield), which was carried through to the next step without further purification. N-Boc-l-amino-cyclopropane carboxaldehyde

N-Boc-l-amino-cyclopropyl-methanol (0.051 g, 0.27 mmol) was submitted to Procedure 11 for oxidation to the corresponding N-Boc-l-amino- cyclopropane carboxaldehyde, which was carried through to the next step without further purification.

N-Boc-l(ii)-amiiio-2(5)-/ert-butyldimethylsilyloxy-cyc)op eiitane-4(5)-carbox lic acid

To a stirring solution of N-Boc-l(i?)-amino-2(5)-hydroxy-cyclopentane- 4 S -carboxylic acid methyl ester (0.622 g, 2.40 mmol) in DCM (1.9 mL) was added imidazole (0.164 g, 2.41 mmol), DMAP (0.047 g, 0.35 mmmol) and TBSC1 (0.363 g, 2.40 mmol) and the reaction was stirred at room temperature for 18 hours, followed by heating at 40°C for 1 hour. The reaction mixture was cooled to room temperature, and was quenched with ¾0 (3 mL). The organic layer was separated and was concentrated to dryness to yield a residue, which was dissolved in isopropanol (6 mL) and 1M NaOH (2.9 mL), and the reaction was heated at 60°C for 1 hour. The reaction was cooled to 0°C and slowly acidified to pH 3 with 1M HC1 (3 mL). After adding chloroform (18 mL), the organic layer was separated, dried over Na ₂ S04, and concentrated to dryness to yield the desired acid (0.75 g, 2.09 mmol, 87.1 % yield). N-Boc-l(i?)-amino-2(5)-tert-butyldiinethylsilyloxy-4(5)-hydr oxyinethyl- cyclopentane

N-Boc- 1 (/?)-amino-2(S)-iert-butyldimethylsilyloxy-cyclopentane-4(5 - carboxylic acid (0.53 g, 1.47 mmol) was submitted to Procedure 12 for reduction to the corresponding N-Boc- 1 (/?)-amino-2(5)-iert-butyldimethylsilyloxy-4(5)- hydroxymethyl-cyclopentane (0.44 g, 1.27 mmol, 86.4 % yield):Ή NMR (250 MHz, CDC1 ₃ ) δ 4.69-4.79 (m, 1 H), 4.08-4.13 (m, 1 H), 3.88 (bs, 1 H), 3.52-3.61 (m, 2 H), 2.16-2.30 (m, 2 H), 1.96-2.14 (m, 2 H), 1.48-1.53 (m, 2 H), 1.47 (s, 9 H), 0.91 (s, 9 H), 0.09 (s, 6 H).

N-Boc-l(/i)-amino-2(5)-ter/-buryldimethylsilyloxy-cyclope ntane-4(5)- carboxaldehyde

N-Boc-l(ii)-amino-2(S)-iert-butyldimethylsilyloxy-4(5}-hy droxymethyl- cyclopentane (0.44 g, 1.27 mmol) was submitted to Procedure 11 for oxidation to the corresponding N-Boc-l(^)-amino-2(5)-<ert-butyldimethylsilyloxy-cyclopen tane-4(S)- carboxaldehyde (0.42 g, 1.22 mmol, 96.1 % yield). teri-Buryl-2-( -Boc-3-hydroxy-azetidin-3-yl)acetate

To a stirring solution of N-Boc-3-azetidinone (0.45 g, 2.64 mmol) in THF (5 mL) was slowly added a 0.5 M solution of 2-fcrt-butoxy-2-oxoethyl-zinc chloride in Et ₂ 0 (10 mL, 5.0 mmol), and the reaction mixture was stirred for 5 h. The reaction was then quenched with sat. aq. NH ₄ C1 (10 mL), and the aqueous layer was separated and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with 5% aq. NaHC(¾ (2 x 10 mL), brine (15 mL), dried over Na2S0 , filtered and concentrated to dryness to yield iert-butyl-2-(N-Boc-3-hydroxy-azetidin-3- yl)-acetate (MS m/e [M+H] ⁺ calcd 288.2, found 287.7).

2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetic acid

To a stirring solution of ier/-butyl-2-(N-Boc-3-hydroxy-azetidin-3-yl)- acetate (0.86 g, 2.99 mmol) in dioxane (18 mL) was added 3M HCl (5 mL), and the mixture was heated at 70°C for lh. The reaction mixture was then cooled to 0°C and it was basified with 2 M NaOH (8 mL), followed by addition of BOC ₂ 0 (1.0 g, 4.6 mmol). The reaction mixture was allowed to warm to room temperature for 2 h, and was then concentrated to half its total volume on the rotary evaporator. Isopropanol (3 mL) and chloroform (12 mL) were then added and the mixture was cooled to 0°C and slowly acidified to pH 3 with 1M HCl. The organic layer was then separated, dried over Na ₂ S0 ₄ , and concentrated to dryness to yield 2-(N-Boc-3-hydroxy-azetidin-3-yl)- acetic acid (0.65 g, 2.81 mmol, 94.0 % yield).

N-Boc-3-(2-hydroxy-ethyl)-azetidin-3-ol

2-(N-Boc-3-hyaVoxy-azetidin-3-yl)-acetic acid (0.44 g, 1.90 mmol) was submitted to Procedure 12 for reduction to yield the corresponding N-Boc-3-(2- hydroxy-ethyl)-azetidin-3-ol (0.29 g, 1.33 mmol, 70.0 % yield).

2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetaldehyde

N-Boc-3-(2-hydroxy-ethyl)-azetidin-3-ol (0.29 g, 1.33 mmol) was submitted to Procedure 11 for oxidation to the corresponding 2-(N-Boc-3-hydroxy- azetidin-3-yl)-acetaldehyde, which was carried through to the next step without further purification.

N-Boc-3-hydroxymethyl-azetidine

N-Boc-azetidine-3-carboxylic acid (1.94 g, 9.64 mmol) was submitted to Procedure 12 for reduction to the corresponding N-Boc-3-hydroxymethyl-azetidine, which was carried through to the next step without further purification. N-Boc-azetidine-3-carboxaldehyde

N-Boc-3-hydroxymethyl-azetidine (9.64 mmol) was submitted to Procedure 11 for oxidation to the desired N-Boc-azetidine-3-carboxaldehyde, which was carried through to the next step without further purification.

2-(N-Boc-azetidin-3-yl)-2-hydroxy-acetic acid

N-Boc-azetidine-3-carboxaldehyde (1.60 g, 8.64 mmol) was submitted to Procedure 9 to yield the desired 2-(N-Boc-azetidin-3-yl)-2-hydroxy-acetic acid (MS m/e [M+H] ⁺ calcd 232.1 , found 231.8).

Synthesis of (2 ?.3 t)-4-a¾ido-2-benz _\ loxy-3-fluorobutanoic acid (51

Molecular sieves (4 A, 4 g) were added to a round bottom flask, and were activated by heating with a Bunsen burner under high vacuum. DCM (100 mL) was then added and the flask was cooled to -35°C with a cryocooler. Titanium 5 tetraisopropoxide (1.75 mL, 5.95 mmol) and (J?,i?)-(-)-diisopropyl tartrate (1.65 mL, 7.75 mmol) were added and the reaction was stirred for 30 min. Penta-l,4-dienol (5 g, 59.4 mmol) and excess cumene hydroperoxide (80%, 17.5 mL) were added in small portions, and stirring was continued at -35°C for 48 hr. The reaction was quenched by addition of sat. aq. Na ₂ S0 ₄ (5 mL) immediately followed by Et ₂ 0 (50 mL) and the

10 reaction was stirred for 2 hr with warming to rt. The reaction mixture was filtered through Celite, and washed with Et ₂ 0. Solvent removal under vacuum without heating resulted in approximately 30 mL of a yellow solution. Excess cumene alcohol and hydroperoxide were removed by flash chromatography (silica gel, 40% Et ₂ 0/hex). Finally solvent removal under vacuum without heating yielded a mixture of (2S, 3R)-

15 l,2-epoxy-4-penten-3-ol (1) (Rf= 0.47, 1 :1 EtOAc/hex) and diisopropyl tartrate (Rf = 0.6), which was used in the next step without further purification.

To a stirring solution of epoxide (1) in THF (100 mL) under an argon atmosphere was added tetrabutylammonium iodide (2.2 g, 5.96 mmol), followed by benzyl bromide (8.6 mL, 71.9 mmol) and the reaction was cooled to -15°C. Sodium

20 hydride (60% in mineral oil, 2.65 g, 66.1 mmol) was added in small portions and the reaction was stirred overnight with warming to rt. The reaction was quenched with MeOH, filtered through Celite, and washed with Et ₂ 0. Solvent removal gave an oily residue which was purified by flash chromatography (silica gel, 5→ 10% Et ₂ 0/hex) to yield (2S, 3i?)-l,2-epoxy-3-benzyloxy-4-pentene (2) as a clear non-volatile liquid (5.3g,

25 47.6% yield): Bf= 0.69 (1:4 EtOAc hex); [ot] _D = -36.7° (c 1.52, CHC1 ₃ ); HRMS (ESI) (M+H) ⁺ calc. for C12H14O2 191.1067, obs. 191.1064; ¾ NMR (CDCI3, 300 MHz) δ 7.38-7.33 (m, 5H), 5.92-5.78 (m, 1H), 5.41-5.39 (m, 1H), 5.37-5.33 (m, 1H), 4.66 (d, J = 11.95 Hz, 1H), 4.49 (d, J = 11.96 Hz, 1H), 3.83 (dd, J = 7.34, 4.20 Hz, 1H), 3.10 (dt, J = 4.07, 4.06, 2.70 Hz, 1H), 2.79 (dd, J = 5.21, 4.00 Hz, 1H), 2.70 (dd, J = 5.23, 2.64 Hz, 1H). ¹³ C NMR (CDCI3, 100 MHz) δ 138.32, 134.67, 128.56 (2C), 127.87 (2C), 127.82, 119.73, 79.54, 70.83, 53.41, 45.00.

NaN ₃ (3.38 g, 52 mmol) and NH ₄ C1 (2.78 g, 52 mmmol) in H ₂ 0 (10 mL) were heated until a clear solution was obtained. This solution was then added dropwise to a solution of (2S, 3-ft)-l,2-epoxy-3-benzyloxy-4-pentene (2) (3.3 g, 17.4 mmol) in MeOH (200 mL) and the reaction mixtiure was stirred for 4 days. The organic solvent was removed under vacuum, and the aqueous layer was extracted with DCM (3 x). The combined organic layers were dried over Na ₂ SC filtered and reduced under vacuum to yield a crude, which was purified by flash chromatography (silica gel, 10→ 20% Et ₂ 0 hex) to yield (2S,3if)-l-azido-3-benzyloxy-4-penten-2-ol (3) (2.66 g, 66% yield) as a non-volatile clear liquid: R = 4.8 (1 :4 EtOAc hex); HRMS (ESI) (M+Na) ⁺ calc. for C ₁₂ H ₁₅ N302 256.1056, obs. 256.1057; [a] _D = -46.3° (c 1.50, CHCI3); Ή NMR (CDC1 ₃ , 300 MHz)D δ 7.42-7.28 (m, 5H), 5.91-5.76 (m, 1H), 5.46 (dd, J = 17.16, 1.42 Hz, 1H), 5.42 (dd, J = 24.00, 1.37 Hz, 1H), 4.65 (d, J = 11.67 Hz, lH), 4.39 (d, J = 11.67 Hz, 1H), 3.88-3.80 (m, 2H), 3.44-3.40 (m, 2H), 2.22 (d, J = 3.60 Hz, 1H); ¹³ C NMR (CDCI3, 100 MHz) δ 137.88, 134.60, 128.66 (2C), 128.08 (2C), 128.05, 121.40, 81.39, 72.61, 70.70, 53.0; FTIR (NaCl): 3435, 2870, 2102, 1642, 1454, 1070 cm ^"1 .

To a stirring solution of DAST (900 μί, 6.87 mmol) in benzene (3.2 mL) and pyridine (400 L) in a plastic container at -10°C was added (25',3i?)-l-azido-3- benzyloxy-4-penten-2-ol (3) (750 mg, 3.21 mmol) in small portions, and the reaction was stirred at this temperature for 48 hr followed by 6 hr at rt. The reaction mixture was slowly added to sat. aq. NaHC0 ₃ (20 mL) at 0°C and was stirred for 10 min. The resulting aqueous mixture was extracted with DCM (3 x) and the combined organic layers were washed with 2 N HC1, dried over MgSC>4, filtered and reduced under vacuum to yield a crude, which was purified by flash chromatography (silica gel, 1% Et ₂ 0 hex) to yield (3 ?,4 ?)-5-azido-4-fluoro-3-benzyloxy-pent-l-ene (4) (128 mg, 16.9% yield) as a nonvolatile clear liquid: R/= 0.63 (1 :9 EtOAC/Hex); [a] _D = -11.9° (c

I.50, CHC1 ₃ ); ^] H NMR (CDCI ₃ , 400 MHz) Ώδ D7.44-7.29 (m, 5H), 4.63 (dddd, J = 47.64, 7.07, 4.99, 3.32 Hz, 1H), 5.49-5.42 (m, 2H), 4.70 (d, J = 11.95 Hz, 1H), 4.57 (ddd, J = 7.07, 4.99, 3.32 Hz, 1H), 4.44 (d, J = 11.90 Hz, 1H), 4.03 (ddd, J = 16.87, 7.57, 5.04 Hz, 1H), 3.64-3.52 (m, 1H), 3.45 (ddd, J = 27.45, 13.63, 3.27 Hz, 1H). ¹⁹ F NMR (CDCI ₃ , 282 MHz) -196.66 (dddd, J = 47.27, 27.08, 19.84, 16.89 Hz); ¹³ C NMR (CDCI ₃ , 100 MHz) δ 137.80, 133.09 (d, J = 5.30 Hz), 128.70 (2C), 128.09 (3C), 121.04, 93.33 (d, J = 181.54 Hz), 79.08 (d, J = 20.39 Hz), 70.92, 51.46 (d, J = 22.25 Hz). FTIR (NaCl): 2930, 2104, 1643, 1454, 1281, 1115, 1069 cm ^-1 .

OBn „„ „„„ OBn

- 1) 0 ₃ , DCM - ₀

p 2) NaH ₂ P0 ₄ , NaCI0 ₂ F OH

-^y 5

(3i?,4i?)-5-azido-4-fluoro-3-benzyloxy-pent-l-ene (4) (128 mg, 0.543 mmol) was submitted to Procedure 13, followed by recrystallization from hot hexanes (2 x) to yield (2i?,3i?)-4-azido-2-benzyloxy-3-fluorobutanoic acid (5) (120 mg, 90%): [a] _D = -56.9° (c 0.68, CHC1 ₃ ); HRMS (ESI negative mode) (M-H) calc. for C11H12FN3O3 252.0790, obs. 252.0782; Ή NMR (CDCI ₃ , 400 MHz) Ώδ □ 10.55 (s, 1H), 7.46-7.34 (m, 5H), 4.98 (dddd, J = 46.40, 7.57, 4.91, 2.92 Hz, 1H), 4.94 (d, J =

I I.47 Hz, 1H), 4.55 (d, J = 11.51 Hz, 1H), 4.17 (dd, J = 27.26, 2.86 Hz, 1H), 3.77 (dt, J = 13.89, 13.66, 7.27 Hz, 1H), 3.42 (ddd, J = 24.28, 13.20, 4.92 Hz, 1H); ¹⁹ F NMR (CDCI ₃ , 376 MHz)D 8D-198.36 (dddd, J = 46.28, 27.22, 24.46, 14.15 Hz); ¹³ C NMR (CDCI ₃ , 100 MHz) δ 174.63 (d, J = 4.21 Hz), 136.37, 129.15 (2C), 129.07, 128.98 (2C), 91.53 (d, J = 182.59 Hz), 76.40 (d, J = 19.90 Hz), 73.96 (s), 50.87 (d, J = 25.13 Hz); FTIR (NaCl): 3151, 2098, 1753, 1407, 1283, 1 112 cm ^"1 . Synthesis of ent-5

Starting from penta-l,4-dienol (5 g, 59.4 mmol) and using {S,S)-(+)- diisopropyl tartrate under the same reaction conditions as described above the enantiomer ent-2 was obtained (4.9 g, 43% yield): [a] _D = +35.7° (c 1.76, CHC1 ₃ ). (2R, 3S)-l,2-Epoxy-3-benzyloxy-4-pentene (ent-2, 3.9g, 20.5 mmol) was submitted to the same reaction conditions described above to yield the enantiomer (2Z?,3S)-l-azido-3- benzyloxy-4-penten-2-ol (ent-3, 2.75 g, 57% yield): [o] _D = +47.3° (c 1.30, CHC1 ₃ ). (27?,35)-l-Azido-3-benzyloxy-4-penten-2-ol (ent-3) (500 mg, 2.14 mmol) was submitted to the same reactions as described above to yield the enantiomer (35,45)-5- azido-4-fluoro-3-benzyloxy-pent-l-ene (ent-4, 75.5 mg, 0.32 mmol, 15% yield, [α]ο = +10.7°,c 1.50, CHCI3), which was submitted to the same reaction conditions as described above to yield ent-5 (59 mg, 73% yield): [<x] _D = +58.6° (c 0.73, CHC1 ₃ ).

Synthesis of (j?)-4-Azido-3.3-difluoro-2-benzyloxy-butanoic acid (3)

To a stirring solution of DMSO (690 ί, 9.65 mmol) in DCM (25 mL) at -78"C was added oxalyl chloride (3.21 mL of a 2.0 M solution in DCM, 6.43 mmol) and the reaction was stirred for 1 hr. A solution of (25',3J?)-l-azido-3-benzyloxy-4- penten-2-ol (1) (750 mg, 3.21 mmol) in DCM (1 mL) was added dropwise and the reaction mixture was stirred for 1 hr at -78°C. ΛΓ-Methyl morpholine (1.41 mL, 12.9 mmol) was added dropwise, and the reaction was stirred at -15°C for 2 hr. The reaction was quenched with phosphate buffer (0.1 M, pH 6.0) and the aqueous layer was 5 separated. The organic layer was washed with the phosphate buffer (3 x), dried over Na2SC filtered and reduced under vacuum to give a brown residue. The residue was dissolved in Et ₂ 0, dried over MgSC^, filtered through a cotton plug, and reduced under vacuum to yield the crude ketone, which was dissolved in DCM (1 mL) and was added to a stirring solution of DAST (2 mL, 15.3 mmol) in DCM (3 mL) in a plastic vial at -

10 25°C. The reaction was allowed to slowly warm to it and was stirred for 48 hr. The reaction mixture was then slowly poured into stirring sat. aq. NaHC(¾ (20 mL) at 0°C, and was stirred for 10 min. The resulting aqueous mixture was extracted with DCM (3x), and the combined organic layers were dried over Na ₂ SC filtered and reduced under vacuum to yield a crude, which was purified by flash chromatography (silica gel,

15 1% Et ₂ 0/hex) followed by preparative TLC purification (silica gel, 0.5 mm, 5% Et ₂ 0/hex) to yield (J?)-5-azido-4,4-difluoro-3-benzyloxy-pent-l-ene (2, 193 mg, 0.76 mmol, 24% yield), as a non-volatile clear liquid: R/= 0.72 (1:4 EtOAc/hex); [a] _D = - 23.8° (c 1.52, CHC1 ₃ ); Ή NMR (CDCI3, 300 ΜΗζ)Π 5Π 7.44-7.31 (m, 5H), 5.89 (dddd, J = 16.88, 10.61, 7.11, 0.62 Hz, 1H), 5.59-5.56 (m, 1H) ₍ 5.53 (d, J = 10.74 Hz,

20 1H), 4.71 (d, J = 11.67 Hz, 1H), 4.50 (d, J = 11.66 Hz, 1H), 4.14 (td, J = 14.25, 7.13, 7.13 Hz, 1H), 3.64 (tq, J = 13.67, 13.67, 13.67, 11.19, 11.19 Hz, 2H); ¹⁹ F NMR (CDCI3, 282 MHz) 5 -116.63 (dtd, J = 257.62, 13.91, 13.90, 8.72 Hz), -111.27 (dtd, J = 257.59, 16.18, 16.16, 7.04 Hz); ¹³ C NMR (CDCI3, 75 MHz) δ 137.14, 130.33 (t, J = 3.06, 3.06 Hz), 128.71 (2C), 128.27, 128.20 (2C), 122.78, 120.69 (dd, J = 249.89,

25 246.83 Hz), 78.87 (dd, J = 30.35, 25.35 Hz), 71.48 (d, J = 0.48 Hz), 51.47 (dd, J =

30.26, 25.92 Hz); FTIR (NaCl): 2928, 2108, 1455, 1292, 1091 cm ^"1 . (i?)-5-Azido-4,4-difiuoro-3-benzyloxy-pent-l-ene (2, 193 mg, 0.76 mmol) was submitted to Procedure 13, followed by washing with cold hexanes (3x) at -20°C to yield (3) (139 mg, 67.6% yield): [<x] _D = -32.4° (c 0.80, CHC1 ₃ ); HRMS (ESI negative mode) (M-H) for enHnF ₂ N ₃ 0 ₃ 270.0696, obs. 270.06924; Ή NMR (CDC1 ₃ , 400 MHz)D δ Π7.46-7.32 (m, 5H), 6.48 (s, 1H), 4.84 (d, J = 11.30 Hz, 1H), 4.67 (d, J = 1 1.30 Hz, 1H), 4.37 (dd, J = 12.23, 9.78 Hz, 1H), 3.75 (dd, J = 14.67, 12.35 Hz, 2H); ¹⁹ F NMR (CDC1 ₃ , 376 MHz)D δ -112.61 (qd, J = 260.95, 12.30, 12.29, 12.29 Hz), - 109.68 (dtd, J = 260.79, 14.75, 14.68, 9.94 Hz); ¹³ C NMR (CDC1 ₃ , 100 MHz) δ 170.84, 135.48, 129.01, 128.94 (2C), 128.78 (2C), 119.59 (t, J = 251.58, 251.58 Hz), 76.56 (dd, J = 29.86, 27.24 Hz), 74.34, 51.58 (dd, J = 28.94, 26.76 Hz). FTIR (NaCl): 3337, 2929, 2112, 1738, 1455, 1292, 1210, 1119 cm ^"1 .

Synthesis of ent-3

ent-1 ent-2 -^ ^ ent-3

(2J?,3S)-l-Azido-3-benzyloxy-4-penten-2-ol (ent-1, 500 mg, 2.14 mmol) was submitted to the same reaction conditions described above to yield (S)-5- azido-4,4-difluoro-3-benzyloxy-pent-l-ene (ent-2, 114 mg, 21% yield, [a] _D = +27.9° (c 3.14, CHC1 ₃ )). Ent-2 (75.5 mg, 0.32 mmol) was submitted to Procedure 13 to yield (S)-4-azido-2-benzyloxy-3,3-difluorobutanoic acid (ent-3, 34.8 mg, 43% yield, [α]ο = +36.4° (c 0.80, CHC1 ₃ ).

Synthesis of (2 3£)-4-azido-2.3-bis-benzyloxybutanoic acid (3)

1 2 To a stirring solution of (2S,3.R)-l-azido-3-benzyloxy-4-penten-2-ol (1) (250 μΐ., 1.07 mmol) in THF (50 mL) under argon was added tetrabutylammonium iodide (42 mg, 0.11 mmol) followed by benzyl bromide (155 μ∑, 1.27 mmol) and the reaction was cooled to 0°C. Sodium hydride (60% in mineral oil, 47 mg, 1.18 mmol) was added in small portions and the reaction was stirred overnight with warming to rt. The reaction was quenched with MeOH, filtered through Celite, and washed with Et ₂ 0. The organic solvent was removed under vacuum to give an oily residue, which was purified by flash chromatography (silica gel, 2% Et ₂ 0 hex) to yield (3J?,4»S)-5-azido- 3,4-bisbenzyloxy-pent-l-ene (2, 237 mg, 65% yield) as a clear non-volatile liquid: R = 0.62 (1:4 EtOAc/hex); [a] _D = -6.1 ° (c 1.50, CHC1 ₃ ); ^l H NMR (CDC1 ₃ , 300 MHz)D δ □7.35-7.24 (m, 10H), 5.81 (ddd, J = 17.15, 10.58, 7.45 Hz, 1H), 5.37 (ddd, J = 5.70,

I.65, 0.86 Hz, 1H), 5.33 (ddd, J = 12.07, 1.44, 0.81 Hz, 1H), 4.63 (s, 2H), 4.61 (d, J =

I I.87 Hz, 1H), 4.35 (d, J = 11.78 Hz, 1H), 3.90 (tdd, J = 7.37, 5.65, 0.79, 0.79 Hz, 1H), 3.60 (ddd, J = 6.39, 5.69, 3.64 Hz, 1H), 3.43 (dd, J = 12.93, 6.42 Hz, 1H), 3.35 (dd, J = 12.93, 3.60 Hz, 1H); ¹³ C NMR (CDC1 ₃ , 75 MHz) δ 138.25, 138.01, 135.43, 128.60 (4C), 128.29 (2C), 128.02, 127.99 (2C), 127.87, 1 19.97, 80.76, 80.23, 73.33, 70.79, 51.69; FTIR (NaCl): 2867, 2100, 1606, 1454, 1286, 1095, 1073. (3.R,4,S 5-azido-3,4-bis-benzyloxy-pent-l-ene (2, 237 mg, 0.69 mmol) was submitted to Procedure 13 to yield (2S,35)-4-azido-2,3-bis-benzyloxybutanoic acid (3, 187.7 mg, 75% yield): [a] _D = -15.1 ⁰ (c 1.05, CHC1 ₃ ); HRMS (ESI negative mode) (M-H) calc. for C18H19N3O4 340.1303, obs. 340.1296; Ή NMR (CDC1 ₃ , 300 MHz)D δ Π7.24 (s, 1H), 7.38-7.33 (m, 10H), 4.79 (d, J = 11.61 Hz, 1H), 4.66 (s, 2H), 4.56 (d, J = 11.61 Hz, 1H), 4.20 (d, J = 4.24 Hz, 1H), 3.98 (td, J = 6.56, 4.30, 4.30 Hz, 1H), 3.58 (dd, J = 13.04, 6.62 Hz, 1H), 3.42 (dd, J = 13.04, 4.31 Hz, 1H); ¹³ C NMR (CDCI3, 75 MHz) δ 175.57, 137.92, 137.34, 129.44 (2C), 129.36 (2C), 129.15, 129.04 (2C), 128.98 (2C), 128.94, 79.71, 77.651

2918, 2103, 1722, 1455, 1284, 1110 cm ¹ .

Synthesis of ent-3

(2i?,35)-l-azido-3-benzyloxy-4-penten-2-ol (ent-1, 250 mg, 1.07 mmol) was submitted to the same reaction conditions as described above to yield (3S,4R)-5- azido -3,4-bis-benzyloxy-pent-l-ene (ent-2, 322mg, 59% yield): [a] _D = +7.9° (c 1.50, CHC1 ₃ ). Ent-2 (178 mg, 0.55 mmol) was submitted to Procedure 13 to yield ent-3 (144 mg, 77% yield): [o] _D = +15.2° (c 0.81, CHC1 ₃ ).

Synthesis of Compound 6

Synthesis of Compound 2 A 2-L three-necked round-bottomed flask equipped with a reflux condenser was charged with epoxide 1 (60 g, 315 mmol), phthalimide (69.6 g, 473 mmol), pyridine (5.1 mL, 63.1 mmol, 20 mol %) and ΓΡΑ (600 mL) and the resulting solution was stirred at 80 - 82 °C for 8 hrs. The reaction mixture was then cooled to ambient temperature and concentrated on a rotatory evaporator to dryness. The residue was adsorbed on silica gel (100 g), dried under high vacuum and then purified by flash column chromatography on silica gel (10 - 40% MTBE/heptanes) to afford the desired phthalimide protected amino alcohol 2 as a white solid (73.5 g, 69%): Ή NMR (CDC1 ₃ , 500 MHz) δ 7.83-7.82 (m, 2H), 7.71-7.69 (m, 2H), 7.32-7.31 (m, 4H), 7.28-7.25 (m, 1H), 5.91 (ddd, J = 17.4, 10.5, 7.6 Hz, 1H), 5.46-5.40 (m, 2H), 4.65 d, J = 11.7 Hz, 1H), 4.40 (d, J = 11.7 Hz, 1H), 3.99-3.97 (m, 1H), 3.95-3.90 (m, 2H), 3.86 (dd, J = 14.0, 3.3 Hz, 1H), 2.61 (d, J= 6.5 Hz, 1H).

Synthesis of Compound 3

A 2-L three-necked round-bottomed flask equipped with an addition funnel, an overhead mechanical stirrer, and a nitrogen inlet/outlet was charged with a solution of alcohol 2 (70 g, 208 mmol) in anhydrous tetrahydrofuran (840 mL). The solution was cooled to -10 to -15 °C, then B114NI (7.66 g, 20.8 mmol, 10 mol %) was charged into the reactor followed by benzyl bromide (29.6 mL, 249 mmol). The resulting solution was stirred for 20 min, then sodium hydride (9.2 g, 228 mmol, 1.1 equiv, 60% mineral oil dispersion) was added to the batch in portions such that the batch temperature was maintained at -10 to -15 °C. Once the addition of sodium hydride was complete, the reaction mixture was stirred for additional 30 min and then brought to ambient temperature and further stirred for 18 h. The reaction was quenched with aqueous NaHC0 ₃ (280 mL) while maintaining the reaction mixture at -5 to 0 °C (ice bath). The reaction mixture was then diluted with MTBE (1.4 L mL) and the phases separated. The organic layer was washed with water (2 χ 210 mL), brine (210 mL), dried (MgSC>4), filtered, and concentrated to obtain the crude product as an oil. The crude product was purified by flash column chromatography on silica gel (5 - 25% MTBE/heptanes) to obtain the desired product 3 as a semi solid (75.7 g, 85%): ¾ NMR (CDCI3, 300 MHz) δ 7.75-7.74 (m, 2H), 7.67-7.66 (m, 2H), 7.34-7.21 (m, 5H), 7.15- 7.13 (m, 2H), 7.07-7.02 (m, 3H), 5.98-5.91 (m, 1H), 5.43 (s, 1H), 5.39 (td, J = 5.9, 1 Hz, 1H), 4.66 (dd, J = 12.0, 5.7 Hz, 2H), 4.49 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 11.8 Hz, 1H), 3.95-3.89 (m, 3H), 3.77-3.72 (m, 1H). Synthesis of Aldehyde 4 and Carboxylic Acid 5

A solution of alkene 3 (30 g, 70.2 mol) in DCM (1.8 L) was sparged with ozone at <-70 °C (dry ice-acetone) for 1 min using oxygen source to generate the ozone. Once the reaction was deemed compete (TLC, 1:1 MTBE heptanes), the solution was sparged with nitrogen for 35 min to remove residual ozone. The reaction was quenched with dimethyl sulfide (52 mL, 702 mmol) while maintaining the reaction mixture at <-70 °C (dry ice-acetone). The cold bath was removed and the mixture was allowed to warm to ambient temperature. The reaction mixture was concentrated under reduced pressure and further dried under high vacuum to obtain the crude aldehyde 4, as a thick oil (35.5 g, >99%). R _f = 0.38 (1:1 MTBE/heptanes). The reaction was repeated at 30 g scale of 3 to afford crude aldehyde 4 (33.4 g, >99%). The two lots of crude aldehyde were combined and subjected to the Pinnick oxidation without further purification.

The crude aldehyde 4 (30.1 g) was taken into a mixture of tetrahydrofuran, iBuOH, and water (226 mL, 226 mL, 151 mL, 3:3:2) along with NaH ₂ P0 ₄ (33.7 g, 281 mmol) and 2-methyl-2-butene (149 mL, 1.4 mol). The solution was cooled (15 ± 5 °C, water bath). Sodium chlorite (12.7 g, 140 mmol) was added to the batch and the resulting solution was stirred at ambient temperature for 4 hr. The completion of the reaction was confirmed by TLC analysis (1:1 MTBE/heptanes and 5% MeOH in DCM). The reaction was then quenched with brine (602 mL) and the product extracted into DCM (3 x 602 mL). The organic layers were dried (MgSO^, concentrated under reduced pressure to obtain the crude acid 5 as a thick oil (42.5 g, >99%). The synthesis was repeated on 30.1 g scale of 4 to afford crude acid 5 (44.2 g, >99%). The both lots of crude acids were combined and purified by flash column chromatography over silica (5 - 100% MTBE/heptanes). Fractions containing the acid were combined and concentrated under reduced pressure to afford acid 5 as a white solid (29.1 g, 47%): R _f = 0.39 (5:95 MeOH/DCM); ¾ NMR (CDC1 ₃ , 500 MHz) δ 7.76 (dd, J= 6.8, 3.7 Hz, 2H), 7.68 (dd, J= 5.5, 3.0 Hz, 2H), 7.35-7.34 (m, 2H), 7.31-7.26 (m, 3H), 7.18-7.16 (m, 2H), 7.11-7.05 (m, 3H), 4.75 (d, J = 11 Hz, 1H), 4.65 (d, J = 12.8, 2H), 4.59 (d, J= 11.9 Hz, 1H), 4.22 (d, J= 3.65, 1H), 4.17 (m, lH), 4.08 (dd, 7 = 14.3, 6.8 Hz, 1H), 3.86 (dd, J= 14.3, 4.7 Hz, 1H).

Synthesis of Compound 6

A round bottomed flask equipped with a magnetic stirring bar, and a thermocouple probe was charged with a solution of phthalimide-protected amino acid 5 (29.0 g, 65.1 mmol) in THF (350 mL). To the clear, yellow solution was added deionized water (175 mL) and the resulting mixture cooled to 5 °C. Methylamine solution in water (58.0 mL, 40 wt %, 665 mmol) was then added to the batch, which was warmed to ambient temperature (21 - 23 °C) and stirred for 26 hours. Analysis of an aliquot from the reaction mixture by LCMS indicated the reaction was complete. The reaction mixture was then concentrated in vacuo to a yellow solid residue, removing all excess methylamine. The residue was taken up in THF (700 mL) and water (350 mL), cooled to 0 - 5 °C, and to the crude amino acid solution was added potassium carbonate (45 g, 326 mmol), followed by benzylchloroformate (17.2 mL, 114 mmol). The batch was warmed to ambient temperature and the reaction allowed to proceed for 28 hours. Analysis of an aliquot at this time point by LCMS indicated a complete conversion of the amino acid to the carbamate. The reaction mixture was concentrated under reduced pressure to remove most of THF, the aqueous residue was diluted with water (320 mL) and the pH adjusted with 2N HC1 to approximately pH 5 (pH paper strip). The crude product was extracted with methylene chloride (3 x 500 mL), the extracts washed with water (60 mL), brine (60 mL), dried (MgS0 ₄ ), and concentrated in vacuo to a yellow oil (40.34 g) which was purified by flash column chromatography on silica gel (400 g; elution with 0 - 5% MeOH in CH ₂ CI ₂ ) to afford compound 6 as a yellow oil (27.5 g, 92% yield over two steps). ^l H NMR (DMSCWtf, 500 MHz) δ 12.93 (s, 1H), 7.36 - 7.23 (m, 16H), 5.01 (s, 2H), 4.63 (d, J= 11.8 Hz, 1H), 4.56 (dd, J = 22.9, 11.7 Hz, 2H), 4.45 (d, J= 11.7 Hz, 1H), 4.14 (d, J = 4.0 Hz, 1H), 3.81 (td, J= 7.3, 4.1 Hz, 1H), 3.31-3.24 (m, 2H). Synthesis of Compound 9

Nl

Synthesis of Epoxy Alcohol Ent-2

A 3 -neck, 5 liter round bottomed flask equipped with an overhead mechanical stirrer, a thermocouple probe and a nitrogen inlet/outlet was charged with powdered, freshly activated molecular sieves (4 A, 84 g, 0.8 wt. equiv), followed by anhydrous dicnloromethane (2.1 L, 20 vol). The resulting suspension was cooled to approximately - 42 °C using an acetonitrile/C0 ₂ bath, then titanium tetraisopropoxide (37 mL, 0.125 mol, 10 mol%) was charged into the batch, followed by (S,S)-(+)- diisopropyl tartrate (35 mL, 0.166 mol, 13.3 mol%). The reaction mixture was stirred for 30 minutes, then divinyl alcohol 1 (105 g, 1.25 mol, 1.0 equiv) was added over 3 minutes using an addition funnel (minor exotherm, 2 °C). Cumene hydroperoxide (370 mL, 80% titer, 1.99 mol, 1.59 equiv) was then added to the batch over 5 minutes using an addition funnel (10 °C exotherm). The reaction was allowed to proceed for 18 hours, holding the temperature between - 45 and - 30 °C. When complete as determined by TLC analysis (R _f 0.42 for divinyl alcohol, and 0.18 for epoxy alcohol, 50% MTBE in Heptanes), the reaction was quenched with saturated aqueous sodium sulfate (105 mL, 1 vol), diluted with MTBE (1.05 L, 10 vol) and the batch allowed to warm to ambient temperature, with vigorous stirring. Diatomaceous earth, Celite ^® 5 (105 g, 1 wt. equiv) was added to the batch, which was then filtered through a pad of Celite ^® . The filter cake was washed with MTBE (0.5 L) and the filtrate concentrated in vacuo on a rotary evaporator (with water bath held at 10 - 20 °C) to afford a yellow/brownish oil. A portion of the crude product [311 g] was subjected to silica plug (1 kg silica gel) using 0-60% MTBE/petroleum ether. The fractions containing

10 the product were collected and concentrated to obtain a colorless oil (48.3 g). This material was then purified via column chromatography (300 g silica gel, 5-30% MTBE/petroleum ether) to afford ent-2 as a clear liquid [22.6 g, 36% overall mass recovery]: R _f = 0.59 (1:1 MTBE/petroleum ether); Ή NMR (CDC1 ₃ , 500 MHz) δ 5.85 (ddd, J = 17.0, 10.5, 6.2 Hz, 1H), 5.40 (dt, J = 17.3, 1.3 Hz, 1H), 5.27 (dt, J =

15 10.5, 1.3 Hz, 1H), 4.36^1.33 (m, lH), 3.10 (ddd, J= 3.8, 3.8, 3.0 Hz, 1 H), 2.81 (dd, / = 2.9, 5.0 Hz, 1H), 2.76 (dd, 4.1, 5.0 Hz, 1H), 2.07 (d, J= 3.0 Hz, 1H).

Synthesis of Compound 3

The reaction was carried out at 20-g scale of alcohol following a 20 literature procedure (J. Org. Chem. 2009, 74(15), 5758-5761). A 2-L round-bottomed flask equipped with a mechanical stirrer, a thermocouple probe, and an addition funnel was charged with a solution of epoxy alcohol ent-2 [20 g, 200 mmol, 1 equiv] in tetrahydrofuran (400 mL, 20 vol) along with Ph ₃ P (105 g, 400 mmol, 2 equiv), and 4- nitrobenzoic acid (67 g, 400 mmol, 2 equiv) under a nitrogen atmosphere. DIAD (81 g, 25 400 mmol, 2 equiv) was added to the reaction mixture using an addition funnel while maintaining the reaction mixture at 0 °C (ice bath). Once the addition of DIAD was complete, the cold bath was removed and the reaction mixture was allowed to come to ambient temperature (23 °C). The reaction mixture was stirred for 1.5 h (all starting material consumed) and then quenched with aqueous NaHCC solution (100 ml, 5 vol) 30 followed by the addition of MTBE (1000 mL, 50 vol). The resulting solution was transferred into a separatory funnel. Brine (100 mL, 5 vol) was added to obtain phase separation. The organic phase was washed with brine (2 x 20 vol), dried (MgSC and concentrated under vacuum to obtain an oil (296 g). The oil was passed through a silica plug (1 kg) using 10-20% MTBE/heptanes. The crude solid (46 g) was then dissolved into MTBE (20 vol) and washed with NaHC0 ₃ (3 5 vol), water (2 x 2 vol), brine (2 2 vol), dried (MgS0 ), concentrated, and further dried to obtain the benzoate ester as a white solid [29 g, 59%: R _f = 0.56 (1:1 MTBE heptanes)]; ¾ NMR (CDC1 ₃ , 500 MHz) δ 8.35(d, J= 10.8 Hz, 2H), 8.25 (d, J= 10.8 Hz, 2H), 5.97 (ddd, J= 17.2, 10.6, 6.2 Hz, 1H), 5.48 (td, J= 17.3, 1.2 Hz, lH), 5.40 (td, J = 10.7, 1.1 Hz, 1H), 5.34 (dd, J= 5.0, 1.3 Hz, 1H), 3.31 (ddd, J = 6.5, 4.1, 2.6 Hz, 1H), 2.93 (dd, J = 4.2, 4.2 Hz, 1H), 2.76 (dd, J = 4.8, 2.6 Hz, lH).

The hydrolysis of the benzoate ester was carried out following the literature procedure (J. Org. Chem. 2009, 74(15), 5758-5761). Thus solution of the ester (22.7 g, 91 mmol, 1 equiv) in methanol (340 mL, 15 vol) was treated with an aqueous solution of K ₂ C0 ₃ (13.8 g, 100 mmol, 1.1 equiv, in 34 mL, 1.5 vol water) at 10 - 15 °C. The solution immediately turned into a thick slurry. The slurry was stirred at ambient temperature (23 °C) for 3 h (starting material consumed). The reaction mixture was concentrated on a rotary evaporator (at ambient water bath temperature) to ~2 vol (45 mL). The thick solution was then reslurried in DCM (454 mL, 20 vol). The slurry was filtered and the solids were washed with DCM (2 5 vol, 2 114 mL). The combined organic filtrate was dried (MgSC ), filtered, and concentrated to obtain a solid (31 g). The crude material was then purified by column chromatography (silica gel, 10—30% MTBE/petroleum ether) to obtain the desired alcohol 3 as a clear oil [9.24 g, quantitative yield, R _f = 0.31 (1:1 MTBE/heptanes)]; Ή NMR (CDCI3, 300 MHz) δ 5.94 (ddd, J= 16.2, 10.6, 5.5, 1H), 5.40 (d, J= 17.3 Hz, 1H), 5.26 (d, J= 10.6 Hz, 1H), 4.0 (t, J= 5.3 Hz, 1H), 3.07 (m, 1 H), 2.84 (t, J= 4.8 Hz, 1H), 2.11-2.1 (m, 1H), 2.57 (br s, 1H).

Synthesis of Compound 4 A 1-L three-necked round-bottomed flask equipped with an addition funnel, an overhead mechanical stirrer, a nitrogen inlet/outlet, was charged with alcohol 3 [9.24 g, 92.3 mmol, 1 equiv] in anhydrous tetrahydrofuran (166 mL, 18 vol). The solution was cooled to -10 to -15 °C. The catalyst BiuNI (3.41 g, 9.23 mmol, 10 mol %) was charged into the reactor followed by benzyl bromide (19.1 g, 112 mmol, 1.2 equiv). The resulting solution was stirred for 20 min. Sodium hydride (4.1 g, 1.1 equiv, 60% mineral oil dispersion) was then added to the batch in portions such that the batch temperature was maintained at -10 to -15 °C. Once the addition of sodium hydride was complete, the reaction mixture was stirred for an additional 30 min and then the cold bath was removed and reaction mixture brought up to ambient temperature and further stirred for 18 h. The reaction was quenched with aqueous NaHC03 (37 mL, 4 vol) while maintaining the temperature at -5 to 0 °C (ice bath). The resulting solution was diluted with MTBE (185 mL, 20 vol), the organic layer was washed with water (2 x 18 mL, 2 x 3 vol), brine (1 18 mL, 1 ^χ 3 vol), dried (MgS0 ₄ ), filtered, and concentrated under reduced pressure to obtain crude product as an oil. The synthesis was repeated on 1.98 g scale of alcohol 3. The crude from both the reactions were combined and purified via column chromatography (silica gel column, 2.5— 10% MTBE/heptanes) to obtain the desired benzylated product 4 as an oil [13.96 g, 65%: R _f = 0.61 (3:7 MTBE/heptanes)]; ¾ NMR (CDC1 ₃ , 500 MHz) δ 7.36-7.32 (m, 4H), 7.29- 7.26 (m, 1H), 5.83 (ddd, J = 17.3, 10.5, 6.7, 1H), 5.36 (td, J= 17.3, 1.4 Hz, 1H), 5.31 (td, J= 10.5, 1.2 Hz, 1H), 4.63 (ABq, J= 12.0 Hz, 2H), 3.62 (ddd, /= , 1H), 3.11-3.08 (m, 1 H), 2.78 (t, J= 4.4 Hz, 1H), 2.60 (dd, J= 5.0, 2.7 Hz, 1H).

Synthesis of Compound 5

A 250-mL round-bottomed flask equipped with a reflux condenser was charged with alcohol 4 [10 g, 52.5 mmol, 1 equiv], phfhalimide (11.6 g, 78.8 mmol, 1.5 equiv), pyridine (0.85 mL, 10.5 mmol, 20 mol %) and IPA (100 mL, 10 vol) and the resulting solution was stirred at 80 - 82 °C for 8 hrs. The reaction mixture was then cooled to ambient temperature and concentrated on a rotatory evaporator to dryness. The residue was adsorbed on silica gel (20 g), dried under high vacuum and then purified by flash column chromatography on silica gel (10 - 40% MTBE/heptanes) to afford the desired phthalimide protected amino alcohol 5 as a white tacky solid [15.85 g, 89%] : R _f = 0.34 (1 :1 MTBE/heptanes); Ή NMR (DMSO-d«, 500 MHz) δ 7.84-7.82 (m, 4H), 7.36-7.31 (m, 4H), 7.28-7.25 (m, 1H), 5.93 (ddd, J= 17.5, 10.5, 10.1 Hz, 1H), 5.38-5.35 (m, 2H), 5.12 (d, = 5.5 Hz, 1H), 4.53 (d, J= 11.9 Hz, 1H), 4.40 (d, / = 11.9 Hz, 1H), 3.98 (dddd, J= 9.0, 4.5, 4.5, 4.5 Hz 1H), 3.86 (dd, J = 5.8, 4.6 Hz, 1H), 3.67 (dd, J= 13.7, 8.9 Hz, 1H), 3.59 (dd, = 13.7, 4.4 Hz, lH).

Synthesis of Compound 6

A 1-L three-necked round-bottomed flask equipped with an addition funnel, an overhead mechanical stirrer, and a nitrogen inlet/outlet was charged with a solution of alcohol 5 [15 g, 44.5 mmol, 1 equiv] in anhydrous tetrahydrofuran (270 mL, 18 vol). The solution was cooled to -10 to -15 °C, then B114NI (1.64 g, 4.45 mmol, 10 mol %) was charged into the reactor followed by benzyl bromide (9.2 g, 53.8 mmol, 1.2 equiv). The resulting solution was stirred for 20 min, then sodium hydride (1.97 g, 1.1 equiv, 60% mineral oil dispersion) was added to the batch in portions such that the batch temperature was maintained at -10 to -15 °C. Once the addition of sodium hydride was complete, the reaction mixture was stirred for an additional 30 min and then brought to ambient temperature and further stirred for 18 h. The reaction was quenched with aqueous NaHC(¾ (60 mL, 4 vol) while maintaining the reaction mixture at -5 to 0 °C (ice bath). The reaction mixture was then diluted with MTBE (300 mL, 20 vol) and the phases separated. The organic layer was washed with water (2 ^χ 45 mL, 2 x 3 vol), brine (1 ^χ 45 mL, 1 3 vol), dried (MgS0 ₄ ), filtered, and concentrated to obtain the crude product as an oil. The synthesis was repeated on 1.75 g scale of alcohol 5. The combined crude products from both reactions were purified by flash column chromatography on silica gel (5 - 25% MTBE/heptanes) to obtain the desired product 6 as a semi solid [15.1 g, 71%: R _f = 0.61 (1 :1 MTBE/heptanes)]; ¾ NMR (CDCI3, 300 MHz) δ 7.74-7.71 (m, 2H), 7.67-7.64 (m, 2H), 7.37-7.27 (m, 5H), 7.10- 7.07 (m, 2H), 6.98-6.93 (m, 3H), 5.97 (ddd, J = 17.5, 10.4, 10.0 Hz, 1H), 5.42 (d, J = 4.38 Hz, 1H), 5.38 (s, 1H), 4.68 (dd, J= 12.3, 12.3 Hz, 2H), 4.45 (d, /= 5.37 Hz, 1H), 4.41 (d, J= 5.58 Hz, 1H), 3.99-3.82 (m, 3H), 3.65 (dd, J = 13.6, 3.2 Hz, 1H).

Synthesis of Aldehyde 7 and Carboxylic Acid 8

A solution of alkene, 6 [1 g, 2.34 mol] in DCM (60 mL, 60 vol) was sparged with ozone at <-70 °C (dry ice-acetone) for 25 min using house air as oxygen source to generate the ozone. Once the reaction was deemed compete (TLC, 1 :1 MTBE/heptanes), the solution was sparged with nitrogen for 20 min to remove residual ozone. The reaction was quenched with dimethyl sulfide (1.7 mL, 23.4 mmol, 10 equiv) while maintaining the reaction mixture at <-70 °C (dry ice-acetone). The cold bath was removed and the mixture was allowed to warm to ambient temperature. The reaction mixture was concentrated under reduced pressure and further dried under high vacuum to obtain the crude aldehyde as a thick oil (1.12 g, >99%, _f = 0.36, 1 :1 MTBE heptanes). The reaction was repeated at 13 g scale of 6. The two lots of crude aldehyde were combined and subjected to the Pinnick oxidation without further purification.

The crude aldehyde 7 [14.06 g], was taken into a mixture of tetrahydrofuran, iBuOH, and water (105 mL, 105 mL, 70 mL, 3:3:2, 20 vol) along with NaH ₂ P0 (15.6 g, 130 mmol, 4 equiv) and 2-methyl-2-butene (34.4 mL, 324 mmol, 10 equiv). The solution was cooled (15 ± 5 °C, water bath). Sodium chlorite (3.9 g, 43 mmol, 1.33 equiv) was added to the batch and the resulting solution was stirred at ambient temperature for 4 nr. The completion of the reaction was confirmed by TLC analysis (1:1 MTBE/heptanes and 5% MeOH in DCM). The reaction was then quenched with brine (280 mL, 20 vol) and the product extracted into DCM (3 * 280 mL, 3 x 20 vol). The organic layers were dried (MgSC ), concentrated under reduced pressure to obtain the crude acid as a thick oil. The crude acid was purified by flash column chromatography over silica (5 - 100% MTBE/heptanes followed by 5 - 20% MeOH/DCM). Fractions containing the acid were combined and concentrated under reduced pressure to afford acid 8 as a white solid [2.64 g, 18%: R _f = 0.33, 5:95 MeOH/DCM)]; ¾ NMR (CDC1 ₃ , 500 MHz) δ 7.78 (dd, J= 5.5, 3.0 Hz, 2H), 7.70 (dd, J= 5.5, 3.0 Hz, 2H), 7.43-7.40 (m, 2H), 7.37-7.29 (m, 3H), 7.20-7.19 (m, 2H), 7.14- 7.11 (m, 2H), 7.09-7.05 (m, 1H), 4.76 (d, J = 11 Hz, 1H), 4.65 (dd, J = 10.9, 9.4 Hz, 2H), 4.55 (d, J= 11.8 Hz, 1H), 4.13 (ddd, J = 6.2, 6.2, 3.1 Hz, 1H), 4.1 (d, /= 3.0 Hz, 1H), 3.98 (dd, J = 14.2, 6.2 Hz, 1H), 3.89 (dd, J = 14.2, 6.2 Hz, 1H).

Synthesis of Compound 9

A round bottomed flask equipped with a magnetic stirring bar, and a thermocouple probe was charged with a solution of phthalimide-protected amino acid 8 [2.5 g, 5.61 mmol, 1.0 equiv] in THF (28 mL, 11 vol, bulk solvent grade). To the clear, yellow solution was added deionized water (15 mL, 6 vol) and the resulting mixture cooled to 5 °C. Methylamine solution in water (5.0 mL, 40 wt%, 56.1 mmol, 10 equiv) was then added to the batch, which was warmed to ambient temperature (21 - 23 °C) and stirred for 22.5 hours. Analysis of an aliquot from the reaction mixture by LCMS indicated the reaction was complete. The reaction mixture was then concentrated in vacuo to a yellow solid residue, removing all excess methylamine. The residue was taken up in THF (60 mL, 24 vol) and water (30 mL, 12 vol), cooled to 0 - 5 °C, and to the crude amino acid solution was added potassium carbonate (3.9 g, 28.26 mmol, 5.0 equiv), followed by benzylchloroformate (1.4 mL, 9.81 mmol, 1.75 equiv). The batch was warmed to ambient temperature and the reaction allowed to proceed for 25.5 hours. Analysis of an aliquot at this time point by LCMS indicated a complete conversion of the amino acid to the carbamate. The reaction mixture was concentrated under reduced pressure to remove most of THF, the aqueous residue was diluted with water (30 mL, 12 vol) and the pH adjusted with 2N HC1 to approximately pH 5 (pH paper strip). The crude product was extracted with chloroform (3 x 60 mL), the extracts washed with water (1 x 60 mL) and with aqueous NaCl (1 x 60 mL), dried (MgS0 ₄ ) and concentrated in vacuo to a yellow, mobile oil (3.52 g) which was purified by flash column chromatography on silica gel (50 wt. equiv; elution with 0 - 5% MeOH in CHCI3) to afford 9 as a yellow oil, which partially solidified upon further drying under high vacuum [2.22 g, 88.1% yield over two steps]. Ή NMR (DMSO, 500 MHz) δ 12.92 (s, 1H), 7.43 - 7.23 (m, 15H), 5.04 (s, 2H), 4.67 (d, J= 11.10 Hz, 1H), 4.58 (d, J = 11.10 Hz, 1H), 4.48 (d, J= 11.05 Hz, 1H), 4.42 (d, J= 11.05 Hz, 1H), 4.09 (d, J -- 2.95 Hz, 1H), 3.96 (ddd, J = 6.30, 6.30, 3.15 Hz, 1H), 3.29 (dd, J = 6.30, 6.30, 2H).

Synthesis of Cvclopropyl Amino Acids

2 3a,3b

™ .

CH ₂ C1 ₂

5b K ₂ CQ ₃ _

water, THF

6b 7b

Ethyl-2-(tert-Butyldimethylsilyloxy)acrylate (2)

A solution of ester 1 (4.00 g, 34.4 mmol) and triethylamine (4.79 mL, 34.4 mmol) in anhydrous dichloromethane (170 mL) was cooled to 0 °C under nitrogen and iert-butyldimethylsilyltrifluoromethane sulfonate (8.31 mL, 36.2 mmol) was added dropwise. The resulting solution was stirred vigorously at reflux for 4 h. The solvent was then carefully evaporated, the residue was dissolved in Et^O (170 mL), and the organic phase was washed with water (3 χ 50 mL). The organic phase was dried (Na ₂ S0 ), filtered, and concentrated. The residue was purified by silica gel chromatography eluting with 0-20% diethyl ether/hexanes to afford 2 (4.89 g, 62%) as a clear oil: ¾ NMR (500 MHz, CDC1 ₃ ) δ 5.50 (d, J =1.0 Hz, 1H), 4.85 (d, J= 1.0 Hz, 1H), 4.21 (q, J= 7.0 Hz, 2H), 1.31 (t, J= 7.0 Hz, 3H), 0.95 (s, 9H), 0.16 (s, 6H). 2-/^Butyl-l-Ethyl-l-(/er^butyldimethylsilyloxy)cyclopropane- l,2-dicarboxylate (3a and 3b)

A mixture of ethyl-2-(iert-butyldimethylsilyloxy)acrylate (2, 500 mg, 2.17 mmol) and Cu(acac) ₂ (0.011 g, 0.043 mmol) was heated at 80 °C. A solution of tot-butyl diazoacetate (463 mg, 3.25 mmol) in benzene (5 mL) was added to the reaction mixture over 2 h. After this time, the reaction mixture was cooled to room temperature and concentrated. The residue was purified by silica gel chromatography eluting with 0-10% diethyl ether hexanes to afford both diastereomers 3a (0.119 g, 16%) and 3b (0.235 g, 31%) as clear oils. 3a: Ή NMR (500 MHz, CDC1 ₃ ) δ 4.25-4.13 (m, 2H), 2.28 (dd, J= 7.5, 2.0 Hz, 1H), 1.73 (dd, J= 7.5, 2.0 Hz, 1H), 1.59 (dd, J= 9.5, 4.0 Hz, 1H), 1.46 (s, 9H), 1.29 (t, J = 7.5 Hz, 3H), 0.90 (s, 9H), 0.18 (s, 3H), 0.12 (s, 3H); ESI MS m/z 367 [M + Na] ⁺ ; 3b: ¾ NMR (500 MHz, CDCI ₃ ) δ 4.23 (dq, 7= 11.0, 7.0 Hz, 1H), 4.13 (dq, J= 11.0, 7.0 Hz, 1H), 2.11 (dd, J= 10.0, 1.5 Hz, 1H), 1.85 (dd, / = 5.5, 2.5 Hz, 1H), 1.43 (s, 9H), 1.54 (dd, 7 = 10.0, 4.0 Hz, 1H), 1.28 (t, J = 7.5 Hz, 3H), 0.86 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); ESI MS m/z 367 [M + Na] ⁺ .

2-(tert-Bulyldimethylsilyloxy)-2-(ethoxycarbonyl)cyclopro panecarboxylic Acid (4a and 4b)

A mixture of dicarboxylate 3a and 3b (0.385 g, 1.12 mmol, 1:2 ratio of 3a/3b), trifluoroacetic acid (0.43 mL), and dichloromethane (0.5 mL) was stirred overnight at room temperature. The solids were filtered, and the filtrate was concentrated. The residue was purified by silica gel chromatography eluting with 0- 100% diethyl ether/hexanes to afford both diastereomers 4a (0.050 g, 15%) and 4b (0.078 g, 24%) as off-white solids. 4a: Ή NMR (500 MHz, CDCI ₃ ) δ 4.25^1.17 (m, 2H), 2.38 (dd, J= 7.5, 1.5 Hz, 1H), 1.81-1.76 (m, 2H), 1.30 (t, J= 7.0 Hz, 3H), 0.90 (s, 9H), 0.21 (s, 3H), 0.13 (s, 3H); ESI MS m/z 289 [M + H] ⁺ ; 4b: Ή NMR (500 MHz, CDCI3) δ 4.22 (q, J= 7.0 Hz, 1H), 2.21 (dd, J= 10.0, 1.5 Hz, 1H), 1.93 (dd, J= 8.0, 2.0 Hz, 1H), 1.52 (dd, J = 6.0, 3.5 Hz, 1H), 1.28 (t, J= 7.0 Hz, 3H), 0.87 (s, 9H), 0.19 (s, 3H), 0.17 (s, 3H); ESI MS m/z 287 [M - H]\ Ethyl-2-(BenzyIoxycarbonylamino)-l-('ert- butyldimethylsilyloxy)cyclopropanecarboxylate (5b)

A mixture of 2-(fert-butyldimethylsilyloxy)-2-

(ethoxycarbonyl)cyclopropanecarboxylic acid (4b, 0.335 g, 1.16 mmol) in toluene (5 mL) under nitrogen was treated with Hiinig's base (0.260 mL, 1.51 mmol) and the mixture was cooled to 0 °C. After this time, DPPA (0.324 mL, 1.51 mmol) was added and the mixture was heated at 90 °C for 30 min, followed by the addition of benzyl alcohol (0.155 mL, 1.51 mmol). After 15 h, the mixture was cooled, diluted with ethyl acetate (75 mL), and washed sequentially with 10% citric acid (2 ^χ 50 mL), water (50 mL), and saturated NaHC<¾ (50 mL). The organic phase was dried (MgS0 ₄ ), filtered, and concentrated. The residue was purified by silica gel chromatography eluting with 10% EtOAc/hexanes to 100% EtOAc to afford the title compound as a clear oil (0.146 g, 30%): ^l H NMR (300 MHz, CDC1 ₃ ) δ 7.34-7.30 (m, 5H), 5.40-5.38 (m, 1H), 5.21- 5.00 (m, 2H), 4.29- 18 (m, 2H), 4.16-4.09 (m, 1H), 1.50-1.47 (m, 2H), 1.30 (t, J= 7.2 Hz, 3H), 0.88 (s, 9H), 0.26-0.07 (m, 6H); Multimode (APCI+ESI) MS m/z 295 [M + H] ⁺ .

Ethyl 2-(Benzyloxycarbonylamino)-l-hydroxycyclopropanecarboxylate (6b)

To a solution of ethyl 2-(benzyloxycarbonylamino)-l-(ferf- butyldimethylsilyloxy)cyclopropanecarboxylate (1.45 g, 3.69 mmol) in THF (35 mL) under N2 was added HF ^« pyridine (1.0 mL, 38 mmol). The reaction mixture was stirred for 5 h. After this time, additional HF»pyridine (1.0 mL, 38 mmol) was added and stirring was continued for 19 h. The reaction mixture was then cooled to 0 °C and diluted with Et ₂ 0 (150 mL). The mixture was then carefully quenched with saturated aqueous NaHC(¾ until gas evolution ceased. At this time, the organic layer was separated and the remaining aqueous layer was extracted with EtiO (300 mL). The combined organic layers were washed with brine (200 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated in vacuo. Purification by silica gel chromatography eluting with 20%-50% EtOAc/hexanes afforded the title compound (0.960 g, 93%): ¾ NMR (300 MHz, CDCI3) δ 7.34-7.30 (m, 5H), 5.11-t.83 (m, 3H), 4.21 (q, J= 7.2 Hz, 2H), 3.37- 3.25 (m, 2H), 1.73-1.68 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 1.14-1.06 (m, lH); ESI MS m z 280 [M + H] ⁺ .

2-(Benzylox carbonylamino)-l-hydrox cyclopropanecarboxylic acid (7b)

To a 0 °C solution of ethyl 2-(benzyloxycarbonylamino)-l- hydroxycyclopropanecarboxylate (6b, 12.5 g, 44.7 mmol) in THF (100 mL) was added K2CO3 (24.7 g, 179.0 mmol) as a solution in ¾0 (300 mL). The reaction was allowed to warm to room temperature and stirred for 4 h and then additional ¾0 (200 mL) was added. After stirring an additional 18 h at room temperature the reaction was concentrated to remove most of the THF. The remaining aqueous solution was washed with Et ₂ 0 (2 x 500 mL), acidified with 2 N HC1 to pH 2, and then extracted with EtOAc (5 200 mL). The combined EtOAc layers were washed with brine (500 mL), dried (Na2SC> ), filtered and concentrated in vacuo to afford the title compounds (7.75 g, 69%) as a mixture of diastereomers. The mixture was triturated with Et ₂ 0 to afford a white solid as mostly the major diastereomers. The supernatant was concentrated and then triturated with Et ₂ 0 to afford a clean mixture of both diastereomers. Major Diastereomer: Ή NM (300 MHz, MeOD) δ 7.50-7.14 (m, 5H), 5.22^i.96 (m, 2H), 3.23-3.10 (m, 1H), 1.60 (dd, J = 8.9, 6.3 Hz, 1H), 1.10 (t, J= 6.2 Hz, 1H); Multimode (APCI + ESI) MS m/z 250 [M - Hf. Mixture of Diastereomers: *H NMR (300 MHz, MeOD) δ 7.45-7.14 (m, 5H), 5.24-5.01 (m, 2H), 3.25-3.15 (m, 0.46H), 3.14-3.01 (m, 0.54H), 1.71-1.53 (m, 1H), 1.42 (dd, J = 9.1, 6.4 Hz, 0.54H), 1.12 (t, = 6.2 Hz, 0.46H); Multimode (APCI + ESI) MS m/z 250 [M - H] ^~

REPRESENTATIVE COMPOUNDS

Example 1

Sodium carbonate (55.0 g, 0.523 mol) and Cbz-Cl (20.00 mL, 0.139 mol) were added to paromomycin sulfate 1 (30.00 g, 0.0271 mol) in water (500 mL). After 35 hours under vigorous stirring, the water was decanted and the white precipitate was washed with water (2 x). A solution of triethylamine (97.00 mL, 0.697mol) in methanol (600 mL) was added, followed by Cbz-Cl (25.00 mL, 0.174 mol). After 24 hours, dimethylamine (100 mL of a 40% aqueous solution) was added to quench the remaining Cbz-Cl. The solvents were evaporated and the oil was washed with 3% methanol in ether (2 x) and water. The resulting sticky solid was co-distilled with pyridine (200 mL) three times and at ½ of the volume of the third co-distillation, toluene (200 mL) was added and the solvents were evaporated to dryness. Another co- distillation with toluene (300 mL) was done before heating the flask at 60°C under 10 mm Hg vacuum for 12 hours. Freshly distilled benzaldehyde (400 mL) was added to the resulting white solid and sonication was used to form a solution. To the stirred mixture was added 4 A molecular sieves (15 g) and formic acid (20.00 mL, 0.530 mol). After stirring for 12 hours at room temperature, the mixture was added dropwise to a stirred ice-cold solution of saturated aqueous a ₂ C0 ₃ , extracted with ethyl acetate (3 times), and the organic layer was washed with water, brine and dried over Na ₂ SO<|. The solvent was evaporated to dryness and excess benzaldehyde was removed under vacuum to afford a crude solid, which was purified by flash column chromatography over silica gel (3% MeOH/CH ₂ Cl ₂ ) to obtain pure 2 (23.89 g, 63%). The spectroscopic analysis of the resulting material was consistent with data reported in the literature for the identical material (Hanessian S., Takamoto T., Masse R., Patil G.; Aminoglycoside antibiotics: Chemical conversion of neomycin B, paromomycin, and lividomycin B into bioactive pseudosaccharides; Can. J. Chem., 1978, 56, 1482).

To a stirred solution of 2 (1.35g, 0.98 mmol) in dry dichloromethane (20 mL) was added 2,4,6-collidine (1.07g, 8.82 mmol) and TBSOTf (1.811g, 6.86 mmol) at 0°C. The reaction mixture was slowly brought to room temperature and stirred for 12 hours. A few drops of water were added to quench the excess TBSOTf, followed by extraction with dichloromethane. The organic layer was washed with brine and dried over anhydrous Na2S04, followed by concentration of the solvent to give the corresponding crude product, which was purified by flash column chromatography to give 3 (1.048g, 55 %): [a] _D = + 16° (c 0.6, CHC1 ₃ ). ESI/MS calcd for C ₁₀ oHi49N ₅ 024Si5 (M+H ^4" ) 1944.94; found 1946.

To a stirred solution of 3 (330 mg, 0.17 mmol) in dry DMF (6 mL) was added 60 % NaH in mineral oil (8 mg) at 0°C with stirring continued for an additional 6 hours at 0°C. A few drops of saturated ammonium chloride solution were added, followed by extraction with ethyl acetate. The organic layer was washed with brine and dried over anhydrous a2SC>4, followed by concentration of the solvent to yield the corresponding crude product, which was purified by flash column chromatography to yield 4 (180 mg, 58 %): [a] _D = + 18°(c 0.5, CHC1 ₃ ). ESI/MS calcd for CsHniNsOzsSij (M+H ⁺ ) 1836.89; found 1837.6.

To a stirred solution of 4 (190 mg, 0.1 mmol) in DMF (7 mL) was added aqueous LiOH (0.7 mL, 9 mg, 0.21 mmol) with stirring continued for an additional 3 hours at room temperature. A few drops of saturated ammonium chloride solution were added, followed by extraction with ethyl acetate. The organic layer was washed with brine and dried over anhydrous Na2SC>4, followed by concentration of the solvent to yield the corresponding crude product, which was purified by flash column chromatography to yield 5 (100 mg, 53 %): [a] _D = + 13°(c 0.3, CHC1 ₃ ). ESI/MS calcd for C _M H ₁₄ 3N ₅ 0 ₂₂ Si ₅ (M+H ⁺ ) 1810.91; found 1811.3.

To a stirred solution of benzyloxy 4-hydroxy aminobutric acid (27 mg, 0.11 mmol) and N-hydroxy succinimide (12 mg, 0.11 mmol) in dry THF (2 mL) was added DCC (22 mg, 0.11 mmol) with stirring continued for an additional 1 hour at room temperature. To this reaction mixture 5 (95 mg, 0.053 mmol) in dry THF (2 mL) and triethyl amine (15 μί, 0.11 mmol) were added with stirring continued for 12 hours at room temperature. Evaporation of the solvent followed by purification by flash column chromatography yielded 6 (80 mg, 74 %): [<x] _D = + 1 °(c 0.4, CHC1 ₃ ).

Compound 6 (90 mg, 0.044 mmol) was dissolved in dry pyridine (2 mL), HF Pyr (2 mL) was added at 0°C, and the reaction was slowly brought to room temperature and stirred for 2 days. Water was added and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SC and evaporated to give the crude product, which was purified by column chromatography to yield 7 (50 mg, 77 %): [a] _D = + 20°(c 0.6, CHC1 ₃ ). ESI/MS calcd for C ₇ 4Hg _{6 6} 026 (Μ+ΤΓ) 1475.56; found 1475.7.

To a solution of 7 (270 mg, 0.183 mmol) in pyridine (2 mL) was added acetic anhydride (1 mL) with stirring maintained for 24 hours at room temperature. Water (10 mL) was added and the precipitated product was filtered. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated CUSO , brine, dried over anhydrous a2SC combined with the precipitated product and evaporated to provide the crude material, which was purified by column chromatography to yield 8 (300 mg, 93%): [a] _D = + 7.5°(c 0.2, CHC1 ₃ ). ESI/MS calcd for C ₈ gHiooN ₆ 0 ₃ 3 (Μ+ί ) 1768.63; found 1769.8.

Compound 8 (300 mg, 0.17 mmol) was stirred in an acetic acid/water mixture (20 mL, 4:1) at room temperature for 4 days. Water was added and the precipitated product was filtered. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over anhydrous Na2S(¾, combined with the precipitated product and evaporated to yield the crude material, which was purified by column chromatography to yield 9 (280 mg, 98%): [a] _D = + 10.7°(c 0.3, CHCI3). HRMS calcd for C ₈ iH ₉₇ N ₆ 0 (M+H*) 1681.60911; found 1681.60830.

To a solution of 9 (290 mg, 0.17 mmol) in pyridine (2 mL) was added TsCl (36 mg, 0.19 mmol) and DMAP (5 mg, 0.041 mmol) with stirring maintained for 12 hours at room temperature. Additional TsCl (36 mg, 0.19 mmol, 1.1 eq) was added and the reaction was stirred for an additional 8 hours at room temperature. Water was added and the precipitated product was filtered. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over anhydrous Na2SC combined with the precipitated product and evaporated to yield the crude material, which was purified by column chromatography to yield 10 (300 mg, 96%): [a] _D = + 14.8°(c 0.25, CHCI3). HRMS calcd for C ₈₈ Hi02N ₆ O35S (M+H ⁴ ) 1835.61796; found 1835.61976.

To a solution of 10 (320 mg, 0.175 mmol) in dry DMF (3 mL) was added NaN3 (113 mg, 1.74 mmol) and stirring was maintained for 24 hours at 70°C. Water was added and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over anhydrous Na ₂ SC>4 and evaporated under reduced pressure to yield the crude, which was purified by column chromatography to yield Compound 11 (252 mg, 84%): [a] _D = + 11.3°(c 0.3, CHC1 ₃ ). ESI/MS calcd for C ₈ iH ₉₅ N<>0 ₃₂ (M+H ⁺ ) 1705.61; found 1707.0.

To a stirred solution of 11 (115 mg, 0.067 mmol) in pyridine (2 mL) at 0°C was added MsCl (10 μί, 0.13 mmol) and the reaction mixture was slowly brought to room temperature and stirred for 3 hours. Few drops of water were added to quench the reaction, which was extracted with ethyl acetate. The organic layer was washed with saturated aq. C SO4, water, brine and dried over anhydrous Na2SC>4, followed by concentration of the solvent to yield the corresponding crude product, which was dissolved in methanolic NaOMe (pH = 10-11) and stirred at room temperature for 12 hours. Dry ice was added to quench the reaction, which was extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na2SC>4, followed by concentration of the solvent to yield the corresponding crude product, which was dissolved in pyridine (2 mL) and acetic anhydride (2 mL) and stirred at room temperature for 12 h. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated NaHC<¾, water and brine. Solvent evaporation yielded the crude material, which was purified by flash column chromatography to yield 12 (85 mg, 77 %): [o] _D = + 30.5°(c 0.8, CHC1 ₃ ). ESI/MS calcd for C ₇₉ H ₉₁ N ₉ 0 ₃ o (M+H ⁺ ) 1646.61; found 1647.5.

To a stirred solution of 12 (80 mg, 0.049 mmol) in acetone (5 mL) was added Nal (36 mg, 0.24 mmol), followed by NaOAc (2 mg, 0.024 mmol) and AcOH (0.1 mL) and the reaction was refluxed at 75° C for 24 hours. The solvent was evaporated and the reaction was extracted with ethyl acetate. The organic layer was washed with water, saturated NaHCC , brine, dried over anhydrous Na ₂ S0 ₄ , and concentrated to yield the crude product. This was dissolved in pyridine, cooled to 0°C and MsCl (7 μ ) was added. The reaction mixture was stirred at room temperature for 3 h. Then one drop of methanol was added and the reaction was heated at 70°C for 24 h. Usual work-up followed by column chromatography yielded 13 (56 mg, 76 %): [α]ο = + 15.2 ⁰ (c 0.5, CHC1 ₃ ). ESI/MS calcd for C ₇₉ H ₉₁ N ₉ 02 ₉ (M+H ⁺ ) 1630.61; found 1631.5.

Compound 13 (56 mg, 0.034 mmol) was dissolved in methanolic NaOMe (10 mL, pH = 10-11) and stirred at room temperature for 12 hours. Dry ice was added to quench the reaction, which was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over anhydrous Na2SC concentrated to a crude, which was purified by column chromatography to yield 14 (31 mg, 66 %):[( + 30.3 ⁰ (c 1, CHC1 ₃ ). ESI/MS calcd for C67H79N9O23 (M+H ⁺ ) 1378.39; found 1379.1.

Compound 14 (50mg, 0.036 mmol) was dissolved in 80% AcOH/water

(20 mL) and palladium hydroxide on carbon (10 mg, 20% Pd) was added. The reaction was stirred under 1 atm hydrogen at room temperature, monitoring closely by LC/MS. The stirring was periodically stopped while awaiting LC/MS data. The reaction was judged to be complete when the majority of the benzyl carbamates had been removed, but the double bond had not been fully reduced. At this point there was an approximately 1 :1 ratio (by mass spec) of reduced to unreduced double bond. The catalyst was removed by filtration and washed with water, and the combined washings were dried on the lyophilizer. The resulting solid was taken up in water, basified with aqueous ammonia and purified by reverse-phase HPLC (Method 1) to yield 15 (2 mg, 0.0029 mmol, 8%): ESI/MS calcd for C27H51N7O13 682.4 (M+H ⁺ ); found: 682.2.

To a stirring solution 15 (0.56g, 0.83 mmol) in H ₂ 0 (8 mL) was added a solution of Na ₂ C0 ₃ (0.263g, 2.48 mml) in H ₂ 0 (5 mL), followed by THF (12 mL). NaHC0 ₃ (0.208g, 2.48mmol) was added, followed by Cbz-OSu (1.68g, 6.62 mmol), and the reaction was stirred overnight. The THF was evaporated off, and the reaction was diluted with ethyl acetate. The organic layer was separated and washed with sat. NH4CI, sat. aq. NaHC(¾, brine, dried over Na ₂ SC filtered and concentrated to a crude, which was purified on a 2-inch reverse phase HPLC column (Method 2) to yield the desired product 16 (0.876g, 0.59 mmol, 71% yield): MS m/z calcd for C ₇ 5Hg7N ₇ 0 (M+Na ⁺ ) 1507.6, found 1508.7.

To a stirring solution of 16 (0.032 g, 0.022 mmol) in acetone (0.9 mL) and H ₂ 0 (0.06 mL) was added hydroquinine (0.28 mg, 0.861 μπιοΐ), followed by NMO (2.5 μΐ,, 0.024 mmol) and Os0 ₄ (4% w/w in ¾0, 5.47 μL, 0.861 μπιοΐ), and the reaction was stirred overnight. Additional NMO (for a total of 3.3 eq) and Os0 ₄ (for a total of 0.4 eq) were added portionwise over 4 days. The reaction mixture was then purified on a 1-inch reverse phase HPLC column (Method 2) to yield 17 (MS m/z calcd for C ₇ 5H8 _{9 7} 027 (M+Na^ 1542.6, found 1542.6), and 18 (MS m/z calcd for C ₇₅ H ₈₉ N ₇ 027 (M+Na ⁺ ) 1542.6, found 1542.6).

To a stirring solution of 17 (13.2 mg, 0.0087 mmol) in AcOH (2 mL) and ¾0 (0.5mL) was added 5% weight Pd/C (5 mg) and the reaction was stirred under a hydrogen atmosphere for 3 hr. The catalyst was removed by filtration through Celite, and washed with ¾0, and the combined washings were dried on the lyophilizer, to yield compound 19 as the acetate salt (8mg, 0.0074 mmol, 85%): MS m/z calcd for C27H53N7O15 (M+H ⁺ ) 716.4, found 716.4; CLND 98.6% purity.

To a stirring solution of 18 (19.5 mg, 0.013 mmol) in AcOH (2 mL) and H2O (0.5mL) was added 5% weight Pd/C (5 mg) and the reaction was stirred under a hydrogen atmosphere for 3 hr. The catalyst was removed by filtration through Celite, and washed with H2O, and the combined washings were dried on the lyophilizer, to yield 20 as the acetate salt (12mg, O.Ol lmmol, 84%): MS m/z calcd for C27H53N7O15 (M+H ) 716.4, found 716.3; CLND 93.8% purity.

Example 2

To stirring THF (5 mL) at 0°C were added Lil (0.746g) and AcOH (0.16 mL), and the resulting solution was added to a solution of compound 1 (1.53g, 0.929mmol) in THF (8.5 mL). The reaction was placed in a 60 °C oil bath for 105 min, it was then allowed to cool to room temp. The reaction mixture was partitioned between water (25 mL) and EtOAc (25 mL) (very slow), and the aqueous layer was separated. The organic layer was washed with Na ₂ SC>3 (1.3 M, lOmL), sat. aq.

NaHCC (20 mL), brine (10 mL), dried over MgSC filtered and concentrated to a crisp foam 2 (992 mg, 0.559 mmol): MS m/z calcd. for C79H92IN9O30 1796.5 (M+Na*), found 1796.5.

To a stirring solution of compound 2 (992 mg, 0.559 mmol) in dioxane (15 mL) was added TEA (0.117 mL, 0.839 mmol), followed by Raney nickel and the reaction was stirred under a hydrogen atmosphere for 30min, with its progress monitored by HPLC. More Raney nickel and TEA (0.117 mL, 0.839 mmol) were 5 added and the reaction was stirred under a hydrogen atmosphere for 1 hour. The reaction was filtered using a 0.2 μπι syringe filter and rinsed with dioxane (2 x 5 mL). The filtrate was concentrated and the residue was dried under high vacuum. The residue was redissolved in EtOAc (50 mL) and washed with 1M citric acid (50 mL), brine, sat. aq. NaHC(¾ (50 mL), brine, dried over MgSC filtered and concentrated to 10 yield compound 3 (417 mg, 0.257 mmol, 30%): MS m/z calcd for C79H95N7O30 1622.6 (M+H ⁺ ), found 1622.6.

15 To a stirring solution of compound 3 (415 mg, 0.256 mmol) in water (10 mL) and acetonitrile (10 mL) at 0°C, was added benzyl 2,5-dioxopyrrolidin-l-yl carbonate (76 mg, 0.307 mmol) followed by NaHC0 ₃ (27.9 mg, 0.332 mmol) and the reaction was stirred at rt for 2 hr. The reaction was cooled to 0°C and quenched with 3- (dimethylamino)-l -propylamine (19 μΐ), followed by the addition of EtOAc (35 mL).

20 The reaction mixture was washed with brine, and the aqueous layer was extrated with EtOAc (20 mL). The combined organic layers were washed with 1 M citric acid, brine, sat. aq. NaHC(¾, brine, dried with MgSC filtered and concentrated to a crude, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield compound 4 (248mg, 0.141 mmol, 55%): MS m/z calcd for C87H _I0 iN ₇ O32 1778.7 (M+Na ⁺ ), found

25 1778.7.

To a stirring solution of compound 4 (86 mg, 0.049 mmol) in pyridine (5 mL) was added methanesulfonyl chloride (0.057 ml, 0.734 mmol) and the reaction was stirred at room temperature overnight. The reaction was then heated at 50 °C for 2 hours and at 100°C overnight. DMF (1.0 mL) was added and the reaction was heated at 120°C overnight. The reaction was partitioned between water (25mL) and EtOAc (25 mL). The aqueous layer was back extracted with EtOAc (20 mL) and the combined organic layers were washed with 1M citric acid/brine (4:1, 2 x 50 mL), brine (10 mL), sat. aq. NaHCC (50 mL), brine, dried over MgSC filtered and concentrated to a brown oil, which was purified on a 1-inch reverse phase HPLC (Method 2) to yield compound 5 (28 mg, 0.017mmol, 35%) as a white solid: MS m/z calcd for C ₈₀ H ₉₃ N ₇ O3i:1648.6 (M+H ⁺ ), found 1648.3.

To a stirring solution of compound 5 (45 mg, 0.027 mmol) in methanol (1.5 mL) was added methylamine (40% in water, 0.381 mL), and the reaction was stirred at room temperature for 6 hours. The reaction mixture was concentrated to a crude aqueous slurry of 6, which was carried through to the next step without further purification. MS m/z calcd for C68H81N7O25 1418.5 (M+Na ⁺ ), found 1418.6

To a stirring solution of compound 6 (37.7 mg, 0.027 mmol) in acetic acid (1.6 ml) and water (0.400 ml) was added Pd/C (22 mg), and the reaction was stirred under a hydrogen atmosphere for 35min. The Pd/C was removed by filtration and rinsed with water. The resulting filtrate was diluted with water (30 mL) and lyophilized to yield compound 7 (32 mg, 0.027 mmol, 100%): MS m/z calcd for C ₂₈ H5iN ₇ Oi5 726.3 (M+H ⁺ ), found 726.3.

To a stirring solution of compound 7 (32 mg, 0.031 mmol) in water (3 ml) was added 2M NaOH (0.624 ml, 1.248 mmol) and the reaction was placed in a 50 °C heating block for 3.5 hours. The reaction mixture was acidified with HFBA (190 μΐ/) and was purified on a 1-inch reverse phase HPLC (0.1% HFBA/H ₂ 0, 0.1% HFBA/CH ₃ CN) to yield 8 (5 mg, 0.0071mmol, 23%yield, CLND 100% purity):MS m/z calcd for C ₂ 7H53 ₇ 0 ₁₄ 700.4 (M+H ⁺ ), found 700.3, as its HFBA salt.

Example 3

To a stirring solution of 1 (500mg, 0.359 mmol) in methanol (8 mL) was added 1% TFA (0.08 mL, 1 mmol) and the reaction was heated to 60 °C for 75min. Cone, ammonium hydroxide (80 μί) was added and the reaction was concentrated to a crude, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield 2 (297 mg, 0.221 mmol, 62%): MS m/z calcd for C ₆ oH ₇₃ Nc,024 (M+H ⁺ ) 1304.5, found: 1304.1.

To a stirring solution of 2 (210mg, 0.161mmol) in AcOH/water (4 mL, 4:1) was added Pd/C (139 mg) and the reaction was stirred under a hydrogen atmosphere for 65min. The Pd/C was removed by filtration and rinsed with water (3 x lmL). The filtrate was diluted with water (40 mL) and lyophilized to yield 3 as its acetate salt (106mg, 0.096 mmol, 60%): MS m/z calcd for C ₂ 8H ₅ iN ₇ 0i ₆ (M+H ^4" ) 742.3, found: 742.3

To a stirring solution of 3 (100 mg, 0.096mmol) in ¾0 (6.25 mL) was added 2M NaOH (0.432 mL, 0.864 mmol) and the reaction was heated to 50 °C overnight. Additional 2M NaOH (0.05 mL) was added and the reaction was heated at 50°C overnight. Additional 2M NaOH (0.05 mL) was added and the reaction was heated at 50°C overnight. Additional 2M NaOH (0.05 mL) was added and the reaction was heated at 50°C overnight. The reaction mixture was acidified with HFBA, and was purified on a 1-inch reverse phase HPLC (0.1% HFBA/¾0 and 0.1% HFBA/CH ₃ CN), followed by purification on a 1-inch reverse phase HPLC column (50 mM NH ₄ OH/ H ₂ 0 and 50 mM NH ₄ OH/CH ₃ CN) to yield 4 (21mg, 0.029 mmol, 30% yield): MS m/z calcd for C ₂ 7H5 ₃ N ₇ Oi5 (M+H ⁴ ) 716.4 found 716.1 CLND 100% purity.

Example 4

To a stirring solution of (5)-4-(benzyloxycarbonylamino)-2- hydroxybutanoic acid (1, 612 g, 2.41 mol) in THF (6 L) at 0°C was added DMAP (2.4 g) and pyridine (196 mL, 2.41 mol). After stirring for 20 minutes, benzoyl chloride (280 mL) was added slowly and the reaction was allowed to stir overnight at room temperature. The resulting precipitate was filtered and the filtrate was acidified with 1 M citric acid (1 L). The organic solvent was removed by rotary evaporation and the desired product was extracted with EtOAc (2 x 2L). The combined organic layers were washed with 1 M citric acid (1 L), brine (1 L) and then dried over MgS0 ₄ . Solvent removal under vacuum resulted in a viscous oil, which was purified on a 6-inch reverse- phase HPLC column (Method 2) to yield (5 2-(benzoyloxy)-4- (benzyloxycarbonylamino)butanoic acid (2) as a non-flowing viscous oil (439 g, 51% yield).

To a stirring solution of (5)-2-(benzoyloxy)-4- (benzyloxycarbonylamino)butanoic acid (2, 196 g, 0.548 mol) and pentafluorophenol (111 g, 0.603 mol) in 1.3 L DMF at 0°C was added EDC (126 g, 0.658 mol) and the reaction was stirred overnight. The reaction mixture was diluted with hexanes:EtOAc (1.5 L, 1:1) and with brine/water (1 L, 1:1), and the organic layer was separated and washed with 1 M citric acid (3 x 600 mL), followed by sat. NaHC0 ₃ :lM NaOH (3 x 500 mL, 1:1) and brine (500 mL). The organic layer was dried over MgS0 ₄ , filtered and concentrated under vacuum to yield (S)-4-(benzyloxycarbonylamino)-l-oxo-l- (perfluorophenoxy)butan-2-yl benzoate (3, 255 g, 88% yield) as a white waxy solid that was used in the next step without further purification.

To a stirring solution of nitrophenol (4, 150 g, 1078 mmol) in ethyl acetate (2 L) at 0 ^° C was added Cbz-CI (161.6 mL, 1132 mmol). DIPEA (221.5 mL, 1294 mmol) was then dripped into the reaction over 45 minutes. The reaction was allowed to warm to room temperature and was stirred for 30 minutes. The reaction was then cooled to 0°C and the precipitate was removed by filtration. The organic layer was washed with 1 M citric acid (1.5 L), 1:1 NaHC0 ₃ :lM NaOH (1.5 L), and brine (1.5 L), dried over Na ₂ SC>4 and concentrated to a yellow solid (5, 298.8 g, 100% yield).

Paromomycin sulfate (6, 685 g free base, 1113 mmol) was dissolved in water (4 L) and then ACN (4 L) was slowly added to the reaction. The reaction was cooled to 0°C and then DIPEA (1525 mL, 8910 mmol) was added. O-Cbz-2- nitrophenol (5, 304 g, 1113 mmol) was dissolved in ACN (1 L) and added to the reaction via an addition funnel in one hour. The reaction was stirred for 18 hours and then was concentrated to a red viscous liquid, which was dripped into ethanol (1 mL reaction mixture: 6 mL ethanol) to yield a pale yellow precipitate, which was collected by filtration, washed with ethanol and dried under high vacuum to yield a pale yellow solid (1073 g). MTO Dowex exchange resin (11.5 kg) was suspended in methanol (11.5 L). Next the yellow solid was added to the suspended resin, and the mixture was stirred for 3 hours. The resin was removed by filtration and washed with methanol (14 L). The organic layer was concentrated to 5 L and dripped into ethyl acetate (30 L) to yield 6"'-Cbz-paromomycin (7, 663 g, 79% yield) as a white solid.

To a stirring solution of 6"'-Cbz-paromomycin (7, 98.3 g, 131 mmol) in

850 mL DMF under N ₂ was added 160 mL DIPEA (918 mmol) and the flask was cooled to -50°C (CO2/ACN). A solution of (5)-4-(benzyloxycarbonylamino)-l-oxo-l- (perfluorophenoxy)butan-2-yl benzoate (3, 50 g, 95 mmol) in 340 mL DMF was then added dropwise and the reaction progress was monitored by HPLC. Solvent evaporation under high vacuum yielded a viscous liquid, which was dissolved in ethyl acetate:IPA (500 mL, 3:2), and washed with water : acetic acid (800 ml :34 mL). The aqueous layer was extracted with ethyl acetate (400 mL), followed by EtOAc:IPA (2 x 960 mL, 85:15). The combined organic layers were washed with H2O (500 mL), and the combined aqueous layers were washed with EtOAc:IPA (800 mL 1:1) and then with EtOAc:IPA (200 mL, 1:1). The combined organic layers were then concentrated under vacuum to 175 mL. This solution was diluted with ¾0 (25 mL) and was purified on a 6-inch reverse phase HPLC column (Method 2) to yield (8) (35.6 g, 25% yield): MS: m/z (M+Na ⁺ ) calcd 1111.4, found 1111.4.

6'"-Cbz-l-(W-Cbz-4-amino-2(5)-benzoyloxy-butyryl)-paromomyci n (8, 41.4 g, 28.93 mmol) was dissolved in DMF (300 mL), and the reaction was cooled to 0 ^° C. Af-(benzyloxycarbonyloxy)succinimide (28.84 g, 115.72 mmol) was then added, followed by DIPEA (40.3 mmol, 231.4 mmol) and the reaction was stirred for two 5 hours and then quenched by the addition of iV.iV-dimethylaminopropylamine (4.59 mL, 36.34 mmol, 1.26 eq). The reaction was stirred for 1 hour and then was diluted with ethyl acetate (1.5 L). The organic layer was washed with 5% acetic acid: water (2 x 1L), water (2 x 1L), and brine (1 x 1L), dried over Na2SC>4 and concentrated to a white foam (9, 44.4 g).

To a stirring solution of 9 (7.0g, 4.7mmol) in pyridine (35 mL) was added DMAP (570mg, 4.7mmol) followed by iert-butylchlorodiphenylsilane (4.8mL,

15 18.8mmol) and the reaction was heated at 80°C for 17 hours. The reaction mixture was cooled to room temperature and was partitioned between EtOAc and 1 M citric acid. The organic layer was washed with 1M citric acid, sat. aq. NaHCC>3, brine, dried over MgSC>4, and concentrated to a residue, which was redissolved in EtOAc (25 mL) and dripped into vigorously stirring Et ₂ 0 (350 mL) and precipitation was observed. The

20 precipitate was collected by filtration, washed with Et ₂ 0 and dried under vacuum (5.1g). The filtrate was concentrated and purified by flash chromatography (silica gel, 0% to 10% MeOH/DCM) to yield 10, which was combined with the solid obtained by precipitation to give a total yield of 7.2 g, (3.7 mmol, 78%): MS m/z calcd for Ci06Hi22N ₆ O ₂₇ Si ₂ (M+Na ⁺ ) 1989.8, found 1989.4.

5

To a stirring solution of cycloheptanone (72mL) and triethyl orthoformate (72mL) at room temperature (water bath) was added PTSA (720mg, 3.8mmol) and the reaction was stirred for 20 min. Compound 10 (7.2g, 3.7mmol) was added and the reaction was stirred until complete dissolution was observed. The reaction was then cooled to 0°C for 10 min, imidazole (300 mg, 4.4 mmol) was added, and the reaction was stirred for 5 min. The reaction was quenched with sat. aq. NaHCC and was partitioned with the addition of minimal EtOAc. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine and dried over MgSC The EtOAc was removed by evaporation to give a solution, which was diluted with hexanes (until almost out of solution) and was purified by flash chromatography (silica gel, 100% hexanes, 100% DCM, 0% to 10% MeOH/DCM) to yield 11 (6.0 g, 2.8 mmol, 75%): MS m/z calcd for CnsH^NeOzySia (M+Na*) 2083.9, found 2083.8.

To a stirring solution of 11 (6.0 g, 2.9 mmol) in pyridine (60 mL) was added acetic anhydride (60mL) and the reaction was stirred for 17 hr. Solvent evaporation gave a crude, which was evaporated twice from toluene, to yield 12 (6.8 g): MS m/z calcd for Ci ₂ iH ₁₄ oN ₆ 0 ₃ iSi ₂ (M+Na ⁺ ) 2251.9, found 2251.8.

To a stirring solution of 12 (6.8 g, 3.1 mmol) in THF (68 mL) was added TREAT-HF (2.0 mL, 12 mmol) and the reaction was placed in the refrigerator (4°C) for 3 days. The reaction mixture was partitioned between water and EtOAc. The organic layer was washed with sat. aq. NaHCCb, brine, dried over MgSC and concentrated under vacuum to yield 13 (6.5 g): MS m/z calcd for CiosHmNeOaiSi (M+Na*) 2013.8, found 2013.6.

To a stirring solution of 13 (5.3 g, 2.66 mmol) in benzaldehyde (21.24 mL, 210 mmol) was added TFA (0.205 mL, 2.66 mmol) and the reaction was stirred at rt overnight. The reaction was poured into hexane/Et ₂ 0 (2:1, 450 mL) and the precipitate was removed by filtration and washed with hexane/Et ₂ 0 (2:1). The solid was dissolved in EtOAc and washed with sat. aq. NaHC0 ₃ , brine, dried over MgSC filtered and concentrated under vacuum to yield 14 (4.5g, 2.27 mmol, 85%): MS m/z calcd for CiosHn ₆ N ₆ 0 ₃ iSi (M+Na ⁺ ) 2009.1, found 2008.6.

To a stirring solution of 14 (1.86 g, 0.936 mmol) in pyridine (30 ml) at 0 ^° C was added triflic anhydride (0.316 ml, 1.873 mmol) and the reaction was stirred for 45 min. The reaction was diluted with EtOAc (100 mL) and washed with 1 M citric acid (2 x 100 mL), sat. aq. NaHC0 ₃ (50 mL), brine, dried over MgSC filtered and concentrated under vacuum to yield 15 (2.07 g, 0.977 mmol, MS m/z calcd for Ci06Hii5F3 ₆ O33SSi (M+Na ⁺ ) 2139.7, found: 2139.6), which was carried through to the next step without further purification.

To a stirring solution of 15 (1.984 g, 0.937 mmol) in DMF (50 ml) at 0°C was added lithium iodide (11.28 g, 84 mmol) and the reaction was heated at 60 °C for 5 hr. The reaction was then cooled to it and diluted with EtOAc (200 mL). The organic layer was washed with brine/water (3:1, 200 mL) and the aqueous layer was back extracted with EtOAc (150 ml). The combined organic layers were washed with aq. Na ₂ S0 ₃ (150 mL), sat. aq. NaHC0 ₃ , brine, dried over MgS0 ₄ , filtered and concentrated in vacuum to yield 16 (2.1 g, MS m/z calcd for CiosHnsINeOsoSi 2117.7, found: 2117.6), which was carried through to the next step without further purification.

To a stirring solution of 16 (1.94 g, 0.937 mmol) in AcOH (4 mL), water (3.5 mL), and dioxane (3.5 mL) was added TFA (0.433 ml, 5.62 mmol) and the reaction was stirred at 55 °C for 2.5 hr. The reaction mixture was purified on a 2-inch reverse phase HPLC (Method 2) to yield 17 (350 mg, 0.174 mmol, 18.6%): MS m/z calcd for CesHmlNsOsoSi (M+Na ⁺ ) 2029.6, found: 2029.5.

To a stirring solution of 17 (0.015 g, 0.0075 mmol) in pyridine (0.150 mL) was added tosyl chloride (7.12 mg, 0.037 mmol) and the reaction was stirred for 4hr. Additional TsCl (7 mg) was added and the reaction was stirred for 4 hr. Addditional TsCl (10 mg) was added and the reaction was stirred overnight. The reaction was quenched with water and was partitioned between 1M citric acid and EtOAc. The organic layer was washed with sat. aq. NaHC03, brine, dried over MgS04, filtered and concentrated under vacuum to yield 18 (23 mg), which was carried through to the next step without further purification.

To a stirring solution of 18 (345 mg, 0.160 mmol) in dioxane (9 mL) was added TEA (44 μΐ _^ , 0.319 mmol), followed by Raney Ni (1.5 mL) and the reaction was stirred under a hydrogen atmosphere for 120 min. The reaction mixture was filtered through a 0.45 μπι filter and washed with EtOAc. The organic layer was washed with 0.2 M citric acid, sat. aq. NaHC03, brine, dried over MgS0 ₄ and concentrated under vacuum to yield 19 (354 mg): MS m/z calcd for CiosHnsNeC^SSi (M+Na ⁺ ) 2057.7, found 2057.5.

To a stirring solution of 19 (304 mg, 0.149 mmol) in DMA (3 mL) was added sodium azide (58.2 mg, 0.90 mmol) and the reaction was heated to 70°C for 2 hr. The reaction was cooled to room temperature and was partitioned between EtOAc and water/brine (1 :1). The organic layer was washed with brine, dried over MgSC>4, filtered and concentrated under vacuum to yield 20 (346 mg), which was carried throught to the next step without further purification. MS miz calcd for CgsHinNgOzsSi (M+Na ⁺ ) 1928.7, found 1928.5.

To a stirring solution of 20 (346 mg, 0.149 mmol) in THF (3.5 mL) was added H ₂ 0 (0.35 mL) and ΓΡΑ (0.35 mL), followed by tris(2-carboxyethyl)phosphine hydrochloride (156 mg, 0.54 mmol) and the reaction was vigorously stirred for 3 hr. Additional IPA (0.35 mL) was added and the reaction was stirred overnight. The reaction mixture was partitioned between EtOAc and dilute aq. NaHC<¾. The organic layer was washed with brine, dried over MgSC>4, filtered and concentrated under vacuum to a crude, which was purified on a 1-inch reverse phase HPLC (Method 2) to yield 21 (76 mg, 0.040 mmol, 27%): MS m/z calcd for CSSHIBNTOZ SI (M+H ⁺ ) 1880.7, found 1880.7.

To a stirring solution of 21 (76 mg, 0.040 mmol) in H ₂ 0 (1 mL) and CH ₃ CN (1 mL) at 0°C was added benzyl 2,5-dioxopyrrolidin-l-yl carbonate (12.08 mg, 0.048 mmol) followed by NaHC<¾ (4.41 mg, 0.053 mmol) and the reaction was stirred at it for 3.5 hr. The reaction was cooled in an ice bath and quenched with 3- (dimethylamino)-l -propylamine (3.05 μΐ., 0.024 mmol), followed by the addition of EtOAc (25 mL). The organic layer was washed with brine, 1 M citric acid, brine, sat. aq. NaHC0 ₃ , brine, dried over Na ₂ S0 ₄ , filtered and concentrated under vacuum to yield 22 (58 mg, 0.029 mmol, 71.2%).

To a stirring solution of 22 (58 mg, 0.029 mmol) in DMSO (0.4 ml) was added acetic anhydride (0.4 ml) and the reaction was stirred at room temperature for 4 days. The reaction was diluted with EtOAc (25 mL) and washed with LbO/sat. aq. NaHC0 ₃ (1:1, 2 x 25 mL) and brine (25 mL), dried over MgS0 ₄ , filtered and concentrated under vacuum to an oil, which was evaporated with toluene and dried on the high-vacuum to yield 23 (61.3 mg): MS m/z calcd for Cio Hii ₇ N ₇ 03iSi (M+Na ⁺ ) 2034.8, found 2034.5

To a stirring solution of 23 (60 mg, 0.030 mmol) in THF (1 ml) at 0°C was added L-selectride (0.596 ml, 0.060 mmol) and the reaction was stirred for 10 min. The reaction mixture was partitioned bwtween EtOAc (25 mL) and water/brine (1 : 1, 25 mL). The organic layer was washed with brine, dried over MgS0 ₄ , filtered and concentrated under vacuum to a crude, which was dissolved in methanol (1 ml) and methylamine (40% in H ₂ 0, 0.250 ml) and the reaction was stirred at room temperature for 18 hrs. The organic solvent was removed by evaporation, then brine was added and the reaction mixture was extracted with EtOAc (10 mL, then 5 mL). The combined organic layers were dried over MgS0 ₄ , filtered and concentrated under vacuum to yield 24 (59 mg): MS m/z calcd for C91H107N7O26S1 (M+Na ^" ") 1764.7, found: 1764.6.

To a stirring solution of 24 (59 mg, 0.030 mmol) in THF (1.1 ml) in a plastic tube at 0°C was added HF-pyridine (0.053 ml, 0.609 mmol) and the reaction was stirred for 10 min, then at room temperature overnight. The reaction was partitioned between EtOAc (15mL) and waten¾rine (1 :1, lOmL). The organic layer was washed with sat. aq. NaHC(½ (1 x 10 mL), brine (lOmL), dried over MgS04, filtered and concentrated under vacuum to a crude, which was purified on a 1-inch reverse phase HPLC (Method 2) to yield 25 (1.5 mg, 0.997 μιηοΐ, 3.3% yield): MS m/z calcd for C75H89N7O26 (M+Na ⁺ ) 1526.6, found: 1526.6.

To a stirring solution of 25 (1.5 mg, 0.997 μιηοΐ) in AcOH (0.4 ml) and H2O (0.10 ml), was added Pd/C (4.24 mg) and the reaction was stirred under a hydrogen atmosphere for 3 hours. The Pd/C was removed by filtration and washed with water. The filtrate was diluted with water (10 mL) and lyophilized to give 26 as its acetate salt (0.45 mg): MS m/z for C ₂ 7Hs ₃ N ₇ Oi4 (M+LT) 700.4, found: 700.4; CLND 96.3% purity.

Example 5

To a stirring solution of 1 (161 g, 108 mmol) in benzaldehyde (625 mL) was added TFA (6.20 mL, 80.5 mmol) dropwise over 10 min, and the reaction was stirred at room temp for 14 hr. The reaction mixture was dripped into a stirred solution of diethyl ether (5.8 L) and hexanes (3.8 L), and the slurry was stirred for 5 hours. The resulting precipitate was collected by filtration, and the solid was purified by flash chromatography (silica gel/EtOAc/hexanes) to yield 2 (104 g, 65.8 mmol, 61.0% yield): MS m/z calcd for C ₈ iH9oN ₆ 0 (M+Na ⁴ ) 1601.6, found 1601.6

To a stirring solution of 2 (104, 65.8 mmol) in pyridine (550 mL) was added acetic anhydride (550 mL), and the reaction was stirred for 43 hours. The solvent was removed by rotary evaporation and the residue was re-concentrated from toluene. The solid was then dissolved in EtOAc (1 L) and washed with 1 M citric acid (3 x 650 mL), sat. aq. NaHC<¾ (3 x 600 mL), brine (500 mL), dried over MgS0 ₄ , filtered and concentrated to yield 3 as a yellow foam (121.5 g, 65.6 mmol, 99.7% yield): MS m/z calcd for C93H102N6O33 (M+Na ⁺ ) 1853.7, found 1853.5

To a stirring solution of 3 (61.5g, 33.6 mmol) in acetic acid (480 mL) and water (120 mL) at 55 °C was added TFA (6.00 mL, 77.9 mmol) and the reaction was stirred for 70 min. The reaction was then cooled to 15°C and DIPEA (27.0 mL, 156 mmol) was added. The reaction mixture was diluted with EtOAc (1.5 L), washed 5 with water (3 x 1.5 L), brine (400 mL), sat. aq. NaHC0 ₃ (2 x 1 L), brine (400 mL), dried over MgSC filtered and concentrated under vacuum to give a crude, which was purified by reverse phase HPLC (Method 2) to yield 4 (40.4g, 23.2 mmol, 69.0% yield): MS m/z calcd for CseHssNeOas (M+Na*) 1765.6, found 1765.5

To a stirring solution of 4 (40.4g 23.2 mmol) in pyridine (375 mL) at 0°C was added methanesulfonyl chloride (18.0 mL, 232 mmol), and the reaction was stirred at room temp for 3.5 nr. The reaction mixture was cooled at 0°C and stirred for

15 an additional 2 hr. DIPEA (58.5 mL, 465 mmol) was then added dropwise over 20 minutes. The reaction was quenched with water (600 mL) and extracted with EtOAc (2 x 500 mL). The combined oraganic layers were washed with 1 M citric acid/brine (4:1, 3 x 500 mL), sat. aq. NaHC0 ₃ (3 x 700 mL), brine (500 mL), dried over MgS0 ₄ , filtered and concentrated to yield 5 (44.7 g): MS m/z calcd for C88H98 6O37S2 (M+Na ⁺ )

20 1921.6, found 1921.2

To a stirring solution of 5 (44.7 g, 23.2 mmol) in DMPU (325 mL) was added sodium azide (4.5 g, 69 mmol), and the reaction was stirred at 70 °C for 4 hours. The reaction mixture was cooled to room temp and was dripped into stirring water (2.5 L) at 0°C. The resulting precipitate was filtered off and washed with water (500 mL). The solid was dissolved in EtOAc (1 L) and washed with water brine (1 :1, 800 mL). The aqueous layer was back-extracted with EtOAc (500 mL). The combined organic layers were washed with water brine (1 :1, 2 x 600 mL), brine (500 mL), dried over MgSC filtered and concentrated to yield 6 (45.9 g): MS m/z calcd for C87H99N9O34S (M+Na ⁺ ) 1868.6, found 1868.3

To a stirring solution of zinc acetate dihydrate (18.2g, 82.9 mmol) in methanol (400 mL) at 10°C was added 25% NaOMe in methanol (131 mL), and the reaction was stirred for 15 min. A solution of 6 (45.9 g, 23.2 mmol) in methanol (550 mL) was added, and the reaction was stirred at room temp for 3 hr. The reaction was cooled to 0°C, diluted with EtOAc (1.5 L), washed with 1 M citric acid/brine (3.75:1, 1.9 L). The aqueous layer was back-extracted with EtOAc (200 mL) and the combined organic layers were washed with 1M citric acid/brine (3:1, 400 mL), water brine (2:1, 300 mL), sat. aq. NaHC0 ₃ (2 x 250 mL), brine (350 mL), dried over MgS0 ₄ , filtered and concentrated to yield 7 (33.6 g, 23.2 mmol) MS m z calcd for C67H79N9O24 (M+Na ⁺ ) 1416.5, found 1416.4

To a stirring solution of 7 (33.6 g, 23 mmol) in THF (335 mL) were added H ₂ 0 (33.5 mL), diisopropylethylamine (42 mL, 245 mmol, 10.7 eq) and tris(2- carboxyethyl)phosphine hydrochloride (20.8g, 72.7 mmol, 3.2 eq) and the reaction was warmed in a 20 °C water bath. After 1 hr the reaction was partitioned between EtOAc (800 mL) and sat. aq. NaHC0 ₃ (400 mL). The aqueous layer was back extracted with EtOAc (200 mL). The combined organic layers were washed with sat. aq. NaHC03 (400 mL), brine (350 mL), dried over MgS0 ₄ , filtered and concentrated in vacuo to yield 8 (33.2 g), which was carried through to the next step without further purification. MS m/z calcd for C ₆ 7H _g iN ₇ 0 ₂₄ (M+Na ⁺ ) 1368.5, found: 1368.3

To a stirring solution of 8 (29.8 g, 22 mmol) in dry acetone was added potassium carbonate (12 g, 88 mmol) followed by bromoethanol (5.1 mL, 73 mmol) and the reaction was heated in a 35 °C oil bath for 18 hr. The reaction was quenched with sat. aq. NH ₄ CI (1.0 L) and the product was extracted with EtOAc (600 mL, then 300 mL). The combined organic layers were washed with sat. aq. NaHCC (500 mL, then 250 mL), brine (350 mL), dried over MgS0 ₄ , filtered and concentrated in vacuo to yield 9 (32 g, MS m/z calcd for CesHgs jOis (M+Na ⁺ ) 1412.6, found 1412.3), which was carried through to the next step without further puririfcation.

To a stirring solution of 9 (33 g, 23 mmol) in acetonitrile (320 mL) and H2O (80 mL) was added diisopropylethylamine (8.1 mL, 47 mmol) and the reaction mixture was stirred at 0°C for 10 min. N-(benzyloxycarbonyloxy)-succinimide (5.9 g, 24 mmol) was then added and the reaction was stirred at 0°C for 5 min and at room temperature for 21 hr. The reaction was then cooled to 0°C for 10 min and 1 M citric acid was added (600 mL) followed by brine (240 mL). The product was extracted with EtOAc (800 mL, then 200 mL). The combined organic layers were washed with brine (400 mL), sat. aq. NaHC0 ₃ (600 mL), brine (350 mL), dried over MgS0 ₄ , filtered and concentrated under vacuum to yield 10 (33 g, MS m/z calcd for C77H91N7O27 (M+Na ⁺ ) 1568.6, found: 1568.5), which was carried through to the next step without further puririfcation.

To a stirring solution of 10 (33 g, 22 mmol) in pyridine (330 mL) and acetic anhydride (330 mL) was added dimethylaminopyridine (1.5g, 13 mmol) and the reaction was stirred for 1.5 hr. The reaction was cooled to 5 °C and methanol (200 mL) was added dropwise over 2 hr, while the internal temperature was kept at 8 - 12 °C. The reaction was diluted with ¾0 (800 mL) and brine (100 mL) and the product was extracted with EtOAc (1000 mL, then 200 mL). The combined organic layers were washed with 1 M citric acid (3 x 800 mL), brine (400 mL), sat. aq NaHC0 ₃ (800 mL), brine (400 mL), dried over MgSC>4, filtered and concentrated under vacuum to yield 11 (39 g, MS m/z calcd for C91H105N7O34 (M+Na ⁺ ) 1862.7, found 1862.6), which was carried through to the next step without further purification.

To a stirring solution of THF (200 mL) at 0°C was added lithium iodide (17 g, 128 mmol) followed by acetic acid (3.7 mL, 64 mmol) and this reaction mixture was added to a stirring solution of 11 (39 g, 21 mmol) in THF (200 mL). The reaction was placed in a 60 °C oil bath for 2 hr. The reaction was cooled to room temperature and was quenched with H ₂ 0 (800 mL) and brine (200 mL), and the product was extracted with EtOAc (800 mL). The organic layer was washed with brine (400 mL), aqueous sodium sulfite (300 mL), sat. aq. NaHCC>3 (600 mL), brine (400 mL), dried over MgSC>4, filtered and concentrated under vacuum to yield a crude, which was purified on a 6-inch reverse phase HPLC column (Method 2) to yield 12 (19 g, 9.65 mmol, 46% yield): MS m/z calcd for C91H106IN7O34 (M+Na ⁺ ) 1990.6, found 1990.4.

To a stirring solution of 12 (605 mg, 0.307 mmol) in dioxane (30 mL), was added TEA (64 μί, 0.461 mmol) followed by Raney nickel (2.5mL) and the reaction was stirred under a hydrogen atmosphere for 75 min, then more Raney nickel (2.5 mL) was added and the reaction was stirred under a hydrogen atmosphere for 40min. The reaction was filtered with a 0.2 μπι syringe filter and rinsed with dioxane (2 x 7 mL). The filtrate was concentrated to a crude, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield 13 (279 mg, 0.149 mmol, 49%): MS m/z calcd for C ₉ iH,07N ₇ O34 (M+H ⁺ ) 1842.7, found 1842.1

To a stirring solution of 13 (210 mg, 0.114 mmol) in toluene (7 mL) was added PPh ₃ (179 mg, 0.684 mmol), followed by benzoic acid (83 mg, 0.684 mmol) and the reaction was cooled to 0°C for 10 min. DIAD (0.133 ml, 0.684 mmol) was added and the reaction was stirred at 55 °C for 3 hr. Additional PPbj (360mg) and benzoic acid (170mg) were added and the reaction was cooled to 0°C. After ten minutes, DIAD (275μί) was added slowly and the reaction was returned to 55°C for 1 hr. The addition was repeated and the reaction was heated at 55 °C for 1 hr. The reaction was stirred at room temp overnight. The addition was repeated and the reaction was at 55°C for 75 min. The reaction was concentrated and dried on the high vac overnight yielding a crude, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield 14 (95 mg, 0.049 mmol, 43%): MS m/z calcd for C9SH111 7O35 (M+H* 1946.7, found 1 46.2.

To a stirring solution of 14 (95 mg, 0.049 mmol) in MeOH (2.6 mL) was added methylamine (0.73 mL) and the reaction was stirred at room temperature. After 5 hr additional methylamine (0.73 mL) was added and the reaction was stirred overnight. Solvent evaporation gave a crude, which was purified on a 1-inch reverse phase HPLC (Method 2) to yield 15 (28 mg, 0.018 mmol, 37%): MS m/z calc for C77H93N7O27 (M+Na ⁺ ) 1570.6, found 1570.7.

To a stirring solution of 15 (28 mg, 0.018 mmol) in 80% aqueous AcOH (1 mL) was added Pd(OH) ₂ /C (20% Pd, 28mg) and the reaction was stirred under a hydrogen atmosphere for 1 r. The reaction mixture was filtered, diluted with water (lOmL) and lyophilized to yield 16 as its acetate salt (7.4mg, 0.0067 mmol, 37%): MS m/z calc for C ₂ 9H57N70 ₁₅ (M+H ⁺ ) 744.4, found 744.3. CLND 94.4%

Example 6

To a stirring solution of paromomycin sulfate 1 (76 g, 84 mmol) in ¾0 (209 mL) and THF (1084 mL) at 0 ^C C was added an aqueous solution of sodium carbonate (254 mL, 218 mmol, 0.86 M), followed by the dropwise addition of benzyl chloroformate (120 mL, 840 mmol). NaHC0 ₃ (70.6 g, 840 mmol) was then added and the reaction was stirred for 3 hr. The organic layer was separated and concentrated (to about 800 mL), diluted with EtOAc (400 mL) and dripped into hexane (9 L). The resulting precipitate is collected by filtration to yield 2 (69.85g, 54.36 mmol, 65%): MS m/z calcd for C63H75N5O24 (M+Na ⁺ ) 1308.48, found 1308.6.

Zinc chloride (59.2 g, 434 mmol) was dissolved in benzaldehyde (440 mL, 4344 mmol) to give a yellow solution, and the reaction was stirred for 5 min. A solution of 2 (69.85 g, 54.3 mmol) in benzaldehyde (440 mL) was then added and the reaction was stirred for 7 hr. The reaction mixture was diluted with EtOAc (2 L) and washed with 0.1M EDTA disodium salt dihydrate (3 x 2L), H ₂ 0 (2 L), brine (2 L), dried over Na ₂ S0 ₄ , concentrated (to about 900mL) and dripped into Et ₂ 0: hexane (1:1, 4 L). The resulting precipitate was collected by filtration and dried under vacuum to yield 3 (93.41g, 63.94 mmol 118: MS m/z calcd for C77H83N5O24 (M+Na ⁺ ) 1484.54, found 1484.7.

To a stirring suspension of sodium hydride (dry 95%, 9.64 g, 382 mmol) in DMA (501 mL) at -10°C was added a solution of 3 (93 g, 63.6 mmol) in DMA (500 mL) and the reaction was stirred for 4 hours. The reaction was quenched with acetic acid (lOOmL) and stirred for 30 minutes. The reaction mixture was then diluted with EtOAc (2 L), and washed with NaHC0 ₃ (2 2 L), H ₂ 0 (2 x 2 L), brine (2 L), dried over Na ₂ SC>4, filtered and concentrated to a yellow-brown solid, which was purified by flash chromatography (silica gel, MeOH/DCM) to yield 4 (30 g, 22.17 mmol, 35%): MS m/z calcd for C ₇ oH ₇₅ N ₅ 023 (M+Na ⁺ ) 1376.5, found 1376.7.

To a stirring solution of 4 (30 g, 22.15 mmol) in pyridine (201 mL) was added DMAP (2.71 g, 22.15 mmol), followed by TBDPS-Cl (65.4 mL, 255 mmol) and the reaction was heated at 80°C for 6 days. The reaction mixture was dripped into Et ₂ 0: hexane (1 :1, 9 L) and the resulting precipitate was collected by filtration and redissolved in THF (130 mL) and MeOH (40 mL). This solution was then dripped into Et20: hexane (1 :1, 9 L) and the resulting precipitate was collected by filtration and dried under vacuum. The white solid was dissolved in ethyl acetate (600mL), washed with 1M citric acid (2 x 500mL), brine (500mL), NaHC0 ₃ (2 x 500 mL), brine (500mL), dried over Na ₂ S04, filtered and concentrated to yield 5 (36.45g, 19.93 mmol, 90%): MS m/z calcd for C102H111N5O23S12 (M+Na ⁺ ) 1852.7, found 1852.8.

To a stirring solution of 5 (2 g, 1.092 mmol) in DMSO (10.92 mL) was added sulfur trioxide-pyridine (1.825 g, 11.47 mmol), followed by TEA (3.20 mL, 22.94 mmol) and the reaction was stirred for 2 hr. The reaction mixture was diluted with EtOAc (50mL), washed with NaHC0 ₃ (2 x 50 mL), brine (50 mL), dried over Na ₂ S04, filtered and concentrated under vacuum to yield 6 (1.78 g, 0.974 mmol, 89%): MS: m/z calcd for C ₁ 02H ₁ 09N ₅ O ₂₃ Si ₂ (M+Na*) 1850.7, found 1850.7.

To a stirring solution of 6 (1.78 g, 0.973 mmol) in DMF (18.71 mL) and methanol (18.71 mL) at 0°C was added NaBEU (0.368 g, 9.73 mmol) and the reaction was stirred for 1 hr. The reaction mixture was diluted with EtOAc (75mL), washed with water: brine (1:1, 75 mL), brine (3 x 75 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to a white solid, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield compound 7 (0.62 g, 0.339 mmol, 35%): MS m/z calcd for Cio2H _m N ₅ 0 ₂₃ Si ₂ (M+Na ⁺ ) 1852.7, found 1852.9.

To a stirring solution of 7 (0.35 g, 0.191 mmol) in THF (3 mL) was added TBAF (1.91 mL, 1 M in THF, 1.91 mmol) and the reaction was stirred for 48 hr. The reaction mixture was quenched with sat. aq. NH ₄ CI, and diluted with EtOAc. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried over Na ₂ SC filtered and concentrated to a crude, which was triturated with Et ₂ 0 to yield 8 (0.191 mmol, MS m/z calcd for C69H77 5O22 (M+H ⁴ ) 1328.3, found 1328.2, which was carried through to the next step without further purification.

To a stirring solution of 8 (0.045 g, 0.034 mmol) in DMF (0.565 mL) at 0°C was added DIPEA (0.018 mL, 0.102 mmol), followed by N-Cbz-2(S)-hydroxy- butyric acid (10.29 mg, 0.041 mmol), and PyBOP (0.019 g, 0.037 mmol) and the reaction was stirred for 1 hour. The reaction mixture was purified on a 2-inch reverse phase HPLC column (Method 2) to yield 9 (0.0375 g, 0.024 mmol, 71%): MS m/z calcd for C8iH ₉ oN ₆ 0 (M+Na ⁴ ) 1585.6, found 1585.6.

To a stirring solution of 9 (37.5 mg, 0.024 mmol) in methanol (4.00 mL) was added TFA (0.022 mL, 0.288 mmol), followed by Pd(OH) ₂ /C (0.034 g, 0.240 mmol) and the reaction was stirred under a hydrogen atmosphere for 18 hours. The Pd was removed by filtration and washed with water (2 x 5mL), and the combined washings were lyophilized to yield 10 as its TFA salt (7.7 mg, 0.006 mmol, 24%): MS m z calcd for C ₂₇ H ₅₂ N ₆ Oi ₆ (M+Na ⁺ ) 739.3, found 739.4; CLND 97.1%.

Example 7

To a stirring solution of 1 (0.150 g, 0.096 mmol) in pyridine (1 mL) was added acetic anhydride (1 mL) and the reaction was stirred for 3 days. The reaction was quenched with MeOH, diluted with EtOAc, washed with sat. aq. NH ₄ CI, 1M citric acid, sat. aq. NaHCC brine, dried over Na ₂ S0 ₄ , filtered and concentrated under vacuum to yield 2 (150 mg, 0.083 mmol, 86%): MS m/z calcd for C93H102N6O32 +) 1837.6, found 1837.6.

To a stirring solution of 2 (150 mg, 0.083 mmol) in AcOH (1.47 mL) and H ₂ 0 (0.295 mL) was added 5% aqueous TFA (1 mL) and the reaction was heated at 50°C for 30 min. The reaction was cooled to 0°C and was quenched with DIPEA (35 μί, 0.198 mmol), diluted with ethyl acetate (50 mL), washed with water, brine, aHC0 ₃ , and brine, dried over Na ₂ SC filtered and concentrated to yield 3 (121 mg, 0.070 mmol, 84%): MS m/z calcd for Cgeftw eCfe (M+Na*) 1749.6, found 1749.6.

To a stirring solution of 3 (121 mg, 0.070 mmol) in pyridine (2.3 mL) was added TsCl (107 mg, 0.56 mmol) and the reaction was stirred overnight. The reaction was quenched with MeOH (2 mL), diluted with EtOAc (100 mL), washed with 1 M citric acid (2 x 100 mL), sat. aq. NaHC0 ₃ (2 x 75 mL), brine (100 mL), dried over Na ₂ SC filtered and concentrated under vacuum to yield a crude, which was purified on a 1-inch reverse phase HPLC column (Method 2) to yield 4 (0.053 g, 0.028 mmol, 40%): MS m/z calcd for C93H104N6O3 ₄ S (M+Na ⁺ ) 1903.6, found 1903.4.

To a stirring solution of 4 (50 mg, 0.027 mmol) in DMA (44.3 μί) was added ethanolamine (89 μί, 1.47 mmol) and the reaction was heated at 40°C overnight. The reaction was diluted with EtOAc (50 mL), washed with 1 M citric acid (50 mL), sat. aq. NaHC(¾ (2 x 50 mL), brine, dried over Na ₂ SC filtered and concentrated under vacuum to give a crude, which was purified on a 1 -inch reverse phase HPLC column (Method 2) to yield 5 (9.5 mg, 0.0063 mmol, 23%): MS m/z calcd for C76H ₉ iN ₇ 0 (M+H ⁺ ) 1518.6, found 1518.6.

To a stirring solution of 5 (9.5 mg, 0.0063 mmol) in MeOH (1 mL) was added TFA (2.9 μΧ, 0.038 mmol), followed by Pd(OH) ₂ /C and the reaction was stirred under a hydrogen atmosphere for 2 days. The reaction was filtered through a 0.2 um syringe filter, diluted with H ₂ 0 and lyophilized to yield 6 as its TFA salt: MS mJz calcd for (M+Ff) 760.4, found 760.4.

Example 8

To a stirring solution of neomycin sulfate (1, 120 g, 0.130 mole) in H2O (430 mL) was added a solution of K ₂ C<¾ (63 g, 0.456 mole, 3.5 eq.) in H ₂ 0 (700 mL) followed by THF (1.46 L). To this vigorously stirred biphasic solution was added drop- wise over 30 min a solution of Cbz-succinimide (292 g, 1.174 mole) in TFIF (820 mL), and the reaction mixture was stirred for 18 hr. The reaction was quenched with the addition of 3-(dimethylamino)-propylamine (148 mL, 1.174 mole), and diluted with EtOAc (1 L) and ¾0 (1 L). The reaction mixture was partitioned between EtOAc (1 L) and 1M citric acid (2 L)/brine (1 L). The aqueous layer was diluted with brine (500 mL) and extracted with EtOAc (500 mL). The combined organic layers were washed with 1 M citric acid (1 L), brine (500 mL). The organic layer was then stirred with saturated NaHC(½ (2 L) and ¾0 (600 mL) until off-gassing ceased. The layers were partitioned, and the organic layer was washed with ½ sat. NaHC(½ (1 L), brine (2 L) dried over Na ₂ SC>4, concentrated (to 660 mL) and dripped into vigorously stirring Et20 (5.5 L). The resulting precipitate was dried under high vacuum for 72 hours at 30°C to yield 2 (172 g, 0.121 mmol, 93% yield) as a white solid: MS m/z calcd for C71H82 6O25 (M+H ) 1418.5, found 1418.9.

To a stirring solution of per-Cbz-neomycin B (2, 50 g, 35.2 mmol) in benzaldehyde (2000 ml, 19.7 mol) was added aluminum chloride (30.5 g, 229 mmol) and the reaction mixture turned from yellow to dark orange with an increase in the internal temperature from 22°C to 27°C. After 45min, the reaction mixture was poured into vigorously stirring ice/sat NH4CI (1 :1, 800 mL) and the off-white slurry was extracted with EtOAc (800 mL). The organic layer was washed with sat. aq. NH4CI (800 mL), 0.1M EDTA (400 mL), brine (400 mL), sat. aq. NaHC0 ₃ (800 mL), brine (400 mL), dried over MgS0 ₄ , filtered and concentrated (to about 2 L). The resulting benzaldehyde solution was dripped into hexanes/Et ₂ 0 (2:1, 18 L) and stirred overnight. The resulting fine white precipitate was collected by filtration, washed with hexanes/Et ₂ 0 (2:1, 1000 mL) and dried under vacuum to yield 3 (54.9 g, 32.6, 93% yield): MS m/z calcd for C ₇ iHs ₂ N ₆ 0 ₂ 5 (M+Na ⁺ ) 1705.6, found 1705.4.

To a stirring suspension of sodium hydride (4.68 g, 195 mmol) in DMA (400 ml) at 0°C was added a cold solution of 3 (54.7 g, 32.5 mmol) in DMA (400 ml) and the reaction was stirred at 0°C for 4 hours. AcOH (53.9 ml, 942 mmol) was then added and the reaction was allowed to warm to rt overnight. The reaction mixture was diluted with EtOAc (1000 mL), washed with water/brine (1:1 , 1000 mL), sat. aq. NaHC0 ₃ (2 x 800 mL), water/brine (4:1, 2 x 1000 mL), brine (1 x 400 mL), dried over MgSC>4, filtered and concentrated under vacuum to yield 4 (52.1 g, 195 mmol, 100% yield): MS m/z calcd for C ₈ 5H ₈₆ N ₆ 024 (M+Na ⁺ ) 1597.6, found 1597.4.

To a stirring solution of 4 (51.2 g, 32.5 mmol) in dioxane (600 ml) was added a solution of TFA (16.02 ml, 208 mmol) in water (200 ml) and the reaction was heated at 50°C for 17 hours. The reaction mixture was diluted with EtOAc (800 mL) and washed with sat. aq. NaHC0 ₃ (2 x 800 mL), brine (400 mL), dried over MgS0 ₄ , filtered and concentrated under vacuum to yield 5 (49.9 g, 32.5 mmol, 100 % yield): MS m z calcd for C ₇₈ H ₈₂ N ₆ 0 ₂₄ (M+Na ⁺ ) 1509.5, found 1509.3.

To a stirring solution of 5 (48.3 g, 32.5 mmol) in pyridine (350 ml) was added TBDPS-C1 (83 ml, 325 mmol) followed by DMAP (3.97 g, 32.5 mmol) and the reaction was heated at 85°C for 5 days. The reaction was allowed to cool to rt and was slowly dripped into hexanes/Et ₂ 0 (1 :1, 11 L). The resulting precipitate was collected by filtration and washed with hexanes/I¾0 (1 :1, 50 mL), followed by purification by flash chromatography (silica gel/ EtOAc/hexanes) to yield 6 (17.6 g, 8.05 mmol, 24.8% yield): MS m/z calcd for CiioH ₁₁₈ N ₆ 0 ₂₄ Si ₂ (M+Na ⁺ ) 1985.8, found 1985.6.

To a stirring solution of 6 (2.52 g, 1.283 mmol) in anhydrous DCM (25 ml) was added DMSO (0.455 ml, 6.41 mmol) and the reaction was cooled to -78°C and stirred for 15 min. Oxalyl chloride (2.0M in DCM, 1.090 ml, 2.181 mmol) was slowly added and the reaction was stirred for an additional 20 min. TEA (1.788 ml, 12.83 mmol) was then added over 5 min and the reaction was stirred for 10 min. The reaction mixture was then warmed to 0°C and stirred for 30min. The reaction was quenched with 1M citric acid (40 mL) and the organic layer was separated and washed with brine (40 mL), dried over MgS0 ₄ , filtered and concentrated under vacuum to yield 7 (2.54 g, 1.283 mmol, 100 % yield): MS m/z calcd for Cii ₀ H _I i ₆ N ₆ O ₂₄ Si ₂ (M+Na ⁴ ) 1983.8, found 1983.9.

To a stirring solution of 7 (9.95 g, 5.07 mmol) in THF (60 ml) at 0°C was added LiBH ₄ (2 M solution in THF, 9.20 ml, 18.41 mmol) and the reaction was stirred for 25 min. The reaction mixture was partitioned between EtOAc (300mL) and water brine (1:1, 300mL). The organic layer was washed with brine (200mL), dried over MgSC>4, filtered and concentrated to a crude, which was purified on a 6-inch reverse phase HPLC (Method 2) to yield 8 (5.8 g, 2.95 mmol, 58.2 % yield): MS m/z calcd for CnoHii8N ₆ 024Si2 (M+Na") 1985.8, found 1985.9.

To a stirring solution of 8 (2.96 g, 1.507 mmol) in THF (21.29 mL) was added TBAF (1 M solution in THF, 16.58 mL, 16.58 mmol) and the reaction was heated at 40°C for 5 hours. The reaction mixture was partitioned between EtOAc (300 mL) and brine/lM citric acid (1 :1, 200 mL). The organic layer was washed with sat. aq. NaHC0 ₃ (200 mL), brine (100 mL), dried over MgS0 ₄ , filtered and concentrated under vacuum to yield 9 (1.5 g, 1.026 mmol, 68 % yield): MS m/z calcd for

1483.6, found 1483.6.

To a stirring solution of 9 (1 mg, 0.684 μί) in DMF (9.77 μί) was added DIPEA (0.215 μί, 1.23 μιηοΐ) followed by N-Cbz-2(S)-hydroxy-propionic acid (0.172 mg, 0.718 umol) and the reaction was cooled to 0°C for 10 minutes. PyBOP (0.392 mg, 0.753 μπιοΐ) was then added and the reaction was stirred at room temp for 1 hour. The reaction was diluted with EtOAc, washed with 1M citric acid, sat. aq. NaHC03, brine, dried over Na ₂ SC>4, filtered and concentrated to yield 10: MS m/z calcd for C88H95N7O27 (M+Na ⁺ ) 1704.6, found 1704.7.

To a stirring solution of 10 (41 mg, 0.024 mmol) in MeOH (4 mL) was added TFA (11 i , 0.147 mmol) followed by Pd(OH) ₂ /C (40 mg) and the reaction was stirred under a hydrogen atmosphere overnight. The reaction mixture was filtered through a 0.45 um syringe filter, and was slowly dripped into vigorously stirring Et ₂ 0 (80 mL). The resulting precipitate was collected by filtration and dried under high vacuum to yield 11 (29 mg, 0.021 mmol, 87%): MS m/z calcd for C26H51N7O15 (M+Na ⁺ ) 724.3, found 724.3. CLND 94.2% purity.

Example 9

Neomycin sulfate (1, 240 g, 391 mmol) was suspended in MeOH (10 L), diluted with H ₂ 0 (400 mL), cooled to 0°C, and K ₂ C0 ₃ (1080 g, 7818 mmol) was slowly added. Benzyl chloroformate (550 mL, 3910 mmol) was then added to the reaction mixture via a dropping funnel over 3 hours. The mixture was stirred vigorously for 18 hours at room temperature, then was filtered and concentrated (to 2 L). The concentrate was diluted with ethyl acetate (2 L), washed with water (2 x 2L), brine (2L), dried over Na2S04 and concentrated to a yellow foam, which was dissolved in MeOH (1 L) and dripped into Et ₂ 0 (24 L). The resulting precipitate was collected by filtration and dried under high vacuum to yield a crude, which was purified on a 6-inch reverse phase HPLC column (Method 2) to yield l,3,2',6',2"',6"'-hexa-Cbz-neomycin 2 (171 g, 31%). MS: m/z (M+Na ⁺ ) calcl441.53 found 1441.6.

To a stirring solution of l,3,2',6',2'",6'"-hexa-Cbz-neomycin (2, 239 g, 169 mmol) in pyridine (1.2 L) was added 4-dimemylammopyridine (20.6 g, 168.6 mmol). The reaction was heated at 80°C and t-butyl-diphenylsilyl chloride (175.2 mL, 673.7 mmol) was added via addition funnel over one hour. The reaction was heated at 5 80°C for 18 hours and then was cooled to room temperature. The reaction mixture (1.4 L) was dripped into vigorously stirring diethyl ether (30 L). The resulting precipitate was filtered, washed with ether (1 L), dried under vacuum to yield a crude, which was purified on a 6-inch reverse-phase HPLC column (Method 2) to yield 1,3,2',6',2"',6"'- hexa-Cbz-5"-OTBDPS-neomycin (3, 238 g, 144 mmol, 85 % yield) : MS: m/z 10 (M+Na ⁺ ) calc. 1679.65 found 1679.6.

To a stirring solution of l,3,2',6',2"',6"'-hexa-Cbz-5"-OTBDPS- 15 neomycin (3, 80 g, 48.3 mmol) in anhydrous toluene (2.7 L) and acetonitrile (0.67 L) was added PPh ₃ (151.9 g, 580 mmol), followed by triiodoimidazole (103.3 g, 232 mmol), and imidazole (19.73 g, 290 mmol), and the reaction was refluxed for 4 hours and then allowed to cool to room temperature. The reaction was quenched by the addition of aq. Na ₂ S ₂ 03 (200 g in 1 L of water) and stirred for 1 hour. The layers were 20 partitioned and the organic layer was concentrated (to 600 mL). The reaction mixture was seeded with triphenyl phosphine oxide and placed in the freezer for 18 hours. The solids were removed by filtration, and the filtrate was concentrated to an orange-brown oil, which was purified on a 6-inch reverse-phase HPLC column (Method 2) to yield 3',4',3'",4"'-tetradehydro-l,3,2',6 ^, ,2"',6"'-hexa-Cbz-5"-OTBDPS-neomycin (4, 38 g, 25 24 mmol, 50% yield): MS: /«/z (M+Na ⁺ ) calc. 1611.64, obs. 1611.5.

To a stirring solution of 3\4',3"',4"'-tetta-dehydro-5"-TBDPS- l,3,2',6',2"',6"'-hexa-Cbz-neomycin (4, 96.3 g, 60.6 mmol) in THF (90 mL) was added TBAF (1 M in THF, 606 mL, 606 mmol) and the reaction was stirred for 4 hours. The reaction was quenched with H ₄ CI (1.6 L) and stirred for 10 minutes. The reaction was then diluted with ethyl acetate (1.5 L) and the layers were partitioned. The organic layer was washed with NH ₄ C1 (1.5 L), dried over Na ₂ SC>4, concentrated (to 400 mL), and dripped into ethenhexane (2:1, 7.3L: 3.7L). The resulting precipitate collected by filtration and dried under vacuum to yield 3',4',3"',4"'-tetra-dehydro-l,3,2',6',2'",6'"- hexa-Cbz-neomycin (5, 78.24 g, 57.9 mmol, 94% yield): MS: m/z (M+Na ⁺ ) calc. 1373.52, obs. 1373.5.

To a stirring suspension of NaH (6.26 g, 260.6 mmol) in anhydrous DMA (1 L) at 0°C was added a solution of 3',4',3'",4'"-tetra-dehydro- 6',2',3,l,2"',6"'-hexa-Cbz-neomycin (5, 78.24 g, 57.9 mmol) in anhydrous DMA (1 L) dropwise over 30 minutes. After 2 hr, the reaction mixture was diluted with ethyl acetate (2 L), washed with water (2 x 2L), dried over Na2SC>4, filtered, and concentrated to an orange oil, which was purified on a 6-inch reverse-phase HPLC column (Method 2) to yield 3',4',3"',4"'-tetra-dehydro-6',2',3,2"',6"'-penta-Cbz-l,6-ox azolidinone- neomycin as a white solid (6, 44.5 g, 36 mmol, 62% yield): MS: mJz (M+Na ⁺ ) calc. 1265.46, obs. 1265.4.

To a stirring solution of 3',4',3"',4"'-tetra-dehydro-l,6-oxazolidinone- 3,2',6',2"',6"'-penta-Cbz-neomycin (6, 44.5 g, 36 mmol) in NMP (1.19 L) was added 0.5 M aq. LiOH (250 mL, 125.27 mmol) dropwise and the reaction was stirred overnight. The reaction was quenched with 1 M citric acid (100 mL) and diluted with EtOAc (1 L) and brine (2 L), and the organic layer was separated. The aqueous layer was washed with ethyl acetate (200 mL) and the combined organic layers were washed with sat. aq. NaHC0 ₃ (600 mL), water/brine (1:1, 600 mL), brine (600 mL), dried over MgS04, filtered and concentrated under vacuum to yield a crude, which was purified on a 6-inch reverse-phase HPLC column (Method 2) to yield 3',4',3"',4"'-tetra-dehydro- 3,2',6',2"',6"'-penta-Cbz-neomycin as a white solid 7 (23.5 g): MS: m/z (M+Na*) calc. 1239.49; obs. 1239.3.

To a stirring solution of 7 (21.6 g, 17.75 mmol) in DMF (800 mL) at 0 ^° C and was added PyBOP (11.25 g, 21.29 mmol), followed by N-Cbz-4-amino-2(S)- bydroxy-butanoic acid (5.40 g, 21.29 mmol) and DIPEA (4.56 mL, 26.62 mmol) with the reaction progress monitored by HPLC. After 3 hours additional DIPEA (1.5 mL) was added and the reaction was stirred for 1 hr. The reaction mixture was neutralized by the addition of sat. aq. NH4CI (100 mL). Citric acid (1 M, 100 mL) was then added, followed by brine (500 mL), and the aqueous layer was extracted with EtOAc (500 mL). The organic layer was washed with 1M citric acid (500 mL), sat. aq. NaHC(¾ (500 mL), brine (500 mL), dried over MgSO filtered and concentrated to dryness under high vacuum to yield a crude, which was purified on a 6-inch reverse-phase HPLC column (Method 2) to yield 3',4',3"',4"'-tetra-dehydro-3,2',6',2"',6"'-penta- Cbz-l-(N-Cbz-2(5)-hydroxy-butyryl)-neomycin as an off-white solid 8 (29.1 g): MS m/z (M+Na ⁺ ) calc. 1474.57; obs. 1474.3.

To a stirring solution of 8 (0.11 g, 0.076 mmol) in acetone (2 mL) and

H ₂ 0 (0.133 mL) was added quinuclidine (0.85 mg, 7.64 umol), followed by NMO (0.048 mL, 0.46 mmol) and Os0 ₄ (4% w/w in ¾0, 0.038 ml, 6.06 umol), and the reaction was stirred overnight. The reaction was quenched by the addition of 6% aqueous Na ₂ S0 ₃ , and allowing the reaction to stir for 30 min. The reaction mixture was diluted with EtOAc and water. The organic layers were washed with brine, dried over Na ₂ S0 ₄ , filtered and concentrated to a crude, which was purified on a 1-inch reverse phase HPLC (Method 2) to yield 9a (11.6 mg, 7.63 μπιοΐ, 10%, MS m/z calcd for C _T sHggNyOj _? (M+Na ⁺ ) 1542.6, found 1542.5) and 10a (23.4 mg, 15.4 umol, 20.6%): MS m/z calcd for C75H89N7O27 (M+Na") 1542.6, found 1542.5; and 9b and 10b.

To a stirring solution of 9a (11.6 mg, 7.63 umol) in AcOH (2 mL) and H ₂ 0 (0.5mL) was added 5% weight Pd/C (5 mg) and the reaction was stirred under a hydrogen atmosphere for 3 hr. The catalyst was removed by filtration through Celite, and washed with ¾0, and the combined washings were lyophilized to yield compound 11 (4.5 mg, 4.19 μπι, 55%) as the acetate salt: MS m/z calcd for C ₂ 7H ₅ 3N ₇ 0, _S (M+¥f) 716.4, found 716.4; CLND 97.9% purity.

To a stirring solution of 10a (23.4 mg, 15.4 umol) in AcOH (2 mL) and ¾0 (0.5mL) was added 5% weight Pd/C (5 mg) and the reaction was stirred under a hydrogen atmosphere for 3 hr. The catalyst was removed by filtration through Celite, and washed with ¾0, and the combined washings were lyophilized to yield compound 12 (16 mg, 14.9 umol, 97%) as the acetate salt: MS m z calcd for C27H53N7O15 (M+H*) 716.4, found 716.4; CLND 96.7% purity. Example 10

To a stirring solution of 1 (0.475 g, 0.39 mmol) in DMF (7 mL) at 0°C was added DIPEA (0.204 mL, 1.71 mmol), followed by N-Cbz-2(5)-hydroxy-propionic acid (0.187 g, 0.78 mmol) and PyBOP (0.325 g, 0.62 mmol) and the reaction was stirred overnight at room temperature. The reaction was diluted with EtOAc, and washed with sat. aq. NH4CI, sat. aq. NaHCC>3, brine (2 x), dried over Na2S<¾, filtered and concentrated to a crude, which was purified on a 2-inch reverse phase HPLC column (Method 2) to yield 2: MS m/z calcd for C ₇ 4H ₈ 3N ₇ 023 (M+Na ⁺ ) 1460.5, found

To a stirring solution of 2 (5.24 g, 3.64 mmol) in acetone (75 mL) and water (5 mL) was added quinuclidine (0.081 g, 0.73 mmol), followed by 4- methylmorpholine-4-oxide (2.30 mL, 21.84 mmol) and Os0 ₄ (4% w/w in H ₂ 0, 1.85 mL, 0.29 mmol) and the reaction was stirred overnight. The reaction was quenched with a2S ₂ 0 ₃ (50 ml, 6% aq.) and was stirred for 25 min. The reaction mixture was diluted with EtOAc, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SC>4, filtered and concentrated to a crude, which was purified on a 2-inch reverse phase HPLC (Method 2) to yield 3a (0.36 g, 0.24 mmol, 7%) and 4a (0.14 g, 0.09 mmol, 3%): MS m/z calcd for C ₇ 4H _S7 N ₇ 027 (M+Na ⁺ ) 1528.6, found 1528.6, and 3b and 4b.

To a stirring solution of 3a in AcOH (2 mL) and ¾0 (0.5 niL) was added Pd C and the reaction was stirred under a hydrogen atmosphere for 4 hr. The reaction mixture was filtered through Celite, and washed with ¾0 and the combined washings were lyophilized to yield 5 (3.3 mg, 3.11 μπιοΐ) as its acetate salt: MS m/z calcd for C26H ₅ 1 ₇ O15 (M+H*) 702.3, found 702.3; CLND 97.5% purity.

To a stirring solution of 4a in AcOH (2 mL) and ¾0 (0.5 mL) was added Pd C and the reaction was stirred under a hydrogen atmosphere for 4 hr. The reaction mixture was filtered through Celite, and washed with H2O and the combined washings were lyophilized to a crude, which was purified on a 1-inch reverse phase HPLC (Method 1) to yield 6 (3.5 mg, 4.99 umol): MS m/z calcd for C26H51N7O15 (M+H ⁺ ) 702.3, found 702.4; CLND 93.2% purity.

Example 11

To a stirring solution of 1 (1.064 g, 0.87 mmol) in DMF (15 mL) at 0°C was added DIPEA (0.457 mL, 2.62 mmol), followed by N-Boc-3 -amino- 1- hydroxycyclobutiric acid (0.303 g, 1.311 mmol) and PyBOP (0.591 g, 1.136 mmol) and the reaction was stirred overnight at room temperature. The reaction was diluted with EtOAc, and washed with sat. aq. NH4CI, sat. aq. NaHCC>3, brine (2 x), dried over Na2SC>4, filtered and concentrated to a crude, which was purified on a 2-inch reverse phase column (Method 2) to yield 2 (0.92 g, 0.65 mmol, 74%): MS m/z calcd for (M+Na ) 1452.6, found 1452.4.

To a stirring solution of 2 (0.925 g, 0.647 mmol) in acetone (15 mL) and water (1 mL) was added quinuclidine (2.88 mg, 0.026 mmol), followed by 4- methylmorpholine-4-oxide (0.273 mL, 2.59 mmol) and Os0 ₄ (4% w/w in H ₂ 0, 0.164 mL, 0.026 mmol) and the reaction was stirred overnight. Additional OSO4 (0.164 mL) was added and the reaction was stirred overnight. Then additional NMO (0.273 mL) was added and the reaction was stirred overnight. The reaction was quenched with Na2S20 ₃ in ¾0 (4 mL) and was stirred for 20 min. The reaction mixture was diluted with EtOAc and water. The organic layer was washed with brine. The combined aqueous layers were extracted with EtOAc. The combined organic layers were dried over Na2S0 ₄ , filtered and concentrated to a crude, which was purified by flash chromatography (silica gel/ 5% to 10%MeOH/DCM) to give a crude, which was purified on a 2-inch reverse phase HPLC column to yield 3a (0.159 g, 0.11 mmol, 17%) and 4a (0.117 g, 0.078 mmol, 12%): MS m/z calcd for C73H91N7O27 (M+Na ⁺ ) 1520.6, found 1520.6; and 3b and 4b.

To a stirring solution of 4a (0.117 g, 0.078 mmol) in AcOH (40 mL) and H ₂ 0 (10 mL) was added Pd/C and the reaction was stirred under a hydrogen atmosphere for 2 hr. The reaction mixture was filtered through Celite, and washed with H2O and the combined washings were lyophilized to yield 5 as its acetate salt (65 mg,

+) 828.4, found 828.4.

Compound 5 (65 mg, 0.054 mmol) was dissolved in cold TFA/DCM (1:1, 2 mL) and the reaction was stirred at 0°C for 10 min. The reaction mixture was then dripped into vigorously stirring ΕΪ ₂ Ο (50 mL) and the resulting precipitate was collected by filtration. The solid was dissolved in water and lyophilized to yield 6 as its TFA salt (0.072 g, 0.051 mmol, 94%): MS m/z calcd for C28H53N7O15 728.4, found 728.3; CLND 96.2% purity.

Example 12

To a stirring solution of 1 (3 g, 1.58 mmol) in DMA (30 mL) was added sodium butanoate (1.5 g, 0.014 mmol) and the reaction was heated to 80°C. The reaction mixture was partitioned between water and EtOAc. The organic layer was washed with sat. aq. NaHC(½, brine, dried over MgSC , filtered and concentrated to yield 2 (3.1g), which was carried through to the next step without further purification.

To a stirring solution of zinc acetate (0.32 g, 1.74 mmol) in MeOH (9 mL) at 10°C was added sodium methoxide (25% in methanol, 2.74 mL, 12.69 mmol). This solution was allowed to stir for 10 minutes, then a solution of 2 (1.0 g, 0.529 mmol) in MeOH (9 mL) was added dropwise over 5 minutes and the reaction was allowed to stir at 10°C for 1 hour. The reaction was partitioned between EtOAc (50 mL) and citric acid:brine (5:1, 60 mL). The organic layer was washed with citric acid:brine (3:1, 200 mL), watenbrine (2:1, 150mL), sat. aq. NaHC0 ₃ (2 x 50 mL), brine (50 mL), dried over MgSC filtered and concentrated under vacuum to yield 3 (700 mg) as a white powder.

To a stirring solution of 3 (0.5 g, 0.365 mmol) in MeOH (8 mL) was added trifluoroacetic acid (80 uL, 1.038 mmol) and the reaction was heated to 60°C for 40 minutes. The reaction was cooled in an ice bath, quenched with Ν¾ΟΗ (80 uL) and concentrated under reduced pressure to yield a crude, which was purified on a 2-inch reverse phase HPLC column (Method 2) to yield 4 (246 mg) as a white powder.

To a stirring solution of 4 (0.225 g, 0.176 mmol) in AcOH:H ₂ 0 (4:1, 8 mL) at ambient temperature was added palladium hydroxide on carbon (20% by weight, wet, 200 mg) and the reaction was stirred under a hydrogen atmosphere for 1 hr. The reaction mixture was filtered and washed with water (20 mL) and the combined aqueous layers were further diluted with water (200 mL) and lyophilized to afford 5 (200 mg) as an off-white powder. MS m/z calcd for C ₂ gH ₅ oN ₆ Oi7 (M+H ¹ ) 743.3, found 743.3.

To a stirring solution of 5 (0.2 g, 0.203 mmol) in water (5 mL) at ambient temperature was added sodium hydroxide (2M, 3.385 mL, 6.77 mmol) and the reaction was heated to 50°C for 3 hours. The reaction mixture was lyophilized to yield a crude, which was purified on a 1-inch reverse phase HPLC column (0.1% HFBA) to yield 6 (26 mg) as its penta-HFBA salt. MS m/z calcd for C ₂ 7H ₅ 2N ₆ 0 ₁₆ (M+H ⁴ ) 717.3, found 717.3.

Compound 1 (200 mg, 0.137 mmol) was treated with N-Boc-l-hydroxy- azetidin-3-yl carboxylic acid following Procedure 14 to yield compound 2 (191 mg, 0.115 mol, 83.9%): MS m/z calcd for C86H97N7O27 (M+H) ⁺ 1661.7, found 1662.1.

Compound 2 (191 mg, 0.115 mmol) was submitted to hydrogenolysis following Procedure 16 to yield 3 as its TFA salt, which was carried through to the next step without further purification: 75.8 mg, 98.0 %): MS m/z calcd for C32H59N7O17 (M+H) ⁺ 814.8, found (M+H)/2 407.9

Compound 3 (128 mg, 0.093 mmol) was dissolved in 0.5 M HCl (1 mL) and the reaction was stirred for 3 hours. The reaction mixture was lyophilized to yield 4 as its HCl salt (86 mg, 99.8%): MS m/z calcd for C27H51N7O15 (M+H) ⁺ 714.7, found 714.3; CLND 97.2%.

Example 14

Compound 1 (200 mg, 0.137 mmol) was treated with iraws-N-Cbz-l- hydroxy-3-aminocyclobutyl carboxylic acid following Procedure 14 to yield compound 2 (197 mg, 0.115 mol, 83.9%): MS m/z calcd for C ₉ oH97N ₇ 0 (M+H) ⁺ 1709.8, found 1710.4,

Compound 2 (197 mg, 0.115 mmol) was submitted to hydrogenolysis following Procedure 16 to yield 3 as its TFA salt, which was converted to its sulfate salt according to Procedure 19 (90.2 mg, 0.088 mol, 76.5 %): MS m/z calcd for C ₂ 8H53N ₇ Oi5 (M+H) ⁺ 728.8, found 728.4; CLND 99.1 %.

Example 15

Compound 1 (200 mg, 0.137 mmol) was treated with czs-N-Cbz-1- hydroxy-3-aminocyclobutyl carboxylic acid following Procedure 14 to compound 2 (193 mg, 0.113 mol, 82.5%): MS m/z calcd for (M+H) ⁺ 1709.8, found 1709.3.

Compound 2 (193 mg, 0.113 mmol) was submitted to hydrogenolysis following Procedure 16 to yield 3 as its TFA salt, which was converted to its sulfate salt according to Procedure 19 (106 mg, 0.104 mol, 92.0 %): MS m/z calcd for C28H53N7O15 (M+H) ⁺ 728.8, found 728.4; CLND 100 %.

Example 16

Compound 1 (200 mg, 0.137 mmol) was treated with (25,3i?)-N-Cbz-2, 3-bisbenzyloxy-4-amino-butyric acid following Procedure 14 to yield compound 2 (214 mg, 0.113 mol, 82.5%): MS m/z calcd for C103H109 7O28 (M+H) ⁺ 1894.0, found 1894.6.

Compound 2 (214 mg, 0.113 mmol) was submitted to hydrogenolysis following Procedure 18 to yield 3 as its acetate salt, which was converted to its sulfate salt according to Procedure 19 (109 mg, 0.106 mol, 93.8 %): MS m/z calcd for C ₂₇ H ₅₃ N ₇ Oi ₆ (M+H) ⁺ 732.7, found 732.3; CLND 98.5 %.

Example 17

Compound 1 (200 mg, 0.137 mmol) was treated with (2S,5S -N-Cbz-2, 3-bisbenzyloxy-4-amino-butyric acid following Procedure 14 to yield compound 2 (188 mg, 0.099 mol, 72.3%): MS m/z calcd for C103H109N7O28 (M+H) ⁺ 1894.0, found 1895.2.

Compound 2 (188 mg, 0.099 mmol) was submitted to hydrogenolysis following Procedure 17 to yield 3 as its acetate salt, which was converted to its sulfate salt according to Procedure 19 (82 mg, 0.080 mol, 80.8 %): MS m/z calcd for C ₇ Oi ₆ (M+H) ⁺ 732.7, found 732.3; CLND 97.5 %.

Example 18

Compound 1 (200 mg, 0.137 mmol) was treated with (2i?,3i?)-2- benzyloxy-3-fluoro-4-azide-butyric acid following Procedure 15 to yield compound 2 (160 mg, 0.094 mol, 68.6%): MS m/z calcd for C88H94FN9O25 (M+H) ⁺ 1697.7, found

Compound 2 (160 mg, 0.094 mmol) was submitted to hydrogenolysis following Procedure 17 to yield 3 as its acetate salt, which was purified by RP HPLC (Method 4) and converted to its sulfate salt (36 mg, 0.035 mol, 37.2 %): MS m/z calcd for C ₂ 7H ₅ 2FN ₇ Oi5 (M+H) ⁺ 734.7, found 734.3; CLND 97.4 %.

Example 19

Compound 1 (79 mg, 0.054 mmol) was treated with 2(/?)-benzyloxy-3,3- bisfluoro-4-azide-butyric acid following Procedure 15 to yield compound 2 (53 mg, 0.031 mol, 57.4%): MS m/z calcd for C88H93F2N9O25 (M+H) ⁺ 1715.7, found 1716.3.

Compound 2 (53 mg, 0.031 mmol) was submitted to hydrogenolysis following Procedure 18 to yield a crude, which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (4 mg, 0.004 mol, 12.9 %): MS m/z calcd for C27H51F2N7O15 (M+H) ⁺ 752.7, found 752.3; CLND 98.9 %.

To a stirring solution of 1 (14.3 g, 7.81 mmol) in pyridine (46 mL) was added acetic anhydride (110 ml) and the reaction was stirred for 2 days. Methanol (100 mL) was added (careful exothermic reaction!), followed by EtOAc (1 L). The organic layer was washed with 1 M citric acid (1 L), 5% NaHC0 ₃ (2 x 1 L), brine : water (1:1, 1 L), brine (1 L), dried over Na ₂ SC>4, filtered and concentrated to dryness to yield compound 2 (8.2 g), which was carried through to the next step without further purification. MS m/z calcd for Cio6Hn5 ₅ 0 ₂₅ Si ₂ (M+Na ⁺ ) 1936.7, found 1936.4.

To a stirring solution of 2 (9.1 g, 4.76 mmol) in AcOH (19.8 mL) was added TFA (1.8 mL of a 3:1 TFA: water solution), and the reaction was heated at 40 ^° C for 45 min. Additional TFA (0.45 mL of a 3:1 TFA: water solution) was added and the reaction was heated for 1.5 hr. The reaction was then cooled to room temperature, diluted with EtOAc (200 mL) and washed with sat. aq. NaHC<¾ (3 x 200 mL) and brine (200 mL). The organic layer was dried over Na ₂ S(¾, filtered and concentrated to dryness to yield compound 3 (8.7 g, 3.43 mmol), which was carried through to the next step without further purification. MS m/z calcd for C HmN ₅ 025Si2 (M+Na ⁺ ) 1848.7, found 1848.3.

To a stirring solution of 3 (8.7 g, 4.76 mmol) in pyridine (95 mL) was added p-toluenesulfonyl chloride (5.45 g, 28.6 mmol) and the reaction was stirred for 2 hours. Additional p-toluenesulfonyl chloride (1.82 g, 9.52 mmol) was added and the reaction was stirred for another 7 hours at room temperature. The reaction was diluted with ethyl acetate (900 mL) and washed with 0.5 M citric acid (2 x 1 L) , 5% NaHC0 ₃ (1 L), brine (500 mL), dried over Na ₂ S0 ₄ and concentrated under vacuum to approximately 80 mL. This solution was dripped into a vigorously stirring mixture of 8:1 hexanes: Et ₂ 0 (900 mL). The precipitate was filtered off and washed with hexanes (200 mL) and dried under high vacuum overnight to provide a crude (8.6 g), which was purified by RP-HPLC (Method 2) to yield compound 4 (4.5 g, 2.158mmol, 64% yield): MS m/z calcd for C106H117N5O27SS12 (M+Na ⁺ ) 2002.7, found 2002.4.

To a stirring solution of 4 (7.7g, 3.88 mmol) in toluene (78 mL) at room temperature was added a solution of TCDI (5.4g, 30.3mmol) in toluene (78 mL) and the reaction was stirred overnight. The reaction was diluted with EtOAc and washed with 1M citric acid (3 x 1 L), brine:water (1 :1 v,v, 3 x 250 mL), dried over Na ₂ S0 ₄ , and concentrated to dryness to yield 5 (8.3 g), which was carried through to the next step without further purification: MS πιίζ calcd for C ₁ ioHii9N ₇ 0 ₂₇ S ₂ Si2 (M+Na ⁺ ) 2112.7, found 2112.4.

To a stirring solution of 5 (8.1g, 3.87 mmol) in 1,4-dioxane (100 mL) was added 2,2'-azobisisobutyronitrile (32 mg, 0.174 mmol) followed by tris(trimethylsilyl)silane (34.3 uL, 7.67 mmol) and the reaction was heated to an internal temperature of 85°C for one hour. The reaction was allowed to cool to room temperature, was diluted with EtOAc (1 L) washed with 0.5M citric acid (2 x 1 L), sat.aq. NaHC0 ₃ (lxlL), brine (500 mL), dried over Na ₂ S0 ₄ and concentrated to dryness to yield a crude, which was dissolved in EtOAc (80 mL) and dripped into a mixture of vigorously stirring hexanes (1.125 L) and diethyl ether (0.125 L). Filtration of the resulting precipitate provided compound 6 (6.6 g), which was carried through to the next step without further purification. MS m/z calcd for (M+H) ⁺ 1964.7, found 1964.5.

To a stirring solution of 6 (3.0 g, 1.53 mmol) in DMPU (52 mL) was added sodium azide (0.794 g, 12.21 mmol) and the reaction was heated to 70 °C. After 2 hours, the reaction was cooled to room temperature, diluted with EtOAc (500 mL), washed with water (2 x 800 mL), brine : water (1:1 v/v, 1 L), brine (500mL), dried over Na ₂ S0 ₄ and concentrated to dryness to yield 7 (2.85 g), which was carried through to the next step without further purification. MS m/z calcd for C Hno g02 ₃ Si2 (M+Na ⁺ ) 1857.7, found 1857.6.

To a stirring solution of 7 (2.2 g, 1.20 mmol) in DMF (9 mL) was added water (0.19 mL) followed by TBAF (75% in water, 3.14 mL, 8.58 mmol) and the reaction was heated at 45 °C overnight. Methylamine (40% in water, 8.4 mL, 50 mmol) was then added and the reaction was stirred for 4 hours. The reaction was diluted with EtOAc (500mL), washed with 1 M citric acid (1 L), brine : water (1:5 v/v, 600 ml), sat. aq. NaHC<¾ (150mL), brine (300mL), dried over Na2S<¾ and concentrated to provide a crude, which was dissolved in EtOAc (30 mL) and dripped into a mixture of vigorously stirring MTBE (250 mL) and hexanes (250 mL). The resulting precipate was filtered, washed with hexanes (3 x 60 mL) and dried under vacuum to provide a crude, which was purified by RP-HPLC (Method 2) to yield 8 (0.582 g, 0.447 mmol, 52% yield): MS m/z calcd for C62H72 8O20 (M+Na ⁺ ) 1271.5, found 1271.3.

Compound 8 (50 mg, 0.040 mmol) was treated with N-Cbz-2(S)- hydroxy-3-aminopropionic following Procedure 14 to yield compound 9 (48 mg, 0.033 mol, 82.5%): MS m/z calcd for C ₇ 3H83N ₉ 024 (M+H) ⁺ 1471.5, found 1471.0.

Compound 9 (48 mg, 0.033 mmol) was submitted to hydrogenolysis following Procedure 18 to yield 10 as its acetate salt, which was converted to its sulfate salt following Procedure 19 (20 mg, 0.020 mol, 60.6 %): MS m/z calcd for C ₂₆ H ₅ iN ₇ Oi4 (M+H) ⁺ 686.7, found 686.3; CLND 92.4 %.

Example 21

Compound 1 (50 mg, 0.040 mmol) was treated with N-Cbz-l-hydroxy- azetidin-3-yl carboxylic acid following Procedure 14 to yield compound 2 (40 mg, 0.027 mol, 67.5%): MS m/z calcd for C74H83N9O24 (M+H) ⁺ 1483.5, found 1482.8.

Compound 2 (40 mg, 0.027 mmol) was submitted to hydrogenolysis following Procedure 18 to yield the crude acetate salt (28 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (9 mg, 0.009 mol, 33.3 %): MS m/z calcd for 0 ₂ τΆ^ _Ί Οη (M+H) ⁺ 698.7, found 698.3; CLND 98.9 %.

Example 22

Compound 1 (50 mg, 0.040 mmol) was treated with iraKs-N-Cbz-l- hydroxy-3-aminocyclobutyl carboxylic acid following Procedure 14 to compound 2 (50 mg, 0.033 mol, 82.5%): MS m/z calcd for C75H85N9O2 ₄ (M+H) ⁺ 1497.5, found 1497.9.

Compound 2 (50 mg, 0.033 mmol) was submitted to hydrogenolysis following Procedure 18 to yield the crude acetate salt (36 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (11 mg, 0.011 mol, 33.3 %): MS m/z calcd for C27H51N7O14 (M+H) ⁺ 712.8, found 712.4; CLND 99.6 %.

Example 23

Compound 1 (50 mg, 0.040 mmol) was treated with cw-N-Cbz-1- hydroxy-3-aminocyclobutyl carboxylic acid following Procedure 14 to yield compound 2 (49 mg, 0.033 mol, 82.5%): MS m/z calcd for C75H85N9O24 (M+H) ⁺ 1497.5, found 1497.8.

Compound 2 (49 mg, 0.033 mmol) was submitted to hydrogenolysis following Procedure 18 to yield the crude acetate salt (35 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (11 mg, O.Ol lmol, 33.3 %): MS m/z calcd for ¾7Η ₅₁ Ν ₇ 0 ₁₄ (M+H) ⁺ 712.8, found 712.4; CLND 98.5 %.

Example 24

Compound 1 (50 mg, 0.040 mmol) was treated with (25,3i)-N-Cbz-2, 3- bisbenzyloxy-4-arnino-butyric acid following Procedure 14 to yield compound 2 (59 mg, 0.035 mol, 87.5%): MS m/z calcd for C88H97N9O25 (M+H) ⁺ 1681.8, found 1682.0.

Compound 2 (59 mg, 0.035 mmol) was submitted to hydrogenolysis following Procedure 18 to yield the crude acetate salt (33 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (9 mg, 0.009 mol, 25.7 %): MS m/z calcd for C27H53N7O15 (M+H) ⁺ 716.7, found 716.4; CLND 95.9 %. Example 25

Compound 1 (50 mg, 0.040 mmol) was treated with (2S,3S)-N-Cbz-2, 3- bisbenzyloxy-4-amino-butyric acid following Procedure 14 to yield compound 2 (52 mg, 0.031 mol, 77 %): MS m/z calcd for C88H97N9O25 (M+H) ⁺ 1681.8, found 1682.0.

Compound 2 (52 mg, 0.031 mmol) was submitted to hydrogenolysis following Procedure 18 to yield the crude acetate salt (38 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (9 mg, 0.009 mol, 29%): MS m/z calcd for C27HS3N7O15 (M+H) ⁺ 716.7, found 716.4; CLND 99.4 %. Example 26

Compound 1 (50 mg, 0.040 mmol) was treated with (2R,3R)-2- benzyloxy-3-fluoro-4-azide-butyric acid following Procedure 15 to yield compound 2 (47 mg, 0.032 mol, 80.0 %): MS m/z calcd for CJSHKFNHC^ (M+H) ⁺ 1485.5, found 1484.8.

Compound 2 (47 mg, 0.032 mmol) was submitted to hydrogenolysis following Procedure 17 to yield the crude acetate salt (36 mg), which was purified by RP HPLC (Method 4) to yield 3 as its sulfate salt (1 1 mg, 0.01 1 mol, 33.7 %): MS m/z calcd for C ₂ 7H52FN7O14 (M+H) ⁺ 718.7, found 718.4; CLND 96.7 %.

Example 27

Compound 1 (81 mg, 0.065 mmol) was treated with 2(if)-benzyloxy-3,3- bisfluoro-4-azide-butyric acid following Procedure 14 to yield compound 2 (70 mg, 0.047 mol, 72.3%): MS m/z calcd for C ₇ 3H ₈₁ F _{2 U} 0 ₂₂ (M+H) ⁺ 1503.5, found 1503.0.

Compound 2 (70 mg, 0.047 mmol) was submitted to hydrogenolysis following Procedure 17 to yield the crude acetate salt (76 mg), which was purified by RP HPLC (Method 4) to yield 3 (5 mg, 0.005 mol, 10.2%): MS m/z calcd for C ₂ 7H5,F2N ₇ Oi4 (M+H) ⁺ 736.7, found 736.3; CLND 99%.

Example 28

To a stirring solution of 1 (275 g, 140 mmol) in toluene (2000 ml) was added TCDI (59.9 g, 336 mmol) and the reaction was stirred for 2 days. Water (3.78 mL, 210 mmol) was added, followed by DMAP (103 g, 840 mmol) and the reaction was stirred for 6 hrs. Additional water (1.26 mL, 0.5 eq) was added and the reaction was stirred overnight. The reaction mixture was washed with 1 M citric acid: brine (4: 1 v/v, 2 x 2000 mL), brine (1000 mL), dried over MgSC , filtered and concentrated to dryness to yield 2 (268 g), which was carried through to the next step without further purification : MS m/z calcd for (M+Na ⁺ ) 2095.8, found 2096.4.

To a stirring solution of 2 (134.6 g, 64.9 mmol) in 1,4-dioxane (1300 mL) in a 2-L two-neck RBF was added l,l ,l,3,3,3-hexamethyl-2- (trimethylsilyl)trisilane (46 mL, 149 mmol) followed by AIBN (1.07 g, 6.49 mmol), and the reaction was stirred for one hour at 100°C. The reaction was allowed to cool to room temperature and water (90 mL) was added, followed by TFA (7.5 mL, 97 mmol) and the reaction was allowed to stir overnight. The reaction mixture was basified by the slow addition of concentrated NH4OH (10 mL) and was then concentrated under vacuum to a sticky solid, which was dissolved in ethyl acetate (1300 mL) and washed with saturated NaHC0 ₃ (1300 mL), 5% NaHC0 ₃ (1300 mL), brine (600 mL), dried over MgSC>4 (10 g), filtered and concentrated under vacuum. The resulting solids were dissolved in ethyl acetate (370 mL) and dripped into a vigorously stirring solution of hexanes: MTBE (3:1 v/v, 7.4 L). The resulting precipitate was filtered and dried under high- vacuum for 3 days to provide 3 (11 1.8 g), which was carried through to the next step without further purification. MS m/z calcd for CiioHii ₈ N ₆ 0 ₂ 3Si2 (M+Na ⁺ ) 1971.3, found 1971.3.

To a stirring solution of 3 (54.5 g, 28 mmol) in DMF (295 mL) was added TBAF (103 mL, 75% in H ₂ 0, 280 mmol) followed by water (7.6 mL, 420 mmol), and the reaction was heated to 50°C and stirred overnight. The reaction mixture was diluted with ethyl acetate (1250 mL), washed with 1M citric acid (1000 mL), brine (1000 mL), sat. aq. NaHC0 ₃ (1000 mL), brine (750 mL), dried over Na ₂ S0 (lOg), concentrated to dryness to yield a crude, which was dissolved in ethyl acetate (90 mL) and dripped into a vigorously stirring solution of MTBE : hexanes (1 :1 v/v, 1.8 L). The resulting precipitate was filtered and dried under high-vacuum to provide a crude (35.9 g), which was purified by RP HPLC (Method 2) to yield 4 (15.5 g, 10.7 mmol, 38.2%): MS wJz calcd for 0 ₇ 7¾ ₄ Ν ₆ θ22 (M+H ⁺ ) 1445.6, found 1445.3.

To a stirring solution of 4 (5.4 g, 3.74 mmol) in DMF (62.3 mL) was added N-Cbz-l-hydroxy-azetidin-3-yl carboxylic acid (0.844 g, 3.36 mmol), followed by DIPEA (1.96 mL, 11.21 mmol) and PyBOP (1.26 g, 3.36 mmol) and the reaction was stirred for 2 hours. Additional N-Cbz-l-hydroxy-azetidin-3-yl carboxylic acid

(0.188 g, 0.747 mmol) and PyBOP (0.280 g, 0.747 mmol) were added and the reaction stirred for 1 hour. The reaction was diluted with ethyl acetate (200 ml), washed with 1M citric acid (200 ml), brine (200 mL), sat. aq. NaHC0 ₃ (200 mL), brine (200 mL), dried over Na ₂ S0 ₄ , filtered, and concentrated to yield compound 5 (6.58g), which was carried through to the next step without further purification. MS: mfz calcd for C89H95N7O26 (M+Na ⁺ ) 1700.6, found 1700.5.

To a stirring solution of compound 5 (6.58 g, 3.92 mmol) in pyridine (47.6 mL, 588 mmol) was added acetic anhydride (55.5 mL, 588 mmol) and the reaction was stirred for 17 hours. The reaction mixture was diluted with ethyl acetate (200 mL), washed with 1M citric acid (3 x 200 mL), brine (200 mL), sat. aq. NaHC0 ₃ (2 x 200 mL), brine (200 mL), dried over Na ₂ SC>4, filtered and concentrated to yield compound 6 (7.6 g), which was carried through to the next step without further purification. MS: m/z calcd for C99H ₁₀ 5 ₇ O3i 1910.7 (M+Na ⁺ ), found 1910.4.

To a stirring solution of compound 6 (6.5 g, 3.44 mmol) in toluene (108 mL) was added 4 M HC1 in dioxane (4.30 mL, 17.21 mmol), followed by ethylene glycol (0.230 mL, 4.13 mmol) and the reaction was stirred for 5 hours. The reaction mixture was diluted with ethyl acetate (100 mL), washed with sat. aq. NaHCCb (2 x 150 mL), brine (100 mL), dried over Na ₂ SC>4, filtered and concentrated to a solid, which was purified by RP HPLC(Method 2) to yield compound 7 (2.69 g, 1.49 mmol, 43%). MS: m/z calcd for C92H101N7O31 (M+Na^ 1822.6, found 1822.4.

To a stirring solution of compound 7 (2.31 g, 1.29 mmol) in pyridine (28.6 mL) was added methanesulfonyl chloride (0.200 mL, 2.57 mmol) and the reaction was heated at 110 °C for 24 hours. The reaction was diluted with ethyl acteate (100 mL), washed with 1M citric acid (3 x 100 mL), sat. aq, NaHC0 ₃ (2 x 100 mL), brine (100 mL), dried over Na ₂ SC>4, filtered and concentrated to a solid, which was purified by RP HPLC (Method 2) to yield compound 8 (0.715 g, 0.422 mmol, 33%). MS: m/z calcd for Ces^N^o (M+lT) 1692.6, found 1692.4.

To a stirring solution of compound 8 (0.775 g, 0.458 mmol) in NMP (12.11 mL) was added 0.5 M LiOH (14.53 mL, 7.27 mmol) and the reaction was heated at 40 ^° C for 27 hours. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water (100 mL), sat. aq. NaHC0 ₃ (100 mL), brine (100ml), dried over Na2S0 ₄ , filtered, and concentrated to dryness yield compound 9 (0.88 g), which was taken through to the next step without further purification. MS: m/z calcd for C ₈ sN ₇ 0 (M+H ⁺ ) 1456.6, found 1456.4.

To a stirring solution of compound 9 (0.88 g, 0.603 mmol) in acetone (13.35 mL) was added ₂ C0 ₃ (0.128 g, 2.14 mmol), followed by 2-bromoethanol (0.124 mL, 1.76 mmol) and the reaction was heated at 35 °C for 17 hours. Additional 2-bromoethanol (0.496 mL, 7.05 mmol) was added and the reaction was heated at 35 °C for 4 days. The reaction mixture was diluted with ethyl acetate (100 mL), washed with brine: water (1:1 v/v, 100 mL), brine (100 ml), dried over Na ₂ SC>4, filtered and concentrated to dryness to yield compound 10 (0.89g), which was carried through to the next step without further purification. MS: m/z calcd for C76H ₈ 9N7C¾(M+H^) 1500.6, found 1500.5.

To a stirring solution of compound 10 (0.89 g, 0.596 mmol) in THF (10.66 mL) and ACN: water (4:1 v/v, 10.66 mL) at 0°C was added N- (benzyloxycarbonyloxy)-succinimide (0.27 g, 1.066 mmol) and the reaction was stirred for 2 hours. DIPEA (0.102 mL, 0.586 mmol) was then added and the reaction was stirred for 2 hours. Additional N-(benzyloxycarbonyloxy)-succinimide (0.133 g, 0.533 mmol) and DIPEA (0.051 mL, 0.293mmol) were added and the reaction was stirred for 1 hour. The reaction was quenched with N,N-dimethylpropane- 1,3 -diamine (0.403 mL, 3.20 mmol) and the reaction was stirred for 30 minutes. The reaction mixture was diluted with ethyl acetate (150 mL), washed with 1 M citric acid (100 mL), brine (50 mL), sat. aq. NaHC0 ₃ (100 mL), brine (50 mL), dried over Na ₂ S0 ₄ , filtered and concentrated to a crude, which was purified by RP HPLC (Method 2) to yield compound 11 (0.132 g, 0.081 mmol, 14%). MS: m/z calcd for C84H95N7O27 (M+Na ⁺ ) 1656.6, found 1656.6.

To a stirring solution of compound 11 (130 mg, 0.080 mmol) in acetic acid (3.18 mL) and water (0.8 mL) was added Pd(OH)2/C (134 mg) and the reaction was stirred under a hydrogen atmosphere for 4 hours. The reaction mixture was filtered, washed with water, and lyophilized. The resulting solid was purified by RP HPLC (Method 4) to yield compound 12 as its sulfate salt (50 mg, 0.048 mmol, 60%). MS: m/z calcd for CIQHSSNTOIS (M+H ⁺ ) 742.4, found 742.3; CLND 99%.

Example 29

To a stirring solution of compound 1 (0.85 g, 0.452 mmol) in DMA (2.258 mL) was added methyl amine (40% aq solution) (1.93 g, 24.84 mmol) and the reaction was heated at 40°C for 18 hours. The reaction was diluted with ethyl acetate (100 mL), washed with sat. aq. NH ₄ C1 (100 mL), sat. aq. NaHC0 ₃ (2 x 100 mL), brine (100 mL), dried over Na ₂ S0 ₄ , filtered, and concentrated to a solid, which was purified by RP HPLC (Method 2) to yield compound 2 (0.431g, 0.29 mmol, 64%). MS: m z calcd for 0 ₇₅ α^ ₇ 0 ₂₅ (M+If) 1488.6, found 1488.4.

To a stirring solution of compound 2 (0.431 g, 0.290 mmol) in methanol (48.3 mL) was added TFA (0.268 mL, 3.47 mmol), followed by Pd(OH) ₂ /C (0.407 g) and the reaction was stirred under a hydrogen atmosphere for 18 hours. The palladium was filtered off, washed with water, and the filtrate was lyophilized. The resulting solid was dissolved in water (5mL) and pH adjusted to 7.5 using concentrated NH4OH. Ammonium sulfate (0.225 g, 1.706 mmol) was then added and the solution was dripped into anhydrous methanol (80 mL). The solids were filtered off, and washed with methanol (20 mL) to yield compound 3 (0.158 g, 0.154 mmol, 53%). MS: m/z calcd for C28H55N7O1S (M+H ⁺ ) 730.4, found 730.3.

Example 30

To a stirring solution of compound 1 (0.338 g, 0.180 mmol) in DMA (3.27 mL) was added Ι,Γ-thiocarbonyldiimidazole (0.298 g, 1.670 mmol) and the reaction was heated at 40°C for 25 hours. The reaction was diluted with ethyl acetate (100 mL), washed with 1 M citric acid (3 xlOO mL), brine (3 x lOOmL), dried over Na2SC filtered, and concentrated to dryness to yield compound 2 (0.35g), which was carried through to the next step without further purification. MS: m/∑ calcd for C97H106N8O34S2 (M+H ⁺ ) 1991.6, found 1991.2.

To a stirring solution of compound 2 (0.35 g, 0.176 mmol) in dioxane (12.76 mL) was added tris(trimethylsilyl)silane (0.162 mL, 0.471 mmol) followed by AIBN (11.49 mg, 0.061 mmol) and the reaction was heated at 100°C for one hour. The reaction was diluted with ethyl acetate (50 mL), washed with sat. aq. NaHC0 ₃ (50 mL), brine (50 mL), dried over a ₂ SC> , filtered and concentrated to dryness to yield compound 3 (0.452g), which was carried through to the next step without further

+) 1887.6, found 1887.5.

To a stirring solution of compound 3 (0.452 g, 0.242 mmol) in DMA (2.42 mL) was added methyl amine (40% aq solution) (2.069 g, 26.6 mmol) and the reaction was heated at 40°C for 2 days. The crude reaction was purified by RP HPLC column (Method 2) to yield compound 4 (0.102g, 0.069 mmol, 29%). MS: m/z calcd for C ₇ 5H _g9 N ₇ 024 (M+H ⁺ ) 1472.6, found 1472.4.

To a stirring solution of compound 4 (0.102 g, 0.069 mmol) in methanol (11.54 mL) was added TFA (0.038 mL, 0.499 mmol) followed by Pd(OH) ₂ /C (0.097 g) and the reaction was stirred under a hydrogen atmosphere for 18 hours. The palladium was filtered off and washed with water. Methanol was removed by evaporation and the resulting aqueous solution was lyophilized. The solid was dissolved in water (1.5 mL), and the pH was adjusted to 7.5 using concentrated NH ₄ OH. Ammonium sulfate (0.051 g, 0.383 mmol) was then added and the solution was dripped into anhydrous methanol (25 ml). The resulting precipitate was collected by centrifugation and washed with methanol. The solid was then dissolved in water (5 ml) and lyophilized to yield compound 5 (0.03g, 0.030 mmol, 43.5%). MS: m/z calcd for C28H55N7O1 (M+rT) 714.4, found 714.2.

Other Representative Compounds

The following representative compounds may be prepared according to the foregoing procedures.

242

IJ44









251

IJ53

IJ54

IJ55







260

261

IJ64

IJ66





270

271

272

IJ73

IJ74

IJ75



IJ83

IJ85

MIC ASSAY PROTOCOL

Minimum inhibitory concentrations (MIC) were determined by reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution methods per M7-A7 [2006]. Quality control ranges utilizing E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 29213, and interpretive criteria for comparator agents were as published in CLSI M100-S17 [2007]. Briefly, serial two-fold dilutions of the test compounds were prepared at 2X concentration in Mueller Hinton Broth. The compound dilutions were mixed in 96-well assay plates in a 1 :1 ratio with bacterial inoculum. The inoculum was prepared by suspension of a colony from an agar plate that was prepared the previous day. Bacteria were suspended in sterile saline and added to each assay plate to obtain a final concentration of 5x10^ CFU/mL. The plates were incubated at 35C for 20 hours in ambient air. The MIC was determined to be the lowest concentration of the test compound that resulted in no visible bacterial growth as compared to untreated control. Data for certain representative compounds is shown in Table 1 below. In addition, comparative data for certain representative 374'-epi compounds and certain non-374'-epi compounds is shown in Table 2 below.

Table 1 Representative

Compound

Example # 1 Compound # AECOOOl APAE001

1/19 B A

1 /20 A A

2/8 A A

3/4 C A

4/26 A A

5/16 B A

6/10 C C

7/6 B B

8/11 B A

9/11 B B

9/12 B A

10/5 B A

10/6 B A

11/6 B A

12/6 C C

13/4 B A

14/3 B A

15/3 B A

16/3 B A

17/3 B A

18/3 B A

19/3 B A

20/10 B A

21/3 B A

22/3 B A

23/3 B A

24/3 B A

25/3 B A

26/3 A A

27/3 B A

28/12 ND B

29/3 B A

Table 2

* AECOOOl is ATCC25922 and APAEOOl is ATCC27853, AABA1082 is an Acinetobacter Baumannii clinical isolate expressing the APH(3')-VI aminoglycoside modifying enzyme, AECL004 is an Enterobacter cloacae clinical isolate expressing the APH(3')-I aminoglycoside modifying enzyme, ASAU003 is a Staphylococcus aureus isolate expressing the ANT(4')-I.

** MIC Key:

MIC's of 1.0 μg/mL or less = A

MIC's of greater than 1.0 μ^ιηΐ, but less than 8.0 μg mL = B

MIC's of greater than or equal to 8.0 μg/mL but less than or equal to 16 μg mL = C

MIC's of greater than 16.0 μ^Γηί = D

All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.