COMPOSITIONS AND METHODS FOR TARGETING RECEPTORS EXPRESSED

IN THE GUT

Related Applications

This application claims the benefit of priority to United States Patent Application serial numbers 62/028,533, filed July 24, 2014; 62/028,521 , filed July 24, 2014;

62/028.538. filed July 24. 2014: 62/059.354, filed October 3, 2014: 62/059.357. filed October 3, 2014; and 62/059,460, filed October 3, 2014, the contents of which arc hereby incorporated by reference.

Background Gut mucosal surfaces bear many receptors, sensitive to nutrients and other signaling molecules that reflect changing conditions in the environment of the gut. Modulation of these receptors can have effects ranging from local (modulating intestinal inflammation) or intuitive (modulating hunger and satiety) to systemic (diabetes) or neurological

(depression). However, many compounds that effectively modulate these receptors to beneficial effect in the gut cause adverse effects when these compounds arc absorbed and affect receptors elsewhere in the body. Improved methods for selectively targeting gut receptors arc needed.

Summary of Invention

The present invention provides compounds and methods for selectively modulating a gut-expressed biological target, such as a receptor, by displaying receptor modulators on scaffolds that arc not absorbed into the bloodstream.

In certain embodiments, the invention prov ides compounds comprising:

a scaffold having a plurality of branches, wherein each of a plurality of branches terminates in a pharmacophore having an affinity for a receptor expressed in a gastrointestinal tract: or a pharmaceutically acceptable salt thereof;

w herein bonds linking the pharmacophores to the scaffold arc not hydrolyzcd under

physiologic conditions; and

wherein the compound weighs less than about 10.000 Daltons.

The invention also provides pharmaceutical compositions of the compounds of the invention. In certain embodiments, the invention prov ides mctliods for treating metabolic syndrome or a disorder associated with metabolic syndrome in a subject in need thereof, comprising administering to the subject a compound of the invention. The invention also provides methods of administering the compound to a subject suffering from metabolic syndrome or a disorder associated with metabolic syndrome.

In certain embodiments, the invention also provides methods of making a compound of the invention, comprising contacting a scaffold having a plurality of branches, wherein each of a plurality of branches terminates in a first reactive moiety, with a plurality of linking molecules, each linking molecule comprising a pharmacophore linked to a second reactive moiety, under conditions sufficient to induce a coupling reaction, such as a cycloaddition. between the first reactive moiety and the second reactive moiety.

Figures 1 -3 describe various embodiments of the invention and methods of preparing compounds of the invention, but arc intended to be exemplary and not limiting in any way. Brief Description of the Drawings

Figure 1 depicts a general structure of a pharmacophorc-appcndcd scaffold of the invention.

Figure 2 depicts characterization data for a compound of the invention.

Figure 3 depicts characterization data for a compound of the invention.

Figure 4 is a table containing in vitro data for exemplary gut-restricted compounds of the invention.

Figure 5 contains tables of w hole cell in vitro data for reduction of cytokine IL- Ι β using exemplary gut-rcstrictcd TLR4 antagonists.

Figure 6 is a tabic showing in vitro data for reduction of cytokine IL- l p using exemplary gut-rcstrictcd compounds of the invention.

Figure 7 is a table showing in vitro data for reduction of cytokines IL-6 and TNF-a using exemplary gut-restricted compounds of the invention.

Figure X contains a scries of bar graphs that show reduction of pro-inflammatory cytokines I L- 1 β and I L-6 using a gut-rcstrictcd TGR-5 agonist compound of the invention.

Figure 9 is a bar graph demonstrating damage scores in a TNBS/DSS-induccd colitis model. Figure 1 0 contains charts that show in vitro results of administration of a gut- rcstrictcd compound of the inv ention in a DSS-induccd col itis model.

Figure 1 1 contains bar graph that show in vitro results of administration of a gut- rcstrictcd compound of the invention in a DSS-induccd colitis model.

Figure 12 contains a bar graph that demonstrates the in vivo reduction of proinflammatory cytokines I L-6 using a gut-restricted TLR-4 atagonist compound of the invention.

Figure 1 3 contains a bar graph that demonstrates the in vivo reduction of proinflammatory cytokines I L- 1 ß using a gut-restricted TLR-4 antagonist compound of the invention.

Figure 14 is a chart that shows in; vivo efficacy of a colitis treatment regimen using a gut-restricted TLR-4 antagonist and a 5HT ₇ antagonist compound of the invention.

Figure 15 is a chart that shows in vivo efficacy of a colitis treatment regimen using a gut-rcstrictcd TGR5 agonist and a 5HT- antagonist compound of the invention.

Figure 16 is a bar graph that provides the weight effects of administration of a TLR4 antagonist, a TGR-5 agonist and a GPR- 1 20 agonist.

Figure 1 7 is a chart that shows the effects of oral-administration of a gut-rcstrictcd compound of the inv ention on plasma glucose levels.

Figure 1 8 is a chart that shows the effects of oral-administration of a gut-rcstrictcd compound of the inv ention on plasma insulin lev els.

Figure 19 is a chart that shows the effects of oral-administration of a gut-rcstrictcd compound of the invention on plasma glucose levels.

Figure 20 contains bar graphs that demonstrate the potential for the gut-rcstrictcd compounds of the invention to be applied to treating colorectal cancer

Detailed Description of the Invention

In one aspect, the invention prov ides a compound weighing less than about 10,000 Daltons (preferably less than about 7500 Da. less than about 5000 Da. less than about 3000 Da, or ev en less than about 2000 Da), comprising a scaffold having a plurality of branches (preferably at least 3. or 4, 5. or more branches), wherein some or all of the branches

(preferably at least 3. or 4, 5, or more branches) terminate in a pharmacophore having an affinity for a biological target expressed in a gastrointestinal tract, or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound is dendrimcr. e.g.. where the branches arc identical or similar (e.g., differing primarily in the terminal

pharmacophore).

The scaffold is a molecular framework for joining the various branches and pharmacophores, and may be oligomcric, dendritic, cyclic, acyclic, branched, linear, or any other suitable form for covalcntly linking the various elements of the compound together. Similarly, the branches may be long or short, cyclic or acyclic, or any other suitable configuration for covalcntly linking the pharmacophores to the scaffold. The primary function of the scaffold and branches is to present the pharmacophores in an environment that is stcrically unhindered enough for the pharmacophore to interact with its target receptor. Thus, a compact scaffold (e.g., a glucose ring, w here branches can be coupled to neighboring hydroxyl groups) may be paired with longer branches to avoid stcric crowding of the pharmacophores, while a scaffold with greater spacing betw een branching points (e.g., an oligomcric chain formed by reacting cpichlorohydrin and ethylene glycol) may be able to utilize shorter branches without compromising interaction of the pharmacophores with their target receptors. In certain embodiments, each branch terminates in a pharmacophore. In certain embodiments, at least one branch docs not terminate in a pharmacophore.

The pharmacophores arc chemical moieties capable of interacting w ith (e.g., activating or inactivating, agonizing or antagonizing) a biological receptor, preferably on a selective basis. In some embodiments, a pharmacophore is essentially a drug molecule that is covalcntly attached to the rest of the compound: that is. a pharmacophore, severed from its branch and scaffold, retains its ability to interact with the receptor that it targets as part of the compound and has biological activity of its own.

For any given molecule of the compound, the pharmacophores can be identical for all branches that terminate in a pharmacophore, or some branches may terminate in one pharmacophore while other branches terminate in a different pharmacophore. In some embodiments, each branch terminates in a different pharmacophore. Different

pharmacophores of a molecule may target the same receptor or different receptors expressed in the gut. In certain embodiments, pharmacophores arc displayed on the scaffold in an arrangement that permits two or more pharmacophores of a compound to

simultaneously interact with tw o or more target receptors on the surface of a single cell. When a compound comprises at least tw o different pharmacophores, administering the compound may provide an additive tlicrapeutic cfTcct (or even a synergistic therapeutic effect) relative to the therapeutic effect of administering a mixture of analogous compounds where each pharmacophore of a compound is the same, but the mixture provides pharmacophores in the same ratio present in the multi-pharmacophorc compound (in other words, as though the pharmacophores of the multi-pharmacophorc compound were redistributed among scaffolds such that the pharmacophores of a single scaffold were all identical, but the mixture of scaffolds comprises the same pharmacophores in

corresponding proportions and amounts as in the multi-pharmacophorc compound).

In certain embodiments, one or more pharmacophores affect the expression level of one or more inflammatory cytokines in gut mucosal cells. Inflammatory cytokines include, but are not limited to IL- l beta, IL-6, TNF-alpha, or IL- 1 U. In certain embodiments, the precise nature of the specific gut-lumen expressed molecular target is not defined. In certain embodiments, one or more pharmacophores decrease the expression of one or more inflammatory cytokines such as IL- l bcta. IL-6, or TNF-alpha.

In certain embodiments, one or more pharmacophores have an affinity for a biological target. Such biological targets include, but arc not limited to receptors, ion channels, and transporters. In certain embodiments, the receptor is a PRR-typc receptor (Pattern Recognition Receptor), such as a TLR-type receptor. In certain embodiments, one or more pharmacophores have an affinity for a bile acid receptor, taste receptor, olfactory receptor. TGR-typc. or G PR-type receptor.

In certain embodiments, one or more pharmacophores have an affinity for receptor such as Bradykinin B _: . GPR40. GPR43, GPR 109A. GPR 120. a taste receptor such as T1 R 1 , T1 R2, T 1 R3, T2R 13, an olfactory receptor such as OR2A4, a-Gustducin,

GPCRC6A, GPR55, and GPR92.

In certain embodiments, one or more pharmacophores have an affinity for receptor such as TLR-4. Bradykinin B _: , GPR40, GPR43. GPR l 09A, GPR 120, a taste receptor such as T l R l , Tl R2. T l R3, T2R 13, an olfactory receptor such as OR2A4. a-Gustducin.

GPCRC6A, GPR55, and G PR92.

In certain embodiments, one or more pharmacophores have an affinity for receptor such as Bradykinin B _: . 5HT-. GPR40. GPR43, GPR 109 A. GPR 120. a taste receptor such as T I R 1 , T1 R2. T 1 R3. T2R 13. an olfactory receptor such as OR2A4, a-Gustducin.

GPCRC6A, GPR55, and GPR92. In certain embodiments, one or more pliarmacophorcs have an affinity for receptor such as Bradykinin B:. GPR40, GPR43. GPR I 09A, GPR 120, Histamine H ₄ , a taste receptor such as T 1 R 1. T 1 R2, Tl R3, T2R 13. an olfactory receptor such as OR2A4. a-Gustducin. GPCRC6A, GPR55, and G PR92.

In certain embodiments, one or more pharmacophores have an affinity for receptor such as TLR-4. Bradykinin B _: , 5HT ₇ , GPR40, GPR43. GPR 109A, GPR 120, Histamine H.,, a taste receptor such as T 1 R 1 , T 1 R2, T I R3, T2R 13, an olfactory receptor such as OR2 A4, α-Gustducin. GPCRC6A, GPR55, and GPR92.

In certain embodiments, one or more pharmacophores hav e an affinity for surface- attached enzymes such as a-glucosidasc and Ghrclin O-acyl Trasfcrasc. ion channels such as TRPV4. or transporters such as SGLT-3 and GLUT-2.

In certain embodiments, one or more pharmacophores have an affinity for a TLR-4 receptor.

In certain embodiments, one or more pharmacophores have an affinity for a 5HT- receptor.

In certain embodiments, one or more pharmacophores have an affinity for a Histamine H ₄ receptor.

Particular embodiments of the compounds of the inv ention are described herein and in Figures 1 -3. In certain embodiments, the pharmacophores that arc incorporated into the compounds of the invention arc derived from bioactivc drug compounds. In certain embodiments, the pharmacophore moieties arc related to the parent structure of the bioactivc drug compounds by chemical modification (e.g.. substitution) of the parent structure. In example embodiments, a parent structure is be modified by a linking moiety that effectively attaches the pharmacophore to a scaffold, as described herein.

Modifications of bioactiv c drug compound include substitution, truncation, stcrcoccntcr inversion, isomcrization, or hybridization change. In certain embodiments, a bioactivc drug compound is substituted at any substitutablc position, including a hctcroatom (e.g., O, N, S, Sc. P), a hydrogen-bearing sp ^' -hybridized carbon, or a hydrogen-bearing sp'- hybridized CH.

A person of ordinary skill in the art would appreciate that a bioactiv c drug compound can be incorporated as a pharmacophore into the compounds of the invention in any one of a variety of spatial orientations, and at any one of a variety of substitutablc positions, as described herein. A person of ordinary skill in the art can readily synthesize a number of such compounds in order to determine which orientations and connectivities exhibit acceptable biological activity for a given application. In example embodiments, a compound with a pharmacophore hav ing appropriate orientation and connectivity retains the type of biological activ ity of the parent bioactivc daig compound, though the activ ity of the compound bearing the pharmacophore may be stronger or weaker than that of the parent bioactivc drug compound. In certain embodiments, a suitable orientation and connectivity is determined when the pharmacophore exhibits at least 99%. 98%. 95%, 90%, 85%. 80 %, 75% 70%. 65%, 60%. 55%. or 50% of the biological activity of parent compound.

In example embodiments, the pharmacophore moiety is derived from olopatadine or aminptinc. Schemes 1 A and I B, below, show synthetic routes for exemplary compounds of the present invention. These compounds illustrate the conceptual approach for utilizing drug molecules as pharmacophores in the compounds and methods described herein.

Scheme 1A:

Scheme 1C provides further exemplary compounds of the invention.

In example embodiments of the compounds of the invention, at least one

pharmacophore is . wherein indicates a point of attachmen In example embodiments of the comp

to a branch;

R ¹ , R ² , R ³ , and R ⁴ arc selected from H. (C _1-6 , )alkyl, and (C _{6 -10} )aryl; and

R ⁵ is H or (C _{1 - 10} )alkyl. optionally substituted with hydroxyl. carboxyl, amide, or amido.

In example embodiments, the compounds of the invention arc TLR-4 antagonists.

In further example embodiments of th n, at least one

In example embodiments, the compounds of the present inv ention arc 5HT- antagonists.

Typically, the compound is configured to be substantially stable to the conditions of the gastrointestinal tract (including pH and digestive enzymes), e.g., such that the pharmacophores arc not released from the scaffold during passage through the

gastrointestinal tract. For example, bonds that link the pharmacophores to the scaffold arc preferably not hydrolyzed under physiologic conditions (e.g.. lack ester, thiocstcr, acetal, ketal, or other acid- or base-labile bonds).

In some embodiments, it is advantageous to selectively target receptors in one portion of the gut and not in other portions of the gut. e.g., to target receptors in the intestines but not the stomach. While this can be accomplished by formulating the compound in an enteric coating that is resistant to stomach acid but dissolved in the basic environment of the intestine, it can also be accomplished by cov alcntly modifying the pharmacophores with a masking moiety that a) blocks interaction of the pharmacophore with its target receptor and b) is resistant to the acid environment of the stomach but readily cleav ed in the basic conditions of the intestine. Modification of the pharmacophore renders the compound a prodrug. For example, a hydroxyl group of a pharmacophore could be cstcri ficd with 4-amino-butanoic acid. In the acidic environment of the stomach, the amino group w ould be protonatcd, masking its nuclcophi licity; in the basic environment of the intestine, the amino group would be deprotonated. leading to intramolecular cyclization, generating a lactam and revealing the pharmacophore hydroxyl. Compounds that undergo reactions of this sort, without cleaving the pharmacophore from the scaffold, arc still considered to be substantially stable in the gastrointestinal tract.

While molecular size is one factor that can contribute to a molecule ^' s resistance to absorption into the bloodstream from the gastrointestinal tract, charge and polarity can play an important role as well. A hydrophobic surface of the compound helps inhibit uptake of the compounds into the bloodstream, and so preferably the pharmacophores arc selected to be relatively non-polar or hydrophobic, i.e.. lacking ionizable moieties (such as carboxylic acid or amino groups). However, to assist in solubilizing the compound in the digestive tract, the scaffold and/or branches may be hydrophilic or even ionizable, and preferably include one or more amino, guanidino. or other moieties (e.g., nitrogen-containing heterocyclic or hctcroaryl rings (such as morpholino, pipcridino, pipcrazino, pyridyl. pyrimidyl. imidazolyl. pyrrolyl. pyrazinyl, oxazolyl, thiazolyl, isoxazolyl. triazolyl, etc. ) that arc protonatcd at least under the acidic conditions of the stomach and at neutral pH. if not also in the comparatively basic environment of the intestine. Thus, in certain embodiments, each branch comprises an optionally substituted hctci ocyclc. such as a nitrogen-containing hctcrocyclc. In certain such embodiments, each branch independently comprises - h i

X. independently for each occurrence, represents O S SO SO NH N(G l) N(G- ) lk l) N((C ) lk l(C l ))

Y is, independently for each occurrence, an optionally substituted hctcrocyclc, such as a nitrogen-containing hctcrocyclc; and

n is an integer from 1 to 20.

Suitable hctcrocyclcs include optional ly substituted isoxazolinc. isoxazol idinc. pyrazolinc, imidazoline, or triazinc.

In certain particular embodiments, the compound has a structure of the formula :

Z repre ₁₀ )alkyl, (C _{6 14} )aryl, (C ₆ -

1 ₄ )aryl(C ₁ - ₁₀ )alkyl, (C-- ₁₀ )alkcnyl, or (C _{2- 10} )alkynyl:

B represents a branch, optionally terminating in a pharmacophore: and

in is an integer from 0 to 10.

In another aspect, the invention provides pharmaceutical compositions of the compounds described herein, optional ly in admixture with one or more pharmaceutically acceptable excipicnts. Preferably, the compositions arc suitable for oral administration, whether solids (tablets, capsules, caplcts. etc. ), liquids (suspensions, solutions, etc. ), or other orally administered formulations (dissolving films, lozenges, chewing gum, etc.).

In yet another aspect, the invention provides methods for administering the compounds and compositions described herein, e.g., for the treatment of metabolic syndrome or a disorder associated with metabolic syndrome in a subject in need thereof.

In yet another aspect, the invention provides methods for making the compounds and compositions disclosed herein, e.g.. by:

contacting a scaffold hav ing a plurality of branches, wherein each of a plurality of branches terminates in a first reactive moiety, with a plurality of linking molecules, each linking molecule comprising a pharmacophore linked to a second reactive moiety, under conditions sufficient to induce a coupling reaction, such as a cycloaddition, between the first reactive moiety and the second reactiv e moiety. Thus, the first reactive moiety may comprise an optionally substituted alkene or alkync (preferably an alkync) and the second reactive moiety may comprize a diazoalkanc. a nitronc, a nitrile oxide, an azidc, or a nitrile inline (preferably a nitrile oxide or an azidc). Alternatively, the first reactive moiety may comprize a diazoalkanc, a nitronc, a nitrile oxide, an azidc. or a nitrile iminc (preferably a nitrile oxide or an azidc) and the second reactive moiety may comprise an optionally substituted alkene or alkync (preferably an alkync). Similarly, one of the first and second reactiv e moieties may comprise a nuclcophilc and the other an clcctrophi le. such that reaction between the nuclcophilc and clcctrophile forms a covalcnt bond.

In embodiments for preparing compounds with different pharmacophores, this method may be readily adapted by, for example:

1 ) contacting the scaffold ( simultaneously or successively) w ith two or more linking molecules, wherein some linking molecules comprise a first pharmacophore and other linking molecules comprise a second pharmacophore, such that the linking molecules randomly react with the first reactive moieties (in this fashion the relative dispositions and even proportions of the different pharniacopliorcs may differ from molecule to molecule); or

2) using a scaffold w here some branches terminate with a first reactive moiety and other branches terminate with a second reactive moiety, and contacting the scaffold (simultaneously or successively) with a first linking molecule comprising a first pharmacophore linked to a third reactive moiety that selectively reacts with the first reactive moiety and a second linking molecule comprising a second pharmacophore linked to a fourth reactive moiety that selectively reacts with the second reactive moiety ( for example, the first and third reactive moieties may comprise a nuclcophilc and an clcctrophilc. while the second and fourth reactive moieties may undergo a |3+2 | cycloaddition with each other).

In certain embodiments, compounds of the invention may have one or more chiral centers, whether in the pharmacophore, the scaffold, or the branches. In certain such embodiments, compounds of the invention may be enriched in one or more diastcrcomcrs or one or more cnantiomcrs. For example, a compound of the invention may have greater than 30% dc. 40% dc. 50% dc, 60% dc, 70% dc, 80% dc, 90% dc, or even 95% or greater dc. A diastcrco-cnrichcd composition or mixture may comprise, for example, at least 60 mol percent of one diastcrcomcr, or more preferably at least 75, 90, 95, or even 99 mol percent. In certain embodiments, the compound enriched in one diastcrcomcr is substantially free of the other diastcrcomcrs, wherein substantially free means that the other diastcrcomcrs make up less than 10%, or less than 5%, or less than 4%, or less than 3%. or less than 2%, or less than 1 % as compared to the amount of the primary diastcrcomcr. e.g.. in the composition or compound mixture. For example, if a composition or compound mixture contains 98 grams of a first diastcrcomcr and 2 grams of a second diastcrcomcr. it would be said to contain 98 mol percent of the first diastcrcomcr and only 2% of the second diastcrcomcr. In certain embodiments, a compound of the invention may hav e greater than 30% cc, 40% cc, 50% cc, 60% cc, 70% cc, 80% cc, 90% cc, or even 95% or greater cc. An cnantio-cnrichcd composition or mixture may comprise, for example, at least 60 mol percent of one cnantiomcr. or more preferably at least 75, 90. 95. or even 99 mol percent. In certain embodiments, the compound enriched in one cnantiomcr is substantially free of the other cnantiomcr. wherein substantially free means that the other cnantiomcr makes up less than 10%, or less than 5%. or less than 4%, or less than 3%, or less than 2%, or less than 1 % as compared to the amount of the primary cnantiomcr, e.g., in the composition or compound mixture. For example, if a composition or compound mixture contains 9X grams of a first cnaiitiomcr and 2 grams of a second cnantiomcr, it would be said to contain S ⁾ X mol percent of the first cnantiomcr and only 2% of the second cnantiomcr. The compounds of the invention may also be raccmic mixtures of cnantiomcrs.

Compounds of any of the structures described herein and any composition of these compounds may be used in the manufacture of medicaments for the treatment of any diseases or conditions disclosed herein.

Definitions

The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term "acylamino" is art-rccognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula

hydrocarbylC(0)NH-.

The term "acyloxy" is art-rccognized and refers to a group represented by the general formula hydrocarbylC(0)0-, preferably aIkylC(0)0-.

The term "alkoxy ^" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include mcthoxy. cthoxy, propoxy, tert-butoxy and the like.

The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl .

The term "alkenyl ^" . as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstitutcd alkcnyls" and "substituted alkeny Is", the latter of which refers to alkenyl moieties having substitucnts replacing a hydrogen on one or more carbons of the alkenyl group. Such substitucnts may occur on one or more carbons that arc included or not included in one or more double bonds.

Moreover, such substitucnts include all those contemplated for alkyl groups, as discussed below . except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl. aryl, hcterocyclyl, or hctcroaryl groups is contemplated.

An "alkyl ^" group or "alkanc" is a straight chained or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from I to about 10 unless otherwise defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n- propyl, iso-propyl. n-butyl, sec-butyl, tcrt-butyl. pciityl, hcxyl. pentyl and octyl. A C ₁ -C ₆ straight chained or branched alkyl group is also referred to as a "lower alkyl" group.

Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstitutcd alkyls" and "substituted alkyls". the latter of which refers to alkyl moieties having substitucnts replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substitucnts, if not otherwise specified, can include, for example, a halogen, a hydroxyl. a carbonyl (such as a carboxyl. an alkoxycarbonyl. a formyl, or an acyl). a thiocarbonyl (such as a thiocstcr, a thioacctatc, or a thioformatc), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidinc, an iminc. a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a hctcrocyclyl. an aralkyl. or an aromatic or hctcroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substitucnts of a substimtcd alkyl may include substituted and unsubstitutcd forms of amino, azido, imino, amido, phosphoryl ( including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios. carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF ₃ , -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkcnyls, alkoxys. alkylthios. aminoalkyls, carbonyl-substitutcd alkyls, -CF ₃ . -CN, and the like.

The term "C when used in conjunction w ith a chemical moiety, such as, acyl. acyloxy. alkyl. alkcnyl. alkynyl. or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "C _N-y alkyl" refers to substituted or unsubstitutcd saturated hydrocarbon groups, including straight-chain alkyl and branched- chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as tri fliioronicthyl and 2,2.2-trifluorocthyl, etc. C ₀ alkyl indicates a hydrogen w here the group is in a terminal position, a bond if internal. The terms "'C:2-yalkcnyl and "C ₂ - _v alkynyl" refer to substituted or unsubstitutcd unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "alkylamino ^" , as used herein, refers to an amino group substituted with at least one alkyl group. The term "alkylthio". as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term "alkynyl". as used herein, refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstitutcd alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substitucnts replacing a hydrogen on one or more carbons of the alkynyl group. Such substitucnts may occur on one or more carbons that arc included or not included in one or more triple bonds

Moreover, such substitucnts include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl. aryl, hctcrocyclyl, or hctcroaryl groups is contemplated.

The term "amide", as used herein, refers to a group

wherein each R ⁱⁿ independently represent a hydrogen or hydrocarbyl group, or tw o R'° arc taken together w ith the N atom to which they arc attached complete a hctcrocyclc hav ing from 4 to X atoms in the ring structure.

The terms "amine" and "amino" are art-recognized and refer to both unsubstitutcd and substituted amines and salts thereof, e.g., a moiety that can be represented by

wherein each R ¹⁰ independently represents a hydrogen or a hydrocarbyl group, or two R ¹⁰ arc taken together with the N atom to which they are attached complete a hctcrocyclc having from 4 to X atoms in the ring structure.

The term "aminoalkyl". as used herein, refers to an alkyl group substituted with an amino group.

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group.

The term "aryl" as used herein include substituted or unsubstitutcd single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- mcmbcrcd ring, more preferably a 6-mcmbcrcd ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons arc common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls. cycloalkcnyls, cycloalkynyls, aryls, hctcroaryls, and or hctcrocyclyls. Aryl groups include benzene, naphthalene, phenanthrenc. phenol, aniline, and the like.

The term "carbamate" is art-rccognized and refers to a group

wherein R'' and R ¹⁰ independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, or R ⁹ and R ¹⁰ taken together with the intervening atom(s) complete a heterocyele having from 4 to 8 atoms in the ring structure.

The terms "carbocycle ^" , and "carbocyclic", as used herein, refers to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic carbocyclcs and non-aromatic carbocyclcs. Non-aromatic carbocyclcs include both cycloalkanc rings, in which all carbon atoms arc saturated, and cycloalkcnc rings, which contain at least one double bond. "Carbocycle ^" includes 5-7 mcmbcrcd monocyclic and 8- 12 mcmbcrcd bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms arc shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms w ith the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g.. phenyl, may be fused to a saturated or unsaturated ring, e.g.. cyclohexanc, cyclopcntanc. or cyclohcxcnc. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary "carbocyclcs ^" include cyclopcntanc, cyclohexanc. bicyclo| 2.2. 1 jheptanc. 1 .5- cyclooctadicnc, 1 ,2,3.4-tctrahydronaphthalcnc, bicyclo| 4.2.0 |oct-3-cnc. naphthalene and adamantane. Exemplary fused carbocyclcs include dccalin. naphthalene, 1.2,3,4- tetrahydronaphthalenc. bicyclo| 4.2.0 |octane, 4,5.6.7-tetrahydro- l H-indcne and bicyclo| 4. 1 ⁽⁾ |hept-3-cne. "Carbocyclcs" may be susbstitutcd at any one or more positions capable of bearing a hydrogen atom.

A "cycloalkyl" group is a cyclic hydrocarbon which is completely saturated.

"Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, more typically 3 to X carbon atoms unless otherwise defined. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. Cycloalkyl includes bicyclic molecules in which one. two or three or more atoms arc shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. A "cycloalkcnyl ^" group is a cyclic hydrocarbon containing one or more double bonds.

The term "carbocyclylalkyl", as used herein, refers to an alkyl group substituted with a carbocyclc group.

The term "carbonate ^" is art-recognized and refers to a group -OCO2-R ¹⁰ , w herein R ^H1 represents a hydrocarbyl group.

The term "carboxy". as used herein, refers to a group represented by the

formula -CO ₂ H.

The term ^" ester ^" , as used herein, refers to a group -C(0)0R"' wherein R ¹ " represents a hydrocarbyl group.

The term "ether", as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substitucnt of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be cither symmetrical or unsymmctrical.

Examples of ethers include, but arc not limited to, hctcrocyclc-O-hctcrocyclc and aryl-O- hctcrocyclc. Ethers include "alkoxyalkyl ^" groups, which may be represented by the general formula alkyl-O-alkyl.

The terms "halo" and "halogen" as used herein means halogen and includes chloro, fluoro. bromo, and iodo.

The terms "hetaralkyl" and "hctcroaralkyl", as used herein, refers to an alkyl group substituted with a hctaryl group.

The term "hcteroalkyl", as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one hctcroatom, wherein no tw o hctcroatoms arc adjacent.

The terms "hctcroaryl ^" and "hctaryi ^" include substituted or unsubstitutcd aromatic single ring structures, preferably 5- to 7-mcmbcrcd rings, more preferably 5- to 6- mcmbcrcd rings, whose ring structures include at least one hctcroatom, preferably one to four hctcroatoms, more preferably one or two hctcroatoms. The terms "hctcroaryl" and "hctaryl" also include polycyclic ring systems hav ing two or more cyclic rings in which two or more carbons arc common to two adjoining rings wherein at least one of the rings is hctcroaromatic. e.g.. the other cyclic rings can be cycloalkyls. cycloalkcnyls, cycloalkynyls. aryls. hctcroaryls. and/or hetcrocyclyls. Hctcroaryl groups include, for example, pyrrole, furan, thiophenc. imidazole, oxazolc. thiazolc, pyrazolc, pyridine, pyrazinc. pyridazinc. and pyrimidinc. and the like.

The term "hctcroatom" as used herein means an atom of any clement other than carbon or hydrogen. Preferred hctcroatoms arc nitrogen, oxygen, and sulfur.

The terms "hctcrocyclyl ^" . "hctcrocyclc ^" , and "heterocyclic ^" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to I O-mcmbcrcd rings, more preferably 3- to 7-mcmbcrcd rings, whose ring structures include at least one hctcroatom, preferably one to four hctcroatoms, more preferably one or tw o hctcroatoms. The terms "hctcrocyclyl" and "heterocyclic ^" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons arc common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkcnyls. cycloalkynyls. aryls, hctcroaryls, and or hetcrocyclyls. Hctcrocyclyl groups include, for example, pipcridinc, pipcrazinc. pyrrolidine, morpholinc. lactones, lactams, and the like.

The term "hetcrocyclylalkyr, as used herein, refers to an alkyl group substituted with a hctcrocyclc group.

The term "hydrocarbyl", as used herein, refers to a group that is bonded through a carbon atom that docs not have a =0 or =S substitucnt, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include hctcroatoms. Thus, groups like methyl, cthoxycthyl, 2-pyridyl, and trifluoromcthyl arc considered to be hydrocarbyl for the purposes of this application, but substitiicnts such as acetyl (which has a =0 substitucnt on the linking carbon) and cthoxy (which is linked through oxygen, not carbon) arc not. Hydrocarbyl groups include, but arc not limited to aryl, hctcroaryl. carbocyclc. hctcrocyclyl. alkyl, alkenyl, alkynyl, and combinations thereof.

The term "hydroxyalkyl". as used herein, refers to an alkyl group substituted with a hydroxy group.

The term "lower" w hen used in conjunction w ith a chemical moiety, such as, acyl, acyloxy, alkyl. alkenyl. alkynyl, or alkoxy is meant to include groups w here there arc ten or fewer non-hydrogen atoms in the substitucnt, preferably six or fewer. A "low er alkyl", for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl. acyloxy, alkyl. alkcnyl. alkynyl. or alkoxy substitucnts defined herein arc respectiv ely lower acyl, lower acyloxy, lower alkyl, lower alkcnyl. lower alkynyl. or lower alkoxy. whether they appear alone or in combination with other substitucnts, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group arc not counted when counting the carbon atoms in the alkyl substitucnt).

The terms "polycyclyl", "polycyclc". and "polycyclic" refer to tw o or more rings (e.g., cycloalkyls, cycloalkcnyls. cycloalkynyls, aryls, hctcroaryls, and/or hctcrocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycyclc can be substituted or unsubstitutcd. In certain embodiments, each ring of the polycyclc contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The term "silyl" refers to a silicon moiety with three hydrocarbyl moieties attached thereto.

The term "substituted" refers to moieties having substitucnts replacing a hydrogen on one or more carbons of the backbone. It w ill be understood that "substitution ^" or "substituted with ^" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substitucnt. and that the substitution results in a stable compound, e.g., which docs not spontaneously undergo transformation such as by rearrangement, cyclization. elimination, etc. As used herein, the term

"substituted ^" is contemplated to include all permissible substitucnts of organic compounds. In a broad aspect, the permissible substitucnts include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substitucnts of organic compounds. The permissible substitucnts can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the hctcroatoms such as nitrogen may have hydrogen substitucnts and/or any permissible substitucnts of organic compounds described herein which satisfy the v alences of the hctcroatoms. Substitucnts can include any substitucnts described herein, for example, a halogen, a hydroxy!, a carbonyl (such as a carboxyl. an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thiocster. a thioacctatc. or a thioformatc). an alkoxyl. a phosphoryl, a phosphate, a phosphonatc. a phosphinatc. an amino, an amido, an amidinc, an iminc, a cyano. a nitro, an azido, a sulfhydryl, an alkylthio. a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a hctcrocyclyl, an aralkyl, or an aromatic or hctcroaroniatic moiety. It will be understood by those skilled in the art that substitucnts can themselves be substituted, if appropriate. Unless specifically stated as "unsubstitutcd." references to chemical moieties herein arc understood to include substituted variants. For example, reference to an "aryf ^" group or moiety implicitly includes both substituted and unsubstitutcd variants.

The term "sulfate' ^* is art-recognized and refers to the group -OSO-,Η, or a pharmaceutically acceptable salt thereof.

The term "sulfonamide ^" is art-rccognized and refers to the group represented by the general formulae

wherein R ⁹ and R ¹⁰ independently represents hydrogen or hydrocarbyl. such as alkyl. or R ⁹ and R ¹⁰ taken together with the interv ening atom(s) complete a hctcrocyclc having from 4 to 8 atoms in the ring strucmrc.

The term "sulfoxide" is art-rccognized and refers to the group -S(0)-R'", wherein R" ¹ represents a hydrocarbyl.

The term "sulfonate" is art-recognized and refers to the group SCKH, or a pharmaceutically acceptable salt thereof.

The term "sulfonc ^" is art-rccognized and refers to the group -S(Ob-R'", wherein R ¹ " represents a hydrocarbyl.

The term ^" thioalkyl". as used herein, refers to an alkyl group substituted with a thiol group.

The term "thiocstcr", as used herein, refers to a group -CiOiSR ¹⁰ or -SC(0)R ¹⁰ w herein R ¹⁰ represents a hydrocarbyl.

The term "thiocthcr", as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

The term "urea" is art-rccognized and may be represented by the general formula

wherein R ^l> and R ^l(J independently represent hydrogen or a hydrocarbyl. such as alkyl, or cither occurrence of R ^y taken together with R"' and the intervening atom(s) complete a hctcrocyclc having from 4 to 8 atoms in the ring strucmrc. "Protecting group ^" refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts. Protective Groups in Organic Chemistry. 3 ^rJ Ed.. 1999. John Wiley & Sons. NY and Harrison ct al.. Compendium of Synthetic Organic Methods, Vols. 1 -8, 1971 - 1996, John Wiley & Sons. NY. Representative nitrogen protecting groups include, but arc not limited to, formyl. acetyl, trifluoroacctyl, benzyl, bcnzyloxycarbonyl ("CBZ ^" ). tcrt-butoxycarbonyl ("Boc"), trimcthylsilyl ("TMS"), 2-trimeihylsilyl-ethanesulfonyl ("TES ^" ), trityl and substituted trityl groups, al lyloxycarbonyl. 9-fluorcnylmcthyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC") and the like. Representative hydroxy 1 protecting groups include, but arc not limited to, those where the hydroxyl group is cither acylatcd (cstcrificd) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers,

tctrahydropyranyl ethers, rrialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

The term "healthcare providers" refers to individuals or organizations that provide healthcare services to a person, community, etc. Examples of "healthcare providers" include doctors, hospitals, continuing care retirement communities, ski lled nursing facilities, subacute care facilities, clinics, multispccialty cl inics, freestanding ambulatory centers, home health agencies, and HMO's.

As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

The term "treating" includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic ^" treatment is art-rccognized and includes administration to the host of one or more of the sub ject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g.. disease or other unwanted state of the host animal) then the treatment is prophylactic ( i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, ( i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). The term "prodrug" is intended to encompass compounds which, under physiologic conditions, arc converted into the therapeutically active agents of the present invention (e.g.. a compound of formula I). A common method for making a prodrug is to include one or more selected moieties which arc hydrolyzcd under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g.. esters or carbonates of alcohols or carboxylic acids) arc preferred prodrugs of the present inv ention. In certain

embodiments, some or all of the compounds of formula I in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxy! in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.

The term ^" masking moiety ^" as used herein, refers to the chemical moiety that is a covalcntly bound modification of a pharmacophore that renders the compounds of the present invention to which it is attached prodrugs. A masking moiety is clcavablc under, for example, acidic conditions, basic conditions, or physiologic conditions. When the masking moiety is cleaved, the prodrugs arc converted to the therapeutically active agents of the present invention. Esters and carbonates can be used to mask hydroxy Is, carbamates and amides can be used to mask amines, carboxyls can be masked as esters, etc., and in certain embodiments the precise masking moiety can be selected to be cleaved under conditions particular to a region of the digestive tract. For example, an amine or hydroxyl can be acylatcd by a 4-aminobutanoyl group, to form a prodrug that can be administered as a salt of the amine. In the acidic conditions of the stomach, the amino group will remain protonatcd, masking its nuclcophilicity. In the more basic conditions of the small intestines, the ammonium group will be deprotonated, revealing the nuclcophi lic amine, which can nuclcophilicly attack the amide or ester formed by the butanoyl group, ultimately revealing the amide or ester w ith the concomitant release of the protecting group as a lactam.

Pharmaceutical ( ^' (impositions

The compositions and methods of the present invention may be util ized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers arc w ell known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil. or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e.. routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-frec, or substantially pyrogen- free. The excipicnts can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule. lyophile for rcconstitution. powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the inv ention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dcxtrans. antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipicnts. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a sclfcmulsifying drug delivery system or a sclfmicrocmulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention.

Liposomes, for example, which comprise phospholipids or other lipids, arc nontoxic, physiologically acceptable and mctabolizable carriers that arc relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and or dosage forms which arc, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate w ith a reasonable benefit risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipicnt. solvent or encapsulating material. Each carrier must be

"acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as

pharmaceutically acceptable carriers include: ( I ) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymcthyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (X) excipicnts, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil. safflowcr oil, sesame oil. olive oil, corn oil and soybean oil; ( 10) glycols, such as propylene glycol; ( 1 1 ) polyols. such as glycerin, sorbitol, mannitol and polyethylene glycol; ( 12) esters, such as ethyl oleate and ethyl lauratc; ( 13) agar; ( 14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; ( 15) alginic acid; ( 16) pyrogen-frec water; ( 17) isotonic saline; ( 18) Ringer's solution; ( 19) ethyl alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqucous solutions or suspensions, tablets, capsules

(including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue): absorption through the oral mucosa (e.g., sublingually); anally, rcctally or vaginally (for example, as a pessary, cream or foam): parcntcrally (including intramuscularly, intravenously, subcutancously or intrathccally as, for example, a sterile solution or suspension); nasally: intraperitoneal ly; subcutancously: transdcrmally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in. for example. U.S. Pat. Nos. 6, 1 10,973. 5,763,493, 5.73 I .OOO.

5,541 ,231 , 5.427.798. 5,358,970 and 4, 1 72,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well know n in the art of pharmacy. The amount of active ingredient w hich can be combined w ith a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound w hich produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, w ith the carrier and, optionally, one or more accessory ingredients. In general, the formulations arc prepared by uniformly and intimately bringing into association a compound of the present invention w ith liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophilc. powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-watcr or watcr-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth w ashes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules ( including sprinkle capsules and gelatin capsules), tablets, pills, dragecs, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: ( 1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid: (2) binders, such as, for example, carboxymcthylccllulosc, alginates, gelatin, polyv inyl pyrrolidonc, sucrose and/or acacia; (3) humcctants. such as glycerol: (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate: (5) solution retarding agents, such as paraffin: (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as. for example, cctyl alcohol and glycerol monostcaratc; (S) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stcaratc, magnesium stcaratc. solid polyethylene glycols, sodium laiiryl sulfate, and mixtures thereof; ( 10) completing agents, such as. modified and unmodified cyclodcxtrins; and ( I I ) coloring agents. In the case of capsules ( including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipicnts as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder ( for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintcgrant ( for example, sodium starch glycolatc or cross-linked sodium carboxymcthyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragecs. capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by. for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or sonic other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingrcdicnt(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding

compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipicnts.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilcs for rcconstitution. microcmulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as. for example, water or other solvents, cyclodcxtrins and derivatives thereof, solubilizing agents and cmulsificrs, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoatc. propylene glycol. l .3-butylcnc glycol, oils ( in particular, cottonseed, groundnut, com. germ, olive, castor and sesame oils), glycerol, tctrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservativ e agents.

Suspensions, in addition to the active compounds, may contain suspending agents as. for example, cthoxylated isostcaryl alcohols, polyoxycthylcnc sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth. and mixtures thereof.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more activ e compounds with one or more suitable nonirritating excipicnts or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations which arc suitable for v aginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as arc known in the art to be appropriate.

Alternatively or additionally, compositions can be formulated for deliv ery via a catheter, stent, wire, or other intraluminal dev ice. Delivery v ia such devices may be especially useful for deliv ery to the bladder, urethra, ureter, rectum, or intestine.

Dosage forms for the topical administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The activ e compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipicnts, such as animal and vegetable fats, oils, waxes, paraffins, starch. tragacaiith. cellulose deriv atives, polyethylene glycols, sil icones, bentonites, si licic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipicnts such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidc powder. or mixtures of these substances. Sprays can additionally contain customary propcllants. such as chlorofliiorohydrocarbons and v olati le unsubstitutcd hydrocarbons, such as butane and propane.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the l ike), and suitable mixtures thereof, v egetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsi fying agents and dispersing agents. Prev ention of the action of

microorganisms may be ensured by the inclusion of v arious antibacterial and anti fungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the in jectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostcaratc and gelatin.

For use in the methods of this invention, active compounds can be given per sc or as a pharmaceutical composition containing, for example, 0. 1 to 99.5% (more preferably. 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Actual dosage levels of the activ e ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the activ e ingredient that is effectiv e to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex. weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is ach ieved. By "therapeutically effective amount" is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex. age, and medical history of the subject. Other factors which influence the effective amount may include, but arc not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and. if desired, another type of therapeutic agent being administered w ith the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage arc known to those skilled in the art ( Issclbachcr cl al. ( 1996) Harrison ^' s Principles of Internal Medicine 13 cd., 1 X 14- 18X2, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in the compositions and methods of the invention w ill be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose w ill generally depend upon the factors described above.

If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times dai ly. In preferred embodi ments, the activ e compound will be administered once daily.

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equincs. cattle, sw ine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase "con joint administration" refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body {e.g., the two compounds arc simultaneously effective in the patient, which may include synergistic effects of the tw o compounds). For example, the different therapeutic compounds can be administered cither in the same formulation or in a separate formulation, cither concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 1 2 hours, 24 hours, 36 hours, 4X hours, 72 hours, or a week of one another. Thus, an individual w ho receives such treatment can benefit from a combined effect of different therapeutic compounds.

In certain embodiments, con joint administration of compounds of the invention with one or more additional therapeutic aycnt(s) (e.g., one or more additional chcmothcrapcutic agcnt( s)) provides improved efficacy relative to each individual administration of the compound of the invention or the one or more additional therapeutic agcnt(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of indiv idual administration of the compound of the inv ention and the one or more additional therapeutic agcnt(s).

This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but arc not l imited to, alkyl, dialkyl, trialkyl or tctra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but arc not limited to, L-argininc. bencnthaminc. benzathine, bctainc. calcium hydroxide, choline, dcanol. dicthanolaminc, dicthylaminc. 2- (dicthylamino)cthanol, cthanolaminc, cthylcncdiaminc, N-mcthylglucaminc. hydrabaminc, I H-imidazolc, lithium, L-lysinc. magnesium, 4-( 2-hydroxycthyl)morpholinc, pipcrazinc, potassium, I -( 2-hydroxycthyl )pyrrolidinc, sodium, tricthanolaminc. tromcthaminc, and zinc salts. I n certain embodiments, contemplated salts of the invention include, but arc not limited to, Na, Ca, K. Mg. Zn or other metal salts.

The pharmaceutically acceptable acid addition salts can also ex ist as various solvates, such as with water, methanol, cthanol. dimcthyl formamidc, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adv entitious to such solvent. Wetting agents, cmulsificrs and lubricants, such as sodium lauryl sul fate and magnesium stcaratc. as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: ( l ) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfatc. sodium mctabisulfitc. sodium sulfite and the like; (2 ) oil-soluble antioxidants, such as ascorbyl palmitatc. butylatcd hydroxyanisolc ( BHA ), butylatcd hydroxytolucnc ( BHT). lec ithin, propyl gallate, alpha-tocopherol, and the like; and (3 ) metal-chelating agents, such as citric acid, cthylcncdiaminc tctraacctic acid ( EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In certain embodiments, the invention relates to a method for conducting a pharmaceutical business, by manufacturing a formulation of a compound of the invention, or a kit as described herein, and marketing to healthcare providers the benefits of using the formulation or kit for treating or preventing any of the diseases or conditions as described herein.

In certain embodiments, the invention relates to a method for conducting a pharmaceutical business, by prov iding a distribution network for selling a formulation of a compound of the invention, or kit as described herein, and prov iding instruction material to patients or physicians for using the formulation for treating or preventing any of the diseases or conditions as described herein.

In certain embodiments, the invention comprises a method for conducting a pharmaceutical business, by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the diseases or conditions as described herein, conducting therapeutic profiling of identified formulations for efficacy and toxicity in animals, and providing a distribution network for selling an identified preparation as having an acceptable therapeutic profile. In certain embodiments, the method further includes providing a sales group for marketing the preparation to healthcare providers.

In certain embodiments, the invention relates to a method for conducting a pharmaceutical business by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the disease or conditions as described herein, and licensing, to a third party, the rights for further development and sale of the formulation.

Examples

Overview of Svnitivitv Platform™ Technology

Based upon the in vivo disease model data obtained, the platform described herein allows oral administration, imparts gut-restriction, and provides access to a multi-mediator approach to the treatment of metabolic diseases. Furthermore, the platform allow s introduction of precisely defined ratios of one to four distinct pharmacologically active agents suitable for oral delivery and imparts and maintains gut-restriction.

Gut-restriction provides a means to utilize validated molecular targets, w hich have failed by virtue of undesired toxicity associated with the expression of the specific molecular target in cell-types in tissue, organs, or organ systems not associated with disease treatment. Thus, gut-rcstriction of the pharmacologically active agent via this approach allows one to "rc-purposc" ( 1 ) agents previously deemed "undesired" by virtue of toxicity unrelated to the disease-modifying mode of action, and also (2) those molecular targets that demonstrate a disease treatment utility and concomitant toxicity-rclatcd pharmacology in cells, tissue, organs, or organ systems unrelated to disease treatment. Lastly, as relates to potential for the rc-purposing and clinical development of previously disclosed

pharmacologically active agents, the gut-rcstriction platform described herein and drug discovery approach also provides a means to re-purpose existing drugs, as well as those molecules demonstrating clinical efficacy with undesired toxicity safety pharmacology signals.

Specifically, with respect to the general IBD indication, namely. Crohn ^' s and ulcerative colitis patient populations, the gut-rcstriction approach outlined herein is applicable to drug-naive patient populations. The platform may also be applied in combination therapy via its addition to presently prescribed agents in patient populations utilizing existing agents. A significant feature of the drug platform described herein is that it is uniquely applicable to I BD patient populations cither refractory' to. or unresponsive to, any agent, whether administered orally or rectally. that is operative via an antiinflammatory mode of action |c.g., a drug from steroid class of drugs prescribed for IBD|, antibodies to the receptors of a range of pro-inflammatory cytokines, including but not limited to, IL- l bcta, IL-6, TNF-alpha, as well as antibodies to those pro-inflammatory cytokines, per sc.

The agents described herein agonize or antagonize the molecular target. The agent's required mode of action (e.g., agonization or antagonization) is specifically determined by the nature of the molecular target selected. The molecular target is specified intrinsically, with the platform acting via an orthostcric or allostcric mode of action to a wide range of known nutrient-sensing gut lumcn-cxprcsscd. lamina propria-cxprcsscd molecular targets. In animal disease model experiments generally accepted to be predictive of outcomes in humans, the molecules described herein demonstrate dose-responsive lowering of pro- inflammatory cytokines in circulation and directly in gut mucosal cells. More specifically, the compounds of the gut-restricted drug discovery platform disclosed herein demonstrate reduction of IL- l bcta levels in samples of rodent gut-microbiota. For example, in the rodent TNBS/DSS-induccd colitis model, the orally administered gut-rcstrictcd compounds of the invention demonstrated dose responsive and statistically significant reductions in IL- Ibcta, IL-6, TNF-alpha, as well as improved mucosal healing determined by scoring gross morphology.

Another application of the gut-rcstrictcd drug discovery platform disclosed herein is for the treatment of cancers localized to various regions of the Gl-tract driven by underlying gut-localized inflammation. An exemplary cancer is colorectal cancer, which can be modeled in rodents utilizing a variant of the DSS-induced colitis model. A gut- rcstrictcd compound, structurally unrelated to compounds useful for treating metabolic disease, was recognized by a specife gut lumcn-cxprcsscd. lamina propria-cxprcsscd receptor, and demonstrated dosc-rcsponsivc effects in vivo in the colorectal cancer variant of the DSS-induced colitis rodent disease model.

Applicability of Selected Molecular Targets to I BD

Structural Characteristics of the Agents Designed For Use in the Synitiritr Platform Isee Figure 11.

There arc a range of preferred qualities exhibited by the pharmacophores applied to the gut-restriction drug discovery Syniriviry Platform described herein. The platform accommodates a wide range of pharmacological agents. For example, novel NCEs, re- purposed know n clinical candidates, designed and biologically profiled small molecules, re- purposed new chemical entities, as well as re-purposed known drugs affecting gut lumen- expressed, lamina propria-cxprcssed molecular targets can be utilized with the platform described herein.

The synthetic approach described herein utilizes unique modifications to known techniques. This modified methodology is designed to allow ready access to the wide range of elaborated matrix, dendrimer compounds described herein. For example, the synthetic routes to various gut-restriction platforms arc convergent, in that the routes utilize a "poly- ync" synthesis that affords a tctra-triazinc dendrimer and provides access to different linker chain lengths. Features such as linker chain length, atom identity, and atom hyridization state contribute to the underlying conformations most necessary for the designed pharmacological agent's observed intrinsic potency, its in vivo disease model activity, and the imposed gut-restriction quality.

Although there is no intrinsic limitation to the nature of the linkers selected, in certain embodiments, preferred linkages include ether, amine, sulfide, and sulfoxide groups. In certain embodiments, the sulfoxide is chiral at the sulfur atom center, or achiral at the sulfur atom center. Further N-aryl or Ν,Ν-alkyl amides arc acceptable linkers.

Materials

The follow ing reagents are commercially av ailable or were synthesized from reported procedures, as follows.

X-azido-3.6-dioxa- l -octanol was purchased from FuturcChcm Company. LTD., or was synthesized according to Chem. C omm. 2005. 34. 43 1 5. Reagents 1 1 -azido-3,6.9- trioxa- l -undccanol and 14-azido-3.6.9.12-tctraoxa- l -tctradccanol. mesylate reagents X- azido-3,6-dioxa- l -octanol mesylate, 1 l -azido-3,6,9-trioxa- l -undccanol mesylate, and 1 -N- /Boc-amino-3,6-dioxa-X-octanol mesylate, amino azidc reagents l -amino-X-azido-3.6- dioxaoctanc and I -amino- 1 I -azido-3,6.9 -trioxaundccanc were also cither purchased from FuturcChcm Company, LTD., or else synthesized according to known procedures. 5-azido- 3-oxa- l -pcntanol tosylatc was prepared according to the procedure found in U. S. patent application publication 2013/02 16475.

The follow ing indo!e-dcrivcd carboxaldchydcs were used in the invention and arc commercially available: I H-indole-3-carboxaldchyde. 5-chloro- l H-indolc-3- carboxaldchydc. 1 -(4-chlorobcnzyloxy )-indolc-3-carboxaldchydc. 1 -bcnzyloxy-indolc-3- carboxaldchydc. l -phcnylsulfonyl-indolc-3-carboxaldchydc. and l -bcnzyl-indolc-3- carboxaldchydc. The coppcr-bascd reagents, catalysts, and copper affinity purification reagents (e.g.. Cuprisorb) utilized in the experimental section arc commercially available.

TBTA (Tris-| ( 1 -bciizyl- I H- 1 .2.3-triazol-4-yl)methyl |aminc. also known as tris- (bcnzyltriazolylmcthyl)aminc). a stabilizing ligand for Cu( I), was synthesized according to a known procedure in 40% yield (sec Lewis, ct al., J. Am. Chem. Soc. 2004, 126. 9152-3 ).

General Synthetic methods

Incorporation of pharmacological aucnt via dendrimer azidc side-chain synthesis Ethcrification introduces the "pharmacological agent" and provides access to variable chain-length polycthcr and azidc precursors. The installation of hctcroatoms can be completed in a flexible synthetic manner v ia Mitsunobu reaction and variants ( Schemes 2 and 3 ). Typically, no matter the relative molecular complexity of the side chain one may select, a Mitsunobu variant is ususally a compatible reaction. Thus, the conv ersion to the azidc provides a dendrimer "side-chain" precursor suitable for use in "click" cycloaddition.

Scheme 3

Amine Incorporation

Svnilictic methods

/. Synthesis of aeetylene-snhstituleii amino reagents

i. Synthesis of N ,Ν ,λ ^' ,N '-tetrapropargyl ethylene diamine

The tctra-substitutcd ethylene diamine was prepared as reported in EP 0 43 1 700 B l . Specifically, into a reaction vessel fitted with a stirrer, a reflux condenser and a nitrogen gas inlet tube, w ere placed ethylene diamine (30.05 g. 0.5 mol). sodium hydroxide (84.21 g). deionized water ( 180 g), methylene chloride ( 180g) and tctrabutyl-ammonium bromide ( 1 .0 g) and the mixture was stirred and propargyl bromide (249.81 g, 2.1 mol) w as added dropwisc at room temperature. After complete addition, the mixture was heated to 50 °C and maintained at 50 °C for 6 hours. Thereafter, the mixture was allowed to cool to room temperature, the methylene chloride layer separated, w ashed w ith deionized water (3X), dried of anhydrous sodium sulfate, filtered and concentrated at reduced pressure affording tctra-alkylatcd diamine. The product w as subjected to silica gel column chromatography to obtain purified Ν,Ν.Ν',Ν'-tctrapropargyl ethylene diamine. X (92% yield). II. Synthesis of triethanolamino-iripropargyl ether (2) via etherification of triethanolamine

To an anhydrous THF ( 15 niL) solution of tricthanolaminc ( 1.4 mmol) was added NaH ( 100.80 nig, 4.2 mmol ) at 0 °C and the resulting mixture was maintained at 0 °C for 20 minutes. A catalytic amount (0.42 mmol) of tctrabutylammonium iodide (TBAI) was added in one portion followed by rapid, dropwisc addition of propargyl bromide ( 1 .3 mL, 14.4 mmol) over 60 seconds. The mixture was warmed to room temperature and stirred at room temperature for an additional 18 h. The reaction was quenched by addition of ice cold distilled water (20 mL). the resulting mixture allowed to stir for 1 5 min, methylene chloride (40 mL) was then added and the pH adjusted to pH = 2.0 via addition of aqueous 1 N HCI. The layers were separated and the aqueous layer was extracted (2x ) with methylene chloride ( 1 5 mL). The aqueous layer was then added to methylene chloride (25 mL), the pH adjusted to pH = 9.5 by addition of 2N NaOH and the layers were separated. The aqueous layer was extracted with methylene chloride (2 x 20mL). the organic layers combined, dried ov er anhydrous sodium sulfate and concentrated at reduced pressure affording the desired tripropargyl ether (2) used without further purification (81 % mass balance). Mass spec: HRMS calc'd: 263.3321 : found: 263.3302; M+ l 264. 1556.

2. Synthesis of I N-substiiuted indole- 3-carboxaldehyde intermediates

Exemplary synthetic procedures for the N-functionalization of indole derivatives may be found in Vartale ct al., Int J I'harm J 'harm ScL 2012, Vol 4, Suppl 4, 635-641.

Intermediate 3 was synthesized utilizing the procucdurc reported in Hu, L. el ul. Tetrahedron 2014, 70. 5626-563 1 . Specifically, an anhydrous DMF (2 mL) solution of 1 H- indolc-3-carboxaldchyde ( 1 mm nyl iodide ( 1 .5 mmol). Cul (0. 1 mmol). metformin hydrochloride (0.2 mmol), and C8 ₂ CO ₃ (2 mmol) was heated to 130 °C under NK After 24 h the mixture was allowed to warm to room temperature and diluted with EtOAc ( 10 mL). The solids removed by filtering through a bed of cclitc:cuprisorb ( 1 : 1 ), the filtrate w ashed w ith w ater (2 x 5mL) and the organic layer concentrated under reduced pressure. The residue was added to watencthyl acetate ( 1 : 1 , 20 mL). the pH adjusted to pH=5.5. the layers separated, and the ethyl acetate layer added to an equal volume of distilled water. The pH of the ethyl acetate water mixture was adjusted to pH=9 by addition of aqueous 2N NaOH, the layers separated, the aqueous layer extracted with an equal volume of methylene chloride (2X). the ethyl acetate and methylene chloride extracts w ere combined, dried over anhydrous sodium sulfate and concentrated at reduced pressure to afford l -phcnyl-indolc-3 carboxaldchydc (3) 47% yield. Mass spec: HRMS calc ^' d: C, ₅ H, ,NO. 221.2539; found: 221 .0841 : 222.0802.

ii. Representative procedure for the conversion of 1 H-indoIe-3-carhoxaldehyde into the corresponding I -phenyl stilfonyl-indole-3-carboxaldehyde (4)

A solution of 1 H-indolc-3-carboxaldchydc ( 1.26 mmol) in methylene chloride (4.5 mL), TEA (0.26 g, 2.5 mmol), DMAP ( 1 5 my, 0. 13 mmol) and phenyl sulfonyl chloride ( 1 .3 mmol) was stirred at room temperature overnight. An equal volume of water then was added, the pH adjusted to pH = 2.5 by addition of I N aqueous HCI, the layers separated, the organic layer added to an equal volume of fresh water, the pH adjusted to pH = 9. by addition of I N aqueous NaOH. the layers separated, the aqueous layer extracted (2x) with an equal volume of methylene chloride, the methylene chloride extracts combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford the 1 - phcnyl sulfonyl-indolc-3 carboxaldchydc (4) (65% mass balance) used w ithout further purification. Mass spec: HRMS calc'd: C ₁₅ ΗC ₁₅ 11 ΝΟ ₃ S, 285.31 77; found: 285.0460.

Hi. Representative procedure for the conversion of 1 H-indole-3-carhoxaIdehyde into the corresponding l-benzyloxy-indole-3 -carhoxakiehyde (5)

A solution of I H-indolc-3-carboxaldchydc ( 1 .26 mmol) in methylene chloride (4.5 ml), TEA (0.26 g, 2.5 mmol). DMAP ( 15 mg, 0. 13 mmol ) and bcnzyloxy chloride ( 1.3 mmol) w as stirred at room temperature overnight. An equal volume of water then was added, the pH adjusted to pH = 2.5 by addition of I N aqueous HCI, the layers separated, the aqueous layer extracted ( I X) with an equal v olume of methylene chloride, the organic layers combined, added to an equal volume of fresh water, the pH adjusted to pH=9. by addition of I N aqueous NaOH. the layers separated, the aqueous layer extracted (2x) with an equal volume of methylene chloride, the methylene chloride extracts combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 1- bcnzyloxy-indolc-3 carboxaldchydc (5) (72% mass balance) used without further purification Mass spec: HRMS calc ^' d: C _u ,H _n NO _: , 249 2640: found: 249.0790.

(5a): l -(4-chloro-bcnzyloxy)-indolc-3-carboxaldchydc was also made according to the procedure, above; (X6% mass balance) used w ithout further purification. Mass spec: HRMS calc'd: Cir,H ₁₀ ,,ClNO ₂ , 283.0400: found: 2X3.0497.

iv. Representative procedure for the conversion of 5-chloro- 1 H-indole-3-curhoxaldehyde into the corresponding 5-chloro- 1 -phenyl sitlfonyl-indole-3-carboxaldehydes (6)

A solution of 5-chloro- l H-indolc-3-carboxaldchydc ( 1 .26 mmol) in methylene chloride (4.5 ml), TEA (0.26 g, 2.5 mmol), DMAP ( 15 mg, 0.13 mmol) and phenyl sulfonyl chloride ( 1 .3 mmol) was stirred at room temperature overnight. An equal volume of water then was added, the pH adjusted to pH = 2.5 by addition of I N aqueous HC1, the layers separated, the aqueous layer extracted ( 1 x) w ith an equal volume of methylene chloride, the organic layers combined, added to an equal volume of fresh water, the pH adjusted to pH = 9, by addition of I N aqueous NaOH, the layers separated, the aqueous layer extracted (2x) with an equal volume of methylene chloride, the methylene chloride extracts combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 5- chloro-( I -phcnylsulfonyl)-indolc-3-carboxaldchydc (6) (55% mass balance) used w ithout further purification. Mass spec: HRMS calc'd: C| ₅ Hi„NO _¾ S, 319.7627: found: 319.0070; 321 002X

v. Representative procedure for the conversion of 5-chloro- 1 H-indole-3-carho.xaldehyde into the corresponding 5-chloro-( l-henzyloxy)-indole-3-carboxaldehyde (7)

A solution of 5-chloro- 1 H-indolc-3-carboxaldchydc ( 1 .26 mmol) in methylene chloride (4.5 ml), TEA (0.26 g, 2.5 mmol), DMAP ( 15 mg, 0. 13 mmol) and bcnzyloxy chloride ( 1 .3 mmol) was stirred at room temperature overnight. An equal v olume of water then was added, the pH adjusted to pH = 2.5 by addition of I N aqueous HCI, the layers separated, the aqueous layer extracted ( 1 x) with an equal v olume of methylene chloride, the organic layers combined, added to an equal volume of fresh water, the pH adjusted to pH = 9, by addition of 1 N aqueous NaOH, the layers separated, the aqueous layer extracted (2x) with an equal v olume of iiicthylcnc chloride, the methane chloride extracts combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 5-chloro- ( l -benzyloxy)-indolc-3 carboxaldchydc ( 7) (80% mass balance) used without further purification. Mass spec: HRMS calc'd: C ₁₆ ,H _{1 0} C1NO;. 283.7090; found: 283.0400;

285.0371.

(7a): 5-chloro- l -(4-chloro-bcnzyloxy)-indolc-3-carboxaldchydc; (93% mass balance) used without further purification. Mass spec: HRMS calc ^' d: C ₁₆ .H ₉ Cl ₂ NO ₂ , 3 1 7.0009; found: 3 1 7.0045 : 3 18.9108.

3. Synthesis of I N-substituted indo/e-3-mefliylenehydroxy intermediates and their conversion into pegylated azido ethers i. Representative procedure for the conversion of I -snbsiiuiied-indole-3-carboxaldehydes into their corresponding 1 -substituted indole-3-meihylenehydroxy carhinol derivatives

To a mcthanol THF ( 1 : 1 , 20 mi L) solution of 5-chloro- l -(4-chloro-bcnzylo.xy)- indolc-3-carbaldchydc. 7a. (0.75 mmol) maintained at 0 °C was slowly added NaBH ₄ ( 32 mg. 0.825 mmol ). After addition is complete, the reaction, maintained at 0 °C. is allowed to stir for additional 30 minutes and then allow ed to w arm to room temperature. Reaction progress was assessed by thin layer chromatography (tic) | silica plate;

CH ₂ Cl ₂ : Hcxanc: McOH (90:9: 1 ) | and, upon indication of total consumption of the indolc-3- carboxaldchdyc. 10 ml of I N HCI was added, the mcthanol/THFwas removed under reduced pressure, and fresh water (20 ml ) was added to the resulting residue, which was extracted with ethyl acetate (3x 10 ml ). The combined organic extracts were washed with water, 10% NaHCO ₃ , brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue w as purified by flash chromatography (silica) affording 5-chloro- l -(4-chloro- bcnzyloxy)-indolc-3-mcthylcnchydroxy carbinol (8) (85% yield). Mass spec: HRMS calc'd: C ₁₆ ,H ₁₁ Cl ₂ :NO ₂ . 3 19.0167; found: 3 19.0162; 32 1.0136; 323.0107.

The following substrates were made according to the representative procedure. above.

( 8a): 5-chloro- l -(bcnzyloxy)-indolc-3-mcthylcnchyxdxroxy carbinol; (79% yield): Mass spec: HRMS calc'd: C ₁₆ H ₁₂ ClNO ₂ , 285.0556; found: 285.0498; 287.0527.

( 8b): l -bcnzyloxy-indolc-3-mcthylcnchydroxy carbinol: (90% yield); Mass spec : HRMS calc ^' d: C ₁₆ .H 1.,3 ₁ NO ₂ , 251 .0947; found: 25 1 .0915. (8c): 5-chloro-( l -phcnylsulfonyl )-indolc-3-mcthylcnchydroxy carbinol; (80%. yield): Mass spec: HRMS calc ^' d: Ci jH i .CINO.-.S, 32 1 .0226; found: 32 1 .02 16; 323.0 I 97.

( 8d): l -phcnylsul fonyl-indolc-3-mcthylcnchydroxy carbinol; ( 80% yield); Mass spec: HRMS calc'd 287.0616; found: 2X7.06X6.

(8e): I -phcnyl-indolc-3-mctliylcnchydroxy carbinol ; (97% yield); Mass spec: H RMS calc'd: CijH i jNO, 223.0997; found: 223.0957.

(8f): 1 -(4-chloro-bcnzyloxy)-indolc-3-mcthylcnchydroxy carbinol; (X4% yield); Mass spec: HRMS calc ^' d: ;. 2X5.0556; found: 285.05 1 7; 2X7.0527.

ii. Representative procedure for the conversion of 1 -stihstitiited-indole-3 methylenehydroxy carhinols into their corresponding J -substituted indole-3 methylenehydnm -pegylated azido ether derivatives via azido-losylate PEG - reagents.

To an anhydrous THF/DMF (4: 1 , 1 5 mL) solution of 5-chloro-(4-chloro- bcnzyloxy)-indolc-3-mcthylcnchydroxy carbinol | (8), 446 mg, 1 .4 mmol | was added NaH (33.6 nig, 1 .4 mmol ) at 0 °C, and the resulting reaction mixture maintained at 0 "C for 20 minutes. To the TH F/DM F solution maintained at 0 "C was added tctrabutylammonium iodide (TBA I ) (0.03 mmol ) in one portion, fol lowed by drop- wise addition of an anhydrous THF/DMF (4: 1 , 7mL ) solution of 5-azido-3-oxa- l -pentanol tosylatc (420 mg, 1 .4 mmol ). After completion of the tosylatc addition, the mixture was allowed to warm to room temperature and maintained at room temperature for 10 h. The reaction was quenched by addition of ice cold water (20 mL). the resulting mixture allowed to stir for 15 min, methylene chloride ( 30 mL) added, and the pH adjusted to pH = 2.0 v ia addition of aqueous I N HCI. The layers were separated and the aqueous layer extracted with fresh methylene chloride (2x 10 mL). The combined methylene chloride layers were added to freshly distilled water (20 mL), the pH ad justed to pH = 9.5 by addition of aqueous 2N NaOH and the layers separated. The aqueous layer was extracted w ith fresh methylene chloride

(2x8mL), the methylene chloride fractions combined, dried over anhydrous sodium sulfate, concentrated at reduced pressure, and isolated by Filtration through silica gel clutcd with methylene chloride to afford the desired 5-chloro-(4-chloro-bcnzyloxy)-indolc-3- mcthylcnc-oxa-azido ether (9) ( 70% yield). Mass spec: HRMS calc'd: C ₂₂ H ₂₃ CI ₂ N ₄ O ₃ , 432.0756; found: 432.0754: 434.0724. ( 9a ): 5-chloro- l -(bcnzyloxy)-indolc-3-mcthylcnc-oxa-azido ether: (76% yield); Mass spec: HRMS calc'd: C_oHiyCuN ₄ Ch, 398. 1 146; found: 398. 1 I OS; 400. 1 I I X.

( 9b): l -Bcnzyloxy-indolc-3-Mcthylciic-oxa-azido ether; (91 % yield): Mass spec: HRMS calc'd: Ca)H; _u N ₄ 0 ₃ , 364. 1535; found: 364. 1569.

(9c): 5-chloi o-( l -phcnylsulfonyD-indolc-3-nicthytcnc-oxa-azido ether; (73% yield); Mass spec: HRMS calc ^' d: Ci9H _w ClN ₄ 0 ₄ S. 434.0815; found: 434.0803; 436.0784.

(9d): l -phcnylsulfonyl-indolc-3-mcthylcnc-oxa-azido ether; (62% yield): Mass spec:

HRMS calc'd: Ci >H _2(l N ₄ 0 ₄ S, 400. 1205: found: 400. 1 191.

(9e): I -phcnyl-indolc-3-mcthylcnc-oxa-azido ether; (90% yield); Mass spec: HRMS calc ^' d: C,.;H;„N ₄ 0 _: . 336. 1586; found: 336. 15 1 8.

(9f): 1 -(4-chloro-bcnzyloxy)-indolc-3-mcthylenc-oxa-azido ether; (74% yield); Mass spec: HRMS calc'd: C:,,H _iy ClN ₄ 0... 398.1 146; found: 398.1098; 400. 1 106.

Hi. Representative procedure for the conversion of 1 -substituted indole-3-meihylenehydroxy carbinols into their corresponding I -substituted indoie-3-methylenehydroxy-peg\ kited azido ether derivatives via azido-mesykite PI-Xi-reagenls.

To an anhydrous THF/DMF (4: 1. 5 ml_) solution of 5-chloro-(4-chloro-bcnzyloxy)- indolc-3-mcthylcnchydroxy carbinol (446 mg. 1.4 mmol) was added NaH (33.6 mg. 1 .4 mmol) at 0 "C. the resulting mixture maintained at 0"C for 20 minutes. To this mixture, maintained at 0 °C. was added tctrabutylammonium iodide (TBAI ) (0.014 mmol) in one portion, followed by drop wise addition of an anhydrous TH F/DMF (4: 1 , 5 niL) solution of 8-azido-3,6-dioxa- l -octanol mesylate (354.2 mg, 1 .4 mmol). After addition complete, the mixture was allowed to warm to room temperature and maintained at room temperature for 4.5 h. The reaction was quenched by addition of ice cold water (20 niL), the resulting mixture allowed to stir for 15 min. methylene chloride (30 niL) added, and the pH adjusted to pH = 2.0 via addition of aqueous IN HC1. The layers were separated and the aqueous layer extracted with fresh methylene chloride (2x10 niL). The combined methylene chloride layers were added to fresh water (20mL), the pH adjusted to pH=9.5 by addition of aqueous 2N NaOH and the layers separated. The aqueous layer was extracted with fresh methylene chloride (2x8mL), the methylene chloride fractions combined, dried over anhydrous sodium sulfate, concentrated at reduced pressure, and isolated by Filtering through silica gel clutcd with methylene chloride to afford the desired 5-chloro-(4-chloro- benzyloxy)-indole-3-mcthylenc-oxa-azido ether ( 10) (80% yield). Mass spec: Mass spec: HRMS calc'd: C.:H-CI:N ₄ 0 ₄ , 476.1018: found: 476.1015:478.0986.

(10a): 5-chloro-l-(bcnzyloxy)-indolc-3-mcthylcne-oxa-azido ether; (X3% yield): Mass spec: HRMS calc ^' d: C ₂₂ H ₂₃ CI ₂ N ₄ O.,, ₅ 442.1408; found: 442.1368; 444.1370.

(10b): l-bcnzyloxy-indolc-3-mcthylcnc-oxa-azido ether; (90% yield): Mass spec: HRMS calc ^' d: C ₂₂ H ₂₄ N ₄ O ₄ 408.1797; found: 408.1691.

(10c): 5-chloro-( I -phcnylsulfonyl)-indolc-3-mcthylcnc-oxa-azido ether; (68% yield): Mass spec: HRMS calc ^' d: C ₂₁ H ₂₃ ClN ₄ O ₅ S.478.1077; found: 478.091 I; 480.1041.

(10d): l-phcnylsulfonyl-indolc-3-mcthylcnc-oxa-azido ether; (65% yield): Mass spec: HRMS calc ^' d: C ₂₁ H ₂₄ N ₄ O, ₅ S 444.1467: found: 444.1423.

( 10e): l-phcnyl-indolc-3-mcthylcnc-oxa-azido ether; (85% yield); Mass spec: HRMS calc'd: C ₂₁ H _:4 N ₄ 0.,.380.01848; found: 380.1788.

(10f): l-(4-chloro-bcnzyloxy)-indolc-3-mcthylcnc-oxa-azido ether: (81% yield): Mass spec: HRMS calc ^' d: C ₂₂ H ₂₃ CI ₂ N ₄ O 442.1408: found: 442.1372; 444.1346.

iV. Reaction of I -substituted intlole-3-inetliylenehydroxy carbinols with azido-mesylate reagents,

Substituted indolc-3-mcthylcnc-oxa-azido ethers resulted from indole carbinol addition to the 1 l-azido-3,6.9-trioxa-l-undccanol mesylate reagents. Refer to Section 3.iii. for an analogous synthetic procedure.

( IQg): 5-chloro-l-(4-chloro-bcnzyloxy)-indolc-3-mcthylcnc-oxa-azido ether; (59% yield): Mass spec: HRMS calc'd: C ₂₄ H ₂₆ CI ₂ N ₄ O ₅ .520.1281 ; found: 520.1221; 522.1250. ( 1 Oh): 5-chIoro-l(bcnzyloxy)-indolc-3-mctiiylcnc-oxa-azido ether; (S3% yield); Mass spec; HRMS calc'd: C^H-ClNjO _* .4X6.1670; found: 4X6.1602; 4XX.I635.

( IQi): l-bcnzyloxy-indolc-3-mcthylcnc-o.\a-azido ether; (X2% yield); Mass spec: HRMS calc'd: GMH^N _J O,, 452.2059; found: 452.2003.

(IQi): 5-chloro-( l-phcnylsulfonyl)-indolc-3-mcthylcnc-o\a-azido ether; (55% yield); Mass spec: HRMS calc'd: C^H^ClN+OfiS.522.1339; found: 522.12X9; 524.1301.

(10k): l-phcnylsiilfonyl-indolc-3-mcthylcnc-oxa-azido ether; (43% yield); Mass spec: HRMS calc'd: C;,H ₂ sN ₄ 06S, 4XX.1729; found: 4XX.I647.

(101): l-phenyl-indolc-3-mcthylcnc-oxa-azido ether; (91% yield); Mass spec: HRMS calc'd: Ο,Η^Ν,,Ο.,, 424.21 I I : found: 424.2055.

(10m): I -(4-chloro-bcnzyloxy)-indolc-3-mcthylcnc-oxa-azido ether; (X9% yield); Mass spec: HRMS calc'd: CuH^ClN-tOs.4X6.1670; found: 4X6.1614; 4XX.1593.

4. Synthesis of gift-restricted dendrimers utilizing the copper catalyzed /3 · 2/ Huisgen cycloaddition I CtiAAC Reaction J.

/. Method A: General procedure for a CuAAC reaction utilizing letraproparg\l ethylenediamine and 1 -substituted indole-3-niethy/ene-oxa-pegy/ated azido ethers, affording tetra-triazole-l inked cycloaddition products

A THF:DMF (2: 1, 45 niL) solution of tctrapropargyl ethylenediamine (0.2 mmol) and a 1 -substituted indolc-3-mcthylcncoxa-pcgylatcd azido ether (0.X mmol) was treated with an 8M aqueous solution of Cii(OAc):*H:0 (X0 niL, 0.16 mmol). freshly prepared XM aqueous solution of sodium ascorbatc ( 160 mL, 0.32 mmol) and TBTA ( 15 mol %). The reaction mixture was allowed to stir at room temperature for 18 h, then diluted with EtOAc ( 1 X0 inL), filtered through CupriSorb, the filtrates collected, added to an equal volume of water, the pH adjusted to pH = 9. the layers separated, and the aqueous layer extracted with fresh methylene chloride (2 x 60 niL). The organic layers w ere combined, washed with brine (3 x 40 niL), dried over anhydrous Na ₂ S0 ₄ , and concentrated under reduced pressure. This material was flash chromatographed on a C l X-rcvcrsc phase column to provide the desired tetra-triazole ether.

Yield and the H R MS for representative examples:

( 1 l a ): I -(benzyloy)-indole-3-methylencoxa-(3-oxa-pentyl)-tctra-triaz ole-ethylencdiamine; (61 % yield ); Mass spec: HRMS calc'd: 166X.7445; found: I 66X.7434:

1669.7468.

( l i b): l -(4-chlorobcnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tc tra-triazolc- cthylcncdiaminc; (58% yield); Mass spec: HRMS calc ^' d: . 1 X04.5XX7; found: 1 X04.5X25: 1806.5848: 1807.5833; I X0X.579X.

( 1 l c): 5-chloro- 1 -(4-chlorobcnzyloxy)-indolc-3-mcthy lcncoxa-(3-oxa-pcntyl)-tctra-triazolc- cthylcncdiaminc; ( 74% yield); Mass spec: HRMS calc ^' d: 1940.4330;

found: 1940.4290; 1944.4200; 1945.4292; 1947.4246.

( l i d): 5-chIoro-( 143cnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pentyl)-tetra-tria zole- cthylcncdiaminc; (48% yield); Mass spec: H RMS calc ^' d: 1804.58X7; found: 1 X04.5X2 1 ; 1 X06.5202; I 807.585X.

( l i e): 5-chloro-( l -phcnylsulfonyl)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tctra- triazolc- cthylcncdiaminc: (80% yield); Mass spec: H RMS calc ^' d: 1948.4562: found: 1948.4518; 1950.4568; 195 1 .451 3. ii, Method H: (reneral procedure of microwave-bused ( uAA( ' reaction utilizing

tetrapropargyl ethylenediatnine and 1 -substituted indole-3-methylene-oxa-pegyIated azido ethers, affording letra-triazole-linked cyc/oaddilion products

A DMF solution of a 1 -substituted indole 3-pegylated azido ether (4.4 cquiv) and tetrapropargyl-cthylcncdiaminc ( 1 . 1 cquiv) was added to TBTA ( 1 5 mol%). CuSOj (20 mol%) and sodium ascorbatc (30 mol%) and the resulting mixture heated to 50 °C in a 120W microwave oven for l hr. The reaction mixture w as allowed to cool to room temperature, then diluted w ith distilled w ater, EtOAc, and filtered through CupriSorb. The filtrates were collected, added to an equal volume of w ater, the pH adjusted to pH = 9, the layers separated, and the aqueous layer extracted with fresh methylene chloride (2 x 60 mL). The organic layers were combined, washed with brine (3 x 40 niL), dried over anhydrous NarSOu. and concentrated under reduced pressure. This material was flash chromatographed on a ClX-rcvcrsc phase column to provide the desired tetra-triazole ether. Yield and the HR MS for representative examples:

(11a): l-(bcnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tctratria zolc-cthylcncdiaminc; (77% yield); Mass spec: HRMS calc ^' d: 166X 7445; found: 166X 7404;

1669.7426.

( 11f): l-(benzyloxy)-indole-3-methyleneo\a-(3,6-dioxa-octyl)-tetrat riazole- ethylene-diamine; (64% yield); Mass spec: HRMS calc'd: 1844.84X9; found: 1X44.8427; 1X46.8546.

( 11g): l-(4-chlorobcnzyloxy)-indolc-3-mcthylcncoxa-(3.6-dioxa-octyl )-tctratriazolc- cthylcncdiaminc; (38% yield); Mass spec: HRMS calc ^' d: C . 19X0.6931; found: 1980.6897; 1983.9686; 1984.6910.

( 11fh): 5-chloro-l-(4-chlorobcnzyloxy)-indolc-3-mcthylcncoxa-(3,6-di oxa-octyl)- tctratriazolc-cthylcncdiaminc: (Xl% yield); Mass spec: HRMS calc ^' d:

2116.5371; found: 2116.5311; 2121.5309; 2124.5314.

(11i) 5-chloro-( 143cnzyloxy)-indole-3-methylencoxa-(3,6-dioxa-octyl)-tetratr iazolc- cthylcncdiaminc; (70% yield); Mass spec: HRMS calc ^' d: 19X0.6931;

found: 1980.6923; I9X3.6X9X; 1984.69(H).

(11i): 5-chloro-( l-phcnylsulfonyl)-indolc-3-mcthylcncoxa-(3,6-dioxa-octyl)-tc tratriazolc- cthylcncdiaminc: (60% yield); Mass spec: HRMS calc ^' d: Ο .2124.5608;

found: 2124.5600; 2126.5569; 2127.5615; 2128.5542. iii. Method ( ^' : CuAAC reaction utilizing tetrapropargyl elhylenediamine and ! -substituted iiidole-3-methyIene-oxa-peg\lated azido ethers, affording tetra-triazole- linked

cycloaddition products

An anhydrous acetonitrile (1.0 mL) solution of (7.5 mg, 0.0X mmol) was added via cannula to an anhydrous acetonitrile ( I mL) solution of tctrapropargyl- cthylcncdiaminc (0.02 mmol). a I -substituted indolc-3-mcthylcnc-oxa-pegylated azidc (0.0X mmol).2,6-lutidinc (10 uL.0.08 mmol) and TBTA (8.0 mg.0.016 mmol) that was maintained at 20 °C. The resulting mixture was allowed to stir at room temperature for 24 hours, water was added (4 mL). the resulting mixture was slurried with CupriSorb and filtered through a cclitc/Cuprisorb mixture with additional methylene chloride. The filtrates were collected, added to an equal volume of water, the pH adjusted to pH = 9, the layers were separated, and the aqueous layer was extracted with fresh methylene chloride (2 x 25 niL). The organic layers arc combined, washed with brine (3 x 40 niL), dried over anhydrous Na:S0 ₄ , and concentrated under reduced pressure. The resulting material was flash chromatographed on a C18-rcvcrsc phase column to provide the desired tetra-triazole ether.

Yield and the HR MS for representative examples:

( 1 la): IKbciizyloxy)-indolc-3-Methyleneoxa-(3-oxa-pcntyl)-tetratria zolc-ethylenediamine; (70% yield); Mass spec: HRMS calc'd: 1668.7445: found: 1668.7412:

1669.7438.

(Ilk): l-(bcnzyloxy)-indolc-3-mcthylcncoxa-(3.6.9-trioxa-undccyl)-t ctratriazolc- cthylcncdiaminc; (78% yield); Mass spec: HRMS calc ^' d: 2020.9535; found: 2020.9521: 2021.9554; 2022.9592; 2023.9622.

(111): 1 -(4-chlorobcnzyloxy)-indolc-3-mcthylcncoxa-(3.6,9-trioxa-und ccyl)-tctratriazolc- cthylcncdiaminc; (72% yield); Mass spec: HRMS calc ^' d: 2156.7986; found: 2156.7922; 2159.7971; 2160.8025.

(11m): 5-chloro-l -{4-chlorobenzylo.\y)-indole-3-methylencoxa-(3,6,9-trioxa-un decyl)- tctratriazolc-cthylcncdiaminc; (70% yield); Mass spec: HRMS calc'd:

2292.6423; found: 2292.6465; 2297.6462; 2299.6395.

( 1 In): 5-chIoro-( l-bcnzyloxy)-indolc-3-mcthylcncoxa-(3.6.9-trioxa-undccyl)-tc tratriazolc- cthylcncdiaminc; (78% yield); Mass spec: HRMS calc ^' d: 2156.7986; found: 2156.7936; 2159.7900; 2160.8005.

(1 lo): 5-chloro-( l-phcnylsulfonyl)-indolc-3-mcthylcncoxa-(3.6,9-trioxa-undccy l)- tctratriazolc-cthylcncdiaminc: (63% yield); Mass spec: HRMS calc ^' d:

, 2300.6667: found: 2300.6647; 2302.6619; 2303.6633; 2304.6647.

/V. Method I): General procedure for a CuAAC reaction utilizing triethanolamin- tripropargylether and I -substituted indole-3 methy/ene-oxa-pegvfated azido ethers, affording tri-triazole linked cvc/oaddition products A TH F:Acctonitri lc ( l : l , 45 niL) solution of tri-propargyl tricthanolamino tri-cthcr

( 0.2 mmol ) and I -substituted indolc-3-mcthylcnc-oxa-pcgylatcd azido ether ( 0.6 mmol) is treated with an XM aqueous solution of Cu(OAc )2H ₂ O (X0 niL. 0. l ii mmol ), freshly prepared XM aqueous solution of sodium ascorbate ( 160 mL, 0.32 mmol ) and TBTA ( 15 mol%). The reaction mixture is allowed to stir at room temperature for 24 h, diluted with ethyl acetate ( 180 mL), and filtered through CupriSorb. Distilled water (50 mL) is added to the filtrate and the pH adjusted to pH = 2 by addition of aqueous I N HC1. The layers were separated and the aqueous layer extracted with methylene chloride (2 x 75 mL). The aqueous layer is then added to fresh ethyl acetate ( 100 mL), the pH adjusted to pH = 9 by addition of aqueous I N NaOH. and the layers separated. The aqueous layer is extracted with half its volume of methylene chloride (2x), all the organic extracts combined, dried over anhydrous sodium sul fate, filtered, concentrated at reduced pressure, and isolated by filtering through silica gel to afford the desired l -substitutcd-indolc-3-mcthylcnc-oxa- pcgylatcd-tricthanolamino tri-triazole ether.

In certain embodiments, the compound may be found in another layer during extraction due to the basicity of the compound. In such a case, the pH of the layers may be adjusted accordingly, and the compound may be collected from the desired layer.

Yield and the H R MS for representative examples: SPH-001 25

12a: I -(bcnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tri-triazo lc-(tri-(3-oxa)butyl)- 1 - amine: (81% yield); Mass spec: HRMS calc'd: OSHKINUOI-.. 1355.6121 : found:

1355.61 10: 1356.6144.

12b: Kbcnzyloxy)-indolc-3-mcthylcncoxa-(3.6-dioxa-octyl)-tri-tria zolc-(tri-(3-oxa)butyl^ 1 -amine: (75% yield): Mass spec: HRMS calc'd:α,Η^Ν,,Ο,,. 1487.6902: found:

1487.6X95; 1488.6927; 1489.6980.

12c: l-(bcnzyloxy)-indolc-3-mcthylcncoxa-(3.6,9-trioxa-undccyl)-t ri-triazolc-(tri-(3- oxa)butyl)-l -amine; (75% yield); Mass spec: HRMS calc ^' d: GCHNJN IJOIN. 1619.7687; found: 1619.7637; 1620.7702; 1621.7744; 1622.7718. v. Method /: ^' : General procedure for a CuAAC reaction utilizing trielhanolantino- tripropargylelher and 1 -substituted indale-3-melhylene-oxa-pegylaled azido ethers affording Iri-lriazole linked cyclaadditian prottuefs

An anhydrous acctonitrile ( 1 mL) solution of Cu(McCN).|PFi, (0.06 mmol) was added via cannula to an anhydrous acctonitrile ( 1 mL) solution of tri-propargyl

tricthanolamino tri-cthcr (0.02 mmol). a 1 -substituted indolc-3-mcthylcnc-oxa-pcgylatcd azidc (0.06 mmol), 2,6-lutidinc ( 10 uL, 0.08 mmol) and TBTA (6.0 mg, 0.012 mmol), maintained at 20 "C. The resulting mixture was allowed to stir at room temperature for 24 hours, water was added (4 mL). the resulting mixture was slurried with CupriSorb and filtered through a cclitc/Cuprisorb mixture with additional methylene chloride. The nitrates were collected, added to an equal volume of water, the pH adjusted to pH = 9, the layers separated, and the aqueous layer extracted with fresh methylene chloride (2 x 25 mL). The organic layers arc combined, washed with brine (3 x 40 mL), dried over anhydrous NaiSQj. and concentrated under reduced pressure. The resulting material was flash

chromatographed on a C18-rcvcrsc phase column to provide the desired tetra-triazole ether. Yield and the HR MS for representative examples:

12a: 1 -(bcnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tri-triazo lc-(tri-(3-oxa)butyl)- 1 - amine: (65% yield); Mass spec: HRMS calc'd: 1355.6121 ; found:

1355.6090; 1356.6103.

12d: 5-chloro- 1 -(4-chlorobcnzyloxy)-indolc-3-mcthylcncoxa-(3-oxa-pcntyl)-tr i-triazolc- (tri-(3-oxa)butyl)-I-aminc: (65% yield): Mass spec: HRMS calc'd: C C O

1559.3774; found: 1559.3712; 1561.3738; 1563.3705; 1565.3786. 12e: 5-chloro-l-(plicnylsulfonyl)-iiidolc-3-mcthylcncoxa-(3,6-dio xa-octyl)-Tri-triazolc-(tri- (3-oxa)butyl)-l-aminc: (X0% yield); Mass spec: HRMS calc'd:

.1697.4741; found: 1697.4711; I69X.4704; 1699.4702; 1700.4702. 12f: 5-chloro-l-(phcnylsulfonyl)-indolc-3-mcthylcncoxa-(3.6,9-tri oxa-undccyl)-tri-triazolc- (tri-(3-oxa)butyl)-l -amine: (78% yield); Mass spec: HRMS calc ^' d: ,

1X29.5526; found: 1X30.5551; 1X31.5424; 1X32.551X.

17. Representative procedure for a Huisgen f3 i 2/ cycl ootid it ion methodology allowing incorporation of two different types of nut-restricted pharmacological agents in a 1:1 ratio in a gnl-reslricled pegyktled dendrimer:

Synthesis of compound (lia): The known N-tBoc protected prolyl acetylene reagent (15) was prepared according to the procedure detailed in Tetrahedron Lett., 35, 3715 (1992). It was then utilized in the |3~2| cycloaddition reaction with azide lOh under reaction conditions described here as "Method C ^" affording J3: (95% mass yield); Mass spec: HRMS calc'd: , 6X1.2930; found: 6X1.2924; 6X2.2964: 6X3.2X90; 6X4.2919. Deprofection of l-Boc N-Prolyl-2-iriazole-3,6,9-tio.xa-undecyl 5-chloro-I-henzyIo.xy indole intrermediate. An ethyl acetate ( 10 niL) solution of N-tcrt-butyl carbamatc-prolyl-2- substituted undccyl-triazolc ( 0.32 mmol ) prc-coolcd to 0 °C was sparged with HCI gas for I inin. The ice-bath was removed, the acidic solution allowed to warm to 23°C. and the mixture stirred for 2 li. The mixture was then concentrated to dryness to afford the de- protected intermediate .16 as its hydrochloride salt (98% mass yield), which was used without further purification.

Conversion of intermediate 16 to 13a. Intermediate 16 was first treated with

propargylbromidc affording the corresponding propargyl amine ( 17), as described in EP-0 43 1 700 B I , and used without further purification. Intermediate .17 was then utilized in the 13+21 cycloaddition reaction with the 5-chloro- l -phenylsulfonyl indole undecyl azide, ( 10i ) under reaction conditions described here as "Method C" affording the desired, single dendrimer containing two different indolc-3-pcgylated triazole ethers. 5-choro-( 1 - (bcnzyloxy)-indolc-3-mcthylcncoxa-(3,6.9-oxa-iindccyl)-triaz olc( 2-prolyl)-5-chloro-( 1 - (phcnylsulfonyl)-indolc-3-mcthylcncoxa-( 3.6.9-oxa-undccyl )-triazolc( 1 -prolyl) di-triazolc in a 1 : 1 ratio ( 13a): (80% yield); Mass spec: HRMS calc ^' d: 1 141 .3898; found: 1 141 .3890; 1 143.3860. Ή and ¹³ C NMR arc shown in Figure 2.

Scheme 5:

vii. Representative procedure for a Huisgen [3 t 2/ cycloaddition methodology alllowing incorporation of two different types oj ' gut-restricted pharmacological agents in a 2: 1 ratio: Synthesis of compound (14a): The known N-tBoc protected phcnylalanyl acetylene reagent ( 18) w as prepared according to the procedure detailed in Tetrahedron Lett., 33, 3715 ( 1992). It was then utilized in the | 3+2 | cycloaddition reaction with azidc 9a under reaction conditions described here as "Method C ^" affording mono triazolc 14 ( Scheme 5): (89% mass yield); Mass spec: HRMS calc ^' d: C ₃₅ H ₃₈ CIN ₅ O ₅ 643.2560 ; found: 643.255 1 ;

644.2551 .

Dcproteclion oft-Hoc X-Phrenylalanyl-2-triazole-3-oxa-pcntyl-5-chloro- 1-benzyloxy indole intrermediaie 14. An ethyl acetate ( 10 niL) solution of N-tcrt-butyl carbamatc- phcnylalanyl-2-substitiitcd pentyl-triazolc (0.32 mmol) pre-coolcd to 0 °C was sparged with HCI gas for 1 min. The ice-bath was removed, the acidic solution allowed to warm to 23°C. and the mixture stirred for 2 h. The mixture was then concentrated to dryness to afford the dc-protcctcd intermediate 19 as its hydrochloride salt (92% mass yield), which was used without further purification.

Conversion of intermediate 19 to 14a: Intermediate .19 w as first treated with propargyl bromide affording the corresponding propargyl amine (20 ), as described in EP-0 43 1 700 B l , and used without further purification. I ntermediate 20 was then uti lized in the | 3+2 | cycloaddition reaction with the 5-cliloro- l -phenyl sul fonyl-indolc pentyl azidc, (9c) under reaction conditions described here as "Method C ^" affording the desired, single dendrimcr containing two different indolc-3-pcgylatcd triazolc ethers. 5-choro-( l -(bcnzyloxy)-indolc- 3-mcthyIcncoxa-(3-oxa-pcntyl)-triazolc(2-phenylalanyl)-5-chl oro-( l -(phcnylsulfonyl)- indolc-3-mcthylcncoxa-( 3-oxa-pcntylyl)-2-amino-ditriazolc in a 1 :2 ratio | ( 14a) |: (87% yield); Mass spec: H RMS calc'd: H RMS calc ^' d: 1487 3972: found: 1487.3946; 1489.3926. Ή and ¹³ C NM R arc shown in Figure 3.

6. Results from the in vivo rodent (rat) distribution experiment designed to monitor, and assess, peripheral and C.V.V levels of an orally administered gut-restricted agent, as well as its potential metabolites.

Method

The standards for quantification of 1 1 a were prepared in matching matrices, via spiking blank plasma samples or brain homogenates, with the 1 1a apiking solution at a ratio of 9: 1 . Standards were treated identical ly as the samples. Spraguc Daw ley rats were dosed 15 rats per dosc | po at 50, KM ⁾ and 200 m»'l<g. Plasma and brain were sampled at 30, 60 and I SO min after each dose and monitored via mass spectroscopy (CI , TS, and El ).

When exposed to a battery of cytochrome P450 enzymes in vitro, mass spectra of the resulting samples indicated the primary 1 TV" metabolites were the result of oxygen addition. Specifically, the most abundant metabolites were M I = M + 16; and M2= M + 32. Less abundant, although mcasurcablc, was a metabolite resulting from oxidation and "effective ^" loss of "2H ^' s ^" , which is typically observed in conversion of a methylene carbon atom into its corresponding Όχο. ^' or carbonyl, oxidation state.

l -( Benzyloxy)-indole-3-methylencoxa-(3-oxa-pcntyl)-tetra-triazo le-ethylenediamine ( I la ): Mass spec: H RMS calc'd: C94 H ₉₆ N ₁₈ O _{12 ,} 1668.7445: found: 1668.741 2: 1669.7438.

Table I . Quantification results for / /</ and relative quantification of its two major metabolites M l and M2 in brain and plasma samples. 30 minutes post dose.

Tabic 2. Quantification results for 1la and relative quantification of its two major metabolites M 1 and M2 in brain and plasma samples. 60 minutes post dose.

Tabic 3. Quantification results for 11a and relative quantification of its tw o major metabolites M 1 and M2 in brain and plasma samples. 1 X0 minutes post dose.

Summary of Results

Neither the parent compound, nor any predicted metabolites resulting from liver- based metabolism of the parent compound, were observ ed at any time point of the experiment. This was found to be true for all plasma and brain samples assessed.

Exemplary Synthetic Schemes

3-Methylenehydroxy indole initially was utilized to assess synthetic scheme, since it contains an N-protcctcd ring nitrogen, a substituted 5-position and the mcthylcnchydroxy moiety allows systematic investigation of the side-chain. For example, we were readily able to explore the tether "repeat" length and the ether linkage formation. Additionally, conversion of mcthylcnchydroxy to nitrogen and sulfur nuclcophilcs enables variation of side-chain stability. Ultimately, the synthetic method described herein proceeded in high yield, with facile dendrimer formation, affording compound l_ isolated in X>% overall yield. It is noteworthy copper reagent A is preferred over B, although the overall yield using B was an acceptable | 7 ⁽⁾ % |.

Dcndrimcr 2 was synthesized to test the possibility of incorporating more than one pharmacological agent into one dcndrimcr matrix. In principle, this approach is applicable to the synthesis of dendrimers containing up to four distinct pharmacologically active components and versions containing additional multiple displays by starting from a tri- or tetra-aminc starting material. This is a sign ificant example of the uniqueness of the platform and drug discovery approach, since it exemplifies dendrimers containing multiple "active agents" necessary for detailed exploration of additivity and synergy resulting from agents recognizing different, surface-expressed molecular targets.

Dcndrimcr 2 also contains two different, protected indoles, which allow s determination of the compatibility of various protecting group blocking/deblocking sequences, the relative case of incorporating aza-cthcr tether, the overall yield for 1 : 1 ratio of two different agents synthesized by the variety of procccdurcs reported herein, and the possibility of "rcgioisomcr ^" preferences as a function of the specific tethers, core structures and pharmacological agent designed. /// Vivo and In Vitro Experimental Protocols and Results

In Vitro P roiocols

I, In vitro Phcnotvpic Screen Measurinu IL- l bcta

1. Test compound and Or vehicle is pre-incubated with human peripheral blood

mononuclear leukocyte ( PBML. 5 x 10 /ml) in AIM-V medium pH 7.4 for 2 hours.

Lipopolysaccharidc ( LPS, 25 ng 'ml) is then added to stimulate the cells overnight in 5% C0 ₂ at 37°C.

2. Alamar Blue reagent is then added to the cells in RPMI- 1640 at 37°C for an additional 16 hour incubation period.

3. Living cells will take up Alamar Blue and emit fluorescence. Fluorescence intensity is measured using a SpcctroFluor Plus plate reader with excitation at 530 nm and emission at 590 nm.

4. Decrease of 50 percent or more (50%) in fluorescence intensity relative to vehicle treated controls indicates significant cytotoxicity.

5. Compounds arc screened at 10. 1. 0. 1 , 0.01 and 0.001 μΜ.

LPS control: LPS + Vch

Cells negative control (Vch): Vch only ' No LPS

Inhibition control (DEX): Dcxamcthasonc ( 100 nM, n=2)

(Vehicle = DMSO. 0.05% v/v i. At 24 hours harvest supcrnatants by spinning plates at 1200 rpm for 10 min. collect. and store at -80 C.

ii. Thaw supcrnatants and assay for cytokine secretion w ith Lumincx beads.

iii. LPS-2: IL- Ι β.

iv. Cytokine levels arc determined using a nonlinear five point parameter curve where fit = (A+((B-A)/( l +(((B-E)/(E-A))*((x/C) ^A D))))) for interpolation of data. EC50 values arc then calculated off of interpolated data using nonl inear regression to fit data to the Dose-Response, One-Site Model where: y = A + | (B - A) I + ((Ox ) ^Λ D))|.

I I. TLR4 In vitro Functional Screen Inhibition and/or Antagonism

TI.R4 Reporter Assay Protocol

1 . To specifical ly evaluate the inhibitory activity of the selected compounds towards TLR4 signaling a HEK-BlucTM-hTLR4 reporter cell line ( InvivoGcn) is used, which selectively expresses the TLR4 receptor and activates secreted embryonic alkaline phosphatase ( SEAP) as a reporter gene via TLR4 agonists.

2. The antagonism of these compounds is assessed as indicated by the manufacturer.

The negative control comprised only the TLR4 agonist lipopolysaccharidc ( LPS) ( Sigma Aldrich, St Louis, MO, USA). For the positive control, the commercially available TLR4 inhibitor TAK-242 is selected and used according to the manufacturer (InvivoGcn) instructions.

3. To evaluate the antagonistic effects of compounds, the cells arc cultured as specified by the prov ider ( InvivoGcn) . The cells arc then prc-incubated with these compounds for 2 h at 37 C at various concentrations. Afterward, the cells are challenged with LPS for 20 h. III. TGR5 in vitro assay protocol

1 . KTGR5 CRE HEK293 or mTGR5/CRE/H EK293 stable cell l ine is obtained by transfection of H EK293 cells w ith human or mouse TG R5 expression plasmid (hTGR5- pcDNA 3. 1 or mTGR5-pcDNA 3. 1 ) and CRE-driven luci ferasc reporter plasmid (pGL 4.29. Promcga, Madison, Wl ), and employed to assess the activ ity of test compounds by reporter gene assay.

2. Cel ls arc seeded into 96-wcll plates and incubated overnight in DMEM

supplemented with 10% FBS in 5% CO , at 37 C. Then, cells arc incubated w ith fresh medium containing different concentrations of test compounds (20 inM oleanolic acid as positive control) for 5.5 h. Luciferasc activity in cel l lysatc is determined using the Stcady-Glo Luciferasc Assay System (Promcga, Madison. Wl ) according to the manufacturer's instructions. In Vivo Protocols

IV. TNBS/DSS protocol utilizinu anti-inflammatorv boscntan as positiv e control

1. At 24 and 2 h prior to induction of colitis, rats arc treated with the oral preparation of boscntan ( 10, 30 or 60mg/kg p.o.) suspended in 5% gum arabic (vehicle). Boscntan is administered twice prior to induction of colitis to ensure that adequate plasma levels of boscntan arc present (Clozcl ct al., 1W). All rats arc treated once daily thereafter for 5 days. To determine the efficacy of the compound after the initiation of inflammation, boscntan ( 10, 30 or 60 mg/kg p.o.) is administered I h after the induction of colitis and once daily for 5 days. In both treatment regimens, appropriate vehicle controls are administered in the same manner.

2. Uninflamed rats arc also treated with boscntan (60mg/kg p.o.) in a manner similar to that employed in the prc-dosc groups receiving trinitrobenzene sulphonic acid in cthanol. These rats arc monitored over the dosing period for any changes in fecal consistency.

V. Induction and macroscopic evaluation of inflammation

I . Male Spraguc-Dau Icy rats (250-260 g) arc lightly anesthetized with ether. A rubber catheter (OD, 2mm) is inserted rectal ly into the colon such that the tip is 8 cm proximal to the anus, approximately at the splenic flcxturc. TNBS dissolved in 50% (v'v ) aqueous cthanol is instilled into the colon via the rubber cannula (2 ⁽⁾ mg/ 0.35mL rat). For macroscopic ev aluation of inflammation, colonic damage score (CDS) and

myeloperoxidase (MPO) activity arc measured as described previously (Hongctal.. 2012).

2 The modified scoring system for CDS is as follows:

i. Normal appearance. 0:

ii. Localized hyperemia but no ulcer. I ;

iii. Linear ulcers without significant inflammation, 2;

iv. 2-Λ cm site of inflammation and ulceration with scab, 3;

v. Serosal adhesion to other organs, 2-A cm site of inflammation and ulceration with scab. 4;

vi. Stricture, serosal adhesion involving several bowel loops, <4 cm site of

inflammation and ulceration with scab, 5. VI. DIP Rodent Model Protocol

1 . Sixty-five C57BL/6J mice (4-6 weeks of age) will be ordered from Charles River UK Limited. Margate. Kent, UK. Mice will be group housed at Charles River UK Limited with free access to a high fat diet (D 12451 45% of Kcal derived from fat; Research Diets, New Jersey. USA) and tap water at all times. Animals will be maintained on a normal phase 12 h light-dark cycle (lights on 07:00). During this time body weight will be recorded weekly.

2. Animals arc exposed to the high fat diet for 14 w eeks. They arc transferred to facilities where they w ill be singly housed in polypropylene cages for a further three week period and placed on reverse phase lighting (lights off for X h from 09:30 - 17:30 h) during which time the room will be illuminated by red light. During the third week, animals undergo daily handling (animals arc handled as if to be dosed but are actually not weighed or dosed). This improves the stability of the body w eight response during the baseline phase. Animals are dosed w ith vehicle orally once daily for a 7 day baseline period. Body weight and food and water intake arc recorded daily. On Day -3 during this baseline period (after the completion of dosing on that day), a blood sample (approx. 40uL) is taken from the lateral tail vein using lithium heparin coated collection tubes ( Sarstcdt CB300LH). The sample is spun in a cooled centrifuge (4"C) and the plasma fraction collected and frozen. The sample is assayed for glucose and insulin content. Towards the end of the baseline treatment, animals arc weighed and allocated into 5 treatment groups matched, as closely as possible, for body weight and baseline glucose and insulin. At this stage fifteen animals arc set aside as spares and will not progress to the dosing phase of this particular study. The criteria for withdrawing the animals include general condition, the body weight response to baseline dosing (e.g. poor condition, excessiv e weight loss), and outlying plasma insulin and glucose values. Subsequently, mice arc dosed once daily for 6 days with vehicle or test drug.

3. All treatments arc dosed orally by gavagc. During the baseline and treatment period food intake, water intake and body weight arc recorded daily. At the completion of dosing animals arc examined and any overt behavior is recorded. Dosing begins at approximately 0S:45 each day.

4. On day 6, all the mice arc fasted at 16:00. On Day 7 the mice undergo an OGTT. Each animal is dosed with vehicle or test compound and 60 minutes later is dosed with D-glucosc (2 g kg po). A baseline blood sample is taken immediately before the glucose load and further blood samples arc taken 1 5. 30 and 60 minutes post glucose administration. All blood samples (approximately 30 niL. with the exception of the baseline samples which arc slightly larger) arc taken from the tail vein. Blood samples arc taken into lithium heparinised tubes (Sarstcdt Microvcttc CB300) and plasma separated by ccntrifugation. Plasma samples arc frozen at -X0°C and subsequently they arc assayed for glucose and insulin using commercial ly available kits and reagents. Food is rc-prcscntcd subsequent to the OGTT and final readings arc taken on the morning of Day X. If necessary, animals are re-used after a suitable interval (a minimum of I week).

5. Results: body weights, food intake and water intake are expressed as mean values ± SEM. Body weight and food and water intake data are analysed by ANCOVA followed by appropriate comparisons (two-tailed) to determine significant differences from the control group. P<0.()5 is considered to be statistically significant. The exact statistical tests depend upon the data: however, the effects of drugs on plasma levels of insulin and glucose in OGTT arc normally determined by robust regression.

VII. Gut-Restriction platform applicability to the treatment of CRC

DSS rodent utility as a CRC disease model

There is now a great deal of data supporting the notion intestinal tumors likely originate from normal ISC ^' s | Intestinal Stem Cells ] and chronic inflammation is a predisposing factor to CRC. For example, TLR-dcficicnt mice hav e been shown to exhibit a reduced rate of tumorigencsis in mouse models of colitis-associated cancer and CRC. Furthermore, intestinal epithelial cel l-specific constitutive activ ation of NF-κΒ signi ficantly accelerates A PC-driven tumor formation. Since NF-κΒ is a major pathway downstream of the cell surface-expressed TLRs, it is reasonable to propose antagonists versus several of the TLR's, and particularly TLR-4. as potential CRC treatments. Inflammation is an underlying causation of disease or at the very least the primary driver for predisposition to disease. Since a range of gut-expressed molecular targets, many of which arc nutrient sensing, clearly effect the overall concentration of pro-inflammatory cytokines not only locally in the intestine and in the intestinal microbiota, but also systcmically, it is v ery reasonable to hypothesize anticipated disease modifying effects resulting from treating patients with agonists, or antagonists, of several gut-expressed GPCR's. Finally, the potential disease-relevant effects, which arc attributable to dysrcgulation of a number of pro-inflammatory cytokines ultimately controlled by the gut-expressed nutrient sensing GPCR molecular targets, may optimally observed for combinations of agents and not a single agent. Thus. ISC transplantation, antagonism of several Pattern Recognition

Receptors, an example of the PRR's is TLR-4. agonism of specific nutrient sensing

GRCR's, one example is the nicotinic acid receptor GPR l ( ⁾ 9a. antagonism of the GPCR H4 receptor, and microbiota engineering arc al l potential clinical approaches to the treatment of CRC

VIII. GPR 109a aaonist in vivo experiment

Agonists of the surface-expressed molecular target GPR l O9a protect against weight loss and diarrhea caused by DSS treatment of mice previously exposed to antibiotics. The mouse in vivo experiment determines the efficacy of gut restricted GPR KWa agonists in protecting against DSS colitis in the absence of endogenous l igand.

Protocol

1 . GROUP I ( S mice) : No antibiotic treatment + Drug ( 20 ⁽⁾ mg/kg)

GROUP 2 ( K mice): Antibiotic treatment - Vehicle

GROUP 3 ( 8 mice): Antibiotic treatment ~ Drug ( 200mg/kg)

All mice ciiv exposed to DSS.

2. Antibiotic treatment is as follows: Ampicillin, Metronidazole, Neomycin.

Gcntamicin (all doses I g/ L), and Vancomycin (dose 0.5 g'L) arc administered once daily by oral gavagc.

3. A preliminary experiment determines precise kinetics of antibiotic DSS colitis model prior to drug study initiation.

4. Data in the GPR I 09- - mouse suggests a mechanism involv ing both hematopoietic cells (Trcgs, IL- 10. DCs) and epithelial cells (I L- 18 ).

5. In the DSS injury model the known GPR 109 agonist, niacin, had a dramatic effect when mice were prctrcatcd with antibiotics.

6. To observe a full effect in the DSS model (including recovery phase) it is necessary to treat for 3-4 weeks total. References

Kolb ct al (2001 ) Angcw. Chcm. Int. Ed. 40:2004-2021 ;

Kolb ct al (2003) Drug Discovery Today X: 1 128- 1 137;

Rostovtscv ct al (2002) Angcw. Chcm. Int. Ed. 41 :2596-2599;

Tornoe ct al (2002) Jour, of Org. Chcm. 67:3057-3064;

Wang ct al (2003) Jour, of the Am. Chcm. Soc. 1 25:3192-3 193:

Lcc ct al (2003) Jour, of the Am. Chcm. Soc. 125:9588-9589;

Lewis ct al (2002) Angcw. Chcm.. Int. Ed. 41 : 1053- 1057:

Manctsch ct al (2004) Jour, of the Am. Chcm. Soc. 126: 12809- 12818;

Mocharla ct al (2005) Angcw. Chcm. Int. Ed. 44: 1 16- 120;

Whiting ct al (2006) Angcw. Chcm. I 18: 1463- 1467;

Whiting ct al (2006) Angcw. Chcm. Int. Ed. Engl. 45: 1435- 1439;

McDonaugh and Murphy ( 2013) Tetrahedron, web edition;

Henning and Wittman (2007) Organic Letters 9: 1 ;

Pcctz. ct al (2008) Tetrahedron Letters 49:6386-6389.

Incorporation by Reference

All publications and patents mentioned herein arc hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application.

including any definitions herein, will control. The compounds, synthetic methods, and experimental protocols and results of U.S. Application Number 13/680,582, filed

November 19. 2012, arc hereby incorporated by reference.

Equivalents

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention w ill become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along w ith such variations.