Attorney Docket No. 47351 -0004

HSP-90 BINDING COMPOUNDS, COMPOSITIONS THEREOF, AND THEIR USE ΓΝ THE TREATMENT AND PREVENTION OF FUNGAL INFECTIONS

Background of the Invention

The heat shock protein 90 family, including HSP90 alpha, HSP90 beta, HSP90 N, GRP94 and TRAP-1 (herein generically referred to as HSP90), are essential molecular chaperones that participate in folding of newly synthesized proteins and stabilization and refolding of stress-denatured client proteins. A large number of HSP90 client proteins have been identified many of which are involved in cellular signaling. Consequently, inhibition of HSP90 is widely regarded as a promising therapeutic approach for the treatment of diseases and conditions characterized by inappropriate cellular signaling and stress responses including cancer, neurodegeneration, inflammation and autoimmune disease. Additionally, the use of HSP90 inhibitors in the inhibition of fungal and parasitic infections is expected to be a useful approach to treating pathogenic fungal and parasitic infections.

HSP90 Structure, Sub-cellular Localization and Function

HSP90 and analogues are relatively large proteins that have characteristic Bergerat ATP binding folds and belong to the GHKL protein superfamily of ATPases and protein kinases (Dutta, R. and Inouye, M., Trends Biochem Sci., 2000, 25( 1): 24-8). Crystallography studies revealed a pocket in the N-terminal domain with sequence homology to type II topoisomerases and MutL mismatch repair proteins which co-crystallized with ATP/ADP and established that the N-terminal region is a binding site for adenine nucleotides

(Prodromou, C. et al., Cell, 1997, 90(1): 65-75). Additionally, HSP90 has a middle segment that participates in client protein binding and a c-terminal domain that is responsible for dimerization and binding of some natural products including novobiocin (Pearl, L. H. and Prodromou, C, Annu Rev Biochem, 2006, 75: 271-94).

HSP90 is one of the most abundant cellular proteins comprising 1-2% of total cellular protein (Iwasaki, M. et al., Biochim Biophys Acta, 1989, 992(1): 1-8). HSP90 alpha and HSP90 beta are dimeric cytosolic proteins whereas GRP94 and TRAP- 1 reside in the endoplasmic reticulum and mitochondria respectively. HSP90 binds to a series of co- chaperones in an ATP dependent manner, thereby modulating the structure of client proteins.

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ATP binding regulates the conformation of HSP90 modulating interactions with client proteins (Richter, K., et al., J Biol Chem, 2008, 283(26): 17757-65).

HSP90 exerts its cellular function through selectively chaperoning, or promoting conformational changes and domain rearrangements in a wide range of client proteins including nuclear hormone receptors (Pratt, W. B. and Toft, D. O., Exp Biol Med (Maywood), 2003, 228(2): 1 1 1-33) and protein kinases (Pearl, L. H., Curr. Opin. Genet. Dev., 2005, 15(1): 55-61). Pharmacological inhibition of HSP90 results in induction of a heat shock response as well as destabilization of HSP90 client proteins. In contexts where pathology is driven by proteins that are HSP90 clients it is expected that HSP90 inhibition will result in destabilization of key proteins leading to therapeutic benefit. It is also expected that induction of a heat shock response, including enhanced synthesis of heat shock protein 70 (HSP70) (Lu, A. et al., J. Neurochem., 2002, 81 (2): 355-64), following inhibition of HSP90, may protect normal cells from inappropriate toxicity.

Various natural product small molecules have been identified which bind to the N- terminal ATP binding site of HSP90, including geldanamycin (Stebbins, C. E. et al., Cell, 1997, 89(2): 239-50 and Whitesell, L. et al., Proc. Natl. Acad. Sci. U S A, 1994, 91 (18): 8324-8) and related benzoquinone ansamycins.

Anti-Fungal Applications of HSP90 Inhibitors

Diverse fungal species express HSP90 (Finkelstein DB, 1983 J Biol

Chem. 258(3): 1908- 13, Swoboda RK et al 1995 Infect Immun. 63(1 1):4506- 14). Preclinical data demonstrate that inhibition of fungal HSP90 may be therapeutically useful for the treatment of fungal infections. HSP90 inhibition has been shown to block emergence of drug resistance in fungal cultures (Cowen LE et al. 2005 Science 309:2185-2189). Anasamycin- based HSP90 inhibitors have been shown to synergize with fluconazole and caspofungin in vitro. However these compounds were toxic to fungal infected mice (Cowen LE et al. Proc Natl Acad Sci U S A. 2009; 106(8):2818-23).

Summary of the Invention

The present inventors teach a novel class of HSP90 inhibitors that demonstrate antifungal activity as single agents and in combination with standard anti-fungal agents such as

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24987688_8.DOC Attorney Docket No. 47351 -0004 fluconazole and caspofungin. Compounds of the present invention demonstrate good pharmaceutical properties, are orally available, and are well tolerated in mice and rats. These HSP90 inhibitors demonstrate improved synergy with known anti-fungal treatments. These HSP90 inhibitors demonstrate surprising oral bioavailability compared to known HSP90 inhibitors. Unlike other known HSP90 inhibitors, compounds of the present invention are surprisingly well tolerated in multiple species. Thus, the compounds of the instant invention are useful in the treatment of fungal infections.

In certain embodiments, the invention relates to compounds having a structure of Formula 1 , or a pharmaceutically acceptable salt thereof

A B

Formula 1

wherein;

A is selected from Al an

each X is independently selected from CR and N, preferably selected such that no more than two occurrences, and more preferably no more than one occurrence, of X are N;

R is selected from -CN and C(0)NH ₂ ;

R ¹ is NH ₂ ;

each R is independently selected from hydrogen halogen, -N0 ₂ , -CN, alkyl, alkenyl, alkynyl, -OR ³ , -NR ⁴ R ⁵ , -S(0) _m R ³ , -C(0)R ³ , -C(0)OR ³ , -C(0)NR ⁴ R ⁵ , -S(0) ₂ NR ⁴ R ⁵ , aryl, heteroaryl, carbocyclyl, and heterocyclyl;;

each R\ R ⁴ , and R^ is independently selected from hydrogen, -alkyl-R ⁶ , carbocyclyl, heterocyclyl, heteroaryl, and aryl; or

R ⁴ and R^ together are alkylene, thereby forming a ring;

24987688_8.DOC Attorney Docket No. 47351 -0004

R ⁶ is selected from hydrogen, hydroxy, alkoxy, -NHC(0)alkyl, -NHSC^alkyl, amino, and heterocyclyl;

m is an integer from 0 to 2;

D is selected from aryl, heteroaryl, carbocyclyl and heterocyclyl; and

E is selected from carbocyclyl and heterocyclyl.

In certain embodiments, the invention relates to pharmaceutical compositions comprising a compound of Formula 1 and a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the invention relates to methods for the treatment of a disease or condition selected from fungal infection comprising administering a compound of Formula 1.

Brief Description of Figures

Figure 1 is a graph showing HSP90 inhibitors block growth of Candida albicans and synergize with Fluconazole and Caspofungin

Figure 2 is a graph showing HSP90 inhibitors are fungocidal alone and in combination with Fluconazole and Caspofungin.

Figure 3 is a graph showing plasma levels of compound 34 in mice dosed with 100 mg/kg compound 34 formulated in cremophor ELP by oral route.

Detailed Description of the Invention

In certain embodiments, the invention relates to compounds having a structure of Formula 1 , or a pharmaceutically acceptable salt thereof

A B

Formula 1

wherein

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24987688_8.DOC Attorney Docket No. 47351 -0004

A is selected from Al (wherein 1-6 and 1-8, respectively refer to positions on the ring);

each X is independently selected from CR and N;

R is selected from -CN and -C(0)NH ₂ ;

R ¹ is NH ₂ ;

each R is independently selected from hydrogen, halogen, -N0 ₂ , -CN, alkyl, alkenyl, alkynyl, -OR ³ , -NR ⁴ R ⁵ , -S(0) _m R ³ , -C(0)R ³ , -C(0)OR ³ , -C(0)NR ⁴ R ⁵ , -S(0) ₂ NR ⁴ R ⁵ , aryl, heteroaryl, carbocyclyl, and heterocyclyl;

each R\ R ⁴ , and R^ is independently selected from hydrogen, -alkyl-R ⁶ , carbocyclyl, heterocyclyl, aryl and heteroaryl; or

R ⁴ and R^ together are alkylene, thereby forming a ring;

R ⁶ is selected from hydrogen, hydroxy, alkoxy, -NHC(0)alkyl, -NHS0 ₂ alkyl, amino, and heterocyclyl;

m is an integer from 0 to 2;

D is selected from aryl, heteroaryl, carbocyclyl and heterocyclyl; and

E is selected from carbocyclyl and heterocyclyl.

In certain embodiments, A is Al , wherein each occurrence of X is independently CR In certain alternative embodiments, A is Al , wherein one or two occurrences of X are

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain preferred embodiments, wherein A is Al and X ¹ (i.e., X at the 3-position of

Al) is CR 2 , R 2 is other than hydrogen.

In certain preferred embodiments, X ¹ is CR ² wherein R ² is -NR ⁴ R\ R ⁴ is hydrogen and R^ is carbocyclyl or heterocyclyl.

In certain preferred embodiments, X ¹ is CR ² wherein R ² is -NR ⁴ R\ R ⁴ is hydrogen and R^ is heterocyclyl.

In certain preferred embodiments, X ¹ is CR ² wherein R ² is -NR ⁴ R\ R ⁴ is hydrogen and R^ is carbocyclyl-R ⁷ wherein

R ⁷ is selected from -OR ¹⁴ , -NR ^I2 R ¹³ , -C(0)NR ^l2 R ¹³ , -S0 ₂ NR ^l2 R ¹³ ,

-NR ^l4 C(0)R ¹⁵ , -NR ^l4 S(0) ₂ R ¹⁵ , -NR ^l4 C(0)NR ^l2 R ¹³ , -OC(0)NR ^l2 R ¹³ , NR ^l4 C(0)OR'\ -C(0)OR ¹⁵ , -OC(0)R ¹⁵ or heterocyclyl;

each R ¹² and R ^{1 1} is independently selected from hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl and heteroaryl; or

R ¹² and R ^{1 1} together form a substituted or unsubstituted heterocyclyl ring system; and R ^l4 and R ¹⁵ are selected from hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl and heteroaryl.

In preferred embodiments, the carbocyclyl group of carbocyclyl-R is a trans 1-4 disubstituted 6-membered ring.

In certain embodiments as described above, the carbocyclyl group of carbocyclyl-R is a six-membered ring. In certain such embodiments, R is preferably located at the 4- position of the carbocyclyl ring relative to the point of attachment to A.

In certain embodiments as described above where X ¹ is CR ² , wherein R ² is -NR ⁴ R\

4 5 7 2

R is hydrogen and R is carbocyclyl-R , other occurrences of R in Formula 1 are independently selected from hydrogen or halogen.

In certain embodiments as described above where X ¹ is CR ² , wherein R ² is -NR ⁴ R\

R ⁴ is hydrogen and R^ is heterocyclyl, other occurrences of R ² in Formula 1 are

independently selected from hydrogen or halogen. In certain embodiments, A is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments, A is A2, wherein each occurrence of X is independently CR . In certain embodiments, A is A2, wherein one or two occurrences of X are N.

In certain preferred embodiments, the R 2 attached to X 6 , X 7 , or X 8 , preferably the R 2 attached to X of A2 is other than hydrogen.

In certain embodiments A is selected from

In certain embodiments, R ² is selected from hydrogen, halogen, -CN, alkyl, -OR\ - NR ⁴ R\ -S(0) _m R ^J , and -C(0)NR ⁴ R\ In certain such embodiments, R ² is selected from hydrogen and NR ⁴ R\

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

Attorney Docket No.47351-0004

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

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In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments wherein A is Al and X ¹ is CR , R is selected from

H

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments wherein A is Al and X ¹ is CR ² , R ² is selected from

In certain embodiments, when A is A2 at least one occurrence of R , preferably when

X 6", X 7', or X 8° is CR 2 more preferably when X 8 is CR 2 , is selected from alkyl, halogen, -OR 3 , and -S(0) _m R\ In certain such embodiments, R ² is alkyl, preferably methyl, ethyl, propyl, isopropyl, butyl, or isobutyl. In certain alternative such embodiments, at least one occurrence of R is halogen, preferably Br or F. In certain alternative such embodiments, at least one occurrence of R ² are -OR\ In certain such embodiments, R ² is selected from - 0(CH ₂ ) ₂ OCH ₃ , -0(CH ₂ ) ₂ N(CH ₃ ) ₂ , -0(CH ₂ ) ₂ OH, and OEt. In certain alternative such embodiments, at least one occurrence of R ² is -S(0) _m R\ In certain such embodiments, R ² is selected from

H _c H H

^S h , ^S . ^S I , ^S^ ^N - , ^s^ ^H ^ , and ^s^ .

In certain embodiments, B is

14

24987688_8.DOC Attorney Docket No. 47351 -0004

wherein Q is selected from -C(O)-, and -S(0) _n -; and n is an integer from 1 to 2.

In certain embodiments D is aryl or heteroaryl, preferably phenyl, pyridyl, thienyl, thiazolyl, oxazole, or isoxazole. In certain other embodiments, D is carbocyclyl, preferably cyclopentyl or cyclohexyl.

In certain embodiments, B is selected from

In certain embodiments, B is selected from

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24987688_8.DOC Attorney Docket No. 47351 -0004

In certain embodiments, B is selected such that the D ring is one of the D rings specified in the previous paragraph and the E ring is one of the E rings specified in the paragraph prior to that.

Another aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula 1 and a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect of the present invention provides a method of treating a proliferative disorder or a disease state, the method comprising administering to a subject in need thereof an amount of a compound or pharmaceutical composition as described above sufficient to treat the proliferative disorder or disease state.

Another aspect of the present invention provides a method of modulating HSP function, the method comprising contacting a cell with a compound of the present invention in an amount sufficient to modulate the binding of a HSP client protein to HSP, thereby modulating the HSP function.

Another aspect of the present invention provides a method of modulating HSP function, the method comprising contacting a cell with a compound of the present invention in an amount sufficient to modulate HSP90 function affecting protein folding, stability and aggregation.

In certain embodiments, the invention relates to methods for the treatment of a disease or condition selected from the treatment of fungal infections. Examples of pathogenic fungal infections that may be treated with compounds of the instant invention include species of: Candida, Aspergillus, Cryptococcus, Trichophyton, Microsporum, Epidermophtyon, and Malassezia.

Infections which may be treated with compounds of the instant invention include: Candida albicans, Aspergillus fumigatus, Aspergillus terreus, Trichophyton rubrum and Cryptococcus neoformans.

Compounds of the instant invention may be used to treat fungal infections anywhere in the body including vaginal, dermal, nail, meningial, as well as systemic infections.

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24987688_8.DOC Attorney Docket No. 47351 -0004

Compounds of the instant invention may be used for the treatment of fungal infections either alone or in combination with other agents used in the treatment or prophylaxis of fungal infections. For example, Table 1 summarizes therapeutic agents used in the treatment of fungal infections and is provided to be illustrative of potential

combination therapies.

Table 1

i) Administration

Compounds prepared as described herein can be administered in various forms, depending on the disorder to be treated and the age, condition, and body weight of the patient, as is well known in the art. For example, where the compounds are to be administered orally, they may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories. For application by the ophthalmic mucous membrane route, they may be formulated as eye drops or eye ointments. These formulations can be prepared by conventional means, and if desired, the active ingredient may be mixed with any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, a cyclodextrin, and/or a buffer. Although the dosage will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2000 mg of the compound is recommended for an adult human patient, and this may be administered in a single dose or in divided doses. The amount of active ingredient which can be combined with a carrier material to produce a

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24987688_8.DOC Attorney Docket No. 47351 -0004 single dosage form will generally be that amount of the compound which produces a therapeutic effect.

The precise time of administration and/or amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound,

physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), route of administration, etc. However, the above guidelines can be used as the basis for fine-tuning the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

The phrase "pharmaceutically acceptable" is employed herein to refer to those ligands, materials, compositions, and/or dosage forms which are, 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 with 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, excipient, 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: ( 1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower 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 laurate; ( 13) agar; ( 14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's

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24987688_8.DOC Attorney Docket No. 47351 -0004 solution; ( 19) ethyl alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, pharmaceutical compositions of the present invention are non-pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient. The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of the compound(s). These salts can be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting a purified compound(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate salts, and amino acid salts, and the like. (See, for example, Berge et al. ( 1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1 - 19.)

In other cases, the compounds useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming

pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term

"pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound(s). These salts can likewise be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting the purified compound(s) in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening,

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24987688_8.DOC Attorney Docket No. 47351 -0004 flavoring, and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert matrix, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes, and the like, each containing a predetermined amount of a compound(s) as an active ingredient. A composition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, 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, cyclodextrins, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,

carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, 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, acetyl alcohol and glycerol

monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a

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24987688_8.DOC Attorney Docket No. 47351 -0004 similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients 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, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound(s) moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, 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 which can be dissolved in sterile water, or some 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 ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, 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, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut,

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24987688_8.DOC Attorney Docket No. 47351 -0004 corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl 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 preservative agents.

Suspensions, in addition to the active compound(s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, 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 agent.

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

Dosage forms for the topical or transdermal administration of a compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required or beneficial.

The ointments, pastes, creams, and gels may contain, in addition to compound(s), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or

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24987688_8.DOC Attorney Docket No. 47351 -0004 mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the composition. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular composition, but typically include nonionic surfactants (Tweens, Pluronics, sorbitan esters, lecithin, Cremophors), pharmaceutically acceptable co-solvents such as polyethylene glycol, innocuous proteins like serum albumin, oleic acid, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlled delivery of a compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the compound(s) across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound(s) in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compound(s) in combination with one or more pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof,

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24987688_8.DOC Attorney Docket No. 47351 -0004 vegetable 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, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include tonicity-adjusting agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms can be made by forming microencapsulated matrices of compound(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically, or rectally. They are, of course, given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, infusion; topically by lotion or ointment; and rectally by suppositories. Oral administration is preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial,

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24987688_8.DOC Attorney Docket No. 47351 -0004 intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a ligand, drug, or other material other than directly into the central nervous system, such that it enters the patient's system and thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compound(s) may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally, and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

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

The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration. In general, the compositions of this invention may be provided in an aqueous solution containing about 0.1 -10% w/v of a compound disclosed herein, among other substances, for parenteral administration. Typical dose ranges are from about 0.01 to about 50 mg/kg of body weight per day, given in 1-4 divided doses. Each divided dose may contain the same or different compounds of the invention. The dosage will be an effective amount depending on several factors including the overall health of a patient, and the formulation and route of administration of the selected compound(s).

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24987688_8.DOC Attorney Docket No. 47351 -0004

Definitions

As used herein, the term "affinity tag" means a ligand or group, linked either to a compound of the present invention or to an HSP domain, that allows the conjugate to be extracted from a solution.

The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, cyclopropylmethyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The terms "alkenyl" and "alkynyl" refer to substituted or unsubstituted 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. Representative alkenyl groups include vinyl, propen-2-yl, crotyl, isopenten-2-yl, l ,3-butadien-2-yl), 2,4-pentadienyl, and l ,4-pentadien-3-yl.

Representative alkynyl groups include ethynyl, 1- and 3-propynyl, and 3-butynyl. In certain preferred embodiments, alkyl substituents are lower alkyl groups, e.g., having from 1 to 6 carbon atoms. Similarly, alkenyl and alkynyl preferably refer to lower alkenyl and alkynyl groups, e.g., having from 2 to 6 carbon atoms. As used herein, "alkylene" refers to an alkyl group with two open valencies (rather than a single valency), such as -(CH ₂ )i-io- and substituted variants thereof.

The term "alkoxy" refers to an alkyl group having an oxygen attached thereto.

Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxy.

The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, thereby forming an ether.

The terms "amide" and "amido" are art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

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24987688_8.DOC Attorney Docket No. 47351 -0004 o

N

R ⁹

wherein R ⁹ , R ¹⁰ are as defined above. Preferred embodiments of the amide will not include imides, which may be unstable.

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

wherein R ⁹ , R ^l() and R ^l() each independently represent a hydrogen, an alkyl, an

alkenyl, -(CH ₂ ) _m -R ⁸ , or R ⁹ and R ^l() taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or an integer from 1 to 8. In preferred embodiments, only one of R ⁹ or R ¹⁰ can be a carbonyl, e.g., R ⁹ , R ^l() , and the nitrogen together do not form an imide. In even more preferred embodiments, R ⁹ and R ^l() (and optionally R ^l() ) each independently represent a hydrogen, an alkyl, an alkenyl, or - (CH ₂ ) _m -R · In certain embodiments, the amino group is basic, meaning the protonated form has a pK _a > 7.00.

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

The term "aryl" as used herein includes 5-, 6-, and 7-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, anthracene, and phenanthrene.

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24987688_8.DOC Attorney Docket No. 47351 -0004

The terms "carbocycle" and "carbocyclyl", as used herein, refer to a non-aromatic substituted or unsubstituted ring in which each atom of the ring is carbon. The terms "carbocycle" and "carbocyclyl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is carbocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Representative carbocyclic groups include cyclopentyl, cyclohexyl, 1-cyclohexenyl, and 3- cyclohexen- l-yl, cycloheptyl.

The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R ^{1 1} represents a hydrogen, an alkyl, an alkenyl, -(CH ₂ ) _m -R or a pharmaceutically acceptable salt. Where X is an oxygen and R ^{1 1} is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R ^{1 1} is a hydrogen, the formula represents a "carboxylic acid".

The terms "heteroaryl" includes substituted or unsubstituted aromatic 5- to 7- membered ring structures, more preferably 5- to 6-membered rings, whose ring structures include one to four heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The terms "heterocyclyl" or "heterocyclic group" refer to substituted or unsubstituted non-aromatic 3- to 10-membered ring structures, more preferably 3- to 7-membered rings,

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24987688_8.DOC Attorney Docket No. 47351 -0004 whose ring structures include one to four heteroatoms. The term terms "heterocyclyl" or "heterocyclic group" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine, lactones, and lactams.

The term "hydrocarbon", as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The terms "polycyclyl" or "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted.

The term "preventing" is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount. Prevention of an infection includes, for example, reducing the number of diagnoses of the infection in a treated population versus an

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24987688_8.DOC Attorney Docket No. 47351 -0004 untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population. Prevention of pain includes, for example, reducing the magnitude of, or alternatively delaying, pain sensations experienced by subjects in a treated population versus an untreated control population.

As used herein, the term "probe" means a compound of the invention which is labeled with either a detectable label or an affinity tag, and which is capable of binding, either covalently or non-covalently, to an HSP domain. When, for example, the probe is non- covalently bound, it may be displaced by a test compound. When, for example, the probe is bound covalently, it may be used to form cross-linked adducts, which may be quantified and inhibited by a test compound.

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will 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 substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched,

carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents 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 thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic 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.

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24987688_8.DOC Attorney Docket No. 47351 -0004

As used herein, the term "treating" or "treatment" includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a subject's condition.

As used herein, the term "well tolerated" refers to a substance, such as a

pharmaceutical compound or composition, that is not poisonous or harmful to humans or other higher life forms when used in a therapeutically effective dose.

As used herein, the term "orally bioavailable" refers to a substance, such as a pharmaceutical compound or composition, that can be taken orally for therapeutic efficacy.

Compounds of the invention also include all isotopes of atoms present in the intermediates and/or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.

Examples

Schemes 1 , 2, 3, 4, 5 and 6 illustrate general synthetic procedures for the preparation of compounds of formula 1.

Method A

Sonogashira coupling reaction of intermediate 1-a with terminal alkyne 1-b affords intermediate 1-c which is then converted to the corresponding trifluoroacetamide

intermediate 1-d. Intermediate 1-f may be prepared by reacting o-alkynyltrifluoroacetanilide intermediate 1-d with aryl halide 1-e in the presence of a ligand, a catalyst and a base.

Treatment of intermediate 1-f with an appropriate base provides intermediate 1-g. Cyclized intermediate 1-g can also be obtained from intermediate 1-d in a one step-method under suitable conditions. Treatment of intermediate 1-g with an amine of formula R ⁴ R ⁵ NH provides intermediate 1 -h. Nitrile hydrolysis of intermediate 1-h provides compounds of formula 1.

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24987688_8.DOC Attorney Docket No. 47351 -0004

Scheme 1

Method B

Treatment of commercially available aryl halide 1-e with an amine of formula R ⁴ R^NH provides nitrile intermediate 2-a which is then converted to the corresponding amide intermediate 2-b. Intermediates 2-c may be prepared by reacting o-alkynyltrifluoroacetanilide intermediates 1-d with aryl halide 2-b in the presence of a ligand, a catalyst and a base.

Treatment of intermediate 2-c with an appropriated base provides compounds of formula 1. Compounds of formula 1 may also be obtained from intermediates 2-b and 1-d in a one step- method under appropriated conditions.

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24987688_8.DOC Attorney Docket No. 47351 -0004

2-c 1

Scheme 2

Method C

Treatment of intermediate 1-a, preferentially a chlorine derivative, with intermediate 1-b afford intermediate 3-a. Sonogashira coupling reaction of aryl halide 1 -e with terminal alkyne intermediate 3-a provides intermediate 3-b. Treatment of intermediate 3-b with an appropriated base, ligand and catalyst provides polycyclic intermediate 1-g.

Scheme 3

Method D

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24987688_8.DOC Attorney Docket No. 47351 -0004

Sonogashira coupling reaction of aryl halide 2-b with terminal alkyne intermediate 3-a provides intermediate 4-a. Treatment of intermediate 4-a with an appropriate base, ligand and catalyst provides a compound of formula 1.

Scheme 4

Method E

Sonogashira coupling reaction of aryl halide 1-e with an alkyne intermediate 1-b provides intermediate 5-a. Treatment of intermediate 1-a with intermediate 5-a affords intermediate 3-b.

Scheme 5

Method F

Sonogashira coupling reaction of aryl halide 2-b with an alkyne intermediate 1-b provides intermediate 6-a. Treatment of intermediate 1-a with intermediate 6-a affords intermediate 4-a.

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24987688_8.DOC Attorney Docket No. 47351 -0004

Scheme 6

SYNTHETIC METHODS

Preparation of representative examples:

Synthesis of Compound 1

7-e Compound 1

Scheme 7

Step 1: 7-b

To a solution of 2-iodoaniline 7-a (5.0 g, 22.83 mmol) in DMF cooled to 0 °C were sequentially added diethylamine (5 mL, 22.83 mmol), copper(I) iodide (870 mg, 4.57 mmol) and dichlorobis(triphenylphosphine) palladium (II) (1.60 g, 2.28 mmol). After complete dissolution of copper(I) iodide, methyl hex-5-ynoate (3.17 g, 25.1 mmol) was added

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24987688_8.DOC Attorney Docket No. 47351 -0004 dropwise at 0 °C and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHC(¾ and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 7-b as colorless oil. MS (m/z) M+H= 218.1

Step 2: 7-c

To a solution of intermediate 7-b (4.8 g, 22.09 mmol) in DCM cooled to 0 °C was sequentially added DIPEA ( 15.48 mL, 88.00 mmol), DMAP (135 mg, 1.10 mmol) and TFAA (6.87 mL, 48.60 mmol) and the reaction was then stirred at room temperature for 18 hours. Water and ethylacetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 7-c as colorless oil. MS (m/z) M+H= 314.3

Ste 3: 7-d

To a solution of intermediate 7-c (1.0 g, 3.19 mmol) in acetonitrile was added 2- fluoro-4-iodobenzonitrile ( 1.18 g, 4.79 mmol), Cs ₂ C0 ₃ (1.56 g, 4.79 mmol) and Pd(PPh ) ₄ (184 mg, 0.16 mmol) and the reaction was stirred at 100 °C for 18 hours and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 7-d as a beige solid. MS (m/z) M+H= 305.9

Step 4: 7-e

To a suspension of intermediate 7-d (440 mg, 1.44 mmol) in DMSO were

sequentially added DIPEA ( 1.26 mL, 7.23 mmol) and trans-4-aminocyclohexanol (666 mg, 5.78 mmol) and the mixture was then heated at 100 °C for 18 hours and then cooled to room temperature. Water and ethyl acetate were added, the organic layer was separated, washed

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24987688_8.DOC Attorney Docket No. 47351 -0004 with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 7-e as a beige solid. MS (m/z) M+H= 400.6

Step 5: Compound 1

To a solution of intermediate 7-e (50 mg, 0.12 mmol) in 1 ,4-dioxane was sequentially added NaOH IN (38 uL, 0.038 mmol) and 30% aqueous hydrogen peroxide (0.40 mL, 3.92 mmol) and the reaction was stirred at room temperature for 15 hours. Water and ethyl acetate were added; the organic layer was separated, washed with saturated ammonium chloride, 10% aqueous Na ₂ S ₂ 0:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 1 as a white solid. MS (m/z) M+H= 418.3

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24987688_8.DOC Attorney Docket No. 47351 -0004

Synthesis of compound 2

8-f Compound 2

Scheme 8

Step 1: 8-b

A solution of intermediate 2-fluoro-4-iodobenzonitrile 8-a (2.50 g, 10.12 mmol), trans-4-aminocyclohexanol (3.50 g, 30.40 mmol) and DIPEA (7.07 mL, 40.50 mmol) in DMSO was heated at 150 °C for 2 hours and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 8-b as a white solid. MS (m/z) M+H= 343.1

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24987688_8.DOC Attorney Docket No. 47351 -0004

Step 2: 8-c

To a solution of intermediate 8-b (750 mg, 2.19 mmol) in DMSO/EtOH (4: 1 , 10 mL) was sequentially added NaOH IN (3.0 mL, 3.0 mmol) and 30% aqueous hydrogen peroxide (2.0 mL, 19.58 mmol) and the reaction was stirred at room temperature for 4 hours. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 8-c as beige solid. MS (m/z) M+H= 361.1

Step 3: 8-e

To a solution of 4-chloro-2-iodoaniline 8-d (2.0 g, 7.89 mmol) in DMF cooled to 0 °C was sequentially added diethylamine (5 mL, 22.83 mmol), copper (I) iodide (301 mg, 1.57 mmol) and dichlorobis(triphenylphosphine) palladium (II) (554 mg, 0.78 mmol). After complete dissolution of copper (I) iodide, methyl hex-5-ynoate (1.09 g, 8.68 mmol) was added dropwise at 0 °C and the reaction was then stirred at room temperature overnight.

Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 8-e as a beige solid. MS (m/z) M+H= 252.2

Step 4: 8-f

To a solution of intermediate 8-e (1.78 g, 7.07 mmol) in DCM cooled to 0 °C were sequentially added DIPEA (4.94 mL, 28.3 mmol), DMAP (43 mg, 0.35 mmol) and TFAA (2.19 mL, 15.56 mmol) and the reaction was then stirred at room temperature for 18 hours. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 8-f as yellow oil. MS (m/z) M+H= 349.0

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24987688_8.DOC Attorney Docket No. 47351 -0004

Step 5: Compound 2

To a solution of intermediate 8-c (544 mg, 1.51 mmol) in acetonitrile was added intermediate 8-f (350 mg, 1.0 mmol), CS ₂ CO (492 mg, 1.51 mmol) and Pd(PPh ₃ ) ₄ (23 mg, 0.02 mmol) and the reaction was stirred at 1 10 °C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 2 as a beige solid. MS (m/z) M+H= 452.4

Synthesis of compound 3

9-f Compound 3

Scheme 9

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24987688_8.DOC Attorney Docket No. 47351 -0004

Step 1: 9-b

A solution of intermediate 9-a ( 1.0 g, 4.05 mmol), 2-methoxyethanamine (912 mg, 12.15 mmol) and DIPEA (2.83 mL, 16.19 mmol) in DMSO was heated at 150 °C for 2 hours and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 9-b as a white solid.

Step 2: 9-c

To a solution of intermediate 9-b (1.10 g, 3.64 mmol) in DMSO (8.0 mL) and MeOH

(8.0 mL) was sequentially added NaOH IN (3.0 mL, 3.0 mmol) and 30% aqueous hydrogen peroxide (2.0 mL, 19.58 mmol) and the reaction was stirred at room temperature for 4 hours. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 9-c as a white solid. MS (m/z) M+H= 321.5

Step 3: 9-e

To a solution of hex-5-ynoic acid (1.0 g, 8.92 mmol) in THF was added DMF (1 19 uL) and oxalyl chloride (820 uL, 9.36 mmol) and the mixture was stirred for 1 hour at room temperature. 2-chloroaniline 9-d ( 1.13 g, 8.92 mmol) and TEA (1.30 mL, 9.36 mmol) were added; the reaction was stirred at reflux for 2 hours and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 9-e as a white solid. MS (m/z) M+H= 222.2

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24987688_8.DOC Attorney Docket No. 47351 -0004

Step 4: 9-f

A mixture of dichlorobis(triphenylphosphine) palladium (II) (21 mg, 0.03 mmol), copper(I) iodide ( 1 1 mg, 0.06 mmol), intermediate 9-c (500 mg, 1.56 mmol) and intermediate 9-e (330 mg, 1.48 mmol) in TEA (4.96 mL) was heated at 50 °C for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 9-f as a white solid. MS (m/z) M+H= 414.5

Step 5: Compound 3

A mixture of intermediate 9-f ( 100 mg, 0.24 mmol), palladium (II) acetate (5.59 mg, 0.02 mmol), bis(di-tert-butylphosphino)ferrocene (23.9 mg, 0.05 mmol), and K ₂ CO:, (86 mg, 0.62 mmol) in NMP was heated at 130 °C overnight and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 3 as a beige solid. MS (m/z) M+H= 378.4

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24987688_8.DOC Attorney Docket No. 473 1 -0004

Synthesis of compound 13

10-h Compound 13

Scheme 10

Step 1: 10-b

To a solution of 3,5,5-trimethylcyclohex-2-enone, 10-a (25.0 mL, 181 mmol), in

MeOH cooled to 15 °C were sequentially added hydrogen peroxide (1 1.5 mL, 181 mmol) and 5N NaOH (4 mL, 20.0 mmol) and the reaction was then stirred at 15 °C for 20 minutes and then at room temperature for 3 hours. Water and ether were added, the organic layer was separated, the aqueous phase was extracted twice with diethyl ether, the combined organic extracts were washed with water, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 10-b as colorless oil.

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Step 2: 10-c

To a solution of intermediate 10-b (3.0 g, 19.45 mmol) in EtOH was added 4- methylbenzenesulfonohydrazide (3.62 g, 19.45 mmol) and the reaction was then stirred at room temperature for 20 minutes and 55 °C for 2 hours. Water and CH ₂ CI ₂ were added; the organic layer was separated, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 10-c as colorless oil.

Step 3: 10-d

To a solution of 3N NaOH (53.3 mL, 160 mmol) in 1 ,4-dioxane: water (200 mL) cooled to -5 °C was added bromine (2.09 mL, 40.6 mmol). The solution was stirred at -5 °C for 5 minutes and then diluted with 1 ,4-dioxane (40 mL). The resulting hypobromite solution was then slowly added to a solution of intermediate 10-c ( 1.70 g, 12.30 mmol) in 1 ,4- dioxane:water 3: 1 (200 mL) and the reaction was then stirred at 0 °C for 2 hours. Sodium thiosulfate (3 g) was added and the mixture was stirred for 15 minutes at room temperature. Concentrated HC1 and CH ₂ CI ₂ were added, the organic layer was separated, the aqueous phase was extracted with CH ₂ CI ₂ , the combined organic extracts were dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 10-d as yellow oil.

Step 4: 10-e

To a solution of intermediate 10-d (600 mg, 4.28 mmol) in MeOH was added acetyl chloride (304 uL, 4.28 mmol) and the reaction was then stirred at 40 °C for 2 hours. Water and CH ₂ CI ₂ were added, the organic layer was separated, the aqueous phase was extracted with CH ₂ CI ₂ , and the combined organic layer was separated, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 10-e as yellow oil.

Step 5: 10-g

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To a solution of 2-iodoaniline, 10-f (383 mg, 1.75 mmol), in DMF cooled to 0 °C were sequentially added diethylamine ( 10 mL, 96.2 mmol), copper(I) iodide (13.3 mg, 0.07 mmol) and dichlorobis(triphenylphosphine) palladium (II) (24.5 mg, 0.03 mmol). After complete dissolution of copper(I) iodide, methyl 3,3-dimethylhex-5-ynoate (270 mg, 1.75 mmol) was added dropwise at 0 °C and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 10-g as a yellow oil. MS (m/z) M+H= 246.1

Step 6: 10-h

To a solution of intermediate 10-g (1 10 mg, 0.44 mmol) in CH2CI2 cooled to 0°C were sequentially added DIPEA (313 uL, 1.79 mmol), DMAP (2.7 mg, 0.02 mmol) and TFAA ( 139 uL, 0.98 mmol) and the reaction was then stirred at room temperature for 18 hours. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 10-h as yellow oil. MS (m/z) M+H= 342.3

Step 7: Compound 13

To a solution of intermediate 10-h (154 mg, 0.45 mmol) in acetonotrile were added intermediate 8-c ( 162 mg, 0.45 mmol), CS2CO3 (220 mg, 0.67 mmol) and Pd(PPh:,) ₄ (26 mg, 0.02 mmol) and the reaction was stirred at 100 °C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 13 as a yellow solid. MS (m/z) M+H= 446.4

Synthesis of compound 50

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11-h Compound 50

Scheme 11

Step 1: 11-b

A solution of 4-bromo-2-fluorobenzonitrile 1 1-a (2.91 g, 14.56 mmol), tert-butyl 4- aminocyclohexylcarbamate (3.12 g, 14.56 mmol) and TEA (6.13 mL, 43.70 mmol) in DMSO was heated at 130°C for 5 hours and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Diethyl ether was added to the residue and intermediate 1 1-b was collected by filtration as a white solid.

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Step 2: 11-c

To a solution of intermediate 1 1-b (4.30 g, 10.91 mmol) in DMSO (15.0 mL) and MeOH (15 mL) were sequentially added IN NaOH ( 10.91 mL) and 30% aqueous hydrogen peroxide ( 1.67 mL, 16.36 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 1 1-c as a white solid. MS (m/z) M+H= 413.9

Step 3: 11-d

To a solution of intermediate 10-d (15.2 g, 108.0 mmol) in DMF cooled to 0°C were sequentially added cesium carbonate (42.4 g, 130.0 mmol) and 1-iodopropane (31.70 mL, 325.0 mmol) and the reaction was then stirred at room temperature overnight. Water and DCM were added, the organic layer was separated, the aqueous phase was extracted with DCM, the combined organic extracts were dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 1 1 -d as yellowish oil.

Step 4: 11-f

To a solution of 5-fluoro-2-iodoaniline 1 1-e (4.29 g, 18.1 1 mmol) in DMF, cooled to 0°C, were sequentially added diethylamine (2.50 mL, 24.0 mmol), copper (I) iodide ( 125 mg, 0.65 mmol) and dichlorobis(triphenylphosphine) palladium (II) (231 mg, 0.32 mmol). After complete dissolution of copper(I)iodide, propyl 3,3-dimethylhex-5-ynoate (3.0 g, 16.46 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature for 3 hours. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 1 1-f as a yellow oil. MS (m/z) M+H=292.2

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Step 5: 11-g

To a solution of intermediate 1 1-f (4.80 g, 16.47 mmol) in DCM cooled to 0°C were sequentially added TEA (4.59 mL, 32.9 mmol) and TFAA (3.49 mL, 24.71 mmol) and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 1 1-g as yellow oil. MS (m/z) M+H=388.3

Step 6: 11-h

To a solution of intermediate 1 1-g (458 mg, 1.18 mmol) in acetonitrile were added intermediate 1 1-c (443 mg, 1.07 mmol), CS2CO3 (700 mg, 2.14 mmol) and Pd(PPh ₃ ) ₄ (37 mg, 0.03 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 1 1-h as a yellow solid. MS (m/z) M+H= 563.4

Step 7: Compound 50

To a solution of intermediate 1 1-h (600 mg, 1.06 mmol) in DCM ( 1 mL) cooled to 0°C was added 2,2,2-trifluoroacetic acid ( 1 mL) and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 50-TFA as an off-white solid. MS (m/z) M+H= 463.4

Synthesis of compound 57

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Compound 57

Scheme 12

Step 1: 12-b

A solution of 4-bromo-2,6-difluorobenzonitrile 12-a (587 mg, 2.69 mmol), 4- aminotetrahydro-2H-thiopyran-l , l-dioxyde (500 mg, 2.69 mmol) and TEA (831 uL, 5.92 mmol) in DMSO was stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 12-b as a white solid.

Step 2: 12-c

To a solution of intermediate 12-b (950 mg, 2.74 mmol) in DMSO (15.0 mL) and MeOH (15 mL) were sequentially added NaOH IN (2.74 mL) and 30% aqueous hydrogen

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24987688_8.DOC Attorney Docket No. 47351 -0004 peroxide (419 uL, 4.10 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 12-c as a white solid.

Step 3: 12-d

To a solution of 2-iodoaniline 7-a (14.54 g, 66.4 mmol) in DMF cooled to 0°C were sequentially added diethylamine ( 12.61 mL, 121.0 mmol), copper(I) iodide (460 mg, 2.41 mmol) and dichlorobis(triphenylphosphine) palladium (II) (847 mg, 1.20 mmol). After complete dissolution of copper(I)iodide, propyl 3,3-dimethylhex-5-ynoate ( 1 1.0 g, 60.4 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 12-d as a yellow oil. MS (m/z) M+H=274.2

Step 4: 12-e

To a solution of intermediate 12-d (7.10 g, 26.0 mmol) in DCM cooled to 0°C were sequentially added DIPEA ( 18.14 mL, 104.0 mmol) and TFAA (8.07 mL, 51.1 mmol) and the reaction was then stirred at room temperature for 4 hours. Water and ethylacetate were added; the organic layer was separated, washed with 10% citric acid, saturated aqueous NaHC(¾ and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo.

Purification by silica gel chromatography provided intermediate 12-e as colorless oil. MS (m/z) M+H=370.5

Step 5: Compound 57

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To a solution of intermediate 12-e (223 mg, 0.60 mmol) in acetonitrile were added intermediate 12-c (200 mg, 0.54 mmol), CS ₂ CO ₃ (357 mg, 1.09 mmol) and Pd(PPh:,)4 (19 mg, 0.01 mmol) and the reaction was stirred at 1 10°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 57 as a yellow solid. MS (m/z) M+H= 498.4

Synthesis of compound 65

Compound 65

Scheme 13

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Step 1: 13-b

To a solution of (R)-5-methoxy-3-methyl-5-oxopentanoic acid (1.0 g, 6.24 mmol) in THF, cooled to - 15°C, was added a IN THF solution of borane-methyl sulfide complex (624 uL, 6.24 mmol) and the reaction was stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated aqueous NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 13-b as colorless oil.

Step 2: 13-c

To a solution of intermediate 13-b (900 mg, 6.16 mmol) in DCM cooled to 0°C were sequentially added DMSO (1.96 mL, 27.7 mmol), DIPEA (4.29 mL, 24.63 mmol) and pyridine-SO:, complex (2.94 g, 18.47 mmol) in DMSO (2 mL) and the reaction was stirred at 0°C for 2 hours. Diethyl ether and saturated aqueous ammonium chloride were added; the organic layer was separated, washed with IN HC1, saturated aqueous NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 13-c as colorless oil.

Step 3: 13-d

To a solution of intermediate 13-c (880 mg, 6.10 mmol) in MeOH cooled to 0°C were sequentially added dimethyl l-diazo-2-oxopropylphosphonate (2.34 g, 12.21 mmol) and K ₂ C0 ₃ (2.10 g, 15.26 mmol) and the reaction was stirred at room temperature overnight. Ether and saturated aqueous ammonium chloride were added; the organic layer was separated, washed with saturated aqueous NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 13-d as colorless oil.

Step 4: 13-e

To a solution of 2-iodoaniline 7-a (1.07 g, 4.89 mmol) in DMF cooled to 0°C were sequentially added diethylamine (561 uL, 5.38 mmol), copper(I) iodide (37 mg, 0.19 mmol)

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24987688_8.DOC Attorney Docket No. 47351 -0004 and intermediate 13-d (720 mg, 5.14 mmol). After complete dissolution of Copper (I) iodide, dichlorobis(triphenylphosphine) palladium (II) (69 mg, 0.09 mmol) was added and the reaction was then stirred at room temperature for 2 days. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 13-e as a yellow oil. MS (m/z) M+H=232.1

Step 5: 13-f

To a solution of intermediate 13-e (600 mg, 2.59 mmol) in DCM cooled to 0°C were sequentially added TEA (723 uL, 5.19 mmol) and TFAA (550 uL, 3.89 mmol) and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide

intermediate 13-f as yellow oil.

Step 6: Compound 65

To a solution of intermediate 13-f (274 mg, 0.83 mmol) in acetonitrile were added intermediate 8-c (250 mg, 0.79 mmol), CS2CO (520 mg, 1.59 mmol) and Pd(PPh:,) ₄ (28 mg, 0.02 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 65 as a yellow solid. MS (m/z) M+H= 432.4

Synthesis of compound 67

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Compound 67

Scheme 14

Step 1: 14-a

To a solution of compound 50-TFA (100 mg, 0.17 mmol) were sequentially added 1- (tert-butoxycarbonyl)azetine-3-carboxylic acid (70 mg, 0.34 mmol), HATU (99 mg, 0.26 mmol) and DIPEA (91 uL, 0.52 mmol) and the reaction was then stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 14-a as a yellow solid.

Step 2: Compound 67

To a solution of intermediate 14-a (85 mg, 0.13 mmol) in DCM ( 1 mL) was added 2,2,2-trifluoroacetic acid (1 mL) and the reaction was then stirred at room temperature overnight. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 67.TFA as a white solid. MS (m/z) M+H= 546.4

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Synthesis of compound 54

Compound 50 Compound 54

Scheme 15

To a solution of compound 50-TFA (100 mg, 0.17 mmol) in DCM were sequentially added TEA (100 uL, 0.72 mmol) and methanesulfonic anhydride (33 mg) and the reaction was stirred for 2 hours. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Diethyl ether was added to the residue and compound 54 was isolated by filtration. MS (m/z) M+H= 541.4

Synthesis of compound 44

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Compound 50 Compound 44

Scheme 16

To a solution of compound 50-TFA (200 mg, 0.34 mmol) in DCM were sequentially added butyric acid (61 mg, 0.69 mmol), DMAP (4 mg) and EDC (133 mg, 0.69 mmol) and the reaction was then stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 44 as a white solid. MS (m/z) M+H= 533.4

Synthesis of compound 72

Compound 50 Compound 72

Scheme 17

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To a solution of compound 50-TFA (100 mg, 0.17 mmol) in DCM were sequentially added 4-methylpiperazine- l-carbonyl chloride (69 mg, 0.35 mmol) and DIPEA ( 121 uL, 0.69 mmol) and the reaction was then stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. The residue was dissolved in IN HC1, ethyl acetate was added, the aqueous layer was separated and the PH was adjusted to 10 with aqueous NaHCO:,. Compound 72 was collected by filtration as a white solid. MS (m/z) M+H= 589.5

Synthesis of compound 23

Compound 13 18-a

Compound 23

Scheme 18

Step 1: 18-a

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To a solution of Boc-Gly-OH ( 1.99 g, 1 1.38 mmol) in DCM cooled to 0°C were sequentially added EDC (2.34 g, 12.25 mmol), DMAP (53 mg, 0.43 mmol) and compound 13 (3.9 g, 8.75 mmol) and the reaction was then stirred overnight at room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 18-a as an off white solid.

Step 2: Compound 23

To a solution of intermediate 18-a (2.0 g, 3.32 mmol) in DCM (7.70 mL) was added 2,2,2-trifluoroacetic acid (7.70 mL) and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 23-TFA as a white solid. MS (m/z) M+H= 503.5

A suspension of compound 23-TFA (2.05 g, 3.32 mmol) in ethyl acetate was washed twice with saturated aqueous NaHCO:, and brine. The organic layer was separated, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide compound 23 as a white solid. MS (m/z) M+H= 503.5

To a solution of compound 23 ( 1.67g, 3.32 mmol) in 1 ,4-dioxane was added 4N HC1 in 1,4-dioxane (2.49 mL, 9.97 mmol) and the solution was stirred for 10 minutes at room temperature. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 23-HC1 as a white solid. MS (m/z) M+H= 503.5

Synthesis of compound 60

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24987688_8.DOC

Scheme 19

Step 1: 19-b

To a solution of 4-fluoro-2-iodoaniline 19-a (1.50 g, 6.33 mmol) in DMF cooled to 0°C were sequentially added diethylamine (2.5 mL, 6.33 mmol), copper(I) iodide (241 mg, 1.26 mmol) and dichlorobis(triphenylphosphine) palladium (II) (444 mg, 0.63 mmol). After complete dissolution of copper(I)iodide, propyl 3,3-dimethylhex-5-ynoate ( 1.26 g, 6.96 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 19-b as a yellow oil. MS (m/z) M+H=292.4 Step 2: 19-c

To a solution of intermediate 19-b (1.84 g, 6.33 mmol) in DCM cooled to 0°C were sequentially added DIPEA (4.42 mL, 25.3 mmol) , DMAP (39 mg, 0.31 mmol) and TFAA (1.96 mL, 13.92 mmol) and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 19-c as a yellow oil.

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Step 3: Compound 60

To a solution of intermediate 19-c (2.0 g, 5.16 mmol) in acetonitrile were added intermediate 8-c ( 1.54 g, 4.92 mmol), Cs ₂ C0 ₃ (2.40 g, 7.38 mmol) and Pd(PPh ₃ ) ₄ (170 mg, 0.15 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 60 as a white solid. MS (m/z) M+H=464.4

Synthesis of Compound 15

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Compound 15

Scheme 20

Step 1: 20-a

To a solution of 2-iodoaniline 7-a (5.0 g, 22.83 mmol) in DMF cooled to 0°C were sequentially added diethylamine ( 1.0 mL, 35.7 mmol), copper(I) iodide (870 mg, 4.57 mmol) and dichlorobis(triphenylphosphine) palladium (II) (1.60 g, 2.28 mmol). After complete dissolution of copper(I)iodide, 5-hexyne-l-ol (2.80 mL, 25.1 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with 10% aqueous citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo.

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Purification by silica gel chromatography provided intermediate 20-a as a white solid. MS (m/z) M+H= 190.1

Step 2: 20-b

To a solution of intermediate 20-a (3.60 g, 19.0 mmol) in DCM cooled to 0°C were sequentially added DIPEA ( 13.29 mL, 76.0 mmol), DMAP ( 1 16 mg, 0.95 mmol) and TFAA (5.91 mL, 41.8 mmol) and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. The residue was dissolved in MeOH (20.0 mL), the solution was cooled to 0°C, IN LiOH in MeOH (0.5 mL) was added and the reaction was then stirred at room temperature for 4 hours. Water and ethyl acetate were added; the organic layer was separated, washed with 10% aqueous citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 20-b as a yellow oil. MS (m/z) M+H=286.6

Step 3: 20-c

To a solution of intermediate 20-b (3.63 g, 12.73 mmol) in DCM cooled to 0°C were sequentially added imidazole (1.04 g, 15.27 mmol) and fcrf-butylchlorodimethylsilane (2.1 1 g, 14.0 mmol) and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 20-c as a yellow oil.

Step 4: 20-d

To a solution of intermediate 20-c (2.50 g, 6.26 mmol) in acetonitrile were added intermediate 4-bromo-2-fluorobenzonitrile ( 1.87 g, 9.39 mmol), CS2CO3 (3.06 g, 9.39 mmol) and Pd(PPh:,) ₄ (362 mg, 0.31 mmol) and the reaction was stirred at 100°C overnight and then

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24987688_8.DOC Attorney Docket No. 47351 -0004 cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. The residue was dissolved in THF (20.0 mL), the solution was cooled to 0°C, IN tetrabutylammonium fluoride in THF (20 mL, 20 mmol) was added and the reaction was then stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with 10% aqueous citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 20-d as a white solid.

Step 5: 20-e

To a solution of intermediate 20-d (1.0 g, 3.24 mmol) in DCM cooled to 0°C were sequentially added DIPEA ( 1.69 mL, 9.73 mmol), DMAP (20 mg, 0.16 mmol) and methanesulfonyl chloride (278 uL, 3.57 mmol) and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 20-e as a white solid.

Step 6: 20-f

To a solution of intermediate 20-e (200 mg, 0.51 mmol) in DMF cooled to 0°C was added sodium hydride (20.70 mg, 0.51 mmol) and the reaction was then stirred at room temperature for 1 hour. Water was added and after stirring for 30 minutes intermediate 20-f was collected by filtration as a tan solid.

Step 7: 20-g

To a solution of intermediate 20-f ( 127 mg, 0.43 mmol) in DMSO were sequentially added DIPEA (382 uL, 2.18 mmol) and trans-4-aminocyclohexanol (202 mg, 1.75 mmol)

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24987688_8.DOC Attorney Docket No. 47351 -0004 and the mixture was then heated at 100°C for 18 hours and then cooled to room temperature. Water and ethyl acetate were added, the organic layer was separated, washed with saturated NaHC(¾ and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo.

Purification by silica gel chromatography provided intermediate 20-g as a white solid. MS (m/z) M+H=386.5

Step 8: Compound 15

To a solution of intermediate 20-g (40 mg, 0.10 mmol) in DMSO were sequentially added K ₂ CO ₃ (28.7 mg, 0.20 mmol) and 30% aqueous hydrogen peroxide (0.40 mL, 3.92 mmol) and the reaction was stirred at room temperature for 1 hour. Water and ethyl acetate were added; the organic layer was separated, washed with saturated ammonium chloride, 10% aqueous Na ₂ S ₂ 0 ₃ and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 15 as a beige solid. MS (m/z) M+H= 404.4

Synthesis of compound 80

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Compound 80

Scheme 21

Step 1: 21-b

To a solution of ( lr,4r)-4-aminocyclohexanecarboxylic acid (2.0 g, 13.97 mmol) in 2N NaOH ( 14 mL) was added dropwise benzyl chloroformate (2.12 mL, 14.88 mmol) and the reaction was then stirred at room temperature for 1.5 hours. The mixture was acidified to PH=3 with IN HCl and then diluted with water. Intermediate 21-b was collected by filtration as a white solid.

Step 2: 21-c

To a solution of intermediate 21-b (2.0 g, 7.21 mmol) in DMF were sequentially added tBuOH (2.76 mL, 28.8 mmol), DMAP (352 mg, 2.88 mmol) and 1 ,3- diisopropylcarbodiimide (2.48 mL, 15.87 mmol) and the reaction was stirred at room

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24987688_8.DOC Attorney Docket No. 47351 -0004 temperature for 4 days. Water and ethyl acetate were added; the organic layer was separated, washed with 10% citric acid, saturated NaHCO, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 21-c as a white solid.

Step 3: 21-d

To a solution of intermediate 21-c ( 1.2 g, 3.60 mmol) in MeOH and stirred under N ₂ was added 10% Pd/C (38 mg). The reaction mixture was purged with H ₂ and stirred for 2 hours. The reaction was then filtered through celite and the filtrate was concentrated in vacuo to provide intermediate 21-d as a white solid. MS (m/z) M+H= 200.0

Step 4: 21-f

To a suspension of intermediate 21-d (357 mg, 1.79 mmol) and 4-bromo-2,6- diflurobenzonotrile (391 mg, 1.79 mmol) in DMSO was added TEA (553 uL, 3.94 mmol) and the reaction was then stirred at room temperature for 2 days. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 21-f as a white solid.

Step 5: 21-g

To a solution of intermediate 21-f (700 mg, 1.76 mmol) in DMSO (7 mL) and MeOH (7 mL) were sequentially added NaOH IN ( 1.76 mL, 1.76 mmol) and 30% aqueous hydrogen peroxide (270 uL, 2.64 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 21-g as a white solid. MS (m/z) M+H= 416.8

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Step 6: Compound 80

To a solution of intermediate 1 1-g (585 mg, 1.51 mmol) in acetonitrile were added intermediate 21-g (570 mg, 1.37 mmol), Cs ₂ C0 ₃ (894 mg, 2.75 mmol) and Pd (PPh ₃ ) ₄ (48 mg, 0.04 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 80 as a yellow solid. MS (m/z) M+H= 566.4

Synthesis of compound 85

Compound 80 Compound 85

Scheme 22

To a solution of Compound 80 (23 mg, 0.04 mmol) in DCM (1 mL) was added 2,2,2- trifluoroacetic acid (1 mL) and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 85 as a white solid. MS (m/z) M+H= 510.3

Synthesis of compound 90

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Compound 90

Scheme 23

Step 1: 23-a

To a solution of Compound 85 (80 mg, 0.16 mmol) in DCM cooled to 0°C were sequentially added EDC (45 mg, 0.23 mmol), DMAP (1.9 mg, 0.01 mmol) and tert-butyl-2- aminoethyl(methyl)carbamate (55 mg, 0.31 mmol) and the reaction was then stirred overnight at room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel

chromatography provided intermediate 23-a as a white solid.

Step 2: Compound 90

To a solution of intermediate 23-a (60 mg, 0.09 mmol) in DCM ( 1 mL) was added 2,2,2-trifluoroacetic acid (1 mL) and the reaction was then stirred at room temperature for 1

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24987688_8.DOC Attorney Docket No. 47351 -0004 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 90-TFA as a white solid. MS (m/z) M+H= 566.4

Synthesis of compound 87

Compound 87

Scheme 24

Step 1: 24-a

To a solution of Boc-P-Ala-OH (93 mg, 0.49 mmol) in DMF cooled to 0°C were sequentially added HATU ( 188 mg, 0.49 mmol), DIPEA (314 uL, 1.79 mmol), DMAP (catalytic) and compound 13 (200 mg, 0.44 mmol) and the reaction was then stirred for 4 hours at room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered

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24987688_8.DOC Attorney Docket No. 4735 1 -0004 and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 24-a as an off white solid.

Step 2: Compound 87

To a solution of intermediate 24-a (200 mg, 0.32 mmol) in DCM (2 mL) was added 2,2,2-trifluoroacetic acid (2 mL) and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 87-TFA as a white solid. MS (m/z) M+H= 517.5

Synthesis of compound 111

25-a

Compound 87

Compound 1 1 1

Scheme 25

Step 1: 25-a

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To a solution of Boc-Lys(Boc)-OH (327 mg, 0.94 mmol) in DMF cooled to 0°C were sequentially added HOBt (19.2 mg, 0.12 mmol), HBTU (310 mg, 0.82 mmol) and DIPEA (220 uL, 1.25 mmol). After stirring for 10 minutes, compound 87-TFA (325 mg, 0.63 mmol) was added and the reaction was then stirred overnight at room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 25-a as an off white solid.

Step 2: Compound 111

To a solution of intermediate 25-a (516 mg, 0.61 mmol) in DCM (2 mL) was added

2,2,2-trifluoroacetic acid (2 mL) and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure and the residue was triturated with diethyl ether to provide compound 1 1 1 -2TFA as a white solid. MS (m/z) M+H= 645.5

Synthesis of compound 102

Compound 13 Compound 102

Scheme 26

To a solution of compound 13 (4.0 g, 8.98 mmol) in THF were sequentially added

DIPEA (4.70 mL, 26.9 mmol) and Ι , -carbonyldiimidazole (2.91 g, 17.96 mmol) and the reaction was stirred at 55°C for 3 hours. N,N-dimethylethylenediamine (4.90 mL, 44.9 mmol) was then added, the reaction was stirred at 55°C for 4 hours and at room temperature

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24987688_8.DOC Attorney Docket No. 47351 -0004 overnight. Water and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Methanol was added to the residue and compound 102 was isolated by filtration as a white solid. MS (m/z) M+H= 560.4

To a solution of compound 102 (3.27 g, 5.86 mmol) in CH ₂ CI ₂ cooled to 0°C was added IN HC1 in diethyl ether (15 mL) and the mixture was stirred for 5 minutes. Volatiles were removed in vacuo, diethyl ether was added to the residue and compound 102-HCl was collected by filtration as a white solid. MS (m/z) M+H= 560.4

Synthesis of compound 104

Compound 104

Scheme 27

To a solution of N,N-dimethylethylenediamine (22 mg, 0.25 mmol) in THF cooled to 0°C were sequentially added Ι , -carbonyldiimidazole (3 mg, 0.22 mmol) and DIPEA (9 uL, 0.52 mmol), the reaction was then stirred for 1 hour at 0°C and at room temperature overnight. Compound 50 (100 mg, 0.17 mmol) and DMAP (cat) were then added and the reaction was heated at reflux overnight and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and

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24987688_8.DOC Attorney Docket No. 47351 -0004 concentrated in vacuo. Purification by silica gel chromatography provided compound 104 as an off-white solid. MS (m/z) M+H=577.5

Synthesis of compound 105

Compound 85 Compound 105

Scheme 28

To a solution of compound 85 (415 mg, 0.81 mmol) in DMF were sequentially added HATU (310 mg, 0.81 mmol), and 2-morpholinoethanamine (212 mg, 1.62 mmol), HOAT (136 μί, 0.81 mmol) ant TEA (454 μί, 3.26 mmol) and the reaction was then stirred at room for 30 minutes. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide compound 105 as a white solid. MS (m/z) M+H=622.4

To a solution of compound 105 (503 mg, 0.80 mmol) in CH ₂ CI ₂ cooled to 0°C was added IN HC1 in diethyl ether (5 mL) and the mixture was stirred for 5 minutes. Volatiles were removed in vacuo, diethyl ether was added to the residue and compound 105 -HQ was collected by filtration as a white solid. MS (m/z) M+H= 622.4

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Synthesis of compound 112

Compound 1 12

Scheme 29

Step 1: 29-a

To a solution of intermediate 1 1 -a (1.65 g, 6.66 mmol) in DMSO were sequentially added TEA (2.80 mL, 19.97 mmol) and 4-aminothiomorpholine 1, 1 -dioxide ( 1.0 g, 6.66 mmol) and the mixture was then heated at 130°C for 7 days and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 29-a as a beige solid.

Step 2: 29-b

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To a solution of intermediate 29-a (1.10 g, 2.92 mmol) in DMSO (15.0 mL) and MeOH (15 mL) were sequentially added NaOH IN (2.92 mL, 2.92 mmol) and 30% aqueous hydrogen peroxide (447 μί, 4.37 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 29-b as a white solid. MS (m/z) M+H= 321.5

Step 3: Compound 112

To a solution of intermediate 1 1-g (196 mg, 0.51 mmol) in acetonitrile were added intermediate 29-b (200 mg, 0.51 mmol), Cs ₂ C0 ₃ (330 mg, 1.0 mmol) and Pd (PPh ₃ ) ₄ (29 mg, 0.02 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 1 12 as an off-white solid. MS (m/z) M+H= 499.4

Synthesis of compound 143

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Compound 143

Scheme 30

Step 1: 30-b

A solution of intermediate 4-bromo-2-fluorobenzonitrile 30-a (25.0 g, 125.0 mmol), trans-4-aminocyclohexanol ( 14.4 g, 125.0 mmol) and TEA (52.3 mL, 375.0 mmol) in DMSO was heated at 150 °C overnight and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Diethyl ether and hexane were added to the residue and intermediate 30-b was collected by filtration as an off-white solid.

Step 2: 30-c

To a solution of intermediate 30-b (31.4 g, 106.0 mmol) in DMSO/MeOH ( 1 : 1 , 304 mL) was sequentially added NaOH IN (106.0 mL, 106.0 mmol) and 30% aqueous hydrogen

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24987688_8.DOC Attorney Docket No. 47351 -0004 peroxide ( 16.30 mL, 160.0 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 30-c an off-white solid. Step 3: 30-e

To a solution of 2-iodo-4-methoxyaniline 30-d ( 1.00 g, 4.02 mmol) in DMF cooled to 0°C were sequentially added diethylamine (419 uL, 4.02 mmol), copper(I) iodide (143 mg, 0.80 mmol) and dichlorobis(triphenylphosphine) palladium (II) (282 mg, 0.40 mmol). After complete dissolution of copper(I)iodide, propyl 3,3-dimethylhex-5-ynoate 1 1-d (805 mg, 4.42 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 30-e as a beige oil. MS (m/z) M+H=304.4 Step 4: 30-f

To a solution of intermediate 30-e (1.21 g, 4.0 mmol) in DCM cooled to 0°C were sequentially added DIPEA (2.80 mL, 16.06 mmol) , DMAP (25 mg, 0.20 mmol) and TFAA (1.24 mL, 8.83 mmol) and the reaction was then stirred at room temperature for 4 hours. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, saturated aqueous NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 30-f as a beige solid.

Step 5: Compound 143

To a solution of intermediate 30-f ( 1.10 g, 2.75 mmol) in acetonitrile were added intermediate 30-c (821 mg, 2.62 mmol), Cs ₂ C0 ₃ (1.28 g, 3.93 mmol) and Pd(PPh ₃ ) ₄ (91 mg, 0.08 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethylacetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and

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24987688_8.DOC Attorney Docket No. 47351 -0004 concentrated in vacuo. Purification by silica gel chromatography provided compound 143 as a white solid. MS (m/z) M+H=476.5

Synthesis of compound 145

Compound 145

Scheme 31

Step 1: 31-b

A solution of bromine (1.82 mL, 35.4 mmol) in DCM was added dropwise to a suspension of 3-fluoro-4-methoxyaniline (5.0 g, 35.4 mmol) and potassium carbonate (5.14 g, 37.2 mmol) in DCM at -15°C and the reaction was then stirred at -15°C for 30 minutes.

Water was added, the organic layer was separated, the aqueous phase was extracted with DCM, the combined organic extracts were washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 31-b as an orange solid. MS (m/z) M+H=221.7

Step 2: 31-c

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To a solution of intermediate 31-b (4.0 g, 18.18 mmol) in DMF cooled to 0°C were sequentially added TEA (36.4 mL), copper(I) iodide (143 mg, 0.80 mmol) and

dichlorobis(triphenylphosphine) palladium (II) (282 mg, 0.40 mmol). After complete dissolution of copper(I)iodide, propyl 3,3-dimethylhex-5-ynoate 1 1-d (3.64 g, 20.0 mmol) was added dropwise at 0°C and the reaction was then stirred at room temperature overnight. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 31-c as a yellow oil. MS (m/z) M+H=322.3 Step 3: 31-d

To a solution of intermediate 31-c (2.60 g, 8.09 mmol) in DCM cooled to 0°C were sequentially added DIPEA (5.65 mL, 32.4 mmol) , DMAP (49 mg, 0.40 mmol) and TFAA (2.51 mL, 17.80 mmol) and the reaction was then stirred at room temperature for 2 hours. Water and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, saturated aqueous NaHCO:, and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 31-d as a yellow oil.

Step 5: Compound 143

To a solution of intermediate 31-d (1.00 g, 2.39 mmol) in acetonitrile were added intermediate 30-c (715 mg, 2.28 mmol), Cs ₂ C0 ₃ (1.1 1 g, 3.42 mmol) and Pd(PPh ₃ ) ₄ (79 mg, 0.07 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethylacetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 145 as a white solid. MS (m/z) M+H=494.4

Synthesis of compound 94

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Compound 94

Scheme 32

Step 1: 32-b

A solution of intermediate 4-bromo-2-fluorobenzonitrile 30-a (4.89 g, 24.46 mmol), (s)-3-aminotetrahydrofuran tosylate (6.0 g, 24.46 mmol) and DIPEA (17.09 mL, 98.0 mmol) in DMSO was heated at 100 °C overnight and then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided intermediate 32-b as an off-white solid.

Step 2: 32-c

To a solution of intermediate 32-b (4.70 g, 17.59 mmol) in DMSO/MeOH ( 1 : 1 , 352 mL) was sequentially added NaOH IN (17.59 mL, 17.59 mmol) and 30% aqueous hydrogen peroxide ( 1.70 mL, 26.4 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was

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24987688_8.DOC Attorney Docket No. 47351 -0004 separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo to provide intermediate 32-c as an off-white solid.

Step 3: Compound 94

To a solution of intermediate 1 1-g (3.0 g, 7.74 mmol) in acetonitrile were added intermediate 32-c (2.10 mg, 7.38 mmol), Cs ₂ C0 (4.81 g, 14.75 mmol) and Pd(PPh ₃ ) ₄ (25 mg, 0.22 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethylacetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 94 as a pale yellow solid. MS (m/z) M+H=436.8

Synthesis of compound 144

Compound 144

Scheme 33

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24987688_8.DOC Attorney Docket No. 47351 -0004

Step 1: 33-b

A solution of intermediate 4-bromo-2,6-difluorobenzonitrile 33-a (5.04 g, 23.14 mmol), (s)-3-aminotetrahydrofuran tosylate (6.0 g, 23.14 mmol) and DIPEA (12.12 mL, 69.4 mmol) in DMSO was stirred at room temperature for 2 days. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride and brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Hexane was added and intermediate 33-b was collected by filtration as pale yellow solid.

Step 2: 33-c

To a solution of intermediate 33-b (1.20 g, 4.21 mmol) in DMSO/MeOH ( 1 : 1 , 84.2 mL) was sequentially added NaOH IN (4.21 mL, 4.21 mmol) and 30% aqueous hydrogen peroxide (645 uL, 6.31 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed with brine, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Hexane was added and intermediate 33-b was collected by filtration as an off-white solid.

Step 3: Compound 144

To a solution of intermediate 1 1-g (671 mg, 1.73 mmol) in acetonitrile were added intermediate 33-c (500 mg, 1.64 mmol), CS2CO (1.0 g, 3.30 mmol) and Pd(PPh:,) ₄ (57 mg, 0.049 mmol) and the reaction was stirred at 120°C overnight and then cooled to room temperature. Water and ethylacetate were added; the organic layer was separated, washed with saturated aqueous ammonium chloride, dried over anhydrous MgS0 ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography provided compound 144 as an off-white solid. MS (m/z) M+H=454.2

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Representative compounds of the instant inventions are listed below:

TABLE 2

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-98-

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Other compounds of the instant invention include the following:

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Fluorescence polarization HSP90 binding assay

Fluorescence polarization based HSP90 binding assay was performed using modifications to previously described methods using full length HSP90 and a geldanamycin- FITC probe (see Llauger-Bufi, L. et al., Bioorg. Med. Chem. Lett. 13 (2003) 3975-3978).

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Briefly, geldanamycin-FITC probe was diluted into HFB buffer [20 mM HEPES (K) pH 7.3, 50 mM KC1, 1 mM DTT, 5 mM MgCl ₂ , 20 mM Na ₂ Mo0 ₄ , 0.01 % NP40, 0.1 mg/mL of Bovine gamma-globuline] to obtain a working concentration of 8 nM. HSP90 protein (Hsp90 Native Protein, Stressgen, SPP-770) was diluted in order to obtain a 4X stock protein solution into HFB buffer. The final amount of protein used in the assay corresponds to the amount of protein necessary to obtain 80% of the maximum FP value in a 2 nM probe saturation experiment. Assay were carried out in duplicates, into not treated black 96-well plate (Corning #3915), in a total volume of 100 μΐ, for a final concentration of 2 nM of geldanamycin-FITC probe, various concentrations of compound and Hsp-90 protein into HFB buffer. Buffer only (blank) or probe only in buffer (G-factor) were also added to be used as controls for calibration. The plate was left on a shaker at 4 °C for 3 hours and the FP values in mP were recorded using Genios Pro FP reader (TECAN). The measured FP values (mP) were then plotted against compound concentration and EC ₅₀ , corresponding to the competitor concentrations where 50% of the tracer was displaced, calculated based on a sigomoidal dose-response (variable-slope) curve fit using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com.

Several representative compounds of the invention are listed below and their respective magnitude of potency is provided.

IC ₅₀ A: less than 100 nM; B between 100 and 1000 nM; C greater than 1000 nM.

Compound ic ₅₀

FP assay (nM)

1 B

2 -

3 -

4 B

5 -

6 -

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7 -

8 -

9 -

10 c

11 -

12 -

13 B

14 -

15 -

16 B

17 B

18 B

19 B

20 -

21 C

22 B

23 A

24 B

25 C

26 B

27 C

28 B

29 C

30 B

31 B

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32 B

33 B

34 B

35 B

36 B

37 B

39 C

40 -

41 B

42 A

43 -

44 B

45 A

46 -

47 A

48 B

49 B

50 B

51 A

52 A

53 -

54 B

55 -

56 B

57 A

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58 A

59 A

62 B

63 -

64 B

65 B

66 B

67 A

69 -

70 A

71 B

72 A

73 -

74 B

75 B

76 B

77 B

78 B

79 B

80 -

81 B

82 A

83 B

84 B

85 B

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86 B

87 A

88 A

89 A

90 A

91 -

92 A

93 B

94 B

95 A

96 -

97 A

98 B

99 A

101 B

102 A

103 A

104 A

105 A

106 B

107 B

108 B

109 A

110 B

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112 A

113 B

114 B

115 A

116 A

117 A

118 B

119 A

120 C

121 A

122 B

123 A

124 A

125 A

126 A

127 A

128 A

129 B

130 A

131 B

132 A

133 B

134 A

135 B

136 B Attorney Docket No. 47351 -0004

Anti-Fungal Activities of HSP90 Inhibitors

Anti-fungal activity was determined in vitro by incubation of Candida albicans cultures with serial two fold dilutions of HSP90 inhibitors alone or in combination with serial two fold dilutions of fluconazole or caspofungin. Fungal growth was measured by absorbance at 600 nM (Figure 1).

Fungicidal activity was demonstrated by plating cells from the above liquid cultures on solid media in the absence of HSP90 inhibitor, fluconazole or caspofugin and allowing fungal growth to occur before photography (Figure 2).

Figure 1 shows that HSP90 inhibitors block growth of Candida albicans and synergize with Fluconazole and Caspofungin. Candida albicans cultures were treated with 2 fold dilutions of compound 34 or compound 1 17 in combination with 2 fold dilutions of either fluconazole or caspofungin. Cell growth was determined by light absorbance.

Figure 2 shows that HSP90 inhibitors are fungocidal alone and in combination with

Fluconazole and Caspofungin. On the left side, Candida albicans cultures were treated with 2 fold dilutions of compound 1 17 or compound 34 in the absence of drug or in the presence

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24987688_8.DOC Attorney Docket No. 47351 -0004 of fixed concentrations of Fluconazole or Caspofungin. Fungal growth was determined by light absorption. On the right, culture aliquots were then plated on solid drug and compound free media and allowed to grow before photography.

Data indicate that Compound 34 and compound 1 17 blocked fungal growth as single agents (Figure 1). Both compounds were synergistic when applied in combination with either

Fluconazole or Caspofungin. Compound 34 was fungocidal as demonstrated by failure of treated cells to grow on compound free media (Figure 2 upper right). The combination of either compound 34 or compound 1 17 with Fluconazole or Caspofungin was fungocidal (Figure 2 middle and bottom right).

These data show that compounds of the instant invention are fungostatic and fungocidal and that they synergize with anti-fungal drugs to increase their potency.

Pharmacokinetics (PK) of Compound in Plasma

The PK of compounds of formula 1 , such as compounds 33, 45 and 102, were determined in the plasma of mice after treating CD-I mice with compound via IV or oral administration. Compound concentrations were determined using acetonitrile extraction of compound from the appropriate matrix and calculation of drug concentrations using a spiked standard curve from the same matrix (ie. plasma).

Compounds of formula 1 demonstrated good oral absorption (10-50 % F) and exposure in plasma which can exceed the concentration required to have anti-fungal activity. These results indicate that compounds of formula 1 are orally available and can cross the blood brain barrier. Therefore compounds of formula 1 may be useful in the treatment of fungal infections in a variety of compartments.

Figure 3 shows the plasma levels of compound 34 in mice dosed with 100 mg/kg compound 34 formulated in cremophor ELP by the oral route. Data indicate that oral administration to mice can deliver active concentrations of compound to plasma. Following oral dosing exposure of compound 34 was in excess of 2 uM which is above the

concentration required to affect fungal growth in vitro for at least 8 hours.

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24987688_8.DOC Attorney Docket No. 47351 -0004

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

All of the above-cited references and publications are hereby incorporated by reference.

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