MACROCYCLIC GRB2 SH2 DOMAIN-BINDING INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/867,307, filed November 27, 2006, which is incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The pharmaceutical industry is in search for new classes of compounds for the therapy and prophylaxis of proliferative diseases such as cancer, autoimmune diseases, and hyperproliferative skin disorders such as psoriasis. These diseases or disorders affect a large portion of the population, leading to suffering and possibly death.

[0003] Some of these diseases or disorders may involve signal transduction. Signal transduction is critical to normal cellular homeostasis and is the process of relaying extracellular messages, e.g., chemical messages in the form of growth factors, hormones and neurotransmitters, via receptors, e.g., cell-surface receptors, to the interior of the cell. Protein-tyrosine kinases play a central role in this biological function. Among others, these enzymes catalyze the phosphorylation of specific tyrosine residues to form tyrosine phosphorylated residues.

[0004] Protein-tyrosine phosphorylation is known to be involved in modulating the activity of some target enzymes as well as in generating specific complex networks involved in signal transduction via various proteins containing a specific amino acid sequence called an Src homology region or SH2 domain (see, e.g., Proc. Natl. Acad. Sd. USA, 90, 5891 (1990)). A malfunction in this protein-tyrosine phosphorylation through tyrosine kinase overexpression or deregulation is manifested by various oncogenic and (hyper-) proliferative disorders such as cancer, inflammation, autoimmune disease, hyper-proliferative skin disorders, such as psoriasis, and allergy/asthma. SH2- and/or SH3- comprising proteins that play a role in cellular signaling and transformation include, but are not limited to, the following: Src, Lck, Eps, ras GTPase-activating protein (GAP), phospholipase C, phosphoinositol-3 (PI-3) kinase, Fyn, Lyk, Fgr, Fes, ZAP-70, Sem-5, p85, SHPTPl, SHPTP2, corkscrew, Syk, Lyn, Yes, Hck, Dsrc, Tec, Atk/Bpk, Itk/Tsk, Arg, Csk, tensin, Vav, Emt, Grb2, BCR-AbI, She, Nek, Crk, CrkL, Syp, BIk, 113TF, 91TF, Tyk2, especially

Src, phospholipase c, phoshoinositol-3 (PI-3) kinase, Grb2, BCR-AbI, She, Nek, Crk, CrkL, Syp, BIk, 113TF, 91TF, and Tyk2. A direct link has been established between activated receptor kinases and Ras with the finding that the mammalian Grb2 protein, a 26 kilo Dalton (kD) protein comprising a single SH2 and two SH3 domains, bind to proline-rich sequences present in the Sos exchange factor.

[0005] The foregoing shows that there exists a need for molecules that have an ability to mimic the structure of the phosphotyrosine peptide binding site, as well as a need for compounds that have the ability to disrupt the interaction between SH2 domains of proteins (e.g., regulatory proteins) for example that of Grb2, and proteins with phosphorylated moieties. There further exists a need for compounds suitable for use in the therapy or prophylaxis of proliferative diseases or conditions. The present invention provides such compounds.

[0006] These and other advantages of the present invention will be apparent from the description as set forth below.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides macrocyclic compounds that inhibit the binding of a phosphoprotein with a Grb2 SH2 domain-containing protein. Thus, the invention provides macrocyclic compounds of formula (I):

wherein R and R are the same and are hydrogen or aryl which is optionally substituted; R ² , in combination with the phenyl ring, is a phenylphosphate mimic group or a protected phenylphosphate mimic group; R ³ is hydrogen, azido, amino, oxalylamino, carboxy C ₁ -Ce alkyl, Ci-C ₆ alkoxycarbonyl Ci-Cβ alkyl, aminocarbonyl Cj-C ₆ alkyl, or Ci-C ₆ alkyl carbonylamino; wherein the alkyl portion of R ³ is optionally substituted; R ⁶ is a linker; AA is an amino acid; m is 1 to 6; and n is 1 to 6; or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof.

[0008] The invention further provides pharmaceutical compositions and methods of use of such compounds, for example, in the treatment of cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] Figure 1 illustrates a method of preparing achiral alkenyl amines 9a and 9f, which are intermediates in the synthesis of compounds in accordance with an embodiment of the invention. Reagents and conditions: (i) CH ₃ SO ₂ Cl, NEt ₃ , CH ₂ Cl ₂ , room temp, 3 h; then NaN ₃ , DMF-H ₂ O, 50 ⁰ C, overnight; (ii) LiAlH ₄ , Et ₂ O, 0 ⁰ C, 1 h then (Boc) ₂ O, Et ₂ O - H ₂ O, room temp, overnight; (iii) CF ₃ CO ₂ H, Et ₃ SiH, CH ₂ Cl ₂ , room temp, 2 h; (iv) NaH, DMF 0 ⁰ C to room temp, overnight; (vii) LiAlH ₄ , Et ₂ O.

[0010] Figure 2 illustrates a method of preparing N-Boc protected asparagine alkenylamides 10a and 1Of, which are intermediates in the synthesis of compounds in accordance with an embodiment of the invention. Reagents and conditions: (i) 4- methylmorpholine, Z-BuOC(O)Cl, DMF, 0 ⁰ C 10 min then (ii) ra-Boc-L-Asn(Trt), room temperature, overnight.

[0011] Figure 3 illustrates a method of preparing compound 18, which is an intermediate in the synthesis of compounds in accordance with an embodiment of the invention. Reagents and conditions: (i) AUyI bromide, K ₂ CO ₃ , DMF, room temp, 2 days; (ii) CF ₃ CO ₂ H, Et ₃ Si, CH ₂ Cl ₂ , room temp, 2 h; (iii) HOAt, EDCI, NEt(Z-Pr) ₂ , DMF, room temp, 2 days; (iv) Pd(PPh ₃ ) ₄ , morpholine, THF, room temp, 30 min.

[0012] Figure 4 illustrates a method of preparing compounds of formula (Ilia) and (HIb) (5a and 5b, respectively) in accordance with an embodiment of the invention. Reagents and conditions: (i) HOAt, EDCI, NEt(Z-Pr) ₂ , DMF, room temp, 2 days; (ii) 1,2-dichloroethane, reflux, 2 days; (iii) CF ₃ CO ₂ H, Et ₃ Si, CH ₂ Cl ₂ , room temp, 2 h.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention provides macrocyclic compounds of formula (I):

wherein R ¹ and R ¹ are the same and are hydrogen or aryl which is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-C ₆ alkyl, Ci-C ₆ alkyl, and Ci-C ₆ alkoxy; R ² , in combination with the phenyl ring, is a phenylphosphate mimic group or a protected phenylphosphate mimic group; R ³ is hydrogen, azido, amino, oxalylamino, carboxy Ci-C ₆ alkyl, Ci-C ₆ alkoxycarbonyl Ci-C ₆ alkyl, aminocarbonyl Ci-C ₆ alkyl, or Ci-C ₆ alkyl carbonylamino; wherein the alkyl portion of R ³ is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-C ₆ alkyl, C]-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl; R is a linker; AA is an amino acid; m is 1 to 6; and n is 1 to 6; or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof.

[0014] In accordance with embodiments of the invention, the macrocyclic compounds of formula (I) can be of a suitable ring size. In preferred embodiments, m is from 1 to 4, more preferably, m is 1 or 2, and in a particular embodiment, m is 1.

[0015] In any of the embodiments of the invention, n is from 2 to 4, more preferably, n is 2 or 3, and in particularly, n is 2.

[0016] In accordance with the invention, the amino acids of (AA) _n are incorporated, to arrive at the compounds of formula I, such that the N- and C-termini of (AA) _n form amide linkages. For example, as depicted in Figure 4, n is 2 and the amino acids of AA are α-aminocyclohexane carboxylic acid and asparagine, wherein each of the amino and carboxyl groups of the N- and C-termini of AA have been reacted to form amide linkages.

[0017] In accordance with the invention, the amino acid or amino acids of (AA) _n can be any suitable amino acids, natural or synthetic. For example, the amino acids can be selected from the group consisting of glycine, alanine, valine, norvaline, leucine, iso-leucine, norleucine, α-amino n-decanoic acid, serine, homoserine, threonine, methionine, cysteine, S-acetylaminomethyl-cysteine, proline, trans-3- and trans-4-hydroxyproline, phenylalanine,

tyrosine, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, tryptophan, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aspartic acid, asparagine, aminomalonic acid, aminomalonic acid monoamide, glutamic acid, glutamine, histidine, arginine, lysine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid and α,β-diaminopropionic acid, homophenylalanine, and α-ter^-butylglycine, and any combination thereof, and in any sequence. In a preferred embodiment, the amino acids are selected from the group consisting of asparagine and α-aminocyclohexane carboxylic acid.

[0018] In accordance with the invention, R ¹ and R ¹ are the same and can be either hydrogen or aryl. Thus, the carbon atom to which R ¹ and R ¹ are attached is achiral. Further, R ¹ and R ¹ can be introduced, to prepare compounds of the invention, using any suitable method such as, for example, using achiral alkenyl amines as depicted in Figures 1-4.

[0019] In accordance with the invention, R and R of formula (I) can be any suitable aryl group. Aryl groups are well-known to the skilled artisan and include, for example, phenyl, naphthyl, anthracenyl, fluorenyl, biphenyl, dihydronaphthyl, tetrahydronaphthyl, heteroaryl, and the like. Heteroaryl groups are well-known to the skilled artisan and include, for example, pyrrolyl, furanyl, pyrenyl, thienyl, pyridyl, pyrazinyl, pyrazolyl, imidazolyl, pyradazinyl, pyrimidinyl, triazinyl, pyranyl, thiazolyl, isothiazolyl, pteridinyl, piperonyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, and the like. The aryl and heterocyclyl moieties may be fused, such as, e.g., indole, isoindole, benzimidazole, quinoline, isoquinolinyl, benzoxazolyl, benzofuranyl, isobenzofuranyl, carbazolyl, benzodioxolyl, and the like.

[0020] In certain embodiments, R ¹ and R ¹ are aryl, which is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-Ce alkyl, Ci-C ₆ alkyl, and Ci-C ₆ alkoxy. The term "halo" refers to any suitable halogen atom, including, for example, fluoro, chloro, bromo, iodo, and any combinations thereof. When the aryl groups are substituted, the substituent can be located at any suitable position

which is available for substitution, for example, in phenyl, the phenyl ring can be substituted at, for example, the 2, 3, 4, and/or 5 positions. In some embodiments, the aryl groups may be substituted by more than one substituent as appropriate.

[0021] In accordance with the invention, R ² in formula (I), in combination with the phenyl ring, is a phenylphosphate mimic group or protected phenylphosphate mimic group. A phenylphosphate mimic group can be one that has the functional property of the phosphorylated side chain of tyrosine-phosphorylated sequences, e.g., it can replicate the interaction of phenylphosphate side chain with proteins. The interaction may involve any number of mechanisms, including geometry, size, and/or charge. A protected phenylphosphate mimetic is a phenylphosphate mimic that contains a protecting group that releases the mimetic, e.g., in a biological environment, such as due to chemical or enzymatic hydrolysis. In embodiments, the protecting groups can be esters or amides.

[0022] In embodiments, R ² can be hydroxyl, carboxyl, formyl, carboxy Ci-C ₆ alkyl, carboxy Ci-C ₆ alkoxy, dicarboxy Ci-C ₆ alkyl, dicarboxy C]-C ₆ alkyloxy, dicarboxyhalo Ci-C ₆ alkyl, dicarboxyhalo Ci-C ₆ alkyloxy, phosphono, phosphono Cj-C ₆ alkyl, phosphonohalo Ci-C ₆ alkyl, phosphoryl, phosphoryl Ci-C ₆ alkyl, and phosphoryl Ci-C ₆ alkoxy, carboxy Ci-C ₆ alkylamino, oxalylamino, C ₆ -Ci ₄ aryl Ci-C ₆ alkyl, phosphino Cj-C ₆ alkyl, Ci-C ₆ alkyl phosphino C]-C ₆ alkyl, C ₆ -Ci ₄ aryl, or RSO ₂ NH- wherein R can be Ci-C ₆ alkyl, halo C]-C ₆ alkyl, C ₆ -Ci ₄ aryl, C ₆ -Ci ₄ aryl Ci-C ₆ alkyl, or trifluoro Ci-C ₆ alkyl, wherein the alkyl or alkoxy portion of R ² is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl. In a preferred embodiment, R ² is dicarboxy Ci-C ₆ alkyl, e.g., dicarboxymethyl. In another preferred embodiment R ² is phosphono Ci-C ₆ alkyl, e.g., phosphonomethyl.

[0023] In accordance with the invention, R ³ of formula (I) can be hydrogen, azido, amino, oxalylamino, carboxy Ci-C ₆ alkyl, Ci-C ₆ alkoxycarbonyl Ci-C ₆ alkyl, aminocarbonyl Ci-C ₆ alkyl, or Ci-C ₆ alkyl carbonylamino; wherein the alkyl portion of R ³ is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-C ₆ alkyl, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl. Preferably, R ³ is carboxy Ci-C ₆ alkyl, e.g., carboxy methyl.

[0024] In accordance with an embodiment of formula (I), the invention provides compounds of formula (II) or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof:

[0025] wherein R ¹ and R ¹ are the same and are hydrogen or aryl which is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-C ₆ alkyl, Ci-C ₆ alkyl, and Ci-C ₆ alkoxy; R ² , in combination with the phenyl ring, is a phenylphosphate mimic group or a protected phenylphosphate mimic group; R ³ is hydrogen, azido, amino, oxalylamino, carboxy Ci-C ₆ alkyl, Ci-C ₆ alkoxycarbonyl Ci-C ₆ alkyl, aminocarbonyl C]-C ₆ alkyl, or Ci-C ₆ alkyl carbonylamino; wherein the alkyl portion of R ³ is optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, amino Ci-C ₆ alkyl, Cj-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl; R ⁴ and R ⁵ , independently, are hydrogen, CpC ₆ alkyl, C ₄ -C ₈ cycloalkyl, or heterocyclyl, or R ⁴ and R ⁵ together form a C ₄ -C ₈ cycloalkyl or heterocyclyl; R ⁶ is a linker; and m is 1 to 6.

[0026] R and R , independently, can be hydrogen, Ci-C ₆ alkyl, C ₄ -C ₈ cycloalkyl, or heterocyclyl, or R ⁴ and R ⁵ together can form a C ₄ -C ₈ cycloalkyl or heterocyclyl. Preferably, heterocyclyl is heterocycloalkyl. Preferably, R and R together form a C ₄ -C ₈ cycloalkyl, e.g., a cyclohexyl group.

[0027] In accordance with the invention, R ⁶ is a linker. The linker R ⁶ connects the benzylic carbon of the phenylphosphate mimic or protected phenylphosphate mimic group (i.e., the carbon atom of the macrocycle to which the R ² -phenyl group is attached) to the carbon atom bearing R ¹ and R ¹ . The bond connecting the linker to the linking sites can have any suitable configuration (R, S, or R/S). In a preferred embodiment, the linking site at R ¹ and R ¹ has a R/S configuration.

[0028] In accordance with the invention the linker R ⁶ can be a bond or a group having 1-6 carbon atoms and can be optionally substituted with a substituent selected from the group

consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl. Preferably, R ⁶ is a C ₂ -CO alkenylenyl or C ₂ -C ₆ alkynylenyl group, optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl. In a more preferred embodiment, R ⁶ is a C ₂ -C ₆ alkenylenyl. In a further preferred embodiment, R ⁶ is propenylenyl.

[0029] When R ¹ and R ^1' are hydrogen, R ⁶ can be a C ₂ -C ₆ alkenylenyl or C ₂ -C ₆ alkynylenyl group, preferably C ₃ alkenylenyl or C ₃ alkynylenyl. R ⁶ can be optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, C]-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl. In a particular embodiment, m can be 1 or 2.

[0030] When R ¹ and R ¹ are hydrogen and m is 1 , R ⁶ can be C ₃ alkenylenyl or

C ₃ alkynylenyl optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, Cj-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl.

[0031] When R and R ¹ are hydrogen and m is 2, R can be C ₃ alkenylenyl or

C ₃ alkynylenyl optionally substituted with a substituent selected from the group consisting of halo, hydroxyl, carboxyl, amino, aminoalkyl, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, and formyl.

[0032] In certain embodiments, the present invention provides compounds of formula (II), wherein R ¹ and R ¹ are hydrogen, R ² is dicarboxymethyl, R ³ is carboxy methyl, R ⁴ and R ⁵ together form a cyclohexyl, R is propenylenyl, and m is 1.

[0033] In certain embodiments, the present invention provides compounds of formula (II), wherein R ¹ and R ¹ are hydrogen, R ² is phosphonomethyl, R ³ is carboxy methyl, R ⁴ and R ⁵ together form a cyclohexyl, R ⁶ is propenylenyl, and m is 1.

[0034] In certain embodiments, the present invention provides compounds of formula (II), wherein R ¹ and R ¹ are phenyl, R ² is dicarboxymethyl, R ³ is carboxy methyl, R ⁴ and R ⁵ together form a cyclohexyl, R ⁶ is propenylenyl, and m is 1.

[0035] In certain embodiments, the present invention provides compounds of formula (II), wherein R ¹ and R ¹ are phenyl, R ² is phosphonomethyl, R ³ is carboxy methyl, R ⁴ and R ⁵ together form a cyclohexyl, R ⁶ is propenylenyl, and m is 1.

[0036] In a specific embodiment, the invention provides a compound of formula (Ilia):

[0037] In yet another specific embodiment, the invention provides a compound of formula (HIb):

[0038] In all of the embodiments, the present invention also provides pharmaceutically acceptable salts, stereoisomers, solvates, or hydrates of the inventive compounds, as appropriate, including alkali or amine salts. Suitable pharmaceutically acceptable salts, stereoisomers, solvates, or hydrates of the inventive compounds are known to the skilled artisan. For example, the acidic groups, e.g., carboxylic, phosphoric, or phosphonic groups, of the compound can be converted to salts known to those skilled in the art, for example, a salt of an alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium), or ammonium salt. Other examples of pharmaceutically-acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, />-toluenesulphonic acids, and arylsulphanic, for example.

[0039] Solvates and hydrates of the present invention can be prepared using any suitable technique known in the art, for example, by crystallizing compounds of the invention in the presence of a suitable solvent, using conditions such that a solvate is formed. Similarly, hydrates of the present invention can be prepared, for example, by crystallizing compounds of the invention in the presence of water using conditions such that a hydrate is formed.

[0040] The present invention further provides compositions comprising a pharmaceutically acceptable carrier and an effective (e.g., therapeutically or prophylactically effective) amount of at least one of the compounds described above. The present invention further provides a method of inhibiting an SH2 domain from binding with a phosphoprotein comprising contacting a sample or substance containing an SH2 domain with a compound of the present invention.

[0041] The present invention discloses the use of above compounds in the manufacture of a medicament for the treatment of a condition that responds to the inhibition of phosphoprotein binding to an SH2 domain of a mammal. The present invention further provides the use of the above compounds in medicine. The compounds can find use as an SH2 domain binding inhibitor. Examples of SH2 domain-containing proteins are Grb2, Shp2, and STAT3 proteins.

[0042] The pharmaceutically acceptable (e.g., pharmacologically acceptable) carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.

[0043] The choice of carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, interperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

[0044] Formulations suitable for oral administration can comprise (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or

orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations can include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0045] The compounds of the present invention, alone or in combination with other suitable components can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

[0046] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane- 4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or

glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0047] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0048] The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants. The quantity of surfactant in such formulations typically ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0049] The compounds of the present invention may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art; see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4 ^th ed., pages 622- 630 (1986).

[0050] Additionally, the compounds of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. In proper doses and with suitable administration of certain compounds, the present invention provides for a wide range of responses. Typically the dosages range from about 0.001 to about 1000 mg/kg body weight of the animal being treated/day. Preferred dosages range from about 0.01 to about 10 mg/kg body weight/day, and further preferred dosages range from about 0.01 to about 1 mg/kg body weight/day.

[0051] Embodiments of the compounds have the advantage that they are stable to or in presence of enzymes encountered during in vivo use. Embodiments of the compounds can find use in in vitro and in vivo applications. For example, the compounds can find use as molecular probes as well as in assays to identify, isolate, and/or quantitate receptor or binding sites in a cell or tissue. The compounds also can find use in vivo for studying the efficacy in the treatment of various diseases or conditions involving SH2 domains.

[0052] The present invention further provides a method of preventing or treating a disease, state, or condition in a mammal by the use of the compounds of the present invention. In an embodiment, the method involves preventing a disease, state, or condition. In another embodiment, the method involves treating an existing disease, state, or condition.

[0053] In an embodiment, the method involves inhibition of SH2 domain binding with a phosphoprotein. The SH2 domain may involve one or more of the following proteins: Shp2, STAT3, Src, Lck, Eps, ras GTPase-activating protein (GAP), phospholipase C, PI-3 kinase, Fyn, Lyk, Fgr, Fes, ZAP-70, Sem-5, p85, SHPTPl, SHPTP2, corkscrew, Syk, Lyn, Yes, Hck, Dsrc, Tec, Atk/Bpk, Itk/Tsk, Arg, Csk, tensin, Vav, Emt, Grb2, BCR-AbI, She, Nek, Crk, CrkL, Syp, BIk, 113TF, 91TF, and Tyk2, especially Grb2, Shp2, and STAT3.

[0054] Grb2 is an adaptor protein with N- and C-terminal src homology 3 (SH3) domains and a central src homology 2 (SH2) domain. The SH2 domain can bind to phosphoTyr residues of receptors or other adaptor proteins, such as SHC. The SH3 domains bind the Ras exchange factor SOS, but can also bind to other adaptor proteins such as GABl and GAB2. Thus, Grb2 is involved in activation of Ras but can also play a role in other signaling pathways in mammalian cells.

[0055] Shp2 is a tyrosine phosphatase that is recruited into tyrosine kinase signaling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs. Shp2 is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Shp2 contains two tandem Src homology-2 domains, which function as phosphotyrosine binding domains and mediate the interaction with its substrates. Shp2 is widely expressed in most tissues and plays a regulatory role in various cell-signaling events that are important for a diversity of cell functions, such as mitogenic activation, metabolic control, transcription regulation, and cell migration.

[0056] Signal Transducers and Activators of Transcription (STATs) are transcription factors that are phosphorylated by JAK kinases in response to cytokine activation of a cell surface receptor tyrosine kinases. Upon activation, the STATs dimerize and are localized to the nucleus where they activate transcription of cytokine-responsive genes. There are at least three JAK kinases and at least six STAT proteins involved in this complex signaling pathway. Cytokines that activate STAT3 include growth hormone, IL-6 family cytokines, and G-CSF. STAT3 induces progression through the cell cycle, prevents apoptosis and upregulates oncogenes, such as c-myc and bcl-X and may play a role in oncogenesis. STAT3 has been shown to play a critical role in hematopoiesis. The importance of STAT3 is

underscored by the failure of mice lacking STAT3 to survive embryogenesis. Crosstalk from pathways other than JAK kinases also leads to phosphorylation and activation of STAT3 as indicated by a role of mTOR (mammalian target of rapamycin, or p70 S 6 kinase) and MAP kinase pathways in STAT3 activation and signaling.

[0057] The method of treatment or prevention of a diseases comprises administering to the mammal one or more compounds of the present invention. The disease, state, condition can be a cancer, e.g., a breast cancer or an ovarian cancer, or a tumor such as a solid tumor, e.g., a brain tumor, a prostate tumor, and the like, leukemia including chronic myelocytic leukemia, lymphoma, an autoimmune disease, an inflammatory disease, a metabolic disease, diabetes, obesity, or cardiovascular disease.

[0058] The present invention further provides a method of enhancing the therapeutic effect of a treatment rendered to a mammal comprising administering a compound in conjunction with the treatment. By conjunction, it is meant that the inhibitor can be used in any suitable manner, for example, prior to, simultaneous with, or post- administration of the therapeutic agent. Synergistic effects are observed when the SH2 domain binding inhibitor is used in combination with other treatments known to those skilled in the art. The inhibitor enhances the cytotoxicity of the chemotherapeutic treatments. Cancer treatment is particularly suitable for this combination treatment.

[0059] The cancer may involve any number of mechanisms. A majority of human breast cancers are dependent upon activation of the Ras signaling pathways through activation of growth factor receptor as the means to achieve continuous cellular proliferation. For example, the cancer may involve overexpression of Her-2/neu. The cancer can be mediated through BCR-AbI or the expression of erbB-2 receptor. In cells transformed by pl85 erbB-2 overexpression, therapeutic agents affecting Grb2 function at its SH2 domain may interrupt the flow of signal transduction to the Ras pathway and thus result in reversal of the cancer phenotype.

[0060] The therapeutic treatment can include chemotherapy, radiation therapy, and/or a biological therapy. Examples of chemotherapy include the use of cancer treatment agents such as alkylating agents, hormonal agents, antimetabolites, natural products, and miscellaneous agents. Particular examples of cancer treatment agents include paclitaxel, 5-

fluoruracil, and doxorubicin. Examples of biological therapy include the use of a protein such as an antibody (monoclonal or polyclonal) or a recombinant protein. An example of an antibody is herceptin, which is targeted for inhibiting the erbB-2 receptor. In embodiments, the enhancement of the therapeutic effect comprises blocking of a cell survival factor in the mammal and/or triggering, e.g., enhancing or speeding up, of cell apoptosis. The treatment can be carried out in vivo and/or in vitro.

[0061] The Grb2 SH2 binding inhibitors are effective in inhibiting the association or binding of Grb2 with activated receptor PTKs. Interaction of native Grb2 protein with phosphotyrosinylated proteins including receptor PTKs can be monitored by immunoprecipitating Grb2 and detecting the amount of phosphotyrosinylated proteins which are coprecipitated using anti-phosphotyrosine Western Blotting.

[0062] The compounds of the present invention can be prepared by any suitable method, for example, a method that advantageously utilizes achiral alkenyl amines in a synthesis involving ring closing metathesis (RCM) reaction of asparagine pentenylamides onto a β-vinyl-containing residue; see, e.g., Figures 1-4. For examples of RCM reactions, see, Gao et al., Org. Lett. 2001, 3, 1617-1620; Reichwein et al., Angew. Chem., Int. Ed. 1999, 38, 3684-3687, J. Org. Chem., 2000, 65, 6187-6195; andJ. Org. Chem., 2000, 65, 2335-2344; Stymiest et al., Org. Lett, 2003, 5, 47-49; Miller et al., J. Am. Chem. Soc, 1995, 117, 5855- 5856; and Dekker et al., Org. Biomol Chem., 2003, 1, 3297-3303. The RCM reaction advantageously allows ring closure with retention of desired functional groups, e.g., phenylphosphate functionality or the chemical (e.g., aryl groups of R and R ) functionality at or near the site of ring juncture(s). In addition to solution chemistries, the preparation of multiple analogues may be made possible through the use of solid-phase chemistries.

[0063] In accordance with the invention, macrocyclic compounds of any suitable size can be prepared. In an exemplary approach, homologs of compounds 9a and 9f, which have one or more additional methylene units between the amino group and the carbon atom bearing R ¹ and R ¹ (i.e., m is greater than 1), can be prepared using any suitable chemistry known in the art. These homologs can then be used to prepare macrocyclic compounds of the invention, wherein m is greater than 1, using similar chemistry as depicted in Figures 2-4. Another exemplary approach is varying the number of amino acids of (AA) _n . For example, analogs of compounds 10a, 1Of, and/or 18 can be prepared, which comprise one or more additional

amino acids (i.e., n of (AA) _n is greater than 2). These analogs can then be used to prepare macrocyclic compounds of the invention, wherein n is greater than 2, using similar chemistry as depicted in Figure 4. Both of these approaches can be used to vary the ring size while producing macrocyclic compounds of the invention. In accordance with the invention, one or both of these approaches can be used to prepare macrocyclic compounds of the invention with various ring sizes.

[0064] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0065] This example demonstrates a method of preparing compounds 5a and 5f in accordance with an embodiment of the invention.

[0066] Reactions are carried out under argon. Anhydrous solvents are purchased from Aldrich Chemical Corporation and used without further drying. H NMR spectra are obtained using a Varian 400 MHz spectrometer and are reported in ppm relative to TMS and referenced to the solvent in which they were run. Fast atom bombardment mass spectra (FABMS) are acquired with a VG analytical 7070E mass spectrometer. HPLC separations are conducted using a Waters Prep LC4000 system with photodiode array detection and either a J-sphere ODS-H80 column (20 x 250 mm) with a solvent system consisting of 0.1% aqueous TFA (v/v, solvent A) / 0.1% TFA in MeCN (v/v, solvent B).

[0067] Achiral 4-penten-l -amines are prepared as depicted in Figure 1.

[0068] 4-pentene-l -amine (9a). To a solution of 4-penten-l -ol (5.0 g, 58.1 mmol) and triethylamine (9.0 mL, 63.9 mmol) in dichloromethane (150 mL) at 0 ⁰ C, is added methanesulfonyl chloride (5.0 mL, 63.9 mmol) dropwise, the mixture is stirred for another 3 hours, then washed by water (50 mL x 2), brine (50 mL), dried over sodium sulfate and concentrated to a pale yellow liquid which is dissolved in DMF/water (100 mL /10 mL), sodium azide (9.44 g, 145.3 mmol) is added, and the mixture is heated to 50 ⁰ C overnight. The reaction mixtue is diluted with water (200 mL) and extracted with ether (300 mL). The ether layer is washed with water (50 mL x 2) and brine (50 mL), dried over sodium sulfate and concentrated in vacuo. The concentrated oil is dissolved in anhydrous ether (100 mL),

LiAlH ₄ (2.2 g, 58.1 mmol) is added in several portions at 0 ⁰ C, and the suspension is stirred for 1 hour. Water (5.0 rnL) is carefully added to the reaction mixture at 0 ⁰ C, which is stirred vigorously until white. (Boc) ₂ O (14.0 g, 64.9 mmol) is added and the reaction mixture is stirred overnight before being washed with water and brine, dried, and purified by short silica gel column. The concentrated oil is treated with trifluoroacetic acid (72 mL) and triethylsilane (30 mL) in dichlorornethane (138 mL) for 2 hours. Evaporation of solvent yields product 9a as a pale yellow oil (6.0 g, 52% total). ¹ H NMR (CDCl ₃ ) δ 7.73 (bis, 2 H), 7.00 (brs, 1 H), 5,74 (m, 1 H)/5.05 (m, 2 H), 2.97 (m, 2 H), 2.15 (m, 2 H), 1.70 (m, 2 H).

[0069] β-Phenyl-β-(2-propenyl)-benzeneethanamine (9f). To a suspension of sodium hydride 95% in oil (0.70 g, 27.7 mmol) in DMF (50 mL), is slowly added a solution of α-phenyl-benzeneacetonitrile 6f (5.0 g, 25.8 mmol) and the mixture is stirred at room temperature (1 h). The mixture is cooled to 0 ⁰ C and allyl bromide (3.50 g, 28.9 mmol) is added then the reaction is brought to room temperature and stirred overnight. The reaction mixture is poured into ice-water, extracted with benzene (2 xlOO mL), dried (Na ₂ SO ₄ ) and solvent evaporated to yield 8f as a viscous oil. [ ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.42 - 7.28 (m, 10 H), 5.70 (m, 1 H), 5.20 (m, 2 H), 3.13 (d, J = 6.8 Hz, 2 H).] The crude 8f is dissolved in ether (20 mL) and added dropwise to a suspension OfLiAlH ₄ (4.O g, 103 mmol) in anhydrous ether (50 mL) at 0 ⁰ C and the mixture is warmed to room temperature and stirred overnight. The reaction mixture is cooled to 0 ⁰ C and quenched by the careful addition of H ₂ O (10 mL). The mixture is vigorously stirred until white, dried (Na ₂ SO ₄ ) and solvent evaporated to afford 9f as a colorless oil (4.00 g, quantitative yield from 6f).η NMR (400 MHz, CDCl ₃ ) δ 7.30 - 7.16 (m, 10 H), 5.39 (m, 1 H), 5.07 - 4.95 (m, 2 H), 3.32 (s, 2 H), 2.92 (d, J= 6.4 Hz, 2 H), 0.88 (brs, 2 H). FAB-MS (+VE) m/z 238.2 (M+H) ⁺ .

[0070] N-Boc protected asparagines alkenylamides are prepared as depicted in Figure 2.

[0071] λ^-Boc-ν-^riphenylmethy^-L-asparagine (4-pentenyl)amide (10a). To a solution of λ^-Boc-L-AsnζTrfl-OH (950 mg, 2.00 mmol) in DMF (10 mL) at 0 ⁰ C with 4-methylmorpholine (1.00 mL, 9.10 mmol) is added isobutylformate (0.32 mL, 2.45 mmol) and the mixture is stirred at 0 ⁰ C (10 minutes) then amine 9a (478 mg, 2.40 mmol) is added and the reaction mixture is warmed to room temperature and stirred overnight. The mixture is diluted with ethyl acetate (150 mL), washed with H ₂ O (2 x 50 mL), brine (50 mL), dried

(Na ₂ SO ₄ ) and solvent removed. Purification by silical gel column chromatography (hexane and ethyl acteate) affords 10a as a white solid (325 mg, 30% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.32 - 7.15 (m, 15 H), 6.98 (bra, 1 H), 6.70 (bra, 1 H), 6.18 (bra, 1 H), 5.77 (m, 1 H), 5.00 (m, 2 H), 4.42 (m, 1 H) ₅ 3.21 (m, 2 H), 3.08 (dd, J= 15.0, 3.4 Hz, 1 H), 2.53 (dd, J = 15.0, 2.2 Hz, 1 H), 2.05 (q, J= 7.2 Hz, 2 H), 1.55 (m, 2 H), 1.43 (s, 9 H). FAB-MS (+VE) m/z 542.4 (M + H) ⁺ . HR-FABMS calcd for C ₃₃ H ₄₀ N ₃ O ₄ (M + H) ⁺ : 542.3019. Found: 542.3009.

[0072] λ^-Boc-N^triphenylmethyty-L-asparagine β-phenyl-β-(2-propenyl)- benzeneethanamide (1Of). Treatment of 9f as described above for the preparation of 10a provides 1Of in quantitative yield. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.35 - 7.10 (m, 25 H), 6.94 (bra, 1 H), 6.32 (m, 1 H), 5.95 (d, J= 7.6 Hz, 1 H), 5.38 (m, 1 H), 4.90 (m, 2 H), 4.30 (m, 1 H), 4.13 (m, 1 H), 3.73 (m, 1 H), 3.00 (dd, J= 14.8, 3.6 Hz, 1 H), 2.79 (m, 2 H), 2.44 (dd, J= 15.0, 5.0 Hz, 1 H),1.40 (s, 9 H). FAB-MS (+VE) m/z: 694.2 (M + H) ⁺ . HR-FABMS calcd for C ₄₅ H ₄₇ N ₃ NaO ₄ (M + Na) ⁺ : 716.3464. Found: 716.3492.

[0073] Compound 18 containing a β-vinyl unit is prepared as depicted in Figure 3.

[0074] λ^-Boc-α-aminocyclohexanecarboxylic acid 2-propenyl ester (15). A mixture of λ^-Boc-l-aminocyclohexanecarboxylic acid (1.70 g, 7.00 mmol), allyl bromide (0.89 mL, 10.5 mmol) and Na ₂ CO ₃ (881 mg, 10.5 mmol) in DMF (20 mL) is stirred at room temperature (2 days). The reaction mixture is diluted with ethyl acetate (150 mL), washed with H ₂ O (2 x 50 mL) and brine (50 mL), dried (Na ₂ SO ₄ ) and solvent evaporated. Purification by silica gel column chromatography (hexane and ethyl acetate) affords 15 as a colorless oil (1.00 g, 50% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.92 (m, 1 H), 5.25 (m, 1 H), 5.20 (m, 1 H), 4.72 (brs, 1 H), 4.61 (m, 2 H), 2.00 (m, 2 H), 1.84 (m, 2 H), 1.65 - 1.53 (m, 3 H), 1.53 - 1.43 (m, 11 H), 1.30 (m, 1 H). FAB-MS (+VE) m/z: 284.2 (M + H) ⁺ . HR- FAB (M + H) ⁺ : 284.1873, CaIc: 284.1862.

[0075] Dipeptide allyl ester 17. Treatment of λ^-Boc-protected 15 (384 mg, 1.35 mmol) with CF ₃ CO ₂ H (1.56 mL) and triethylsilane (0.64 mL) in CH ₂ Cl ₂ (3.00 mL) at room temperature (2 h) and removal of volatiles provides the free amine as its CF ₃ CO ₂ H salt (16). This is dissolved in DMF (2.0 mL) and added to a pre-formed active ester solution obtained by reacting pTyr mimetic 12 (450 mg, 0.90 mmol) in DMF (4.0 mL) along with HOAt (183

mg, 1.35 mmol), EDCI (258 mg, 1.35 mmol) and NEt(Z-Pr) ₂ (0.78 mL, 4.50 mmol) with stirring at room temperature (15 minutes). The combined reaction mixture is stirred at room temperature (2 days). The mixture is diluted with ethyl acetate (150 mL), washed with H ₂ O (2 x 50 mL) and brine (50 mL), dried (Na ₂ SO ₄ ) and solvent evaporated. Purification by silica gel column chromatography (hexane and ethyl acetate) provides 17 as a white solid (540 mg, 90% combined yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22 - 7.10 (m, 4 H), 5.94 (s, 1 H), 5.83 - 5.77 (m, 2 H), 5.25 - 5.00 (m, 4 H), 4.46 (m, 2 H), 3.52 (m, 1 H), 3.00 (m, 1 H), 2.92 (d, J= 21.2 Hz, 2 H), 2.52 (m, 2 H), 1.70 - 1.20 (m, 36 H), 0.85 (m, 1 H). FAB-MS (+VE) m/z: 662 A (M + H) ⁺ . HR-FABMS calcd for C ₃₆ H ₅₆ NNaO ₄ P (M + Na) ⁺ : 684.3641. Found: 684.3644.

[0076] Dipeptide acid 18. To a solution of dipeptide allyl ester 17 (540 mg, 0.82 mmol) in anhydrous THF (30 mL) which has been degassed under argon for 5 minutes is added Pd(PPh ₃ ) ₄ (93 mg, 0.080 mmol) and morpholine (0.70 mL, 8.00 mmol). The mixture is stirred at room temperature (30 minutes) then 0.1 N HCl (100 mL) is added, THF is removed in vacuuo and the remaining residue is extracted with ethyl acetate (3 x 50 mL). The combined ethyl acetate extracts are washed with brine (50 mL), dried (Na ₂ SO ₄ ) and purified by silica gel column chromatography (CH ₂ Cl ₂ and methanol) to afford 18 as a white solid (460 mg in 91% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 - 7.12 (m, 4 H), 5.91 (m, 1 H), 5.75 (s, 1 H), 5.15 (m, 2 H), 3.49 (m, 1 H), 3.10 - 2.90 (m, 3 H), 2.72 - 2.55 (m, 2 H), 1.70 (m, 2 H), 1.60 - 1.25 (m, 33 H), 1.15 (m, 2 H). FAB-MS (+VE) m/z: 644 (M + Na) ⁺ . HR-FABMS calcd for C ₃₃ H ₅₂ NNaO ₈ P (M + Na) ⁺ : 644.3328. Found: 644.3359.

[0077] Compounds 5a and 5f are synthesized as depicted in Figure 4.

[0078] Metathesis precursor 13a. λ^-Boc-protected asparagine amide 10a (94 mg, 0.174 mmol) is treated with CF ₃ CO ₂ H (1.60 mL) and triethylsilane (0.30 mL) in CH ₂ Cl ₂ (1.0 mL) at room temperature (4 h). Volatiles are then removed in vaccuo and the residue is placed in vacuum (30 minutes) to yield the CF ₃ CO ₂ H amine salt 19a. This is dissolved in DMF (2.0 mL) and added to a pre-formed active ester solution prepared by stirring dipeptide acid 18 (70 mg, 0.116 mmol) in DMF (2.0 mL) with HOAT (19 mg, 0.140 mmol), EDCI (28 mg, 0.140 mmol) and NEt(Z-Pr) ₂ (91 uL, 0.522 mmol) at room temperature (15 minutes). The resulting reaction mixture is stirred at room temperature (2 days) then diluted with ethyl acetate (150 mL), washed with H ₂ O (2 x 50 mL) and brine (50 mL) and dried (Na ₂ SO ₄ ).

Purification by silical gel column chromatography (CH ₂ Cl ₂ and methanol) provides 13a (85 mg, 91% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.35 (t, J= 6.0 Hz, 1 H), 7.24 (dd, J= 8.4, 2.0 Hz, 2 H), 7.19 (d, J= 8.4 Hz, 2 H), 7.11 (d, J= 8.4 Hz, 1 H), 6.78 (brs, 1 H), 6.28 (s, 1 H), 5.89 - 5.76 (m, 2 H), 5.56 (brs, 1 H), 5.12 - 4.97 (m, 4 H), 4.49 (dd, J= 13.4, 7.0 Hz, 1 H), 3.46 (t, J= 9.4 Hz, 1 H), 3.25 (m, 1 H), 3.18 (m, 1 H), 3.07 (m, 1 H), 3.00 (dd, J= 21.4, 3.4 Hz, 2 H), 2.79 (m, 2 H), 2.64 (m, 2 H), 2.14 - 2.08 (m, 4 H), 1.78 - 1.57 (m, 5 H), 1.52 - 1.27 (m, 29 H), 1.13 (m, 2 H), 0.60 (m, 1 H). FAB-MS (+VE) m/z: 803.5 (M + H) ⁺ . HR- FABMS calcd for C ₄₂ H ₆₇ N ₄ NaO ₉ P (M + Na) ⁺ : 825.4543. Found: 825.4568.

[0079] Metathesis precursor 13f. Deprotection of λ^-Boc-protected asparagine amide 1Of yields the CF ₃ CO ₂ H amine salt 19f, which is coupled with dipeptide acid 18 as in 13a to provide 13f in 85% yield. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.30 - 7.15 (m, 15 H), 6.68 (t, J= 5.8 Hz, 1 H), 6.63 (brs, 1 H), 5.87 - 5.78 (m, 2 H), 5.46 (m, 1 H), 5.33 (brs, 1 H), 5.12 - 2.00 (m, 3 H), 4.94 (dd, J= 10.4, 2.4 Hz, 1 H), 4.41 (dd, J= 13.8, 5.8 Hz, 1 H), 4.05 (dd, J= 9.2, 6.4 Hz, 1 H), 3.93 (dd, J= 13.0, 5.4 Hz, 1 H), 3.48 (t, J= 9.8 Hz, 1 H), 3.00 - 2.89 (m, 5 H), 2.60 - 2.53 (m, 3 H), 2.45 (dd, J= 15.6, 5.6 Hz, 1 H), 1.66 - 1.56 (m, 3 H), 1.50 - 1.35 (m, 30 H), 1.29 (m, 1 H), 1.09 (m, 2 H), 0.62 (m, 1 H). FAB-MS (+VE) m/z: 955.7 (M + H) ⁺ . HR-FABMS calcd for C ₅₄ H ₇₅ N ₄ NaO ₉ P (M + Na) ⁺ : 977.5169. Found: 977.5144.

[0080] Macrocycle 5a. A solution of tripeptide 13a (50 mg, 0.063 mmol) in 1,2- dichloroethane (20 mL) is degassed under argon (5 minutes) then Grubbs 2 ^nd generation catalyst [((PCy ₃ )(Im(MeS) ₂ )Ru=CHPh) 20] (26 mg, 0.032 mmol) is added and the mixture is refluxed (2 days). The mixture is concentrated and purified by silica gel column chromatography (CH ₂ Cl ₂ and methanol) to provide the brown crude product 21a. This is treated with a mixture Of CF ₃ CO ₂ H (9.0 mL), triethylsilane (0.50 mL) and H ₂ O (0.50 mL) at room temperature (2 h). The solvent is removed and the residue is purified by reverse phase preparative HPLC using a Phenomenex Ci ₈ column (21 mm dia x 250 mm, cat. no: 00G- 4436-PO) using a linear gradient from 0% aqueous acetonitrile (0.1% CF ₃ CO ₂ H) to 100% acetonitrile (0.1% CF ₃ CO ₂ H) over 35 minutes at a flow rate of 10.0 mL/minute (detection at 225 nm). Lyophilization provides the macrocyclic final product 5a as a white solid (15 mg, 39% yield from 13a). ¹ H NMR (400 MHz, DMSO-J ₆ ) δ 8.50 (s, 1 H), 8.32 (d, J= 8.0 Hz, 1 H), 7.57 (brs, 1 H), 7.31 (d, J= 8.4 Hz, 2 H), 7.20 - 7.13 (m, 4 H), 5.78 (dd, J= 15.0, 10.0 Hz, 1 H), 5.56 (m, 1 H), 4.26 (m, 1 H), 4.09 (d, J= 8.4 Hz, 1 H), 3.55 (m, 1 H), 3.28 (d, J=

11.6 Hz, 1 H), 2.93 (d, J= 21.2 Hz, 2 H), 2.83 (dd, J= 15.8, 5.0 Hz, 1 H), 2.76 (m, 1 H), 2.53 (m, 1 H), 2.33 (dd, J = 15.2, 4.8 Hz, 1 H), 2.20 - 1.95 (m, 4 H), 1.90 - 1.70 (m, 3 H), 1.60 - 1.40 (m, 7 H), 1.20 (m, 1 H). FAB-MS (-VE) m/z: 605.2 (M - H) ^" . HR-FABMS calcd for C ₂₈ H ₄₀ N ₄ O ₉ P (M + H) ⁺ : 607.2533. Found: 607.2558.

[0081] Macrocycle 5f. Ring-closing metathesis of 13f to 21f followed by deprotection and HPLC purification as reported above for the conversion of 13a to 5a yields the macrocyclic final product 5f in 4% yield. ¹ H NMR (400 MHz, DMSCM ₆ ) δ 8.12 (s, 1 H), 7.88 (d, J= 7.2 Hz, 1 H), 7.28 - 7.08 (m, 15 H), 6.84 (s, 1 H), 6.68 (dd, J= 10.2, 2.2 Hz, 1 H), 6.01 (dd, J= 15.0, 9.8 Hz, 1 H), 5.70 (m, 1 H), 4.50 (m, 1 H), 4.22 (q, J= 6.4 Hz, 1 H), 4.06 (d, J= 7.2 Hz, 1 H), 3.40 (m, 1 H), 3.20 (m, 1 H), 2.95 - 2.84 (m, 3 H), 2.70 - 2.64 (m, 2 H), 2.05 (m, 2 H), 2.00 - 1.70 (m, 5 H), 1.50 - 1.35 (m, 5 H), 1.20 (m, 1 H). FAB-MS (- VE) m/z: 757.2 (M - H) ^" . HR-FABMS calcd for C ₄₀ H ₄₇ N ₄ NaO ₉ P (M + Na) ⁺ : 781.2978. Found: 781.2998.

EXAMPLE 2

[0082] This example demonstrates the Grb2 SH2 domain-binding affinities of compounds 5a and 5f (compounds of formula Ilia and HIb, respectively) using surface plasmon resonance (SPR).

[0083] The steady state values are determined using a Biacore 2000 and S51 instruments using amine coupled surfaces as described in Oishi et al. ChemBioChem, 2005, 6, 668-674 and Oishi et al. Bioorg. Med. Chem., 2005, 13, 2431-2438. The binding experiment directly measures the binding of 5a and 5f to biotinylated Grb2 SH2 domain protein immobilized onto a sensor chip. Immobilization of protein is achieved either by amine coupling or by streptavidin capturing of the biotin functionality.

[0084] The binding data is provided in Table 1.

TABLE l

[0085] As depicted in Table 1 , 5a and 5f display binding affinities of 320 nM and 72 nM, respectively.

[0086] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0087] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0088] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible

variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.