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Definitions

• the present invention relates to the fields of pharmaceuticals, medicine and cell biology. More specifically, it relates to pharmaceutical agents (compounds) which are useful as integrin receptor antagonists, with particularly exceptional biological activity as antagonists of a group of integrins that mediate the pathologic processes of angiogenesis and fibrosis. As such, these compounds are useful in pharmaceutical compositions and in methods for treating conditions mediated by such integrins by inhibiting or antagonizing these integrins.
• Integrins are a family of integral cytoplasmic membrane proteins that mediate cell interactions with other cells and with the extracellular matrix. Approximately one third of the members of the integrin family directly bind to a specific amino acid motif, arginine-glycine-asparate (RGD), that is contained within the sequence of their cognate protein ligands. It has been established in the art that peptides containing the RGD sequence, and synthetic small molecule compounds that mimic the RGD sequence, are capable of binding to these integrin receptors with varying degrees of specificity, and thereby inhibit the binding to normal physiologic ligands (Millard et al, 201 1.). The biological effects of treatment with such agents is dependent on intrinsic molecular properties, reflected in the structure, that determine to what degree a particular integrin, or combination of integrins, is inhibited in a body tissue over a period of time.
• RGD arginine-glycine-asparate
• Integrins which have been shown to have a role in promoting angiogenesis include, ⁇ 3, ⁇ 5, and ⁇ 5 ⁇ 1.
• ⁇ 3 and ⁇ 5 were initially described as mediators of bFGF- and VEGF-induced angiogenesis, respectively, in corneal or choriallantoic models. More recently, data from studies using mice lacking these integrins also support an important functional role for ⁇ 5 ⁇ 1.
• the integrin ⁇ 5 ⁇ 1 (also known as VLA-5) is often referred to as the 'classic fibronectin receptor' reflecting its well characterized interaction with this extracellular matrix protein.
• ⁇ 5 ⁇ 1 binds to fibronectin in a region that incorporates the ninth and tenth type III fibronectin repeats, the latter of which contains the RGD motif critical for integrin binding.
• ⁇ 5 ⁇ 1 has been reported to interact with other RGD-containing extracellular matrix proteins including fibrinogen, denatured collagen, and fibrillin- 1 (Bax et al, 2003; Perdih, 2010; Suehiro et al, 2000).
• These ligands are components of the provisional matrix that is laid down by cells as part of the wound healing response in tissues. Key components of this response are angiogenesis (new blood vessel formation) and fibrosis (scar formation) which are beneficial for healing of acute injuries, but can be deleterious in many disease contexts.
• Antagonists of RGD-binding integrins should be useful for treatment of human diseases having angiogenesis or fibrosis as a principal part of their pathology.
• angiogenesis a major histone deacetylase
• fibrosis a major histone deacetylase derived from a sarcoma.
• ⁇ 5 ⁇ 1 in angiogenesis is supported by numerous studies. For example, mice lacking this integrin exhibit embryonic lethality at day 10- 11 with a phenotype that includes defects in both the embryonic and extraembryonic vasculature (Yang et al, 1993).
• Angiogenic cytokines such as bFGF, IL-8, ⁇ , and TNFa upregulate ⁇ 5 ⁇ 1 expression on endothelial cells in vitro and in vivo, and immunohistochemistry shows coordinated increases in both ⁇ 5 ⁇ 1 and fibronectin staining in blood vessels from various types of human tumor biopsies and xenograft tumors in animals (Collo, 1999; Kim et al, 2000).
• ⁇ 5 ⁇ 1 expression is not confined to the endothelium, it has other functional roles in addition to angiogenesis. It is expressed to varying degrees in many cell types including fibroblasts, hematopoietic and immune cells, smooth muscle cells, epithelial cells, and tumor cells. Expression on tumor cells has been implicated in the progression of tumor growth and metastasis (Adachi et al, 2000; Blase et al, 1995; Danen et al, 1994; Edward, 1995). In human fibroblasts, ⁇ 5 ⁇ 1 promotes motility and survival (Lobert et al, 2010).
• pancreatic stellate cells In pancreatic stellate cells, it interacts with connective tissue growth factor to stimulate adhesion, migration, and fibrogenesis (Gao and Brigstock, 2006). It has been shown that pharmacologic antagonism of ⁇ 5 ⁇ 1 inhibits the attachment migration, and proliferation of human retinal epithelial cells in vitro, and reduces retinal cell proliferation and scarring when administered intravitreally to rabbits with retinal detachment (Li et al, 2009; Zahn et al, 2010).
• RGD-binding integrins of the alpha v family have been implicated in promoting the biological activation of the latent pro-fibrotic cytokine TGFp. This is mediated by binding to the latency associated peptide (LAP), particularly by ⁇ and ⁇ 8, but also by ⁇ , ⁇ 3, and ⁇ 5. These integrin interactions are all critically dependent upon the amino acid sequence arg-gly-asp (RGD) contained in LAP. Indeed, mice containing a mutation in the RGD sequence are incapable of cytokine activation and phenocopy ⁇ - ⁇ mice. It is anticipated that simultaneous inhibition of multiple integrins with the potential to activate ⁇ may have particular utility to prevent or treat a range of fibrotic conditions. In addition, such broad spectrum integrin antagonists may be particularly useful for simultaneous modulation of both angiogenesis and fibrosis.
• the present disclosure provides novel integrin receptor antagonists, pharmaceutical compositions, and methods for their manufacture, and methods for their use.
• the present invention provides a compound of the formula:
• RB is -H, -OH, -NH 2 , -F, -CN, or alkoxy(c ⁇ 8), wherein if R A is -F, then R B is -H or -F; and m is 0-3 ;
• R4 and R5 are each independently alkyl( C ⁇ 8), substituted alkyl( C ⁇ 8), or -CH 2 0-alkyl( C ⁇ 8) ;
• Re is -OH, alkoxy( -NH 2 ; n is 1 or 2 and Xi is
• the compound is further defined as:
• RB is -H, -OH, -NH 2 , -F, -CN, or alkoxy(c ⁇ 8), wherein if R A is -F, then R B is -H or -F; and m is 0-3;
• R4 and R5 are each independently alkyl( C ⁇ 8) or substituted alkyl( C ⁇ 8);
• the compound is further defined as:
• W is wherein: R A is -H or -F;
• RB is -H, -OH, -NH 2 , -F, -CN, or alkoxy(c ⁇ 8), wherein if R A is -F, then R B is -H or -F; and m is 0-3 ;
• R4 and R5 are each independently alkyl( C ⁇ 8), substituted alkyl (c ⁇ 8), or - ⁇ 2 0- 3 ⁇ 4 1 (0 ⁇ 8 );
• R 9 are each -CF 3 , then R 10 is -OH, alkoxy(c ⁇ 8) -NH 2 ;
• the compound is further defined as:
• W is A is -H or -F; RB is
• R A is -F, then R B is -H or -F;
• A is C-R" or N, wherein:
• R 5 are each independently alkyl( C ⁇ 8) or substituted alkyl( C ⁇ 8);
• t en X an Y are not ot eac t- uty ; an urt er prov e t at
• A is C-OH, Z is hydrogen, and X is bromo or iodo, then Y is not t-butyl; or a pharmaceutically acceptable salt or tautomer thereof.
• the carbon atom labeled ⁇ is in the R configuration. In other embodiments, the carbon atom labeled ⁇ is in the S configuration. In some embodiments, Y is:
• Rs and R9 are each independently alkyl(c ⁇ s), substituted alkyl(c ⁇ s), or - CH 2 0-alkyl (C ⁇ 8); and R 10 is -OH, -CF 3 , " CF 2 H, -CFH 2 , -C0 2 -alkyl (C ⁇ 8), -CH 2 OH, -CH 2 0-alkyl(c ⁇ 8), or alkoxy(c ⁇ 8), provided that where Rs and R9 are each -CF 3 , then Rio is -OH, alkoxy(c ⁇ 8) or -NH 2 .
• W is .
• A is C-OH.
• A is N.
• X is halo.
• X is bromo.
• X is chloro.
• X is alkyl(c ⁇ 8) or substituted alkyl(c ⁇ 8 ).
• X is t-butyl.
• X is 2-hydroxy-isopropyl.
• X is -CF 3 .
• X is cyano.
• X is heteroaryl.
• X is pyrimidyl.
• X is pyridyl.
• L is hydrogen.
• Y is t-but l.
• Y is 2-
• Rs and R9 are alkyl( C ⁇ s). In some embodiments, Rs and R9 are methyl. In some embodiments, Rio is -CN, -CH OH -CH 0-alk l ⁇ or -CF . In other embodiments, wherein R 10 is other embodiments, Y is In other embodiments, Y is In other embodiments, Y is ⁇ CF
• Rs is alkyl(c ⁇ 8). In other embodiments, Rs is methyl. In other embodiments, Rg is substituted alkyl( C ⁇ 8). In other embodiments, Rg is -CF 3 . In other emb In other embodiments, Y
• A" is a covalent bond.
• A" is alkoxydiyl(c ⁇ 8). In some embodiments, A" is -CH 2 OCH 2 -. In other embodiments, A" is alkanediyl(c ⁇ 6> In some embodiments, A" is -CH 2 - In other embodiments, Rn is -CN, -CH 2 OH, -CH 2 0-alkyl( C ⁇ 8), -CF 2 H, or -CFH 2 . In some embodiments, Rn is -CH 2 OCH 3 . In
• Y is . In other embodiments, Y In other
• Y is In other embodiments, Y is . In other
• Y is . in other embodiments, Y is .
• Z is hydrogen.
• Z is hydroxy and attached to carbon atom 2.
• Z is hydroxy and attached to carbon atom 6.
• Z is fluoride and attached to carbon atom 2.
• Z is fluoride and attached to carbon atom 6.
• R' is hydrogen.
• A is C ⁇ H. In other embodiments, A is N. In some embodiments, R' is hydrogen. In some embodiments, L is hydrogen. In some embodiments, Z is hydrogen. In some embodiments, Y is alkyl( C ⁇ 8). In some embodiments, Y is t-butyl. In some embodiments, X is halo. In some embodiments, X is bromo. In other embodiments, X is alkyl ( c ⁇ 8> In other embodiments, X is t-butyl.
• W is .
• A is C-OH.
• A is C ⁇ H.
• R' is hydrogen.
• L is hydrogen.
• Z is hydrogen.
• Y is alkyl( C ⁇ 8).
• Y is t-butyl.
• X is halo. In some embodiments, X is bromo.
• the compound is further defined as:
• the compound of claim 1 of the formula shown Table A or a pharmaceutically acceptable salt or tautomer thereof.
• the present invention provides a pharmaceutical
• composition comprising: a) the compound of the present invention; and b) an excipient.
• the present invention provides a method of treating and/or preventing a disease or a disorder in a patient in need thereof, comprising administering to the patient a compound of the present invention in an amount sufficient to treat and/or prevent the disease or disorder.
• the disease or disorder is associated with angiogenesis.
• the disease or disorder is associated with fibrosis.
• the disease or disorder is associated with fibrosis and/or angiogenesis.
• the disease or disorder is pulmonary, liver, renal, cardiac, and pancreatic fibrosis, scleroderma, scarring, retinopathy of prematurity, familial exudative vitreoretinopathy, proliferative vitreoretinopathies, macular degeneration, diabetic retinopathy, cancer, osteoporosis, autoimmune diseases, humoral hypercalcemia of malignancy, Paget' s disease, periodontal disease, psoriasis, arthritis, restenosis, and infection.
• the disease or disorder is pulmonary fibrosis.
• the disease or disorder is liver fibrosis.
• the disease or disorder is cardiac fibrosis.
• the disease or disorder is renal fibrosis. In other embodiments, the disease or disorder is pancreatic fibrosis. In other embodiments, the disease or disorder is scleroderma. In other embodiments, the disease or disorder is scarring. In some embodiments, the scarring is dermal scarring. In other embodiments, the scarring is retinal scarring. In other embodiments, wherein the scarring is corneal scarring. In other embodiments, the disease or disorder is retinopathy of prematurity. In other embodiments, the disease or disorder is familial exudative vitreoretinopathy. In other embodiments, the disease or disorder is proliferative vitreoretinopathies. In other embodiments, the disease or disorder is macular degeneration.
• the disease or disorder is diabetic retinopathy.
• the disease or disorder is cancer.
• the cancer includes solid tumor growth or neoplasia.
• the cancer includes tumor metathesis.
• the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid.
• the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.
• the disease or disorder is osteoporosis.
• the disease or disorder is an autoimmune disease.
• the autoimmune disorder is multiple sclerosis.
• the disease or disorder is humoral hypercalcemia of malignancy.
• the disease or disorder is Paget' s disease.
• the disease or disorder is periodontal disease.
• the disease or disorder is psoriasis.
• the disease or disorder is arthritis.
• the arthritis is rheumatoid arthritis.
• the disease or disorder is restenosis.
• the disease or disorder is an infection.
• the patient is a human, monkey, cow, horse, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
• the patient is a monkey, cow, horse, sheep, goat, dog, cat, mouse, rat, or guinea pig.
• the patient is a human.
• the present invention provides a compound of the fomula:
• R' is -H, alkyl( C ⁇ 8) substituted alkyl( C ⁇ 8), alkylaryl( C ⁇ i2), and silyl;
• R" and R" are each independently -H, alkyl( C ⁇ 8) substituted alkyl( C ⁇ 8), alkylaryl( C ⁇ i2), substituted alkylaryl ( c ⁇ i2), acyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, carbamate, carbobenzyloxy, or benzoyl;
• X is: hydrogen, halo, or cyano; alkyl(c ⁇ i2), alkoxy(c ⁇ i2), aryl(c ⁇ i2), aralkyl (c ⁇ i2), heteroaryl (c ⁇ 8), heterocycloalkyl (c ⁇ i2), aryloxy (c ⁇ i2), acyloxy(c ⁇ i2), or a substituted version of any of the
• n" is 1 or 2 and Yi is -H or alkyl( C ⁇ 8); or
• the present invention provides a compound of the formula:
• R' is -H, alkyl( C ⁇ 8) substituted alkyl( C ⁇ 8), alkylaryl( C ⁇ i 2 ), and silyl;
• R" and R" are each independently -H, alkyl( C ⁇ 8) substituted alkyl( C ⁇ 8), alkylaryl( C ⁇ i 2 ), substituted alkylaryl(c ⁇ i 2) , acyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, carbamate, carbobenzyloxy,or benzoyl;
• X is: hydrogen, halo, or cyano; alkyl( C ⁇ i 2) , alkoxy(c ⁇ i 2) , aryl(c ⁇ i 2 ), aralkyl (c ⁇ i 2 ), heteroaryl (c ⁇ 8), heterocycloalkyl (c ⁇ i 2 ), aryloxy (c ⁇ i 2 ), acyloxy(c ⁇ i 2 ),
• n" is 1 or 2 and Yi is -H or alkyl(c ⁇ 8); or
• the compound is limited by the provisio that X and Y are not both each ?-butyl; and further provided that Z is hydrogen, and X is bromo or iodo, then Y is not ?-butyl.
• the carbon atom labeled ⁇ is in the R configuration. In other embodiments, the carbon atom labeled ⁇ is in the «S configuration.
• R' is -H. In other embodiments, R is alkyl( C ⁇ 8). In some embodiments, R' is ethyl.
• R" is -H. In some embodiments, R" is -H. In other embodiments, R" and R'" are both -H.
• L is hydrogen.
• Z is hydrogen.
• Z is hydroxy.
• Z is hydroxyl and attached to the carbon labeled 2.
• Z is hydroxyl and attached to the carbon labeled 6.
• X is halo.
• X is chloro.
• X is bromo.
• X is alkyl( C ⁇ i2) or substituted alkyl( C ⁇ i2).
• X is alkyl(c ⁇ i2).
• X is t-butyl.
• X is substituted alkyl( C ⁇ i2 ) .
• X is trifluoromethyl. In other embodiments, X is heteroaryl( C ⁇ s). In some embodiments, X is 3-pyridinyl. In other embodiments, X is 3-pyrimidyl. In other embodiments X is cyano. In some
• Y is t-butyl.
• Rs is alkyl( C ⁇ 8). In some embodiments, Rs is methyl. In some embodiments, Rg is alkyl( C ⁇ 8) or substituted alkyl( C ⁇ s). In some embodiments, Rg is alkyl( C ⁇ s). In some embodiments, Rg is methyl. In other embodiments, Rg is substituted alkyl( C ⁇ s ) . In some embodiments, Rg is trifluoromethyl. In some embodiments, Rio is -OH, -CN, -CF 3 , -CH 2 OH, or -CH 2 0-alkyl(c ⁇ s). In some embodiments, Rio is -OH. In other embodiments, Rio is -CN. In other embodiments, Rio is -CH 2 OH. In other embodiments, Rio is -CF 3 . In other embodiments, Rio is -CH20-alkyl( C ⁇ s). In other
• A" is a covalent bond, thereby forming a cyclopropane ring.
• A" is alkanediyl(c ⁇ 6>
• A" is -CH 2 -
• A" is alkoxydiyl(c ⁇ 8).
• A" is -CH 2 -0-CH 2 -.
• Rn is -CN.
• Rn is -CHF 2 .
• Rn is -CH 2 F.
• Rn is -CH 2 OH.
• Rn is -CH 2 0 .
• Rn is -CH 2 0-CH 3 .
• Y is In
• Y is . In other embodiments, Y is . in other
• the present disclosure contemplates the fact that the bond between the phenyl ring and the amino acid backbone on the ⁇ -amino acid is freely rotating.
• the structure may rotate such that the X group is on the oriented towards the backbone and the Y is oriented away form the backbone as well as the manner drawn in most commonly in the specification showing the X group on the oriented towards the backbone and the Y oriented away from the backbone as shown in the structures below.
• R may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise.
• Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed.
• R may reside on either the 5-membered or the 6-membered ring of the fused ring system.
• (Cn) defines the exact number (n) of carbon atoms in the group/class.
• (C ⁇ n) defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl( C ⁇ 8)” or the class “alkene(c ⁇ 8)” is two.
• alkoxy(c ⁇ io) designates those alkoxy groups having from 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms).
• Cn-n' defines both the minimum (n) and maximum number ( ⁇ ') of carbon atoms in the group.
• alkyl( C2 -io) designates those alkyl groups having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms)).
• saturated means the compound or group so modified has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below.
• the term does not preclude carbon-heteroatom multiple bonds, for example a carbon oxygen double bond or a carbon nitrogen double bond. Moreover, it does not preclude a carbon-carbon double bond that may occur as part of keto-enol tautomerism or imine/enamine tautomerism.
• aliphatic when used without the "substituted” modifier signifies that the compound/group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group.
• the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic).
• Aliphatic compounds/groups can be saturated, that is joined by single bonds (alkanes/alkyl), or unsaturated, with one or more double bonds (alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -NO2, -CO2H, -C0 2 CH 3 , -CN, -SH, -OCH3, -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , - C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• alkyl when used without the "substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, and no atoms other than carbon and hydrogen.
• cycloalkyl is a subset of alkyl.
• the groups -CH 3 (Me), -CH 2 CH 3 (Et), -CH 2 CH 2 CH 3 (w-Pr), -CH(CH 3 ) 2 (fco-Pr), -CH(CH 2 ) 2 (cyclopropyl), -CH 2 CH 2 CH 2 CH 3 (w-Bu), -CH(CH 3 )CH 2 CH 3 (sec-butyl), -CH 2 CH(CH 3 ) 2 (iso-butyl), -C(CH 3 ) 3 (tert-butyl), -CH 2 C(CH 3 ) 3 (weo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups.
• alkanediyl when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Th (methylene), -CH 2 CH 2 -, -CH 2 C(CH 3 )2CH 2 -,
• CH 2 CH 2 CH 2 CH 2 - are non-limiting examples of alkanediyl groups.
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -NO2, -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• the following groups are non- limiting examples of substituted alkyl groups: -CH 2 OH, -CH 2 C1, ⁇ CF 3 , -CH 2 CN, -CH 2 C(0)OH, -CH 2 C(0)OCH 3 , -CH 2 C(0)NH 2 , -CH 2 C(0)CH 3 , -CH 2 OCH 3 , -CH 2 OC(0)CH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , and -CH 2 CH 2 C1.
• haloalkyl is a subset of substituted alkyl, in which one or more hydrogen atoms has been substituted with a halo group and no other atoms aside from carbon, hydrogen and halogen are present.
• the group, -CH 2 C1 is a non-limiting examples of a haloalkyl.
• An “alkane” refers to the compound H-R, wherein R is alkyl.
• the term “fluoroalkyl” is a subset of substituted alkyl, in which one or more hydrogen has been substituted with a fluoro group and no other atoms aside from carbon, hydrogen and fluorine are present.
• the groups, -CH 2 F, -CF 3 , and -CH 2 CF 3 are non-limiting examples of fluoroalkyl groups.
• An “alkane” refers to the compound H-R, wherein R is alkyl.
• alkenyl when used without the "substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
• alkenediyl when used without the "substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
• alkynyl when used without the "substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen.
• alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds.
• the groups, -C ⁇ CH, -C ⁇ CCH 3 , and -CH 2 C ⁇ CCH 3 are non-limiting examples of alkynyl groups.
• substituted modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• An "alkyne” refers to the compound H-R, wherein R is alkynyl.
• aryl when used without the "substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present.
• Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C 6 H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and the monovalent group derived from biphenyl.
• aromaticiyl when used without the "substituted” modifier refers to a divalent aromatic group, with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen.
• the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused.
• alkyl group carbon number limitation permitting
• arenediyl groups include:
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , - C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• An "arene” refers to the compound H-R, wherein R is aryl.
• aralkyl when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above.
• Non-limiting examples of aralkyls are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
• substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl.
• heteroaryl when used without the "substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
• the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or unfused.
• heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.
• heteroarenediyl when used without the "substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
• the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or unfused.
• Non-limiting examples of heteroarenediyl groups include:
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , - C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• heterocycloalkyl when used without the "substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur.
• the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. If more than one ring is present, the rings may be fused or unfused.
• heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl.
• heterocycloalkyl used with the "substituted" modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• acyl when used without the “substituted” modifier refers to the group -C(0)R, in which R is a hydrogen, alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above.
• the groups, -CHO, -C(0)CH 3 (acetyl, Ac), -C(0)CH 2 CH 3 , -C(0)CH 2 CH 2 CH 3 , -C(0)CH(CH 3 ) 2 , -C(0)CH(CH 2 ) 2 , -C(0)C 6 H 5 , -C(0)C 6 H 4 CH 3 , -C(0)CH 2 C 6 H 5 , -C(0)(imidazolyl) are non-limiting examples of acyl groups.
• a “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(0)R has been replaced with a sulfur atom, -C(S)R.
• one or more hydrogen atom (including the hydrogen atom directly attached the carbonyl or thiocarbonyl group) has been independently replaced by-OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , - C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• the groups, -C(0)CH 2 CF 3 , -C0 2 H (carboxyl), -C0 2 CH 3 (methylcarboxyl), -C0 2 CH 2 CH 3 , -C(0)NH 2 (carbamoyl), and -CON(CH 3 ) 2 are non-limiting examples of substituted acyl groups.
• alkoxy when used without the "substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above.
• alkoxy groups include: -OCH 3 (methoxy), -OCH 2 CH 3 (ethoxy), -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 (isopropoxy), -OCH(CH 2 ) 2 , -C-cyclopentyl, and -O-cyclohexyl.
• alkenyloxy when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively.
• alkoxydiyl refers to the divalent group -O-alkanediyl-, -O-alkanediyl-0-, or -alkanediyl-O-alkanediyl-.
• alkylthio and acylthio when used without the “substituted” modifier refers to the group -SR, in which R is an alkyl and acyl, respectively.
• R is an alkyl and acyl, respectively.
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• alcohol corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group.
• alkylamino when used without the "substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above.
• alkylamino groups include: -NHCH 3 and -NHCH 2 CH 3 .
• dialkylamino when used without the "substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl.
• Non-limiting examples of dialkylamino groups include: -N(CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), and N-pyrrolidinyl.
• dialkylamino groups include: -N(CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), and N-pyrrolidinyl.
• alkoxyamino refers to groups, defined as -NHR, in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and alkylsulfonyl, respectively.
• a non-limiting example of an arylamino group is -NHC 6 H 5 .
• a non-limiting example of an amido group is -NHC(0)CH 3 .
• alkylaminodiyl refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-.
• alkylsulfonyl and “alkylsulfinyl” when used without the “substituted” modifier refers to the groups -S(0) 2 R and -S(0)R, respectively, in which R is an alkyl, as that term is defined above.
• alkenylsulfonyl alkynylsulfonyl
• arylsulfonyl arylsulfonyl
• aralkylsulfonyl and “heteroarylsulfonyl”
• one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, - H 2 , -N0 2 , -C0 2 H, -C0 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(0)CH 3 , -N(CH 3 ) 2 , -C(0)NH 2 , -OC(0)CH 3 , or -S(0) 2 NH 2 .
• a "chiral auxiliary” refers to a removable chiral group that is capable of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with such compounds, and many are commercially available.
• hydrate when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.
• IC5 0 refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
• An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
• the term "patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, horse, sheep, goat, pig, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
• the patient may also comprise avian, reptilian, amphibian, fish, and insect animals.
• the patient may also comprise a zoo animal or an animal raised as a pet such as a dog, cat, mouse, rat, guinea pig, lizard, snake, bird, turtle, frog, or fish.
• Non-limiting examples of avian subjects include chickens, turkeys, ducks, geese, game birds such as quail and pheasants, and pet birds, such as parakeets, cockatiel, lovebirds, parrots, and macaws. Turtles, terrapins, tortoises, snakes and lizards represent non-limiting examples of reptilian subjects or patients.
• Frogs, toads, newts and salamanders represent non-limiting examples of amphibian subjects
• the fish subject is represented by the following non-limiting examples: freshwater fish such as tilapia, salmon, catfish, carp, eel, and trout, marine fish such as tuna, cod, herring, sardine, anchovy, flounder, sole, and shark, as well as mollusk and crustacean such as shrimp, prawn, octopus, squid, lobsters, crabs, oysters, krill, and mussels.
• the patient is an insect including the non-limiting examples of honey bees.
• the patient or subject is a primate.
• Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.
• pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
• “Pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity.
• Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinna
• Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
• Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
• Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002).
• pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
• Prevention includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
• Prodrug means a compound that is convertible in vivo metabolically into an inhibitor according to the present invention.
• the prodrug itself may or may not also have activity with respect to a given target protein.
• a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound.
• esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-P-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, -toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids, and the like.
• a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.
• saturated when referring to an atom means that the atom is connected to other atoms only by means of single bonds.
• a “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs.
• “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands.
• Diastereomers are stereoisomers of a given compound that are not enantiomers.
• Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer.
• the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds.
• a molecule can have multiple stereocenters, giving it many stereoisomers.
• n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture.
• a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%.
• enantiomers and/or diasteromers can be resolved or separated using techniques known in the art.
• stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, 5 * form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures.
• the phrase "substantially free from other stereoisomers” means that the composition contains ⁇ 15%, more preferably ⁇ 10%, even more preferably ⁇ 5%, or most preferably ⁇ 1% of another stereoisomer(s).
• Effective amount means that amount which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.
• Treatment includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
• treatment of a patient afflicted with one of the pathological conditions described herein comprises administering to such a patient an amount of compound described herein which is therapeutically effective in controlling the condition or in prolonging the survivability of the patient beyond that expected in the absence of such treatment.
• inhibitor of the condition also refers to slowing, interrupting, arresting or stopping the condition and does not necessarily indicate a total elimination of the condition. It is believed that prolonging the survivability of a patient, beyond being a significant advantageous effect in and of itself, also indicates that the condition is beneficially controlled to some extent.
• ⁇ - MR is proton nuclear magnetic resonance
• AcOH is acetic acid
• Ar is argon
• ACN or CH 3 CN is acetonitrile
• CHN analysis is carbon/hydrogen/nitrogen elemental analysis
• CHNC1 analysis is carbon/hydrogen/nitrogen/chlorine elemental analysis
• CHNS analysis is carbon/hydrogen/nitrogen/sulfur elemental analysis
• DI water is deionized water
• DIC diisopropyl carbodiimide
• DMA is N,N-dimethylacetamide
• DMAP is 4-( ⁇ , ⁇ - dimethylamino)pyridine
• DMF is N,N-dimethylformamide
• EDC1 is l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
• EtOAc is ethyl acetate
• EtOH is ethanol
• FAB MS fast atom bombardment mass spectroscopy
• g is gram(s
• dilute HCI Scheme I illustrates general methodology useful for preparing the cyclic guanidine substituted left hand side aromatic acid portion of Formula I of the present invention which can then be coupled to a gly-p-amino acid ester, or to gly ester first, followed by (after ester hydrolysis) coupling to the appropriate ⁇ -amino acid ester.
• the appropriate amino benzoic (or pyridine) acid is reacted with ammonium thiocyanate in hot dilute hydrochloric to give the resulting 3 -thiourea benzoic (or pyridine) acid after normal work-up.
• the starting amino benzoic (or pyridine) acids are either commercially available or can be converted to such amino benzoic (or pyridine) acids via reduction of the corresponding nitro benzoic (or pyridine) acid, which can be obtained commercially or synthesized by nitration of the appropriate benzoic (or pyridine) acid, followed by reduction to the desired amino benzoic (or pyridine) acid, or by other reported methodologies that are known to those skilled in the art.
• This thiourea intermediate is converted to the S-methyl derivative by reaction with methyl iodide in ethanol at reflux.
• the appropriate l,3-diamino-2- substituted propane, or ethylene diamine is reacted with this resulting intermediate in hot DMA (or DMF).
• the HC1 salt may be obtained by lyophilizing from dilute hydrochloric acid.
• the product may be isolated from the original reaction mixture by removing volatiles and concentrating.
• the resulting product is taken up in water and pH adjusted to about 5-7 where zwitterionic product precipitates and is isolated by filtration.
• the HC1 salt may be obtained as previously stated or by simply dissolving in dilute hydrochloric acid and concentrating to a solid and drying.
• Scheme IA is illustrative of methodology useful for preparing the simple guanidine substituted left hand side aromatic acid portion of Formula I, which can then be coupled to a gly- -amino acid ester, or to gly ester first, followed by (after ester hydrolysis) coupling to the appropriate ⁇ -amino acid ester. This can also be accomplished using other appropriate guanidating reagents known to those skilled in the art, for example using pyrazole-carboxamidine. HCl.
• the methodology of Scheme IA can be modified using conventional techniques and methods to prepare alternate compounds useful for coupling to the ⁇ -amino acids.
• Scheme IB is illustrative of methodology useful for preparing a cyclic amidine substituted left hand side aromatic acid portion of Formula I, which can then be coupled to a gly- -amino acid ester, or to gly ester first, followed by (after ester hydrolysis) coupling to the appropriate ⁇ -amino acid ester.
• Scheme II illustrates methodology useful for preparing a preferred tetrahydropyrimidinobenzoic acid portion of Formula I or II of the present invention which can then be coupled to a gly-p-amino acid ester, or to gly ester first, followed by (after ester hydrolysis) coupling to the appropriate ⁇ -amino acid ester.
• 3,5-dihydroxybenzoic acid is converted to 3 -amino-5 -hydroxy -benzoic acid using the procedure described in Austr. J. Chem. (1981) or Becker et ah, (1983), which are incorporated herein by reference.
• the product is reacted with methyl isothiocyanate in DMF at room temperature taught by Organic Process Research & Development, 2004, which is incorporated herein by reference, to give 3-N-methyl thiourea-5-hydroxybenzoic acid after normal work-up.
• This thiourea intermediate is converted to the S-methyl derivative by reaction with methyl iodide neat at below 40 °C. l,3-diamino-2-hydroxypropane is reacted with this resulting intermediate in hot DMA (or DMF).
• the HC1 salt may be obtained by lyophilizing from dilute hydrochloric acid.
• the product may be isolated from the original reaction mixture by removing volatiles and concentrating.
• the resulting product is taken up in water and pH adjusted to about 5-7 where zwitterionic product precipitates and is isolated by filtration.
• the HCl salt may be obtained as previously stated or by simply dissolving in dilute hydrochloric acid and concentrating to a solid and drying.
• This beta amino acid ester can then be coupled to Boc-glycine followed by (after removal of the Boc protecting group) coupling to the appropriate benzoic acid described in Schemes I and II, or to the benzoic acid that has been coupled to glycine.
• To the appropriate benzaldehyde in isopropanol is added ammonium acetate followed by malonic acid. The reaction mixture is stirred at reflux, the resulting precipitate filtered and washed with hot isopropanol and dried to yield the desired racemic beta amino acid.
• the ethyl ester is synthesized by heating this acid in excess ethanol in the presence of excess HCl gas.
• These racemic beta amino acid esters can be resolved into the (R) and the preferred (S) enantiomers via chiral chromatographic separation, or via enzymatic resolution as described in Faulconbridge et al. (2000) or Landis et al. (2002), which are incorporated herein by reference.
• This beta amino acid ester can then be coupled to Boc-glycine followed by (after removal of the Boc protecting group) coupling to the appropriate benzoic acid described in Schemes I and II (preferred method), or to the benzoic acid that has been coupled to glycine.
• coumarins are readily prepared from salicylaldehydes using a modified Perkin reaction taught for example by Vogel's Textbook of Practical Organic Chemistry, 1989, which is incorporated herein by reference.
• racemic beta amino acid esters can be resolved into the (R) and the preferred (5) enantiomers via chiral chromatographic separation (for example, via the CBZ derivative of the racemic ester, which is separated on a reverse phase chiral column, providing, after deprotection with, for example, TMSI, the pure (5) and (R) beta amino acid ester enantiomers) or via enzymatic resolution as described in Faulconbridge et al. (2000) or Landis et al. (2002), which are incorporated herein by reference.
• Scheme V illustrates an alternate general methodology for the synthesis of the beta amino acid ester portion of Formula I or II of the present invention, starting from an appropriate benzaldehyde.
• This beta amino acid ester can then be coupled to Boc- glycine followed by (after removal of the Boc protecting group) coupling to the appropriate benzoic acid described in Schemes I and II, or to the benzoic acid that has been coupled to glycine.
• the appropriate benzaldehyde is converted to the corresponding cinnamate via the Wittig reaction. Michael addition of hydroxylamine to the resulting cinnamate affords the N-hydroxylated beta-amino acid ester.
• Scheme VI illustrates an alternate general methodology for the chiral synthesis of the beta amino acid ester portion of Formula I or II of the present invention, wherein Z is OH, starting from an appropriate benzaldehyde, and using a chiral auxiliary.
• This beta amino acid ester can then be coupled to Boc-glycine followed by (after removal of the Boc protecting group) coupling to the appropriate benzoic acid described in Schemes I and II (particular method), or to the benzoic acid that has been coupled to glycine.
• Scheme VI illustrates the chiral synthesis of the preferred (S) enantiomer of the desired beta amino acid ester using S-phenylglycinol as the chiral auxiliary (synthesis of the (R) isomer is afforded by utilizing R- phenylglycinol instead).
• Literature references describing such reactions include: Organic Process Research & Development (2004); Awasthi et al. (2005); U.S. Patent 6,414,180; U.S. Patent 5,840,961, which are incorporated herein by reference. Briefly, the appropriate salicylaldehyde is first treated with MEM chloride and potassium carbonate to afford the MEM protected salicylaldehyde.
• the MEM ether protected salicylaldehyde is then reacted with S-phenylglycinol in the presence of magnesium sulfate in THF to afford the imine.
• the Reformatsky reagent, tert-butyl zinc bromoacetate, is then added to the imine in N-methylpyrrolidine.
• the chiral auxiliary of the resulting beta amino acid ester is cleaved off by treatment with lead tetraacetate. Basic workup of the reaction mixture followed by heating at reflux with -toluenesulfonic acid in ethanol affords the desired PTSA salt of the (5)-beta amino acid ester.
• Scheme VII illustrates a general methodology for preparing the ethyl-N-gly- beta amino acid portion of Formula I of the present invention, which can be coupled to the benzoic acid portion of Formula I or II described in Schemes I and II).
• This method describes coupling a beta amino acid ester to glycine. Briefly, the desired beta amino acid ester (example methodologies described in Schemes III- VI above) is treated with activated Boc glycine. Removal of the Boc protective group (by treatment with ethanol/HCl, for example) affords the glycine amide of the corresponding beta amino acid ester (the preferred (S) enantiomer is afforded by utilizing the (S) -beta amino acid ester, described in the above schemes).
• the desired beta amino acid ester (example methodologies described in Schemes III- VI above) is treated with activated Boc glycine.
• Scheme IX illustrates a general methodology useful for preparing various compounds of the present invention.
• the appropriate left hand side aromatic acid (described for example in Schemes I, IA, IB, and II) is activated for coupling using known methods.
• a suitable solvent such as DMA
• NMM an equivalent of NMM is added.
• the reaction mixture is cooled to ice-bath temperatures and IBCF added.
• the gly-p-amino acid ester and NMM is added to the gly-p-amino acid ester and NMM.
• the product is purified by prep HPLC and the ester hydrolyzed to the acid by treating with a base, such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water).
• a base such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water).
• a suitable acid such as TFA can be used.
• TFA a suitable acid
• the product is isolated by prep HPLC or by isolating the zwitterion at pH 5-7 and converting to the desired salt by standard procedures. (The preferred (5) enantiomer is afforded by utilizing the (5)
• Scheme X illustrates a general methodology useful for preparing various compounds of the present invention.
• 3-Hydroxy-5-[(l,4,5,6-tetrahydro-5- hydroxy-2-pyrimidinyl)amino]benzoic acid (described for example in Scheme II) is activated for coupling using known methods.
• a suitable solvent such as DMA an equivalent of NMM is added.
• the reaction mixture is cooled to ice-bath temperatures and IBCF added.
• To the mixed anhydride intermediate is added the gly-p-amino acid ester and NMM.
• the product is purified by prep HPLC and the ester hydrolyzed to the acid by treating with a base, such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water).
• a suitable acid such as TFA can be used.
• the product is isolated by prep HPLC or by isolating the zwitterion at pH 5-7 and converting to the desired salt by standard procedures. (The preferred (5) enantiomer is afforded by utilizing the (5) - beta amino acid ester, described in the above schemes).
• Scheme XI illustrates a general methodology useful for preparing various compounds of the present invention.
• the appropriate left hand side aromatic acid (described for example in Schemes I, IA, IB and II) is activated for coupling using known methods.
• a suitable solvent such as DMA an equivalent of NMM is added.
• the reaction mixture is cooled to ice-bath temperatures and IBCF added.
• ethyl glycinate HC1 and NMM is added to the mixed anhydride intermediate.
• the product is purified by prep HPLC and the ester hydrolyzed to the acid by treating with a base, such as NaOH in a suitable solvent (water, dioxane/water or acetonitrile/water), followed by acidification.
• a base such as NaOH
• a suitable solvent water, dioxane/water or acetonitrile/water
• This gly adduct is then activated for coupling using known methods.
• a suitable solvent such as DMA an equivalent of NMM is added.
• the reaction mixture is cooled to ice-bath temperatures and IBCF added.
• the appropriate beta amino acid ester salt described, for example, in Schemes III- VI above
• the product is purified by prep HPLC and the ester hydrolyzed to the acid by treating with a base, such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water).
• a suitable acid such as TFA can be used.
• the product is isolated by prep HPLC or by isolating the zwitterion at pH 5-7 and converting to the desired salt by standard procedures (the particular (5) enantiomer is afforded by utilizing the (5) - beta amino acid ester, described in the above schemes).
• Scheme XII illustrates general synthetic methodologies for benzaldehyde starting materials that may not be readily available from commercial sources and that are useful for preparing various compounds of the present invention as described in the previous schemes.
• known methods of aromatic chlorination can be substituted for the bromination reactions depicted, thus yielding the corresponding chlorine substituted benzaldehydes.
• Such methods are well known in the art. See Kurahashi et al. (2011) in the supporting information section; Nomura et al. (2007); and March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which are all incorporated by reference herein.
• Scheme XIII illustrates general synthetic methodologies for benzaldehyde starting materials that may not be readily available from commercial sources and that are useful for preparing various compounds of the present invention as described in the previous schemes.
• known methods of aromatic chlorination can be substituted for the bromination reactions depicted, thus yielding the corresponding chlorine substituted benzaldehydes.
• Such methods are well known
• Scheme XIII illustrates general synthetic methodologies for benzaldehyde starting materials that may not be readily available from commercial sources and that are useful for preparing various compounds of the present invention as described in the previous schemes.
• it illustrates general methodologies for benzaldehyde analogues that utilize an appropriate aldehyde protected aromatic Br or I reagent, whereas the Br or I can be displaced using cross coupling or other aromatic Br or I facilitated derivatizations widely known to those skilled in the art and that yield benzaldehyde starting materials useful for preparing various compounds of the present invention as described in previous schemes.
• Z is OH
• the hydroxyl group can be protected with various protecting groups known to those skilled in the art as needed to efficiently execute the synthetic procedures depicted.
• the protecting group can subsequently be removed with known de-protecting reagents.
• Scheme XIIIA further illustrates general synthetic methodologies for benzaldehyde starting materials that may not be readily available from commercial sources and that are useful for preparing various compounds of the present invention as described in the previous schemes.
• Z is OH
• the hydroxyl group can be protected with various protecting groups known to those skilled in the art as needed to efficiently execute the synthetic procedures depicted.
• the protecting group can subsequently be removed with known de-protecting reagents. See, for example, Greene & Wuts (1999), which is incorporated herein by reference.
• a trifluoromethyl or other amenable group as defined for X in the general formula can be substituted for the substituent depicted as (Cl)Br- in the above schemes.
• Scheme XV illustrates a convenient synthetic method for the introduction of a cyano substituent in the synthesis of beta amino ester reagents wherein X is cyano as defined in the general formula and Y can be multiple amenable substituents as defined in the general formula and characterized in the above schemes and subsequent examples.
• This scheme illustrates one method for synthesizing compounds where X is cyano and is not intended to be limiting in nature and can be further adapted and modified in ways that are known to those skilled in the art.
• Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diasiereomeric, racemic form, epimerie form. and ail geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
• the (S)-enantiomer of the beta amino acid portion of formula I is the preferred enatiomer.
• Compounds may occur as racemates and racemic mixtures, single enantiomers, diastcreomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
• the chiral centers of the compounds of the present, invention can have the S or the R configuration, as defined by the lUPAC 1974 Recommendations.
• mixtures of stereoisomers may be separated using the techniques taught in the Examples section below, as well as modifications thereof.
• Tautomeric forms are also included as well as pharmaceutically acceptable salts of such isomers and tautomers.
• Atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms.
• Compounds of the present invention include those with one or more atoms that have been isotopically modified or enriched, in particular those with pharmaceutically acceptable isotopes or those useful for pharmaceutically research.
• Isotopes include those atoms having the same atomic number but different mass numbers.
• isotopes of hydrogen include deuterium and tritium
• isotopes of carbon include 13 C and 14 C.
• one or more carbon atom(s) of a compound of the present invention may be replaced by a silicon atom(s).
• one or more oxygen atom(s) of a compound of the present invention may be replaced by a sulfur or selenium atom(s).
• Compounds of the present invention may also exist in prodrug form. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g. , solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
• prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a subject, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.
• ⁇ should be recognized thai the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and us are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
• the compounds of the present invention include those that have been further modified to comprise substituents that are convertible to hydrogen in vivo.
• hydro lyzable groups such as acyl groups, groups having an oxycarbonyl group, amino acid residues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl, diphenylphosphinyl, and the like.
• acyl groups include formyl, acetyl, trifluoroacetyl, and the like.
• groups having an oxycarbonyl group include ethoxycarbonyl, tert- butoxycarbonyl (-C(0)OC(CH 3 ) 3 , Boc), benzyloxycarbonyl, / methoxy- benzyloxycarbonyl, vinyloxycarbonyl, -(p-toluenesulfonyl)ethoxycarbonyl, and the like.
• Suitable amino acid residues include, but are not limited to, residues of Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), He (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn (ornithine) and ⁇ -Ala.
• suitable amino acid residues also include amino acid residues that are protected with a protecting group.
• suitable protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethoxycarbonyl groups (such as benzyloxycarbonyl and p- nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (-C(0)OC(CH 3 ) 3 , Boc), and the like.
• Suitable peptide residues include peptide residues comprising two to five amino acid residues. The residues of these amino acids or peptides can be present in stereochemical configurations of the D-form, the L-form or mixtures thereof.
• amino acid or peptide residue may have an asymmetric carbon atom.
• suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr.
• Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom.
• suitable amino acid protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethoxycarbonyl groups (such as benzyloxycarbonyl and -nitrobenzyloxycarbonyl), tert- butoxycarbonyl groups (-C(0)OC(CH 3 ) 3 ), and the like.
• acyl groups such as formyl and acetyl
• arylmethoxycarbonyl groups such as benzyloxycarbonyl and -nitrobenzyloxycarbonyl
• tert- butoxycarbonyl groups tert- butoxycarbonyl groups
• Suitable reductively eliminable hydrogenolyzable groups include, but are not limited to, arylsulfonyl groups (such as o-toluenesulfonyl); methyl groups substituted with phenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such as ⁇ , ⁇ , ⁇ -trichloroethoxycarbonyl and ⁇ -iodoethoxycarbonyl).
• arylsulfonyl groups such as o-toluenesulfonyl
• methyl groups substituted with phenyl or benzyloxy such as benzyl, trityl and benzyloxymethyl
• arylmethoxycarbonyl groups such as benzyloxy
• Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
• a better pharmacokinetic profile e.g., higher oral bioavailability and/or lower clearance
• Such compounds and compositions are useful in inhibiting or antagonizing integrins, and therefore in another embodiment, the present invention relates to a method of inhibiting or antagonizing the ⁇ 5 ⁇ 1 integrin in particular, and additionally inhibiting or antagonizing the ⁇ and ⁇ 8 integrins.
• Such compounds and compositions may be used to inhibit or antagonize additional integrins, such as ⁇ 3, ⁇ 5 and ⁇ (herein defined as related integrins).
• the invention further involves treating or inhibiting pathological conditions associated therewith such as angiogenesis, including tumor angiogenesis, fibrosis and fibrotic diseases such as pulmonary fibrosis, renal, cardiac, and liver fibrosis, scleroderma, scarring, such as retinal, corneal and dermal scarring, retinopathy, including diabetic retinopathy and macular degeneration, vitreoretinopathy, including retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR), osteoporosis, humoral hypercalcemia of malignancy, Paget' s disease, tumor metastasis, solid tumor growth (neoplasia), arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, smooth muscle cell migration and restenosis in a mammal in need of such treatment.
• pathological conditions associated therewith such as angiogenesis, including tumor angiogenesis, fibrosis and fibrotic diseases such as pulmonary
• Such pharmaceutical agents are useful as antiviral agents, and antimicrobials. Further, such pharmaceutical agents are useful as immune system modulators via inhibition of TGF- ⁇ activation resulting from inhibiting or antagonizing the targeted integrins. Such immune modulation affects the immune activity and functions of T regulatory and T effector cells, and as such can be useful in the treatment of immune related pathologies, including autoimmune diseases such as multiple sclerosis, as well as in the treatment of tumors and infectious pathogens.
• the present invention relates to the fields of pharmaceuticals, medicine and cell biology. More specifically, it relates to pharmaceutical agents (compounds) which are useful as integrin receptor antagonists, with particularly exceptional biological activity as antagonists of the integrin a5bl, and additionally as exceptional antagonists of the integrins avb6 and avb8. As such, these compounds are useful in pharmaceutical compositions and in methods for treating conditions mediated by such integrins by inhibiting or antagonizing these integrins.
• Certain compounds of the invention may combine ⁇ 5 ⁇ 1 antagonism with antagonism of other RGD-binding integrins.
• Such mixed antagonists may be especially useful in treating or preventing diseases in which more than one integrin promotes aberrant angiogenesis. They may also be useful when a second disease process, which is either co-dependent or independent of angiogenesis, is mediated by RGD integrins that can be simultaneously affected with the anti-angiogenic antagonist.
• tumors are critically dependent on the formation of new blood vessels to sustain growth beyond a few millimeters in diameter.
• Aberrant angiogenesis in the retina is a characteristic of many blinding disorders such as wet age-related macular degeneration, vitreoretinopathies, retinopathy of prematurity, and diabetic retinopathy.
• Angiogenesis has been associated with progression of pulmonary and liver fibrosis, and with growth of the synovial pannus in rheumatoid arthritis.
• the integrins ⁇ 3 and ⁇ 5 have been implicated in promoting angiogenesis (Avraamides et al, 2008), so that their antagonism in addition to ⁇ 5 ⁇ 1 may be predicted to provide superior blockade of this process.
• Integrin ⁇ 3 is also known to play a role in tumor cell metastasis, and in the elevated bone resorption associated with osteoporosis and some cancers.
• the antagonists of the invention possess varying activity against at least five integrins that have been reported to bind the latent cytokine ⁇ complex in vitro: ⁇ , ⁇ 3, ⁇ 5, ⁇ , and ⁇ 8. See (Asano et al, 2005; Mu et al, 2002; Munger et al, 1999; Wipff et al, 2007; and Munger et al, 1998), which are incorporated herein by referenc.
• ⁇ is frequently co- expressed with the angiogenic cytokine VEGF and induces its synthesis (Ferrari et al, 2006). Aside from having vascular regulatory activity, ⁇ is a powerful inducer of fibrosis in many tissues such as lung, liver, kidney, and skin (Nishimura, 2009). Virtually all ⁇ is secreted from cells in a complex which contains the latency associated peptide (LAP).
• LAP latency associated peptide
• the integrins ⁇ 3, ⁇ 5, and ⁇ interact with the RGD motif contained within LAP, producing a conformational change in the complex which allows ⁇ to bind cellular receptors that activate pro-fibrotic pathways. Integrin ⁇ 8 also activates ⁇ in an RGD-dependent manner, but utilizes a protease-dependent mechanism distinct from the other integrins.
• Latent ⁇ is ubiquitously present in tissues, and is activated by integrins in a spatially and temporally restricted manner. Therefore, upregulation of the epithelial integrin ⁇ in the lungs or liver may promote localized collagen deposition and scarring, as has been observed in patients with idiopathic pulmonary fibrosis (Horan et al, 2008) or hepatic fibrosis (Popov et al, 2008). Similarly, ⁇ 5, and to a lesser extent ⁇ 3, are present on mesenchymal cells and are able to activate mesenchymal ⁇ (Wipff et al, 2007; Scotton et al, 2009).
• Integrin ⁇ 8 is expressed on subsets of epithelial, neural, immune, and mesenchymal cell types. In the skin, the ⁇ activation that accompanies the wound healing process mediates matrix deposition and promotes the formation of scars.
• Compounds of this invention by virtue of their ability to simultaneously inhibit several ⁇ integrins, have potential for greater efficacy in treatment of fibrosis than any previously described compounds having more restricted inhibitory profiles. Furthermore, these compounds which have exceptional ⁇ 5 ⁇ 1 potency, have unique potential for benefit in diseases characterized by both aberrant angiogenic and fibrotic pathologies
• TGFP is an important inducer of the formation of FoxP3 + regulatory T cells (T reg ) (Yoshimura, 201 1). Therefore, compounds of the present invention that inhibit the activation of TGFP may reduce T reg activity, and in turn relieve immune suppression in disease states such as cancer, when administered alone or in combination with existing therapies. Mitigation of T reg activity with such compounds also has the potential to enhance the activity of vaccines which are intended to prevent or treat cancer and infectious diseases.
• TGFP in the presence of IL-6, promotes the conversion of naive T cells to TH17 cells (Yoshimura, 2011). These cells promote a variety of autoimmune diseases.
• mice lacking all ⁇ 8 expression on dendritic cells have near complete protection from experimental autoimmune encephalitis, a model of multiple sclerosis (Melton et ah, 2010). Therefore, compounds of the present invention that inhibit the activation of TGFP may reduce Thl7 activity, and be useful in preventing or treating autoimmune disease when administered alone or in combination with existing therapies.
• Antagonism of the integrin ⁇ 3 ⁇ 4 ⁇ 3 (also known as the fibrinogen receptor), is known to block platelet aggregation as part of the blood coagulation process.
• integrin ⁇ 5 ⁇ 1 and other integrins it would be beneficial to utilize compounds which selectively spare ⁇ 3 ⁇ 4 ⁇ 3.
• a role for ⁇ 5 in normal maintenance of the retina has also been described (Nandrot et ah, 2006). Therefore, in some uses of compounds, it may be desirable to spare ⁇ 5 inhibition.
• integrins are a family of integral cytoplasmic membrane proteins that mediate cell interactions with other cells and with the extracellular matrix (ECM). They also play a role in cell signaling and thereby regulate cellular shape, motility, and the cell cycle. Not only do integrins perform "outside-in” signaling typical of receptors, but they also operate an "inside-out” mode. Thus, they transduce information from the ECM to the cell as well as reveal the status of the cell to the outside, allowing rapid and flexible responses to changes in the environment, for example to allow blood coagulation by platelets.
• ECM extracellular matrix
• Integrins are of vital importance to all animals and have been found in all animals investigated, from sponges to mammals. As such compounds which target integrins have found numerous uses in different animals including companion animals, livestock animals, zoo animals as well as wild animals. Integrins have been extensively studied in humans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins and syndecans to mediate cell-cell and cell-matrix interaction and communication. Integrins bind cell surface and ECM components such as fibronectin, vitronectin, collagen, and laminin.
• integrin dimers When released into the cell membrane, newly synthesized integrin dimers are speculated to be found in the same "bent" conformation revealed by the structural studies described above. One school of thought claims that this bent form prevents them from interacting with their ligands, although bent forms can predominate in high-resolution EM structures of integrin bound to an ECM ligands. Therefore, integrin dimers must apparently not be 'unbent' in order to prime them and allow their binding to the ECM. In cells, the priming is accomplished by a protein named Talin, which binds to the ⁇ tail of the integrin dimer and changes its conformation.
• talin proteins are able to dimerize and thus are thought to intervene in the clustering of integrin dimers which leads to the formation of a focal adhesion.
• the kindlin-1 and Kindlin-2 proteins have also been found to interact with integrin and activate it.
• Each integrin is formed by the non-covalent heterodimerization of alpha and beta glycoprotein subunits, the combination of which conveys distinct biological activities such as cell attachment, migration, proliferation, differentiation, and survival.
• 24 integrins have been described in mammals that are formed by pairing of 18 a subunits and 8 ⁇ subunits:
• variants of some of the subunits are formed by differential splicing; for example, four variants of the beta-1 subunit exist. Through different combinations of these a and ⁇ subunits, some 24 unique integrins are generated, although the number varies according to different studies.
• Integrin subunits span the plasma membrane and in general have very short cytoplasmic domains of about 40-70 amino acids. The exception is the beta-4 subunit, which has a cytoplasmic domain of 1088 amino acids, one of the largest known cytoplasmic domains of any membrane protein. Outside the cell plasma membrane, the a and ⁇ chains lie close together along a length of about 23 nm; the final 5 nm N-termini of each chain forms a ligand-binding region for the extracellular matrix (ECM).
• ECM extracellular matrix
• the molecular mass of the integrin subunits can vary from 90 kDa to 160 kDa.
• Beta subunits have four cysteine-rich repeated sequences. Both a and ⁇ subunits bind several divalent cations. The role of divalent cations in the a subunit is unknown, but may stabilize the folds of the protein. The cations in the ⁇ subunits are more interesting: they are directly involved in coordinating at least some of the ligands that integrins bind.
• integrins carrying this domain either bind to collagens (e.g., integrins al ⁇ , and 2 ⁇ ), or act as cell-cell adhesion molecules (integrins of the ⁇ 2 family).
• This a-I domain is the binding site for ligands of such integrins.
• Those integrins that don't carry this inserted domain also have an A-domain in their ligand binding site, but this A-domain is found on the ⁇ subunit.
• the A-domains carry up to three divalent cation binding sites.
• One is permanently occupied in physiological concentrations of divalent cations, and carries either a calcium or magnesium ion, the principal divalent cations in blood at median concentrations of 1.4 mM (calcium) and 0.8 mM (magnesium).
• the other two sites become occupied by cations when ligands bind - at least for those ligands involving an acidic amino acid in their interaction sites.
• An acidic amino acid features in the integrin-interaction site of many ECM proteins, for example as part of the amino acid sequence Arginine-Glycine-Aspartic acid ("RGD").
• the invention also relates to a method of inhibiting or antagonizing the ⁇ 5 ⁇ 1 integrin in particular, as well as ⁇ and ⁇ 8 and related integrins. More specifically, it relates to a method of inhibiting pathological conditions associated therewith such as angiogenesis, including tumor angiogenesis, fibrosis and fibrotic diseases such as pulmonary, renal, cardiac and liver fibrosis, scarring, such as retinal, corneal and dermal scarring, retinopathy, including diabetic retinopathy and macular degeneration, vitreoretinopathy, including retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR), osteoporosis, humoral hypercalcemia of malignancy, Paget's disease, tumor metastasis, solid tumor growth (neoplasia), arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, smooth muscle cell migration and restenosis, autoimmune disease, such as multiple
• compounds of the present invention may be administered orally, parenterally, or by inhalation spray, or topically in unit dosage formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles.
• parenteral as used herein includes, for example, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
• the compounds of the present invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended.
• Therapeutically effective doses of the compounds required to prevent or arrest the progress of or to treat the medical condition are readily ascertained by one of ordinary skill in the art using preclinical and clinical approaches familiar to the medicinal arts.
• the present invention provides a method of treating conditions mediated by inhibiting or antagonizing the a5bl, avb6, avb8 and related cell surface integrin receptors, which method comprises administering a therapeutically effective amount of a compound selected from the class of compounds described above, wherein one or more compounds is administered in association with one or more non- toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as "carrier" materials) and if desired other active ingredients. More specifically, the present invention provides a method for inhibition of the a5bl, avb6, avb8 and related cell surface integrin receptors.
• the present invention provides a method for inhibiting angiogenesis, including tumor angiogenesis, inhibiting and treating fibrosis and fibrotic diseases such as pulmonary fibrosis and liver fibrosis, inhibiting and treating scarring, such as retinal, corneal and dermal scarring, inhibiting and treating retinopathy, including diabetic retinopathy and macular degeneration, inhibiting and treating vitreoretinopathy, including retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR), inhibiting bone resorption, treating osteoporosis, treating humoral hypercalcemia of malignancy, treating Paget' s disease, inhibiting tumor metastasis, inhibiting solid tumor growth (neoplasia), treating arthritis, including rheumatoid arthritis, treating periodontal disease, treating psoriasis, inhibiting smooth muscle cell migration and restenosis, treating autoimmune disease, such as multiple sclerosis, and inhibiting and treating infectious pathogens
• angiogenesis including tumor
• the compounds described above can be used in the treatment of patients suffering from the above pathological conditions.
• selection of the most appropriate compound of the invention is within the ability of one with ordinary skill in the art and will depend on a variety of factors including assessment of results obtained in standard assay and animal models.
• the compounds of the invention can be used in a variety of biological, prophylactic or therapeutic areas. It is contemplated that these compounds are useful in prevention or treatment of any disease state or condition wherein the a5bl, avb6, avb8 and related integrins plays a role. V. Pharmaceutical Formulations and Routes of Administration
• the compounds in a therapeutically effective amount are ordinarily combined with one or more excipients appropriate to the indicated route of administration.
• the compounds of the present invention are contemplated to be formulated in a manner ameniable to treatment of a veterinary patient as well as a human patient.
• the veterinary patient may be a companion animal, livestock animals, zoo animals, and wild animals
• the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and tableted or encapsulated for convenient administration.
• the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.
• Other excipients and modes of administration are well and widely known in the pharmaceutical art and may be adapted to the type of animal being treated.
• compositions useful in the present invention may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional pharmaceutical carriers and excipients such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.
• the compounds of the present disclosure may be administered by a variety of methods, e.g., orally or by injection (e.g. subcutaneous, intravenous, intraperitoneal, etc.).
• the active compounds may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. They may also be administered by continuous perfusion/infusion of a disease or wound site.
• the therapeutic compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent.
• suitable diluents include saline and aqueous buffer solutions.
• Liposomes include water- in-oil-in- water CGF emulsions as well as conventional liposomes.
• the therapeutic compound may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally.
• Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
• compositions may be suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
• Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the therapeutic compound into a sterile carrier which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., the therapeutic compound) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the therapeutic compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
• the therapeutic compound and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
• the therapeutic compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• the percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient.
• the therapeutic compound may also be administered topically to the skin, eye, or mucosa. Alternatively, if local delivery to the lungs is desired the therapeutic compound may be administered by inhalation in a dry-powder or aerosol formulation.
• Active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient.
• the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.
• HED human equivalent dose
• HED Animal dose (mg/kg)
• X Animal K m /Human K m
• K m values for humans and various animals are well known. For example, the K m for an average 60 kg human (with a BSA of 1.6 m 2 ) is 37, whereas a 20 kg child (BSA 0.8 m 2 ) would have a K m of 25.
• mice K m of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster K m of 5 (given a weight of 0.08 kg and BSA of 0.02); rat K m of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey K m of 12 (given a weight of 3 kg and BSA of 0.24).
• HED dose Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment and the potency, stability and toxicity of the particular therapeutic formulation.
• the actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors may be determined by a skilled artisan.
• the practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage may be adjusted by the individual physician in the event of any complication.
• An effective amount typically will vary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, from about 10.0 mg/kg to about 150 mg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above).
• Other suitable dose ranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500 mg to 10000 mg per day, and 500 mg to 1000 mg per day. In some particular embodiments, the amount is less than 10,000 mg per day with a range of 750 mg to 9000 mg per day.
• the effective amount may be less than 1 mg/kg/day, less than 500 mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It may alternatively be in the range of 1 mg/kg/day to 200 mg/kg/day.
• the unit dosage may be an amount that reduces blood glucose by at least 40% as compared to an untreated subject.
• the unit dosage is an amount that reduces blood glucose to a level that is ⁇ 10% of the blood glucose level of a non-diabetic subject.
• a dose may also comprise from about 1 micro- gram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
• a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
• a pharmaceutical composition of the present disclosure may comprise, for example, at least about 0.1% of a compound of the present disclosure.
• the compound of the present disclosure may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
• Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, subjects may be administered two doses daily at approximately 12 hour intervals. In some embodiments, the agent is administered once a day.
• the agent(s) may be administered on a routine schedule.
• a routine schedule refers to a predetermined designated period of time.
• the routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined.
• the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between.
• the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc.
• the invention provides that the agent(s) may taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the subject has eaten or will eat.
• the compounds of the present invention may also find use in combination therapies.
• Effective combination therapy may be achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, administered at the same time, wherein one composition includes a compound of this invention, and the other includes the second agent(s).
• the therapy may precede or follow the other agent treatment by intervals ranging from minutes to months.
• Non-limiting examples of such combination therapy include combination of one or more compounds of the invention with another anti-inflammatory agent, a chemotherapeutic agent, radiation therapy, an antidepressant, an antipsychotic agent, an anticonvulsant, a mood stabilizer, an anti-infective agent, an antihypertensive agent, a cholesterol-lowering agent or other modulator of blood lipids, an agent for promoting weight loss, an antithrombotic agent, an agent for treating or preventing cardiovascular events such as myocardial infarction or stroke, an antidiabetic agent, an agent for reducing transplant rejection or graft-versus-host disease, an anti-arthritic agent, an analgesic agent, an anti-asthmatic agent or other treatment for respiratory diseases, or an agent for treatment or prevention of skin disorders.
• Compounds of the invention may be combined with agents designed to improve a patient's immune response to cancer, including (but not limited to) cancer vaccines.
• the above crude reaction mixture was cooled to 10 °C (ice-bath) and a 2.5 N NaOH solution (90.0 mL) was added slowly with stirring, the solution temperature was kept below 20 °C, to give a pale yellow solution/suspension.
• the reaction mixture was stirred at room temperature for 1.5 h.
• the reaction mixture was acidified with 5N HCl with stirring to pH 5 to give a colorless precipitate and the mixture was stirred at room temperature for another 15 min and filtered to give a colorless solid.
• the solid was washed with water (1 x25 mL) and then with acetonitrile (1 x25 mL). The solid was dried in-vacuo to give a colorless powder (9.686 g, yield 88%).
• 5-guanidino benzoic acid was prepared according to the following procedure:
• step 2 70% 3 Into a stirred solution of compound 2 (83 g, 0.28 mol) in anhydrous CH 3 OH
• Example C A mixture of compound 4 (14.5 g, 0.069 mol) in NH 4 OH (100 ml) was heated to reflux and stirred for 3 h. The solid formed was filtered and dried. 5-guanidino benzoic acid (Example C) (5.68 g, 46%) was obtained as a white solid.
• step 2 48%
• Example D A solution of compound 2 (11.6 g , 0.054 mol) and compound 3 (7.0 g, 0.05 mol) in CH 3 CN/DMF (50 ml/20 ml) was stirred at room temperature for 2d. The solid formed was filtered and dried. Example D (7.28 g, 48%) was obtained as a white solid.
• Example E A solution of compound 2 from Example D (15.5 g , 0.072 mol) and 3- amino-5 -hydroxy benzoic acid (10 g, 0.065 mol) in CH 3 CN/DMF (50 ml/20 ml) was stirred at room temperature for 2d. The solid formed was filtered, dried. Then the solid was purified by Prep-HPLC to give Example E (2.1 g, 10%), obtained as a 0 white solid.
• 5-guanidino nicotinic acid was prepared according to the following procedure:
• Example G A mixture of compound 4 from example F (15.5 g, 0.074 mol) and the hydroxy diamino propane (20 g, 0.22 mol) in DMF (100 ml) was heated to reflux and stirred for 5 h. The solid formed was filtered and dried. Example G (5.2 g, 30%) was obtained as a white solid.
• Step-2 Preparation of 3-Amino-3 utyl-phenyl)-pro ionic acid
• Step-4 Preparation of (S)-3-Amino-3-(3-bromo-5-tert-butyl-phenyl)-propionic acid
• Step-6 Preparation of (S)-3-(3-Bromo-5-tert-butyl-phenyl)-3-(2-tert- butoxycarbonylamino-acetylamino)-propionic acid ethyl ester
• Step-7 Preparation of (S)-3-(2-Amino-acetylamino)-3-(3-bromo-5-tert-butyl- phenyl)-propi ochloride salt
• Step-2 Preparation of (S)-3-(3-cyano-5-tert-butyl-phenyl)-3-(2-tert- butoxycarbonylam acid ethyl ester
• step 1 The product from step 1 (250 mg, 0.515 mmol), and Zn (6.7 mg, 0.103 mmol) were taken in DMF (5 ml) and the resulting suspension was degassed with argon for 10 mins.
• Zn(CN) 2 (60.4 mg, 0.515 mmol) followed by Pd(OAc) 2 (11.56 mg, 0.052 mmol) and Di-ter-butyl-phosphino-l, l-binapthyl (20.52 mg, 0.052 mmol) were added to it and further degassed with argon for 15 min.
• reaction mass was cooled to room temperature, 2N NH 4 OH solution (20 mL) was added to it.
• Step-3 Preparation of (S)-3-(2-Amino-acetylamino)-3-(3-tert-butyl-5-cyano- phenyl)-propi rochloride salt
• step 2 The product from step 2 (100 mg, 0.232 mmol) was treated with 4N Dioxane- HCl (2 mL) at 0 °C for 2 hrs. Reaction mixture was concentrated and the residue was triturated with pentane (2 x 5 mL) to afford the desired compound as the HCl salt (150 mg) as an off-white sticky solid.
• Step-1 Preparation of 2-(3-bromo-5-trifluoromethyl-phenyl)-[l,3]dioxolane
• Step-3 Preparation of 3-(l-Hydroxy-l-methyl-ethyl)-5-trifluoromethyl- benzaldehyde
• the reaction mixture was added 80 mL of NH 4 C1 and the reaction mixture was stirred for 15 min.
• the ether layer was separated, washed with water (2 x 250 mL), dried over anhydrous NaS0 4 , filtered and evaporated in vacuo,
• the product was purified by silica-gel flash chromatography to give the desired product (16 g, 67%).
• step 2 the product from step 2 (3 g, 11 mmol), Pd 2 (dba) 3 (0.20 g, 0.22 rnmol) and P(tBu) 3 (0.083 g, 0.22 mmol) are added and the reaction mixture is stirred for 1 h.
• the reaction mixture is quenched with 1 IN HCT in Et 2 0 to precipitate the dicyelobexylarnine as HQ salt.
• the reaction mixture is filtered and concentrated.
• the desired compound is obtained after purification by flash-cnromatography on silica gel to give the desired product (2.3g, 70%).
• step 5 The product from step 5 (2.2 g, 8 mmol) and PTS (0.3 g, 1.6 mmol) were dissolved in anhydrous acetone (100.0 mL) in a dried flask under nitrogen. Then, RT stirred, TLC traced, TLC showed the reaction finished. A satd. NaHC0 3 soln. (10 mL) was added, EtOAc (100 mL x 2) extraction, dried over anhydrous Na 2 S0 4 , filtered and evaporated in vacuo to give the desired crude product. (1.7 g, crude) The crude products was used as such in the next reaction.
• Steps 1 to 9 were repeated to yield additional amounts of each of the desired
• step 1 the product from step 1 (3 g, 1 1 mmol), Pd 2 (dba) 3 (0.20 g, 0.22 mmol) and P(t-Bu) 3 (0.083 g, 0.22 mmol) are added and the reaction mixture is stirred for 1 h.
• the reaction mixture was quenched with 1 N HC3 in Et?0 to precipitate the dicyclohexylamine as HC3 salt.
• the reaction mixture is filtered and concentrated.
• the desired compound is obtained after purification by flash-chroraatography on silica gel to give the desired product (2.3 g, 70%).
• Step 5 To a solution of the crude product from step 3 (19 g, 63 mmol) in THF (10 mL) at 25 °C under argon is added NaH. After 10 min stirring at 25 °C, Mel is added and solution is stirred at 80 °C for 1 h. The reaction mixture is extracted with EtOAc. The organic layers are washed with water and brine, dried over NazSO filtered and concentrated. The crude desired product (20 g, crude) is used as such in the next reaction. Step 5
• step 4 The crude product from step 4 (20 g, 63 mmol) and PTS (2.3 g, 12 mmol) were dissolved in anhydrous acetone (200.0 mL) in a dried flask under nitrogen and stirred at RT for several hours. TLC showed the reaction was finished. Saturated aHC0 3 soln. (50 mL) was added, the mixture was extracted with EtOAc (200 mL x 2),dried over anhydrous a2S0 4 , filtered and evaporated in vacuo to give the desired product (17 g, quant).
• step 5 The product from step 5 (17 g, 63 mmol), mono-ethyl malonate (16 g, 126 mmol) and ammonium acetate (24 g, 315 mmol) in anhydrous ethanol (50.0 mL) were heated at reflux for 7 h to give a pale yellow solution.
• the reaction mixture was cooled to room temperature and the solvent was evaporated in vacuo to give a yellow viscous liquid.
• the residue was partitioned between aqueous saturated aHC0 3 solution (100 mL) and ethyl acetate (100 mL). The organic layer was removed, dried over anhydrous sodium sulfate, filtered and evaporated in vacuo.
• the product was purified by Silica-gel flash chromatography to give the desired product (3.5 g, 17%) as a racemate.
• the racemic BOC protected product from step 7 was separated by SFC to give the individual (S) and (R) BOC enantiomers ( ⁇ 1 gram each enantiomer).
• step 3 To a solution of the product from Example L, step 3 (15g, mmol) in DCM (300 mL) at -60 °C under argon is added DAST (24g, I SOmmoI). After 1 hour stirring at -60 °C, water was added and solution is stirred at 20 °C for 1 h. The reaction mixture is extracted with DCM. The organic layers were washed with water and brine, dried over Na 2 SC>4, filtered and concentrated. The crude desired product (12 g, crude) is used as such in the next reaction.
• step 1 The product from step 1 (12 g, 40 mmol) and PTS (2.3 g, 12 mmol) were dissolved in anhydrous acetone (120.0 mL) in a dried flask under nitrogen and stirred at RT for several hours. TLC showed the reaction was finished. Saturated aHC0 3 soln., 50mL, was added and the mixture was extracted with EtOAc (200ml x 2), dried over anhydrous a 2 S0 4 , filtered and evaporated in vacuo to give the crude desired product (2.3 g, 18.7% yield).
• step 2 The product from step 2 (2.3 g, 8.9 mmol), mono-ethyl malonate (5.2 g, 40 mmol) and ammonium acetate (5.6 g, 80 mmol) in anhydrous ethanol (50.0 mL) was heated at reflux for 7 h to give a pale yellow solution.
• the reaction mixture was cooled to room temperature and the solvent was evaporated in vacuo to give a yellow viscous liquid.
• the residue was partitioned between aqueous saturated aHC0 3 solution (100 mL) and ethyl acetate (100 mL). The organic layer was removed, dried over anhydrous sodium sulfate, filtered and evaporated in vacuo.
• the product was purified by Silica-gel flash chromatography to give the desired product (0.7 g).
• the racemic BOC protected product from step 4 was separated by SFC to give the individual (S) and (R) BOC enantiomers (- 1 10 mg each enantiomer).
• 1, 1,1-trifluoro acetone (2.2 g, 20.0 mmol) was added to above reaction mixture dropwise, keeping the reaction mixture below -30 °C. After addition is complete, the reaction mixture was warmed slowly to -30 °C. (30 min) and then stirring at room temperature. The reaction mixture was poured into 40 mL of aqueous NH 4 C1 and the reaction mixture was stirred for 15 min. The isopropyl ether was separated, dried over anhydrous MgS0 4 , filtered and evaporated in vacuo to give the compound 4 (4.0 g, crude) as a pale yellow viscous liquid. Step 4
• reaction mixture was poured into 40 mL of aqueous NH 4 CI and the reaction mixture was stirred for 15 min.
• the organic phase was separated, dried over anhydrous MgS0 4 , filtered and evaporated in vacuo to give compound 6 (10 g, crude) as a liquid.
• reaction mixture was poured into 40 mL of aqueous NH 4 CI and the reaction mixture was stirred for 15 min.
• the organic phase was separated, dried over anhydrous MgSC , filtered and evaporated in vacuo to give compound 4 (10 g, crude) as a liquid.
• 1,3-Dibromo-benzyl alcohol (1) (20 g, 75.2 mmol) was dissolved in anhydrous DCM (200 mL) in a dried flask under nitrogen. The reaction mixture was cooled to 0 °C and stirred under nitrogen atmosphere. DIPEA (25.8 mL, 150.4 mmol) was added drop wise to the above solution, after 10 minutes of stirring at 0 °C, mesyl cholride (8.7 mL, 112.8 mmol) was added drop-wise to the above reaction mixture. Finally, the reaction mixture was allowed to stir at RT for 2 hrs.
• Benzyl triethyl ammonium chloride (0.35 gm, 1.55 mmol) in 50% NaOH (50 mL) was added to a (3,5-Dibromo-phenyl)-acetonitrile (3) (8.5 gm, 30.9 mmol), 1,2-dibromoethane (7.9 mL, 91.5 mmol) solution at 0 °C.
• TEBAC Benzyl triethyl ammonium chloride
• reaction mixture was concentrated under reduced pressure and the resulting residue was dissolved in water and neutralized with saturated aHC0 3 solution, extracted with ethyl acetate (4 x 75 mL), the organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford 4.5 gm of crude product which was purified by column chromatography [silica (100- 200 mesh) and 0.5 % MeOH in DCM as eluent] to afford 1.3 gm of desired compound 8 as pale brown liquid.
• Zinc metal (3.84 g, 58.80 mmol) and anhydrous THF (30 mL) were placed in a 150 mL flask that was fitted with a condenser under nitrogen atmosphere. With magnetic stirring, 1,2-dibromoethane (152 ⁇ , 1.76 mmol) was added and the suspension of zinc in THF was heated to reflux (65-70 °C) for 1 h at this temperature. The reaction mixture was cooled to 50 °C before charging the /er/-butyl bromoacetate (9.0 mL, 60.95 mmol) dropwise by a syringe in the following sequence: 2.0 mL, 2.0 mL and 2.0 mL. The reaction mixture was heated with stirring under nitrogen atmosphere.
• reaction mixture was warmed to 58 °C and within a few minutes of heating at 58 °C (15-20 min), an exotherm was observed and the reaction mixture starting to boiling, zinc started to disappear.
• the reaction mixture was charged with remaining tert-butyl bromoacetate (3.00 mL) after that exotherm allowed to subside, a colorless solid started to precipitate and increased with time.
• the reaction mixture was heated for another 1 h at 60 °C with stirring to give a pale yellow-green solution. After 1 h, the reaction mixture was cooled to room temperature and then at 0 °C to give a colorless precipitate.
• the reaction mixture was stored in a freezer (-20 °C) overnight under nitrogen atmosphere.
• reaction mixture was cooled to -10 °C (salt/ice bath).
• a mixture of cone. HC1 (1.0 mL) in 100 mL of a saturated ammonium chloride solution was added slowly to above cold reaction mixture in 10 min and the reaction mixture was stirred at 0 °C (ice-bath) to give a yellow-orange solution.
• the reaction mixture was warmed to room temperature.
• MTBE 80 mL was added and the reaction mixture was stirred at room temperature for 30 min. Stirring was stopped and the layers were separated. The aqueous layer was extracted with MTBE (20 mL).
• a dirty cream gummy solid precipitated in a yellow-orange heptane mother liquor was decanted off and the residue was triturated with heptane (10 mL).
• the heptane layer was removed and combined with the decanted heptane layer.
• a colorless precipitate started to deposit in the combined heptane layer and the amount of the solid increased significantly after standing at room temperature for 30 min.
• the precipitated solid was filtered, washed with heptane and dried in vacuo to afford a white solid (6.24 g).
• Acetonitrile was evaporated on a rotary evaporator to give a pale yellow aqueous residue.
• the residue was extracted with dichloromethane (2 x 25 mL) to remove HOBt and DIPU.
• the aqueous layer was neutralized with TFA (3.0 mL in 5.0 mL CH3CN) and the mixture was evaporated in vacuo to give a pale viscous residue.
• the crude product was purified by reverse-phase HPLC on Biotage SP1 system using a gradient 10-40% CH 3 CN in water containing 0.05% TFA to afford a colorless glassy solid.
• the purified product was dissolved in water containing a few drops of acetonitrile and lyophilized to afford a colorless powder (778.0 mg).
• Zinc metal (3.44 g, 52.60 mmol) and anhydrous THF (30 mL) were placed in a 150 mL flask that was fitted with a condenser under nitrogen atmosphere. With magnetic stirring, 1,2-dibromoethane (150 ⁇ , 1.76 mmol) was added and the suspension of zinc in THF was heated to reflux (65-70 °C) for 1 h at this temperature. The reaction mixture was cooled to 50 °C before charging the /er/-butyl bromoacetate (7.80 mL, 52.80 mmol) dropwise by a syringe in the following sequence: 2.0 mL and 2.0 mL. The reaction mixture was heated with stirring under nitrogen atmosphere.
• reaction mixture was warmed to 58 °C and within a few minutes of heating at 58 °C (15-20 min), an exotherm was observed and the reaction mixture starting to boiling, zinc started to disappear.
• the reaction mixture was charged with remaining tert-butyl bromoacetate (3.80 mL) after that exotherm allowed to subside, a colorless solid started to precipitate and increased with time.
• the reaction mixture was heated for another 1 h at 60 °C with stirring to give a pale yellow-green solution. After 1 h, the reaction mixture was cooled to room temperature and then at 0 °C to give a colorless precipitate.
• the reaction mixture was stored in a freezer (-20 °C) overnight under nitrogen atmosphere.
• a colorless to white crystalline solid with a pale yellow- green mother liquor was removed by a double-tipped cannula to give a colorless to dirty white solid, still contains the residual THF (2-3 mL).
• the solid precipitate was triturated with anhydrous THF (10 mL) and the THF wash was removed by a cannula to give a cream cake.
• the crude tert- butoxycarbonylmethylzinc bromide will be used as such in the next step.
• Solid Reformatsky reagent ⁇ 2 0000( ⁇ 3 ) ⁇ 3 . ⁇ (52.0 mmol) from the Step # 4-1 was dissolved in anhydrous l-methyl-2-pyrrolidinone (NMP) (12.0 mL) at -10 to -15 °C (ice/salt bath) under nitrogen to give a pale yellow solution.
• NMP l-methyl-2-pyrrolidinone
• the crude imine from Step # 3 (9.40 g, 20.24 mmol) was dissolved in anhydrous NMP (15.0 mL) under nitrogen atmosphere separately.
• the solution of the imine in NMP was added slowly to a solution of the Reformatsky reagent in NMP at -15 °C in 30 min under nitrogen atmosphere.
• the reaction mixture was first let stirred at -10 °C for 2 h and then at -5°C for another 1 h.
• the reaction mixture was cooled to -10 °C (salt/ice bath).
• a mixture of cone. HC1 (1.0 mL) in 100 mL of a saturated ammonium chloride solution was added slowly to above cold reaction mixture in 10 min and the reaction mixture was stirred at 0 °C (ice-bath) to give a yellow-orange solution.
• MTBE 80 mL was added and the reaction mixture was stirred at room temperature for 30 min. Stirring was stopped and the layers were separated.
• Acetonitrile was evaporated on a rot-vap to give a pale yellow aqueous residue.
• the residue was diluted with water (10 mL) and filtered to rempve precipitated urea.
• the aqueous layer was neutralized with TFA (1.0 mL in 5.0 mL CH 3 CN) and the mixture was evaporated in vacuo to give a pale yellow-cream foamy solid.
• the crude product was purified by reverse- phase HPLC using a gradient 10-40% CH 3 CN in water containing 0.05% TFA to afford a colorless glassy solid.
• the purified product was dissolved in water containing a few drops of acetonitrile and lyophilized to afford a colorless powder (407.0 mg).

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