Patent Application
20110034521
The present invention provides compounds and methods for treating zinc matrix metalloprotease dependent diseases.
Cancer (neoplasia) is characterized by deregulated cell growth and cell division. Cancers include carcinomas which are tumors arising in a tissue originating from endoderm or exoderm, and sarcomas which originate from mesoderm (Darnell, J., Molecular Cell Biology, Third Ed., W.H. Freeman, NY, 1990). Solid tumors are found in nervous system, breast, retina, lung, skin, kidney, liver, pancreas, genito-urinary tract, gastrointestinal tract, cancers of bone, and cancers of hematopoietic origin include various types of leukemia and lymphoma.
Tumor cell invasion and metastasis are associated with destruction of the basement membrane and extracellular matrix by various secreted proteinases from malignant and stromal cells. Thus a variety of matrix metalloproteases (MMP), enzymes that degrade extracellular matrix proteins, have been associated with tumor growth, invasion, and metastasis, and have important roles at several stages in progression of metastatic cancer. Zinc proteases are an example of matrix metalloproteases that contain zinc at the active site of the enzyme.
Extracellular matrix components, such as collagen, proteoglycan, fibronectin, vitronectin and laminin that are degraded by zinc matrix metalloproteases facilitate detachment of tumor cells and invasiveness. For example, proteolytic degradation of structural protein in the basal membrane is involved with expansion of a tumor at a primary site, evasion from this site, and homing and invasion of metastatic cells at distant secondary sites. Further, tumor induced angiogenesis that is required for tumor growth is dependent on proteolytic tissue remodeling.
Inhibitors of MMPs have been studied for potential therapeutic effects on cancer cells and metastasis. One class of MMP inhibitors are compounds which contain a hydroxamate group, i.e., a nitrogen atom bonded to a hydroxyl group, and the nitrogen is also bonded to a carbonyl group. Hydroxamate groups interact with metal ions such as zinc in active pocket of enzymes to disrupt the functionality of the enzyme. However, a hydroxamate reacts in general with metal ions, therefore such a compound can have undesirable non-specific side effects.
Other inhibitors of MMPs that have been widely studied for anti-cancer activities include Batismastat and Marimastat. Batismastat's usefulness has been limited by poor water solubility, requiring intraperitoneal administration of the drug as a detergent emulsion (Wojtowwicz-Praga et al., Investigational New Drugs 15:61-75, 1997). Marimastat's toxicity, particularly musculoskeletal toxicity, makes this compound less attractive as an anti-cancer drug (Sparano et al., J Clin Oncol 22(23):4683-4690, 2004).
There is a need for active inhibitory compounds that are suitable for treating MMP dependent diseases, such as but not limited to cancerous tumors and metastasis, and that are stable, efficacious, and specific with minimal side effects.
The present invention provides compositions that are useful for treating MMP dependent diseases, such as cancer and metastasis. An aspect of the invention provides a compound of formula I,
in which: X is a C3-C6 heterocycloalkenyl, wherein the atoms of the ring are optionally substituted by R6, and wherein when one or more heteroatoms are nitrogen, the nitrogens are each independently unsubstituted or substituted by R7; R1 is present at n occurrences, n is an integer from 0 to 1, and R1 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R2 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R3 is present at m occurrences, m is an integer from 0 to 1, and R3 is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is present at p occurrences, p is an integer from 0 to 1, and R4 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs, H, —C(═O)R9, and —C(═O)OR9; R6 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R9; R7 is selected from H, C1-C6 alkyl optionally substituted by R8; C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and aryl optionally substituted by halo or C1-C6 alkyl; R8 is selected from C(═O)OR9, OR9, and halo; R9 is a C1-C6 alkyl optionally substituted by aryl; R10 is selected from H, halo, OR9, OH, NO2, NH2, alkoxy, cyano, SO2CH3, SO2NH2, COCH3, COCH3, CONH2, CHO and C1-C6 alkyl optionally substituted by halo; R11 is an aryl optionally substituted by halo; or pharmaceutically acceptable salts and prodrugs thereof.
In a related embodiment of the compound of formula I: X is a C3-C6 heterocycloalkenyl, wherein carbon atoms of the ring are optionally substituted by R6, and wherein when one or more heteroatoms are nitrogen, the nitrogens are each independently unsubstituted or substituted by R7; R1 and R2 are independently selected from H or methyl; is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R6 and R7 are independently selected from H or methyl; or pharmaceutically acceptable salt and prodrugs thereof.
In certain embodiments, X is at least one compound selected from the group consisting of pyrazole, thiazole, and thiadiazole. In a related embodiment, the pyrazole is a 1,2 pyrazole. In another related embodiment, the thiazole is a 1,3 thiazole. In yet another related embodiment, the thiadiazole is a 4-thia-1,2 diazole.
In another embodiment of the compound of formula I: R1 and R2 are both H. In another embodiment of the compound of formula I: R6 and R7 are both H. In another embodiment of the compound of formula I: R3 is at least one compound selected from the group consisting of phenyl, furyl, pyridyl, and thiophene. In a related embodiment, the thiophene is 2-thiophene.
Another aspect of the invention provides a compound of formula II,
in which: R1 is present at m occurrences, m is an integer from 0 to 1, and R1 is C1-C6 alkyl or C—R8; R2 is present at n occurrences, n is an integer from 0 to 1, and R2 is selected from a proton, C1-C6 alkyl, C(═O)OR7, alkyl-ORS, C—R8, and alkyl-NR9; is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is present at p occurrences, p is an integer from 0 to 1, and R4 is C1-C6 alkyl or C—R8; R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs, H, and —C(═O)R10; R6 is present at q occurrences, q is an integer from 0 to 1, and R6 is aryl; R7 is selected from C—R8; R8 is selected from C3-C6 cycloalkenylaryl, C3-C6 heterocycloalkenylaryl, and aryl optionally substituted by OH, aryl, or ORM; R9 is C(═O)OR10; R10 is C1-C6 alkyl; or pharmaceutically acceptable salts and prodrugs thereof.
Another aspect of the invention provides a compound of formula III,
in which: Y is selected from C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkenylaryl, C3-C6 heterocycloalkenylaryl, C3-C6 cycloalkylaryl, C3-C6 heterocycloalkylaryl, aryl, heteroaryl, C3-C6 heterocycloalkenyl, C3-C6 arylcycloalkylaryl, any of which is optionally substituted at each carbon atom by R1, and wherein when one or more heteroatoms are nitrogen, the nitrogens are each independently unsubstituted or substituted by R2; R1 is selected from OH, cyano, SH, halo, alkyl-NR2R3, OR2, aryl, oxo, C—R3, OR3, C2-C6 alkynyl, C3-C6 heterocycloalkenylaryl, C1-C6 alkyl optionally substituted by halo; and C3-C6 heterocycloalkenyl which is optionally substituted at each carbon atom by R4; R2 is selected from C1-C6 alkyl; is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is C(═O)OR2; R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs; or pharmaceutically acceptable salts and prodrugs thereof.
In certain embodiments of the above compounds, R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs.
Another aspect of the invention provides a compound of formula IV,
in which: R1 is present at n occurrences, n is an integer from 0 to 5 and R1 is selected from halo and C1-C6 alkyl optionally substituted by halo.
Another aspect of the invention provides a compound of formula V,
in which: R1 is present at n occurrences, n is an integer from 0 to 5 and R1 is selected from halo and C1-C6 alkyl optionally substituted by halo.
Another aspect of the invention provides a method for treating a zinc matrix metalloprotease (MMP) dependent disease involving administering to a mammal in need thereof at least one of the above described compounds. In a related embodiment of the method, the zinc metalloprotease dependent disease is cancer or metastasis.
Another aspect of the invention provides a method of purifying a zinc matrix metalloprotease from a sample. The method involves: immobilizing at least one compound according to Formulas I-V to a substrate surface to form an immobilized compound matrix; contacting the matrix with sample, wherein a component of the sample includes a zinc matrix metalloprotease, wherein the zinc matrix metalloprotease binds to the at least one compound on the matrix to form at least one complex with the compound on the matrix; and washing the matrix to separate unbound components of the sample from the complex, to purify the zinc matrix metalloprotease.
Compounds are provided for use in the treatment of MMP dependent diseases, such as cancer and metastasis, and for the manufacture of pharmaceutical compositions for use in the treatment of these diseases. Methods of use of exemplary compounds of the present invention in the treatment of these diseases, or pharmaceutical preparations having compounds of the present invention for the treatment of these diseases are also provided.
In certain embodiments, the compounds of the present invention are compounds of Formula I,
in which: X is a C3-C6 heterocycloalkenyl, wherein carbon atoms of the ring are optionally substituted by R6, and wherein when one or more heteroatoms are nitrogen, the nitrogens are each independently unsubstituted or substituted by R7; R1 is present at n occurrences, n is an integer from 0 to 1, and R1 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R2 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R3 is present at m occurrences, m is an integer from 0 to 1, and R3 is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is present at p occurrences, p is an integer from 0 to 1, and R4 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs, H, —C(═O)R9, and —C(═O)OR9; R6 is selected from H, C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and C1-C6 alkyl optionally substituted by R8; R7 is selected from H, C1-C6 alkyl optionally substituted by R8; C2-C6 alkenyl, C2-C6 alkynyl, C(═O)OR9, and aryl optionally substituted by halo or C1-C6 alkyl; R8 is selected from C(═O)OR9, OR9, and halo; R9 is a C1-C6 alkyl optionally substituted by aryl; R10 is selected from H, halo, OR9, NO2, alkoxy, cyano, SO2CH3, SO2NH2, COCH3, COCH3, CONH2, CHO and C1-C6 alkyl optionally substituted by halo; and R11 is an aryl optionally substituted by halo.
In alternative embodiments, the present invention provides compounds having Formula II,
in which: R1 is present at m occurrences, m is an integer from 0 to 1, and R1 is C1-C6 alkyl or C—R8; R2 is present at n occurrences, n is an integer from 0 to 1, and R2 is selected from a proton, C1-C6 alkyl, C(═O)OR7, alkyl-ORS, C—R8, and alkyl-NR9; R3 is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is present at p occurrences, p is an integer from 0 to 1, and R4 is C1-C6 alkyl or C—R8; R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs, H, and —C(═O)R10; R6 is present at q occurrences, q is an integer from 0 to 1, and R6 is aryl; and R7 is selected from C—R8; R8 is selected from C3-C6 cycloalkenylaryl, C3-C6 heterocycloalkenylaryl, and aryl optionally substituted by OH, aryl, or OR10; R9 is C(═O)OR10; R10 is C1-C6 alkyl.
In another embodiment, the present invention provides compounds having Formula III,
in which: Y is selected from C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkenylaryl, C3-C6 heterocycloalkenylaryl, C3-C6 cycloalkylaryl, C3-C6 heterocycloalkylaryl, aryl, heteroaryl, C3-C6 heterocycloalkenyl, C3-C6 arylcycloalkylaryl, any of which is optionally substituted at each carbon atom by R1, and wherein when one or more heteroatoms are nitrogen, the nitrogens are each independently unsubstituted or substituted by R2; R1 is selected from OH, cyano, SH, halo, alkyl-NR2R3, OR2, aryl, oxo, C—R3, OR3, C2-C6 alkynyl, C3-C6 heterocycloalkenylaryl, C1-C6 alkyl optionally substituted by halo; and C3-C6 heterocycloalkenyl which is optionally substituted at each carbon atom by R4; R2 is selected from C1-C6 alkyl; R3 is selected from a proton, C(═O)OR10, C(═O)OR7, C(═O)NR7, C3-C6 heterocycloalkylaryl, C3-C6 cycloalkenylaryl, C—R8, heteroaryl, and aryl optionally substituted at each carbon atom by halo, OH, OCH3, O-alkyl, amino, substituted amino, —SO2NH2, substituted sulfonamide, —SO2CH3, substituted sulfoxies, CONH2, substituted amido, COCH3, substituted ketones, CHO, cyano, NO2, C(═O)OR10, and C1-C6 alkyl which is further optionally substituted by halo, amino, or hydroxyl; R4 is C(═O)OR2; and R5 is a hydrogen atom, or a bond such that the molecule formed a symmetrical dimer at the disulfide bond, a mixed disulfide with other monosulfide compounds such as ethanethiol, or functional groups such as acetyl to form esters which can be used as prodrugs.
In yet another embodiment, the invention provides compounds of Formula IV,
in which: R1 is present at n occurrences, n is an integer from 0 to 5 and R1 is selected from halo and C1-C6 alkyl optionally substituted by halo.
In still another embodiment, the invention provides compounds of Formula V,
in which: R1 is present at n occurrences, n is an integer from 0 to 5 and R1 is selected from halo and C1-C6 alkyl optionally substituted by halo.
The following are exemplary compounds of Formulas I-V:
Yet another embodiment provided herein is use of a compound above in preparation of a pharmaceutical composition. Yet another embodiment is a pharmaceutical composition that includes a compound according to the above. In certain embodiments, the pharmaceutical composition has at least one of the above a compounds and an acceptable pharmaceutical carrier. Another embodiment provides use of a compound above in preparation of a pharmaceutical composition for use in treatment of an MMP dependent disease.
The terms below shall have the following meanings herein and in the claims, unless otherwise required by the context.
A compound having a plurality of tautomeric forms is not limited to any one specific tautomer. The compound includes the full range of tautomeric forms of the compound. Further, as is evident to those skilled in the art, the compounds herein contain asymmetric carbon atoms. It should be understood, therefore, that the full range of stereoisomers are within the scope of this invention.
The term, “unsubstituted” refers to an atom absent a substituent at the designated atom, or that has a substituent that is a hydrogen atom.
The term, “substituted” refers to one or more hydrogen atoms covalently bonded to the designated atom is replaced by a specified group, provided that the valence on the designated atom is not exceeded, and that a chemically stable compound results from the substitution.
The term “heteroatom” refers to an oxygen, a sulfur, or a nitrogen atom substituted at a designated atom.
The term, “C1-C6 alkyl”, “lower alkyl” or “alkyl” refer to a straight or branched chain alkyl group having 1-6 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. The term “higher alkyl” refers to a straight or branched chain alkyl group having 6-12 carbon atoms.
The term, “C1-C6 heteroalkyl” refers to a C1-C6 alkyl group in which one or more of the carbon atoms have been replaced with a heteroatom, for example O, N, or S.
The term, “C2-C6 alkenyl” refers to a hydrocarbon chain having 2 to 6 carbon atoms in a straight or a branched arrangement and containing one or more unsaturated carbon-carbon double bonds that occur between two adjacent carbon atoms at any stable point in the chain, such as, for example, ethenyl (vinyl), allyl, isopropenyl, and the like.
The term, “C2-C6 alkynyl” refers to a hydrocarbon chain that has 2 to 6 carbon atoms in a straight or branched arrangement and containing one or more unsaturated carbon-carbon triple bonds that occur between two carbon atoms at any stable point in the chain, such as, for example, ethynyl, propargyl, and the like.
The term, “C3-C6 cycloalkyl” refers to an alkyl group that has 3-6 carbon atoms that form a monocyclic ring system, such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
The term, “C3-C6 heterocycloalkyl” refers to a C3-C6 cycloalkyl group in which one or more of the ring carbon atoms have been replaced with a heteroatom, for example O, N, or S. Examples of such compounds include tetrahydropyran, tetrahydropyrrole, tetrahydrothiophene, piperidine, dioxane, dithiane, and piperazine.
The term, “C3-C6 cycloalkenyl” refers to an alkyl group that has 3-6 carbon atoms that form a monocyclic ring system and contain one or more carbon-carbon double bonds between two carbon atoms, preferably in a stable position, in the ring, such as, for example, cyclopentenyl, cyclohexenyl, or cycloheptenyl.
The term, “C3-C6 heterocycloalkenyl” refers to C3-C6 cycloalkenyl group in which one or more of the ring carbon atoms have been replaced with a heteroatom, for example O, N, or S. Examples of such compounds include pyrazole, pyrazoline, thiazole, thiadiazole, isothiazole, oxazole, imidazole, furan, and thiophene.
The term, “aryl” refers to a monocyclic aromatic group that has 6 to 10 carbon atoms, such as, for example, phenyl, naphthyl, indenyl, azulenyl, and anthryl.
The term, “heteroaryl” refers to an aryl group in which one or more of the ring carbon atoms have been replaced with a heteroatom, for example O, N, or S. Examples of such compounds include pyridine, pyrimidine, pyrazine, and pyridazine. It also includes fused ring systems including indole, benzimidazole, phenothiazinyl, and the like.
The term “C1-C6 cycloalkylaryl” refers to a cycloalkyl group that has 3-6 carbon atoms that are fused to an aryl group. Examples of such compounds include indane and tetrahydronaphthalene. The C1-C6 cycloalkylaryl functional group is attached to the remaining atoms in the structure at a carbon atom in the cycloalkyl group or at a carbon atom in the aryl group.
The term, “heterocycloalkylaryl” refers to a cycloalkyl group that has 3-6 carbon atoms that are fused to an aryl group in which one or more of the ring carbon atoms in the cycloalkyl group have been replaced with a heteroatom, for example O, N, or S. Examples of such compounds include isoindoline, benzodioxane, and indoline. The heterocycloalkylaryl functional group is attached to the remaining atoms in the structure at an atom in the heterocycloalkyl group or at a carbon atom in the aryl group.
The term, “C3-C6 cycloalkenylaryl” refers to a cycloalkenyl group having 3-6 carbon atoms that are fused to an aryl group. Examples of such compounds include indene, isoindene and naphthalene. The C3-C6 cycloalkenylaryl functional group is attached to the remaining atoms in the structure at a carbon atom in the cycloalkenyl group or at a carbon atom in the aryl group.
The term, “C3-C6 heterocycloalkenylaryl” refers to a cycloalkenyl group having 3-6 carbon atoms that are fused to an aryl group in which one or more of the ring carbon atoms in the cycloalkenyl group have been replaced with a heteroatom, for example O, N, or S. Examples of such compounds include indole, benzothiophene, benzimidazole, indazole, isoquinoline, quinoline, benzofuran, and phthalazine. The C3-C6 heterocycloalkenylaryl functional group is attached to the remaining atoms in the structure at an atom in the cycloalkenyl group or at a carbon atom in the aryl group.
The term “C3-C6 arylcycloalkylaryl” refers to a first aryl group fused to a cycloalkyl group having 3-6 carbon atoms which is fused to a second aryl group. The C3-C6 arylcycloalkylaryl functional group is attached to the remaining atoms in the structure at a carbon atom in the cycloalkyl group or at a carbon atom in either of the aryl groups. Examples include compounds of Formula VI:
The term, “alkoxy” refers to a straight or branched chain alkoxy group having 1-6 carbon atoms, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
The term, “oxo” (indicated herein as ═O) refers to a double-bond oxygen group that is formed by replacing two geminal hydrogen atoms on a carbon atom with a double-bond oxygen group.
The term “halo” refers to any of fluoro, chloro, bromo and iodo.
The term “cyano” refers to a carbon atom joined to a nitrogen atom by a triple bond.
The term “salts” includes for example, pharmaceutically acceptable salts of a compound herein. Such salts are formed, for example, as acid addition salts, including organic or inorganic acids, from compounds herein with a basic nitrogen atom, including pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, and other organic protonic acids, such as ascorbic acid.
In the presence of a negatively charged ion, such as a carboxy or a sulfo, salts may also be formed with bases, e.g. metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethylamine or tri(2-hydroxyethyl)amine, or heterocyclic bases, for example N-ethyl-piperidine or N,N′-dimethylpiperazine.
When a basic group and an acidic group are present in the same molecule, a compound of the present invention may also form an internal salt, a zwitterion.
For purposes of isolation or purification salts that are not necessarily pharmaceutically acceptable, for example picrates or perchlorates, are within the scope of the invention. For therapeutic use, pharmaceutically acceptable salts or free compounds are employed (in the form of pharmaceutical preparations).
Reference to the compounds herein before and hereinafter is to be understood as referring also to the corresponding tautomers of these compounds, tautomeric mixtures of these compounds, or salts of any of these, as appropriate and expedient and if not mentioned otherwise, in view of the close relationship between the compounds in free form and those in the form of their salts, including those salts that can be used as intermediates, for example in the purification or identification of the compounds, tautomers or tautomeric mixtures and their salts.
The present invention relates also to a pro-drug of a compound provided herein, that is converted in vivo to a compound provided herein. Reference to a compound of the present invention therefore encompasses a corresponding pro-drug of the compound of the present invention, as appropriate and expedient.
The present invention relates also to active metabolites that are biologically generated after administration of one or more of the claimed analogs into a mammal. It is conceivable that the active metabolite could be isolated and identified and subsequently used as a drug itself.
The present invention relates also to a pharmaceutically acceptable substituent of a compound of the present invention. The term, “pharmaceutically acceptable substituent” refers to a structural modification that is made to a compound herein that does not materially alter the structure-activity relationship of the compound. For example, a successful bioisosteric replacement or substitution of a functional group or system in the compounds of Formulas I-V, as is well known in the art, provides a clinically useful compound (structural homolog, analog, and/or congener) with similar biopharmaceutical properties and activities against zinc matrix metalloproteases. Examples of pharmaceutically acceptable substituents and methods of obtaining such compounds are found in Foye et al. (Principals of Medicinal Chemistry, 4th edition, Lea & Febiger/Williams and Wilkins, Philadelphia, Pa., 1995).
The compounds of the present invention have valuable pharmacological properties and are useful in the treatment of MMP dependent diseases, e.g., as drugs to treat MMP diseases, such as cancer and metastasis. Examples of cancers are brain, kidney, liver, adrenal gland, bladder, breast, stomach (for example gastric tumors), ovaries, esophagus, colon, rectum, prostate, pancreas, lung, vagina, thyroid, sarcoma, glioblastomas, multiple myeloma or gastrointestinal cancer, for example, colon carcinoma or colorectal adenoma, or a tumor of the neck and head, an epidermal hyperproliferation, for example, psoriasis, prostate hyperplasia, a neoplasia, including a neoplasia of epithelial character, including mammary carcinoma, or a leukemia.
Tumor invasion and metastases are major causes of morbidity and death for cancer patients. As used herein, the term “metastasis” refers to a condition of spread of cancer from an organ of origin to additional distal sites in a patient. An important event of tumor invasion that signals initiation of a metastatic cascade is interaction of a tumor cell with a basement membrane. Basement membranes are barriers to tumor cell invasion at multiple points in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also in angiogenesis, is degradation of basement membrane components.
Steps involved in metastasis include: attachment to the extracellular matrix (ECM), mediated for example by pre-existing or newly formed contact sites; creation of a proteolytic defect in the ECM; and migration through the proteolytically modified matrix (Ray et al., Eur Respir J. 7:2062-2072, 1994 and Wojtowitz-Praga et al., Investigational New Drugs 15:61-75, 1997).
Zinc matrix metalloproteases (MMP) are zinc-ion dependent endopeptidases with specific and selective activities against components of the extracellular matrix. Different MMPs have been described which are membrane associated, or are secreted as zymogens and are activated extracellularly. MMPs have been classified into three subgroups based on substrate preference: interstitial collagenases, stromelysins, and gelatinases. These enzymes have overlapping substrate specificity, and the compounds of the present invention are found herein to have activity against a plurality of each of these subgroups.
A common primary amino acid consensus sequence of the family has five modular domains, including a signal sequence; a profragment activation locus; a Zn-ion binding site, catalytic domain; a proline-rich hinge region; and a haemopexin- or vitronectin-like C-terminal domain (Ray et al., Eur Respir J. 7:2062-2072, 1994). The gelatinases additionally contain a fibronectin-like gelatin-binding domain immediately upstream of the Zn-binding domain.
An MMP dependent disease is a pathology associated with expression of one or more genes encoding an MMP protein or an MMP-associated protein, or an activity of such a protein, such that inhibition of the protein results in remediation of the pathology. MMP genes and proteins are as described in the Online Mendelian Inheritance in Man (O.M.I.M). Without being limited by any particular theory or mechanism of action, inhibition of an MMP protein is here envisioned to provide remediation of an MMP dependent disease. Table 1 lists MMP proteins and locus of each on the human genome.
MMP1 known as a collagenase is one of a few enzymes able to initiate breakdown of interstitial collagen types I, II, and III. MMP1 is a matrix metalloprotease that is secreted as a zymogen. Collagens are abundant proteins and MMP1 is important in remodeling occurring under both normal and diseased conditions. For example, MMP1 has been implicated in malignant melanoma invasion (Iida et al., Melanoma Res. 17(4):205-213, 2007).
MMP2 known as Type IV collagenase or gelatinase is a 72-kD protein that specifically cleaves type IV collagen, the major structural component of basement membranes. MMP2 is a matrix metalloprotease that is secreted as a zymogen. The metastatic potential of tumor cells has been correlated with MMP activity. For example, MMP2 has been implicated in the progression of colorectal carcinomas, breast cancer, and non-small cell lung cancer (Wojtowicz-Praga et al., Investigational New Drugs 15:61-75, 1997).
MMP3 known as human fibroblast stromelysin or transin is a proteoglycanase having 477 amino acid residues. MMP3 has 54% sequence identity with MMP1 (Koklitis et al., Biochem. J. 276: 217-221, 1991). MMP3 is a secreted metalloprotease produced predominantly by connective tissue cells, and is involved in degradation of major components of the extracellular matrix, such as proteoglycan, fibronectin, laminin, and type IV collagen. Invasion and metastasis of colorectal cancer is associated with overexpression of MMP3 (Woo et al., J Gastroenterol Hepatol. 22(7):1064-1070, 2007).
MMP7 known as putative metalloproteinase I (PUMP1) or matrilysin is a 28-kD zymogen having 267 amino acids, and is secreted as a zymogen. MMP7 possesses catalytic activities against a broad range of extracellular matrix substrates including proteoglycans, gelatin, fibronectin, laminin, and elastin. Metastasis of ovarian cancer is associated with overexpression of MMP7 (Shigemasa et al., Med. Oncol. 17(1):52-58, 2000).
MMP8 known as neutrophil collagenase is a protein having 467 amino acid residues, and is a matrix metalloprotease that is secreted as a zymogen. MMP8 is produced mainly by neutrophils in inflammatory reactions and is detected in some malignant tumors (Balbin et al., Nature Genet. 35: 252-257, 2003).
MMP9 known as 92-kD gelatinase or type V collagenase is a 92-kD protein produced by normal alveolar macrophages and granulocyte, and is secreted as a zymogen. MMP9 has been associated with the progression of colorectal carcinomas, breast cancer, and non-small cell lung cancer (Wojtowicz-Praga et al., Investigational New Drugs 15:61-75, 1997). A functional relationship has been shown among the hyaluronan receptor CD44, MMP9, and transforming growth factor-beta (TGFB) in control of tumor-associated tissue remodeling (Yu et al., Genes Dev. 14:163-176, 2000).
MMP10 known as stromelysin II is structurally related to MMP3, and degrades various components of the extracellular matrix. MMP10 is a matrix metalloprotease that is secreted as a zymogen, and is associated with development of lymphoma (Van Themsche et al., J. Immunol. 15; 173(6):3605-3611, 2004).
MMP11 known as stromelysin III is secreted as a zymogen, and invasive breast carcinomas are associated with overexpression of MMP11 by stromal cells (Decock et al., Dis Markers. 23(3):189-196, 2007).
MMP12 known as macrophage metalloelastase is a 470 amino acid protein that is secreted as a zymogen. MMP12 is produced by human alveolar macrophages, and has the capacity to degrade elastin. Hepatocellular carcinoma and metastasis thereof is associated with overexpression of MMP12 (Gorrin-Rivas, et al., Ann Surg 231(1):67-73, 2000).
MMP13 known as collagenase 3 is a 471 amino acid protein that is secreted as a zymogen. MMP-13 is a factor associated with tumor aggressiveness in cutaneous malignant melanoma, both in tumoral invasion and in proliferation (Corte et al., Int J Biol Markers. 20(4):242-248, 2005).
MMP14 is a 582 amino acid residue membrane associated zinc matrix metalloprotease, expressed at the surface of invasive tumor cells (Sato et al., Nature 370: 61-65, 1994).
MMP15 is a 669 amino acid residue membrane associated zinc matrix metalloprotease, with 74% sequence identity with MMP14. Prostate cancer is associated with increased expression of MMP15 (Riddick et al., British Journal of Cancer 92:2171-2180, 2005).
MMP16 is a 604 amino acid residue membrane associated zinc matrix metalloprotease and is involved in the Wnt signaling pathway. Upregulation of MMP16 is found in invasive human tumors, particularly gastric cancer (Lowy et al., Cancer Res. 1; 66(9):4734-4741, 2006).
MMP17 is a 518 amino acid residue membrane associated zinc matrix metalloprotease. Upregulated MMP17 expression has been found in breast carcinomas and breast cancer cell lines (Puente et al., Cancer Res. 56: 944-949, 1996).
MMP19 is a 508 amino acid residue protein that in contrast to other MMPs, is widely expressed in human tissues under normal quiescent conditions. However, deregulation of MMP19 is associated with diverse pathological conditions such as rheumatoid arthritis and cancer (Pendas et al., Mol Cell Biol. 24(12):5304-5313, 2004).
MMP20 known as enamelysin is a matrix metalloprotease secreted as a zymogen, and is involved in tooth enamel formation. Formation and metastasis of odontogenic is associated with upregulation of MMP20 (Vaananen et al., Matrix Biol., 23(3):153-161, 2004).
MMP23A (formerly called MMP21) is a membrane associated zinc matrix metalloprotease, and is involved in epithelial tumor progression. MMP23A has been detected in cancer cells and inflammatory cells at the invasive front, and is associated with invasion, inflammation, apoptotic and well-differentiated areas of tumors (Ahokas et al., Tumour Biol. 27(3):133-141, 2006).
MMP23B (formerly called MMP22) is a membrane associated zinc matrix metalloprotease that is down regulated in metastatic cancer (Chinnaiyan et al., U.S. patent application number 20070128639, published Jun. 7, 2007).
MMP24 is a membrane associated zinc matrix metalloprotease found overexpressed in a variety of brain tumors, including astrocytomas, anaplastic astrocytomas, glioblastomas, mixed gliomas, oligodendrogliomas, ependymomas, neurocytomas, and meningiomas (Llano et al., Cancer Res. 59: 2570-2576, 1999).
MMP25 is a membrane associated zinc matrix metalloprotease overexpressed in different cancers, for example, leukocytes, lung, spleen, primary colon carcinomas, anaplastic astrocytomas, and glioblastomas (Pei, Cell Res. 9:291-303, 1999 and Velasco et al., Cancer Res. 60:877-882, 2000).
MMP26 known as matrilysin 2 is a matrix metalloprotease that is secreted as a zymogen, and expressed in placenta and uterus. MMP26 is associated with many malignant tumors and tissue remodeling events associated with tumor progression (Uria et al., Cancer Res. 60:4745-4751, 2000).
MMP28 known as epilysin is a 520 amino acid residue matrix metalloprotease secreted as a zymogen and overexpressed in tumor growth and metastasis (Marchenko et al., Gene. 7; 265(1-2):87-93, 2001).
The compounds provided herein are selectively effective against rapidly proliferating cells compared to normal cells, including, for example, human cancer cells, e.g., cancerous tumors. Compounds of the present invention thereby cause regression of tumors and prevent the formation of tumor metastases and the growth of (also micro)metastases.
The phrase “treatment of zinc matrix metalloprotease dependent diseases” refers to the prophylactic or therapeutic (including palliative and/or curing) treatment of these diseases, including for example, cancer and metastasis.
The term “use” includes any one or more of the following embodiments of the invention, respectively: use in the treatment of MMP dependent diseases; use for the manufacture of pharmaceutical compositions for use in the treatment of these diseases; methods of use of derivatives of Formulas I-V in the treatment of these diseases; pharmaceutical preparations having derivatives of Formulas I-V for the treatment of these diseases; and derivatives of Formulas I-V for use in the treatment of these diseases, as appropriate and expedient, if not stated otherwise.
In particular, diseases to be treated by a compound of the present invention are selected from MMP dependent (“dependent” meaning also “supported” or “associated”) diseases, including those corresponding MMP dependent diseases, and those diseases that depend on MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23A, MMP23B, MMP24, MMP25, MMP26, and MMP28, can therefore be used in the treatment of MMP dependent diseases.
Tumors that grow beyond a maximum diameter of about 1-2 mm require angiogenesis for further growth. Up to this limit, oxygen and nutrients are supplied to the tumor cells by diffusion.
Mechanisms involved in blocking angiogenesis include: inhibition of growth of vessels, especially capillaries, into avascular resting tumors, so that tumor growth is inhibited and both apoptosis and proliferation are occurring; prevention of migration of tumor cells associated with absence of blood flow to and from tumors; and inhibition of endothelial cell proliferation, reducing or eliminating paracrine growth-stimulating effect exerted on the surrounding tissue by endothelial cells that line the vessels.
The term “use” further includes embodiments of compounds herein that bind to an MMP protein sufficiently to serve as tracers or labels, so that when coupled to a fluorophore or tag, or in a radioactive form, are research reagents or as diagnostics or imaging agents. Thus in another embodiment, the compounds of the present invention are also useful as probes.
Working examples provided herein demonstrate in vivo antitumor activity of compounds provided herein.
Embodiments of the compounds of the present invention have pharmacological properties useful in the treatment of MMP dependent diseases, for example, cancer or metastasis. Other embodiments of the compounds of the present invention have binding properties useful in diagnostic and labeling capacities and as imaging agents. Other embodiments of the compounds of the present invention are useful in protein purification capacities, i.e., purifying a zinc matrix metalloprotease from a mixture of components in a sample.
Cloning and expression of MMP: The baculovirus donor vector pFB-GSTX3 is used to generate a recombinant baculovirus that expresses the MMP polypeptide. Transfer vectors containing the MMP coding region are transfected into the DH10Bac cell line (GIBCO) and plated on selective agar plates. Colonies without insertion of the fusion sequence into the viral genome (carried by the bacteria) are blue. Single, white colonies are picked and viral DNA (bacmid) are isolated from the bacteria by standard plasmid purification procedures. Sf9 cells or Sf21 (American Type Culture Collection) cells are then transfected in 25 cm2 flasks with the viral DNA using Cellfectin reagent.
Determination of small scale protein expression in Sf9 cells: Virus-containing media is collected from the transfected cell culture and used for infection to increase its titer. Virus-containing media obtained after two rounds of infection is used for large-scale protein expression. For large-scale protein expression 100 cm2 round tissue culture plates are seeded with 5×107 cells/plate and infected with 1 mL of virus-containing media (at an approximately MOI of 5). After 3 days, the cells are scraped off the plate and centrifuged at 500 rpm for 5 minutes. Cell pellets from 10-20, 100 cm2 plates, are re-suspended in 50 mL of ice-cold lysis buffer (25 mM tris-HCl, pH 7.5, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM P MSF). The cells are stirred on ice for 15 minutes and then centrifuged at 5,000 rpms for 20 minutes.
Purification of GST-tagged proteins: The centrifuged cell lysate is loaded onto a 2 mL glutathione-sepharose column (Pharmacia) and is washed 3× with 10 mL of 25 mM tris-HCl, pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCl. The GST-tagged proteins are then eluted by 10 applications (1 mL each) of 25 mM tris-HCl, pH 7.5, 10 mM reduced-glutathione, 100 mM NaCl, 1 mM DTT, 10% glycerol and stored at −70° C.
Measurement of enzyme activity: MMP assays with purified GST-MMP protein are carried out in a final volume of 30 μL containing 15 ng of GST-MMP protein, 20 mM tris-HCl, pH 7.5, 1 mM MnCl2, 10 mM MgCl2, 1 mM DTT, 3 μg/mL poly(Glu,Tyr) 4:1, 1% DMSO, 2.0 μM ATP (γ-[33P]-ATP 0.1 μCi). The activity is assayed in the presence or absence of inhibitors. The assay is carried out in 96-well plates at ambient temperature for 15 minutes under conditions described below and terminated by the addition of 20 μL of 125 mM EDTA. Subsequently, 40 μL of the reaction mixture are transferred onto Immobilon-PVDF membrane (Millipore) previously soaked for 5 minutes with methanol, rinsed with water, then soaked for 5 minutes with 0.5% H3PO4 and mounted on vacuum manifold with disconnected vacuum source. After spotting all samples, a vacuum is connected and each well-rinsed with 200 μL 0.5% H3PO4. Membranes are removed and washed 4× on a shaker with 1.0% H3PO4, once with ethanol. Membranes are counted after drying at ambient temperature, mounting in Packard TopCount 96-well frame, and addition of 10 μL/well of Microscint™ (Packard). IC50 values are calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at 4 concentrations (usually 0.01, 0.1, 1 and 10 μM).
IC50 values are calculated by logarithmic regression analysis of the percentage inhibition of each compound at 4 concentrations (usually 3- or 10-fold dilution series starting at 10 μM).
In each experiment, the actual inhibition by reference compound is used for normalization of IC50 values to the basis of an average value of the reference inhibitor:
Normalized IC50=measured IC50 average ref. IC50/measured ref. IC50
Example: Reference inhibitor in experiment 0.4 μM, average 0.3 μM
For example, staurosporine or a synthetic staurosporine derivative are used as reference compounds.
Using this protocol, the compounds provided herein are found to have IC50 values for MMP inhibition in the range from about 0.005 to about 100 μM, or about 0.002 to about 50 μM, including, for example, the range from about 0.001 to about 2 μM or lower.
Compounds provided herein are prepared from commonly available starting materials using procedures known to those skilled in the art, including any one or more of the following procedures without limitation.
Within the scope of this text, only a readily removable group that is not a constituent of the particular desired end product of the compounds of the present invention is designated a “protecting group”, unless the context indicates otherwise. The protection of functional groups by such protecting groups, the protecting groups themselves, and their cleavage reactions are described for example in standard reference works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie” (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15/I, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jeschkeit, “Aminosauren, Peptide, Proteine” (Amino acids, Peptides, Proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate” (Chemistry of Carbohydrates: Monosaccharides and Derivatives), Georg Thieme Verlag, Stuttgart 1974. A characteristic of protecting groups is that they can be removed readily (i.e. without the occurrence of undesired secondary reactions) for example by solvolysis, reduction, photolysis or alternatively under physiological conditions (e.g. by enzymatic cleavage).
Salts of compounds of the present invention having at least one salt-forming group may be prepared in a manner known by one of ordinary skill in the art of chemistry. For example, salts of compounds of the present invention having acid groups may be formed, for example, by treating the compounds with metal compounds, such as alkali metal salts of suitable organic carboxylic acids, e.g. the sodium salt of 2-ethylhexanoic acid, with organic alkali metal or alkaline earth metal compounds, such as the corresponding hydroxides, carbonates or hydrogen carbonates, such as sodium or potassium hydroxide, carbonate or hydrogen carbonate, with corresponding calcium compounds or with ammonia or a suitable organic amine, stoichiometric amounts or only a small excess of the salt-forming agent preferably being used. Acid addition salts of compounds of the present invention are obtained in customary manner, e.g. by treating the compounds with an acid or a suitable anion exchange reagent. Internal salts of compounds of the present invention containing acid and basic salt-forming groups, e.g. a free carboxy group and a free amino group, may be formed, e.g. by the neutralization of salts, such as acid addition salts, to the isoelectric point, e.g. with weak bases, or by treatment with ion exchangers.
Salts can be converted in a customary manner into the free compounds; metal and ammonium salts can be converted, for example, by treatment with suitable acids, and acid addition salts, for example, by treatment with a suitable basic agent.
Mixtures of isomers are obtained according to methods provided herein and are separated in a manner known to one of ordinary skill in the art of chemistry into the individual isomers; diastereoisomers are separated, for example, by partitioning between polyphasic solvent mixtures, recrystallisation and/or chromatographic separation, for example over silica gel or by e.g. medium pressure liquid chromatography over a reversed phase column, and racemates are separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by fractional crystallisation, or by chromatography over optically active column materials.
Intermediates and final products are further purified according to standard methods, e.g. using chromatographic methods, distribution methods, (re-) crystallization, and the like.
The following applies in general to all processes mentioned herein before and hereinafter. All the above-mentioned process steps are carried out under reaction conditions that are well known in the art, including those mentioned specifically, in the absence or, customarily, in the presence of solvents or diluents, including, for example, solvents or diluents that are inert towards the reagents used and dissolve them, in the absence or presence of catalysts, condensation or neutralizing agents, for example ion exchangers, such as cation exchangers, e.g. in the H+ form, depending on the nature of the reaction and/or of the reactants at reduced, normal or elevated temperature, for example in a temperature range of from about −100° C. to about 190° C., including, for example, from about −80° C. to about 150° C., for example at from about −80 to about 60° C., at room temperature, at from about −20 to about 40° C. or at reflux temperature, under atmospheric pressure or in a closed vessel, where appropriate under pressure, and/or in an inert atmosphere, for example under an argon or nitrogen atmosphere.
At each stage of the reactions, mixtures of isomers that are formed can be separated into the individual isomers, for example diastereoisomers or enantiomers, or into any desired mixtures of isomers, for example racemates or mixtures of diastereoisomers.
The solvents include solvents suitable for a particular reaction that are selected among, for example, water, esters, such as lower alkyl-lower alkanoates, for example ethyl acetate, ethers, such as aliphatic ethers, for example diethyl ether, or cyclic ethers, for example tetrahydrofurane or dioxane, liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol, ethanol or 1- or 2-propanol, nitriles, such as acetonitrile, halogenated hydrocarbons, such as methylene chloride or chloroform, acid amides, such as dimethylformamide or dimethylacetamide, bases, such as heterocyclic nitrogen bases, for example pyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides, such as lower alkanoic acid anhydrides, for example acetic anhydride, cyclic, linear or branched hydrocarbons, such as cyclohexane, hexane or isopentane, or mixtures of those solvents, for example aqueous solutions, unless otherwise indicated in the description of the processes. Such solvent mixtures may also be used in purifying or isolating the compounds herein, for example by chromatography or partitioning.
The compounds, including their salts, may also be obtained in the form of hydrates, or their crystals may, for example, include the solvent used for crystallization. Different crystalline forms may be present.
The invention encompasses also those forms of the process in which a compound obtainable as an intermediate at any stage of the process is used as starting material and the remaining process steps are carried out; or in which a starting material is formed under reaction conditions; or is used in the form of a derivative, for example, in a protected form or in the form of a salt; or a compound obtainable by the process according to the invention is produced under the process conditions and is processed further in situ.
A compound described above is, in certain embodiments of the invention, provided and used in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, when appropriate, pharmaceutically acceptable base addition salts and acid addition salts, for example, metal salts, such as alkali and alkaline earth metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts, and sulfonate salts. Acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as alkyl sulfonate, arylsulfonate, acetate, maleate, fumarate, tartrate, citrate and lactate. Examples of metal salts are alkali metal salts, such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of ammonium salts are ammonium salt and tetramethylammonium salt. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. Sulfonate salts include mesylate, tosylate and benzene sulfonic acid salts.
The invention provides pharmaceutical compositions comprising a compound of the present invention, and use of pharmaceutical compositions in therapeutic or prophylactic treatment, or use in a method of treatment of an MMP dependent disease, including, for example, cancer or metastasis, and provides compounds for use and preparation of pharmaceutical preparations.
The present invention provides also pro-drugs of a compound of the present invention that are converted in vivo to the compound of the present invention. Any reference to a compound of the present invention is therefore to be understood as referring also to a corresponding pro-drug of the compound of the present invention, as appropriate and expedient. The pharmacologically acceptable compounds of the present invention may be used, for example, for the preparation of pharmaceutical compositions that comprise an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, as active ingredient together or in admixture with an amount of one or more inorganic or organic, solid or liquid, pharmaceutically acceptable carriers.
The invention relates also to a pharmaceutical composition that is suitable for administration to a warm-blooded animal, including, for example, a human (or to cells or cell lines derived from a warm-blooded animal, including for example, a human cell, e.g. a lymphocytes, for the treatment or, in another aspect of the invention, prevention of (i.e. prophylaxis against) a disease that responds to inhibition of an MMP, comprising an amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, which is effective for this inhibition, including inhibition of activity of an MMP or inhibition of an MMP protein interacting with a transcriptional effector protein, together with at least one pharmaceutically acceptable carrier.
The pharmaceutical compositions according to the invention are formulated for administration, for example, by a route that is enteral, such as nasal, rectal or oral, or parenteral, such as intramuscular or intravenous, the composition formulated for administration to a warm-blooded animal (including, for example, a human), formulated in an effective dose of the pharmacologically active ingredient, alone or together with an amount of a pharmaceutically acceptable carrier. The dose of the active ingredient is formulated in an amount that is suitable for the species of warm-blooded animal, using parameters such as the body weight, the age and the individual condition, individual pharmacokinetic data, the disease to be treated and the mode of administration as is well-known to one of ordinary skill in the art of pharmacology.
The dose of a compound of the present invention or a pharmaceutically acceptable salt thereof to be administered to a warm-blooded animal, for example a human of about 70 kg body weight, is for example, from about 3 mg to about 10 g, from about 10 mg to about 1.5 g, from about 100 mg to about 1000 mg/person/day. Further, the dose is divided into 1 to 3 single doses, which may, for example, be of the same size. Usually, children receive half of an adult dose.
The pharmaceutical compositions have active ingredient, for example, from about 1% to about 95%, or from about 20% to about 90% of the full amount administered, by weight. Pharmaceutical compositions according to the invention are formulated in an amount that is in unit dose form in a container, such as in the form of an ampoule, a vial, a suppository, a dragée, a tablet or a capsule.
The pharmaceutical compositions are prepared by conventional processes herein such as dissolving, lyophilizing, mixing, granulating or confectioning processes or any combination of these processes.
The compound provided herein as the active ingredient is formulated as a solution or as a suspension, and an isotonic aqueous solution or suspension. The active ingredient in certain embodiments is formulated with a carrier, for example mannitol, prior to further processes such as lyophilization. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are further prepared in a manner well-known in the pharmaceutical arts, such as conventional dissolving or lyophilizing processes. The solution or suspension may include a viscosity-increasing substance, such as sodium carboxymethylcellulose or carboxymethylcellulose in another form, dextran, polyvinylpyrrolidone or gelatin.
Suspensions of a compound herein formulated in oil comprise as the oil component a vegetable, synthetic or semi-synthetic oil customary for injection purposes. Oils include without limitation, liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, or from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brasidic acid or linoleic acid, and further mixed if desired by addition of one or more antioxidants, for example vitamin E, β-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of the fatty acid ester has a maximum of 6 carbon atoms and is a mono- or poly-hydroxy, for example a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, glycol and glycerol. The following are examples of fatty acid esters: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethylene glycerol trioleate, Gattefossé, Paris), “Miglyol 812” (triglyceride of saturated fatty acids with a chain length of C8 to C12, Hüls AG, Germany), and vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil.
The injection compositions are prepared in customary manner under sterile conditions, and are introduced into ampoules or vials and sealed into containers under sterile conditions.
Pharmaceutical compositions for oral administration are in certain embodiments obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragée cores or capsules. Alternatively the composition is incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
Suitable carriers are for example, fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders such as starch pastes using for example corn, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators such as the above-mentioned starches, and/or carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Excipients are flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Dragée cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations such as ethylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Capsules include dry-filled capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The dry-filled capsules may comprise the active ingredient in the form of granules, for example with fillers such as lactose; binders such as starches, and/or glidants such as talc or magnesium stearate, and if desired with stabilizers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable oily excipients such as fatty oils, paraffin oil or liquid polyethylene glycols, and stabilizers and/or antibacterial agents can be added. Dyes or pigments may be added to the tablets or dragée coatings or the capsule casings, for example for identification purposes or to indicate different doses of active ingredient.
The invention having now been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not meant to be further limiting. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are within the scope of the present invention and claims. The contents of all references, including issued patents and published patent applications cited throughout this application, are hereby incorporated by reference.
Starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume 21). Further, the compounds provided herein are produced by organic synthesis methods known to one of ordinary skill in the art as illustrated in the following Examples.
Starting materials to synthesize the compounds of Formulas I-V are commercially available from, for example, Sigma-Aldrich (Millwaukee, Wis.). Table 2 below provides exemplary starting materials that were used to synthesize the compounds of Formulas Ito V, and the commercial supplier of these materials.
Reactions were monitored by TLC (Silica Gel 60 F254, EMD Chemicals) or HPLC(HP 1090). Compounds of Formulas I-V and their intermediates were purified by crystallization or silica gel flash chromatography. Characterization of compounds and intermediates were done with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS).
The above list of starting materials was used to prepare the following chemical structures:
To each of eighty reaction wells of a 96-well FlexChem® Synthesis Reaction Block (Scigene, Corp., Sunnyvale, Calif.) was added 2-carboxyethanethiol 4-methoxytrityl resin (44 mg, 0.0792 mmol, loading 1.8 mmol/g; obtained from Novabiochem, San Diego, Calif.). The resin was swollen by adding CH2Cl2 (1 mL/well) to each well and shaking the reaction block for 30 min. The 96-well reaction block was then drained and the resin was washed first with CH2Cl2 (1 mL×80 wells) and then with DMF (1 mL×80 wells).
To each well was added a solution of diisopropylcarbodiimide (DIC; 24.5 μL, 0.158 mmol) and hydroxybenzotriazole-hydrate (24.3 mg, 0.158 mmol) in dimethylformamide (DMF, 0.5 mL). The reaction block was shaken for 3 h at room temperature to pre-form the HOBt-activated ester. Stock solutions of eighty different amines (0.15 mmol) in DMF (0.5 mL) were added to each well and the reactor was shaken at room temperature for 18 h. The reaction block was then drained using a vacuum manifold and each well containing resin was washed with DMF (2×1 mL), MeOH (2×1 mL), H2O (2×1 mL), MeOH (2×1 mL), and CH2Cl2 (3×1 mL).
The thiol-bound mercaptoacetamide products were then cleaved from the resin. First the resin in each well was swollen by shaking with CH2Cl2 (1 mL×80 wells) for 15 min. The solvent was then removed using a vacuum manifold. A cleavage cocktail of 5% trifluoroacetic acid (TFA) and 5% triethylsilane (TIS) in CH2Cl2 (1 mL×80 wells) was added to each well, and the reactor was then shaken for 15 min. Next the reactor was placed over the top of a clean deep-well (2 mL) 96-well plate in a collection manifold, vacuum was applied and the product (in solution) was collected. The solvent was allowed to evaporate in a ventilation hood for 8 h, and then placed in a vacuum desiccator (20-100 mm Hg) overnight to remove the trace amounts of TFA and TIS.
Benzylamine substituted analogs were synthesized using a two-step reaction sequence starting from commercially available phenylketones. The phenylketones were reductively aminated using ammonia and sodium borohydride in the presence of a dehydrating agent to provide benzylamine analogs. The mercaptoacetamides were obtained by refluxing the benzylamine analogs with thioglycolic acid in toluene.
To a solution of 2,4-dichloropropiophenone (1.58 mL, 10 mmol) and titanium IV isopropoxide (6 mL, 20 mmol) was added an ice-cold solution of ammonia in methanol (7N, 7 mL, 49 mmol). Sodium borohydride (600 mg, 15 mmol) was added and the reaction was stirred for 3 days. The reaction mixture was poured into 25 mL of 2% NH4OH, prepared from concentrated NH4OH (1.7 mL) diluted with H2O (23 mL). The resulting white solid was removed by filtration, washed with diethyl ether (2×50 mL), and the aqueous layer was extracted with ether. The combined organic layers were extracted with 1N HCl (2×25 mL). The acidic layer was made basic with concentrated NH4OH and the product was extracted with dichloromethane and dried to provide 900 mg, 44% of a white solid.
A solution of 1-(2,4-dichlorophenyl)propan-1-amine (900 mg, 4.43 mmol), thioglycolic acid (308 μL, 4.43 mmol), and toluene (30 mL) were heated to reflux under argon for 18 h and the resulting water was removed using a Dean-Stark apparatus. The reaction solution was then cooled to room temperature and the volatiles were removed by rotary-evaporation. The resulting oil was chromatographed on flash column silica gel using a gradient of hexane to 50% ethyl acetate/hexane to provide after evaporation 670 mg, 54.4% yield of product as a white powder.
A solution of 2,4-dichlorobenzylamine (1200 mg, 6.82 mmol), thiolactic acid (604 μL, 6.82 mmol), and toluene (40 mL) were heated to reflux under argon for 18 h and the resulting water was removed using a Dean-Stark apparatus. The reaction solution was then cooled to room temperature and the volatiles were removed by rotary-evaporation. The resulting oil was chromatographed on flash column silica gel using a gradient of hexane to 50% ethyl acetate/hexane to provide after evaporation 1.12 g, 62.24% yield of product as a white powder.
The amine (2 mmol) was weighed into a 10 mL vial, and toluene (2 mL) and thioglycolic acid (4 mmol, 277 uL) were added to the vial. The vial was purged with Argon, capped, and placed in an aluminum reaction block heated to 100° C. for 24 h. After cooling the reaction mixture to room temperature, work up varied based upon the presence or lack of filterable solid. In the situation in which the product precipitated out as a solid, the solid was transferred to a 2 ml, glass fritted filter using toluene, rinsed with toluene, H2O, saturated aqueous NaHCO3 solution, H2O, 1N HCl, a minimum amount of acetonitrile, and a minimum amount of diethyl ether. In the situation in which the product remained in solution, the solution was diluted with 6 mL diethyl ether (6 mL), washed 3× with saturated aqueous NaHCO3, H2O, 3× with 1N HCl, and brine. The organic solution was dried over anhydrous Na2SO4 and evaporated in vacuo. The resulting solid was tritrated with a minimum amount of diethyl ether to yield a solid.
N-(2-chloro-5-hydroxyphenyl)-2-mercaptoacetamide was synthesized using the above general procedure by the reaction of a substituted aniline with mercaptoacetic acid at elevated temperature.
Under argon a solution of 3-amino-4-chlorophenol (287 mg, 2 mmol), thioglycolic acid (695 uL, 10 mmol), and toluene (2 mL) were heated to 100° C. in a sealed tube using an aluminum reaction block heater-stirrer. After 24 h the reaction was cooled to room temperature, and the resulting precipitate was collected on fritted glass and washed with toluene. The solid was dried under high vacuum (1 mm) at room temperature to provide 335 mg, 46% yield of a light-gray powder.
N-(3-(4-chlorophenyl)-1H-pyrazol-5-yl)-2-mercaptoacetamide was synthesized using the above general procedure by the reaction of an aminopyrazole with mercaptoacetic acid at elevated temperature.
Under argon a solution of 5-amino-3-(4-chlorophenyl)pyrazole (1.93 g, 10 mmol), thioglycolic acid (1.4 mL, 20 mmol), and toluene (10 mL) were heated to 100° C. in a sealed tube using an aluminum reaction block heater-stirrer. After 24 h the reaction was cooled to room temperature, and the resulting precipitate was collected on fritted glass and washed with toluene, saturated aqueous NaHCO3, H2O, acetonitrile, and diethyl ether. The solid was dried under high vacuum (1 mm) at room temperature to provide 2.30 g, 86% yield of a white powder.
2-Mercapto-N-(3-(thiophen-2-yl)-1H-pyrazol-5-yl)acetamide was synthesized using the above general procedure by the reaction of an aminopyrazole with mercaptoacetic acid at elevated temperature.
Under argon a solution of 5-amino-3-(2-thienyl)pyrazole (330 mg, 2 mmol), thioglycolic acid (554 uL, 8 mmol), and toluene (2 mL) were heated to 100° C. in a sealed tube using an aluminum reaction block heater-stirrer. After 30 h the reaction was cooled to room temperature, and the resulting precipitate was collected on fritted glass and washed with toluene, saturated aqueous NaHCO3, acetonitrile, and diethyl ether. The solid was dried under high vacuum (1 mm) at room temperature to provide 248 mg, 52% yield of a light-gray powder.
2-Mercapto-N-((R)-1-(naphthalen-7-yl)ethyl)acetamide was synthesized using the above general procedure by the reaction of a primary amine with mercaptoacetic acid at elevated temperature.
Under argon a solution of 5-amino-3-(4-chlorophenyl)pyrazole (1.93 g, 10 mmol), thioglycolic acid (1.4 mL, 20 mmol), and toluene (10 mL) were heated to 100° C. in a sealed tube using an aluminum reaction block heater-stirrer. After 24 h the reaction was cooled to room temperature, and the resulting precipitate was collected on fritted glass and washed with toluene, saturated aqueous NaHCO3, H2O, acetonitrile, and diethyl ether. The solid was dried under high vacuum (1 mm) at room temperature to provide 2.30 g, 86% yield of a white powder.
A four-step reaction sequence starting from commercially available 4-chlorobenzoic acid was used to synthesize 4-Substituted analogs. After esterification, the methyl ester was condensed with an appropriately substituted nitrile such as propionitrile to give, for example, an alpha-methyl-beta-ketonitrile. The 4-methylpyrazole was obtained by cyclization with hydrazine. The mercaptoacetamide analog was obtained by refluxing the 3-aminopyrazole with thioglycolic acid in toluene.
To 4-chlorobenzoic acid (15.6 g, 100 mmol) dissolved in methanol (100 mL) was added concentrated H2SO4 (1 mL). The reaction was then heated to reflux for 18 h. The reaction mixture was then cooled in an ice bath, and the crystalline product was collected on fritted glass, washed with water, saturated aqueous NaHCO3, and water. The material was further dried under high vacuum to give 15.3 g, 90% yield of a white crystalline solid.
Methyl 4-chlorobenzoate (1.70 g, 10 mmol) was dissolved in propionitrile (10 mL, dried over 3 Å molecular sieves), and NaOCH3 (1.08 g, 20 mmol) was added and the reaction was stirred at room temperature under argon for 18 h. The reaction was heated to 100° C. for 1 h, and the reaction mixture was then cooled to ambient temperature and the volatiles were removed by rotary evaporation leaving a residue. The residue was dissolved in water (10 mL) and washed with ether (3 times). The aqueous layer was then acidified to pH 6.4 with citric acid. The resulting precipitate was collected on fitted glass, washed with water, saturated aqueous NaHCO3, and water. The solid on the filter was dried under high vacuum over night to provide the beta-ketonitrile (428 mg, 48% yield).
The beta-ketonitrile, prepared above, 3-(4-chlorophenyl)-2-methyl-3-oxopropanenitrile (353 mg, 2 mmol), was dissolved in abs. ethanol (2 mL). To this solution was added anhydrous hydrazine (75 μL, 2.4 mmol), and the reaction was stirred for 1 h at room temperature allowing the hydrazone to form and precipitate. The mixture was then heated to 100° C. for 45 min., and the progress of the reaction was monitored by HPLC. After heating for 45 min., water (1 mL to 5 mL) was added to precipitate the heterocyclic product as a solid. This material was collected on a fitted glass funnel, washed with water, then dried overnight under high vacuum to provide 267.5 mg, 644% yield of a white powder.
A solution of 3-(4-chlorophenyl)-4-methyl-1H-pyrazol-5-amine (207.5 mg, 1 mmol), thioglycolic acid (104 μL, 1.5 mmol), and toluene (0.5 mL) were heated in a sealed tube under argon for 24 h. The reaction solution was then cooled to room temperature to precipitate the crude product as a solid. The solid was collected on a fitted glass funnel, and washed with water (2×2 mL), saturated aqueous NaHCO3 (3×2 mL), water (3×2 mL), 5% HCl (3×2 mL), water (2×2 mL), acetonitrile (1 ml), and diethyl ether (1 mL). The washed product was dried overnight under high vacuum to provide 201.0 mg, 71% yield of a white powder.
A four-step reaction sequence starting from commercially available 4-chlorobenzoic acid was used to synthesize 4-Substituted analogs. After esterification, the methyl ester was condensed with an appropriately substituted nitrile such as butyronitrile to give, for example, an alpha-ethyl-beta-ketonitrile. The 4-ethylpyrazole was obtained by cyclization with hydrazine. The mercaptoacetamide analog was obtained by refluxing the 3-aminopyrazole with thioglycolic acid in toluene.
To 4-chlorobenzoic acid (15.6 g, 100 mmol) dissolved in methanol (100 mL) was added concentrated H2SO4 (1 mL), and the reaction was then heated to reflux for 18 h. The reaction mixture was then cooled in an ice bath, and the crystalline product was collected on fritted glass, washed with water, saturated aqueous NaHCO3, and water. The material was further dried under high vacuum to give 15.3 g, 90% yield of a white crystalline solid.
Methyl 4-chlorobenzoate (1.70 g, 10 mmol) was dissolved in butyronitrile (10 mL, dried over 3 Å molecular sieves). NaOCH3 (1.08 g, 20 mmol) was added and the reaction was stirred at room temperature under argon for 18 h, and the reaction was then heated to 10° C. for 1 h. The reaction mixture was cooled to ambient temperature and the volatiles were removed by rotary evaporation. The residue was dissolved in water (10 mL) and washed with ether (3 times), and the aqueous layer was then acidified to pH 6.4 with citric acid. The resulting precipitate was collected on fitted glass, washed with water, saturated aqueous NaHCO3, and water. The solid on the filter was dried under high vacuum over night to provide the beta-ketonitrile (841 mg, 41% yield).
The beta-ketonitrile, prepared above, 3-(4-chlorophenyl)-2-ethyl-3-oxopropanenitrile (415 mg, 2 mmol), was dissolved in abs. ethanol (2 mL). To this solution was added anhydrous hydrazine (75 μL, 2.4 mmol), and the reaction was stirred for 1 h at room temperature allowing the hydrazone to form and precipitate. The mixture was then heated to 10° C. for 45 min, and the progress of the reaction was monitored by HPLC. After heating for 45 min, water (1 mL to 5 mL) was added to precipitate the heterocyclic product as a solid. This material was collected on a fritted glass funnel, washed with water, and dried overnight under high vacuum to provide 303.1 mg, 68.4% yield.
A solution of 3-(4-chlorophenyl)-4-ethyl-1H-pyrazol-5-amine (221.5 mg, 1 mmol), thioglycolic acid (104 μL, 1.5 mmol), and toluene were heated in a sealed tube under argon for 24 h. The reaction solution was then cooled to room temperature to precipitate the crude product as a solid. The solid was collected on a fritted glass funnel, and washed with water (2×2 mL), saturated aqueous NaHCO3 (3×2 mL), water (3×2 mL), 5% HCl (3×2 mL), water (2×2 mL), acetonitrile (1 ml), and diethyl ether (1 mL). The washed product was dried overnight under high vacuum to provide 221.3 mg, 74.8% yield of a white powder.
A four-step reaction sequence starting from commercially available 4-chlorobenzoic acid was used to synthesize 4-Substituted analogs. After esterification, the methyl ester was condensed with acetonitrile to give the 3-aminopyrazole. The N1-benzylpyrazole was obtained by cyclization with benzylhydrazine. The mercaptoacetamide analog was obtained by refluxing the N1-substituted pyrazole with thioglycolic acid in toluene.
To 4-chlorobenzoic acid (15.6 g, 100 mmol) dissolved in methanol (100 mL) was added concentrated H2SO4 (1 mL). The reaction was then heated to reflux for 18 h, and the reaction mixture was cooled in an ice bath. The crystalline to product was collected on fritted glass, washed with water, saturated aqueous NaHCO3, and water. The material was further dried under high vacuum to give 15.3 g, 90% yield of a white crystalline solid.
Methyl 4-chlorobenzoate (3.40 g, 20 mmol) was dissolved in toluene (16 mL). Acetonitrile (1.32 mL, 25 mmol) and NaOCH3 (1.08 g, 20 mmol) were added and the reaction was stirred at room temperature under argon for 18 h. The reaction was heated to 100° C. for 1 h, and the reaction mixture was cooled to ambient temperature and the volatiles were removed by rotary evaporation leaving a residue. The residue was dissolved in water (10 mL) and washed with diether (3 times). The aqueous layer was then acidified to pH 6.4 with citric acid. The resulting precipitate was collected on fitted glass, washed with water, saturated aqueous NaHCO3, and water. The solid on the filter was dried under high vacuum over night to provide the beta-ketonitrile (692 mg, 20% yield).
The beta-ketonitrile, prepared above, 3-(4-chlorophenyl)-3-oxopropanenitrile (177 mg, 1 mmol), was dissolved in abs. ethanol (1 mL). To this solution was added benzyl hydrazine (146, 1.2 mmol, prepared by free basing commercial benzylhydrazine hydrochloride). The reaction mixture was heated in a sealed tube to 100° C. for 1 h, and the reaction was cooled to room temperature. Water (2 mL) was added dropwise to precipitate the heterocyclic product as a solid. This material was collected on a fritted glass funnel, washed with water, then dried overnight under high vacuum to provide 238.8 mg, 85% yield of a fluffy powder.
A solution of 1-benzyl-3-(4-chlorophenyl)-1H-pyrazol-5-amine (238.0 mg, 0.85 mmol), thioglycolic acid (117 μL, 2.0 mmol), and toluene (850 μL) were heated in a sealed tube under argon for 48 h. The reaction solution was cooled to room temperature to precipitate the crude product as a solid. The solid was collected on a fritted glass funnel, and washed with H2O (2×2 mL), saturated aqueous NaHCO3 (3×2 mL), H2O (3×2 mL), 5% HCl (3×2 mL), water (2×2 mL), acetonitrile (1 ml), and diethyl ether (1 mL). The washed product was dried overnight under high vacuum to provide 173.5 mg, 57% yield of a white powder.
An alternative method to synthesize mercaptoacetamide analogs involved coupling of amines and anilines with the para-nitrophenylester (PNP) of S-trityl-mercaptoacetic acid. The PNP-ester was synthesized in three steps from triphenylthiomethanol. The mercaptan was reacted with ethyl bromoacetate, the ethyl ester was cleaved under basic conditions to provide the free carboxylic acid. The acid was coupled with para-nitrophenol to give the PNP-activated, trityl-protected, mercaptoacetic acid.
A solution of triphenylmethanethiol (20.0 g, 72.3 mmol), ethyl bromoacetate (8.83 mL, 79.6 mmol), diisopropylethylamine (15.1 mL, 86.8 mmol) and dimethylformamide (60 mL) was stirred at room temperature for 3 h. The reaction was diluted with ethyl acetate (300 mL) and washed with H2O (3×80 mL), saturated aqueous citric acid (3×80 mL), saturated aqueous NaHCO3 (3×80 mL), and brine (2×80 mL). The organic solution was dried (Na2SO4) and rotary-evaporated (30 min with a bath temperature of 45° C.) to remove the excess ethyl bromoacetate. High vacuum (1 mm Hg) was applied for 48 h to provide the desired product as light-yellow crystals (24.09 g, 92% yield).
A solution of ethyl 2-(tritylthio)acetate (24.09 g, 66.5 mmol), 2N NaOH (66 mL, 133 mmol), and dioxane (66 mL) was refluxed for 2 h. The reaction was cooled to room temperature and diluted with ethyl acetate (150 mL) and H2O (150 mL). The basic aqueous layer was collected, and the organic layer was extracted once with H2O (50 mL). The combined aqueous layers were made acidic with solid citric acid (−40 g) with stirring to pH 2-4. The product was extracted with dichloromethane (3×80 mL), dried (Na2SO4) and rotary-evaporated to provide 20.49 g, 92.1% yield of a white solid.
A solution of 2-(tritylthio)acetate (20.49 g, 61.3 mmol), 4-nitrophenol (10.2 g, 73.5 mmol), N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.9 g, 67.4 mmol) in ethyl acetate (200 mL), dichloromethane (100 mL), and dimethylformamide (50 mL) was stirred 3 days under argon at room temperature. The reaction was diluted with diethyl ether (400 mL) and washed with saturated aqueous citric acid (3×100 mL), saturated aqueous NaHCO3 (3×100 mL), saturated aqueous K2CO3 (100 mL), and brine (100 mL). The organic layer was then dried (Na2SO4) and rotary-evaporated to a yellow solid. This material was further purified by silica gel chromatography, gradient elution from hexane to 4:1 ethylacetate:hexane to provide 18.81 g, 67% yield of a faintly yellow powder that was subsequently stored under argon.
To a solution of 2-amino-5-fluoropyridine (235.4 mg, 2.1 mmol) and 4-nitrophenyl-2-(tritylthio)acetate (957.6 mg, 2.1 mmol) in DMF (10 mL) was added triethylamine (725 μL, 4.2 mmol). The reaction was stirred for 18 h at 50 C, and the reaction mixture was poured into diethyl ether (100 mL), washed with 2N NaOH (4×20 mL), H2O (2×20 mL), and brine (20 mL). The organic layer was dried (Na2SO4) and rotary-evaporated to give 674 mg, 75% yield of a solid.
Under argon tri-isopropyl silane (500 μL, 4.08 mmol) was added to a solution of N-(5-fluoropyridin-2-yl)-2-(tritylthio)acetamide (674 mg, 1.58 mmol) dissolved in dichloromethane (10 mL). Trifluoroacetic acid (10 mL) was added slowly over 5 min., and the reaction was stirred at room temperature for 15 min. after the addition was complete. The volatiles were removed on the rotary-evaporator, and the crude product was votexed twice with hexane (5 mL) to remove the triphenylmethane byproduct. The insoluble product was collected on fitted glass and washed with hexane (5 mL) to provide 291 mg, 99% yield of a powder.
An alternative route to mercaptoacetamide analogs is a three step procedure in which an amine is first reacted with chloroacetylchloride. The resulting chloride is then reacted with potassium thioacetate. The mercaptoacetamide is formed after aqueous hydrolysis of the thioacetate ester.
A solution of 2,4-dichlorobenzylamine (352 mg, 2.0 mmol) in THF (5 mL) was cooled in an ice bath, and chloroacetyl chloride (191 μL, 2.4 mmol) was added followed by the dropwise addition of triethylamine (418 μL, 3.0 mmol). The reaction was tested for completion using Ninhydrin spray reagent on a TLC plate. The reaction was quenched by the addition of 1N HCl (10 mL) and ethyl acetate (50 mL). The organic layer was washed with 1N HCl (3×10 mL), saturated aqueous NaHCO3 (2×10 mL), and brine (10 mL). The organic layer was then washed (Na2SO4) and dried under high vacuum to provide 444.6 mg, 88% yield of a tan solid.
To a solution of N-(2,4-dichlorobenzyl)-2-chloroacetamide (440 mg, 1.74 mmol) in DMF (3 mL) was added potassium thioacetate (398 mg, 3.48 mmol). The reaction mixture was heated to reflux for 1 min. and cooled to room temperature. The reaction was diluted with ethyl acetate (50 mL) and washed with saturated aqueous NaCl (2×20 mL), saturated aqueous citric acid (2×20 mL), and brine (20 mL). The organic layer was dried (Na2SO4), rotary-evaporated, and chromatographed on silica gel, gradient elution with 25% ethyl acetate/hexane to 50% ethyl acetate/hexane to provide 353 mg (70% yield) of a pink solid. NMR was consistent with the product.
A solution of S-(2,4-dichlorobenzylcarbamoyl)methyl ethanethioate (150 mg, 0.517 mmol) was dissolved in methanol (4 mL), and the solution was repeatedly degassed with vacuum/argon. An aqueous solution of 2N NaOH (1.3 mL, 2.58 mmol) was added through a septum and the reaction mixture was stirred at room temperature for 30 min. to cleave the acetate ester. The reaction was then quenched with 1N HCl (3.0 mL) while still under an inert atmosphere. The methanol was evaporated under vacuum and the resulting solution was extracted with dichloromethane (2×5 mL). The organic layer was washed with brine (2 mL), dried over Na2SO4 and evaporated under vacuum to give 122.5 mg (95% yield) of the desired mercaptoacetamide as a solid.
This example describes an exemplary procedure expression of MMP enzymes and purification from lysed cells. Equivalent procedures are within the scope of the invention.
The cell line used is a derivative of 293 cells overexpressing a fusion of the gene encoding each MMP protein with a nucleotide sequence encoding the Flag marker.
Cells are grown in Optimem, 2% Fetal Calf Serum, Pen/Strep. For enzyme preparation, Lysis buffer (IPLS) is 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5 mM EDTA and 0.5% Nonidet P-40, to which is added one tablet of Protease inhibitors (Roche 11836170001) per 10 ml buffer. Other buffers are IPHS, which is IPLS containing 1 M NaCl; TBS (Sigma #T5912) dilute 10× stock to 1× with dH2O; HD buffer: 10 mM Tris pH 8.0 (1M Stock) 10 mM NaCl (5M Stock), 10% glycerol, and for dialysis: 400 μM PMSF is added (for 2 L: use 8 ml 100 mM Stock). Protease inhibitors (Complete mini, Boehringer Mannheim), 1 tablet/10 mL are added to all buffers but not used in buffers for enzyme assays.
Cells are grown in 500 cm2 trays, from which about half of the media is aspirated (50 ml total). Cells are harvested in PBS without trypsin, and most cells are readily recovered with gentle striking or agitation of flasks if necessary. Remaining adhering cells are scraped in PBS. Cells are scraped in additional medium and are transferred to a centrifuge tube Trays are washed with 25 ml cold PBS, scraped again to collect additional cells, and cells are centrifuged at 1500 rpm at 4° C. for 5 min. Cells are washed at least three times in PBS to remove medium, pelleting cells after each wash by centrifugation at 1500 rpm for 5 minutes. After each washing, the cells are recovered, PBS is removed, and the resulting cell pellet is frozen at −80° C. for storage prior to purification.
Prior to MMP purification, cells are resuspended in lysis buffer, in an amount of 12 mL of IPLS for the amount of cells collected from ten 500 cm trays. Cells are lysed at 4° C. for 3 hrs with rocking, and debris is removed by centrifugation for 20 min at 17,000 rpm in 30 ml centrifuge tubes. A clear supernatant which is a resulting cell lysate is obtained. Protein concentration of the cell lysate is determined (generally in the range of about 2-5 mg/ml).
Immunoprecipitation of the cell lysate is performed to affinity purify the MMP. For immunoprecipitation per mg of protein, 15 μL of anti-Flag M2-Agarose Affinity beads (Sigma #A2220) is used. Prior to mixing with the lysate, beads are prepared by washing three times with 10× bead volume of PBS and once with IPLS, centrifuging each of the washes at 1500 rpm for 5 min and combining the bead pellets. The cell lysate is incubated with the Ab-beads overnight at 4° C., and beads are centrifuged to collect the MMP bound to the beads. The MMP bound to the beads is washed in 5× volume of each of the following buffers: three times in IPLS (30 sec at 4° C., spin at 1500 RPM for 5 min); three times in IPHS; and three times in TBS buffer. After each centrifugation, the supernatant is removed by aspiration.
MMP is recovered by elution from the beads, by resuspending the beads in 5× bead volume of TBS with protease inhibitor (Roche 11836170001 1 tablet/10 mL). Enzyme is eluted with 400 μg/ml Flag peptide (Sigma #F-3290) in TBS for three hrs at 4° C. with rotary mixing. After elution of MMP, the beads are removed by centrifugation, and the supernatant with the MMP is transferred to a new tube to which is added 1/10 volume of glycerol. The supernatant is transferred to a dialysis cassette (Pierce #66410) using a 3 cc syringe and 18 G needle, and is dialyze against 2 L HD buffer for 2 hrs at 4° C. (1 L/hour). The resulting purified MMP is divided into aliquots (300 μL/tube), is snap frozen in a dry ice bath, and is stored at −80° C.
The compounds provided herein are tested for inhibitory activity with each of a plurality of different MMPs. For assay of MMP, MMP Fluorescent Activity Assay/Drug Discovery Kit (BioMol # AK500) is used. However any equivalent MMP assay is within the scope of the invention.
The kit uses the following reagents: Fluorescent Assay Buffer (FAB) having 25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl2. Developer: the 20× solution contains 27 mg/mL Trypsin (Sigma #T-8003), is dissolved in Fluorescent Assay Buffer, and divided into aliquots and stored at −80 C (250 μL/96-well plate). Prior to use, the Developer is diluted to 1× and added 10 μL/mL 0.2 mM TSA. Final assay concentrations are: up to 15 μL MMP, 25 μL of substrate (25 μM of rhodamine, 50 μM Fluor de lys substrate, BIOMOL, Plymouth Meeting PA available as kit AK-500), and ±10 μL inhibitor diluted in FAB. The final reaction volume of 50 μL is obtained by adding FAB.
All reaction components are prepared in Fluorescent Assay Buffer; MMP and diluted inhibitors (total volume is 25 μL) are added to each well of a clear bottom 96-well ISOPLATE (Wallac #1450-514). The reactions are initiated by adding 25 μL of 100 μM substrate. Negative control wells contain buffer and substrate only or with potent levels of a known inhibitor such as Bastimastat and Marimastat. Enzyme reactions with DMSO are used as positive controls.
The reaction is incubated for 1-2 hours at 37° C., and reactions are stopped with 50 μL/well of 1× developer containing TSA. Reactions are developed at room temperature for 10 min, and are read with a pre-warmed lamp of Cytofluor Fluorescence Reader. For Fluor de Lys: plates are read at Excitation 360 nm, Emission 460 nm, Gain 65. For Rhodamine: plates are read at Excitation 485 nm, Emission 530 nm, Gain 60.
The general procedure to determine the ability of a compound to inhibit growth of cells to a 50% extent (IC50 of the compound) uses an in vitro cell based assay. Cells are seeded into wells of 96-well plates as described above, and are incubated for growth for 24 hours, after which an aliquot of the compound is added at a variety of dilutions to the cells in each well. After further incubation of 72 hours, plates are read to determine extent of growth.
In general, a set of dilutions of each compound is made to cell growth medium, and 100 samples of dilutions of the compounds are added to the cells, in triplicate (3 rows). Plates are incubated at 37° C. for 72 hours. For determination of activity, CellTiter 96® AQueous One Solution Reagent (Promega) is used. This reagent is stored frozen, and is then thawed, prior to use it is protected from light. A sample of 100 of CellTiter 96® AQueous One Solution Reagent is added into each well of the 96-well assay plate. Plates are incubated for 3 hours at 37° C. in a humidified, 5% CO2 atmosphere, and the absorbance at 490 nm is recorded using a 96-well plate reader.
Compounds herein are determined to be active inhibitors of selected MMP proteins tested, with some having nanomolar activities. A specific pattern of inhibition is observed for each compound, for example, a compound is found that inhibits MMPs 1, 2, 3, 7, 9, and 15, and compounds herein are provided that include inhibitors of each of the MMP species.
This application claims the benefit of U.S. provisional patent application Ser. No. 60/995,769 which was filed in the U.S. Patent and Trademark Office Sep. 28, 2007, the contents of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/77073 | 9/19/2008 | WO | 00 | 10/26/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60995769 | Sep 2007 | US |
