Non-hydrophobic compounds for use in treating metastasis and/or cartilage defect
10828281 · 2020-11-10
Assignee
Inventors
- Anja-Katrin Bosserhoff (Regensburg, DE)
- Alexander Riechers (Babensham, DE)
- Burkhard König (Lappersdorf, DE)
- Manuel Bause (Michelstadt, DE)
- Fabian Rauscher (Steinhöring, DE)
Cpc classification
A61K31/513
HUMAN NECESSITIES
A61K31/4025
HUMAN NECESSITIES
A61K31/53
HUMAN NECESSITIES
A61K31/4184
HUMAN NECESSITIES
A61K31/4402
HUMAN NECESSITIES
A61K31/498
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/517
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61K31/435
HUMAN NECESSITIES
A61K31/4985
HUMAN NECESSITIES
A61K31/40
HUMAN NECESSITIES
A61K31/341
HUMAN NECESSITIES
A61K31/197
HUMAN NECESSITIES
A61K31/4453
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
A61K31/496
HUMAN NECESSITIES
A61K31/4152
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/138
HUMAN NECESSITIES
International classification
A61K31/4184
HUMAN NECESSITIES
A61K31/4152
HUMAN NECESSITIES
A61K31/4402
HUMAN NECESSITIES
A61K31/4453
HUMAN NECESSITIES
A61K31/498
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K31/55
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/138
HUMAN NECESSITIES
A61K31/341
HUMAN NECESSITIES
A61K31/496
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K31/40
HUMAN NECESSITIES
A61K31/517
HUMAN NECESSITIES
A61K31/197
HUMAN NECESSITIES
A61K31/4985
HUMAN NECESSITIES
A61K31/435
HUMAN NECESSITIES
A61K31/53
HUMAN NECESSITIES
Abstract
The present invention is directed to compounds, tautomers, stereoisomers, and chemically modified compounds thereof, and their use in preventing and/or treating tumors of metastasis and/or cartilage defect, and to a pharmaceutical composition comprising such compound.
Claims
1. A method of preventing the occurrence of and/or treating a subject for metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein or of a cartilage defect, wherein regeneration is inhibited by MIA dimerization, the method comprising: administering to the subject a composition comprising a compound that is non-hydrophobic, wherein the compound is selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide.
2. The method according to claim 1, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole.
3. The method according to claim 1, wherein the metastasis is based on a primary tumor that is an MIA-expressing tumor.
4. The method according to claim 3, wherein the primary tumor is selected from the group consisting of melanoma, breast cancer, glioma, pancreas carcinoma, and colon carcinoma.
5. The method according to claim 1, wherein the metastasis is located in the liver, lung, bone, colon, stomach, nerves, lymph nodes, skin and/or brain.
6. The method according to claim 1, wherein the composition is administered orally.
7. The method according to claim 1, further comprising: administering a chemotherapeutic to the subject.
8. The method according to claim 7, wherein the chemotherapeutic is selected from the group consisting of Vermurafenib, Ipilimumab, Trametinib, Dabradenib, Dacarbazine, Paclitaxel, Carboplatin, Interferon-alpha and Aldesleukin.
9. The method according to claim 7, wherein the composition and the chemotherapeutic are administered at the same time.
10. A method for preventing the occurrence of and/or treating a subject for a metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, the method comprising: administering to the subject a pharmaceutical composition comprising: at least one compound selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide, and a pharmaceutically acceptable carrier and/or solvent.
11. The method according to claim 10, further comprising: administering a chemotherapeutic to the subject.
12. The method according to claim 10, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole.
13. The method according to claim 7, wherein the chemotherapeutic is selected from the group consisting of vermurafenib, ipilimumab, trametinib, dabradenib, dacarbazine, paclitaxel, carboplatin, interferon-, and aldesleukin.
14. The method according to claim 10, wherein the pharmaceutical composition further comprises a chemotherapeutic.
15. The method according to claim 7, wherein the composition and the chemotherapeutic are administered consecutively.
16. The method according to claim 8, wherein the composition and the chemotherapeutic are administered at the same time.
17. The method according to claim 8, wherein the composition and the chemotherapeutic are administered consecutively.
18. A method of treating a subject in need thereof by inhibiting melanoma inhibitory activity protein (MIA)MIA dimerization, the method comprising: administering to the subject a composition comprising a non-hydrophobic compound selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide.
19. The method according to claim 18, wherein the subject has a primary tumor.
20. The method according to claim 18, wherein the compound is administered orally.
21. The method according to claim 18, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole.
Description
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION
(7) As a general treatment concept, the selective inhibition of MIA dimerization is an attractive therapy concept since, apart from its expression in malignant melanoma, MIA is only expressed by differentiating chondrocytes. Furthermore, the inhibition strategy of the present invention targets secreted extracellular MIA; thus the need for cell permeability of the compounds of the present invention is avoided. The compounds of the present invention are specifically developed and used for preventing and/or treating metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein or a cartilage defect which results for example in mechanical or immunological destruction and suppression of regeneration of the cartilage by MIA dimerization. MIA avoids the dedifferentiation of chondrocytes which would allow the proliferation of the chondrocytes. Without (dimerized) MIA, the chondrocytes transform into a certain type of precursor cells which are ale to grow and to regenerate, i.e., close a cartilage defect. MIA blocks this regeneration.
(8) The compounds of the present invention interact and bind respectively, with/to the dimerization site of MIA proteins. Thus, the compounds of the present invention avoid the dimerization of MIA proteins and/or break up existing MIA dimers.
(9) The MIA dimerization site which is the site where MIA proteins interact comprises or is formed by at least three amino acid residues of said MIA protein selected from cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, valine 64, tyrosine 69, aspartate 87, lysine 91, glycine 54, leucine 58, phenylalanine 59, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85 and lysine 94. In one embodiment, the dimerization site of a MIA protein comprises or consists of cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85, and lysine 94. In another embodiment, the MIA dimerization site comprises or consists of cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103 and cysteine 106.
(10) Via the dimerization site even more than two MIA proteins may interact and form an aggregate comprising or consisting of three or more MIA proteins.
(11) TABLE-US-00001 TABLE 1 Com- Reference pound of x Structure No. Tautomere/Stereoisomer Variant Com- pound 1
(12) Table 1 presents compounds of the present invention as well as examples of tautomers and stereoisomers, respectively, the number of potential tautomer or stereoisomers is indicated in brackets in column 5, reference of variant.
(13) A chemical modification of compounds 1 to 270 is any modification that results in a compound characterized by a specific and effective interaction with or binding to the dimerization site of MIA. Chemical modifications are for example selected from the group consisting of H, alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or alkyl, arylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryalkyl, and alkoxyalkyl, each of which is optionally substituted one, two or three times with independently selected non-polar or polar groups.
(14) Alkyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. Lower alkyl as used herein, is a subset of alkyl, in some embodiments preferred, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The term alkyl or loweralkyl is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(0)m, haloalkyl-S(0)m, alkenyl-S(0)m, alkynyl-S(0)m, cycloalkyl-S(0)m, cycloalkylalkyl-S(0)m, aryl-S(0)m, arylalkyl-S(0)m, heterocyclo-S(0)m, heterocycloalkyl-S(0)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxy acylamino, aminoacyloxy, nitro or cyano where m=0, 1, 2 or 3.
(15) Alkenyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in lower alkenyl 1 to 4 carbon atoms) which include 1 to 4 double bonds in the normal chain.
(16) Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term alkenyl or lower alkenyl is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.
(17) Alkynyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triple bond in the normal chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term alkynyl or loweralkynyl is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
(18) Cycloalkyl as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below). Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or lower alkyl. The term cycloalkyl is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.
(19) Heterocyclic group or heterocyclo as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or a bicyclic-ring system. Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(O)m, haloalkyl-S(O)m, alkenyl-S(O)m, alkynyl-S(O)m, cycloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocyclo-S(O)m, heterocycloalkyl-S(O)m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m=0, 1, 2 or 3.
(20) Aryl as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system. having one or more aromatic rings. Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
(21) Arylalkyl as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.
(22) Heteroaryl as used herein is as described in connection with heterocyclo above.
(23) Alkoxy as used herein alone or as part of another group, refers to an alkyl or loweralkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
(24) Halo as used herein refers to any suitable halogen, including F, CI, Br, and I.
(25) Compound 1 is represented by the following general Formula (I)
(26) ##STR00533##
(27) wherein,
(28) R.sub.1, R.sub.2 and R.sub.4 represent, independent of each other, hydrogen, halogens, alkyls, alkenyls, alkynyls, cycloalkyls or aryls;
(29) R.sub.3 represents a linear n.sub.1-7 alkyl, a branched n.sub.3-7 alkyl, a linear n.sub.1-7 alkenyl, a branched n.sub.3-7 alkenyl, a linear n.sub.1-7 alkynyl, a branched n.sub.3-7 alkynyl, a n.sub.5-7 cycloalkyl, a n.sub.5-7 cycloalkenyl, a n.sub.5-7 cycloalkynyl, a n.sub.5-7 aryl;
(30) X represents, independent of each other, a substituted or unsubstituted heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom;
(31) Y represents a substituted or unsubstituted amine which may form a double bond with a neighboring carbon atom; and
(32) Z represents a halogen or a substituted or unsubstituted heteroatom selected from O, N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.
(33) Compound 1 comprises for example the following modifications:
(34) ##STR00534## ##STR00535##
(35) Compound 2 is represented by the following general Formula (II)
(36) ##STR00536##
(37) wherein,
(38) R.sub.1 represents a hydrogen, alkyl, alkenyl, alkynyl, amin, aminoalkyl, aminoalkenyl, halogens, alkyls, alkenyls, alkynyls, cycloalkyls or aryl;
(39) R.sub.2 represents, independent from each other, a hydrogen, alkyl, alkenyl, or alkynyl;
(40) X represents a substituted or unsubstituted heteroatom selected from N and S; and
(41) Y represents a linear n.sub.3-5 alkyl, a linear n.sub.3-5 alkenyl, or a linear n.sub.3-5 alkynyl.
(42) Compound 2 comprises for example the following modifications:
(43) ##STR00537##
(44) Compound 3 is represented by the following general Formula (III)
(45) wherein,
(46) ##STR00538##
(47) R.sub.1 represents a hydrogen, linear n.sub.3-5 alkyl, a linear n.sub.3-5 alkenyl, a linear n.sub.3-5 alkynyl, a linear n.sub.3-5 hydroxlylalkyl, a linear n.sub.3-5 hydroxlylalkenyl, a linear n.sub.3-5 hydroxlylalkynyl, a linear
(48) n.sub.3-5 ethoxylylalkyl, a linear n.sub.3-5 ethoxylylalkenyl, or a linear n.sub.3-5 ethoxylylalkynyl;
(49) R.sub.2 represents a hydrogen, halogen, alkyl, alkenyl, or alkynyl;
(50) X represents a hydroxyl or a O group;
(51) Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.
(52) Compound 3 comprises for example the following modifications:
(53) ##STR00539##
(54) Compound 4 is represented by the following general Formula (IV)
(55) ##STR00540##
(56) wherein,
(57) R.sub.1 represents, independent from each other, a hydrogen, halogen, alkyl, alkenyl, or alkynyl;
(58) R.sub.2 represents, independent from each other, a hydrogen, halogen, alkyl, alkenyl, alkynyl or wherein the R.sub.2 residues together form a n.sub.5-7 cycloalkyl or an n.sub.5-7 aryl;
(59) X represents a hydrogen, a hydroxyl or a O group;
(60) Y represents, independent from each other, a carbon atom or a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.
(61) Compound 4 comprises for example the following modifications:
(62) ##STR00541##
(63) Compound 5 is represented by the following general Formula (V)
(64) ##STR00542##
(65) wherein,
(66) R.sub.1 represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl, or wherein two R.sub.1 form a cycloalkyl or an aryl;
(67) R.sub.2 represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl;
(68) R.sub.3 represents a hydrogen, halogen, ethoxyl, alkyl, alkenyl, or alkynyl;
(69) Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom; and
(70) Z represents a carbon atom or N.
(71) Compound 5 comprises for example the following modifications:
(72) ##STR00543##
(73) Compound 6 is represented by the following general Formula (VI)
(74) ##STR00544##
(75) wherein,
(76) R.sub.1 represents a linear n.sub.2-6 alkyl, a branched n.sub.3-6 alkyl, a linear n.sub.2-6 alkenyl, a branched n.sub.3-6 alkenyl, a linear n.sub.2-6 alkynyl, a branched n.sub.3-6 alkynyl, a n.sub.5-7 cycloalkyl, a n.sub.5-7 cycloalkenyl, a n.sub.5-7 cycloalkynyl, a n.sub.5-7 arylalkyl, a n.sub.1-3 alkylcarboxyl;
(77) R.sub.2 represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl;
(78) Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom; and
(79) Z represents, independent of each other, a carbon atom which may form a double bond with a neighboring carbon atom.
(80) Compound 6 comprises for example the following modifications:
(81) ##STR00545##
(82) The present invention is not only directed to the non-hydrophobic compounds of Table 1, to the tautomers and stereoisomers thereof, but also to chemically modified forms of these compounds as mentioned for example above.
(83) The metastasis preventable and/or treatable by use of a compound of the present invention is based on any type of primary tumor for example melanoma, breast cancer, glioma, pancreas carcinoma, and colon carcinoma. In one embodiment, the primary tumor is a late stage tumor. The metastasis is for example located in the liver, lung, bone, colon, stomach, nerves, lymph nodes, skin and/or brain.
(84) The cartilage defect preventable and/or treatable by use of a compound of the present invention is for example a degenerative disorder of cartilage selected from rheumatoid arthritis, degeneration of cartilage in a joint, degenerative disc disease, meniscus tears, anterior crucial ligament (ACL) injury, arthritis, osteoarthritis, psoriatic arthritis, juvenile chronic arthritis, rhizomelic arthritis, rheumatoid poly-arthritis, synovitis and villonodular synovitis.
(85) The dimerization of the MIA protein is for example measured by a heterogeneous transition metal-based fluorescence polarization (HTFP) assay, wherein the ratio P/P.sub.0 is measured. P is the fluorescence polarization signal of a MIA protein labeled with a transition metal complex in the presence of substrate bound MIA-protein and of a compound to be tested. P.sub.0 is the fluorescence polarization signal of free MIA-protein labeled with said luminescent transition metal complex in the absence of substrate bound MIA-protein and in the absence of the compound. In the absence of the compound, usually, the labeled MIA-protein would interact with the substrate bound MIA-protein, which, in turn, would contribute to a reduction in rotational mobility of the labeled MIA-protein, and therefore, the fluorescence polarization signal would increase upon such interaction. If, additionally, a compound is present that interferes with such interaction, no or little dimerization/aggregation occurs and no or little increase in fluorescence polarization signal would be detected. The smaller or even more negative P/P.sub.0 is, the stronger such interference with dimer formation and aggregation is, and the better such compound prevents or breaks up dimerization/aggregation of MIA protein. In an alternative embodiment, binding of the compound to MIA protein is determined by NMR, e.g., heteronuclear NMR, e.g., .sup.15N-.sup.1H-HSQC-NMR.
(86) In the present invention at least one compound of 1 to 270 of Table 1, a tautomer, a stereoisomer or a chemically modified compound thereof is administered to a subject, or in another embodiment two or more of these compounds, tautomers, stereoisomers and/or chemically modified compounds thereof are administered to a subject for use in preventing and/or treating of metastasis or a cartilage defect. The compounds are administered at the same time or consecutively.
(87) In one embodiment the size of compounds of the present invention is increased by compound growing or compound linking. Regarding fragment growing an initial compound is steadily build up to explore favorable interactions with adjacent regions of the dimerization site; and regarding compound linking compounds of the present invention (e.g., two or more for example 3, 4, 5, 6, 7, 8, 9, 10) are connected to each other, i.e., are coupled with or without a linker.
(88) In an embodiment of the present invention the compounds are hydrophobic, in another embodiment the compounds are hydrophilic or neutral.
(89) In another embodiment one or more compounds of the present invention are administered to a subject in combination with a chemotherapeutic which is any chemical agent, e.g., naturally occurring or synthesized, effective in the treatment of cancer such as Vermurafenib, Ipilimumab, Trametinib, Dabradenib, Dacarbazine, Paclitaxel, Carboplatin, Interferon-alpha and Aldesleukin, TGF alpha, TGF beta, interleukin etc. The compound and the chemotherapeutic are administered at the same time or consecutively.
(90) The present invention further refers to a pharmaceutical composition for use in preventing and/or treating of metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, wherein the pharmaceutical composition comprises at least one compound selected from the group consisting of compound 1 to 270 of Table 1, tautomers, stereoisomers and/or chemically modified compounds thereof, and a pharmaceutically acceptable carrier and/or solvent. The pharmaceutical composition may further comprise a chemotherapeutic or any substance, including cells such as chondrocytes, positively influencing cartilage growth and regeneration, respectively.
EXAMPLES
(91) The following examples show the present invention in more detail, however, the invention is not limited to these examples.
Example 1
(92) Fragment-based in silico screening was applied to identify the small, fragment-sized compounds of the present invention that inhibit MIA. The suggested structures were screened in vitro and modular synthesis strategies were developed for the most promising molecules.
(93) NMR Titration Experiment
(94) Derived from HSQC titrations of .sup.15N-labeled MIA with AR71 (a peptide having the amino acid sequence FHWRYPLPLPGQ (SEQ ID NO:1)), the amino acids CYS17, SER18, TYR47, GLY66, ASP67, LEU76, TRP102, ASP103, and CYS106 were found to exhibit strong shift perturbations (Schmidt, J., A. Riechers, and A. K. Bosserhoff, MIAa new target protein for malignant melanoma therapy. Histol Histopathol, 2013. 28(4): p. 421-6) and were defined as interacting residues in an in silico protein-peptide docking of MIA and AR71. The resulting model shows the binding of the peptide in the hydrophobic cleft, which forms part of the dimerization domain.
(95) Virtual Screening
(96) The 1.4 resolution crystal structure of human MIA protein 1I1J (Lougheed, J. C., et al., Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins. Proceedings of the National Academy of Sciences, 2001, 98(10): p. 5515-5520) and the first model of each of the NMR solution structures 1K0X (Lougheed, J., P. Domaille, and T. Handel, Solution structure and dynamics of melanoma inhibitory activity protein. Journal of Biomolecular NMR, 2002, 22(3): p. 211-223) and 1HJD (Stoll, R., et al., The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold. 2001. 20(3): p. 340-349) were used as distinct MIA protein receptor conformations for the virtual screening experiment. The same amino acid residues that were used in the protein-peptide docking were provided as input to the molecular docking software in order to guide the automated detection and definition of putative binding sites in the MIA protein.
(97) In each of the three MIA protein conformations the same single putative binding site was detected. The site is located in a cleft near the distal loop (residues 69 to 75) and is framed by the solvent exposed disulfide loop (residues 13 to 19), the turn of the RT loop (residues 35-38), and the C-terminal residues 102 to 106. Most of the amino acid residues identified to interact with the AR71 peptide by induced chemical shift changes constitute to the putative binding site, with the exception of TYR48, GLY66, and ASP67, which are located on the distal loop and thus too far away. Differences in the arrangement of the structural elements surrounding the cleft within each of the three distinct MIA receptor conformations result in a different volume and shape of the putative binding site.
(98) Fragment library preparation commenced with 28751 fragment structures that were contained within the unprocessed fragment library catalogue obtained from the compound provider. Filtering for salts, unwanted chemistry and functional groups, as well as duplicate structures led to the removal of 1, 2577 and 127 structures, respectively. Each of the remaining 26046 structures was subjected to complete enumeration of its protonation and tautomeric states, which led to 87270 protomer variants for all fragment structures. Subsequently, all stereoisomers were enumerated for the protomer variants, which resulted in 124590 fragment variants. Generation of 3D conformations allowed for up to five alternate ring conformations per fragment variant and produced a final library of 217176 fragment variant conformations, which were docked into the three MIA receptor conformations with their respective putative binding sites.
(99) The resulting docking poses were subjected to a filtering and ranking workflow, which, for each MIA protein conformation, determined a list of fragments ranked by the empirical scoring function of the docking software. The ten highest-ranking fragments for each MIA receptor conformation were selected for experimental testing.
(100) All listed compounds that were screened for their ability to interfere with the MIA-MIA interaction (Table 1) in a heterogeneous transition metal-based fluorescence polarization (HTFP) assay previously developed (Riechers, A., et al., Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions. Biotechniques, 2009. 47(4): p. 837-44). This screening revealed significant decreases in the fluorescence polarization of Ru-(bpy).sub.3, labelled MIA for compounds 1, 2, 3, 4, 5, and 6; the results are shown in
Example 2
(101) Boyden chamber migration assays (Stoll, R., Lodermeyer, S. & Bosserhoff, A. K. Detailed analysis of MIA protein by mutagenesis. Biol Chem 387, 1601-1606, (2006)) using human Mel-Im melanoma cells demonstrated a reduction in the MIA activity on melanoma cell migration in the presence of compounds 1, 2, 3, 4, 5, and 6 at a concentration of 1 M as shown in
(102) The melanoma cell line Mel-Im, established from a human metastatic bioptic sample (generous gift from Dr. Johnson, University of Munich, Germany) was used for the Boyden chamber migration experiments. All cells were maintained in DMEM (PAA, Pasching, Germany) supplemented with penicillin (400 U/mL), streptomycin (50 g/mL), L-glutamine (300 g/mL) and 10% fetal calf serum (Pan Biotech GmbH, Aidenbach, Germany) and split in a 1:6 ratio every three days. Migration assays were performed in Boyden Chambers containing polycarbonate filters with 8-nm pore size (Neuro Probe, Gaithersburg, Md., USA) essentially as described. MIA was added to the cell suspension at a final concentration of 200 ng/mL. Selected compounds were used at a final concentration of 1 M. Experiments were carried out in triplicates and repeated at least three times.
Example 3
(103) In order to assess whether compounds of the present invention show any adverse effects on normal cells, human fibroblasts and kidney cells were treated with the compounds 1, 2, 3, 4, 5, and 6, respectively, in vitro at a concentration of 7.8 M in analogy to previous studies (Schmidt, J., et al., Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition. PLoS One, 2012. 7(5): p. e37941; Riechers, A., et al., Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions. Biotechniques, 2009. 47(4): p. 837-44). There were no negative effects on the proliferation of human fibroblasts (
(104) A significant decrease in proliferation of the human melanoma cell line Mel Im after treatment with compound 1 was observed as presented in (