Patent Grant
8580815
The present invention relates to compounds that are inhibitors of protein kinases, the method of preparation thereof and the therapeutic application thereof.
Dysfunction of protein kinases (PK) is the cause of a large number of pathologies. In fact, a high proportion of the oncogenes and proto-oncogenes involved in human cancers code for PK.
It has also been discovered that increased activity of the PK is involved in many non-malignant diseases such as benign prostatic hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, proliferation of smooth cells of blood vessels associated with atherosclerosis, pulmonary fibrosis, glomerulonephritis and stenoses and post-operative restenoses.
The PK are involved in the inflammatory response and the multiplication of viruses and parasites. The PK are also known to have a major role in the pathogenesis and development of neurodegenerative disorders.
Chronic myeloid leukaemia (CML) is due to the malignant transformation of a haematopoietic stern cell characterized by an acquired genetic abnormality: the oncogene BCR-ABL, produced by the chromosome Philadelphia (Ph) 22q (Deininger et al., Blood, 2000, 96, 3343-3356). This fusion gene codes for a chimeric protein Bcr-AbI displaying constitutively activated tyrosine kinase AbI activity.
Expression of a Bcr-AbI fusion protein arising from a different translocation of the BCR gene is also the cause of Ph-positive acute lymphoblastic leukaemia (ALL Ph+), for Philadelphia-chromosome positive (Chan et al., Nature, 1987, 325, 635-637; Melo et al., Leukemia, 1994, 8, 208-211; Ravandi et al., Br. J. Haematol., 1999, 107, 581-586). CML represents about 15 to 20% of leukaemias in adults. This disease, characterized by excessive proliferation of the cells of the myeloid line, is regarded by many specialists as a reference model for understanding the mechanisms of proliferation of haematopoietic stem cells, as well as for the development of new medicinal products against cancers.
One of the first inhibitors discovered to be active against kinase AbI is Imatinib (Gleevec/Glivec, STI-571; Novartis Pharma AG), a molecule of the phenylaminopyrimidine type. Imatinib was developed for the therapeutic treatment of cancers (Druker et al., Nat. Med., 1996, 2, 561-566). Druker demonstrated selective inhibition of the kinase AbI by Imatinib with an IC50 between 0.038 and 0.025 μM.
It was demonstrated that this molecule not only inhibits the kinase AbI, but also plays the role of inhibitor of c-KIT, PDGFR (platelet-derived growth factor receptor) or also of other proteins (Bantscheff et al., Nat. Biotechnol., 2007, 25, 1035-1044; Okuda et al., Blood, 2001, 97, 2440-2448; Dewar et al., Blood, 2005, 105, 3127-3332). Clinical trials have shown that Imatinib induces rapid remission in patients in the chronic phase, expressing the Bcr-AbI fusion protein, while displaying negligible toxicity (O'Brien et al., N. Engl. J. Med, 2003, 384, 994-1004; Apperley et al., N. Engl. J. Med, 2002, 347, 471-487).
Imatinib has also been tested successfully in phase II clinical trials on gastrointestinal stromal tumours (GIST) expressing KIT and PDGFRα and has shown promising efficacy on a certain number of diseases in which deregulation of the kinase activity of PDGFR has been demonstrated including a dermatofibrosarcoma, a hypereosinophilic syndrome and chronic myelomonocytic leukaemia; (Rubin et al., J. Clin. Oncol, 2002, 20, 3586-3591; Maki et al., Int. J. Cancer, 2002, 100, 623-6262; Apperley et al., N. Engl. J. Med, 2002, 347, 471-487; Gotlib et al., Blood, 2004, 103, 2879-2891).
However, some patients with CML or ALL Ph+ at an advanced stage develop resistance to Imatinib, generally reflected in the appearance of mutations at the catalytic site or in the regions near Bcr-AbI or in amplification of the BCR-ABL gene (Gorre et al., Science, 2001, 293, 876-80; Le Coutre et al., Blood, 2000, 95, 1758-66; Weisberg et al., Blood, 2000, 95, 3498-505; Mahon et al., 2000, Blood, 96, 1070-9; Campbell et al., Cancer Genet Cytogenet, 2002, 139, 30-3; Hochhaus et al., Leukemia, 2002, 16, 2190-6).
The protein kinases of the Src family are involved in leukaemias mediated by Bcr-AbI and have been implicated in several cases of resistance to Imatinib. In this connection, several Src/AbI inhibitors have been developed, such as 4-anilo-3-quinolinecarbonitrile, 2,6,9 trisubstituted purine analogues, and pyrido[2,3-d]pyrimidines, to offer alternative treatments for patients with Chronic Myeloid Leukaemia or Acute Lymphoblastic Leukaemia (Martinelli et al., Leukemia, 2005, 19, 1872-1879).
The kinases of the Src family also regulate key routes of metastatic progression of malignant tumours including adhesion, invasion, cellular motility and angiogenesis (Brunton et al., Curr Opin Pharmacol., 2008, 8, 427-432). Overexpression of the protein c-Src as well as increase in its activity were observed in several types of cancers including colorectal cancer, various gastrointestinal cancers (hepatic, pancreatic, gastric and oesophageal) as well as breast, ovarian and lung cancers (Yeatman et al., Nat Rev Cancer, 2004, 4, 470-80).
Moreover, crystallographic studies have revealed that Imatinib binds to the kinase domain of AbI only when the latter assumes an inactive conformation (Nagar et al., Cancer Res, 2002, 62, 4236-43; Schindler et al., Science, 2000, 289, 1938-42). These results were at the origin of the development of alternative therapies in order to overcome resistance to Imatinib but also at the origin of the design of new molecules capable of binding specifically to the kinase domain of AbI in active conformation (Cowan-Jacob et al., Acta Crystallogr D Biol Crystallogr, 2007, 63, 80-93; Weisberg et al., Nat Rev Cancer, 2007, 7, 345-56). The classes of new inhibitors developed include selective inhibitors of AbI, inhibitors of the AbI kinases and of the kinases of the Src family, inhibitors of Aurora kinases and inhibitors of Bcr-AbI non-competitive for ATP.
Among the new classes of inhibitors we may mention (Weisberg et al., Nat Rev Cancer, 2007, 7, 345-56): Nilotinib which mainly inhibits AbI as well as KIT and PDGFRβ, Dasatinib which inhibits kinases of the Src family as well as KIT, Bcr-AbI, PDGFR, Bosutinib which inhibits kinases of the Src family and Bcr-AbI and Dasatinib which attaches, in contrast to the other inhibitors, on the active conformation of the AbI kinase domain. However, side effects are observed with Dasatinib, of fluid retention (including pleural effusion), diarrhoea, etc.
However, as suggested by Weisberg et al. (Weisberg et al., Nat Rev Cancer, 2007, 7, 345-56), it can be assumed, as for Imatinib, Nilotinib or some other inhibitors of kinases, that some patients may develop resistance to the new classes of inhibitors. It is therefore necessary to find new molecules that are inhibitors of protein kinases.
The object of the present invention is to offer novel inhibitors of kinases, having an original backbone, which can be used therapeutically in the treatment of pathologies associated with deregulation of protein kinases.
The inhibitors of the present invention can be used for the treatment of numerous cancers and more particularly in the case of chronic or acute myeloproliferative disorders such as certain leukaemias and in the case of colorectal cancer, various gastrointestinal cancers (hepatic, pancreatic, gastric and oesophageal), as well as breast, ovarian and lung cancers.
Another object of the invention is to offer novel selective inhibitors of the kinases AbI and Src.
Yet another objective of the invention is to offer a method of preparation of said inhibitors.
The present invention relates to compounds of general formula I:
in which R represents:
a group NHCOR1, or
a group NR3R4
an aryl, preferably phenyl, group optionally mono- or polysubstituted with:
a heteroaryl group, preferably
a cycloalkyl group, preferably cyclopropyl,
a linear or branched C1-C6 alkyl group, preferably ethyl, isopropyl, or
a C1-C6 aralkyl group, preferably phenylalkyl, preferably phenylmethyl, optionally mono- or poly-substituted with an alkoxy group, preferably methoxy, and/or a halogen atom, preferably bromine.
R2 represents:
an ester group COOR14,
an alkyl group CH2R9, CH2OCOR10, CH2NR11R12,
an amide group CONR7R8, or
a group COR13,
R7 and R8, which may be identical or different, represent:
a hydrogen atom,
a C1-C6 aminoalkyl group, preferably N,N-dimethylaminopropyl, or
a C1-C6 morpholinoalkyl group, preferably N-morpholinoethyl,
R9 represents:
a heteroaryl group, preferably imidazyl or pyrryl,
a heterocyclic group, preferably N-morpholinyl or tetrahydrofuranyl,
an alkoxy group, preferably methoxy, or
a hydroxyl group,
R10 represents:
a heteroaryl group, preferably quinoxaline,
R11 and R12, which may be identical or different, represent:
a hydrogen atom,
a linear or branched C1-C6 alkyl group, preferably tert-butyl,
an aralkyl group, preferably phenylalkyl, preferably phenylmethyl,
a C1-C6 alkoxyalkyl group, preferably methoxyethyl,
a cycloalkyl group, preferably cyclohexyl, optionally mono- or polysubstituted with a C1-C6 alkyl group, preferably methyl,
an aryl, preferably phenyl, group, optionally mono- or polysubstituted with:
or a heteroaryl group, preferably a pyridyl group,
R13 represents a heterocyclic group, preferably N-morpholyl,
R14 represents:
a linear or branched C1-C6 alkyl group, preferably methyl, or
an aryl group, preferably phenyl, optionally substituted with an alkoxy, preferably methoxy, group,
R3, R4, which may be identical or different, represent:
a hydrogen atom,
a group CH2R15,
a heteroaryl group, preferably pyridyl, indyl, benzimidazyl, or pyrazyl optionally substituted with a C1-C6 alkyl group, preferably methyl, or
an aryl, preferably phenyl, group optionally mono- or polysubstituted with:
an aryl, preferably phenyl, group optionally mono- or poly-substituted with:
or a heteroaryl group, preferably:
In one embodiment the present invention relates to compounds of general formula I:
in which R represents:
a group NHCOR1, or
a group NR3R4
an aryl, preferably phenyl, group optionally mono- or polysubstituted with:
a heteroaryl group, preferably
a cycloalkyl group, preferably cyclopropyl,
a linear or branched C1-C6 alkyl group, preferably ethyl, isopropyl, or
a C1-C6 aralkyl group, preferably phenylalkyl, preferably phenylmethyl, optionally mono- or poly-substituted with an alkoxy group, preferably methoxy, and/or a halogen atom, preferably bromine,
R2 represents:
an ester group COOR14,
an alkyl group CH2R9, CH2OCOR10, CH2NR11R12,
an amide group CONR7R8, or
a group COR13,
R7 and R8, which may be identical or different, represent:
a hydrogen atom,
a C1-C6 aminoalkyl group, preferably N,N-dimethylaminopropyl, or
a C1-C6 morpholinoalkyl group, preferably N-morpholinoethyl,
R9 represents:
a heteroaryl group, preferably imidazyl or pyrryl,
a heterocyclic group, preferably N-morpholinyl or tetrahydrofuranyl,
an alkoxy group, preferably methoxy, or
a hydroxyl group,
R10 represents:
a heteroaryl group, preferably quinoxaline,
R11 and R12, which may be identical or different, represent:
a hydrogen atom,
a linear or branched C1-C6 alkyl group, preferably tert-butyl,
an aralkyl group, preferably phenylalkyl, preferably phenylmethyl,
a C1-C6 alkoxyalkyl group, preferably methoxyethyl,
a cycloalkyl group, preferably cyclohexyl, optionally mono- or polysubstituted with a C1-C6 alkyl group, preferably methyl,
an aryl, preferably phenyl, group, optionally mono- or polysubstituted with:
or a heteroaryl group, preferably a pyridyl group,
R13 represents a heterocyclic group, preferably N-morpholyl,
R14 represents:
a linear or branched C1-C6 alkyl group, preferably methyl, or
an aryl group, preferably phenyl, optionally substituted with an alkoxy, preferably methoxy, group,
R3, R4, which may be identical or different, represent:
a hydrogen atom,
a group CH2R15,
a heteroaryl group, preferably pyridyl, indyl, benzimidazyl, or pyrazyl optionally substituted with a C1-C6 alkyl group, preferably methyl, or
an aryl, preferably phenyl, group optionally mono- or polysubstituted with:
an aryl, preferably phenyl, group optionally mono- or poly-substituted with:
or a heteroaryl group, preferably:
According to one embodiment, the compounds of the present invention are represented by the compounds of formula II:
in which R1 and R2 represent the groups as previously defined, with respect to formula I or to its first embodiment presented above.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula III:
in which R2, R3 and R4 represent the groups as previously defined, with respect to formula I or to its first embodiment presented above.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula IV:
in which Ra represents:
a cycloalkyl group, preferably cyclopropyl,
a linear or branched C1-C6 alkyl group, preferably ethyl, isopropyl,
an aryl group, preferably phenyl, optionally mono- or polysubstituted with:
a heteroaryl group, preferably:
or a CH2-phenyl group, optionally substituted with an alkoxy group, preferably methoxy; a halogen atom, preferably bromine,
According to one embodiment, the compounds of the present invention are represented by the compounds of formula V:
in which Rb represents:
an ester group, preferably methyl ester,
a group CH2E,
a group CH2OCOG, or
an amide group CONHI,
E represents:
a hydroxyl group,
an alkoxy, preferably methoxy, group,
a secondary amine group substituted with:
a tertiary amine group disubstituted with an alkoxyalkyl group, preferably methoxypropyl,
a heterocyclic group, preferably morpholyl, tetrahydrofuranyl,
or a heteroaryl group, preferably pyrryl, imidazyl,
G represents a heteroaryl group, preferably quinoxaline,
I represents an aminoalkyl group, preferably N-dimethylaminopropyl.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula VI:
in which
Rd and Rc, which may be identical or different, represent:
a hydrogen atom,
a nitro group,
a cyano group,
a halogen atom, preferably fluorine, bromine or chlorine,
a methylthiazyl group,
a linear or branched C1-C6 alkyl group, preferably methyl or isopropyl,
a trifluoroalkyl, preferably trifluoromethyl, group,
a substituted or unsubstituted sulphonamide group, preferably N-methyl sulphonamide,
a heteroaryl group, preferably pyridazinyl optionally mono- or polysubstituted with a halogen atom, preferably chlorine,
an alkoxy, preferably methoxy, group,
a trifluoroalkoxy, preferably trifluoromethoxy, group,
a group selected from the groups A, B, C or D as defined above, or
an aryloxy group, preferably phenyloxy,
or Rd and Rc form, with the phenyl, a dihydrobenzofuran or indole ring.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula VII:
in which
Re represents:
an alkoxy, preferably methoxy, group, or
a halogen atom, preferably bromine.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula VIII:
in which
Rf represents:
a hydrogen atom, or
a sulphenyl group, preferably propylsulphenyl.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula IX:
in which
Rg represents:
a halogen atom, preferably bromine,
a nitro group,
a cyano group,
a sulphonamide group,
a hydrogen atom,
a C1-C6 alkyl group, preferably methyl,
a hydroxyl group, or
an alkoxy group, preferably methoxy.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula X:
in which
Ri and Rj, which may be identical or different, represent:
a halogen atom, preferably bromine, fluorine or chlorine,
a nitro group,
a methylthiazyl group,
a hydrogen atom,
an aryloxy, preferably phenyloxy, group,
an alkoxy, preferably methoxy, group,
a trifluoroalkoxy, preferably trifluoromethoxy, group,
a C1-C6 alkyl group, preferably methyl,
a trifluoroalkyl group, preferably trifluoromethyl,
a substituted or unsubstituted sulphonamide group, preferably N-methyl sulphonamide,
a heteroaryl group, preferably pyridazinyl, optionally mono- or polysubstituted with a halogen atom, preferably chlorine,
a group selected from the groups A, B, C or D as defined above, or
a cyano group,
or Ri and Rj form, with the phenyl, a dihydrobenzofuran, indole or quinoxaline ring.
Rh represents:
an aminoalkyl group, preferably N,N-dimethylaminopropyl, or
an N-morpholinoalkyl group, preferably N-morpholinoethyl,
K represents:
an aryl group, preferably phenyl, optionally substituted with an alkoxy, preferably methoxy, or
a C1-C6 alkyl group, preferably methyl.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XI:
in which
Rm represents:
a hydroxyl group,
a heteroaryl group, preferably pyrryl or imidazyl,
a heterocyclic group, preferably trihydrofuranyl or N-morphoiyl,
an alkoxy group, preferably methoxy,
a secondary amine group substituted with:
a tertiary amine group N,N-dimethoxypropylamine, or
a group OCOL
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XII:
in which
Rl and Rk, which may be identical or different, represent:
an alkoxyalkyl group, preferably methoxypropyl,
a hydrogen atom,
a cycloalkyl group, preferably cyclohexyl, optionally substituted with a C1-C6 alkyl group, preferably methyl,
an aryl group, preferably phenyl, optionally substituted with:
a heteroaryl group, preferably pyridyl,
an aralkyl group, preferably a phenylmethyl, or
a linear or branched C1-C6 alkyl group, preferably tert-butyl; or
Rl and Rk form, with N, a morpholyl group.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XIII:
in which
Rn represents:
an alkoxy, preferably methoxy, group, or
a secondary amine group, preferably N,N-dimethylpropylamine.
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XIV:
In which Ro represents:
an aryl group, preferably phenyl, optionally mono- or polysubstituted, preferably with:
a heteroaryl group, preferably:
In one embodiment the compound of formula XIV are chosen among:
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XV:
In which Rp represents:
a heteroaryl group, preferably a pyridyl group,
an aryl group, optionally mono- or polysubstituted, preferably with a:
According to one embodiment, the compounds of the present invention are represented by the compounds of formula XVI:
In which Rq represents:
an aryl group, preferably phenyl, optionally mono- or poly substituted, preferably with:
In one embodiment the compounds of formula XVI are chosen among:
In general, the following definitions are used:
In one embodiment, the invention relates to compounds chosen from the group consisting of: ND0009, ND0019, ND0020, ND0021, ND0029, ND0031, ND0033, ND0037, ND0038, ND0045, ND0047, ND0057, ND0072, ND0074, ND0076, ND0077, ND0082, ND0085, ND0087, ND0088, ND0089, ND0090, ND0091, ND0093, ND0094, ND0096, ND0098, ND0101, ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an inhibition percentage greater than 50% for kinase.
In one embodiment, the invention relates to compounds chosen from the group consisting of: ND0009, ND0019, ND0020, ND0021, ND0029, ND0031, ND0033, ND0037, ND0038, ND0045, ND0047, ND0057, ND0072, ND0074, ND0076, ND0077, ND0082, ND0085, ND0087, ND0088, ND0089, ND0090, ND0091, ND0093, ND0094, ND0096, ND0098, ND0101, ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an inhibition percentage greater than 50% for the AbI WT kinase.
In one embodiment, the invention relates to compounds chosen from the group consisting of ND0118, ND0119, ND0096. Those compounds, which are represented below are characterized in that they present an inhibition percentage greater than 50% for the AbI T315I kinase.
In one embodiment, the invention relates to compounds chosen from the group consisting of: ND009, ND0020, ND0021, ND0037, ND0038, ND0047, ND0057, ND0072, ND0074, ND0076, ND0077, ND0087, ND0088, ND0089, ND0090, ND0091, ND0093, ND0094, ND0096, ND0098, ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an inhibition percentage greater than 50% for the Src kinase.
In one embodiment, the invention relates to compounds chosen from the group consisting of: ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 inferior to 1·10−9M for the kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 inferior to 1·10−9M for AbI WT kinase.
In one embodiment the invention relates to compounds is ND0118. This compound, which is represented below is characterized in that it presents an IC50 inferior to 1·10−9M for Src kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0072, ND0074, ND0077, ND0087, ND0089, ND0090, ND0096, ND0117, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−7 to 1·10−9M for the kinase.
In one In one embodiment the invention relates to compounds chosen from the group consisting of: ND0072, ND0074, ND0077, ND0087, ND0089, ND0090, ND0096. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−7 to 1·10−9M for the AbI WT kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0072, ND0074, ND0077, ND0087, ND0089, ND0090, ND0117, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−7 to 1·10−9M for the Src kinase.
In one In one In one embodiment the invention relates to compounds chosen from the group consisting of: ND0009, ND0019, ND0020, ND0021, ND0031, ND0037, ND0038, ND0047, ND0057, ND0076, ND0082, ND0085, ND0088, ND0091, ND0093, ND0094, ND0098, ND0096, ND0118, ND0119, ND0077. Those compounds, which are represented below are characterized in that they present an 1050 comprised between 1·10−5 to 1·10−7M for the kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0009, ND0019, ND0020, ND0021, ND0031, ND0037, ND0038, ND0047, ND0057, ND0076, ND0082, ND0085, ND0088, ND0091, ND0093, ND0094, ND0098. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−5 to 1·10−7M for the AbI WT kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0096, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−5 to 1·10−7 M for the AbI T315I kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0009, ND0057, ND0077, ND0088, ND0091, ND0093, ND0094, ND0096, ND0098. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−5 to 1·10−7 M for the Src T315I kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0006, ND0010, ND0011, ND0029. Those compounds, which are represented below are characterized in that they present an IC50 greater than 1·10−5M for the AbI WT kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0117, ND0118, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 inferior to 1·10−8 M at 72 hours for the K562 Bcr-AbI kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0117, ND0119, ND0076, ND0087, ND0090, ND0096, ND0072, ND 0074, ND0089. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−8 and 1·10−8 M for the K562 Bcr-AbI kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0117, ND0119. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−8 and 1·10−8 M for the K562 Bcr-AbI kinase at 24 h.
In one embodiment the invention relates to compounds chosen from the group consisting of: N00076, ND0087, ND0090, ND0096, ND0072, ND 0074, ND0089. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−8 and 1·10−6 M for the K562 Bcr-AbI kinase at 72 h.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0020, ND0076, ND0087, ND0090, ND0096, ND0118. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·106 and 1·10−5 M for the K562 Bcr-AbI kinase.
In one embodiment the invention relates to compounds chosen from the group consisting of: ND0020, ND0076, ND0087, ND0090, ND0096, ND0118. Those compounds, which are represented below are characterized in that they present an IC50 comprised between 1·10−6 and 1·10 M for the K562 Bcr-AbI kinase at 24 h.
In one embodiment the invention relates to compound ND0020. This compound, which is represented below are characterized in that it presents an IC50 comprised between 1·10−6 and 1·10−5 M for the K562 Bcr-AbI kinase at 72 h.
In one embodiment the present invention relates to compound chosen from the group consisting of: ND0072, ND0074, ND0089, ND0009. Those compounds, which are represented below are characterized in that they present an IC50 greater than 1·10−5 for the K562 Bcr-AbI kinase.
In one embodiment the present invention relates to compound chosen from the group consisting of: ND0072, ND0074, ND0089. Those compounds, which are represented below are characterized in that they present an IC50 greater than 1·10−5 for the K562 Bcr-AbI kinase at 24 h.
In one embodiment the present invention relates to compound ND0009. This compound, which is represented below is characterized in that it presents an IC50 greater than 1·10−5 for the K562 Bcr-AbI kinase at 72 h.
In one embodiment the present invention relates to compound ND0009. This compound, which is represented below is characterized in that it presents an IC50 greater than 1·10−5 for the U937 Bcr-AbI kinase at 72 h.
In one embodiment, the invention relates to compounds, characterized in that they are selected from the group constituted by methyl 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, methyl 5-(2-fluoro-6-methoxybenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, N-(2-((4-hydroxyphenylamino)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-bromobenzamide, N-(2-((1H-pyrrol-2-yl)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-bromobenzamide, 2-bromo-N-(2-(methoxymethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide, or 5-(2-bromobenzamido)-N-(3-(dimethylamino) propyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide.
When the compounds according to the invention comprise one or more asymmetric carbons, the compounds according to the invention can be obtained in the form of isomers and the invention relates to the compounds in all their forms, namely the enantiomeric, diastereoisomeric, racemic forms or mixtures.
When the compounds comprise bonds that can give geometric isomers, the present invention relates to all the geometric isomers, isolated or as a mixture of the compounds according to the invention.
The compounds according to the invention can be in solvated form and in non-solvated form.
According to the invention, all the pharmaceutically acceptable salts of the compounds according to the invention are included within the scope of the invention, in particular the salts of weak acids and of weak bases.
The present invention also relates to the use of the compounds according to the invention as inhibitors of protein kinases.
In one embodiment, the compounds according to the invention are used as inhibitor of protein kinase AbI.
In another embodiment, the compounds according to the invention are used as inhibitors of protein kinases Src.
The compounds of the invention can be used in the treatment of pathologies associated with deregulation of protein kinases:
According to another aspect, the invention relates to a medicinal product comprising a compound according to the invention as active principle. Thus, the compounds according to the invention can be used as medicinal products in the treatment of pathologies associated with deregulation of protein kinases:
The present invention also relates to pharmaceutical compositions comprising, as active principle, a compound of the invention and a pharmaceutically acceptable excipient.
“Pharmaceutical composition” means any composition comprising an effective dose of a compound of the invention and at least one pharmaceutically acceptable excipient. Said excipients are selected, depending on the pharmaceutical form and the desired method of administration, from the usual excipients known by a person skilled in the art.
The compositions according to the invention can be used in the treatment of pathologies associated with deregulation of protein kinases:
In a first embodiment, the method according to the invention is represented by scheme 1.
In one embodiment the method according to the invention is represented by scheme 1bis.
The method comprises at least the stages of:
In a second embodiment, the method is represented by scheme 2
Wherein
represents an aryl or heteroaryl group.
In one embodiment
represents
In one embodiment
represents
In one embodiment
represents
The process comprises the following steps:
In a third embodiment, the method is represented by scheme 3.
The method comprises at least the stages of:
In a fourth embodiment of the invention, the method is represented by scheme 4.
The method comprises at least the stages of:
In a fifth embodiment the method of the invention is represented by scheme 5.
The method comprises at least the stages of:
In a sixth embodiment, the method of the invention is represented by scheme 6.
The method comprises at least the stages of:
In a seventh embodiment, the method of the invention is represented by scheme 7.
Wherein
represents various aromatic amines carrying an ester function on the aromatic ring.
In one embodiment
represents:
The method comprises at least the stages of:
In a eighth embodiment, the method of the invention is represented by scheme 8.
The method comprises at least the stages of:
In a ninth embodiment, the method of the invention is represented by scheme 9.
The method comprises at least the stages of:
The invention will be better understood on reading the following examples.
The compounds of the invention were obtained from methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (commercially available from the company Azasynth) in multi-stage synthesis, if necessary employing parallel synthesis apparatus (“Synthesis 1”, Heidolph). The various synthesis protocols are detailed below together with the physicochemical characteristics of the compounds of the 7-azaindole type obtained.
The syntheses and analyses were carried out in the following conditions:
1H and 13C Nuclear Magnetic Resonance:
Equipment:
Bruker Avance 300 (300 MHz); Bruker DPX 200 (200 MHz)
Conditions of Use:
Room temperature, chemical shifts expressed in parts per million (ppm), internal reference trimethylsilane (TMS), multiplicity of the signals indicated by lower-case letters (singlet s, doublet d, triplet t, quadruplet q, multiple m), dimethylsulphoxide d6, methanol d4, chloroform d1 as deuterated solvents.
High-Performance Liquid Chromatography (HPLC):
Equipment:
Waters Alliance 2790 chromatographic system, UV 996 detector
Conditions of Use:
Thermo Hypersil C1 column (50×2.1 mm), Water/Acetonitrile/Trifluoroacetic acid elution gradient (99.9%/0%/0%/0.1% to 19.9%/80%/0.1%)
Mass Spectrometry (MS):
Equipment:
Micromass Q-T of
Conditions of Use:
ElectroSpray (ESI) in positive mode.
Weighings:
Equipment:
Denver Instrument TP214 (precision 0.1 mg)
Conditions of Use:
Weighings carried out to the nearest milligram.
Parallel Synthesis:
Equipment:
Heidolph Synthesis 1 (16 reactors)
Conditions of Use:
16 reactions in parallel, room temperature, multiple evaporation.
Reactions Under Pressure:
Equipment:
Parr 300 mL autoclave.
Conditions of Use:
Hydrogenation under 20 bar of hydrogen.
Scheme 10 represents a general method of synthesis of inhibitors of protein kinases.
The first stage is a catalytic hydrogenation of methyl 5-nitro-1H-pyrrob[2,3-b]pyridine-2-carboxylate, in the presence of palladium on charcoal and under hydrogen atmosphere (Seela, F.; Gumbiowski, R., Heterocycles 1989, 29 (4), 795-805). The product is obtained at a raw yield of 95%. The second stage was carried out by parallel synthesis. The amine formed during the first stage is reacted with various acyl chlorides to give the corresponding amides at yields ranging from 15 to 74% after purification on silica gel (Mouaddib, A.; Joseph, B. et al., Synthesis 2000, (4), 549-556).
An autoclave is charged with 2.9 g (13.12 mmol) of methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (Azasynth), 290 mg of palladium on charcoal 10% and 200 mL of methanol. The reaction mixture is placed under 20 bar of hydrogen, and stirred for 18 h at room temperature. The solution is then filtered on Celite, and the Celite is rinsed with 3×100 mL of hot methanol. The filtrate is evaporated under reduced pressure. 2.38 g of a yellow solid is obtained at a yield of 95%.
1H NMR (300 MHz, DMSO d6): 11.98 (br s, 1H), 7.95 (d, 1H), 7.14 (d, 1H), 6.90 (s, 1H), 3.84 (s, 3H).
A reactor is charged with 50 mg of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (0.26 mmol), the reagent (acyl chlorides) and 5 mL of dichloromethane. 56 μL (0.39 mmol) of triethylamine is added to the reaction mixture and it is stirred at room temperature for 18 hours. Then 60 mL of a saturated solution of sodium hydrogen carbonate is added to the reaction mixture. The precipitate that forms is filtered and rinsed with a little water. The filtrate is extracted with 3×50 ml of dichloromethane. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. A precipitate is obtained, which is combined with the filtered precipitate, then purified by silica gel chromatography (ethyl acetate/petroleum ether eluent).
Table 1 shows the various inhibitors synthesized according to the synthesis scheme described above.
31 mg of a beige solid (46%); 1H NMR (300 MHz, DMSO d6): 12.43 (br s, 1H), 10.41 (br s, 1H), 8.48 (d, 1H), 8.41 (d, 1H), 7.14 (s, 1H), 3.87 (s, 3H), 1.81 (m, 1H), 0.87 (m, 4H); HPLC: 97%; MS (ESI): 260.0 (M + 1)
38 mg beige solid (54%). HPLC: 97%. MS (ESI): 262.2 (M + 1)
22 mg of white solid (54%). HPLC: 83%. MS (ESI): 247.2 (M + 1)
18 mg of yellow solid (22%). 1H NMR (300 MHz, DMSO d6): 12.54 (br s, 1H), 10.67 (br s, 1H), 8.68 (d, 1H), 8.56 (d, 1H), 8.46 (s, 1H), 8.30 (m, 1H), 8.10 (m, 1H), 7.78 (m, 1H), 7.22 (s, 1H), 3.89 (s, 3H). HPLC: 96%. MS (ESI): 321.2 (M + 1)
19 mg of yellow solid (23%). HPLC: 100%. MS (ESI): 314.2 (M + 1)
33 mg of beige solid (74%). 1H NMR (300 MHz, DMSO d6): 12.52 (br s, 1H), 10.65 (br s, 1H), 8.58 (s, 2H), 7.74 (m, 1H), 7.62-7.41 (m, 3H), 7.22 (s, 1H), 3.89 (s, 3H). HPLC: 95%. MS (ESI): 373.8; 375.8 (M + 1) 338.2 (M + 1)
26 mg of beige solid (59%). 1H NMR (300 MHz, DMSO d6): 12.61 (br s, 1H), 10.76 (br s, 1H), 8.65 (d, 1H), 8.62 (d, 1H), 7.75-7.58 (m, 2H), 7.48-7.35 (m, 1H), 7.28 (s, 1H), 3.95 (s, 3H). HPLC: 100%. MS (ESI): 332.2 (M + 1)
26 mg of beige solid (25%). HPLC: 75%. MS (ESI): 393.2 (M + 1)
24 mg of brown solid (15%). HPLC: 100%. MS (ESI): 338.2 (M + 1)
30 mg of white solid (33%). 1H NMR (300 MHz, DMSO d6): 12.50 (br s, 1H), 10.67 (br s, 1H), 8.60 (d, 1H), 8.53 (d, 1H), 7.47 (m, 1H), 7.21 (s, 1H), 7.00- 6.90 (m, 2H), 3.39 (s, 3H), 3.84 (s, 3H). HPLC: 99%. MS (ESI): 344.2 (M + 1)
28 mg of brown solid (28%). HPLC: 91%. MS (ESI): 388.2 (M + 1)
15 mg of brown solid (20%). HPLC: 100%. MS (ESI): 297.2 (M + 1)
60 mg of beige solid (28%). HPLC: 98%. MS (ESI): 373.1 (M + 1)
22 mg of beige solid (27%). HPLC: 100%. MS (ESI): 340.2 (M + 1)
19 mg of brown solid (24%). HPLC: 93%. MS (ESI): 388.1; 390.1 (M + 1)
12 mg of product (12%); 1H NMR (400 MHz, DMSO d6): 12.55 (br s, 1H), 10.92 (br s, 1H), 8.57 (d, 1H), 8.52 (d, 1H), 7.63-7.50 (m, 3H), 7.23 (s, 1H), 3.90 (s, 3H); HPLC: 97%; MS: 365.9 (M + 1)
30 mg of product (35%); 1H NMR (400 MHz, DMSO d6): 12.48 (br s, 1H), 10.21 (br s, 1H), 8.6 (s, 2H), 7.69 (m, 1H), 7.52 (m, 1H), 7.21 (m, 2H), 7.09 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H); HPLC: 97%; MS: 326.0 (M + 1)
10 mg of product (20%); 1H NMR (400 MHz, DMSO d6): 12.53 (br s, 1H), 10.70 (br s, 1H), 8.56 (m, 2H), 8.39 (m, 2H), 7.32 (m, 1H), 7.21 (s, 1H), 3.88 (s, 3H), 2.32 (s, 3H); HPLC: 96%; MS: 343.6 (M + 1)
30 mg of product (30%); 1H NMR (400 MHz, DMSO d6): 12.50 (br s, 1H), 10.48 (br s, 1H), 8.65 (d, 1H), 8.54 (d, 1H), 8.19 (s, 1H), 8.00 (d, 1H), 7.82 (d, 1H), 7.53 (t, 1H), 7.21 (s, 1H), 3.90 (s, 3H); HPLC: 95%; MS: 375.9 (M + 1)
40 mg of product (49%); 1H NMR (400 MHz, DMSO d6): 12.50 (br s, 1H), 10.43 (br s, 1H), 8.60 (s, 2H), 7.52 (m, 1H), 7.41 (m, 1H), 7.32 (m, 2H), 7.20 (s, 1H), 3.91 (s, 3H); HPLC: 95%; MS: 310.1 (M + 1)
20 mg of product (21%); 1H NMR (400 MHz, DMSO d6): 12.53 (br s, 1H), 10.68 (br s, 1H), 8.54 (s, 2H), 7.90-7.70 (m, 4H), 7.21 (s, 1H), 3.92 (s, 3H); HPLC: 97%; MS: 364.0 (M + 1)
40 mg of product (40%); 1H NMR (400 MHz, DMSO d6): 12.52 (br s, 1H), 10.62 (br s, 1H), 8.55 (s, 2H), 7.78 (m, 1H), 7.67 (m, 1H), 7.54 (m, 2H), 7.22 (s, 1H), 3.90 (s, 3H); HPLC: 98%; MS: 380.0 (M + 1)
30 mg of product (40%); 1H NMR (400 MHz, DMSO d6): 12.50 (br s, 1H), 10.40 (br s, 1H), 8.63 (d, 1H), 8.46 (d, 1H), 8.04 (d, 1H), 7.88 (d, 1H), 7.25 (t, 1H), 7.20 (s, 1H), 3.87 (s, 3H); HPLC: 95%; MS: 302.1 (M + 1)
Scheme 11 represents a general method of synthesis of inhibitors of protein kinases.
Wherein
represents an aryl or heteroaryl group.
In one embodiment
represents
In one embodiment
represents
In one embodiment
represents
The first stage is a catalytic hydrogenation of methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, in the presence of palladium on charcoal and under hydrogen atmosphere (Seela, F.; Gumbiowski, R., Heterocycles 1989, 29 (4), 795-805). The product is obtained at a raw yield of 95%. The second stage was carried out by parallel synthesis and could be either the formation of amidoazaindol by peptidic coupling, or the formation of arylaminoazaindol or hétéroarylaminoazaindol by nucleophilic aromatic substitution, or the formation of, benzylaminoazaindol by reductive amination. The following examples will enable to better understand the invention.
The first stage is the same as in previous example A.
A reactor is charged with 25 mg (0.13 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, one equivalent of a carboxylic acid, 1.5 equivalents of hydroxybenzotriazole, 1.5 equivalents of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrogen carbonate, 2 equivalents of N,N-diisopropylethylamine into 1 ml of dimethylformamide. The solution is mixed at room temperature for 12 hours. The solvent is then evaporated under reduced pressure and the raw product is purified directly on preparative HPLC.
The following table 2 regroups the inhibitor synthesized following this protocol.
A reactor is charged with 25 mg (0.13 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, 1 equivalent of aldehyde and 1 ml of methanol, 1 equivalent of acetic acid is added and the reaction mixture is then mixed at room temperature for 2 hours. Once the Shiff base is confirmed by MS analysis, 1.5 equivalents of sodium cyanoborohydride is added and the mixture is then mixed at room temperature for 12 hours. The reacting medium is then purified by preparative HPLC.
The following table 3 regroups the inhibitor synthesized following this protocol.
A reactor is charged with 25 mg (0.13 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, 0.2 equivalents of copper iodide and 6 equivalents of potassium carbonate in 2 ml of dioxane. 1.5 equivalents of a halogenated reagent, 0.2 equivalents of trans-N,N-dimethylcyclohexane-1,2-diamine are then added and the mixture is stirred at 130-140° C. for 2 days. The reacting medium is then filtered on celite, evaporated and then purified by preparative HPLC to give the expected product. The following table 4 regroups the inhibitor synthesized following this protocol.
Scheme 12 shows the reaction scheme of Example B.
The first stage is selective reduction of the ester function by lithium aluminium hydride at room temperature to give the corresponding alcohol at a yield of 89% (Karrer, P., Boettcher, Augusta, Helvetica Chimica Acta, 1953, 36, 570-2). The alcohol formed is then converted to alkane bromide by PBr3, at room temperature for 18 h in THF (raw yield of 81%) (Ku, Jin-Mo., J. et al., Journal of Organic Chemistry, 2007, 72 (21), 8115-8118). The intermediate alkane bromide is very reactive and cannot be purified without decomposition. When dissolved in methanol, it leads to the methoxyazaindole obtained by reaction of the solvent on the halogenated function, at room temperature (yield of 17%). To obtain the desired amines, the alkane bromide is therefore reacted directly without purification during the third and last stage. The latter is carried out by parallel synthesis using Heidolph's synthesis 1. Two equivalents of primary or secondary amine are added to the alkane bromide in anhydrous dimethylformamide, at room temperature for 18 h, to give the corresponding 7-azaindoles at yields ranging from 9 to 96% after purification on silica gel (Nagarathnam D., Journal of Heterocyclic Chemistry, 1992, 29 (6), 1371-3).
A flask is charged with 4.6 g (12.3 mmol) of methyl 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (example 6) in 180 mL of anhydrous tetrahydrofuran under argon atmosphere. The reaction mixture is kept at 0° C. by means of a cold ethanol bath and 0.7 g (18.5 mmol) of lithium aluminium hydride in powder form is added cautiously, in several portions. It is stirred at 0° C., under argon, for 15 minutes and the cold bath is removed. The reaction mixture is stirred for a further 2 h, at room temperature. Then 3 mL of saturated aqueous ammonium chloride solution is added very slowly, and it is stirred for 15 min. The precipitate formed is then filtered on Celite, and the Celite is rinsed with 3×50 mL of hot tetrahydrofuran. The filtrate is evaporated under reduced pressure and 2.38 g of yellow solid is obtained (raw yield 95%).
1H NMR (300 MHz, DMSO d6): 11.50 (br s, 1H), 10.46 (br s, 1H), 8.34 (d, 1H), 8.28 (d, 1H), 7.80-7.37 (m, 5H), 6.32 (s, 1H), 5.30 (m, 1H), 4.60 (m, 2H).
HPLC: 95%. MS (ESI): 345.8; 347.8 (M+1).
A flask is charged successively with 1 g (2.89 mmol) of 2-bromo-N-(2-(hydroxymethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Example 16), 100 mL of tetrahydrofuran and 408 μL (4.34 mmol) of phosphorus bromide (PBr3) under argon atmosphere. It is stirred at room temperature for 18 hours. The solvent is then evaporated under reduced pressure, and the residue is taken up in 150 mL of ethyl acetate. The organic phase is washed with 3×50 mL of water, dried over sodium sulphate and evaporated under reduced pressure. The precipitate obtained is dried under vacuum for 1 h, finally obtaining 957 mg of a brown solid (raw yield 81%).
MS (ESI): 408.0; 410.0; 412.0 (M+1).
A reactor is charged with 50 mg (0.12 mmol) of the 2-bromo-N-(2-(bromomethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide formed during the preceding stage, 0.5 mL of dimethylformamide (distilled on calcium hydride) and the reagent, under argon atmosphere. The solution is stirred at room temperature for 18 hours and then diluted in 30 mL of ethyl acetate, washed with 4×10 mL of water, dried over sodium sulphate and evaporated under reduced pressure. The raw product is then purified by silica gel chromatography (ethyl acetate/methanol eluent).
Table 5 shows the various inhibitors synthesized as a result of the second stage.
A flask is charged with 200 mg (0.49 mmol) of 2-bromo-N-(2-(bromomethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (stage 1) and 5 mL of methanol. The solution is stirred at room temperature for 18 hours and then diluted in 100 mL of ethyl acetate, washed with 3×30 mL of water, dried over sodium sulphate and evaporated under reduced pressure. The raw product is then purified by silica gel chromatography (ethyl acetate/petroleum ether eluent). 30 mg of a yellow solid is obtained (17%).
1H NMR (300 MHz, DMSO d6): 11.75 (br s, 1H), 10.54 (br s, 1H), 8.43 (d, 1H), 8.38 (d, 1H), 7.80-7.45 (m, 4H), 6.47 (s, 1H), 4.58 (s, 2H), 3.36 (s, 3H).
HPLC: 96%. MS (ESI): 360.1; 362.1 (M+1).
A flask is charged successively with 50 mg (0.14 mmol) of 2-bromo-N-(2-(hydroxymethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide AT2-2BF2 (example 16), 5 mL of dichloromethane, 30 mg (0.15 mmol) of 6-quinoxalinecarbonyl chloride and 30 μL (0.22 mmol) of triethylamine. The solution is stirred at room temperature for 18 hours and then diluted in 60 mL of ethyl acetate, washed with 10 mL of saturated solution of sodium bicarbonate, 10 mL of water, and 10 mL of saturated solution of sodium chloride. The organic phase is then dried over sodium sulphate and evaporated under reduced pressure. The raw product is then purified by silica gel chromatography (ethyl acetate/petroleum ether eluent). 14 mg of a yellow solid is obtained (24%).
1H NMR (300 MHz, DMSO d6): 12.00 (br s, 1H), 10.53 (br s, 1H), 9.10 (s, 2H), 8.77 (s, 1H), 8.45-8.24 (m, 4H), 7.76-7.41 (m, 4H), 6.66 (s, 1H), 5.59 (s, 2H).
HPLC: 97%. MS (ESI): 502.1; 504.1 (M+1).
In the first stage, the ester is saponified with lithium hydroxide to give the corresponding carboxylic acid at a raw yield of 52% (Kanth, Sribhashyam R. et al., Heterocycles, 2005, 65 (6), 1415-1423). This carboxylic acid is then converted to amide by peptide coupling using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCl) hydrochloride and morpholine. The amide obtained is then hydrogenated with catalytic palladium, leading quantitatively to the corresponding aminoazaindole. The latter is then reacted with various acyl chlorides, in the presence of triethylamine in dimethylformamide, to obtain the desired 7-azaindoles.
A flask is charged successively with 5 g (22.62 mmol) of methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (Azasynth), 50 mL of 95% ethanol, 50 mL of water and 1.63 g (67.92 mmol) of lithium hydroxide. The reaction mixture is refluxed for 30 minutes and then left to return to room temperature. The solution is concentrated under reduced pressure and then diluted in 200 mL of water. The aqueous phase is extracted with 3×60 mL of ethyl acetate, then acidified with 32% hydrochloric acid until the pH is between 2 and 3. The precipitate formed is filtered, rinsed with 50 mL of ethyl acetate and then dried under vacuum for 6 h. 2.45 g of a white solid is obtained (52%).
1H NMR (300 MHz, DMSO d6): 13.60 (br s, 1H), 13.20 (br s, 1H), 9.25 (d, 1H), 9.26 (d, 1H), 7.38 (s, 1H).
A flask is charged successively with 600 mg (2.90 mmol) of the 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid formed during the preceding stage, 10 mL of dimethylformamide (distilled on calcium hydride), 0.5 mL (5.80 mmol) of morpholine and 1.11 g (5.80 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCl) hydrochloride under argon atmosphere. The solution is stirred at room temperature for 18 hours. The solvent is then evaporated under reduced pressure and the raw product is purified directly by silica gel chromatography (ethyl acetate/methanol eluent). 313 mg of a white solid is recovered (39%).
1H NMR (300 MHz, DMSO d6): 13.04 (br s, 1H), 9.20 (d, 1H), 8.97 (d, 1H), 7.05 (s, 1H), 3.70 (m, 8H).
An autoclave is charged with 313 mg (1.13 mmol) of the morpholino(5-nitro-1H-pyrrolo[2,3-b]pyridin-2-yl)methanone formed during the preceding stage, 100 mL of methanol and 31 mg of palladium on charcoal at 10%. The autoclave is placed under 20 bar of hydrogen and the solution is stirred at room temperature for 18 hours, The solution is then filtered on Celite, the Celite is rinsed with 50 mL of methanol and the filtrate is evaporated under reduced pressure. 280 mg of a white solid is recovered, and is used in the next stage without further purification (quantitative raw yield).
1H NMR (300 MHz, DMSO d6): 11.60 (br s, 1H), 7.86 (d, 1H), 7.13 (d, 1H), 6.52 (s, 1H), 4.84 (br s, 2H), 3.68 (m, 8H).
A flask is charged successively with 100 mg (0.40 mmol) of H-pyrrolo[2,3-b]pyridin-2-yl)(morpholino)methanone formed during the preceding stage, 5 mL of dimethylformamide (distilled on calcium hydride), 90 mg (0.44 mmol) of 2-fluoro-5-nitrobenzoyl chloride and 85 μL (0.60 mmol) of triethylamine under argon atmosphere. The solution is stirred at room temperature for 4 hours. The solvent is evaporated under reduced pressure and the residue is taken up in 100 mL of ethyl acetate. The organic phase is rinsed with 30 mL of saturated solution of sodium bicarbonate, 30 mL of water and 30 mL of saturated solution of sodium chloride. The organic phase is dried over sodium sulphate and evaporated under reduced pressure. The raw product obtained is purified by silica gel chromatography (ethyl acetate/methanol eluent), obtaining 90 mg of yellow solid (53%).
1H NMR (300 MHz, DMSO d6): 12.12 (br s, 1H), 10.30 (br s, 1H), 8.51-8.43 (m, 2H), 8.41 (d, 1H), 8.36 (d, 1H), 7.85 (m, 1H), 6.81 (s, 1H), 3.673 (m, 8H).
HPLC: 74%. MS (ESI): 414.3 (M+1).
5-(3-Fluorobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Scheme 12) was obtained in 3 stages (General scheme 14) starting from methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (Azasynth). The methyl ester of the azaindole is converted directly to primary amide by reaction of aqueous ammonia on the ester function (Ramasamy, K. et al., Tetrahedron, 1986, 42 (21), 5869-78). The reaction is conducted at room temperature for 24 h and the expected product is obtained at a yield of 10% after purification. The nitroazaindole thus obtained is hydrogenated, giving the corresponding aminoazaindole quantitatively. The latter leads to the compound of example 38 (ND0005) by reaction of the amine function on 3-fluorobenzoyl chloride, in dimethylformamide in the presence of triethylamine.
A flask is charged with 300 mg (1.36 mmol) of methyl 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (Azasynth), 7.5 mL of methanol and 15 mL of 28% ammonia solution. The reaction mixture is stirred at room temperature for 24 hours. The solution is concentrated under reduced pressure and then diluted with 100 mL of saturated solution of sodium chloride. The aqueous phase is extracted with 3×30 mL of ethyl acetate. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. The raw product obtained is purified by silica gel chromatography (ethyl acetate/petroleum ether eluent). 30 mg of a yellow solid is obtained (10%).
An autoclave is charged with 30 mg (0.14 mmol) of the 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-carboxamide that formed previously, 100 mL of methanol and 10 mg of palladium on charcoal at 10%. The autoclave is placed under 20 bar of hydrogen and the solution is stirred at room temperature for 2 hours. The solution is then filtered on Celite. The Celite is rinsed with 50 mL of methanol and the filtrate is evaporated under reduced pressure. 25 mg of a yellow solid is recovered, and is used in the next stage without further purification (quantitative raw yield).
A flask is charged successively with 25 mg (0.14 mmol) of the 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxamide that formed previously, 3 mL of dimethylformamide (distilled on calcium hydride), 19 μL (0.16 mmol) of 3-fluorobenzoyl chloride and 30 μL (0.21 mmol) of triethylamine under argon atmosphere. The solution is stirred at room temperature for 2.5 hours. The solvent is evaporated under reduced pressure and the residue is taken up in 100 mL of saturated solution of sodium bicarbonate. The aqueous phase is extracted with 3×30 mL of ethyl acetate. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. The raw product obtained is purified by silica gel chromatography (ethyl acetate eluent), obtaining 14 mg of beige solid (33%).
1H NMR (300 MHz, DMSO d6): 12.04 (br s, 1H), 10.44 (br s, 1H), 8.57 (d, 1H), 8.45 (d, 1H), 7.98 (br s, 2H), 7.89-7.43 (m, 4H), 7.12 (s, 1H).
HPLC: 56%. MS (ESI): 299.2 (M+1).
Scheme 15 shows the general synthesis scheme.
The aminoazaindole is converted to amides by reacting different acyl chlorides on the amine function. The amides thus obtained are saponified by the action of potassium hydroxide under reflux in a water-methanol mixture, and lead to the compounds described in examples 39 to 43 by reacting the acid function on various alcohols and amines.
A flask is charged successively with 425 mg (1.14 mmol) of methyl 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (example 6), 20 mL of methanol, 20 mL of water and 127 mg of potassium hydroxide. The reaction mixture is refluxed for 2 hours and then left to return to room temperature. The solution is concentrated under reduced pressure and then diluted in 200 mL of water. The aqueous phase is extracted with 3×60 mL of ethyl acetate, and acidified with 32% hydrochloric acid, until the pH is between 2 and 3. The aqueous phase is extracted again with 3×60 mL of ethyl acetate. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. 250 mg of a beige solid is obtained, and is used directly without purification.
A flask is charged successively with 250 mg (0.70 mmol) of the solid formed previously, 25 mL of dimethylformamide (distilled on calcium hydride), 96 μL (0.76 mmol) of 3-(dimethylamino)-1-propylamine, 3.28 mL (23.05 mmol) of triethylamine and 94 mg (0.70 mmol) of hydroxybenzotriazole. It is stirred for 15 minutes at room temperature and then 160 mg (0.83 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCl) hydrochloride is added. The reaction mixture is stirred for a further 18 h. The solvent is then evaporated under reduced pressure and the residue is taken up in 100 mL of water. The aqueous phase is extracted with 6×50 mL of dichloromethane, the organic phases are combined and are then dried over sodium sulphate and evaporated under reduced pressure. The raw product is purified by silica gel chromatography (ethyl acetate/methanol eluent). 98 mg of beige solid is obtained (20% from methyl 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate).
1H NMR (300 MHz, CD3OD): 8.40 (s, 7.62-7.27 (m, 4H), 6.97 (s, 1H), 3.34 (t, 2H), 2.33 (t, 2H), 2.17 (s, 6H), 1.74 (m, 2H).
HPLC: 95%. MS (ESI): 442.2; 446.2 (M+1).
The compound is prepared according to the protocol described in example 39 using 150 mg of methyl 5-(4-(2-methylthiazol-4-yl)benzamido)-1H-pyrrolo[2,3-b]pyridine-2-methyl carboxylate (example 8) instead of 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate AT10-3A. After treatment, 73 mg of beige solid is obtained (22% over 2 stages),
1H NMR (300 MHz, CD3OD): 8.50-8.39 (m, 2H), 7.97 (m, 4H), 7.74 (s, 1H), 7.01 (s, 1H), 3.40 (t, 2H), 2.69 (s, 3H), 2.60 (t, 2H), 2.06 (s, 6H), 1.85 (m, 2H).
HPLC: 80%. MS (ESI): 463.3 (M+1).
The compound is prepared according to the protocol described in example 1 (stage 2) using 55 mg of quinoxaline-6-carbonyl chloride instead of cyclopropanecarbonyl chloride. After treatment, 42 mg of beige solid is obtained (60%).
The compound is prepared according to the protocol described in example 39 using 42 mg of methyl 5-(quinoxaline-6-carboxamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate described previously instead of methyl 5-(2-bromobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate. After treatment, 7 mg of beige solid is obtained (15% over 2 stages).
1H NMR (300 MHz, CD3OD): 8.89 (m, 2H), 8.65 (s, 1H), 8.52 (m, 1H), 8.45 (m, 1H), 8.30 (d, 1H), 8.14 (d, 1H), 6.99 (s, 1H), 3.36 (t, 2H), 2.40 (t, 2H), 2.22 (s, 6H), 1.74 (m, 2H). HPLC: 83%. MS (ESI): 418.3 (M+1).
The compound is prepared according to the protocol described in example A (stage 2) using 200 mg (1.04 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate and 121 μL (1.04 mmol) of benzoyl chloride instead of cyclopropanecarbonyl chloride. After treatment, 170 mg of beige solid is obtained (55%).
A flask is charged with 100 mg (0.34 mmol) of methyl 5-(benzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate described previously, 20 mL of methanol, 20 mL of water and 200 mg (3.40 mmol) of potassium hydroxide. The reaction mixture is heated to 60° C. for 30 minutes and then left to return to room temperature. The solution is concentrated under reduced pressure and then diluted in 100 mL of water. The aqueous phase is extracted with 3×30 mL of ethyl acetate, then acidified with 32% hydrochloric acid until the pH is between 2 and 3. The aqueous phase is extracted again with 3×30 mL of ethyl acetate. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. 85 mg of white solid is obtained, and is used directly without purification.
A flask is charged successively with 85 mg (0.30 mmol) of the solid formed previously, 20 mL of tetrahydrofuran, 68 mg (0.38 mmol) of p-methoxyphenol, 90 mg (0.30 mmol) of N,N′-dicyclohexylcarbodiimide (DCC) and 27 mg (0.15 mmol) of 4-dimethylaminopyridine (DMAP). It is stirred for 18 hours. The solvent is evaporated under reduced pressure and the residue is taken up in 100 mL of water. The aqueous phase is extracted with 3×30 mL of ethyl acetate, the organic phases are combined and are dried over sodium sulphate and evaporated under reduced pressure. The raw product is purified by silica gel chromatography (ethyl acetate/petroleum ether eluent). 17 mg of beige solid is obtained (10% starting from methyl 5-(benzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate).
1H NMR (300 MHz, DMSO d6): 12.40 (br s, 1H), 10.52 (br s, 1H), 8.77 (d, 1H), 8.67 (d, 1H), 8.08 (d, 2H), 7.68-7.60 (m, 3H), 7.49 (s, 1H), 7.32 (d, 2H), 7.08 (d, 2H).
A flask is charged with 865 mg (2.76 mmol) of methyl 5-(3-fluorobenzamido)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (example 5), 20 mL of ethanol, 20 mL of water and 464 mg (8.30 mmol) of potassium hydroxide. The reaction mixture is heated at 60° C. for 30 minutes and then left to return to room temperature. The solution is concentrated under reduced pressure and then diluted in 200 mL of water. The aqueous phase is extracted with 3×60 mL of ethyl acetate and acidified with 32% hydrochloric acid until the pH is between 2 and 3. The aqueous phase is extracted again with 5×50 mL of ethyl acetate. The organic phases are combined, dried over sodium sulphate and evaporated under reduced pressure. 590 mg of white solid is obtained, and is used directly without purification (raw yield of 71%).
A flask is charged with 280 mg (0.93 mmol) of the solid formed previously together with 5 mL of thionyl chloride under argon atmosphere. The reaction is carried out under reflux for 1 hour and then left to return to room temperature. The solvent is then evaporated under reduced pressure and 10 mL of dimethylformamide (distilled on calcium hydride), 0.2 mL (1.40 mmol) of triethylamine and 135 μL (1.03 mmol) of 4-(2-aminoethyl)morpholine are added to the mixture. It is stirred at room temperature for 2 hours and the solvent is evaporated under reduced pressure. The residue is taken up in 100 mL of ethyl acetate. The organic phase is rinsed with 30 mL of saturated solution of sodium bicarbonate, then with 3×30 mL of water. The organic phase is dried over sodium sulphate and the solvent is evaporated under reduced pressure. 48 mg of yellow solid is obtained (13%).
1H NMR (300 MHz, DMSO d6): 12.09 (br s, 1H), 10.43 (br s, 1H), 8.55 (d, 1H), 8.46 (d, 1H), 8.44 (br s, 1H), 7.87-7.78 (m, 2H), 7.65-7.58 (m, 1H), 7.50-7.43 (m, 1H), 7.09 (s, 1H), 3.58 (t, 4H), 3.42 (m, 2H), 3.31 (m, 2H), 2.42 (t, 4H).
HPLC: 85%. MS (ESI): 412.2 (M+1).
The Inhibitors of Example F are Obtained as Represented in Scheme 16.
Wherein
represents various aromatic amines carrying an ester function on the aromatic ring.
In one embodiment
represents:
The synthesis starts with the use of various aromatic amines carrying an ester methylic or ethylic function (Liu, Y., Wang, C et al, Organic Process Resarch & Development, 2008, 12(3), 490-495). This amine reacts with 4-chloromethylbenzoyle chloride to give an intermediate amide. The amide obtained is substituted with N-methylpiperazine in acetone at reflux. The intermediate ester obtained is saponified and then coupling with methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate or reduced into aldehyde to then undergo a reductive amination with methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate. Some examples are given below to illustrate the present invention.
A reactor is charged with 1 g (6.06 mmol) of methyl 3-amino-6-methylbenzoate and 1.2 g (11.9 mmol) of triethylamine in 25 ml of dichloromethane. 1.145 g (6.05 mmol) of 4-chloromethylbenzoyle chloride is then added and the mixture is stirred at room temperature for 2 hours. The reacting medium is then washed with 10 ml of a saturated NaHCO3 solution, 10 ml of water and 10 ml of a saturated NaCl solution. The organic phase is dried, evaporated and the obtained residue is triturated in ethylic ether and then in pentane to give a white solid (1.2 g, 66%).
A reactor is charged with 1 g (3.3 mmol) of the solid obtained in step 1, 2.28 g (16.5 mmol) of potassium carbonate, 30 ml of acetone, 0.39 g (3.96 mmol) of N-methylpiperazine and 55 mg (0.33 mmol) of potassium iodide. The mixture is heated at reflux for 6 hours. The solvent is then evaporated under reduce pressure and the residue is taken off into 100 ml of a saturated sodium bicarbonate solution. The aqueous phase is extracted by 3*30 ml ethyl acetate. The organic phases are then combined, dried on sodium sulphate and evaporated under reduced pressure. The obtained product is triturated in ethylic ester and then dried to give a white solid (700 mg, 56%).
A reactor is charged with 0.5 g (1.3 mmol) of the solid obtained in step 2, 20 ml of a mixture water/THF (1/1) and 0.55 g (13 mmol) of lithium hydroxide. The mixture is stirred at room temperature for 6 hours. The solution is concentrated under reduced pressure and then diluted in 100 ml of water. The aqueous phase is extracted by 3*30 ml of ethyl acetate and acidified with chlorhydric acid 32% until pH comprised between 2 and 3. the aqueous phase is extracted with 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and evaporated under reduced pressure. 250 mg of a solid is obtained (52%).
A reactor is charged successively with 150 mg (0.4 mmol) of the solid obtained in step 3, 15 ml of anhydrous dichloromethane, 90 mg (0.4 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, 0.12 g (0.49 mmol) of triethylamine and 93 mg (0.61 mmol) of hydroxybenzotriazole. The mixture is stirred at room temperature for 15 min and then 90 mg (0.49 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrogen carbonate (EDCl) is added. The mixture is stirred for 16 hours. The solvent is then evaporated under reduced pressure and the residue is taken off in 100 ml of water. The aqueous phase is extracted by 6*50 ml dichloromethane. The aqueous phases are combined, dried on sodium sulphate and then evaporated under reduced pressure. The raw product obtained is purified by preparative HPLC and 18 mg (8%) of a solid is obtained.
1H NMR (400 MHz, CD3OD): 8.63 (d, 1H), 8.57 (d, 1H), 7.98 (d, 2H), 7.96 (m, 1H), 7.70 (m, 1H), 7.56 (d, 2H), 7.36 (m, 1H), 7.23 (s, 1H), 3.98 (s, 3H), 3.80 (s, 2H), 3.60-3.00 (m, 8H), 2.95 (s, 3H), 2.55 (s, 3H); HPLC: 95%. MS: 541.4 (M+1).
A reactor is charged with 100 mg (0.26 mmol) of methyl 5-(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2-methylbenzoate (example 82, step 2) in 5 ml anhydrous tetrahydrofuran and 38 mg (1.75 mmol) of lithium borohydride is added. The mixture is stirred at room temperature for 16 hours. The mixture is then concentrated under reduced pressure, taken off into 60 ml of ethyl acetate, washed with 20 ml of a saturated NaHCO3 solution, 20 ml of water and 20 ml of a saturated NaCl solution. The organic phase is dried, evaporated and the residue is taken off and triturated into ethylic ester to give a solid (80 mg, 86%)
A reactor is charged with 150 mg (0.42 mmol) of the solid obtained in step 1 and 216 mg (0.5 mmol) of the Dess-Martin reagent in 5 ml acetonitrile. The mixture is stirred at room temperature for 16 hours. The insoluble compounds are filtered and the filtrate is concentrated under reduced pressure. The obtained solid is washed with ethylic ether and pentane to give the required aldehyde (120 mg, 82%).
A reactor is charged with 120 mg (0.34 mmol) of the solid obtained in step 2, 15 ml of anhydrous dichloromethane, 78 mg (0.41 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, 216 mg (1.02 mmol) of sodium acetoxyborohydride and a catalytic amount of acetic acid. The mixture is stirred at room temperature for 24 hours. The solvent is then evaporated under reduced pressure and the residue is taken off in 100 ml of water. The aqueous phase is extracted by 3*30 ml ethyl acetate. The aqueous phases are combined, dried on sodium sulphate and then evaporated under reduced pressure. The raw product obtained is purified by preparative HPLC and 18 mg of a solid is obtained (10%).
1H NMR (400 MHz, DMSO d6): 12.02 (br s, 1H), 10.10 (br s, 1H), 8.10 (d, 1H), 7.86 (d, 2H), 7.69 (d, 1H), 7.64 (d, 1H), 7.39 (d, 2H), 7.18 (d, 1H), 6.95 (s, 1H), 6.90 (s, 1H), 6.05 (br s, 1H), 4.21 (s, 2H), 3.82 (s, 3H), 3.45 (s, 2H), 2.38 (m, 8H), 2.08 (s, 3H); HPLC: 95%. MS: 527.4 (M+1).
The compound is obtained by using the procedure of example 83 replacing methyl 3-amino-6-methylbenzoate by ethyl 5-aminothiazole-2-carboxylate.
The compound is obtained by using the procedures of example 82 replacing methyl 3-amino-6-methylbenzoate by methyl 6-aminopyridine-2-carboxylate.
The compound is obtained by using the procedures of example 83 replacing methyl 3-amino-6-methylbenzoate by methyl 2-aminopyrimidine-4-carboxylate.
The compound is obtained by using the procedures of example 82 replacing 3-amino-6-methylbenzoate by methyl 4-aminopyrimidine-2-carboxylate.
The synthesis of example G is represented by the general scheme 17.
The 3-(trifluoromethyl)-4-methylbenzoic acid is transformed into methylic ester in methanol in the presence of sulfuric acid and then functionalized with bromine by the action of NBS in the presence of benzoic peroxide (Asaki, T. et al, Bioorg. Chem. Med. Lett, 2006, 16, 1421-1425). The halogenated compound is then substituted by 3-(N,N-dimethyl)pyrrolidine with acetone at reflux to give the expected product with a yield of 61%. The obtained compound is saponified and coupled to methyl 5-amino-2-methylbenzoate in the presence of EDCl, hydroxybenzotriazole and triethylamine. The intermediate amide obtained can be used in two different manner: it can be saponified and coupled with azaindolic compound (e.g: methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate or 5-amino-2-methoxymethyl-1H-pyrrolo[2,3-b]pyridine) or it can be reduced in aldehyde and then transformed in amine via a reductive amination. The following examples enable to well understand the invention.
A reactor is charged with 1 g (4.9 mmol) of 3-(trifluoromethyl)-4-methylbenzoic acid in 10 ml methanol in the presence of a catalytic amount of sulphuric acid. The mixture is heated for 6 hours at reflux. The solvent is then evaporated under reduced pressure and 100 ml of a saturated sodium bicarbonate solution is added to the mixture. The solution is extracted by 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and evaporated under reduced pressure to give the expected product (1 g, 95%).
A reactor is charged with 1 g (4.6 mmol) of the compound obtained in step 1, 0.97 g (5.5 mmol) of N-bromosuccinimide, 133 mg (0.55 mmol) of benzyl peroxide and 25 ml of carbon tetrachloride. The mixture is heated for 4 hours at reflux. The solvent is evaporated under reduced pressure and 100 ml of a saturated sodium bicarbonate solution is added. The solution obtained is extracted with 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and evaporated under reduced pressure to give the expected product (1.2 g, 88%).
A reactor is charged with 1.2 g (4.06 mmol) of the compound obtained in step 2, 0.55 g (4.88 mmol) of 3-dimethylaminopyrrolidine, 2.8 g (20.3 mmol) of potassium carbonate and 25 ml acetone. The mixture is heated for 6 hours at reflux. The solvent is evaporated under reduced pressure and 100 ml of water is then added. The obtained solution is extracted with 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and then evaporated under reduced pressure to give the expected product (0.8 g, 61%),
A reactor is charged with 0.8 g (2.42 mmol) of the compound obtained in step 3, 0.5 g (12.12 mmol) of lithium hydroxide and 20 ml of a mixture water/THF (1/1). The mixture is stirred at room temperature for 16 hours. The solution is concentrated under reduced pressure and then diluted in 100 ml water. The aqueous phase is extracted with 3*30 ml ethyl acetate, and acidified with chlorhydric acid 32% until a pH of between 2 and 3. The aqueous phase is extracted with 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and evaporated under reduced pressure. 750 mg of a solid is obtained which is charged in a reactor and 25 ml anhydrous dichloromethane, 460 mg (2.84 mmol) of methyl 3-amino-6-methylbenzoate, 0.71 g (7.02 mmol) of triethylamine and 540 mg (3.52 mmol) of hydroxybenzotriazole are successively added. The mixture is stirred at room temperature for 15 minutes and then 540 mg (2.82 mmol) of EDCl are added. The reaction is stirred for 16 hours. The solvent is then evaporated under reduced pressure and the residue is taken off in 100 ml ethyl acetate. The organic phase is washed with 30 ml of a saturated sodium bicarbonate solution and 2*30 ml water. The organic phase is dried on sodium sulphate and evaporated under reduced pressure. After purification on a silica column 620 mg (52%) of the expected compound are obtained.
A reactor is charged with 620 mg (1.52 mmol) of the compound obtained in step 4, 180 mg (7.6 mmol) of lithium hydroxide and 20 ml of a mixture water/THF (1/1). The mixture is stirred at room temperature for 16 hours. The solution is concentrated under reduced pressure and then diluted in 100 ml water. The aqueous phase is extracted with 3*30 ml ethyl acetate and acidified with chlorohydric acid 32% until a pH of between 2 and 3. The aqueous phase is extracted again with 3*30 ml ethyl acetate. The organic phases are combined, dried on sodium sulphate and then evaporated under reduced pressure. 600 mg of a solid is obtained which is charged in a reactor and 25 ml of anhydrous dichloromethane, 350 mg (1.83 mmol) of methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate, 0.46 g (4.56 mmol) of triethylamine and 350 mg (2.28 mmol) of hydroxybenzotriazole are successively added, The mixture is stirred for 15 minutes at room temperature and then 540 mg (350 mmol) of EDCl are added. The reaction is stirred for 16 hours. The solvent is evaporated under reduced pressure and the residue is taken off in 100 ml ethyl acetate. The organic phase is washed with 30 ml of a saturated sodium bicarbonate solution and then with 2*30 ml of water. The organic phase is dried on sodium sulphate and evaporated under reduced pressure. After purification by preparative HPLC 110 mg of the expected compound are obtained (12%).
1H NMR (400 MHz, CD3OD): 8.59 (d, 1H), 8.53 (s, 1H), 8.26 (s, 1H), 8.18 (d, 1H), 7.95 (m, 2H), 7.70 (d, 1H), 7.34 (d, 1H), 7.21 (s, 1H), 3.95 (s, 3H), 3.87 (m, 2H), 3.49 (m, 1H), 2.95 (m, 1H), 2.79 (m, 2H), 2.53 (m, 1H), 2.48 (s, 3H), 2.25 (s, 6H), 2.06 (m, 1H), 1.78 (m, HPLC: 95%. MS: 623.4 (M+1).
The compound is obtained by using the procedures of example 83 (step 3) replacing the methyl 5-(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2-methylbenzoate with the methyl 5-(4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)-benzamido)-2-methylbenzoate.
The compound is obtained by using the procedures of example 88 (step 5) replacing the methyl 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylate with the 5-amino-2-methoxymethyl-1H-pyrrolo[2,3-b]pyridine which is obtained according to the example 35.
The synthesis according to example H are represented in scheme 18.
The 3-(trifluoromethyl)-5-(4-methyl-1H-imidazol-1-yl)benzoic acid is coupled with the methyl 5-amino-2-methylbenzoate in the presence of EDCl, hydroxybenzotriazole and triethylamine (Shakespeare W. C, WO2007133562). The intermediate amide obtained is then reduced into aldehyde in 2 steps and then transformed into amine via a reductive amination reaction with aminoazaindoles. The following examples will enable to better understand the invention.
The compound is obtained using the procedures of example 88 (step 4) replacing the 4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)-benzoic acid (Shakespeare W. C, WO2007133562) by the 3-(trifluoromethyl)-5-(4-methyl-1H-imidazol-1-yl)benzoic acid.
The compound is obtained by using the procedures of examples 83 (steps 1 and 2) replacing the methyl 5-(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2-methylbenzoate with the methyl 5-(3-(trifluoromethyl)-5-(4-methyl-1H-imidazol-1-yl)benzamido)-2-methylbenzoate.
The composed is obtained according to example 83 (step 3) replacing N-(3-formyl-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)-benzamide with the 3-(trifluoromethyl)-N-(3-formyl-4-methylphenyl)-5-(4-methyl-1H-imidazol-1-yl)benzamide.
The compound is obtained according to example 91 (step 3) replacing 5-amino-1H-pyrrolo[2,3-b]pyridine-2-methyl carboxylate with 5-amino-2-methoxymethyl-1H-pyrrolo[2,3-b]pyridine
The results are expressed as percentage of the specific activity of the control obtained in the presence of the tested compound. The IC50 and Hill coefficient (nH) obtained are noted above each curve.
The inhibitory activity of the compounds of the chemical series on different protein kinases was evaluated by Cerep (Cerep Kinase Profiling Service; www.cerep.com). The kinases used in the tests in vitro are human recombinant proteins expressed in human cells, insect cells or in bacteria. The references of the kinases tested are given in the following table.
Each recombinant protein kinase was incubated with its appropriate biotinylated substrate in the presence of 0.05-20 μM of ATP (0.3 to 3 times the Km of the individual kinase) at 22° C. for 15 to 90 minutes depending on the kinase tested. The activity of the kinase was then detected by an HTRF assay.
The HTRF assay (CisBio International) is based on fluorescence transfer, using Europium (Eu3+) cryptate and XL665 respectively as donor and acceptor. When the biotinylated or tagged substrate is phosphorylated by the kinase, it reacts with a cryptate-labelled phosphospecific antibody. Addition of Streptavidin-XL665, SA-XL665, (or anti-tag antibody XL665) leads to juxtaposition of the cryptate and of the fluorophore XL665, which is reflected in fluorescence resonance energy transfer (FRET). The intensity of FRET depends on the quantity of cryptate-labelled antibody bound to the substrate, which is proportional to the quantity of phosphorylated substrate (Mathis, G. Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin Chem 41, 1391-7 (1995)).
To determine the inhibitory capacity of a compound on a kinase, the compound was incubated at a concentration of 10 μM with the kinase activity to be evaluated. The results obtained (Tables 6 and 7) therefore show the inhibitory power of a compound, at 10 μM on the kinase, expressed as percentage inhibition.
The evaluation of the activity of compounds ND0057, ND0058, ND0072, ND0073, ND0074, ND0075, ND0076, ND0077, ND0078, ND0079, ND0082, ND0085, ND0086, ND0087, ND0088, ND0089, ND0090, ND0091, ND0092, ND0093, ND0094, ND0095, ND0096, ND0098, ND0101, ND0102, ND0103, ND0104, ND0107, ND0108, ND0109, ND0117, ND0118, ND0119 at the concentration of 10 μM in kinase trial on AbI WT, AbI T315I or Src as well as the 1050 determination were carried out by the Reaction Biology Corp. (USA) society.
In Vitro Kinase Assays on Mutated AbI:
IC50 values (concentration of the compound inhibiting 50% of the kinase activity) of compound ND0006, ND0009, ND0010, ND0011, ND0019, ND0020, ND0021, ND0029, ND0031, ND0037, ND0038 et ND0047 were determined by Cerep incubating 5 increasing concentrations between 10−10 M and 10−4 M of the compound, The results are presented in Table 6 and in
IC50 values of compounds ND0057, ND0072, ND0074, ND0076, ND0077, ND0082, ND0085, ND0087, ND0088, ND0089, ND0090, ND0091, ND0093, ND0094, ND0096, ND0098, ND0117, ND0118 et ND0119 were determined by the company Reaction Biology Corp. (USA) on savage kinase AbI, mutants of kinase AbI (Q252H, Y253F and T3151) and/or Src kinase. Five increasing concentrations between 10−8 M and 10−4 M of each compound were incubated with one of the kinases in the presence of ATP at 10 μM according to the protocol of the company Reaction Biology Corp. The results are presented in Table 7.
In Vitro Assays of Inhibition of Cellular Proliferation
Cellular tests were carried out at the Montpellier Cancer Research Centre. The inhibition of proliferation by our compounds was analysed on 2 cell lines (U937 and K562) by calculating the IC50, testing 6 concentrations in triplicate for each of the compounds and for each line.
Imatinib, the known activity of which is described in the following table, was used as positive control in the tests.
The compounds (Imatinib and compound(s) of the present invention) were diluted at 10 mM in DMSO. Cells in exponential phase were seeded in 96-well plates at a concentration of 104 cells/100 μL/well. The final concentrations of compound in the well in contact with the cells were 0.1; 0.3; 0.9; 3.3; 10; 30; 90 μM. The negative and positive controls were DMSO (whose final concentration must not exceed 5% of the final volume of the well) and Imatinib, respectively. The cells were treated with the compound for 72 h (the cells are treated 3 times, once every 24 h) before being washed and analysed using an MTT kit (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) for determining cellular proliferation.
Biological Results
Percentage Inhibition of Kinase AbI by Different Compounds of the Chemical Class Invented
The results, presented in Table 6, show the percentage inhibition of kinase AbI activity as a function of the compound incubated.
The tests of kinase inhibition in vitro reveal several kinase-inhibiting molecular structures: 11 compounds inhibit kinase AbI activity at a level of at least 50%. It should be noted that 11 compounds also display inhibitory activity on kinase between 25 and 50% at a concentration of 10 μM.
The inhibition percentages of the kinase activity of savage AbI, mutated AbI T315I or Src kinase are classified in 3 categories according to table 7
The reference product for the treatment of CML, ALL and GISTs, imatininib, is capable of inhibiting, in addition to Bcr-AbI, the kinases c-kit and PDGFRα. The second and third generation compounds (Dasatinib, Nilotinib, Bosutinib) are, for their part, capable of inhibiting, in addition to Bcr-AbI, the kinases Aurora-A and/or c-Src. An analysis was performed for several of the compounds of the invention concerning their capacity for inhibiting one or more of these kinases. The results are presented in the following Table 8.
These tests show a strong activity of some compounds towards the kinases AbI and c-Src.
Determination of the IC50 Values of the Compounds of Interest in Kinase Tests In Vitro
Evaluation of IC50 was carried out for each compound having a molecular structure as well as an inhibitory activity of interest. The results obtained are presented in the form of tables (Tables 9 and 10) and of curves presented in
These results confirm the results previously obtained and reveal the inhibitory potential of several compounds of this chemical class.
Kinase Tests In Vitro on Mutated AbI
4 compounds were tested for their capacity to inhibit various mutated kinases AbI (American company, Reaction Biology Corp.).
Determination of the IC50 Values of the Compounds of Interest in Tests of Cellular Proliferation
The capacity of compound ND0009 for inhibiting the proliferation of malignant cells expressing kinase Bcr-AbI (K562), or not (U937), was analysed by determining the IC50 corresponding to the concentration of compound inhibiting cell growth at a level of 50%.
The results obtained are presented in Table 12 below and in
Compound ND0009 is active on the Bcr-AbI+ cells (K562) and not on the Bcr-AbI− cells (U937), and it displays cellular specificity similar to that of Imatinib.
Another trial was carried out: increasing concentrations of compound ND0009, ND0072, ND0074, ND0076, ND0087, ND0089, ND0090, ND0096, ND0117, ND0118, ND0119 were incubated for 24 h and/or 72 h on K562 and on U937 cells in order to determine their IC50. Imatinib was tested as a positive control. (table 13)
Most of the compounds tested are more active than the reference product (Imatinib).
| Number | Date | Country | Kind |
|---|---|---|---|
| 09 00631 | Feb 2009 | FR | national |
This is a §371 National Stage of International Application No. PCT/IB2010/000593 filed Feb. 12, 2010 (which is hereby incorporated by reference), which claims benefit of U.S. Provisional Application No. 61/202,279, filed Feb. 12, 2009 (which is hereby incorporated by reference).
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2010/000593 | 2/12/2010 | WO | 00 | 8/11/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/092489 | 8/19/2010 | WO | A |
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| 61202279 | Feb 2009 | US |
