Patent Application
20100069409
This invention relates to novel compound and their use as pharmaceuticals, particularly as p38 kinase inhibitors, for the treatment of certain diseases and conditions.
Intracellular signal transduction is the means by which cells respond to extracellular stimuli. Regardless of the nature of the cell surface receptor (e.g. protein tyrosine kinase or seven-transmembrane G-protein coupled), protein kinases and phosphatases along with phospholipases are the essential machinery by which the signal is further transmitted within the cell [Marshall, J. C. Cell, 80, 179-278 (1995)]. Protein kinases can be categorized into five classes with the two major classes being tyrosine kinases and serine/threonine kinases, depending upon whether the enzyme phosphorylates its substrate(s) on specific tyrosine(s) or serine/threonine(s) residues [Hunter, T., Methods in Enzymology (Protein Kinase Classification) p. 3, Hunter, T.; Sefton, B. M.; eds. vol. 200, Academic Press; San Diego, 1991].
Three major related intracellular pathways, the mitogen-activated kinases, or MAPKs, are now understood to transduce signals from many extracellular stimuli such as environmental stress, infectious agents, cytokines and growth factors. The MAPKs modulate the activity of numerous cell functions such as translocation and activation of transcription factors that control transcription of effector molecules such as cytokines, COX-2, iNOS; the activity of downstream kinases that effect translation of mRNAs; and cell cycle pathways through transcription or modification of enzymes. One of these three major pathways is the p38 MAPK pathway, which refers in most cell types to the isoform p38a which is ubiquitously expressed. The role of p38 in a multitude of functions, particularly related to inflammatory response has been elucidated using selective p38 inhibitors in numerous in vitro and in vivo studies. These functions have been extensively reviewed and a summary can be found in Nature Reviews [Kumar, S, Nature Rev. Drug Discovery, 2:717 (2003)]
Extracellular stimuli such as those described above are generated in a number of chronic diseases which are now understood to have a common underlying pathophysiology termed inflammation. An environmental insult or local cell damage activates cellular response pathways, including but not limited to p38; local cells then generate cytokines and chemokines, in turn recruiting lymphocytes such as neutrophils and other granulocytes. In a secondary response, the consequences include recruitment of additional lymphocytes such as additional phagocytic cells or cytotoxic T cells, and ultimately the adaptive immune response is initiated through activation of T cells. It is not currently fully understood how this acute inflammatory response becomes a chronic response leading to diseases such as rheumatoid arthritis (RA), athersclerosis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), etc. Nevertheless, the features of inflammation are recognized to contribute to a large number of chronic diseases and pathways such as the p38 pathway are accepted to contribute to the initiation of inflammatory diseases.
Small molecule synthetic inhibitors have been designed in an attempt to treat pain, multiple myeloma and rheumatoid arthritis (Peiffer et al., 2006. Curr. Top. Med. Chem. 6: 113-149). However their utility has not yet been extended to explore other conditions, particularly in the area of neuropsychiatry, where evidences of inflammatory mechanisms are apparent in depression, anxiety, schizophrenia and sleep disorders.
For example, atherosclerosis is regarded as a chronic inflammatory disease, which develops in response to injury of the vessel wall and is characterized by the complex development of an occlusive and prothrombotic atheroma. The pathogenesis of this lesion generally involves endothelial dysfunction (reduced bioavailable NO), adhesion molecule expression, adhesion and infiltration of leukocytes, cytokine and growth factor generation, accumulation of foam cells, expansion of extracellular lipid and matrix, activation of matrix metalloproteases (MMPs) and proliferation of vascular smooth muscle cells.
The discovery of p38 (initially termed CSBP, now p38; the isoforms p38α and p38β are the targets of the compounds described) provided a mechanism of action of a class of anti-inflammatory compounds for which SK&F 86002 was the prototypic example. These compounds inhibited IL-1 and TNF synthesis in human monocytes at concentrations in the low uM range [Lee, et al., Int. J. Immunopharmac. 10(7), 835 (1988)] and exhibited activity in animal models which are refractory to cyclooxygenase inhibitors [Lee; et al., Annals N.Y. Acad. Sci., 696, 149 (1993)].
The mechanism by which stress signals (including bacterial and viral infection, pro-inflammatory cytokines, oxidants, UV light and osmotic stress) activate p38 is through activation of kinases upstream from p38 which in turn phosphorylate p38 at threonine 180 and tyrosine 182 resulting in p38 activation. MAPKAP kinase-2 and MAPKAP kinase-3 have been identified as downstream substrates of CSBP/p38 which in turn phosphorylate heat shock protein Hsp27 and other substrates. Additional downstream substrates known to be phosphorylated by p38 include kinases (Mnk1/2, MSK1/2 and PRAK) and transcription factors (CHOP, MEF2, ATF2 and CREB). While many of the signaling pathways required for transduction of stress stimuli remain unknown it appears clear that many of the substrates for p38 listed above are involved. [Cohen, P. Trends Cell Biol., 353-361 (1997) and Lee, J. C. et al, Pharmacol. Ther. vol. 82, nos. 2-3, pp. 389-397, 1999]. There is also emerging evidence that p38 is involved in modulation of the activity of the NF-kB signaling pathway through a role in histone phosphorylation or acetylation, or through reduction of transcription competence of the NF-kB complex [Saccini, S. Nature Immunol., 3: 69-75, (2002); Carter, A B et al J Biol Chem 274: 30858-63 (1999)]. Finally, a role for p38 in generation of response to IFNs through activation by the Type I IFN receptor has been described [Platanias, Pharmacol. Therap. 98:129-142 (2003)]. Activation of p38 is involved in the transcriptional regulation of IFN sensitive genes through modification of specific transcription factors binding to promotor elements in these genes. Direct phosphorylation of STATs by p38 has not been conclusively demonstrated.
In addition to inhibiting IL-1 and TNF upregulation in response to inflammatory stimuli, p38 kinase inhibitors (e.g., SK&F 86002 and SB-203580) are effective in a number of different cell types in decreasing the synthesis of a wide variety of pro-inflammatory proteins including, IL-6, IL-8, GM-CSF, RANTES and COX-2. Inhibitors of p38 kinase have also been shown to suppress the TNF-induced expression of VCAM-1 on endothelial cells, the TNF-induced phosphorylation and activation of cytosolic PLA2 and the IL-1-stimulated synthesis of collagenase and stromelysin. These and additional data demonstrate that p38 is involved not only cytokine synthesis in response to stress, but also in propagating the consequent cytokine signaling [CSBP/P38 kinase reviewed in Cohen, P. Trends Cell Biol., 353-361 (1997)].
Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) are important inflammatory cytokines produced by a variety of cells, such as monocytes, macrophages, and smooth muscle cells. IL-1 has been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and other physiological conditions such as inflammation [See, e.g., Dinarello et al., Rev. Infect. Disease, 6, 51 (1984)]. The myriad of known biological activities of IL-1 include the activation of T helper cells, induction of fever, stimulation of prostaglandin or collagenase production, neutrophil chemotaxis, induction of acute phase proteins and the suppression of plasma iron levels.
There are many disease states in which excessive or unregulated IL-1 production is implicated in exacerbating and/or causing the disease. These include rheumatoid arthritis, osteoarthritis, endotoxemia and/or toxic shock syndrome, other acute or chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease; tuberculosis, atherosclerosis, muscle degeneration, cachexia, psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumatic arthritis, rubella arthritis, and acute synovitis. Evidence also links IL-1 activity to diabetes and pancreatic β cells [review of the biological activities which have been attributed to IL-1 Dinarello, J. Clinical Immunology, 5 (5), 287-297 (1985)].
Excessive or unregulated TNF production has been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic obstructive pulmonary disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusion injury, graft vs. host reaction, allograft rejections, fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
Inflammatory diseases are also marked by increases in IL-6 and C-reactive protein (CRP), both of which are sensitive to inhibition by p38 inhibitors. IL-6 stimulation of CRP production is directly inhibited by p38 inhibitors in human vascular endothelial cells, and CRP is produced by hepatocytes in response to IL-6. CRP is considered a major risk factor for cardiovascular disease [Circulation 2003.107: 363-369] and may be a significant independent risk factor for chronic obstructive pulmonary disease [Circulation 2003. 107:1514-1519]. IL-6 is also upregulated in endometriosis [Bedaiwy et al., 2002, Human Reproduction 17:426-431; Witz, 2000, Fertility and Sterility 73: 212-214].
Interleukin-8 (IL-8) and RANTES are chemotactic factors produced by several cell types including mononuclear cells, fibroblasts, endothelial cells, epithelial cells, neutrophils and T cells. Chemokine production is induced by pro-inflammatory stimuli such as IL-1, TNF, or lipopolysachharide (LPS), or viral infection. IL-8 stimulates a number of functions in vitro. It has been shown to have chemoattractant properties for neutrophils, T-lymphocytes, and basophils. In addition it induces histamine release from basophils from both normal and atopic individuals as well as lysozomal enzyme release and respiratory burst from neutrophils. IL-8 has also been shown to increase the surface expression of Mac-1 (CD11b/CD18) on neutrophils without de novo protein synthesis, which may contribute to increased adhesion of the neutrophils to vascular endothelial cells. Many diseases are characterized by massive neutrophil infiltration.
Conditions such as chronic obstructive pulmonary disease associated with an increase in IL-8 production would benefit by compounds which are suppressive of IL-8 production. RANTES is produced by cells such as epithelial cells and airway smooth muscle in response to infection or cytokine stimulation. Its main chemoattraction is for T cell subtypes and blood-borne monocytes.
IL-1, TNF and other cytokines affect a wide variety of cells and tissues and these cytokines as well as other leukocyte derived cytokines are important as critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
In addition to the involvement of p38 signaling in the production of IL-1, TNF, IL-8, IL-6, GM-CSF, COX-2, collagenase and stromelysin, signal transduction via CSBP/p38 is required for the effector functions of several of these same pro-inflammatory proteins plus many others. For example, growth factors such as VEGF, PDGF, NGF signal through surface receptors which in turn activate cellular signaling pathways including p38 MAPK [Ono, K. and Han, J., Cellular Signalling, 12 1-13 (2000); Kyriakis, J M and Avruch, J. Physiol Rev 81: 807-869 (2001)]. TGFχ, a key molecule in the control of inflammatory response, also activates p38 as a consequence of engagement of the TGFβ receptor. The involvement of CSBP/p38 in multiple stress-induced signal transduction pathways provides additional rationale for the potential utility of CSBP/p38 in the treatment of diseases resulting from the excessive and destructive activation of the immune system, or chronic inflammation. This expectation is supported by the potent and diverse activities described for CSBP/p38 kinase inhibitors [Badger, et al., J. Pharm. Exp. Thera. 279 (3): 1453-1461. (1996); Griswold, et al, Pharmacol. Comm. 7, 323-229 (1996); Jackson, et al., J. Pharmacol. Exp. Ther. 284, 687-692 (1998); Underwood, et al., J. Pharmacol. Exp. Ther. 293, 281-288 (2000); Badger, et al., Arthritis Rheum. 43, 175-183 (2000)].
Chronic inflammation is also characterized by ongoing remodeling and repair of affected tissue, leading in some cases to excess fibrotic tissue. A role for p38 MAPK in fibrosis is supported by findings that this enzyme mediates signaling of transforming growth factor beta (TGF-β) on markers and proteins of fibrosis. For example, it has been shown that TGF-β increases the kinase activity of p38 MAPK through the TGF-β activated kinase TAK-1 (Hanafusa et al., 1999, J. Biol. Chem. 274:27161-27167). Furthermore, the p38 inhibitor SB-242235 inhibited the TGF-β-induced increases in fibronectin and thrombospondin (Laping et al., 2002, Molec. Pharmacol. 62:58-64). These results show that p38 MAPK is a key signaling intermediate for the effect of the pro-fibrotic cytokine TGF-β on components of the extracellular matrix and markers of fibrosis.
P38 also plays a role in directing survival and apoptosis of cells in response to various stimuli. Both survival and apoptosis can be p38 regulated depending on the stimulus and the cell type [Morin and Huot, Cancer Research. 64:1893-1898 (2004)]. For example, TGF-beta can stimulate apoptosis in murine hepatocytes through activation of gadd45b, a protein involved in cell-cycle control, in a p38 mediated process [Yoo et al, J. Biol. Chem. 278:43001-43007, (2003)]. In a different response pathway, UV-stress can activate p38 and trigger apoptosis of a damaged cell. P38 has also been shown to promote survival of lymphocytes in response to stress, including neutrophils and CD8+ T cells.
There remains a need for treatment, in this field, for compounds which are cytokine suppressive anti-inflammatory drugs, i.e. compounds which are capable of inhibiting the CSBP/p38/RK kinase. The present invention is directed to such novel compounds which are inhibitors of p38 kinase.
This invention relates to the novel compounds of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof; and pharmaceutical compositions comprising a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, in admixture with a pharmaceutically acceptable diluent or carrier.
This invention relates to a method of treating a CSBP/RK/p38 kinase mediated disease in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
This invention also relates to a method of inhibiting cytokines and the treatment of a cytokine mediated disease, in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
This invention also relates to a method of inhibiting the production of IL-1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
This invention also relates to a method of inhibiting the production of IL-6 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
This invention also relates to a method of inhibiting the production of IL-8 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
This invention also relates to a method of inhibiting the production of TNF in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) and (Ia), and a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.
Accordingly, the present invention provides for a compound of Formula (I) and (Ia) having the structure:
wherein
The present invention provides for an alternative process for preparation of compounds having a pyrido[2,3-d]pyrimidin-7-one template or a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one derivatives.
This novel process provides for facile variation of the C4 position in the both of these templates, and therefore ease of use in the making of a combinatorial array.
The process as will be described herein provides for different (R1) substituents to be introduced at the C4 position of the compounds with the general structure of Formula I, late in a synthetic sequence after the pyrido[2,3-d]pyrimidin-7-one or the 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one template has already been substituted with a substituent (“X” in Formula I) at the C2 position and with another substituent (“R3” in Formula I) at the N8 position. For purposes herein, the term Formula (I) will be used to refer to both compounds of Formula (I) and (Ia) unless specified otherwise.
Generically the reaction is:
wherein W is a leaving group such as chlorine, bromine, iodine, OS(O)CF3; and R1, R3 and X are as defined above in Formula (I).
Compounds of Formula (I) are represented by the structure:
wherein
Compounds of Formula (I) having a similar template are described in WO 01/64679, WO 02/059083, and WO 03/088972 whose disclosures are incorporated by reference in their entirety herein.
It should be noted that the difference between compounds of Formula (I) and (Ia), lie in the unsaturation of the ring at the C5 position and the 6-position of the ring which may be a carbon or a nitrogen. The remaining variables on the ring are the same otherwise, e.g. Rx, R1, R3, etc. for each formula. Unless otherwise specified, the substitution applicable to Formula (I) is also applicable to Formula (Ia), etc.
For all of the formulas herein having an R1 substitutent, R1 is suitably an aryl, aryl C2-10 alkyl, heteroaryl, heteroaryl C2-10 alkyl; aryl C2-10 alkenyl, arylC2-10 alkynyl, heteroaryl C2-10 alkenyl, heteroaryl C2-10 alkynyl, C2-10 alkenyl, or C2-10 alkynyl moiety, which moieties may be optionally substituted.
In one embodiment R1 is an optionally substituted aryl, or an optionally substituted heteroaryl ring. Preferably, R1 is an optionally substituted aryl, more preferably an optionally substituted phenyl.
R1 may be substituted one or more times, suitably 1 to 4 times, independently at each occurrence by halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, aryl, arylC1-4 alkyl, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, (CR10R20)v′OR13, (CR10R20)vC(Z)NR4R14, (CR10R20)vC(Z)OR8, (CR10R20)vCORa′, (CR10R20)vC(O)H, ZC(Z)R11, N(R10′)C(Z)R11, N(R10′)S(O)2R7, C(Z)N(R10′)(CR10R20)vRb, C(Z)O(CR10R20)vRb, N(R10′)C(Z)(CR10R20)vRb; N(R10′)C(Z)N(R10′)(CR10R20)vRb; or N(R10)OC(Z)(CR10R20)vRb.
Suitably, Rb is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, aryl, arylC1-10alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety, which moieties excluding hydrogen, may all be optionally substituted.
In one embodiment of the invention when the R1 moiety is phenyl, and the phenyl ring is substituted by the moiety (R1″) wherein R1″ is selected from C(Z)N(R10′)(CR10R20)vRb, C(Z)O(CR10R20)vRb, N(R10′)C(Z)(CR10R20)vRb, N(R10′)C(Z)N(R10′)(CR10R20)vRb, or N(R10′)OC(Z)(CR10R20)vRb. The phenyl ring may also be additionally substituted by the substituent (R1′)g, wherein g is 0 or an integer having a value of 1, 2, 3, or 4. In one embodiment of the invention, g is 0, 1 or 2. When the R1 moiety is substituted by R1″ then these substituents are preferably in the 3- or 4-position of the phenyl ring.
Suitably, the R1′ moiety is independently selected at each occurrence from halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SR5, S(O)R5, S(O)2R5, or (CR10R20)v′OR13.
In one embodiment of the invention, R1 is substituted by C(Z)N(R10′)(CR10R20)v Rb, or N(R10′)C(Z)(CR10R20)vRb, and R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine, or halo-substituted-C1-4 alkyl, such as CF3. In a further embodiment R1 is an aryl moiety, preferably a phenyl ring.
In another embodiment of the invention R1 is substituted by C(Z)N(R10′)(CR10R20)v Rb′ and R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine, chlorine or bromine.
In one embodiment, R1′ is independently selected at each occurrence from halogen, C1-4 alkyl, or halo-substituted-C1-4 alkyl. In another embodiment, R1′ is independently selected at each occurrence from fluorine, chlorine, methyl, or CF3. In a further embodiment R1 is an aryl moiety, preferably a phenyl ring.
In one embodiment, R1 is an aryl moiety, preferably a phenyl ring, optionally substituted one or more times by halogen, C1-4 alkyl, or halo-substituted-C1-4 alkyl. More preferably, the phenyl ring is substituted in the 2, 4, or 6-position, or di-substituted in the 2,4-position, such as 2-fluoro, 3-fluoro, 4-fluoro, 2,4-difluoro, or 2-methyl-4-fluoro; or tri-substituted in the 2,4,6-position such as 2,4,6-trifluoro.
In another embodiment R1 is an aryl moiety, preferably a phenyl ring, optionally substituted one or more times by halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, SRS, S(O)R5, S(O)2R5, (CR10R20)v′OR13, (CR10R20)vC(Z)NR4R14, C(Z)N(R10′)(CR10R20)vRb, and (CR10R20)vC(Z)OR8. In one embodiment, R8 is hydrogen, or C1-4 alkyl, R13 is hydrogen, or C1-4 alkyl, such as methyl; Rb is suitably hydrogen, C1-4 alkyl, aryl, or heteroaryl. Preferably, R1 is a phenyl substituted by 2-methoxy, 3-methoxy, 4-methoxy, 2-chloro, 3-chloro, 4-chloro, 2-fluoro, 3-fluoro, 4-fluoro, 4-difluoro, 2,4,6-trifluoro, 3,4-difluoro, 3,5-difluoro, 2-methyl-4-fluoro, 2-methyl-4-chloro, 2-methylsulfanyl, 3-methylsulfanyl, 4-methylsulfanyl, 2-phenyl, 3-phenyl, 4-phenyl, 2-methyl, 3-methyl, 4-methyl, 3-fluoro-4-phenyl, 2-hydroxy, 3-hydroxy, 4-hydroxy, 2-methylsulfonyl, 3-methylsulfonyl, 4-methylsulfonyl, 3-N-cyclopropylamide, 2-methyl-3-fluoro-5-N-cyclopropylamide, 2-C(O)OH, 3-C(O)OH, 4-C(O)OH, 2-methyl-5-C(O)OH, 2-methyl-3-C(O)OH, 2-methyl-4-C(O)OH, 2-methyl-3-F-5-C(O)OH, 4-F-phenyl1-amide, 2-ethyl-5-C(O)OH, 2-ethyl-3-C(O)OH, 2-ethyl-4-C(O)OH, 2-methyl-5-dimethylamide, 2-methyl-4-dimethylamide, 5-dimethylamide, and 4-dimethylamide.
A preferred R1 moiety is 4-methyl-N-1,3-thiazol-2-ylbenzamide, N-(4-fluorophenyl)-4-methylbenzamide, 4-methyl-N-propylbenzamide, 4-methyl-N-isopropylbenzamide, 2-methyl-4-fluorophenyl, or 2-methyl-3-fluorophenyl, and 2-methyl-4-chlorophenyl.
Suitably, when R1 is a heteroaryl moiety, the ring is not attached to the pharmacophore via one of the heteroatoms, such as nitrogen to form a charged ring. For instance, a pyridinyl ring would be attached through a carbon atom to yield a 2-, 3- or 4-pyridyl moiety, which is optionally substituted.
If R1 is substituted by C(Z)N(R10′)(CR10R20)vRb, C(Z)O(CR10R20)vRb, or N(R10′)C(Z)(CR10R20)vRb; N(R10′)C(Z)N(R10′)(CR10R20)vRb; N(R10′)OC(Z)(CR10R20)vRb; it is preferably in the 4 or 5 position of the ring. If the ring is additionally substituted by R1′, and R1 is a phenyl ring, then the additional substituents are present in the ortho position, if a second R1′ moiety is also substituted on the ring, then preferably, this second R1′ substitution is not in the other ortho position.
Suitably, Ra′ is C1-4 alkyl, halo-substituted C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, heterocyclylC1-4 alkyl, (CR10R20)vOR7, (CR10R20)O(O)mR7, (CR10R20)v N(R10′)S(O)2R7, or (CR10R20)vNR4R14; and wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted.
Suitably, Rd and Rd′ are each independently selected from hydrogen, C1-4 alkyl, C3-5 cycloalkyl, C3-5 cycloalkylC1-4alkyl, or the Rd and Rd′ together with the nitrogen which they are attached form an optionally substituted heterocyclic ring of 5 to 6 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9′, and wherein the Rd and Rd′ moieties which are C1-4 alkyl, C3-6cycloalkyl, C3-6cycloalkylC1-4 alkyl, and the R4 and R14 cyclized ring are optionally substituted, 1 to 4 times, independently by halogen; halosubstituted C1-4 alkyl; hydroxy; hydroxy substituted C1-4alkyl; C1-4 alkoxy; halosubstituted C1-4 alkoxy; S(O)mRf; C(O)Rj; C(O)ORj; C(O)NR4′R14′, NR4′C(O)C1-4alkyl; S(O)2NR4′R14′C1-4 alkyl; NR4′R14′S(O)2C1-4 alkyl; or NR4′R14′.
Suitably R9′ is independently selected at each occurrence from hydrogen, or C1-4 alkyl.
Suitably, Z is independently at each occurrence selected from oxygen or sulfur.
Suitably, m is independently selected at each occurrence from 0 or an integer having a value of 1 or 2.
Suitably, v is 0 or an integer having a value of 1 to 2.
Suitably, v′ is 0 or an integer having a value of 1 or 2.
Suitably, R10 and R20 are independently selected at each occurrence from hydrogen or C1-4 alkyl.
Suitably, R10′ is independently selected at each occurrence from hydrogen or C1-4 alkyl.
Suitably, R11 is independently selected at each occurrence from hydrogen, or C1-4 alkyl.
Suitably, R12 is independently selected at each occurrence from hydrogen, C1-4 alkyl, halo-substituted C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkyl C1-4 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenyl C1-4 alkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or heterocyclylC1-4 alkyl, and wherein these moieties, excluding hydrogen, may be optionally substituted.
Suitably, R13 is independently selected at each occurrence from hydrogen, C1-4 alkyl, halo-substituted C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylC1-4 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenyl C1-4 alkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or a heterocyclylC1-4 alkyl moiety, and wherein each of these moieties, excluding hydrogen, may be optionally substituted and wherein these moieties, excluding hydrogen, may be optionally substituted 1 to 4 times by halogen; halosubstituted C1-4 alkyl; C1-4 alkyl; hydroxy; hydroxy substituted C1-4alkyl; C1-4alkoxy; halosubstituted C1-4 alkoxy; S(O)mC1-4 alkyl; —C(O), C(O)C1-4 alkyl; or NR21′R31′.
Suitably, R21′ and R31′ are each independently selected from hydrogen or C1-4 alkyl, or R21′ and R31′ together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally contains an additional heteroatom selected from oxygen, nitrogen or sulfur.
The Rb moieties, excluding hydrogen, may be optionally substituted, one or more times, preferably 1 to 4 times independently at each occurrence by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy, such as methoxy or ethoxy; halosubstituted C1-10 alkoxy; OR8, such as methoxy, ethoxy or phenoxy; SR5, S(O)R5, S(O)2R5, such as methyl thio, methylsulfinyl or methyl sulfonyl; C(O)Rj; C(O)ORj; C(O)NR4″R14″; cyano; nitro; NR15R25; —Z′—(CR10R20)s-Z′; C1-10alkyl; such as methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, etc.; C3-7cycloalkyl or a C3-7cycloalkyl C1-10 alkyl group, such as cyclopropyl, or cyclopropyl methyl, or cyclopropyl ethyl, etc.; halosubstituted C1-10 alkyl, such CF2CF2H, CH2CF3, or CF3; an optionally substituted aryl, such as phenyl, or an optionally substituted aryl C1-10alkyl, such as benzyl or phenethyl; an optionally substituted heterocyclic or heterocyclic C1-10alkyl, or an optionally substituted heteroaryl or heteroaryl C1-10alkyl, and wherein these aryl, heteroaryl, and heterocyclic containing moieties may also be substituted one to two times by halogen, hydroxy, hydroxy substituted alkyl, C1-10 alkoxy, S(O)malkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, when Rb is an optionally substituted C1-10alkyl, the moiety includes but is not limited to a methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, heptyl, 2-methylpropyl; a halosubstituted alkyl, such as 2,2,2-trifluoroethyl, trifluoromethyl, 2-fluoroethyl; a cyano substituted alkyl, such as cyanomethyl, cyanoethyl; an alkoxy, thio or hydroxy substituted alkyl, such as 2-methoxyethyl, 2-hydroxy propyl or serinol, or an ethylthioethyl.
In an alternative embodiment, when Rb is an optionally substituted C1-10alkyl the moiety is a methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, or 2,2-dimethylpropyl or 2-hydroxy propyl group.
Suitably, when Rb is an optionally substituted heteroaryl, heteroaryl alkyl they are as defined in the definition section, and include but are not limited, to furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and uracil, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, purinyl, and phthalazinyl.
Suitably, when Rb is an optionally substituted heterocyclic, heterocyclic alkyl, they are as defined in the definition section,
In one embodiment of the invention, when Rb is an optionally substituted heteroaryl, heteroaryl alkyl, heterocyclic or heterocyclic alkyl, the moiety is a 1,3-thiazol-2-yl, 5-methyl-1,3-thiazol-2-yl, isoquinoline, 3-thiophene, indol-5yl, pyridin-3-yl, pyridine-4-yl, indazolyl, benzothiazolyl, 2-methyl-1,3-benzothiazol-5-yl, pyrazol-3-yl, 4-morpholino, 2-furanyl, 2-furanylmethyl, 2-thienyl, 2-thienylmethyl, tetrahydro-2H-pyran-4-yl, tetrahydro-2H-pyran-4-yl methyl, tetrahydro-2-furanyl, or tetrahydro-2-furanylmethyl, 1H-imidazol-4-yl or 1H-imidazol-4-ylethyl.
In an alternative embodiment, when Rb is an optionally substituted heteroaryl the moiety is a 1,3-thiazol-2-yl or 5-methyl-1,3-thiazol-2-yl, isoquinolinyl, thiophene, pyridinyl, indazolyl, benzothiazolyl, e.g. 2-methyl-1,3-benzothiazol-5-yl.
In another embodiment, the heteroaryl ring is an optionally substituted thiazolyl, pyridyl, or thiophene ring.
Suitably, when Rb is an optionally substituted aryl or arylalkyl moiety, the aryl containing is unsubstituted or substituted independently at each occurrence one or more times by halogen, alkyl, cyano, OR8, SRS, S(O)2R5, C(O)Rj, C(O)ORj, —Z′—(CR10R20)s-Z′, halosubstituted C1-10 alkyl, or an optionally substituted aryl.
In one embodiment, Rb is a phenyl, or napthylene, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorphenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3-chloro-4-fluorophenyl, 2-methyl phenyl, 3-methylphenyl, 4-methylphenyl, 6-methyl phenyl, 2-methyl phenyl, 3-amino phenyl, 3,4-dimethyl phenyl, 4-methyl-3-fluorophenyl, 4-trifluorophenyl, 4-ethoxyphenyl, 4-methoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, 4-thiomethylphenyl, 4-acetylphenyl, 4-dimethylaminophenyl, benzyl, phenethyl, phenylpropyl, 2,3-difluoro-benzyl, 3,5-difluoro-benzyl, biphenyl, 4′-fluorobiphenyl, 4-sulfonamindo-2-methylphenyl, or 3-phenyloxyphenyl, 4-phenyloxyphenyl, 4-(1-piperidinylsulfonyl)-phenyl, or 3-(aminocarbonyl)phenyl.
In another embodiment, Rb is a phenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3-chloro-4-fluorophenyl, 4-methyl-3-fluorophenyl, 4-trifluorophenyl, 2-methylphenyl, 3-methylphenyl, 4-ethoxyphenyl, 4-methoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, 4-thiomethylphenyl, 4-acetylphenyl, 4-dimethylaminophenyl, biphenyl, 4′-fluorobiphenyl, 4-sulfonamindo-2-methylphenyl, 3-phenyloxyphenyl, benzyl, or phenethyl.
Suitably, when Rb is an optionally substituted cycloalkyl or cycloalkyl alkyl moiety, the moiety is a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, or a cyclopentylmethyl. In another embodiment, Rb is a cyclopropyl or cyclopropylmethyl group.
In another embodiment, Rb is hydrogen, or an optionally substituted alkyl.
In another embodiment, Rb is C1-10 alkyl, heteroaryl, or aryl, all optionally substituted.
The moiety —Z′—(CR10R20)s-Z′ forms a cyclic ring, such as a dioxalane ring.
Suitably Z′ is independently selected at each occurrence from oxygen, or sulfur.
Suitably, s is independently selected at each occurrence from 0 or an integer having a value of 1, 2, or 3.
For each of the integer variables where appropriate, e.g. n, n′, m, q′, s, t, or v′, etc. they are independently chosen at each occurrence.
Suitably, R5 is independently selected at each occurrence from hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR4′R14′, excluding the moieties SR5 being SNR4′R14′, S(O)2R5 being SO2H and S(O)R5 being SOH.
Suitably, Rf is hydrogen, C1-10alkyl, aryl, aryl C1-10alkyl, heteroaryl, heteroaryl C1-10alkyl, heterocyclic, or a heterocyclic C1-10alkyl moiety, and wherein these moieties, excluding hydrogen, may be optionally substituted.
Suitably, Rj is C1-10alkyl, aryl, aryl C1-10alkyl, heteroaryl, heteroaryl C1-10alkyl, heterocyclic, or a heterocyclic C1-10alkyl moiety.
Suitably, R8 is independently selected at each occurrence from hydrogen, C1-4 alkyl, halo-substituted C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkyl C1-4 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenyl C1-4 alkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or a heterocyclylC1-4 alkyl moiety, and wherein these moieties, excluding hydrogen, may be optionally substituted independently at each occurrence, 1 to 4 times, by halogen; halosubstituted C1-4 alkyl; C1-4 alkyl; C3-5cycloalkyl; C3-5cycloalkyl C1-4alkyl; halosubstituted C1-4 alkyl; hydroxy; hydroxy substituted C1-4alkyl; C1-4alkoxy; halosubstituted C1-4 alkoxy; S(O)mC1-4 alkyl; —C(O), C(O)C1-4 alkyl; NR21′R31′; or an aryl or aryl C1-4 alkyl, and wherein these aryl containing moieties may also be substituted one to two times independently at each occurrence, by halogen, hydroxy, hydroxy substituted alkyl, C1-4 alkoxy, S(O)mC1-4alkyl, amino, mono & di-substituted C1-4 alkylamino, C1-4 alkyl, or CF3.
Suitably, R15 and R25 are each independently selected at each occurrence from hydrogen, C1-4 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-4alkyl, aryl, or aryl-C1-4 alkyl, heteroaryl or heteroaryl C1-4 alkyl moiety, and wherein these moieties, excluding hydrogen may be optionally substituted; or R15 and R25 together with the nitrogen which they are attached form an optionally substituted heterocyclic ring of 4 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9; and wherein these moieties are optionally substituted 1 to 4 times, independently at each occurrence by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; SR5, S(O)R5, S(O)2R5; C(O)Rj; C(O)ORj; C(O)NR4′R14′; NR4′C(O)C1-10alkyl; NR4′C(O)aryl; NR4′R14; cyano; nitro; C1-10 alkyl; C3-7cycloalkyl; C3-7cycloalkyl C1-10 alkyl; halosubstituted C1-10 alkyl; aryl, arylC1-4 alkyl, heteroaryl, or heteroC1-4 alkyl, heterocyclic and heterocyclicC1-4 alkyl and wherein these aryl, heterocyclic and heteroaryl containing moieties may also be substituted one to two times independently at each occurrence by halogen, C1-4 alkyl, hydroxy, hydroxy substituted C1-4 alkyl, C1-10 alkoxy, S(O)malkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, R4 and R14 are each independently selected at each occurrence from hydrogen, C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-4alkyl, aryl, aryl-C1-4 alkyl, heterocyclic, heterocyclic C1-4 alkyl, heteroaryl or heteroaryl C1-4 alkyl; or the R4 and R14 together with the nitrogen which they are attached form an unsubstituted or substituted heterocyclic ring of 4 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or nitrogen; and wherein the C1-10 alkyl, C3-7cycloalkyl, C3-7cycloalkylC1-4 alkyl, aryl, aryl-C1-4 alkyl, heteroaryl and heteroaryl C1-4 alkyl moieties, and the R4 and R14 cyclized ring are optionally substituted, 1 to 4 times, independently at each occurrence, by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; C1-10 alkyl; halosubstituted C1-10 alkyl; SRS; S(O)R5; S(O)2R5; C(O)Rj; C(O)ORj; C(O)NR4′R14; (CR10R20)nN(R10′)C(Z)OR7; (CR10R20)nN(R10′)C(Z)NRdRd′; NR4′C(O)C1-10alkyl; NR4′C(O)aryl; NR4′R14′; cyano; nitro; C3-7cycloalkyl; C3-7cycloalkyl C1-10 alkyl; C1-10 alkyl substituted one or more times by an optionally substituted aryl; an unsubstituted or substituted aryl, or arylC1-4 alkyl; an unsubstituted or substituted heteroaryl, or heteroaryl C1-4 alkyl; an unsubstituted or substituted heterocyclic, or heterocyclic C1-4 alkyl, and wherein these aryl, heterocyclic and heteroaryl containing moieties are substituted one to two times independently at each occurrence by halogen; C1-4 alkyl, hydroxy; hydroxy substituted C1-4 alkyl; C1-4 alkoxy; S(O)malkyl; amino, mono & di-substituted C1-4 alkyl amino, or CF3.
Suitably, when R4 and R14 together with the nitrogen cyclize to form an optionally substituted ring, such as described above, such rings include, but are not limited to pyrrolidine, piperidine, piperazine, diazepine, azepine, morpholine, and thiomorpholine (including oxidizing the sulfur).
Suitably, R4′ and R14′ are each independently selected at each occurrence from hydrogen or C1-4 alkyl, or R4′ and R14′ can cyclize together with the nitrogen to which they are attached to form an optionally substituted 5 to 7 membered ring which optionally contains an additional heteroatom from oxygen, sulfur or NR9′. Suitably, when R4′ and R14′ cyclize to form an optionally substituted ring, such rings include, but are not limited to pyrrolidine, piperidine, piperazine, morpholine, and thiomorpholine (including oxidizing the sulfur).
Suitably, R4″ and R14″ are each independently selected from hydrogen or C1-10 alkyl, or R4″ and R14″ can cyclize together with the nitrogen to which they are attached to form an optionally substituted 5 to 7 membered ring which optionally contains an additional heteroatom selected from oxygen, sulfur or NR9′. Suitably, when R4″ and R14′ cyclize to form an optionally substituted ring, such rings include, but are not limited to pyrrolidine, piperidine, piperazine, diazepine, azepine, morpholine, and thiomorpholine (including oxidizing the sulfur).
Suitably, R6 is independently selected from hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl C1-10alkyl, aryl, arylC1-10 alkyl, heteroaryl or a heteroarylC1-10 alkyl moiety, and wherein these moieties, excluding hydrogen may be optionally substituted independently, one or more times, suitably 1 to 2 times, by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; S(O)m alkyl; C(O); NR4′R14′; C1-10 alkyl; C3-7cycloalkyl; C3-7cycloalkyl C1-10 alkyl; halosubstituted C1-10 alkyl; an unsubstituted aryl or arylalkyl, or an aryl or arylalkyl substituted one or two times by halogen, hydroxy, hydroxy substituted alkyl, C1-10 alkoxy, S(O)malkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, R9 is hydrogen, C(Z)R6, optionally substituted C1-10 alkyl, optionally substituted aryl or optionally substituted aryl-C1-4 alkyl. The alkyl, aryl and arylalkyl moieties may be optionally substituted 1 or 2 times, independently by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; S(O)m alkyl; —C(O); NR4′R14′; C1-10 alkyl, C3-7cycloalkyl; C3-7cycloalkyl C1-10 alkyl; halosubstituted C1-10 alkyl; an aryl or aryl C1-4 alkyl, and wherein these aryl containing moieties may also be substituted one or two times independently by halogen, hydroxy, hydroxy substituted alkyl, C1-10 alkoxy, S(O)mC1-4 alkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, R3 is a C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkyl C1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or heterocyclylC1-10 alkyl moiety, which moieties may be optionally substituted 1 to 4 times, independently at each occurrence by hydrogen, halogen, nitro, C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-7cycloalkyl, C3-7cycloalkylC1-10 alkyl, C5-7cycloalkenyl, C5-7cycloalkenylC1-10 alkyl, (CR10R20)nOR6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nN(R10′)S(O)2R7, (CR10R20)nNR16R26, (CR10R20)nCN, (CR10R20)nS(O)2NR16R26, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nC(Z)NR16R26, (CR10R20)nN(R10′)C(Z)R6, (CR10R20)nN(R10′)C(═N(R10′))NR16R26, (CR10R20)nOC(Z)NR16R26, (CR10R20)nN(R10′)C(Z)NR16R26, or (CR10R20)nN(R10′)C(Z)OR7.
In one embodiment, the R3 moieties are optionally substituted 1 to 4 times, independently at each occurrence by halogen, nitro, C1-4 alkyl, halo-substituted C1-4 alkyl, C2-4 alkenyl, C2-4alkynyl, C3-6cycloalkyl, C3-6cycloalkylC1-4 alkyl, C5-6cycloalkenyl, C5-6cycloalkenylC1-4 alkyl, (CR10R20)nOR6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nNHS(O)2R7, (CR10R20)nS(O)2NR16R26, (CR10R20)nNR16R26, (CR10R20)nCN, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nN(R10′)C(Z)R6, or (CR10R20)nC(Z)NR16R26.
In one embodiment the R3 moieties are optionally substituted independently, one or more times, suitably 1 to 4 times, independently at each occurrence by halogen, C1-10alkyl, (CR10R20)nOR6, cyano, nitro, (CR10R20)nNR16R26, or halosubstituted C1-10alkyl. Further to this embodiment, R3 is a phenyl ring, optionally substituted independently, one or more times, suitably 1 to 4 times, independently at each occurrence by halogen, C1-10 alkyl, hydroxy, C1-10 alkoxy, cyano, nitro, amino, or halosubstituted C1-10 alkyl. In another embodiment, the R3 substituents are selected independently from halogen, such as fluorine, chlorine, bromine or iodine, or C1-10 alkyl, such as methyl.
In one embodiment the R3 moieties are an optionally substituted C1-10 alkyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkylalkyl, or optionally substituted aryl. In another embodiment, the R3 moiety is an optionally substituted C1-10 alkyl, or an optionally substituted aryl. In another embodiment, R3 is an optionally substituted phenyl.
Suitably, in one embodiment when R3 is an aryl moiety, it is an optionally substituted phenyl ring. The phenyl is optionally substituted one or more times, independently at each occurrence, suitably 1 to 4 times by halogen, C1-4 alkyl, or halo-substituted-C1-4 alkyl. The phenyl ring may be substituted in the 2, 4, or 6-position, or di-substituted in the 2,4-position or 2,6-position, such as 2-fluoro, 4-fluoro, 2,4-difluoro, 2,6-difluoro, or 2-methyl-4-fluoro; or tri-substituted in the 2,4,6-position, such as 2,4,6-trifluoro.
In one embodiment of the invention, the R3 optional substituents are independently selected from halogen, alkyl, hydroxy, alkoxy, cyano, nitro, amino, or halosubstituted alkyl.
In another embodiment, the optional substituents are independently selected from halogen, or alkyl.
Suitably, R7 is C1-6alkyl, aryl, arylC1-6alkyl, heterocyclic, heterocyclylC1-6 alkyl, heteroaryl, or heteroarylC1-6alkyl; and wherein each of these moieties may be optionally substituted one or two times independently, by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; S(O)m alkyl; C(O); NR4′R14′; C1-10 alkyl; C3-7cycloalkyl; C3-7cycloalkylC1-10 alkyl; halosubstituted C1-10 alkyl; an aryl or arylalkyl moiety, and wherein these aryl containing moieties may also be substituted one to two times by halogen, hydroxy, hydroxy substituted alkyl, C1-10 alkoxy, S(O)malkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, R16 and R26 are each independently selected from hydrogen, or C1-4 alkyl; or the R16 and R26 together with the nitrogen which they are attached form an unsubstituted or substituted heterocyclic ring of 4 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9′.
Suitably, n is 0 or an integer having a value of 1 to 10, and is independently selected at each occurrence.
Suitably, X is R2, OR2′, S(O)mR2′, (CH2)n′N(R11)S(O)mR2′, (CH2)n′N(R11)C(O)R2′, (CH2)n′NR4R14, (CH2)n′N(R2′)(R2″), or N(R10′)RhNH—C(═N—CN)NRqRq′.
Suitably, n′ is independently selected at each occurrence from 0 or an integer having a value of 1 to 10;
Suitably, Rh is selected from an optionally substituted C1-10 alkyl, CH2CH2—O—CH2—CH2—, —CH2—O—CH2—, —CH2—C(O)N(R10′)CH2—CH2—, —CH2—N(R10′)C(O)CH2—, —CH2—CH(OR10′)—CH2—, —CH2—C(O)O—CH2—CH2—, or —CH2—CH2—O—C(O)CH2—.
Suitably, Rq and Rq′ are independently selected from hydrogen, C1-10 alkyl, C3-7cycloalkyl, C3-7cycloalkylC1-10alkyl, C5-7 cycloalkenyl, C5-7 cycloalkenyl-C1-10alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety, wherein all of the moieties are optionally substituted, or Rq and Rq′ together with the nitrogen to which they are attached form a 5 to 7 membered optionally substituted ring, which ring may contain an additional heteroatom selected from oxygen, nitrogen or sulphur.
Suitably, X1 is N(R10′), O, S(O)m, or CR10R20. In one embodiment of the invention, X1 is N(R10′), or O.
Suitably, R2 is independently selected from hydrogen, optionally substituted C1-10 alkyl, optionally substituted C3-7 cycloalkyl, optionally substituted C3-7cycloalkylalkyl, optionally substituted aryl, optionally substituted arylC1-10alkyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-10 alkyl, optionally substituted heterocyclic, optionally substituted heterocyclylC1-10alkyl moiety; or R2 is the moiety (CR10R20)q′X1(CR10R20)qC(A1)(A2)(A3), or (CR10R20)q′C(A1)(A2)(A3).
Suitably q′ is 0, or an integer having a value of 1 to 6.
Suitably q is 0, or an integer having a value of 1 to 10.
The R2 moieties, excluding hydrogen, may be optionally substituted one or more times, preferably 1 to 4 times, independently at each occurrence by C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylC1-10alkyl, C5-7cycloalkenyl, C5-7 cycloalkenyl C1-10 alkyl, halogen, —C(O), cyano, nitro, aryl, aryl C1-10 alkyl, heterocyclic, heterocyclic C1-10 alkyl, heteroaryl, heteroaryl C1-10 alkyl, (CR10R20)n0R6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nN(R10′)S(O)2R7, (CR10R20)nNReRe′, (CR10R20)nNReRe′ C1-4alkyl NReRe′, (CR10R20)nCN, (CR10R20)nS(O)2NReRe′, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nC(Z)NReRe′, (CR10R20)nN(R10′)C(Z)R6, (CR10R20)nN(R10′)C(═N(R10′))NReRe′, (CR10R20)nC(═NOR6)NReRe′, (CR10R20)nOC(Z)NReRe′, (CR10R20)nN(R10′)C(Z) NReRe′, or (CR10R20)nN(R10′)C(Z)OR7.
Suitably, Re and Re′ are each independently selected at each occurrence from hydrogen, C1-4 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-4alkyl, aryl, aryl-C1-4 alkyl, heterocyclic, heterocyclic C1-4 alkyl, heteroaryl or a heteroaryl C1-4 alkyl moiety, which moieties may be optionally substituted; or Re and Re′ together with the nitrogen which they are attached form an optionally substituted heterocyclic ring of 4 to 7 members, which ring optionally contains an additional heteroatom selected from oxygen, sulfur or nitrogen; and wherein each of these moieties, including the cyclized ring and excluding hydrogen, may be substituted 1 to 4 times, independently at each occurrence by halogen; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy; halosubstituted C1-10 alkoxy; C1-10 alkyl; halosubstituted C1-4 alkyl; S(O)mRf; C(O)Rj; C(O)ORj; (CR10R20)nN(R10′)C(Z)OR7; (CR10R20)nN(R10′)C(Z)NRdRd′; C(O)NR4′R14′; NR4′C(O)C1-10alkyl; NR4′C(O)aryl; cyano; nitro; C1-10 alkyl; C3-7cycloalkyl; C3-7cycloalkyl C1-10 alkyl; halosubstituted C1-10 alkyl; aryl, arylC1-4alkyl, heterocyclic, heterocyclic C1-4 alkyl, heteroaryl, or hetero C1-4 alkyl, and wherein these aryl, heterocyclic or heteroaryl containing moieties may be optionally substituted one to two times independently at each occurrence by halogen, C1-4 alkyl, hydroxy, hydroxy substituted C1-4 alkyl, C1-10 alkoxy, S(O)malkyl, amino, mono & di-substituted C1-4 alkyl amino, C1-4 alkyl, or CF3.
Suitably, Rf′ is independently selected at each occurrence from hydrogen, C1-10alkyl, aryl, aryl C1-10alkyl, heteroaryl, heteroaryl C1-10alkyl, heterocyclic, heterocyclic C1-10alkyl or NR4′R14′; and wherein these moieties, excluding hydrogen, and NR4′R14′, may be optionally substituted.
In one embodiment of the present invention X is R2, OR2′, (CH2)n'NR4R14, or (CH2)n′N(R2′)(R2″). In another embodiment of the present invention, X is R2, and R2 is (CR10R20)q′X1(CR10R20)qC(A1)(A2)(A3), or (CR10R20)q′C(A1)(A2)(A3).
When X is R2 and R2 is an optionally substituted heterocyclic or heterocyclic alkyl, the heterocyclic containing moiety is suitably selected from tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-1-pyrrolidinyl, 3-oxo-1-pyrrolidinyl, 1,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur moiety).
In one embodiment, R2 is an optionally substituted piperidinyl or piperazinyl ring.
In another embodiment, when R2 is an optionally substituted heterocyclic or heterocyclic alkyl ring the ring is substituted one or mores times independently by an optionally substituted heterocyclic, heterocyclic alkyl, aryl, arylalkyl, alkyl, (CR10R20)nNReRe′, or (CR10R20)nN(R10′)C(Z)OR7. The second heterocyclic ring is suitably selected from an optionally substituted tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-1-pyrrolidinyl, 3-oxo-1-pyrrolidinyl, 1,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, diazepine, morpholino or thiomorpholino (including oxidized versions of the sulfur moiety). Suitably, the second heterocyclic ring is selected from morpholino, piperidine, or pyrrolidinyl.
In one embodiment, R2 is a 4-amino-1-piperidinyl, 1,1-dimethylethyl)oxy]-carbonyl}amino)-1-piperidinyl, 4-methyl-1-piperazinyl, 4-ethyl-1-piperazinyl, 4-propyl-1-piperazinyl, 4-butyl-1-piperazinyl, 4-(methylamino)-1-piperidinyl, 1,1-dimethylethyl-4-piperidinyl}methylcarbamate, 4-phenyl-1-piperazinyl, 1,4′-bipiperidin-1′-yl, 4-(1-pyrrolidinyl)-1-piperidinyl, 4-methyl-1,4′-bipiperidin-1′-yl, 4-(4-morpholinyl)-1-piperidinyl, 4-(diphenylmethyl)-1-piperazinyl, or 4-methylhexahydro-1H-1,4-diazepin-1-yl.
Suitably, R2′ is independently selected at each occurrence from hydrogen, C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylalkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety, and wherein each of these moieties, excluding hydrogen, may be optionally substituted 1 to 4 times, independently, at each occurrence, by C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylC1-10alkyl, C5-7cycloalkenyl, C5-7 cycloalkenylC1-10 alkyl, halogen, —C(O), cyano, nitro, aryl, aryl C1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, heterocyclylC1-10 alkyl, (CR10R20)nOR6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nN(R10′)S(O)2R7, (CR10R20)nNReRe′, (CR10R20)nNReRe′C1-4alkylNReRe′, (CR10R20)nCN, (CR10R20)nS(O)2NReRe′, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nC(Z)NReRe′, (CR10R20)nN(R10′)C(Z)R6, (CR10R20)nN(R10′)C(═N(R10′))NReRe′, (CR10R20)nC(═NOR6) NReRe′, (CR10R20)nOC(Z) NReRe′, (CR10R20)nN(R10′)C(Z)NReRe′, or (CR10R20)nN(R10′)C(Z)OR7.
In one embodiment, when X is (CH2)nN(R2′)(R2″), one of R2′, or R2″ is hydrogen, or methyl.
In one embodiment, when R2′ is an optionally substituted heterocyclic or heterocyclylC1-10 alkyl the heterocyclic containing moiety is substituted one or more time independently by C1-10 alkyl, aryl, heterocyclic, (CR10R20)nNReRe′, (CR10R20)nN(R10′)C(Z)OR7, or (CR10R20)nC(Z)OR6. More specifically, methyl, ethyl, NHC(O)O—CCH3, N(CH3)C(O)O—CCH3, amino, methylamino, dimethylamino, phenyl, piperidine, pyrrolidine, 1-ethylpropyl, 4-methyl-1,4′-bipiperidin-1′-yl, 1,4′-bipiperidin-1′-yl, morpholino,
In one embodiment, when X is (CH2)nN(R2′)(R2″), R2′ is an optionally substituted C1-10 alkyl, cycloalkyl, heterocyclic, heterocyclyl C1-10 alkyl, heteroarylalkyl. Suitably, when R2′ is an optionally substituted cycloalkyl it is an a cyclohexyl ring. In one embodiment the cyclohexyl ring is optionally substituted one or more times by (CR10R20)nNReRe′. Suitably, when R2′ is an optionally substituted heterocyclic, or a heterocyclylC1-10 alkyl, the ring is selected from tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-1-pyrrolidinyl, 3-oxo-1-pyrrolidinyl, 1,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, diazepine, hexahydro-1-H-azepine, morpholino or thiomorpholino (including oxidized versions of the sulfur moiety). Preferably, the ring is a piperidine, piperazine, pyrrolidinyl, 2-oxo-1-pyrrolidinyl, morpholino, hexahydro-1-H-azepine ring. In one embodiment, the rings are substituted one or more times, suitably 1 to 4 times, independently by C1-10 alkyl, aryl, arylalkyl, (CR10R20)nNReRe′, or (CR10R20)nN(R10′)C(Z)OR7.
In one embodiment, (CH2)nN(R2′)(R2″) is 1-(phenylmethyl)-4-piperidinamine, 2-[4-(phenylmethyl)-1-piperazinyl]ethylamine, 2-(1-piperidinyl)ethylamine, 2-(1-methyl-2-pyrrolidinyl)ethylamine, 1-[(phenylmethyl)-3-pyrrolidinyl]amine, 3-[(1-pyrrolidinyl)propyl]amine, 3-[(hexahydro-1H-azepin-1-yl)propyl]amine, (1-methyl-4-piperidinyl)amine, 3-[(4-morpholinyl)propyl]amine, 3-[(2-oxo-1-pyrrolidinyl)propyl]-amine, 2-[(4-morpholinyl)ethyl]amine, 2-[(1-pyrrolidinyl)ethyl]-amine, or [(1-ethyl-2-pyrrolidinyl)methyl]amino.
In one embodiment when X is (CH2)nN(R2′)(R2″), and R2′ is an optionally substituted C1-10 alkyl, the alkyl is substituted one or more times independently by (CR10R20)nNReRe′ or (CR10R20)nNReRe′C1-4alkylNReRe′. In one embodiment Re and Re′ are independently an optionally substituted C1-4 alkyl, such as methyl, ethyl, isopropyl, n-butyl, or t-butyl. Preferably, (CH2)nN(R2′)(R2″) is 3-(dimethylamino)propyl(methyl)amine, 3-(diethylamino)propylamine, propylamine, (2,2-dimethylpropyl)amine, (2-hydroxypropyl)amino, 2-(dimethylamino)ethylamine, 2-(dimethylamino)ethyl(methyl)amine, 3-(dimethylamino)propylamine, 2-(dimethylamino)ethyl(methyl)amine, 3-(diethylamino)propylamine, 2-(methylamino)ethylamine, [(1-methylethyl)amino]ethylamine, 3-(diethylamino)propylamine, 3-(dibutylamino)propylamine, 3-[(1-methylethyl)amino]propylamine, 3-(1,1-dimethylethyl)aminopropylamine, 3-(dimethylamino)-2,2-dimethylpropylamine, 4-(diethylamino)-1-methylbutylamine, or 3-[[3-(dimethylamino)propyl]-(methyl)amino]propyl(methyl)amine.
Suitably R2″ is selected from hydrogen, C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylalkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety, and wherein these moieties, excluding hydrogen, may be optionally substituted 1 to 4 times, independently at each occurrence, by C1-10 alkyl, halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylC1-10alkyl, C5-7cycloalkenyl, C5-7 cycloalkenyl C1-10 alkyl, halogen, —C(O), cyano, nitro, aryl, aryl C1-10 alkyl, heteroaryl, heteroarylC1-10 alkyl, heterocyclic, heterocyclylC1-10 alkyl, (CR10R20)nOR6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nN(R10′)S(O)2R7, (CR10R20)nNReRe′, (CR10R20)nNReRe′C1-4alkyl NReRe′, (CR10R20)nCN, (CR10R20)nS(O)2 NReRe′, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nC(Z)NReRe′, (CR10R20)nN(R10′)C(Z)R6, (CR10R20)nN(R10′)C(═N(R10′)) NReRe′, (CR10R20)nC(═NOR6) NReRe′, (CR10R20)nOC(Z)NReRe′, (CR10R20)nN(R10′)C(Z)NReRe′, or (CR10R20)nN(R10′)C(Z)OR7; or wherein R2″ is the moiety
(CR10R20)tX1(CR10R20)qC(A1)(A2)(A3);
Suitably, t is an integer having a value of 2 to 6.
Suitably, q is 0 or an integer having a value of 1 to 10.
Suitably, A1 is an optionally substituted C1-10 alkyl, heterocyclic, heterocyclic C1-10 alkyl, heteroaryl, heteroaryl C1-10 alkyl, aryl, or aryl C1-10 alkyl.
Suitably, A2 is an optionally substituted C1-10 alkyl, heterocyclic, heterocyclic C1-10 alkyl, heteroaryl, heteroaryl C1-10 alkyl, aryl, or aryl C1-10 alkyl.
Suitably, A3 is hydrogen or is an optionally substituted C1-10 alkyl.
The A1, A2, and A3 C1-10 alkyl moieties may optionally substituted one or more times independently at each occurrence, preferably from 1 to 4 times, with halogen, such as chlorine, fluorine, bromine, or iodine; halo-substituted C1-10alkyl, such as CF3, or CHF2CF3; C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylC1-10alkyl, C5-7cycloalkenyl, C5-7 cycloalkenylC1-10 alkyl, (CR10R20)nOR6, (CR10R20)nSH, (CR10R20)nS(O)mR7, (CR10R20)nN(R10′)S(O)2R7, (CR10R20)nNR4R14, (CR10R20)nCN, (CR10R20)nS(O)2NR4R14, (CR10R20)nC(Z)R6, (CR10R20)nOC(Z)R6, (CR10R20)nC(Z)OR6, (CR10R20)nC(Z)NR4R14, (CR10R20)nN(R10′)C(Z)R6, (CR10R20)nN(R10′)C(═N(R10′))NR4R14, (CR10R20)nOC(Z)NR4R14, (CR10R20)nN(R10′)C(Z)NR4R14, or (CR10R20)nN(R10′)C(Z)OR7.
In another embodiment of the present invention, X is R2, and R2 is (CR10R20)q′X1(CR10R20)qC(A1)(A2)(A3), or (CR10R20)q′C(A1)(A2)(A3). In a further embodiment, q′ is 0.
In another embodiment when R2 is the moiety (CR10R20)q′X1(CR10R20)qC(A1)(A2)(A3), q' is 0, X1 is nitrogen, q is 0 or 1, A1 is an optionally substituted heterocyclic or heterocyclic alkyl, and A2 is an optionally substituted aryl. More specifically, R2 is 2-phenyl-2-(1-pyrrolidinyl)ethyl]amino, or 1-phenyl-2-(1-pyrrolidinyl)ethyl]amino. In another embodiment, A1 is an optionally substituted aryl or arylalkyl, and A2 is an optionally substituted aryl or arylalkyl.
In one embodiment of the invention, one or more of the A1, A2 and A3 moieties are substituted with (CR10R20)nOR6. In another embodiment of the invention, the R6 substituent Fin (CR10R20)nOR6 is hydrogen.
In yet another embodiment of the present invention, X is R2 and R2 is C(A1)(A2)(A3), such as CH(CH2OH)2, or C(CH3)(CH2OH)2; or X1(CR10R20)qCH(CH2OH)2, or X1(CR10R20)qC(CH3)(CH2OH)2; and further wherein X1 is oxygen or nitrogen.
In another embodiment X is S(O)mR2′, (CH2)nNR4R14, or (CH2)nN(R2)(R2′). In yet another embodiment, X is (CH2)nNR4R14, or (CH2)nN(R2)(R2′).
Suitably, when X is (CH2)nNR4R14, and R4 and R14 are C1-10 alkyl, aryl, aryl-C1-4 alkyl, heterocyclic, heterocyclic C1-4 alkyl, heteroaryl or heteroaryl C1-4 alkyl, the C1-4 alkyl may be substituted one or more times, independently at each occurrence with NR4′R14′; halogen, hydroxy, alkoxy, C(O)NR4′R14; or NR4C(O)C1-10alkyl. Preferably, the C1-4 alkyl is substituted with NR4′R14′.
In one embodiment at least one of R4 and R14 may be hydrogen when R4 and R14 are not cyclized. In another embodiment neither R4 nor R14 is hydrogen.
In one embodiment when X is (CH2)nNR4R14, one of R4 and R14 are hydrogen, and the other is an optionally substituted heteroaryl C1-4 alkyl. Suitably, the optionally substituted heteroaryl alkyl is an imidazolyl alkyl, such as a 1H-imidazol-2-yl-methyl group.
In one embodiment when X is (CH2)nNR4R14 and one of R4 and R14 is a heteroaryl C1-4 alkyl moiety, then the heteroaryl ring is selected from thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, triazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzoxazolyl, benzimidazolyl, and benzothiazolyl. Suitably, the heteroaryl C1-4 alkyl is selected from pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, imidazolyl, benzoxazolyl, benzimidazolyl, and benzothiazolyl.
In another embodiment when X is (CH2)nNR4R14 and one of R4 and R14 is a heterocyclic C1-4 alkyl moiety, then the heterocyclic ring is selected from tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, and morpholino. Suitably, the heterocyclic C1-4 alkyl moiety is selected from pyrrolinyl, pyrrolidinyl, piperidinyl, piperazinyl, and morpholino.
In another embodiment when X is (CH2)nNR4R14 and R4 and R14 together with the nitrogen cyclize to form an optionally substituted ring, such as described above, such rings include, but are not limited to pyrrolidine, piperidine, piperazine, diazepine, and morpholine.
In one embodiment when X is (CH2)nNR4R14, the R4 and R14 substituents cyclize to form a heterocyclic 5 or 6 membered ring, which ring is optionally substituted as defined herein. When the R4 and R14 substituents cyclize to form a 4 to 7 membered ring, the optional substitutents are suitably selected from an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, optionally substituted heterocyclic, (CR10R20)nN(R10′)C(Z)OR7, NR4′R14′, or a C1-10 alkyl substituted one or more times by an optionally substituted aryl. Such substitutents more specifically include phenyl, pyrrolidinyl, morpholino, piperazinyl, 4-methyl-1-piperazinyl, piperidinyl, 2-oxo-2,3-dihydro-1H-benzimidazol-1-yl, 5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl, diphenylmethyl, methyl, ethyl, propyl, butyl, amino, methylamino, and dimethylamino.
In one embodiment the X substituent is a 1,4′-bipiperin-1-yl ring which may be optionally substituted such as in 4-methyl-1,4′-bipiperin-1-yl; 4-piperidinylamino, 4-amino-1-piperidinyl, 2,2,6,6-tetramethyl-4-piperidinyl)amino, 4-methyl-1-piperazinyl, (4-morpholinyl)-1-piperidinyl, (4-methyl-1-piperazinyl)-1-piperidinyl, 4-ethyl-1-piperazinyl, (2-oxo-2,3-dihydro-1H-benzimidazol-1-yl-1-piperidinyl, 5-chloro-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1-piperidinyl, 4-(1-pyrrolidinyl)-1-piperidinyl, 4-(diphenylmethyl)-1-piperazinyl, 4-methylhexahydro-1H-1,4-diazepin-1-yl, 4-propyl-1-piperazinyl, or 4-butyl-1-piperazinyl.
In another embodiment, when X is (CH2)nN(R2′)(R2″), and R2′ is an optionally substituted C1-10 alkyl moiety, and the alkyl is substituted by (CR10R20)nNReRe′, and Re and Re′ are hydrogen, or an optionally substituted C1-10 alkyl. Suitably, the X moiety is 3-(diethylamino)propylamino, 3-(dimethylamino)propyl(methyl)amino, 3-(dimethylamino)propyl(methyl)amino, 2-(dimethylamino)ethylamino, 1-methylethyl)amino-propylamino, (1,1-dimethylethyl)aminopropylamino, (1-methylethyl)aminoethylamino, 2-(methylamino)ethylamino, 2-aminoethyl(methyl)amino, or a 2-(dimethylamino)ethyl(methyl)amino.
In another embodiment when X is (CH2)nN(R2′)(R2″), and R2′ moiety is an optionally substituted heteroarylC1-10 alkyl, the heteroaryl moiety is suitably an optionally substituted imidazole.
In one embodiment at least one of R4 and R14 may be hydrogen when R4 and R14 are not cyclized. In another embodiment neither R4 and R14 is hydrogen.
In one embodiment R3 is a 2,6-difluoro phenyl, R1 is a phenyl ring substituted by and R1 is selected from C(Z)N(R10′)(CR10R20)vRb, or C(Z)O(CR10R20)vRb, or N(R10′)C(Z)(CR10R20)vRb and also substituted by R1′ independently selected at each occurrence from hydrogen, fluorine, or methyl; g is 1 or 2. Preferably, R1 is substituted by C(Z)N(R10′)(CR10R20)vRb and R1′ independently selected at each occurrence from hydrogen, fluorine, or methyl. In another embodiment, the Rb moiety is selected from thiazolyl, C1-10 alkyl or an optionally substituted aryl. In another embodiment the Rb moiety is propyl or 4-fluorophenyl.
In another embodiment, X is suitably selected from (1H-imidazol-2-ylmethyl)amino or 4-methyl-1,4′-bipiperidin-1′-yl, 2,2,6,6-tetramethyl-4-piperidinyl)amino, 4-amino-1-piperidinyl, 3-(diethylamino)propylamino, 3-(dimethylamino)propyl(methyl)amino, 3-(dimethylamino)propyl(methyl)amino, 2-(dimethylamino)ethylamino, 1-methylethyl)amino-propylamino, (1,1-dimethylethyl)aminopropylamino, (1-methylethyl)aminoethylamino, 2-(methylamino)ethylamino, 2-aminoethyl(methyl)amino, or 2-(dimethylamino)ethyl(methyl)amino.
In one embodiment, R3 is a 2,6-difluoro phenyl, R1 is phenyl, R1′ is independently selected at each occurrence from hydrogen, fluorine, or methyl; g is 1 or 2; and the phenyl ring is also substituted in the 3- or 4-position by C(Z)N(R10′)(CR10R20)vRb, Rb moiety is C1-10 alkyl or an optionally substituted aryl, preferably propyl or 4-fluorophenyl, X is (CH2)nN(R2′)(R2″), and n is 0. In another embodiment, X is (CH2)nN(R2′)(R2″), R2″ is hydrogen, n is 0, and R2′ is an alkyl substituted by (CR10R20)nNReRe′. In a further embodiment, Re and Re′ are independently selected from an optionally substituted C1-4 alkyl, such as methyl, ethyl, isopropyl, n-butyl, or t-butyl, preferably ethyl.
In another embodiment of the present invention, for compounds of Formula (I) the X term may also be the B-Non-Ar-cyc moiety as disclosed in U.S. Pat. No. 6,809,199 whose disclosure is incorporated by reference herein.
As represented by the disclosure in U.S. Pat. No. 6,809,199, Non-Ar-Cyc is suitably selected from;
wherein
d is an integer having a value of 1, 2, 3, or 4;
d′ is 0, or an integer having a value of 1, 2, or 3;
d″ is 0, or an integer having a value of 1, 2, or 3;
e is 0, or is an integer having a value of 1, 2, 3, or 4;
e′ is 0, or an integer having a value of 1, 2, or 3;
e″ is 0, or an integer having a value of 1, 2, or 3;
f is 0, or is an integer having a value of 1, 2, or 3;
d+e is 2, 3, 4, 5, or 6;
d′+e″=d
e′+e″=m
Suitably, R7′, R77 and R77″ are each independently selected from hydrogen, C1-6 alkyl-group, C2-6 alkenyl-group, C4-6 cycloalkyl-C0-6 alkyl-group, N(C0-4 alkyl)(C0-4 alkyl)-C1-4 alkyl-N(C0-4 alkyl)-group, —N(C0-4 alkyl)(C0-4 alkyl) group, Cl—3 alkyl-CO—C0-4 alkyl-group, C0-6 alkyl-C—O—C(O)C0-4 alkyl-group, C0-6alkyl-C(O)—O—C0-4alkyl-group, N(C0-4 alkyl)(C0-4 alkyl)-(C0-4 alkyl)C(O)(C0-4 alkyl)-group, phenyl-C0-4 alkyl-group, pyridyl-C0-4 alkyl-group, pyrimidinyl-C0-4 alkyl-group, pyrazinyl-C0-4 alkyl-group, thiophenyl-C0-4 alkyl-group, pyrazolyl-C0-4 alkyl-group, imidazolyl-C0-4 alkyl-group, triazolyl-C0-4 alkyl-group, azetidinyl-C0-4 alkyl-group, pyrrolidinyl-C0-4 alkyl-group, isoquinolinyl-C0-4alkyl-group, indanyl-C0-4 alkyl-group, benzothiazolyl-C0-4 alkyl-group, any of the groups optionally substituted with 1-6 substituents, each substituent independently being —OH, —N(C0-4 alkyl)(C0-4alkyl), C1-4alkyl, C1-6 alkoxyl, C1-6 alkyl-CO—C0-4 alkyl-, pyrrolidinyl-C0-4 alkyl-, or halogen; or R7′ together with a bond from an absent ring hydrogen is ═O.
Suitably, B is —C1-6alkyl-, —C0-3 alkyl-O—C0-3 alkyl-, —C0-3 alkyl-NH—C0-3alkyl-, —C0-3alkyl-NH—C3-7 cycloalkyl-, —C0-3 alkyl-N(C0-3 alkyl)-C(O)—C0-3 alkyl-, —C0-3 alkyl-NH—SO2—C0-3 alkyl-, —C0-3 alkyl-, —C0-3alkyl-S—C0-3 alkyl-, —C0-3 alkyl-SO2—C0-3 alkyl-, —C0-3 alkyl-PH—C0-3 alkyl-, —C0-3 alkyl —C(O)—C0-3 alkyl, or a direct bond.
Suitably, E1 is CH, N, or CR66; or B and E1 together form a double bond, i.e., CH═C.
Suitably, E2 is CH2, CHR77, C(OH)R77NH, NR77, O, S, —S(O)—, or —S(O)2—.
Suitably, R66 is independently selected from at each occurrence from halogen, C0-4 alkyl, —C(O)—O(C0-4 alkyl), or C(O)—N(C0-4 alkyl)-(C0-4 alkyl).
In an alternative embodiment of this invention, Non-Ary Cyc is:
In another embodiment of the present invention, for compound of Formula (I) herein, the X term may also be the X moiety as disclosed in WO 2004/073628, published September 2004, Boehm et al., whose disclosure is incorporated by reference herein.
The term “halo” or “halogens” is used herein to mean the halogens, chloro, fluoro, bromo and iodo.
As used herein, the term “alkyl” refers to straight or branched hydrocarbon chains containing the specified number of carbon atoms. For example, C1-6alkyl means a straight or branched alkyl containing at least 1, and at most 6, carbon atoms. Examples of “alkyl” as used herein include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, isopropyl, sec-butyl, tent-butyl or t-butyl and hexyl and the like. If unspecified, the term alkyl shall mean C1-10alkyl.
As used herein, the term “alkenyl” refers to straight or branched hydrocarbon chains containing the specified number of carbon atoms and containing at least one double bond. For example, C2-6alkenyl means a straight or branched alkenyl containing at least 2, and at most 6, carbon atoms and containing at least one double bond. Examples of “alkenyl” as used herein include, but are not limited to ethenyl, 2-propenyl, 3-butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, 3-methylbut-2-enyl, 3-hexenyl, 1,1-dimethylbut-2-enyl and the like.
As used herein, the term “alkoxy” refers to straight or branched chain alkoxy groups containing the specified number of carbon atoms. For example, C1-6alkoxy means a straight or branched alkoxy containing at least 1, and at most 6, carbon atoms. Examples of “alkoxy” as used herein include, but are not limited to, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2-oxy, 2-methylprop-1-oxy, 2-methylprop-2-oxy, pentoxy and hexyloxy.
As used herein, the term “cycloalkyl” refers to cyclic radicals, such as a non-aromatic hydrocarbon ring containing a specified number of carbon atoms. For example, C3-7cycloalkyl means a non-aromatic ring containing at least three, and at most seven, ring carbon atoms. Representative examples of “cycloalkyl” as used herein include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl and the like.
The term “cycloalkenyl” is used herein to mean cyclic radicals, such as a non-aromatic hydrocarbon ring containing a specified number of carbon atoms preferably of 5 to 7 carbons, which have at least one bond including but not limited to cyclopentenyl, cyclohexenyl, and the like.
The term “alkenyl” is used herein at all occurrences to mean straight or branched chain radical of 2-10 carbon atoms, unless the chain length is limited thereto, including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like.
The term “aryl” is used herein to mean phenyl, naphthyl, and indene.
The terms “heteroaryl ring”, “heteroaryl moiety”, and “heteroaryl” are used herein to mean a monocyclic five- to seven-membered unsaturated hydrocarbon ring containing at least one heteroatom selected from oxygen, nitrogen and sulfur. Examples of heteroaryl rings include, but are not limited to, furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and uracil. The terms “heteroaryl ring”, “heteroaryl moiety”, and “heteroaryl” shall also used herein to refer to fused aromatic rings comprising at least one heteroatom selected from oxygen, nitrogen and sulfur. Each of the fused rings may contain five or six ring atoms. Examples of fused aromatic rings include, but are not limited to, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, purinyl, and phthalazinyl.
The terms “heterocyclic rings”, “heterocyclic moieties”, and “heterocyclyl” is used herein to mean a monocyclic three- to seven-membered saturated or non-aromatic, unsaturated hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(O)m, and m is 0 or an integer having a value of 1 or 2. The terms “heterocyclic rings”, “heterocyclic moieties”, and “heterocyclyl” shall also refer to fused rings, saturated or partially unsaturated, and wherein one of the rings may be aromatic, or heteroaromatic. Each of the fused rings may have from four to seven ring atoms. Examples of heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-1-pyrrolidinyl, 3-oxo-1-pyrrolidinyl, 1,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur moiety).
The term “arylalkyl” or “heteroarylalkyl” or “heterocyclicalkyl” is used herein to mean a C1-4 alkyl (as defined above) attached to an aryl, heteroaryl or heterocyclic moiety (as also defined above) unless otherwise indicated.
The term “sulfinyl” is used herein to mean the oxide S(O) of the corresponding sulfide, the term “thio” refers to the sulfide, and the term “sulfonyl” refers to the fully oxidized S(O)2 moiety.
The term “aroyl” is used herein to mean C(O)Ar, wherein Ar is as phenyl, naphthyl, or aryl alkyl derivative such as defined above, such group include but are not limited to benzyl and phenethyl.
The term “alkanoyl” is used herein to mean C(O)C1-10 alkyl wherein the alkyl is as defined above.
As used herein, the term “optionally” means that the subsequently described event(s) may or may not occur, and includes both event(s) which occur and events that do not occur.
As used herein, the term “substituted” refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
As used herein, “optionally substituted” unless specifically defined shall mean such groups as halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy substituted C1-10alkyl; C1-10 alkoxy, such as methoxy or ethoxy; halosubstituted C1-10 alkoxy; S(O)m alkyl, such as methyl thio, methylsulfinyl or methyl sulfonyl; a ketone (—C(O)), or an aldehyde (—C(O)R6′), such as C(O)C1-10 alkyl or C(O)aryl, wherein R6′ is hydrogen, C1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl C1-10alkyl, aryl, arylC1-10 alkyl, heteroaryl or heteroarylC1-10 alkyl (and wherein the R6′ moieties, excluding hydrogen, may themselves be optionally substituted 1 or 2 times, independently by halogen; hydroxy; hydroxy substituted alkyl; C1-4 alkoxy; S(O)mC1-4 alkyl; amino, mono & di-substituted C1-4 alkyl amino; C1-4 alkyl, or CF3); C(O)OR6′; NR4′R14′, wherein R4′ and R14′ are each independently hydrogen or C1-4 alkyl, such as amino or mono or -disubstituted C1-4 alkyl or wherein the R4′R14′ can cyclize together with the nitrogen to which they are attached to form a 5 to 7 membered ring which optionally contains an additional heteroatom selected from O/N/S; C1-10 alkyl, C3-7cycloalkyl, or C3-7cycloalkyl C1-10 alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc. or cyclopropyl methyl; halosubstituted C1-10 alkyl, such CF2CF2H, or CF3; an optionally substituted aryl, such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl containing moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; C1-4 alkoxy; S(O)m C1-4 alkyl; amino, mono & di-substituted C1-4 alkyl amino; C1-4 alkyl, or CF3.
It is to be understood that the present invention covers all combinations of particular and preferred groups described hereinabove. It is also to be understood that the present invention encompasses compounds of formula (I) in which a particular group or parameter, for example R5, R6, R9, R10, R11, R12, R13, p, n, or q, etc. may occur more than once. In such compounds it will be appreciated that each group or parameter is independently selected from the values listed. When any variable occurs more than one time in a Formula (as described herein), its definition on each occurrence is independent of its definition at every other occurrence.
Particular compounds according to the invention include those mentioned in the examples and their pharmaceutically derivatives.
As used herein, the term “pharmaceutically acceptable” means a compound which is suitable for pharmaceutical use. Salts and solvates of compounds of the invention which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.
As used herein, the term “pharmaceutically acceptable derivative”, means any pharmaceutically acceptable salt, solvate or prodrug e.g. ester, of a compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. In one embodiment of the present invention the pharmaceutically acceptable derivatives are salts, solvates, esters, carbamates and phosphate esters. In another embodiment pharmaceutically acceptable derivatives are salts, solvates and esters. In yet another embodiment, pharmaceutically acceptable derivatives are salts and esters, in particular salts.
The compounds of the present invention may be in the form of and/or may be administered as a pharmaceutically acceptable salt. For a review on suitable salts see Berge et al., J. Pharm. Sci., 1977, 66, 1-19.
Typically, a pharmaceutical acceptable salt may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
Salts of the compounds of the present invention may, for example, comprise acid addition salts resulting from reaction of an acid with a nitrogen atom present in a compound of formula (I). Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention. Suitable addition salts are formed from acids which form non-toxic salts and examples are acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, ethanesulphonate, formate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydrogen phosphate, hydroiodide, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, oxaloacetate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, piruvate, polygalacturonate, saccharate, salicylate, stearate, subacetate, succinate, sulphate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate and valerate.
Pharmaceutically acceptable base salts include ammonium salts such as a trimethylammonium salt, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases, including salts of primary, secondary and tertiary amines, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine and N-methyl-D-glucamine.
Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. As used herein, the term “solvate” refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of Formula (I), or a salt thereof) and a solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Examples of suitable solvents include water, methanol, ethanol and acetic acid. Preferably the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include water, ethanol and acetic acid. Most preferably the solvent used is water. A complex with water is known as a “hydrate”. Solvates of the compound of the invention are within the scope of the invention.
As used herein, the term “prodrug” means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; and in D. Fleisher, S. Ramon and H. Barbra “Improved oral drug delivery: solubility limitations overcome by the use of prodrugs”, Advanced Drug Delivery Reviews (1996) 19(2) 115-130, each of which are incorporated herein by reference.
Prodrugs are any covalently bonded carriers that release a compound of formula (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this invention wherein hydroxy or amine groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy or amine groups. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups of the compounds of formula (I). Further, in the case of a carboxylic acid (—COOH), esters may be employed, such as methyl esters, ethyl esters, and the like. Esters may be active in their own right and/or be hydrolysable under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those which break down readily in the human body to leave the parent acid or its salt.
Cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the compound of the invention and where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. A stereoisomeric mixture of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
Furthermore, some of the crystalline forms of the compounds of the Formulas herein may exist as polymorphs, which are included in the present invention.
Exemplified compounds of the compounds of this invention include the racemates, or optically active forms of the compounds of the working examples herein, and pharmaceutically acceptable salts thereof.
The compounds of this invention may be made by a variety of methods, including standard chemistry. Any previously defined variable will continue to have the previously defined meaning unless otherwise indicated. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working Examples.
Compounds of Formula (II) are represented by the structure:
wherein
The optional substituents for the R3 moiety are as defined herein for compounds of Formula (I).
In one embodiment of the invention, Rx is chloro. In another embodiment, Rg is methyl.
In a further embodiment, m is 0. In another embodiment m is 1, and R3 is an optionally substituted phenyl (as defined in Formula (I)).
In another embodiment, m is O, Rg is methyl, Rx is chloro and R3 is an optionally substituted phenyl (as defined in Formula (I)).
Compounds of Formula (III) are represented by the structure:
wherein
X is R2, ORT, S(O)mR2′, (CH2)n'N(R11)S(O)mR2′, (CH2)n′N(R11)C(O)R2′, (CH2)n′NR4R14, or (CH2)n′N(R2′)(R2″), or N(R10′)—Rh—NH—C(═N—CN)NRqRq′;
Suitably, Rx is chloro, bromo, iodo, or O—S(O)2CF3. In an embodiment of the invention, Rx is chloro.
The X term moieties and their substituent groups, etc. are as defined herein for compounds of Formula (I).
Compounds of Formula (IV) are represented by the structure:
wherein
Suitably R1 and R3 are substituted as defined herein for compounds of Formula (I). In one embodiment, R1 is an optionally substituted aryl or heteroaryl ring, preferably and optionally substituted aryl.
In another embodiment Rg is methyl. In a further embodiment, m is 0 or 2.
Compounds of Formula (V) are represented by the formula:
wherein
Ry is chloro, bromo, iodo, O—S(O)2CF3; and
Rg is a C1-10 alkyl.
In one embodiment of the invention, Ry is bromo, iodo, or O—S(O)2CF3.
In another embodiment of the invention, Rg is methyl.
The general preparation of analogs around the pyrido[2,3-d]pyrimidin-7-one template is shown in the Schemes, Schemes 1 to 4 below. While a particular formula with particular substituent groups is shown herein, e.g. Rg as methyl, or Rx or LG2 as chloro, the synthesis is applicable to all formulas and all substituent groups as described herein.
The synthesis described herein, Schemes 1 to 4, start with a 4,6-Ry substituted-2-methylsulfanyl-pyrimidine-5-carboxaldehyde (1), such as described in Formula (V). Treatment of 1, Scheme 1, with an optionally substituted aniline in the presence of an olefin forming agent, such as bis(2,2,2-trifluoroethyl)-(methoxycarbonylmethyl)-phosphonate or an acylating agent, such as acetic acid anhydride affords the pyrido[2,3-d]pyrimidin-7-one, 2. Oxidation of 2 with a peracid, such as 3-chloroperoxybenzoic acid (m-CPBA) yields compound 3. This is followed by substitution of 3 in C2 position with a suitable X moiety as described in Formula (I) herein. In this scheme, substitution of the C2 position is demonstrated with serinol to furnish compound 4. Palladium (0) mediated Suzuki cross-coupling affords compound 5. Other cross coupling reactions known in the art may also be suitable for use herein.
In Routes 1 & 3; Schemes 1 & 3, Compound 1 to Compound 2, while the olefin forming reagent Bis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)phosphonate is used, alternative cyclization reagents, include, but are not limited to Bis(2,2,2-trifluoroethyl)-(ethoxycarbonylmethyl)phosphonate, Bis(2,2,2-trifluoroethyl)-(isopropoxycarbonylmethyl)phosphonate, (Diethoxy-phosphoryl)-acetic acid methyl ester, (Diisopropoxy-phosphoryl)-acetic acid methyl ester, (Diphenyloxy-phosphoryl)-acetic acid methyl ester, (Diethoxy-phosphoryl)-acetic acid ethyl ester, (Diisopropoxy-phosphoryl)-acetic acid ethyl ester, or (Diphenyloxy-phosphoryl)-acetic acid ethyl ester.
While this reaction as shown in Scheme 1 and Scheme 3 uses triethylamine as a base, suitable alternative bases can include, but are not limited to pyridine, diisopropyl ethyl amine, or pyrrolidone, or combinations thereof.
Further, while the reaction scheme as shown in Scheme 1 and Scheme 3 utilizes tetrahydrofuran as a solvent, it is recognized that suitable alternative organic solvents can be used. Such solvents include, but are not limited to chloroform, methylene chloride, acetonitrile, toluene, DMF, or n-methylpyrrolidine, or combinations thereof.
The reaction temperature of this particular step in the reaction scheme can be varied from room temperature to >100° C., i.e. reflux temperature of the solvent. Alternatively, this reaction process step may be performed under suitable microwave conditions.
In Routes 2 & 4, Schemes 2 & 4, Compound 1 to Compound 2, while the reagent acetic anhydride is shown, this reagent can be replaced with acetyl chloride, or any other suitably acylating reagent.
Further, while the reaction scheme as shown in Scheme 2 and Scheme 4 utilizes chloroform as a solvent, it is recognized that suitable alternative organic solvents can be used.
Such solvents include, but are not limited to tetrahydrofuran, methylene chloride, acetonitrile, toluene, DMF, n-methylpyrrolidine, or dioxane, or combinations thereof.
The reaction temperature of this particular step in the reaction scheme can be varied from room temperature to >100° C., i.e. reflux temperature of the solvent. Alternatively, this reaction process step may be performed under suitable microwave conditions.
In Routes 1 & 2; Schemes 1 & 2, Compound 2 to Compound 3, or for Routes 3 & 4, Schemes 3 & 4, Compound 6 to Compound 7, while the oxidizing reagent 3-chloroperoxybenzoic acid (m-cPBA) is used, alternative reagents, include but are not limited to hydrogen peroxide, sodium periodinate, potassium periodinate, Oxone, OsO4, catalytic tertiary amine N-oxide, peracids, such as aryl peracids, i.e. perbenzoic and the aforementioned m-cPBA, or alkylperacids, as such peracetic acid and pertrifluoroacetic acid, oxygen, ozone, organic peroxides, peroxide (H2O2), and inorganic peroxides, potassium and zinc permanganate, potassium persulfate. It is recognized that the peroxide agents can be used in combination with sodium tungstate, acetic acid or sodium hyperchlorite.
It is recognized that the oxidation process may in fact yield Compound 3, or Compound 7, but may also result in the corresponding sulfone, as well as the sulfoxide, or mixtures thereof.
This reaction step, while demonstrated in the schematics with methylene chloride as the solvent, may use alternative organic solvents other than primary amines or alcohols which include, but are not limited to chloroform, acetone, DMF, THF, acetonitrile, dioxane, or DMSO, or combinations thereof. This reaction step may be conducted at about 0° C. to room temperature.
In Routes 1 & 2; Schemes 1 & 2, Compound 3 to Compound 4, or for Route 3 & 4, Schemes 3 & 4, Compound 7 to Compound 5, the reaction solvent of DMF may alternatively be replaced with other suitable anhydrous organic solvents, which does not contain a nucleophile, which include but are not limited to THF, methylene chloride, acetone, acetonitrile, toluene, chloroform, n-methyl-pyrrolidine, or dioxane, or combinations thereof.
This temperature step may be conducted at room temperature to >100° C., i.e. reflux temperature of the solvent. Alternatively, this reaction process step may be performed under suitable microwave conditions.
In Routes 1 & 2, Schemes 1 & 2, Compound 4 to Compound 5, or for Routes 3-4, Schemes 3-4, Compound 2 to Compound 6 wherein Compound 2 or 4 are coupled to arylboronic acids, heteroarylboronic acids or the corresponding boronic acid esters under standard Suzuki coupling conditions. These reaction conditions utilize a palladium catalyst, such as tetrakis(triphenylphosphine)palladium (0), which has been shown to provide good yields of either compound 5 or 6. The reaction conditions may be from room temperature to about 250° C., by heating in an oil bath, or with microwave irradiation. If desired, these Suzuki coupling reactions may be run under microwave conditions.
The aryl or heteroaryl boronic acid or ester intermediates can be synthesized either by the palladium catalyzed coupling of an aryl halide and 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane or the transmetalation of an aryl halide with a Grignard reagent, e.g., isopropylmagnesium bromide followed by a trialkylborate (e.g., triethylborate) in a suitable solvent like THF.
Alternatively, the coupling reaction of 2 or 4 may be performed utilizing aryl or heteroaryl organozinc, organocopper, organotin, or other organometallic reagents known to afford cross-coupling products such as 5 or 6 [See for example: Solberg, J.; Undheim, K. Acta Chemica Scandinavia 1989, 62-68, whose disclosure is incorporated by reference herein].
Using the reaction procedures described in the aforementioned WO 02/059083, it has been found that compounds of Formula (III) as described herein, were unable to be synthesized following those procedures. The present invention provides for an alternative method to synthesize compounds of Formula (I) having differing R1 substituents on the C4 position of the pyrido[2,3-d]pyrimidin-7-one pharmacophore. These substituents may be introduced to this position after the pyrido[2,3-d]pyrimide-7-one pharmacophore is substituted with functional groups at the C2 and Ng position. This particular substitution has not previously been available using the reaction conditions as set forth in WO 02/059083.
While it is possible to produce individual compounds using the process illustrated herein, a benefit for these reaction pathways lies in its ability to optimize leads and to make arrays for combinatorial chemistry, with various R1, R2, and R3 substituents.
Another aspect of the invention is a process to make compounds of Formula (I) as defined herein, which comprises reacting a compound of Formula (III), as defined herein with a coupling agent selected from an arylboronic acid, or a heteroarylboronic acid or their corresponding boronic acid esters, with a suitable palladium catalyst to yield a compound of Formula (I). This coupling process takes place under standard Suzuki conditions.
Suitably the arylboronic acids, heteroarylboronic acids, or their corresponding boronic acid esters are R1-boronic acid or an R1-boronic acid ester; e.g. R1B(OH)2, R1B(O—C1-4 alkyl)2, or
wherein R1, R10, and R20 is as defined for compounds of Formula (I) herein; and r is an integer having a value of 2 to 6.
The coupling conditions include the use of appropriate solvents. These solvents include, but are not limited to dioxane, THF, DMF, DMSO, NMP, acetone, water, or a combination or a mixture thereof. Preferably, the solvent is THF/H2O, or dioxane/H2O.
The coupling conditions also include the presence of catalytic amount of catalysts and these catalysts include, but not limited to tetrakis(triphenylphosphine)-palladium (0), PdCl2, Pd(OAc)2, (CH3CN)2PdCl2, Pd(dppf)2, or [1,1′-bis(diphenylphosphino)-ferrocene]-dichloropalladium(II).
The coupling reaction may or may not require the presence of a base. Suitable bases include, but are not limited to NaHCO3, KHCO3, Na2CO3, K2CO3, KOAc or combination or mixture thereof. Preferably, the base is K2CO3 and KOAc.
The coupling reaction may or may not require heating. The heating can be carried out with a regular oil bath or microwave irrediations and the temperature can be varied from room temperature to >100° C., i.e. reflux temperature of the solvent. The coupling reaction may or may not require a sealed reaction vessal and the internal pressure can be varied from one atmosphere to 100 atmospheres.
The aryl or heteroaryl boronic acid or ester intermediates containing the R1 moiety, used in the Suzuki coupling reactions may or may not be commercially available and they can be prepared by utilizing proper methods in the literature known to those with appropriate training Examples of these methods include, but not limited to palladium catalyzed coupling of an aryl halide and 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane or the transmetalation of an aryl halide with a Grignard reagent, e.g., isopropylmagnesium bromide followed by a trialkylborate (e.g., triethylborate) in a suitable solvent. These solvents include, but not limited to CH2Cl2, chloroform, CH3CN, benzene, THF, hexane, ethyl ether, tent-butyl methyl ether, DMSO, DMF, toluene, n-methyl-pyrrolidine, dioxane. The reaction temperature can be varied from −78° C. to >100° C., i.e. reflux temperature of the solvent. Alternatively, this reaction process step may or may not be performed under suitable microwave irradiation conditions. This reaction may or may not require a sealed reaction vessal and the internal pressure can be varied from one atmosphere to 100 atmospheres.
One embodiment of the invention are the arylboronic acids and esters which are generically referred to as R1B(OH)2, R1B(O—C1-4 alkyl)2, or
wherein
R10 and R20 are independently selected from hydrogen or C1-4 alkyl;
r is an integer having a value of 2 to 6;
R1 is an optionally substituted phenyl, as defined according to Formula (I).
Suitably, the phenyl ring is substituted by one or more times independently at each occurrence by halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, or (CR10R20)v' 0R13, (CR10R20)vC(Z)NR4R14, (CR10R20)vC(Z)OR8, (CR10R20)vCORa′, (CR10R20)vC(O)H, SRS, S(O)R5, S(O)2R5, (CR10R20)vOR8, ZC(Z)R11, N(R10C)C(Z)R11, N(R10′)S(O)2R7, C(Z)N(R10′)(CR10R20)vRb, C(Z)O(CR10R20)vRb, N(R10′)C(Z)(CR10R20)vRb; N(R10′)C(Z)N(R10′)(CR10R20)vRb; or N(R10′)OC(Z)(CR10R20)vRb; and wherein
C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 cycloalkyl, C3-7 cycloalkyl C1-4 alkyl, C5-7 cycloalkenyl, C5-7cycloalkenyl C1-4 alkyl, aryl, arylC1-4 alkyl, heteroaryl, heteroarylC1-4 alkyl, heterocyclyl, or a heterocyclylC1-4 alkyl moiety, and wherein these moieties, excluding hydrogen, may be optionally substituted independently at each occurrence;
In one embodiment, the phenyl ring is substituted by C(Z)N(R10′)(CR10R20)vRb, or N(R10′)C(Z)(CR10R20)vRb, and optionally another substituent (R1′)g, and g is 1 or 2. Rb is suitably as defined in Formula (I) herein. Suitably, R1′ is independently selected at each occurrence from halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, or (CR10R20)v′OR13. Preferably R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine, or halo-substituted-C1-4 alkyl, such as CF3.
In one embodiment, the C(Z)N(R10′)(CR10R20)v Rb, is substituted on the phenyl ring in the 4-position or the 5-position, preferably the 5-position. If an R1′ moiety is present, it is preferably in the 2-position, and R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine. Preferably the aryl is 4-methyl-N-1,3-thiazol-2-ylbenzamide, N-(4-fluorophenyl)-4-methylbenzamide, or 4-methyl-N-propylbenzamide.
In another embodiment the phenyl ring is substituted one or more times, preferably 1 to 4 times by R1′ and R1′ is independently selected at each occurrence from halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, or (CR10R20)v′OR13. Preferably, the phenyl ring is di-substituted in the 2,4-position. In another embodiment R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine, or halo-substituted-C1-4 alkyl, such as CF3. Preferably, the aryl is phenyl, 2-methyl-4-fluorophenyl, 2-methylphenyl, 2-chlorophenyl, 2-fluorophenyl, or 2-methyl-3-fluorophenyl.
Another aspect of the invention is another process to make compounds of Formula (I) as defined herein, which comprises reacting a compound of Formula (III), as defined herein utilizing aryl or heteroaryl organozinc, organocopper, organotin, or other organometallic reagents known in the art to afford a cross-coupling product of the desired R1 moiety in the C4 position of the template yielding a compound of Formula (I).
This coupling reaction may be performed utilizing aryl or heteroaryl organozinc (e.g., R1—ZnBr, R1—ZnCl, R1—Zn—R1), organocopper [e.g., (R1)2—CuLi], organotin (e.g., R1—Sn(CH3)3, R1—Sn(CH2CH2CH2CH3)3], or other organometallic reagents to afford the cross-coupling product. The R1 aryl and heteroaryl moiety is as defined for Formula (I) herein. If the desired aryl or hetero aryl organozinc (e.g., R1—ZnBr, R1—ZnCl, R1—Zn—R1), organocopper [e.g., (R1)2—CuLi], organotin (e.g., R1—Sn(CH3)3, R1—Sn(CH2CH2CH2CH3)3], or other organometallic reagent is not commercially available, they can readily be prepared by utilizing proper methods, known in the literature.
These types of coupling reactions require the use of appropriate solvents. Such solvents include, but are not limited to dioxane, THF, methylene chloride, chloroform, benzene, hexane, ethyl ether, tent-butyl methyl ether or a combination or a mixture thereof.
The coupling reaction may, or may not, require the presence of catalytic amount of a catalyst. Such catalysts include, but are not limited to tetrakis(triphenylphosphine)palladium (0), PdCl2, Pd(OAc)2, (CH3CN)2PdCl2, Pd(dppf)2.
The reaction temperature can be varied from −78° C. to >100° C., i.e. reflux temperature of the solvent. Alternatively, this reaction process step may be performed under suitable microwave irradiation conditions, if needed. This reaction may, or may not, require a sealed reaction vessel and the internal pressure can be varied from one atmosphere to 100 atmospheres.
Suitably, the R1 moiety is as defined for compounds of Formula (I) herein.
In one embodiment, the R1 moiety is an optionally substituted aryl ring, preferably a phenyl ring. In another embodiment, the phenyl ring is substituted by C(Z)N(R10′)(CR10R20)vRb, or N(R10′)C(Z)(CR10R20)vRb, and optionally another substituent (R1′)g, and g is 1 or 2. Rb is suitably as defined in Formula (I) herein. Suitably, R1′ is independently selected at each occurrence from halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, or (CR10R20)v′OR13. Preferably R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine, or halo-substituted-C1-4 alkyl, such as CF3.
In one embodiment, the C(Z)N(R10′)(CR10R20)v Rb, is substituted on the phenyl ring in the 4-position or the 5-position, preferably the 5-position. If an R1′ moiety is present, it is preferably in the 2-position, and R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine. Preferably the aryl is 4-methyl-N-1,3-thiazol-2-ylbenzamide, N-(4-fluorophenyl)-4-methylbenzamide, or 4-methyl-N-propylbenzamide.
In another embodiment the phenyl ring is substituted one or more times, preferably 1 to 4 times by R1′ and R1′ is independently selected at each occurrence from halogen, C1-4 alkyl, halo-substituted-C1-4 alkyl, cyano, nitro, (CR10R20)v′NRdRd′, (CR10R20)v′C(O)R12, SRS, S(O)R5, S(O)2R5, or (CR10R20)v′OR13. Preferably, the phenyl ring is di-substituted in the 2,4-position. In another embodiment R1′ is independently selected at each occurrence from C1-4 alkyl, such as methyl, or halogen, such as fluorine or chlorine or bromine, or halo-substituted-C1-4 alkyl, such as CF3. Preferably, the aryl moiety is a 2-methyl-4-fluorophenyl.
Additional methods to produce compounds of Formula (II), wherein G1 is NH, are shown in Scheme 5 below.
Method A is for conversion of 1 to 2. Examples of the methods include, but are not limited to condensation with NH2OH followed by treatment with thionyl chloride (SOCl2) [e.g., Santilli et al., J. Heterocycl. Chem. (1971), 445-53] or oxidation of —CHO group to —COOH followed by formation of a primary amide (—CONH2) and treatment with POCl3. Suitable Method A can also be utilized to furnish the conversion of 4 to 3-Scheme 5.
Leaving groups (LG, described as Leaving group 1 (LG1) & LG2) in 1 (or 2), or elsewhere, can be independently selected from —Cl, —Br, —I, or —OTf and these groups can be installed through the transformation of another functional group (e.g. —OH) by following the methods well known in the art (e.g., treatment of the —OH compound with POCl3).
Method B is for selective displacement of suitable aldehyde 1 or nitrile 2 with an amine (R3—NH2). This type of displacement may be achieved using triethylamine and the desired amine R3NH2 in chloroform at room temperature for 10 minutes. The reaction was very effective for a range of alkyl amines (78-95% yield). For aryl or heteroaryl amines, elevated temperatures (reflux), longer reaction time (24 hours) and presence of NaH (or Na) may be necessary for reaction completion. Use of the base could be omitted when 3 or more equivalent of the desired amine were used. Other suitable bases include but are not limited to pyridine, diisopropyl ethylamine or pyrrolidine, which may also be used in an appropriate organic solvent, including but not limited to THF, diethyl ether, DCM, DMF, DMSO, toluene or dioxane.
Method C is for the reduction of nitrile 3 to amine 5. 5 may be considered a primary amine (NH2), a secondary amine (because of —NH(R3)) or an amine (as it contains basic nitrogen). This method includes, but is not limited to BH3 in appropriate organic solvent, such as THF, DCM, toluene, DMSO, diethyl ether or dioxane. Other suitable reduction reagents, include but are not limited to NaBH4, LAH or DIBAL. Method C may require elevated temperatures (e.g., heating, refluxing or irradiating with microwave). Another example of the method is hydrogenation (H2) in the presence of transition metals (e.g., Pd/C, Raney-Ni, PdCl2).
Method D is for the cyclization of 5 to 6. This method requires the presence of a cyclization reagent (e.g., CDI, COCl2, tri-phosgene, or phenyl chloroformate methyl chloroformate). Presence of a suitable base may help the reaction to go to completion and examples of the base include, but not limited to triethyl amine, diisopropylethylamine or pyrrolidine. Reaction solvent can be DCM, THF, toluene, DMSO, or DMF.
Compounds of Formula (VI) are represented by the formula:
wherein
LG2 is chloro, bromo, iodo, or O—S(O)2CF3;
LG1 is chloro, bromo, iodo, or O—S(O)2CF3; and
Rg is an optionally substituted C1-10 alkyl.
In one embodiment, LG2 is chloro. IN a further embodiment, LG1 is chloro. In another embodiment, Rg is methyl.
Compounds of Formula (VII) are represented by the formula:
wherein
Suitably R3 is substituted as defined herein for compounds of Formula (I).
In one embodiment, Rg is methyl. In another embodiment, LG2 is chloro.
Another aspect of the invention are compounds of Formula (VIII) represented by the formula:
wherein
Suitably R3 is substituted as defined herein for compounds of Formula (I).
In an embodiment of the invention LG2 is chloro. In another embodiment, Rg is methyl. In another embodiment, LG2 is chloro, Rg is methyl, and R3 is an optionally substituted phenyl.
Another aspect of the present invention is the novel process, shown in Scheme-6 below, to make the transformation of a compound of Formula (VIII) to a compound of Formula (II) wherein Rx is now defined as LG2, and m=0.
Method J is for imine formation to convert compound 13 to a compound of Formula (II) wherein m is 0, compound 14. This can be achieved by following various strategies known in the art. Strategies include, but are not limited to treatment with an acid including TFA, HOAc, HCl, H2SO4 or a Lewis acid (e.g., AlCl3). This conversion may require elevated temperatures (e.g., heat, solvent reflux, microwave irradiation) in appropriate organic solvents (e.g., THF, CH2Cl2, toluene, DMSO, CH3CN or dioxane).
Compounds of Formula (VIII) (compound 13-Scheme 7) may be made by reacting the compound 4 using Method I as described below. Compound 4 may be obtained from compound 1 using Method B as described above.
Method I is for urea formation to convert 4 to 13. This can be achieved by following strategies well-established in the art. Strategies include, but are not limited to reaction with suitably substituted isocyanate, such as C1SO2NCO (or Me3SiNCO) in a aprotic organic solvent, such as toluene, methylene chloride, chloroform, benzene, THF, hexane, optionally with a non-nucleophilic base, such as triethylamine, diisopropyl ethylamine, pyridine, followed by reaction with ammonia or H2O; or by reaction with COCl2 (CDI, or triphosgene) or methylchloroformate or other chloroformates in an aprotic organic solvent, such as toluene, methylene chloride, chloroform, benzene, THF, hexane, optionally with a non-nucleophilic base, such as triethylamine, diisopropyl ethylamine, pyridine, followed by treatment with NH3 (or NH4OH); or by reaction with ClCO2Me (or ClCO2Et) in a aprotic organic solvent, such as toluene, methylene chloride, chloroform, benzene, THF, hexane, optionally with a non-nucleophilic base, such as triethylamine, diisopropyl ethylamine, pyridine followed by treatment with NH3 (or NH4OH) or reaction with NH2CO2(t-Bu), followed by reaction with ammonia. This reaction may, or may not, require heating (e.g, temperature between r.t. and 250° C.). The heating can be carried out in any manner and may include the use of an oil bath or microwave irradiation.
Another aspect of the invention is a process for making a compound of Formula (III):
wherein
wherein
Y is hydrogen, a metal, a boronic acid derivative, or a trialkyl tin derivative, in an anhydrous organic solvent which does not contain a nucleophile to yield a compound of Formula (III).
In the transformation of (II) to (III), when Y is hydrogen then X is the following:
In the transformation of (II) to (III), when Y is a metal, such as Li, Mg, or any other appropriate metal or metal complex; then X is the following:
a) X is R2, and R2 is C1-10 alkyl, C3-7 cycloalkyl, C3-7 cycloalkylC1-10 alkyl, aryl, arylC1-10 alkyl, heteroaryl, heteroaryl C1-10 alkyl, heterocyclic, or a heterocyclylC1-10 alkyl moiety.
In the transformation of (II) to (III), when Y is a boronic acid, (B(OH)2) or boronic ester derivatives; then X is the following
a) X═R2, and R2=aryl, or heteroaryl.
When Y is a trialkyl tin derivative, such as (C1-4 alkyl)3 Sn, then
a) X═R2, and R2=aryl, or heteroaryl.
It is recognized that for compounds of Formula (II) or (III) when G1 is NH, the nitrogen may need to be protected under standard conditions, and then deprotected after the transformation, as desired.
The anhydrous organic solvents include, but are not limited to CH2Cl2, chloroform, CH3CN, benzene, THF, hexane, ethyl ether, tent-butyl methyl ether, DMSO, DMF and toluene.
This reaction may or may not require heating (e.g., temperature between r.t. and 300° C.) and the heating can be carried out with, but not limited to a regular oil bath or microwave irradiations;
This reaction may or may not require the presence of bases, and the bases include, but are not limited to triethyl amine, diisopropyl ethyl amine, NaH, n-Buli, tert-BuLi, tert-BuOK, Li2CO3, Cs2CO3 and pyridine. It is recognized that some of these bases will be incompatible with the organic solvents specified above.
This reaction may or may not be carried out in a sealed reaction vessel and the internal pressure may be higher than one atmosphere (e.g., between 1 and 100 atmospheres).
This reaction may or may not require the presence of catalytic amount of catalysts containing transition metals (e.g., Pd, Cu, Ni or W). These catalysts include but are not limited to Pd/C, Pd(PPh3)4 and PdCl2.
Another aspect of the invention is a process for making a compound of Formula (III),
wherein
wherein
The anhydrous organic solvents include, but are not limited to CH2Cl2, chloroform, CH3CN, benzene, THF, hexane, ethyl ether, tent-butyl methyl ether, DMSO, DMF and toluene, DMF, acetone, toluene, n-methyl-pyrrolidine, or dioxane, or a combination or mixture thereof.
This reaction may or may not require heating (e.g., temperature between room temperature and 300° C.) and the heating can be carried out with, but not limited to a regular oil bath or microwave irradiations;
This reaction may or may not require the presence of bases, and the bases include, but are not limited to triethyl amine, diisopropyl ethyl amine, NaH, n-Buli, tert-BuLi, tert-BuOK, Li2CO3, Cs2CO3 and pyridine. It is recognized that some of these bases will be incompatible with the organic solvents specified above.
This reaction may or may not be carried out in a sealed reaction vessel and the internal pressure may be higher than one atmosphere (e.g., between 1 and 100 atmospheres).
This reaction may or may not require the presence of catalytic amount of catalysts containing transition metals (e.g., Pd, Cu, Ni or W). These catalysts include but are not limited to Pd/C, Pd(PPh3)4 and PdCl2. It is recognized that use of these metals is generally not needed for simple transformations.
The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. All temperatures are given in degrees centigrade, all solvents are highest available purity and all reactions run under anhydrous conditions in an Ar atmosphere where necessary.
System: Shimadzu LC system with SCL-10A Controller and dual UV detector
Autosampler: Leap CTC with a Valco six port injector
Inj. Vol. (uL): 2.0
Gradient: linear
Acquisition mode: Profile
Nuclear magnetic resonance spectra were recorded at 400 MHz using on a Bruker AC 400 spectrometer. CDCl3 is deuteriochloroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and CD3OD (or Me0D) is tetradeuteriomethanol. Chemical shifts are reported in parts per million (6) downfield from the internal standard tetramethylsilane (TMS) or the NMR solvent. Abbreviations for NMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad. J indicates the NMR coupling constant measured in Hertz. Mass spectra were taken on a instruments, using electrospray (ES) ionization techniques. All temperatures are reported in degrees Celsius. Other abbreviations are as described in the ACS Style Guide (American Chemical Society, Washington, D.C., 1986).
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Both flash and gravity chromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel. Preparative hplc were performed using a Gilson Preparative System using a Luna 5u C18(2) 100A reverse phase column eluting with a 10-80 gradient (0.1% TFA in acetonitrile/0.1% aqueous TFA) or a 10-80 gradient (acetonitrile/water). The CombiFlash system used for purification in this application was purchased from Isco, Inc. CombiFlash purification was carried out using a prepacked SiO2 column, a detector with UV wavelength at 254 nm and mixed solvents.
Heating of reaction mixtures with microwave irradiations was carried out on either a Smith Creator (purchased from Personal Chemistry, Forboro/MA, now owned by Biotage), a Emrys Optimizer (purchased from Personal Chemistry) or an Explorer (provided by CEM Discover, Matthews/NC) microwave.
A solution of 4,6-dichloro-2-methylsulfanyl-pyrimidine-5-carbaldehyde (1.0 g, 4.5 mmol) and Et3N (1.26 mL, 9.0 mmol) in THF (25 mL) was mixed with 4-trifluoromethylaniline (0.62 mL, 4.9 mmol). The resultant mixture was stirred at room temperature for 2 hours before bis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)-phosphonate (0.95 mL, 4.5 mmol) was added. After stirring at room temperature for additional 12 hours, the mixture was diluted with dichloromethane (50 mL) and washed with H2O (2×25 mL). The organic layer was dried over Na2SO4, filtered and concentrated. This crude product was further purified by washing with a mixture of THF/Hexane (1:3, 2×10 mL) to provide the title compound (1.17 g, 70%): MS (ES) m/z 372 (M+H)+; 1H-NMR (CDCl3) δ 2.18 (s, 3H), 6.79 (d, J=9.8 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.83 (d, J=8.4 Hz, 2H), 8.03 (d, J=9.8 Hz, 1H).
A solution of 4,6-dichloro-2-methylsulfanyl-pyrimidine-5-carbaldehyde (1.0 g, 4.5 mmol) and Et3N (1.26 mL, 9.0 mmol) in THF (25 mL) was mixed with 2,4-difluoroaniline (0.50 mL, 4.9 mmol). The resultant mixture was stirred at room temperature for 2 hours before bis(2,2,2-trifluoroethyl) (methoxycarbonyl-methyl)phosphonate (0.95 mL, 4.5 mmol) was added. After stirring at room temperature for additional 48 hours, the mixture was diluted with dichloromethane (50 mL) and then washed with H2O (2×25 mL). The organic layer was dried over Na2SO4, filtered and concentrated. This crude product was applied to flash chromatography (EtOAc/Hexane, 1:5) to provide the title compound (0.79 g, 52%): MS (ES) m/z 340 (M+H)+; 1H-NMR (CDCl3) δ 2.24 (s, 3H), 6.79 (d, J=9.8 Hz, 1H), 7.06 (m, 2H), 7.29 (m, 1H), 8.03 (d, J=9.8 Hz, 1H).
A solution of 4-Chloro-6-(2,6-difluoro-phenylamino)-2-methylsulfanyl-pyrimidine-5-carbaldehyde (200 mg, 0.63 mmol) in DMF (4.0 mL) and Ac2O (2.0 mL) was heated with “Smith Creator” (microwave, 160° C.) for 30 minutes. The mixture was concentrate under vacuum. Flash chromatography (EtOAc/Hexane, 1:5) then provided the title compound (50%): MS (ES) m/z 340 (M+H)+; 1H-NMR (CDCl3) δ 2.24 (s, 3H), 6.80 (d, J=9.8 Hz, 1H), 7.12 (m, 2H), 7.49 (m, 1H), 8.04 (d, J=9.8 Hz, 1H).
A solution of 4-Chloro-2-methylsulfanyl-8-(4-trifluoromethyl-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (1.0 g, 2.7 mmol) in dichloromethane (50 mL) was mixed with m-CPBA (0.63 g, 4.0 mmol). The resultant mixture was stirred at room temperature for 10 minutes and concentrated under vacuum. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (0.86 g, 82%): MS (ES) m/z 388 (M+H)+; 1H-NMR (CDCl3) δ 2.80 (s, 3H), 7.03 (d, J=9.9 Hz, 1H), 7.39 (d, J=8.0 Hz, 2H), 7.86 (d, J=8.0 Hz, 2H), 8.19 (d, J=9.9 Hz, 1H).
4-Chloro-2-methylsulfinyl-8-(2,4-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one A solution of 4-Chloro-2-methylsulfanyl-8-(2,4-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (1.0 g, 2.9 mmol) in dichloromethane (50 mL) was mixed with m-CPBA (0.69 g, 4.4 mmol). The resultant mixture was stirred at room temperature for 10 minutes and concentrated under vacou. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (1.02 g, 97%): MS (ES) m/z 356 (M+H)+; 1H-NMR (CDCl3) δ 2.84 (m, 3H), 7.01 (d, J=9.9 Hz, 1H), 7.09 (m, 2H), 7.29 (m, 1H), 8.16 (d, J=9.9 Hz, 1H).
A solution of 4-Chloro-2-methylsulfanyl-8-(2,6-difluorophenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (1.0 g, 2.9 mmol) in dichloromethane (50 mL) was mixed with m-CPBA (0.69 g, 4.4 mmol). The resultant mixture was stirred at room temperature for 10 minutes and concentrated under vacou. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (0.91 g, 87%): MS (ES) m/z 356 (M+H)+; 1H-NMR (CDCl3) δ 2.85 (s, 3H), 7.03 (d, J=9.6 Hz, 1H), 7.15 (m, 2H), 7.53 (m, 1H), 8.18 (d, J=9.6 Hz, 1H).
A solution of 4-Chloro-2-methylsulfinyl-8-(4-trifluoromethyl-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (0.29 g, 0.75 mmol) in dichloromethane (30 mL) was mixed with a solution of serinol (0.075 g, 0.82 mmol) in DMF (0.75 mL). The resultant mixture was stirred at room temperature for 1 hour before concentrated under vacuum. Flash chromatography (EtOAc:Hexane, 3:1) then provided the title compound (0.14 g, 45%): MS (ES) m/z 415 (M+H)+; 1H-NMR (CDCl3) δ 2.25 (s, br, 2H), 3.66 (m, br, 5H), 6.15 (m, br, 1H), 6.55 (d, J=9.2 Hz, 1H), 7.36 (m, 2H), 7.81 (d, J=8.1 Hz, 2H), 7.92 (d, J=9.2 Hz, 1H).
A solution of 4-Chloro-2-methylsulfinyl-8-(2,4-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (0.24 g, 0.67 mmol) in dichloromethane (24 mL) was mixed with a solution of serinol (0.065 g, 0.71 mmol) in DMF (0.65 mL). The resultant mixture was stirred at room temperature for 1 hour before concentrated under vacuum. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (0.12 g, 46%): MS (ES) m/z 383 (M+H)+; 1H-NMR (CDCl3) δ 2.15 (s, br, 2H), 3.75 (m, br, 5H), 6.10 (m, br, 1H), 6.55 (m, 1H), 7.04 (m, 2H), 7.28 (m, 1H), 7.90 (m, 1H).
A solution of 4-Chloro-2-methylsulfinyl-8-(2,6-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (0.90 g, 2.53 mmol) in dichloromethane (90 mL) was mixed with a solution of serinol (0.24 g, 2.66 mmol) in DMF (2.0 mL). The resultant mixture was stirred at room temperature for 1 hour before concentrated under vacuum. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (0.40 g, 42%): MS (ES) m/z 383 (M+H)+; 1H-NMR (CDCl3) δ 1.95 (s, br, 2H), 3.90 (m, br, 5H), 6.05 (m, br, 1H), 6.56 (d, J=9.6 Hz, 1H), 7.10 (m, 2H), 7.48 (d, J=8.1 Hz, 2H), 7.94 (d, J=9.6 Hz, 1H).
A solution of 4-Chloro-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(4-trifluoromethyl-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (50 mg, 0.12 mmol) in dioxane/H2O (3:1, 4.8 mL) was mixed with 2-methylthiophenyl boronic acid (30.4 mg, 0.18 mmol) and K2CO3 (50.1 mg, 0.36 mmol). The resultant mixture was bubbled with argon for 5 minutes, and added by Pd(PPh3)4 (2.8 mg, 0.0024 mmol). The reaction tube was sealed and heated with “Smith Creator” (microwave, 150° C.) for 15 minutes. The mixture was concentrated under vacuo. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (88%): MS (ES) m/z 503 (M+H)+; 1H-NMR (CDCl3) δ 2.48 (s, 3H), 2.65 (s, br, 2H), 3.70 (m, br, 5H), 6.20 (m, br, 1H), 6.45 (m, 1H), 7.43 (m, 6H), 7.68 (m, 1H), 7.83 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except 3-methylthiophenyl boronic acid was used in the coupling step (76%): MS (ES) m/z 503 (M+H)+; 1H-NMR (CDCl3) δ 2.49 (s, br, 2H), 2.54 (s, 3H), 3.68 (m, br, 5H), 5.90 (s, br, 1H), 6.47 (s, b, 1H), 7.45 (m, 6H), 7.65 (m, 1H), 7.82 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except 4-methylthiophenyl boronic acid was used in the coupling step (56%): MS (ES) m/z 503 (M+H)+; 1H-NMR (CDCl3) δ 2.40 (s, br, 2H), 2.58 (s, 3H), 3.69 (m, br, 5H), 5.85 (s, br, 1H), 6.48 (m, 1H), 7.40 (m, 2H), 7.48 (m, 2H), 7.56 (m, 2H), 7.67 (m, 1H), 7.83 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except phenyl boronic acid was used in the coupling step (82%): MS (ES) m/z 457 (M+H)+; 1H-NMR (CDCl3) δ 1.81 (s, br, 2H), 3.68 (m, br, 5H), 6.10 (m, br, 1H), 6.47 (m, 1H), 7.40 (m, 2H), 7.59 (m, 5H), 7.82 (m, 3H).
The title compound was prepared by following the procedure in Example 10 except 3-chlorophenyl boronic acid was used in the coupling step (76%): MS (ES) m/z 491 (M+H)+; 1H-NMR (CDCl3) δ 1.66 (s, br, 2H), 3.73 (m, br, 5H), 6.15 (m, br, 1H), 6.50 (m, 1H), 7.52 (m, 6H), 7.75 (m, 1H), 7.84 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except 4-chlorophenyl boronic acid was used in the coupling step (72%): MS (ES) m/z 491 (M+H)+; 1H-NMR (CDCl3) δ 1.61 (s, br, 2H), 3.73 (m, br, 5H), 6.05 (m, br, 1H), 6.50 (m, 1H), 7.41 (m, 2H), 7.58 (m, 4H), 7.76 (m, 1H), 7.84 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except 3,4-difluorophenyl boronic acid was used in the coupling step (65%): MS (ES) m/z 493 (M+H)+; 1H-NMR (CDCl3) δ 2.06 (s, br, 2H), 3.72 (m, br, 5H), 6.05 (m, br, 1H), 6.50 (m, 1H), 7.42 (m, 5H), 7.76 (m, 1H), 7.83 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except 2-chlorophenyl boronic acid was used in the coupling step (72%). MS (ES) m/z 491 (M+H)+; 1H-NMR (CDCl3) δ 3.60 (m, br, 5H), 6.10 (m, br, 1H), 6.45 (m, 1H), 7.32 (m, 2H), 7.50 (m, 5H), 7.80 (m, 2H).
The title compound was prepared by following the procedure in Example 10 except [4-(methyloxy)phenyl]boronic acid was used in the coupling step (66%). MS (ES) m/z 487 (M+H)+; 1H-NMR (CDCl3) δ 1.85 (s, br, 2H), 3.69 (m, br, 5H), 3.92 (s, 3H), 6.10 (m, br, 1H), 6.47 (m, 1H), 7.07 (m, 2H), 7.40 (m, 2H), 7.60 (m, 2H), 7.84 (m, 3H).
The title compound was prepared by following the procedure in Example 10 except [3-(methyloxy)phenyl]boronic acid was used in the coupling step (65%): MS (ES) m/z 487 (M+H)+; 1H-NMR (CDCl3) δ 2.90 (s, br, 2H), 3.61 (m, br, 5H), 3.88 (s, 3H), 6.05 (m, br, 1H), 6.45 (m, 1H), 7.09 (m, 2H), 7.45 (m, 4H), 7.80 (m, 3H).
The title compound was prepared by following the procedure in Example 10 except [2-(methyloxy)phenyl]boronic acid was used in the coupling step (75%): MS (ES) m/z 487 (M+H)+; 1H-NMR (CDCl3) δ 2.20 (s, br, 2H), 3.69 (m, br, 5H), 3.84 (s, 3H), 6.05 (m, br, 1H), 6.41 (m, 1H), 7.12 (m, 2H), 7.47 (m, 5H), 7.83 (m, 2H).
A solution of 4-Chloro-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,4-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (50 mg, 0.13 mmol) in dioxane/H2O (3:1, 4.8 mL) was mixed with 3-methylthiophenyl boronic acid (33.8 mg, 0.20 mmol) and K2CO3 (54.3 mg, 0.39 mmol). The resultant mixture was bubbled with argon for 5 minutes followed by the addition of Pd(PPh3)4 (3.0 mg, 0.0026 mmol). The reaction tube was sealed and heated with “Smith Creator” (microwave, 150° C.) for 15 minutes. The mixture was concentrated under vacuo. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (90%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.40 (s, br, 2H), 2.40 (s, 3H), 3.90 (m, br, 5H), 6.00 (m, br, 1H), 6.45 (m, 1H), 7.15 (m, 2H), 7.40 (m, 5H), 7.85 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 4-methylthiophenyl boronic acid was used in the coupling step (95%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.35 (s, br, 2H), 2.57 (s, 3H), 3.76 (m, br, 5H), 6.05 (m, br, 1H), 6.46 (m, 1H), 7.05 (m, 2H), 7.27 (m, 1H), 7.39 (m, 2H), 7.55 (m, 2H), 7.81 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 2-methylthiophenyl boronic acid was used in the coupling step (72%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.45 (s, 3H), 2.55 (s, br, 2H), 3.72 (m, br, 5H), 6.05 (m, br, 1H), 6.40 (m, 1H), 7.05 (m, 2H), 7.40 (m, 6H).
The title compound was prepared by following the procedure in Example 21 except 3,4-difluorophenyl boronic acid was used in the coupling step (56%): MS (ES) m/z 461 (M+H)+; 1H-NMR (CDCl3) δ 2.25 (s, br, 2H), 3.77 (m, br, 5H), 6.15 (m, br, 1H), 6.48 (m, 1H), 7.06 (m, 2H), 7.49 (m, 4H), 7.74 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 2-methoxyphenyl boronic acid was used in the coupling step (89%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 2.85 (s, br, 2H), 3.67 (m, br, 5H), 3.81 (s, 3H), 6.10 (m, br, 1H), 6.38 (m, 1H), 7.07 (m, 5H), 7.30 (s, 1H), 7.38 (m, 1H), 7.50 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 4-methoxyphenyl boronic acid was used in the coupling step (70%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 2.60 (s, br, 2H), 3.73 (m, br, 5H), 3.91 (s, 3H), 6.15 (m, br, 1H), 6.45 (m, 1H), 7.05 (m, 4H), 7.38 (s, 1H), 7.58 (m, 2H), 7.83 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 2-biphenyl boronic acid was used in the coupling step (89%): MS (ES) m/z 501 (M+H)+; 1H-NMR (CDCl3) δ 2.06 (s, br, 2H), 3.73 (m, br, 5H), 6.15 (m, br, 1H), 6.18 (s, br, 1H), 7.00 (m, 2H), 7.25 (m, 7H), 7.58 (m, 4H).
The title compound was prepared by following the procedure in Example 21 except 3-biphenyl boronic acid was used in the coupling step (92%): MS (ES) m/z 501 (M+H)+; 1H-NMR (CDCl3) δ 2.10 (s, br, 2H), 3.72 (m, br, 5H), 6.10 (m, br, 1H), 6.47 (m, 1H), 7.06 (m, 2H), 7.60 (m, 8H), 7.82 (m, 3H).
The title compound was prepared by following the procedure in Example 21 except 2-tolyl boronic acid was used in the coupling step (66%): MS (ES) m/z 439 (M+H)+; 1H-NMR (CDCl3) δ 2.24 (s, 3H), 2.96 (s, br, 2H), 3.68 (m, br, 5H), 6.10 (m, br, 1H), 6.39 (m, 1H), 7.04 (m, 2H), 7.40 (m, 6H).
The title compound was prepared by following the procedure in Example 21 except 3-fluoro-4-biphenyl boronic acid was used in the coupling step (48%): MS (ES) m/z 519 (M+H)+; 1H-NMR (CDCl3) δ 2.15 (s, br, 2H), 3.78 (m, br, 5H), 6.05 (m, br, 1H), 6.50 (m, 1H), 7.06 (m, 2H), 7.28 (m, 1H), 7.52 (m, 8H), 7.86 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 4-chlorophenyl boronic acid was used in the coupling step (70%): MS (ES) m/z 459 (M+H)+; 1H-NMR (CDCl3) δ 2.83 (s, br, 2H), 3.72 (m, br, 5H), 6.15 (m, br, 1H), 6.46 (m, 1H), 7.04 (m, 2H), 7.28 (m, 1H), 7.53 (m, 4H), 7.72 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 3-chlorophenyl boronic acid was used in the coupling step (49%). MS (ES) m/z 459 (M+H)+; 1H-NMR (CDCl3) δ 2.55 (s, br, 2H), 3.74 (m, br, 5H), 6.10 (m, br, 1H), 6.47 (m, 1H), 7.05 (m, 2H), 7.28 (m, 1H), 7.53 (m, 4H), 7.73 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 3-fluorophenyl boronic acid was used in the coupling step (64%): MS (ES) m/z 443 (M+H)+; 1H-NMR (CDCl3) δ 2.05 (s, br, 2H), 3.79 (m, br, 5H), 6.10 (m, br, 1H), 6.49 (m, 1H), 7.07 (m, 2H), 7.42 (m, 5H), 7.77 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 3-methoxyphenyl boronic acid was used in the coupling step (89%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 2.07 (s, br, 2H), 3.83 (m, br, 5H), 3.91 (s, 3H), 6.15 (m, br, 1H), 6.47 (m, 1H), 7.10 (m, 5H), 7.28 (m, 1H), 7.47 (m, 1H), 7.82 (m, 1H).
The title compound was prepared by following the procedure in Example 21 except 3,5-difluorophenyl boronic acid was used in the coupling step (89%): MS (ES) m/z 461 (M+H)+; 1H-NMR (CDCl3) δ 2.05 (s, br, 2H), 3.89 (m, br, 5H), 6.10 (m, br, 1H), 6.51 (m, 1H), 7.08 (m, 5H), 7.28 (m, 1H), 7.75 (m, 1H).
A solution of 4-Chloro-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,6-difluoro-phenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (50 mg, 0.13 mmol) in dioxane/H2O (3:1, 4.8 mL) was mixed with 2-methylthiophenyl boronic acid (33.8 mg, 0.20 mmol) and K2CO3 (54.3 mg, 0.39 mmol). The resultant mixture was bubbled with argon for 5 minutes, and added by Pd(PPh3)4 (3.0 mg, 0.0026 mmol). The reaction tube was sealed and heated with “Smith Creator” (microwave, 150° C.) for 15 minutes. The mixture was concentrated under vacuo. Flash chromatography (EtOAc/Hexane, 3:1) then provided the title compound (89%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.45 (s, 3H), 2.55 (s, br, 2H), 3.72 (m, br, 5H), 6.25 (m, br, 1H), 6.41 (m, 1H), 7.11 (m, 2H), 7.30 (m, 2H), 7.45 (m, 2H), 7.51 (m, 2H).
The title compound was prepared by following the procedure in Example 36 except 3-methylthiophenyl boronic acid was used in the coupling step (71%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.50 (s, br, 2H), 2.55 (s, 3H), 3.72 (m, br, 5H), 6.25 (m, br, 1H), 6.47 (m, 1H), 7.11 (m, 2H), 7.35 (m, 1H), 7.46 (m, 4H), 7.77 (m, 1H).
The title compound was prepared by following the procedure in Example 36 except 4-methoxyphenyl boronic acid was used in the coupling step (70%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 1.80 (s, br, 2H), 3.77 (m, br, 5H), 3.92 (s, 3H), 6.10 (m, br, 1H), 6.47 (m, 1H), 7.12 (m, 4H), 7.50 (m, 1H), 7.62 (m, 2H), 7.86 (m, 1H).
The title compound was prepared by following the procedure in Example 36 except 3-methoxyphenyl boronic acid was used in the coupling step (79%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 2.25 (s, br, 2H), 3.75 (m, br, 5H), 3.90 (s, 3H), 6.15 (m, br, 1H), 6.46 (m, 1H), 7.15 (m, 5H), 7.47 (m, 2H), 7.82 (m, 1H).
The title compound was prepared by following the procedure in Example 36 except phenyl boronic acid was used in the coupling step (89%): MS (ES) m/z 425 (M+H)+; 1H-NMR (CDCl3) δ 2.16 (s, br, 2H), 3.83 (m, br, 5H), 6.15 (m, br, 1H), 6.47 (m, 1H), 7.13 (m, 2H), 7.55 (m, 6H), 7.80 (m, 1H).
The title compound was prepared by following the procedure in Example 36 except 4-methylthiophenyl boronic acid was used in the coupling step (67%): MS (ES) m/z 471 (M+H)+; 1H-NMR (CDCl3) δ 2.50 (s, 3H), 2.57 (s, br, 2H), 3.72 (m, br, 5H), 6.20 (m, br, 1H), 6.46 (m, 1H), 7.11 (m, 2H), 7.39 (m, 2H), 7.45 (m, 1H), 7.56 (m, 2H), 7.81 (m, 1H).
The title compound was prepared by following the procedure in Example 36 except 2-methoxyphenyl boronic acid was used in the coupling step (91%): MS (ES) m/z 455 (M+H)+; 1H-NMR (CDCl3) δ 2.70 (s, br, 2H), 3.68 (m, br, 5H), 3.82 (s, 3H), 6.20 (m, br, 1H), 6.40 (m, 1H), 7.08 (m, 4H), 7.48 (m, 4H).
A solution of 4-Chloro-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,6-difluorophenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (50 mg, 0.13 mmol) in dioxane/H2O (3:1, 4.8 mL) was mixed with 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (44.0 mg, 0.20 mmol) and K2CO3 (71.9 mg, 0.52 mmol). The resultant mixture was bubbled with argon for 5 minutes, and added by Pd(PPh3)4 (3.0 mg, 0.0026 mmol). The reaction tube was sealed and heated with “Smith Creator” (microwave, 150° C.) for 15 minutes. The mixture was concentrated under vacuo. Flash chromatography (EtOAc/Hexane, 3:1) then afforded the title compound (82%): MS (ES) m/z 441 (M+H)+; 1H-NMR (CDCl3) δ 1.65 (s, br, 2H), 3.80 (m, br, 5H), 6.05 (m, br, 1H), 6.54 (m, 1H), 7.15 (m, 4H), 7.48 (m, 3H), 7.98 (m, 1H).
The title compound was prepared by following the procedure in Example 43 except 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol was used in the coupling step (13%): MS (ES) m/z 441 (M+H)+; 1H-NMR-(CD3OD) δ 2.18 (m, br, 5H), 4.92 (m, 1H), 5.50 (m, 1H), 5.65 (m, 4H), 5.86 (m, 1H), 6.08 (m, 1H), 6.44 (m, 1H).
The title compound was prepared by following the procedure in Example 43 except 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol was used in the coupling step (56%): MS (ES) m/z 441 (M+H)+; 1H-NMR (CD3OD) δ 2.18 (m, br, 5H), 4.91 (m, 1H), 5.48 (m, 2H), 5.71 (m, 2H), 6.07 (m, 3H), 6.51 (m, 1H).
A solution of 4-(4-Methylsulfanyl-phenyl)-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,6-difluorophenyl)-8H-pyrido[2,3-d]pyrimidin-7-one (50 mg, 0.11 mmol) in methylene chloride (3 mL) was mixed with mCPBA (80.0 mg, 0.33 mmol). After stirring at room temperature for 30 minutes, the mixture was concentrated under vacuo. Flash chromatography (EtOAc/Hexane, 10:1) then provided the title compound (61%): MS (ES) m/z 502 (M+H)+; 1H-NMR (CDCl3) δ 2.06 (s, br, 2H), 3.16 (s, 3H), 3.77 (m, br, 5H), 6.20 (m, br, 1H), 6.51 (m, 1H), 7.13 (m, 2H), 7.51 (m, 1H), 7.68 (m, 1H), 7.85 (m, 2H), 8.15 (m, 2H).
The title compound was prepared from 4-(3-methylsulfanyl-phenyl)-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,6-difluorophenyl)-8H-pyrido[2,3-d]pyrimidin-7-one by following the procedure in Example 46 (64%): MS (ES) m/z 503 (M+H)+; 1H-NMR (CDCl3) δ 2.09 (s, br, 2H), 3.16 (s, 3H), 3.78 (m, br, 5H), 6.35 (m, br, 1H), 6.55 (m, 1H), 7.14 (m, 2H), 7.50 (m, 1H), 7.70 (m, 1H), 7.81 (m, 1H), 7.95 (m, 1H), 8.17 (m, 1H), 8.30 (s, br, 1H).
The title compound was prepared from 4-(2-methylsulfanyl-phenyl)-2-(2-hydroxy-1-hydroxymethyl-ethylamino)-8-(2,6-difluorophenyl)-8H-pyrido[2,3-d]pyrimidin-7-one by following the procedure in Example 46 (62%): MS (ES) m/z 503 (M+H)+; 1H-NMR (CDCl3) δ 2.27 (s, br, 2H), 3.28 (s, 3H), 3.77 (m, br, 5H), 6.20 (s, br, 1H), 6.41 (m, 1H), 7.13 (m, 2H), 7.25 (m, 1H), 7.48 (m, 2H), 7.81 (m, 2H), 8.26 (m, 1H).
3-Fluoro-4-methylbenzoic acid (1.54 g, 0.01 mol) is dissolved in trifluoromethanesulfonic acid (10 mL) and cooled to about 0° C. NIS (2.25 g. 0.01 mol) is added in several portions over a 6 h period while maintaining the reaction temperature at about 0° C. The mixture is allowed to warm to rt. overnight. The reaction mixture is then poured over ice and extracted with ethyl acetate (3×). The organic layers are washed (Na2S2O5) and concentrated. The material is carried on crude.
The crude acid from above (˜1.5 g) is dissolved in thionyl chloride (75 mL) and heated to 80° C. for about 2 h. The mixture is then cooled to room temperature and stirred under N2 overnight. The mixture is concentrated in vacuo and dissolved in 15 mL DCM. Na2CO3 (3 g) is added along with the cyclopropyl amine (0.69 mL, 0.01 moles (hereinafter “mol”)). The mixture is allowed to stir overnight and purified via flash chromatography (5% MeOH/CH2Cl2) to afford 0.904 g of N-cyclopropyl-3-fluoro-5-iodo-4-methylbenzamide
N-cyclopropyl-3-fluoro-5-iodo-4-methylbenzamide (0.904 g, 2.83 mmol) is dissolved in DMF (30 mL). Bis-pinicalato-diborane (1.44 g, 2.83 mmol) is added followed by PdCl2.dppf (55 mg) and potassium acetate (1.38 g, 14.15 mmol). The mixture are stirred for about 18 h, concentrated in vacuo and purified via flash chromatography to afford N-cyclopropyl-3-fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (60 mg).
4-Chloro-8-(2,6-difluorophenyl)-2-{[2-hydroxy-1-(hydroxymethyl)ethyl]-amino}pyrido[2,3-d]pyrimidin-7(8H)-one (0.056 g, 0.17 mmol), N-cyclopropyl-3-fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (0.065 g, 0.17 mmol), K2CO3 (0.07 g, 0.51 mmol) and tetrakis triphenyl phosphine palladium (10 mg, 0.05 eq) are dissolved in dioxane/water (3:1, 10 mL) and heated to about 100° C. for about 3 h. The mixture is concentrated and purified via reverse phase HPLC to afford the title compound (9 mg, yellow powder, mp 214.2-217.5): LC-MS m/z 540 (M+H)′, 1.69 min (ret time). HPLC indicates 96% pure.
To the compound 4-chloro-8-(2,6-difluorophenyl)-2-(methylsulfinyl)-pyrido[2,3-d]pyrimidin-7(8H)-one (1.59 g, 4.47 mmol) in dichloromethane (89.4 mL) were added N,N-diethyl-1,3-propanediamine (0.845 mL, 5.36 mol) and triethylamine (1.26 uL, 8.94 mmol). The mixture was stirred at rt overnight. Some white precipitate was formed during the reaction. Filtration followed by wash with ethyl acetate/dichoromethane/methanol afforded the title compound (1.028 g, 60%). LC-MS m/z 383 (M+H)+.
To the compound 4-chloro-8-(2,6-difluorophenyl)-2-(methylsulfinyl)pyrido[2,3-d]pyrimidin-7(8H)-one (1.39 g, 3.9 mmol) in dichloromethane (80 mL) were added 4-methyl-1,4′-bipiperidine (0.75 g, 5.85 mol) and triethylamine (1.03 mL, 11.7 mmol). The mixture was stirred at about −20° C. overnight. Filtration followed by concentration, the crude was purified with flash chromatography to afford the title compound (0.904 g, 51%). LC-MS m/z 474 (M+H)+.
To a stirring solution of 3-iodo-4-methylbenzoic acid (60 g, 0.22 mol, 1 eq) in degassed DMF (1400 mL, 23.3 vol.) was charged 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (81.4 g, 0.32 mol, 1.4 eq) followed by potassium acetate (112 g, 1.14 mole, 5 eq) and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) (18.7 g, 0.02 mole, 0.1 eq). The resulting mixture was placed under a nitrogen atmosphere and was heated to 80° C. with the exclusion of light overnight. The mixture was then concentrated under high vacuum and the residue partitioned between EtOAc and 2M HCl. The mixture was then filtered and the layers separated. The aqueous phase was re-extracted with EtOAc. The combined organics were then washed with brine, dried and evaporated to yield a brown solid that was applied to a silica plug then eluted with 2:1 cyclohexane:ethyl acetate. Fractions were then combined and evaporated to yield a brown foam that was triturated with cyclohexane, collected by filtration then dried in vacuo to yield 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid. (CDCl3) 8.50-8.49 (1H, d), 8.04-8.02 (1H, dd), 7.27-7.25 (1H, d), 2.61 (3H, s), 1.36 (12H, s).
To the compound 4-chloro-8-(2,6-difluorophenyl)-2-(4-methyl-1,4′-bipiperidin-1′-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (47.5 mg, 0.1 mmol) in dioxane (3 mL) and water (1 mL) were added 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (38.4 mg, 0.15 mol), potassium carbonate (83 mg, 0.6 mmol) and tetrakis(triphenylphosphine)palladium(0) (4.6 mL, 0.005 mmol). The mixture was heated with microwave at about 150° C. for about 15 min. The mixture was concentrated & then mixed with DMSO (0.75 mL) and water (0.25 mL). Separation by HPLC afforded the title compound (39 mg, 68%). LC-MS m/z 574 (M+H)+.
To the compound 4-chloro-2-{[3-(diethylamino)propyl]amino}-8-(2,6-difluorophenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (176 mg, 0.418 mmol) in dioxane (4.5 mL) and water (1.5 mL) was added 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (173 mg, 0.626 mol), potassium carbonate (289 mg, 2.09 mmol) and tetrakis(triphenylphosphine)palladium(0) (24.2 mg, 0.0259 mmol). The mixture was heated with microwave at about 150° C. for about 15 min. The mixture was filtered. Separation by HPLC with TFA afforded the title compound (238 mg, 99%). LC-MS m/z 522 (M+H)+.
To the compound 4-chloro-2-{[3-(diethylamino)propyl]amino}-8-(2,6-difluorophenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (600 mg, 1.422 mmol) in dioxane (15 mL) and water (5 mL) were added 1,1-dimethylethyl 3-fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (542 mg, 2.132 mol), potassium carbonate (590 mg, 4.26 mmol) and tetrakis(triphenylphosphine)-palladium(0) (82 mg, 0.071 mmol). The mixture was heated with microwave at 150° C. for 15 minutes. The mixture was filtered. Separation by HPLC with TFA afforded the crude title compound.
To the compound 4-chloro-2-{[3-(diethylamino)propyl]amino}-8-(2,6-difluorophenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (168.75 mg, 0.40 mmol) in dioxane (12 mL) and water (4 mL) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (148.85 mg, 0.60 mol), potassium carbonate (208 mg, 1.20 mmol) and tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02 mmol). The mixture was heated with microwave at 150° C. for 15 min. The mixture was concentrated. It was mixed with DMSO (0.75 mL) and water (0.25 mL). Separation by HPLC afforded the title compound (147 mg, 72%). LC-MS m/z 508 (M+H)+.
To the compound 4-chloro-2-{[3-(diethylamino)propyl]amino}-8-(2,6-difluorophenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (210.5 mg, 0.50 mmol) in dioxane (15 mL) and water (5 mL) were added 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (125 mg, 0.75 mol), potassium carbonate (210 mg, 1.20 mmol) and tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol). The mixture was heated with microwave at about 150° C. for about 15 min. The mixture was concentrated, then mixed with DMSO (0.75 mL) and water (0.25 mL). Separation by HPLC afforded the title compound (467 mg, 32%). LC-MS m/z 508 (M+H)+.
To the solution of phosphorus oxychloride (65 mL, 0.70 mol) in trichloroethylene (46.5 mL) was added DMF (25 mL, 0.32 mol) slowly to keep the temperature between 5° C. to 10° C. The solution was then warmed up to room temperature before 6-hydroxy-2-(methylthio)-4(1H)-pyrimidinone (25 g, 0.16 mol) was added in portions. The resultant reaction mixture was heated at 80° C. overnight followed by concentration under vacuum. The resulting slurry like residue was poured into ice, stirred for about 2 hours then filtered to afford the crude product. The crude product was further purified by recrystallization with hexane to afford 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbaldehyde (21.3 g, 61%). 1H-NMR (CDCl3) δ 2.66 (s, 3H), 10.4 (s, 1H).
To the mixture of hydroxylamine hydrochloride (139 mg, 2.0 mmol), HOAc (0.113 mL, 2.0 mmol) and EtOH (5 mL) was added 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbaldehyde (223 mg, 1.0 mol) to room temperature. The solution was then heated at 50° C. for about 1 hour, 60° C. for about 30 minutes and 70° C. for about 30 minutes before it was concentrated under vacuum and washed with H2O (10-20 mL) to afford 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbaldehyde oxime (190 mg, 80%). LC-MS m/z 238 (M+H)+ 1.57 minute, 1.65 minute; 1H-NMR (CDCl3) δ 2.62, 2.65 (3H), 7.53, 8.30 (1H).
To 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbaldehyde oxime (2.38 g, 10 mmol) was added SOCl2 (21.8 mL, 0.30 mol) slowly at room temperature. The solution was then heated at 75° C. for about 3 hours before it was concentrated under vacuum. The residue SOCl2 was removed by evaporation with toluene (5 mL) under vacuum. The resulting solid was washed with EtOH/H2O (10 mL, 1:1) to afford 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbonitrile (2.04 g, 93%). LC-MS m/z 220 (M+H)+ 1.99 minute; 1H-NMR (CDCl3) δ 2.64 (3H).
To the solution of 4,6-dichloro-2-(methylthio)-5-pyrimidinecarbonitrile (2.20 g, 10.0 mmol) in DMF (10 mL) was added 2,6-difluoroaniline (2.17 mL, 20.0 mmol). The solution was stirred at 50° C. for about 60 minutes. The mixture was slowly added into a solution of MeOH (20 mL) and water (30 mL). The resultant solid was filtered and washed with MeOH/H2O (20 mL, 1:1) to give 4-chloro-6-[(2,6-difluorophenyl)amino]-2-(methylthio)-5-pyrimidinecarbonitrile as a white solid (2.82 g, 90%). LC-MS m/z 313 (M+H)+; 1H-NMR (CDCl3) δ 2.33 (s, 3H), 6.94 (s, 1H), 7.04 (m, 2H), 7.35 (m, 1H).
To the solution of 4-chloro-6-[(2,6-difluorophenyl)amino]-2-(methylthio)-5-pyrimidinecarbonitrile (0.938 g) was added borane.THF complex (1.0 M, 15 mL). The reaction mixture was then heated at reflux for about 4 h until all the starting material disappeared. The solution was cooled to r.t., mixed with HCl solution (6 M, 5 mL), and stirred at room temperature for about 30 minutes. The solution was then mixed with NaOH solution (3 M) to pH 9.0-10.0. The organic phase was separated and the aqueous layer was extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (50 mL), collected, dried over Na2SO4 and concentrated to afford the title compound 0.97 g (quantitative). LC-MS m/z 317 (M+H)+, 1.5 min (ret. time).
To the solution of 5-(aminomethyl)-6-chloro-N-(2,6-difluorophenyl)-2-(methylthio)-4-pyrimidinamine (0.317 g) in CH2Cl2 (5 mL) was added the mixture of carbonyl diimidazole (0.178 g) in CH2Cl2 (5 mL). The resultant mixture was stirred for about 3 hours at r.t., mixed with CH2Cl2 (10 mL) and washed with HCl (1 N, 2×10 mL) and H2O (20 mL). The organic layers were collected, dried over Na2SO4, filtered and concentrated to provide the title compound (0.279 g, 81%). LC-MS m/z 343 (M+H)+, 1.75 min (ret. time); 1H-NMR (400 MHz, CDCl3) δ 7.44-7.40 (m, 1H), 7.07-7.03 (m, 2H), 5.84 (br, 1H), 4.62 (s, 2H), 2.19 (s, 3H).
To a solution of 3-[8-(2,6-difluorophenyl)-2-(methylthio)-7-oxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-4-yl]-4-methylbenzoic acid (1.71 g, 5 mmol) in CH2Cl2 (60 mL) was added m-CPBA (1.17 g, 5.2 mmol). The mixture was stirred at room temperature for 10 minutes, then directly loaded onto a column. Flash chromatography (mobile phase EtOAc/Hexane) afforded the title compound as a white solid 1.58 g (88%). LC-MS m/z 358 (M+H)+.
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-(methylsulfinyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (250 mg, 0.70 mmol) in DCM (10 mL) were added 4-(1-pyrrolidinyl)piperidine (323 mg, 2.1 mmol) and N,N-diisopropylethylamine (0.3 mL, 1.7 mmol). The resultant solution was stirred at room temperature over night. The result mixture was concentrated. CombiFlash chromatography (mobile phase DCM/DCM[90]+MeOH[7]+NH4OH[3]) provided the title compound as a white solid (253 mg, 81%). LC-MS m/z 449 (M+H)'
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-[4-(1-pyrrolidinyl)-1-piperidinyl]-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (18 mg, 0.04 mmol) in dioxane (1.5 mL)/water (0.5 mL) were added potassium carbonate (34 mg, 0.25 mmol), tetrakis(triphenylphosphine)palladium(0) (2.3 mg, 0.002 mmol) and N-(4-fluorophenyl)-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (22 mg, 0.062 mmol). The reaction mixture was bubbled with N2 for 5 mins, then microwaved at about 150° C. for about 30 mins. The reaction mixture was concentrated. CombiFlash chromatography (mobile phase DCM/DCM[90]+MeOH[7]+NH4OH[3]) provided the title compound as a white solid (14 mg, 54%). LC-MS m/z 642 (M+H)+; 1H-NMR (CD3OD) δ 1.32 (m, 2H), 1.80 (m, 4H), 1.88 (m, 2H), 2.24 (m, 1H), 2.33 (s, 3H), 2.62 (m, 4H), 2.74 (t, 2H), 4.17 (m, 2H), 4.40 (m, 2H), 7.12 (m, 4H), 7.52 (m, 2H), 7.72 (m, 2H), 7.82 (s, 1H), 7.98 (d, 1H).
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-(methylsulfinyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (200 mg, 0.56 mmol) in DCM (10 mL) were added 1,4′-bipiperidine (270 mg, 1.61 mmol) and N,N-diisopropylethylamine (0.3 mL, 1.7 mmol). The resultant solution was stirred at room temperature over night. The result mixture was concentrated. CombiFlash chromatography (mobile phase DCM/DCM[90]+MeOH[7]+NH4OH[3]) provided the title compound as a white solid (298 mg, 83%). LC-MS m/z 463 (M+H)+.
To a solution of compound 7-(1,4′-bipiperidin-1′-yl)-5-chloro-1-(2,6-difluorophenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (18 mg, 0.04 mmol) in dioxane (1.5 mL)/water (0.5 mL) were added potassium carbonate (34 mg, 0.25 mmol), tetrakis(triphenylphosphine)palladium(0) (2.3 mg, 0.002 mmol) and N-(4-fluorophenyl)-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (22 mg, 0.062 mmol). The reaction mixture was bubbled with N2 for 5 mins, then microwaved at about 150° C. for about 30 mins. The reaction mixture was concentrated. CombiFlash chromatography (mobile phase DCM/DCM[90]+MeOH [7]+NH4OH[3]) provided the title compound as a white solid (13 mg, 51%). LC-MS m/z 656 (M+H)+; 1H-NMR (CD3OD) δ 1.37 (m, 2H), 1.48 (m, 2H), 1.60 (m, 4H), 1.80 (m, 2H), 2.33 (s, 3H), 2.56 (m, 5H), 2.72 (t, 2H), 4.17 (m, 2H), 4.46 (m, 2H), 7.12 (m, 4H), 7.52 (m, 2H), 7.72 (m, 2H), 7.82 (s, 1H), 7.98 (d, 1H).
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-(methylsulfinyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (800 mg, 2.23 mmol) in DCM (45 mL) were added N,N-dimethylethylenediamine (0.36 mL, 3.23 mmol) and triethylamine (0.63 mL, 4.5 mmol). The resultant solution was stirred at room temperature over night. The result mixture was concentrated. CombiFlash chromatography (mobile phase DCM/DCM[90]+MeOH[7]+NH4OH[3]) provided the title compound as a white solid (730 mg, 85%). LC-MS m/z 383 (M+H)+.
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-{[2-(dimethylamino)-ethyl]amino}-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (100 mg, 0.26 mmol) in dioxane (9 mL)/water (3 mL) were added potassium carbonate (217 mg, 1.57 mmol), tetrakis(triphenylphosphine)palladium(0) (15 mg, 0.013 mmol) and 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (65 mg, 0.39 mmol). The reaction mixture was bubbled with N2 for 5 mins, then microwaved at about 150° C. for about 30 mins. The reaction mixture was concentrated. To the concentrated mixture were added DMSO (2 mL), H2O (0.5 mL) and AcOH (0.05 mL). Separation via a HPLC then provided the title compound as a white solid (120 mg, 98%). LC-MS m/z 469 (M+H)+.
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-{[2-(dimethylamino)-ethyl]-amino}-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (150 mg, 0.39 mmol) in dioxane (12 mL)/water (4 mL) were added potassium carbonate (325 mg, 2.36 mmol), tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.019 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (146 mg, 0.59 mmol). The reaction mixture was bubbled with N2 for about 5 mins, then microwaved at about 150° C. for about 30 mins. The reaction mixture was concentrated. To the concentrated mixture were added DMSO (2 mL), H2O (0.5 mL) and AcOH (0.05 mL). Separation via a HPLC then provided the title compound as a white solid (142 mg, 77%). LC-MS m/z 469 (M+H)+.
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-{[2-(dimethylamino)ethyl]-amino}-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (200 mg, 0.52 mmol) in dioxane (15 mL)/water (5 mL) were added potassium carbonate (433 mg, 3.14 mmol), tetrakis(triphenylphosphine)palladium(0) (31 mg, 0.027 mmol) and 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (205 mg, 0.78 mmol). The reaction mixture was bubbled with N2 for about 10 mins, then microwaved at 150° C. for about 30 mins. The reaction mixture was concentrated. To the concentrated mixture were added DMSO (2 mL), H2O (0.5 mL) and AcOH (0.05 mL). Gilson with TFA provided the title compound as a white solid (310 mg, 99%). LC-MS m/z 483 (M+H)+.
To a solution of 5-chloro-1-(2,6-difluorophenyl)-7-{[2-(dimethylamino)ethyl]amino}-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (200 mg, 0.52 mmol) in dioxane (15 mL)/water (5 mL) were added potassium carbonate (433 mg, 3.14 mmol), tetrakis(triphenylphosphine)palladium(0) (31 mg, 0.027 mmol) and (5-{[(1,1-dimethylethyl)oxy]carbonyl}-3-fluoro-2-methylphenyl)boronic acid (159 mg, 0.63 mmol). The reaction mixture was bubbled with N2 for 10 mins, then microwaved at 150° C. for 30 mins. The reaction mixture was concentrated. To the concentrated mixture were added DMSO (2 mL), H2O (0.5 mL) and AcOH (0.05 mL). Separation via a HPLC then provided the title compound as a white solid (270 mg, 88%). LC-MS m/z 587 (M+H)+.
To the solution of 5-chloro-1-(2,6-difluorophenyl)-7-{[2-(dimethylamino)ethyl]amino}-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (191 mg, 0.50 mmol) in dioxane (15 mL) and water (5 mL) were added 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (197 mg, 0.75 mmol), K2CO3 (415 mg, 3.0 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (23 mg, 0.025 mmol). The reaction mixture was heated to 150° C. for about 15 minutes with microwave. The reaction mixture was concentrated to dry then was added DMSO (2 mL), water (0.5 mL) and HOAc (1 drop). The solution was filtered and applied to the reverse phase HPLC to afford the titled compound 0.24 g (quantitative). LC-MS m/z 483 (M+H)+; 1H-NMR (CD3OD) 2.36 (s, 3
H), 2.76 (s, 6H), 3.16 (s, 2H), 3.56 (s, 2H), 4.13 (s, 2H), 7.21 (m, 1H), 7.53 (m, 2H), 7.95 (s, 1H), 8.09 (d, J=7.6 Hz, 1H).
The title compound was prepared by following the procedure in Example 68 except 3-(dihydroxyboranyl)-4-ethylbenzoic acid was used in the coupling reaction (yield: 38%). LC-MS m/z 497 (M+H)+; 1H-NMR (CD3OD) 1.22 (t, J=7.6 Hz, 3H), 2.68 (s, 2H), 2.77 (s, 6H), 3.16 (m, 2H), 3.53 (m, 2H), 4.13 (m, 2H), 7.18 (m, 2H), 7.55 (m, 2H), 7.86 (s, 1H), 8.10 (m 1H).
To 5-chloro-1-(2,6-difluorophenyl)-7-(methylsulfinyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (275 mg, 0.767 mmol) in dichloromethane (15 mL) was added N,N-diethyl-1,3-propyldiamine (0.181 mL, 1.15 mmol) and triethylamine (0.215 mL, 1.53 mmol). The mixture was stirred over night. The mixture was concentrated and separated by Gilson HPLC (with 0.1% TFA) to afford the title compound (207 mg, 64%).
To 5-chloro-7-{[3-(diethylamino)propyl]amino}-1-(2,6-difluorophenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (207 mg, 0.488 mmol) in 1,4-dioxane (7.5 mL) and water (2.5 mL) was added 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (0.192 g, 0.733 mmol), tetrakis(triphenylphosphine)-palladium(0) (28.3 mg, 0.024 mmol), and potassium carbonate (270 mg, 1.95 mmol). The mixture was heated with microwave for about 15 min at 150° C., and then allowed to cool to room temperature. The mixture was concentrated and separated by HPLC to afford the title compound (66 mg, 26%). LC-MS m/z 525 (M+H)+.
The solution of 4-chloro-8-(2,6-difluorophenyl)-2-(methylthio) pyrido[2,3-d]pyrimidin-7(8H)-one (1.70 g, 5.00 mmol) in DME (150 mL) and H2O (50 mL), in a pressure flask (500 mL, Chemglass), was added 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxa borolan-2-yl)benzoic acid (1.97 g, 7.50 mmol) and K2CO3 (4.15 g, 30.0 mmol). The resulting mixture was degassed with Argon for 5 minutes, mixed with Pd(PPh3)4 (0.232 g, 0.20 mmol) and heated with a preheated oil bath (160° C.) under vigorous stirring for 30 minutes. The reaction mixture was filtered through celite, concentrated under vacuum to remove DME. It was then mixed with EtOAc (200 mL) and AcOH (2.5 mL), and shaked. The layers were separated. The organic layer was collected, further washed with brine (70 mL), dried over Na2SO4, filtered, concentrated and purified via a flash chromatography (load column with DCM, mobile phase EtOAc/Hexane) to afford the title compound as a white solid 2.15 g (98%). LC-MS (ES) m/z 440 (M+H)+; 1H-NMR (CD3OD) δ 2.27 (s, 3H), 2.31 (s, 3H), 6.71 (d, J=9.6 Hz, 1H), 7.28 (t, J=8.2 Hz, 2H), 7.57 (d, J=8.4 Hz, 1H), 7.64 (m, 2H), 8.00 (d, J=1.6 Hz, 1H), 8.14 (dd, J1=7.6 Hz, J2=1.6 Hz, 1H).
4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (1 g, 3.8 mmoles) was taken up in CH2Cl2 (200 mL) and was treated with oxalyl chloride (0.44 mL, 5 mmol) and DMF (1 drop). One hour after gas evolution had ceased, the solvents were pumped off in vacuo, and the residue stripped from toluene. This was again taken up in CH2Cl2 (200 mL), and excess dimethyl amine was bubbled into the mixture, which was then sealed off and stirred overnight at room temperature. The solvents were pumped off to give the crude N,N,4-trimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide, which was used without further purification in the next step.
5-chloro-1-(2,6-difluorophenyl)-7-(methylthio)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (0.102 g, 0.298 mmol), N,N,4-trimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide from above, (0.129 g, 0.447 mmol), and K2CO3 (0.123 g, 0.894 mmol), were taken up in dioxane (6 mL) and water (1.2 mL). The mixture was degassed with argon for 30 min and tetrakis(triphenyl-phosphine)palladium(0) (0.026 g, 0.022 mmol) was added. The mixture was then heated under argon at 95° C. for 18 h. The solvents were pumped off, and after aqueous workup, the crude material was flashed on silica gel (15 g), eluted with a EtOAc/CH2Cl2 gradient to give the title compound as a white amorphous solid. mp 144-147° C. LC-MS m/z 470 (M+H)+, 2.02 min (ret time).
The compounds of (I) and (Ia), or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of any disease state in a human, or other mammal, which is exacerbated or caused by excessive or unregulated cytokine production by such mammal's cell, such as but not limited to monocytes and/or macrophages.
For purposes herein, compounds of Formula (I) and (Ia), will all be referred to as compounds of Formula (I) herein unless otherwise indicated.
Compounds of Formula (I) are capable of inhibiting proinflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF and are therefore of use in therapy. IL-1, IL-6, IL-8 and TNF affect a wide variety of cells and tissues and these cytokines, as well as other leukocyte-derived cytokines, are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these pro-inflammatory cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
Accordingly, the present invention provides a method of treating a cytokine-mediated disease which comprises administering an effective cytokine-interfering amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
Pro-inflammatory cytokines, such as IL-1, IL-6 & TNF-are commonly elevated in the plasma of depressed patients (Elenkov I J et al., 2005. Neuroimmunomod. 12: 255-269, Hayley S et al., 2005. Neurosci. 135: 659-678, Raison C L et al., 2006. Trends in Immuno. 27: 24-31) and bipolar patients in both the depressed and mania phases (O′Brien S M. et al., 2006. J. Affective Disorders. 90: 263-267). In animals, systemic injection of such pro-inflammatory cytokines result in a sickness like behaviour that can mimic some of the symptoms observed in depression in man which can be reversed by antidepressant drugs (Simen, B. B. et al., 2006 Biol Psychiatry. 59: 775-785). These cytokines can increase the activity of monoamine transporters, known molecular targets of antidepressants, through a P38 dependent mechanism (Zhu et al., 2006, Neuropsychopharmacol. ahead of print, Prasad H. C. et al., 2005, PNAS 102: 11545-11550). P38 inhibitors, or mechanisms that have the potential to decrease pro-inflammatory mediators can stabilise monoamine transporter activity and could therefore be antidepressant drugs. The soluble TNF-receptor, etanercept, which sequesters TNF-signalling, has demonstrated efficacy in alleviating clinical symptoms of psoriasis on fatigue and symptoms of depression associated with the condition (Tyring S. et al., 2006 Lancet. 367: 29-35). Like depression, anxiety, commonly apparent under stressful conditions is also regulated by the immune system and pro-inflammatory cytokines (Holden R. J., 1999 Med. Hypotheses. 52: 155-162; Pitsavos C. et al., 2006 Atherosclerosis. 185: 320-326). P38 inhibitors, by blocking the signalling of pro-inflammatory cytokines, therefore have the potential to treat multiple facets of depressive and anxiety disorders.
P38 inhibitor effects in depression can be assessed using randomised, double-blind, placebo-controlled studies compared to an active clinically effective comparator in patients with Major Depressive Disorders with elevated pro-inflammatory cytokine levels initially, enriched for loss of energy, pleasure, interest and with psychomotor retardation.
TNF-levels have also been reported to be elevated in animal models of schizophrenia and in schizophrenic patients. These elevated levels of pro-inflammatory cytokines can be normalized by antipsychotic drugs (Paterson G. J. et al., 2006 J. Psychopharmacol. ahead of print, Zhang X. Y., et al., 2005 Neuropsychopharmacol 30:1532-1538). Despite a lack of a genetic association between TNF and schizophrenia (Shirts B. H. et al., 2006 Schizophr. Res. 83: 7-13), however p38 inhibitors may still have utility in this psychiatric disorder when inflammatory signalling pathways are altered in the pathophysiology of the disease.
TNF- and IL-6 are also increased in normal subjects with sleep deprivation (Vgontzas A. N., et al. 2004 J Clin Endo Metab. 89: 2119-2126), in subjects with insomnia (Vgontzas A. N., et al 2002 Metabolism 7: 887-892.) and in subjects with sleep apnea (see Hatipoglu U., et al. 2003 Respiration 70: 665-671; Alberti A., et al. 2003 J Sleep Res. 12: 305-311; and Yokoe T., et al. 2003 Circulation. 107: 1129-1134). Etanercept has also been used to demonstrate decreases in sleepiness in patients with sleep apnea (Vgontzas A. N., et al. 2004 J Clin Endocrinol Metab. 89: 4409-4413) suggesting that drugs that inhibit pro-inflammatory cytokines may return sleep architecture back to normal.
In an embodiment of the invention, diseases or conditions that may be mediated by P38 inhibitors are selected from the list consisting of [the numbers in brackets after the listed diseases below refer to the classification code in Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, published by the American Psychiatric Association (DSM-IV) and/or the International Classification of Diseases, 10th Edition (ICD-10)]:
i) Depression and mood disorders including Major Depressive Episode, Manic Episode, Mixed Episode and Hypomanic Episode; Depressive Disorders including Major Depressive Disorder, Dysthymic Disorder (300.4), Depressive Disorder Not Otherwise Specified (311); Bipolar Disorders including Bipolar I Disorder, Bipolar II Disorder (Recurrent Major Depressive Episodes with Hypomanic Episodes) (296.89), Cyclothymic Disorder (301.13) and Bipolar Disorder Not Otherwise Specified (296.80); Other Mood Disorders including Mood Disorder Due to a General Medical Condition (293.83) which includes the subtypes With Depressive Features, With Major Depressive-like Episode, With Manic Features and With Mixed Features), Substance-Induced Mood Disorder (including the subtypes With Depressive Features, With Manic Features and With Mixed Features) and Mood Disorder Not Otherwise Specified (296.90):
ii) Schizophrenia including the subtypes Paranoid Type (295.30), Disorganised Type (295.10), Catatonic Type (295.20), Undifferentiated Type (295.90) and Residual Type (295.60); Schizophreniform Disorder (295.40); Schizoaffective Disorder (295.70) including the subtypes Bipolar Type and Depressive Type; Delusional Disorder (297.1) including the subtypes Erotomanic Type, Grandiose Type, Jealous Type, Persecutory Type, Somatic Type, Mixed Type and Unspecified Type; Brief Psychotic Disorder (298.8); Shared Psychotic Disorder (297.3); Psychotic Disorder Due to a General Medical Condition including the subtypes With Delusions and With Hallucinations; Substance-Induced Psychotic Disorder including the subtypes With Delusions (293.81) and With Hallucinations (293.82); and Psychotic Disorder Not Otherwise Specified (298.9).
iii) Anxiety disorders including Panic Attack; Panic Disorder including Panic Disorder without Agoraphobia (300.01) and Panic Disorder with Agoraphobia (300.21); Agoraphobia; Agoraphobia Without History of Panic Disorder (300.22), Specific Phobia (300.29, formerly Simple Phobia) including the subtypes Animal Type, Natural Environment Type, Blood-Injection-Injury Type, Situational Type and Other Type), Social Phobia (Social Anxiety Disorder, 300.23), Obsessive-Compulsive Disorder (300.3), Posttraumatic Stress Disorder (309.81), Acute Stress Disorder (308.3), Generalized Anxiety Disorder (300.02), Anxiety Disorder Due to a General Medical Condition (293.84), Substance-Induced Anxiety Disorder, Separation Anxiety Disorder (309.21), Adjustment Disorders with Anxiety (309.24) and Anxiety Disorder Not Otherwise Specified (300.00):
iv) Sleep disorders including primary sleep disorders such as Dyssomnias such as Primary Insomnia (307.42), Primary Hypersomnia (307.44), Narcolepsy (347), Breathing-Related Sleep Disorders (780.59), Circadian Rhythm Sleep Disorder (307.45) and Dyssomnia Not Otherwise Specified (307.47); primary sleep disorders such as Parasomnias such as Nightmare Disorder (307.47), Sleep Terror Disorder (307.46), Sleepwalking Disorder (307.46) and Parasomnia Not Otherwise Specified (307.47); Sleep Disorders Related to Another Mental Disorder such as Insomnia Related to Another Mental Disorder (307.42) and Hypersomnia Related to Another Mental Disorder (307.44); Sleep Disorder Due to a General Medical Condition, in particular sleep disturbances associated with such diseases as neurological disorders, neuropathic pain, restless leg syndrome, heart and lung diseases; and Substance-Induced Sleep Disorder including the subtypes Insomnia Type, Hypersomnia Type, Parasomnia Type and Mixed Type; sleep apnea and jet-lag syndrome:
In general a human patient population which has an increase of peripheral and or central pro-inflammatory cytokines may be associated with depressive type symptoms such as loss of energy and interest, psychomotor retardation and fatigue, and is a candidate for treatment with a p38kinase inhibitor of this invention.
Increased circulating levels of pro-inflammatory cytokines, acute phase proteins and chemokines are known to be associated with symptoms of depression and fatigue in humans and preclinical animal models [Elenkov I J., et al., Neuroimmunomodulation. 12(5):255-69, 2005] [Raison, et al., Trends in Immunology. 27(1):24-31, 2006]. A consistent dataset suggests that peripheral pro-inflammatory cytokine (i.e, IL-1, IL-6, interferon and TNFα) can activate the production of the same cytokine in the brain acting via the blood-brain barrier in human and preclinical species. Stress, anxiety or autonomic hyperarousal can transiently activate the production of pro-inflammatory cytokines, with stronger effects in susceptible individuals [Maes et al., Cytokine, Vol. 10, No. 4, 313-318, 1998] [Dunn A. J., et al., Neuroscience & Biobehavioral Reviews. 29(4-5):891-909, 2005]. Elevated cytokines may in turn sensitise the limbic system and hypothalamus to stress, increasing the risk of depression [Anisman 2005]. Increased circulating levels of IL-6 and TNFα were consistently described in populations of subjects suffering from Major Depressive Disorders (MDD) during the symptomatic episode [Mikova, et al., European Neuropsychopharmacology. 11(3): 203-8, 2001] [Hestad, et al., Journal of ECT. 19(4):183-8, 2003] [Dunn A. J., et al., Neuroscience & Biobehavioral Reviews. 29(4-5):891-909, 2005]. This was more apparent in subjects with severe symptoms, psychomotor retardation and loss of energy. There is evidence that these symptoms can be generated, at least in part, by increases in the plasma cytokines levels. Correlation between the high levels of plasma IL-6 in the morning and depressive symptoms were found in MDD subjects and healthy volunteers (HV) by Alesci et al. Journal of Clinical Endocrinology & Metabolism. 90(5):2522-30, 2005. In addition, treatments with interferon α in subjects who did not have mood disorders produced fatigue, motor retardation and depression in more than 50% of individuals [Wichers et al., Biological Psychiatry. 60(1): 77-9, 2006] [Raison et al., supra 2006]. Fatigue and loss of energy are the most common behavioural changes associated with elevated cytokines in chronic inflammatory diseases [Raison et al., supra 2006] [Tyring et al., Lancet 367, 29-35, 2006].
A relationship between pro-inflammatory cytokine and excessive daytime sleepiness (EDS) or disturbed sleep was recently described [Vgonzas et al., Metabolism: Clinical & Experimental. 51(7):887-92, 2002], while a very high incidence of EDS was found in subject in treatment for MDD [Bixler et al., Journal of Clinical Endocrinology & Metabolism. 90(8):4510-5, 2005] [Chellappa et al., Revista Brasileira de Psiquiatria. 28(2):126-9, 2006], suggesting a possible link between high plasma cytokine levels and sleepiness in MDD subjects.
Successful antidepressant treatments of MDD episodes with SSRIs or TCAs have been associated with the reduction of circulating cytokine levels, in particular TNFα [Tuglu et al., Psychopharmacology. 170(4):429-33 (2003)] [Narita et al., Progress in Neuro-Psychopharmacology & Biological Psychiatry. 30(6):1159-62, 2006] and IL-6 [Lanquillon et al. Neuropsychopharmacology. 22(4):370-9, 2000]. This evidence indicates a state-dependent relationship between elevated cytokines and depression symptoms, suggesting a causal link. Therefore, inhibition of pro-inflammatory cytokines production are believed to be a novel method of treatment for depression.
Experimental evidence is available in the literature to support this view. For example, increased response rate and improvement of depression symptoms in MDD subjects has been reported in one add-on study using the anti-inflammatory COX inhibitors [Muller et al., Molecular Psychiatry. 11(7): 680-4, 2006]. Support for this approach was recently obtained in populations suffering from primary inflammatory disorders associated with high incidence of depression, such as Rheumatoid Arthritis (RA) or Psoriasis. For example, in patients suffering from Psoriasis the anti-TNFα agent etanercept was found to reduce symptoms of depression (scored with HAMD and BDI score) independently from the clinical improvement of the primary disorder. These effects were seen as early as 4 weeks of treatment and persisted for 12 weeks [Tyring et al., Lancet 367, 29-35, 2006].
Recent observations suggest that bupropion, a marketed antidepressant, can also reduce circulating levels of TNFα in mice [Brustolim et al., International Immunopharmacology. 6(6):903-7, 2006] and in subjects with inflammatory disorders associated with increase in fatigue, i.e., Crohn's disease and aplastic anemia [Kast et al., Archivum Immunologiae et Therapiae Experimentalis. 53(2):143-7, 2005]. Interestingly, bupropion produced a strong antidepressant signal of antidepressant effects in a population of MDD subjects selected for loss of energy, interest and pleasure (AK130913). In addition, chronic treatment with SSRI has been shown to significantly reduce plasma TNFα in MDD subjects below the control healthy volunteer (HV) values [Narita et al., Progress in Neuro-Psychopharmacology & Biological Psychiatry. 30(6):1159-62, 2006].
Compounds of Formula (I) are capable of inhibiting inducible proinflammatory proteins, such as COX-2, also referred to by many other names such as prostaglandin endoperoxide synthase-2 (PGHS-2) and are therefore of use in therapy. These proinflammatory lipid mediators of the cyclooxygenase (CO) pathway are produced by the inducible COX-2 enzyme. Regulation, therefore of COX-2 which is responsible for the these products derived from arachidonic acid, such as prostaglandins affect a wide variety of cells and tissues are important and critical inflammatory mediators of a wide variety of disease states and conditions. Expression of COX-1 is not effected by compounds of Formula (I). This selective inhibition of COX-2 may alleviate or spare ulcerogenic liability associated with inhibition of COX-1 thereby inhibiting prostaglandins essential for cytoprotective effects. Thus inhibition of these pro-inflammatory mediators is of benefit in controlling, reducing and alleviating many of these disease states. Most notably these inflammatory mediators, in particular prostaglandins, have been implicated in pain, such as in the sensitization of pain receptors, or edema. This aspect of pain management therefore includes treatment of neuromuscular pain, headache, cancer pain, and arthritis pain. Compounds of Formula (I) or a pharmaceutically acceptable salt thereof, are of use in the prophylaxis or therapy in a human, or other mammal, by inhibition of the synthesis of the COX-2 enzyme.
Accordingly, the present invention provides a method of inhibiting the synthesis of COX-2 which comprises administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. The present invention also provides for a method of prophylaxis treatment in a human, or other mammal, by inhibition of the synthesis of the COX-2 enzyme.
Another aspect of the invention is the co-administration, sequentially or contemporaneously with a COX-2 inhibitor, such as Celebrex®, and Vioxx®. e.g., celecoxib, rofecoxib, valdecoxib, paracoxib, etoricoxib and lumiracoxib.
In particular, compounds of Formula (I) or a pharmaceutically acceptable salt thereof are of use in the prophylaxis or therapy of any disease state in a human, or other mammal, which is exacerbated by or caused by excessive or unregulated IL-1, IL-6, IL-8 or TNF production by such mammal's cell, such as, but not limited to, monocytes and/or macrophages.
Accordingly, in another aspect, this invention relates to a method of inhibiting the production of IL-1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
There are many disease states in which excessive or unregulated IL-1 production is implicated in exacerbating and/or causing the disease. These include rheumatoid arthritis, osteoarthritis, meningitis, ischemic and hemorrhagic stroke, neurotrauma/closed head injury, stroke, endotoxemia and/or toxic shock syndrome, other acute or chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, multiple sclerosis, cachexia, bone resorption, psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis, rubella arthritis and acute synovitis. Recent evidence also links IL-1 activity to diabetes, pancreatic β cell diseases and Alzheimer's disease.
Use of a CSAID inhibitor compound for the treatment of CSBP mediated disease states, can include, but not be limited to neurodegenerative diseases, such as Alzheimer's disease (as noted above), Parkinson's disease and multiple sclerosis, etc.
In a further aspect, this invention relates to a method of inhibiting the production of TNF in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
Excessive or unregulated TNF production has been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, chronic pulmonary inflammatory disease and chronic obstructive pulmonary disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, such as osteoporosis, cardiac, brain and renal reperfusion injury, graft vs. host reaction, allograft rejections, fever and myalgias due to infection, such as influenza, brain infections including encephalitis (including HIV-induced forms), cerebral malaria, meningitis, ischemic and hemorrhagic stroke, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation, inflammatory bowel disease, Crohn's disease, ulcerative colitis and pyresis.
Compounds of Formula (I) are also useful in the treatment of viral infections, where such viruses are sensitive to upregulation by TNF or will elicit TNF production in vivo. The viruses contemplated for treatment herein are those that produce TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirectly, by the TNF inhibiting-compounds of Formula (I). Such viruses include, but are not limited to HIV-1, HIV-2 and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex. Accordingly, in a further aspect, this invention relates to a method of treating a mammal afflicted with a human immunodeficiency virus (HIV) which comprises administering to such mammal an effective TNF inhibiting amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
It is also recognized that both IL-6 and IL-8 are produced during rhinovirus (HRV) infections and contribute to the pathogenesis of common cold and exacerbation of asthma associated with HRV infection (Turner et al. (1998), Clin. Infec. Dis., Vol. 26, p 840; Teren et al. (1997), Am. J. Respir. Crit. Care Med., Vol. 155, p 1362; Grunberg et al. (1997), Am. J. Respir. Crit. Care Med. Vol. 156, p 609 and Zhu et al, J. Clin. Invest (1996), 97:421). It has also been demonstrated in vitro that infection of pulmonary epithelial cells with HRV results in production of IL-6 and IL-8 (Subauste et al., J. Clin. Invest. 1995, 96:549.) Epithelial cells represent the primary site of infection of HRV. Therefore another aspect of the present invention is a method of treatment to reduce inflammation associated with a rhinovirus infection, not necessarily a direct effect on virus itself.
Compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than in humans, in need of inhibition of TNF production. TNF mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples of such viruses include, but are not limited to, lentivirus infections such as, equine infectious anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections, such as but not limited to feline immunodeficiency virus (FIV), bovine immunodeficiency virus, or canine immunodeficiency virus or other retroviral infections.
The compounds of Formula (I) may also be used topically in the treatment or prophylaxis of topical disease states mediated by or exacerbated by excessive cytokine production, such as by IL-1 or TNF respectively, such as inflamed joints, eczema, psoriasis and other inflammatory skin conditions such as sunburn; inflammatory eye conditions including conjunctivitis; pyresis, pain and other conditions associated with inflammation. Periodontal disease has also been implemented in cytokine production, both topically and systemically. Hence use of compounds of Formula (I) to control the inflammation associated with cytokine production in such peroral diseases such as gingivitis and periodontitis is another aspect of the present invention.
Compounds of Formula (I) have also been shown to inhibit the production of IL-8 (Interleukin-8, NAP). Accordingly, in a further aspect, this invention relates to a method of inhibiting the production of IL-8 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
There are many disease states in which excessive or unregulated IL-8 production is implicated in exacerbating and/or causing the disease. These diseases are characterized by massive neutrophil infiltration such as, psoriasis, inflammatory bowel disease, asthma, cardiac, brain and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and glomerulonephritis. All of these diseases are associated with increased IL-8 production which is responsible for the chemotaxis of neutrophils into the inflammatory site. In contrast to other inflammatory cytokines (IL-1, TNF, and IL-6), IL-8 has the unique property of promoting neutrophil chemotaxis and activation. Therefore, the inhibition of IL-8 production would lead to a direct reduction in the neutrophil infiltration.
The compounds of Formula (I) are administered in an amount sufficient to inhibit cytokine, in particular IL-1, IL-6, IL-8 or TNF, production such that it is regulated down to normal levels, or in some case to subnormal levels, so as to ameliorate or prevent the disease state. Abnormal levels of IL-1, IL-6, IL-8 or TNF, for instance in the context of the present invention, constitute: (i) levels of free (not cell bound) IL-1, IL-6, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) any cell associated IL-1, IL-6, IL-8 or TNF; or (iii) the presence of IL-1, IL-6, IL-8 or TNF mRNA above basal levels in cells or tissues in which IL-1, IL-6, IL-8 or TNF, respectively, is produced.
The discovery that the compounds of Formula (I) are inhibitors of cytokines, specifically IL-1, IL-6, IL-8 and TNF is based upon the effects of the compounds of Formulas (I) on the production of the IL-1, IL-8 and TNF in in vitro assays which are described herein.
As used herein, the term “inhibiting the production of IL-1 (IL-6, IL-8 or TNF)” refers to:
a) a decrease of excessive in vivo levels of the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels by inhibition of the in release of the cytokine by all cells, including but not limited to monocytes or macrophages;
b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels;
c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL-1, IL-6, IL-8 or TNF) as a postranslational event; or
d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels.
As used herein, the term “TNF mediated disease or disease state” refers to any and all disease states in which TNF plays a role, either by production of TNF itself, or by TNF causing another monokine to be released, such as but not limited to IL-1, IL-6 or IL-8. A disease state in which, for instance, IL-1 is a major component, and whose production or action, is exacerbated or secreted in response to TNF, would therefore be considered a disease stated mediated by TNF.
As used herein, the term “cytokine” refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response. A cytokine includes, but is not limited to, monokines and lymphokines, regardless of which cells produce them. For instance, a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte. Many other cells however also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-lymphocytes. Lymphokines are generally referred to as being produced by lymphocyte cells. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-α) and Tumor Necrosis Factor beta (TNF-β).
As used herein, the term “cytokine interfering” or “cytokine suppressive amount” refers to an effective amount of a compound of Formula (I) which will cause a decrease in the in vivo levels of the cytokine to normal or sub-normal levels, when given to a patient for the prophylaxis or treatment of a disease state which is exacerbated by, or caused by, excessive or unregulated cytokine production.
As used herein, the cytokine referred to in the phrase “inhibition of a cytokine, for use in the treatment of a HIV-infected human” is a cytokine which is implicated in (a) the initiation and/or maintenance of T cell activation and/or activated T cell-mediated HIV gene expression and/or replication and/or (b) any cytokine-mediated disease associated problem such as cachexia or muscle degeneration.
As TNF-β (also known as lymphotoxin) has close structural homology with TNF-α (also known as cachectin) and since each induces similar biologic responses and binds to the same cellular receptor, both TNF-α and TNF-β are inhibited by the compounds of the present invention and thus are herein referred to collectively as “TNF” unless specifically delineated otherwise.
A member of the MAP kinase family, alternatively termed CSBP, p38, or RK, has been identified independently by several laboratories. Activation of this novel protein kinase via dual phosphorylation has been observed in different cell systems upon stimulation by a wide spectrum of stimuli, such as physicochemical stress and treatment with lipopolysaccharide or proinflammatory cytokines such as interleukin-1 and tumor necrosis factor. The cytokine biosynthesis inhibitors, of the present invention, compounds of Formula (I) have been determined to be potent and selective inhibitors of CSBP/p38/RK kinase activity. It has been found that some of the compounds of Formula I exhibit reversible time-dependent inhibition of the p38 kinase due to the kinetics of slow binding and/or slow dissociation, resulting in an improved apparent IC50 when a compound has been preincubated with the enzyme or with cells. This slow, tight binding property may contribute to enhanced potency of such compounds both in vitro and in vivo.
These inhibitors are of aid in determining the signaling pathways involvement in inflammatory responses. In particular, for the first time a definitive signal transduction pathway can be prescribed to the action of lipopolysaccharide in cytokine production in macrophages. In addition to those diseases already noted, treatment of stroke, neurotrauma, cardiac and renal reperfusion injury, congestive heart failure, coronary arterial bypass grafting (CABG) surgery, chronic renal failure, angiogenesis & related processes, such as cancer, thrombosis, glomerulonephritis, diabetes and pancreatic β cells, multiple sclerosis, muscle degeneration, eczema, psoriasis, sunburn, and conjunctivitis are also included.
The CSBP inhibitors were subsequently tested in a number of animal models for anti-inflammatory activity. Model systems were chosen that were relatively insensitive to cyclooxygenase inhibitors in order to reveal the unique activities of cytokine suppressive agents. The inhibitors exhibited significant activity in many such in vivo studies. Most notable are its effectiveness in the collagen-induced arthritis model and inhibition of TNF production in the endotoxic shock model. In the latter study, the reduction in plasma level of TNF correlated with survival and protection from endotoxic shock related mortality. Also of great importance is the compounds effectiveness in inhibiting bone resorption in a rat fetal long bone organ culture system. Griswold et al., (1988) Arthritis Rheum. 31:1406-1412; Badger, et al., (1989) Circ. Shock 27, 51-61; Votta et al., (1994) in vitro. Bone 15, 533-538; Lee et al., (1993). B Ann. N.Y. Acad. Sci. 696, 149-170.
Chronic diseases which have an inappropriate angiogenic component are various ocular neovasularizations, such as diabetic retinopathy and macular degeneration, including age related macular degeneration. Other chronic diseases which have an excessive or increased proliferation of vasculature are tumor growth and metastasis, atherosclerosis, and certain arthritic conditions. Therefore CSBP kinase inhibitors will be of utility in the blocking of the angiogenic component of these disease states.
Additional ophthalmic disorders include retinitis, retinopathies, uveitis, ocular photophobia, acute injury to the eye tissue, corneal graft rejection, ocular neovascularization, retinal neovascularization (including neovascularization following injury or infection, retrolental fibroplasias, neovascular glaucoma, optic neuropathy, optic neuritis, retinal ischemia, laser induced optic damage, and surgery or trauma induced proliferative vitroretinopathy.
The term “excessive or increased proliferation of vasculature inappropriate angiogenesis” as used herein includes, but is not limited to, diseases which are characterized by hemangiomas and ocular diseases.
The term “inappropriate angiogenesis” as used herein includes, but is not limited to, diseases which are characterized by vesicle proliferation with accompanying tissue proliferation, such as occurs in cancer, metastasis, arthritis and atherosclerosis.
Accordingly, the present invention provides a method of treating a CSBP kinase mediated disease in a mammal in need thereof, preferably a human, which comprises administering to said mammal, an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
In order to use a compound of Formula (I) or a pharmaceutically acceptable salt thereof in therapy, it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice. This invention, therefore, also relates to a pharmaceutical composition comprising an effective, non-toxic amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.
Compounds of Formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation. The compounds of Formula (I) may be administered in conventional dosage forms prepared by combining a compound of Formula (I) with standard pharmaceutical carriers according to conventional procedures. The compounds of Formula (I) may also be administered in conventional dosages in combination with a known, second therapeutically active compound. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
A wide variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg. to about 1 g. When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
Compounds of Formula (I) may be administered topically, that is by non-systemic administration. This includes the application of a compound of Formula (I) externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. The active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It may however comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1% to 1% w/w of the formulation.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100° C. for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
Compounds of Formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. Compounds of Formula (I) may also be administered by inhalation, that is by intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
In one embodiment of the present invention, the agents of the present invention are delivered via oral inhalation or intranasal administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
For administration by inhalation the compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as tetrafluoroethane or heptafluoropropane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of for example gelatine or blisters of for example laminated aluminium foil, for use in an inhaler or insufflator. Powder blend formulations generally contain a powder mix for inhalation of the compound of the invention and a suitable powder base (carrier/diluent/excipient substance) such as mono-, di or poly-saccharides (e.g. lactose or starch). Use of lactose is preferred.
Each capsule or cartridge may generally contain between 20 μg-10 mg of the compound of formula (I) optionally in combination with another therapeutically active ingredient. Alternatively, the compound of the invention may be presented without excipients.
Suitably, the packing/medicament dispenser is of a type selected from the group consisting of a reservoir dry powder inhaler (RDPI), a multi-dose dry powder inhaler (MDPI), and a metered dose inhaler (MDI).
By reservoir dry powder inhaler (RDPI) it is meant an inhaler having a reservoir form pack suitable for comprising multiple (un-metered doses) of medicament in dry powder form and including means for metering medicament dose from the reservoir to a delivery position. The metering means may for example comprise a metering cup, which is movable from a first position where the cup may be filled with medicament from the reservoir to a second position where the metered medicament dose is made available to the patient for inhalation.
By multi-dose dry powder inhaler (MDPI) is meant an inhaler suitable for dispensing medicament in dry powder form, wherein the medicament is comprised within a multi-dose pack containing (or otherwise carrying) multiple, define doses (or parts thereof) of medicament. In a preferred aspect, the carrier has a blister pack form, but it could also, for example, comprise a capsule-based pack form or a carrier onto which medicament has been applied by any suitable process including printing, painting and vacuum occlusion.
In the case of multi-dose delivery, the formulation can be pre-metered (e.g. as in Diskus, see GB 2242134, U.S. Pat. Nos. 6,632,666, 5,860,419, 5,873,360 and 5,590,645 or Diskhaler, see GB 2178965, 2129691 and 2169265, U.S. Pat. Nos. 4,778,054, 4,811,731, 5,035,237, the disclosures of which are hereby incorporated by reference) or metered in use (e.g. as in Turbuhaler, see EP 69715 or in the devices described in U.S. Pat. No. 6,321,747 the disclosures of which are hereby incorporated by reference). An example of a unit-dose device is Rotahaler (see GB 2064336 and U.S. Pat. No. 4,353,656, the disclosures of which are hereby incorporated by reference).
The Diskus inhalation device comprises an elongate strip formed from a base sheet having a plurality of recesses spaced along its length and a lid sheet hermetically but peelably sealed thereto to define a plurality of containers, each container having therein an inhalable formulation containing a compound of formula (I) or (Ia) preferably combined with lactose. Preferably, the strip is sufficiently flexible to be wound into a roll. The lid sheet and base sheet will preferably have leading end portions which are not sealed to one another and at least one of the said leading end portions is constructed to be attached to a winding means. Also, preferably the hermetic seal between the base and lid sheets extends over their whole width. The lid sheet may preferably be peeled from the base sheet in a longitudinal direction from a first end of the said base sheet.
In one aspect, the multi-dose pack is a blister pack comprising multiple blisters for containment of medicament in dry powder form. The blisters are typically arranged in regular fashion for ease of release of medicament there from.
In one aspect, the multi-dose blister pack comprises plural blisters arranged in generally circular fashion on a disc-form blister pack. In another aspect, the multi-dose blister pack is elongate in form, for example comprising a strip or a tape.
In one aspect, the multi-dose blister pack is defined between two members peelably secured to one another. U.S. Pat. Nos. 5,860,419, 5,873,360 and 5,590,645 describe medicament packs of this general type. In this aspect, the device is usually provided with an opening station comprising peeling means for peeling the members apart to access each medicament dose. Suitably, the device is adapted for use where the peelable members are elongate sheets which define a plurality of medicament containers spaced along the length thereof, the device being provided with indexing means for indexing each container in turn. More preferably, the device is adapted for use where one of the sheets is a base sheet having a plurality of pockets therein, and the other of the sheets is a lid sheet, each pocket and the adjacent part of the lid sheet defining a respective one of the containers, the device comprising driving means for pulling the lid sheet and base sheet apart at the opening station.
By metered dose inhaler (MDI) it is meant a medicament dispenser suitable for dispensing medicament in aerosol form, wherein the medicament is comprised in an aerosol container suitable for containing a propellant-based aerosol medicament formulation. The aerosol container is typically provided with a metering valve, for example a slide valve, for release of the aerosol form medicament formulation to the patient. The aerosol container is generally designed to deliver a predetermined dose of medicament upon each actuation by means of the valve, which can be opened either by depressing the valve while the container is held stationary or by depressing the container while the valve is held stationary.
Where the medicament container is an aerosol container, the valve typically comprises a valve body having an inlet port through which a medicament aerosol formulation may enter said valve body, an outlet port through which the aerosol may exit the valve body and an open/close mechanism by means of which flow through said outlet port is controllable.
The valve may be a slide valve wherein the open/close mechanism comprises a sealing ring and receivable by the sealing ring a valve stem having a dispensing passage, the valve stem being slidably movable within the ring from a valve-closed to a valve-open position in which the interior of the valve body is in communication with the exterior of the valve body via the dispensing passage.
Typically, the valve is a metering valve. The metering volumes are typically from 10 to 100 μl, such as 25 μl, 50 μl or 63 μl. Suitably, the valve body defines a metering chamber for metering an amount of medicament formulation and an open/close mechanism by means of which the flow through the inlet port to the metering chamber is controllable. Preferably, the valve body has a sampling chamber in communication with the metering chamber via a second inlet port, said inlet port being controllable by means of an open/close mechanism thereby regulating the flow of medicament formulation into the metering chamber.
The valve may also comprise a ‘free flow aerosol valve’ having a chamber and a valve stem extending into the chamber and movable relative to the chamber between dispensing and non-dispensing positions. The valve stem has a configuration and the chamber has an internal configuration such that a metered volume is defined there between and such that during movement between is non-dispensing and dispensing positions the valve stem sequentially: (i) allows free flow of aerosol formulation into the chamber, (ii) defines a closed metered volume for pressurized aerosol formulation between the external surface of the valve stem and internal surface of the chamber, and (iii) moves with the closed metered volume within the chamber without decreasing the volume of the closed metered volume until the metered volume communicates with an outlet passage thereby allowing dispensing of the metered volume of pressurized aerosol formulation. A valve of this type is described in U.S. Pat. No. 5,772,085. Additionally, intra-nasal delivery of the present compounds is effective.
To formulate an effective pharmaceutical nasal composition, the medicament must be delivered readily to all portions of the nasal cavities (the target tissues) where it performs its pharmacological function. Additionally, the medicament should remain in contact with the target tissues for relatively long periods of time. The longer the medicament remains in contact with the target tissues, the medicament must be capable of resisting those forces in the nasal passages that function to remove particles from the nose. Such forces, referred to as ‘mucociliary clearance’, are recognised as being extremely effective in removing particles from the nose in a rapid manner, for example, within 10-30 minutes from the time the particles enter the nose.
Other desired characteristics of a nasal composition are that it must not contain ingredients which cause the user discomfort, that it has satisfactory stability and shelf-life properties, and that it does not include constituents that are considered to be detrimental to the environment, for example ozone depletors.
A suitable dosing regime for the formulation of the present invention when administered to the nose would be for the patient to inhale deeply subsequent to the nasal cavity being cleared. During inhalation the formulation would be applied to one nostril while the other is manually compressed. This procedure would then be repeated for the other nostril.
In one embodiment, the means for applying a formulation of the present invention to the nasal passages is by use of a pre-compression pump. Most preferably, the pre-compression pump will be a VP7 model manufactured by Valois SA. Such a pump is beneficial as it will ensure that the formulation is not released until a sufficient force has been applied, otherwise smaller doses may be applied. Another advantage of the pre-compression pump is that atomisation of the spray is ensured as it will not release the formulation until the threshold pressure for effectively atomising the spray has been achieved. Typically, the VP7 model may be used with a bottle capable of holding 10-50 ml of a formulation. Each spray will typically deliver 50-100 μl of such a formulation, therefore, the VP7 model is capable of providing at least 100 metered doses.
Spray compositions for topical delivery to the lung by inhalation may for example be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant. Aerosol compositions suitable for inhalation can be either a suspension or a solution and generally contain the compound of Formula (I) optionally in combination with another therapeutically active ingredient and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, especially 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxide or other suitable gas may also be used as propellant. The aerosol composition may be excipient free or may optionally contain additional formulation excipients well known in the art such as surfactants, e.g., oleic acid or lecithin and cosolvents, e.g. ethanol. Pressurised formulations will generally be retained in a canister (e.g. an aluminium canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.
Medicaments for administration by inhalation desirably have a controlled particle size. The optimum particle size for inhalation into the bronchial system is usually 1-10 μm, preferably 2-5 μm. Particles having a size above 20 μm are generally too large when inhaled to reach the small airways. To achieve these particle sizes the particles of the active ingredient as produced may be size reduced by conventional means e.g., by micronization. The desired fraction may be separated out by air classification or sieving. Suitably, the particles will be crystalline in form. When an excipient such as lactose is employed, generally, the particle size of the excipient will be much greater than the inhaled medicament within the present invention. When the excipient is lactose it will typically be present as milled lactose, wherein not more than 85% of lactose particles will have a MMD of 60-90 μm and not less than 15% will have a MMD of less than 15 μm.
Intranasal sprays may be formulated with aqueous or non-aqueous vehicles with the addition of agents such as thickening agents, buffer salts or acid or alkali to adjust the pH, isotonicity adjusting agents or anti-oxidants.
Solutions for inhalation by nebulization may be formulated with an aqueous vehicle with the addition of agents such as acid or alkali, buffer salts, isotonicity adjusting agents or antimicrobials. They may be sterilised by filtration or heating in an autoclave, or presented as a non-sterile product.
For all methods of use disclosed herein for the compounds of Formula (I), the daily oral dosage regimen will preferably be from about 0.05 to about 80 mg/kg of total body weight, preferably from about 0.1 to 30 mg/kg, more preferably from about 0.5 mg to 15 mg/kg, administered in one or more daily doses. The daily parenteral dosage regimen about 0.1 to about 80 mg/kg of total body weight, preferably from about 0.2 to about 30 mg/kg, and more preferably from about 0.5 mg to 15 mg/kg, administered in one or more daily doses. The daily topical dosage regimen will preferably be from 0.01 mg to 150 mg, administered one to four times daily. The daily inhalation dosage regimen will preferably be from about 0.05 microgram/kg to about 1 mg/kg per day, more preferably from about 0.2 microgram/kg to about 20 microgram/kg, administered in one or more daily doses. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of Formula (I) or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
The novel compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than humans, in need of inhibition of CSBP/p38 or cytokine inhibition or production. In particular, CSBP/p38 mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted herein in the Methods of Treatment section, but in particular viral infections. Examples of such viruses include, but are not limited to, lentivirus infections such as, equine infectious anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections, such as but not limited to feline immunodeficiency virus (FIV), bovine immunodeficiency virus, or canine immunodeficiency virus or other retroviral infections.
Another aspect of the present invention is a method of treating, the common cold or respiratory viral infection caused by human rhinovirus (HRV), other enteroviruses, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus in a human in need thereof which method comprises administering to said human an effective amount of a CBSP/p38 inhibitor.
Another aspect of the present invention is a method of treating, including prophylaxis, of influenza induced pneumonia in a human in need thereof which method comprises administering to said human an effective amount of a CBSP/p38 inhibitor
The present invention also relates to the use of the CSBP/p38 kinase inhibitor for the treatment, including prophylaxis, of inflammation associated with a viral infection of a human rhinovirus (HRV), other enteroviruses, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus.
In particular, the present invention is directed to the treatment of a viral infection in a human, which is caused by the human rhinovirus (HRV), other enterovirus, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or an adenovirus. In particular the invention is directed to respiratory viral infections that exacerbate asthma (induced by such infections), chronic bronchitis, chronic obstructive pulmonary disease, otitis media, and sinusitis. While inhibiting IL-8 or other cytokines may be beneficial in treating a rhinovirus may be known, the use of an inhibitor of the p38 kinase for treating HRV or other respiratory viral infections causing the common cold is believed novel.
It should be noted that the respiratory viral infection treated herein may also be associated with a secondary bacterial infection, such as otitis media, sinusitis, or pneumonia.
For use herein treatment may include prophylaxis for use in a treatment group susceptible to such infections. It may also include reducing the symptoms of, ameliorating the symptoms of, reducing the severity of, reducing the incidence of, or any other change in the condition of the patient, which improves the therapeutic outcome.
It should be noted that the treatment herein is not directed to the elimination or treatment of the viral organism itself but is directed to treatment of the respiratory viral infection that exacerbates other diseases or symptoms of disease, such as asthma (induced by such infections), chronic bronchitis, chronic obstructive pulmonary disease, otitis media, and sinusitis.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents, or those for inhalation may include carriers, such as lactose.
The compounds and pharmaceutical formulations according to the invention may be used in combination with or include one or more other therapeutic agents, for example selected from anti-inflammatory agents, anticholinergic agents (particularly an M1, M2, M1/M2 or M3 receptor antagonist), β2-adrenoreceptor agonists, antiinfective agents (e.g. antibiotics, antivirals), or antihistamines. The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof together with one or more other therapeutically active agents, for example selected from an anti-inflammatory agent (for example a corticosteroid or an NSAID), an anticholinergic agent, β2-adrenoreceptor agonist, an antiinfective agent (e.g. an antibiotic or an antiviral), or an antihistamine. One aspect of the present invention are combinations comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof together with a corticosteroid, and/or an anticholinergic, and/or a PDE-4 inhibitor. Preferred combinations are those comprising one or two other therapeutic agents.
It will be clear to a person skilled in the art that, where appropriate, the other therapeutic ingredient(s) may be used in the form of salts, (e.g. as alkali metal or amine salts or as acid addition salts), or prodrugs, or as esters (e.g. lower alkyl esters), or as solvates (e.g. hydrates) to optimise the activity and/or stability and/or physical characteristics (e.g. solubility) of the therapeutic ingredient. It will be clear also that where appropriate, the therapeutic ingredients may be used in optically pure form.
One suitable combination of the present invention comprises of compound of the invention together with a β2-adrenoreceptor agonist.
Examples of β2-adrenoreceptor agonists include salmeterol (which may be a racemate or a single enantiomer, such as the R-enantiomer), salbutamol, formoterol, salmefamol, fenoterol or terbutaline and salts thereof, for example the xinafoate salt of salmeterol, the sulphate salt or free base of salbutamol or the fumarate salt of formoterol. Long-acting β2-adrenoreceptor agonists are preferred, especially those having a therapeutic effect over a 24 hour period, such as salmeterol or formoterol.
Suitable long acting β2-adrenoreceptor agonists include those described in WO02/66422A, WO02/270490, WO02/076933, WO03/024439, WO03/072539, WO 03/091204, WO04/016578, WO04/022547, WO04/037807, WO04/037773, WO04/037768, WO04/039762, WO04/039766, WO01/42193 and WO03/042160, whose disclosures are incorporated by reference herein.
Preferred long-acting β2-adrenoreceptor agonists are:
Suitable anti-inflammatory agents include corticosteroids. Suitable corticosteroids which may be used in combination with the compounds of the invention are those oral and inhaled corticosteroids and their pro-drugs which have anti-inflammatory activity. Examples include methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, 6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-(1-methylcylopropylcarbonyl)oxy-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-(2,2,3,3-tetramethylcyclopropylcarbonyl)oxy-androsta-1,4-diene-17β-carboxylic acid cyanomethyl ester, beclomethasone esters (such as the 17-propionate ester or the 17,21-dipropionate ester), budesonide, flunisolide, mometasone esters (such as the furoate ester), triamcinolone acetonide, rofleponide, ciclesonide, (16α,17-[[(R)-cyclohexylmethylene]bis(oxy)]-11β,21-dihydroxy-pregna-1,4-diene-3,20-dione), butixocort propionate, RPR-106541, and ST-126. Preferred corticosteroids include fluticasone propionate, 6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester and 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester, more preferably 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester.
Non-steroidal compounds having glucocorticoid agonism that may possess selectivity for transrepression over transactivation and that may be useful in combination therapy include those covered in the following patents: WO03/082827, WO01/10143, WO98/54159, WO04/005229, WO04/009016, WO04/009017, WO04/018429, WO03/104195, WO03/082787, WO03/082280, WO03/059899, WO03/101932, WO02/02565, WO01/16128, WO00/66590, WO03/086294, WO04/026248, WO03/061651, WO03/08277.
Suitable anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAID's).
Suitable NSAID's include sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (for example, theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors), leukotriene antagonists, inhibitors of leukotriene synthesis (for example, montelukast), iNOS inhibitors, tryptase and elastase inhibitors, beta-2 integrin antagonists and adenosine receptor agonists or antagonists (for example, adenosine 2a agonists), cytokine antagonists (for example, chemokine antagonists, such as a CCR3 antagonist) or inhibitors of cytokine synthesis, or 5-lipoxygenase inhibitors. Suitable other β2-adrenoreceptor agonists include salmeterol (for example, as the xinafoate), salbutamol (for example, as the sulphate or the free base), formoterol (for example, as the fumarate), fenoterol or terbutaline and salts thereof. An iNOS (inducible nitric oxide synthase inhibitor) is preferably for oral administration. Suitable iNOS inhibitors include those disclosed in WO93/13055, WO98/30537, WO02/50021, WO95/34534 and WO99/62875. Suitable CCR3 inhibitors include those disclosed in WO02/26722.
Another embodiment of the invention is the use of the compound of a Formula (I) or (Ia) in combination with a phosphodiesterase 4 (PDE4) inhibitor or a mixed PDE3/PDE4 inhibitor. The PDE4-specific inhibitor useful in this aspect of the invention may be any compound that is known to inhibit the PDE4 enzyme or which is discovered to act as a PDE4 inhibitor, and which are only PDE4 inhibitors, not compounds which inhibit other members of the PDE family as well as PDE4. Generally it is preferred to use a PDE4 inhibitor which has an IC50 ratio of about 0.1 or greater as regards the IC50 for the PDE4 catalytic form which binds rolipram with a high affinity divided by the IC50 for the form which binds rolipram with a low affinity. For the purposes of this disclosure, the cAMP catalytic site which binds R and S rolipram with a low affinity is denominated the “low affinity” binding site (LPDE 4) and the other form of this catalytic site which binds rolipram with a high affinity is denominated the “high affinity” binding site (HPDE 4). This term “HPDE4” should not be confused with the term “hPDE4” which is used to denote human PDE4.
A method for determining IC50s ratios is set out in U.S. Pat. No. 5,998,428 which is incorporated herein in full by reference as though set out herein. See also PCT application WO 00/51599 for another description of said assay. In one embodiment, PDE4 inhibitors of use in this invention will be those compounds which have a salutary therapeutic ratio, i.e., compounds which preferentially inhibit cAMP catalytic activity where the enzyme is in the form that binds rolipram with a low affinity, thereby reducing the side effects which apparently are linked to inhibiting the form which binds rolipram with a high affinity. Another way to state this is that the compounds will have an IC50 ratio of about 0.1 or greater as regards the IC50 for the PDE4 catalytic form which binds rolipram with a high affinity divided by the IC50 for the form which binds rolipram with a low affinity.
A further refinement of this standard is that of one wherein the PDE4 inhibitor has an IC50 ratio of about 0.1 or greater; said ratio is the ratio of the IC50 value for competing with the binding of 1 nM of [3H]R-rolipram to a form of PDE4 which binds rolipram with a high affinity over the IC50 value for inhibiting the PDE4 catalytic activity of a form which binds rolipram with a low affinity using 1 μM[3H]-cAMP as the substrate.
Suitable PDE compounds are cis 4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylic acid, 2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one and cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol]; these are examples of compounds which bind preferentially to the low affinity binding site and which have an IC50 ratio of 0.1 or greater.
Other compounds of interest include: Compounds set out in U.S. Pat. No. 5,552,438 issued 3 Sep. 1996; this patent and the compounds it discloses are incorporated herein in full by reference. The compound of particular interest, which is disclosed in U.S. Pat. No. 5,552,438, is cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid (also known as cilomalast) and its salts, esters, pro-drugs or physical forms; AWD-12-281 from elbion (Hofgen, N. et al. 15th EFMC Int. Symp. Med. Chem. (September 6-10, Edinburgh) 1998, Abst. P.98; CAS reference No. 247584020-9); a 9-benzyladenine derivative nominated NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plough; a benzodiazepine PDE4 inhibitor identified as CI-1018 (PD-168787) and attributed to Pfizer; a benzodioxole derivative disclosed by Kyowa Hakko in WO99/16766; K-34 from Kyowa Hakko; V-11294A from Napp (Landells, L. J. et al. Eur Resp J [Annu Cong Eur Resp Soc (September 19-23, Geneva) 1998] 1998, 12 (Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3) and a pthalazinone (WO99/47505, the disclosure of which is hereby incorporated by reference) from Byk-Gulden; Pumafentrine, (−)-p-[(4aR*,10bS*)-9-ethoxy-1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[c][1,6]naphthyridin-6-yl]-N,N-diisopropylbenzamide which is a mixed PDE3/PDE4 inhibitor which has been prepared and published on by Byk-Gulden, now Altana; arofylline under development by Almirall-Prodesfarma; VM554/UM565 from Vernalis; or T-440 (Tanabe Seiyaku; Fuji, K. et al. J Pharmacol Exp Ther, 1998, 284(1): 162), and T2585. Other possible PDE-4 and mixed PDE3/PDE4 inhibitors include those listed in WO01/13953, the disclosure of which is hereby incorporated by reference.
Suitable anticholinergic agents are those compounds that act as antagonists at the muscarinic receptor, in particular those compounds which are antagonists of the M1 and M2 receptors. Exemplary compounds include the alkaloids of the belladonna plants as illustrated by the likes of atropine, scopolamine, homatropine, hyoscyamine; these compounds are normally administered as a salt, being tertiary amines. These drugs, particularly the salt forms, are readily available from a number of commercial sources or can be made or prepared from literature data via, to with:
Atropine—CAS-51-55-8 or CAS-51-48-1 (anhydrous form), atropine sulfate—CAS-5908-99-6; atropine oxide—CAS-4438-22-6 or its HCl salt—CAS-4574-60-1 and methylatropine nitrate—CAS-52-88-0; Homatropine—CAS-87-00-3, hydrobromide salt—CAS-51-56-9, methylbromide salt—CAS-80-49-9; Hyoscyamine (d,l)—CAS-101-31-5, hydrobromide salt—CAS-306-03-6 and sulfate salt—CAS-6835-16-1; and Scopolamine—CAS-51-34-3, hydrobromide salt—CAS-6533-68-2, methylbromide salt—CAS-155-41-9.
Suitable anticholinergics for use herein include, but are not limited to, ipratropium (e.g. as the bromide), sold under the name Atrovent, oxitropium (e.g. as the bromide) and tiotropium (e.g. as the bromide) (CAS-139404-48-1). Also of interest are: methantheline (CAS-53-46-3), propantheline bromide (CAS-50-34-9), anisotropine methyl bromide or Valpin 50 (CAS-80-50-2), clidinium bromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamide iodide (CAS-71-81-8), mepenzolate bromide (U.S. Pat. No. 2,918,408), tridihexethyl chloride (Pathilone, CAS-4310-35-4), and hexocyclium methylsulfate (Tral, CAS-115-63-9). See also cyclopentolate hydrochloride (CAS-5870-29-1), tropicamide (CAS-1508-75-4), trihexyphenidyl hydrochloride (CAS-144-11-6), pirenzepine (CAS-29868-97-1), telenzepine (CAS-80880-90-9), AF-DX 116, or methoctramine, and the compounds disclosed in WO 01/04118, the disclosure of which is hereby incorporated by reference.
Other suitable anticholinergics may be found in WO 2004/012684; WO2004/091482; WO2005/009439; WO2005/009362; WO2005/009440; WO2005/009362; WO2005/037224; WO2005/046586; WO2005/055940; WO2005/055941; WO2005/067537; WO2005/087236; WO2005/086873; WO2005/094835; WO2005/094834; WO2005/094251; WO2005/095407; WO2005/099706; WO2005/104745; WO2005/112644; WO2005/118594; WO2006/005057; WO2006/017768; WO2006/017767; WO2006/050239; WO2006/055553; WO2006/055503; WO2006/065755; WO2006/065788; WO2007/018514; WO2007/018508; WO2007/016650; WO2007/016639; and WO2007/022351.
Suitably, this includes the following exemplifications:
Suitable antihistamines (also referred to as Hi-receptor antagonists) include any one or more of the numerous antagonists known which inhibit Hi-receptors, and are safe for human use. All are reversible, competitive inhibitors of the interaction of histamine with Hi-receptors. The majority of these inhibitors, mostly first generation antagonists, have a core structure, which can be represented by the following formula:
This generalized structure represents three types of antihistamines generally available: ethanolamines, ethylenediamines, and alkylamines. In addition, other first generation antihistamines include those which can be characterized as based on piperizine and phenothiazines. Second generation antagonists, which are non-sedating, have a similar structure-activity relationship in that they retain the core ethylene group (the alkylamines) or mimic the tertiary amine group with piperizine or piperidine. Exemplary antagonists are as follows:
Ethanolamines: carbinoxamine maleate, clemastine fumarate, diphenylhydramine hydrochloride, and dimenhydrinate.
Ethylenediamines: pyrilamine amleate, tripelennamine HCl, and tripelennamine citrate.
Alkylamines: chloropheniramine and its salts such as the maleate salt, and acrivastine.
Piperazines: hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl, cyclizine lactate, meclizine HCl, and cetirizine HCl.
Piperidines: Astemizole, levocabastine HCl, loratadine or its descarboethoxy analogue, and terfenadine and fexofenadine hydrochloride or another pharmaceutically acceptable salt.
Azelastine hydrochloride is yet another H1 receptor antagonist which may be used in combination with a PDE4 inhibitor.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a physiologically acceptable diluent or carrier represent a further aspect of the invention.
The individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.
The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
The cytokine-inhibiting effects of compounds of the present invention may be determined by the following in vitro assays:
Assays for Interleukin-1 (IL-1beta), Interleukin-8 (IL-8), and Tumour Necrosis Factor (TNFalpha) are well known in the art, and may be found in a number of publications, and patents. Representative suitable assays for use herein are described in Adams et al., U.S. Pat. No. 5,593,992, whose disclosure is incorporated by reference in its entirety.
It is recognized that the respective assays herein may have been run multiple times for particular compounds of Formula (I) or (Ia), etc. as described herein. The determination of activity, as reported in these assays, will be based upon a mean or median of these values.
Human peripheral blood monocytes are isolated and purified from either fresh blood preparations from volunteer donors, or from blood bank buffy coats, according to the procedure of Colotta et al, J Immunol, 132, 936 (1984), or another suitable procedure such as positive selection selection using MACS CD14+ beads. These monocytes (1×106) are plated in 24, 48, 96 or 384-well plates at a concentration of 1-2 million/ml per well. The cells are allowed to adhere for 2 hours, after which time non-adherent cells can be removed by gentle washing. Test compounds are then added to the cells for 1 h before the addition of lipopolysaccharide (50±200 ng/ml), and the cultures are incubated at 37° C. for an additional 24 h. At the end of this period, culture supernatants are removed and clarified of cells and all debris. IL-1beta levels in the cell-free supernatant are then determined by enzyme-linked immunoassay (ELISA) or other antibody based procedure.
In order to evaluate in vivo inhibition of LPS-induced TNFα production in rodents, both mice or rats are injected with LPS.
Male Balb/c mice from Charles River Laboratories are pretreated (30 minutes) with compound or vehicle. After the 30 min. pretreat time, the mice are given LPS (lipopolysaccharide from Esherichia coli Serotype 055-B5, Sigma Chemical Co., St Louis, Mo.) 25 ug/mouse in 25 ul phosphate buffered saline (pH 7.0) intraperitoneally. Two hours later the mice are killed by CO2 inhalation and blood samples are collected by exsanguination into heparinized blood collection tubes and stored on ice. The blood samples are centrifuged and the plasma collected and stored at −20° C. until assayed for TNFα by ELISA.
Male Lewis rats from Charles River Laboratories are pretreated at various times with compound or vehicle. After a determined pretreat time, the rats are given LPS (lipopolysaccharide from Esherichia coli Serotype 055-B5, Sigma Chemical Co., St Louis, Mo.) 3.0 mg/kg intraperitoneally. The rats are killed by CO2 inhalation and heparinized whole blood is collected from each rat by cardiac puncture 90 minutes after the LPS injection. The blood samples are centrifuged and the plasma collected for analysis by ELISA for TNFα levels.
TNFα levels are measured using a sandwich ELISA, Olivera et al., Circ. Shock, 37, 301-306, (1992), whose disclosure is incorporated by reference in its entirety herein, using a hamster monoclonal antimurine TNFα (Genzyme, Boston, Mass.) as the capture antibody and a polyclonal rabbit antimurine TNFα (Genzyme) as the second antibody. For detection, a peroxidase-conjugated goat antirabbit antibody (Pierce, Rockford, Ill.) is added, followed by a substrate for peroxidase (1 mg/ml orthophenylenediamine with 1% urea peroxide). TNFα levels in the plasma samples from each animal are calculated from a standard curve generated with recombinant murine TNFα (Genzyme).
Assay: Test compound concentrations are prepared at 10× concentrations and LPS prepared at 1 ug/ml (final conc. of 50 ng/ml LPS) and added in 50 uL volumes to 1.5 mL eppendorf tubes. Heparinized human whole blood is obtained from healthy volunteers and was dispensed into eppendorf tubes or multiwell plates containing compounds and LPS in 0.2-0.4 mL volumes and the tubes incubated at 37 C. In some studies, compound is incubated with blood for up to 30 min prior to addition of LPS. Following a 4 hour incubation, the tubes or plates are centrifuged to remove cells and plasma is withdrawn and frozen at −80 C.
Cytokine measurement: IL-1beta and/or TNFalpha are quantified using a standardized ELISA, or similar technology. Concentrations of IL-1beta or TNFalpha are determined from standard curves of the appropriate cytokine and IC50 values for test compound (concentration that inhibited 50% of LPS-stimulated cytokine production) are calculated by linear regression analysis.
Compounds would be considered active in this assay if they demonstrated a IC50 of less than 10 uM up to about an IC50 of less than 0.0001 uM.
This assay measures the CSBP/p38-catalyzed transfer of 32P from [α-32P]ATP to threonine residue in an epidermal growth factor receptor (EGFR)-derived peptide (T669) with the following sequence: KRELVEPLTPSGEAPNQALLR (residues 661-681). (See Gallagher et al., “Regulation of Stress Induced Cytokine Production by Pyridinyl Imidazoles: Inhibition of CSBP Kinase”, BioOrganic & Medicinal Chemistry, 1997, 5, 49-64).
Reactions are carried in round bottom 96 well plate (from Corning) in a 30 ml volume. Reactions contained (in final concentration): 25 mM Hepes, pH 7.5; 8 mM MgCl2; 0.17 mM ATP (the Km[ATP] of p38 (see Lee et al., Nature 300, n72 pg. 639-746 (December 1994)); 2.5 uCi of [g-32P]ATP; 0.2 mM sodium orthovanadate; 1 mM DTT; 0.1% BSA; 10% glycerol; 0.67 mM T669 peptide; and 2-4 nM of yeast-expressed, activated and purified p38. Reactions are initiated by the addition of [gamma-32P]Mg/ATP, and incubated for 25 min. at 37° C. Inhibitors (dissolved in DMSO) are incubated with the reaction mixture on ice for 30 minutes prior to adding the 32P-ATP. Final DMSO concentration was 0.16%. Reactions are terminated by adding 10 ul of 0.3 M phosphoric acid, and phosphorylated peptide is isolated from the reactions by capturing it on p81 phosphocellulose filters. Filters are washed with 75 mM phosphoric acids, and incorporated 32P was quantified using beta scintillation counter. Under these conditions, the specific activity of p38 has been measured previously at 400-450 μmol/μmol enzyme, and the activity is linear for up to 2 hours of incubation. The kinase activity values are obtained after subtracting values generated in the absence of substrate which were 10-15% of total values.
The kinase enzyme, fluorescent ligand and a variable concentration of test compound are incubated together to reach thermodynamic equilibrium under conditions such that in the absence of test compound the fluorescent ligand is significantly (>50%) enzyme bound and in the presence of a sufficient concentration (>10×Ki) of a potent inhibitor the anisotropy of the unbound fluorescent ligand is measurably different from the bound value.
The concentration of kinase enzyme should preferably be >2×Kf. The concentration of fluorescent ligand required will depend on the instrumentation used, and the fluorescent and physicochemical properties. The concentration used must be lower than the concentration of kinase enzyme, and preferably less than half the kinase enzyme concentration.
The fluorescent ligand is the following compound:
which is derived from 5-[2-(4-aminomethylphenyl)-5-pyridin-4-yl-1H-imidazol-4-yl]-2-chlorophenol and rhodamine green.
Recombinant human p38a was expressed as a GST-tagged protein. To activate this protein, 3.5 μM unactivated p38a is incubated in 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% 2-mercaptoethanol, 0.1 mM sodium vanadate, 10 mM MgAc, 0.1 mM ATP with 200 nM MBP-MKK6 DD at 30 degrees for 30 mins. Following activation p38a is re-purified and the activity assessed using a standard filter-binding assay.
Protocol: All components are dissolved in buffer of composition 62.5 mM HEPES, pH 7.5, 1.25 mM CHAPS, 1 mM DTT, 12.5 mM MgCl2 with final concentrations of 12 nM p38a and 5 nM fluorescent ligand. 30 μl of this reaction mixture is added to wells containing 1 μl of various concentrations of test compound (0.28 nM-16.6 μM final) or DMSO vehicle (3% final) in NUNC 384 well black microtitre plate and equilibrated for 30-60 mins at room temperature. Fluorescence anisotropy is read in Molecular Devices Acquest (excitation 485 nm/emission 535 nm).
Ki=dissociation constant for inhibitor binding
Kf=dissociation constant for fluorescent ligand binding
This assay is the same as the standard volume assay but for the amount used in the protocol, for volume used and the plate type. It has been demonstrated that there is no difference in potency between the two formats, and that the assays are considered to be equivalent. The results described herein may have been performed in either assay format and are not differentiated as to which.
Low volume Protocol:
Protocol: All components are dissolved in buffer of composition 62.5 mM HEPES, pH 7.5, 1.25 mM CHAPS, 1 mM DTT, 12.5 mM MgCl2 with final concentrations of 12 nM p38a and 5 nM fluorescent ligand. 30 μl of this reaction mixture is added to wells containing 0.1 μl of various concentrations of test compound (0.02 nM-25 μM final) or DMSO vehicle (1.7% final) in Greiner low volume 384 well black microtitre plate and equilibrated for 30-60 mins at room temperature. Fluorescence anisotropy is read in Molecular Devices Acquest (excitation 485 nm/emission 535 nm).
Compounds are considered active in this assay if they demonstrate a pIC50 of greater than 4.6 up to about a pIC50 of 9.0.
Recombinant human p38a is expressed as a His-tagged protein. To activate this protein, 3 uM unactivated p38a is incubated in 200 mM Hepes pH 7.4, 625 mM NaCl, 1 mM DTT with 27 nM active MKK6 (Upstate), 1 mM ATP and 10 mM MgCl2. The activity of the MKK6-activated p38a is assessed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
Biotinylated-GST-ATF2 (residues 19-96, 400 nM final), ATP (125M final) and MgCl2 (5 mM final) in assay buffer (40 mM HEPES pH 7.4, 1 mM DTT) are added to wells containing 1 ul of various concentrations of compound or DMSO vehicle (3% final) in NUNC 384 well black plate. The reaction is initiated by the addition of MKK6-activated p38 (100 μM final) to give a total volume of 30 ul. The reaction is incubated for 120 minutes at room temperature, then terminated by the addition of 15 μl of 100 mM EDTA pH 7.4. Detection reagent (15 μl) in buffer (100 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w/v BSA, 1 mM DTT) containing antiphosphothreonine-ATF2-71 polyclonal antibody (Cell Signalling Technology, Beverly Mass., USA) labelled with W-1024 europium chelate (Wallac OY, Turku, Finland), and APC-labelled streptavidin (Prozyme, San Leandro, Calif., USA) is added and the reaction was further incubated for 60 minutes at room temperature. The degree of phosphorylation of GST-ATF2 is measured using a Packard Discovery plate reader (Perkin-Elmer Life Sciences, Pangbourne, UK) as a ratio of specific 665 nm energy transfer signal to reference europium 620 nm signal.
Recombinant human p38a is expressed as a His-tagged protein. To activate this protein, 3 uM unactivated p38a was incubated in 200 mM Hepes pH7.4, 625 mM NaCl, 1 mM DTT with 27 nM active MKK6 (Upstate), 1 mM ATP and 10 mM MgCl2. The activity of the MKK6-activated p38a is assessed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
Biotinylated-GST-ATF2 (residues 19-96, 400 nM final), ATP (125 uM final) and MgCl2 (5 mM final) in assay buffer (40 mM HEPES pH 7.4, 1 mM DTT) are added to wells containing 0.1 μl of various concentrations of compound or DMSO vehicle (1.7% final) in Greiner low volume 384 well black plate. The reaction is initiated by the addition of MKK6-activated p38a (100 μM final) to give a total volume of 6 μl. The reaction is incubated for 120 minutes at room temperature, then terminated by the addition of 3 μl of detection reagent in buffer (100 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w/v BSA, 1 mM DTT, 100 mM EDTA) containing antiphosphothreonine-ATF2-71 polyclonal antibody (Cell Signalling Technology, Beverly Mass., USA) labelled with W-1024 europium chelate (Wallac OY, Turku, Finland), and APC-labelled streptavidin (Prozyme, San Leandro, Calif., USA). The reaction is further incubated for 60 minutes at room temperature. The degree of phosphorylation of GST-ATF2 is measured using a BMG Rubystar plate reader (BMG, UK) as a ratio of specific 665 nm energy transfer signal to reference europium 620 nm signal.
It is noted that there are two assay formats shown above for the Time-resolved fluorescence resonance energy transfer kinase assay. The only difference between these two assays is the volume used and the plate type. It has been demonstrated that there is no difference in potency between the two formats, and that the assays are considered to be equivalent. The results described herein may have been performed in either assay format and are not differentiated as to which.
Compounds would be considered active in this assay if they demonstrated a pIC50 of greater than 4.6 up to about a pIC50 of greater than 10.0.
For purposes herein for the HTRF assay and the Fluorescence anisotropy kinase binding assay:
TNF-Stimulated IL-8 Production from Human Neutrophils
The effect of test compounds on TNF-stimulated IL8 production by human neutrophils is measured as follows. Neutrophils are prepared from blood obtained from consenting donors, using standard methods. Blood is collected in heparinized syringes and layered over histopaque (30 ml/20 ml). Following centrifugation, the red cell pellet is resuspended in PBS and purified over a dextran gradient. Red blood cells are lysed with water for 40 sec, remaining granulocytes collected by centrifugation and resuspended at 1.5×10̂6 cells/ml. Cells are added (0.5-1 ml) to 48 well plates already containing compound at 1000× final concentration in neat DMSO or 10% DMSO in RPMI1640 with 10% FBS. TNF (final concentration 100 ng/ml) is used as the stimulus. Cells incubated for approximately 20 hrs at 37° C., 5% CO2. Levels of IL-8 in the cell free supernatant are determined by sandwich ELISA, and inhibition relative to a control with DMPO but no compound is calculated.
Compounds would be considered active in this assay if they demonstrated an IC50 of less than 10 uM up to about an IC50 of less than 0.0001 uM, and are screened at concentrations up to 100 nM.
The effect of compounds on the influx of neutrophils to the lung in LPS-challenged rats is evaluated as follows. The test compound is suspended in one of the following solutions: 0.5% tween 80/PBS, 0.5% tween 80/saline, 10% EtOH/saline (with pH adjusted to 2.0, or 8.0 with HCl, or unadjusted), Saline @ pH 2.0, 6.5 or 8.0, 0.5% Tragacanth, 1% DMSO/20% Encapsin/Saline, or acidified 5% Tragacanth. The suspension process may be aided by the use of a glass homogenizer. For intratracheal administration, the animals are anesthetized with inhaled isoflurane and placed in a supine position, the trachea is intubated with a steel gavage needle (1.5 inch, 22 gauge, small ball) or a Penn-Century Microsprayer Aerosolizer (model IA-1B) and 200 ul of dosing solution is delivered. The animals are visually monitored during the recovery process, which typically occurs within two minutes.
Rats treated with compound or vehicle (15 min-24 hours pretreatment) are exposed to an LPS aerosol (100 ug/ml) for 15 min. Four hours later the rats are euthanized with pentobarbital (100 mg/kg, i.p.) and the airways are lavaged with 5 washes of 5 ml of phosphate buffered saline. The harvested cells are stained (Diffquick) and counted to determine total and differential cell data. In a typical study, macrophages represent 40-70% of the total cells, and polymorphonuclear cells 30-60% of the total cells. Inhibition of neutrophil levels relative to no compound controls is calculated based on the differential counts.
The assay has varying conditions, such as concentration, pretreat time, form of the compound (crystalline, amorphous, salts, micronised), and a wet or dry application of the compound. The data is obtained as % inhibition using a particular concentration and pretreat time. While a number of the compounds were found to be statistically nonsignificant (p>0.05), it is expected that upon retesting with either increasing concentrations, and/or a change in pretreat time that some of them may reach statistical significance (p<0.05).
Compounds would be considered active in this assay if they demonstrate statistically significant inhibition of neutrophilia in at least one of the range of conditions tested in this assay.
This assay provides for examination of the expression of tumor necrosis factor mRNA in specific brain regions which follow experimentally induced lateral fluid-percussion traumatic brain injury (TBI) in rats. Since TNF-α is able to induce nerve growth factor (NGF) and stimulate the release of other cytokines from activated astrocytes, this post-traumatic alteration in gene expression of TNF-α plays an important role in both the acute and regenerative response to CNS trauma. A suitable assay may be found in WO 97/35856 whose disclosure is incorporated herein by reference.
CNS Injury Model for IL-b mRNA
This assay characterizes the regional expression of interleukin-1β (IL-1β) mRNA in specific brain regions following experimental lateral fluid-percussion traumatic brain injury (TBI) in rats. Results from these assays indicate that following TBI, the temporal expression of IL-1β mRNA is regionally stimulated in specific brain regions. These regional changes in cytokines, such as IL-1β play a role in the post-traumatic pathologic or regenerative sequelae of brain injury. A suitable assay may be found in WO 97/35856 whose disclosure is incorporated herein by reference.
Described in WO 97/32583, whose disclosure is incorporated herein by reference, is an assay for determination of inflammatory angiogenesis which may be used to show that cytokine inhibition will stop the tissue destruction of excessive or inappropriate proliferation of blood vessels.
Cell lines, rhinovirus serotype 39, and influenza virus A/PR/8/34 are purchased from American Type Culture Collection (ATCC). BEAS-2B cells were cultured according to instructions provided by ATCC using BEGM (bronchial epithelial growth media) purchased from Clonetics Corp. HELA cell cultures, used for detection and titration of virus, are maintained in Eagle's minimum essential media containing 10% fetal calf serum, 2 mM 1-glutamine, and 10 mM HEPES buffer (MEM).
A modification of the method reported by Subauste et al., Supra, for in vitro infection of human bronchial epithelial cells with rhinovirus is used in these studies. BEAS-2B cells (2×105/well) were cultured in collagen-coated wells for 24 hours prior to infection with rhinovirus. Rhinovirus serotype 39 is added to cell cultures for one hour incubation at 34° C. after which inoculum is replaced with fresh media and cultures are incubated for an additional 72 hours at 34° C. Supernatants collected at 72 hours post-infection are assayed for cytokine protein concentration by ELISA using commercially available kits (R&D Systems). Virus yield was also determined from culture supernatants using a microtitration assay in HELA cell cultures (Subauste et al., supra 1995). In cultures treated with p38 kinase inhibitors, drug was added 30 minutes prior to infection. Stocks of compounds were prepared in DMSO (10 mM drug) and stored at −20° C.
For detection of p38 kinase, cultures are incubated in basal media without growth factors and additives to reduce endogenous levels of activated p38 kinase. Cells are harvested at various time points after addition of rhinovirus. Detection of tyrosine phosphorylated p38 kinase by immunoblot was analyzed by a commercially available kit and is performed according to the manufacturer's instructions (PhosphoPlus p38 MAPK Antibody Kit: New England BioLabs Inc.).
In some experiments, BEAS-2B cells are infected with influenza virus (strain A/PR/8/34) in place of rhinovirus. Culture supernatant is harvested 48 and 72 hour post-infection and tested by ELISA for cytokine as described above.
Cells and Virus: Influenza A/PR/8/34 sub type H1N1 (VR-95 American Type Culture Collection, Rockville, Md.) is grown in the allantoic cavity of 10 day old chicken eggs. Following incubation at 37° C., and refrigeration for 2½ hours at 4° C., allantoic fluid was harvested, pooled, and centrifuged (1,000 rcf; 15 min; 4° C.) to remove cells. Supernatent is aliquoted and stored at −70° C. The titer of the stock culture of virus is 1.0×1010 Tissue Culture Infective Dose/ml (TCID50)
Inoculation procedure: Four-six week old female Balb/cAnNcrlBr mice are obtained from Charles River, Raleigh, N.C. Animals were infected intranasally. Mice are anesthetized by intraperitioneal injection of Ketamine (40 mg/kg; Fort Dodge Labs, Fort Dodge, Ia) and Xylazine (5 mg/kg; Miles, Shawnee Mission, Ks) and then inoculated with 100 TCID50 of PR8 diluted in PBS in 20 ul. Animals are observed daily for signs of infection. All animal studies will be approved by the appropriate GSK Committee.
Virus titration: At various times post infection, animals are sacrificed and lungs are aseptically harvested. Tissues are homogenized, in vials containing 1 micron glass beads (Biospec Products, Bartlesville, Okla.) and 1 ml. of Eagles minimal essential medium. Cell debris is cleared by centrifugation at 1,000 rcf for 15 minutes at 4° C., and supernatants are serially diluted on Madin-Darby canine kidney (MDCK) cells. After 5 days of incubation at 37° C. (5% CO2), 50 μl of 0.5% chick red blood cells were added per well, and agglutination is read after 1 hour at room temperature. The virus titer is expressed as 50% tissue culture infective dose (TCID50) calculated by logistic regression.
ELISA: Cytokine levels are measured by quantitative ELISA using commercially available kits. Ear samples are homogenized using a tissue minser in PBS. Cell debris is cleared by centrifugation at 14,000 rpm for 5 minutes. The cytokine concentrations and thresholds are determined as described by the manufacturer; IL-6, IFN-γ, and KC(R&D Systems, Minneapolis, Minn.).
Myeloperoxidase Assay Myeloperoxidase (MPO) activity is determined kinetically as described by Bradley et al. (1982). Briefly, rabbit cornea are homogenized in Hexadecyl Trimethyl-Ammonium Bromide (HTAB) (Sigma Chemical Co. St. Louis, Mo.) which is dissolved in 0.5 m Potassium phosphate buffer (J. T. Baker Scientific, Phillipsburg, N.J.). Following homogenization, the samples are subjected to freeze-thaw-sonication (Cole-Parmer 8853, Cole-Parmer, Vernon Hills, Ill.) 3 times. Suspensions are then cleared by centrifugation at 12,500×g for 15 minutes at 4° C. MPO enzymatic activity is determined by colormetric change in absorbance during a reaction of O-Dianisidine dihydrochloride (ODI) 0.175 mg/ml (Sigma Chemical Co. St. Louis, Mo.) with 0.0002% Hydrogen peroxide (Sigma Chemical Co. St. Louis, Mo.). Measurements are performed by using a Beckman Du 640 Spectrophotometer (Fullerton, Calif.) fitted with a temperature control device. 50 ul of material to be assayed is added to 950 ul of ODI and change in absorbance is measured at a wave length of 460 nm for 2 minutes at 25° C.
Whole Body Plethysomography: Influenza virus infected mice are placed into a whole body plethysomograph box with an internal volume of approximately 350-ml. A bias airflow of one l/min is applied to the box and flow changes are measured and recorded with a Buxco XA data acquisition and respiratory analysis system (Buxco Electronics, Sharon, Conn.). Animals are allowed to acclimate to the plethysmograph box for 2 min. before airflow data is recorded. Airway measurements are calculated as Penh (enhanced pause). Penh has previously been shown as an index of airway obstruction and correlates with increased intrapleural pressure. The algorithm for Penh calculation is as follows: Penh=[(expiratory time/relaxation time)−1]×(peak expiratory flow/peak inspiratory flow) where relaxation time is the amount of time required for 70% of the tidal volume to be expired.
Determination of arterial oxygen saturation. A Nonin veterinary hand held pulse oximeter 8500V with lingual sensor (Nonin Medical, Inc., Plymouth Minn.) is used to determine daily arterial oxygen saturation % SpO2 as described (Sidwell et al. 1992 Antimicrobial Agents and Chemotherapy 36:473-476).
Suitably, a clinical trial to assess the use of a p38 kinase inhibitor for use in depression will be a randomised, double-blind, parallel-group with dosing of the active agent at a suitably acceptable dose against a placebo.
A second, higher dosage trial of the active agent can be initiated if a stronger cytokine level decrease is desired, and/or no-minimal clinical signal seen in the first dosing.
The duration of treatment should be approximately 6-weeks with visits: Weeks 0, 1, 2, 4, and 6. The patient population should be enriched at screening for symptoms of loss of energy & interest, fatigue, psychomotor retardation, and increased cytokine levels.
Follow up visits: 1 week after last dose (Week 7)
Sample size: approximately 30-40:20-30 (active:placebo)
Study assessment should be done using a Bayesian approach
One desired primary outcome is to assess if treatment with a p38 kinase inhibitor will reduce cytokine levels in the patient during a Major Depressive Episode. The desired secondary outcome is to evaluate the relationship between plasma cytokine levels, efficacy endpoints and drug exposure on:
Depression symptoms (HAM-D Bech, IDS-C and QIDS-SR)
Psychomotor retardation (Digit Symbol Test, item analysis from scales)
Fatigue (FACIT-F1) and on Sleep parameters (scores from scales plus LSEQ).
Additional data and assay modifications may be found in PCT/US00/25386, (WO 01/19322) filed 15 Sep. 2000, whose disclosure is incorporated herein by reference in its entirety.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Therefore, the Examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/071319 | 6/15/2007 | WO | 00 | 12/4/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60805033 | Jun 2006 | US |
