CN1304375C - Pyrazoloanthrone and derivatives thereof as JNK inhibitors and their compositions - Google Patents

Abstract

Compounds having activity as selective inhibitors of JNK are disclosed. The compounds of this invention are pyrazoloanthrone and derivatives thereof having structure (I) wherein R1 and R2 are as defined herein. Such compounds have utility in the treatment of a wide range of conditions that are responsive to JNK inhibition. Thus, methods of treating such conditions are also disclosed, as are pharmaceutical compositions containing one or more compounds of the above compounds.

Description

Pyrazoloanthrones and their derivatives as JNK inhibitors and their compositions

technical field

The present invention relates to pyrazoloanthrone and derivatives thereof, which have broad indications, including use as Jun N-terminal activating enzyme inhibitors, and related compositions and methods.

Background of the invention

The Jun N-terminal kinase (JNK) pathway is activated by exposure of cells to a stressful environment or cell therapy with pro-inflammatory cytokines, and the targets of the JNK channel include the transcription factors c-jun and ATF2 (Whitmarsh A.J. and Davis R.J.J Mol. Med. Journal of Molecular Medicine 74:589-607, 1996), these transcription factors are members of the basic leucine zipper family (bZIP), which binds to many AP-1 and AP-1-like sites of gene promoters (Karin M., Liu Z.G. and Zandi E. Curr Opin Cell Biol 9:240-246, 1997); within the activation region of each transcription factor, JNK binds to c-jun and the N-terminal site of ATF-2, and phosphorylate both sites (HibiM., Lin A., Smeal T., Minden A., Karin M. Genes Dev.7: 2135-21481993; Mohit A.A. , Martin M.H., and Miller C.A. Neuron 14:67-75, 199)]. Three JNK enzymes have been identified as products of different genes (Hibi et al., supra; Mohit et al., supra); 10 different isoforms of JNK have been identified, and these represent other spliced forms of three different genes: JNK1, JNK2 and JNK3. JNK1 and 2 are ubiquitously expressed in human tissues, while JNK3 is selectively expressed in brain, heart and testis tissues (Dong, C., Yang, D., Wysk, M., Whitmarsh, A., Davis, R., Flavell, R. Science, 270:1-4, 1998). When gene transcription is spliced in other ways, four JNK1 isoforms, four JNK2 isoforms and two JNK3 isoforms are produced. JNK1 and 2 are widely expressed in mammalian tissues, while JNK3 is almost exclusively expressed in the brain organization. Selectivity of JNK signaling is achieved through specific interactions of JNK pathway components and through the use of scaffolding proteins that selectively engage various components of the signaling cascade. JIP-1 (JNK-interaction-1) selectively engages the MAPK module, MLK→JNKK1→JNK.12,13. It has no binding affinity for other classes of MAPK cascade enzymes. For other MAPK signaling cascades, there are also different scaffolding proteins that maintain matrix specificity.

JNK classes are activated by dual phosphorylation on Thr-183 and Tyr-185. JNKK1 (also known as MKK 4) and JNKK2 (MKK7), two MAPKK-class enzymes that regulate the activation of JNK in cells (Lin A., Minden A., Martinetto H., Claret F.-Z., Lange-Carter C., Mercurlo F., Johnson G.L., and Karin M. Science, Science, 268:286-289, 1995; Tournier C., Whitmarsh A.J., Cavanagh J., Barrett T., and Davis R.J. Proc. Nat .Acad Sci.USA 94:7337-7342,1997). JNKK2 specifically phosphorylates JNK, while JNKK1 can also phosphorylate and activate p38. Both JNKK1 and JNKK2 are widely expressed in mammalian tissues. JNKK1 and JNKK2 are activated by the MAPKKK enzymes MEKK1 and 2 (Lange-Carter C.A., Pleiman C.M., Gardner A.M., Blumer K.J., and Johnson G.L. Science, Science, 260:315-319, 1993; Yan M., Dai J.C., Deak J.C., Kyriakis J.M., Zon L.I., Woodgett J.R., and Templeton D.J. Nature 372:798-781, 1994). Both MEKK1 and MEKK2 are widely expressed in mammalian tissues.

Activation of the JNK pathway has been documented in many disease settings, providing a rationale for targeting this pathway for drug discovery. In addition, molecular genetic approaches have identified the pathogenic role of this pathway in several diseases. For example, autoimmune and inflammatory diseases are caused by excessive activation of the immune system. Activated immune cells express many genes encoding inflammatory molecules, including cytokines, growth factors, cell surface receptors, cell adhesion molecules and degradative enzymes. Many of these genes are controlled through the JNK pathway, that is, by the transcription factors AP-1 and ATF-2, including TNFα, IL-2, E-selectin, and matrix metalloproteinases such as collagenase-1 ( Manning A.M. and Mercurio F. Exp Opin Invest Drugs 6:555-567, 1997). Monocytes, tissue macrophages, and tissue mast cells are the major sources of TNFa production, and in bacterial lipopolysaccharide-stimulated macrophages and FceRII receptor-stimulated mast cells, the JNK pathway controls TNFa production (Swantek J.L., Cobb M.H. , Geppert T.D.Mol.Cell.Biol.17:6274-6282, 1997; Ishizuka, T., Tereda N., Gerwins, P..Hamelmann E., Oshiba A., Fanger G.R., JohnsonG.L., and Gelfland E.W. Proc. Nat. Acad. Sci. USA 94:6358-6363, 1997). Inhibition of JNK activation effectively controls TNFa secretion in these cells, and thus the JNK pathway controls the production of this major pro-inflammatory cytokine. Matrix metalloproteinases (MMPs) promote cartilage and bone erosion in rheumatoid arthritis and tissue destruction in other autoimmune diseases. Inducible expression of MMPs, including MMP-3 and MMP-9, II and IV Collagenase, regulated by JNK pathway and activation of AP-1 (Gum, R., Wang.H..Lengyel, E..Juarez.J., and Boyd, D.Oncogene 14:1481-1493, 1997) . In human rheumatic synoviocytes activated by TNFa, IL-1 or Fas ligand, the JNK pathway is activated (Han Z., Boyle D.L.. Aupperle K.R.. Bennett B., Manning A.M., Firestein G.S.J Pharm.Exp.Therap.291:1-7, 1999; Okamoto K., Fujisawa K., Hasunuma T., Kobata T., Sumida T., and Nishioka K. Arth & Rheum40:919-92615, 1997), Inhibition Activation of JNK reduces AP-1 activation and collagenase-1 expression (Han et al., supra). The JNK pathway is thus able to control MMP expression in rheumatoid arthritis cells.

Inappropriate activation of T lymphocytes initiates and sustains many autoimmune diseases, including asthma, inflammatory bowel disease, and multiple sclerosis. The JNK pathway is activated in T cells due to antigen stimulation and CD28 receptor co-stimulation (co-stimulation), and controls the production of growth factor IL-2 and cell proliferation (Su B., Jacinto E..Hibi M .Kallunki T., Karin M., Ben-Neriah Y. Cell 77:727-736, 1994; Faris M., Kokot N., Lee L., and Nel A.E.J Biol. Chem. 271:27366-27373, 1996) . Peripheral T cells from mice genetically deficient in JNKK1 showed reduced proliferation and IL-2 production following CD28 co-stimulation and PMA/Ca2+ electrolyte activation, providing insight into the role of the JNK pathway in these cells. important evidence. (Nishina H., Bachmann M., Oliveria-dos-Santos A.J., et al. JExp. Med 186:941-953, 1997). It is known that T cells activated by antigen receptors lose the ability to synthesize IL-2 in the absence of helper cell-derived co-stimulatory signals, a state known as clonal anergy. This is an important method by which auto-reactive T cell populations are removed from the peripheral circulation. Apparently, anergic T cells fail to activate the JNK pathway in response to CD3- and CD28-receptor co-stimulation, even though JNK enzyme expression is unchanged (Li W., Whaley C.D., Mondino A., and Mueller D.L. Science, 271:1272-1276, 1996). Recently, tests of JNK-deficient mice have shown that the JNK pathway plays a key role in T cell activation and differentiation into type 1 and type 2 T helper cells. JNK1 or JNK2 disrupt normal development in mice without being apparent outwardly. Activated naive CD4+ T cells from these mice failed to produce IL-2 and did not proliferate well (Sabanathy, K, Hu, Y, Kallunki, T, Schreiber, M, David, J-P. Jochum, W, Wagner, E, Karin, M. Curr Biol 9:116-125, 1999). T cells from these mice can induce the differentiation of T cells to generate Th1 cells (generators of IFN-g and TNFβ) and Th2 effector cells (IL-4, IL-5, IL-6, IL-10 and Generator of IL-13) [22, 23]. Deletion of either JNK1 or JNK2 in mice results in a selective defect in the ability of Th1 effector cells to express IFNg, suggesting that JNK1 and JNK2 have no redundant functions in T cells, and they play a role in controlling cell growth, differentiation and death. with different effects. Therefore, the JNK pathway is important for regulating T cell responses to antigens.

Cardiovascular disease (CVD) accounts for nearly a quarter of all annual deaths worldwide. Vascular diseases such as atherosclerosis and restenosis are due to irregular growths in vessel walls that restrict blood flow to vital organs. The JNK pathway is activated by atherosclerosis-promoting factors and controls local cytokine and growth factor production in vascular cells (Yang, DD, Conze, D, Whitmarsh, AJ, et al., Immunity, 9:575, 1998) . In addition, changes in blood flow, hemodynamics, and blood flow lead to activation of JNK in the vascular endothelium, causing AP-1 activation and expression of pro-atherosclerotic genes (Aspenstrom P., Lindberg U., and Hall A Curr. Biol 6:70-77, 1996). Ischemia of the heart, kidney, and brain, and ischemia associated with reperfusion, leads to cell death and scarring, which can eventually lead to congestive heart failure, renal failure, and brain dysfunction. During organ transplantation, reperfusion of a previously ischemic donor organ results in acute leukocyte-mediated tissue injury and delays graft function. JNK pathway is activated by ischemia and reperfusion (Li Y., Shyy J., Li S., Lee J., Su B., Karin M.. Chien S Mol. Cell. Biol. 16:5947-5954, 1996), leading to activation of JNK-responsive genes and leukocyte-mediated tissue damage. In many different contexts, JNK activation can be pro-apoptotic or anti-apoptotic (pro-or anti-apoptotic), in cardiac tissue after ischemia and reperfusion, JNK activation Associated with increased apoptosis (Pombo CM. Bonventre JV, Avruch J, Woodgett JR, Kyriakis J.M, Force T.J. Biol Chem. 269:26546-26551, 1994).

Cancer is characterized by the uncontrolled growth, proliferation and migration of cells. Cancer is the second leading cause of death with 500,000 deaths in the United States in 1996 and an estimated 1.3 million new patients. It is generally accepted that signaling pathways play a role in cellular transformation and cancer. The JNK pathway leading to AP-1 appears to play a decisive role in cancer. c-jun expression is altered in early lung cancer and may mediate growth factor signaling in non-small cell lung cancer (Yin T., Sandhu G., Wolfgang C.D., Burrier A., Webb R.L.. Rigel D.F. Hai T., and Whelan J.J BioL Chem. 272:19943-19950, 1997). Indeed, over-expression of c-jun in cells leads to transformational variation, and blocking c-jun activity inhibits MCF-7 colony formation (Szabo E., Riffe M., Steinberg SM., Birrer M.J., Linnoila R.I. Cancer Res. Cancer Research, 56:305-315.1996). DNA destructive agents, ionizing radiation, and tumor necrosis factor can all activate the JNK pathway. In addition to controlling the production and activation of c-jun, activation of JNK can control the phosphorylation of p53 and thus regulate the cell cycle progression (Chen T.K., Smith L.M., Gebhardt D.K.. Birrer M.J., Brown P.H.Mol. Carcinogenesis 15:215-226, 1996). The oncogene BCR-Ab1, associated with the t(9,22) Philadelphia chromosome translocation of chronic myelogenous leukemia, can activate JNK and lead to transformational mutation of hematopoietic cells (Milne D.M., Campbell L.E., Campbell D.G., Meek D.W.J BioLChem.270 : 5511-5518, 1995). Selective inhibition of JNK activation by a naturally-occurring JNK inhibitory protein, termed JIP-1, blocks cell transformation induced by BCR-Ab1 expression (Raitano A.B., Halpern J.R., Hambuch T.M., Sawyers C.L. Proc. Nat . Acad. Sci USA 92: 11746-11750, 1995). Thus JNK inhibitors can block transformational mutations and tumor cell growth.

Accordingly, there is a need in the art for selective inhibitors of JNK, as well as methods for their preparation, pharmaceutical compositions containing these inhibitors, and methods of inhibiting JNK and of treating diseases in mammals responsive to JNK inhibition. The present invention fulfills these needs and further provides related advantages.

Summary of the invention

Briefly, the present invention relates to compounds having JNK selective inhibitor activity and related compositions and methods, the compounds of the present invention (also referred to herein as "JNK inhibitors") can be generally classified as Ketone derivatives" have the following structure (I):

Wherein R ₁ and R ₂ are as defined below, including their pharmaceutically acceptable salts.

The present invention also relates to methods of treating various diseases by administering an effective amount of a JNK inhibitor to animals and subjects in need thereof (referred to herein as "patients"), typically warm-blooded animals (including humans). Prior to administration, the compounds of the present invention are preferably formulated as pharmaceutical compositions containing effective doses of one or more JNK inhibitors in admixture with one (or more) pharmaceutically acceptable carriers. With the compound of the present invention or the pharmaceutical composition containing the compound of the present invention, the diseases that can be treated include any disease that can benefit from the administration of JNK inhibitors, and the various diseases that are particularly suitable for prevention and/or treatment include (but not limited to) rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gout, asthma, bronchitis, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, mucous colitis, ulcerative colitis, Rohn's disease, gastritis, esophagitis, hepatitis, multiple sclerosis, atherosclerosis, restenosis after angioplasty, left ventricular hypertrophy, myocardial infarction, stroke, cardiac, renal, hepatic or Brain injury, transplant rejection, endotoxic shock, psoriasis, eczema, dermatitis, epilepsy, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, peripheral neuropathy, spinal cord injury, Parkinson's disease, and cancer.

With reference to the following detailed description, these contents and other aspects of the present invention will be clearer. For this reason, some patents and other documents are cited herein to more specifically illustrate various aspects of the present invention. Therefore, these documents are incorporated by reference in their entirety. This article.

Brief description of the drawings

Figure 1 illustrates the ability of representative compounds of the invention to inhibit IL-2 in Jurkat T-cells.

Figure 2 illustrates the ability of representative compounds of the invention to inhibit TNF-α in a mouse model of endotoxic shock.

Figure 3 illustrates the ability of representative compounds of the invention to inhibit leukocyte recruitment in a mouse model of pneumonia.

Accompanying drawing 4 illustrates that representative compound of the present invention inhibits paw swelling (accompanying drawing 4A), joint damage (accompanying drawing 4B), activation of transcription factor AP-1 (accompanying drawing 4C) in adjuvantarthrisis (adjuvantarthrisis) mouse model ) and MMP-13 expression (Fig. 4D).

Figure 5 illustrates the ability of representative compounds of the invention to reduce kainic acid-induced seizure responses.

Detailed description of the invention

As mentioned above, the present invention relates to a compound having activity as a selective inhibitor of JNK and compositions and methods related to the compound. The compound of the present invention has the following structure (I):

and their pharmaceutically acceptable salts, of which:

R _{and R} are optional identical or different substituents and independently represent hydrocarbyl, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl, hydrocarbyloxycarbonyl, hydrocarbyloxy, aryl, aryloxy Alkyl, aryloxy, arylalkyl, cycloalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyloxy, aminoalkoxy, mono- or di-hydrocarbylaminoalkoxy , or a group represented by formula (a), (b), (c) or (d):

R ₃ and R ₄ together represent an alkylene group or an alkylene group containing a heteroatom, or R ₃ and R ₄ are the same or different and each independently represents hydrogen, hydrocarbyl, cyclohydrocarbyl, aryl, aryl hydrocarbyl, cyclohydrocarbyl hydrocarbyl , aryloxyalkyl, alkoxyalkyl, alkoxyamino or alkoxy (mono- or di-hydrocarbylamino); and

_R represents hydrogen, hydrocarbyl, cycloalkyl, aryl, arylhydrocarbyl, cyclohydrocarbyl, hydrocarbyloxy, amino, mono- or di-hydrocarbylamino, arylamino, arylhydrocarbylamino, cyclohydrocarbylamino or cyclohydrocarbyl Hydrocarbylamino.

The terms used above shall have the following meanings herein,

"Hydrocarbyl" refers to a straight or branched, saturated or unsaturated hydrocarbon chain of 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , tert-butyl, propenyl, 1-butenyl, propynyl, etc.

"Halogen" means fluorine, chlorine, bromine or iodine.

"Trifluoromethyl" refers to _-CF3 .

"Sulfonyl" means _-SO3H .

"Carboxy" means -COOH.

"Alkyloxy" means -O-(hydrocarbyl), such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and the like.

"Alkyloxyalkoxy" refers to -O-( _hydrocarbyl )-O-( _hydrocarbyl ), such as _-OCH2CH2OCH3 and the like.

"Oxycarbonyl" refers to -C(=O)O(hydrocarbyl), such as -C(=O) _OCH3 , -C(=O) _{OCH2CH3 ,} and the like.

"Oxyalkyl" refers to -(hydrocarbyl)-O-(hydrocarbyl), such as -CH ₂ OCH ₃ , -CH ₂ OCH ₂ CH ₃ and the like.

"Aryl" means a carbocyclic or heterocyclic aromatic group containing 5-10 ring atoms. The ring atoms of the carbocyclic aromatic group are all carbon atoms, including phenyl and naphthyl; the ring atoms of the heterocyclic aromatic group contain at least one heteroatom selected from nitrogen, oxygen and sulfur, including pyridyl, pyrimidyl, furyl, Thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl and indolyl.

"Aryloxy" means -O-(aryl), such as -O-phenyl, -O-pyridyl, and the like.

"Arylalkyl" means -(hydrocarbyl)-(aryl), such as benzyl (ie, _-CH2phenyl ), _-CH2 -pyridyl, and the like.

"Aryloxy" means -O-(hydrocarbyl)-(aryl), eg -O-benzyl, -O- _CH2 -pyridyl, and the like.

"Cycloalkyl" means a cyclic hydrocarbon group having 3 to 7 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl and the like.

"Cycloalkyloxy" means -O-(cycloalkyl), such as -O-cyclohexyl and the like.

"Cycloalkyloxy" refers to -O-(hydrocarbyl)-(cycloalkyl), such as -O- _CH2 -cyclohexyl and the like.

"Alkylene" refers to a divalent group -C _n H _2n -, wherein n is an integer from 1 to 8, such as -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, - _{CH2CH2CH2CH2- , -CH2CH2CH2CH2CH2- , etc. _ _ _ _}

"Heteroatom-containing alkylene" refers to an alkylene group in which at least _{one carbon} atom is replaced by a heteroatom selected from nitrogen, oxygen and sulfur, such as _{-CH2CH2OCH2CH2-} and the like _.

"Aminoalkoxy" refers to -O-(hydrocarbyl) _-NH2 , such as _-OCH2NH2 , _-OCH2CH2NH2 , and _{the like .}

"Mono- or di-hydrocarbylamino" refers to -NH(hydrocarbyl) or -N(hydrocarbyl)(hydrocarbyl), respectively, such as -NHCH ₃ , -N(CH ₃ ) ₂ and the like.

"One- or two-hydrocarbyl aminooxy" means -O-(hydrocarbyl)-NH(hydrocarbyl) or -O-(hydrocarbyl)-N(hydrocarbyl)(hydrocarbyl), respectively, such as -OCH ₂ NHCH ₃ ,- OCH ₂ CH ₂ N(CH ₃ ) ₂ and so on.

"Arylamino" means -NH(aryl), such as -NH-phenyl, -NH-pyridyl, and the like.

"Arylalkylamino" means NH-(hydrocarbyl)-(aryl), such as -NH-benzyl, _-NHCH2 -pyridyl, and the like.

"Hydroxyamino" means -NH(hydrocarbyl), such as _-NHCH3 , _{-NHCH2CH3 ,} and the like.

"Cycloalkylamino""means -NH-(cycloalkyl), such as -NH-cyclohexyl and the like.

"Cycloalkylalkylamino" means -NH-(hydrocarbyl)-(cycloalkyl), such as -NHCH ₂ cyclohexyl and the like.

In embodiments where R _{and R} are absent, compounds of the invention have the following structure (II) (also referred to herein as "Compound 1"):

This compound is commercially available from Pfaltz-Bauer (Conn., U.S.).

In embodiments where only one _of R and _R is present, the compounds of the invention have one of the following structures (III) or (IV):

In embodiments where _both R and _R are present, the compounds of the invention have one of the following structures (V), (VI) or (VII):

The pharmaceutically acceptable salts of the compounds of formula (I) are also within the scope of the present invention. For this reason, the compounds are usually used in the form of free bases, and the compounds can also be used in the form of acid addition salts. The acid addition salts of the free base amino compounds of this invention may be prepared by methods known in the art and may be formed from organic or inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamon acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid and benzenesulfonic acid; suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric and nitric acids. Accordingly, the term "pharmaceutically acceptable salt" of a compound of formula (I) includes any and all acceptable salt forms.

The compounds of the present invention can generally be prepared according to organic synthesis methods known to those skilled in the art, and can also be prepared according to the following general methods or the methods described in the examples. therefore,

The compounds of the present invention can be prepared according to the following reaction schemes 1-7.

Reaction Line 1

In reaction scheme 1, appropriate anthraquinone and hydrazine are condensed in a suitable solvent (such as pyridine, dimethylformamide, dichloromethane, chloroform or dioxane), and the pyrazoloanthracene of the present invention can be prepared Ketone, the anthraquinone has a leaving group (such as fluorine, chlorine, bromine, iodine, nitro, methanesulfonyloxy, tosyloxy or phenoxy) at the 1-position. The reaction is carried out at a temperature ranging from 0°C to 200°C for 1-16 hours. Suitable anthraquinone starters are commercially available from various sources, having _R1 and/or _R2 groups at various positions on the anthraquinone ring. For illustrative purposes, the following reaction schemes describe only the synthesis of 5- and/or 7-substituted pyrazoloanthrones, those skilled in the art will recognize that pyrazoloanthrones substituted at other positions can also Prepared in an analogous manner from appropriately substituted anthraquinone starting materials.

Reaction Line 2

In reaction scheme 2, 5-chloropyrazoloanthrone and mono- or di-substituted amine are subjected to condensation reaction at 0-250°C for 1-16 hours without or in the presence of a solvent to prepare Pyrazoloanthrones with 5-amino substituents. Commonly used solvents are pyridine, dimethylformamide in the presence of excess amine, or in the presence of acid quenchers such as triethylamine, diisopropylethylamine, sodium bicarbonate, potassium carbonate, or sodium hydroxide , dimethylsulfoxide, dichloroethane, chloroform, tetrahydrofuran, dioxane, diglyme or triglyme.

Reaction Line 3

In reaction scheme 3, 7-chloropyrazoloanthrone and mono- or di-substituted amine are subjected to condensation reaction at 0-250 °C for 1-16 hours without or in the presence of a solvent to prepare Pyrazoloanthrones with 7-amino substituents. Commonly used solvents are pyridine, dimethylformamide in the presence of excess amine, or in the presence of acid quenchers such as triethylamine, diisopropylethylamine, sodium bicarbonate, potassium carbonate, or sodium hydroxide , dimethylsulfoxide, dichloroethane, chloroform, tetrahydrofuran, dioxane, diglyme or triglyme.

Reaction Line 4

In reaction scheme 4, 5-amino-2-(2-methoxyethoxymethyl)pyrazoloanthrone is condensed with acid chloride or sulfonyl chloride, and then deprotected to prepare 5-acyl- or Pyrazoloanthrones with Sulfonylamino Substituents. Condensation reaction of 5-amino-2-(2-methoxyethoxymethyl)pyrazoloanthrone with appropriate acid chloride R ₆ COCl or sulfonyl chloride R ₆ SO ₂ Cl in the presence of an acid quencher such as triethyl In the presence of amines, diisopropylethylamine, sodium bicarbonate, potassium carbonate or sodium hydroxide, in a solvent at -20°C-50°C for 0.5-16 hours, solvents such as dichloromethane, chloroform, tetrahydrofuran, Dioxane, Dimethylformamide and Ethyl Acetate. To carry out the deprotection step, the above product can be treated with an acid such as trifluoroacetic acid, aqueous hydrochloric acid, aqueous hydrobromic acid or aqueous sulfuric acid.

The starting material can be prepared from two steps. The 2-position of 5-nitropyrazoloanthrone can be protected with a protecting group, such as methoxymethyl (MOM), methoxyl, with the assistance of a base. ethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM) or 4-methoxybenzyl (PMB), bases used such as triethylamine, diisopropyl Ethylamine, pyridine, sodium hexamethyldisilazide, potassium hexamethyldisilazide, or lithium diisopropylamide; when using a tertiary amine as a base, 4-(N , N-dimethylamino)pyridine (DMAP) as catalyst. The reaction is generally carried out at -40-60°C in a solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane or dimethoxyethane for 1-16 hours. As nitrogen protecting group, the MEM group is preferred.

Then, the N-protected 5-nitropyrazoloanthrone was reduced to its 5-amino derivative with various reducing agents such as Sn or Fe metal under acidic medium such as acetic acid or aqueous hydrochloric acid. The reaction is generally carried out at 20-160°C for 1-16 hours. The same conversion can also be achieved by hydrogenation in the presence of a transition metal catalyst, for example, palladium, platinum, rhodium or iridium, with or without a support such as carbon, in a solvent such as ethanol, ethyl acetate, tetrahydrofuran, Dioxane or dimethoxyethane, hydrogen at 1-20 atmospheres, 1-16 hours at 20-60°C. A hydrogenation method using palladium/charcoal as a catalyst is preferred.

Reaction Line 5

In reaction scheme 5, condensing 7-amino-2-(2-methoxyethoxymethyl)pyrazoloanthrone with acid chloride or sulfonyl chloride, followed by deprotection, can prepare 7-acyl- or Pyrazoloanthrones with Sulfonylamino Substituents. The condensation reaction of 7-amino-2-(2-methoxyethoxymethyl)pyrazoloanthrone with the appropriate acid chloride R ₆ COCl or sulfonyl chloride R ₆ SO ₂ Cl is in the presence of an acid quencher such as triethyl In the presence of amines, diisopropylethylamine, sodium bicarbonate, potassium carbonate or sodium hydroxide, in a solvent at -20°C-50°C for 0.5-16 hours, solvents such as dichloromethane, chloroform, tetrahydrofuran, Dioxane, dimethylformamide or ethyl acetate. To carry out the deprotection step, the above product can be treated with an acid such as trifluoroacetic acid, aqueous hydrochloric acid, aqueous hydrobromic acid or aqueous sulfuric acid.

The starting material can be prepared in two steps by protecting the 2-position of 7-nitropyrazoloanthrone with the assistance of a base, such as methoxymethyl (MOM), methoxy ethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM) or 4-methoxybenzyl (PMB), bases used such as triethylamine, diisopropyl Ethylamine, pyridine, sodium hexamethyldisilazide, potassium hexamethyldisilazide, or lithium diisopropylamide; when using a tertiary amine as a base, 4-(N,N-di Methylamino)pyridine (DMAP) as catalyst. The reaction is generally carried out at -40-60°C in a solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane or dimethoxyethane for 1-16 hours. As nitrogen protecting group, the MEM group is preferred.

Then, the N-protected 7-nitropyrazoloanthrone was reduced to its 7-amino derivative with various reducing agents such as Sn or Fe metal under acidic medium such as acetic acid or aqueous hydrochloric acid. The reaction is generally carried out at 20-160°C for 1-16 hours. The same conversion can also be achieved by hydrogenation in the presence of a transition metal catalyst, for example, palladium, platinum, rhodium or iridium, with or without a support such as carbon, in a solvent such as ethanol, ethyl acetate, tetrahydrofuran, Dioxane or dimethoxyethane, hydrogen at 1-20 atmospheres, 1-16 hours at 20-60°C. A hydrogenation method using palladium/charcoal as a catalyst is preferred.

Reaction Line 6

In reaction scheme 6, condensing 5-hydroxy-2-(2-methoxyethoxymethyl)pyrazoloanthrone with hydrocarbyl halide or sulfonate R ₇ -X, followed by deprotection, can prepare Pyrazoloanthrone with 5-oxyl substituent, X as leaving group, chloride, bromide, iodide, mesylate, tosylate, benzenesulfonate or trifluoro mesylate. The condensation reaction of 5-hydroxy-2-(2-methoxyethoxymethyl)pyrazoloanthrone with a suitable hydrocarbyl halide or sulfonate is in the presence of an acid quencher such as triethylamine, diisopropyl In the presence of ethylamine, sodium bicarbonate, potassium carbonate or sodium hydroxide, carry out at -20°C-50°C in a solvent for 0.5-16 hours, such as dichloromethane, chloroform, tetrahydrofuran, dioxane, Dimethylformamide or ethyl acetate. The deprotection step is carried out by treating the above product with an acid such as trifluoroacetic acid, aqueous hydrochloric acid, aqueous hydrobromic acid or aqueous sulfuric acid.

The starting material can be prepared in two steps by protecting the 2-position of 5-benzyloxypyrazoloanthrone with the assistance of a base, such as methoxymethyl (MOM), methyl Oxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM) or 4-methoxybenzyl (PMB), bases used such as triethylamine, diisopropyl ethylamine, pyridine, sodium hexamethyldisilazide, potassium hexamethyldisilazide or lithium diisopropylamide; when using a tertiary amine as a base, 4-(N,N- Dimethylamino)pyridine (DMAP) as catalyst. The reaction is generally carried out at -40-60°C in a solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane or dimethoxyethane for 1-16 hours. As nitrogen protecting group, the MEM group is preferred.

Then, the N-protected 5-benzyloxypyrazoloanthrone is reduced to its 5-hydroxy derivative by hydrogenation over a transition metal catalyst such as palladium, platinum, In the presence of rhodium or iridium, in a solvent such as ethanol, ethyl acetate, tetrahydrofuran, dioxane or dimethoxyethane, hydrogen at 1-20 atmospheres at 20-60°C for 1-16 hours. A hydrogenation method using palladium/charcoal as a catalyst is preferred.

Reaction Line 7

In reaction scheme 7, condensing 7-hydroxy-2-(2-methoxyethoxymethyl)pyrazoloanthrone with hydrocarbyl halide or sulfonate R ₇ -X, followed by deprotection, can prepare Pyrazoloanthrone with 5-oxyl substituent, X as leaving group, chloride, bromide, iodide, mesylate, tosylate, benzenesulfonate or trifluoro mesylate. The condensation reaction of 7-hydroxy-2-(2-methoxyethoxymethyl)pyrazoloanthrone with a suitable hydrocarbyl halide or sulfonate is in the presence of an acid quencher such as triethylamine, diisopropyl In the presence of ethylamine, sodium bicarbonate, potassium carbonate or sodium hydroxide, carry out at -20°C-50°C in a solvent for 0.5-16 hours, such as dichloromethane, chloroform, tetrahydrofuran, dioxane, Dimethylformamide or ethyl acetate. The deprotection step is carried out by treating the above product with an acid such as trifluoroacetic acid, aqueous hydrochloric acid, aqueous hydrobromic acid or aqueous sulfuric acid.

The starting material can be prepared in two steps by protecting the 2-position of 7-benzyloxypyrazoloanthrone with the assistance of a base, such as methoxymethyl (MOM), methyl Oxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM) or 4-methoxybenzyl (PMB), bases used such as triethylamine, diisopropyl ethylamine, pyridine, sodium hexamethyldisilazide, potassium hexamethyldisilazide or lithium diisopropylamide; when using a tertiary amine as a base, 4-(N,N- Dimethylamino)pyridine (DMAP) as catalyst. The reaction is generally carried out at -40-60°C in a solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane or dimethoxyethane for 1-16 hours. As nitrogen protecting group, the MEM group is preferred.

Then, the N-protected 7-benzyloxypyrazoloanthrone is reduced to its 7-hydroxy derivative by hydrogenation over a transition metal catalyst such as palladium, platinum, In the presence of rhodium or iridium, in a solvent such as ethanol, ethyl acetate, tetrahydrofuran, dioxane or dimethoxyethane, hydrogen at 1-20 atmospheres at 20-60°C for 1-16 hours. A hydrogenation method using palladium/charcoal as a catalyst is preferred.

Compounds of formula (V), (VI) and (VII) can be prepared in the same manner as above, using starting materials having multiple reactive sites at the corresponding positions of the desired product.

In another embodiment of the present invention, pharmaceutical compositions containing one or more compounds of the present invention are disclosed. For administration purposes, the compounds of formula (I) are preferably formulated as pharmaceutical compositions. The pharmaceutical composition of the present invention contains the compound of the present invention and a pharmaceutically acceptable carrier, wherein the amount of the compound in the composition is an effective amount for treating related diseases. According to different administration routes, the pharmaceutical composition of the present invention preferably contains 0.1 mg-250 mg of the compound of structural formula (I) per unit dose, more preferably 1 mg-60 mg. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

Pharmaceutically acceptable carriers are familiar to those skilled in the art. For liquid solution preparation compositions, applicable carriers include physiological saline and sterile water, and may optionally contain antioxidants, buffers, bacteriostats and other conventional additives. The compositions can also be formulated as pills, capsules, granules or tablets, which contain, in addition to the compound of the invention, diluents, dispersants and surfactants, binders and lubricants. Those skilled in the art can further formulate the compounds of the present invention in an appropriate manner, and formulate according to general practice, such as the methods disclosed in Remington's Pharmaceutical Sciences, [Gennaro, Ed., Mack Publishing Co., Easton, PA 1990].

In another embodiment, the present invention provides methods of treating various diseases by administering an effective amount of a JNK inhibitor to a patient in need thereof. Diseases that may be treated using the compounds of the present invention or pharmaceutical compositions containing the same include any disease that responds to JNK inhibition and thus would benefit from the use of a JNK inhibitor. Representative diseases in this regard include (but are not limited to) rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gout, asthma, bronchitis, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Mucinous colitis, ulcerative colitis, Crohn's disease, gastritis, esophagitis, hepatitis, multiple sclerosis, atherosclerosis, restenosis after angioplasty, left ventricular hypertrophy, myocardial infarction, stroke, Heart, kidney, liver, or brain injury due to ischemia, graft rejection (eg, kidney, liver, heart, lung, etc.), endotoxic shock, psoriasis, eczema, dermatitis, epilepsy, Alzheimer's disease, Huntington chorea, amyotrophic lateral sclerosis, peripheral neuropathy, spinal cord injuries, Parkinson's disease, and cancer.

The methods of the invention include systemic administration of a compound of the invention, preferably in the form of a pharmaceutical composition. The systemic administration mentioned here includes both oral and parenteral administration methods. For oral administration, suitable pharmaceutical compositions include powders, pills, granules, tablets and capsules, as well as liquids, syrups, suspensions and emulsions, which compositions may also contain flavoring agents, preservatives, Suspending agents, thickening and emulsifying agents, and other pharmaceutically acceptable additives. For parenteral administration, the compounds of the present invention can be formulated into aqueous injection solutions, which may contain buffers, antioxidants, bacteriostats and additives commonly used in such solutions.

The following examples are given for purposes of illustration, not limitation.

Example

Example 1

Synthesis of representative compounds

A. Anthra[1,9cd]pyrazol-6(2H)-one (“Compound 1”)

Hydrazine hydrate was added to a solution of 2-chloroanthraquinone (Aldrich) in 10 mL of pyridine, and the mixture was heated at 100° C. for 16 hours. The mixture was cooled, the solvent was evaporated in vacuo, the residue was taken into hot 6N HCI and the solid was collected by filtration. The crude product was flash chromatographed on silica gel to afford anthra[1,9cd]pyrazol-6(2H)-one ("compound 1") as a yellow solid.

Due to the limited solubility of compound 1, its purification can be carried out by first derivatizing compound 1 to a more soluble intermediate, such as the corresponding acetate, recrystallizing the intermediate, and then converting the intermediate to obtain in good yield Pure compound 1. More specifically, to a solution of pyrazoloanthrone (9.67 g, 43.9 mmol) in acetic acid (700 mL) was added acetic anhydride (12.4 mL.132 mmol), and the solution was heated to 80° C. for 5 hours, then cooled to room temperature for 16 hours The reaction mixture was then cooled to 0 °C for 2 hours. Filtration then yielded the intermediate N-acetylpyrazoloanthrone, which was recrystallized in acetic acid to give the pure intermediate (5.96 g, 52%).

¹ H NMR (CDCL ₃ ) δ10.6(br s, 1H), 8.46(d, 1H), 8.33(d, 1H), 8.26(d, 1H), 8.08(d, 1H), 7.96-7.87(m , 2H), 7.78 (t, 1H), 2.83 (s, 3H); ES-MS (m/z) 263 [M+1] ⁺ . Ammonium hydroxide (60mL) was added to the methanol (600mL) solution of the pure intermediate (5.96g, 23mmol), stirred and reacted at Baowen for 16 hours, then filtered, dried in a vacuum oven, and the second crop of crystals was recovered to obtain the total It is 4.8 g of compound 1 with a purity greater than 98%, ES-MS (m/z) 221 [M+1] ⁺ .

B. 5-Chloroanthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1,4-dichloroanthraquinone (commercially available) in the same manner.

C.7-Chloroanthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1,5-dichloroanthraquinone (commercially available) in the same manner.

D.5-Nitroanthracene[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1,4-dinitroanthraquinone (Krapcho, A.P.; Avery, K.L., Jr. J. Org. Chem. Journal of Organic Chemistry, 55, 5562-4, 1990).

E. 7-Nitroanthracene[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1,5-dichloroanthraquinone (commercially available) in the same manner.

F.5-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1-nitro-4-benzyloxyanthraquinone in the same manner. The starting material can be prepared by adding benzyl bromide to a mixture of 1-nitro-4-hydroxyanthraquinone (Aldrich) and potassium carbonate in dimethylformamide, stirring the mixture for 16 hours, adding water, and washing the mixture with ethyl acetate Ester extraction (×2), the combined organic layers were washed successively with sodium bicarbonate solution, water, 1N hydrochloric acid and brine, dried and evaporated, and the residue was chromatographed on silica gel to give 1-nitro-4-benzyloxyanthraquinone .

G.7-Benzyloxyanthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 1-nitro-5-benzyloxyanthraquinone in the same manner. Its starting material can be prepared according to the method disclosed in the German patent DE 2254199 of Reubke, Hohmann and Bien.

H.5-(Acetamido)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared from 4-acetamido-1-chloroanthraquinone in the same manner. The starting material can be prepared by adding 4-amino-1-chloroanthraquinone to pyridine and treating with acetic anhydride. The mixture was stirred for 1 hour, poured into water, and the solid was collected by filtration, washed with water, and dried in vacuo to give 4-acetamido-1-chloroanthraquinone as a colorless solid.

Example 2

Synthesis of representative compounds

A. 5-(Dimethylamino)anthra[1,9cd]pyrazol-6(2H)-one

A mixture of 5-chloroanthra[1,9cd]pyrazol-6(2H)-one (Example 1-B) and dimethylamine in pyridine was heated at 100°C for 16 hours. The mixture was cooled and evaporated and the residue was chromatographed on silica gel to give the desired compound as a yellow solid.

B. 5-(1-piperidinyl)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using piperidine as the amine.

C. 5-(1-morpholinyl)anthracene[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using morpholine as the amine.

D. 5-(Benzylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using benzylamine as the amine.

E. 5-{(4-pyridylmethyl)amino}anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 4-pyridylmethylamine as the amine.

F. 5-(2-(1-piperidinyl)ethylamino}anthracene[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-(1-piperidinyl)ethylamine as the amine.

Example 3

Synthesis of representative compounds

A. 7-(Dimethylamino)anthra[1,9cd]pyrazol-6(2H)-one

A mixture of 7-chloroanthra[1,9cd]pyrazol-6(2H)-one (Example 1-C) and dimethylamine in pyridine was heated at 100°C for 16 hours. The mixture was cooled and evaporated and the residue was chromatographed on silica gel to give the desired compound as a yellow solid.

B. 7-(1-piperidinyl)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using piperidine as the amine.

C. 7-(1-morpholinyl)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using morpholine as the amine.

D. 7-(Benzylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using benzylamine as the amine.

E. 7-{(4-pyridylmethyl)amino}anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 4-pyridylmethylamine as the amine.

F.7-{2-(1-piperidinyl)ethylamino}anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-(1-piperidinyl)ethylamine as the amine.

Example 4

Synthesis of representative compounds

A. 5-(Benzamido)anthra[1,9cd]pyrazol-6(2H)-one

Add benzoyl chloride to 2-(methoxyethoxymethyl)-5-aminoanthra[1,9cd]pyrazol-6(2H)-one and triethylamine in dichloromethane at 0 °C In solution, the mixture was stirred for 16 hours, quenched with water, and extracted with ethyl acetate (x2). The combined organic layers were washed with sodium bicarbonate and brine, dried and evaporated. The crude reaction mixture was then transferred into 6N hydrochloric acid and heated at 80°C for 4 hours. After cooling, the mixture was extracted with ethyl acetate (×2), washed with brine, dried and evaporated. The residue was chromatographed on silica gel to give a yellow The desired amide as a solid.

The starting material was prepared by adding sodium hexamethyldisilazide to cooled (0 °C) 5-nitroanthraceno[1,9cd]pyrazol-6(2H)-one (Example 1-D ) in THF, and the mixture was stirred at 0°C for 30 minutes, MEM-chlorine was added, the mixture was stirred at room temperature for 16 hours, water was added and extracted with ethyl acetate (×2), the combined organic layers were washed with sodium bicarbonate Washed with aqueous solution, water, 1N hydrochloric acid and brine, dried and evaporated, the residue was chromatographed on silica gel to give 2-MEM-5-nitroanthra[1,9cd]pyrazol-6(2H)-one as an oil.

Palladium (10%)/charcoal and 2-MEM-5-nitroanthra[1,9cd]pyrazol-6(2H)-one in ethanol were placed under 1 atm of hydrogen, and the mixture was stirred for 6 hours. The catalyst was filtered off with diatomaceous earth (celite), and the filtrate was evaporated to dryness to obtain 2-(methoxyethoxymethyl)-5-aminoanthra[1,9cd]pyrazol-6(2H)-one, This compound was used without further purification.

B. 5-(isonicotamido)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using isonicotinoyl chloride as the acid chloride.

C. 5-(nicotinylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using nicotinoyl chloride as the acid chloride.

D. 5-(2-Thienoylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-thiophenecarboxylic acid as the acid chloride.

E. 5-(3-Methylbutyrylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using isovaleryl chloride as the acid chloride.

F.5-(methylsulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using methanesulfonyl chloride as the sulfonyl chloride.

G. 5-(Benzenesulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using benzenesulfonyl chloride as the sulfuryl chloride.

Example 5

Synthesis of representative compounds

A. 7-(Benzamido)anthra[1,9cd]pyrazol-6(2H)-one

Add benzoyl chloride to 2-(methoxyethoxymethyl)-7-aminoanthra[1,9cd]pyrazol-6(2H)-one and triethylamine in dichloromethane at 0 °C In solution, the mixture was stirred for 16 hours, quenched with water, and extracted with ethyl acetate (x2). The combined organic layers were washed with sodium bicarbonate and brine, dried and evaporated. The crude reaction mixture was then transferred into 6N hydrochloric acid and heated at 80°C for 4 hours. After cooling, the mixture was extracted with ethyl acetate (×2), washed with brine, dried and evaporated. The residue was chromatographed on silica gel to give a yellow The desired amide as a solid.

The starting material was prepared by adding sodium hexamethyldisilazide to cooled (0 °C) 7-nitroanthraceno[1,9cd]pyrazol-6(2H)-one (Example 1-E ) in tetrahydrofuran, and the mixture was stirred at 0°C for 30 minutes, MEM-chlorine was added, the mixture was stirred at room temperature for 16 hours, water was added and the mixture was extracted with ethyl acetate (×2), and the combined organic layers were washed with Washed with aqueous sodium bicarbonate, water, 1N hydrochloric acid and brine, dried and evaporated, the residue was chromatographed on silica gel to give 2-MEM-7-aminoanthra[1,9cd]pyrazole-6(2H)- ketone.

Palladium (10%)/charcoal and 2-MEM-7-nitroanthra[1,9cd]pyrazol-6(2H)-one in ethanol were placed under 1 atm of hydrogen, and the mixture was stirred for 6 hours. The catalyst was filtered off with diatomaceous earth (celite), and the filtrate was evaporated to dryness to obtain 2-(methoxyethoxymethyl)-7-aminoanthra[1,9cd]pyrazol-6(2H)-one, This compound was used without further purification.

B. 7-(isonicotamido)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using isonicotinoyl chloride as the acid chloride.

C. 7-(nicotinylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using nicotinoyl chloride as the acid chloride.

D. 7-(2-Thienoylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-thiophenoyl chloride as the acid chloride.

E. 7-(3-Methylbutyrylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using isovaleryl chloride as the acid chloride.

F. 7-(methylsulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using methanesulfonyl chloride as the sulfonyl chloride.

G. 7-(Benzenesulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using benzenesulfonyl chloride as the sulfuryl chloride.

Example 6

Synthesis of representative compounds

A. 5-(3-Methylbutoxy)anthra[1,9cd]pyrazol-6(2H)-one

At room temperature, isoamyl bromide was added to 2-(methoxyethoxymethyl)-5-hydroxyanthra[1,9cd]pyrazol-6(2H)-one and potassium carbonate in dimethyl In the mixture in formamide, after the mixture was stirred for 16 hours, water was added, and the mixture was extracted with ethyl acetate (×2). The combined organic layers were washed with aqueous sodium bicarbonate, water, 1N hydrochloric acid and brine, dried and evaporated. Then the residue was transferred into 6N hydrochloric acid, heated at 80°C for 4 hours, after cooling, the mixture was extracted with ethyl acetate (×2), the combined organic layers were washed with brine, dried and evaporated, and the residue was purified by column chromatography , to give the title compound as a yellow solid.

The starting material was prepared by adding sodium hexamethyldisilazide to cooled (0° C.) 5-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one (Example 1- F) solution in tetrahydrofuran, and the mixture was stirred at 0°C for 30 minutes, MEM-chlorine was added, the mixture was stirred at room temperature for 16 hours, water was added and extracted with ethyl acetate (×2), the combined organic layers were washed with bicarbonate Washed with aqueous sodium solution, water, 1N hydrochloric acid and brine, dried and evaporated, the residue was chromatographed on silica gel to give 2-MEM-5-benzyloxyanthra[1,9cd]pyrazole-6(2H)- ketone.

Palladium (10%)/charcoal and 2-MEM-5-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one in ethanol were placed under 1 atm of hydrogen, and the mixture was stirred for 6 hours. The catalyst was filtered off with diatomaceous earth (Gelite), and the filtrate was evaporated to dryness to obtain 2-(2-methoxyethoxymethyl)-5-hydroxyanthra[1,9cd]pyrazole-6(2H)- Ketone, this compound was used without further purification.

B. 5-(4-pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using chloromethyl-4-pyridine as the hydrocarbyl halide.

C. 5-(3-pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using chloromethyl-3-pyridine as the hydrocarbyl halide.

D. 5-(2-methoxyethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-methoxyethyl bromide as the hydrocarbyl halide.

E. 5-(2-Dimethylaminoethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-dimethylaminoethyl chloride as the hydrocarbyl halide.

Example 7

Synthesis of representative compounds

A. 7-(3-Methylbutoxy)anthra[1,9cd]pyrazol-6(2H)-one

At room temperature, isoamyl bromide was added to 2-(methoxyethoxymethyl)-7-hydroxyanthra[1,9cd]pyrazol-6(2H)-one and potassium carbonate in dimethyl In the mixture in formamide, after the mixture was stirred for 16 hours, water was added, and the mixture was extracted with ethyl acetate (×2). The combined organic layers were washed with aqueous sodium bicarbonate, water, 1N hydrochloric acid and brine, dried and evaporated. Then the residue was transferred into 6N hydrochloric acid, heated at 80°C for 4 hours, after cooling, the mixture was extracted with ethyl acetate (×2), the combined organic layers were washed with brine, dried and evaporated, and the residue was purified by column chromatography , to give the title compound as a yellow solid.

The starting material was prepared by adding sodium hexamethyldisilazide to cooled (0° C.) 7-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one (Example 1- F) solution in tetrahydrofuran, and the mixture was stirred at 0°C for 30 minutes, MEM-chlorine was added, the mixture was stirred at room temperature for 16 hours, water was added and extracted with ethyl acetate (×2), the combined organic layers were washed with bicarbonate Washed with aqueous sodium solution, water, 1N hydrochloric acid and brine, dried and evaporated, the residue was chromatographed on silica gel to give 2-MEM-7-benzyloxyanthra[1,9cd]pyrazole-6(2H)- ketone.

Palladium (10%)/charcoal and 2-MEM-7-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one in ethanol were placed under 1 atm of hydrogen, and the mixture was stirred for 6 hours. The catalyst was filtered off with diatomaceous earth (celite), and the filtrate was evaporated to dryness to obtain 2-(2-methoxyethoxymethyl)-7-hydroxyanthra[1,9cd]pyrazole-6( 2H)-one, this compound was used without further purification.

B. 7-(4-pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using chloromethyl-4-pyridine as the hydrocarbyl halide.

C. 7-(3-pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using chloromethyl-3-pyridine as the hydrocarbyl halide.

D. 7-(2-methoxyethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-methoxyethyl bromide as the hydrocarbyl halide.

E. 7-(2-Dimethylaminoethoxy)anthra[1,9cd]pyrazol-6(2H)-one

This compound can be prepared in the same manner using 2-dimethylaminoethyl chloride as the hydrocarbyl halide.

Example 8

Activity of representative compounds

The activity of the compound of the present invention is determined according to the following method

JNK assay

Add 10 μL of test compound in 20% DMSO/80% dilution buffer (which consists of 20 mM HEPES (pH 7.6), 0.1 mM EDTA, 2.5 mM magnesium chloride, 0.004% Triton×100, 2 μg/mL leupeptin, 20 mM glycerophosphate , 0.1 mM sodium vanadate and 2 mM DTT in water), add 30 μL of 50-300 ng His6-JNK1, JNK2 or JNK3 in the same dilution buffer. The resulting mixture was preincubated at room temperature for 30 minutes, and 60 μl of 10 μg GST-c-Jun (1-79) was added in assay buffer (which consisted of 20 mM HEPES (pH 7.6), 50 mM NaCl, 0.1 mM EDTA, 24 mM MgCl, 1 mM DTT, 25 mM PNPP, 0.05% Triton×100, 11 μM ATP and 0.5 μCi γ-32P ATP in water) and let the reaction proceed at room temperature for 1 hour. Add 150 μL of 12.5% trichloroacetic acid to terminate c-Jun phosphorylation. After 30 minutes, the precipitate was collected on a filter plate, diluted with 50 μL of scintillation fluid, and counted and measured. _IC50 values were calculated as the concentration of test compound at which phosphorylation of c-Jun decreased to 50% of the control value. Preferred compounds of the invention have _IC50 values in the range of 0.01-10 [mu]M in this test. Thus, the preferred compound of the invention is Compound 1, which according to this assay has _{an IC50} of 0.11 μM for JNK1 and JNK2 and 0.15 μM for JNK3.

Selectivity to JNK

According to methods known to those skilled in the art (for example, see ProteinPhosphorylation.Sefton & Hunter, Eds., Academic Press.pp.97-367, 1998), the inhibitory activity of compound 1 to the following protein activating enzymes was also determined:

Enzyme _IC50

p38-2 >30,000nM

ERK1 >30,000nM

MEKK1 ＞30,000nM

IKK1 ＞30,000nM

IKK2 ＞30,000nM

PKA ＞30,000nM

PKC ＞10,000nM

EGF-TK ＞10,000nM

Jurkat T-cell IL-2 production assay

Jurkat T cells (clone E6-1) were purchased from the American Tissue Culture Collection and maintained in growth medium (RPMI 1640 medium containing 2 mM L-glutamine (Mediatech), and 10% fetal bovine serum (Hyclone) and penicillin/streptomycin), all cells were cultured at 37°C in 95% air and 5% _CO2 . At a density of 0.2×10 ⁶ cells per well, the cells were plated with 200 μL of culture medium. The compound stock solution (20 mM) was diluted with growth medium, equivalent to 10-fold dilution of the concentrated solution in a volume of 25 μL, added to each well, mixed and pre-incubated for 30 minutes. The compound excipient (dimethylsulfoxide) was maintained at a final concentration of 0.5% in all samples. After 30 minutes, cells were activated with PMA (phorbol myristate acetate, final concentration 50 ng/mL) and PHA (phytohemagglutinin, final concentration 2 μg/mL). Add PMA and PHA, which is equivalent to a 10-fold dilution of the concentrated solution in the growth medium, and add a volume of 25 μL to each well. The cells were plated for 10 hours, centrifuged to pellet the cells, the culture medium was removed and stored at -20°C, and an aliquot of the culture medium was analyzed for the presence of IL-2 by sandwich ELISA according to the manufacturer's instructions (Endogen) . _IC50 values were calculated as the concentration of test compound at which IL-2 production was reduced to 50% of the control value. Preferred compounds of the invention have _IC50 values in the range of 0.1-30 [mu]M in this test. Figure 1 shows the dose-dependent relationship between the inhibitory effect of compound 1 on IL-2 in Jarkat T-cells according to this method, and its IC ₅₀ is 5 μM.

Determination of LPS-induced TNF-α production in mice

Non-fasted mice were acclimatized for at least 7 days. Before injecting 0.5 mg/kg Bacto LPS (from E.coli 055: B5 (Difco Labs)), 4-6 female BALB/c or CD-1 mice (8-10 weeks old, provided by Charles River laboratories) in each group Pre-treatment with test compound was administered intravenously or by oral gavage for 15-180 minutes. 90 minutes after LPS challenge, blood was regularly collected from the abdominal vein, and the blood was collected in a micro-serum separator tube (Microtainer serum separator tube) for 30 minutes at room temperature. After centrifugation, the serum was stored frozen at -80°C. The thawed diluted samples (1:10-1:20) were subjected to ELIZA analysis with the mouse TNF-α kit (Biosource International), and the _ED50 value was calculated as the concentration of the test compound when TNF-α production was reduced to 50% of the control value. dose. Preferred compounds of the invention have _ED50 values in the range of 1-30 mg/kg in this test. Figure 2 illustrates the results obtained in this experiment using compound 1 administered intravenously (IV) at 15 and 30 mg/kg and orally (PO) at 7.5, 15 and 30 mg/kg. Separate excipients (PEG-400, propylene glycol, cremophor EL and ethanol in normal saline solution, "PPCES") and dexamethasone-21 acetate ('DEX") (1 mg/kg P.O.) were used as As usual (n=6, *=p 0.01). Compound 1 administration was performed 15 minutes before LPS challenge and blood was drawn 90 minutes after LPS.

Inhibition of Leukocyte Recruitment in Inflamed Rat Lung

Aerosol administration of ovalbumun to Brown Norway mice previously sensitized by ovalbumin (OA) injections resulted in allergic tracheitis, manifested by eosinophil-rich and T-lymphocyte-rich lungs Leukocyte infiltration (see Richards et al., Am. J. Physiol, 271: 2Pt1, L267-76, 1996). Compound 1 was injected subcutaneously at a dose of 30 mg/kg, b.i.d., for 3 days, and then challenged by aerosol ovalbumin. Cell counts obtained from bronchoalveolar lavage samples are shown in Figure 3 (V = PPCES vehicle).

Adjuvant arthritis in mice

From day 0, male Lewis mice were immunized with complete Freund's adjuvant to induce erosive arthritis, characterized by joint damage and paw swelling. From day 8 to day 20, Compound 1 was administered subcutaneously once a day. Paw swelling was determined by water displacement plethysmometry (see Figure 4A; *=p<0.01). A radiograph of the right hind paw was obtained, and bone changes were evaluated using a semiquantitative scoring system: degree of demineralization (0-2+), calcaneal erosion (0-1+), and ectopic bone formation (0-1+), Highest score = 6 (see Figure 4B). Activation of AP-1 was measured by DNA binding activity using the electrophoretic mobility shift assay (EMSA) (see Figure 4C) (Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., John Wiley & Sons Publisher, New York .1992). Expression of matrix metalloproteinase-13 (see Figure 4D), as measured by northern blot analysis of MMP-13 mRNA (Ausebel et al., supra) (see also Winter et al., Arthritis and Rheumatism 9(3) : 394-404, 1966; Weichman et al., Pharmacological Methods in the Control of Inflammation, Chang and Lewis Eds., Alan R. Liss, Inc., PubI., New York, 1989).

Kainic acid-induced seizure response

Compound 1 was administered to male CD rats intravenously via a tail vein catheter at a dose of 10 mg/kg and immediately thereafter at a dose of 30 mg/kg subcutaneously. The vehicle control group received the same injected volume of PPCES vehicle only. Thirty minutes later, the animals were injected with kainic acid in normal saline at a dose of 1-mg/kg i.p. It has been previously reported that this dose of kainic acid is capable of inducing a seizure syndrome in mice (Maj et al., Eur. J. Pharm. 359:27-32, 1992). Seizure behavior was monitored for 4 hours after kainic acid injection. As shown in Figure 5, behavior was assessed on the basis of the following cumulative scoring system: 1 point = cessation of movement; 2 points = myoclonic reflexes of the head and neck (moderate); 3 points = unilateral or bilateral Forelimb clonic activity; 4 points = generalized clonus; 5 points = clonic-tonic seizures; 6 points = seizure status (see also Mathis and Ungerer, Exp. Brain Res. 88: 277-282 , 1992; Rong et al., Proc. Natl. Acad. Sci. USA 96: 9897-9902, 1999; Yang et al., Nature 389: 865-870, 1997).

It will be understood that, although only specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to what is defined in the appended claims.

Claims (17)

1. the compound or pharmaceutically acceptable salt thereof that has following structure:

Wherein:

R ₁And R ₂Be identical or different substituting group, and represent nitro, pyridyl-oxyl,-oxyl-oxyl, amino-oxyl ,-or amino-oxyl of two-alkyl independently, perhaps by formula (a), (c) or (d) group of expression:

R ₃And R ₄Represent piperidyl or morpholinyl together, perhaps R ₃And R ₄Identical or different and represent phenyl alkyl or pyridyl alkyl, perhaps R independently of one another ₃And R ₄One of expression piperidyl alkyl, another represents H; And

R ₅Expression alkyl, pyridyl or thienyl;

And has only R ₁Or R ₂One of them exists,

Wherein said alkyl has 1-8 carbon atom, and described-oxyl is-O-C _1-8Alkyl.

2. the compound of claim 1 has one of following structure:

Or its pharmacologically acceptable salt.

3. the compound of claim 1, wherein R ₁And R ₂Be:

4. the compound of claim 1, wherein R ₁And R ₂Be:

5. the compound of claim 1, wherein R ₁And R ₂Be:

6. the compound of claim 1 has one of following structure:

Or its pharmacologically acceptable salt,

Wherein

R ₁Be by formula (c) or (d) expression group;

R ₃And R ₄Identical or different and represent phenyl alkyl, pyridyl alkyl, perhaps R independently of one another ₃And R ₄One of expression piperidyl alkyl, another represents H; And

R ₅Expression pyridyl, thienyl or alkyl,

Wherein said alkyl has 1-8 carbon atom.

7. contain the compound or pharmaceutically acceptable salt thereof of claim 1 and the medicinal compositions of pharmaceutically acceptable carrier.

8. contain following formula: compound

Or the medicinal compositions of its pharmacologically acceptable salt and pharmaceutically acceptable carrier.

9. the purposes of the compound or pharmaceutically acceptable salt thereof of each of claim 1-6 aspect preparation can be used for suppressing the medicine of JNK in the body.

10. the purposes of claim 9, disease wherein is a cancer.

11. the purposes of claim 9, disease wherein are rheumatoid arthritis, similar rheumatism spondylitis, osteoarthritis, gout, asthma, bronchitis, Cysticfibrosis, inflammatory bowel disease, irritable bowel syndrome, mucous colitis, ulcerative colitis, Crohn disease, gastritis, esophagitis, hepatitis, multiple sclerosis, endotoxin shock, psoriatic, eczema or dermatitis.

12. the purposes of claim 9, disease wherein are restenosis, left ventricular hypertrophy or the myocardial infarction of atherosclerosis, postangioplasty.

13. the purposes of claim 9, disease wherein is an apoplexy, perhaps the ischemic injury of heart, kidney, liver or brain.

14. the purposes of claim 9, disease wherein are the transplantability rejections.

15. the application of claim 9, disease wherein are nervus centralis or peripheral nerve degeneration disease.

16. the purposes of claim 15, wherein said nervus centralis or peripheral nerve degeneration disease are epilepsy, alzheimer's disease, Parkinson's disease, Huntington Chorea, amyotrophic lateral sclerosis, peripheral neurophaty or chorda dorsalis injury.

17., comprise that each the compound or pharmaceutically acceptable salt thereof of the claim 1-6 that makes described cell and significant quantity contacts in the external method that in the cell that can express JNK, suppresses JNK.

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