TW200524869A - Benzamide kind histon deacetylase inhibiting agent having dissociation and antibred activity and its medicinal preparation - Google Patents

Abstract

A benzamide type histone deacetylase inhibitor with differential and anti-reproduction activity, and its application in preparing medicines for treating the diseases associated with differentiation and reproduction, such as cancer and psoriasis, are disclosed.

Description

200524869 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to the synthesis of a new type of small molecule compound with therapeutic effect and its clinical application in the treatment of diseases related to differentiation and proliferation. [Previous technology] Abnormal gene expression plays an important role in the pathogenesis of many diseases, including tumors, endocrine disorders, immune system diseases, genetic diseases and neurological diseases. The human genome exists as a chromatin structure packed with DNA, histones, and non-histone proteins. The chromatin structure plays an important role in determining whether a particular gene is expressed. In general, concentrated chromatin inhibits transcription, while transcriptionally active genes are often located in open chromatin. The basic repeating unit of chromatin, the nucleosome, is surrounded by a double-stranded DNA with a histone core containing four histones Make up. This histone core contains one H3-H4 tetramer and two H2A-H2B dimers. Histones attach to the junctions between nucleosomes, through which the carboxy terminus of the positively charged nuclei neutralizes the negative charge on the DNA strand to maintain the stability of the chromatin structure. This highly ordered structure of nucleosomes determines the relationship between chromatin composition and gene activation (Ricky W. Johnstone, uHistone deacetylase inhibitors: novel drugs for the treatment of cancer55, Nature Reviews Drug Dansuanhu 7). The N-terminus of histones can be post-translationally modified, 200524869 and thus can change the structure and function of chromatin. One such modification is the reversible acetylation and deacetylation of lysine residues at the histone tail. Histone acetylation levels are controlled by both histone acetylases (HATs) and histone deacetylases (HDACs). In addition to histone N-terminal modifications, histones can also be pot acidified, methylated, and ADP-ribosylated. These modifications affect the electrical properties of histones and their functions, which in turn alters the structure and gene expression of chromatin {Current Opinion in Oncology 2001, 13.477-483). Research in recent years has revealed a close link between histone acetylation and chromatin rebuilding and gene regulation. Many transcriptional promoter complexes have inherent histone acetate enzyme activity. Conversely, transcriptional repression complexes have the activity of recruiting histone deacetylase to the target gene promoter (Wan 1998, 20: 615). Some specific transcription initiation factors' such as nuclear receptor superfamily, cAMP effector binding protein (CREB), signaling activation transcription factor 1 (STAT-1), etc. can interact with various co-promoters and co-suppressors in different tissues and Selective role in genes constitutes a regulatory network for the selective expression of genes. These regulatory networks control the balance of our bodily functions. Interfering with these networks will cause disease or affect the course of disease. In addition, regulating the interactions between these transcription complex proteins provides a new approach for the treatment of tumors, endocrine disorders, immune system diseases, genetic diseases and neurological diseases (E. Korzus, Transcription Factor-specific

Requirements for Coactivator and Their

Acetyl transferase Functions. Science 1998, 279: 200524869 703-707; N. J. McKenna and BW O'Malley, Combinatorial Control of Gene Expression by Nuclear Receptors and Coregulators. Cell 2002, 108 (4): 465-474; MJ Pazin and JT Kadonaga, What's Up and Down with Histone Deacetylation and Transcription? 1997, 89 (3): 325-328; H. Zhong, RE Vol 1 and S. Ghosh, Phosphorylation of NF-B p65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP / p300. Molecular Cell 1 (5): 661-671; JS

Stef fan, Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila, Nature 2001, 413: 691-694; US20020115716A1, W00056153A1). For example, cell development and differentiation are regulated by gene programming, which is the regulation of chromatin structure levels. Genetic mutations or mutations that cause constitutive initiation of oncogenes such as RAS, or inactivation of tumor suppressor genes such as P53, will affect a range of molecular processes including transcription. In addition, some genetic mutations that cause abnormal effects of histone beta-enzyme and deacetate ^, such as misaligning their target genes, or inactivating the function of histone acetate, or over-expressing histone deacetylase Enzymes and the like will disrupt the normal development and differentiation of cells and cause tumorigenesis and development (Opinion Genet. Development 1999, 9: 40-48 and 200524869 175-184). Some human tumors are related to dysregulation of histone beta enzyme and deacetylase activity, an example of which is ectopic and ectopic chromosomes 15 and 17 in human acute myeloid leukemia patients. The result is a fusion protein containing three protein molecules, RARa, PML, and PLZF. This abnormal fusion protein can bind to the cis-acting element of RAR, and through the strong binding recruitment with SMRT co-repressor, a high-affinity histone deacetylase & enzyme, so that the target gene expression of RAR is sustained Inhibits, cripples, and loses response to vitamin A acid (2 01, * 20: 7204-7215). Vitamin A acid receptor (RAR) is a transcription factor that depends on the activation of ligands, and it plays a very important role in bone marrow differentiation. The heterodimer composed of RAR and RXR can bind to the vitamin A acid response element in the promoter region of the target gene. When vitamin A acid is lacking, RAR / RXR can recruit SIN / HDAC through the cosuppressors NCOR and SMRT to suppress transcription; and when the ligand is added, HDAC is released, and then RAR / RXR can have HAT with TIF2, CBP, etc. Active cofactors initiate transcription. φ Therefore, activation or inhibition of genes containing vitamin A acid response elements is important for the differentiation of bone marrow cells. Moreover, the addition of HDAC inhibitors can restore the ability of acute myeloid leukemia cells to induce vitamin A acid differentiation, suggesting that abnormal histone deacetylation is a key factor in the pathogenesis of leukemia. It has been reported that 'when histone deacetylation " over-expression of the enzyme inhibits the expression of some tumor suppressor genes, such as p53. p53 is a key regulator of cell proliferation, it does not transmit signals to genes that control the cell cycle, but induces cell death in the presence of external pressure. The function of p53 is mainly because it can directly bind to specific DNA sequences and start transcription. If its DNA binding region is mutated to inactivate the function, it often leads to cancer. There is evidence that CBP / p300 can up-regulate p53 by acetylating histones and p53 (W. Gu and RG Roeder, Activation of p53 Sequence -Specific DNA Binding by Acetylation of the p53 C-Terminal Domain · 6W / 1997, 90 ( 4): 595-606 ·). In contrast, mammalian HDAC-1, HDAC-2, and HDAC-3 can down-regulate p53 by deacetylating histones and p53 (L.-J. Juan, et al., Histone Deacetylases Specifically Down-regulate p53 -dependent Gene Activation. The Journal of Biological Chemistry 275 (27): 20436-20443) 〇 The above experiments show that abnormal transcription inhibition by HDACs can change the structure of chromatin, interfere with normal cell differentiation, and cause tumors And other proliferative diseases. Therefore, inhibition of HDAC activity may be an effective method for treating tumors and other proliferative diseases. Several classes of histone deacetylase inhibitors have been discovered, including (1) short-chain fatty acids such as butyric acid and phenylbutyric acid; and (2) organic hydroxamic acids such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); (3) cyclic tetrapeptides containing 2-amino-8-oxy-9,10-epoxydecanoyl, such as trapoxin and HC-toxon; (4) free of 2-amino-8-oxy-9 , 10-epoxydecanoyl cyclic tetrapeptides, such as Apicidin and FK228; (5) stupid formamide compounds, such as MS-275 (EP0847992A1, 200524869 US2002 / 0103192A Bu W002 / 26696A Bu W001 / 70675A2, WOOl / 18171A2) . Butyric acid, as an inhibitor of cell proliferation and an inducer of cell differentiation, mainly derives its activity from the inhibition of histone deethylase (A. Nudelman and A. Rephaeli, Novel Mutual Prodrug of Retinoic and Butyric Acids with Enhanced Anticancer Activity. / · 2000, 43 (15): 2962-2966 ·). Phenylbutyric acid can be used alone to treat thalassemia, toxoplasmosis, malaria and other diseases, and can also be used in combination with vitamin A acid to treat acute myeloid leukemia (RP WarrelL et al., Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J. Natl · / Still 1998, 90 (21): 1621-1625.). Valproic acid is an anticonvulsant and has also been found to work by directly inhibiting histone deacetylase (C.J. Phiel etal., Histone Deacetylase Is a Direct Target of Valproic Acid, a Potent Anticonvulsant, Mood Stabilizer , and Teratogen. The Journal of Biological Chemistry 2001, 276 (39): 36734-36741; EP1170008A1). Some benzamides have histone deacetylase inhibitor activity at lower micromolar concentrations. Among them, the lead compound MS-275 developed by Mitsui Chemicals is the first histone deacetylase > enzyme inhibitor that has been shown to have oral anticancer activity in animals, and has no serious side effects of 200524869 (A · Saito et a 1., A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in antitumor activity against human tumors. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (8): 4592-4597; EP 0847992A1). Currently, MS-275 is conducting clinical research on leukemia at the Greenebaum Cancer Center at the University of Maryland, and clinical research on solid tumors at the National Cancer Institute (E.B. Levit, Clinical Trials in Leukemia focus on New Treatment Approaches. 2001 Release-University of Maryland Medical News 2001 Maryland http://www.umm.edu/ news / releases / karp · html, A Phase I Study of an Oral Histone Deacetylase Inhibitor, MS-275, in Refractory Solid Tumors and Lymphomas. 2001, National Cancer Institute). However, some new compounds with better performance still need to be developed in order to obtain drugs with stronger HDAC inhibitory activity and lower side effects. [Summary of the Invention] One of the objectives of the present invention is to disclose a class of compounds designed to treat differentiation and proliferation-related diseases such as cancer and psoriasis with inducing differentiation and antiproliferative activity based on histone deacetylases; The second is to disclose the method of preparing the compounds described in this class. 200524869; The second purpose of the present invention is to disclose the clinical application of the compounds described in this class to treat diseases such as cancer and psoriasis. A compound of the child should be represented by the general formula (1) · A—Z—C = c—Υ—B \ NJ χΐ

(I) where 'A is a benzene ring or a heterocyclic ring, and its substituent may be a halogen atom, m containing V to 4 substituents, an alkyl group, a residue, an alkyl group, an alkyl group, and an alkyl group of one carbon atom. ,! Alkyl groups with 4 to 4 carbon atoms, 4 groups with 1 to 4 carbon atoms, amino groups with 1 to 4 carbon atoms, acyl groups with 2 to 4 carbon atoms, 1 to 4 carbon atoms Thioxanyl with 1f4 carbon atoms perfluorine, ί to ⑽ carbon atoms — =, / to 4 carbon atoms fluorenyl,] to 4 rhyme heterooxy groups, base or heterocyclic substitution Group, β is a benzene ring or heterocyclic ring, and may contain 1 to 3 substituents, and the substituents may be functional groups, amino groups, meridian groups, nitro groups, cyano groups, and so on. Carbo group to 4 carbon atoms, 1 i 4 carbon atom oxo group,] to 4 carbon atom ammonium group 1 to 4 & atom cyano group, 2 to 4 carbon atom acid group , Amido with 2 to 4 carbon atoms, Thioalkyl with 1 to 4 carbon atoms,! Perfluoro records of 4 to 4 carbon atoms, perfluoroalkyloxy groups of 1 to 4 carbon atoms, counties of 1 to 4 carbon atoms, 1 to 4 carbon material oxygen silk groups, benzene 200524869 group or heterocyclic substitution Base; covalent bond ,! Posts containing up to 4 carbon atoms or containing linear structures, cyclic structures, or linear structures and rings of ♦-丽-, -C0-,-"ς, cn η ^ cs-, -so ... s〇2— The combination of structures, · γ is a combination containing -c〇——cs—, _so_, _s structure or a combination of a linear structure and a cyclic structure, and the central point of the ring B 中 " The distance between the center point (3, 0, ^ ', (W3) as a hydrogen bond acceptor in the chip atm / at 3) satisfies the following conditions: W1-W2 6 · 〇 ~ 12 · 〇Α > — ZZ W3 A; The preferred distance between them is W3 = 3. 0 ~ 5 · 〇A, W2, uo 3 · 〇 ~ 6 · 〇A, W2] 3 = 4 · 0 ~ 8 ·.

W2 8 · 〇 ～ 10 · 〇A, W3 2 5.0 ~ 8 · 〇A; R, R2 are hydrogen or a alkynyl group containing 1 to 4 carbon atoms, r1r can form a covalent bond together; R is hydrogen or an alkyl group containing 1 to 4 carbon atoms;

R4 is hydrogen, i element, amino group, honesty, Wei, cyano group, alkyl group of 4 to 4 carbon atoms, alkyl group of 1 to 4 carbon atoms, alkyl group of i to 4 carbon atoms, 1 to Alkylamino groups of 4 carbon atoms, acyl groups of 2 to 4 carbon atoms, amido groups of 2 to 4 carbon atoms, thioalkyl groups of 4 carbon atoms, perfluoroalkyl groups of up to 4 private atoms , All i-oxyl groups of 4 to 4 carbon atoms, t-alkyl groups of 1 to 4 carbon atoms, i-oxyl groups of i to 4 carbon atoms;-one of X1, X2, X3, X4 is halogen , Amino, hydroxy, nitro, cyano, alkyl of 1 to 4 carbon atoms, alkoxy of 4 to 4 carbon atoms, aminoalkyl of 1 to 4 carbon atoms, of 4 to 4 carbon atoms Alkylamino, 2 to 13 200524869 acyl with 4 carbon atoms, amido with 2 to 4 carbon atoms, thioalkyl with 4 to 4 carbon atoms, perfluoroalkyl with 1 to 4 carbon atoms, Perfluoroalkoxy group of 4 carbon atoms, carboxyl group of 1 to 4 carbon atoms, alkoxycarbonyl group of 4 to 4 carbon ^; the rest are hydrogen, halogen, amino, hydroxyl, nitro, cyano, Alkyl of 1 to 4 carbon atoms, to 4 carbons Alkoxy, 1 to 4 carbon atoms, aminoalkyl, 1 to 4 carbon atoms, alkylamino, 2 to 4 carbon atoms, acyl, 2 to 4 carbon atoms, amido,] to * atoms Thioxanyl, perfluorinated wire with i to 4 carbon atoms ,! A total aerosyl group of 4 to 4 carbon atoms, a radical of 4 to 4 carbon atoms, and an alkoxycarbonyl group of 1 to 4 carbon atoms.杂环 "Heterocycle" according to the present invention refers to a saturated or unsaturated heterocyclic ring containing one or more heteroatoms (nitrogen, oxygen or sulfur), such as tetrahydropyrrole, dihydropyrrole, dihydropyrazole, 呱Pyridine, morpholine, imidazole, pyridine, etc. · The "halogen" according to the present invention is fluorine, chlorine, molybdenum, iodine; the alkyl group of 1 to 4 carbon atoms according to the present invention "includes methyl, I Ethyl, n-propyl, isopropyl, n-butyl, isobutyl " The "alkoxy group of 1 to 4 carbon atoms" in the present invention includes methyllactyl, ethoxy, n-propoxy Group, isopropoxy group, n-butoxy group, isobutoxy group, etc .; "Amino group of 1 to 4 carbon atoms" according to the present invention, including aminoethyl, aminopropyl, aminopropyl The "!! to 4 carbon atom alkyl group" according to the present invention includes N-methylamino, 1 ethylamino, N-isopropylamino and the like; 14 200524869 The " "Acyl groups of 2 to 4 carbon atoms" include acetyl, propionyl, isobutyryl, etc .; "amide groups of 2 to 4 carbon atoms" according to the present invention, include acetamido and propionyl , Butyryl, isopropyl, etc .; the "thioalkyl group of 1 to 4 carbon atoms" according to the present invention includes methylthio, ethylthio, propylthio and the like; "Perfluoroalkyl groups of 1 to 4 carbon atoms", including difluorofluorenyl, pentafluoroethyl, etc .; "Perfluoroalkyl groups of 1 to 4 carbon atoms" according to the present invention, including difluoromethyl Oxygen, pentafluoroethoxy, etc .; "alkylene of 4 to 4 carbon atoms" according to the present invention includes methylene, ethylene, etc .; "the center point of the ring" according to the present invention refers to the constitution The average value of the X, Y, and Z axis values corresponding to the atoms of the ring. The method for synthesizing the compound of the present invention is as follows: (a) A compound of the general formula (II) and a compound of the general formula (111) are subjected to a condensation reaction to obtain a general compound. Compounds of formula (IV) · a—Z—c = c—R5 (II) 15 200524869

R6—B—COOH (III) R1 R2

A I I

A—Z—C = C—YB—CO〇H (IV) where 'eight ^^ 咄 ^ is the same as above ^ is a hawk eagle (c is o or s atom) or a surname consisting of __NH r5… Wu 9 sessions;, ... "'' 马 吒 (, _ is 0,52%, 舄 contains a structure of -NH2; when V is a structure containing -guan 2, R6 is -C (= Q) 〇H (Q (0 or s atom). (B) A compound of the general formula (IV) is subjected to a condensation reaction with a compound of the general formula ("to obtain the target compound (I): R3 | R4 HN X4" 9 ^ χ2 (V) where R3, R4, Xi, X2, X > rism ^. The above condensation reactions (a) and (b) are based on a catalyst such as dicyclohexylcarbonimide, N, N-carbonyldiimidazole, etc. The above condensation The reaction temperature of the reactions (a) and (b) is 0 ~ 8 (rc, the reaction time is 4 ~ 72 hours. The solvent used in the reaction is a common solvent, such as stupid, A 16 200524869, the present four rat widows, one oxygen six Clothing, monochrysene, chloroform, N, N-dimethylmethanamine, etc. If necessary, you can add domains such as sodium hydroxide, triethylamine, hydrazine, etc., or add acids such as hydrochloric acid, acetic acid, Sandun acetic acid, etc. The compound of the general formula (I) can be used See the separation methods for purification, such as extraction, recrystallization, column chromatography, etc. The compounds of the present invention have the activity of inducing differentiation and anti-proliferation, and can be used to treat diseases related to differentiation and proliferation, such as cancer and psoriasis, In particular, it has excellent curative effect on blood cancer and solid tumor. ▲ The compound of the present invention can be made into common medicinal preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, injections, can be added = spices, Common carrier materials such as sweeteners, liquid or solid fillers or diluents (see the Handbook of Medicinal Excipients, American Pharmaceutical Association, October 1986). = Preparations usually contain 7% active ingredient, preferably content It is 5 ~ 50%, and the component is a carrier filler, diluent or solvent. 〃 For mammals (including It ::: or injection Fangjia. Use Hua called Dan.) To order Le 'Zhongyou The most individual by oral method is 体重 2-body weight. The optimal dose depends on the initial 蚪 dose, and then gradually increases the dose. The gene shows that the compound and its pharmaceutical preparations are caused by the treatment capacity and wide ^. Tumor Endocrine disorders, dysentery, dysentery, and neurological diseases have very good curative effects. [Embodiment] 200524869 In order to make your reviewing committee understand the purpose, features, and effects of the present invention, the following are provided. Specific embodiments, together with the accompanying drawings, make a detailed description of the present invention, which will be described later: The content of the present invention will be further explained with reference to the examples below, but the scope of protection of the present invention is not limited to these examples. The present invention Unless otherwise stated, the percentages are all weight percentages. Example 1

* Preparation of 4- [N-(3 · -pyridineacryloyl) aminomethyl] benzoic acid Add 0.33 g (2.11 mmol) of N, N-carbonyldiimidazole and 10 ml of tetrahydrofuran to the reaction flask. After cooling to 0 ° C, 10 ml of a tetrahydrofuran solution containing 0.30 g (2.01 mmol) of 3-pyridine acrylic acid was added dropwise, and the reaction was stirred at room temperature for 3 hours, and then 2 ml (2.00 mmol) of the solution was added dropwise. There was 0. 30 g (2.00 mmol) of a 1N sodium hydroxide solution of 4-aminofluorenylbenzoic acid, and the reaction was continued with stirring at room temperature for 8 hours. The reaction mixture was concentrated in vacuo, and 2 ml of a saturated sodium gas solution was added to the residue, and the pH was adjusted to 5 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with ice water, and dried to obtain 0.46 g (82%) of the target compound. HRMS (C16H14N2O3) Calculated value (%): 282. 2988; Measured value (%): 282 · 2990. Elemental analysis (Ci6HuN2 03) Calculated value (%): C, 68.07%; Hf, 5.0%; N, 9.92%; Found C, 68.21%; Hf, 5.03%; 18 200524869 N, 9.90%. Example 2 Preparation of N- (2-amino-5 -fluorophenyl) -4- [N- (3- ° Specific Acryl ifcyl) aminomethyl] Benzoamide

Add 0.29 g (1.88 mraol) of N, N-carbon amidamizol and 0.50 g (1.88 mmol) of 4- [N- (3-pyridylpropenyl) to the reaction flask. Aminomethyl] phenylarsinic acid and 15 ml of tetrahydrosound squeak '45 ° C for 1 hour. After the reaction mixture was cooled, it was added to another solution containing 10 ml of tetrahydrofuran, 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine, and 0.20 g (1.78 mmol) of trifluoro In the reaction flask of acetic acid, the reaction was stirred at room temperature for 24 hours. The precipitated solid was washed with hydrazone, tetrahydroan, and dried to obtain 0.40 g (57%) of the target compound.屮 NMR (300 MHz, DMSO-de): 6ppm: 4.49 (2H, d), 4.84 (2H, br · s), 6.60 (1H, t), 6.80 (2H, m) , 6.96 (1H, t), 7.18 (1H, d), 7.42 (2H, d), 7.52 (1H, d), 7.95 (2H, d), 8.02 ( 1H, d), 8.56 (1H, d), 8.72 (1H, br · t), 8.78 (1H, s), 9.60 (1H, br · s. IR (KBr) cnf1: 3310, 1655, 163, 1524, 130, 750. HRMS (C22HnN4〇2F) Calculated value (%): 390. 4170; Found: 390. 4172. Elemental analysis 19 200524869 Analysis (C22Hi9N4 2F) Calculated value (° / 〇: C, 67.68%; H, 4.40%; N 14.35; Found (%): C, 67.52%; H, 4.38%; N, 14.42 / 0 Example 3 4- (N-cinnamoyl ammonium Preparation of methyl) benzoic acid

Into the reaction flask, 0.33 g (2.01 mm) of n, N-carbon ugamizole and 10 ml of tetrahydrofuran were added and cooled to 0. 〇10 ml of a tetrahydrofuran solution containing 0.30 g (2.01 mol) of 3-pyridine acrylic acid was added dropwise, and the reaction was allowed to proceed at room temperature for 3 hours, and then 2 ml (2.00 mm) of the solution was added dropwise. There was 0.30 g (2.00 mmol) of a 1N sodium hydroxide solution of 4-aminomethylbenzoic acid, and the reaction was stirred at room temperature for 8 hours. The reaction mixture was concentrated in vacuo, and 2 ml of a saturated sodium gasified solution was added to the residue, and the pjj value was adjusted to 7 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with ice water, and dried to obtain 0.51 g (91%) of the target compound. HRMS (C) 7Hi5NO3) Calculated value (%): 281 · 3242; Measured value (%) · 281 · 3240. Elemental analysis (CnH 5NO3) Calculated value (%): C, 72.58%; Η, 5.38%; N, 4.98%; Measured value (%): C, 72.42%; Η , 5.37%; N, 4.87% 〇 Example 4 Preparation of N- (2-amino-5 -fluorophenyl) -4- (N-cinnamoylaminomethyl) 20 200524869 Preparation of benzamidine

Add 0.29 g (1.88 ol) of N, N-carbonyldiimidazole, 0.50 g (1.88 mmol) of 4- (N-cinnamoylamino) phenylbenzoic acid and 15 ml to the reaction flask Tetrahydrofuran was stirred at 45 ° C for 1 hour. After the reaction mixture was cooled, it was added to another solution containing 10 ml of tetrahydrofuran, 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine, and 0.20 g (1.88 mmol) of trifluoroacetic acid. In the reaction flask, stir the reaction at room temperature for 16 hours. The precipitated solid was filtered, washed with tetrahydrofuran, and dried to obtain 0.45 g (64%) of the target compound. 4 NMR (300 MHz, DMS0-d6): &lt; 5ppm: 4.42 (2H, d), 4.92 (2H, br.s), 6.62 (1H, t), 6.78 (2H , M), 7.01 (1H, t), 7.32 (5H, m), 7.54 (5H, m), 8.76 (1H, br · t), 9.58 (1H, br · s). IR (KBr) cm1: 3306, 1618, 1517, 1308, 745. HRMS (C23H2NO2〇2F) Calculated (%): 389.4292; Found (%): 389.4294. Elemental analysis (C23H2 () N3〇2F) Calculated value (%): C, 70 · 94%; Η, 5.18%; N, 10 · 79; Measured value (%): C, 70.72%; Η, 5 180 / 〇; N, 10.88%. Example 5 Compounds N- (2-amino-5-fluorophenyl) -4- [N- (3-σΛσfiximidoyl) aminomethyl] benzamide (CS02100055), N- (2-aminobenzene Group) -4- [N- (4-fluorophenylacryloyl 200524869 group) aminomethyl] benzamide (CS021_9: ^ N— (2_aminophenyl) _4— [N # The in vitro inhibitory activity of histone deacetylases of amino) aminomethyl] benzamide (MS275, Ep 0847992) was performed using HDAC colorimetric analysis kit (BI0M0] L Research Laboratories, PA, USA). All operations were performed in accordance with the laboratory manual. First add the tested compounds to 96-well plates at different concentrations, then mix with the nuclear extract of HeLa cells, add the histone deacetate substrate, and leave it at 3 7 C for 10 minutes, then stop the color development solution. The reaction was stopped and the results were read on a microplate reader at a wavelength of 405 nm. Calculation of the inhibition rate was performed with reference to the instructions. The experimental results are shown in Table 1. Table 1.Inhibition percentage of in vitro inhibitory compounds of histone deacetylase by MS275, CS02100055, and CS02100019 at different concentrations IC50 1 μM 5 μM 10 μM 50 μM (μM) MS-275 14. 7 19.1 64 · 1 82.3 8.4 CS02100055 17.5 37.3 62 · 1 78 · 4 7.2 CS02100019 1 · 3 24.9 28.4 29.8 &gt; 50 · 0 Example 6 Compound N- (2-amino-5-fluorophenyl) -4- [N- (3- ° Than bityl) aminomethyl] benzyl 22 200524869 formamide (CS02100055), N- (2-aminophenyl) -4- [N- (4-fluorophenylacryloyl) aminomethyl] benzoyl Amide (CS02100019) and N- (2-aminophenyl) -4- [N- (3-pyridinyloxycarbonyl) aminomethyl] benzamide (MS275, EP0847992) cancer cell growth inhibitory activity in vitro using MTS method The growth inhibition rate was measured. In a 96-well plate, 5000 cells were tested per well (the amount of cells seeded at different growth rates was different). After 24 hours of incubation, test compounds of different concentrations were added. After 48 hours of incubation, 20 plMTS of detection substrate (Promega) was added to each well. After 2 hours at 37 ° C, the results were read on a microplate reader at a wavelength of 490 nm. The relative viable cell volume was calculated as the experimental / control group X 100%. The concentration of the compound that inhibits cell growth by 50% is labeled Gl50. All compounds were dissolved in DMS0, and 幷 was diluted 1: 1 at the time of dosing so that the final concentration of DMS0 was 0.1%. Each experiment was repeated three times independently. The experimental results are shown in Table 2. Table 2. MS-275, CS2100055 and CS02100019 on tumor cell growth compounds. GI50 (μM) of different tumor cells * U20S HeLa DU-145 SMMC-7721 HepG2 293 MCF-7 231 H292 MS-275 1.0 25 13 20 3.2 16 6.3 5.0 16 CS02100055 2.0 40 25 16 4.0 50 5.0 7.9 50 CS02100019 2.5 50 50 50 3.2 25 5.0 7.9 50 Compounds <3 丨 5 «(μΜ) * LNCaP SK-N-SH PANC-1 SK- OV-3 SGC-7901 Raji HL-60 28SC Jurkat MS-275 2.5 50 5.0 50 50 6.3 0.32 4.0 1.6 CS02100055 4.0 50 6.3 50 50 4.0 0.4 5.8 1.5 CS02100019 10 50 5.0 50 50 2.0 0.5 2.0 1.0 CS02I00019 10 50 5.0 50 50 2.0 0.5 2.0 1.0 23 200524869 * Cell source: U20S: human osteosarcoma cells DI [-145: human prostate cancer cells SMMC-7721: human liver cancer cells 293 · human embryonic kidney cells MDA-MB-231 · human breast cancer cells LNCaP : Human prostate cancer cell PANC-1: Human pancreatic cancer cell 28SC: Human monocyte macrophage HL-60: Human myeloid leukemia cell

HeLa: human cervical cancer cell SGC-7901: human gastric cancer cell HepG2 · human liver cancer cell MCF-7: human breast cancer cell H292: human lung cancer cell SK-N-SH · human neuroblastoma SK-0V-3: human Ovarian cancer cells Raji: Human Burkitt lymphoma Jurkat: Human T lymphocytic leukemia Example 7 Compound N- (2-amino-5-fluorophenyl) -4- [N- (3-pyridylpropenyl) aminomethyl ] Benzylamide (CS02100055), N- (2-aminophenyl) -4- [N- (3- ° pyridylmethoxycarbonyl) aminomethyl] benzamide (MS275, EP0847992) • and Trichostatin A (TSA) Nuclear Hormone Receptor Transcription Initiation The nuclear receptor transcription initiation experiment was performed using a reporter gene system. The specific operation is as follows: U20S cells are seeded into a 96-well plate the day before transfection, so that the density at the time of transfection is 50-80%. Cells were transfected with FuGene6 transfection reagent (Roche) cDNA expression vectors for the following nuclear receptors, which are glucocorticoid receptor (GR), peroxide-promoted receptor estrogen receptor a (ERa), and estrogen Receptor β (ERp), and also co-transfected the corresponding fluorescein 24 200524869 photonase reporter gene plasmid and internal reference plasmid yS-ga 1. Among them, GR and ρρΑΚγ were also co-transfected with retinoid X receptor (RXR). Various compounds and solvent controls (DMSO) were added 24 hours after transfection. Cells were lysed 24 hours after drug action to detect luciferase activity, and dot-galactosidase activity was also measured to correct transfection efficiency. The experimental results are shown in Figure 1. 30.0 25. 0 20.0 10. |

GR ERa • 50.0 40.0 30.0 20.0 10.0 0.0 80.0 60.0 40.0 20.0 〇.〇

PPAR ERp Figure 1. 1 &gt; 1 heart (^ 31: 111 VIII, elbow 8-275, and 匸 82100055 start transcription of nuclear receptors [LD stands for per acceptor ligand, GS55 stands for W ordinate represents relative induction multiple The concentration of the compound in all experiments is as follows: TSAG.2mM, Ms-275 i ^ cs55i ^ dexamethasone (0.1 μM), Rogglico (10 μM), and estradiol (0.01 μM, respectively As a ligand of GR, PPARY and £ R] / 疋 The invention is a highly developed invention of the technical idea of using natural law 200524869, which is simpler than traditional rehabilitation therapy, and also has substantial efficacy improvement and high industrial availability , Which is in full compliance with the statutory requirements of an invention patent, and filed an application for an invention patent according to law.

[Description of main component symbols] None

26

Claims (1)

200524869 10. Scope of patent application: Benzoamide histone deethylation S # inhibitors with anti-proliferative and anti-proliferative activity, which are characterized by enantiomers, non-oppositions, stereoisomers of compounds, The general formula for the structure of +, structure, hydrate and its salt is as follows: R1 R2 Λ I | A ~ Z ~ C = C—Υ — Β Wherein, A is a benzene ring or a heterocyclic ring, which may contain i to 4 substituents, and the serino group may be _ prime, amino, silk, nitro, Cyano, 1 to 4 carbon atoms, fluorenyl, i to 4 carbon atoms, oxyl, i 1 to 4 carbon atoms, weilyl, 1 to 4 carbon atoms, carbamino, 2 to 4 carbon atoms Cyl radical, amido group of 2 to 4 carbon atoms, thiocarbon of 1 to 4 carbon atoms, f 1 to 4 &amp; atom full radical, 丨 to 4 carbon atom of full gas roasted milk radical, 1 to 4 carbon atoms minus, 1 to 4 carbon atoms alkoxy groups, phenyl or heterocyclic substituents; B is a benzene ring or heterocyclic ring and may contain 1 to 3 substituents, and the substituents may be Halogen, amino, mesityl, nitro, aryl, alkyl with 4 to 4 carbon atoms, alkoxy with 1 to 4 carbon atoms, aminoalkyl with 4 to 4 carbon atoms, 1 to 4 carbons Atomic alkylamino groups, acyl groups of 2 to 4 carbon atoms, amido groups of 2 to 4 carbon atoms, thioalkyl groups of 4 to 4 carbon atoms, perfluoroalkyl groups of i to 4 carbon atoms ,! Total gas oxy groups of 4 carbon atoms, 27 200524869 carboxyl groups of 4 carbon atoms,] to groups or heterocyclic substituents; alkoxycarbonyl groups of 1 carbon atom, benzene z is a covalent bond, to 4 Burning of one carbon atom or containing a residual structure such as j or a linear structure and a cyclic combination Γ2, a cyclic structure, —SG—, a prepared linear structure, a cyclic 冓, or —D structure and The combination of the ring structure, the center point of the towel ring A (W3 3 · 0 ~ 5. 0 A, the center point (W2) of W2 ^ _) and the 0 "or S contained in γ as a hydrogen bond acceptor The atomic ⑽) distance satisfies $ · 〇 ~ ^ A'wi— W3mGA, f2 — w3 = 4〇82〇 The better distance between the daggers is: W1 — W2 = 8 〇ι〇〇 入, 扪 — W3 = 5. 0 ~ 8 · 〇A; w · V / J ^ X y can also form a covalent bond R1 and P together are hydrogen or a alkynyl group containing 1 to 4 carbon atoms, R1, r VJ- For example, if U,. IR is hydrogen or an alkyl group containing 1 to 4 carbon atoms; R4 is hydrogen, halogen, amino, mesityl, nitro, cyano, [] to 4 carbon atoms, 1 4 to 4 carbon atoms, 1 to 4 carbon atoms Ammonia group 1 to 4 stone counter-atom carbamo group, 2 to 4 carbon atom acyl group, 2 to 4 carbon atom amido group, to 4 carbon atom thioalkyl group, to 4 carbon A perfluoroalkyl radical, a perfluoroalkyloxy radical of 4 to 4 carbon atoms, a radical of 1 to 4 carbon atoms, a radical of 4 to 4 carbon atoms; one of 乂 ^^^^ ^^ amino ~ via radical "Shoryl, cyano, alkyl of 1 to 4 carbon atoms, alkoxy of 4 to 4 carbon atoms, 200524869 aminoalkyl of 1 to 4 carbon atoms, 1 to 4 Alkylamino groups of 2 carbon atoms, acyl groups of 2 to 4 carbon atoms, amido groups of 2 to 4 carbon atoms, thioalkyl groups of 1 to 4 carbon atoms, perfluoroalkyl groups of 1 to 4 carbon atoms,丨 Four fluorinated oxy groups of 4 to 4 carbon atoms, unrefined groups of 1 to 4 carbon atoms, alkoxycarbonyl groups of 1 to 4 carbon atoms; the rest are hydrogen, halide, amino, hydroxyl, nitro, Cyano, alkyl of 1 to 4 carbon atoms, alkoxy of 4 to 4 carbon atoms, aminoalkyl of 1 to 4 carbons, alkylamino of 4 to 4 carbons, 2 to 4 carbons Atomic acyl, 2 to 4 carbon atoms, amido, 丨 to 4 A fluorene atom of a thion, a linear residue of 4 to 4 carbon atoms, a perfluorosilyl group of 4 to 4 carbon atoms, a fluorene of 4 to 4 carbon atoms, an alkoxycarbonyl group of 4 to 4 carbon atoms. 2 The benzylamine histone deacetylase inhibitor with differentiation and antiproliferative activity according to item 1 of the declared patent scope, characterized in that the stereoisomers of The structural formulas of enantiomers, diastereomers, hydrates and their salts are as follows: /, R1 R2 Y-B-COOH where 'A is a benzene ring or a heterocyclic ring and may contain] to 4 Substituent, the 4th generation group may be amino group, amino group, perylene group, cyano group, 4 to 4 dialkyl group, 1 to 4 carbon atom alkoxy group, 1 to 4 carbon orthocarbonyl group :: 1 ΐ 4 carbon atom alkylamino group, 2 to 4 carbon atom '4 carbon atom amido group, 1 to 4 carbon atom, all 1 to 4 carbon atoms all in the United States 15 &quot; anti-atomic halogen burned silk, 1 to Perfluoroalkanes of 4 carbon atoms &quot; Silk of carbon atoms, alkoxy groups of 1 to 4 carbon atoms 29 200524869 group, phenyl or heterocyclic substituent; β is a benzene ring or heterocyclic ring, and may contain 3 substituents, the substituents of which can be i-prime, amino, minus "shoryl, cyano," to 4 carbon atoms of silk, 1 i 4 carbon atoms of alkoxy, to 4 Atomic aminoalkyl 1 to 4 carbon atoms, alkylamino, 2 to 4 carbon atoms acyl, 2 to 4 carbon atoms_amino, thiocarbons to 4 carbon atoms, to 4 carbons Atomic perfluoroalkyl group, perfluoroalkoxy group of 4 to 4 carbon atoms, fluorenyl group of 1 to 4 carbon atoms, alkoxy group of 1 to 4 carbon atoms, phenyl or heterocyclic substituent; Z is a covalent bond, an alkylene group of 1 to 4 carbon atoms, or a linear structure, cyclic structure, or line containing -〇 ___ s-, NH, -C〇-, -CS S0 --- S〇2- The combination of sexual structure and cyclic structure; the last name y is 3 with —C0-, -CS-, -S0-, -S〇2- a linear structure, a cyclic structure, or a combination of a linear structure and a cyclic structure, The distance between the center point (W1) of ring A, the center point (W2) of ring B, and the 0 or S atom (W3) contained as a hydrogen bond acceptor in γ satisfies the following conditions. Η — W2 = 6 · 〇12 · 〇A, W1-W3 = 3. 0 ~ 6. 0 A, W2-W3 = 4. 0 ~ 8 · 0 A 'The better distance between them is: W1-W2 2 8. 0 ～ 10. 〇A, W1-W3 = 3. 〇 ~ 5. 〇A, W2 — W3 5.0 ~ 8.0 A; only, R F or R2 can form a covalent bond together; 3 · ~ • a benzamide histone deacetylase with differentiation and antiproliferative activity Inhibitors are characterized by: stereoisomers of the compounds, 30 200524869 enantiomers, diastereomers, hydrates and their salts are prepared by the following two-step reaction: (a) general formula (Π) The compound is subjected to a condensation reaction with a compound of the general formula (II) to obtain a compound of the general formula (IV): R1 R2 azc = c-r5 (II) R6—b-cooh (III) R1 R2 AIIA—z—c two C — Y—B—COOH (IV) where 'eight, two, y, 6, 1 ^ 1, 1 ^ 2 is as described in claim 1; | ^ is — (: (= ^) 诎 (Q is 0 or S atom ) Or a structure containing -NH2; when R5 is -c (; = q) 0h (Q is 0 or S atom), r6 is a structure containing -NH2; when ^ is a structure containing -NH2, r6 is — (Q is 0 or s atom). (b) The compound of general formula (iv) is subjected to a condensation reaction to obtain the target compound (I): 31 200524869 R3 R4 h &gt; sA ^ x1 I Γ x4 ^ V ^ x2 X3 (V) where 'R3 , R4, X1, X2, X3, and X4 as described in claim 1. • 4. The benzamidamine histone deacetylation a) as described in the third top of the scope of the patent application. The enzyme inhibitor is characterized in that the condensation reactions (a) and (b) use the existing condensation agent as a catalyst. 5. The benzyl 6¾ amines with differentiation and anti-proliferative activity as described in the top 4 of the scope of patent application. Histone deacetate inhibitor, the peptide condensing agent, can be a 3 hexyl carboimine 'N, N-carbo diimidal salivary. 6 · Has differentiation as described in the top 3 of the scope of the patent application And antiproliferative styryl &gt; amine histone deacetylase inhibitor preparation method, characterized in that the reaction temperature of the condensation reaction (a) and (b) is 0 ~ ⑼◦ [, reaction time It is 4 to 72 hours. 7. A benzamide histone deacetylation s # having a cell differentiation and antiproliferative activity is a commercially available agent, which It is characterized in that its preparation consists of a derivative of the active ingredient benzamide or a salt thereof and a pharmaceutically acceptable carrier adjuvant or diluent. 8 As described in claim 7 of the patent scope, it has a cell differentiation and anti-proliferative activity. A medicinal preparation of formamide histone deacetylase is characterized in that the preparation is used to treat diseases related to cell differentiation and proliferation, including psoriasis, blood cancer, and solid tumors. 0001 ～ 200 mg 之间。 Pharmaceutical preparations of benzamide histone deacetylases with cell differentiation and anti-proliferative 200524869 activity, characterized in that the content of the effective components it contains is between 0.001 ~ 200 mg. 1 0 · 如The medicinal preparation of benzamide histone deacetylase with cell differentiation and anti-proliferative activity as described in the top 7 of the scope of the patent application, which is characterized in that the medicinal method can be oral, nasal, transdermal, meridian Lung, blue injection or intravenous injection, etc. 1 1 · The pharmaceutical preparation for treating diseases related to cell differentiation and proliferation, as described in the eighth top of the scope of patent application, characterized in that said effective The amount is 0.0001 to 200 mg per kilogram of body weight per day. 12. The benzophenone cream histone deacetylating enzyme inhibitor with differentiation and antiproliferative activity as described in item 1 of the scope of the patent application, which is characterized by The pharmaceutical preparation of the compound can be used in combination with commonly used tumor chemotherapeutic drugs or fluorenyltransferase inhibitors for the treatment of cell proliferation diseases, and one or more pharmaceutically acceptable carrier excipients or diluents can be added. The benzylamine histone deacetate zeolase inhibitor having differentiation and antiproliferative activity according to item 1, characterized in that the preparation comprises a derivative of a benzamide active ingredient or a salt thereof, and a medicament Composed of carrier materials or diluents. 14. The phenylamidine histone deacetylated 1-enzyme inhibitor with differentiation and anti-proliferative activity as described in item 13 of the scope of application for a patent, characterized in that the content of the effective component it contains is between 0. Between 0001 and 200 mg. 15. The benzoate histone deacetate A enzyme inhibitor with differentiation and anti-proliferation as described in item 13 of the scope of application patent 33 200524869, which is characterized in that it can be administered orally, nasally, Percutaneous, pulmonary, sub-injection or intravenous injection. 16 · A benzylamine histone deacetylase inhibitor with differentiation and antiproliferative activity as described in item 1 of the scope of the patent application, characterized in that it can treat nuclear receptors at its effective dose. Regulates diseases caused by depression. 1 7 The benzyl &amp; amine histone deacetylase inhibitor with differentiation and antiproliferative activity as described in item 1 of the scope of the patent application, characterized in that it can treat Diseases caused by decreased receptor activity. 18 · The phenylpyrene late histone deacetylating enzyme inhibitor with differentiation and anti-proliferative activity according to item 16 of the scope of the patent application, wherein the diseases include diseases related to endocrine , Immune / inflammatory diseases, genetic diseases, neurodegenerative diseases, etc. φ 1 9 · The benzamide histone deacetylation enzyme inhibitor with differentiation and antiproliferative activity according to item 17 of the scope of the patent application, wherein the diseases include endocrine-related diseases, Immune / inflammatory diseases, genetic diseases, neurodegenerative diseases, etc. 2 0. The benzoyl A amine histone deethylating enzyme inhibitor with differentiation and anti-proliferative activity according to item 16 of the scope of the patent application, characterized in that the compound is a pharmaceutical preparation which is effective 0001〜200 mg。 Dosage is 0. 0001 ~ 200 mg per kg of body weight per day. 34 200524869 21 · The phenylpyrene 1,2 amine histone deacetylase inhibitor with differentiation and antiproliferative activity according to item 17 of the scope of application for patent, characterized in that the effective dose of the pharmaceutical preparation of the compound is 0.001 to 200 mg per kg of body weight per day. 35 200524869 VII. Designated representative map (1) The designated representative map in this case is: (none) map. (2) Brief description of the component symbols in this representative picture: 8. If there is a chemical formula in this case, please disclose the chemical that best shows the characteristics of the invention • Formula: R1 R2 I I A-Z-C two C one Y—B 4 1