Indane amine derivatives, processes for their preparation and uses thereof

Abstract

The application discloses an indane amine derivative, a preparation method and application thereof. The application provides an indane amine compound as shown in formula I or a pharmaceutically acceptable salt thereof. The compound of the application has HDAC6 inhibitory activity, good selectivity to HDAC1, HDAC3 or HDAC8, and good metabolic stability.

Description

Technical Field

[0001] This invention relates to a dihydroindeneamine derivative, its preparation method, and its uses. Background Technology

[0002] Epigenetic modifications play a crucial role in gene expression regulation, and histone deacetylases (HDACs), as important functional proteins in epigenetic regulation, have attracted widespread attention from scientists in recent decades. On the one hand, HDACs can mediate the deacetylation of histone substrate lysine, thereby promoting the formation of a more compact chromatin structure. Furthermore, some HDACs can interact with other chromatin regulatory proteins to form co-repressive complexes, thereby regulating life processes such as gene expression, cell cycle, and cell differentiation. On the other hand, some HDACs can also catalyze the deacetylation of non-histone substrate lysine, thus playing an important role in a wider range of cellular regulatory pathways (Nat Biotechnol, 2015, 10.1038 / nbt.3130; Int J Cancer, 2019, 10.1002 / ijc.32169). Current research indicates that examples of histone deacetylase-mediated diseases associated with HDAC inhibition include proliferative disorders such as malignant neoplasms like cancer, inflammatory diseases such as inflammatory bowel disease, Crohn's disease, or ulcerative colitis, autosomal dominant disorders such as Huntington's disease, Down syndrome, Edwards syndrome, or Pataus syndrome, and inherited metabolic disorders such as diabetes, Niemann Pick disease, and Gaucher's disease. Diseases such as phenylketonuria, Wilson's disease, or fibrotic diseases, such as cystic fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis, or skin fibrosis; autoimmune diseases such as rheumatoid arthritis, asthma, lupus, psoriasis, psoriatic arthritis, multiple sclerosis, Behçet's disease, or organ transplant rejection; acute / chronic neurological and systemic diseases such as stroke or polycystic kidney disease; hypertrophy such as cardiac hypertrophy; heart failure such as congestive heart failure or hemorrhagic heart failure; eye diseases such as glaucoma and dry eye; syndromes such as dry macular degeneration, wet macular degeneration, diabetic retinopathy or uveitis; neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, progressive peroneal muscular atrophy, or spinal muscular atrophy; and conditions and diseases caused by abnormal function of HDAC enzymes; as well as peripheral neuropathy, such as chemotherapy-induced peripheral neuropathy, diabetic peripheral neuropathy, and peripheral neuropathy caused by viral infections.

[0003] Currently, 18 HDACs have been identified in the human body. Based on evolutionary and sequence homology analysis, they are classified into four types (Strahl BD, Allis CD. Nature. 2000 Jan 6; 403(6765):41-5.): Type I consists of HDACs 1-3, 8, which are highly homologous to the Rpd3 protein in yeast. They are mainly located in the nucleus, with HDAC 3 also present in the cytoplasm. Type II consists of HDACs 4-7, 9, 10, which are highly homologous to the Hda1 (histone 0.1% diethylamine (DEA)cetylase-1) protein in yeast. They can respond to different cellular signaling by shuttling between the nucleus and cytoplasm, exhibiting cell and tissue specificity. Type III consists of HDACs 4-7, 9, 10, which are highly homologous to the Sir2 (silent information regulator) protein in yeast. 2) The sirtuin protein family, which shares protein homology, includes SIRT1-7; class IV contains only one member, HDAC11, whose sequence homology is intermediate between Rpd3 and Hda1, and it is mainly located in the cell nucleus. Classes I, II, and IV HDACs can be further classified as the Rpd3 / Had1 deacetylase family, all of which contain a highly homologous catalytic core domain, and their catalytic activity depends on the participation of zinc ions. Currently, HDAC inhibitors in clinical trials can be divided into four types based on their chemical structure: hydroxamic acids, cyclic peptides, benzamides, and short-chain fatty acids. SAHA and PXD101 belong to the hydroxamic acid class, while FK228 is a member of the cyclic peptide class. None of these compounds have shown any HDAC subtype selectivity (Tomaselli et al. Med Res Rev. 2019 Jun 20. doi:10.1002 / med.21600).

[0004] Because subtype-selective inhibitors can address the biological functions of different HDAC subtypes separately, they tend to have fewer potential side effects. However, designing HDAC subtype-selective inhibitors is currently very difficult, mainly for two reasons: existing HDAC structural data are limited to a few subtypes (HDAC1, HDAC2, HDAC3, HDAC4, HDAC7, and HDAC8); secondly, the high sequence conservation within a certain class of HDACs results in high structural similarity, making it extremely difficult to design inhibitors targeting a specific HDAC within these subtypes. To date, there are very few reports on HDAC subtype-selective inhibitors. Only non-selective HDAC inhibitors have been identified as anticancer agents.

[0005] However, it is known that non-selective HDAC inhibitors often cause side effects, such as fatigue and nausea, at high doses (Piekarz et al. Pharmaceuticals (Basel). 2010 Sep; 3(9):2751–2767). These side effects have been reported as being due to the inhibition of class I HDACs. Because of these side effects, the use of non-selective HDAC inhibitors in drug development, other than anticancer drugs, is limited (Witt et al., Cancer Lett. 2009 May 8; 277(1):8-21.).

[0006] Meanwhile, it has been reported that selective inhibition of class II HDACs does not exhibit the toxicity shown in the inhibition of class I HDACs. Therefore, selective HDAC inhibitors have the potential to be developed as effective therapeutic agents for various diseases (Matthias et al. MolCell Biol. 2008 Mar; 28(5):1688-70).

[0007] Selective HDAC6 inhibitors are currently a research hotspot in this field, and are expected to overcome the shortcomings of broad-spectrum HDAC inhibitors, such as poor selectivity and large side effects.

[0008] HDAC6 primarily catalyzes the deacetylation of α-tubulin, heat shock protein Hsp90, cortical actin, and peroxide reductase. Due to its unique structure, HDAC6 possesses a variety of distinctive biological functions, regulating multiple cellular pathways related to cell growth, metastasis, and apoptosis through deacetylase-dependent and deacetylase-independent mechanisms. Studies have also shown that HDAC6 plays a significant role in the transfer and accumulation of misfolded proteins into deposits and in responding to misfolded proteins to prevent apoptosis. HDAC6 is closely related to the pathophysiological processes of many diseases, including tumors, neurodegenerative diseases, inflammation, autoimmune responses, bacterial infections, and heart disease, making it a highly promising drug target.

[0009] Currently, very few HDAC6 inhibitors have been discovered, mainly including Tubacin, Tubastatin A, ACY-1215, and Citarinostat (ACY-241). Among them, Tubacin is a highly effective, selective, reversible, cell-permeable HDAC6 inhibitor with an IC50 of 4 nM in cell-free assays, which is 350 times more selective than that for HDAC1 (Butler KV, et al. J Am Chem Soc. 2010, 132(31), 10842-0846.). Tubastatin A is an effective, selective HDAC6 inhibitor with an IC50 of 4 nM in cell-free assays. 50The selectivity is 15 nM, which is much higher than other subtypes (1000 times) except for HDAC8 (57 times). These two compounds have not been clinically developed due to their high toxicity (Gradilone et al. Cancer Res, 2013, 73(7), 2259-2270). ACY-1215 is an orally bioeffective specific inhibitor of histone deacetylase 6 (HDAC6) with potential antitumor activity. Compared with non-selective HDAC inhibitors, ACY-1215 can reduce the toxicity to normal and healthy cells. ACY-1215 is an isohydroxamic acid derivative that is 12, 10, and 11 times less effective against HDAC1, HDAC2, and HDAC3 (type I HDACs), respectively. ACY-1215 exhibits minimal activity against HDAC4, HDAC5, HDAC7, HDAC9, HDAC11, Sirtuin1, and Sirtuin2 (IC50 > 1 μM), and shows slight activity against HDAC8 (IC50 = 0.1 μM). The IC50 value for ACY-1215's T-cell cytotoxicity is 2.5 μM. ACY-1215 acts on the bone marrow (BM) environment, overcoming the growth and survival of tumor cells embodied by BMSCs and cytokines. ACY-1215, in combination with Bortezomib, induces synergistic anti-MM activity. At very low doses, ACY-1215 induces potent α-tubulin acetylation; only at higher doses does it trigger the acetylation of histone H3 and H4 histone lysines, confirming its specific inhibitory effect on HDAC6 activity. ACY-1215 selectively targets and binds to HDAC6, thereby disrupting the Hsp90 chaperone system through Hsp90 hyperacetylation, preventing subsequent aggregate degradation. ACY-1215 is currently undergoing phase II clinical trials for hematologic malignancies and solid tumors, such as alone and in combination with bortezomib (Velcade). (Santo L, et al. Blood, 2012, 119(11), 2579-2589.) and dexamethasone for the treatment of relapsed / refractory multiple lesions, combined with Pd-1 antibody for the treatment of breast cancer, lung cancer, etc. (Santo L, et al. Blood, 2012, 119(11), 2579-2589.). ACY-241 is a selective HDAC6 inhibitor with oral activity that is structurally similar to ricolinostat (ACY-1215), and its IC50 of HDAC6 and HDAC3 is high. 50The effective doses were 2.6 nM and 46 nM, respectively. The selectivity for HDAC6 was 13-18 times that for HDAC1-3. Niesvizky et al. conducted the first Ia / Ib clinical study of ACY-241, which included 40 patients with RRMM. Patients in three groups received a single dose of 180, 360, and 480 mg of ACY-241 for 3 weeks, with each cycle lasting 28 days. From the second cycle onwards, POM and DEX were added. From June 2015, 34 cases were evaluable for safety. Grade 3 and 4 hematologic toxicities were mainly neutropenia (10 cases, 30%). The median follow-up period was 3.5 months. 22 patients were evaluable for efficacy: 1 achieved very good partial remission (VGPR), 10 achieved PR, 2 achieved minimal remission (MR), 8 had stable disease (SD), and 1 had progressive disease (PD). The median PFS and median duration of remission were not reached. Based on comprehensive pharmacodynamics, pharmacokinetics, and safety, 360 mg once daily is recommended as the dose for future trials. The combination of ACY-241 and Pom+Dex showed good tolerability and limited adverse reactions, and further clinical trials are still underway. In addition to hematologic malignancies, there are also Phase 1 clinical trials currently underway for melanoma and lung cancer indications (Huang P, et al. Oncotarget. 2017, 8(2):2694-2707.).

[0010] However, ACY-1215 and ACY-241 have poor cellular metabolism and their half-life of only 3 hours, as shown in human trials, bring many inconveniences to clinical use.

[0011] Therefore, there is an urgent need to develop a novel selective HDAC6 inhibitor with few side effects for the treatment of cancer, inflammatory diseases, autoimmune diseases, nervous system diseases and neurodegenerative diseases. Summary of the Invention

[0012] The technical problem this invention aims to solve is the limited variety of selective HDAC6 inhibitors in existing technologies. To address this, this invention provides a dihydroindoleamine derivative, its preparation method, and its uses. The compounds of this invention exhibit high inhibitory activity against HDAC6, good selectivity for HDAC1, HDAC3, or HDAC8, and good metabolic stability.

[0013] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0014] This invention provides a dihydroindoleamine compound as shown in Figure I, or a pharmaceutically acceptable salt thereof:

[0015]

[0016] Among them, R1 R 2 R 3 and R 4 Independently, it is hydrogen, halogen, hydroxyl, cyano, C1-C6 alkyl, halogen-substituted C1-C6 alkyl (C1-C6 alkyl is substituted only by halogen), C1-C6 alkoxy, halogen-substituted C1-C6 alkoxy (C1-C6 alkoxy is substituted only by halogen), C3-C6 cycloalkyl or C3-C6 heterocycloalkyl, wherein the heteroatom in the C3-C6 heterocycloalkyl is selected from one or more of O, S and N, and the number is 1 to 4;

[0017] R 5 It is hydrogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl (C1-C6 alkyl is only substituted by halogen), C3-C6 cycloalkyl or C3-C6 heterocycloalkyl, wherein the heteroatom in the C3-C6 heterocycloalkyl is selected from one or more of O, S and N, and the number is 1 to 4.

[0018] Y is -O(CH2) n1 -、-(CH2) n2 -or

[0019] R a It is hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl or C3-C6 heterocycloalkyl, wherein the heteroatom in the C3-C6 heterocycloalkyl is selected from one or more of O, S and N, and the number is 1 to 4;

[0020] n1, n2, and n3 are independently 0, 1, 2, or 3.

[0021] In this invention, the definitions of certain substituents in the dihydroindoleamine compounds of Formula I or their pharmaceutically acceptable salts are as follows, and the definitions of substituents not mentioned are as described in any embodiment of this application (hereinafter referred to as "in a certain technical embodiment"):

[0022] In a certain technical solution, when R 1 When the halogen is halogen, the halogen is preferably F, Cl, Br or I, and more preferably Cl.

[0023] In a certain technical solution, when R 2 When the halogen is halogen, the halogen is preferably F, Cl, Br or I, and more preferably Cl.

[0024] In a certain technical solution, when R 3 When the halogen is halogen, the halogen is preferably F, Cl, Br or I, and more preferably Cl.

[0025] In a certain technical solution, when R 4When the halogen is halogen, the halogen is preferably F, Cl, Br or I, and Cl is also preferred.

[0026] In a certain technical solution, when R 1 R 2 R 3 R 4 R 5 or R a When the alkyl group is C1-C6, the C1-C6 alkyl group is preferably C1-C3 alkyl group.

[0027] In a certain technical solution, when R 1 R 2 R 3 R 4 or R 5 When the C1-C6 alkyl group is halogen-substituted, the C1-C6 alkyl group is preferably a C1-C3 alkyl group.

[0028] In a certain technical solution, when R 1 R 2 R 3 R 4 or R 5 When the halogen is a C1-C6 alkyl group substituted with a halogen, the halogen is preferably F, Cl, Br or I.

[0029] In a certain technical solution, when R 1 R 2 R 3 R 4 or R 5 When the alkoxy group is C1-C6, the C1-C6 alkoxy group is preferably C1-C3 alkoxy.

[0030] In a certain technical solution, when R 1 R 2 R 3 R 4 or R 5 When the C1-C6 alkoxy group is halogen-substituted, the C1-C6 alkoxy group is preferably a C1-C3 alkoxy group.

[0031] In a certain technical solution, when R 1 R 2 R 3 R 4 or R 5 When the halogen is a C1-C6 alkoxy group substituted with a halogen, the halogen is preferably F, Cl, Br or I.

[0032] In a certain technical solution, when R 1 R 2 R 3 R 4R 5 or R a When the alkyl group is C3-C6 cycloalkyl, the C3-C6 cycloalkyl group is preferably cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0033] In a certain technical solution, when R 1 R 2 R 3 R 4 R 5 or R a When the heterocyclic alkyl group is C3-C6, the C3-C6 heterocyclic alkyl group is preferably a 3-6 member heterocyclic alkyl group.

[0034] In a certain technical solution, when Y is -O(CH2) n1 When Y is connected to the benzene ring via a C atom or via an O atom, it is more preferably connected to the benzene ring via an O atom.

[0035] In one technical solution, in the dihydroindoleamine compound or its pharmaceutically acceptable salt as shown in I, one or more atoms are atoms existing in a non-natural abundance form, or all atoms are atoms existing in a natural abundance form.

[0036] In one technical solution, among the dihydroindoleamine compound as shown in Figure I or its pharmaceutically acceptable salt, the dihydroindoleamine compound as shown in Figure I is preferably... and / or

[0037] In a certain technical solution, R 1 Preferably hydrogen or halogen, more preferably halogen.

[0038] In a certain technical solution, R 2 Preferably hydrogen or halogen, more preferably halogen.

[0039] In a certain technical solution, R 3 Preferably hydrogen or halogen, more preferably halogen.

[0040] In a certain technical solution, R 4 Preferably hydrogen or halogen, more preferably halogen.

[0041] In a certain technical solution, R 5 Hydrogen is preferred.

[0042] In a certain technical solution, Y is preferably -O(CH2). n1 -or-(CH2) n2 -, more preferably -O(CH2) n1 -

[0043] In a certain technical solution, n1 is preferably 0.

[0044] In a certain technical solution, n1 is preferably 1.

[0045] In a certain technical solution, n2 is preferably 0 or 1.

[0046] In a certain technical solution, Preferred More preferably

[0047] In a certain technical solution, R 1 It is hydrogen or halogen;

[0048] R 2 It is hydrogen or halogen;

[0049] R 3 It is hydrogen or halogen;

[0050] R 4 It is hydrogen or halogen;

[0051] R 5 It is hydrogen;

[0052] Y is -O(CH2) n1 -or-(CH2) n2 ;

[0053] n1 is 0;

[0054] And n2 is 0 or 1.

[0055] In a certain technical solution, R 1 It is hydrogen or halogen;

[0056] R 2 It is hydrogen or halogen;

[0057] R 3 It is hydrogen or halogen;

[0058] R 4 It is hydrogen or halogen;

[0059] R 5 It is hydrogen;

[0060] Y is -O(CH2) n1 -;

[0061] And n1 is 0.

[0062] In a certain technical solution, R 1 It is a halogen;

[0063] R 2It is a halogen;

[0064] R 3 It is a halogen;

[0065] R 4 It is a halogen;

[0066] R 5 It is hydrogen;

[0067] Y is -O(CH2) n1 -;

[0068] And n1 is 0.

[0069] In a certain technical solution, R 1 It is hydrogen or halogen;

[0070] R 2 It is hydrogen or halogen;

[0071] R 3 It is hydrogen or halogen;

[0072] R 4 It is hydrogen or halogen;

[0073] R 5 It is hydrogen;

[0074] Y is -O(CH2) n1 -or-(CH2) n2 ;

[0075] n1 is either 0 or 1;

[0076] And n2 is 0 or 1.

[0077] In a certain technical solution, R 1 It consists of hydrogen and halogens;

[0078] R 2 It consists of hydrogen and halogens;

[0079] R 3 It consists of hydrogen and halogens;

[0080] R 4 It is hydrogen and halogen; and R 1 R 2 R 3 and R 4 At least one of them is halogen;

[0081] Y is -O(CH2) n1 -

[0082] In a certain technical solution, R 1 It is hydrogen or halogen;

[0083] R 2It is hydrogen or halogen;

[0084] R 3 It is hydrogen or halogen;

[0085] R 4 It is hydrogen or halogen;

[0086] R 5 It is hydrogen;

[0087] Y is -O(CH2) n1 -or-(CH2) n2 ;

[0088] n1 is 0;

[0089] And n2 is 0.

[0090] In a certain technical solution, among the dihydroindoleamine compound or its pharmaceutically acceptable salt as shown in I, the dihydroindoleamine compound as shown in I is preferably any one of the following compounds:

[0091]

[0092] In a certain technical solution, among the dihydroindeneamine compound or pharmaceutically acceptable salt shown in I, the dihydroindeneamine compound shown in I is any one of the following compounds:

[0093]

[0094] Under the following HPLC conditions, the retention time is 2.096 min or 2.286 min. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: ethanol; Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 20 min; Flow rate: 2.8 ml / min; Column temperature: 35℃;

[0095]

[0096] The retention times under the following HPLC conditions are 11.53 min or 10.637 min. Chromatographic conditions: CHIRALCEL OJ column 250*4.6mm 5μm; mobile phase A: n-hexane; mobile phase B: ethanol solution containing 0.1% trifluorohexanoic acid; elution conditions: elution with 80% mobile phase A and 20% mobile phase B for 30 min; flow rate: 1 ml / min; column temperature: 35℃;

[0097] The content of trifluorohexanoic acid in mobile phase B is a volume percentage.

[0098] The present invention also provides a method for preparing the dihydroindeneamine compound shown in Figure I above, which includes the following steps:

[0099] In a solvent, under alkaline conditions, the compound shown in Formula II is reacted with hydroxylamine as shown to obtain the dihydroindeneamine compound shown in Formula I above.

[0100]

[0101] Among them, R 6 It is a C1-C4 alkyl group, R 1 R 2 R 3 R 4 R 5 The definitions of Y and Y are the same as those described above.

[0102] In a certain scheme, when R 6 When the alkyl group is C1-C4, the C1-C4 alkyl group is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and may also be preferably ethyl.

[0103] In a certain scheme, when R 6 When the alkyl group is C1-C4, the C1-C4 alkyl group is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and may also be preferably methyl.

[0104] In the reaction, the solvent can be a conventional solvent in the art, preferably an alcohol solvent (e.g., methanol) and a chlorinated hydrocarbon solvent (e.g., dichloromethane). The volume ratio of the alcohol solvent to the chlorinated hydrocarbon solvent can be 1:1 to 1:3, for example, 1:1.

[0105] In the reaction described, the base can be a conventional base in the art, preferably an inorganic base, and more preferably an alkali metal base (e.g., sodium hydroxide).

[0106] In the reaction, the molar ratio of the base to the compound as shown in Formula II can be a conventional molar ratio in the art, preferably 10:1 to 100:1.

[0107] In the reaction described, the concentration of hydroxylamine can be a concentration conventional in the art, such as a 50% aqueous solution of hydroxylamine.

[0108] In the reaction, the molar ratio of the hydroxylamine to the compound shown in Formula II can be a conventional molar ratio in the art, preferably 10:1 to 100:1.

[0109] The reaction temperature can be a temperature conventional in the art, preferably 0-30°C, for example 0°C.

[0110] The progress of the reaction can be monitored using conventional monitoring methods in the art (e.g., TLC, HPLC, HNMR), and the reaction endpoint is generally defined as the disappearance or cessation of the reaction of the compound shown in Formula II, for example, 2 hours.

[0111] After the reaction is completed, a post-processing step is further included. This post-processing step can be a conventional post-processing step in organic reactions. Preferably, the post-processing step includes the following steps: pH adjustment, extraction, drying, concentration, and column chromatography.

[0112] The reagent used in the pH adjustment step is preferably hydrochloric acid, such as 2N dilute hydrochloric acid.

[0113] The column chromatography is preferably reversed-phase column chromatography (e.g., using 0.1% formic acid (FA) as the eluent).

[0114] The present invention also provides a compound as shown in Formula II:

[0115]

[0116] Among them, R 1 R 2 R 3 R 4 R 5 R 6 The definitions of Y and Y are the same as those described above.

[0117] In a certain technical solution, the compound shown in Formula II is preferably any one of the following compounds:

[0118]

[0119] In a certain technical solution, the compound shown in Formula II is preferably any one of the following compounds:

[0120]

[0121] The retention time is 0.793 min or 0.999 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: methanol solution containing 0.1% diethylamine; Elution conditions: elution with 85% mobile phase A and 15% mobile phase B for 6 min; Flow rate: 1.5 ml / min; Column temperature: 35℃;

[0122]

[0123] The retention time is 2.550 min or 4.303 min under the following HPLC conditions. Chromatographic conditions: CHIRALCEL OJ column 250*4.6mm 5μm; mobile phase A: supercritical CO2; mobile phase B: ethanol solution containing 0.1% diethylamine; elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 8 min; flow rate: 2.8 ml / min; column temperature: 35℃;

[0124] The content of diethylamine in mobile phase B is a volume percentage.

[0125] The present invention also provides a pharmaceutical composition comprising substance A and one or more pharmaceutically acceptable carriers, wherein substance A is a dihydroindoleamine compound as shown in I above, or a pharmaceutically acceptable salt thereof.

[0126] The pharmaceutical compositions described in this invention can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols, etc.

[0127] The pharmaceutical composition described in this invention can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0128] The routes of administration for the compounds or pharmaceutically acceptable salts thereof or pharmaceutical compositions described in this invention include, but are not limited to, oral, rectal, transmucosal, enteral, or local, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration. The preferred route of administration is oral administration.

[0129] For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compounds of the present invention to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients. For example, a pharmaceutical composition for oral administration can be obtained as a tablet by combining the active ingredient with one or more solid carriers, granulating the resulting mixture if necessary, and adding a small amount of excipients to process it into a mixture or granules to form a tablet or tablet core. The tablet core can be combined with a coating material optionally suitable for enteric coating to process it into a coated formulation more favorable for absorption by the organism (e.g., human).

[0130] The present invention also provides a pharmaceutical combination comprising substance A and substance X, wherein substance A is a dihydroindoleamine compound as shown in I above or a pharmaceutically acceptable salt thereof; and substance X is bortezomib or a pharmaceutically acceptable salt thereof.

[0131] The present invention also provides the use of substance A in the preparation of a drug, wherein substance A is a dihydroindeneamine compound as shown in I above, or a pharmaceutically acceptable salt thereof; wherein the drug is a drug for the prevention or treatment of HDAC6-related diseases, or, wherein the drug is a drug for the prevention or treatment of cancer or autoimmune diseases.

[0132] The drug is used in combination with bortezomib or a pharmaceutically acceptable salt thereof.

[0133] The present invention also provides the use of substance B in the preparation of HDAC6 inhibitors, wherein substance B is a dihydroindoleamine compound as shown in I above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

[0134] The present invention also provides the use of substance B in the preparation of a drug, wherein substance B is a dihydroindoleamine compound as shown in I above or a pharmaceutically acceptable salt thereof, the above-described pharmaceutical composition or the above-described pharmaceutical combination, and the drug is a drug for the prevention or treatment of HDAC6-related diseases.

[0135] The HDAC6-related diseases mentioned above can be cancer and / or autoimmune diseases.

[0136] The cancers mentioned can be one or more of the following: lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal cell carcinoma, head and neck cancer, leukemia, lymphoma, myeloma, multiple myeloma, melanoma, and hematologic malignancies. They can also be one or more of the following: melanoma, breast cancer, ovarian cancer, multiple myeloma, lymphoma, lung cancer, and degenerative cancer cells.

[0137] The autoimmune diseases mentioned include rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, psoriasis, post-ischemic perfusion injury, inflammatory bowel disease, chronic inflammatory lung disease, eczema, asthma, psoriasis, ulcerative colitis, acute respiratory distress syndrome, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, degenerative arthritis, femoral arthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, polychondritis, acute synovitis and spondylitis, glomerulonephritis, hemolytic anemia, and aplastic anemia. Blood, idiopathic thrombocytopenic purpura, neutropenia, ulcerative colitis, Crohn's disease, graft-versus-host disease, allogeneic graft rejection, chronic thyroiditis, Graves' disease, scleroderma, active hepatitis, primary biliary cirrhosis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, allergic dermatitis, contact dermatitis, chronic renal insufficiency, Schroeder-Johnson syndrome, idiopathic steatorrhea, sarcoidosis, Guillain-Barré syndrome, pulmonary fibrosis, and chronic inflammatory lung disease, one or more of the following:

[0138] Among them, the HDAC6-related diseases mentioned can be peripheral neuropathy.

[0139] The peripheral neuropathy mentioned may include one or more of the following: chemotherapy-induced peripheral neuropathy, diabetic peripheral neuropathy, and peripheral neuropathy caused by viral infection.

[0140] The present invention also provides the use of substance B in the preparation of a drug, wherein substance B is a dihydroindoleamine compound as shown in I above or a pharmaceutically acceptable salt thereof, the above pharmaceutical composition or the above pharmaceutical combination, and the drug is a drug for the prevention or treatment of cancer or autoimmune diseases.

[0141] The cancers and autoimmune diseases mentioned are the same as those described above.

[0142] The present invention also provides the use of substance B in the preparation of a drug, wherein substance B is a dihydroindoleamine compound as shown in I above or a pharmaceutically acceptable salt thereof, the above-described pharmaceutical composition or the above-described pharmaceutical combination, and the drug is a drug for the prevention or treatment of peripheral neuropathy.

[0143] The peripheral neuropathy described above is the same as previously mentioned.

[0144] When a group in this invention is substituted by a substituent, the term "substituent-substituted group" is used, indicating that it is substituted by only one substituent. For example, in this invention, "halogen-substituted C1-C6 alkyl" in "..., cyano, ..., halogen-substituted C1-C6 alkyl" indicates that the C1-C6 alkyl is substituted only by a halogen.

[0145] The term "compound" refers to a compound that, if stereoisomers exist, can exist as a single stereoisomer or a mixture thereof (e.g., a racemic mixture, or a mixture of unequal amounts of enantiomers).

[0146] The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0147] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group composed of carbon and hydrogen atoms, such as C1-C6 alkyl groups, including methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylhexyl, etc.

[0148] The term “cycloalkyl” refers to a monocyclic, fused, spirocyclic, or bridged ring consisting entirely of carbon, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, spiro[3.4]octyl, and bicyclo[3.1.1]hexyl.

[0149] The term "alkoxy" refers to a cyclic or acyclic alkyl group having the stated number of carbon atoms connected by an oxygen bridge. "Alkoxy" encompasses the definitions of alkyl and cycloalkyl groups above.

[0150] The term "heterocyclic alkyl" refers to a monocyclic or fused ring containing one or more heteroatoms of N, O, or S. Typically, it is a 5- or 6-membered heterocyclic group containing one or more heteroatoms of N, O, or S, such as piperazine, morpholino, piperidinium, pyrrolidinyl, and their derivatives.

[0151] “Treatment” means any treatment of disease in a mammal, including: (1) preventing disease, i.e. causing the symptoms of clinical disease to not develop; (2) suppressing disease, i.e. preventing the development of clinical symptoms; and (3) alleviating disease, i.e. causing the clinical symptoms to subside.

[0152] The term "pharmaceutically acceptable carrier" refers to a substance that is co-administered with the active ingredient and facilitates the administration of the active ingredient. This includes, but is not limited to, any flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the State Food and Drug Administration for use in humans or animals (e.g., livestock). Examples include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0153] The atoms in the terms "compound" and "pharmaceuticalally acceptable salt" can exist in either their natural abundance or non-natural abundance forms. For example, the hydrogen atom in its natural abundance form is approximately 99.985% protium and approximately 0.015% deuterium; in its non-natural abundance form, it is approximately 95% deuterium. That is, one or more atoms in the terms "compound" and "pharmaceuticalally acceptable salt" can be atoms existing in a non-natural abundance form. Alternatively, all atoms in the terms "compound" and "pharmaceuticalally acceptable salt" can also be atoms existing in their natural abundance form.

[0154] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0155] The reagents and raw materials used in this invention are all commercially available.

[0156] The positive and progressive effects of this invention are as follows: the compounds of this invention have good inhibitory activity and good metabolic stability against HDAC6, and further, compared with ACY-241, they also have better selectivity for HDAC1, HDAC3 or HDAC8. Detailed Implementation

[0157] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0158] Column chromatography was performed using silica gel (100-200 mesh) produced by Rushan Shangbang New Materials Co., Ltd.; thin-layer chromatography was performed using GF254 (0.3-0.4 mm); nuclear magnetic resonance chromatography (NMR) was performed using a Bruker-400 NMR spectrometer; liquid chromatography-mass spectrometry (LC / MS) was performed using a Waters UPLC-QDa LC / MS system, a Shimadzu LCMS2020 LC / MS system, and an Agilent Technologi ESI 6120 LC / MS system.

[0159] Furthermore, all operations involving easily oxidized or hydrolyzed raw materials are performed under nitrogen protection. Unless otherwise stated, the raw materials used in this invention are commercially available and can be used directly without further purification.

[0160] Example 1: N-hydroxy-2-((2,3-dihydro-1H-inden-1-yl)amino)pyrimidine-5-carboxamide

[0161]

[0162] Step 1: Synthesis of ethyl 2-((2,3-dihydro-1H-inden-1-yl)amino)pyrimidine-5-carboxylate

[0163] Ethyl 2-chloropyrimidine-5-carboxylate (200 mg, 1.07 mmol) and 1-aminoindenhydride (214 mg, 1.61 mmol) were dissolved in 1,4-dioxane (3 mL). Triethylamine (326 mg, 3.22 mmol) was added dropwise to the reaction mixture under ice bath conditions. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure, and methanol (2 mL) was added. The mixture was filtered, and the filter cake was dried under reduced pressure to give the target compound (174 mg, 57.3% yield, white solid). LC-MS (ESI) m / z [M+H] + 284.2.

[0164] Step 2: Synthesis of N-hydroxy-2-((2,3-dihydro-1H-inden-1-yl)amino)pyrimidine-5-carboxamide

[0165] Ethyl 2-((2,3-dihydro-1H-inden-1-yl)amino)pyrimidine-5-carboxylate (100 mg, 0.353 mmol) was dissolved in a mixture of methanol and dichloromethane (10 mL, 1:1), and hydroxylamine aqueous solution (50%, 2 mL) was added at room temperature. A saturated methanol solution of sodium hydroxide (2 mL) was added dropwise under ice bath conditions. The reaction mixture was stirred at room temperature for 3 hours. The organic solvent was removed by vacuum concentration, and the mixture was adjusted to pH 1–2 by adding dilute hydrochloric acid (2N). The mixture was filtered, and the filter cake was washed with water (10 mL), saturated NaHCO3 solution, and water (5 mL), respectively. After drying, the target compound was given (21.7 mg, yield 22.7%, white solid). LC-MS (ESI) m / z [M+H] + 271.2. 1 H NMR(400MHz,DMSO-d6)δ11.04(br s,1H),8.91(br s,1H),8.63(s,2H),8.02-8.00(m,1H),7.26-7.12(m,4H),5.58(q,J=8.0Hz,1H ),3.00-2.93(m,1H),2.86-2.78(m,1H),2.47-2.41(m,1H),1.98-1.88(m,1H).

[0166] Example 2: N-hydroxy-2-((1,2,3,4-tetrahydronaphth-1-yl)amino)pyrimidine-5-carboxamide

[0167]

[0168] Step 1: Synthesis of ethyl 2-((1,2,3,4-tetrahydronaphth-1-yl)amino)pyrimidine-5-carboxylate

[0169] Ethyl 2-chloropyrimidine-5-carboxylate (100 mg, 0.537 mmol), 1,2,3,4-tetrahydronaphthyl-1-amine (79.0 mg, 0.537 mmol), and potassium carbonate (222 mg, 1.612 mmol) were added to N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (20 mL), extracted with dichloromethane (30 mL x 2), and the combined organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the target compound (136 mg, crude, yellow solid), which was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + 298.2.

[0170] Step 2: Synthesis of N-hydroxy-2-((1,2,3,4-tetrahydronaphth-1-yl)amino)pyrimidine-5-carboxamide

[0171] Ethyl 2-((1,2,3,4-tetrahydronaphth-1-yl)amino)pyrimidine-5-carboxylate (106 mg, 0.356 mmol) was dissolved in a mixed solvent of methanol (2 ml) and dichloromethane (2 ml). Hydroxylamine aqueous solution (50%, 2 ml) was added at 0 °C, and after stirring for five minutes at 0 °C, a saturated methanol solution of sodium hydroxide (2 ml) was added dropwise. The reaction mixture was stirred at 0 °C for 2 hours. Dilute hydrochloric acid (2N) was added to the mixture to adjust the pH to 2–3. The mixture was extracted with dichloromethane (30 ml x 2), and the combined organic phases were washed with water (30 ml x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was obtained by lyophilization with (NH4OH) to give the target compound (42.88 mg, two-step yield 35.9%, white solid). LC-MS (ESI) m / z [M+H] + 285.2. 1 H NMR(400MHz,DMSO-d6)δ10.85(br s,1H),9.07(br s,1H),8.61(s,2H),7.93(d,J=9.2Hz,1H),7.16-7.10(m,4H),5.30-5.26(m,1H),2.78-2.72(m,2H),1.98-1.92(m,2H),1.80-1.75(m,2H).

[0172] Example 3; N-hydroxy-2-(benzodihydropyran-4-yl-amino)pyrimidine-5-carboxamide

[0173]

[0174] Step 1: Synthesis of ethyl 2-(benzodihydropyran-4-yl-amino)pyrimidine-5-carboxylate

[0175] Ethyl 2-chloropyrimidine-5-carboxylate (200 mg, 1.08 mmol) and benzodihydropyran-4-ylamine (240 mg, 1.61 mmol) were dissolved in 1,4-dioxane (5 mL). Triethylamine (326 mg, 3.23 mmol) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure, and methanol (2 mL) was added. The mixture was filtered, and the filter cake was dried under reduced pressure to give the target compound (265 mg, yield 82.1%, white solid). LC-MS (ESI) m / z [M+H] + 300.1.

[0176] Step 2: Synthesis of N-hydroxy-2-(benzodihydropyran-4-yl-amino)pyrimidine-5-carboxamide

[0177] Ethyl 2-(benzodihydropyran-4-yl-amino)pyrimidine-5-carboxylate (130 mg, 0.435 mmol) was dissolved in a mixture of methanol and dichloromethane (13 mL, 1:1), and an aqueous solution of hydroxylamine (50%, 2.60 mL) was added at room temperature. A saturated methanol solution of sodium hydroxide (2.60 mL) was added dropwise under ice bath conditions. The reaction mixture was stirred at room temperature for 3 hours. The organic solvent was removed by concentration under reduced pressure, and the mixture was adjusted to pH 4–5 by adding dilute hydrochloric acid (2N). The mixture was filtered, and the filter cake was washed with saturated NaHCO3 solution and water (5 mL). After drying under reduced pressure, the target compound was given (48.6 mg, yield 39.1%, white solid). LC-MS (ESI) m / z [M+H] + 287.3. 1 HNMR (400MHz, DMSO-d6) δ8.63 (s, 2H), 8.02 (d, J = 8.8Hz, 1H), 7.16-7.11 (m, 2H), 6.85-6. 81(m,1H),6.77(d,J=8.0Hz,1H)5.31-5.29(m,1H),4.28-4.21(m,2H),2.11-2.00(m,2H).

[0178] Example 4; N-hydroxy-(2-(2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0179]

[0180] Step 1: Synthesis of (Z)-benzofuran-3(2H)-ketooxime

[0181] Benzofuran-3(2H)-one (500 mg, 3.73 mmol) and hydroxylamine hydrochloride (257 mg, 3.73 mmol) were dissolved in pyridine (10 mL) at room temperature. After stirring the reaction mixture at room temperature for 3 hours, it was diluted with a saturated aqueous solution of copper sulfate pentahydrate (30 mL), extracted with dichloromethane (30 mL × 2), and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the target compound (500 mg, 90.8% yield, deep yellow solid). LC-MS (ESI) m / z [M+H] + 150.0.

[0182] Step 2; Synthesis of 2,3-dihydrobenzofuran-3-amine

[0183] (Z)-benzofuran-3(2H)-ketooxime (200 mg, 1.34 mmol) was dissolved in methanol (10 mL), and wet palladium on carbon (10%, 100 mg) was added. The reaction mixture was stirred under hydrogen atmosphere at 1 atm for 24 hours. The palladium on carbon was removed by diatomaceous earth filtration. The filtrate was concentrated under reduced pressure to give the target compound (150 mg, 82.8% yield, yellow solid).

[0184] Step 3: Synthesis of ethyl 2-(2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate

[0185] Ethyl 2-chloropyrimidine-5-carboxylate (150 mg, 0.806 mmol) was dissolved in N,N-dimethylformamide (5 mL), and potassium carbonate (334 mg, 2.42 mmol) and 2,3-dihydrobenzofuran-3-amine (109 mg, 0.806 mmol) were added sequentially at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature, diluted with water (30 mL), and extracted with dichloromethane (30 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the crude product. Purification by column chromatography (silica gel, ethyl acetate: petroleum ether = 0–10%) gave the target compound (90 mg, yield 39.3%, yellow solid). LC-MS (ESI) m / z [M+H] + 286.1.

[0186] Step 4: Synthesis of N-hydroxy-(2-(2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0187] Ethyl 2-(2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid (90 mg, 0.315 mmol) was dissolved in a mixed solvent of methanol (1 ml) and dichloromethane (1 ml). Hydroxylamine (1 ml, 50% aqueous solution) and a saturated sodium hydroxide methanol solution (1 ml) were added sequentially at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. The pH was adjusted to 2–3 at room temperature by adding dilute hydrochloric acid (2N). The mixture was extracted with dichloromethane (30 ml × 2), the combined organic phases were washed with water (30 ml × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by reverse-phase preparation (0.1% FA), and lyophilized to give the target compound (10.69 mg, yield 12.4%, white solid). LC-MS (ESI) m / z [M+H] + 273.1. 1 H NMR(400MHz,DMSO-d6)δ11.02(br s,1H),9.04(br s,1H),8.66(s,2H),8.35(d,J=7.2Hz,1H),7.33(d,J=7.2Hz,1H),7.23-7.19(m, 1H),6.88-6.83(m,2H),5.81-5.77(m,1H),4.77-4.72(m,1H),4.36-4.32(m,1H).

[0188] Examples 5 and 6: (S)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide or (R)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide / (R)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide or (S)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0189]

[0190] Note: The meaning of "or 1" in structural formulas 5 and 6 is that the compound has a single stereoconfiguration, but its configuration is uncertain, that is, the compound may have a single R configuration or a single S configuration.

[0191] Step 1: Preparation of (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (R)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 5-1 and (R)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 6-1

[0192] Ethyl 2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (60 mg, 0.210 mmol) was isolated by Waters SFC 80 (room temperature, 100 bar, 214 nm) and 250*25 mm 10 μm Daicel Chiral-AS (supercritical carbon dioxide:methanol (0.1% ammonia), 80:20, 6 min, 70 ml / min) to obtain compounds (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 5-1 (20 mg, colorless oil, retention time (Ret. time) = 0.793 min, ee 100%). LC-MS (ESI) m / z [M+H] was used to separate the compounds (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 5-1 (20 mg, colorless oil, retention time (Ret. time) = 0.793 min, ee 100%). + 286.1; and compounds (R)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 6-1 (25 mg, colorless oil, retention time (Ret.time) = 0.999 min, ee 98.9%). LC-MS (ESI) m / z [M+H] + 286.1

[0193] Chiral analysis method: Chiral column: CHIRALCEL AS 100*3.0mm 3μm; Mobile phase A: Supercritical CO2; Mobile phase B: MeOH (0.1% diethylamine (DEA)); Elution conditions: Elution with 85% mobile phase A and 15% mobile phase B for 6 min; Flow rate: 1.5 ml / min; Column temperature: 35℃.

[0194] Step 2: Synthesis of (S)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide or (R)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide 5

[0195] Ethyl (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate or (R)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 5-1 (20 mg, 0.0706 mmol) was dissolved in a mixed solvent of methanol (1 ml) and dichloromethane (1 ml). Hydroxylamine (0.5 ml, 50% aqueous solution) was added at 0 °C, followed by dropwise addition of saturated sodium hydroxide methanol solution (0.5 ml). The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated and diluted with water (10 ml). The pH was adjusted to 6 with 2N hydrochloric acid, the solid was collected by filtration, washed with water (10 ml), and dried under reduced pressure to give target compound 5 (9.98 mg, yield 51.9%, white solid, retention time (Ret. time) = 2.096 min, ee 100%). Chiral analysis method: Chiral column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: EtOH; Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 20 min; Flow rate: 2.8 ml / min; Column temperature: 35℃.

[0196] LC-MS(ESI)m / z[M+H] + 273.3. 1 H NMR (400MHz, DMSO-d6) δ11.07(s,1H),9.01(s,1H),8.66(s,2H),8.34(d,J=7.2Hz,1H),7.33(d,J=7.6Hz ,1H),7.22-7.18(m,1H),6.88-6.83(m,2H),5.80-5.77(m,1H),4.75(t,J=8.4Hz,1H),4.36-4.32(m,1H).

[0197] Step 3: Synthesis of (R)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide or (S)-N-hydroxy-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide 6

[0198] Ethyl (R)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate or (S)-2-((2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 6-1 (25 mg, 0.0877 mmol) was dissolved in a mixed solvent of methanol (1 ml) and dichloromethane (1 ml). Hydroxylamine (0.5 ml, 50% aqueous solution) was added at 0 °C, followed by dropwise addition of a saturated sodium hydroxide methanol solution (0.5 ml). The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated and diluted with water (10 ml). After adjusting the pH to 6 with 2N hydrochloric acid, the mixture was extracted with ethyl acetate (20 ml × 3). The organic phases were combined and concentrated under reduced pressure to obtain the crude product. After purification by reverse-phase (0.1% FA) and lyophilization, target compound 6 was obtained (12.4 mg, yield 51.9%, white solid, retention time (Ret. time) = 2.286 min, ee 100%). Chirality analysis method: consistent with Example 5. LC-MS (ESI) m / z [M+H] + 273.3. 1 H NMR (400MHz, DMSO-d6) δ11.08(s,1H),9.05(s,1H),8.67(s,2H),8.36(d,J=7.6Hz,1H),7.34(d,J=7.2Hz ,1H),7.23-7.19(m,1H),6.88-6.83(m,2H),5.82-5.77(m,1H),4.75(t,J=9.2Hz,1H),4.36-4.32(m,1H).

[0199] Example 7: N-hydroxy-2-((6-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0200]

[0201] Step 1: Synthesis of methyl 2-(2-ethoxy-2-oxoethoxy)-4-chlorobenzoate

[0202] Methyl 2-hydroxy-4-chlorobenzoate (3.30 g, 17.7 mmol) was dissolved in N,N-dimethylformamide (20 mL), and ethyl bromoacetate (3.55 g, 21.3 mmol) and potassium carbonate (4.89 g, 35.5 mmol) were added. The reaction mixture was stirred at 60 °C for 3 hours. The reaction mixture was cooled to room temperature, and water (60 mL) was added to the mixture. Extraction was performed with ethyl acetate (30 mL × 3). The combined organic phases were washed with water (20 mL), saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (5.10 g, yellow oil). This crude product was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + 273.1.

[0203] Step 2: Synthesis of 2-(carboxymethoxy)-4-chlorobenzoic acid

[0204] Methyl 2-(2-ethoxy-2-oxoethoxy)-4-chlorobenzoate (5.10 g, crude, 17.7 mmol) was added to water (50 mL), followed by sodium hydroxide (4.0 g, 100 mmol). The reaction mixture was stirred overnight at room temperature. The pH of the reaction solution was adjusted to 4 with 1 N dilute hydrochloric acid. The solid was filtered, the filter cake was washed with water (100 mL), and the solid was dried under reduced pressure to give the target compound (3.1 g, 76.6% yield in two steps, white solid). LC-MS (ESI) m / z [MH] - 229.1.

[0205] Step 3: Synthesis of 6-chlorobenzofuran-3-ylacetic acid ester

[0206] 2-(carboxymethoxy)-4-chlorobenzoic acid (700 mg, 3.04 mmol) was added to acetic anhydride (5 mL), followed by pyridine (24 mg, 0.30 mmol). The reaction mixture was stirred overnight at 145 °C. The reaction mixture was cooled to room temperature, and water (30 mL) was added to the mixture. Extraction was performed with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (650 mg, brown oil). This crude product was used directly in the next reaction without purification. 1 H NMR (400MHz, CDCl3) δ8.01(s,1H),7.48-7.46(m,2H),7.26-7.24(m,1H),2.37(s,3H).

[0207] Step 4: Synthesis of 6-chlorobenzofuran-3(2H)-one

[0208] 6-Chlorobenzofuran-3-ylacetate (650 mg, crude product, 3.04 mmol) was added to water (10 ml), followed by concentrated hydrochloric acid (1 ml). The reaction mixture was stirred at 100 °C for 2 hours. The reaction solution was cooled to room temperature and extracted with ethyl acetate (20 ml × 2). The combined organic phases were washed with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:10) to give the target compound (300 mg, two-step yield 58.7%, yellow solid). 1 H NMR (400MHz, DMSO-d6) δ7.66 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 1.6 Hz, 1H), 7.21-7.19 (m, 1H), 4.86 (s, 2H).

[0209] Step 5: Synthesis of 6-chloro-2,3-dihydrobenzofuran-3-amine

[0210] 6-Chlorobenzofuran-3(2H)-one (100 mg, 0.595 mmol) was added to isopropanol (5 mL), followed by ammonium acetate (916 g, 11.90 mmol) and sodium cyanoborohydride (187 mg, 2.97 mmol). The reaction mixture was stirred overnight at 80 °C. The reaction solution was cooled to room temperature, and the mixture was added to water (30 mL). The pH was adjusted to 13 with 2 N sodium hydroxide solution, and then extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with water (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (100 mg, crude product, yellow oil). This crude product was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + No signal.

[0211] Step 6: Synthesis of ethyl 2-((6-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate

[0212] 6-Chloro-2,3-dihydrobenzofuran-3-amine (100 mg, crude, 0.591 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of potassium carbonate (163 mg, 1.18 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (110 mg, 0.591 mmol). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative chromatography (silica gel, ethyl acetate: petroleum ether = 1:5) to give the target compound (25 mg, two-step yield 13.2%, yellow oil). LC-MS (ESI) m / z [M+H] + 320.1.

[0213] Step 7: Synthesis of N-hydroxy-2-((6-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0214] Ethyl 2-((6-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (25 mg, 0.078 mmol) was dissolved in a mixed solvent of methanol (1 mL) and dichloromethane (1 mL). Hydroxylamine (0.5 mL, 50% aqueous solution) and a saturated sodium hydroxide methanol solution (0.5 mL) were added sequentially at 0 °C. The reaction mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reverse-phase preparation (0.1% FA), followed by lyophilization to give the target compound (7.14 mg, yield 30.0%, white solid). LC-MS (ESI) m / z [M+H] + 307.1. 1 H NMR (400MHz, DMSO-d6) δ11.07(s,1H),9.01(s,1H),8.66(s,2H),8.36(d,J=7.2Hz,1H),7.40(d,J=8.4Hz ,1H),6.97-6.96(m,1H),6.92-6.89(m,1H),5.78-5.73(m,1H),4.81(t,J=8.4Hz,1H),4.43-4.39(m,1H).

[0215] Example 8: N-hydroxy-2-((7-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0216]

[0217] Step 1: Synthesis of methyl 3-chloro-2-hydroxybenzoate

[0218] 3-Chloro-2-hydroxybenzoic acid (1.30 g, 7.53 mmol) was dissolved in methanol (15 mL), followed by the slow addition of thionyl chloride (2 mL). The reaction mixture was stirred overnight at 70 °C. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give the target compound (1.40 g, crude product, yellow oil). It was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + 187.1.

[0219] Step 2: Synthesis of methyl 3-chloro-2-(2-ethoxy-2-oxoethoxy)benzoate

[0220] Methyl 3-chloro-2-hydroxybenzoate (1.40 g, 7.53 mmol) was dissolved in N,N-dimethylformamide (15 mL), and ethyl bromoacetate (1.50 g, 9.03 mmol) and potassium carbonate (2.07 g, 15.06 mmol) were added. The reaction mixture was stirred at 60 °C for 3 hours. The reaction solution was cooled to room temperature, and water (30 mL) was added to the mixture. Extraction was performed with ethyl acetate (20 mL × 3). The combined organic phases were concentrated under reduced pressure to give the crude product. Purification by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:3) yielded the target compound (1.50 g, two-step yield 73.5%, colorless oil). LC-MS (ESI) m / z [M+H] + 273.1.

[0221] Step 3: Synthesis of 2-(carboxymethoxy)-3-chlorobenzoic acid

[0222] Methyl 3-chloro-2-(2-ethoxy-2-oxoethoxy)benzoate (1.50 g, 5.51 mmol) was added to a mixture of water (20 mL) and methanol (10 mL), followed by sodium hydroxide (1.0 g, 25 mmol). The reaction mixture was stirred overnight at room temperature. The pH of the reaction solution was adjusted to 5 with 1 N dilute hydrochloric acid. The solid was filtered, the filter cake was washed with water (20 mL), and the solid was dried under reduced pressure to give the target compound (1.2 g, 95.3% yield, white solid). LC-MS (ESI) m / z [MH] - 228.9.

[0223] Step 4: Synthesis of 7-chlorobenzofuran-3-ylacetic acid ester

[0224] 2-(carboxymethoxy)-4-chlorobenzoic acid (1.20 g, 5.21 mmol) was added to acetic anhydride (10 mL), followed by pyridine (40 mg, 0.521 mmol). The reaction mixture was stirred overnight at 140 °C. The reaction mixture was cooled to room temperature, and water (50 mL) was added to the mixture. Extraction was performed with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (1.5 g, yellow oil). This crude product was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + No signal.

[0225] Step 5: Synthesis of 7-chlorobenzofuran-3(2H)-one

[0226] 1.50 g of crude 7-chlorobenzofuran-3-ylacetate (5.21 mmol) was added to water (10 ml), followed by 2 ml of concentrated hydrochloric acid. The reaction mixture was stirred at 100 °C for 3 hours. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (20 ml × 3). The combined organic phases were washed with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:10) to give the target compound (500 mg, two-step yield 57.1%, yellow solid). 1 H NMR (400MHz, DMSO-d6) δ7.86-7.84 (m, 1H), 7.63-7.61 (m, 1H), 7.17 (t, J = 8.0Hz, 1H), 4.93 (s, 2H).

[0227] Step 6: Synthesis of 7-chloro-2,3-dihydrobenzofuran-3-amine

[0228] 7-Chlorobenzofuran-3(2H)-one (300 mg, 1.78 mmol) was added to isopropanol (10 mL), followed by ammonium acetate (2.75 g, 35.6 mmol) and sodium cyanoborohydride (559 mg, 8.90 mmol). The reaction mixture was stirred overnight at 80 °C. The reaction solution was cooled to room temperature, and water (30 mL) was added to the mixture. The pH was adjusted to 14 with 2 N sodium hydroxide solution, and then extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound (310 mg, crude product, yellow oil). This unpurified product was used directly in the next reaction. LC-MS (ESI) m / z [M+H] + No signal.

[0229] Step 7: Synthesis of ethyl 2-((7-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate

[0230] 7-Chloro-2,3-dihydrobenzofuran-3-amine (310 mg, crude, 1.82 mmol) was dissolved in N,N-dimethylformamide (4 mL), followed by the addition of potassium carbonate (502 mg, 3.64 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (340 mg, 1.82 mmol). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:5) to give the target compound (130 mg, two-step yield 22.9%, yellow solid). LC-MS (ESI) m / z [M+H] + 320.1.

[0231] Step 8: Synthesis of N-hydroxy-2-((7-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0232] Ethyl 2-((7-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (40 mg, 0.125 mmol) was dissolved in a mixed solvent of methanol (1 mL) and dichloromethane (1 mL). Hydroxylamine (1 mL, 50% aqueous solution) and a saturated sodium hydroxide methanol solution (1 mL) were added sequentially at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reverse-phase preparation (0.1% FA), followed by lyophilization to give the target compound (12 mg, yield 31.3%, white solid). LC-MS (ESI) m / z [M+H] + 307.1. 1 HNMR(400MHz,DMSO-d6)δ11.09(s,1H),9.04(s,1H),8.67(s,2H),8.42(d,J=7.6Hz,1H),7.31-7 .29(m,2H),6.89(t,J=7.8Hz,1H),5.92-5.86(m,1H),4.86(t,J=9.4Hz,1H),4.47-4.43(m,1H).

[0233] Example 9: N-hydroxy-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0234]

[0235] Step 1: Synthesis of methyl 2-(2-ethoxy-2-oxoethoxy)-5-chlorobenzoate

[0236] Methyl 2-hydroxy-5-chlorobenzoate (2.10 g, 11.3 mmol) was dissolved in N,N-dimethylformamide (20 mL), and ethyl bromoacetate (2.50 g, 14.7 mmol) and potassium carbonate (3.10 g, 22.6 mmol) were added. The reaction mixture was stirred at 50 °C for 3 hours. The reaction mixture was cooled to room temperature, and water (100 mL) was added to the mixture. Extraction was performed with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (3.50 g, colorless oil). This crude product was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + 273.1.

[0237] Step 2: Synthesis of 2-(carboxymethoxy)-5-chlorobenzoic acid

[0238] Methyl 2-(2-ethoxy-2-oxoethoxy)-5-chlorobenzoate (3.50 g, 11.30 mmol) was added to water (30 mL), followed by sodium hydroxide (2.0 g, 80.0 mmol). The reaction mixture was stirred overnight at room temperature. The pH of the reaction solution was adjusted to 4 with 1 N dilute hydrochloric acid. The solid was filtered, the filter cake was washed with water (20 mL), and the solid was dried under reduced pressure to give the target compound (2.1 g, 68.3% yield in two steps, white solid). LC-MS (ESI) m / z [MH] - 228.9.

[0239] Step 3: Synthesis of 5-chlorobenzofuran-3-ylacetic acid ester

[0240] 2-(carboxymethoxy)-5-chlorobenzoic acid (2.0 g, 8.70 mmol) was added to acetic anhydride (20 mL), followed by pyridine (100 mg, 1.26 mmol). The reaction mixture was stirred overnight at 140 °C. The reaction mixture was cooled to room temperature, and water (100 mL) was added to the mixture. Extraction was performed with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product (2.8 g, black oil). This crude product was used directly in the next reaction without purification. 1 H NMR (400MHz, CDCl3) δ 8.03 (s, 1H), 7.54 (d, J = 2.0Hz, 1H), 7.39-7.37 (m, 1H), 7.29-7.27 (m, 1H), 2.37 (s, 3H).

[0241] Step 4: Synthesis of 5-chlorobenzofuran-3(2H)-one

[0242] 2.8 g of crude 5-chlorobenzofuran-3-ylacetate (8.70 mmol) was added to water (10 ml), followed by concentrated hydrochloric acid (3 ml). The reaction mixture was stirred at 100 °C for 3 hours. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (20 ml × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:7) to give the target compound (800 mg, two-step yield 54.0%, brown solid). 1 HNMR (400MHz, CDCl3) δ7.63 (d, J = 2.0 Hz, 1H), 7.57-7.54 (m, 1H), 7.11 (d, J = 8.8 Hz, 1H), 4.68 (s, 2H).

[0243] Step 5: Synthesis of 5-chloro-2,3-dihydrobenzofuran-3-amine

[0244] 5-Chlorobenzofuran-3(2H)-one (500 mg, 2.97 mmol) was added to isopropanol (20 mL), followed by ammonium acetate (4.54 g, 59.4 mmol) and sodium cyanoborohydride (942 mg, 15.0 mmol). The reaction mixture was stirred overnight at 80 °C. The reaction solution was cooled to room temperature, and water (50 mL) was added to the mixture. The pH was adjusted to 14 with 2N sodium hydroxide solution, and then extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with water (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (520 mg, yellow oil). This crude product was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + No signal.

[0245] Step 6: Synthesis of ethyl 2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate

[0246] 5-Chloro-2,3-dihydrobenzofuran-3-amine (520 mg, crude, 2.97 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of potassium carbonate (1.38 g, 10.0 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (554 mg, 2.97 mmol). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. Purification by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:5) yielded the target compound (180 mg, two-step yield 19.0%, yellow solid). LC-MS (ESI) m / z [M+H] + 320.0.

[0247] Step 7: Synthesis of N-hydroxy-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0248] Ethyl 2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (50 mg, 0.516 mmol) was dissolved in a mixed solvent of methanol (2 mL) and dichloromethane (3 mL). Hydroxylamine (1 mL, 50% aqueous solution) and a saturated sodium hydroxide methanol solution (1 mL) were added sequentially at 0 °C. The reaction mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to give the crude product. Purification was achieved by reverse-phase preparation (0.1% FA), followed by lyophilization to give the target compound (24 mg, 50.2% yield, white solid). LC-MS (ESI) m / z [M+H] + 307.1. 1 H NMR (400MHz, DMSO-d6) δ11.08(s,1H),9.03(s,1H),8.67(s,2H),8.42(d,J=7.6Hz,1H),7.35(d,J=1.2Hz,1 H),7.26-7.23(m,1H),6.88(d,J=8.8Hz,1H),5.80-5.75(m,1H),4.79(t,J=9.2Hz,1H),4.41-4.37(m,1H).

[0249] Example 10: N-hydroxy-2-((4-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0250]

[0251] Step 1: Synthesis of methyl 2-chloro-6-hydroxybenzoate

[0252] 2-Chloro-6-hydroxybenzoic acid (2.0 g, 11.6 mmol) was dissolved in methanol (30 mL), followed by the slow addition of thionyl chloride (5 mL). The reaction mixture was stirred at 70 °C for 16 hours. The organic solvent was removed by concentration under reduced pressure. Water (50 mL) was added to the mixture, and extraction was performed with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound (2.20 g, colorless oil). This unpurified compound was used directly in the next reaction. LC-MS (ESI) m / z [M+H] + 187.2.

[0253] Step 2: Synthesis of methyl 2-chloro-6-(2-ethoxy-2-oxoethoxy)benzoate

[0254] Methyl 2-chloro-6-hydroxybenzoate (2.2 g, 11.6 mmol) was dissolved in N,N-dimethylformamide (20 mL), and ethyl bromoacetate (1.94 g, 11.6 mmol) and potassium carbonate (3.20 g, 23.2 mmol) were added. The reaction mixture was stirred at 50 °C for 3 hours. The reaction solution was cooled to room temperature, and water (50 mL) was added to the mixture. The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound (2.70 g, red oil). This compound was used directly in the next reaction without purification. LC-MS (ESI) m / z [M+H] + 273.1.

[0255] Step 3: Synthesis of 2-(carboxymethoxy)-6-chlorobenzoic acid

[0256] Methyl 2-chloro-6-(2-ethoxy-2-oxoethoxy)benzoate (2.7 g, 11.6 mmol) was dissolved in methanol (30 mL), and an aqueous sodium hydroxide solution (5 N, 20 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After removing the organic solvent by concentration under reduced pressure, the pH was adjusted to 5 with dilute hydrochloric acid (6 N). The mixture was filtered, the filter cake was washed with water (10 mL), and the solid was dried under reduced pressure to give the target compound (1.6 g, 59.9% yield in three steps, white solid). LC-MS (ESI) m / z [MH] - 228.9.

[0257] Step 4: Synthesis of 4-chlorobenzofuran-3-ylacetic acid ester

[0258] 1.6 g (6.96 mmol) of 2-(carboxymethoxy)-6-chlorobenzoic acid was added to acetic anhydride (20 mL), followed by 5 drops of pyridine. The reaction mixture was stirred at 140 °C for 16 hours. The reaction mixture was cooled to room temperature, and 50 mL of water was added to the mixture. Extraction was performed with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL), saturated sodium bicarbonate (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (silica gel, petroleum ether: ethyl acetate = 10:1) yielded the target compound (1.4 g, 95.7% yield, off-white solid). LC-MS (ESI) m / z [MH] - 208.9.

[0259] Step 5: Synthesis of 4-chlorobenzofuran-3(2H)-one

[0260] 1.4 g (6.67 mmol) of 4-chlorobenzofuran-3-ylacetate was added to methanol (20 mL), followed by 5 mL of concentrated hydrochloric acid. The reaction mixture was stirred at 100 °C for 2 hours. The reaction mixture was cooled to room temperature, concentrated under reduced pressure to remove the organic solvent, filtered, washed with water (10 mL), and dried under reduced pressure to give the target compound (1.1 g, 98.1% yield, red solid). 1 H NMR (400MHz, CDCl3) δ7.65-7.58 (m, 2H), 7.07 (t, J = 7.6Hz, 1H), 4.74 (s, 2H).

[0261] Step 6: Synthesis of 4-chloro-2,3-dihydrobenzofuran-3-amine

[0262] 4-Chlorobenzofuran-3(2H)-one (500 mg, 2.97 mmol) was added to isopropanol (15 mL), followed by ammonium acetate (4.62 g, 60.0 mmol) and sodium cyanoborohydride (942 mg, 15.0 mmol). The reaction mixture was stirred overnight at 80 °C. The reaction mixture was cooled to room temperature, and water (50 mL) was added to the mixture. The pH was adjusted to 14 with 2N sodium hydroxide solution, and then extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with water (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product (530 mg, black oil). This crude product was used directly in the next reaction without purification.

[0263] Step 7: Synthesis of ethyl 2-((4-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate

[0264] 4-Chloro-2,3-dihydrobenzofuran-3-amine (530 mg, crude, 2.97 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of potassium carbonate (1.38 g, 10.0 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (552 mg, 2.97 mmol). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. Purification by column chromatography (silica gel, ethyl acetate: petroleum ether = 1:5) yielded the target compound (250 mg, two-step yield 26.3%, yellow solid). LC-MS (ESI) m / z [M+H] + 320.0.

[0265] Step 8: Synthesis of N-hydroxy-2-((4-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxamide

[0266] Ethyl 2-((4-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (75 mg, 0.235 mmol) was dissolved in a mixed solvent of methanol (2 mL) and dichloromethane (2 mL). Hydroxylamine (1 mL, 50% aqueous solution) and a saturated sodium hydroxide methanol solution (1 mL) were added sequentially at 0 °C. The reaction mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to give the crude product. Purification was achieved by reverse-phase preparation (0.1% FA), followed by lyophilization to give the target compound (33 mg, 45.9% yield, white solid). LC-MS (ESI) m / z [M+H] + 307.3. 1 H NMR(400MHz,DMSO-d6)δ11.09(s,1H),9.03(s,1H),8.67(br s,2H),8.42(d,J=7.6Hz,1H),7.31-7.29(m,2H),6.88(t,J=7.6Hz,1H),5.92-5.88(m,1H),4.86(t,J=9.2Hz,1H),4.47-4.43(m,1H).

[0267] Examples 11 and 12: (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidin-5-methyl amide or (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide / (R)-2-((5- Chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide or (S)-2-((5-chloro-2,3-dihydrobenzo[a]) (Furan-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide

[0268]

[0269] Note: The meaning of "or 1" in structural formulas 11 and 12 is that the compound has a single stereoconfiguration, but its configuration is uncertain, that is, the compound may have a single R configuration or a single S configuration.

[0270] Step 1: Preparation of (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 11-1 / (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 12-1

[0271] Ethyl 2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate (500 mg, 1.56 mmol) was subjected to a Waters SFC 80 (room temperature, 100 bar, 214 nm) and a 250*25 mm 10 μm Daicel microscope. The compounds (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 11-1 (180 mg, yield 36.0%, white solid, retention time (Ret. time) = 2.550 min, ee 100%) were isolated by Chiral-AS (supercritical carbon dioxide: ethanol (0.1% ammonia), 60:40, 12 min, 70 ml / min). LC-MS (ESI) m / z [M+H] + 320.4; and (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester or (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylic acid ethyl ester 12-1 (180 mg, yield 36.0%, white solid, retention time (Ret. time) = 4.303 min, ee 100%), LC-MS (ESI) m / z [M+H] + 320.4.

[0272] Chiral analysis method: Chiral column: CHIRALCEL AY 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: EtOH (0.1% diethylamine (DEA)); Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 8 min; Flow rate: 2.8 ml / min; Column temperature: 35℃.

[0273] Step 2: Preparation of (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide or (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide 11

[0274] Ethyl (S)- or (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 11-1 (180 mg, 0.56 mmol) was dissolved in a mixed solvent of methanol (3 ml) and dichloromethane (6 ml). Hydroxylamine (50% aqueous solution, 1.5 ml) was added at 0 °C, followed by dropwise addition of a saturated sodium hydroxide methanol solution (1.5 ml). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, diluted with water (80 ml), and the pH was adjusted to 5 with 2 N hydrochloric acid. The solid was collected by filtration, washed with water (10 ml × 6), and the filter cake was dried under reduced pressure to give target compound 11 (140 mg, yield 81.1%, off-white solid, retention time (Ret. time) = 11.53 min, ee 100%). Chiral analysis method: Chiral column; CHIRALCEL OJ250*4.6mm 5μm; Mobile phase A: n-Hexane; Mobile phase B: EtOH (0.1% trifluorohexanoic acid (TFA)); Elution conditions: A 80%, B 20% for 30 min; Flow rate 1 mL / min; Column temperature: 35℃. LC-MS (ESI) m / z [M+H] + 307.3. 1 HNMR(400MHz,DMSO-d6)δ11.09(s,1H),9.03(s,1H),8.68(s,2H),8.41(d,J=6.8Hz,1H),7.36(s,1H), 7.27-7.25(m,1H),6.89(d,J=8.4Hz,1H),5.79-5.78(m,1H),4.79(t,J=9.2Hz,1H),4.42-4.38(m,1H).

[0275] Step 3: Preparation of (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide or (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)-N-hydroxypyrimidine-5-carboxamide 12

[0276] Ethyl (R)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate or ethyl (S)-2-((5-chloro-2,3-dihydrobenzofuran-3-yl)amino)pyrimidine-5-carboxylate 12-1 (180 mg, 0.56 mmol) was dissolved in a mixed solvent of methanol (3 ml) and dichloromethane (6 ml). Hydroxylamine (1.5 ml, 50% aqueous solution) was added at 0 °C, followed by dropwise addition of a saturated sodium hydroxide methanol solution (1.5 ml). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated. It was diluted with water (80 ml), and the pH was adjusted to 5 with 2N hydrochloric acid. The solid was collected by filtration and washed with water (10 ml × 6). The filter cake was dried under reduced pressure to give target compound 12 (140 mg, yield 81.1%, off-white solid, retention time (Ret.time) = 10.637 min, ee 100%). Chiral analysis method: consistent with Example 11. LC-MS (ESI) m / z [M+H] + 307.3. 1 H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.03(s,1H),8.68(s,2H),8.40(d,J=7.2Hz,1H),7.36(s,1H),7 .27-7.25(m,1H),6.89(d,J=8.4Hz,1H),5.79-5.78(m,1H),4.80(t,J=9.2Hz,1H),4.42-4.38(m,1H).

[0277] Effect Example

[0278] Example 1: HDAC enzyme activity and subtype selectivity test

[0279] The HDAC Caliper Assay was used to determine the inhibitory activity of each HDAC isoform of the compounds. All HDAC enzymes were purchased from BPS Bioscience, and all general reagents not otherwise specified were purchased from Sigma.

[0280] Reagent preparation: 1. Store HDAC enzyme stock solution at -80℃; 2. Substrate stock (2.5mM) (sequence: 5-FAM-TSRH-Lys(Ac)-Lys(Ac)-M-NH2) at -80℃; 3. Prepare sodium chloride solution (1M) at room temperature; 4. Prepare potassium chloride solution (1M) at room temperature; 5. Prepare reaction buffer (1000ml) at room temperature, consisting of: Trizma hydrochloride 25ml (Sigma), sodium chloride solution 137ml, potassium chloride solution 2.7ml, magnesium chloride solution 1ml, bovine serum albumin (BSA) 0.1g. The substrate working solution (2X), HDAC enzyme working solution (2X), and stop solution (1000 ml) consisted of: 100 ml HEPES buffer; 20 ml 0.5 M EDTA solution; 1.33 ml Brij 35 solution; and 0.5 ml 5 mM Trichostatin A (Sigma). The test compound was dissolved in 1 mM DMSO and diluted semi-logarithmically to the desired concentration.

[0281] Experimental protocol: 1. Add 5 μL of substrate working solution to each well of a 384-well plate; 2. Mix at 1000 rpm for 1 minute; 3. Add 5 μL of HDAC enzyme working solution (ZPE, without test sample) or reaction buffer (HPE) to each well to start the reaction; 4. Mix at 1000 rpm for 1 minute; 5. Incubate at 30°C for 60 minutes; 6. Add stop solution to each well to stop the reaction; 7. Mix at 1000 rpm for 2 minutes; 8. Place the 384-well plate in a Caliper EZ Reader II to test the results.

[0282] Data Analysis: Inhibition rate calculation, Inhibition% = 100 - ((ZPE conversion% - test compound conversion%) / (ZPE conversion% - HPE conversion%) x 100)(Inhibition% = 100 - ((Conversion% ZPE - Conversion% test compound) / (Conversion% ZPE - Conversion% HPE)) x 100); IC 50 The values ​​were calculated using GraphPad Prism 5 or XLFit, and the specific results are shown in Table 1.

[0283] Table 1: Compound activity against HDAC6 enzyme and selectivity for HDAC subtypes

[0284]

[0285] Note: “——” in the table indicates that no test was conducted.

[0286] The structural formula of ACY-241 is as follows:

[0287] The results showed that the compounds in this invention have good inhibitory activity against HDAC6 enzyme, and compared with ACY-241, the compounds in this invention have certain selectivity for HDAC1, HDAC3 or HDAC8, especially for HDAC1 and HDAC3, which are better than the positive control ACY-241.

[0288] Example 2: Cell Viability Test

[0289] Experimental protocol: A549, Calu-6 and A375 cells were purchased from the CAS cell bank, and the remaining cells were purchased from the ATCC cell bank. The compound was prepared into a 20 mM stock solution using DMSO. (1) Day 1: Plating: Digest the cells with trypsin, resuspend them in culture medium, and count them using an automated cell counter. Dilute the cell suspension to the required density according to the seeding density. Soak 100 μL of cell suspension in each well of a 96-well plate and incubate overnight at 37°C. (2) Day 2: Compound preparation: Prepare the compound into a 200-fold final concentration solution using DMSO, and perform serial dilutions of 3-fold. Dilute the prepared compound with culture medium to prepare a compound with a final concentration of 3-fold, i.e., take 3 μL of the prepared compound and add it to 197 μL of culture medium to make a total volume of 200 μL. Add 50 μL of the compound to each well, and use the well with the same volume of DMSO as a control. Incubate at 37°C for 72 hours. (3) Day 4: Detection: Equilibrate the cell plate to room temperature. Add 40ul of Cell Titer to each well. The reagent was shaken for 2 minutes, allowed to stand for 60 minutes, and then detected using EnVision.

[0290] Data analysis: (1) Data were processed using GraphPad Prism 5 software; (2) %Inh = (Max signal - Compound signal) / (Max signal - Min signal) x 100; (3) Max signal is the result of DMSO treatment; (4) Min signal is the result of culture medium treatment. Specific results are shown in Tables 2 and 3.

[0291] Table 2: Inhibitory activity of the compounds against multiple myeloma (MM.1S) cells

[0292] Compound numbering <![CDATA[MM.1S(IC 50 ,μM)]]> Example 1 10.4 Example 2 40.0 Example 3 23.6 Example 4 6.4 Example 9 0.72 Example 11 2.60 Example 12 0.53 ACY-241 13.1

[0293] Table 3: Inhibitory activity of the compounds in Example 4 against 11 types of tumor cells

[0294] Tumor cell lines <![CDATA[Example 4; IC 50 , μM]]> <![CDATA[ACY-241;IC 50 ,μM]]> Melanoma (SK-MEL-5) 6.557 5.402 Breast cancer (MCF-7) 7.29 5.088 Ovarian cancer (SKOV-3) 15.69 11.8 Multiple myeloma (RPMI-8226) 1.494 2.402 Mantle cell lymphoma (REC-1) 1.129 2.541 Mantle cell lymphoma (Mino) 6.521 7.197 Lung cancer (A549) 7.363 12.02 Lung cancer (Calu-6) 10.64 15.21 Ovarian cancer (OVCAR-3) 4.059 4.008 Malignant melanoma (A375) 7.383 6.277 Breast cancer (MDA-MB-231) 13.43 14.73

[0295] The results showed that the compounds of the present invention have certain inhibitory activities against a variety of human tumor cell lines, including MM.1S cells, such as melanoma, breast cancer, ovarian cancer, lymphoma, and lung cancer cell lines, and showed better cell inhibitory activity than the positive control drug ACY-241 in most cell lines.

[0296] Example 3: In vivo antitumor efficacy test in animals

[0297] Experimental protocol: MM.1S cells were cultured and expanded in vitro. Cells in the logarithmic growth phase were collected and resuspended in serum-free RPMI 1640 medium. Martigel was added at a 1:1 ratio to adjust the cell concentration to 6.67 × 10⁷ / ml. The cell suspension was injected subcutaneously into the axilla of the right forelimb of BALB / c nude mice using a 1ml syringe, with 0.15ml injected into each animal. The growth of the animals and the transplanted tumors was observed regularly. When the average tumor volume grew to about 100-200 mm³, animals with excessively large or small tumors or irregular tumor shapes were culled. The animals were then randomly divided into groups of 3-6 animals each using a randomized block design.

[0298] After grouping, the animals were given medication as per Table 4, with a trial period of 3 weeks. During the medication period, the tumor diameter was measured twice a week, the animal weight was weighed, the animal's living condition was observed, and any abnormal conditions were recorded. After 3 weeks of medication, the experiment was terminated, the animals were euthanized with CO2, the tumor tissue was removed, photographed, weighed, and the tumor weight inhibition rate was calculated.

[0299] Dosage regimen:

[0300] Blank control group (vehicle): physiological saline.

[0301] Preparation of the positive control drug bortezomib (0.5 mg / kg): Weigh an appropriate amount of Bortezomib sample powder and place it in a 5 ml centrifuge tube. Add an appropriate amount of physiological saline, vortex to mix, and prepare a solution with a Bortezomib sample concentration of 0.05 mg / ml. Prepare immediately before use. Protect from light during preparation and use.

[0302] Preparation of drug formulations for the test compound: Weigh an appropriate amount of sample powder and place it in two 5ml centrifuge tubes. Add appropriate amounts of DMSO and PEG 400 sequentially, and vortex to completely dissolve and mix the sample. Add appropriate amounts of physiological saline to each tube, vortex to mix, and prepare solutions with sample concentrations of 3.0 mg / ml and 6.0 mg / ml, respectively. The solvent ratios are 4% DMSO, 30% PEG 400, and 66% physiological saline. Prepare solutions fresh for each use.

[0303] Table 4: Dosing Regimen

[0304]

[0305] Note: ip, intraperitoneal injection; iv, tail vein injection; administration volume is 10 ml / kg; days / wk, days / week; BIW, twice a week.

[0306] Observe and record the animal's condition each time it is administered the medication; if the animal dies, perform a gross necropsy, visually inspect the internal organs for any abnormalities, and record the findings.

[0307] During the experiment, the tumor diameter was measured twice a week, and the animals' weight was also measured.

[0308] Data processing: Tumor volume (TV): The calculation formula is TV = 1 / 2 × a × b 2 Where a represents the long diameter of the tumor and b represents the short diameter of the tumor. The average tumor volume data after 24 days of drug administration are shown in Table 5.

[0309] Animal weight change rate: The calculation formula is weight change rate = 100% × (BWinitial - BWfinal) / Bwinitial. Where BWinitial represents the animal's weight at the time of administration; BWfinal represents the animal's weight at the end of the experiment. Data on the animal weight change rate over 24 days of administration are shown in Table 6.

[0310] Statistical analysis methods

[0311] Experimental data were calculated and statistically processed using Microsoft Office Excel 2003 software. Unless otherwise specified, data are expressed as mean ± standard error (Mean ± SE). The t-test was used for comparisons between two groups. A p-value < 0.05 was considered statistically significant; the smaller the p-value, the more significant the difference.

[0312] Table 5: Average tumor volume in animal experiments (mm) 3 )

[0313]

[0314] 1. Compared with the blank control group, P < 0.005; 2. Compared with the blank control group, P < 0.05.

[0315] Table 6: Animal Relative Weight Data

[0316]

[0317] As shown in Table 5, the relative tumor volume curve after 24 days of administration indicates that the compound in Example 4 has good inhibitory activity against xenografts of MM (multiple myeloma) in animals. The treatment of Example 4 and the combination of Example 4 and Bortezomib showed significant differences from the control group (P < 0.05). It also showed advantages compared with ACY-241 and Bortezomib monotherapy.

[0318] Table 6 shows that the animals' body weight did not decrease significantly by more than 5% after 24 days, indicating that the compound has good safety. The compound of this invention possesses antitumor efficacy, has good safety, and has the potential for application in the treatment of cancer diseases.

[0319] Example 4: Liver microsomal stability test

[0320] Experimental protocol: Liver microsomes were suspended in 0.1 M pH 7.4 phosphate buffer, and 5 mM MgCl2, 0.1 μM of the test compound, 0.01% DMSO, 0.005% fetal bovine serum albumin, and 1 mM NADPH were added. The mixture was incubated at 37 °C for 60 min. The reaction was then terminated by adding methanol at 4 °C. The metabolic stability (MF%) of the test compound was analyzed and calculated by LC-MS / MS. The results are shown in Table 7. The calculation process is as follows:

[0321] Internal clearance rate In vivo clearance rate

[0322] Liver clearance rate metabolic stability

[0323] Where: slope is the slope of the LC-MS / MS compound concentration-time regression curve;

[0324] P represents the concentration of liver microsomal protein (mg / ml);

[0325] Houston: The Houston parameter is liver microsomal protein mg / liver g, set to 45 mg / g;

[0326] LW: Liver weight; HBF: Liver blood flow (ml / min); fu: Free fraction set to 1.

[0327] Table 7: Results of liver microsomal stability

[0328] compound Human liver microsomal stability (MF%) Mouse liver microsomal stability (MF%) Example 1 92.7 94.7 Example 3 86.6 -- Example 4 85.2 80.5 Example 6 98.5 93.5 Example 7 100.0 100.0 Example 8 100.0 100.0 Example 9 100.0 100.0 Example 10 100.0 100.0 ACY-241 74.1 50.9

[0329] Note: "--" in the table indicates that no test was conducted.

[0330] As shown in Table 7, the compounds of the present invention exhibit good metabolic stability in both human liver microsomes and mouse microsomes in in vitro metabolic models.

[0331] In human liver microsomal assays, the metabolic stability (MF%) of the compounds of the present invention was at least 11.1% higher than that of the positive compound ACY-241; in mouse microsomal assays, the metabolic stability (MF%) of the compounds of the present invention was at least 29.6% higher than that of the positive compound ACY-241. Therefore, the metabolic stability of the compounds of the present invention is significantly higher than that of the positive compound ACY-241.

Claims

1. A dihydroindoleamine compound as shown in I, or a pharmaceutically acceptable salt thereof: in, R 1 R 2 R 3 and R 4 It is independently hydrogen, halogen, hydroxyl, cyano, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy or halogen-substituted C1-C6 alkoxy; R 5 It is hydrogen; Y is -O-; All atoms in the dihydroindoleamine compounds or their pharmaceutically acceptable salts, as shown in I, are atoms that exist in natural abundance.

2. The dihydroindeneamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, When R 1 When the halogen is halogen, the halogen is F, Cl, Br or I; And / or, when R 2 When the halogen is halogen, the halogen is F, Cl, Br or I; And / or, when R 3 When the halogen is halogen, the halogen is F, Cl, Br or I; And / or, when R 4 When the halogen is halogen, the halogen is F, Cl, Br or I; And / or, when R 1 R 2 R 3 or R 4 When the alkyl group is C1-C6, the C1-C6 alkyl group is a C1-C3 alkyl group; And / or, when R 1 R 2 R 3 or R 4 When the alkyl group is a halogen-substituted C1-C6 alkyl group, the halogen is F, Cl, Br or I; And / or, when R 1 R 2 R 3 or R 4 When the alkoxy group is C1-C6, the C1-C6 alkoxy group is C1-C3 alkoxy; And / or, when R 1 R 2 R 3 or R 4 When the C1-C6 alkoxy group is halogen-substituted, the C1-C6 alkoxy group is a C1-C3 alkoxy group; And / or, when R 1 R 2 R 3 or R 4 When the C1-C6 alkoxy group is halogen-substituted, the halogen is F, Cl, Br or I; And / or, the dihydroindeneamine compound shown in I is and / or 3. The dihydroindoleamine compound as shown in claim 2, or a pharmaceutically acceptable salt thereof, characterized in that, When R 1 When the halogen is halogen, the halogen is Cl; And / or, when R 2 When the halogen is halogen, the halogen is Cl; And / or, when R 3 When the halogen is halogen, the halogen is Cl; And / or, when R 4 When the halogen is halogen, the halogen is Cl.

4. The dihydroindeneamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R 1 It is hydrogen or halogen; And / or, R 2 It is hydrogen or halogen; And / or, R 3 It is hydrogen or halogen; And / or, R 4 It is hydrogen or halogen.

5. The dihydroindoleamine compound as shown in claim 4, or a pharmaceutically acceptable salt thereof, characterized in that, R 1 It is a halogen; And / or, R 2 It is a halogen; And / or, R 3 It is a halogen; And / or, R 4 It is a halogen.

6. The dihydroindoleamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, for 7. The dihydroindoleamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It is any of the following schemes: Option 1: R 1 It is hydrogen or halogen; R 2 It is hydrogen or halogen; R 3 It is hydrogen or halogen; R 4 It is hydrogen or halogen; Option 2: R 1 It is a halogen; R 2 It is a halogen; R 3 It is a halogen; R 4 It is a halogen; R 5 It is hydrogen; Option 3: R 1 It is hydrogen or halogen; R 2 It is hydrogen or halogen; R 3 It is hydrogen or halogen; R 4 It is hydrogen or halogen; and R 1 R 2 R 3 and R 4 At least one of them is a halogen.

8. The dihydroindeneamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The dihydroindeneamine compound shown in I is any of the following compounds:

9. The dihydroindeneamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The dihydroindeneamine compound shown in I is 10. The dihydroindoleamine compound as shown in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The dihydroindeneamine compound shown in I is any of the following compounds: The retention time was 2.096 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: ethanol; Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 20 min; Flow rate: 2.8 ml / min; Column temperature: 35℃; The retention time was 2.286 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: ethanol; Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 20 min; Flow rate: 2.8 ml / min; Column temperature: 35℃; The retention time was 11.53 min under the following HPLC conditions. Chromatographic conditions: CHIRALCEL OJ column 250*4.6mm 5μm; mobile phase A: n-hexane; mobile phase B: ethanol solution containing 0.1% trifluorohexanoic acid; elution conditions: elution with 80% mobile phase A and 20% mobile phase B for 30 min; flow rate: 1 ml / min; column temperature: 35℃; The retention time was 10.637 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OJ 250*4.6mm 5μm; Mobile phase A: n-hexane; Mobile phase B: ethanol solution containing 0.1% trifluorohexanoic acid; Elution conditions: elution with 80% mobile phase A and 20% mobile phase B for 30 min; Flow rate: 1 ml / min; Column temperature: 35℃.

11. A method for preparing a dihydroindeneamine compound as shown in claim 1-10, comprising the following steps: In a solvent, under basic conditions, the compound shown in Formula II is reacted with hydroxylamine as shown below to obtain the dihydroindeneamine compound shown in Formula I: Among them, R 6 It is a C1-C4 alkyl group, R 1 R 2 R 3 R 4 R 5 The definitions of Y and Y are as described in any one of claims 1-10.

12. A compound as shown in Formula II: in, R 6 It is a C1-C4 alkyl group, R 1 R 2 R 3 R 4 R 5 Both Y and Y are defined as described in any one of claims 1-10.

13. The compound of formula II as claimed in claim 12, characterized in that, The compound represented by Formula II is any one of the following compounds:

14. The compound of formula II as claimed in claim 13, characterized in that, The compound represented by Formula II is any one of the following compounds: The retention time was 0.793 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: methanol solution containing 0.1% diethylamine; Elution conditions: elution with 85% mobile phase A and 15% mobile phase B for 6 min; Flow rate: 1.5 ml / min; Column temperature: 35℃; The retention time is 0.999 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OZ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: methanol solution containing 0.1% diethylamine; Elution conditions: elution with 85% mobile phase A and 15% mobile phase B for 6 min; Flow rate: 1.5 ml / min; Column temperature: 35℃; The retention time was 2.550 min under the following HPLC conditions. Chromatographic conditions: CHIRALCEL OJ column 250*4.6mm 5μm; mobile phase A: supercritical CO2; mobile phase B: ethanol solution containing 0.1% diethylamine; elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 8 min; flow rate: 2.8 ml / min; column temperature: 35℃; The retention time was 4.303 min under the following HPLC conditions. Chromatographic conditions: Column: CHIRALCEL OJ 250*4.6mm 5μm; Mobile phase A: supercritical CO2; Mobile phase B: ethanol solution containing 0.1% diethylamine; Elution conditions: elution with 60% mobile phase A and 40% mobile phase B for 8 min; Flow rate: 2.8 ml / min; Column temperature: 35℃.

15. A pharmaceutical composition comprising substance A and one or more pharmaceutically acceptable carriers, wherein substance A is a dihydroindeneamine compound as shown in claim 1-10 or a pharmaceutically acceptable salt thereof.

16. A pharmaceutical combination comprising substance A and substance X, wherein substance A is a dihydroindoleamine compound as shown in claim 1-10 or a pharmaceutically acceptable salt thereof; and substance X is bortezomib or a pharmaceutically acceptable salt thereof.

17. The use of substance B in the preparation of HDAC6 inhibitors or drugs, wherein, The substance B is a dihydroindoleamine compound as shown in any one of claims 1-10, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition as described in claim 15, or a pharmaceutical composition as described in claim 16; the drug is a drug for the prevention or treatment of HDAC6-related diseases, or the drug is a drug for the prevention or treatment of cancer; the cancer is one or more of lung cancer, breast cancer, ovarian cancer, lymphoma, multiple myeloma, and melanoma.

18. The application as described in claim 17, characterized in that, When the drug is used to prevent or treat HDAC6-related diseases, the HDAC6-related diseases are one or more of cancer, autoimmune diseases, and peripheral neuropathy.

19. The application as described in claim 18, characterized in that, When the drug is used to prevent or treat diseases related to HDAC6, the cancer is one or more of the following: lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal cell carcinoma, head and neck cancer, leukemia, lymphoma, myeloma, multiple myeloma, melanoma, and hematologic malignancies; the autoimmune disease is rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, psoriasis, post-ischemic perfusion injury, inflammatory bowel disease, chronic inflammatory lung disease, eczema, asthma, psoriasis, acute respiratory distress syndrome, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, deforming arthritis, femoral arthritis, traumatic arthritis, gouty arthritis, and Reiter's syndrome. The condition includes one or more of the following: polychondritis, acute synovitis, spondylitis, glomerulonephritis, hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, neutropenia, ulcerative colitis, Crohn's disease, graft-versus-host disease, allogeneic graft rejection, chronic thyroiditis, Graves' disease, scleroderma, active hepatitis, primary biliary cirrhosis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, allergic dermatitis, contact dermatitis, chronic renal insufficiency, Schwarz-Jones syndrome, idiopathic steatorrhea, sarcoidosis, Guillain-Barré syndrome, pulmonary fibrosis, and chronic inflammatory lung disease; the peripheral neuropathy is one or more of the following: chemotherapy-induced peripheral neuropathy and pain, diabetic peripheral neuropathy and pain, and viral infection-induced peripheral neuropathy and pain.

20. The application as described in claim 18, characterized in that, When the drug is used to prevent or treat diseases related to HDAC6, the cancer is one or more of melanoma, breast cancer, ovarian cancer, multiple myeloma, lymphoma, lung cancer, and degenerative cancer cells.

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