WO2022188846A1 - 一种三氮唑类衍生物及其制备方法和应用 - Google Patents

一种三氮唑类衍生物及其制备方法和应用 Download PDF

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WO2022188846A1
WO2022188846A1 PCT/CN2022/080255 CN2022080255W WO2022188846A1 WO 2022188846 A1 WO2022188846 A1 WO 2022188846A1 CN 2022080255 W CN2022080255 W CN 2022080255W WO 2022188846 A1 WO2022188846 A1 WO 2022188846A1
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crm1
disorder
pharmaceutically acceptable
triazole derivative
compound
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PCT/CN2022/080255
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English (en)
French (fr)
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朱永强
雷萌
苗航
王雪源
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深圳济康医药科技有限公司
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Priority to EP22766368.9A priority Critical patent/EP4306517A4/en
Priority to AU2022231918A priority patent/AU2022231918B2/en
Priority to CN202280015399.3A priority patent/CN116897153A/zh
Priority to JP2023552234A priority patent/JP2024507596A/ja
Priority to US18/277,099 priority patent/US20240140934A1/en
Priority to CA3210944A priority patent/CA3210944A1/en
Priority to KR1020237028581A priority patent/KR20230134571A/ko
Publication of WO2022188846A1 publication Critical patent/WO2022188846A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a triazole derivative and a preparation method and application thereof.
  • malignant tumor is still one of the major diseases that threaten people's life.
  • the treatment of cancer has made great progress, it has not been able to fundamentally cure cancer.
  • the anticancer drugs currently on the market have a certain curative effect, most of them are cytotoxic drugs with serious toxic and side effects. Therefore, how to study new targeted anticancer drugs from effective tumor targets has become a top priority for medical workers.
  • CRM1 inhibition may offer particularly interesting applications in familial syndromes (eg, Li-Fraumeni syndrome, BRCA1 or BRCA2 cancer syndromes caused by deletion of one p53 allele) in which specific tumor suppressor proteins (TSPs) ) is cleared or dysfunctional, and wherein increased TSP levels achieved by systemic (or local) administration of a CRM1 inhibitor can help restore normal tumor suppressor function.
  • familial syndromes eg, Li-Fraumeni syndrome, BRCA1 or BRCA2 cancer syndromes caused by deletion of one p53 allele
  • TSPs tumor suppressor proteins
  • RNAs are imported into and exported from the nucleus by specific transport molecules, which are classified as import proteins if they import the molecule into the nucleus, and as export proteins if they export the molecule out of the nucleus. Proteins that are imported or exported from the nucleus contain nuclear import localization (NLS) or nuclear export (NES) sequences that allow them to interact with associated transport factors. Chromosomal region stabilization protein 1 (CRM1), also known as exportin-1 or Xpol, is the major export protein.
  • NLS nuclear import localization
  • NES nuclear export
  • CCM1 Chromosomal region stabilization protein 1
  • Inhibitors of CRM1 block arrestin and growth regulators such as p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27, E2F4, KLF5, YAP1, ZAP, KIF5, HDAC4, HDAC5 or forkhead proteins (e.g. Nuclear export of FOXO3a), which are associated with gene expression, cell proliferation, angiogenesis, and epigenetics. It has been shown that CRM1 inhibitors can induce apoptosis in cancer cells, even in the presence of activating or growth-activating signals, without affecting normal (untransformed) cells. Most of the functional studies on CRM1 have been performed using the natural product lepramycin B (LMB).
  • LMB lepramycin B
  • Lepramycin itself is highly toxic to tumor cells, but it is difficult to be used clinically due to its greater gastrointestinal toxicity.
  • Derivation of LMB to improve drug-like properties results in compounds that retain antitumor activity and are better tolerated by animal models of tumor.
  • nuclear export inhibitors may have beneficial effects on neoplastic disorders and other proliferative disorders.
  • small molecule drug-like CRM1 inhibitors for in vitro and in vivo use still have certain deficiencies.
  • CRM1 exports several key proteins involved in many inflammatory processes. These proteins include IkB, NF-kB, Cox-2, RXRa, Commal, HIFI, HMGBI, FOXO, FOXP, etc.
  • a NF-kB protein extractant called IkB, binds to NF-kB in the nucleus, and the IKB-NF-kB complex inactivates the transcriptional function of NF-kB.
  • IkB In response to inflammatory stimuli, IkB dissociates from the IkBNF-kB complex, releasing NF-kB while restoring its underlying transcriptional activity. Many signals that activate NF-kB do this by targeting IkB proteolysis (IkB Phosphorylation of 'tagged' it for ubiquitination followed by proteolysis).
  • the nuclear IkBa-MF-kB complex can be exported by CRM1 to the cytoplasm, where it dissociates, thereby reactivating NF-kB. Ubiquitinated IkB can also dissociate from the NF-kB complex, restoring the transcriptional activity of NF-kB.
  • Inhibition of CRM1-induced export by IMB in human neutrophils and macrophage-like cells (U937) not only results in accumulation of transcriptionally inactive nuclear IkBa-NF-kB complexes, but also prevents initial NF-kB activation , even under cellular stimulation.
  • treatment with LMB inhibited IL-1 ⁇ -induced F-KB DNA binding (the first step in NF-kB transcriptional activation), 1L-8 expression, and intercellular Adhesion molecule expression.
  • COMMD1 is another nuclear inhibitor of the transcriptional activity of both NF-kB and hypoxia-inducible factor 1 (HIF1). Blocking the nuclear export of COMMD1 by inhibiting CRM1 results in increased inhibition of the transcriptional activity of NF-kB and HIF1.
  • CRM1 also mediates retinoid X receptor alpha (RXRa) trafficking.
  • RXRa is highly expressed in the liver and plays a central role in the regulation of bile acid, cholesterol, fatty acid, steroid and xenobiotic transduction and homeostasis.
  • nuclear RXRa levels were significantly reduced, mainly attributable to inflammation-mediated nuclear export of RXRa through CRM1.
  • LepB prevented L-1B-induced increases in cytoplasmic RXRa levels.
  • vasculature vasculature
  • arteritis polymyalgia rheumatica, atherosclerosis
  • skin diseases rheumatism (rheumatoid and related arthritis, psoriatic arthritis, spondyloarthropathy, conjunctivitis, systemic lupus erythematosus
  • mixed connective tissue disease inflammatory syndrome, dermatomyositis, inclusion body myositis, undifferentiated connective tissue disease, Sjögren's syndrome, scleroderma, overlap syndrome, etc.
  • CRM1 can affect gene expression by inhibiting activation of a series of transcription factors like ICp27, E2F4, KL5, YAP1, and ZAP.
  • CRM1 has potential therapeutic consequences for many dermatological syndromes, including inflammatory skin diseases (atopic, allergic dermatitis, chemical dermatitis, psoriasis), sun damage (external/V damage) and infections.
  • the best-studied CRM1 inhibition with LMB exhibited minimal effect on normal keratinocytes and exhibited anti-inflammatory activity on keratinocytes under UV, TNFa or other inflammatory stimuli.
  • Inhibition of CRM1 also upregulates the activity of NRF2 (nuclear factor 2-related factor 2), which protects keratinocytes from chlorination damage.
  • LMB induces apoptosis in keratinocytes infected with a lethal human papillomavirus (PV) strain such as IPV16, but not in uninfected keratinocytes.
  • PV human papillomavirus
  • CRM1 also mediates the transport of key neuroprotective proteins that may be useful in neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease and ALS.
  • PD Parkinson's disease
  • ALS Alzheimer's disease
  • key neuroprotective regulators such as NRF2, docking in neural cells
  • sequestering 1XB in the nucleus of glial cells to achieve transcriptional activity of NF-kB Inhibiting knocking down CRM1 slows or prevents the neuronal cell death found in these disorders.
  • abnormal glial cell proliferation is associated with abnormalities in CRM1 levels or CRM1 function.
  • HIV human immunodeficiency virus
  • adenovirus adenovirus
  • simian type I retrovirus Borna disease virus
  • influenza virus conventional strains and HINL
  • poultry HN1 strain hepatitis B virus
  • HCV hepatitis C virus
  • HPV human papilloma virus
  • RY respiratory syncytial virus
  • SV herpes simplex virus
  • CMV cytomegalovirus
  • MMV Merkel cell polyoma virus
  • the HIV-1 Rev protein which traverses the nucleolus and shuttles between the nucleus and the cytoplasm, facilitates the export of unspliced and singly spliced HIV transcripts containing Rev response element (RRE) RNA through the CRM1 export pathway.
  • RRE Rev response element
  • Inhibition of Rev-mediated RA transport using CRM1 inhibitors such as LepB or PKF050-638 can block HIV-1 transcription, inhibit the production of new HIV-1 virions, and thereby reduce HIV-1 levels.
  • Dengue virus is the causative agent of the common arthropod-borne viral disease Dengue Fever (DF) and its more severe and potentially fatal Dengue Hemorrhagic Fever (DHF). DHF appears to result from an overactive inflammatory response to DENV, the largest and most conserved protein of DENV.
  • CRM1 regulates the transport of NS5 from the nucleus to the cytoplasm, where most of the functions of NS5 are mediated.
  • CRM1-mediated export of NS5 results in altered virion kinetics and reduces induction of the inflammatory chemokine interleukin-8 (IL-8), a promising candidate for the treatment of DENV as well as other medically important flaviviruses including type C Diseases caused by hepatitis viruses provide a new avenue.
  • IL-8 inflammatory chemokine interleukin-8
  • virus-encoded RNA-binding proteins that use CRM1 to export the nucleus include the HS VI type lamina protein (P13/14 or HUA7), the human CMV protein pp65, the SARS coronavirus ORF3b protein, and the RSV matrix (M) protein.
  • HCC hepatocellular carcinoma
  • cervical cancer due to HPV
  • CRM1 controls nuclear localization and thus the activity of various DNA metabolizing enzymes, examples of which include histone deacetylase (HDAC), histone acetyltransferase (HAT) and histone methyltransferase (HMT).
  • HDAC histone deacetylase
  • HAT histone acetyltransferase
  • HMT histone methyltransferase
  • CRM1 is also associated with other disorders.
  • Lebers disorder a genetic disorder characterized by degeneration of retinal ganglion cells and visual loss, is associated with ineffectiveness of the CRM1 switch.
  • neurodegenerative disorders are associated with abnormal nuclear transport.
  • the purpose of the present invention is to provide a triazole derivative, a preparation method and application thereof, and the triazole derivative can be used as a CRM1 inhibitor to prepare a therapeutic drug for diseases related to CRM1 activity.
  • a triazole derivative or a pharmaceutically acceptable salt thereof the structure of the triazole derivative is shown in formula I,
  • R 1 and R 2 are independently hydrogen, C1-6 alkyl, haloC1-6 alkyl or C1-6 alkoxy.
  • R 1 and R 2 are independently hydrogen or C1-4 alkyl.
  • triazole derivatives are selected from the following compounds:
  • a pharmaceutical composition for treating diseases, disorders or symptoms related to CRM1 activity comprising the above triazole derivatives or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.
  • disorders related to CRM1 activity are proliferative disorders, cancer, inflammatory disorders, autoimmune disorders, viral infections, ophthalmic disorders, neurodegenerative disorders, abnormal tissue growth disorders, disorders related to food intake, Allergies and breathing disorders.
  • the disorder associated with CRM1 activity is cancer.
  • the disorder associated with CRM1 activity is multiple myeloma.
  • the cell activity results show that the compounds I and IV of the present invention are basically equivalent to KPT8602.
  • the oral and intravenous half-lives of Compound I are longer than those of KPT-8602, especially the oral half-life is about 2 times longer, and the intravenous and oral AUCs of Compound I are much higher than KPT-8602, showing Better pharmacokinetic properties.
  • Compound I showed a good safety profile.
  • Figure 1 shows the results of the BALB/C in vivo toxicity test of compound I.
  • the compounds of the present invention include those generally described above, and are further described by the classes, subclasses and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of the present invention, these chemical elements are identified according to the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics, 75th Edition.
  • the compounds of the present invention may possess asymmetric centers, chiral axes, and chiral planes, and exist as racemates, racemic mixtures, and individual diastereomers or enantiomers, all possible Isomers and mixtures thereof (including optical isomers) are included in the present invention.
  • halogen includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Particularly preferred are fluorine atoms and chlorine atoms.
  • alkyl group includes a linear or branched chain having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. Hydrocarbyl.
  • alkoxy group means a group in which the above-mentioned "alkyl group” is bonded to an oxygen atom.
  • alkoxy group means a group in which the above-mentioned "alkyl group” is bonded to an oxygen atom.
  • alkoxy group examples include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, and a tert-butoxy group.
  • haloalkyl group includes a group obtained by substituting one or more of the above-mentioned "halogen” for a hydrogen atom bonded to a carbon atom of the above-mentioned "alkyl group”.
  • halogen for a hydrogen atom bonded to a carbon atom of the above-mentioned "alkyl group”.
  • monofluoromethyl, monofluoroethyl, monofluoropropyl, 2,2,3,3,3-pentafluoropropyl monochloromethyl, trifluoromethyl, trichloromethyl, 2 , 2,2-trifluoroethyl, 2,2,2-trichloroethyl, 1,2-dibromoethyl, 1,1,1-trifluoropropan-2-yl, etc.
  • haloalkyl group trifluoromethyl and trichloromethyl may be mentioned.
  • the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable biologic thereof and a pharmaceutically acceptable body, adjuvant or carrier.
  • the amount of the compound in the composition of the present invention is such that it is effective to moderately inhibit CRW1 in a biological sample or.
  • a composition of the invention is formulated for administration to a patient in need of such a composition.
  • the term "person" means an animal.
  • the animal is a dairy animal.
  • the patient is a veterinary patient (ie, a non-human dairy animal patient).
  • pharmaceutically acceptable carrier refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • a pharmaceutically acceptable carrier that can be used in the compositions of the present invention
  • Acceptable carriers, adjuvants or vehicles include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg, acid salts), glycine, sorbitan acid, potassium sorbate, partial glycerol mixture of saturated vegetable fats to prevent acid, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, trisodium phosphate Magnesium silicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid, wax
  • the preparation method of the triazole derivative or its pharmaceutically acceptable salt is synthesized according to the following route:
  • each group R in the reaction formula is as described above; formula (1) is reacted under the action of sodium hydrosulfide to obtain formula (2), and formula (2) undergoes nucleophilic substitution to generate (3).
  • Formula (3) reacts with bromine water and triethylamine to generate formula (4), formula (4) undergoes a coupling reaction to generate (5), and formula (5) is hydrolyzed to carboxylic acid formula (6) under the action of LiOH ), the formula (6) and the amine substrate are condensed to obtain the compound of formula I.
  • the cyano group of compound 1 generates thioformamide under the action of sodium hydrosulfide and magnesium chloride, and then reacts under the conditions of hydrazine hydrate and formic acid to generate triazole 2, 2 is then reacted with (Z under the catalysis of triethylenediamine.
  • Extract with ethyl acetate (100 mL ⁇ 3), combine the organic phases and wash with saturated sodium chloride solution (100 mL ⁇ 1), dry over anhydrous Na 2 SO 4 , filter and evaporate the solvent under reduced pressure to obtain crude 3,5-bis( Trifluoromethyl)benzthionamide (10.9 g, yield 95.1%, purity 84%) as yellow oily liquid, MS (ESI) m/z 274.35 [M+H]+. used directly in the next step.
  • Extract with ethyl acetate (100 mL ⁇ 3), combine the organic phases and wash with saturated sodium bicarbonate solution (300 mL ⁇ 3) and saturated sodium chloride solution (100 mL ⁇ 1), dry over anhydrous Na 2 SO 4 , filter and reduce pressure The solvent was evaporated to obtain the crude compound. Washed with n-hexane (200 mL ⁇ 3), filtered and dried to obtain 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole 2 (8.5 g, collected yield 76.0%, purity 90%), white solid.
  • Extract with ethyl acetate 80 mL ⁇ 3), combine the organic phases and wash with saturated sodium chloride solution (100 mL ⁇ 1), dry over anhydrous Na 2 SO 4 , filter and evaporate the solvent under reduced pressure to obtain the crude compound.
  • the reaction was cooled to room temperature, and purified water (50 mL) was added to the reaction solution. Extract with ethyl acetate (15 mL ⁇ 3), combine the organic phases and wash with saturated sodium chloride solution (20 mL ⁇ 1), dry over anhydrous Na 2 SO 4 , filter and evaporate the solvent in vacuo to obtain crude compound.
  • mice 25 BALB/C mice were randomly divided into 5 groups: solvent control group (Control), positive control KPT-8602 (30 mg/kg, once a day) group, KPT-8602 (60 mg/kg, once a day) group , compound I (30mg/kg, once a day) group, compound I (60mg/kg, once a day) group, each group is 5, and each group is given the corresponding concentration of The test substances, KPT-8602 and Compound I were administered continuously daily for 21 days.
  • solvent control group Control
  • positive control KPT-8602 (30 mg/kg, once a day) group
  • KPT-8602 60 mg/kg, once a day
  • compound I 30mg/kg, once a day
  • compound I 60mg/kg, once a day

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Abstract

本发明公开了一种三氮唑类衍生物及其制备方法和应用，属于医药技术领域。该三氮唑类衍生物结构如式I所示，本发明的三氮唑类衍生物可作为CRM1抑制剂，用于制备与CRM1活性相关疾病的治疗药物。

Description

一种三氮唑类衍生物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体涉及一种三氮唑类衍生物及其制备方法和应用。
背景技术
目前，恶性肿瘤仍然是威胁人们生命的主要疾病之一。癌症的治疗目前虽然已经取得了很大的进步，但还未能从根本上治疗癌症。目前上市的抗癌药物虽然具有一定的疗效，但它们大多是细胞毒药物，具有严重的毒副作用。因此，如何从有效的肿瘤靶点出发来研究靶向性的新型抗癌药物成为医药工作者的当务之急。
来自大部分主要人类实体和血液系统恶性肿瘤的细胞表现出多种致癌蛋白、肿瘤抑制蛋白、以及细胞周期调控子的异常细胞定位。例如，某些p53突变可导致细胞基质定位，而非细胞核定位。这导致了正常生长调控的缺失，尽管肿瘤抑制功能是完整的。在其他瘤中，野生型p53被隔离在细胞质中或被迅速降解，再次导致其抑制因子功能的丧失。功能性p53蛋白的恰当的核定位的恢复可使肿瘤性细胞的一些特性正常化，可恢复癌细胞对DNA损伤剂的敏感度，并且可导致长成的肿瘤消退。从其他肿瘤抑制蛋白如叉头和Sullivar和c-Abl中获得了类似数据。此外，一些肿瘤抑制因子和生长调控蛋白的异常定位可能与自身免疫性疾病的发病机理有关。CRM1抑制可在家族性症综合征中提供特別有意义的应用(例如，由一个p53等位基因缺失引起的李一佛美尼综合征、BRCA1或BRCA2癌症综合征)，其中特异的肿瘤抑制蛋白(TSP)被清除或具有功能障碍，并且其中通过系统的(或局部的)CRM1抑制剂的给予实现的TSP水平的提高能够帮助恢复正常的肿瘤抑制功能。
特异的蛋白和RNA由特异的转运分子输入和输出细胞核，若它们将分子输入细胞核，则这被分类为输入蛋白，若它们将分子输出细胞核，则被分类为输出蛋白。被输入或输出细胞核的蛋白质包含核输入定位(NLS)或核输出(NES)序列，使它们与相关的转运因子相互作用。染色体区域稳定蛋白1(CRM1)，也称作输出蛋白-1或Xpol，为主要的输出蛋白。CRMl的抑制剂阻断抑制蛋白和生长调节因子如p53、c-Ab1、p21、p27、pRB、BRCA1、IkB、ICp27、E2F4、KLF5、YAP1、 ZAP、KIF5、HDAC4、HDAC5或叉头蛋白(例如FOXO3a)的核输出，它们与基因表达、细胞增殖、血管生成以及表现遗传相关。有结果表明CRM1抑制因子可诱导癌细胞的凋亡，甚至是在致激活信号或生长激活信号存在的情况下，而不影响正常的(未被转化的)细胞。大多数关于CRM1的功能研究都是使用天然产物来普霉素B(LMB)进行的。来普霉素本身对肿瘤细胞具有很高的毒性，但因较大的胃肠毒性很难用于临床。衍生LMB来改善类药特性可得到保留抗肿活性并且可被动物肿模型更好地耐受的化合物。因此，核输出抑制剂对肿瘤性紊乱及其他增生性素乱可具有有益效果。然而至今，用于在体外和体内使用的小分子的类药CRM1抑制剂仍然有一定的缺陷。
除肿瘤抑制蛋白外，CRM1还输出若干与很多炎性过程相关的关键蛋白。这些蛋白包括IkB、NF-kB、Cox-2、RXRa、Commal、HIFI、HMGBI、FOXO、FOXP等。因为能够引发免疫球蛋白x基因表达的发现而得名的核因子xB(NF-kB/rel)族的转录激活因子可调控各种与炎症、增殖、免疫与细胞存活相关的基因的mRNM表达。在基本情况下，一种称为IkB的NF-kB蛋白抽剂，在核内与NF-kB结合，且IKB-NF-kB复合物使NF-kB的转录功能失活。在炎症刺激的应答中，IkB从IkBNF-kB复合物中解离，释放NF-kB，同时恢复其潜在的转录活性很多激活NF-kB的信号就是通过靶向IkB蛋白水解来做到这些(IkB的磷酸化可“标记”它进行泛素化然后蛋白水解)。核IkBa-MF-kB复合物可被CRM1输出到细胞质，在细胞质中该复合物解离，从而使NF-kB重新激活。泛素化的IkB还可以从NF-kB复合物上解离，恢复NF-kB的转录活性。通过IMB的在人嗜中性细胞和类巨噬细胞(U937)中CRM1诱导的输出的抑制不仅导致无转录活性的核IkBa-NF-kB复合物的累积，还防止了初始的NF-kB激活，甚至是在细胞的刺激下。在一项不同研究中，在肺毛微血管内皮细胞中，用LMB处理抑制了IL-1β诱导的F-KB DNA结合(NF-kB转录激活的第1个步骤)、1L-8表达以及胞间黏附分子表达。COMMD1是NF-kB和缺氧诱导因子1(HIF1)两者的转录活性的另一种核抑制剂。通过抑制CRMl阻断COMMD1的核输出可导致对NF-kB和HIF1的转录活性的抑制的增加。
CRM1还介导类视黄醇X受体a(RXRa)运输。RXRa在肝脏中高度表达并且在调控胆汁酸、胆固醇、脂肪酸、类固醇和异生物质代讲以及内环境稳定中起到 核心作用。在肝炎期间，核RXRa水平显著降低，主要归因于通过CRM1的炎症介导的RXRa核输出。在人肝源性细胞中，LepB能够阻止L-1B诱导的细胞质的RXRa水平增加。
在NF-kB、HIF-1和RXRa信号转导中CRM1介导的核输出的作用表明阻断核输出对于很多跨多组织和器官的炎症过程可以有潜在的益处，包括脉管系统(血管炎、动脉炎、风湿性多肌痛、动脉粥样硬化)、皮肤病、风湿病(类风湿及相关关节炎、银屑病关节炎、脊椎关节病、结品性关节病、系统性红斑狼疮、混合性结缔组织病、炎综合征、皮肌炎、包涵体肌炎、未分化结缔组织病、干燥综合征、硬皮病以及重叠综合征等)
抑制CRM1可通过抑制激活一系列转录因子像ICp27、E2F4、KL5、YAP1、ZAP来影响基因表达。
抑制CRM1对很多皮肤科综合征有潜在的治疗破果，包括炎性皮肤病(特应性、变应性皮炎、化学性皮炎、银屑病)、阳光损伤(外线/V损伤)和感染。用LMB研究的最充分的CRM1抑制对正常的角质化细胞呈现最小的作用，且对在UV、TNFa或其他炎症刺激下的角质化细胞呈现抗炎活性。抑制CRM1还可上调NRF2(核因子2相关因子2)的活性，NRF2可保护角质化细胞免于氯化损伤。LMB诱导被致性人乳头瘤病毒(PV)毒株如IPV16感染的角质化细胞的调亡，但不诱导未被感染的角质化细胞的亡。
CRM1还介导关键神经保护蛋白的转运，这些神经保护蛋白可能对神经退行性疾病包括帕金森氏病(PD)、阿尔茨海默病和肌菱缩性侧索硬化症有用。例如，(1)对关键神经保护调控因子如NRF2的强制核阻留，停泊在神经细胞中，和/或通过(2)将1XB隔离于神经胶质细胞的细胞核实现NF-kB的转录活性的抑制，抽制CRM1可减缓或防止这些素乱中发现的神经细胞死亡。还有证据表明，异常的神经胶质细胞增殖与CRM1水平或CRM1功能的异常相关。
很多病毒的完整的成熟也要求主要由CRM1介导的完整的核输出。生命周期里牵涉到核输出和/或CRM1自身的病毒包括人类免疫缺陷病毒(HIV)、腺病毒、猴I型逆转录病毒、波尔纳病(Borna disease)病毒、流感病毒(常规株以及HINL和禽HN1株)、乙型肝炎病毒(BV)和丙型肝炎病毒(HCV)、人乳头縮病毒(HPV)、呼吸道合胞体病毒(RSY、Dungee、严重急性呼吸器官综合征冠状毒、 黄热病毒、西尼罗病毒、单纯疹病毒(SV)、巨细胞病毒(CMV)以及默克尔(Merke1)细胞多瘤病毒(MCV)。
穿过核仁并穿梭于细胞核与细胞质之间的HIV-1Rev蛋白促使未剪接的和单剪接的含有Rev应答元件(RRE)RNA的HIV转录物的通过CRM1输出通路的输出。使用CRM1抑制剂如LepB或PKF050-638实现的Rev介导的RA转运的抑制可以阻止HIV-1的转录过程，抑制新的HIV-1病毒粒子的产生，并且从而降低HIV-1的水平。
登革热病毒(DENV)为常见的节肢动物传播的病毒性疾病登革热(DF)及其更严重的和潜在致命的登革出血热(DHF)的病原体。DHF似乎是由对DENV的过旺的炎症应答导致的，NS5是DENV的最大的也是最保守的蛋白。CRM1调控NS5从细胞核到细胞质的转运，其中NS5的大部分功能是被介导的。抑制CRM1介导的NS5的输出可导致产病毒动力学改变，并且减少炎性趋化因子白细胞介素-8(IL-8)的诱导，为治疗DENV以及其他医学上重要的黄病毒包括丙型肝炎病毒引发的疾病提供了一个新的途径。
其他使用CRM1输出细胞核的病毒编码的RNA结合蛋白包括HSVI型间层蛋白(P13/14或HUA7)、人CMV蛋白pp65、SARS冠状病毒ORF3b蛋白、以及RSV基质(M)蛋白。
有趣的是，很多这些病与特定类型的人类癌症相关，这些癌症包括归因于慢性HBV或HCV感染的肝细胞癌(HCC)、归因于HPV的宫颈癌以及与MCV相关的默克尔细胞癌。因此CRM1抑制因子对病毒感染过程及这些病毒引起的肿瘤性转化过程均有益。
CRM1控制核定位，并且从而控制多种DNA代谢酶的活性，这些例包括组蛋白脱乙酰基酶(HDAC)、组蛋白乙酰转移酶(HAT)和组蛋白甲基转移酶(HMT)。
CRM1也与其他紊乱相关。一种以视网膜神经节细胞的退化和视觉失为特征的遗传疾病-莱博病(Lebers disorder)与CRM1开关的无效相关。还有证据表明，神经退行性紊乱和异常的核转运有关。
发明内容
本发明的目的是提供一种三氮唑类衍生物及其制备方法和应用，该三氮唑类衍生物可作为CRM1抑制剂，用于制备与CRM1活性相关疾病的治疗药物。
为了实现上述发明目的，本发明采用以下技术手段：
一种三氮唑类衍生物或其药学上可接受盐，所述三氮唑类衍生物结构如式I所示，
Figure PCTCN2022080255-appb-000001
其中：
R选自羟基、C1-6烷基、C1-6烷氧基或-C(OR 1)(＝NR 2)；
R 1和R 2独立地为氢、C1-6烷基、卤代C1-6烷基或C1-6烷氧基。
进一步地，所述R选自羟基、C1-4烷基、C1-4烷氧基或-C(OR 1)(＝NR 2)；
R 1和R 2独立地为氢或C1-4烷基。
进一步地，所述三氮唑类衍生物选自以下化合物：
Figure PCTCN2022080255-appb-000002
上述三氮唑类衍生物或其药学上可接受盐的制备方法，按如下所示路线进行合成：
Figure PCTCN2022080255-appb-000003
一种用于治疗与CRM1活性相关疾病、紊乱或症状的药物组合物，包括上述三氮唑类衍生物或其药学上可接受盐，以及药学上可接受的载体。
上述三氮唑类衍生物或其药学上可接受盐在制备用于治疗与CRM1活性相关的紊乱的药物中的应用。
进一步地，所述与CRM1活性相关的紊乱为增生性紊乱、癌症、炎症性紊乱、自身免疫性紊乱、病毒感染、眼科紊乱、神经退行性紊乱、异常组织生长紊乱、与食物摄取有关的紊乱、过敏以及呼吸紊乱。
进一步地，所述与CRM1活性相关的紊乱为癌症。
进一步地，，所述与CRM1活性相关的紊乱为多发性骨髓瘤。
有益效果：细胞活性结果表明，本发明的化合物I和IV与KPT8602基本相当。化合物I的口服和静脉的半衰期均比KPT-8602的口服及静脉的半衰期长，特别是口服的半衰期长约2倍，而且化合物I的静脉和口服的AUC均远高于KPT-8602，表现出更好的药物动力学性质。此外，化合物I表现出良好的安全性。
附图说明
图1为化合物I的BALB/C体内毒性实验结果。
具体实施方式
化合物的定义
本发明的化合物包括在上文中总体上描述的那些，并且通过在此披露的类别、子类别以及种类进一描述。如如在此使用的，除非另外指明，应当使用下面的定义。出于本发明的目的，根据元素周期表，CAS版本，化学和物理手册(Handbook of Chemistry and Physics)，第75版对这些化学元素进行鉴定。
在本说明书之内除非另外说明，在本说明书中使用的命名法总体上遵循在Nomenclature of Organic Chemistry(有机化学命名法)，A、B、C、D、E、F、和H章节，对于它的示例性的化学结构命化合物的名称。
本发明的化合物可以具有不对称中心、手性轴以及手性平面，并且作为外消旋体、外消旋混合物以及单个的非对映异构体或者对映异构体存在，其中全部可能的异构体及其混合物(包括旋光异构体)被包括在本发明中。
所谓“卤素”，包含氟原子、氯原子、溴原子及碘原子。特别优选为氟原子及氯原子。
所谓“烷基”，包含碳原子数1～15、优选为碳原子数1～10、更优选为碳原子数1～6、进一步优选为碳原子数1～4的直链或支链状的烃基。例如可列举：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基、正己基、异己基、正庚基、异庚基、正辛基、异辛基、正壬基、正癸基等。
所谓“烷氧基”，意指上述“烷基”键合于氧原子而成的基团。例如可列举：甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、叔丁氧基、异丁氧基、仲丁氧基、戊氧基、异戊氧基、己氧基等。
作为“烷氧基”的优选实施方式，可列举：甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基。
所谓“卤代烷基”包括：1个以上的上述“卤素”置换上述“烷基”的与碳原子键合的氢原子而成的基团。例如可列举:单氟甲基、单氟乙基、单氟丙基、2,2,3,3,3-五氟丙基、单氯甲基、三氟甲基、三氯甲基、2,2,2-三氟乙基、2,2,2-三氯乙基、1，2-二溴乙基、1，1，1-三氟丙烷-2-基等。
作为“卤代烷基”的一个实施方式，可列举:三氟甲基、三氯甲基。
本发明提供了一种组合物，该组合物包括本发明的化合物或其药学上可接受的行生物以及一种药学上可接受的体、佐剂或运载体。本发明组合物中的化合物 的量是这样，使得其在生物样品或者中有效地适度抑制CRW1。在某些实施例中，配制用于向需要此类组合物的患者给药的木发明的一种组合物。如在此使用的，术语“者”意指一种动物。在一些实施例中，该动物是一种乳动物。在某些实施例中，该患者是种兽用患者(即，非人类乳动物患者)。
术语药学可接受的载体、佐剂或运载体”是指不会破坏与其一起配的化物的药理学活性的无毒载体、佐剂或运载体。可以在本发明的组合物中使用的药学上可接受的载体，佐剂或运载体包括但不限于离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(如人血清白蛋白)、缓冲物质(如酸盐)、甘氨酸、山梨酸、山梨酸钾、饱和植物脂防酸的偏甘油混合物、水、盐或电解质(如硫酸精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐)、胶体二氧化硅、三硅酸镁，聚乙烯吡咯烷酮、基于纤维素的物质、聚乙二醇、羧甲基纤素钠、聚丙烯酸酸、蜡、聚乙烯一聚氧丙烯嵌段聚合物、乙二醇以及羊毛脂。
所述三氮唑衍生物或其药物可接受盐的制备方法，按如下所示路线进行合成：
Figure PCTCN2022080255-appb-000004
该反应式中各基团R定义如前所述；式(1)在硫氢化钠作用下反应得到式(2)，式(2)发生亲核取代生成(3)。式(3)再和溴水、三乙胺作用下反应生成式(4)，式(4)发生偶联反应生成(5)，式(5)在LiOH的作用下水解成羧酸式(6)，式(6)和胺类底物缩合得到式I化合物。
以下详述本发明化合物的制备方法：
化合物1的氰基在硫氢化钠和氯化镁的作用下生成硫代甲酰胺，再在水合肼和甲酸的条件下反应生成三氮唑2，2再在三乙烯二胺的催化作用下与(Z)-3-碘代丙烯酸异丙酯偶联生成化合物3，然后，化合物3的双键部分与液溴发生加成，再在三乙胺的作用下脱去一分子溴得到关键中间体4，中间体4在二(三苯基膦)二氯化钯的催化作用下，分别与带有硼酸结构的多种含氮芳香基发生Suzuki偶联，生成了连接有不同含氮芳香基的化合物5，最后化合物5的酯键在LiOH的作用下水解成羧酸化合物6，羧酸化合物6进一步进行缩合得到目标式I化合物。
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
一、化合物的合成
本发明的化合物的制备可按照如下过程实施：
化合物的I路线设计与合成
Figure PCTCN2022080255-appb-000005
(Z)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)丙烯酸异丙酯(3)的合成
在250mL茄形瓶中，加入3,5-双(三氟甲基)苯腈1(10g，41.8mmol)，溶于DMF(50mL)溶液中，依次加入NaSH(7.8g，83.7mmol)和MgCl 2(8.5g，41.8mmol)。然后将反应在室温下继续搅拌3h。TLC监测反应完全后将反 应液倒入冰水混合溶液(500mL)中。用乙酸乙酯(100mL×3)萃取，合并有机相并用饱和氯化钠溶液(100mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂得粗品3,5-双(三氟甲基)苯甲硫酰胺(10.9g，收率95.1％，纯度84％)，为黄色油状液体，MS(ESI)m/z 274.35[M+H]+。直接用于下一步使用。
在250mL茄形瓶中，加入3,5-双(三氟甲基)苯甲硫酰胺(10.9g，39.8mmol)，溶于DMF(30mL)溶液中，在室温下向其中滴加80％水合肼(5.1mL，83.6mmol)。将混合物继续搅拌1h，然后向其中滴加甲酸(30mL)。然后将反应在90℃下继续搅拌3h。TLC监测反应完全后，将反应降至室温，将反应液倾倒入纯净水(600mL)中。用乙酸乙酯(100mL×3)萃取，合并有机相并用饱和碳酸氢钠溶液(300mL×3)和饱和氯化钠溶液(100mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂得化合物粗品。用正己烷(200mL×3)搅洗，过滤并干燥后得到3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑2(8.5g，收率76.0％，纯度90％)，白色固体。 1H NMR(400MHz,CDCl 3)δ8.63(s,2H,Ph),8.40(s,1H,NCH),8.02(d,J＝13.8Hz,1H,NCHCH),7.95(s,1H,Ph),6.74(d,J＝13.8Hz,1H,NCHCH)MS(ESI)m/z 279.89[M+H] +。
在250mL茄形瓶中，加入3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑2(8.5g，30.2mmol)，溶于DMF(40mL)中，向其中加入DABCO(8.5g，75.5mmol)。将混合物在室温下继续搅拌30min后，向反应液中滴加(Z)-3-碘代丙烯酸乙酯(7.5g，33.2mmol)。然后将反应在室温下继续搅拌3h。TLC监测反应完全后，将反应液倒入冰水混合溶液(400mL)中。用乙酸乙酯(80mL×3)萃取，合并有机相并用饱和氯化钠溶液(100mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂得化合物粗品。将得到的粗品通过柱色谱(PE/EtOAc＝25：1)纯化，得到化合物(Z)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)丙烯酸乙酯3(7.9g，收率68.2％，纯度98％)，为白色固体。 1H NMR(400MHz,CDCl 3)δ8.63(s,2H,Ph),8.40(s,1H,NCH),8.02(d,J＝13.8Hz,1H,NCHCH),7.95(s,1H,Ph),6.74(d,J＝13.8Hz,1H,NCHCH),4.87(m,1H,CH),1.36(t,J＝7.1Hz,6H,Me).MS(ESI)m/z 394.23[M+H] +。
(Z)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-溴丙烯酸异丙酯(4)的合成
在250mL的茄形瓶中，将之前得到的(Z)-3-(3-(3,5-双(三氟甲基)苯基)-1H- 1,2,4-三唑-1-基)丙烯酸乙酯3(7.9g，20.6mmol)溶于二氯甲烷(40mL)中。在30min内，向其中缓慢滴加液溴(6.6g，41.2mmol)，然后将反应于室温下继续搅拌8h。TLC监测反应完全后，将反应液倒入冰水混合溶液(100mL)中。用二氯甲烷(50mL×3)萃取，合并有机相并用饱和亚硫酸氢钠溶液(100mL×3)和饱和氯化钠溶液(50mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂。柱色谱(PE/EtOAc＝50：1)分离纯化，得到3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2,3-二溴丙酸异丙酯(10.3g，收率92.7％，纯度95％)，为白色固体。MS(ESI)m/z 551.97[M+H] +。
将上一步得到的中间体3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2,3-二溴丙酸异丙酯(10.3g，19.1mmol)称取至250mL茄形瓶中，用四氢呋喃(40mL)溶解后，在冰浴下搅拌10min后，向反应液中滴加三乙胺(3.9g，38.2mmol)并继续搅拌30min。然后将反应移至室温下继续搅拌6h。TLC监测反应完全后，向反应液中加入冰水混合溶液(100mL)。用乙酸乙酯(50mL×3)萃取，合并有机相并用饱和氯化钠溶液(50mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂。柱色谱(PE/EtOAc＝50：1)分离纯化得到(Z)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-溴丙烯酸乙酯4(7.7g，收率88.2％，纯度96％)，为白色固体。1H NMR(400MHz,CDCl3)δ8.75(s,1H,NCH),8.56(s,2H,Ph),7.93(s,1H,Ph),7.65(s,1H,CBrCH),4.38(m,1H,CH),1.37(t,J＝7.1Hz,6H,Me).MS(ESI)m/z 473.09[M+H] +。
(E)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-(嘧啶-5-基)丙烯酸异丙酯(5)的合成
取一个25ml三颈瓶，分别称取重要中间体4(200mg，0.44mmol)和5-嘧啶硼酸(81.9mg，0.66mmol)溶于二氧六环(5mL)和水(1mL)的混合溶液中。随后称取醋酸钠(86.4mg，0.88mmol)加入反应液，将反应置换氮气3次后在室温下搅拌。然后向反应液中加入Pd(PPh 3)Cl 2(30.9mg，0.04mmol)，并再次用氮气置换反应3次后在80℃下搅拌过夜。TLC监测反应完全后，将反应降至室温，向反应液中加入纯净水(50mL)。用乙酸乙酯(15mL×3)萃取，合并有机相并用饱和氯化钠溶液(20mL×1)洗涤，无水Na 2SO 4干燥，过滤并真空蒸除溶剂得化合物粗品。通过柱色谱(PE/EtOAc＝8：1)分离纯化， 得到(E)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-(嘧啶-5-基)丙烯酸乙酯5(125.3mg，收率62.3％，纯度93％)，为白色固体。 1H NMR(400MHz,CDCl 3)δ9.28(s,1H,Pyrimidine),8.89(s,2H,Pyrimidine),8.72(s,1H,NCH),8.59(s,2H,Ph),7.96(s,1H,Ph),7.40(s,1H,COCCH),4.85(m,1H,CH),1.36(t,J＝7.2Hz,6H,Me).MS(ESI)m/z 472.17[M+H] +。
(E)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-(嘧啶-5-基)丙烯羧酸(6)的合成
在25mL的茄形瓶中，将5(125.3mg，0.27mmol)溶于四氢呋喃(3mL)中。在冰浴下搅拌10min后，向反应液中滴加LiOH·H 2O(45.3mg，1.08mmol)的水(1mL)溶液。继续搅拌30min后将反应移至室温下搅拌过夜。TLC监测反应完全后，将冰水混合溶液(10mL)倒入反应液中，用4N盐酸调节溶液的pH至2-3。用乙酸乙酯(5mL×3)萃取，合并有机相并用饱和氯化钠溶液(20mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂。得到较纯的化合物(E)-3-(3-(3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-2-(嘧啶-5-基)丙烯酸(99.4mg，收率85.8％，纯度89％)，为白色固体。直接用于下一步使用。MS(ESI)m/z 427.92[M-H] -。
(E)-3-(3-(3-，3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-N-甲氧基-2-(嘧啶-5-基)丙烯酰胺(I)的合成
25mL的茄形瓶中，将6(125.3mg，0.27mmol)溶于二氯乙烷(3mL)中。在冰浴下搅拌10min后，向反应液中加入EDCI(20.3mg，1.08mmol)和HOBT(70.5mg，1.5mmol)。继续搅拌30min后将反应移至室温下滴加DIPEA(54.5mg，3.0mmol)和甲氧基羟胺盐酸盐(23.4mg,1.0mmol)搅拌过夜。TLC监测反应完全后，将冰水混合溶液(10mL)倒入反应液中。用乙酸乙酯(5mL×3)萃取，合并有机相并用饱和氯化钠溶液(20mL×1)洗涤，无水Na 2SO 4干燥，过滤并减压蒸除溶剂。得到化合物(E)-3-(3-(3-，3,5-双(三氟甲基)苯基)-1H-1,2,4-三唑-1-基)-N-甲氧基-2-(嘧啶-5-基)丙烯酰胺(35.4mg，收率40％，纯度89％)，为白色固体。 1H NMR(400MHz,CDCl3)δ8.78(d,J＝5.9Hz,2H,Pyridine),8.40(s,2H,Ph),8.33(s,1H,NCH),7.92(s,1H,Ph),7.87(s,1H,COCCH),7.28(d,J＝1.6Hz,2H,Pyridine),4.34(t,J＝7.1Hz,3H,Me).MS(ESI)m/z 459.10[M+H] +。
合成的具体化合物及其名称如下表。
Figure PCTCN2022080255-appb-000006
Figure PCTCN2022080255-appb-000007
二、细胞株抑制活性
1.细胞冻存
(1)收获细胞后常温1000转离心5min，PBS漂洗。
(2)含7％DMSO和10％胎牛血清的1640培养基重悬。
(3)分装入冻存管，放入细胞冻存盒中-80℃过夜处理后，液氮保存。
2.细胞复苏与培养：
(1)将冻存管从液氮罐中取出，放入37℃温水中轻轻摇晃，使细胞液解冻。
(2)转入无菌离心管，加入含10％胎牛血清1640培养液，轻轻吹打成悬液。
(3)室温1000转离心5min，弃上清液，加入10％含胎牛血清的1640培养液，轻轻吹打成悬液。
(4)转入培养瓶，于37℃含5％CO 2饱和湿度孵箱培养1-2日换液。
3.细胞传代：
弃掉原培基加入无菌PBS冲洗一遍，加入1mL 0.25％的胰酶孵育1分钟左右，镜下观察，待多数细胞开始变圆后小心吸净胰酶，并加入新鲜的培养基终止消化，将细胞吹打成均匀的细胞悬液，转入细胞孵箱中继续培养。
4.细胞毒性实验
(1)将对数生长期的RPMI8226细胞于1000个/well加入384孔板中，体积为18μL/well，在37℃，5％CO 2下孵育24h。
(2)将储存浓度为10mM的待测化合物用DMSO稀释10倍后，再用不含血清的1640培养基稀释100倍，得到10μM含1％DMSO的工作浓度，然后2倍梯度稀释，用1％DMSO的无血清1640培养基稀10个浓度，第十个浓度点是溶剂对照组(无药物)。将稀释好的化合物每孔吸取2μL加到铺好的细胞板中，得到化合物终浓度为1000nM起，2倍梯度稀释，10个浓度梯度，每个浓度设4个重复。1％DMSO用作溶剂对照，KPT-8602作为阳性对照。
(3)37℃下孵育72h后，每孔加入10μL的Cell-Titer检测试剂，再置于细胞培养箱孵育10min。
(4)震荡混匀后，于酶标仪运行Cell-Titer程序检测，并用GraphPad Prism5计算inhibition％和IC 50值(μM)。
化合物的细胞结果如下表：
编号 RPMI8226 编号 RPMI8226
KPT-8602 0.34 I 0.37
II 6.9 III 7.1
IV 0.54    
细胞活性结果表明，化合物I和IV与KPT8602基本相当。
三、药物在SD大鼠的药代动力学结果
Figure PCTCN2022080255-appb-000008
结论：化合物I的口服和静脉的半衰期均比KPT-8602的口服及静脉的半衰期长，特别是口服的半衰期长约2倍，而且化合物I的静脉和口服的AUC均远高于KPT-8602，表现出更好的药物动力学性质。
四、候选化合物BALB/C体内毒性实验
将25只BALB/C小鼠随机分为5组：溶剂对照组(Control)、阳性对照KPT-8602(30mg/kg，一天1次)组、KPT-8602(60mg/kg，一天1次)组、化合物I(30mg/kg，一天1次)组、化合物I(60mg/kg，一天1次)组，各组均为5只，各组按3mL/kg的给药容量灌胃给予相应浓度的受试物，KPT-8602和化合物I每天连续给药，共给药21天。
化合物溶液配制方法：
10％磺丁基-β-环糊精配制：
称取5.0g的磺丁基-β-环糊精粉末置于烧杯中，用移液器吸取50mL的柠檬酸缓冲液至烧杯，溶解后转移至容器中。
化合物I的配制：
称取24mg的化合物I，加入1.6mL的20％聚乙二醇水溶液，溶清后再加入10％磺丁基-β-环糊精水溶液6.4mL，得到3mg/mL的化合物I受试物溶液。
KPT-8602的配制：
称取24mg的化合物KPT-8602，加入1.6mL的20％聚乙二醇水溶液，溶清后再加入10％磺丁基-β-环糊精水溶液6.4mL，得到3mg/mL的KPT-8602受试物溶液。
连续给药后小鼠体重变化结果如图1所示。
结论：
如图所示，KPT-8602的60mg/kg组在第七天有两只小鼠出现死亡，第10天全部死亡。KPT-8602的30mg/kg组体重出现显著的下降。而化合物I的30mg/kg和化合物I的60mg/kg组在21天后体重仍与空白相当，表现出良好的安全性。

Claims (9)

  1. 一种三氮唑类衍生物或其药学上可接受盐，其特征在于：所述三氮唑类衍生物结构如式I所示，
    Figure PCTCN2022080255-appb-100001
    其中：
    R选自羟基、C1-6烷基、C1-6烷氧基或-C(OR 1)(＝NR 2)；
    R 1和R 2独立地为氢、C1-6烷基、卤代C1-6烷基或C1-6烷氧基。
  2. 根据权利要求11所述的三氮唑类衍生物或其药学上可接受盐，其特征在于：所述R选自羟基、C1-4烷基、C1-4烷氧基或-C(OR 1)(＝NR 2)；
    R 1和R 2独立地为氢或C1-4烷基。
  3. 根据权利要求1所述的三氮唑类衍生物或其药学上可接受盐，其特征在于：所述三氮唑类衍生物选自以下化合物：
    Figure PCTCN2022080255-appb-100002
  4. 权利要求1所述的三氮唑类衍生物或其药学上可接受盐的制备方法，其特征在于：按如下所示路线进行合成：
    Figure PCTCN2022080255-appb-100003
  5. 一种用于治疗与CRM1活性相关的疾病、紊乱或症状的药物组合物，其特征在于：包含权利要求1-3中任一项所述三氮唑类衍生物或其药学上可接受盐，以及药学上可接受的载体。
  6. 权利要求1-3任一项所述三氮唑类衍生物或其药学上可接受盐在制备用于治疗与CRM1活性相关的紊乱的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于：所述与CRM1活性相关的紊乱为增生性紊乱、癌症、炎症性紊乱、自身免疫性紊乱、病毒感染、眼科紊乱、神经退行性紊乱、异常组织生长紊乱、与食物摄取有关的紊乱、过敏以及呼吸紊乱。
  8. 根据权利要求7所述的应用，其特征在于：所述与CRM1活性相关的紊乱为癌症。
  9. 根据权利要求8所述的应用，其特征在于：所述与CRM1活性相关的紊乱为多发性骨髓瘤。
PCT/CN2022/080255 2021-03-12 2022-03-11 一种三氮唑类衍生物及其制备方法和应用 WO2022188846A1 (zh)

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