Triazole derivatives and their preparation and use

Abstract

The present invention discloses a triazole derivative and its preparation and use, which belongs to the field of pharmaceutical technology. The structure of the triazole derivative is represented by Formula I, and the triazole derivative of the present invention is used as a CRM1 inhibitor for the preparation of a therapeutic agent for diseases related to CRM1 activity.

Description

[Technical field]

[0001] The present invention belongs to the field of pharmaceutical technology, specifically to a triazole derivative and its preparation method and use. [Background technology]

[0002] Currently, malignant tumors remain one of the main diseases threatening human life. Although cancer treatment has made great strides, cancer has not been fundamentally cured. Although currently available anticancer drugs have a certain degree of efficacy, most of them are cytotoxic drugs and have severe toxicity and side effects. Therefore, it is an urgent task for medical professionals to research new targeting anticancer drugs from the perspective of effective tumor targeting.

[0003] Many cells, mainly from human solid tumors and hematological malignancies, show various abnormal subcellular localizations of oncogenic proteins, tumor suppressor proteins, and cell cycle regulators. For example, certain p53 mutations lead to cytoplasmic matrix localization instead of nuclear localization. This leads to loss of normal growth regulation despite full tumor suppressor function. In other tumors, wild-type p53 is sequestered in the cytoplasm or rapidly degraded, further leading to loss of its suppressor function. Proper restoration of nuclear localization of functional p53 protein normalizes several properties of neoplastic cells, restores sensitivity of cancer cells to DNA damaging agents, and leads to regression of proliferating tumors. Similar data have been obtained from other tumor suppressor proteins, such as forkhead (Sullivar) and c-Abl. Also, the abnormal localization of some tumor suppressor and growth regulator proteins may be involved in the pathogenesis of autoimmune diseases. CRM1 inhibition offers particularly valuable applications in familial cancer syndromes in which a specific tumor suppressor protein (TSP) is ablated or dysfunctional (e.g., Li-Fraumeni syndrome due to deletion of one p53 allele, BRCA1 or BRCA2 cancer syndromes), and the increase in TSP levels achieved by systemic (or local) administration of CRM1 inhibitors leads to restoration of normal tumor suppressor function.

[0004] Specific proteins and RNAs are transported into or out of the nucleus by specific translocators. Those that transport molecules into the nucleus are classified as importins, whereas those that transport molecules out of the nucleus are classified as exportins. Proteins that are transported into or out of the nucleus contain a nuclear localization signal (NLS) or nuclear export signal (NES). These interact with associated export factors. Chromosome maintenance region 1 (CRM1), also called exportin-1 or Xpol, is the main exportin. Blockade of inhibitory proteins and growth regulators by CRM1 inhibitors, such as p53, c-Ab1, p21, p27, pRB, BRCA1, IkB, ICp27, E2F4, KLF5, YAP1, ZAP, KIF5, HDAC4, HDAC5, or nuclear export of forkhead proteins (e.g. FOXO3a). These are involved in gene expression, cell proliferation, angiogenesis, and epigenetics. Results show that CRM1 inhibitors can induce apoptosis in cancer cells without affecting normal (non-transformed) cells, even in the presence of activation or growth activation signals. Most of the research on CRM1 function has been carried out with the natural product leptomycin B (LMB). Leptomycin itself is highly toxic to tumor cells, but its strong gastrointestinal toxicity makes it difficult to apply in clinical practice. By deriving LMB to improve drug-likeness, compounds that retain antitumor activity and are tolerated in animal tumor models are obtained. Thus, nuclear export inhibitors have beneficial effects on neoplastic and other proliferative disorders. However, currently, small molecule drug-like CRM1 inhibitors used in vitro and in vivo still have certain deficiencies.

[0005] Besides tumor suppressor proteins, several key proteins involved in various inflammatory processes are also exported by CRM1. These proteins include IkB, NF-kB, Cox-2, RXRa, Commal, HIFI, HMGBI, FOXO, FOXP, etc. The nuclear factor xB (NF-kB / rel) family of transcription activators, named for their ability to induce immunoglobulin x gene expression, can regulate the mRNA expression of various genes involved in inflammation, proliferation, immunity, and cell survival. In basic situations, the NF-kB protein inhibitor, called IkB, binds to NF-kB in the nucleus, and the IKB-NF-kB complex inactivates the transcriptional function of NF-kB. In response to inflammatory stimuli, IkB dissociates from the IkBNF-kB complex, releasing NF-kB and restoring its potential transcriptional activity. Many of the signals that activate NF-kB achieve this by targeting IkB protein hydrolysis (they are "tagged" by phosphorylation of IkB, ubiquitinate it, and then hydrolyze the protein). NF-kB is reactivated by export of the nuclear IkBa-MF-kB complex to the cytoplasm by CRM1, where it dissociates. Ubiquitinated IkB dissociates from the NF-kB complex, restoring NF-kB transcriptional activity. Inhibition of CRM1-induced export by IMB in human neutrophil and macrophage-like cells (U937) not only led to the accumulation of transcriptionally inactive nuclear IkBa-NF-kB complexes, but also prevented early NF-kB activation, even in the presence of cell stimulation. In a different study, LMB treatment suppressed IL-1β-induced NF-KB DNA binding (the first step in NF-kB transcriptional activation), 1L-8 expression, and intercellular adhesion molecule expression in pulmonary microvascular endothelial cells. COMMD1 is another nuclear repressor of the transcriptional activity of both NF-kB and hypoxia-inducible factor 1 (HIF1). Inhibition of CRM1 blocks the nuclear export of COMMD1, leading to increased inhibition of the transcriptional activity of NF-kB and HIF1.

[0006] CRM1 further mediates the transport of retinoid X receptor a (RXRa). RXRa is highly expressed in the liver and plays an important role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism as well as stabilizing the internal environment. During hepatitis, nuclear RXRa levels are significantly decreased, mainly due to inflammation-mediated CRM1 nuclear export of RXRa. In human liver-derived cells, L-1B-induced increase in cytoplasmic RXRa levels can be inhibited by LepB.

[0007] According to the role of CRM1-mediated nuclear export in NF-kB, HIF-1 and RXRa signaling, blockade of nuclear export has potential advantages in many inflammatory processes involving multiple tissues and organs, including the vascular system (vasculitis, arteritis, polymyalgia rheumatica, atherosclerosis), dermatology and rheumatology (rheumatoid arthritis and related arthritis, psoriatic arthritis, spondyloarthritis, crystalline arthritis, systemic lupus erythematosus, mixed connective tissue disease, myositis syndrome, dermatomyositis, inclusion body myositis, undifferentiated connective tissue disease, sicca syndrome, scleroderma and overlap syndrome, etc.).

[0008] Suppression of CRM1 affects gene expression by suppressing the activation of a series of transcription factors, including ICp27, E2F4, KL5, YAP1, and ZAP.

[0009] Inhibition of CRM1 has potential therapeutic effects on many dermatological syndromes, including inflammatory skin diseases (atopic, allergic dermatitis, chemical dermatitis, psoriasis), sunburn (ultraviolet (UV) damage), and infection. CRM1 inhibition, the most studied by LMB, shows minimal effects on normal keratinocytes and anti-inflammatory activity on keratinocytes exposed to UV, TNFα, or other inflammatory stimuli. Inhibition of CRM1 can further upregulate the activity of NRF2 (nuclear factor 2-related factor 2), which can protect keratinocytes from oxidative damage. Apoptosis of keratinocytes infected with oncogenic human papillomavirus (PV) strains such as IPV16, but not uninfected keratinocytes, is induced by LMB.

[0010] CRM1 mediates the trafficking of important neuroprotective proteins, which may have potential benefits against neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. For example, CRM1 inhibition can reduce or prevent the neuronal death found in these disorders by (1) forcing nuclear retention of important neuroprotective regulators, such as NRF2, and / or (2) isolating 1XB in the nucleus of glial cells, thereby suppressing the transcriptional activity of NF-kB. Evidence also suggests that abnormal proliferation of glial cells is associated with abnormalities in CRM1 levels or function.

[0011] Full maturation of many viruses requires complete nuclear export, which is primarily mediated by CRM1. Viruses that involve nuclear export in their life cycle and / or that themselves contain CRM1 include human immunodeficiency virus (HIV), adenovirus, simian retrovirus type 1, Borna disease virus, influenza virus (normal, HINL and avian HN1 strains), hepatitis B virus (BV) and hepatitis C virus (HCV), human papillomavirus (HPV), respiratory syncytial virus (RSV), Dungee, severe acute respiratory syndrome coronavirus, yellow fever virus, West Nile virus, herpes simplex virus (SV), cytomegalovirus (CMV), and Merkel cell polyomavirus (MCV).

[0012] The HIV-1 Rev protein, which transits the nucleolus and shuttles between the cell nucleus and the cytoplasm, exports unspliced ​​and single-spliced ​​HIV transcripts that contain the Rev response element (RRE) RNA via the CRM1 nuclear export pathway. Inhibition of Rev-mediated RNA export, achieved by CRM1 inhibitors such as LepB or PKF050-638, blocks the HIV-1 transcription process, suppresses the production of new HIV-1 viral particles, and reduces the levels of HIV-1.

[0013] Dengue virus (DENV) is the causative agent of the common arthropod-borne viral disease dengue fever (DF) and the more severe, potentially fatal dengue hemorrhagic fever (DHF). DHF appears to be the result of an exaggerated inflammatory response to DENV, and NS5 is the largest and most conserved protein of DENV. CRM1 regulates the transport of NS5 from the nucleus to the cytoplasm, where it is mediated through most of its functions. Inhibition of CRM1-mediated NS5 nuclear export leads to altered viral dynamics and reduces induction of the inflammatory chemokine interleukin-8 (IL-8), providing a new route to treat DENV and other medically important flaviviruses, including hepatitis C virus-induced disease.

[0014] Other virally encoded RNA-binding proteins exported to the nucleus by CRM1 include the HSVI type intermediate layer protein (P13 / 14 or HUA7), human CMV protein pp65, the SARS coronavirus ORF3b protein, and the RSV matrix (M) protein.

[0015] Interestingly, many of these diseases are related to certain types of human cancers, including hepatocellular carcinoma (HCC) caused by chronic HBV or HCV infection, cervical cancer caused by HPV, and Merkel cell carcinoma associated with MCV. Therefore, CRM1 inhibitors have an advantage over the viral infection process and the neoplastic transformation process caused by these viruses.

[0016] CRM1 regulates the nuclear localization and activity of various DNA metabolic enzymes, including histone deacetylases (HDACs), histone acetyl transferases (HATs), and histone methyltransferases (HMTs).

[0017] CRM1 has also been implicated in other disorders: Lebers disorder, a genetic disorder characterized by degeneration of retinal ganglion cells and vision loss, is linked to an inactivated CRM1 switch, and evidence suggests that neurodegenerative disorders involve abnormal nuclear transport. Summary of the Invention

[0018] The present invention aims to provide a triazole derivative and a method for producing the same and its use, and the triazole derivative is used as a CRM1 inhibitor in the production of a therapeutic agent for a disease associated with CRM1 activity.

[0019] In order to achieve the above object of the invention, the present invention employs the following technical means: The triazole derivative has the structure represented by Formula I, or a pharma- ceutically acceptable salt thereof. [ka] (however, R is a hydroxyl group, a C1-6 alkyl group, a C1-6 alkoxy group or -C(OR 1 )(=NR 2 ) are selected, R 1 and R 2 are each independently a hydrogen atom, a C1-6 alkyl group, a halogenated C1-6 alkyl group, or a C1-6 alkoxy group.

[0020] Furthermore, the R is a hydroxyl group, a C1-4 alkyl group, a C1-4 alkoxy group, or -C(OR 1 )(=NR 2 ) are selected, R 1 and R 2 are each independently hydrogen or a C1-4 alkyl group.

[0021] Furthermore, the triazole derivative is selected from the following compounds: [ka]

[0022] The method for producing the above-mentioned triazole derivative or a pharma- ceutically acceptable salt thereof is as follows: [ka]

[0023] A pharmaceutical composition for treating a disease, disorder or condition associated with CRM1 activity comprises the above triazole derivative or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier.

[0024] The present invention relates to use of the above-mentioned triazole derivative or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating a disorder associated with CRM1 activity.

[0025] Further, the disorder associated with CRM1 activity is a proliferative disorder, a cancer, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmological disorder, a neurodegenerative disorder, an abnormal tissue growth disorder, a disorder associated with food intake, an allergy, a respiratory disorder.

[0026] Further, said disorder relating to CRM1 activity is cancer.

[0027] Further, said disorder relating to CRM1 activity is multiple myeloma.

[0028] Beneficial effects: According to the results of cell activity, compound I and IV of the present invention are almost equivalent to KPT-8602. The half-life of compound I in oral and intravenous administration is longer than that of KPT-8602 in oral and intravenous administration. In particular, the half-life of oral administration is about twice as long. In addition, the AUC of compound I in intravenous and oral administration is much higher than that of KPT-8602, showing better pharmacokinetic properties. In addition, compound I shows good safety. [Brief description of the drawings]

[0029] [Figure 1] 1 shows the in vivo toxicity test results of Compound I in BALB / C mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Compound definition The compounds of the present invention include those generally described above and are further described according to the classes, subclasses and genus described herein. As used herein, the following definitions shall apply unless otherwise stated. For purposes of the present invention, the chemical elements are identified based on the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition.

[0031] Unless otherwise explained herein, the nomenclature used herein generally follows Chapters A, B, C, D, E, F, and H of the Nomenclature of Organic Chemistry, for which exemplary chemical structure nomenclature and rules for chemical structure nomenclature are incorporated herein by reference.

[0032] The compounds of the present invention have asymmetric centers, asymmetric axes and asymmetric planes and may exist as racemates, racemic mixtures and single diastereomers or enantiomers, and all possible isomers and mixtures thereof (including optical isomers) are included in the present invention.

[0033] "Halogen" includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Particularly preferred are a fluorine atom and a chlorine atom.

[0034] The "alkyl group" refers to a linear or branched hydrocarbon group containing 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. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, an n-octyl group, an isooctyl group, an n-nonyl group, and an n-decyl group.

[0035] The term "alkoxy group" refers to a group formed by bonding the above-mentioned "alkyl group" with an oxygen atom. Examples include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, a tert-butoxy group, an isobutoxy group, a sec-butoxy group, a pentoxy group, an isopentoxy group, and a hexyloxy group.

[0036] Preferred embodiments of the "alkoxy group" include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, and a tert-butoxy group.

[0037] The "halogenated alkyl group" includes a group formed by substituting one or more hydrogen atoms bonded to a carbon atom of the above-mentioned "alkyl group" with the above-mentioned "halogen". Examples thereof include a monofluoromethyl group, a monofluoroethyl group, a monofluoropropyl group, a 2,2,3,3,3-pentafluoropropyl group, a monochloromethyl group, a trifluoromethyl group, a trichloromethyl group, a 2,2,2-trifluoroethyl group, a 2,2,2-trichloroethyl group, a 1,2-dibromoethyl group, and a 1,1,1-difluoropropan-2-yl group.

[0038] An embodiment of the "halogenated alkyl group" is a trifluoromethyl group or a trichloromethyl group.

[0039] The present invention provides compositions comprising a compound of the present invention or a pharma- ceutically acceptable derivative thereof and a pharma- ceutically acceptable carrier, adjuvant or carrier. The amount of compound in the compositions of the present invention is an amount to effectively and appropriately inhibit CRM1 in a biological sample or in a patient. In certain embodiments, the compositions of the present invention are prepared for administration to a patient in need of these compositions. As used herein, the term "patient" refers to an animal. In some embodiments, the animal is a mammal. In certain embodiments, the patient is a veterinary patient (i.e., a non-human mammalian patient).

[0040] The term "pharmaceutically acceptable carrier, adjuvant or carrier" refers to a non-toxic carrier, adjuvant or carrier that does not impair the pharmaceutical activity of the compound with which it is prepared. Pharmaceutically acceptable carriers, adjuvant or carriers that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, glycols, and lanolin.

[0041] The triazole derivative or a pharma- ceutically acceptable salt thereof can be prepared by the steps shown below. [ka]

[0042] In this reaction scheme, each group R is defined as above. Formula (1) is reacted with sodium hydrosulfide to obtain formula (2). Formula (2) undergoes a nucleophilic substitution reaction to produce formula (3). Formula (3) is further reacted with bromine water and triethylamine to produce formula (4). Formula (4) undergoes a conjugation reaction to produce formula (5). Formula (5) is hydrolyzed with LiOH to give the carboxylic acid formula (6). Formula (6) is condensed with an amine substrate to obtain a compound of formula I.

[0043] The process for producing the compound of the present invention will now be described in detail.

[0044] The cyano group of compound 1 is reacted with sodium hydrosulfide and magnesium chloride to generate thioformamide. It is then reacted with hydrazine hydrate and formic acid to generate triazole 2. Triazole 2 is conjugated with isopropyl (Z)-3-iodoacrylate under the catalysis of triethylenediamine to generate compound 3. The double bond of compound 3 then undergoes addition reaction with liquid bromine, and one molecule of bromine is removed under the catalysis of triethylamine to obtain key intermediate 4. Intermediate 4 undergoes Suzuki conjugation reaction with various nitrogen-containing aryl groups with boric acid structure under the catalysis of bis(triphenylphosphine)palladium(II) dichloride to generate compound 5 linked to different nitrogen-containing aryl groups. Finally, the ester bond of compound 5 is hydrolyzed to carboxylic acid compound 6 under the catalysis of LiOH. The carboxylic acid compound 6 is further condensed to obtain the target compound of formula I.

[0045] The present invention will be described in more detail below with reference to the drawings and specific examples, but the present invention is not limited thereto. Modifications or substitutions made to the methods, steps or conditions of the present invention are also included in the scope of the present invention, as long as they do not deviate from the spirit and substance of the present invention. Experimental methods for which specific conditions are not specified in the examples and reagents for which the formulation is not described are in accordance with normal conditions in this field.

[0046] Example 1 1. Compound synthesis The compounds of the present invention are prepared by the following process.

[0047] Step design and synthesis of compound I [ka]

[0048] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)isopropyl acrylate (3) 3,5-Bis(trifluoromethyl)cyanophenyl 1 (10 g, 41.8 mmol) was placed in a 250 mL eggplant flask and dissolved in DMF (50 mL). NaSH (7.8 g, 83.7 mmol) and MgCl 2 (8.5 g, 41.8 mmol) were added in this order, and the reaction was allowed to proceed with stirring at room temperature for 3 h. After monitoring the complete reaction by TLC, the reaction solution was poured into ice-water mixture (500 mL). Extraction was performed with ethyl acetate (100 mL x 3), and the combined organic phase was washed with saturated sodium chloride solution (100 mL x 1), and then washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the solvent was removed by evaporation under reduced pressure to give crude 3,5-bis(trifluoromethyl)benzenesulfonamide (10.9 g, 95.1% yield, 84% purity). Yellow oily liquid. MS (ESI) m / z 274.35 [M+H]+. Used directly in the next step.

[0049] 3,5-Bis(trifluoromethyl)benzenesulfonamide (10.9 g, 39.8 mmol) was placed in a 250 mL eggplant flask and dissolved in DMF (30 mL). 80% hydrazine hydrate (5.1 mL, 83.6 mmol) was added dropwise at room temperature, the mixture was stirred for 1 h, and then formic acid (30 mL) was added dropwise. The mixture was then stirred at 90 °C for 3 h. The reaction was monitored by TLC to ensure complete reaction, and the reaction was allowed to reach room temperature. The reaction solution was poured into pure water (600 mL). Extraction was performed with ethyl acetate (100 mL x 3), and the organic phases were combined and washed with saturated sodium bicarbonate solution (300 mL x 3) and saturated sodium chloride solution (100 mL x 1), and the mixture was washed with anhydrous Na 2 SO 4 The crude product was obtained by evaporating the solvent under reduced pressure, washing with n-hexane (200 mL x 3) under stirring, filtering and drying to obtain 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole 2 (8.5 g, yield 76.0%, purity 90%) as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 8.63 (s, 2H, Ph), 8.40 (s, 1H, NCH), 8.02 (d, J= 13.8 Hz, 1H, NCHCH), 7.95 (s, 1H, Ph), 6.74 (d, J= 13.8 Hz, 1H, NCHCH)MS (ESI) m / z 279.89 [M+H] +

[0050] 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole 2 (8.5 g, 30.2 mmol) was placed in a 250 mL eggplant flask, dissolved in DMF (40 mL), and DABCO (8.5 g, 75.5 mmol) was added. The mixture was stirred at room temperature for 30 min, and then (Z)-3-ethyl iodoacrylate (7.5 g, 33.2 mmol) was added dropwise to the reaction solution. The reaction was then stirred at room temperature for 3 h. After monitoring by TLC to ensure complete reaction, the reaction solution was poured into a mixture of ice and water (400 mL). Extraction was performed with ethyl acetate (80 mL x 3), and the combined organic phase was washed with saturated sodium chloride solution (100 mL x 1), and anhydrous NaCl was added. 2 SO 4 The mixture was dried at 40° C., filtered, and the solvent was removed by evaporation under reduced pressure to obtain a crude product of the compound. The crude product was purified by column chromatography (PE / EtOAc=25:1) to obtain compound (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)ethyl acrylate 3 (7.9 g, yield 68.2%, purity 98%), which is a white solid. 1 HNMR (400 MHz, CDC1 3 ) δ 8.63 (s, 2H, Ph), 8.40 (s, 1H, NCH), 8.02 (d, J= 13.8 Hz, 1H, NCHCH), 7.95 (s, 1H, Ph), 6.74 (d, J = 13.8 Hz, 1H, NCHCH), 4.87 (m, 1H, CH), 1.36 (t, J =7.1 Hz, 6H, Me). MS (ESI) m / z 394.23 [M+H] + .

[0051] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl-triazol-1-yl)-2-bromoacrylate isopropyl (4) In a 250 mL eggplant flask, previously obtained ethyl (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate 3 (7.9 g, 20.6 mmol) was dissolved in dichloromethane (40 mL). Liquid bromine (6.6 g, 41.2 mmol) was slowly added dropwise within 30 min, and the reaction was continued with stirring at room temperature for 8 h. After monitoring the complete reaction by TLC, the reaction solution was poured into ice-water mixture (100 mL). Extraction was performed with dichloromethane (50 mL x 3), and the combined organic phase was washed with saturated sodium bisulfite solution (100 mL x 3) and saturated sodium chloride solution (50 mL x 1), and the mixture was cooled to room temperature for 1 h. The reaction was continued for 1 h at room temperature ... 2 SO 4 The mixture was dried at 40° C., filtered, and the solvent was removed by evaporation under reduced pressure. The product was separated and purified by column chromatography (PE / EtOAc = 50:1) to give 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2,3-dibromoisopropyl acrylate (10.3 g, yield 92.7%, purity 95%). It is a white solid. MS (ESI) m / z 551.97 [M+H] + .

[0052] The intermediate 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2,3-dibromo isopropyl acrylate (103 g, 19.1 mmol) obtained in the previous step was weighed into a 250 mL eggplant flask, dissolved in tetrahydrofuran (40 mL), stirred in an ice bath for 10 min, and triethylamine (3.9 g, 38.2 mmol) was added dropwise to the reaction solution while stirring for 30 min. The reaction was then continued at room temperature for 6 h while stirring. After monitoring by TLC to ensure complete reaction, ice water mixture (100 mL) was added to the reaction solution. Extraction was performed with ethyl acetate (50 mL x 3), and the combined organic phase was washed with saturated sodium chloride solution (50 mL x 1), and anhydrous NaCl was added. 2 SO 4The mixture was dried at 400 rpm, filtered, and the solvent was removed by evaporation under reduced pressure. Separation and purification by column chromatography (PE / EtOAc=50:1) gave (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-bromoethyl acrylate 4 (7.7 g, 88.2% yield, 96% purity). It was a white solid. 1H NMR (400 MHz, CDCl3) δ 8.75 (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=7.1 Hz, 6H, Me). MS (ESI) m / z 473.09 [M+H] + .

[0053] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)acrylate isopropyl ester (5) A 25 ml three-neck flask was taken, and the key intermediate 4 (200 mg, 0.44 mmol) and 5-pyrimidineboronic acid (81.9 mg, 0.66 mmol) were weighed out and dissolved in a mixture of dioxane (5 mL) and water (1 mL). Then, sodium acetate (86.4 mg, 0.88 mmol) was weighed out and added to the reaction solution, and the reaction was carried out at room temperature while stirring after replacing the atmosphere with nitrogen gas three times. Then, Pd(PPh 3 )Cl 2 (30.9 mg, 0.04 mmol) was added, and the mixture was purged with nitrogen gas three times, and then reacted at 80°C overnight with stirring. After monitoring by TLC to ensure a complete reaction, the mixture was reacted until it reached room temperature, and then pure water (50 mL) was added to the reaction solution, which was then extracted with ethyl acetate (15 mL x 3). The organic phases were combined and washed with saturated sodium chloride solution (20 mL x 1), and anhydrous Na 2 SO 4The crude product was obtained by filtration, drying at 400 rpm, and removing the solvent by evaporation under vacuum. The crude product was obtained by separation and purification by column chromatography (PE / EtOAc=8:1) to obtain ethyl (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)acrylate 5 (125.3 mg, yield 62.3%, purity 93%). It is a white solid. PNMR (400 MHz, CDCl 3 ) δ 9.28 (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=7.2 Hz, 6H, Me). MS (ESI) m / z 472.17 [M+H] + .

[0054] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)propylenecarboxylic acid (6) In a 25 mL eggplant flask, 5 (125.3 mg, 0.27 mmol) was dissolved in tetrahydrofuran (3 mL). After stirring for 10 min in an ice bath, the reaction solution was diluted with LiOH H 2 A solution of 1H2O (45.3 mg, 1.08 mmol) in water (1 mL) was added dropwise. After stirring for 30 min, the reaction was stirred at room temperature overnight. After monitoring the complete reaction by TLC, ice-water mixture (10 mL) was poured into the reaction solution, and the pH value of the solution was adjusted to 2-3 with 4N hydrochloric acid. Extraction was performed with ethyl acetate (5 mL x 3), and the combined organic phase was washed with saturated sodium chloride solution (20 mL x 1), and then washed with anhydrous NaCl. 2 SO 4The mixture was dried at 40° C., filtered, and the solvent was removed by evaporation under reduced pressure. The relatively pure compound (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)acrylic acid (99.4 mg, 85.8% yield, 89% purity) was obtained as a white solid. It was used directly in the next step. MS (ESI) m / z 427.92 [MH] - .

[0055] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-methoxy-2-(pyrimidin-5-yl)acrylamide (I) 6 (125.3 mg, 0.27 mmol) was dissolved in dichloroethane (3 mL) in a 25 mL eggplant flask. After stirring in an ice bath for 10 min, EDCI (20.3 mg, 1.08 mmol) and HOBT (70.5 mg, 1.5 mmol) were added to the reaction solution. After stirring for 30 min, DIPEA (54.5 mg, 3.0 mmol) and methoxyhydroxylamine hydrochloride (23.4 mg, 1.0 mmol) were added dropwise at room temperature and the reaction was allowed to proceed with stirring overnight. After monitoring the complete reaction by TLC, ice-water mixture (10 mL) was poured into the reaction solution. Extraction was performed with ethyl acetate (5 mL x 3), and the combined organic phase was washed with saturated sodium chloride solution (20 mL x 1) and anhydrous NaCl was added. 2 SO 4 The mixture was dried at 40° C., filtered, and the solvent was removed by evaporation under reduced pressure to obtain compound (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-methoxy-2-(pyrimidin-5-yl)acrylamide (35.4 mg, yield 40%, purity 89%). It was a white solid. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 5.9 Hz, 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 = 1.6 Hz, 2H, Pyridine), 4.34 (t, J = 7.1Hz, 3H, Me).MS (ESI) m / z 459.10 [M+H] + .

[0056] The specific compounds to be synthesized and their names are shown in the table below. [Table 1-1]

[0057] [Table 1-2]

[0058] 2. Inhibitory activity of cell lines 1. Cell cryopreservation (1) After harvesting the cells, they were centrifuged at 1,000 rpm for 5 min at room temperature and washed with PBS. (2) Resuspended in 1640 medium containing 7% DMSO and 10% fetal bovine serum. (3) The cells were dispensed into cryopreservation tubes, placed in a cell cryopreservation box, and incubated at -80°C overnight before being stored in liquid nitrogen.

[0059] 2. Cell Resuscitation and Culturing (1) Remove the cryopreservation tube from the liquid nitrogen tank, place it in 37°C warm water, and shake it slowly to thaw the cell fluid. (2) Transfer to a sterile centrifuge tube, add 1640 culture medium containing 10% fetal bovine serum, and gently infuse to form a suspension. (3) Centrifuge at room temperature at 1,000 rpm for 5 minutes, discard the supernatant, add 1640 culture medium containing 10% fetal bovine serum, and gently blow in to form a suspension. (4) Transfer to a culture flask and incubate at 37°C and 5% CO 2 The cells were then cultured in a humidity-saturated incubator for 1-2 days, after which the liquid was replaced.

[0060] 3. Passaging of cells The original medium was discarded, and the cells were washed once with sterile PBS, and then 1 mL of 0.25% pancreatin was added and incubated for about 1 minute. After observing under a microscope and most of the cells had rounded up, the pancreatin was thoroughly sucked off, fresh medium was added to terminate the digestion, and the cells were blown evenly to form a cell suspension, and the cells were moved to an incubator and continued to be cultured.

[0061] 4. Cytotoxicity experiments (1) RPMI8226 cells in the logarithmic growth phase were plated at 1,000 cells / well in a 384-well plate and incubated at 37°C, 5% CO in a volume of 18 μL / well. 2 The mixture was incubated at 4°C for 24 h. (2) The target compound with a stock concentration of 10 mM was diluted 10-fold with DMSO, and then further diluted 100-fold with serum-free 1640 medium to obtain a working concentration of 10 μM with 1% DMSO. It was then diluted in a 2-fold gradient and diluted in 10 concentrations with serum-free 1640 medium with 1% DMSO, with the 10th concentration point being the solvent control group (no drug). The diluted compound was aspirated and put into the plated cell plate at 2 μL per well to obtain the compound. The final concentration was 1000 nM, diluted in a 2-fold gradient, and set up a 10 concentration gradient with 4 replicates at one concentration. 1% DMSO was used as the solvent control, and KPT-8602 was used as the positive control. (3) After incubation at 37°C for 72 hours, 10 μL of Cell-Titer measurement reagent was added per well and further incubated in an incubator for 10 minutes. (4) After shaking evenly, measure the titer using a microplate reader with the Cell-Titer program, and calculate the inhibition% and IC 50 Values ​​(μM) were calculated.

[0062] The cellular results for the compounds are shown in the table below. [Table 2]

[0063] According to the cellular activity results, compounds I and IV are roughly comparable to KPT-8602.

[0064] 3. Pharmacokinetics of drugs in SD rats [Table 3]

[0065] Conclusion: The half-life of compound I after oral and intravenous administration is longer than that of KPT-8602 after oral and intravenous administration. In particular, the half-life of compound I after oral administration is about twice as long. In addition, the AUC of compound I after intravenous and oral administration is much higher than that of KPT-8602, indicating better pharmacokinetic properties.

[0066] 4. In vivo toxicity test of candidate compounds in BALB / c 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 (30 mg / kg, once a day) group, and Compound I (60 mg / kg, once a day) group. Each group had 5 mice. The corresponding concentrations of test substances, KPT-8602 and Compound I, were intragastrically administered to each group at a dose of 3 mL / kg, and were administered continuously every day for a total of 21 days.

[0067] Preparation of compound solutions: Preparation of 10% sulfobutyl-β-cyclodextrin: 5.0 g of sulfobutyl-β-cyclodextrin powder was weighed out and placed in a flask, and 50 mL of citrate buffer was sucked up with a pipetter and placed in the flask, and the solution was dissolved before being transferred to a container.

[0068] Preparation of Compound I: 24 mg of compound I was weighed out, and 1.6 mL of 20% aqueous polyethylene glycol solution was added to dissolve it. Then, 6.4 mL of 10% aqueous sulfobutyl-β-cyclodextrin solution was added to obtain a test solution of compound I at 3 mg / mL.

[0069] Preparation of KPT-8602: 24 mg of compound KPT-8602 was weighed out, and 1.6 mL of 20% aqueous polyethylene glycol solution was added to dissolve it, and then 6.4 mL of 10% aqueous sulfobutyl-β-cyclodextrin solution was added to obtain a test solution of 3 mg / mL KPT-8602.

[0070] After continuous administration, the results of the body weight changes of the mice are shown in FIG.

[0071] Conclusion: As shown in the figure, in the KPT-8602 60mg / kg group, 2 mice died on the 7th day, and all died on the 10th day, and the KPT-8602 30mg / kg group showed a significant weight loss. The weights of the compound I 30mg / kg and compound I 60mg / kg groups after 21 days were equivalent to the blank, indicating good safety.

Claims

1. A triazole derivative or a pharma- ceutically acceptable salt thereof, characterized in that the structure is represented by Formula I: 【Chemistry 1】 (however, R is a hydroxyl group, a C1-6 alkoxy group, or -C(OR １ ) (=NR ２ ) were selected, R １ each independently represents hydrogen, a C1-6 alkyl group, or a halogenated C1-6 alkyl group; Each R 2 is independently a hydrogen atom, a C1-6 alkyl group, a halogenated C1-6 alkyl group, or a C1-6 alkoxy group.

2. The R is a hydroxyl group, a C1-4 alkoxy group, or -C(OR １ ) (=NR ２ ) were selected, R １ and R ２ and each independently represents hydrogen or a C1-4 alkyl group, or a pharma- ceutically acceptable salt thereof, according to claim 1.

3. 2. The triazole derivative or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the triazole derivative is selected from the following compounds: 【Chemistry 2】

4. A pharmaceutical composition for treating a disorder associated with CRM1 activity, comprising the triazole derivative or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3 and a pharma- ceutical acceptable carrier, The pharmaceutical composition, characterized in that the disorder relating to CRM1 activity is a proliferative disorder, cancer, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, an abnormal tissue growth disorder, a disorder relating to food intake, an allergy, or a respiratory disorder.

5. The pharmaceutical composition described in claim 4, characterized in that the disorder related to CRM1 activity is cancer.

6. The pharmaceutical composition described in claim 5, characterized in that the cancer is multiple myeloma.

7. Use of the triazole derivative or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 3 in the manufacture of a medicament for treating a disorder associated with CRM1 activity, comprising: The use, wherein the disorder relating to CRM1 activity is a proliferative disorder, cancer, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmological disorder, a neurodegenerative disorder, an abnormal tissue growth disorder, a disorder relating to food intake, an allergy, or a respiratory disorder.

8. The use according to claim 7, characterized in that the disorder relating to CRM1 activity is cancer.

9. The use described in claim 8, characterized in that the cancer is multiple myeloma.

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