A triazole derivative, its preparation method and application

Abstract

This invention discloses a triazole derivative, its preparation method, and its application, belonging to the field of pharmaceutical technology. The structure of the triazole derivative is shown in Formula I. The triazole derivative of this invention can be used as a CRM1 inhibitor for the preparation of therapeutic drugs for diseases related to CRM1 activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a triazole derivative, its preparation method, and its application. Background Technology

[0002] Currently, malignant tumors remain one of the leading causes of death. While significant progress has been made in cancer treatment, a complete cure for cancer has not yet been achieved. Although currently available anticancer drugs have some efficacy, most are cytotoxic and have serious side effects. Therefore, researching novel targeted anticancer drugs based on effective tumor targets has become a pressing task for medical professionals.

[0003] Cells from most major human solid tumors and hematologic malignancies exhibit anomalous cellular localization of various oncogenes, tumor suppressor proteins, and cell cycle regulators. For example, certain p53 mutations can lead to matrix localization rather than nuclear localization. This results in the loss of normal growth regulation, although tumor suppressor function remains intact. In other tumors, wild-type p53 is isolated in the cytoplasm or rapidly degraded, again leading to loss of its suppressor function. Restoration of proper nuclear localization of functional p53 proteins can normalize some characteristics of tumorigenic cells, restore cancer cell sensitivity to DNA damaging agents, and lead to tumor regression. Similar data were obtained from other tumor suppressor proteins such as forkhead and Sullivar and c-Abl. Furthermore, anomalous localization of some tumor suppressor and growth regulatory proteins may be related to the pathogenesis of autoimmune diseases. CRM1 inhibition can provide particularly meaningful applications in familial syndromes (e.g., Livmerniosis syndrome caused by a p53 allele deletion, BRCA1 or BRCA2 cancer syndromes) in which specific tumor suppressor proteins (TSPs) are cleared or have dysfunction, and in which the increase in TSP levels achieved by the administration of a systemic (or local) CRM1 inhibitor can help restore normal tumor suppressor function.

[0004] Specific proteins and RNAs are imported into and exported from the cell nucleus by specific transport molecules. If they import molecules into the nucleus, they are classified as importing proteins; if they export molecules, they are classified as exporting proteins. Proteins imported into or exported from the nucleus contain nuclear import localization (NLS) or nuclear export (NES) sequences, enabling them to interact with associated transport factors. Chromosomal region-stabilizing protein 1 (CRM1), also known as exporting protein-1 or Xpol, is a major exporting protein. Inhibitors of CRM1 block the nuclear export of inhibitory proteins and growth regulators such as p53, c-Ab1, p21, p27, pRB, BRCA1, IkB, ICP27, E2F4, KLF5, YAP1, ZAP, KIF5, HDAC4, HDAC5, or forkhead proteins (e.g., FOXO3a), which are associated with gene expression, cell proliferation, angiogenesis, and phenotypic inheritance. Results have 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 functional studies on CRM1 have used the natural product lepromycin B (LMB). Lepromycin itself is highly toxic to tumor cells, but its significant gastrointestinal toxicity makes it difficult to use clinically. Derivatives of LMB to improve drug-like properties can yield compounds that retain antitumor activity and are better tolerated in animal tumor models. Therefore, nuclear export inhibitors may have beneficial effects on tumor-related disorders and other proliferative disorders. However, to date, small-molecule drug-like CRM1 inhibitors for in vitro and in vivo use still have certain limitations.

[0005] In addition to tumor suppressor proteins, CRM1 also exports several key proteins associated with many inflammatory processes. These proteins include IkB, NF-κB, Cox-2, RXRa, Common, HIFI, HMGBI, FOXO, and FOXP. The nuclear factor xB (NF-κB / rel) family of transcriptional activators, named for their ability to induce immunoglobulin x gene expression, regulates the mRNM expression of various genes related to inflammation, proliferation, immunity, and cell survival. In its basic form, an NF-κB protein called IkB binds to NF-κB in the nucleus, and the IkB-NF-κB complex inactivates NF-κB transcription. In response to inflammatory stimuli, IkB dissociates from the IkBNF-κB complex, releasing NF-κB and restoring its potential transcriptional activity. Many signals that activate NF-κB achieve this by targeting IkB proteolysis (IkB phosphorylation can "label" it for ubiquitination and then proteolysis). The nuclear IkBa-NF-κB complex can be exported to the cytoplasm by CRM1, where it dissociates, thereby reactivating NF-κB. Ubiquitinated IkB can also dissociate from the NF-κB complex, restoring NF-κB transcriptional activity. Inhibition of CRM1-induced export via IMB in human neutrophils and macrophage-like cells (U937) not only leads to the accumulation of transcriptionally inactive nuclear IkBa-NF-κB complexes but also prevents initial NF-κB activation, even upon cellular stimulation. In a separate study, treatment with LMB in lung capillary microvascular endothelial cells inhibited IL-1β-induced F-κB DNA binding (the first step in NF-κB transcriptional activation), IL-8 expression, and intercellular adhesion molecule expression. COMMD1 is another nuclear inhibitor of the transcriptional activity of both NF-κB and hypoxia-inducible factor 1 (HIF1). Blocking COMMD1 nuclear export by inhibiting CRM1 leads to increased inhibition of the transcriptional activity of both NF-κB and HIF1.

[0006] CRM1 also mediates retinoid X receptor α (RXRa) transport. RXRa is highly expressed in the liver and plays a central role in regulating bile acid, cholesterol, fatty acid, steroid, and xenobiotic metabolism, as well as homeostasis. During hepatitis, nuclear RXRa levels are significantly reduced, primarily due to inflammation-mediated nuclear export of RXRa via CRM1. In human liver-derived cells, LepB can prevent L-1B-induced increases in cytoplasmic RXRa levels.

[0007] The role of CRM1-mediated nuclear output in NF-κB, HIF-1, and RXRa signaling suggests that blocking nuclear output may have potential benefits for many inflammatory processes across multiple tissues and organs, including the vascular system (vasculitis, arteritis, polymyalgia rheumatica, atherosclerosis), skin diseases, and rheumatic diseases (rheumatoid arthritis and related arthritis, psoriatic arthritis, spondyloid arthropathy, nodular arthropathy, systemic lupus erythematosus, mixed connective tissue disease, inflammatory syndromes, dermatomyositis, inclusion body myositis, undifferentiated connective tissue disease, Sjögren's syndrome, scleroderma, and overlap syndromes, etc.).

[0008] Inhibition of CRM1 can affect gene expression by inhibiting the activation of a series of transcription factors such as 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), sun damage (UV / UV damage), and infections. The most well-studied CRM1 inhibition using LMB showed minimal effect on normal keratinocytes and exhibited anti-inflammatory activity against keratinocytes stimulated by UV, TNFα, or other inflammatory factors. 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 pathogenic human papillomavirus (PV) strains such as IPV16, but does not induce apoptosis in uninfected keratinocytes.

[0010] 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 amyotrophic lateral sclerosis (ALS). For example, (1) forced nuclear arrest of key neuroprotective regulators such as NRF2, docking them in nerve cells, and / or inhibition of NF-κB transcriptional activity by (2) isolating 1XB in the nucleus of glial cells, CRM1 inhibition can slow or prevent nerve cell death found in these disorders. There is also evidence that abnormal glial cell proliferation is associated with abnormal CRM1 levels or CRM1 function.

[0011] The complete maturation of many viruses also requires complete nuclear export, primarily mediated by CRM1. Viruses involving nuclear export and / or CRM1 itself in their life cycle include human immunodeficiency virus (HIV), adenovirus, simian retrovirus type I, Borna disease virus, influenza virus (common strains as well as HINL and avian HN1 strains), hepatitis B virus (BV) and hepatitis C virus (HCV), human papillomavirus (HPV), respiratory syncytial virus (RSY, Dungeney, 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 passes through the nucleolus and shuttles between the nucleus and cytoplasm, facilitates the export of unspliced ​​and single-spliced ​​HIV transcripts containing Rev response element (RRE) RNA through the CRM1 export pathway. Inhibition of Rev-mediated RA transport using CRM1 inhibitors such as LepB or PKF050-638 can block HIV-1 transcription, suppress the production of new HIV-1 viral particles, and thus reduce HIV-1 levels.

[0013] Dengue virus (DENV) is the pathogen of dengue fever (DF), a common arthropod-borne viral disease, and its more severe and potentially fatal form, dengue hemorrhagic fever (DHF). DHF appears to be caused by an overactive inflammatory response to DENV, and NS5 is the largest and most conserved protein in DENV. CRM1 regulates the transport of NS5 from the nucleus to the cytoplasm, mediating most of NS5's functions. Inhibition of CRM1-mediated NS5 export leads to altered viral dynamics and reduced induction of the inflammatory chemokine interleukin-8 (IL-8), providing a novel avenue for the treatment of DENV and other medically important flaviviruses, including those caused by hepatitis C virus.

[0014] Other virus-encoded RNA-binding proteins that use CRM1 to export cells to the nucleus include HSVI type mesenchymal proteins (P13 / 14 or HUA7), human CMV protein pp65, SARS coronavirus ORF3b protein, and RSV matrix (M) protein.

[0015] Interestingly, many of these diseases are associated with specific types of human cancer, including hepatocellular carcinoma (HCC) attributed to chronic HBV or HCV infection, cervical cancer attributed to HPV, and Merkel cell carcinoma associated with MCV. Therefore, CRM1 inhibitors are beneficial in both the viral infection process and the tumorigenic transformation process induced by these viruses.

[0016] CRM1 controls nuclear localization and thus the activity of various DNA metabolic enzymes, including histone deacetylases (HDAC), histone acetyltransferases (HAT), and histone methyltransferases (HMT).

[0017] CRM1 is also associated with other disorders. Leber's disorder, a genetic disorder characterized by the degeneration of retinal ganglion cells and visual impairment, is associated with the ineffective switching of CRM1. There is also evidence that neurodegenerative disorders are associated with abnormal nuclear transport. Summary of the Invention

[0018] The purpose of this invention is to provide a triazole derivative, its preparation method and application, which can be used as a CRM1 inhibitor to prepare therapeutic drugs for diseases related to CRM1 activity.

[0019] To achieve the above-mentioned objectives, the present invention employs the following technical means:

[0020] A triazole derivative or a pharmaceutically acceptable salt thereof, said triazole derivative having the structure shown in Formula I.

[0021]

[0022] in:

[0023] R is selected from hydroxyl, C1-6 alkyl, C1-6 alkoxy, or -C(OR). 1 (=NR) 2 );

[0024] R 1 and R 2 It is independently hydrogen, C1-6 alkyl, halo-C1-6 alkyl, or C1-6 alkoxy.

[0025] Further, the R is selected from hydroxyl, C1-4 alkyl, C1-4 alkoxy, or -C(OR) 1 (=NR) 2 );

[0026] R 1 and R 2 It is independently hydrogen or C1-4 alkyl.

[0027] Furthermore, the triazole derivatives are selected from the following compounds:

[0028]

[0029] The above-mentioned triazole derivatives or their pharmaceutically acceptable salts are synthesized according to the following route:

[0030]

[0031] A pharmaceutical composition for treating diseases, disorders, or symptoms associated with CRM1 activity, comprising the above-described triazole derivatives or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0032] The use of the above-mentioned triazole derivatives or their pharmaceutically acceptable salts in the preparation of medicaments for treating disorders related to CRM1 activity.

[0033] Furthermore, the disorders associated with CRM1 activity include proliferative disorders, cancer, inflammatory disorders, autoimmune disorders, viral infections, ophthalmic disorders, neurodegenerative disorders, abnormal tissue growth disorders, food intake-related disorders, allergies, and respiratory disorders.

[0034] Furthermore, the disorder associated with CRM1 activity is cancer.

[0035] Furthermore, the disorder associated with CRM1 activity is multiple myeloma.

[0036] Beneficial Effects: Cell activity results showed that compounds I and IV of this invention are substantially equivalent to KPT8602. The oral and intravenous half-lives of compound I are both longer than those of KPT-8602, particularly the oral half-life, which is approximately twice as long. Furthermore, the intravenous and oral AUCs of compound I are significantly higher than those of KPT-8602, indicating better pharmacokinetic properties. In addition, compound I exhibits good safety. Attached Figure Description

[0037] Figure 1 These are the results of the BALB / C in vivo toxicity test for compound I. Detailed Implementation

[0038] Definition of a compound

[0039] The compounds of this invention include those generally described above, and are further described by the categories, subcategories and species disclosed herein. Unless otherwise specified, the following definitions shall apply as used herein. For the purposes of this invention, these chemical elements have been identified according to the periodic table, CAS version, and the Handbook of Chemistry and Physics, 75th edition.

[0040] Unless otherwise stated, the nomenclature used in this specification generally follows the nomenclature of Organic Chemistry, sections A, B, C, D, E, F, and H, for naming compounds with exemplary chemical structures.

[0041] The compounds of the present invention may have an asymmetry center, a chiral axis and a chiral plane, and exist as racemic mixtures, racemic mixtures and individual diastereomers or enantiomers, wherein all possible isomers and mixtures thereof (including optical isomers) are included in the present invention.

[0042] The term "halogen" contains fluorine, chlorine, bromine, and iodine atoms. Fluorine and chlorine atoms are particularly preferred.

[0043] The term "alkyl" refers to a straight-chain or branched hydrocarbon group 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. Examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, n-decyl, etc.

[0044] The term "alkoxy" refers to a group formed by the bonding of an alkyl group to an oxygen atom. Examples include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, sec-butoxy, pentoxy, isopentoxy, hexoxy, etc.

[0045] Preferred embodiments of "alkoxy" include: methoxy, ethoxy, n-propoxy, isopropoxy, and tert-butoxy.

[0046] The term "halogenated alkyl" includes groups formed by replacing one or more hydrogen atoms bonded to carbon atoms of the aforementioned "alkyl" group with one or more of the aforementioned "halogens". Examples include: 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-trifluoropropane-2-yl, etc.

[0047] Examples of embodiments of "halogenated alkyl" include: trifluoromethyl and trichloromethyl.

[0048] This invention provides a composition comprising a compound of the invention or a pharmaceutically acceptable biological agent thereof, and a pharmaceutically acceptable agent, adjuvant, or carrier. The amount of the compound in the composition of the invention is such that it effectively and moderately inhibits CRW1 in a biological sample or in vivo. In some embodiments, a composition of the invention is formulated for administration to a patient requiring such a composition. As used herein, the term "patient" refers to an animal. In some embodiments, the animal is a dairy animal. In some embodiments, the patient is a veterinary patient (i.e., a non-human dairy animal patient).

[0049] The term "pharmaceuticalally acceptable carrier, adjuvant, or transporter" refers to a non-toxic carrier, adjuvant, or transporter that does not impair the pharmacological activity of the compound it is formulated with. Pharmaceutically acceptable carriers, adjuvants, or transporters that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as acid salts), glycine, sorbic acid, potassium sorbate, glyceryl-2-sulfate mixtures of saturated vegetable lipids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylic acid, waxes, polyethylene-polyoxypropylene block polymers, ethylene glycol, and lanolin.

[0050] The preparation method of the triazole derivative or its pharmaceutically acceptable salt is carried out according to the following route:

[0051]

[0052] In this reaction formula, the definitions of each group R are as described above; Formula (1) reacts with sodium hydrosulfide to give Formula (2), and Formula (2) undergoes nucleophilic substitution to generate (3). Formula (3) then reacts with bromine water and triethylamine to generate Formula (4), and Formula (4) undergoes a coupling reaction to generate (5). Formula (5) is hydrolyzed with LiOH to form a carboxylic acid (6), and Formula (6) condenses with an amine substrate to give compound I.

[0053] The preparation method of the compound of the present invention is described in detail below:

[0054] The cyano group of compound 1 reacts with sodium hydrosulfide and magnesium chloride to form thioformamide, which then reacts with hydrazine hydrate and formic acid to form triazole 2. 2 is then coupled with isopropyl (Z)-3-iodoacrylate under the catalysis of triethylenediamine to form compound 3. The double bond of compound 3 then undergoes addition with liquid bromine, followed by the removal of one molecule of bromine under the action of triethylamine to obtain the key intermediate 4. Intermediate 4 undergoes Suzuki coupling with various nitrogen-containing aromatic groups with boric acid structures under the catalysis of bis(triphenylphosphine)palladium dichloride to form compound 5, which is linked with different nitrogen-containing aromatic groups. Finally, the ester bond of compound 5 is hydrolyzed in the action of LiOH to form carboxylic acid compound 6. Carboxylic acid compound 6 is further condensed to obtain the target compound I.

[0055] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0056] Example 1

[0057] I. Synthesis of Compounds

[0058] The compounds of the present invention can be prepared according to the following process:

[0059] I-route design and synthesis of compounds

[0060]

[0061] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)isopropyl acrylate (3)

[0062] In a 250 mL round-bottom flask, 10 g (41.8 mmol) of 3,5-bis(trifluoromethyl)benzonitrile was dissolved in 50 mL of DMF solution. NaSH (7.8 g, 83.7 mmol) and MgCl2 (8.5 g, 41.8 mmol) were then added sequentially. The reaction was stirred at room temperature for 3 h. After the reaction was complete as monitored by TLC, the reaction mixture was poured into an ice-water mixture (500 mL). Extraction was performed with ethyl acetate (100 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (100 mL × 1). The mixture was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain crude 3,5-bis(trifluoromethyl)benzylthionamide (10.9 g, yield 95.1%, purity 84%), a yellow oily liquid, MS (ESI) m / z 274.35 [M+H]+. This was used directly in the next step.

[0063] In a 250 mL round-bottom flask, 10.9 g (39.8 mmol) of 3,5-bis(trifluoromethyl)benzylthionamide was added to a 30 mL DMF solution. 80% hydrazine hydrate (5.1 mL, 83.6 mmol) was added dropwise at room temperature. The mixture was stirred for 1 h, followed by the addition of 30 mL formic acid. The reaction was then continued at 90 °C with stirring for 3 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature and poured into 600 mL of purified water. The mixture was extracted with ethyl acetate (100 mL × 3), and the organic phases were combined and washed with 300 mL × 3 saturated sodium bicarbonate solution and 100 mL × 1 saturated sodium chloride solution. The mixture was dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation to obtain the crude compound. The mixture was 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, yield 76.0%, purity 90%), a white solid. 1 H NMR(400MHz,CDCl3)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] + .

[0064] In a 250 mL round-bottom flask, 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole 2 (8.5 g, 30.2 mmol) was added and dissolved in DMF (40 mL). DABCO (8.5 g, 75.5 mmol) was then added. After stirring the mixture at room temperature for 30 min, ethyl (Z)-3-iodoacrylate (7.5 g, 33.2 mmol) was added dropwise. The reaction was then stirred at room temperature for 3 h. After the reaction was complete as monitored by TLC, the reaction mixture was poured into an ice-water mixture (400 mL). Extraction was performed with ethyl acetate (80 mL × 3), the organic phases were combined and washed with saturated sodium chloride solution (100 mL × 1), dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under reduced pressure to obtain the crude 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%), as a white solid. 1H NMR (400MHz, CDCl3) 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] + .

[0065] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-bromoacrylate isopropyl ester (4)

[0066] In a 250 mL pear-shaped flask, 7.9 g (20.6 mmol) of the previously obtained (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)ethyl acrylate was dissolved in 40 mL of dichloromethane. Liquid bromine (6.6 g, 41.2 mmol) was slowly added dropwise over 30 min, and the reaction was continued at room temperature with stirring for 8 h. After the reaction was complete as monitored by TLC, the reaction mixture was poured into an ice-water mixture (100 mL). The mixture was extracted with dichloromethane (50 mL × 3), and the organic phases were combined and washed with saturated sodium bisulfite solution (100 mL × 3) and saturated sodium chloride solution (50 mL × 1). The mixture was dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation. The solution was purified by column chromatography (PE / EtOAc = 50:1) to give isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2,3-dibromopropionate (10.3 g, yield 92.7%, purity 95%), as a white solid. MS (ESI) m / z 551.97 [M+H] + .

[0067] The intermediate 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2,3-dibromopropionate isopropyl ester (10.3 g, 19.1 mmol) obtained in the previous step was weighed into a 250 mL round-bottom flask, dissolved in tetrahydrofuran (40 mL), and stirred in an ice bath for 10 min. Then, triethylamine (3.9 g, 38.2 mmol) was added dropwise to the reaction solution, and stirring was continued for 30 min. The reaction was then moved to room temperature and stirred for 6 h. After the reaction was monitored by TLC until complete, an ice-water mixture (100 mL) was added to the reaction solution. The mixture was extracted with ethyl acetate (50 mL × 3), the organic phases were combined and washed with saturated sodium chloride solution (50 mL × 1), dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation. Ethyl (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-bromoacrylate 4 (7.7 g, yield 88.2%, purity 96%) was obtained by column chromatography (PE / EtOAc = 50:1) as 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] + .

[0068] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)isopropyl acrylate (5)

[0069] Take a 25 mL three-necked flask and weigh out important intermediate 4 (200 mg, 0.44 mmol) and 5-pyrimidineboronic acid (81.9 mg, 0.66 mmol), respectively, and dissolve them in a mixed solution of dioxane (5 mL) and water (1 mL). Then weigh out sodium acetate (86.4 mg, 0.88 mmol) and add it to the reaction solution. After purging the reaction solution with nitrogen three times, stir at room temperature. Then add Pd(PPh3)Cl2 (30.9 mg, 0.04 mmol) to the reaction solution, and purge the reaction solution with nitrogen three times again. Stir overnight at 80 °C. After the reaction is complete as monitored by TLC, cool the reaction solution to room temperature and add purified water (50 mL). Extract with ethyl acetate (15 mL × 3), combine the organic phases and wash with saturated sodium chloride solution (20 mL × 1), dry with anhydrous Na2SO4, filter and remove the solvent under vacuum to obtain the crude compound. The (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)ethyl acrylate 5 (125.3 mg, yield 62.3%, purity 93%) was obtained by column chromatography (PE / EtOAc = 8:1) as a white solid. 1 H NMR(400MHz,CDCl3)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] + .

[0070] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-2-(pyrimidin-5-yl)propenylic acid (6)

[0071] In a 25 mL round-bottom flask, 5 (125.3 mg, 0.27 mmol) was dissolved in tetrahydrofuran (3 mL). After stirring for 10 min in an ice bath, a solution of LiOH·H₂O (45.3 mg, 1.08 mmol) in water (1 mL) was added dropwise to the reaction mixture. After stirring for another 30 min, the reaction mixture was moved to room temperature and stirred overnight. After the reaction was monitored by TLC until complete, an ice-water mixture (10 mL) was poured into the reaction mixture, and the pH of the solution was adjusted to 2-3 with 4N hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL × 3), the organic phases were combined and washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation. The 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, yield 85.8%, purity 89%) was obtained in relatively pure form as a white solid. It was used directly in the next step. MS (ESI) m / z 427.92 [MH] - .

[0072] Synthesis of (E)-3-(3-(3-,3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-methoxy-2-(pyrimidin-5-yl)acrylamide (I)

[0073] In a 25 mL round-bottom flask, 6 (125.3 mg, 0.27 mmol) was dissolved in 3 mL of dichloroethane. After stirring for 10 min in an ice bath, EDCI (20.3 mg, 1.08 mmol) and HOBT (70.5 mg, 1.5 mmol) were added to the reaction mixture. After stirring for another 30 min, the reaction mixture was moved to room temperature and DIPEA (54.5 mg, 3.0 mmol) and methoxyhydroxylamine hydrochloride (23.4 mg, 1.0 mmol) were added dropwise and stirred overnight. After the reaction was complete as monitored by TLC, an ice-water mixture (10 mL) was poured into the reaction mixture. The mixture was extracted with ethyl acetate (5 mL × 3), the organic phases were combined and washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation. The compound (E)-3-(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%) was obtained as a white solid. 1HNMR(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] + .

[0074] The specific compounds synthesized and their names are shown in the table below.

[0075]

[0076]

[0077] II. Cell line inhibitory activity

[0078] 1. Cell cryopreservation

[0079] (1) After harvesting the cells, centrifuge at 1000 rpm for 5 min at room temperature and rinse with PBS.

[0080] (2) Resuspend in 1640 medium containing 7% DMSO and 10% fetal bovine serum.

[0081] (3) Dispense into cryovials, place in a cell cryopreservation box and incubate overnight at -80°C, then store in liquid nitrogen.

[0082] 2. Cell resuscitation and culture:

[0083] (1) Remove the cryovial from the liquid nitrogen tank and gently shake it in 37°C warm water to thaw the cell fluid.

[0084] (2) Transfer to a sterile centrifuge tube, add 1640 culture medium containing 10% fetal bovine serum, and gently pipette to form a suspension.

[0085] (3) Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 10% 1640 culture medium containing fetal bovine serum, and gently pipette to form a suspension.

[0086] (4) Transfer to a culture flask and incubate at 37°C in a saturated humidity incubator with 5% CO2 for 1-2 days, then change the medium.

[0087] 3. Cell passage:

[0088] Discard the original culture medium, rinse once with sterile PBS, add 1 mL of 0.25% trypsin and incubate for about 1 minute. Observe under a microscope. After most cells begin to round out, carefully aspirate the trypsin and add fresh culture medium to stop digestion. Pipe the cells into a uniform cell suspension and transfer them to a cell incubator for continued culture.

[0089] 4. Cytotoxicity test

[0090] (1) 1000 RPMI8226 cells in logarithmic growth phase were added to a 384-well plate at a volume of 18 μL / well and incubated at 37°C and 5% CO2 for 24 h.

[0091] (2) The 10 mM test compound was diluted 10-fold with DMSO, and then diluted 100-fold with serum-free 1640 medium to obtain a working concentration of 10 μM containing 1% DMSO. This was followed by a 2-fold serial dilution with 1% DMSO in serum-free 1640 medium to obtain 10 concentrations. The tenth concentration point served as the solvent control (no drug). 2 μL of the diluted compound was added to each well of the plated cell culture plate to obtain a final concentration of 1000 nM. A 2-fold serial dilution was performed for 10 concentration gradients, with 4 replicates for each concentration. 1% DMSO was used as the solvent control, and KPT-8602 was used as the positive control.

[0092] (3) After incubating at 37℃ for 72h, add 10μL of Cell-Titer detection reagent to each well and then incubate in a cell culture incubator for 10min.

[0093] (4) After vortexing and mixing, run the Cell-Titer program on the microplate reader for detection, and calculate the inhibition percentage and IC5 using GraphPad Prism5. 50 Value (μM).

[0094] The cellular results for the compounds are shown in the table below:

[0095]

[0096]

[0097] Cell activity results showed that compounds I and IV were essentially equivalent to KPT8602.

[0098] III. Pharmacokinetic Results of the Drug in SD Rats

[0099]

[0100] Conclusion: The oral and intravenous half-lives of compound I are longer than those of KPT-8602, especially the oral half-life, which is about twice as long. Moreover, the AUC of compound I in both the intravenous and oral doses is much higher than that of KPT-8602, indicating better pharmacokinetic properties.

[0101] IV. In vivo toxicity test of candidate compound BALB / C

[0102] Twenty-five BALB / c mice were randomly divided into five groups: a solvent control group (Control), a positive control group (KPT-8602, 30 mg / kg, once daily), a group (KPT-8602, 60 mg / kg, once daily), a group (Compound I, 30 mg / kg, once daily), and a group (Compound I, 60 mg / kg, once daily), with five mice in each group. Each group was administered the corresponding concentration of the test substance by gavage at a dose of 3 mL / kg. KPT-8602 and Compound I were administered daily for a total of 21 days.

[0103] Methods for preparing compound solutions:

[0104] 10% sulfobutyl-β-cyclodextrin formulation:

[0105] Weigh 5.0 g of sulfobutyl-β-cyclodextrin powder into a beaker, pipette 50 mL of citrate buffer into the beaker, dissolve and transfer to a container.

[0106] Preparation of compound I:

[0107] Weigh 24 mg of compound I, add 1.6 mL of 20% polyethylene glycol aqueous solution, dissolve thoroughly, then add 6.4 mL of 10% sulfonyl-β-cyclodextrin aqueous solution to obtain a 3 mg / mL solution of compound I. Preparation of KPT-8602:

[0108] Weigh 24 mg of compound KPT-8602, add 1.6 mL of 20% polyethylene glycol aqueous solution, dissolve, and then add 6.4 mL of 10% sulfobutyl-β-cyclodextrin aqueous solution to obtain a 3 mg / mL KPT-8602 test solution.

[0109] The results of changes in mouse body weight after continuous administration are as follows: Figure 1 As shown.

[0110] in conclusion:

[0111] As shown in the figure, two mice in the 60 mg / kg KPT-8602 group died on day 7, and all mice died on day 10. The 30 mg / kg KPT-8602 group showed a significant decrease in body weight. However, the 30 mg / kg and 60 mg / kg compound I groups maintained body weight comparable to the control group after 21 days, demonstrating good safety.

Claims

1. A triazole derivative or a pharmaceutically acceptable salt thereof, characterized in that: The triazolic derivative is selected from the following compounds:

2. A pharmaceutical composition for treating a disease, disorder, or symptom associated with CRM1 activity, characterized in that: A pharmaceutical composition comprising a triazolic derivative of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of a triazolic derivative of claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disorder associated with CRMl activity.

4. Use according to claim 3, characterized in that: The disorder associated with CRMl activity is a proliferative disorder, a cancer, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, a disorder of abnormal tissue growth, a disorder related to food intake, an allergy, and a respiratory disorder.

5. Use according to claim 4, characterized in that: The disorder associated with CRMl activity is a cancer.

6. Use according to claim 5, characterized in that: The disorder associated with CRMl activity is multiple myeloma.

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