Alkyne-substituted halogenated heterocyclic amide derivatives, their preparation methods and applications

Abstract

This invention relates to the field of medicinal chemistry, specifically to an alkynyl-substituted halogenated heterocyclic amide derivative, its preparation method, and its applications. The compound provided by this invention exhibits excellent PRMT5 inhibitory activity and, relative to the MTAP wild-type (WT) HCT-116 cell line, demonstrates superior selective inhibition against the human MTAP-deficient HCT-116 cell line. It can be used to develop small molecule drugs targeting PRMT5·MTA, and preferentially acts on MTAP-deficient tumor cells.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to an alkynyl-substituted halogenated heterocyclic amide derivative, its preparation method, and its application. Background Technology

[0002] Epigenetics-regulated gene expression plays a vital biological role in protein maturation and cell differentiation, and is crucial in many human diseases. Arginine guanidinomethylation catalyzed by protein arginine methyltransferase (PRMT) is a common post-translational modification in eukaryotic cells, affecting various biological processes such as cell signaling, gene transcription, mRNA translation, DNA recombination, and repair (Cell Mol. Life Sci. 2015. 72(11): 2041-2059).

[0003] PRMT5 is a member of the PRMT family, and its mediated methylation plays an important role in maintaining normal intracellular homeostasis. However, a growing body of research has shown that aberrant expression of PRMT5 is associated with the development of various tumors. It is overexpressed in a variety of tumors and the mechanisms of its development differ in different tumors (Cell Mol. Life Sci. 2015. 72(11):2041-2059).

[0004] Homozygous deletion of tumor suppressor genes is a key driver of tumorigenesis. The deletion of the tumor suppressor gene CDKN2A, located on human chromosome 9p21, is one of the most frequently mutated genes in tumors, occurring in 15% of cases. Due to its close resemblance to CDKN2A, the methionine phosphorylase gene (MTAP) is frequently deleted in tumors, playing a crucial role in the methionine and adenine rescue pathway (Cell Reports, 2016, 15: 574–587). MTAP deletion leads to the accumulation of its substrate, methylthioadenosine (MTA). Because MTA is structurally similar to S-adenosylmethionine (SAM), it selectively competes with SAM for binding to PRMT5, inhibiting some PRMT5 activity and sensitizing further PRMT5 inhibition, i.e., synthetic lethality (Science, 2016, VOL 351 ISSUE 6278: 1214-1217).

[0005] However, PRMT5 is a known essential gene. Knockout or siRNA silencing of PRMT5 in normal tissues leads to abnormal physiological functions, such as reduced blood cell counts, infertility, skeletal muscle loss, and myocardial hypertrophy (Journal of Clinical Investigation, 2015, 125(9):3532-44). Currently, none of the PRMT5 inhibitors in the clinical stage can induce synthetic lethality due to MTAP deficiency. Therefore, new strategies are needed to exploit the metabolic vulnerability caused by MTAP deficiency.

[0006] Developing small molecule inhibitors targeting PRMT5·MTA can preferentially act on MTAP-deficient tumor cells. Since normal cells do not lack MTAP and have low MTA concentrations, they do not have a significant inhibitory effect on normal cells, thereby increasing the therapeutic index (AACR Annual Meeting, 2021, Abstract LB003), providing a new strategy for tumor treatment. Summary of the Invention

[0007] The problem the invention aims to solve:

[0008] Although several patent applications for PRMT5 inhibitors have been published, given the huge market demand from MTAP- / - cancer patients and the less-than-ideal clinical efficacy of existing PRMT5 inhibitors, new compounds still need further development.

[0009] Solution for solving the problem:

[0010] To achieve the above-mentioned objectives, the first aspect of the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0011] .

[0012] Pharmaceutical composition:

[0013] The present invention also provides a pharmaceutical composition for treating and / or preventing diseases associated with abnormal PRMT5 expression, the pharmaceutical composition comprising a therapeutic and / or preventive effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof, and optionally pharmaceutical excipients.

[0014] In some specific implementations, the diseases associated with abnormal PRMT5 expression refer to tumors or cancers with MTAP deficiency.

[0015] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of the present invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc.

[0016] Medical uses:

[0017] The present invention also provides compounds as described above or pharmaceutically acceptable salts thereof, or pharmaceutical compositions as described above, in the preparation of medicaments for treating and / or preventing diseases associated with abnormal PRMT5 expression.

[0018] In some implementations, the diseases associated with abnormal PRMT5 expression described herein refer to tumors or cancers with MTAP deficiency.

[0019] In this invention, "treatment" generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) preventing the disease or symptoms occurring in a patient who has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

[0020] Invention effects:

[0021] The compounds of the present invention have excellent PRMT5 inhibitory activity, and compared with the MTAP wild-type (WT) HCT-116 cell line, the compounds of the present invention have excellent selective inhibitory activity against the human MTAP-deficient HCT-116 cell line. They can be used to develop small molecule drugs targeting PRMT5·MTA and preferentially act on MTAP-deficient tumor cells. Detailed Implementation

[0022] It should be understood that the terminology used herein is intended to describe specific embodiments and is not intended to be limiting. Furthermore, although any methods, apparatus, and materials similar to or equivalent to those described herein may be used to practice or test the invention, preferred methods, apparatus, and materials are described here.

[0023] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker ASCENA-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). Chemical shifts were expressed as 10⁻⁶. -6 (ppm) is given as the unit.

[0024] Reaction monitoring and MS determination were performed using a Thermofisher ESQ (ESI) mass spectrometer.

[0025] The HPLC determination was performed using a Thermo Fisher Scientific U3000 DAD high-performance liquid chromatograph (GL Sciences ODS-HL HP 3μm 3.0*100mm column).

[0026] Thin-layer chromatography (TLC) used Qingdao Ocean GF254 silica gel plates. The silica gel plates used in TLC had a diameter of 0.15–0.2 mm, while the purified products were separated using high-performance thin-layer chromatography (HPLC) preparative plates with a diameter of 0.9–1.0 mm. Column chromatography used Qingdao Ocean 200–300 mesh silica gel as the carrier. The developing solvent systems were A: dichloromethane and methanol; and B: petroleum ether and ethyl acetate. The volume ratio of the solvents was adjusted according to the polarity of the compounds. For medium-pressure preparative liquid chromatography (PLC) purification, a Biotage Isera One preparative PL was used.

[0027] In the following embodiments, unless otherwise specified, all reaction materials can be purchased from suppliers in the SciFinder database. For example, some reagents in the embodiments of this invention were purchased from manufacturers such as Saen Chemical Technology (Shanghai) Co., Ltd., Shanghai Shaoyuan Reagent Co., Ltd., Nanjing Yaoshi Technology Co., Ltd., Jiangsu Aikon Biomedical R&D Co., Ltd., and Shanghai Bid Pharmaceutical Technology Co., Ltd. Furthermore, unless otherwise specified, all raw materials used in the embodiments of this invention are of analytical grade. Unless otherwise specified, all ratios of two liquid substances mentioned herein are volume ratios; all percentages of substances mentioned are mass percentages.

[0028] Example 1: 4-Amino-N-(3,3-difluoro-5-((1-methyl-1H-pyrazol-4-yl)ethynyl)-2,3-dihydro-1H-inden-1-yl)-7-fluoro-N,1-dimethyl-1H-pyrazolo[4,3-c]quinoline-8-carboxamide (Formula I)

[0029]

[0030] Preparation of compound 1a

[0031] Azobisisobutyronitrile (164 mg, 1 mmol) and N-bromosuccinimide (1.78 g, 10 mmol) were added to a solution of 6-bromo-2,3-dihydro-1H-inden-1-one (2.11 g, 10 mmol) in acetonitrile (20 mL). The mixture was stirred at 70 °C for 2 hours. The reaction solution was cooled to room temperature and directly concentrated to obtain a crude product, which was then subjected to column chromatography (petroleum ether / ethyl acetate = 50:1) to obtain 1a (2.05 g).

[0032] Preparation of compound 1b

[0033] To a solution of 1a (1.36 g, 4.69 mmol) in dichloromethane (20 mL), methylamine hydrochloride (950 mg, 14.07 mmol) and triethylamine (950 mg, 9.38 mmol) were added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was directly concentrated to obtain 1b (1.12 g). ESI-MS (m / z): 242.12 [M+H] +

[0034] Preparation of compound 1c

[0035] Triethylamine (950 mg, 9.38 mmol) was added to a solution of 1b (1.12 g, 4.69 mmol) in dichloromethane (20 mL), followed by the addition of a solution of trifluoroacetic anhydride (1.48 g, 7.04 mmol) in dichloromethane (10 mL) at 0 °C, and the mixture was stirred at 0 °C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was then subjected to column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain 1c (800 mg).

[0036] Preparation of compound 1d

[0037] At -15 °C, BF3·Et2O (899 mg, 6.34 mmol) was added to a solution of compound 1c (710 mg, 2.11 mmol) and 1,2-ethylenedithiol (398 mg, 4.22 mmol) in dichloromethane (20 mL). The mixture was stirred at -15 °C for 2 hours, then allowed to warm to room temperature and stirred for another 2 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with 90 mL (30 mL * 3). The organic phase was concentrated to obtain the crude product, which was then subjected to column chromatography (petroleum ether / ethyl acetate = 50:1) to obtain 1d (780 mg).

[0038] Preparation of compound 1e

[0039] Dibromohydantoin (624 mg, 2.18 mmol) was added to a solution of HF / pyridine (824 mg, 5.18 mmol) in dichloromethane (10 mL) under a nitrogen atmosphere at -70 °C. The slurry mixture was stirred at -70 °C for 30 min. Then, a solution of 1d (300 mg, 0.73 mmol) in dichloromethane (10 mL) was added to the mixture. The mixture was stirred at -70 °C for another 1 h, and then at 20 °C for another 3 h. The reaction mixture was quenched with saturated sodium bicarbonate solution at 0 °C and extracted with 90 mL (30 mL * 3). The organic phase was concentrated to give crude product, which was then subjected to column chromatography (petroleum ether / ethyl acetate = 50:1) to give 1e (160 mg).

[0040] Preparation of compound 1f

[0041] Potassium carbonate (185 mg, 1.34 mmol) was added to a solution of 1e (160 mg, 0.45 mmol) in methanol (5 mL), and the mixture was stirred at 45 °C for 2 hours. The mixture was concentrated to obtain a crude product, which was then subjected to column chromatography (dichloromethane / methanol = 30:1) to give 1f (95 mg). ESI-MS (m / z): 261.88 [M+H] + .

[0042] Preparation of 1g of compound

[0043] Compound 1f (60 mg, 0.23 mmol), triethylamine (57.9 mg, 0.57 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (130.6 mg, 0.34 mmol) were added to a solution of compound 1h (59.6 mg, 0.23 mmol, prepared according to WO2022169948A1) in N,N-dimethylformamide (5 mL). The reaction was carried out at 25 °C for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature and poured into ice water, precipitating a solid. The solid was filtered to obtain the crude product, which was then subjected to normal phase column chromatography (dichloromethane / methanol = 30:1) to give compound 1 g (80 mg). ESI-MS (m / z): 505.9 [M+H] +

[0044] Preparation of compound (Formula I)

[0045] To a 1 g (50 mg, 0.10 mmol) solution of N-methylpyrrolidone (2 mL) of the compound, cuprous iodide (3.8 mg, 0.02 mmol), tetrakis(triphenylphosphine)palladium (11.5 mg, 0.01 mmol), triethylamine (40.1 mg, 0.40 mmol) and 4-ethynyl-1-methylpyrazole (21.0 mg, 0.20 mmol) were added, and the mixture was microwaved at 80 °C for 4 hours under nitrogen protection. The reaction solution was filtered through diatomaceous earth, water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 * 10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated. Normal-phase column chromatography (methanol / dichloromethane = 10:1) was performed to obtain the crude compound, followed by reverse-phase column chromatography (acetonitrile / 0.05% ammonium bicarbonate solution = 0:100% – 45%:55%) to give Formula I (28 mg). ESI-MS (m / z): 529.96 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.41 –8.25 (m, 2H), 8.13 – 8.07 (m, 1H), 7.82 – 7.68 (m, 3H), 7.60 – 7.46 (m, 1H),7.41 – 7.33 (m, 1H), 7.32 – 7.22 (m, 2H), 6.41 – 5.42 (m, 1H), 4.42 (s, 3H), 3.88 (s, 3H), 3.20 – 2.92 (m, 2H), 2.71 and 2.63 (s, 3H).

[0046] Experimental Example 1: Inhibitory activity of the compound of the present invention against the proliferation of human colon cancer cells.

[0047] 1. Build MTAP - / - HCT-116 cell line

[0048] HCT-116 colon cancer cell line (purchased from Nanjing Kebai) was co-transfected with gene editing tools CRISPR / Cas9 and sgRNA. MTAP biallelic inactivated MTAP was obtained by Western blotting, Sanger sequencing, and other detection methods. - / - HCT-116 cell line. MTAP - / - The HCT-116 cell line was used for selective screening and evaluation of compounds based on the MTAP deficiency and PRMT5 synergistic mechanism, including symmetric dimethylarginine (SDMA) level assay and cell proliferation assay.

[0049] 2. Cell proliferation

[0050] MTAP was cultured in complete culture medium McCoy's 5A (Gibco, 16600082) / 10% FBS (Gibco, 10099141C) / 1% p / s (Gibco, 15140122). - / - HCT-116 and wild-type HCT-116 cell lines were used to evaluate the effects of compounds on MTAP. - / - The selective inhibition of HCT-116 cell proliferation was enhanced. Day 0: 100 MTAP molecules were added to each well of a 96-well cell culture plate (Corning, 3599). - / - HCT-116 cells or wild-type HCT-116 cells were cultured in a 37 ℃, 5% CO2 incubator. The compound was serially diluted 3x with DMSO (Sigma, D5879) (starting concentration 20 μM, 3X dilution, for a total of 8 concentration points). On Day 1, the compound was diluted to multiple concentration points and used to treat cells, and the cells were cultured for another 10 days in a 37 ℃, 5% CO2 incubator. On Day 11, 20 μL of MTS (CellTiter 96® A) was added to each well. Queous The OneSolution Cell Proliferation Assay (Promega, G3581) was used. After incubation at 37 ℃ and 5% CO2 for 2 hours, data were read using Tecan Spark (OD=490 nM). Data analysis was then performed using GraphPad Prism8 software, employing the equation "log(inhibitor) vs. normalized response -- variable slope" (formula Y=Bottom + (Top-Bottom) / (1+10^(LogIC)). 50 Data analysis was performed using -X)*HillSlope))) to obtain the IC50 of the compound. 50 Value. Where Y is the inhibition rate, X is the logarithm of the compound concentration, Top refers to the maximum response (inhibition rate at maximum compound concentration), Bottom refers to the baseline response (inhibition rate at 0 compound concentration), and Hill Slope refers to the IC50 value. 50 The slope of the curve, IC 50 The concentration of the compound at half-maximal inhibition is given. The experimental results are shown in Table 1.

[0051] Table 1 IC 50 (μM)

[0052]

[0053] The results showed that the specific compounds provided by the present invention have excellent inhibitory activity against the human MTAP-deficient HCT-116 cell line. Compared with the MTAP wild-type (WT) HCT-116 cell line, the compounds of the present invention showed excellent selective inhibition against the human MTAP-deficient HCT-116 cell line, with a selective inhibition of more than 1000 times.

[0054] Industrial applicability

[0055] The compounds of the present invention have excellent PRMT5 inhibitory activity, and compared with the MTAP wild-type (WT) HCT-116 cell line, the compounds of the present invention have excellent selective inhibitory activity against the human MTAP-deficient HCT-116 cell line. They can be used to develop small molecule drugs targeting PRMT5·MTA and preferentially act on MTAP-deficient tumor cells.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition for treating and / or preventing diseases associated with abnormal PRMT5 expression, said pharmaceutical composition comprising a therapeutically and / or preventively effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical excipient; wherein the disease associated with abnormal PRMT5 expression is a tumor with MTAP deficiency, said tumor being colon cancer.

3. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of diseases associated with abnormal PRMT5 expression; wherein the disease associated with abnormal PRMT5 expression is a tumor with MTAP deficiency, wherein the tumor is colon cancer.

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