Crystal form A of compound A as well as preparation method and application of crystal form A

Abstract

The invention belongs to the technical field of chemical drugs, and relates to a crystal form A of a compound A and a preparation method thereof, and the crystal form A prepared by the invention has good stability and good fluidity. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical drugs, and the present invention relates to a crystal form A of compound A and a preparation method thereof. Background Art

[0002] Myeloperoxidase (MPO) is a highly sensitive and specific indicator of vascular inflammatory mediators and has become an important indicator for predicting the risk of cardiovascular and cerebrovascular events. MPO can kill microorganisms in phagocytes by catalyzing the oxidation of chloride ions to produce hypochlorous acid, destroy a variety of target substances, and play a role in the body's production and regulation of inflammatory responses. More importantly, its oxidative modification of low-density lipoprotein (LDL) can cause atherosclerosis, so MPO is believed to be related to the occurrence of cardiovascular diseases.

[0003] At present, MPO is considered to be the most promising cardiovascular marker. Increased MPO levels in the body indicate the risk of arteriosclerosis and coronary heart disease. It is an early warning of myocardial infarction and is more sensitive than other indicators such as troponin T, CK-MB and CRP, allowing for earlier diagnosis and risk assessment. MPO levels can increase significantly within 2 hours of chest pain onset, so for patients with chest pain, MPO will have more important clinical significance in diagnosing acute coronary syndrome (ACS).

[0004] PCT / CN2023 / 136923 discloses a pyrrolo[3,2-d]pyrimidin-4-one derivative, a preparation method and medical use thereof, and specifically discloses (R)-1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one hydrochloride, the structural formula of which is:

[0005] The crystal form of the active pharmaceutical ingredient often affects the chemical stability of the drug. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, and sometimes other forms of crystals may be produced. Generally speaking, amorphous drug products do not have a regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, poor fluidity, etc. The polymorphic form of the drug has different requirements for product storage, production and scale-up. Therefore, it is necessary to conduct in-depth research on the crystal form of the aforementioned compound and improve the various properties of the aforementioned compound. Summary of the invention

[0006] The object of the present invention is to provide a crystalline form A of compound A and a preparation method and application thereof.

[0007] Specifically, the present invention provides a compound A crystal form A, characterized in that: the crystal form A of the compound has characteristic peaks at 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14° and 26.37° in an X-ray diffraction pattern expressed as a 2θ angle, with an error of ±0.2°; the structural formula of the compound A is shown as follows:

[0008]

[0009] Preferably, as a preferred embodiment of the present invention, the crystalline form A of the compound has characteristic peaks at 14.86°, 15.94°, 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14°, 26.37°, 27.14° and 28.84° in the X-ray diffraction pattern in terms of 2θ angle, with an error of ±0.2°;

[0010] More preferably, as a preferred embodiment of the present invention, the crystalline form A of the compound has characteristic peaks at 13.96°, 14.86°, 15.94°, 17.41°, 18.43°, 19.12°, 21.01°, 21.20°, 22.05°, 24.21°, 25.14°, 25.57°, 26.37°, 27.14°, 28.84°, 29.72°, 32.11°, 33.73°, 34.52° and 35.82° in the X-ray diffraction pattern with a 2θ angle, and the error is ±0.2°;

[0011] Further preferably, as a preferred embodiment of the present invention, the X-ray diffraction pattern of the crystalline form A of the compound A is as shown in the attached Figure 2 or attached Figure 4 As shown;

[0012] Further preferably, as a preferred embodiment of the present invention, the crystal form A is a triclinic P1 crystal space group at a temperature of 293 (2) K, and the unit cell dimensions are: a = 7.40910 (10), b = 12.7287 (2), c = 17.0243 (3), α = 90°, β = 90°, γ = 90°.

[0013] It should be noted that different samples of a specific crystal form have the same main XRPD peak, but the minor peaks in the powder diagram may vary. In addition, when a person skilled in the art uses the same instrument and detection method to detect isomorphous samples obtained by the corresponding method, the error of each 2θ angle is usually within ±0.2° (the meaning of the error of each 2θ angle being usually within ±0.2° refers to most of the characteristic peaks, such as more than 80% of the characteristic peaks have errors within this range, and occasionally there are a few individual characteristic peaks whose errors exceed this range, which should be considered as XRPD spectra belonging to the same crystal form); moreover, the characteristic peaks of each shift are medium intensity absorption peaks, while other weak absorption peaks may change significantly due to experimental operation errors. Therefore, the X-ray diffraction absorption peak is not limited to the above-mentioned specific values, and the possible error range should also be considered.

[0014] X-ray powder diffraction conditions:

[0015] The X-ray powder diffraction (XRPD) spectrum shown in the present invention is obtained by using an Empyrean X-ray powder diffractometer. The detection conditions are: Cu-Kα radiation, wavelength The divergence slit was 1 / 4°, the X-ray tube voltage was 45 kV, the X-ray tube current was 40 mA, the scanning range was 3-40° (2θ), the step length was 0.026°, and the dwell time per step was 30 s.

[0016] The DSC spectrum of the present invention is obtained by using a German NETZSCH DSC 200F3 differential scanning calorimeter, and the detection conditions are: aluminum plate, pressure cover (sealed), heating rate of 10°C / min, and heating from room temperature to a specific temperature.

[0017] Further preferably, as a preferred embodiment of the present invention, the crystalline form A of the compound A has a differential scanning calorimetry curve having a starting value of an endothermic peak at 296.6±5°C and a peak value of an endothermic peak at 304.0±5°C.

[0018] More preferably, as a preferred embodiment of the present invention, the crystal form A of the compound A has a DSC spectrum as shown in the attached Figure 3 shown.

[0019] Another object of the present invention is to provide a method for preparing the crystalline form A of the above-mentioned compound A, which has a simple process and can be achieved at room temperature;

[0020] Among them, compound A can be prepared according to the method disclosed in PCT / CN2023 / 136923, and the specific synthesis route and main reaction conditions are as follows:

[0021]

[0022] The preparation method of the crystalline form A of compound A comprises the following steps:

[0023] a) Solvent method: take the refined product of compound A into a reaction bottle, add a small amount of organic solvent, heat to a certain temperature, suspend and stir, filter, wash the filter cake with an organic solvent, and vacuum dry to obtain the crystalline form A of compound A; the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, ethyl acetate, isopropyl acetate, methyl acetate, ethyl formate, isobutyl acetate, or a mixture of more than one thereof; or

[0024] b) Volatilization method: take the refined product of compound A into a reaction bottle, add a small amount of organic solvent, heat to saturation, filter, and evaporate the filtrate at room temperature to obtain the crystalline form A of compound A. The organic solvent is selected from: methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, or a mixture of more than one; or

[0025] c) Diffusion method: Take the refined compound A into a reaction bottle, add DMSO to dissolve it, control the concentration range of the DMSO solution to 50-500 mg / ml, and then diffuse it in an organic solvent at room temperature; the selected organic solvent is selected from: acetone, isopropanol, ethyl acetate, isopropyl acetate, ethanol, methanol, acetonitrile, THF, isopropyl ether, or a mixture of more than one.

[0026] Furthermore, as a preferred technical solution of the present invention, the heating temperature in step a) is 40-60°C, and the heating time is 1-2 days; the heating temperature in step b) is 40-60°C, and the heating time is 1-2 hours.

[0027] Another object of the present invention is to provide a pharmaceutical composition, characterized in that it comprises the above-mentioned crystal form A of compound A and one or more pharmaceutically acceptable carriers.

[0028] Furthermore, the present invention also provides a use of the crystalline form A of compound A described in the present invention or the pharmaceutical composition described in the present invention in the preparation of a medicament for treating a myeloperoxidase-related disease; preferably, the myeloperoxidase-related disease is selected from cardiovascular-related diseases.

[0029] The pharmaceutically acceptable carrier includes various pharmaceutical excipients, packaging materials, delivery vehicles, etc., which are selected according to the needs of the preparation. For example, excipients include fillers, disintegrants, binders, lubricants, etc., and can be suitable for oral administration, inhalation, parenteral administration or topical use; dosage forms include but are not limited to injections, solution preparations, tablets, capsules, granules, etc.

[0030] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical drugs. For additional information about the carrier, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0031] The term "excipient" generally refers to a carrier, diluent and / or vehicle required to formulate an effective pharmaceutical composition.

[0032] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. For oral dosage forms of the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate effective amount in each case can be determined by a person skilled in the art based on routine experiments.

[0033] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0034] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0035] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , is a schematic diagram of the single crystal structure of compound A in Example 1;

[0037] Figure 2 , is the XRPD spectrum of Compound A Form A in Example 2;

[0038] Figure 3 , is the DSC spectrum of Compound A Form A in Example 2;

[0039] Figure 4 , is the XRPD spectrum of Compound A Form A in Example 6;

[0040] Figure 5 , which is a schematic diagram comparing the single crystal simulated XRPD spectrum of compound A in Example 1 and the XRPD spectrum of compound A crystalline form A in Example 2. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). -6 The unit of (ppm) is given. NMR measurements were performed using a Bruker AVANCE-III NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as the internal standard.

[0043] MS was measured using ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).

[0044] High performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC DAD and an Agilent 1260 HPLC.

[0045] The CombiFlash rapid preparation instrument uses CombiFlash Rf+LUMEN (TELEDYNE ISCO).

[0046] Thin layer chromatography silica gel plate using Yantai Yinlong HSGF 254 or GF 254 Silica gel plate: The specifications of silica gel plates used in thin layer chromatography (TLC) are 0.17mm to 0.23mm, and the specifications used for thin layer chromatography separation and purification products are 0.4mm to 0.5mm.

[0047] Silica gel column chromatography generally uses Rushan Shangbang silica gel 100-200 mesh silica gel as the carrier.

[0048] Reagents: NaBH3-sodium borohydride, EtOH-ethanol, NaOAc-sodium acetate, toluene-toluene, rt-room temperature, NaCO3-sodium carbonate, MeOH-methanol, AcOH-acetic acid, AcONa-sodium acetate, H2SO4-sulfuric acid, NaOH-sodium hydroxide, CS2CO3-cesium carbonate, TEMPO-2,2,6,6,-tetramethylpiperidinyl oxide, TMSN3-azidotrimethylsilane, ACN-acetonitrile, NaBH3CN-cyano Sodium borohydride, p-TsOH-p-toluenesulfonic acid, NaH-sodium hydride, THF-tetrahydrofuran, CH3I-iodomethane, Acetone-acetone, CCL4-carbon tetrachloride, NBS-N-bromosuccinimide, AIBN-azobisisobutyronitrile, DIEA-N,N-diisopropylethylamine, DMF-N,N-dimethylformamide, Pd(PPh3)2Cl2-bistriphenylphosphine palladium dichloride, LiCL-lithium chloride, n-BuLi-n-butyllithium, NH2OH HCl-hydroxylamine hydrochloride, AgNO3-silver nitrate.

[0049] abs means absolute configuration.

[0050] Example 1 1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one, Compound 6A and Compound 6B

[0051] Synthesis route:

[0052]

[0053] Step A: Synthesis of 5-bromochroman-4-amine

[0054] 5-Bromo-4-chromanone (4.4 g, 19.37 mmol) was dissolved in anhydrous methanol (44 ml) and isopropanol (55 ml), and ammonium acetate (29.87 g, 387 mmol) and sodium cyanoborohydride (6.08 g, 96.85 mmol) were added, and the mixture was refluxed at 80°C for 12 hours.

[0055] After the reaction was completed, the solvent in the solution was dried by spin drying, the pH was adjusted to 10 with 1 molar sodium hydroxide solution, extracted with ethyl acetate (30 ml × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 0 / 1) to obtain 3.0 g of white solid 5-bromo-4-chroman-amine (yield 68.0%). 1H NMR (400MHz, DMSO) δ7.16–7.01(m,2H),6.86–6.76(m,1H),4.32–4.17(m,2H),4.09–3.98(m,1H),1.96–1.77(m,2H).

[0056] Step B: Synthesis of tert-butyl (5-bromo-4-chroman-amine) carbamate

[0057] 5-Bromo-4-chroman-amine (3.0 g, 13.2 mmol), di-tert-butyl dicarbonate (3.15 g, 14.5 mmol) and triethylamine (4.0 g, 39.6 mmol) were dissolved in dichloromethane (30 ml) and reacted at room temperature for 6 hours.

[0058] The reaction solution was added with 20 ml of water and extracted with dichloromethane (20 ml x 2). The combined organic phases were washed with aqueous NaHCO3 solution, dried over anhydrous sodium sulfate, filtered, and spin-dried. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 3.8 g of tert-butyl (5-bromo-4-chroman) carbamate (yield: 88.3%). LCMS: m / z (ESI), [M+Na] + =350.0; 1 H NMR(400MHz,DMSO)δ7.41(d,J=7.6Hz,1H),7.16-7.09(m,2H),6.81(dd,J=8.0,1.2Hz,1H),4.6 3(s,1H),4.25(d,J=10.8Hz,1H),4.04(dd,J=17.2,6.4Hz,1H),1.98–1.78(m,2H),1.42(s,9H).

[0059] Step C: Synthesis of tert-butyl (5-bromo-4-chroman)(tert-butoxycarbonyl)carbamate

[0060] Tert-butyl (5-bromo-4-chroman) carbamate (3.8 g, 11.58 mmol), di-tert-butyl dicarbonate (5.05 g, 23.16 mmol) and 4-dimethylaminopyridine (2.83 g, 23.16 mmol) were dissolved in dimethyltetrahydrofuran (20 ml) and reacted at 60°C for 12 hours.

[0061] After the reaction was completed, the mixture was cooled to room temperature and then dried by rotation. 20 ml of water was added and the mixture was extracted with ethyl acetate (30 ml x 2). The combined organic phases were washed with an aqueous solution of NaHCO3, dried over anhydrous sodium sulfate, filtered, and dried by rotation. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 3.5 g of tert-butyl (5-bromo-4-chroman)(tert-butoxycarbonyl) carbamate (yield: 71.4%).

[0062] 1 HNMR(400MHz,DMSO)δ7.19–7.08(m,2H),6.87–6.78(m,1H),5.26(t,J=4.2Hz ,1H),4.37-4.32(m,1H),4.27–4.15(m,1H),2.16–2.00(m,2H),1.34(s,18H).

[0063] Step D: Synthesis of tert-butyl (5-vinyl-4-chroman)(tert-butoxycarbonyl) carbamate

[0064] Tert-butyl (5-bromo-4-chroman)(tert-butoxycarbonyl) carbamate (3.5 g, 8.17 mmol), potassium ethylene trifluoroborate (1.42 g, 10.6 mmol), 1,1'-bis (diphenylphosphino) ferrocene palladium dichloride (1.18 g, 1.63 mmol) and potassium carbonate (3.38 g, 24.5 mmol) were dissolved in dimethyl sulfoxide (30 ml), protected by nitrogen, and reacted at 100°C for 12 hours.

[0065] After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was filtered through diatomaceous earth. The filtrate was collected, and then 50 ml of water was added, and the mixture was extracted with ethyl acetate (40 ml x 3). The combined organic phases were washed with an aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and spin-dried. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 1.8 g of yellow oil, tert-butyl (5-vinyl-4-chroman)(tert-butoxycarbonyl) carbamate (yield: 58.8%). 1 HNMR (400MHz, DMSO) δ7.15–7.06(m,1H),7.06(d,J=7.2Hz,1H),6.76-6.69(m,2H),5.65(dd,J=17.2,1.2Hz,1H),5.41 (t,J=5.2Hz,1H),5.30–5.21(m,1H),4.36(dd,J=7.2,3.6Hz,1H),4.17–4.08(m,1H),2.17–2.02(m,3H),1.26(s,18H).

[0066] Step E: Synthesis of tert-butyl (5-formyl-4-chroman)(tert-butoxycarbonyl)carbamate

[0067] Tert-butyl (5-vinyl-4-benzodihydropyran) (tert-butoxycarbonyl) carbamate (1.0 g, 2.67 mmol) was dissolved in a mixture of tetrahydrofuran (16 ml) and water (4 ml), potassium osmate (98.2 mg, 0.267 mmol) was added to the reaction solution, and after stirring at room temperature for 30 minutes, sodium periodate (2.28 g, 10.68 mmol) was added to the reaction solution, and the reaction was allowed to react at room temperature for 2 hours.

[0068] After the reaction was completed, 20 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 ml x 2). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 600 mg of yellow oil, tert-butyl (5-formyl-4-chroman)(tert-butoxycarbonyl) carbamate (yield: 60.0%). LCMS: m / z (ESI), [M+Na] + =400.1; 1 HNMR(400MHz,DMSO)δ10.02(s,1H),7.42–7.39(m,2H),7.14-7.01(m,1H),5.89(t,J =5.6Hz,1H),4.43–4.37(m,1H),4.23–4.18(m,1H),2.2-2.12(m,2H),1.26(s,18H).

[0069] Step F: Synthesis of ethyl 3-(((4-(bis(tert-butoxycarbonyl)amino)pyran-5-yl)methyl)amino)-1H-pyrrole-2-carboxylate

[0070] 3-Amino-2-ethoxycarbonylpyrrole hydrochloride (333 mg, 1.75 mmol) was dissolved in 6 ml of anhydrous ethanol, and then N,N-diisopropylethylamine (190 mg, 1.75 mmol) and glacial acetic acid (270 mg, 4.74 mmol) were added to the mixture. After stirring at room temperature for 10 minutes, tert-butyl (5-formyl-4-benzodihydropyran) (tert-butoxycarbonyl) carbamate (600 mg, 1.58 mmol) was added to the mixture. After stirring at room temperature for 2 hours, sodium cyanoborohydride (180 mg, 3.16 mmol) was added to the mixture, and the mixture was reacted at room temperature for 12 hours.

[0071] After the reaction was completed, the anhydrous ethanol in the reaction solution was dried by spin drying. The mixture was extracted with 10 ml of water and ethyl acetate (20 ml x 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The resulting residue was purified by reverse phase column (eluent: 0.1% aqueous ammonia solution) to obtain 450 mg of ethyl 3-(((4-(bis(tert-butoxycarbonyl)amino)pyran-5-yl)methyl)amino)-1H-pyrrole-2-carboxylate (yield: 81.9%). 1 H NMR (400MHz, DMSO) δ10.78(s,1H),7.13(t,J=8.0Hz,1H),6.86(d,J=7.6Hz,1H),6.73–6.67(m,2H),5.63(s,1H),5.52(t,J=2.4Hz,1H),5.4 3(t,J=4.4Hz,1H),4.47–4.34(m,1H),4.35–4.21(m,1H),4.24–4.08(m,4H),2.13-2.09(m,2H),1.26(m,18H),1.18(dd,J=9.2,5.2Hz,3H).

[0072] Step G: Synthesis of tert-butyl(tert-butoxycarbonyl)(5-((4-oxo-2-thioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-1-yl)methyl)chroman-4-yl)carbamate

[0073] Ethyl 3-(((4-(bis(tert-butoxycarbonyl)amino)pyran-5-yl)methyl)amino)-1H-pyrrole-2-carboxylate (450 mg, 0.87 mmol) and benzoyl isothiocyanate (171 mg, 1.05 mmol) were dissolved in methanol (10 ml). After stirring at room temperature for 3 hours, cesium carbonate (567 mg, 1.74 mmol) was added and the reaction solution was reacted at 65°C for 2 hours.

[0074] After the reaction was completed, the anhydrous methanol in the reaction solution was dried by spin drying. The mixture was extracted with 20 ml of water and ethyl acetate (20 ml x 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The resulting residue was purified by reverse phase column (eluent: 0.1% ammonium bicarbonate solution) to obtain 400 mg of tert-butyl (tert-butoxycarbonyl) (5-((4-oxo-2-thioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-1-yl)methyl)chroman-4-yl)carbamate (yield: 86.9%). LCMS: m / z (ESI), [M+H] + =529.0; 1H NMR (400MHz, DMSO) δ7.33(t,J=2.8Hz,1H),7.04(t,J=8.0Hz,1H),6.71(d,J=8.0Hz,1H),6.24(d,J=7.6Hz,1H),5.91(t,J=2.4Hz,1H),5 .71-5.67(m,1H),5.60(t,J=5.2Hz,1H),5.35(d,J=16.4Hz,1H),4.44-4.41(m,1H),4.21–4.17(m,1H),2.26-2.22(m,1H),1.32(s,18H).

[0075] Step H: Synthesis of 1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one

[0076] Tert-butyl (tert-butoxycarbonyl) (5-((4-oxo-2-thioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-1-yl)methyl)chroman-4-yl)carbamate (200 mg, 0.37 mmol) was dissolved in ethyl acetate (10 ml), and hydrochloric acid ethyl acetate solution (10 ml) was added to the reaction solution and stirred at room temperature for 12 hours. The reaction solution was spin-dried. The solution pH was then adjusted to 8 with sodium bicarbonate aqueous solution, and then extracted with ethyl acetate (15 ml x 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried. The obtained residue was purified by reverse phase column (eluent: 0.1% aqueous ammonia solution) to obtain 93.83 mg of 1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (yield: 68.4%). LCMS: m / z (ESI), [M+H] + =329.1; 1 H NMR (400MHz, DMSO-d6) δ7.29 (d, J = 2.9Hz, 1H),

[0077] 6.96(t,J=7.9Hz,1H),6.64(d,J=8.1Hz,1H),6.21-6.15(m,2H),5.97(d,J=16.4Hz,1H),5.83( d,J=16.4Hz,1H),4.34-4.18(m,2H),4.15-4.05(m,1H),2.07-1.95(m,1H),1.87-1.79(m,1H).

[0078] Step I: Synthesis of compounds I-1 and I-2

[0079] Tert-butyl(tert-butoxycarbonyl)(5-((4-oxo-2-thioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-1-yl)methyl)chroman-4-yl)carbamate (200 mg, 0.38 mmol) was subjected to SFC separation ( OZ column (250*25mm 10μm), using Supercritical CO2 and MEOH (+0.1% 7.0mol / lAmmonia in MEOH) as mobile phases, chiral separation was carried out to prepare isomers I-1 (RT=4.18min, 70mg, yield=34%, ee>99%) and I-2 (RT=5.16min, 65mg, yield=32%, ee>99%).

[0080] Preparation of compound 6A:

[0081] Compound I-1 (200 mg, 0.37 mmol) was dissolved in ethyl acetate (10 ml), and hydrochloric acid ethyl acetate solution (10 ml) was added to the reaction solution and stirred at room temperature for 12 hours. The reaction solution was spin-dried. The pH of the solution was then adjusted to 8 with sodium bicarbonate aqueous solution, and then extracted with ethyl acetate (15 ml x 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried. The resulting residue was purified by reverse phase column (eluent: 0.1% ammonia solution) to obtain 96.5 mg of compound 6A (R)-1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (yield: 77.8%). ee>99%.LCMS: m / z (ESI), [M+H] + =329.1; 1 H NMR (400MHz, DMSO-d6) δ7.28(d,J=2.8Hz,1H),6.95(t,J=7.9Hz,1H),6.64(d,J=8.1Hz,1H),6.21–6.13(m,2H),5.9 7(d,J=16.4Hz,1H),5.83(d,J=16.4Hz,1H),4.34–4.17(m,2H),4.11(s,1H),2.07–1.94(m,1H),1.88–1.76(m,1H).

[0082] Preparation of compound A:

[0083] (R)-tert-butyl(tert-butoxycarbonyl)(5-((4-oxo-2-thioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-1-yl)methyl)chroman-4-yl)carbamate (200 mg, 0.37 mmol) was dissolved in ethyl acetate (10 ml), and a hydrochloric acid ethyl acetate solution (4M, 10 ml) was added to the reaction solution, and stirred at room temperature for 12 hours. The mixture was filtered and washed with ethyl acetate to obtain 94 mg of (R)-1-((4-aminochroman-5-yl)methyl)-2-thioxo-1,2,3,5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4-one hydrochloride as a white solid (yield: 68%).

[0084] LCMS: m / z(ESI),[M+H] + =329.1.

[0085] 1 H NMR (400MHz, DMSO-d6) δ12.52(s,1H),12.38(s,1H),8.63(s,3H),7.36(t,J=3. 0Hz,1H),7.15(t,J=8.0Hz,1H),6.77(d,J=8.2Hz,1H),6.26(d,J=7.7Hz,1H),6 .16(t,J=2.5Hz,1H),5.93(d,J=16.6Hz,1H),5.71(d,J=16.6Hz,1H),4.89(s,1 H),4.45-4.36(m,1H),4.34-4.25(m,1H),2.39-2.31(m,1H),2.26-2.14(m,1H).

[0086] The single crystal structure diagram of compound 6A hydrochloride (compound A) is shown in Figure 1 As shown, the specific crystal parameters are as follows:

[0087]

[0088] Example 2

[0089] Preparation of Form A of Compound A

[0090] Take 1 g of the refined product of compound A in a 25 mL round-bottom flask, add 5 mL of acetone, heat to 50°C, suspend and stir for 1-2 days, cool to room temperature, filter, wash the filter cake with a small amount of acetone, and vacuum dry the filter cake to obtain 900 mg of Form A of compound A with a purity of 99.6%.

[0091] The X-ray diffraction spectrum of the crystal form A of the obtained compound A is as follows: Figure 2As shown, there are characteristic peaks at 13.96°, 14.86°, 15.94°, 17.41°, 18.43°, 19.12°, 21.01°, 21.20°, 22.05°, 24.21°, 25.14°, 25.57°, 26.37°, 27.14°, 28.84°, 29.72°, 32.11°, 33.73°, 34.52° and 35.82°.

[0092] The DSC spectrum of the crystalline form A of the obtained compound A is as follows: Figure 3 As shown, its differential scanning calorimetry curve has an onset value of an endothermic peak at 296.8±5°C and a peak value of an endothermic peak at 304.0±5°C.

[0093] Example 3

[0094] Preparation of Form A of Compound A

[0095] Take 1g of the refined product of compound A into a 25mL round-bottom flask, add 20mL DMSO, stir for 1-2h to dissolve, filter using a 0.22um filter membrane, and then place it in an ethyl acetate atmosphere for diffusion. After about 3 days, needle-shaped crystals precipitate, filter, and vacuum dry the filter cake to obtain Form A of compound A with a purity of 99.5%.

[0096] Example 4

[0097] Preparation of Form A of Compound A

[0098] Take 1g of the refined product of compound A in a 100mL round-bottom flask, add 20mL DMSO, stir for 1-2h to dissolve, then drop 40ml acetonitrile, precipitate solid, filter, and vacuum dry the filter cake to obtain Form A of compound A with a purity of 99.3%.

[0099] Example 5

[0100] Preparation of Form A of Compound A

[0101] Take 100 mg of the refined product of compound A into a 5 mL EP tube, add 2 mL of methanol, stir to form a saturated solution, filter while hot, and let the filtrate stand at room temperature for evaporation. White needle-shaped crystals will precipitate in about 3 days. Filter and vacuum dry the solid to obtain Form A of compound A with a purity of 99.4%.

[0102] The X-ray diffraction spectrum of the crystal form A of the obtained compound A is as follows: Figure 4As shown, there are characteristic peaks at 13.88°, 14.80°, 15.87°, 17.32°, 18.37°, 19.08°, 20.95°, 21.13°, 21.99°, 24.14°, 25.07°, 25.54°, 26.29°, 27.12°, 28.84°, 29.59°, 31.96°, 33.69°, 34.45° and 35.77°.

[0103] in, Figure 2 and Figure 4 The comparison of the absorption peaks is shown in Table 1 below, with an error of ±0.2°.

[0104] Table 1 X-ray diffraction data of Form A of Compound A

[0105]

[0106] Wherein, No. = serial number, Rel. Int. = Relative Intensity, Pos. [° 2θ] = Position [° 2θ], and the error is ± 0.2°. Rel. Int. = Relative Intensity only indicates the approximate intensity of the characteristic peak intensity and should not be used as a limitation on the specific crystal form.

[0107] X-ray powder diffraction conditions:

[0108] The X-ray powder diffraction (XRPD) spectrum shown in the present invention is obtained by using an Empyrean X-ray powder diffractometer. The detection conditions are: Cu-Kα radiation, wavelength The divergence slit was 1 / 4°, the X-ray tube voltage was 45 kV, the X-ray tube current was 40 mA, the scanning range was 3-40° (2θ), the step length was 0.026°, and the dwell time per step was 30 s.

[0109] according to Figure 2 and Figure 4 The XRPD spectrum and characteristic peak data are expressed in 2θ angles as having the strongest characteristic absorption peak at 26.37°, with an error of ±0.2° and a relative absorption intensity of 100%.

[0110] Further, the crystalline form A has characteristic peaks at 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14° and 26.37°, with an error of ±0.2° and a relative absorption intensity greater than 50%.

[0111] Specifically, the crystal form A has characteristic peaks at 14.86°, 15.94°, 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14°, 26.37°, 27.14° and 28.84°, with an error of ±0.2° and a relative absorption intensity greater than 30%, which can be used to distinguish in detail that other substances represent this crystal form. Further specifically, the crystal form A has characteristic peaks at 13.96°, 14.86°, 15.94°, 17.41°, 18.43°, 19.12°, 21.01°, 21.20°, 22.05°, 24.21°, 25.14°, 25.57°, 26.37°, 27.14°, 28.84°, 29.72°, 32.11°, 33.73°, 34.52° and 35.82°, with an error of ±0.2° and a relative absorption intensity greater than 10%, which can distinguish other substances representing the present crystal form in more detail. Other weak absorption peaks may change significantly due to experimental operation errors, and for those skilled in the art, other absorption peaks are all absorption peaks that can be considered unnecessary when characterizing the present crystal form.

[0112] Figure 5 , which is a schematic diagram comparing the single crystal simulated XRPD spectrum of compound A in Example 1 and the XRPD spectrum of compound A crystalline form A in Example 2. Figure 5 From the above table Pos.[°2θ], it can be seen that the crystal forms obtained in Example 1 and Examples 2 and 5 are the same crystal form.

[0113] Example 6: In vitro evaluation of the inhibitory effect of the compounds of the present invention on human myeloperoxidase (hMPO) activity

[0114] 50 μL of 2× substrate (composed of 3'-(p-aminophenyl)fluorescein and H2O2, both 3'-(p-aminophenyl)fluorescein and H2O2 are Km concentrations, purchased from Invitrogen, A36003) was added to a black opaque, low-protein adsorption 96-well plate (purchased from Perkinelmer, 6005270), and 45 μL of 2× compounds of different concentrations (the final DMSO concentration was 1%) were added. After being thoroughly pipetted and mixed, 5 μL of 2μg / mL human myeloperoxidase (purchased from PLANTA NATURAL, 700-03-001) was added and mixed, and the fluorescence value of 0 to 30 min (read every 20 s) was immediately read using a microplate reader at Ex / Em: 488 / 520 nm wavelength using a kinetic mode. Calculate the inhibitors of different concentrations of compounds: Inhibition rate (%) = [1-(SlopeControl-Slopecpd) / SlopeControl–SlopeBlank)]*100, where SlopeBlank is the slope of the control without compound and the MPO well from 0 to 5 min (Ex / Em: 544 / 620nm reading / reaction time t); SlopeControl is the slope of the well without compound (containing 1% DMSO) from 0 to 5 min (linear reaction stage); Slopecpd is the slope of the compound from 0 to 5 min. With the logarithm of the compound concentration as the abscissa and the inhibition rate as the ordinate, a 4-parameter nonlinear regression curve was fitted to calculate the IC 50 Value(Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope)), where: Hillslope represents the slope of the fitting curve, IC 50 The test results are shown in Table 2.

[0115] Table 2 Inhibitory activity of compounds on the peroxidation cycle of human myeloperoxidase (hMPO) in vitro

[0116] Example compounds <![CDATA[IC 50 (nM)]]> 6 8.02 6A 4.64

[0117] Conclusion: The compounds of the present invention have the effect of inhibiting the activity of hMPO.

[0118] Example 7: Pharmacokinetic study of the compounds of the present invention in rats

[0119] 7.1 Experimental Materials

[0120] SD rats: male, 180-350 g, purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0121] Reagents: DMSO (dimethyl sulfoxide), PEG-400 (polyethylene glycol 400), normal saline, acetonitrile, formic acid, propranolol (internal standard) are all commercially available.

[0122] Instrument: Thermo Fisher LC-MS (Ultimate 3000UPLC, TSQ QUANTUMN ULTRA triple quadrupole mass spectrometer).

[0123] 7.2 Experimental methods

[0124] Weigh the compound and dissolve it in DMSO-PEG-400-normal saline (5:60:35, v / v / v) system. After intravenous or oral administration to rats, collect 200μL of venous blood in EDTA-K2 anticoagulant tubes at 15min, 30min, 1h, 2h, 5h, 7h, and 24h (5min for iv group), centrifuge at 10000rpm for 2min, and freeze the plasma at -80℃ for testing. Accurately weigh a certain amount of the test sample and dissolve it in DMSO to 2mg / mL as the stock solution. Accurately draw an appropriate amount of the compound stock solution and add acetonitrile to dilute it to prepare a standard series of solutions. Accurately draw 4μL of each of the above standard series solutions, add it to 36μL of blank plasma, vortex and mix well, and prepare plasma samples equivalent to plasma concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000ng / mL. Perform double sample analysis for each concentration to establish a standard curve. Take 30 μL of plasma (5 minutes, 15 minutes, and 30 minutes of intravenous administration, plasma diluted 5 times), add propranolol acetonitrile solution (internal standard, 50 ng / mL) to precipitate protein, add 100 μL of water, vortex mix, centrifuge at 4000 rpm for 5 minutes, and take the supernatant for LC-MS analysis. LC-MS detection conditions are as follows:

[0125] Chromatographic column: YMC-Triart C 18 50×3.0mmI.DS-3μm,12nm

[0126] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution:

[0127]

[0128]

[0129] 7.3 Data processing

[0130] After LC-MS detection of blood drug concentration, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method. The test results are shown in Table 3.

[0131] Table 3 Pharmacokinetic results of the compounds of the present invention in rats

[0132]

[0133] It can be seen from the experimental results in Table 3 that the compounds of the present invention are metabolized more slowly in rats and have higher oral exposure and bioavailability.

[0134] Example 8: Enzyme induction test study of the compounds of the present invention

[0135] 8.1 Experimental Materials and Instruments

[0136] Materials: Williams'Medium E (without phenol red) was purchased from Sigma-Aldrich Trading Co., Ltd.; Williams'Medium E, human recombinant insulin, GlutaMAX and HEPES were purchased from Life Technologies; Isotonic Percoll was purchased from General Electric; Fetal bovine serum was purchased from Corning; Dexamethasone was commercially available; CellTiter-Fluor TM Cell Viability Assay kit was purchased from Promega; Taqman Gene Expression Assay probe (20×, CYP1A2, FAM labeled), Taqman Gene Expression Assay probe (20×, CYP2B6, FAM labeled), Taqman Gene Expression Assay probe (20×, CYP3A4, FAM labeled) and Taqman Gene Expression Assay probe (20×, ACTB, VIC labeled) were purchased from AB (Applied biosystems); human primary hepatocytes were purchased from BioIVT.

[0137] Instrument: QuantStudio 6.

[0138] 8.2 Experimental Design

[0139] Accurately weigh the test compound, positive inducer, negative inducer, and internal standard to prepare the corresponding concentration of stock solution. Prepare the cell recovery solution and incubation solution, mRNA extraction, reverse transcription, and related reagents and consumables for fluorescence quantitative PCR.

[0140] The frozen primary human hepatocytes were revived and seeded on appropriate cell culture plates at a certain density for monolayer cell culture to meet the cell growth requirements.

[0141] Freshly prepared and preheated positive / negative inducer or test substance solution (10 μM) was added to the corresponding wells, with two replicate wells for each concentration, and incubated for 3 consecutive days with the solution changed every day, and then the mRNA level of CYP enzyme was tested. The extraction, reverse transcription and fluorescence quantitative PCR of mRNA were carried out according to the instructions of the relevant kits.

[0142] 8.3 Experimental Results

[0143] The results of the induction effect of the test compounds on CYP enzyme mRNA levels are shown in Table 4 below.

[0144] Table 4 Experimental results

[0145]

[0146] It can be seen from the experimental results in Table 4 that the compound of the present invention has a lower induction risk than the compound of Example 3 (CN201580065064.2).

[0147] Example 9: Enzyme inhibition test study of the compounds of the present invention

[0148] 9.1 Experimental Materials and Instruments

[0149] Materials: Midazolam and testosterone were purchased from Cerilliant and PANPHY; ketoconazole was purchased from MCE; human liver microsomes were purchased from Corning. Instrument: AB Sciex5500+.

[0150] 9.2 Experimental Design

[0151] The inhibitory potential of the compounds on the cytochrome P450 enzyme CYP3A was evaluated using a phosphate buffered saline (100mM, pH 7.4) system containing 0.2mg / mL human liver microsomes. The test concentrations of the compounds were 0.068, 0.206, 0.62, 1.85, 5.56, 16.67 and 50μM. The positive substrates in the incubation system were 1μM midazolam and 40μM testosterone (CYP3A), respectively, and the concentration of the positive inhibitor ketoconazole (CYP3A) in the incubation system was 0.05μM. The incubation was carried out at 37°C, and the reaction was initiated by adding a NADPH solution with a final concentration of 1mM. The incubation time was 10 minutes. After the incubation, all samples were added with 300μL of acetonitrile solution containing 3% formic acid and internal standard (0.5μM tolbutamide) to precipitate proteins and UPLC-MS / MS was used to detect the amount of metabolites of the labeled substrates to calculate the IC50 value of the compound inhibition.

[0152] 9.3 Experimental Results

[0153] The IC50 values ​​of the test compounds for the inhibition of CYP3A are shown in Table 5 below.

[0154] Table 5 Experimental results

[0155]

[0156] It can be seen from the experimental results in Table 5 that the compounds of the present invention have a lower risk of inhibiting CYP3A than the compound of Example 3 (CN201580065064.2).

[0157] Example 10: Stability Study

[0158] A certain amount of the crystal form A of compound A obtained in Example 2 was placed naked at 95% RH and 50° C. to test the stability of the sample. The sampling time was 0 day and 5 days, and the HPLC purity and crystal form of the sample were investigated. The research results are shown in Table 6.

[0159] Table 6 Stability study of Form A of Compound A

[0160]

[0161] According to the results in Table 6, it can be seen that the crystalline form A of compound A of the present invention has good stability.

[0162] Example 11: Fluidity comparison experiment

[0163] A certain amount of the crystalline form A of compound A obtained in Example 2 was weighed and placed steadily in a 50 ml measuring cylinder to the 20 ml mark, and its bulk density was detected and calculated.

[0164] The bulk density of Form A of Compound A is calculated to be 0.48 g / cm3.

[0165] According to the above results, it can be seen that the crystalline form A of compound A of the present invention has a good bulk density and good fluidity.

[0166] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A crystalline form A of compound A, characterized in that: The crystal form A has characteristic peaks at 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14° and 26.37° in the X-ray diffraction diagram at 2θ angle, with an error of ±0.2°; the structural formula of the compound A is shown below:

2. The crystalline form A of compound A according to claim 1, characterized in that: The crystal form A has characteristic peaks at 14.86°, 15.94°, 17.41°, 18.43°, 21.01°, 21.20°, 24.21°, 25.14°, 26.37°, 27.14° and 28.84° in the X-ray diffraction pattern with a 2θ angle, and the error is ±0.2°; preferably, the crystal form A has characteristic peaks at 13.96°, 14. There are characteristic peaks at .86°, 15.94°, 17.41°, 18.43°, 19.12°, 21.01°, 21.20°, 22.05°, 24.21°, 25.14°, 25.57°, 26.37°, 27.14°, 28.84°, 29.72°, 32.11°, 33.73°, 34.52° and 35.82°, with an error of ±0.2°.

3. The crystalline form A of compound A according to claim 1, characterized in that: The X-ray diffraction pattern of the crystal form A is shown in Figure 2 or Figure 4.

4. The crystalline form A of compound A according to claim 1, characterized in that: The differential scanning calorimetry spectrum of the crystal form A has an endothermic peak at 304.0±5°C.

5. The crystalline form A of compound A according to claim 1, characterized in that: The differential scanning calorimetry spectrum of the crystal form A is shown in FIG3 .

6. The crystalline form A of compound A according to claim 1, characterized in that: The crystal form A is of triclinic P1 space group at a temperature of 293 (2) K, with unit cell dimensions of a = 7.40910 (10), b = 12.7287 (2), c = 17.0243 (3), α = 90°, β = 90°, γ = 90°.

7. The method for preparing the crystalline form A of compound A according to any one of claims 1 to 6, characterized in that: The following steps are involved: a) Solvent method: take the refined product of compound A into a reaction bottle, add a small amount of organic solvent, heat to a certain temperature, suspend and stir, filter, wash the filter cake with an organic solvent, and vacuum dry to obtain the crystalline form A of compound A; the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, ethyl acetate, isopropyl acetate, methyl acetate, ethyl formate, isobutyl acetate, or a mixture of more than one thereof; or b) Volatilization method: take the refined product of compound A into a reaction bottle, add a small amount of organic solvent, heat to saturation, filter while hot, and evaporate the filtrate at room temperature to obtain the crystalline form A of compound A. The organic solvent is selected from: methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, or a mixture of more than one; or c) Diffusion method: Take the refined compound A into a reaction bottle, add DMSO to dissolve it, control the concentration range of the DMSO solution to 50-500 mg / ml, and then diffuse it in an organic solvent at room temperature; the selected organic solvent is selected from: acetone, isopropanol, ethyl acetate, isopropyl acetate, ethanol, methanol, acetonitrile, THF, isopropyl ether, or a mixture of more than one.

8. The preparation method according to claim 7, characterized in that: The heating temperature in step a) is 40-60° C., and the heating time is 1-2 days; the heating temperature in step b) is 40-60° C., and the heating time is 1-2 hours.

9. A pharmaceutical composition, characterized in that Comprising the crystalline form A of compound A according to any one of claims 1 to 6 and one or more pharmaceutically acceptable carriers.

10. Use of the crystalline form A of compound A according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating a myeloperoxidase-related disease; preferably, the myeloperoxidase-related disease is selected from cardiovascular-related diseases.

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