CN119264137A

CN119264137A - Crystal form A of compound A and preparation method and application thereof

Info

Publication number: CN119264137A
Application number: CN202410904224.9A
Authority: CN
Inventors: 赖英杰; 王斌; 乐晓亮; 吴康; 熊晓琳; 邢伟; 李松
Original assignee: Shenzhen Salubris Pharmaceuticals Co Ltd
Current assignee: Shenzhen Salubris Pharmaceuticals Co Ltd
Priority date: 2023-07-06
Filing date: 2024-07-05
Publication date: 2025-01-07

Abstract

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

Description

Crystal form A of compound A and preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical medicines, and relates to a crystal form A of a compound A and a preparation method thereof.

Background

Myeloperoxidase (myeloperoxidase, MPO) is a highly sensitive and specific vascular inflammatory mediator indicator, and has become an important indicator for predicting cardiovascular and cerebrovascular event risk. MPO can kill microorganisms in phagocytes by catalyzing and oxidizing chloride ions to generate hypochlorous acid, destroy various target substances, and play roles in various aspects such as organism generation and regulation of inflammatory reaction. More importantly, oxidative modification of Low Density Lipoproteins (LDL) can cause atherosclerosis, and thus MPO is believed to be involved in the occurrence of cardiovascular disease.

Currently, MPO is considered the most promising cardiovascular marker, and elevated MPO levels in the body are indicative of risk of arteriosclerosis and coronary heart disease, an early warning of myocardial infarction, more sensitive than other indicators such as troponin T, CK-MB and CRP, and earlier diagnosis and risk assessment. MPO levels are significantly elevated within 2 hours of chest pain, so MPO will be of greater clinical significance for diagnosing Acute Coronary Syndrome (ACS) in chest pain patients.

PCT/CN2023/136923 discloses pyrrolo [3,2-d ] pyrimidine-4-ketone derivatives, a preparation method and medical application thereof, and specifically discloses (R) -1- ((4-amino chroman-5-yl) methyl) -2-thio-1, 2,3, 5-tetrahydro-4H-pyrrolo [3,2-d ] pyrimidine-4-ketone hydrochloride, wherein the structural formula is as follows:

The crystal form of the pharmaceutical active ingredient often affects the chemical stability of the drug, and the difference in crystallization conditions and storage conditions may cause a change in the crystal form structure of the compound, and sometimes other forms of crystal form are generated. Generally, amorphous pharmaceutical products have no regular crystalline structure and often have other drawbacks such as poor product stability, finer crystallization, difficult filtration, easy caking, poor flowability, etc. The polymorphic forms of the drug have different requirements for product storage, production and scale-up. Therefore, intensive studies on the crystalline forms of the aforementioned compounds are required to improve the properties of the aforementioned compounds in various aspects.

Disclosure of Invention

The invention aims to provide a crystal form A of a compound A, and a preparation method and application thereof.

Specifically, the invention provides a compound A crystal form A, which is characterized in that the compound A has characteristic peaks at 17.41 degrees, 18.43 degrees, 21.01 degrees, 21.20 degrees, 24.21 degrees, 25.14 degrees and 26.37 degrees in an X-ray diffraction diagram by 2 theta angles, and the error is +/-0.2 degrees, and the compound A has the following structural formula:

Preferably, as a preferred mode of the present invention, 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 terms of 2θ angles in an X-ray diffraction pattern, and the error is ±0.2°;

More optionally, as a preferred embodiment of the present invention, 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 as expressed in terms of 2θ, with an error of ±0.2°;

Further preferably, as a preferred mode of the present invention, the X-ray diffraction pattern of the crystal form a of the compound a is shown in fig. 2 or fig. 4;

Further preferably, as a preferred mode of the present invention, the crystal form a is a triclinic P1 crystal system space group at a temperature of 293 (2) K, and the unit cell size is a= 7.40910 (10), b= 12.7287 (2), c= 17.0243 (3), α=90°, β=90°, γ=90°.

It should be noted that different samples of a particular crystalline form have the same major XRPD peak, but there may be variations in the small peaks in the powder pattern. Furthermore, when the isomorphous samples obtained by the corresponding methods are detected by the same instrument and detection method by a person skilled in the art, each 2 theta angle error is usually within + -0.2 deg. (each 2 theta angle error is usually within + -0.2 deg. meaning that most of the characteristic peaks, such as more than 80% of the characteristic peak error, are within this range, and that there are occasionally few characteristic peaks with errors outside of this range, all should be considered as XRPD patterns belonging to the same crystal form), and the characteristic peaks of each shift are moderate intensity absorption peaks, while other weak absorption peaks may vary significantly due to experimental operation errors. Therefore, the X-ray diffraction absorption peak is not limited to the above specific values, and a possible error range should be considered.

X-ray powder diffraction conditions:

The X-ray powder diffraction (XRPD) spectrum shown in the invention is detected by a Panake sharp (Empyrean) X-ray powder diffractometer under the detection conditions of Cu-K alpha radiation and wavelength The divergence slit is 1/4 degree, the X-ray tube voltage is 45kV, the X-ray tube current is 40mA, the scanning range is 3-40 degrees (2 theta), the step length is 0.026 degree, and the stay time of each step is 30s.

The DSC spectrogram is obtained by detecting a German relaxation-resistant DSC 200F3 differential scanning calorimeter under the detection conditions of an aluminum tray, a gland (tying), and the temperature rising rate is 10 ℃ per minute, and the temperature rises from room temperature to a specific temperature.

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 start value of an endothermic peak at 296.6±5 ℃ and a peak value of an endothermic peak at 304.0±5 ℃.

Still more preferably, as a preferred embodiment of the present invention, the compound a is in the form of form a, the DSC profile of which is shown in figure 3.

The invention also aims to provide a preparation method of the crystal form A of the compound A, which has simple process and can be realized under normal temperature conditions;

Wherein, the compound A can be prepared according to the method disclosed in PCT/CN2023/136923, and the specific synthetic route and main reaction conditions are as follows:

the preparation method of the crystal form A of the compound A comprises the following steps:

a) The solvent method comprises adding refined product of compound A into a reaction bottle, heating to a certain temperature, suspending and stirring, filtering, washing filter cake with organic solvent, vacuum drying to obtain crystal form A of compound A, wherein the organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, ethyl acetate, isopropyl acetate, methyl acetate, ethyl formate and isobutyl acetate, or mixture thereof

B) And volatilizing the refined product of the compound A into a reaction bottle, adding a small amount of organic solvent, heating to saturation, filtering, and volatilizing the filtrate at room temperature to obtain a crystal form A of the compound A. The organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile and acetone, or mixture thereof

C) The diffusion process includes adding refined compound A into reaction bottle, dissolving in DMSO in concentration of 50-500mg/ml, and dispersing in organic solvent selected from one or more of acetone, isopropanol, ethyl acetate, isopropyl acetate, ethanol, methanol, acetonitrile, THF and isopropyl ether.

Further, as a preferable technical scheme of the invention, the heating temperature in the step a) is 40-60 ℃, the heating time is 1-2 days, and the heating temperature in the step b) is 40-60 ℃ and the heating time is 1-2 hours.

It is still another object of the present invention to provide a pharmaceutical composition comprising the above-described form a of compound a and one or more pharmaceutically acceptable carriers.

Furthermore, the invention also provides an application of the crystal form A of the compound A or the pharmaceutical composition of the invention in preparing medicines for treating myeloperoxidase-related diseases, preferably, the myeloperoxidase-related diseases are selected from cardiovascular-related diseases.

The pharmaceutically acceptable carrier comprises various pharmaceutic adjuvants, packaging materials, delivery tools and the like, and is selected according to the preparation requirements, for example, the adjuvants comprise fillers, disintegrants, adhesives, lubricants and the like, and the pharmaceutically acceptable carrier can be suitable for oral administration, inhalation, parenteral administration or surface use, and the dosage forms comprise, but are not limited to, injection, solution preparation, tablets, capsules, granules and the like.

The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active agents of the present invention, which does not interfere with the biological activity of the active agents and which does not have toxic or side effects to the host or patient, representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on the vector, reference may 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.

The term "excipient" generally refers to the carrier, diluent, and/or medium required to make an effective pharmaceutical composition.

For a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For the purposes of the present oral dosage form, an "effective amount" of one active agent in a composition refers to that amount which is required to achieve the desired effect when used in combination with another active agent in the composition. Determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, a suitable effective amount in an individual case can be determined by one skilled in the art according to routine experimentation.

The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a disorder, disease or condition of interest.

"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.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.

Drawings

FIG. 1 is a schematic diagram showing the single crystal structure of Compound A in example 1;

FIG. 2 is an XRPD pattern for Compound A, form A, of example 2;

FIG. 3 is a DSC of form A of compound A of example 2;

FIG. 4 is an XRPD pattern for Compound A, form A, in example 6;

fig. 5 is a schematic diagram of a comparison of single crystal simulated XRPD patterns of compound a in example 1 and XRPD patterns of compound a crystalline form a in example 2.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the invention are not limited thereto.

The structure of the compound is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). The NMR shift (. Delta.) is given in units of 10 ^-6 (ppm). NMR was performed using a Bruker AVANCE-III nuclear magnetic instrument with deuterated dimethyl sulfoxide (DMSO-d ₆), deuterated chloroform (CDCl ₃) and an internal standard of Tetramethylsilane (TMS).

The MS was determined by ISQ EC mass spectrometry (manufacturer: thermo, model: ISQ EC).

High Performance Liquid Chromatography (HPLC) analysis used a Thermo U3000 HPLC DAD HPLC and an Agilent 1260 HPLC.

The CombiFlash rapid preparation instrument uses CombiFlash rf+ LUMEN (TELEDYNE ISCO).

The thin layer chromatography silica gel plate uses a smoke table silver dragon HSGF ₂₅₄ or GF ₂₅₄ silica gel plate, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.17-0.23 mm, and the specification of the thin layer chromatography separation and purification product is 0.4-0.5 mm.

Silica gel column chromatography generally uses 100-200 mesh silica gel of Shangbang silica gel as a carrier.

The reagent is NaBH ₃ -sodium borohydride, etOH-ethanol, naOAc-sodium acetate, tolene-toluene, r.t. -room temperature, naCO ₃ -sodium carbonate, meOH-methanol, acOH-acetic acid, acona-sodium acetate, H ₂SO₄ -sulfuric acid, naOH-sodium hydroxide, CS ₂CO₃ -cesium carbonate, TEMPO-2, 6, -tetramethylpiperidine oxide, TMSN ₃ -azidometrimethylsilane, ACN-acetonitrile, naBH ₃ CN-sodium cyanoborohydride, p-TsOH-p-toluenesulfonic acid, naH-sodium hydride, THF-tetrahydrofuran, CH ₃ I-iodomethane, acetone-acetone, CCL ₄ -carbon tetrachloride, NBS-N-bromosuccinimide, AIBN-azobisisobutyronitrile, DIEA-N, N-diisopropylethylamine, DMF-N, N-dimethylformamide, pd (PPh ₃)₂Cl₂ -bis-triphenylphosphine dichloride, L-lithium chloride, N-BuLi-N-butyllithium nitrate, N-butyl lithium nitrate, N-OH-28-silver nitrate, agHCl-76.

Abs refers to absolute configuration, absolute configuration.

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

The synthetic route is as follows:

Step A, synthesizing 5-bromo chroman-4-amine

5-Bromo-4-chromanone (4.4 g, 19.37 mmol) is dissolved in anhydrous methanol (44 ml) and isopropanol (55 ml), ammonium acetate (29.87 g, 387 mmol) and sodium cyanoborohydride (6.08 g, 96.85 mmol) are added and reacted at 80 ℃ under reflux for 12 hours.

At the end of the reaction, the solvent was dried by spin-drying, pH was adjusted to 10 with 1 mol of sodium hydroxide solution, extracted with ethyl acetate (30 ml. Times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and the resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=0/1) to give 3.0 g of a white solid, yield of 5-bromo-4-chroman-amine 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).

Step B Synthesis of tert-butyl (5-bromo-4-chroman-amine) carbamate

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) are dissolved in dichloromethane (30 ml) and reacted at room temperature for 6 hours.

The reaction solution was added to 20 ml of water, extracted with dichloromethane (20 ml x 2), and the combined organic phases were washed with aqueous NaHCO ₃, dried over anhydrous sodium sulfate, filtered and dried by spinning. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1) to give 3..8 g of tert-butyl (5-bromo-4-chroman) carbamate (yield ：88.3％).LCMS:m/z(ESI),[M+Na]⁺＝350.0;¹H 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),4.63(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).

Step C Synthesis of tert-butyl (5-bromo-4-chroman) (tert-butoxycarbonyl) carbamate

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 ℃ for 12 hours.

After the reaction was completed, it was cooled to room temperature, dried by spin-drying, 20ml of water was added, extracted with ethyl acetate (30 ml×2), and the combined organic phases were washed with aqueous NaHCO ₃, dried over anhydrous sodium sulfate, filtered and dried by spin-drying. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1) to give 3.5 g of t-butyl (5-bromo-4-chroman) (t-butoxycarbonyl) carbamate (yield: 71.4%).

¹HNMR(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).

Step D Synthesis of tert-butyl (5-vinyl-4-chroman) (tert-butoxycarbonyl) carbamate

Tert-butyl (5-bromo-4-chroman) (tert-butoxycarbonyl) carbamate (3.5 g, 8.17 mmol), ethylene potassium trifluoroborate (1.42 g, 10.6 mmol), 1' -bis (diphenylphosphine) 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), nitrogen-blanketed, and reacted at 100 ℃ for 12 hours.

After the reaction was completed, cooled to room temperature, the reaction solution was filtered with celite, and the filtrate was collected, then 50ml of water was added, extracted with ethyl acetate (40 ml×3), and the combined organic phases were washed with aqueous NaCl, dried over anhydrous sodium sulfate, filtered, and dried by spinning. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1) to give 1.8 g of tert-butyl (5-vinyl-4-chroman) (tert-butoxycarbonyl) carbamate (yield ：58.8％).¹HNMR(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).

Step E Synthesis of tert-butyl (5-formyl-4-chroman) (tert-butoxycarbonyl) carbamate

Tert-butyl (5-vinyl-4-chroman) (tert-butoxycarbonyl) carbamate (1.0 g, 2.67 mmol) was dissolved in a mixture of tetrahydrofuran (16 ml) and water (4 ml), potassium osmium (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 reacted at room temperature for 2 hours.

After the completion of the reaction, 20 ml of water was added to the reaction solution, which was extracted with ethyl acetate (20 ml. Times.2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered and dried by spinning. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1) to give 600 mg of tert-butyl (5-formyl-4-chroman) (tert-butoxycarbonyl) carbamate as a yellow oil (yield ：60.0％).LCMS:m/z(ESI),[M+Na]⁺＝400.1;¹HNMR(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).

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

3-Amino-2-ethoxycarbonylpyrrole hydrochloride (333 mg, 1.75 mmol) was dissolved in 6 ml of absolute ethanol, 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-chroman) (tert-butoxycarbonyl) carbamate (600 mg, 1.58 mmol) was then 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.

And after the reaction is finished, spin-drying the absolute ethyl alcohol in the reaction liquid. Extracted with water 10 ml and ethyl acetate (20 ml x 2) and the combined organic phases were dried over anhydrous sodium sulfate, filtered and spun-dried. The resulting residue was purified by reverse phase column (eluent: 0.1% aqueous ammonia solution) to give 450 mg of ethyl 3- (((4- (bis (tert-butoxycarbonyl) amino) pyran-5-yl) methyl) amino) -1H-pyrrole-2-carboxylate (yield) ：81.9％).¹H 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),5.43(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).

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

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), stirred at room temperature for 3 hours, cesium carbonate (567 mg, 1.74 mmol) was added and the reaction solution was reacted at 65 ℃ for 2 hours.

After the reaction, the anhydrous methanol in the reaction solution was dried by spin-drying. Extracted with water 20 ml and ethyl acetate (20 ml x 2) and the combined organic phases were dried over anhydrous sodium sulfate, filtered and spun-dried. The resulting residue was purified by reverse phase column (eluent: 0.1% ammonium bicarbonate solution) to give 400 mg of tert-butyl (tert-butoxycarbonyl) (5- ((4-oxo-2-thio-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).

Step H Synthesis of 1- ((4-amino chroman-5-yl) methyl) -2-thio-1, 2,3, 5-tetrahydro-4H-pyrrolo [3,2-d ] pyrimidin-4-one

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 an ethyl acetate hydrochloride solution (10 ml) was added to the reaction solution and stirred at room temperature for 12 hours. The reaction solution was dried by spin-drying. The solution was then adjusted to pH8 with aqueous sodium bicarbonate, then extracted with ethyl acetate (15 ml x 2) and the combined organic phases were dried over anhydrous sodium sulfate, filtered and spun dry. The resulting residue was purified by reverse phase column (eluent: 0.1% aqueous ammonia) to give 93.83 mg of 1- ((4-aminochroman-5-yl) methyl) -2-thio-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 (400 MHz, DMSO-d 6) δ7.29 (d, J=2.9 Hz, 1H),

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).

Step I Synthesis of Compounds I-1 and I-2

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 sent to SFC resolution ]Chiral resolution on OZ column (250 x 25mm 10 μm) using Supercritical CO ₂, and MEOH (+0.1% 7.0mol/lAmmonia in MEOH) as mobile phase gave isomer I-1 (rt=4.18 min,70mg, yield=34%, ee > 99%) and I-2 (rt=5.16 min,65mg, yield=32%, ee > 99%).

Preparation of compound 6A:

Compound I-1 (200 mg, 0.37 mmol) was dissolved in ethyl acetate (10 ml), and an ethyl acetate hydrochloride solution (10 ml) was added to the reaction solution and stirred at room temperature for 12 hours. The reaction solution was dried by spin-drying. The solution was then adjusted to pH 8 with aqueous sodium bicarbonate, then extracted with ethyl acetate (15 ml x 2) and the combined organic phases were dried over anhydrous sodium sulfate, filtered and spun dry. The resulting residue was purified by reverse phase column (eluent: 0.1% aqueous ammonia) to give 96.5 mg of compound 6A (R) -1- ((4-aminochroman-5-yl) methyl) -2-thio-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;¹H 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),5.97(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).

Preparation of Compound A:

(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 solution of ethyl acetate hydrochloride (4M, 10 ml) was added to the reaction solution and stirred at room temperature for 12 hours. Filtration and washing with ethyl acetate gave 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%).

LCMS:m/z(ESI),[M+H]⁺=329.1.

¹H 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,1H),4.45-4.36(m,1H),4.34-4.25(m,1H),2.39-2.31(m,1H),2.26-2.14(m,1H).

The single crystal structure of the hydrochloride of the compound 6A (compound A) is shown in fig. 1, and specific crystal parameters are as follows:

Example 2

Preparation of form A of Compound A

Taking 1g of refined product of the compound A, adding 5mL of acetone into a 25mL round bottom flask, heating to 50 ℃, suspending and stirring for 1-2 days, cooling to room temperature, filtering, washing a filter cake with a small amount of acetone, and drying the filter cake in vacuum to obtain 900mg of the crystal form A of the compound A, wherein the purity is 99.6%.

The X-ray diffraction pattern of the obtained compound A in the crystal form A is shown in figure 2, and particularly 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 degrees.

The DSC spectrum of the crystal form A of the obtained compound A is shown in FIG. 3, and a differential scanning calorimetry curve of the DSC spectrum has a starting value of an endothermic peak at 296.8 + -5 ℃ and a peak value of the endothermic peak at 304.0 + -5 ℃.

Example 3

Preparation of form A of Compound A

Taking 1g of refined product of the compound A, putting the refined product into a 25mL round-bottom flask, adding 20mL of LDMSO, stirring for 1-2h for dissolution, filtering by using a filter membrane with 0.22um, then putting the mixture into an atmosphere of ethyl acetate for diffusion for about 3 days, precipitating needle-shaped crystals, filtering, and vacuum drying a filter cake to obtain the crystal form A of the compound A, wherein the purity is 99.5%.

Example 4

Preparation of form A of Compound A

Taking 1g of refined product of the compound A, adding 20mL of LDMSO into a 100mL round bottom flask, stirring for 1-2 hours for dissolution, then dripping 40mL of acetonitrile, separating out solid, filtering, and drying a filter cake in vacuum to obtain the crystal form A of the compound A, wherein the purity is 99.3%.

Example 5

Preparation of form A of Compound A

Taking 100mg of refined product of the compound A, adding 2mL of methanol into a 5mLEP pipe, stirring to form saturated solution, filtering while the saturated solution is hot, standing and volatilizing filtrate at room temperature, precipitating white needle-like crystals about 3 days, filtering, and drying the solid in vacuum to obtain the crystal form A of the compound A, wherein the purity is 99.4%.

The X-ray diffraction pattern of the obtained compound A in the form A is shown in figure 4, and particularly has 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 degrees.

The comparison of the absorption peaks of fig. 2 and 4 is shown in table 1 below, with an error of ±0.2°.

Table 1X-ray diffraction data for form a of compound a

Wherein No. =serial number, rel.int. = RELATIVE INTENSITY, pos. [ ° 2θ ] =position °2θ ], error is ±0.2 °. Rel.int.= RELATIVE INTENSITY is only an approximate intensity case representing the intensity of the characteristic peak, and should not be taken as a limitation of the specific crystal form.

X-ray powder diffraction conditions:

According to the XRPD patterns and characteristic peak data of fig. 2 and 4, the strongest characteristic absorption peak at 26.37 ° is shown as a 2θ angle, the error is ±0.2°, and the relative absorption intensity is 100%.

Further, the crystal form A has characteristic peaks at 17.41 degrees, 18.43 degrees, 21.01 degrees, 21.20 degrees, 24.21 degrees, 25.14 degrees and 26.37 degrees, the error is +/-0.2 degrees, and the relative absorption intensity is more than 50 percent.

Specifically, the crystal form A has characteristic peaks at 14.86 degrees, 15.94 degrees, 17.41 degrees, 18.43 degrees, 21.01 degrees, 21.20 degrees, 24.21 degrees, 25.14 degrees, 26.37 degrees, 27.14 degrees and 28.84 degrees, the error is +/-0.2 degrees, the relative absorption intensity is more than 30 percent, and other substances can be distinguished in detail to represent the 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° degrees and 35.82 degrees, the error is +/-0.2 degrees, the relative absorption intensity is more than 10 percent, and other substances can be distinguished in more detail to represent the crystal form. While other weak absorption peaks may vary significantly due to experimental operating errors, other absorption peaks may be considered unnecessary absorption peaks by those skilled in the art when characterizing the present crystalline form.

Fig. 5 is a schematic diagram of a comparison of single crystal simulated XRPD patterns of compound a in example 1 and XRPD patterns of compound a crystalline form a in example 2. According to fig. 5 and the above table Pos [ ° 2θ ] it can be seen that the crystalline forms obtained in example 1 and examples 2, 5 are the same crystalline form.

Example 6 evaluation of the inhibition of the Activity of the Compounds of the invention on human myeloperoxidase (hMPO) in vitro

50. Mu.L of 2 Xsubstrate (composed of 3'- (p-aminophenyl) fluoroscein and H ₂O₂, 3' - (p-aminophenyl) fluoroscein and H ₂O₂ each at Km concentration, from Invitrogen, A36003) were added to a black opaque low protein adsorbed 96 well plate (from Perkinelmer, 6005270), and after thoroughly pipetting and mixing, 5. Mu.L of 2. Mu.g/mL human myeloperoxidase (from PLANTA NATURAL, 700-03-001) was added and mixed immediately using an enzyme-labelling instrument to read fluorescence values (once every 20 s) using kinetic mode at an Ex/Em:488/520nm wavelength. The compound inhibitors were calculated at different concentrations as inhibition (%) = [1- (SlopeControl-Slopecpd)/SlopeControl-SlopeBlank) ]. 100, wherein SlopeBlank is the slope of 0-5 min (Ex/Em: 544/620nm read/reaction time t) for the no-compound control and MPO wells, slopeControl is the slope of 0-5 min (linear reaction stage) for the no-compound wells (containing 1% dmso), and Slopecpd is the slope of 0-5 min for the compounds. A 4-parameter nonlinear regression curve fit was performed with the logarithmic value of the compound concentration as the abscissa and the inhibition ratio as the ordinate, and IC ₅₀ values (y=bottom+ (Top-Bottom)/(1+10 ((LogIC ₅₀ -X) × HillSlope)) were calculated, where Hillslope represents the slope of the fitted curve and IC ₅₀ represents the half-inhibition concentration.

The compounds of Table 2 have inhibitory activity against the peroxidation cycle of human myeloperoxidase (hMPO) in vitro

Example Compounds	IC⁵⁰(nM)
		6	8.02
6A	4.64

Conclusion the compounds of the invention have an inhibitory effect on hMPO activity.

EXAMPLE 7 rat pharmacokinetic Studies of Compounds of the invention

7.1 Experimental materials

SD rats, male, 180-350g, purchased from Zhejiang Vitolihua laboratory animal technologies Co.

Reagents DMSO (dimethyl sulfoxide), PEG-400 (polyethylene glycol 400), physiological saline, acetonitrile, formic acid and propranolol (internal standard) are all commercially available.

Instrument-Siemens LC-MS (Ultimate 3000UPLC,TSQ QUANTUMN ULTRA triple quadrupole mass spectrometry).

7.2 Experimental methods

Weighing the compound, dissolving in DMSO-PEG-400-normal saline (5:60:35, v/v/v) system, taking 200 mu L of venous blood in EDTA-K2 anticoagulant tube at 15min, 30min, 1h, 2h, 5h, 7h and 24h (5 min from group iv), centrifuging at 10000rpm for 2min, and taking blood plasma to be detected by freezing at-80 ℃. A certain amount of test sample was precisely weighed and dissolved to 2mg/mL with DMSO to be used as a stock solution. Accurately absorbing a proper amount of compound stock solution, and adding acetonitrile to dilute the stock solution to prepare a standard series of solution. Accurately sucking 4 mu L of each standard series solution, adding the standard series solution into 36 mu L of blank plasma, uniformly mixing by vortex to prepare plasma samples with the plasma concentrations of 1,3,5, 10, 30, 100, 300, 1000 and 3000ng/mL, carrying out double-sample analysis on each concentration, and establishing a standard curve. Taking 30 mu L of plasma (5 min, 15min, 30min plasma dilution 5 times) and adding propranolol acetonitrile solution (internal standard, 50 ng/mL) to precipitate protein, adding 100 mu L of water, vortex mixing, centrifuging at 4000rpm for 5min, and taking supernatant for LC-MS analysis. LC-MS detection conditions were as follows:

Chromatographic column YMC-Triart C ₁₈ X3.0 mm I.D.S-3 μm,12nm

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

7.3 data processing

After LC-MS detection of blood concentration, the pharmacokinetic parameters were calculated using WinNonlin 6.1 software, non-compartmental model method, and the test results are shown in Table 3.

TABLE 3 pharmacokinetic results of the compounds of the invention on rats

From the experimental results in table 3, the compounds of the present invention are metabolized more slowly in rats, and have higher oral exposure and bioavailability.

EXAMPLE 8 enzyme-induced assay study of Compounds of the invention

8.1 Experimental materials and instruments

The materials were Williams 'Medium E (withoutphenol red) from Sigma Aldrich trade Inc., williams' Medium E, human recombinant insulin, glutamax and HEPES from Life Technologies, isotonic Percoll from GENERAL ELECTRIC, fetal bovine serum from Corning, dexamethasone from commercial sources, cellTiter-Fluor ^TM Cell Viability Assay kit 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 Assayprobe (20×, ACTB, VIC labeled) from AB (Appliedbiosystems), human primary hepatocytes from BioIVT.

Instrument QuantStudio.

8.2 Experimental design

Precisely weighing the tested compound, the positive inducer, the negative inducer and the internal standard to prepare corresponding concentration stock solution. The cell resuscitator and incubation, mRNA extraction, reverse transcription and fluorescent quantitative PCR related reagents and consumables were prepared.

Resuscitates the frozen primary hepatic cells, and inoculates the primary hepatic cells on a proper cell culture plate according to a certain density to carry out monolayer cell culture so as to meet the cell growth requirement.

To the corresponding wells, a solution (10. Mu.M) containing a positive/negative inducer or test substance freshly prepared with an incubation solution and pre-heated, two duplicate wells per concentration, was added, incubation was continued for 3 days and daily exchanges, and then the mRNA level of CYP enzyme was tested. The extraction of mRNA, reverse transcription and fluorescent quantitative PCR procedures were performed according to the relevant kit instructions.

8.3 Experimental results

The test compounds were tested for their induction of CYP enzyme mRNA levels as shown in table 4 below.

Table 4 Experimental results

From the experimental results of table 4, it is seen that the compounds of the present invention have reduced induction risk compared to the compound of example 3 (CN 201580065064.2).

EXAMPLE 9 Compound enzyme inhibition test study of the invention

9.1 Experimental materials and instruments

Materials, midazolam and testosterone were purchased from CERILLIANT and PANPHY, ketoconazole from MCE, and human liver microsomes from Corning. Instrument AB Sciex5500+.

9.2 Experimental design

The inhibition potential of the compounds against cytochrome P450 enzyme CYP3A was evaluated using a phosphate buffer (100 mM, 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 50 μm. The positive substrates in the incubation system were 1. Mu.M midazolam and 40. Mu.M testosterone (CYP 3A), respectively, and the concentration of the positive inhibitor ketoconazole (CYP 3A) in the incubation system was 0.05. Mu.M. Incubation was performed at 37 ℃ and the reaction was initiated by addition of NADPH solution with a final concentration of 1mM for 10 minutes, after the end of incubation all samples were precipitated with 300 μl of acetonitrile solution containing 3% formic acid and internal standard (0.5 μm tolbutamide) and the metabolite production of the labeled substrate was detected using UPLC-MS/MS to calculate the IC50 value for compound inhibition.

9.3 Experimental results

The IC50 values of the test compounds for CYP3A inhibition are shown in table 5 below.

TABLE 5 experimental results

From the experimental results in table 5, it is seen that the compounds of the present invention have a reduced risk of inhibition of CYP3A compared to the compound of example 3 (CN 201580065064.2).

EXAMPLE 10 stability Studies

A certain amount of form a of compound a obtained in example 2 was exposed to a humidity of 95% rh and a temperature of 50 ℃, and the stability of the sample was tested, sampling time was 0 day and 5 days, and HPLC purity and form of the sample were examined. The results of the study are shown in Table 6.

Table 6 study of stability of form a of compound a

As can be seen from the results of table 6, the stability of the crystalline form a of the compound a of the present invention is better.

Example 11 flowability comparison experiment

A certain amount of form a of compound a obtained in example 2 was weighed and placed in a 50ml measuring cylinder smoothly to a 20ml scale position, and the bulk density thereof was detected and calculated.

The bulk density of form A of compound A was calculated to be 0.48g/cm3.

From the above results, it can be seen that the crystalline form a of the compound a of the present invention has a good bulk density and good flowability.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A crystal form A of a compound A is characterized in that the crystal form A has characteristic peaks at 17.41 degrees, 18.43 degrees, 21.01 degrees, 21.20 degrees, 24.21 degrees, 25.14 degrees and 26.37 degrees in an X-ray diffraction diagram according to a 2 theta angle, the error is +/-0.2 degrees, and the structural formula of the compound A is shown as follows:

2. Form a of compound a according to claim 1, characterized in that it has characteristic peaks in the X-ray diffraction pattern at angles of 14.86 °, 15.94 °, 17.41 °, 18.43 °, 21.01 °, 21.20 °, 24.21 °, 25.14 °, 26.37 °, 27.14 ° and 28.84 ° expressed in terms of 2Θ, with an error of ± 0.2 °, preferably that it has characteristic peaks in the X-ray diffraction pattern at angles of 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 ° expressed in terms of 2Θ, with an error of ± 0.2 °.

3. Form a of compound a according to claim 1, characterized by an X-ray diffraction pattern of form a as shown in fig. 2 or fig. 4.

4. Form a of compound a according to claim 1, characterized in that the differential scanning calorimetry spectrum of form a has a peak of the endothermic peak at 304.0±5 ℃.

5. Form a of compound a according to claim 1, characterized by a differential scanning calorimetry spectrum of form a as shown in figure 3.

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

7. Process for the preparation of form a of compound a according to any one of claims 1 to 6, characterized in that it comprises the following steps:

B) And volatilizing the refined product of the compound A into a reaction bottle, adding a small amount of organic solvent, heating to saturation, filtering while the mixture is hot, and volatilizing the filtrate at room temperature to obtain a crystal form A of the compound A. The organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile and acetone, or mixture thereof

8. The preparation method according to claim 7, wherein the heating temperature in the step a) is 40-60 ℃ and the heating time is 1-2 days, and the heating temperature in the step b) is 40-60 ℃ and the heating time is 1-2 hours.

9. A pharmaceutical composition comprising form a of compound a according to any one of claims 1-6 and one or more pharmaceutically acceptable carriers.

10. Use of form a of compound a according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment of a myeloperoxidase-related disease, preferably selected from cardiovascular-related diseases.