Crystal form of alkyl carboxylic acid compound and application thereof

Abstract

The invention discloses a crystal form of an alkyl carboxylic acid compound and application of the crystal form, and particularly discloses a crystal form of a compound shown in a formula (I) and application of the crystal form. # imgabs0 #

Description

A crystal form of an alkyl carboxylic acid compound and its application

[0001] The present invention claims the following priority

[0002] Application number: CN202211394422.2, application date: November 8, 2022. Technical Field

[0003] The present invention relates to a crystal form of an alkyl carboxylic acid compound and applications thereof, and particularly to a crystal form of a compound represented by formula (I) and applications thereof. Background Art

[0004] Soluble guanylate cyclase (sGC) is a receptor enzyme for the second messenger nitric oxide (NO) and is widely present in several cell types, including muscle, epithelium, neurons, and endothelial cells. sGC is a heterodimer composed of an α1 or α2 subunit bound to a β1 subunit. The β1 subunit contains a heme prosthetic group and is a key signal transduction enzyme in the NO-sGC-cGMP signaling pathway. Under physiological conditions, NO binds to the heme prosthetic group of sGC, activating it to catalyze the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine monophosphate (cGMP).

[0005] cGMP is an important secondary messenger molecule that activates multiple downstream effector molecules, such as phosphodiesterases (PDEs), cyclic nucleotide-gated ion channels (CNGs), and protein kinases (PKGs), triggering a series of downstream cascade reactions. These cascades play crucial physiological roles in the gastrointestinal, circulatory, and nervous systems, including promoting vascular and smooth muscle relaxation, inhibiting platelet aggregation, vascular remodeling, apoptosis, and inflammation, and participating in neurotransmission. Therefore, sGC stimulators have potential therapeutic applications for cardiovascular diseases (heart failure, pulmonary hypertension, angina pectoris, myocardial infarction) and fibrotic diseases (renal fibrosis, systemic sclerosis). Under these pathological conditions, prolonged oxidative stress can lead to oxidation of the heme prosthetic group of sGC (from ferrous to ferric). This prevents sGC enzyme activation by NO and may exacerbate disease progression. This can further lead to endothelial dysfunction, atherosclerosis, hypertension, stable or unstable angina pectoris, thrombosis, myocardial infarction, stroke, or worsening erectile dysfunction. Therefore, activation of oxidized sGC to produce cGMP makes it possible to treat and / or prevent such diseases.

[0006] sGC activators are NO-independent and heme-independent, directly activating the sGC-cGMP signaling pathway. This has the potential to provide benefits in many diseases caused by defective NO pathway signaling, particularly following oxidative stress.

[0007] To address the unmet market and clinical demand for such soluble guanylate cyclase stimulators, the present invention provides a novel class of crystalline compounds and methods for their preparation. These compounds can act as activators of soluble guanylate cyclase, exhibiting excellent in vitro stimulatory activity and favorable pharmacokinetic properties.

[0008] Summary of the Invention

[0009] The present invention provides a crystalline form A of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.540±0.200°, 16.100±0.200° and 17.601±0.200°,

[0010] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) above has characteristic diffraction peaks at the following 2θ angles: 11.041±0.200°, 14.381±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200° and 22.903±0.200°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) above has characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, 8.113±0.200°, 8.799±0.200°, 11.041±0.200°, 14.381±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200°, 22.903±0.200° and 23.682±0.200°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, 7.198±0.200°, 8.113±0.200°, 8.799±0.200°, 11.041±0.200°, 13.863±0.200°, 14.38 1±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200°, 19.523±0.200°, 22.903±0.200°, 23.682±0.200° and 24.940±0.200°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, and / or 7.198±0.200°, and / or 8.113±0.200°, and / or 8.799±0.200°, and / or 9.914±0.200°, and / or 11.041±0.200°, and / or 11.8 80±0.200°, and / or 13.863±0.200°, and / or 14.381±0.200°, and / or 15.540±0.200°, and / or 16.100±0.200°, and / or 17.063±0.200°, and / or 17.601±0.200°, and / or 18.281±0.200°, and / or 18.799±0.200°, and / or 19 .523±0.200°, and / or 20.333±0.200°, and / or 21.164±0.200°, and / or 21.761±0.200°, and / or 22.176±0.200°, and / or 22.903±0.200°, and / or 23.682±0.200°, and / or 24.940±0.200°, and / or 25.823±0.200°, and / or Or 26.539±0.200°, and / or 28.124±0.200°, and / or 28.756±0.200°, and / or 30.261±0.200°, and / or 31.477±0.200°, and / or 32.582±0.200°, and / or 36.303±0.200°, and / or 38.141±0.200°, and / or 38.742±0.200°.

[0014] In some embodiments of the present invention, the XRPD pattern of Form A of the compound of formula (I) is shown in FIG1 .

[0015] In some embodiments of the present invention, the XRPD pattern analysis data of Form A of the compound of formula (I) above are shown in Table 1.

[0016] Table 1. XRPD pattern analysis data of Form A of the compound of formula (I)

[0017] In some embodiments of the present invention, the differential scanning calorimetry curve of the crystal form A of the compound of formula (I) has an onset value of an endothermic peak at 164.69°C±5.00°C.

[0018] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (I) is shown in FIG2 .

[0019] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (I) above shows no weight loss before the melting point.

[0020] In some embodiments of the present invention, the TGA spectrum of Form A of the compound of formula (I) is shown in FIG3 .

[0021] The present invention also provides the use of the crystal form A of the compound of formula (I) in the preparation of a drug for treating chronic kidney disease.

[0022] Technical Effects

[0023] The compound of the present invention has significant in vitro stimulating activity on guanylate cyclase and excellent pharmacokinetic properties. The compound of the present invention has a stable crystal form, has no or almost no hygroscopicity, and is less affected by light and heat.

[0024] Definition and Description

[0025] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0026] The intermediate 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 replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0027] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0028] For any given crystalline form, the relative intensity of the diffraction peaks can change due to factors such as the preferred orientation caused by factors such as the crystal morphology, which is well known in the art of crystallography. Where there is a preferred orientation effect, the peak intensity changes, but the position of the diffraction peaks of the crystal form cannot be changed. In addition, for any given crystal form, there may be slight errors in the positions of the peaks, which is also well known in the art of crystallography. For example, due to changes in temperature when analyzing the sample, movement of the sample, or calibration of the instrument, the position of the peak can move, and the measurement error of the 2θ value is sometimes about ± 0.2 degrees. Therefore, it is well known to those skilled in the art that this error should be taken into account when determining each crystalline structure.

[0029] DSC measures the transition temperatures of crystals when they absorb or release heat due to changes in their crystalline structure or melting. For the same crystalline form of the same compound, the error in thermal transition temperatures and melting points in consecutive analyses is typically within about 5°C or 3°C. When we say that a compound has a given DSC peak or melting point, this refers to the DSC peak or melting point ±5°C or ±3°C. DSC provides an auxiliary method for distinguishing different crystalline forms. Different crystalline forms can be identified by their different transition temperature characteristics. It should be noted that for mixtures, their DSC peaks or melting points may vary over a wider range. In addition, since decomposition is associated with the melting process of a substance, the melting temperature is related to the heating rate.

[0030] For the same crystal form, the TGA weight loss temperature may vary due to factors such as the measuring instrument, measuring method / conditions, etc. For any specific crystal form, the weight loss temperature may have an error of about ±5°C, or about ±3°C.

[0031] It should be noted that during the preparation of drug crystal forms, during the contact between drug molecules and solvent molecules, external conditions and internal factors may cause the solvent molecules to form a co-crystal with the compound molecules and remain in the solid material, which is difficult to avoid, thereby forming solvates, specifically including stoichiometric solvates and non-stoichiometric solvates. All such solvates are included within the scope of the present invention.

[0032] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0033] Unless otherwise specified, DSC spectra are exothermic upwards.

[0034] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0035] All solvents used in the present invention were commercially available and used without further purification.

[0036] Compounds are named according to the conventional nomenclature in this field or Software naming, commercially available compounds use supplier catalog names.

[0037] X-ray powder diffractometer (XRPD) method of the present invention

[0038] Instrument model: D2 Phaser X-ray diffractometer

[0039] Test method: Approximately 3 to 10 mg of sample was used for XRPD analysis.

[0040] The detailed XRPD parameters are as follows:

[0041] Light pipe: Cu, kα,

[0042] Light tube voltage: 30kV, light tube current: 10mA

[0043] Divergence slit: 0.6mm

[0044] Detector slit: 0.075mm

[0045] Anti-scatter slit: 1mm

[0046] Scanning range: 3-40 degrees

[0047] Step size: 0.02 degrees

[0048] Scan time: 0.2s

[0049] Differential Scanning Calorimeter (DSC) method of the present invention

[0050] Instrument model: TA DSC 250 differential scanning calorimeter

[0051] A sample (1-5 mg) was placed in a covered aluminum crucible under the protection of 50 mL / min dry nitrogen and tested by heating from 25°C to the set test temperature at a heating rate of 10°C / min.

[0052] Thermogravimetric analysis (TGA) method of the present invention

[0053] Instrument model: TA TGA 550 thermogravimetric analyzer

[0054] Test method: Take a sample (2-5 mg) and place it in an uncovered aluminum crucible under the protection of 60 mL / min dry nitrogen for testing. The method is: room temperature to 300°C, and the heating rate is 10°C / min.

[0055] Dynamic Vapor Sorption (DVS) method of the present invention

[0056] Instrument model: SMS (Intrinsic Plus) dynamic vapor adsorption instrument

[0057] Test conditions: Take a sample (30-50 mg) and place it in the DVS sample tray for testing.

[0058] The detailed DVS parameters are as follows:

[0059] Temperature: 25℃

[0060] Balance: dm / dt<0.002% / min (balance time 1h)

[0061] Drying: Dry at 0% RH for 120 minutes

[0062] Humidity cycle: 0%-95%-0%RH

[0063] Gradient: 0-90% RH, 10% RH; 90%-95% RH, 5% RH

[0064] RH (%) test step range: 0%-95%

[0065] The classification of moisture absorption evaluation is shown in Table 2:

[0066] Table 2. Hygroscopicity evaluation classification Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 is an XRPD spectrum of Form A of the compound of formula (I) using Cu-Kα radiation;

[0068] FIG2 is a DSC spectrum of Form A of the compound of formula (I);

[0069] FIG3 is a TGA spectrum of Form A of the compound of formula (I);

[0070] FIG4 is a DVS spectrum of Form A of the compound of formula (I);

[0071] Figure 5 is a diagram of the molecular structure of the compound of formula (I);

[0072] FIG6 is an ellipsoid diagram of the molecular structure of the compound of formula (I). DETAILED DESCRIPTION

[0073] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0074] Example 1: Preparation of compound of formula (I)

[0075] Step 1: Synthesis of compound SM1-7

[0076] Dissolve compound SM1-6 in dichloromethane (120 mL), cool to 0°C under nitrogen, add oxalyl chloride (21.32 g) dropwise, and then add N,N-dimethylformamide (0.2 mL) dropwise. Stir at 20°C for 1 hour. Quench a drop of the reaction solution with methanol. TLC (petroleum ether / ethyl acetate = 1:1) shows residual starting material and the appearance of new spots. Cool to 0°C, add additional oxalyl chloride (21.32 g), and stir at 20°C for 1 hour. Concentrate the reaction solution under reduced pressure at 40°C, dissolve in dichloromethane (100 mL), and continue concentrating under reduced pressure.

[0077] Methoxymethylamine hydrochloride (9.83 g) was added to dichloromethane (100 mL), followed by diisopropylethylamine (43.42 g). The mixture was cooled to 0°C under nitrogen, and a solution of the above compound in dichloromethane (130 mL) was added dropwise. The mixture was stirred at 20°C for 12 hours. The reaction mixture was poured into water (100 mL), and the organic phase was separated and collected. The aqueous phase was extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0 to 6 / 1, volume ratio) to obtain compound SM1-7.

[0078] 1H NMR (400MHz, CDCl3) δppm 2.22 (s, 6H) 3.19 (s, 3H) 3.31 (s, 3H) 3.66 (s, 3H).

[0079] Step 2: Synthesis of compound SM1-8

[0080] Compound SM1-7 (11.5 g) was added to tetrahydrofuran (120 mL), and the atmosphere was replaced with nitrogen three times. The mixture was cooled to -70°C under nitrogen protection, and diisobutylaluminum hydride (1 M solution in toluene, 74.51 mL) was added dropwise. After the addition was complete, the mixture was stirred at -70°C for 0.5 hours. After the reaction was complete, the reaction mixture was poured into dilute hydrochloric acid (3 mol / L, 100 mL) and stirred for 0.5 hours. The mixture was then extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (200 mL), and dried over anhydrous sodium sulfate. Filtered, the filtrate was concentrated under reduced pressure at 45°C to yield compound SM1-8.

[0081] Step 3: Synthesis of compound SM1

[0082] Tert-butyl diethylphosphonoacetate (15.60 g) was added to tetrahydrofuran (100 mL), the atmosphere was replaced with nitrogen three times, and the mixture was cooled to 0°C under a nitrogen atmosphere. Potassium tert-butoxide (1 M solution in tetrahydrofuran, 68.01 mL) was added dropwise, and the mixture was stirred for 0.5 hours. A solution of compound SM1-8 (7.8 g) in tetrahydrofuran (150 mL) was added dropwise at 0°C, and the mixture was stirred at 20°C for 12 hours. After the reaction was completed, saturated aqueous ammonium chloride (200 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure at 45°C. The crude product was isolated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1 / 0 to 97 / 3, volume ratio) to obtain compound SM1.

[0083] 1 H NMR (400MHz, CDCl3) δ: 6.95 (d, J = 15.6 Hz, 1H), 5.75 (d, J = 15.2 Hz, 1H), 3.31 (s, 3H), 1.99 (s, 6H), 1.49 (s, 9H).

[0084] Step 4: Synthesis of compound 1

[0085] Compounds SM1 (6 g) and SM2 (6.78 g) were added to tetrahydrofuran (100 mL) and isopropanol (50 mL). Potassium hydroxide (1.80 g) and 1,5-cyclooctadiene (289.39 mg) were added, and the atmosphere was replaced with nitrogen three times. The temperature was raised to 60°C under nitrogen protection, and the previously prepared catalyst (1,5-cyclooctadiene rhodium chloride dimer (329.75 mg) and 2,2-bis(diphenylphosphino)-1,1-binaphthyl (95.78 mg) were added to tetrahydrofuran (10 mL) and stirred for 10 minutes) was added. The mixture was stirred at 60°C for 12 hours. After the reaction was completed, the reaction solution was poured into water (200 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure at 45°C. The crude product was separated and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 19 / 1 to 9 / 1, volume ratio) to obtain a racemic compound. Separation by SFC (column type: DAICEL CHIRALCEL OJ (250 mm × 50 mm, 10 μm); mobile phase: [0.1% aqueous ammonia, isopropanol]%: 15%-15%) afforded compound 1.

[0086] 1 H NMR (400MHz, CDCl3) δ: 7.14 (d, J=8.0Hz, 1H), 6.53 (d, J=1.6Hz, 1H), 6.45 (dd, J= 2.0,8.2Hz,1H),3.32-3.11(m,4H),2.62-2.43(m,2H),1.65(s,6H),1.33(s,9H).

[0087] SFC analysis method [column type: Chiralcel OJ-3 (150 mm × 4.6 mm, 3 μm); mobile phase: phase A: CO2, phase B: isopropanol (containing 0.1% isopropylamine); gradient (B%): 10%-50% over 5 min]. The peak time of compound 1 was 1.834 min, and the retention time of its isomer was 1.960 min.

[0088] Step 5: Synthesis of compound 2

[0089] SM3 (2.5 g) was added to dichloromethane (25 mL) and cooled to 0°C under nitrogen. Oxalyl chloride (2.38 g) was added dropwise, followed by N,N-dimethylformamide (0.6 mL). After the addition was complete, the mixture was stirred at 20°C for 1 hour. The reaction solution was concentrated to dryness under reduced pressure at 40°C, dissolved in dichloromethane (10 mL), and concentrated again to dryness under reduced pressure. The resulting oil was dissolved in dichloromethane (30 mL) and added dropwise to a solution of compound 1 (3 g) and N,N-diisopropylethylamine (2.75 g, 21.31 mmol) in dichloromethane (30 mL) at 0°C. After the addition was complete, the mixture was stirred at 20°C for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was isolated and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 19 / 1 to 9 / 1, volume ratio) to obtain compound 2.

[0090] 1 H NMR(400MHz, DMSO-d6)δ:12.44-11.49(m,1H),9.82(s,1H),7.52-7.40(m,4H),7.37-7.26(m,2H),6.95(dd,J=2.0,8.4Hz,1H),4.13(d,J=10.6Hz ,1H),3.47-3.38(m,1H),3.28-3.23(m,1H),3.09(s,3H),2.56(d,J=6.4 Hz, 1H), 2.48-2.40 (m, 1H), 1.54 (q, J = 9.4Hz, 6H), 0.80 (d, J = 7.0Hz, 3H).

[0091] Step 6: Synthesis of compound of formula (I)

[0092] Ethyl acetate hydrochloride (4M, 100 mL) was added to compound 2 (4.7 g) and stirred at 20°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 19 / 1-17 / 3, volume ratio) to obtain the target compound (I).

[0093] Example 2: Preparation of Crystal Form A of the Compound of Formula (I)

[0094] Add n-hexane (20 mL) to a dry three-necked flask and add compound (I) (1.84 g) in batches. After the addition is complete, heat to 50-55°C and stir for 12 hours. Filter the reaction mixture, collect the solid, and dry in vacuo at 45°C to obtain Form A of compound (I).

[0095] 1H NMR(400MHz,DMSO-d6)δppm 12.11(s,1H)9.82(s,1H)7.42-7.51(m,4H)7.30-7.38(m,2H)6.95(dd,J=8.4,2.06Hz,1H)4.13(d,J=10.8Hz,1H)3.37-3.43(m ,1H)3.27(dd,J=8.8,6.44Hz,1H)3.10(s,3H)2.54-2.62(m,1H)2.43-2.48(m,1H)1.54(q,J=9.2Hz,6H)0.80(d,J=6.8Hz,3H).

[0096] Example 3: Hygroscopicity study of Form A of the compound of formula (I)

[0097] Experimental Materials:

[0098] SMS (Intrinsic Plus) Dynamic Vapor Sorption Analyzer

[0099] Experimental methods:

[0100] 10-15 mg of Form A of the compound of formula (I) was placed in a DVS sample tray for testing.

[0101] Experimental results:

[0102] The DVS spectrum of Form A of the compound of formula (I) is shown in FIG4 , with ΔW=0.13%.

[0103] Experimental conclusion:

[0104] At 25° C., the crystal form A of the compound of formula (I) has a moisture absorption weight gain of 0.13% at 80% RH compared to the initial 0% RH, and the sample has no or almost no hygroscopicity.

[0105] Example 4: Solid Stability Test of Form A of the Compound of Formula (I)

[0106] In accordance with the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 9001), the stability of Form A of the compound of formula (I) under high temperature (60°C, open), high humidity (room temperature / relative humidity 92.5%, open) and strong light (5000 lx, sealed) conditions was investigated.

[0107] Weigh 15 mg of Form A of the compound of formula (I) and place it in a thin layer at the bottom of a glass sample vial. For samples stored under high temperature and high humidity conditions, seal the vial with aluminum foil and poke small holes in the foil to ensure sufficient contact between the sample and ambient air. For samples stored under strong light conditions, seal the vial with a screw cap. Samples stored under different conditions were sampled and analyzed (XRPD) on days 5, 10, and 1 month after exposure. The results were compared with the initial test results at day 0. The test results are shown in Table 3 below:

[0108] Table 3. Solid stability test results of Form A of the compound of formula (I)

[0109] Conclusion: Crystal form A of the compound of formula (I) has good stability under high temperature, high humidity and strong light conditions.

[0110] Example 5: Single crystal X-ray diffraction analysis of the compound of formula (I)

[0111] X-ray single crystal diffraction method of the present invention

[0112] Instrument model: Single crystal X-ray diffractometer (SC-XRD) (Rigaku Oxford Diffraction XtaLAB Synergy-S)

[0113] Instrument: Rigaku Oxford Diffraction XtaLAB Synergy-S four-circle diffractometer

[0114] Area detector: HyPix-6000HE

[0115] Cryogenic system: Oxford Cryostream 800

[0116] Light source: Cu, 50W,

[0117] Distance from crystal to CCD detector: d = 35 mm

[0118] Tube voltage: 50kV

[0119] Tube current: 1mA.

[0120] The diffraction experiment collected 95826 diffraction points, of which 5150 were independent diffraction points (Rint=0.1139). The diffraction collection range 2=7.056 to 133.196, and the diffraction index range was -29≤h≤29, -13≤k≤13, -12≤l≤12. The structure was solved using SHELXT (Sheldrick, GM2015. ActaCryst.A71, 3-8), and the structure was refined using SHELXL (against F2) (Sheldrick, GM2015. ActaCryst.C71, 3-8). Among the 5150 independent diffraction points, the parameters involved in the structure refinement were 340. After refinement, R1=0.0566, wR2=0.1252. The residual electron density values ​​were 0.36 and

[0121] Single crystal growth process: 5 mL of a 7:5 methanol / water solution was added to the sample under ultrasonic heating at 50°C. Insoluble solids were removed by filtration. The clarified sample solution was placed in an 8 mL semi-sealed vial and stored in a place protected from light and vibration. The solution slowly evaporated at room temperature. On the 14th day, colorless flaky crystals were obtained.

[0122] Conclusion: The detected crystals are colorless flakes (0.30×0.20×0.05mm3) and belong to the orthorhombic system P21212 space group. α=90°, β=90°, γ=90°, Z = 4. Calculated density Dc = 1.278 g / cm3, number of electrons per unit cell F(000) = 1168.0, linear absorption coefficient μ(Cu Kα) = 2.466 mm–1, diffraction experimental temperature T = 150.01(10) K. Data refinement was performed with solvent subtraction.

[0123] Single crystal X-ray diffraction analysis revealed that each unit cell contains one molecule of the compound of formula (I) and one molecule of solvent (water). The molecular structure of the compound of formula (I) is shown in Figure 5 , and the molecular structure ellipsoid is shown in Figure 6 . Crystal structure data and parameters of the compound of formula (I) are shown in Tables 4, 5, 6, 7, and 8.

[0124] Table 4. Crystal data of compound of formula (I)

[0125] Table 5. Atomic coordinates of the crystals of the compound of formula (I) (×10 4 ) and the equivalent isotropic shift parameter

[0126] Table 6. Bond lengths of compounds of formula (I)

[0127] Table 7. Bond angles (°) of compounds of formula (I)

[0128] Table 8. Torsion angles (°) of compounds of formula (I)

[0129] Biological testing

[0130] Test Example 1. In vitro activity test

[0131] 1. cGMP expression test based on lnCap cells

[0132] 1. Experimental Procedure

[0133] 1) Solution preparation

[0134] 10% BSA (bovine serum albumin)

[0135] 10 g of BSA was dissolved in 100 mL of double distilled water (ddH2O) to obtain 10% BSA.

[0136] 10mM ODQ stock solution

[0137] 1 mg of ODQ powder was weighed and dissolved in 534 μl of DMSO to obtain a 10 mM ODQ solution, which was then aliquoted and stored in a -20°C refrigerator.

[0138] Washing Buffer (50 mL)

[0139] Assay Buffer (50 mL)

[0140] Detection Buffer

[0141] a) Add 50 μL of cGMP-D2 (D2-labeled cyclic GMP) to 1 mL of lysis buffer and mix well.

[0142] b) Add 50 μL of anti-cGMP cryptate (Eu 3+ The cryptate-labeled anti-cyclic GMP antibody was added to 1 mL of lysis buffer and mixed evenly.

[0143] 2) Compound dilution

[0144] (1) Dilute the compound to 10 mM with DMSO.

[0145] (2) Perform a gradient dilution of the compound. Dilute each compound into 10 concentration gradients and add 100 nL to a 96-well microplate.

[0146] 3) Preparation of LNCap cells

[0147] (1) LNCap culture medium: RPMI1640 + 10% fetal bovine serum + 1% double antibody

[0148] (2) Preheat the phosphate buffer, trypsin, and culture medium used in the cell passaging process in a 37°C water bath.

[0149] (3) Remove the cells from the 37°C 5% CO2 incubator and remove the old culture medium from the culture flask with a pipette.

[0150] (4) Pipette 5 mL of phosphate buffer into the culture flask to rinse the cells, and then discard the liquid.

[0151] (5) Pipette 3 mL of pancreatic enzyme into the culture flask, shake it, discard the liquid, and place the culture flask in the incubator.

[0152] (6) After about 2 minutes, remove the culture flask and observe that all cells have separated. Then, pipette 9 mL of culture medium into the culture flask and pipette it several times to transfer the cell suspension to a 50 mL centrifuge tube.

[0153] (7) Pipette 0.7 mL of cell suspension into a counting cup and count on a ViCell XR. Centrifuge the remaining cells at 1000 rpm for 5 min and discard the supernatant.

[0154] (8) Add 10 mL of washing buffer to wash the cells, centrifuge at 1000 rpm for 5 min, and remove the supernatant.

[0155] (9) Add assay buffer and adjust the cell concentration to 3×10 6 / mL.

[0156] 4) Preparation and addition of OQD solution

[0157] (1) Take 10mM ODQ stock solution 1:1000 and add it to the cell solution

[0158] (2) After thorough mixing, add 10 μL / well into the microplate.

[0159] 5) Prepare cGMP standard curve

[0160] (1) Dilute the 1 mM cGMP stock solution to 10 μM with assay buffer. Then perform a 4-fold serial dilution to 11 concentration gradients.

[0161] (2) Add 10 μL / well of the diluted cGMP to the microplate.

[0162] 6) Add detection reagent and read the plate

[0163] (1) Transfer 5 μL / well of cGMP-D2 to a 384-well microplate. Transfer 5 μL / well of anti-cGMP cryptate to a 96-well microplate. Centrifuge at 1500 rpm for 1 min.

[0164] (2) Incubate at room temperature for 1 hour.

[0165] (3) Use Envision to read 665 / 615.

[0166] 7) Data Analysis

[0167] (1) cGMP standard curve: A standard curve was prepared using Graphpad Prism based on the cGMP concentration and the 665 / 615 ratio.

[0168] (2) Conversion of HTRF (homogeneous time-resolved fluorescence) ratio (665 / 615) to cGMP concentration: In Graphpad Prism, copy the HTRF ratio (665 / 615) to the ratio column of the cGMP standard curve, run the analysis "Log inhibitor vs response-variable slope", select "interpolate", and convert the HTRF ratio (665 / 615) to cGMP concentration.

[0169] (3) Compound activation curve: The curve was drawn based on the converted cGMP concentration and the compound concentration using the “Log agonist vs response-variable slope” analysis method in Graphpad Prism.

[0170] Table 9 MEC values ​​of the sGC stimulating activity of the compounds of the present invention

[0171] MEC: Minimum effective concentration that stimulates cGMP production (threefold greater than the basal value) in lnCap cells.

[0172] Experimental conclusion: The compound of the present invention can effectively stimulate sGC and increase cGMP levels.

[0173] Test Example 2. In vitro hepatocyte metabolic stability study

[0174] 1. Purpose: The purpose of this study is to evaluate the stability of the compound in different species of hepatocytes.

[0175] 2. Experimental steps:

[0176] Prepare several 96-well sample precipitation plates, named T0, T15, T30, T60, T90, T0-MC, T90-MC, and blank matrix. Remove the recovery medium and incubation medium in advance and place them in a 37°C water bath to preheat. Remove the frozen hepatocytes of different species from the liquid nitrogen tank and immediately immerse them in a 37°C water bath (about 90 seconds). After the frozen part is thawed and loosened, pour them into centrifuge tubes containing 40mL of recovery medium and gently invert to resuspend the cells in the recovery medium. Centrifuge at 100×g for 5 minutes at room temperature, remove the supernatant, resuspend the hepatocytes with an appropriate volume of incubation medium, and calculate the cell viability using trypan blue staining. Add 198 μL of hepatocyte suspension (0.51×106 cells / mL) to a preheated incubation plate. For the culture medium control group, add 198 μL of incubation medium without hepatocytes to the T0-MC and T120-MC incubation plates. All incubation plates are preincubated in a 37°C incubator for 10 minutes. Then, add 2 μL of the test and control compound working solutions, mix thoroughly, and immediately place the incubation plate on a shaker in the incubator. Start the timer to begin the reaction. Prepare two replicates for each compound at each time point. Incubation conditions are 37°C, saturated humidity, and 5% CO2. In the test system, the final concentration of the test article is 1 μM, the final concentration of the control is 3 μM, the final concentration of the hepatocytes is 0.5×106 cells / mL, and the final concentration of the total organic solvent is 0.96%, of which the final concentration of DMSO is 0.1%. At the end of the incubation at the corresponding time point, the incubation plate was removed and 25 μL of the mixture of compound and control compound and cells was added to the sample plate containing 125 μL of stop solution (acetonitrile solution containing 200 ng / mL tolbutamide and labetalone). For the blank sample plate, 25 μL of incubation medium without hepatocytes was directly added. After sealing all sample plates, shake them on a shaker at 600 rpm for 10 minutes and centrifuge at 3220 × g for 20 minutes. The supernatants of the test and control substances were diluted with ultrapure water at a ratio of 1:3. All samples were mixed and analyzed by LC / MS / MS.

[0177] The experimental results are shown in Table 10

[0178] Table 10 Stability of the compounds of the present invention in different species of hepatocytes

[0179] T 1 / 2 : half-life, CL int(liver) : Hepatic clearance;

[0180] Experimental conclusion: The compound of the present invention has good stability in human hepatocytes, and has moderate clearance rate and half-life.

[0181] Experimental Example 3: In vivo PK study of Form A of the compound of formula (I) in SD male rats

[0182] Experimental purpose: To study the in vivo PK of the test compound in SD male rats

[0183] Experimental materials: Two 7-8 week old male Sprague Dawley rats (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.)

[0184] Experimental Procedure: The pharmacokinetic profile of the compound was tested in rodents following oral administration using a standard protocol. The candidate compound was formulated into a homogenous suspension (the oral administration solvent was 0.5% MC + 0.2% Tween 80 / H2O). A single oral administration of Form A of the compound of Formula (I) at a concentration of 1 mg / mL was performed. Animals were weighed prior to administration, with the initial weight at 230-240 g. The dose volume was calculated based on body weight, and the animals were administered orally (10 mg / kg). Approximately 0.2 mL of whole blood was collected by jugular vein puncture (or other appropriate blood collection site) at the designated time. The actual blood collection time was recorded in the experimental log. The acceptable error for the collection time was ±1 minute for those within 1 hour of administration and ±5% of the theoretical time for other time points. The supernatant was separated and the plasma sample was obtained by centrifugation at 3200 g for 10 minutes at 4°C. The plasma was transferred to a pre-cooled, labeled commercial centrifuge tube containing K2-EDTA, quickly frozen in dry ice, and then stored in an ultra-low temperature freezer at -70±10°C / -60°C or lower. The plasma drug concentration was quantitatively analyzed by LC-MS / MS analysis. The plasma drug concentration data of the metabolites of the compound of the present invention were processed using a non-compartmental model using WinNonlin Version 6.3 or above (Pharsight) pharmacokinetic software. The relevant pharmacokinetic parameters, such as peak concentration (C max ), half-life (T 1 / 2 ), area under the drug-time curve (AUC), time to peak (T max ), etc. The experimental results are shown in Table 11.

[0185] Table 11. In vivo pharmacokinetic results of Form A of the compound of formula (I)

[0186] Experimental conclusion: Crystal form A of the compound of formula (I) has excellent pharmacokinetic properties.

Claims

1. Crystal form A of the compound represented by formula (I), It is characterized in that The X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 15.540±0.200°, 16.100±0.200° and 17.601±0.200°.

2. The crystalline form A of the compound of formula (I) according to claim 1, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 11.041±0.200°, 14.381±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200° and 22.903±0.200°.

3. The crystalline form A of the compound of formula (I) according to claim 2, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, 8.113±0.200°, 8.799±0.200°, 11.041±0.200°, 14.381±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200°, 22.903±0.200° and 23.682±0.200°.

4. The crystal form A of the compound of formula (I) according to claim 3, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, 7.198±0.200°, 8.113±0.200°, 8.799±0.200°, 11.041±0.200°, 13.863±0.200°, 14.381 ±0.200°, 15.540±0.200°, 16.100±0.200°, 17.601±0.200°, 18.281±0.200°, 18.799±0.200°, 19.523±0.200°, 22.903±0.200°, 23.682±0.200° and 24.940±0.200°.

5. The crystal form A of the compound of formula (I) according to claim 4, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.441±0.200°, and / or 7.198±0.200°, and / or 8.113±0.200°, and / or 8.799±0.200°, and / or 9.914±0.200°, and / or 11.041±0.200°, and / or 11.88 0±0.200°, and / or 13.863±0.200°, and / or 14.381±0.200°, and / or 15.540±0.200°, and / or 16.100±0.200°, and / or 17.063±0.200°, and / or 17.601±0.200°, and / or 18.281±0.200°, and / or 18.799±0.200°, and / or 19 .523±0.200°, and / or 20.333±0.200°, and / or 21.164±0.200°, and / or 21.761±0.200°, and / or 22.176±0.200°, and / or 22.903±0.200°, and / or 23.682±0.200°, and / or 24.940±0.200°, and / or 25.823±0.200°, and / or Or 26.539±0.200°, and / or 28.124±0.200°, and / or 28.756±0.200°, and / or 30.261±0.200°, and / or 31.477±0.200°, and / or 32.582±0.200°, and / or 36.303±0.200°, and / or 38.141±0.200°, and / or 38.742±0.200°.

6. Crystal Form A of the compound of formula (I), Its XRPD pattern is shown in Figure 1.

7. The crystal form A of the compound of formula (I) according to any one of claims 1 to 6, wherein the differential scanning calorimetry curve thereof has an endothermic peak onset at 164.69°C ± 5°C.

8. The crystal form A of the compound of formula (I) according to claim 7, whose DSC spectrum is shown in Figure 2.

9. The crystal form A of the compound of formula (I) according to any one of claims 1 to 6, whose TGA spectrum is shown in Figure 3.

10. Use of the crystal form A of the compound of formula (I) according to any one of claims 1 to 9 in the preparation of a drug for treating chronic kidney disease.

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