Crystal form of dihydrothienopyrimidine derivative and use thereof

Abstract

Provided in the present application are a crystal form of a compound as represented by formula (I), a hydrate thereof, specific crystal forms thereof, a pharmaceutical composition containing same, and the use thereof. The crystal form of the compound as represented by formula (I) and the specific crystal forms thereof exhibit excellent crystallinity and chemical purity, are easy to prepare, have high yields, and have a good druggability potential.

Description

Crystallographic forms and uses of dihydrothiophene-pyrimidine derivatives

[0001] This application requires the applicant to have:

[0002] The priority right of the prior application filed with the China National Intellectual Property Administration on August 28, 2024, with patent application number 202411189361.5 and entitled "Crystal form of dihydrothiophenepyrimidine derivative and its use therein";

[0003] The full text of the prior application is incorporated herein by reference. Technical Field

[0004] This invention belongs to the field of medicinal chemistry, specifically relating to the crystalline form and uses of dihydrothiophenepyrimidine derivatives. Background Technology

[0005] Phosphodiesterases (PDEs) hydrolyze intracellular second messengers (cAMP, cyclic adenosine monophosphate or cGMP, cyclic guanosine monophosphate), degrading intracellular cAMP or cGMP and thus terminating the biochemical processes mediated by these second messengers. cAMP and cGMP play crucial regulatory roles in cellular activity, and their concentrations are primarily determined by the balance between the synthesis of nucleotide cyclases and the hydrolytic activity of phosphodiesterases (PDEs). PDEs are widely distributed in the human body, and their physiological functions are studied in multiple fields. Human PDE4 isoenzymes are classified into four subtypes: PDE4A, 4B, 4C, and 4D. PDE4 is involved in the hydrolysis of cAMP in various inflammatory cells; therefore, inhibiting PDE4 can suppress immune and inflammatory cells.

[0006] In addition, according to literature reports, PDE4 plays a role in controlling calcium-induced calcium release, and preferential inhibition of PDE4B can maintain the efficacy of treatment for pulmonary fibrosis.

[0007] Therefore, selective inhibitors of PDE4B are expected to become effective drugs for the treatment of idiopathic pulmonary fibrosis and progressive fibrotic interstitial lung disease by inhibiting both inflammation and fibrosis. Summary of the Invention

[0008] A crystal form of compound (I) with low hygroscopicity, good stability and suitable for industrial production is provided, which is suitable for production, storage and use as a raw material for pharmaceuticals.

[0009] In a first aspect, the present invention provides a compound of formula (I) in crystalline form.

[0010] On the other hand, this application provides hydrates of the compound represented by formula (I) in crystalline form, preferably monohydrates.

[0011] On the other hand, this application provides crystal form A of the compound shown in formula (I), which, when irradiated with Cu-Kα, exhibits characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.9±0.2°, 21.4±0.2°, and 26.3±0.2°.

[0012] In some embodiments of this application, the crystal form A described above, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 10.9±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, and 26.3±0.2°.

[0013] In some embodiments of this application, the crystal form A described above, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, and 26.3±0.2°.

[0014] In some embodiments of this application, the crystal form A described above, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, 26.3±0.2°, 27.3±0.2°, and 28.3±0.2°.

[0015] In some embodiments of this application, the crystal form A described above, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 15.7±0.2°, 16.3±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, 26.3±0.2°, 27.3±0.2°, 28.3±0.2°, and 30.7±0.2°.

[0016] In some embodiments of this application, the crystal form A described above, when subjected to Cu-Kα radiation, exhibits a characteristic diffraction peak in one or more of the following 2θ angles in its X-ray powder diffraction pattern: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 15.7±0.2°, 16.3±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 22.9±0.2°, 24.1±0.2°, 24.4±0.2°, 25.6±0.2°, 26.3±0.2°, 27.3±0.2°, 28.3±0.2°, and 30.7±0.2°.

[0017] In some embodiments of this application, the crystal form A described above, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in Figure 1.

[0018] In some embodiments of this application, the differential scanning calorimetry curve of the aforementioned crystal form A has an endothermic peak at 116±5℃.

[0019] In some embodiments of this application, the crystal form A described above has a DSC pattern substantially as shown in Figure 2.

[0020] In some embodiments of this application, the aforementioned crystal form A exhibits a weight loss of 3.7 ± 0.2% in its thermogravimetric analysis curve between room temperature and 120 ± 5 °C.

[0021] In some embodiments of this application, the crystal form A described above has a TGA pattern substantially as shown in Figure 3.

[0022] In some embodiments of this application, the monohydrate of the compound represented by formula (I) in crystalline form is selected from crystal form A.

[0023] On the other hand, this application provides a method for preparing a monohydrate of the above-mentioned crystal form A or the compound represented by formula (I), comprising the following steps:

[0024] (1) Add solvent I to the compound shown in formula (I) and dissolve it;

[0025] (2) Add solvent II to the solution obtained in step (1), stir, and precipitate solid.

[0026] In some embodiments of this application, solvent I is selected from one or more of acetonitrile, tetrahydrofuran, isopropanol, and ethyl acetate; solvent II is selected from one or two of water and n-heptane.

[0027] In some embodiments of this application, solvent I is selected from acetonitrile and solvent II is selected from n-heptane; or solvent I is selected from tetrahydrofuran and solvent II is selected from water; or solvent I is selected from isopropanol and solvent II is selected from n-heptane; or solvent I is selected from ethyl acetate and solvent II is selected from n-heptane.

[0028] In some embodiments of this application, the preparation method one further includes the following steps: after the solid is precipitated, centrifuge and dry overnight; the drying conditions are preferably vacuum drying at 40°C.

[0029] This application provides a second method for preparing the monohydrate of the compound represented by crystal form A or formula (I) above, comprising the following steps:

[0030] Organic solvent III was added to compound I, and the mixture was stirred, resulting in the precipitation of a solid.

[0031] In some embodiments of this application, organic solvent III is selected from one or more of isopropyl acetate, acetone, water, n-butanol, butyl acetate, n-heptane, tetrahydrofuran, dichloromethane, acetonitrile, butanone, and methyl tert-butyl ether; for example, organic solvent III is selected from a mixture of acetone and water, or a mixture of n-butanol, butyl acetate, and n-heptane, or a mixture of tetrahydrofuran and n-heptane, or a mixture of dichloromethane and n-heptane, or a mixture of butanone and methyl tert-butyl ether.

[0032] In some embodiments of this application, in preparation method two, the mixture is stirred for 2-4 days at a temperature of 10-60°C, or preferably at 20-50°C (e.g., room temperature, 40°C), for example, for 3 days.

[0033] In some embodiments of this application, the second preparation method further includes the following steps: after the solid is precipitated, centrifuge and dry overnight; the drying conditions are preferably vacuum drying at 40°C.

[0034] On the other hand, this application provides a crystalline composition comprising a hydrate of the compound represented by formula (I) in crystalline form, preferably a hydrate, or crystal form A of the compound of formula (I).

[0035] In some embodiments of this application, the hydrate or monohydrate or crystal form A accounts for more than 50%, 60%, 70%, 80%, 90%, or 95% of the weight of the crystalline composition.

[0036] On the other hand, this application provides a pharmaceutical composition comprising a compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, or the above-described crystalline composition.

[0037] In some embodiments of this application, the pharmaceutical composition described above comprises crystal form A of the compound shown in formula (I).

[0038] On the other hand, this application also provides a pharmaceutical composition comprising a compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, or the above-described crystalline composition, and a pharmaceutically acceptable carrier.

[0039] In some embodiments of this application, the above-mentioned pharmaceutical composition comprises crystal form A of the compound of formula (I) and a pharmaceutically acceptable carrier.

[0040] On the other hand, this application provides the use of the compound of formula (I) in its crystalline form, or the hydrate of the compound of formula (I) in its crystalline form, preferably a hydrate, as a drug or in the preparation of a drug.

[0041] In some embodiments of this application, the drug is used to prevent and / or treat PDE4B-mediated diseases, symptoms, or conditions. The PDE4B-mediated diseases, symptoms, or conditions are fibrosis-related diseases or immune-inflammatory-related diseases.

[0042] On the other hand, this application also provides the hydrate of the compound of formula (I) in crystalline form, or the hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the pharmaceutical composition thereof, for the prevention and / or treatment of PDE4B-mediated diseases.

[0043] On the other hand, this application also provides a method for preventing and / or treating PDE4B-mediated diseases, comprising: administering to a patient a therapeutically effective amount of the compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the pharmaceutical composition.

[0044] On the other hand, this application also provides the use of the crystalline form of the compound of formula (I) or the hydrate of the crystalline form of the compound of formula (I), preferably a hydrate, in combination with other therapeutic drugs such as fibrosis or immune inflammation in the preparation of a drug for treating fibrosis or immune inflammation.

[0045] On the other hand, this application also provides the use of the crystalline form of the compound of formula (I) or the hydrate of the crystalline form of the compound of formula (I), preferably a hydrate, in the preparation of drugs to improve other treatments such as fibrosis or immune inflammation.

[0046] On the other hand, this application also provides the use of other therapeutic drugs for treating fibrosis or immune inflammation in the preparation of drugs that improve the therapeutic effects of the compound of formula (I) in the above-mentioned crystalline form, the above-mentioned crystalline composition, crystal form A of the compound of formula (I), or the above-mentioned pharmaceutical composition on treating fibrosis or immune inflammation.

[0047] On the other hand, this application also provides a medicament comprising the compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the medicament composition, and other treatments such as fibrosis or immune inflammation. Preferably, the medicament is used to treat fibrosis or immune inflammation.

[0048] On the other hand, this application also provides a medicament comprising the crystalline form of the compound of formula (I) or a hydrate of the crystalline form of the compound of formula (I), preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the medicament composition, said medicament being used to improve the efficacy of other fibrotic or immune-inflammatory drugs in treating fibrosis or immune-inflammatory diseases.

[0049] On the other hand, this application also provides a medicament comprising other drugs for treating fibrosis or immune inflammation, said medicament being used to improve the effect of the crystalline composition, crystal form A of the compound of formula (I), or the hydrate of the crystalline composition of formula (I), preferably a hydrate, on the treatment of fibrosis or immune inflammation.

[0050] On the other hand, this application also provides a method for treating fibrosis or immune inflammation, comprising: administering a patient or subject a therapeutically effective amount of the compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the pharmaceutical composition.

[0051] On the other hand, this application also provides a method for treating fibrosis or immune inflammation, comprising: administering a patient or subject a therapeutically effective amount of the compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the pharmaceutical composition and other drugs for treating fibrosis or immune inflammation.

[0052] On the other hand, this application also provides a method for improving the efficacy of other drugs for treating fibrosis or immune inflammation, comprising: administering a therapeutically effective amount of the compound of formula (I) in crystalline form, or a hydrate of the compound of formula (I) in crystalline form, preferably a hydrate, the crystalline composition, crystal form A of the compound of formula (I), or the drug composition to a patient or subject.

[0053] On the other hand, this application also provides a method for improving the effect of the crystalline form of the compound of formula (I) or the hydrate of the crystalline form of the compound of formula (I), preferably a hydrate, on treating fibrosis or immune inflammation, comprising: administering a therapeutically effective amount of other drugs for treating such as fibrosis or immune inflammation to a patient or subject.

[0054] In any of the foregoing aspects, the PDE4B-mediated disease, symptom, or condition is a fibrosis-related disease or an immune-inflammatory-related disease; more preferably, the disease is selected from respiratory diseases, gastrointestinal diseases, inflammatory diseases, allergic diseases, autoimmune diseases, and cancer;

[0055] More preferably, the respiratory disease is selected from respiratory or pulmonary diseases accompanied by increased mucus production, respiratory inflammation and / or obstructive diseases; even more preferably, the respiratory disease is selected from idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, interstitial pneumonia, chronic obstructive pulmonary disease (COPD), α-antitrypsin deficiency, chronic sinusitis, asthma or chronic bronchitis.

[0056] Preferably, the gastrointestinal disease is selected from segmental ileitis, ulcerative colitis, or Crohn's disease;

[0057] Preferably, the inflammatory disease is selected from dry eye syndrome or glaucoma;

[0058] Preferably, the autoimmune disease is selected from diffuse connective tissue diseases such as systemic lupus erythematosus, atopic dermatitis, seborrheic dermatitis, psoriasis, urticaria, rheumatoid arthritis, multiple sclerosis, dermatomyositis, polymyositis, vasculitis, or Sjögren's syndrome.

[0059] More preferably, in some embodiments, the PDE4B-mediated disease is chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, asthma, or interstitial lung disease.

[0060] Definitions and Explanations

[0061] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. The phrase "the compound of formula (A) in crystalline form" as used in this application refers to the compound of formula (A) in crystalline form, including its anhydrous and solvent-free form, hydrate form, and solvate form.

[0062] The term "hydrate" refers to an associative compound formed by water molecules in stoichiometric or non-stoichiometric proportions with the compound represented by formula (A) of this application.

[0063] In the context of this application, the characteristic diffraction peaks, diffraction peaks and / or 2θ angle values ​​in X-ray powder diffraction patterns are all in degrees (°).

[0064] The term "crystalline composition" refers to a solid form comprising a hydrate, preferably a monohydrate, of the compound of formula (I) mentioned in this application, or crystal form A of the compound of formula (I). Furthermore, in addition to the crystal form specified in this application, the crystalline composition may optionally contain other crystal forms or other amorphous forms of the compound of formula (A), or impurities other than these substances. Those skilled in the art will understand that the sum of the contents of all components in the crystalline composition should be 100%.

[0065] The term "room temperature" refers to room temperature in the conventional sense of the art, generally 10 to 30°C, preferably 25°C ± 5°C.

[0066] In X-ray powder diffraction (XRD) patterns, the term "substantially" or "substantially as shown" refers to a substantially pure crystal form in which at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the peaks appear in the given pattern. Furthermore, as the content of a crystal form in a product gradually decreases, some diffraction peaks attributable to that crystal form in the XRD pattern may decrease due to factors such as the instrument's detection sensitivity. Additionally, for any given crystal form, the peak positions may have slight errors, which is well known in crystallography. For example, peak positions can shift due to variations in temperature during sample analysis, sample movement, or instrument calibration; the measurement error for the 2θ value is sometimes approximately ±0.3°, and typically approximately ±0.2°. Therefore, this error should be taken into account when determining each crystal structure, and the terms "substantially" or "substantially as shown in the figure" are also intended to cover such differences in diffraction peak positions, referring to ±0.3°, preferably ±0.2°.

[0067] In DSC or TGA spectra, the terms "substantially" or "substantially as shown" refer to the fact that, for the same compound and the same crystal form, the error in thermal transition onset temperature, endothermic peak peak temperature, exothermic peak peak temperature, melting point, weight loss onset temperature, or weight loss endpoint temperature, etc., is typically within about 5°C, and usually within about 3°C, in consecutive analyses. When describing a compound as having a given thermal transition onset temperature, endothermic peak peak temperature, exothermic peak peak temperature, melting point, weight loss onset temperature, or weight loss endpoint temperature, it refers to that temperature ±5°C.

[0068] As used herein, the term "prevention" means, when used for a disease or condition (e.g., cancer), that the compound or drug reduces the frequency of symptoms of the medical condition or delays the onset of the condition in a subject compared to a subject who has not been given the compound or drug (e.g., the combination product claimed in this application).

[0069] As used in this article, the term “treatment” means to reduce, alleviate or improve the symptoms of a disease or condition, improve underlying metabolic symptoms, suppress a disease or condition, such as preventing the development of a disease or condition, alleviating a disease or condition, causing the remission of a disease or condition, relieving the condition caused by a disease or condition, or preventing the symptoms of a disease or condition.

[0070] The terms "pharmaceutical-grade carrier" or "pharmaceuticalally acceptable excipient" refer to carriers or excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound.

[0071] The intermediate compounds of this application can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0072] The chemical reactions in the specific embodiments of this application are carried out in a suitable solvent, which must be suitable for the chemical changes and the reagents and materials required in this application. In order to obtain the compounds of this application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction process based on existing embodiments.

[0073] The present application will be described in detail below through embodiments, which are not intended to limit the present application in any way.

[0074] All solvents used in this application are commercially available and can be used without further purification.

[0075] Technical effect

[0076] The compounds of formula (I) provided in this application, as well as their specific crystal forms, have one or more of the following beneficial effects:

[0077] (1) The crystalline form of the compound shown in formula (I) has good properties and is easy to weigh, transfer, separate, purify and store.

[0078] (2) The compounds and specific crystal forms shown in formula (I) have good crystallinity;

[0079] (3) The crystalline form of the compound shown in formula (I) and its specific crystal form are easy to purify, filter and separate, and the preparation is simple and the yield is high;

[0080] (4) The preferred crystal form has good physical and chemical stability and good medicinal prospects. Attached Figure Description

[0081] Figure 1 shows the X-ray powder diffraction pattern of crystal form A in Example 1.

[0082] Figure 2 shows the DSC spectrum of crystal form A in Example 1.

[0083] Figure 3 shows the TGA spectrum of crystal form A in Example 1. Detailed Implementation

[0084] To better understand the content of this application, further explanation is provided below with reference to specific embodiments. However, the specific implementation methods are not intended to limit the content of this application. Test methods not specifying specific conditions in the following embodiments or test examples should be performed according to conventional methods and conditions, or as selected according to the product instructions. The raw materials used in this application can be prepared using methods in the prior art.

[0085] Preparation Example 1:

[0086] Synthesis of (R)-2-(3-(4-chlorophenyl)-3-methoxyazacyclobutane-1-yl)-4-((1-hydroxymethylcyclobutyl)amino)-6,7-dihydrothiopheno[3,2-d]pyrimidine 5-oxide:

[0087] Step 1: Preparation of (4-chlorophenyl)lithium:

[0088] Under nitrogen protection, 1-chloro-4-iodobenzene (4.18 g, 1 eq) was dissolved in anhydrous THF (50 mL) and stirred until dissolved. A 2.5 M n-butyllithium solution in hexane (1 eq) was added dropwise at -78 °C, and the reaction was continued at -78 °C with stirring for 0.5 hours to obtain the crude target compound. This was used directly in the next step.

[0089] Step 2: Preparation of tert-butyl 3-(4-chlorophenyl)-3-hydroxyazacyclobutane-1-carboxylate:

[0090] Under nitrogen protection, tert-butyl 3-oxoazacyclobutane-1-carboxylate (2 g, 1 eq) was dissolved in anhydrous THF (10 mL) and slowly added to the previous reaction solution at -78 °C. The mixture was stirred at -78 °C for 1 hour and detected by TLC. The reaction was quenched by adding saturated ammonium chloride solution (10 mL) to the reaction solution at 0 °C, and the crude product was purified. The crude product was added to petroleum ether (30 mL) and stirred at room temperature for 1 hour. After filtration, the filter cake was washed with petroleum ether (20 mL) to obtain the target compound (2.56 g). LCMS (ESI) [M+H-56] + =228.1.

[0091] Step 3: Preparation of tert-butyl 3-(4-chlorophenyl)-3-methoxyazacyclobutane-1-carboxylate:

[0092] Under nitrogen protection, tert-butyl 3-(4-chlorophenyl)-3-hydroxyazacyclobutane-1-carboxylate (500 mg, 1 eq) was dissolved in anhydrous DMF (5 mL). 60 wt% sodium hydride (2.05 eq) was added in an ice-water bath, and the mixture was stirred for 10 minutes. Iodomethane (5 eq) was then added, and stirring continued for 30 minutes. LC-MS analysis confirmed complete consumption of the reactants. The reaction was quenched by adding saturated NH4Cl aqueous solution. The mixture was then added to water (30 mL), extracted with methyl tert-butyl ether (20 mL × 2), and the organic phases were combined. The mixture was washed successively with water (20 mL) and saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude target compound (585 mg, which was used directly in the next step).

[0093] Step 4: Preparation of 3-(4-chlorophenyl)-3-methoxyazacyclobutane hydrochloride:

[0094] 585 mg of tert-butyl 3-(4-chlorophenyl)-3-methoxyazacyclobutane-1-carboxylate (the crude product from the previous step) was added to a 4M solution of ethyl acetate (5 mL) and stirred at room temperature for 0.5 hours. LCMS analysis confirmed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure, and 20 mL of methyl tert-butyl ether was added to the residue. The mixture was stirred at room temperature for 30 minutes, filtered, and the filter cake was washed with 10 mL of methyl tert-butyl ether to obtain the target compound (380 mg). LCMS (ESI) [M+H] + =198.1.

[0095] Step 5: Preparation of (R)-2-(3-(4-chlorophenyl)-3-methoxyazacyclobutane-1-yl)-4-((1-hydroxymethylcyclobutyl)amino)-6,7-dihydrothiopheno[3,2-d]pyrimidine 5-oxide:

[0096] (R)-2-chloro-4-((1-(hydroxymethyl)cyclobutyl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-oxide (100 mg, 1 eq), 3-(4-chlorophenyl)-3-methoxyazacyclobutane hydrochloride (1.22 eq), and N,N-diisopropylethylamine (3.98 eq) were added to a mixed solution of water (0.5 mL) and THF (2.5 mL), and the mixture was stirred at 70 °C for 2 hours. LCMS analysis confirmed complete consumption of the starting materials. The reaction mixture was then added to water (20 mL), filtered, and purified by Prep-HPLC (C18, 10 mmol / L ammonium bicarbonate aqueous solution, acetonitrile) to obtain the target compound (82 mg, yield 52%). LCMS (ESI) [M+H] + =449.3; 1 H NMR (400MHz, DMSO-d6) δ7.49 (s, 5H), 4.84 (t, J = 5.7Hz, 1H), 4.22 (s, 3H), 3.77-3.65 (m, 2H), 3.41 (dt, J = 16.6, 8.0Hz, 2H), 3.27-3.1 5(m,2H),2.99-2.82(m,3H),2.34(d,J=10.5Hz,2H),2.26(d,J=10.0Hz,1H),2.14(t,J=10.2Hz,2H),1.74(dq,J=19.6,10.3Hz,2H).

[0097] Example 1: Preparation of crystal form A of compound (I)

[0098] Weigh 30 mg of the compound sample of formula I obtained in Preparation Example 1, add 0.4 mL of acetonitrile to dissolve it, add 5.0 mL of n-heptane dropwise to the acetonitrile solution, stir, and a solid precipitates. Stir for 0.5 h, centrifuge, and vacuum dry at 40 °C overnight. Take the solid for XRPD characterization, which shows that it is a crystalline solid (crystal form A). The obtained solid has good crystallinity. The spectrum is shown in Figure 1, and its XRPD diffraction peak data are shown in Table 1.

[0099] A sample was subjected to DSC-TGA testing. The DSC chromatogram showed an endothermic peak at 116.04℃ (Figure 2). The TGA chromatogram showed a weight loss of 3.702% between room temperature and 120±5℃ (Figure 3), indicating that crystal form A contains one molecule of water of crystallization. HPLC analysis of the sample showed a relative peak area of ​​99.33%.

[0100] Table 1: XRPD diffraction peak data of crystal form A in Example 1 Note: Peaks with a relative intensity >10.0% are listed in the table.

[0101] Example 2: Preparation of crystal form A of compound (1)

[0102] 2-1 Weigh 30 mg of the compound sample of formula I obtained in Preparation Example 1, add a certain amount of solvent, stir the slurry at a certain temperature for 3 days, centrifuge, and vacuum dry at 40°C overnight.

[0103] Table 2-1 Results of Crystal Form A Preparation Experiment in Example 2

[0104] 2-2 Weigh 30 mg of the compound sample of formula I obtained in Preparation Example 1, add solvent 1 and dissolve it. Add solvent 2 dropwise to the sample solution formed after dissolving in solvent 1, stir, precipitate solid, stir for 0.5 h, centrifuge, and vacuum dry at 40 °C overnight.

[0105] Table 2-2 Results of Crystal Form A Preparation Experiment in Example 2

[0106] Test Example 1: Solid stability test of crystal form A of compound (I)

[0107] Weigh an appropriate amount of the sample from Example 1 (Crystal Form A) into a vial. Place it in the open and protected from light for 7 days under high temperature (60℃±2℃, <30%RH), long-term (25℃±5℃, 60%RH±5%), and accelerated (40℃±2℃, 75%RH±5%) conditions. Take samples for purity testing and X-ray powder diffraction to investigate the stability of Example 1 (Crystal Form A) under different conditions. The results are shown in Table 3.

[0108] Table 3. Experimental results of solid stability

[0109] The data show that Example 1 (Crystal Form A) maintained chemical and crystal form stability in the solid stability test.

[0110] Test Example 2: DVS experiment of different crystal forms of compound (I)

[0111] Sample 1 (crystal form A) was placed in the DVS sample chamber for testing. X-ray powder diffraction was performed on the DVS-treated sample, and the results are shown in Table 4.

[0112] Table 4. DVS Experiment Results

[0113] Example 1: In vitro evaluation of the inhibitory activity of the compound against PDE4B2 and PDE4D2 enzymes

[0114] 1. Experimental materials and instruments:

[0115] 2. Experimental steps:

[0116] 2.1 Preparation and treatment of compounds

[0117] 2.1.1 Preparation of DMSO stock solution for compound: Dissolve the compound powder in 100% DMSO to prepare a 10 mM stock solution, and shake with a shaker to completely dissolve the compound.

[0118] 2.1.2 Storage of DMSO stock solution of compound: The DMSO stock solution of compound is stored in a desiccator at room temperature.

[0119] 2.1.3 Preparation of working stock solutions: a) Dilute the reference compound roflumilast in DMSO solution to 60 μM, then perform 4-fold dilutions to create 10 concentration gradients. b) Dilute the analyte compound in DMSO solution to 200 μM, then perform 3-fold dilutions. c) Prepare a 200X positive control (60 μM roflumilast) and a 200X carrier control (100% DMSO). d) Centrifuge the test plate at 1000 rpm for 1 minute.

[0120] 2.2 Compound Testing

[0121] 1) Use an Echo 550 to transfer 20 nL of compound dilution to each well of the detection plate.

[0122] 2) Seal the test plate and centrifuge it at 1000 rpm for 1 minute.

[0123] 3) Prepare 2X PDE4B2 / PDE4D2 in refrigerated PDE detection buffer.

[0124] 4) Add 2 μL of 2X PDE4B2 / PDE4D2 to each well of the detection plate (prepared in step b).

[0125] 5) Seal the test plate and allow it to equilibrate at room temperature for 10 minutes.

[0126] 6) Prepare 2X Cyclic-3',5'-AMP in PDE detection buffer.

[0127] 7) Add 2 μl of 2X Cyclic-3',5'-AMP (prepared in step f) to each well of the detection plate (prepared in step e) to start the reaction. Incubate at room temperature for 60 minutes.

[0128] 8) Add 4 μL of AMP-Glo ​​Reagent I and incubate at room temperature for 60 minutes.

[0129] 9) Add 8 μL of AMP detection solution. Incubate at room temperature for 60 minutes.

[0130] 10) Read the RLU signal on the Envision 2105 tablet reader.

[0131] Experimental results:

[0132] PDE4B2 enzyme inhibitory activity test results

[0133] PDE4B2 enzyme inhibition IC 50 Value: A≤2nM; 2nM <B≤5nM;5nM<C≤20nM;20nM<D≤50nM。

[0134] PDE4D2 / PDE4B2 selectivity (= IC50 of PDE4D2 enzyme inhibition) 50 IC50 value of PDE4B2 enzyme inhibition 50 (Ratio of values): 5 <A≤10;2<B≤5;C≤2。

[0135] Reference 1 is a compound It is publicly available in CN103889970B.

[0136] Experimental Example 2. Test of the inhibitory activity of the compound on LPS-induced secretion of TNFα by human PBMCs

[0137] Frozen hPBMCs were rapidly thawed in a 37°C water bath. Cells were transferred to complete culture medium (RPMI 1640 + 10% FBS + 1% P / S). Centrifuged at 1000 pm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium (RPMI 1640 + 10% FBS + 1% P / S), then gently pipetted to mix. Trypan blue staining was performed for cell counting. Cells were counted at a rate of 5 × 10⁻⁶ cells / mL. 4 Cells were inoculated into 96-well cell culture plates with 100 μL / well of complete culture medium. The compound to be screened was prepared to a final concentration of 4× and added to the cells at 50 μL / well. Pre-incubation was performed for 30 min. Control wells were prepared without the compound. The final concentration of LPS stimulation was 10 ng / ml, diluted 4-fold, and added to the cells at 50 μL / well. Control wells were prepared without LPS. Cells were incubated for another 24 hours, and 10% supernatant was collected for analysis. The collected supernatant was analyzed using the Human TNF-α kit (VAL105G) from R&D Company. The inhibitory activity of the compound provided in this invention against LPS-induced human TNFα secretion was determined according to the above method.

[0138] Test results of the compound's inhibitory activity against LPS-induced TNFα in human PBMCs

[0139] Experimental results show that the compound of formula (I) of this invention has excellent inhibitory activity against the secretion of TNFα by human PBMCs, and can better inhibit the secretion of the inflammatory factor TNFα in human PBMCs, thus exhibiting good anti-inflammatory effects.

[0140] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The crystalline form of the compound shown in formula (I), 2. The compound of formula (I) in crystalline form according to claim 1, characterized in that, It is a hydrate of the compound shown in formula (I), preferably a monohydrate.

3. The compound of formula (I) in crystalline form according to claim 1, characterized in that, It is the crystal form A of the compound shown in formula (I). Using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.6±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.9±0.2°, 21.4±0.2°, 26.3±0.2°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.6±0.2°, 10.9±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 26.3±0.2°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, 26.3±0.2°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, 26.3±0.2°, 27.3±0.2°, 28.3±0.2°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.6±0.2°, 10.9±0.2°, 11.6±0.2°, 15.2±0.2°, 15.7±0.2°, 16.3±0.2°, 16.8±0.2°, 17.6±0.2°, 18.5±0.2°, 19.4±0.2°, 19.9±0.2°, 21.4±0.2°, 24.1±0.2°, 25.6±0.2°, 26.3±0.2°, 27.3±0.2°, 28.3±0.2°, 30.7±0.2°; Alternatively, it has an X-ray powder diffraction pattern that is essentially as shown in Figure 1.

4. The compound of formula (I) in crystalline form according to claim 3, characterized in that, The differential scanning calorimetry curve of crystal form A has an endothermic peak at 116±5℃. Alternatively, it has a DSC spectrum that is essentially as shown in Figure 2; Alternatively, its thermogravimetric analysis curve shows a weight loss of 3.7 ± 0.2% between room temperature and 120 ± 5 °C; Alternatively, it has a TGA pattern that is essentially as shown in Figure 3.

5. A method for preparing the compound of formula (I) in crystalline form according to any one of claims 1-4, wherein the preparation method is selected from preparation method one and includes the following steps: (1) Add solvent I to the compound shown in formula (I) and dissolve it; (2) Add solvent II to the solution obtained in step (1), stir, and precipitate solid; And / or, solvent I is selected from one or more of acetonitrile, tetrahydrofuran, isopropanol, and ethyl acetate; solvent II is selected from one or two of water and n-heptane; And / or, solvent I is selected from acetonitrile and solvent II is selected from n-heptane; or solvent I is selected from tetrahydrofuran and solvent II is selected from water; or solvent I is selected from isopropanol and solvent II is selected from n-heptane; or solvent I is selected from ethyl acetate and solvent II is selected from n-heptane; And / or, the preparation method one further includes the following steps: after the solid is precipitated, centrifuge and dry overnight; the drying conditions are preferably vacuum drying at 40°C; Alternatively, the preparation method may be selected from preparation method two, and include the following steps: Organic solvent III was added to compound I, and the mixture was stirred, resulting in the precipitation of a solid. And / or, Organic Solvent III is selected from one or more of isopropyl acetate, acetone, water, n-butanol, butyl acetate, n-heptane, tetrahydrofuran, dichloromethane, acetonitrile, butanone, and methyl tert-butyl ether; for example, Organic Solvent III is selected from a mixture of acetone and water, or a mixture of n-butanol, butyl acetate, and n-heptane, or a mixture of tetrahydrofuran and n-heptane, or a mixture of dichloromethane and n-heptane, or a mixture of butanone and methyl tert-butyl ether; And / or, in the second preparation method, the mixture is stirred for 2-4 days at a temperature of 10-60°C, or preferably at 20-50°C (e.g., room temperature, 40°C); And / or, the second preparation method further includes the following steps: after the solid is precipitated, centrifuge and dry overnight; the drying conditions are preferably vacuum drying at 40°C.

6. A crystalline composition comprising the compound of formula (I) in the crystalline form as described in any one of claims 1-4; preferably, the crystalline form accounts for more than 50% by weight of the crystalline composition; more preferably more than 60%; more preferably more than 70%; more preferably more than 80%; more preferably more than 90%; more preferably more than 95%.

7. A pharmaceutical composition comprising a compound of formula (I) in crystalline form as described in any one of claims 1-4, or a crystalline composition as described in claim 6; said pharmaceutical composition comprising an optional pharmaceutically acceptable carrier; preferably, said pharmaceutical composition comprising crystal form A as described in claim 3, and an optional pharmaceutically acceptable carrier.

8. The use of the compound of formula (I) in crystalline form according to any one of claims 1-4, the crystalline composition according to claim 6, or the pharmaceutical composition according to claim 7 as a medicament or in the preparation of a medicament; preferably, the medicament is used for the prevention and / or treatment of PDE4B-mediated diseases, symptoms, or conditions; the PDE4B-mediated diseases, symptoms, or conditions are fibrosis-related diseases or immune-inflammatory-related diseases; more preferably, the diseases are selected from respiratory diseases, gastrointestinal diseases, inflammatory diseases, allergic diseases, autoimmune diseases, and cancer; more preferably, the respiratory diseases are selected from respiratory or... Lung diseases; more preferably, the respiratory diseases are selected from idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, interstitial pneumonia, chronic obstructive pulmonary disease (COPD), α-antitrypsin deficiency, chronic sinusitis, asthma, or chronic bronchitis; preferably, the gastrointestinal diseases are selected from segmental ileitis, ulcerative colitis, or Crohn's disease; preferably, the inflammatory diseases are selected from dry eye syndrome or glaucoma; preferably, the autoimmune diseases are selected from systemic lupus erythematosus, atopic dermatitis, seborrheic dermatitis, psoriasis, urticaria, rheumatoid arthritis, multiple sclerosis, dermatomyositis, polymyositis, vasculitis, or Sjögren's syndrome, etc., diffuse connective tissue diseases; more preferably, the PDE4B-mediated diseases are chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, asthma, or interstitial lung disease.

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