A pharmaceutical salt of isoquinolinone compound, a crystal form and a preparation method thereof

Abstract

The present disclosure relates to a pharmaceutically acceptable salt of isoquinolinone compound, a crystal form and a preparation method thereof. Specifically, the present disclosure provides a fumarate salt, a sulfate salt and a hydrochloride salt of a compound as shown in formula (I), and a crystalline form.

Description

Technical Field

[0001] This disclosure relates to a pharmaceutically acceptable salt, crystal form, and preparation method of an isoquinolinone compound. Specifically, it provides fumarate, sulfate, and hydrochloride salts of the compound as shown in Formula I, as well as crystalline forms. Background Technology

[0002] Phosphodiesterases (PDEs) belong to a superfamily of enzymes, comprising 11 families, each involved in different signal transductions and regulating various physiological processes. PDE3 hydrolyzes both cAMP and cGMP, but its hydrolytic capacity for cAMP is approximately ten times that for cGMP. There are two genotypes of PDE3: PDE3A and PDE3B, located on chromosomes 11 and 12, respectively. Due to differences in the start codon, PDE3A can be further divided into three subtypes: PDE3A1, PDE3A2, and PDE3A3. These are mainly distributed in the heart, platelets, vascular smooth muscle, and oocytes, and play a role in regulating myocardial contractility, platelet aggregation, vascular smooth muscle contraction, oocyte maturation, and renin release. PDE3B has only one subtype, PDE3B1, which is mainly distributed in adipocytes, hepatocytes, spermatocytes, and the pancreas. It primarily participates in regulating the signal transduction of insulin, insulin-like growth factor, and leptin, playing a crucial role in metabolic diseases such as obesity and diabetes. Selective PDE3 inhibitors mainly include cilostazol, cilostazolamide, milrinone, amrinone, enoxidone, and cyanidizole.

[0003] For example, amrinone can inhibit PDE3 activity, increase intracellular cAMP concentration in cardiomyocytes, and increase intracellular calcium levels. 2+ The concentration is adjusted to fully exert its positive inotropic effect. Simultaneously, amrinone can directly act on vascular smooth muscle cells, exhibiting excellent vasodilatory effects, increasing myocardial contractility, reducing pulmonary artery pressure, and restoring cardiopulmonary function. It holds significant value in the treatment of chronic cor pulmonale complicated by heart failure. Furthermore, cilostazol is clinically used for the treatment of platelet aggregation, pulmonary hypertension (PAH), chronic obstructive pulmonary disease (COPD), intermittent claudication, and cerebral microvascular diseases.

[0004] On the other hand, PDE4 exhibits high specificity for cAMP, with four subtypes: PDE4A, PDE4B, PDE4C, and PDE4D. PDE4 participates in physiological and pathological processes related to the promotion of monocyte and macrophage activation, neutrophil infiltration, vascular smooth muscle proliferation, vasodilation, and myocardial contraction, influencing central nervous system function, cardiovascular function, the inflammatory / immune system, and cell adhesion. PDE4 plays a major regulatory role in the expression of pro-inflammatory and anti-inflammatory mediators; PDE4 inhibitors can suppress the release of harmful mediators from inflammatory cells.

[0005] Developing new molecules with both PDE3 and PDE4 inhibitory activities would have the bronchodilatory effect of β-adrenergic receptor agonists and the anti-inflammatory effect of inhaled corticosteroids. The dual-target complementary function would be more effective than a single target.

[0006] For example, RPL554 (9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,la]isoquinolin-4-one) is a dual PDE3 / PDE4 inhibitor, disclosed in WO00 / 58308. Recent Phase II clinical data show that it can significantly improve bronchiectasis and symptoms in patients with COPD, while the drug is well tolerated with no significant adverse events, such as cardiac problems, nausea, and diarrhea, which are all mild. The safety profile and its "limited systemic exposure" are encouraging.

[0007]

[0008] Developing new molecules with both PDE3 and PDE4 inhibitory activities would have the bronchodilatory effect of β-adrenergic receptor agonists and the anti-inflammatory effect of inhaled corticosteroids. The dual-target complementary function would be more effective than a single target.

[0009] PCT / CN2022 / 090175 provides a compound as shown in Formula I, chemically named 9,10-dimethoxy-2-[[2-(2-oxo-imidazolin-1-yl)-ethyl]-(2,4,6-trimethyl-phenyl)-amino]-6,7-dihydro-pyrimidinyl[6,1-a]isoquinoline-4-one, which exhibits good PDE3 and PDE4 inhibitory activity.

[0010] Summary of the Invention

[0011] This disclosure provides pharmaceutically acceptable salts of compounds represented by formula (I), wherein the pharmaceutically acceptable salts are selected from fumarates, sulfates, and hydrochlorides, and the chemical name of the compound represented by formula (I) is 9,10-dimethoxy-2-[[2-(2-oxo-imidazolin-1-yl)-ethyl]-(2,4,6-trimethyl-phenyl)-amino]-6,7-dihydro-pyrimidinyl[6,1-a]isoquinoline-4-one.

[0012]

[0013] In some embodiments, the stoichiometry of the compound represented by formula (I) with an acid molecule or acid ion is 1:0.1 to 1:3, preferably 1:0.2 to 1:2, and most preferably 1:0.5, 1:1, or 1:2. In some embodiments, the stoichiometry of the compound represented by formula (I) with fumaric acid is 1:0.5. In some embodiments, the stoichiometry of the compound represented by formula (I) with sulfate is 1:1. In some embodiments, the stoichiometry of the compound represented by formula (I) with chloride ions is 1:1.

[0014] This disclosure provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I), comprising the step of forming a salt of the compound of formula (1) with an acid. In some embodiments, the solvent used in the salt-forming reaction is selected from one or more of isopropanol, acetonitrile, ethanol, and tetrahydrofuran.

[0015] In some embodiments, the method for preparing the aforementioned pharmaceutically acceptable salt also includes steps such as evaporating solvents or stirring to crystallize, filtering, and drying.

[0016] This disclosure provides a pharmaceutical composition prepared from the aforementioned pharmaceutically acceptable salt.

[0017] This disclosure provides a pharmaceutical composition comprising the aforementioned pharmaceutically acceptable salt or a pharmaceutically acceptable salt prepared by the aforementioned method, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] This disclosure provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned pharmaceutically acceptable salt, or a pharmaceutically acceptable salt of a compound of formula (I) prepared by the aforementioned method, with a pharmaceutically acceptable carrier, diluent, or excipient.

[0019] This disclosure provides the use of a pharmaceutically acceptable salt of the compound represented by formula (I) above, or a pharmaceutically acceptable salt prepared by the aforementioned method, or the aforementioned composition, or the composition prepared by the aforementioned method, in the preparation of a medicament for the prevention and / or treatment of PDE-related conditions.

[0020] This disclosure provides the use of a pharmaceutically acceptable salt of the compound shown in formula (I), or a pharmaceutically acceptable salt prepared by the aforementioned method, or the aforementioned composition, or the composition prepared by the aforementioned method, in the preparation of a medicament for the prevention and / or treatment of asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

[0021] This disclosure provides the α-crystal form of the fumarate of formula (I), with X-ray powder diffraction patterns expressed as diffraction angles 2θ, showing characteristic peaks at 11.5, 14.6, 15.3, 16.5, 18.3, 22.2, and 24.3. In some embodiments, the α-crystal form of the fumarate of formula (I) shows characteristic peaks at 11.5, 14.6, 15.3, 16.5, 18.3, 20.7, 21.1, 22.2, 24.3, and 27.1. In some embodiments, the α-crystal form of the fumarate of formula (I) shows characteristic peaks at 8.2, 11.5, 14.6, 15.3, 16.5, 18.3, 19.4, 20.7, 21.1, 22.2, 24.3, 27.1, and 29.1. In some embodiments, the X-ray powder diffraction pattern of the α-crystal form of the fumarate of formula (I), expressed as a diffraction angle 2θ, is as follows: Figure 1 As shown.

[0022] This disclosure further provides a method for preparing the a-crystal form of the fumarate of the compound shown in formula (I), comprising: method 1, a) mixing the compound shown in formula (I), solvent I and fumaric acid, and heating to dissolve, wherein solvent I is selected from one or more of isopropanol, acetonitrile, tetrahydrofuran and ethanol, b) cooling to crystallize.

[0023] In some embodiments, the volume (μl) of the solvent used in this disclosure can be 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments, it can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 200. In some embodiments, the method for preparing the α-crystal form of fumarate described in this disclosure further includes steps such as filtration, washing, or drying.

[0024] This disclosure provides an α-crystal form of the sulfate of formula (I), with an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, showing characteristic peaks at 7.4, 9.1, 11.1, 16.3, 18.1, 23.3, and 24.7. In some embodiments, the α-crystal form of the sulfate of formula (I) shows characteristic peaks at 7.4, 9.1, 11.1, 14.0, 16.3, 17.1, 18.1, 22.4, 23.3, and 24.7. In some embodiments, the α-crystal form of the sulfate of formula (I) shows characteristic peaks at 7.4, 9.1, 11.1, 14.0, 16.3, 17.1, 18.1, 21.8, 22.4, 23.3, 23.7, 24.7, and 28.9. In some embodiments, the X-ray powder diffraction pattern of the sulfate of the compound shown in formula (I), expressed as a diffraction angle 2θ, is as follows: Figure 3 As shown.

[0025] This disclosure further provides a method for obtaining the α-crystal form of the sulfate of the compound shown in formula (I), comprising: a) mixing the compound shown in formula I with isopropanol and sulfuric acid solution, heating to dissolve, and b) cooling to crystallize.

[0026] In some embodiments, the volume (μl) of the solvent used in this disclosure can be 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments, it can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 200. In some embodiments, the method for preparing the α-crystal form of sulfate described in this disclosure further includes steps such as filtration, washing, or drying.

[0027] This disclosure provides the α-crystal form of the hydrochloride salt of the compound shown in formula (I), with X-ray powder diffraction patterns expressed as diffraction angles of 2θ, exhibiting characteristic peaks at 6.7, 10.2, 11.7, 13.3, 15.8, 23.9, and 26.2. In some embodiments, the α-crystal form of the hydrochloride salt of the compound shown in formula (I) exhibits characteristic peaks at 6.7, 10.2, 11.7, 13.3, 15.8, 19.0, 23.9, 26.2, and 27.7. In some embodiments, the α-crystal form of the hydrochloride salt of the compound shown in formula (I) exhibits characteristic peaks at 6.7, 10.2, 11.7, 13.3, 14.6, 15.8, 18.6, 19.0, 23.9, 24.6, 26.2, and 27.7.

[0028] This disclosure further provides a method for obtaining the a-crystal form of the hydrochloride salt of the compound shown in formula (I), comprising the steps of: a) mixing the compound shown in formula (I) with solvent III and hydrochloric acid solution, and heating to dissolve, wherein solvent III is selected from one or more of isopropanol, acetonitrile, ethanol, and tetrahydrofuran; b) cooling to crystallize.

[0029] In some embodiments, the volume (μl) of the solvent used in this disclosure may be 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 200. In some embodiments, the method for preparing the α-crystal form of hydrochloride described in this disclosure further includes steps such as filtration, washing, or drying.

[0030] This disclosure also provides pharmaceutical compositions prepared from the crystal form of the pharmaceutically acceptable salt of the compound shown in formula (I).

[0031] This disclosure also provides a pharmaceutical composition comprising the crystal form of the aforementioned pharmaceutically acceptable salt and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0032] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form of the aforementioned pharmaceutically acceptable salt with a pharmaceutically acceptable carrier, diluent, or excipient.

[0033] This disclosure also provides the use of the crystal form of the aforementioned pharmaceutically acceptable salt, or the aforementioned composition, or the composition prepared by the aforementioned method, in the preparation of a medicament for the prevention and / or treatment of PDE-related conditions.

[0034] This disclosure also provides the use of the crystal form of the aforementioned pharmaceutically acceptable salt, or the aforementioned composition, or the composition prepared by the aforementioned method, in the preparation of medicaments for the prevention and / or treatment of asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

[0035] The determination of the stoichiometry of the compound of formula (I) described in this disclosure with the base or acid molecule is subject to a certain error. Generally, ±10% is within a reasonable error range. The error may vary to a certain extent depending on the context in which it is used, but this variation shall not exceed ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, preferably ±5%.

[0036] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place after rounding), and can be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0037] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "Guiding Principles on Hygroscopicity of Drugs" in Part IV of the 2015 edition of the Chinese Pharmacopoeia,

[0038] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0039] Extremely hygroscopic: the weight gain due to hygroscopic absorption is not less than 15%;

[0040] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0041] Slightly hygroscopic: the weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0042] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0043] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0044] The drying temperature described in this disclosure is generally 25℃~150℃, preferably 40℃~80℃, and can be dried under normal pressure or reduced pressure.

[0045] "Pharmaceutical composition" means a mixture containing one or more compounds of formula (I) described herein, or pharmaceutically acceptable salts thereof, along with other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0046] The crystal forms described in this disclosure include, but are not limited to, solvates of pharmaceutically acceptable salts of compounds of formula (I), wherein the solvents include, but are not limited to, isopropanol, acetonitrile, tetrahydrofuran / ethanol, and isopropanol / ethanol.

[0047] The “solvents” described in this disclosure include, but are not limited to, complexes formed by combining pharmaceutically acceptable salts of compounds of formula (I) with solvents. Attached Figure Description

[0048] Figure 1 XRPD pattern of fumarate a crystal form of the compound shown in formula (I).

[0049] Figure 2 XRPD pattern of the α-crystal form of the compound sulfate shown in formula (I).

[0050] Figure 3 XRPD pattern of the hydrochloride a crystal form of the compound shown in formula (I). Detailed Implementation

[0051] The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0052] Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0053] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0054] MS measurements were performed using an Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector), or a THERMO Ultimate3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0055] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 high-performance liquid chromatograph.

[0056] Chiral HPLC analysis was performed using an Agilent 1260DAD high-performance liquid chromatograph.

[0057] High performance liquid chromatography (HPLC) was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0058] Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.

[0059] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0060] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0061] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0062] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0063] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0064] Unless otherwise specified in the examples, the reactions can be carried out under an argon or nitrogen atmosphere.

[0065] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0066] A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

[0067] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0068] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0069] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0070] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0071] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0072] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0073] The testing conditions of the instruments used in the experiments in this disclosure are as follows:

[0074] 1. Differential Scanning Calorimeter (DSC)

[0075] Instrument Model: Mettler Toledo DSC 3+STARe System

[0076] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / min

[0077] Heating rate: 10.0℃ / min

[0078] Temperature range: 25-350℃ (or 25℃-300℃)

[0079] 2. X-ray Powder Diffraction (XRPD)

[0080] Instrument Model: BRUKER D8 Discover X-ray Powder Diffractometer

[0081] Rays: Monochromatic Cu-Kα rays

[0082] Scanning mode: θ / 2θ, scanning range (2θ range): 3~45°

[0083] Voltage: 40kV, Current: 40mA

[0084] 3. Thermogravimetric Analysis (TGA)

[0085] Instrument model: Mettler Toledo TGA2

[0086] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / min

[0087] Heating rate: 10.0℃ / min

[0088] Temperature range: 30℃-350℃

[0089] 4. DVS is a dynamic moisture adsorption method.

[0090] The test was performed using SMSDVS Advantage at 25°C, with humidity ranging from 0-95% in 10% increments. The judgment criterion was that the mass change dM / dT for each gradient was less than 0.002%, with a TMAX of 360 min and two cycles.

[0091] 5. Anion chromatography

[0092] Instrument Model: DIONEX INTEGRION HPIC Ion Chromatograph (USA)

[0093] Detection method: conductivity; Separation column: Dionex IonPac TM -AS11-HC

[0094] Rinse solution: EGC-500-KOH

[0095] Flow rate: 1.5 ml / min

[0096] Example 1: Preparation of the compound shown in formula (I)

[0097] 9,10-Dimethoxy-2-[[2-(2-oxo-imidazolin-1-yl)-ethyl]-(2,4,6-trimethyl-phenyl)-amino]-6,7-dihydro-pyrimidino[6,1-a]isoquinoline-4-one (Compound 1)

[0098]

[0099] Preparation of intermediate 1a: 1-(2-chloroethyl)imidazolinone

[0100] At 0°C, thionyl chloride (5 ml) was slowly added to 1-(2-hydroxyethyl)imidazolinone (3.5 g, 26.9 mmol), the temperature was raised to 45°C, and the mixture was stirred until the reaction was complete. The reaction was quenched with saturated sodium chloride solution, the pH was adjusted to 7 with 10% NaOH solution, the mixture was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give intermediate 1a (3.5 g, yield 88.4%). MS (ESI) m / z 149.1 [M+H] + .

[0101] Preparation of intermediate 1b: 1-(3,4-dimethoxyphenethyl)urea

[0102] At room temperature, 4.3 g (19.8 mmol) of 2-(3,4-dimethoxyphenyl)ethylamine hydrochloride was dissolved in 25 ml of water. The solution was heated to 50 °C, and potassium cyanate (1.8 g, 21.8 mmol) was added in portions. The mixture was stirred until the reaction was complete, cooled to 0 °C, filtered, and the filter cake was washed with ice water and dried to give intermediate 1b (4.1 g, yield 93.8%). MS (ESI) m / z 225.1 [M+H] + .

[0103] Preparation of intermediate 1c: 1-[2-(3,4-dimethoxy-phenyl)-ethyl]-pyrimidine-2,4,6-trione

[0104] Under ice bath conditions, sodium ethoxide (3.8 g, 55.8 mmol) was added in portions to anhydrous ethanol (50 ml). After the addition was complete, the mixture was heated to reflux, and diethyl malonate (5.9 g, 36.6 mmol) was added dropwise. After the addition was complete, stirring was continued for 0.25 h to 0.5 h. An ethanol solution (30 ml) of intermediate 1b (4.1 g, 18.3 mmol) was added dropwise, and the mixture was stirred until the reaction was complete. The mixture was cooled to 0 °C, and 5% HCl solution was added dropwise until the pH reached 6. 300 ml of water was added, and the mixture was filtered. The filter cake was washed with ice water and dried to obtain intermediate 1c (3.9 g, yield 77.1%). MS (ESI) m / z 293.1 [M+H] + .

[0105] Preparation of intermediate 1d: 2-chloro-9,10-dimethoxy-6,7-dihydropyrimidino[6,1-a]isoquinoline-4-one

[0106] Intermediate B2 (3.9 g, 13.4 mmol) was added to phosphorus oxychloride (120 ml) at room temperature. The mixture was heated to 110 °C and stirred until the reaction was complete. The mixture was then cooled and concentrated. The solid was poured into ice water, and saturated NaOH solution was added dropwise until the pH reached 10. The mixture was filtered, the filter cake was washed with ice water, and dried to give intermediate 1d (2.4 g, yield 62.4%). MS (ESI) m / z 293.1 [M+H] + .

[0107] Preparation of intermediate 1e: 9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-2,3,6,7-tetrahydropyrimidino[6,1-a]isoquinoline-4-one

[0108] At room temperature, intermediate 1d (2.4 g, 8.2 mmol) was suspended in isopropanol (30 ml), and 2,4,6-trimethylaniline (4.5 g, 24.6 mmol) was added. The system was heated to 90 °C and stirred until the reaction was complete. After cooling, the mixture was filtered, the filter cake was washed with ice water, and dried to obtain intermediate 1e (3.0 g, yield 92.1%). MS (ESI) m / z 392.2 [M+H] + .

[0109] Preparation of Compound 1: 9,10-Dimethoxy-2-[[2-(2-oxo-imidazolin-1-yl)-ethyl]-(2,4,6-trimethyl-phenyl)-amino]-6,7-dihydro-pyrimidinyl[6,1-a]isoquinoline-4-one

[0110] At room temperature, intermediate 1e (0.72 g, 1.8 mmol) was dissolved in tetrahydrofuran (20 ml), and potassium tert-butoxide (0.42 g, 3.6 mmol) was added under a nitrogen atmosphere. After the addition was complete, the temperature was raised to 65 °C and stirred for 48 h. The temperature was then lowered to 25 °C and intermediate 1a (0.82 g, 5.5 mmol) was added. After the addition was complete, the temperature was raised to 80 °C and stirred until the reaction was complete. The reaction was quenched by adding saturated sodium chloride solution, extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography (n-heptane / ethyl acetate) to obtain target compound 1 (0.21 g, yield 46.5%).

[0111] 1H NMR(400MHz, CDCl3)δ6.99(s,2H),6.69(s,1H),6.64(s,1H),5.39(s,1H),4.61(s,1H),4.22-4.15(m,2H),4.08-3.99(m,2H),3.93 (s,3H),3.77-3.69(m,5H),3.55-3.46(m,2H),3.38-3.42(t,J=6.8Hz,2H),2.88-2.92(t,J=6.4Hz,2H),2.34(s,3H),2.18(s,6H).

[0112] MS(ESI) m / z 504.4 [M+H] + .

[0113] Comparative Example 1

[0114]

[0115]

[0116] Intermediate 3d was prepared using 2-(3-ethoxy-4-methoxyphenyl)ethylamine hydrochloride as the starting material, following the method in Example 1.

[0117] At room temperature, intermediate 3d (1 g) was dissolved in 1,2-dichloroethane (20 ml), followed by the addition of 2-(2-oxazolidinone-3-yl)ethyl 4-methylbenzenesulfonic acid (844 mg), potassium carbonate (612 mg), and sodium iodide (443 mg). The mixture was heated to 80 °C and stirred until complete. After cooling, the mixture was filtered, concentrated, diluted with water, and extracted with ethyl acetate. The combined organic phases were dried, filtered, concentrated, and subjected to column chromatography to obtain WX001. MS (ESI) m / z 519.0 [M+H]+.

[0118] Biological evaluation

[0119] The following test examples further describe and explain the present disclosure, but these test examples are not intended to limit the scope of the present disclosure.

[0120] Test Example 1 In vitro PDE4B enzyme activity assay: Detection using an IMAP FP-based analytical method.

[0121] 1. Experimental materials

[0122] PDE4B1 BPS 60041 Trequinsin Sigma T2057 384-hole plate Perkin Elmer 6007279 IMAP FP IPP Detection Kit MOLECULAR DEVICES R8124

[0123] 2. Experimental Procedure

[0124] The compound was serially diluted 5-fold with DMSO to obtain different concentrations (10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM, 0.005 nM). Add 200 μL of compounds of different concentrations to 384-well plates (n=2), and simultaneously add two 200 μL aliquots of DMSO to 384-well plates (n=2) as blank controls. Next, add 10 μL of 0.025 μg / mL PDE4B1 enzyme solution (prepared with 1 mM 5*IMAP reaction buffer and 1 mM DTT) to one well of the 384-well plate, and add 10 μL of blank buffer without PDE4B1 enzyme to one of the blank controls. Incubate at room temperature with shaking for 15 minutes. Then add 10 μL of 0.1 μM FAM-cAMP solution (prepared with 1 mM 5*IMAP reaction buffer and 1 mM DTT), and incubate at room temperature with shaking for 30 minutes. Finally, add 60 μL of detection solution (prepared with 0.5625 mM 5*IMAP progression binding buffer A). A) Prepare 0.1875 mM 5*IMAP progression binding buffer and B) and 0.75 mM binding beads. Incubate at room temperature with shaking for 60 minutes, then collect data. The inhibition rate is calculated using the formula: Inhibition rate = M / (MM) 对照 *100; The IC50 value was calculated based on the fitted curve of concentration and inhibition rate. RPL554 was used as a positive control in this experiment.

[0125] The embodiments of this disclosure demonstrate the in vitro inhibition of PDE4B1 enzyme activity through the above-mentioned experiments, and the measured IC50 values ​​are as follows: 50 The values ​​are shown in Table I and Table II.

[0126] Test Example 2 In vitro PDE3A enzyme activity assay: Detection using an IMAP FP-based analytical method.

[0127] 1. Experimental materials

[0128] PDE3A BPS 60030 troquinine Sigma T2057 384-hole plate Perkin Elmer 6007279 IMAP FP IPP Detection Kit MOLECULAR DEVICES R8124

[0129] 2. Experimental Procedure

[0130] The compounds were serially diluted 5-fold with DMSO to obtain different concentrations (10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM, 0.005 nM). 200 μL of each concentration was added to 384-well plates (n=2), and two 200 μL aliquots of DMSO were added to 384-well plates as blank controls. Next, 10 μL of 0.025 μg / mL PDE4B1 enzyme solution (prepared with 1 mM 5*IMAP reaction buffer and 1 mM DTT) was added to the 384-well plates, and 10 μL of blank buffer without PDE3A enzyme was added to one of the blank controls. The plates were incubated at room temperature with shaking for 15 minutes, followed by the addition of 10 μL of 0.1 μM DMSO. FAM-cAMP solution (prepared with 1 mM 5*IMAP reaction buffer and 1 mM DTT) was incubated at room temperature with shaking for 30 minutes. Then, 60 μL of detection solution (prepared with 0.5625 mM 5*IMAP progression binding buffer A, 0.1875 mM 5*IMAP progression binding buffer B, and 0.75 mM binding beads) was added. After incubating at room temperature with shaking for 60 minutes, data was collected. The inhibition rate was calculated using the formula: Inhibition rate = M / (MM) 对照 *100; Calculate IC50 based on the concentration and inhibition rate fitting curve. 50 Value. RPL554 was used as a positive control in this experiment.

[0131] The embodiments of this disclosure demonstrate the in vitro inhibition of PDE3A enzyme activity through the above-mentioned experiments, and the measured IC50 values ​​are as follows: 50 The values ​​are shown in Table I and Table II.

[0132] Table I

[0133]

[0134] Table II

[0135]

[0136]

[0137] Note: N / A not detected.

[0138] Conclusion: Compared with the positive compound RPL554, compound 1 showed good biological activity in in vitro enzyme experiments, and compared with compound WX001, compound 1 showed a 7-fold increase in PDE3A enzyme inhibitory activity, indicating good development prospects.

[0139] Test Example 3: PK Experiment of Intratracheal Drug Administration

[0140] 1. Experimental Objective

[0141] The pharmacokinetic characteristics and distribution in lung tissue of the test sample after intratracheal administration were evaluated in SD rats.

[0142] 2. Test Plan

[0143] 2.1 Test Drugs

[0144] Compound 1 and RPL-554

[0145] 2.2 Experimental Animals

[0146] 198 ICR mice (Shanghai Slack Laboratory Animal Co., Ltd.), half male and half female.

[0147] 2.3 Drug Preparation

[0148] 1) Complete solution:

[0149] Weigh 0.5g of Tween 80 and dissolve it in 50ml of a pH 2.5 citric acid / disodium hydrogen phosphate buffer solution for later use.

[0150] Weigh 1.0 mg of the test drug, dissolve it in an appropriate amount of Tween solution, and prepare a 0.03 mg / ml solution for later use.

[0151] 2) Suspension:

[0152] Weigh 0.5g of CMC-Na and 0.5g of Tween 20, add 50ml of 0.9% physiological saline solution and stir well to obtain a 1% CMC-Na and Tween 20 solution for later use.

[0153] Weigh 1.0 mg of the test drug and add it to 10 ml of the aforementioned solution. Disperse the solution by sonication and stir until homogeneous to obtain a suspension for later use.

[0154] 2.4 Dosing regimen

[0155] Dosage concentration 0.15 mg / mL 0.03 mg / mL Dosage volume 40μL 40μL dose 6μg / each 1.2μg / each

[0156] 3. Experimental Operation / Procedure

[0157] 3.1 Intratracheal administration to mice

[0158] Mice were anesthetized with isoflurane gas and then administered the drug via trachea. Plasma was collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours. 200 μL of whole blood was collected, anticoagulated with EDTA-K2, and centrifuged at approximately 6800g for 6 minutes at 2-8°C. The resulting plasma was transferred to appropriately labeled test tubes within 1 hour of collection / centrifugation and stored at -80°C. Lung tissue was collected at 0.5, 2, 8, and 24 hours. Tissue samples were transferred to appropriately labeled test tubes and stored at -80°C.

[0159] 3.2 Plasma processing and LC-MS / MS analysis

[0160] Take 30.0 μL of plasma sample into a 1.5 mL centrifuge tube, add 150 μL of internal standard working solution, vortex mix for 1 min, centrifuge for 5 min (13000 rpm, 4℃), take 70.0 μL of supernatant into a 96-well plate, add 70.0 μL of deionized water, shake well, and then inject for analysis. Inject 2.00 μL for LC-MS / MS analysis.

[0161] 3.3 Lung tissue processing

[0162] Accurately weigh an appropriate amount of lung tissue sample and place it in a homogenization tube. Add acetonitrile in a volume equivalent to 5 times its weight and homogenize. Sonicate for 5 min. Take 20.0 μL of lung tissue homogenate sample, add 30.0 μL of internal standard working solution and 200 μL of acetonitrile, vortex for 1 min, centrifuge for 10 min (4000 rpm, 4℃), take 100 μL of the supernatant and add 100 μL of deionized water to a 96-well plate. Mix well by shaking (1000 rpm, RT), and inject 1.00 μL for LC-MS / MS analysis.

[0163] 4. Pharmacokinetic Parameter Results

[0164] Compared to RPL-554, compound 1 has a higher in vivo exposure and a longer duration of activity in the lungs. Relevant data are shown in Tables III and IV.

[0165] Table III: Results of PK and Tissue Distribution Experiments for Suspension Formulations

[0166]

[0167] Table IV: Results of PK and tissue distribution experiments for whole-solution formulations

[0168]

[0169] Example 2: Preparation of fumarate crystal form a of the compound shown in formula (I)

[0170] 10 mg of the compound shown in formula (I) was added to 0.5 ml of isopropanol and stirred at 50 °C until dissolved. Then, 2.5 mg of fumaric acid was added, and the temperature was slowly lowered to 5 °C, causing the solid to precipitate. The solid was centrifuged and dried under vacuum to obtain the product. X-ray powder diffraction analysis identified the product as crystal form a, and the XRPD pattern is shown below. Figure 1 As shown in Table 1, the positions of its characteristic peaks are as follows. The DSC spectrum shows an endothermic peak at 255.55℃. The TGA spectrum shows a weight loss of 9.76% from 30℃ to 305℃. Ion chromatography analysis revealed a fumarate ion content of 10.48%.

[0171] Table 1

[0172]

[0173] Example 3: Preparation of sulfate crystal form α of the compound shown in formula (I)

[0174] Approximately 10 mg of the compound shown in formula (I) was added to 0.5 mL of isopropanol and stirred at 50 °C until dissolved. Then, 11 μl of 2 M sulfuric acid aqueous solution was added, and the temperature was slowly lowered to 5 °C, causing the solid to precipitate. The solid was centrifuged and dried under vacuum to obtain the product. X-ray powder diffraction analysis determined the product to be of crystal form α. The XRPD spectrum is shown below. Figure 2 As shown in Table 2, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peaks have peak values ​​of 164.73℃ and 169.09℃. The TGA spectrum shows a weight loss of 3.61% from 30℃ to 175℃. Ion chromatography analysis revealed a sulfate ion content of 14.87%.

[0175] Table 2

[0176]

[0177]

[0178] Example 4: Preparation of crystal form a of the hydrochloride salt of the compound shown in formula (I)

[0179] 10 mg of the compound shown in formula (I) was added to 0.5 ml of isopropanol and stirred at 50 °C until dissolved. Then, 11 μl of 2 M hydrochloric acid aqueous solution was added, and the temperature was slowly lowered to 5 °C, causing the solid to precipitate. The solid was centrifuged and dried under vacuum to obtain the product. X-ray powder diffraction analysis identified the product as crystal form a, and the XRPD pattern is shown below. Figure 3 As shown in Table 3, the positions of its characteristic peaks are as follows. The DSC spectrum shows endothermic peaks at 120.16℃, 195.79℃, 203.30℃, and 249.48℃. The TGA spectrum shows a weight loss of 5.63% from 30℃ to 120℃, 5.16% from 120℃ to 205℃, and 1.56% from 205℃ to 285℃. Ion chromatography analysis revealed a chloride ion content of 6.12%.

[0180] Table 3

[0181]

[0182]

[0183] Example 5: Hygroscopicity study of the crystal form of the salt of the compound shown in formula (I)

[0184] Using Surface Measurement Systems advantage 2, at 25°C and with humidity starting at 50%, the humidity range was investigated from 0% to 95% in 10% increments. The criterion was that the mass change dM / dT for each gradient was less than 0.002%, and the running time TMAX for each humidity gradient was 360 min, with two cycles.

[0185] Table 4

[0186]

[0187] Example 6: Study on factors affecting the crystal form of the salt of the compound shown in formula (I)

[0188] The fumarate a crystal form of the compound shown in formula (I) was laid flat in an open container, and the stability of the samples was investigated under high temperature (40℃, 60℃) and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0189] Table 5

[0190]

[0191] Conclusion: The influencing factor experiment shows that the fumarate a crystal form of the compound shown in formula (I) has good physical and chemical stability after 30 days under high temperature (40℃ and 60℃) and high humidity (75% and 92.5%).

[0192] Example 7: Long-term / accelerated stability of the crystal form of the salt of the compound shown in formula (I)

[0193] The stability of the fumarate a crystal form of the compound salt shown in formula (I) was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH.

[0194] Table 6

[0195]

[0196] Conclusion: Long-term accelerated experiments show that fumarate a crystal form has good physical and chemical stability under conditions of 25℃ / 60RH and 40℃ / 75RH for 6 months.

Claims

1. A pharmaceutically acceptable salt of the compound shown in formula (I), wherein the pharmaceutically acceptable salt is selected from fumarate, sulfate, and hydrochloride. Formula I.

2. The medicinal salt according to claim 1, characterized in that, The chemical ratio of the compound shown in formula (I) to the acid molecule or acid ion is 1:0.1 to 1:

3.

3. The medicinal salt according to claim 1, characterized in that, The chemical ratio of the compound shown in formula (I) to the acid molecule or acid ion is 1:0.2 to 1:

2.

4. The medicinal salt according to claim 1, characterized in that, The chemical ratio of the compound shown in formula (I) to the acid molecule or acid ion is 1:0.5, 1:1 or 1:

2.

5. A method for preparing the pharmaceutically acceptable salt according to any one of claims 1-4, comprising: The steps of the compound described in formula (1) forming a salt with an acid.

6. The method according to claim 5, characterized in that, The solvent used in the salt formation reaction is selected from one or more of isopropanol, acetonitrile, ethanol, and tetrahydrofuran.

7. A pharmaceutical composition prepared from any one of the pharmaceutically acceptable salts according to claims 1-4.

8. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of claims 1-4, or a pharmaceutically acceptable salt prepared by the method of claim 5 or 6, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

9. A method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt according to any one of claims 1-4, or a pharmaceutically acceptable salt prepared by the method of claim 5 or 6, with a pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of any one of the pharmaceutically acceptable salts of claims 1-4, or pharmaceutically acceptable salts prepared by the method of claim 5 or 6, or compositions of claim 7 or 8, or compositions prepared by the method of claim 9, in the preparation of medicaments for the prevention and / or treatment of PDE-related conditions.

11. The use according to claim 10, characterized in that, The conditions associated with PDE include asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

12. The α-crystal form of the fumarate salt shown in formula (I), Formula I, Its features are, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 11.5, 14.6, 15.3, 16.5, 18.3, 22.2, and 24.

3.

13. The α-crystal form of the fumarate of formula (I) according to claim 12, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 11.5, 14.6, 15.3, 16.5, 18.3, 20.7, 21.1, 22.2, 24.3, and 27.

1.

14. The α-crystal form of the fumarate of formula (I) according to claim 12, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 8.2, 11.5, 14.6, 15.3, 16.5, 18.3, 19.4, 20.7, 21.1, 22.2, 24.3, 27.1, and 29.

1.

15. The α-crystal form of the fumarate of formula (I) according to claim 12, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 1.

16. The α-crystal form of the sulfate of the compound shown in formula (I), Formula I, Its features are, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 7.4, 9.1, 11.1, 16.3, 18.1, 23.3, and 24.

7.

17. The α-crystal form of the sulfate of the compound of formula (I) according to claim 16, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 7.4, 9.1, 11.1, 14.0, 16.3, 17.1, 18.1, 22.4, 23.3, and 24.

7.

18. The α-crystal form of the sulfate of the compound of formula (I) according to claim 16, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 7.4, 9.1, 11.1, 14.0, 16.3, 17.1, 18.1, 21.8, 22.4, 23.3, 23.7, 24.7, and 28.

9.

19. The α-crystal form of the sulfate of the compound of formula (I) according to claim 16, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 2.

20. The α-crystal form of the hydrochloride salt of the compound shown in formula (I), Formula I, Its features are, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.7, 10.2, 11.7, 13.3, 15.8, 23.9, and 26.

2.

21. The α-crystal form of the hydrochloride salt of formula (I) according to claim 20, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.7, 10.2, 11.7, 13.3, 15.8, 19.0, 23.9, 26.2, and 27.

7.

22. The α-crystal form of the hydrochloride salt of formula (I) according to claim 20, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.7, 10.2, 11.7, 13.3, 14.6, 15.8, 18.6, 19.0, 23.9, 24.6, 26.2, and 27.

7.

23. The α-crystal form of the hydrochloride salt of formula (I) according to claim 20, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 3.

24. The crystal form according to any one of claims 12-23, characterized in that, The error range of the 2θ angle is ±0.

2.

25. A pharmaceutical composition prepared from the crystal form according to any one of claims 12-24.

26. A pharmaceutical composition comprising the crystal form according to any one of claims 12-24 and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

27. A method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form according to any one of claims 12-24 with a pharmaceutically acceptable carrier, diluent or excipient.

28. Use of the crystal form according to any one of claims 12-24, or the composition according to claim 25 or 26, or the composition prepared by the method of claim 27, in the preparation of a medicament for the prevention and / or treatment of PDE-related conditions.

29. The use according to claim 28, characterized in that, The PDE-related conditions mentioned are asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

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