Substituted thienyl-5-fluoro-1h-pyrazolopyridine compound and use thereof

Abstract

The present invention relates to a substituted thienyl-5-fluoro-1H-pyrazolopyridine compound and a use thereof. The present invention specifically relates to a compound as shown in a formula (I), a preparation method for the compound, a pharmaceutical composition and a combination product containing the compound, and a use of the compound, the pharmaceutical composition or the combination product in preparation of drugs for treating and / or preventing diseases such as heart failure.

Description

Substituted thienyl-5-fluoro-1H-pyrazolopyridine compounds and uses thereof

[0001] This application is based on the application with CN application number 202410323174.5 and application date March 20, 2024, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to substituted thienyl-5-fluoro-1H-pyrazolopyridine compounds, methods for preparing the compounds, pharmaceutical compositions and combined drugs containing the compounds, and uses of the compounds, pharmaceutical compositions or combined drugs in preparing drugs for treating and / or preventing diseases such as heart failure. Background Art

[0003] Heart failure (HF) is a serious and terminal stage of various cardiovascular diseases. It is a syndrome caused by impaired ventricular filling or ejection function, with clinical manifestations including limited exercise tolerance, dyspnea, pulmonary congestion, and peripheral edema. The prevalence in my country is as high as 1.3% among individuals aged 35 years and older, representing approximately 13.7 million patients. Clinically, patients with HF with reduced left ventricular ejection fraction (HFrEF) have an ejection fraction <40%, present with symptoms and / or signs of HF, and are accompanied by elevated brain natriuretic peptide (BNP). Currently, the mortality rate within one year of diagnosis of HFrEF is as high as 20%, the 30-day hospitalization mortality rate is 5%, and the 5-year survival rate is <50%. Despite advances in HF treatment, hospitalization and mortality rates remain high, and quality of life for patients with HF is also poor. Therefore, new treatments are urgently needed to improve the clinical course of the growing number of HF patients worldwide.

[0004] Soluble Guanylate Cyclasey (sGC) is a key signal transduction enzyme in the NO-sGC-cGMP signal transduction pathway. It can be activated by nitric oxide (NO), thereby catalyzing the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). As a second messenger, cGMP can regulate related effectors downstream of the transduction pathway, including protein kinases, phosphodiesterases (PDEs), and certain ion channels, thereby regulating corresponding physiological processes, such as vasodilation, promotion of vascular smooth muscle cell production, regulation of platelet aggregation, and nerve conduction.

[0005] sGC exists in two forms: oxidized and reduced. The reduced form represents its active state. Excessive NO produced under pathological conditions combines with superoxide anions to form peroxynitrite, which inactivates enzymes and other proteins through oxidation and nitration, causing cellular damage. Due to reduced NO bioavailability, sGC converts from its activated reduced form to its inactive oxidized form. This reduces sGC's sensitivity to endogenous NO and NO-releasing drugs, leading to a blockage of the NO-sGC-cGMP signaling pathway. This can lead to conditions such as hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, and myocardial infarction. The most serious of these conditions is increased vascular pressure, resulting in left and right ventricular hypertrophy, ultimately leading to left and right ventricular failure and even death. Therefore, repairing this pathway is particularly important for treating and / or preventing diseases caused by pathway obstruction, such as hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction and myocardial infarction, especially heart failure and even death.

[0006] Currently, the primary approach to repairing the NO-sGC-cGMP signaling pathway is to increase cGMP levels. This is achieved by activating sGC enzyme activity and enhancing its sensitivity to NO, thereby achieving the goal of treating the disease. Treatment approaches can be divided into two types based on whether they are heme-dependent: 1. Heme-dependent sGC stimulators; 2. Heme-independent sGC activators. Potential treatments based on these two categories include NO synthase activators, inhaled NO and NO donor drugs, and phosphodiesterase inhibitors. To date, NO-based compounds, such as organic nitrates, have been ineffective for soluble guanylate cyclase stimulation. Besides side effects, the development of tolerance is a major drawback of this treatment approach.

[0007] One of the most promising therapeutic approaches is to directly activate sGC independently of NO. This approach is generally considered to be highly effective and has few side effects. CN102939289A discloses a substituted 5-fluoro-1H-pyridine soluble guanylate cyclase stimulator that can directly stimulate the sGC-cGMP signaling pathway, promoting cGMP production in a NO-independent and NO-synergistic manner, inhibiting ventricular remodeling, delaying the progression of vascular and ventricular stiffness, and improving myocardial and vascular function. It is the representative compound in this patent. Summary of the Invention

[0008] The present invention aims to identify novel stimulants that directly activate sGC independently of NO. By enhancing sGC's sensitivity to NO, sGC can be directly activated under conditions of extremely low or even absent NO levels, thereby achieving the purpose of treating and / or preventing cardiovascular diseases, including but not limited to hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, and myocardial infarction, particularly heart failure caused by ventricular hypertrophy due to increased vascular pressure. The present inventors have discovered that the substituted thienyl-5-fluoro-1H-pyrazolopyridine compounds of formula (I) provided herein have significantly superior anti-heart failure effects than vericipiguat.

[0009] To this end, the first aspect of the present invention provides a compound represented by formula (I), its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts,

[0010] In certain embodiments, the compound is selected from the compounds represented by formula (I-1) and (I-2),

[0011] In certain embodiments, the compound is selected from:

[0012] The second aspect of the present invention provides a method for preparing the compound of the first aspect of the present invention, comprising:

[0013] 1) reacting the intermediate represented by formula IV with sodium methoxide to obtain the intermediate represented by formula V;

[0014] 2) reacting the intermediate represented by formula V with ammonium chloride to obtain the intermediate represented by formula VI;

[0015] 3) reacting the intermediate represented by formula VI with phenylazomalononitrile to obtain the intermediate represented by formula VII;

[0016] 4) hydrogenating the intermediate represented by formula VII to obtain the intermediate represented by formula VIII;

[0017] 5) reacting the intermediate represented by formula VIII with methyl chloroformate to obtain the compound represented by formula (I).

[0018] In certain embodiments, the intermediate of formula IV is reacted with sodium methoxide in methanol to obtain the intermediate of formula V.

[0019] In certain embodiments, the intermediate of formula VI is reacted with phenylazomalononitrile in the presence of sodium methoxide to obtain the intermediate of formula VII.

[0020] In certain embodiments, the intermediate of formula VII is hydrogenated over a Raney-Ni catalyst to obtain the intermediate of formula VIII. In certain embodiments, the intermediate of formula VII is hydrogenated over a Raney-Ni catalyst at 60° C. to 70° C. (e.g., 65° C.) and a pressure of 60 bar to 70 bar (e.g., 65 bar) to obtain the intermediate of formula VIII.

[0021] In certain embodiments, the intermediate 3-cyano-1-fluorothienylmethyl-1H-pyrazolo[3,4-b]pyridine represented by Formula IV can be prepared using Method A or Method B.

[0022] Method A includes:

[0023] The compound represented by formula II is reacted with the compound represented by formula III to obtain the intermediate represented by formula IV.

[0024] In method A, the compound 3-cyano-1H-pyrazolo[3,4-b]pyridine represented by formula III is a difficult-to-obtain commercial product, which is the limiting step of method A.

[0025] Method B includes:

[0026] 1) reacting the compound represented by formula II with hydrazine hydrate to obtain the intermediate represented by formula IV-1,

[0027] 2) reacting the intermediate represented by formula IV-1 with sodium salt of ethyl cyanoaceruvate to obtain the intermediate represented by formula IV-2,

[0028] 3) reacting the intermediate represented by formula IV-2 with 3-dimethylamino-2-fluoroacrolein to obtain the intermediate represented by formula IV-3,

[0029] 4) aminolyzing the intermediate of formula IV-3 to obtain the intermediate of formula IV-4,

[0030] 5) dehydrating the intermediate represented by formula IV-4 to obtain the intermediate represented by formula IV,

[0031] In certain embodiments, the intermediate represented by formula IV-3 undergoes an aminolysis reaction in a methanolic ammonia solution to obtain the intermediate represented by formula IV-4.

[0032] In certain embodiments, the intermediate represented by formula IV-4 is dehydrated under the action of trifluoroacetic anhydride to obtain the intermediate represented by formula IV.

[0033] In certain embodiments, the compound represented by Formula II, chloromethyl fluorothiophene, can be prepared by the following method:

[0034] The method comprises:

[0035] 1) reducing the compound represented by formula II-1 to obtain the intermediate represented by formula II-2,

[0036] 2) Substituting the hydroxyl group of the intermediate represented by formula II-2 with chlorine to obtain the compound represented by formula II.

[0037] In certain embodiments, the compound of formula II-1 is reduced with sodium borohydride to obtain the intermediate of formula II-2.

[0038] In certain embodiments, the intermediate represented by formula II-2 undergoes a substitution reaction with concentrated hydrochloric acid to obtain the compound represented by formula II.

[0039] The third aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or their pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers and / or excipients.

[0040] In certain embodiments, the pharmaceutical composition described in the present invention comprises a therapeutically and / or prophylactically effective amount of the compound described in the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers and / or excipients.

[0041] The pharmaceutical carriers described in the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, and lanolin.

[0042] The fourth aspect of the present invention provides a combination product comprising the compound of the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts, and one or more NO donors.

[0043] In certain embodiments, the combination product described in the present invention comprises a therapeutically and / or prophylactically effective amount of the compound described in the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotopically labeled compounds, or its pharmaceutically acceptable salts, and a therapeutically and / or prophylactically effective amount of one or more NO donors.

[0044] The NO donor used in the present invention is generally a substance that exhibits its therapeutic effect by releasing NO or NO substances, such as organic nitrates.

[0045] In certain embodiments, the NO donor is selected from sodium nitroprusside, nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, madoxamine, and linsidomine (SIN-1).

[0046] The fifth aspect of the present invention provides a combination product comprising the compound described in the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or pharmaceutically acceptable salts thereof, and one or more guanosine-monophosphate (cGMP) decomposition inhibitors.

[0047] In certain embodiments, the combination product described in the present invention comprises a therapeutically and / or prophylactically effective amount of the compound described in the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotopically labeled compounds, or its pharmaceutically acceptable salts, and a therapeutically and / or prophylactically effective amount of one or more guanosine-monophosphate (cGMP) decomposition inhibitors.

[0048] In certain embodiments, the guanosine-phosphate (cGMP) decomposition inhibitor is selected from phosphodiesterase 1 (PDE1) inhibitors, phosphodiesterase 2 (PDE2) inhibitors and phosphodiesterase 5 (PDE5) inhibitors. In certain embodiments, the guanosine-phosphate (cGMP) decomposition inhibitor is a phosphodiesterase 5 (PDE5) inhibitor. In certain embodiments, the guanosine-phosphate (cGMP) decomposition inhibitor is selected from sildenafil (sildenafil), vardenafil and tadalafil. These inhibitors enhance the effects of the compounds of the present invention and increase the desired pharmacological effects.

[0049] In the present invention, the compound represented by formula (I), its tautomers, polymorphs, solvates, prodrugs, isotope-labeled compounds, or pharmaceutically acceptable salts thereof and the second therapeutic agent (NO donor or guanosine phosphate decomposition inhibitor) are in the same dosage unit or in different dosage units. Different dosage units can be two identical dosage forms or two different dosage forms.

[0050] In use, the compound represented by formula (I), its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt and the second therapeutic agent (NO donor or guanosine-phosphate decomposition inhibitor) can be co-administered to the individual in need of treatment, or can be administered separately to the individual in need of treatment. The compound represented by formula (I), its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt and the second therapeutic agent (NO donor or guanosine-phosphate decomposition inhibitor) can be administered to the individual in need of treatment simultaneously by the same administration route, or can be administered to the individual in need of treatment simultaneously by different administration routes; can be administered to the individual in need of treatment separately in sequence by the same administration route, or can be administered to the individual in need of treatment separately in sequence by different administration routes, for example The compound represented by formula (I), its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt is first administered, and then the second therapeutic agent (NO donor or guanosine-phosphate decomposition inhibitor) is administered at a certain interval; the second therapeutic agent (NO donor or guanosine-phosphate decomposition inhibitor) can also be administered first, and then the compound represented by formula (I), its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt is administered at a certain interval.

[0051] The sixth aspect of the present invention provides the use of the compound described in the first aspect of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect of the present invention, or the combination product described in the fourth aspect of the present invention, or the combination product described in the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing diseases and / or conditions, wherein the diseases and / or conditions are selected from hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, myocardial infarction and high altitude pulmonary edema.

[0052] The sixth aspect of the present invention further provides the compound described in the first aspect of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect of the present invention, or the combination product described in the fourth aspect of the present invention, or the combination product described in the fifth aspect of the present invention, which is used to treat and / or prevent diseases and / or conditions, wherein the diseases and / or conditions are selected from hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, myocardial infarction and high altitude pulmonary edema.

[0053] The sixth aspect of the present invention also provides a method for treating and / or preventing diseases and / or conditions, which comprises administering to an individual in need thereof an effective amount of the compound of the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or their pharmaceutically acceptable salts, or the pharmaceutical composition of the third aspect of the present invention, or the combination product of the fourth aspect of the present invention, or the combination product of the fifth aspect of the present invention, wherein the diseases and / or conditions are selected from hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, myocardial infarction and high altitude pulmonary edema.

[0054] In certain embodiments, the disease and / or condition is selected from hypertension, thromboembolic disease, ischemia, and heart failure.

[0055] In certain embodiments, the disease and / or condition is heart failure.

[0056] The seventh aspect of the present invention provides the use of the compound described in the first aspect of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect of the present invention, or the combination product described in the fourth aspect of the present invention, or the combination product described in the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing a disease and / or condition, wherein the disease and / or condition is cardiovascular disease.

[0057] The seventh aspect of the present invention also provides the compound described in the first aspect of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect of the present invention, or the combination product described in the fourth aspect of the present invention, or the combination product described in the fifth aspect of the present invention, which is used to treat and / or prevent diseases and / or conditions, wherein the diseases and / or conditions are cardiovascular diseases.

[0058] The seventh aspect of the present invention provides a method for treating and / or preventing a disease and / or condition, which comprises administering to an individual in need thereof an effective amount of the compound of the first aspect of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or their pharmaceutically acceptable salts, or the pharmaceutical composition of the third aspect of the present invention, or the combination product of the fourth aspect of the present invention, or the combination product of the fifth aspect of the present invention, wherein the disease and / or condition is cardiovascular disease.

[0059] The active ingredient of the present invention, the compound represented by formula (I), may have systemic and / or local effects and, therefore, may be administered by a suitable route, such as oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, transdermal, conjunctival, topical administration or in the form of an implant.

[0060] The active ingredient can also be administered in administration forms suitable for these administration routes.

[0061] Suitable for oral administration are known administration forms which deliver the active ingredient rapidly and / or in a modified manner, such as tablets (uncoated or coated, e.g. with an enteric coating or a molten coating), capsules, sugar-coated tablets, granules, pellets, powders, emulsions, suspensions and aerosols.

[0062] Parenteral administration may avoid the absorption step (intravenous, intraarterial, intracardial, intraspinal or intramedullary administration) or include absorption (intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal administration). Administration forms suitable for parenteral administration include, in particular, solutions, suspensions, emulsions, lyophilizates and sterile powders for injection and infusion.

[0063] Suitable for other routes of administration are, for example, medicaments for inhalation (particularly powder inhalation, spray), nasal drops / solutions, sprays; tablets or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the ears and eyes, vaginal capsules, aqueous suspensions (lotions, shaken mixtures), lipophilic suspensions, ointments, creams, emulsions, pastes, dusting powders or implants, such as stents.

[0064] The active ingredient can be converted into the described administration form by methods known per se. It can be achieved with inert, non-toxic suitable pharmaceutical excipients. It particularly includes carriers (e.g., microcrystalline cellulose), solvents (e.g., liquid polyethylene glycol), emulsifiers (e.g., sodium lauryl sulfate), dispersants (e.g., polyvinyl pyrrolidone), synthetic and natural biopolymers (e.g., proteins), stabilizers (e.g., antioxidants and ascorbic acid), colorants (e.g., inorganic pigments such as iron oxide), or flavorings and / or taste-masking agents. In suitable cases, the active ingredient can be present in one or more of the above-mentioned carriers in the form of microencapsulation.

[0065] In addition to the compound represented by formula (I) of the present invention, the above-mentioned pharmaceutical preparation may further contain other pharmaceutically active ingredients, such as NO donors and guanosine phosphate decomposition inhibitors.

[0066] In one embodiment, the compound shown in the formula (I) of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound or its pharmaceutically acceptable salt can be used alone, or used in the form of a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient. When used in the form of a pharmaceutical composition, an effective dose of the compound shown in the formula (I) of the present invention, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers or diluents are usually combined to form a suitable form of administration or dosage form, and this procedure includes mixing, granulating, compressing or dissolving the components in a suitable manner. Therefore, the present invention provides a pharmaceutical composition comprising a compound shown in the formula (I), its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound or its pharmaceutically acceptable salt and at least one pharmaceutically acceptable carrier.

[0067] The pharmaceutical composition of the present invention can be administered in any of the following ways: oral, spray inhalation, rectal, nasal, vaginal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, wherein oral, intramuscular, intraperitoneal or intravenous administration is preferred.

[0068] The compound of the present invention, or a pharmaceutical composition or combination product containing the same, can be administered in a unit dosage form. The pharmaceutical composition or combination product can contain 0.01 mg to 1000 mg of the compound of formula (I) of the present invention, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts.

[0069] The pharmaceutical compositions and combination products of the present invention may also contain conventional carriers. Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically accounting for approximately 80% by weight. For convenience, local anesthetics, preservatives, buffers, and the like may be dissolved directly in the carrier.

[0070] Tablets and capsules for oral administration may contain excipients such as binders, such as syrup, gum arabic, sorbitol, tragacanth, or polyvinyl pyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. Tablets may be coated by methods known in the art.

[0071] Oral liquids can be prepared as suspensions, solutions, emulsions, syrups, or elixirs of water and oil, or as dry products that are supplemented with water or other suitable media before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils and fats, emulsifiers such as lecithin, sorbitan monooleate, and gum arabic; or non-aqueous vehicles (which may contain edible oils) such as almond oil, fats such as glycerol, ethylene glycol, or ethanol; preservatives such as methyl or propyl parahydroxybenzoate, and sorbic acid. Flavorings or coloring agents may be added as needed.

[0072] Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

[0073] For parenteral administration, liquid dosage forms are typically prepared by combining the compound with a sterile carrier. Water is the preferred carrier. Depending on the carrier and drug concentration, the compound can be dissolved in the carrier or prepared as a suspension. For injectable solutions, the compound is first dissolved in water, filtered, sterilized, and then dispensed into sealed bottles or ampoules.

[0074] It should be understood that the optimal dosage and interval of administration of the compound of formula (I) is determined by the properties of the compound and external conditions such as the form, route, and site of administration, as well as the specific mammal being treated, and that this optimal dosage can be determined using conventional techniques. It should also be understood that the optimal course of treatment, i.e., the daily dosage of the compound of formula (I) over a specified period of time, can be determined using methods well known in the art.

[0075] The compounds represented by formula (I) of the present invention also include isomers and solvates thereof, such as hydrates, alcoholates, etc. The aforementioned compounds may be in the form of prodrugs or can release the active ingredient after metabolic changes in the body. The selection and preparation of appropriate prodrug derivatives are well known to those skilled in the art. In general, for the purposes of the present invention, solvate forms with pharmaceutically acceptable solvents such as water, ethanol, etc. are comparable to non-solvate forms.

[0076] The actual dosage level of each active ingredient in the pharmaceutical composition or combination product of the present invention can be varied so that the amount of active compound obtained can effectively achieve the desired therapeutic response for a specific patient, composition, and route of administration. The dosage level must be selected based on the activity of the specific compound, the route of administration, the severity of the condition being treated, and the condition and medical history of the patient to be treated. However, it is common practice in the art to start the dosage of the compound at a low level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally speaking, the dosage of the compound of formula (I) of the present invention for mammals, particularly humans, can be between 0.001 and 1000 mg / kg body weight / day.

[0077] Definition of terms

[0078] The various terms and phrases used in this invention have their general meanings as known to those skilled in the art. Nevertheless, the present invention intends to provide a more detailed description and explanation of these terms and phrases. If any term or phrase mentioned herein is inconsistent with the generally known meaning, the meaning as set forth in this invention shall prevail. The following are definitions of various terms used in this invention. These definitions apply to the terms used throughout this specification, unless otherwise specified in specific circumstances.

[0079] Compound of the present invention can exist with two or more mixtures (commonly referred to as tautomers) of structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imines-enamine tautomers etc. It is to be understood that the scope of the application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0080] The present application encompasses all possible crystalline forms or polymorphs of the compounds, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0081] The compounds of the present application may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present application contain a solvent, such as water, methanol, or ethanol, as a structural element of the crystal lattice of the compound. The amount of the solvent may be present in a stoichiometric or non-stoichiometric ratio.

[0082] As used in this application, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or undergo other reactions to provide the compounds of the present invention under biological conditions (in vitro or in vivo). Prodrugs become active compounds only through this reaction under biological conditions, or they do not have or only have relatively low activity in their unreactive form. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178,949-982 (Manfred E. Wolff compiles, 5th edition).

[0083] As used herein, the term "isotopically labeled compound" refers to a compound in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Examples of suitable isotopes for inclusion in the compounds of the present invention include, but are not limited to, hydrogen isotopes such as 2 H, 3 H; carbon isotopes such as 11 C, 13 C and 14 C; chlorine isotopes such as 36 Cl; fluorine isotopes such as 18 F; iodine isotopes such as 123 I and 125 I; Nitrogen isotopes such as 13 N and 15 N; oxygen isotopes such as 15 O, 17 O and 18 O; and sulfur isotopes such as 35 S.

[0084] As used in this application, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" which is used interchangeably therewith, for example, when describing a "pharmaceutically acceptable salt", means that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate in performing chiral resolution. Although the salt of this intermediate cannot be directly administered to a subject, the salt can play a role in obtaining the final product of the present invention.

[0085] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include acid addition salts formed with inorganic or organic acids, such as salts formed with tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-toluenesulfonic acid, ethanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, stearic acid, and tannic acid. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, can be used to prepare salts used as intermediates to obtain the compounds of the present invention and their pharmaceutically acceptable salts. Alternatively, salts formed when the acidic protons present in the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions, may form, for example, sodium salts, potassium salts, magnesium salts, or calcium salts. Alternatively, a coordination compound is formed with an organic base, such as ethanolamine, diethanolamine, triethanolamine or N-methylglucamine, to form an ammonium salt.

[0086] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, pH adjusters, surfactants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0087] As used herein, unless otherwise specified, "%" refers to weight / weight percentages, particularly when describing solid substances. Of course, when describing liquid substances, the "%" may refer to weight / volume percentages (for solids dissolved in liquids) or volume / volume percentages (for liquids dissolved in liquids).

[0088] As used herein, the term "effective amount" refers to a dose that can achieve treatment and / or prevention of the diseases or conditions described herein in a subject. As used herein, the term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives a compound of Formula I or a pharmaceutical composition thereof for treatment and / or prevention of the diseases or conditions described herein.

[0089] As used herein, the term "pharmaceutical composition", which may also refer to a "composition", can be used to achieve treatment and / or prevention of the diseases or disorders described herein in a subject, particularly a mammal.

[0090] As used herein, the term "treatment" is intended to alleviate, mitigate, improve, or eliminate the disease state or condition being treated. If a subject receives a therapeutic amount of the ligand-conjugated drug or its racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture of the foregoing forms according to the methods described herein, and the subject exhibits an observable and / or detectable reduction or improvement in one or more signs and symptoms, the subject is successfully "treated." It should also be understood that the treatment of the disease state or condition includes not only complete treatment, but also achieving some biologically or medically relevant results despite not achieving complete treatment.

[0091] As used herein, the term "prevention" is intended to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms, provided that the disease or disease-related symptoms have not yet appeared before the administration of the relevant drug. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, if the administration of the relevant drug can reduce the risk of a subject developing a particular disease or disease-related symptom, or reduce the severity of related symptoms that later appear, it can be considered to have "prevented" the onset or development of the disease.

[0092] As used herein, the term "disease and / or condition" refers to a physical condition of the subject that is associated with the disease and / or condition described herein. For example, the disease and / or condition described herein may refer to a physical condition, such as a physical condition characterized by higher blood pressure.

[0093] abbreviation: BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 shows the effects of sGC003F on sGC and cGMP activities, where A represents the effect on sGC activity and B represents the effect on cGMP activity;

[0095] Figure 2 shows the dose-effect curves of sGC003F and other compounds on thoracic aorta vasodilation;

[0096] Figure 3 shows the effects of sGC003F on rat cardiac function;

[0097] Figure 4 shows the effects of sGC003F on cardiac function in mice;

[0098] Figure 5 shows the improvement effect of sGC003F on the severity of heart failure in mice;

[0099] Figure 6 shows the improvement effect of sGC003F on mouse cardiac structure and fibrosis;

[0100] Figure 7 shows the average drug-time curves of rats after intravenous and oral administration of different doses of sGC003F;

[0101] Figure 8 shows the cardiac ultrasound results of mice after oral administration of sGC003F;

[0102] Figure 9 shows the results of HE staining and Masson staining after oral administration of sGC003F to mice;

[0103] Figure 10 shows the plasma drug-dose-time curves of normal and TAC mice after a single intravenous injection of 3 mg / kg sGC003F;

[0104] Figure 11 shows the plasma drug-dose-time curves of normal and TAC mice after a single oral administration of 3 mg / kg sGC003F;

[0105] Figure 12 shows the plasma drug-dose-time curves of normal and TAC mice after multiple oral administration of 3 mg / kg sGC003F;

[0106] Figure 13 shows the concentration-time distribution curves of sGC003F in different tissues and blood after a single oral administration of 3 mg / kg sGC003F to normal and TAC mice;

[0107] Figure 14 shows the concentration-time distribution curves of sGC003F in different tissues and blood after multiple oral administration of 3 mg / kg sGC003F to normal and TAC mice;

[0108] Figure 15 shows the AUC and Kp values ​​of sGC003F in different tissues of normal and TAC mice after a single oral administration of 3 mg / kg sGC003F;

[0109] FIG16 shows the AUC and Kp values ​​of sGC003F in different tissues of normal and TAC mice after multiple oral administration of 3 mg / kg sGC003F.

[0110] Note: Unless otherwise specified, in Figures 1 and 3-6, compared with the sham-operated group or solvent control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; compared with the model group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001; compared with the positive control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; in Figures 8, 10, 11, and 15, * indicates P < 0.05, and ** indicates P < 0.01. Beneficial effects

[0111] The compounds provided by the present invention act on the sGC enzyme-related signaling pathway, can effectively activate sGC enzyme activity, and have significant effects in treating and / or preventing cardiovascular diseases, especially anti-heart failure effects. The anti-heart failure effects of the compounds of the present invention are significantly better than those of Vericipiraguat.

[0112] The compounds provided by the present invention also have outstanding pharmacokinetic characteristics suitable for the development of anti-heart failure drugs, such as a long drug metabolic half-life, which enables clinical medication to be taken once a day, reducing the frequency of medication and improving patient compliance. More importantly, the compounds of the present invention can maintain a stable blood drug concentration in the body, which can significantly reduce the risk of hypotension in patients.

[0113] The compound provided by the present invention can improve pulmonary edema induced by plateau environment, reduce tissue inflammation of pulmonary edema induced by plateau environment, and has the effect of treating and / or preventing high altitude pulmonary edema. DETAILED DESCRIPTION

[0114] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0115] General synthesis of starting compounds:

[0116] Example 1A: Ethyl cyanoaceruvate sodium salt

[0117] Place the reaction flask in an ice bath and cool to approximately 0°C. Add 10.7 g (0.073 mol) of diethyl oxalate and 5 mL of anhydrous ether solution dropwise to 20 mL of a 20% sodium ethoxide-ethanol solution. Continue stirring for 30 minutes. Add 2.9 g (0.071 mol) of acetonitrile and 5 mL of anhydrous ether solution dropwise and stir at 30°C for 24 hours. Stop the reaction, filter, and wash the filter cake three times with 10 mL of tert-butyl methyl ether to obtain 10.7 g of the title compound (yield 89.6%). ESI-MS (m / z): 140.2 [M-Na] - .

[0118] Example 2A: 3-Fluoro-2-thiophenemethanol

[0119] Dissolve 9.0 g (0.062 mol) of 3-fluoro-2-thiophenecarboxylic acid in 90 mL of anhydrous THF and slowly add it dropwise to 3.9 g (0.103 mol) of sodium borohydride and 90 mL of anhydrous THF. The reaction will produce a large amount of bubbles. After the addition is complete, continue stirring for 30 minutes until the bubbles disappear. Then, add 11.0 g (0.0433 mol) of iodine and 120 mL of anhydrous THF solution dropwise and react at 50°C for 8 hours. Quench the reaction with water, extract with ether (150 mL × 3), wash with 50 mL of saturated brine, and concentrate under reduced pressure to remove the ether to obtain 7.28 g of the title compound (yield 89.5%). GC-MS (m / z): M + (132.0). The intermediate was directly used for the next reaction.

[0120] Example 3A: 2-Chloromethyl-3-fluorothiophene

[0121] 30 mL of concentrated hydrochloric acid was added to the 3-fluoro-2-thiophene methanol prepared in Example 2A and stirred for 15 minutes. 70 mL of water was added and the mixture was extracted with ether (50 mL x 4). The mixture was washed with dilute sodium bicarbonate solution and then with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to remove the ether, yielding 7.46 g of the title compound (90% yield). GC-MS (m / z): 150.0 (100%), 152.0 (36%). The intermediate was directly used in the next reaction.

[0122] Example 4A: 3-Fluoro-2-(hydrazinemethyl)thiophene

[0123] In a reaction flask, 2-chloromethyl-3-fluorothiophene prepared in Example 3A was dissolved in anhydrous ethanol. The reaction flask was then placed in an ice bath and the temperature was lowered to approximately 0°C. Under nitrogen, 15 g of 85% hydrazine hydrate was added dropwise to the reaction system. After completion of the addition, the temperature was raised to reflux for 6 hours. The solvent was removed by concentration under reduced pressure, and 50 mL of dichloromethane was added to the residue. 50 mL of saturated aqueous sodium carbonate solution was added dropwise at 0°C and extracted. The aqueous phase was further extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous potassium carbonate, filtered, and concentrated to obtain 6.15 g of the product (85% yield). 1 H NMR (400MHz, DMSO-D6) δ7.61–7.53 (m, 1H), 7.00 (d, J = 5.6Hz, 1H), 4.15 (s, 2H).

[0124] Example 5A: 2-Amino-1-(3-fluorothiophen-2-yl)-pyrazole-3-carboxylic acid ethyl ester

[0125] 6.0 g (0.043 mol) of ethyl cyanoaceruvate sodium salt prepared in Example 1A was dissolved in 150 mL of 1,4-dioxane. Under nitrogen, 4.87 g (0.043 mol) of trifluoroacetic acid was added at room temperature. After stirring for 0.5 hour, 6.15 g (0.042 mol) of 3-fluoro-2-(hydrazinemethyl)thiophene prepared in Example 4A was added. After reflux for 18 hours, the mixture was concentrated under reduced pressure to remove the 1,4-dioxane. 60 mL of water was added and the mixture was extracted with ethyl acetate (60 mL x 3). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and eluted by column chromatography (ethyl acetate:petroleum ether = 1:3) to obtain 5.1 g of a yellow solid (yield 45.1%). ESI-MS m / z 270.05 [M+1] + .

[0126] Example 6A: Ethyl 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate

[0127] 5.0 g (0.0186 mol) of ethyl 2-amino-1-(3-fluorothiophen-2-yl)-pyrazole-3-carboxylate prepared in Example 5A was dissolved in 50 mL of 1,4-dioxane. 3.3 g (0.0278 mol) of commercially available 3-dimethylamino-2-fluoroacrolein and 3.2 g (0.0278 mol) of trifluoroacetic acid were added at room temperature. The mixture was reacted in a sealed bottle at 100°C for 3 days. The mixture was concentrated under reduced pressure, 50 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (ethyl acetate:petroleum ether = 1:2) to obtain 2.5 g of the title compound (yield 41.6%). ESI-MS m / z 324.04 [M+1] + .

[0128] Example 7A: 5-Fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

[0129] 2.5 g of ethyl 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate prepared in Example 6A was dissolved in 30 mL of ammonia-methanol solution (7N), stirred at room temperature overnight in a sealed bottle, and concentrated under reduced pressure to remove the solvent to obtain 2.27 g of the title compound (yield 100%).

[0130] Example 8A: 5-Fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

[0131] 2.20 g (7.5 mmol) of 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide, prepared in Example 7A, was dissolved in 30 mL of anhydrous tetrahydrofuran. 1.47 g (18.7 mmol) of pyridine was added, and 3.93 g (18.7 mmol) of trifluoroacetic anhydride was slowly added dropwise. The mixture was stirred at room temperature overnight. 50 mL of ice water was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phase was washed with 20 mL of 1N hydrochloric acid and 20 mL of saturated sodium bicarbonate solution, then washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent, yielding 2.03 g of the title compound (98.3% yield) as a light yellow solid. ESI-MS m / z 277.04 [M+1] + .

[0132] Example 9A: Methyl 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidate

[0133] 3.43 g (0.0124 mol) of 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile prepared in Example 8A was dissolved in 150 mL of anhydrous methanol, and 2.68 g (0.0496 mol) of sodium methoxide was added. After stirring at room temperature for 4 hours, the mixture was directly used for the next reaction.

[0134] Example 10A: 5-Fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide hydrochloride

[0135] 2.98 g (0.0496 mol) of glacial acetic acid and 0.96 g (0.0179 mol) of ammonium chloride were added to the final reaction solution obtained in Example 9A and refluxed under nitrogen for 12 hours. The solvent was removed by concentration under reduced pressure, and 5 mL of acetone was added to the residue, stirred, and filtered. The solid was dissolved in an aqueous sodium carbonate solution (3 g dissolved in 50 mL of water). After stirring for 0.5 hours, it was extracted with ethyl acetate (50 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove 2 / 3 of the solvent. 15 mL of hydrochloric acid-ethyl acetate solution was added, and crystallization was allowed to proceed overnight in an ice bath. The solvent was then removed by concentration under reduced pressure to obtain 3.1 g of the title compound (total yield of the above two steps was 75.8%) as an off-white solid. ESI-MS (m / z): 294.04 [M+1] + .

[0136] Example 11A: 2-[5-Fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4,b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine

[0137] 3.1 g (0.0106 mol) of 5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide hydrochloride, prepared in Example 10A, was dissolved in 100 mL of anhydrous DMF. 0.69 g (0.0127 mol) of sodium methoxide and 2.15 g (0.0127 mol) of phenylazomalononitrile were then added. The mixture was reacted at 110°C under nitrogen for 12 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure and filtered. The resulting solid was added to 5 mL of ethanol and 50 mL of water, stirred for 1 hour, and then filtered. The filter cake was washed with 50 mL of water and then 10 mL of ethanol to obtain 2.9 g of the title compound (yield 66.7%) as a brown solid. ESI-MS (m / z): 464.08 [M+1] + .

[0138] Example 12A: 2-[5-Fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4,b]pyridin-3-yl]-pyrimidine-4,5,6-triamine

[0139] 2.9 g (6.24 mmol) of 2-[5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4,b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine prepared in Example 11A and 1.0 g of T-1 Raney-Ni (wet weight) were added to 150 mL of DMF and hydrogenated at 65°C and 65 bar for 12 hours. The catalyst was removed by filtration through celite, and the solvent was removed by concentration under reduced pressure. After cooling to room temperature, a mixed solvent of water (50 mL) and ethanol (5 mL) was added and stirred for 3 hours. The filter cake was filtered, washed with a mixed solvent of water (50 mL) and ethanol (5 mL), then washed with 5 mL of ethanol, and dried at 50°C to obtain 2.05 g of a brown product (yield 87.6%). ESI-MS (m / z): 375.09 [M+1] + .

[0140] Specific examples of substituted thienyl-5-fluoro-1H-pyrazolopyridine compounds:

[0141] Example 1: 4,6-diamino-2-[5-fluoro-1-(3-fluorothiophen-2-yl)methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylcarbamic acid methyl ester (Compound Ia, i.e., SGC003F or sGC003F or sGC-003F or sGC-003-F or sGC003-F)

[0142] Dissolve 2.05 g (5.47 mmol) of 2-[5-fluoro-1-(3-fluorothiophen-2-yl)-1H-pyrazolo[3,4,b]pyridin-3-yl]-pyrimidine-4,5,6-triamine from Example 12A in 30 mL of anhydrous tetrahydrofuran and add 0.77 g (8.21 mmol) of methyl chloroformate at room temperature. Under nitrogen, react at 60°C for 3 hours, cool to room temperature, add 2 mL of water, and stir for 30 minutes. Filter with suction, add 20 mL of saturated aqueous sodium bicarbonate solution, and stir for 1 hour. Filter with suction, and wash the filter cake with 20 mL of water. Separate by column chromatography using a gradient elution ratio of dichloromethane:methanol (50:1 to 10:1) to obtain 1.2 g of the title compound as a pale yellow solid. 1 H NMR (400MHz, DMSO-d6) δppm: 8.89 (dd, J=8.9, 2.7Hz, 1H), 8.70 (d, J=1.6Hz, 1H), 8.02 (s, 1H) ,7.49(dd,J=5.5,4.3Hz,1H),6.98(d,J=5.6Hz,1H),6.28(s,4H),5.83(s,2H),3.35(s,3H). ESI-MS(m / z): 433.09[M+1] + .

[0143] Example 2: Methyl 4,6-diamino-2-[5-fluoro-1-(2-fluorothien-3-yl)methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylcarbamate (Compound If)

[0144] Methyl 4,6-diamino-2-[5-fluoro-1-(2-fluorothiophen-3-yl)methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylcarbamate was synthesized according to the method of Examples 1A to 12A and Example 1 to obtain 0.21 g of a light yellow solid. 1 HNMR (400MHz, DMSO-d6) δppm: 8.88 (dd, J=8.9, 2.7Hz, 1H), 8.71 (d, J=1.5Hz, 1H), 8.03 (s, 1H ), 7.50 (dd, J = 5.6, 4.2Hz, 1H), 6.67 (d, J = 5.6Hz, 1H), 6.19 (s, 4H), 5.73 (s, 2H), 3.34 (s, 3H). ESI-MS(m / z): 433.08[M+1] + .

[0145] Compounds Ib, Ic, Id and Ie were prepared according to the methods of Examples 1A to 12A and Example 1.

[0146] Example 3: Methyl (4,6-diamino-2-(5-fluoro-1-((3-fluorothiophen-2-yl)methyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)(methyl)carbamate

[0147] 2.01 g (4.62 mmol) of compound sGC-003F was dissolved in 10 mL of DMF. Under nitrogen, 0.68 g (9.26 mmol) of lithium carbonate was added portionwise. The temperature was maintained at 30±5°C. With stirring, 0.85 g (6.01 mmol) of iodomethane was added, and the temperature was maintained at 30±5°C. Stirring continued for 2 hours. The reaction endpoint was determined by HPLC. After the reaction, 120 mL of water was added, stirred for 30 minutes, filtered, rinsed once with 50 mL of water, filtered, rinsed once with 50 mL of water, filtered, and dried at 50°C with air. 1.72 g of product was obtained. 1 H NMR (400MHz, DMSO-d6) δppm: 8.89 (dd, J=8.9, 2.7Hz, 1H), 8.70 (d, J=1.6Hz, 1H), 8.02 (s, 1H), 7.49 (dd, J= 5.5, 4.3Hz, 1H), 6.98 (d, J = 5.6Hz, 1H), 6.28 (s, 4H), 5.83 (s, 2H), 3.66 (s, 1H), 3.53 (s, 2H), 3.00 (s, 3H). ESI-MS(m / z): 447.4[M+1] + .

[0148] In the present application, SGC003F, sGC003F, sGC-003F, sGC003-F, and sGC-003-F have the same meanings, all representing methyl 4,6-diamino-2-[5-fluoro-1-(3-fluorothien-2-yl)methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylcarbamate prepared in Example 1; vericiguat and VAXG both represent vericiguat.

[0149] Experimental Example 1: Effects of sGC003F on sGC activity and function

[0150] Experimental animals: C57 male mice, 18-22 g, were purchased from Beijing Experimental Animal Technology Co., Ltd.

[0151] Test samples: Two test samples, sGC003F and vericiguat, were dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0152] Experimental instrument: microplate reader.

[0153] Experimental Methods: A heart failure model was established in mice using TAC. Four weeks after modeling, the mice were randomly divided into a model group, a vericipiguat group (positive control group), an sGC003F group, and a sham-operated group, with 8-10 mice in each group. Mice in the positive control group were given vericipiguat (3 mg / kg) by gavage; mice in the sGC003F group were given sGC003F (3 mg / kg) by gavage; and mice in the sham-operated and model groups were given an equal volume of normal saline by gavage. After four weeks of continuous administration, serum samples were collected from the mice, and serum sGC and cGMP levels were measured using a kit to evaluate the effects of sGC003F on sGC enzyme activity and function.

[0154] Experimental results: See Figure 1.

[0155] As shown in Figure 1A, compared with the model group, the sGC003F (3 mg / kg) group showed a significant increase in sGC levels (P < 0.01). However, there was no significant difference between the vericipiguat (3 mg / kg) group and the model group. The sGC003F-induced sGC stimulation was significantly superior to that of vericipiguat (P < 0.05). As shown in Figure 1B, compared with the model group, the cGMP level in the sGC003F (3 mg / kg) group was significantly increased (P < 0.001). There was also a significant difference between the vericipiguat (3 mg / kg) group and the model group (P < 0.01). However, the serum cGMP level in the sGC003F group was significantly higher than that in the vericipiguat group (P < 0.05). These results indicate that sGC003F has a stronger selectivity for sGC enzyme and has a stronger sGC activation effect than vericipiguat.

[0156] Experimental Example 2: Vasodilation of the Compound on the Thoracic Aorta of Rats

[0157] Experimental animals: SD rats, male, 200-250 g, purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd.

[0158] The test samples included sGC-003F, sGC-003, sGC-003F+CH3, sGC-003-TF, sGC-003-CH3, VAXG (Vericiguat), VAXG+CH3, sGC-010, and sGC-015. The specific structures of the test samples are shown in Table 1. All samples were dissolved in dimethyl sulfoxide (DMSO).

[0159] Experimental instruments: biological signal acquisition and processing system, iWorx, USA.

[0160] Experimental Methods: Rats were decapitated, and the thoracic aorta was rapidly removed and placed in a culture dish containing ice-cold, oxygen-saturated modified Kreb's-Henseleit solution (NaCl 118 mM, NaHCO3 25 mM, KCl 4.7 mM, MgCl2 1.2 mM, KH2PO4 1.2 mM, CaCl2 2.5 mM, glucose 10 mM, pH 7.4 ± 0.5). Thrombi were carefully washed away, and extravascular connective tissue was removed. The vessel was cut into 3 mm wide loops and placed in a 10 mL Kreb's-Henseleit solution bath at 37°C. The lower end was fixed, and the upper end was connected to a multichannel physiological recorder via a tension transducer. A mixture of 95% oxygen and 5% carbon dioxide was continuously introduced into the tissue bath, and the nutrient solution was replaced every 15 minutes. After the vascular rings were incubated for 20 to 30 minutes, a 2g preload was applied to the vascular rings and allowed to stabilize for 45 to 60 minutes. - 7 mol·L -1 Norepinephrine (NE) induces vascular ring contraction. After contraction reaches maximum and reaches equilibrium (approximately 8-10 minutes), the nutrient solution is replaced to wash out the NE and allow equilibrium to return. This tension value is used as the baseline tension value to begin observing the effects of treatment factors on vascular tension.

[0161] After the blood vessels were incubated to equilibrium, 3 × 10 -7 mol·L -1 NE is injected into the bath to pre-contract the vascular ring. After the contraction reaches the maximum and stabilizes, the test compound is administered in increasing concentrations to observe the relaxation effect of the test compound on the NE-contracted vascular ring; the relaxation percentage and EC value of each compound are calculated. 50 Compound relaxation value = 3 × 10 -7 mol·L -1 The maximum tension value induced by NE is the tension value after administration of the test compound.

[0162] Experimental results: See Table 1 and Figure 2.

[0163] The test compound was administered at multiple doses (1×10 -8 M, 3×10 -8 M, 1×10 -7 M, 3×10 -7 M, 1×10 -6 M) were added cumulatively, and each concentration was incubated for 5 minutes. As shown in Figure 2A, the vasodilation effect of the test compound on the vascular ring gradually increased with the increase of the compound concentration. The EC value of the vasodilation effect of sGC-003F on the vascular ring was obtained from the concentration-effect curve. 50 The EC value of sGC-003 for vascular ring relaxation was 94.8 nM. 50The EC value of VAXG on vascular ring relaxation was 88.5 nM. 50 The EC value of VAXG+CH3 on vascular ring relaxation is 184nM. 50 The value is 171.8nM, which indicates that sGC-003F has a stronger relaxing effect on vascular rings than VAXG and VAXG+CH3, and is comparable to sGC-003. As shown in Figure 2B, the EC value of sGC-003F+CH3 on vascular ring relaxation is 171.8nM. 50 The EC value of sGC-003-CH3 for vascular ring relaxation was 304.4 nM. 50 The value is 245.8nM, and sGC-003F has a stronger relaxing effect on vascular rings than sGC-003F+CH3 and sGC-003-CH3. As shown in Figure 2C, the EC value of sGC-015 on vascular ring relaxation is 245.8nM. 50 The EC value of sGC-010 for vascular ring relaxation was 7943 nM. 50 The EC value of sGC-003-TF on vascular ring relaxation was 1075 nM. 50 The value was 431.5 nM, which indicated that sGC-003F had a stronger relaxing effect on vascular rings than sGC-015, sGC-010, and sGC-003-TF.

[0164] Table 1. EC values ​​of the test samples for vasodilation 50 value

[0165] Experimental Example 3: Adriamycin-induced heart failure rat model, anti-heart failure effect of the compound of Example 1 (sGC003F) Experimental animals: SD rats, male, 180-250 g, purchased from Beijing Sibeifu Experimental Animal Technology Co., Ltd.

[0166] Test samples: sGC003F and vericiguat (positive control drug) were dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0167] Experimental instrument: Vevo small animal ultrasound imaging platform, VisualSonics, USA.

[0168] Experimental Methods: A heart failure model was induced in rats via tail vein injection of doxorubicin. Doxorubicin was administered at doses of 3 mg / kg and 1 mg / kg, alternating every five days. After successful model establishment, the rats were divided into a model group, a drug-treated group (sGC003F group) and a blank group (solvent control group), with eight rats in each group. The drug-treated groups were gavaged with sGC003F (10 mg / kg) and vericipiguat (10 mg / kg), respectively. The blank and model groups were gavaged with an equal volume of normal saline. Dosage was continued for two weeks, and cardiac function was assessed by ultrasound.

[0169] Experimental results: See Figure 3.

[0170] Ultrasound results showed that compared with the model group, sGC003F administration significantly increased left ventricular ejection fraction and fractional shortening in rats. The ejection fraction and fractional shortening in the vericipiguat group were also significantly higher than those in the model group, but the left ventricular ejection fraction in the sGC003F group was significantly higher than that in the vericipiguat group. sGC003F administration significantly increased the rate of ventricular septal thickening and cardiac output, with significant differences compared with the model group (P < 0.05), while there was no difference between the vericipiguat group and the model group. These results indicate that sGC003F can significantly improve doxorubicin-induced heart failure in rats and enhance cardiac function, with a superior effect compared with vericipiguat.

[0171] Experimental Example 4: Anti-heart failure effect of the compound in Example 1 (sGC003F) in a mouse model of heart failure induced by aortic arch constriction

[0172] Experimental animals: C57 male mice, 18-22 g, were purchased from Beijing Experimental Animal Technology Co., Ltd.

[0173] Test samples: Two test samples, sGC003F and Vericipiraguat, were dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0174] Experimental instrument: Vevo small animal ultrasound imaging platform, VisualSonics, USA.

[0175] Experimental Methods: A heart failure model was established in mice by transverse aortic constriction (TAC). Four weeks after model establishment, cardiac ultrasound was performed. A left ventricular ejection fraction (LVEF) <45% was considered a successful model. After excluding mice that died, 60 mice were successfully established and randomly divided into a model group, a vericipiguat group, an sGC003F group, and a sham-operated group, with 10 mice in each group. Mice in the positive control group were given vericipiguat (3 mg / kg) by gavage; mice in the sGC003F group were given sGC003F (3 mg / kg) by gavage; and mice in the sham-operated and model groups were given an equal volume of normal saline by gavage for four consecutive weeks. Cardiac function was then assessed by ultrasound.

[0176] Experimental results: See Figure 4.

[0177] Ultrasound results showed that compared with the model group, sGC003F significantly increased the left ventricular ejection fraction of mice. The left ventricular ejection fraction in the vericipiguat group was also significantly higher than that in the model group, and the left ventricular ejection fraction in the sGC003F group was significantly higher than that in the vericipiguat group. Compared with the model group, sGC003F significantly increased the left ventricular short-axis shortening rate in mice. The left ventricular short-axis shortening rate in the vericipiguat group was also significantly higher than that in the model group, and the short-axis shortening rate in the sGC003F group was significantly higher than that in the vericipiguat group.

[0178] The above results show that sGC003F can significantly improve TAC-induced heart failure in mice and improve cardiac function, and its effect is better than that of vericipiguat.

[0179] Experimental Example 5: Effect of the Compound in Example 1 (sGC003F) on the Severity of Heart Failure in TAC Mice

[0180] Experimental animals: C57 male mice, 18-22 g, were purchased from Beijing Experimental Animal Technology Co., Ltd.

[0181] Test samples: Two test samples, sGC003F and Vericipiraguat, were dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0182] Experimental instrument: microplate reader.

[0183] Experimental Methods: A heart failure model was established in mice using TAC. Four weeks after modeling, the mice were randomly divided into a model group, a vericipiguat group (positive control group), an sGC003F group, and a sham-operated group, with 8-10 mice in each group. Mice in the positive control group were given vericipiguat (3 mg / kg) by gavage; mice in the sGC003F group were given sGC003F (3 mg / kg) by gavage; and mice in the sham-operated and model groups were given an equal volume of normal saline by gavage. After four weeks of continuous administration, serum samples were collected from the mice and measured for the heart failure marker NT-proBNP (amino-terminal pro-brain natriuretic peptide) using a kit (Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) Assay Kit, Catalog No. L230412963, Cloud Clone) to assess the severity of heart failure.

[0184] Experimental results: see Figure 5.

[0185] The results are shown in the figure. Compared with the sham operation group, the NT-proBNP level in the model group was significantly increased. The administration of sGC003F (3 mg / kg) significantly reduced the NT-proBNP level, while there was no significant difference between the vericipiguat (3 mg / kg) group and the model group. These results indicate that sGC003F can improve the severity of heart failure, and the effect of sGC003F is better than that of vericipiguat.

[0186] Experimental Example 6: Effects of sGC003F on the improvement of cardiac structure and fibrosis in mice with heart failure

[0187] Experimental animals: C57 male mice, 18-22 g, were purchased from Beijing Experimental Animal Technology Co., Ltd.

[0188] Test samples: Two test samples, sGC003F and Vericipiraguat, were dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0189] Experimental instrument: Olympus whole slide scanning system.

[0190] Experimental Methods: A heart failure model was established in mice using TAC. Four weeks after modeling, the mice were randomly divided into a model group, a vericipiguat group (positive control group), an sGC003F group, and a sham-operated group, with 8-10 mice in each group. Mice in the positive control group were given vericipiguat (3 mg / kg) by gavage; mice in the sGC003F group were given sGC003F (3 mg / kg) by gavage; and mice in the sham-operated and model groups were given an equal volume of normal saline by gavage. After four weeks of continuous administration, left heart tissues were obtained, fixed in 40% paraformaldehyde, embedded in paraffin, and stained with HE and Masson's stains. The slides were then mounted with gum, and microscopic observation of changes in myocardial tissue morphology and degree of fibrosis was performed.

[0191] Experimental results: See Figure 6.

[0192] HE staining results showed that the myocardial fibers in the model group were loosely arranged and disorganized, even ruptured, with widened and edematous myocardial interstitial space, accompanied by extensive inflammatory cell infiltration. Mice in the vericipiguat (3 mg / kg) group showed partially loosely arranged and disorganized myocardial fibers, accompanied by extensive inflammatory cell infiltration, with significant improvement compared to the model group. Mice in the sGC003F (3 mg / kg) group showed slightly loosely arranged and disorganized myocardial fibers, accompanied by a small amount of inflammatory cell infiltration, with significant improvement compared to the model group. Masson staining results showed that the myocardial fibers in the model group were disorganized, with a significantly larger area of ​​blue-stained collagen fibers. Both vericipiguat (3 mg / kg) and sGC003F (3 mg / kg) significantly improved the degree of cardiac fibrosis, with sGC003F having a greater effect on improving interstitial fibrosis. The above results show that sGC003F can improve the structure and fibrosis of the myocardium in mice with heart failure, and the effect of sGC003F is better than that of vericipiguat.

[0193] Experimental Example 7: Pharmacokinetics in rats

[0194] Experimental methods:

[0195] 1) Drug administration and blood sampling in rats

[0196] Thirty SD rats were randomly divided into five groups (n=6). One group each received intravenous injection of the compound of Example 1 (sGC003F) and the positive control drug, Vericipaguat (VAXG) (0.3 mg / kg), and three groups received oral administration of sGC003F (0.3, 1, and 3 mg / kg). The oral administration groups fasted for 12 hours prior to administration and had free access to water. Approximately 200 μL of venous blood was collected into heparinized EP tubes before administration and at 2 minutes (intravenous group), 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration. The blood was centrifuged at 1000 × g for 10 minutes at 4°C, and the plasma was stored at -40°C for analysis.

[0197] 2) Sample processing

[0198] Rat plasma samples were processed using a protein precipitation method. 20 μL of rat plasma was added to 20 μL of acetonitrile, followed by 160 μL of acetonitrile containing the internal standard riociguat (5 ng / mL). The mixture was vortexed for 1 minute and centrifuged at 14,000 × g for 10 minutes. 60 μL of the supernatant was quantitatively collected and added to 60 μL of 50% ACN in purified water, mixed thoroughly, and injected for analysis.

[0199] Experimental results:

[0200] The mean plasma concentration-time curves and key pharmacokinetic parameters of sGC003F in rats following intravenous administration of 0.3 mg / kg and oral administration of 0.3, 1, and 3 mg / kg are shown in Figure 7 and Table 2, respectively. The results showed that after intravenous administration of sGC003F in rats, clearance was low, the volume of distribution was small, and the terminal elimination half-life was 3-4 hours. Following oral administration, plasma exposure was essentially linear within the 0.3-3 mg / kg dose range, with peak exposure delayed somewhat with increasing dose, from 0.5 hours at low doses to approximately 2 hours at high doses. Peak concentrations increased with increasing dose, but at a lesser rate than the dose-dependent rate. Mean intestinal absorption time (MAT) also increased with increasing dose. Terminal elimination half-lives were consistently 4-5 hours, and mean oral bioavailability was in the 45-50% range.

[0201] Table 2. Pharmacokinetic parameters of sGC003F in rats after intravenous and oral administration of different doses (Mean ± SD, n = 6)

[0202] Experimental Example 8: PK / PD Study of sGC003F in Mice with Chronic Heart Failure

[0203] 1. Experimental Methods

[0204] 1.1 Experimental Animals and Dosing Solutions

[0205] Male C57BL / 6J mice, 6–8 weeks old and weighing 18–20 g, were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Animals were housed in a temperature-controlled environment with a 12-h light / dark cycle and free access to food and water, and acclimated for 1 week. All animal experiments were approved by the Experimental Animal Ethics Committee of the Academy of Military Medical Sciences.

[0206] sGC003F was dissolved in a solvent (11% isopropyl myristate, 74% polyoxyethylene 35 castor oil, 15% diethylene glycol monoethyl ether) to a stock solution concentration of 1 mg / mL. The solution was diluted with saline to obtain a 0.3 mg / mL sGC003F solution for oral and intravenous administration to mice.

[0207] 1.2 Model establishment

[0208] A mouse heart failure model was established using transaortic arch constriction (TAC). Forty male C57BL / 6 mice were randomly divided into a TAC surgery group and a sham surgery group, with 20 mice in each group. Mice in the TAC surgery group were anesthetized with an intraperitoneal injection of sodium pentobarbital solution. The mouse chest was disinfected, and the skin was longitudinally incised along the midline of the neck and chest using ophthalmic scissors. Ophthalmic forceps were used to gently separate the connective tissue and muscles on both sides of the tracheal midline. A midline incision was made at the upper edge of the sternum, extending to the second rib, to expose the aortic arch. The innominate artery and left common carotid artery were identified, and a 27G constriction needle was threaded and placed parallel to the aortic arch. After ligation, the 27G needle was removed, and the sternum and skin were sutured. Chronic heart failure (CHF) developed four weeks after surgery. The sham surgery group underwent the same procedures as the TAC surgery group, but no ligation was performed.

[0209] 1.3 Pharmacokinetic studies

[0210] Single-dose pharmacokinetic study: Control group: 12 normal mice were randomly divided into two groups, 6 mice each; Group I: sGC003F (iv, 3 mg / kg); Group II: sGC003F (po, 3 mg / kg). TAC+sGC003F group: 12 model mice were randomly divided into two groups, 6 mice each; Group III: sGC003F (iv, 3 mg / kg); Group IV: sGC003F (po, 3 mg / kg). Normal mice and model mice were administered sGC003F by different routes on day 28. Oral administration required a 12-hour fasting period and free access to water. Approximately 30 μL of blood was collected from the vein and placed in heparin tubes at 0, 0.033 (intravenous administration), 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h before and after administration. All blood samples were immediately centrifuged at 2,500 × g for 10 min at 4°C, and the plasma was stored at -40°C for testing.

[0211] Multiple-dose pharmacokinetic study: Control group (Group V): 6 normal mice, administered sGC003F (3 mg / kg, po, 28 days); TAC+sGC003F group (Group VI): 6 model mice, administered sGC003F (3 mg / kg, po, 28 days). Both groups of mice were gavage-administered 3 mg / kg sGC003F solution daily for 28 consecutive days, starting 28 days after model establishment. Approximately 30 μL of venous blood was collected into heparin tubes at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours before and after the last dose. All blood samples were immediately centrifuged at 2,500 × g for 10 minutes at 4°C, and plasma was stored at -40°C for analysis.

[0212] 1.4 Tissue distribution studies

[0213] Single-dose tissue distribution study: 18 normal mice and 18 TAC mice with successful modeling were randomly divided into 6 groups, 3 mice per group. Both normal and model mice received a single oral gavage of 3 mg / kg sGC003F on day 28 after modeling. At 0.5 h, 1 h, 2 h, 4 h, 24 h, and 36 h after the single dose, the mice were euthanized and blood was collected by cardiac puncture into heparinized tubes. Plasma was collected by centrifugation at 2500 × g for 10 min at 4°C. Tissue samples of the heart, liver, spleen, lung, kidney, stomach, intestine, brain, fat, testis, and muscle were collected. All tissues were thoroughly rinsed with ice-cold saline. Four volumes of water were added for tissue homogenization. All samples were stored at -40°C until analysis.

[0214] Repeated-dose tissue distribution study: 18 normal mice and 18 TAC mice with successful modeling were randomly divided into 6 groups of 3 mice each. 3 mg / kg sGC003F was administered orally daily for 28 consecutive days starting 28 days after modeling. Euthanasia was performed at 0.5 h, 1 h, 2 h, 4 h, 24 h, and 36 h after the last dose. Blood was collected by cardiac puncture into heparinized tubes, and tissue samples were collected. The specific procedures were the same as above.

[0215] 2. Experimental Results

[0216] 2.1 Pharmacological effects of sGC003F on CHF mice

[0217] The TAC mice with successful modeling were divided into a TAC group and a TAC+sGC003F group, and a sham-operated group was also established. Twenty-eight days after modeling, the TAC+sGC003F group was gavaged daily for 28 consecutive days with 3 mg / kg sGC003F, while the sham-operated and TAC groups were gavaged daily with normal saline for 28 consecutive days.

[0218] Echocardiographic results (Figure 8) of mice undergoing sham surgery, TAC 4 weeks (TAC-28), and 8 weeks (TAC-56) after surgery, and those receiving sGC003F (TAC+sGC003F group) showed that compared with the sham group, the TAC group had significantly decreased EF (ejection fraction) and FS (short axis fraction) (P < 0.01), and significantly increased LVPW;d (left ventricular posterior wall thickness) and LV Mass (ventricular mass) (P < 0.05), indicating decreased cardiac function in the TAC group. Compared with the TAC group, the TAC+sGC003F group had significantly increased EF and FS (P < 0.01), and significantly decreased LVPW;d and LV Mass (P < 0.05), significantly improving the indicators of chronic heart failure mice.

[0219] The histopathological results are shown in Figure 9. HE staining results show that in the sham-operated group, the myocardium was tightly packed and neatly arranged, with normal interstitial tissue. At 4 and 8 weeks after TAC surgery, myocardial cell hypertrophy was observed, with varying degrees of irregular arrangement of myocardial fibers, rupture and dissolution, and increased interfiber spacing. Four weeks after administration (TAC+sGC003F group), myocardial hypertrophy was alleviated, myocardial cell arrangement was slightly neater, and interfiber spacing was reduced. Masson staining results showed that in the sham-operated group, staining was uniform, with no obvious interstitial collagen proliferation. Four weeks after TAC surgery, myocardial collagen fiber content increased, and eight weeks after TAC surgery, myocardial collagen fiber content increased significantly. Myocardial fibrosis was significantly improved after administration of sGC003F (3 mg / kg) (TAC+sGC003F group). These results indicate that 3 mg / kg of sGC003F can effectively improve myocardial tissue morphology in CHF mice and reduce collagen fiber deposition.

[0220] 2.2 Pharmacokinetic studies

[0221] The plasma concentration-time curves and pharmacokinetic parameters of sGC003F following a single intravenous injection of 3 mg / kg in normal mice and successfully established TAC mice are shown in Figure 10 and Table 3. The mean plasma concentration-time curves showed that the TAC+sGC003F group had higher plasma concentrations than the control group at all time points, with statistically significant differences observed after 1 hour (p < 0.05). Compared with the control group, the AUC in TAC mice was significantly increased (approximately 1.29-fold, p < 0.05). Clearance (CL) decreased by 23% (p < 0.01), while Vss did not significantly differ.

[0222] Table 3: PK parameters after a single intravenous injection of 3 mg / kg sGC003F (Mean±SD, n=6) Note: *P<0.05 (TAC+sGC003F group compared with control group)

[0223] The plasma concentration-time curves and pharmacokinetic parameters of sGC003F in normal mice and TAC mice after a single oral dose (po) of 3 mg / kg are shown in Figure 11 and Table 4. The concentration-time curves show that after 1 hour, the plasma concentration in the TAC+sGC003F group was higher than that in the control group, and the difference was statistically significant after 8 hours (p < 0.05). Compared with the control group, the AUC in TAC mice was significantly increased (approximately 1.30-fold, p < 0.05), and the half-life and mean residence time (MRT) were significantly prolonged (p < 0.05), while the Cmax did not change significantly.

[0224] Table 4: PK parameters after single oral administration of 3 mg / kg sGC003F (Mean±SD, n=6) Note: *P<0.05 (TAC+sGC003F group compared with control group)

[0225] The plasma drug-dose curves and pharmacokinetic parameters of 3 mg / kg sGC003F in normal mice and TAC mice with successful modeling were shown in Figure 12 and Table 5 after repeated administration (repeated-po) for 28 days. The results showed that after 28 days of repeated administration, the drug-dose curves of the two groups tended to coincide (no statistical difference), and the C max , AUC,t 1 / 2 There was no statistical difference in MRT.

[0226] Table 5: PK parameters after repeated oral administration of 3 mg / kg sGC003F for 28 days (Mean±SD, n=6)

[0227] The above results indicate that heart failure has no significant effect on the absorption of sGC003F, while its metabolic clearance is significantly inhibited, resulting in increased exposure in mice; after heart failure is corrected, the pharmacokinetic characteristics of sGC003F return to normal.

[0228] 2.3 Tissue distribution studies

[0229] 2.3.1 Single administration:

[0230] Figure 13 shows the concentration-time distribution of sGC003F in various tissues and blood following a single oral administration of 3 mg / kg sGC003F in normal mice and successfully established TAC mice (Mean ± SD, n = 3). As shown, after a single oral administration of 3 mg / kg sGC003F, drug concentrations in the TAC+sGC003F group were higher than those in the control group in most tissues. After 36 hours, drug concentrations in plasma and tissues in both groups were below the limit of quantification, with no significant accumulation.

[0231] Figure 15 shows the AUC and Kp values ​​of sGC003F in different tissues of normal mice and TAC-derived mice after a single oral gavage of 3 mg / kg sGC003F. As shown in the figure, in normal mice, Kp values ​​were higher in the stomach and intestine, likely related to the route of administration. Kp values ​​in other tissues were all below 1, with the lowest exposure in the brain. Compared with the control group, plasma exposure of sGC003F in the TAC+sGC003F group was significantly increased (1.27-fold, p<0.05). Exposure in most tissues was also significantly increased (p<0.05), with increases in the liver, kidney, heart, lung, and spleen exceeding the plasma ratio (p<0.05).

[0232] 2.3.2 Repeated administration:

[0233] Figures 14 and 16 (Mean ± SD, n = 3) show that after repeated oral administration of 3 mg / kg sGC003F for 28 days, the plasma and tissue concentration-time curves of the two groups of mice converged. Furthermore, the successfully established TAC mice exhibited similar tissue and plasma exposures and Kp values ​​to those of normal mice (no statistical differences).

[0234] Experimental Example 9: Evaluation of anti-pulmonary edema effects in a high altitude environment-induced acute pulmonary edema model

[0235] Experimental animals: ICR mice, male, 18-22 g, were purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd.

[0236] Test sample: sGC003F, dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0237] Experimental equipment: low-pressure and low-oxygen environment experimental chamber, Guizhou Fenglei Company.

[0238] Experimental Methods: An acute pulmonary edema model was established by simulating a high-altitude environment. Mice were placed in a hypobaric oxygen chamber simulating an altitude of 7000 meters. The model was successfully established within 6 hours. The modeled mice were divided into a model group, a drug-treated group (a low-dose sGC003F group, a high-dose sGC003F group), and a blank group, with 6-10 mice in each group. The drug-treated groups were gavage-administered sGC003F (5 mg / kg) and sGC003F (10 mg / kg), respectively; the blank and model groups were gavage-administered an equal volume of normal saline. Pre-modeling was initiated with prophylactic medication once daily for 7 days. After successful model establishment, the severity of pulmonary edema in each group was assessed.

[0239] Experimental Results: The drug's anti-pulmonary edema effect was evaluated by measuring the lung tissue wet-to-dry mass ratio (W / D) in mice with pulmonary edema. The results showed that both sGC003F (5 mg / kg) and sGC003F (10 mg / kg) significantly reduced the lung wet-to-dry mass ratio compared to the model group.

[0240] Experimental Example 10: Effect of sGC003F on lung tissue pathology in mice with high altitude induced pulmonary edema

[0241] Experimental animals: ICR mice, male, 18-22 g, were purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd.

[0242] Test sample: sGC003F, dissolved in dimethyl sulfoxide (DMSO) and administered orally.

[0243] Experimental instrument: Olympus whole slide scanning system.

[0244] Experimental Methods: An acute pulmonary edema model was established using a simulated plateau environment. Modeled mice were divided into a model group, a drug-treated group (a low-dose sGC003F group, a high-dose sGC003F group), and a blank control group. The drug-treated groups were gavaged with sGC003F (5 mg / kg) and sGC003F (10 mg / kg), respectively; the blank and model groups were gavaged with an equal volume of normal saline. Pre-modeling prophylaxis was initiated with daily dosing for 7 days. After successful model establishment, the right upper lobe of the lungs of each group was removed, fixed in 40% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin, and mounted with gum. Lung tissue morphology was observed under a microscope.

[0245] Experimental Results: Staining revealed that mice modeling high-altitude pulmonary edema exhibited significant and uneven thickening of the alveolar walls, shrinkage of the alveolar cavities, localized narrowing and deformation of the bronchial lumens, and extensive intraluminal inflammatory cell infiltration and mucus secretion. Compared with the model group, the alveolar cavities in the sGC003F (5 mg / kg) and sGC003F (10 mg / kg) groups were significantly enlarged, with thickened alveolar walls and significantly reduced inflammatory cell infiltration.

[0246] Experimental Example 11: Anti-pulmonary edema effect of sGC003F on acute pulmonary edema induced by acute hypoxia combined with LPS

[0247] Experimental animals: ICR mice, male, 18-22 g, were purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd.

[0248] Test samples: The test samples sGC003F and dexamethasone were dissolved in dimethyl sulfoxide (DMSO) and prepared for oral administration.

[0249] Experimental equipment: low-pressure and low-oxygen environment experimental chamber, Guizhou Fenglei Company.

[0250] Experimental Methods: An acute pulmonary edema model was established by simulating acute high-altitude hypoxia combined with intratracheal instillation of LPS. After intratracheal instillation of LPS (2.5 mg / mL, 50 μL / 25 g), mice were placed in a hypobaric oxygen chamber simulating an altitude of 6000 m. The model was successfully established within 24 hours. The mice were divided into a model group, a drug group (sGC003F group), a positive drug group, and a blank group, with 6-10 mice in each group. The drug group was gavaged with sGC003F (10 mg / kg), while the positive drug group was given dexamethasone (4 mg / kg). Mice in the blank and model groups were gavaged with an equal volume of normal saline. Dexamethasone was administered twice, before model establishment and before sampling. The sGC003F group was given the drug daily for one week before model establishment. Pulmonary edema was assessed after model establishment.

[0251] Experimental Results: The drug's anti-pulmonary edema effect was evaluated by measuring the lung tissue wet-to-dry mass ratio (W / D) in mice subjected to an acute hypoxia-LPS-induced pulmonary edema model. The results showed that sGC003F (10 mg / kg) significantly reduced the lung wet-to-dry mass ratio compared to the model.

[0252] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present invention. They should all be included in the scope of the technical solutions claimed for protection by the present invention.

Claims

1. A compound represented by formula (I), its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts, 2. The compound according to claim 1, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, wherein: The compound is selected from the compounds of formula (I-1) and (I-2), 3. The compound according to claim 1 or 2, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, wherein: The compound is selected from:

4. A method for preparing the compound according to any one of claims 1 to 3, comprising: 1) reacting the intermediate represented by formula IV with sodium methoxide to obtain the intermediate represented by formula V; 2) reacting the intermediate represented by formula V with ammonium chloride to obtain the intermediate represented by formula VI; 3) reacting the intermediate represented by formula VI with phenylazomalononitrile to obtain the intermediate represented by formula VII; 4) hydrogenating the intermediate represented by formula VII to obtain the intermediate represented by formula VIII; 5) reacting the intermediate represented by formula VIII with methyl chloroformate to obtain the compound represented by formula (I).

5. The method of claim 4, further comprising: The intermediate of formula IV is prepared by method A or method B, wherein method A comprises: The compound represented by formula II is reacted with the compound represented by formula III to obtain the intermediate represented by formula IV. Method B includes: a) reacting the compound represented by formula II with hydrazine hydrate to obtain the intermediate represented by formula IV-1, b) reacting the intermediate of formula IV-1 with sodium salt of ethyl cyanoaceruvate to obtain the intermediate of formula IV-2, c) reacting the intermediate represented by formula IV-2 with 3-dimethylamino-2-fluoroacrolein to obtain the intermediate represented by formula IV-3, d) aminolyzing the intermediate of formula IV-3 to obtain the intermediate of formula IV-4, e) dehydrating the intermediate shown in formula IV-4 to obtain the intermediate shown in formula IV, 6. The method of claim 5, further comprising: Prepare the compound represented by formula II, wherein the method for preparing the compound represented by formula II comprises: i) reducing the compound represented by formula II-1 to obtain the intermediate represented by formula II-2, ii) Substituting the hydroxyl group of the intermediate represented by formula II-2 with chlorine to obtain the compound represented by formula II.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3, its tautomer, its polymorph, its solvate, its prodrug, its isotope-labeled compound, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carriers and / or excipients.

8. A combination product comprising the compound according to any one of claims 1 to 3, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts, and one or more NO donors.

9. The combination product of claim 8, wherein the NO donor is an organic nitrate.

10. The combination product of claim 8, wherein the NO donor is selected from the group consisting of sodium nitroprusside, nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, madomine and linsidomine.

11. A combination product comprising the compound of any one of claims 1 to 3, its tautomers, its polymorphs, its solvates, its prodrugs, its isotope-labeled compounds, or its pharmaceutically acceptable salts, and one or more guanosine-phospholysis inhibitors.

12. The combined product of claim 11, wherein The guanosine monophosphate decomposition inhibitor is selected from the group consisting of phosphodiesterase 1 inhibitors, phosphodiesterase 2 inhibitors, and phosphodiesterase 5 inhibitors.

13. The combined product of claim 11, wherein The guanosine-phosphate decomposition inhibitor is a phosphodiesterase 5 inhibitor.

14. The combined product of claim 11, wherein The guanosine-phospholysis inhibitor is selected from sildenafil, vardenafil and tadalafil.

15. A compound according to any one of claims 1 to 3, a tautomer thereof, a polymorph thereof, a solvate thereof, a prodrug thereof, an isotope-labeled compound thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, or a combination product according to any one of claims 8 to 14, for use in the preparation of a medicament for treating and / or preventing a disease and / or condition, wherein the disease and / or condition is selected from hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, heart failure, thrombosis, stroke, sexual dysfunction, myocardial infarction, and high altitude pulmonary edema.

16. Use of a compound according to any one of claims 1 to 3, a tautomer thereof, a polymorph thereof, a solvate thereof, a prodrug thereof, an isotope-labeled compound thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, or a combination product according to any one of claims 8 to 14, in the preparation of a medicament for treating and / or preventing a disease and / or condition, wherein the disease and / or condition is cardiovascular disease.

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