Pyrimidinedione derivative as well as preparation method and application thereof

Abstract

The invention discloses a pyrimidinedione derivative as well as a preparation method and application thereof. Molecules of the pyrimidinedione derivative have better myosin inhibitory activity, also have antioxidant effects of DPPH and / or MDA and the like and anti-ferroptosis activity, and can be applied to treatment / prevention of heart myosin, anti-oxidative stress and anti-ferroptosis mechanism related diseases. The diseases or symptoms mainly comprise hypertrophic cardiomyopathy, diastolic heart failure, left ventricular hypertrophy, heart diseases with pathophysiological characteristics related to the hypertrophic cardiomyopathy, or other disease states and conditions which are obvious to those skilled in the art, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. Or a hydrate or solvate thereof, and a pharmaceutical composition containing the same.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly relates to a class of pyrimidine dione derivatives, their preparation methods and uses, and more particularly to compounds of formula (I) and their applications in the preparation of drugs for preventing or treating heart diseases involving hypertrophic obstructive cardiomyopathy, diastolic heart failure, left ventricular hypertrophy, symptoms related to heart injury, etc. Background Art

[0002] Excessive actin-myosin cross-bridge formation is the root cause of myocardial systolic and diastolic dysfunction in hypertrophic cardiomyopathy. In 2022, the myosin inhibitor Mavacamten was approved by the FDA for the treatment of adult patients with symptomatic NYHA II-II grade obstructive hypertrophic cardiomyopathy. Mavacamten is a reversible allosteric inhibitor of myocardial myosin ATPase, which can promote more myosin heads to form a compact closed structure IHM and stabilize in the OFF state to reduce excessive actin-myosin cross-bridges, thereby reducing the hypercontractility of HCM. During the actual clinical application of Mavacamten, it can cause systolic dysfunction leading to heart failure, and left ventricular ejection fraction must be evaluated by echocardiogram during the application process. Another myosin inhibitor Aficamten submitted for NDA has a short half-life and a shallower exposure-effect relationship than Mavacamten, so it has higher safety and has obtained breakthrough therapy designations from the FDA and NMPA. However, the dose still needs to be adjusted according to echocardiogram during clinical application. Studies have found that redox imbalance caused by sarcomere gene mutations plays a core role in the pathology of hypertrophic cardiomyopathy and leads to the occurrence and development of hypertrophic cardiomyopathy. Excessive cellular ROS is an important link between sarcomere mutations and the phenotypes of hypertrophic cardiomyopathy. In patients with hypertrophic cardiomyopathy and animal models, significant increases in oxidative stress markers (including protein, DNA, and lipid damage) have been found in the heart and serum, and their levels are positively correlated with the severity of the hypertrophic cardiomyopathy phenotype. Antioxidants can inhibit oxidative stress in animal models of hypertrophic cardiomyopathy (Tm-E180G and cTnT-Q92 transgenic mice) and prevent and reverse the occurrence and development of hypertrophic cardiomyopathy. In addition, a large number of basic and clinical studies suggest that ferroptosis is closely related to the pathophysiology of cardiovascular diseases. Mice lacking ferritin heavy chain fed a high-iron diet can lead to severe heart injury and hypertrophic obstructive cardiomyopathy, and ferroptosis-related inhibitors can reverse angiotensin II-mediated myocardial hypertrophy. Summary of the Invention

[0003] The present invention provides a pyrimidine dione derivative represented by formula (I), its pharmaceutically acceptable stereoisomers, salts or solvates:

[0004]

[0005] Wherein: R 1 and R 3 are independently selected from H, D, F, Cl, Br, CN, NO 2 , -CO-NH(OH), -CON(OH)-R’, or form a five-membered heterocyclic group, substituted five-membered heterocyclic group, six-membered heterocyclic group, substituted six-membered heterocyclic group, etc. with R 2 ;

[0006] R 2 is selected from a five-membered heterocyclic group, substituted five-membered heterocyclic group, or forms a five-membered heterocyclic group, substituted five-membered heterocyclic group, six-membered heterocyclic group, substituted six-membered heterocyclic group with R 1 / R 3 ;

[0007] R 4 is selected from H, D or CH 3 ;

[0008] R’: is selected from H, C 1 -C 4 linear alkyl, C 1 -C 4 branched alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0009] The five-membered heterocyclic group is preferably selected from the following substituents, such as:

[0010] R 2 and R 1 / R 3 The five- or six-membered heterocyclic groups formed include: etc.

[0011] In another aspect, the present invention provides a compound as shown below or a pharmaceutically acceptable salt or deuterated analogue thereof:

[0012]

[0013] The compound of formula (I) provided by the present invention has good myosin inhibitory activity.

[0014] The compound of formula (I) provided by the present invention has certain antioxidant activities such as DPPH and / or MDA.

[0015] The compound of formula (I) provided by the present invention has certain ferroptosis resistance activity.

[0016] Use of the pyrimidine dione derivative shown in formula (I), its pharmaceutically acceptable stereoisomers, salts or solvates in the preparation of a myosin inhibitor.

[0017] Furthermore, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above-mentioned compounds or its stereoisomers or pharmaceutically acceptable salts, or a crystalline form of any one of the above-mentioned compounds and a pharmaceutically acceptable carrier. The carrier includes conventional excipient components in the art, such as fillers, binders, diluents, disintegrants, lubricants, colorants, flavoring agents, antioxidants, and wetting agents, etc.

[0018] The pharmaceutical composition can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquid preparations, suspensions, granules, powders, microparticles, pills, mini-tablets, fast-dissolving films, nasal sprays, transdermal patches, injections, or various sustained-release and controlled-release preparations, etc. The pharmaceutical composition can be administered orally, transmucosally, rectally, or parenterally (including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, and intracostally). The dosage can be appropriately adjusted according to the age, gender, and type of disease of the patient.

[0019] For oral administration, the pharmaceutical composition can be in the form of, for example, tablets, capsules, liquid capsules, suspensions, or liquids, and the pharmaceutical composition is preferably prepared in the form of dosage units containing a specific amount of the active ingredient. For example, the pharmaceutical composition can be provided in tablets or capsules containing an amount of the active ingredient within the range of 1 g. The suitable daily dosage for humans or other mammals can vary widely depending on the condition of the patient and other factors, but can be determined using conventional methods.

[0020] In one aspect, the present invention relates to the use of the pyrimidinedione derivative represented by formula (I) or its stereoisomers, pharmaceutically acceptable salts, or solvates, or a pharmaceutical composition comprising the pyrimidinedione derivative represented by formula (I) or its stereoisomers, pharmaceutically acceptable salts, or solvates in the preparation of medicaments for treating / preventing diseases related to cardiac myosin, antioxidant stress, and anti-ferroptosis mechanisms. The diseases or symptoms mainly include hypertrophic cardiomyopathy, diastolic heart failure, left ventricular hypertrophy, and methods for treating heart diseases with pathophysiological characteristics related to hypertrophic cardiomyopathy.

[0021] Furthermore, the use of the pyrimidinedione derivative represented by formula (I), its pharmaceutically acceptable stereoisomers, salts, or solvates in the preparation of medicaments for inhibiting hypertrophic cardiomyopathy.

[0022] Accordingly, the pyrimidine dione derivatives represented by formula (I) or their stereoisomers, pharmaceutically acceptable salts or solvates provided by the present invention can be used as components of combination drugs for preventing / treating heart diseases related to pathophysiological characteristics associated with hypertrophic cardiomyopathy, such as therapies that delay the progression of heart failure and attempt to prevent cardiac remodeling by downregulating neurohormonal stimulation of the heart (e.g., ACE inhibitors, angiotensin receptor blocker (ARB) blockers, aldosterone receptor antagonists or neprilysin inhibitors); therapies that improve cardiac function by stimulating cardiac contractility (e.g., inotropic agents such as β-adrenergic agonist dobutamine or phosphodiesterase inhibitor milrinone); and therapies that reduce cardiac preload (e.g., diuretics such as furosemide) or therapies that reduce cardiac afterload (any class of vasodilators and including but not limited to calcium channel blockers, phosphodiesterase inhibitors, endothelin receptor antagonists, renin inhibitors or smooth muscle myosin modulators).

[0023] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear if not specifically defined and should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0024] The term "pharmaceutically acceptable" as used herein pertains to those compounds, materials, compositions and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0025] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from compounds found in the present invention having specific substituents with relatively non-toxic acids or bases. When a compound of the present invention contains a relatively acidic functional group, the base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When a compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, organic acid salts, salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either base or acid addition salts.

[0026] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of the salts is: in water, an organic solvent or a mixture of both, these compounds in the form of free acids or bases are reacted with a stoichiometric amount of appropriate bases or acids to prepare the salts.

[0027] Some compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereoisomers, geometric isomers and individual isomers are all included within the scope of the present invention.

[0028] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)- enantiomers, R and S enantiomers, diastereoisomers, D-form, L-form isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereoisomer-enriched mixtures. All these isomers and their mixtures are included within the scope of the present invention.

[0029] Optically active R-form and S-form isomers, as well as D-form and L-form isomers can be prepared by chiral synthesis, chiral reagents or other conventional techniques. If an enantiomer of a certain compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereoisomers is separated, and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereoisomeric salts are formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods well-known in the art, and then the pure isomer is recovered. In addition, the separation of enantiomers and diastereoisomers is usually accomplished by using chromatography, which employs a chiral stationary phase and optionally combined with chemical derivatization methods (such as generating carbamates from amines).

[0030] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance and is non-toxic and has no side effects on the host or patient. Representative carriers include, but are not limited to: binders, fillers, lubricants, disintegrants, wetting agents, dispersants, solubilizers, suspending agents, etc.

[0031] For a drug or a pharmacological active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but can achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also depends on the specific active substance. The appropriate effective amount in a particular case can be determined by those skilled in the art according to conventional tests.

[0032] The present invention is intended to include all isotopes of the atoms present in the compounds of the present invention. Isotopes include those atoms having the same atomic number but different mass numbers. As a general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13 C and 14 C. The isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described herein, using appropriate isotopically labeled reagents in place of the unlabeled reagents otherwise employed. Detailed Description of the Invention

[0033] The present invention will be clearly and completely described below in conjunction with specific embodiments. Those skilled in the art will understand that the following embodiments are partial embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention and should not be construed as limiting the protection scope of the present invention.

[0034] In the present invention, unless otherwise specified, the specific test conditions are carried out according to the conventional test conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, conventional products can be obtained by purchasing from the market.

[0035] In the present invention, the test results are expressed as averages.

[0036] The detection indexes in the present invention are: myocardial myosin ATPase inhibitory activity test; DPPH free radical scavenging ability test; MDA anti-lipid peroxidation ability test; intervention effect on ferroptosis process (qPCR).

[0037] Example 1

[0038]

[0039] Synthesis route:

[0040]

[0041] 1) Synthesis of Intermediate 3

[0042]

[0043] Raw material 2 (3.85 g, 20.43 mmol, 2 eq) and raw material 1' (2.6 g, 10.22 mmol, 1 eq, HCl) were added to N-methylpyrrolidone (26 mL) for dissolution. At room temperature, N,N-diisopropylethylamine (3.96 g, 30.65 mmol, 5.34 mL, 3 eq) was added to the reaction solution. Microwave stirring was carried out at 120 °C for 4 hours. After the reaction was completed as detected by LCMS, the reaction mixture was directly purified by reverse-phase column chromatography (C18 column, 0.1% formic acid solution) to obtain intermediate 3 (2.75 g, yield 60.29%, light yellow solid). LCMS: rt = 0.480 min, 368.1 / 370.1 [M+H]+, purity 82.9%. 1 1H NMR (400 MHz, DMSO-d6) δ = 9.93 (br s, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.43 (dd, J = 2.0, 10.0 Hz, 1H), 7.21 (dd, J = 2.0, 8.4 Hz, 1H), 6.67 (br d, J = 7.0 Hz, 1H), 5.03 - 4.86 (m, 1H), 4.57 (br t, J = 6.8 Hz, 1H), 4.36 (s, 1H), 1.43 (d, J = 6.8 Hz, 3H), 1.31 (dd, J = 1.4, 7.0 Hz, 6H).

[0044] 2) Synthesis of intermediate 5

[0045]

[0046] Raw material 3 (150 mg, 387.93 μmol, 1 eq) was added to tert-amyl alcohol (5 mL) for dissolution, then raw material 4 (51.27 mg, 387.93 μmol, 1 eq), cesium carbonate (379.18 mg, 1.16 mmol, 3 eq) and tBuXPhos Pd G3 (30.82 mg, 38.79 μmol, 0.1 eq) were added. The reaction was carried out at 100 °C for 2 hours under nitrogen protection. After the reaction was completed as detected by LCMS, the mixture was concentrated to obtain the crude product. The crude product was purified by flash thin layer chromatography (petroleum ether: ethyl acetate = 1:2, Rf = 0.5) to obtain compound 5 (70 mg, yield 41.21%, yellow solid) LCMS: Rt = 0.492 min, 438.2 [M+H]+, purity 60.89%.

[0047] 3) Synthesis of intermediate 6

[0048]

[0049] Intermediate 5 (400 mg, 949.07 mmol, 1 eq) was added to anhydrous dichloromethane (4 mL) and trifluoroacetic acid (0.8 mL). The mixture was stirred at 25 °C for 1 h. After the reaction was completed as detected by LCMS, the reaction solution was concentrated under reduced pressure to obtain crude product 6. The crude product (400 mg, crude) was directly used for the next reaction. LCMS: rt = 0.349 min, 322.2 [M+H]+.

[0050] 4) Synthesis of Compound 1

[0051]

[0052] Intermediate 6 (400 mg, 1.24 mmol, 1 eq) and Intermediate 7 (323.99 mg, 2.49 mmol, 2 eq) were added to anhydrous acetonitrile (4 mL) and dissolved. The mixture was stirred at 60 °C for 1 h. After the reaction was completed as detected by LCMS, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel plate (dichloromethane:methanol = 10:1) to obtain a mixture. The mixture was purified by chiral separation to obtain a crude product. The crude product was purified by reverse-phase preparation (C18 column, 0.1% ammonium bicarbonate solution) and freeze-dried to obtain Compound 1 (57.2 mg, white solid, yield 11.86%). LCMS: rt = 0.759 min, 388.1 [M+H]+, 100% purity. 1 1H NMR (400 MHz, DMSO-d6) δ = 11.31 - 10.93 (m, 1H), 9.99 (br s, 1H), 7.30 - 7.15 (m, 3H), 5.99 (s, 2H), 5.40 - 5.13 (m, 1H), 5.01 - 4.88 (m, 1H), 4.06 (q, J = 7.0 Hz, 1H), 2.07 (s, 3H), 1.54 (d, J = 7.0 Hz, 3H), 1.29 (dd, J = 1.8, 6.9 Hz, 6H).

[0053] Example 2

[0054]

[0055] Synthetic route:

[0056] 1) Synthesis of Intermediate 3

[0057]

[0058] Raw material 1 (500 mg, 1.85 mmol, 1.5 eq, HCl) was added to 1-methyl-2-pyrrolidone (8 mL) for dissolution, then raw material 2' (232.01 mg, 1.23 mmol, 1 eq) and N,N-diisopropylethylamine (476.93 mg, 3.69 mmol, 642.77 μL, 3 eq) were added. The reaction was carried out at 120 °C under microwave for 2 hours. After the reaction was completed as detected by LCMS, the reaction solution was filtered and concentrated under reduced pressure to obtain a residue. It was purified by reverse-phase column chromatography (C18 column, 0.1% formic acid solution) and freeze-dried to obtain intermediate 3 (360 mg, yield 75.69%, yellow solid). LCMS: Rt = 0.495 min, 386.1 / 388.1 [M+H+]+.

[0059] 2) Synthesis of intermediate 5

[0060]

[0061] Intermediate 3 (150 mg, 387.93 μmol, 1 eq) was added to tert-amyl alcohol (5 mL) for dissolution, then raw material 4 (51.27 mg, 387.93 μmol, 1 eq), cesium carbonate (379.18 mg, 1.16 mmol, 3 eq) and tBuXPhos Pd G3 (30.82 mg, 38.79 μmol, 0.1 eq) were added. The reaction was carried out at 100 °C under nitrogen protection for 2 hours. After the reaction was completed as detected by LCMS, it was concentrated to obtain a crude product. The crude product was purified by flash thin-layer chromatography (petroleum ether: ethyl acetate = 1:2, Rf = 0.5) to obtain compound 5 (70 mg, yield 41.21%, yellow solid) LCMS: Rt = 0.492 min, 438.2 [M+H]+, purity 60.89%.

[0062] 3) Synthesis of intermediate 6

[0063]

[0064] Raw material 5 (50 mg, 114.18 μmol, 1 eq) was added to anhydrous dichloromethane (2 mL) for dissolution, then trifluoroacetic acid (0.4 mL) was added. The reaction was carried out at 25 °C for 1 hour. After the reaction was completed as detected by LCMS, the reaction solution was filtered and concentrated under reduced pressure to obtain a residue. The crude product was directly used for the next step without further purification. Compound 6 (50 mg, crude product, TFA) was a yellow oil. LCMS: Rt = 0.356 min, 338.0 [M+H]+.

[0065] 4) Synthesis of compound 2

[0066]

[0067] The raw materials 6 (50 mg, 110.66 μmol, 1 eq, TFA) and 7 (14.40 mg, 110.66 μmol, 1 eq) were added to anhydrous acetonitrile (1.5 mL) for dissolution, and then reacted at 60 °C for 2 hours. After the reaction was detected by LCMS, the reaction solution was filtered and concentrated under reduced pressure to obtain a residue. It was purified by reverse-phase column chromatography (C 18 column, 0.1% formic acid solution), and after lyophilization, compound 2 (12.4 mg, yield: 27.54%, white solid) was obtained. LCMS: Rt = 0.791 min, 404.1 [M+H] + . HPLC: Rt = 1.005 min. 1 1H NMR (400 MHz, DMSO-d6) δ = 11.40 - 10.76 (m, 1H), 10.21 - 9.91 (m, 1H), 7.47 (d, J = 1.2 Hz, 1H), 7.39 - 7.33 (m, 1H), 7.31 - 7.23 (m, 1H), 6.08 (s, 2H), 5.27 (s, 1H), 4.97 (td, J = 7.2, 13.6 Hz, 1H), 4.15 - 3.98 (m, 1H), 2.09 (s, 3H), 1.56 (d, J = 7.2 Hz, 3H), 1.30 (dd, J = 1.6, 6.8 Hz, 6H)

[0068] Example 3

[0069]

[0070] Synthetic route:

[0071] 1) Synthesis of intermediate 3'

[0072]

[0073] At 0 °C, diisopropylethylamine (1.29 g, 10.00 mmol, 1.74 mL) and 2 (628.47 mg, 3.33 mmol) were added to a solution of 1 (1 g, 5.00 mmol) in 1,4-dioxane (10 mL). The mixture was microwave-heated in a microwave reactor at 120 °C for 2 hours. After the reaction was completed, it was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse-phase preparative column chromatography in a formic acid system to obtain intermediate 3' (0.47 g, yield 40.71%). LCMS: Rt = 0.490 min, m / z: 352.1 / 354.1 [M+H]+. 1HNMR(400MHz, METHANOL-d4) δ=7.50 (d, J=8.4Hz, 2H), 7.26 (d, J=8.4Hz, 2H), 5.02 - 5.00 (m, 1H), 4.86 (s, 1H), 4.50 - 4.45 (m, 1H), 1.48 (d, J=6.8Hz, 3H), 1.37 (d, J=6.6Hz, 6H).

[0074] 2) Synthesis of Intermediate 5

[0075]

[0076] At room temperature, cesium carbonate (10.55 g, 32.37 mmol) and tBuXPhos-Pd-G3 (857.01 mg, 1.08 mmol) were added to a solution of starting material 3’ (3.8 g, 10.79 mmol) and compound 4 (3.76 g, 16.18 mmol) in tert-amyl alcohol (120 mL). The reaction mixture was heated to 100 °C and stirred under nitrogen for 16 hours. After the reaction was completed, the reaction mixture was diluted with ethyl acetate (500 mL) at room temperature. The organic phase was washed with saturated brine (300 mL × 2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was first purified by column chromatography and then by preparative reverse-phase purification to obtain Intermediate 5 (870 mg, 1.73 mmol, yield 16.01%). LCMS: Rt = 0.527 min, m / z: 504.3 [M + H]+.

[0077] 3) Synthesis of Intermediate 6

[0078]

[0079] At room temperature, trifluoroacetic acid (4.8 mL) was added to a solution of starting material 5 (770 mg, 1.53 mmol) in dichloromethane (24 mL). Then the reaction mixture was reacted at 25 °C for 0.5 hour. After the reaction was completed, the reaction mixture was directly concentrated under reduced pressure to obtain Intermediate 6 (770 mg, crude product, TFA). The crude product was used directly in the next step without purification. LCMS: Rt = 0.433 min, 304.2 [M + H]+.

[0080] 4) Synthesis of Compound 3

[0081]

[0082] At room temperature, compound 7 (330.33 mg, 2.54 mmol) was added to a solution of compound 6 (770 mg, 2.54 mmol) in acetonitrile (24 mL). The reaction mixture was heated to 60 °C and stirred for 0.5 h under nitrogen protection. After the reaction was completed, the reaction mixture was directly concentrated under reduced pressure to obtain an 8 mL crude product solution. The crude product solution was purified by preparative chromatography (C 18 column, 0.1% formic acid solution) to obtain compound 3 (292.8 mg, 786.26 μmol, yield 30.98%). LCMS: Rt = 0.776 min, 370.1 [M+H] +.1 1H NMR (400 MHz, DMSO-d6) δ = 11.30 (br s, 1H), 9.95 (s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 5.82 (br s, 2H), 5.33 (br s, 1H), 5.01 - 4.88 (m, 1H), 4.12 - 4.03 (m, 1H), 2.09 (s, 3H), 1.52 (d, J = 7.2 Hz, 3H), 1.33 - 1.17 (m, 6H).

[0083] Example 4, 4-1 and 4-2

[0084]

[0085] Synthetic route:

[0086]

[0087] 1) Synthesis of intermediate 2

[0088]

[0089] Compound 1 (5.3 g, 27.02 mmol, 1 eq) was dissolved in anhydrous methanol (60 mL), then ammonium acetate (20.83 g, 270.17 mmol, 10 eq) and 4A molecular sieve (10.6 g) were added. After stirring at room temperature for 1 hour, sodium cyanoborohydride (3.40 g, 54.03 mmol, 2 eq) was added, and then the reaction solution was heated to 60 °C and stirred for 2 hours. LCMS detected that the raw material reaction was complete and the product was produced. The reaction solution was filtered, and the filter cake was washed with methanol several times until there was no product residue in the filter cake. After concentrating part of the solvent from the filtrate, it was prepared by reverse phase and freeze-dried to obtain Compound 2 (2.38 g, yield 44.67%) as a colorless colloid. Prep-HPLC: column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (HCl)-ACN]; gradient: 5%-30% B over 20 min. LCMS: rt = 0.330 min, 181.0 [M-NH 2 + .

[0090] 2) Synthesis of Intermediate 4'

[0091]

[0092] Compound 2 (1.3 g, 5.56 mmol, 1 eq, HCl) and Compound 3 (1.05 g, 5.56 mmol, 1 eq) were dissolved in N-methylpyrrolidone (13 mL), then diisopropylethylamine (2.88 g, 22.25 mmol, 3.88 mL, 4 eq) was added. The mixture was heated to 120 °C under microwave conditions and reacted for 4 hours. LCMS monitored that there was remaining raw material and the product was produced. The reaction solution was prepared by reverse phase and freeze-dried to obtain Compound 4' (910 mg, yield 41.37%) as an off-white solid. Prep-HPLC: column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (FA)-ACN]; gradient: 25%-55% B over 20 min. LCMS: rt = 0.905 min, 350.1 [M+H] + .

[0093] 3) Synthesis of Compound 4

[0094]

[0095] ​Compound 4’ (1.3 g, 3.29 mmol, 1 eq, FA) was dissolved in N-methylpyrrolidone (130 mL), and then hydrazine hydrate (3.48 g, 68.13 mmol, 3.37 mL, 98% purity, 20.72 eq) was added. The reaction solution was placed in a stainless-steel sealed tube and reacted in a heating box at 200 °C for 10 minutes (flow chemistry). LCMS monitored that the raw material reaction was complete and the product was formed. After reverse-phase preparation and lyophilization, compound 4 (610 mg, yield 56.33%) was obtained as an off-white solid. Prep-HPLC: column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (FA)-ACN]; gradient: 5%-35% B over 20 min. LCMS: rt = 0.885 min, 330.2 [M+H] + . HPLC: rt = 0.909 min. 97.693% purity. 1 H NMR (400 MHz, DMSO-d 6 ) δ = 11.34 (br s, 1H), 10.78 - 10.43 (m, 1H), 9.78 (br s, 1H), 7.56 (s, 1H), 7.33 - 7.23 (m, 2H), 6.52 (br d, J = 6.6 Hz, 1H), 4.89 (quin, J = 6.8 Hz, 1H), 4.55 (quin, J = 6.6 Hz, 1H), 4.38 (s, 1H), 1.42 (d, J = 6.8 Hz, 3H), 1.26 (dd, J = 2.8, 6.8 Hz, 6H).

[0096] 4) Synthesis of compounds 4-1 and 4-2

[0097]

[0098] Compound 4 (610 mg, 1.85 mmol, 1 eq) was chiral resolved and then obtained by reverse-phase preparation and lyophilization to give compound 4-1 (187.4 mg, 562.51 μmol, yield 30.37%) as a white solid and compound 4-2 (210.6 mg, 624.15 μmol, yield 33.70%) as a white solid.

[0099] Spectral information of 4-1 is as follows: LCMS: rt = 0.881 min, 330.1 [M+H]+. HPLC: rt = 0.900 min. 1HNMR(400MHz, DMSO-d6) δ = 11.33 (br s, 1H), 10.78 - 10.39 (m, 1H), 9.79 (br s, 1H), 7.56 (s, 1H), 7.32 - 7.23 (m, 2H), 6.53 (br d, J = 6.6 Hz, 1H), 4.89 (quin, J = 6.8 Hz, 1H), 4.55 (quin, J = 6.6 Hz, 1H), 4.38 (s, 1H), 1.42 (d, J = 6.8 Hz, 3H), 1.26 (dd, J = 2.6, 6.8 Hz, 6H).

[0100] The spectral information of 4-2 is as follows: Chiral SFC: column: DAICEL CHIRALPAK IK (250mm * 30mm, 10um); mobile phase: [CO 2 -i-PrOH (0.1% NH 3 H 2 O)]; B%: 40%, isocratic elution mode. Prep-HPLC: column: Phenomenex luna C18 150 * 25mm * 10um; mobile phase: [water (FA) - ACN]; gradient: 5% - 35% B over 9 min. LCMS: rt = 0.900 min, 330.1 [M + H]+. HPLC: rt = 0.915 min. 1 1H NMR (400MHz, DMSO-d6) δ = 11.32 (br s, 1H), 10.77 - 10.36 (m, 1H), 9.78 (br s, 1H), 7.56 (s, 1H), 7.34 - 7.22 (m, 2H), 6.52 (br d, J = 6.6 Hz, 1H), 4.89 (quin, J = 6.8 Hz, 1H), 4.55 (quin, J = 6.6 Hz, 1H), 4.38 (s, 1H), 1.42 (d, J = 6.8 Hz, 3H), 1.26 (dd, J = 2.6, 6.8 Hz, 6H).

[0101] Example 5

[0102]

[0103] Synthesis route:

[0104]

[0105] 1) Synthesis of intermediate 3

[0106]

[0107] Add material 1 (1 g, 5.55 mmol, 1 eq) and material 2 (807.29 mg, 6.66 mmol, 1.2 eq) to anhydrous tetrahydrofuran (20 mL). Add titanium tetraisopropoxide (2.84 g, 12.21 mmol, 2.58 mL, purity 98%, 2.2 eq) to the reaction solution, and react at 80 °C for 12 hours. After detecting the completion of the reaction of the raw materials by LCMS, add water (20 mL) to the reaction solution and filter. Extract with ethyl acetate (20 mL * 3), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude product is purified by a normal-phase chromatography column (ethyl acetate / petroleum ether = 30 - 50%) to obtain intermediate 3 (1.58 g, crude) as a yellow solid. LCMS: Retention time = 0.472 min, 306.0 [M+Na] + .

[0108] 2) Synthesis of intermediate 4

[0109]

[0110] Add intermediate 3 (1.2 g, 4.24 mmol, 1 eq) to anhydrous tetrahydrofuran (24 mL) and water (0.48 mL), and cool to -50 °C. Add sodium borohydride (480.67 mg, 12.71 mmol, 3 eq) to the reaction solution in portions and slowly warm to room temperature. Stir at room temperature for 0.5 hour. After detecting the complete reaction of the raw materials by LCMS, cool the reaction solution to 5 °C. Add the reaction solution to ice water at 0 °C (24 mL), extract with ethyl acetate (24 mL * 3), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain intermediate 4 (475 mg, crude) as a yellow solid. LCMS: Retention time = 0.455 min, 308.1 [M+Na] + .

[0111] 3) Synthesis of intermediate 5’

[0112]

[0113] The intermediate 4 (475 mg, 1.66 mmol, 1 eq) was dissolved in anhydrous ethanol (25 mL), and then wet palladium carbon (177.14 mg, 166.46 μmol, 10% purity) was added. The reaction flask was purged with hydrogen three times and reacted at 25 °C and 40 Psi for 6 hours. After monitoring the reaction of the raw materials by LCMS and completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by a normal-phase chromatography column (ethyl acetate / petroleum ether = 30%-50%) to obtain intermediate 5' (470 mg, 1.53 mmol, yield 97.77%) as a brown solid. LCMS: Retention time = 0.378 min, 278.3 [M+Na]+. 1 1H NMR (400 MHz, DMSO-d6) δ = 6.65 - 6.58 (m, 1H), 6.58 - 6.44 (m, 2H), 5.09 (d, J = 5.2 Hz, 1H), 4.58 - 4.41 (m, 4H), 4.29 - 4.18 (m, 1H), 1.50 - 1.41 (m, 3H), 1.22 (s, 9H).

[0114] 4) Synthesis of intermediate 6

[0115]

[0116] The intermediate 5' (400 mg, 1.57 mmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (4 mL) and anhydrous dichloromethane (4 mL), and then CDI (380.96 mg, 2.35 mmol, 1.5 eq) was added. The reaction solution was stirred at 60 °C for 2 hours. After monitoring the reaction of the raw materials by LCMS and completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by a reverse-phase column (0.1% formic acid) and then freeze-dried to obtain intermediate 6 (300 mg, 880.68 μmol, yield 56.23%) as a brown solid. LCMS: Retention time = 0.407 min, 282.1 [M+H] + .

[0117] 5) Synthesis of intermediate 7

[0118]

[0119] Compound 6 (300 mg, 1.07 mmol, 1 eq) was dissolved in hydrochloric acid-methanol (2 M, 10 mL). The reaction solution was stirred at 25 °C for 1 hour. After monitoring the reaction of the raw materials by LCMS and completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was triturated with anhydrous methanol (0.5 mL) and filtered to obtain intermediate 7 (120 mg, crude) as a pale yellow solid. LCMS: Retention time = 0.328 min, 161.0 [M-NH2 + .

[0120] 6) Synthesis of Compound 5

[0121]

[0122] Intermediate 7 (120 mg, 677.19 μmol, 1 eq) and Material 8 (127.73 mg, 677.19 μmol, 1 eq) were dissolved in N-methylpyrrolidone (2 mL), and then N,N-diisopropylethylamine (262.57 mg, 2.03 mmol, 353.86 μL, 3 eq) was added. The reaction was carried out under microwave at 120 °C for 2 hours. LCMS was used to monitor the consumption of raw materials and the formation of products. Water (1 mL) was added to the reaction solution, and the mixture was separated and purified by reverse-phase preparative (C 18 column, 0.1% formic acid solution), and then lyophilized to obtain Compound 5 (7 mg, 21.25 μmol, 3.14% yield, 100% purity) as a white solid. LCMS: Retention time = 0.644 min, 330.0 [M+H] + . HPLC: retention time = 0.810 min, 100% purity. 1 1H NMR 1 1H NMR (400 MHz, DMSO-d6) δ = 10.60 (s, 1H), 10.56 (s, 1H), 9.76 (br s, 1H), 6.91 - 6.86 (m, 3H), 6.55 - 6.43 (m, 1H), 4.96 - 4.84 (m, 1H), 4.46 (br t, J = 6.4 Hz, 1H), 4.35 (s, 1H), 1.37 (d, J = 6.8 Hz, 3H), 1.26 (dd, J = 2.4, 7.2 Hz, 6H).

[0123] Example 6

[0124]

[0125] Synthetic route:

[0126]

[0127] 1) Synthesis of Intermediate 2

[0128]

[0129] ​To N,N-dimethylformamide (5 mL), add Material 1 (500 mg, 3.12 mmol, 1 eq) and DBU (475.23 mg, 3.12 mmol, 470.52 μL, 1 eq). The reaction mixture was stirred at 80 °C for 12 h under a carbon dioxide atmosphere of 0.1 MPa. LCMS showed that the reaction was complete. After the reaction mixture was cooled to room temperature, it was poured into 1 M aqueous HCl (15 mL) and stirred at room temperature for 30 min. The resulting precipitate was filtered and dried to obtain Intermediate 2 (470 mg, 1.87 mmol, yield 60.06%, purity 96%) as a yellow solid. LCMS: rt = 0.339 min, 205.2 [M+H] + , 96.070% purity. 1 H NMR (400 MHz, DMSO-d 6 ) δ = 11.47 (s, 2H), 8.41 (d, J = 1.6 Hz, 1H), 8.15 (dd, J = 1.6, 8.8 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 2.58 (s, 3H).

[0130] 2) Synthesis of Intermediate 3

[0131]

[0132] To anhydrous methanol (10 mL) and dimethyl sulfoxide (3 mL), add Intermediate 2 (470 mg, 2.30 mmol, 1 eq), ammonium acetate (3.55 g, 46.04 mmol, 20 eq), and sodium cyanoborohydride (289.30 mg, 4.60 mmol, 2 eq). The reaction mixture was heated at 60 °C for 24 h. LCMS showed that the reaction was complete. After the reaction mixture was cooled to room temperature, it was purified by reverse-phase chromatography: mobile phase: [water (NH 4 ·H 2 O)-acetonitrile], gradient: 12% - 20% B, 30 min, and then lyophilized to obtain Intermediate 3 (400 mg, 1.89 mmol, yield 82.14%, purity 97% purity) as a yellow solid. LCMS: rt = 0.374 min, 204.1 [M-H]-, 97.295% purity. 1 H NMR (400 MHz, DMSO-d6) δ = 8.05 (d, J = 2.0 Hz, 1H), 7.72 (dd, J = 2.0, 8.5 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 4.47 (q, J = 6.8 Hz, 1H), 1.48 (d, J = 6.8 Hz, 3H).

[0133] 3) Synthesis of Compound 6

[0134]

[0135] Add dioxane (1 mL), intermediate 3 (100 mg, 487.30 μmol, 1 eq), material 4 (73.53 mg, 487.30 μmol, 0.8 eq) and diisopropylethylamine (188.94 mg, 1.46 mmol, 254.63 μL, 3 eq) to the microwave tube. The reaction solution was subjected to microwave reaction at 120 °C for 4 hours. LCMS showed that the reaction was completed. DMSO was added to dissolve the reaction mixture and purified by high performance liquid chromatography: column: Waters Xbridge 150*25mm*5um, mobile phase: [water (NH4HCO3)-acetonitrile], gradient: 2%-32% B, 9 min to obtain compound 6 (12.67 mg, 35.38 μmol, yield 7.26%, purity 99.8%) as a white solid. LCMS(6): rt = 0.611 min, 358.0 [M+H]+, 100% purity. HPLC(6): rt = 0.865 min, 99.071% purity. 1 H NMR(400MHz, DMSO-d6) δ=11.28(s, 1H), 11.13(s, 1H), 9.84(br s, 1H), 7.87(d, J=1.6Hz, 1H), 7.62(dd, J=2.0, 8.4Hz, 1H), 7.15(d, J=8.4Hz, 1H), 6.59(br d, J=6.8Hz, 1H), 4.96 - 4.80(m, 1H), 4.57(quin, J=6.8Hz, 1H), 4.35(s, 1H), 1.39(d, J=6.8Hz, 3H), 1.26(dd, J=1.2, 6.8Hz, 6H).

[0136] Example 7

[0137]

[0138] Synthetic route:

[0139]

[0140] 1) Synthesis of intermediate 2

[0141]

[0142] Add material 1 (500 mg, 2.34 mmol, 1 eq), Zn(CN) 2 (411.42 mg, 3.50 mmol, 222.39 μL, 1.5 eq) and Pd(PPh3 ) 4 (269.92 mg, 233.58 μmol, 0.1 eq), and the reaction solution was purged with N 2 three times and stirred at 100 °C for 16 h under a N 2 atmosphere. TLC (petroleum ether:ethyl acetate = 2:1, Rf = 0.4) indicated the completion of the reaction. After the reaction solution was cooled to room temperature, it was filtered to obtain a filtrate, which was poured into an aqueous solution (20 mL), and extracted twice with ethyl acetate (20 mL × 2). The organic layer was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, petroleum ether:ethyl acetate = 10:0 - 1:1) and concentrated to obtain intermediate 2 (350 mg, 2.08 mmol, yield 88.87%, purity 95%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ = 7.53 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 7.10 (dd, J = 1.5, 8.0 Hz, 1H), 6.31 (s, 2H), 2.52 (s, 3H).

[0143] 2) Synthesis of intermediate 3

[0144]

[0145] Compound 2 (350 mg, 2.19 mmol, 1 eq) and DBU (332.66 mg, 2.19 mmol, 329.36 μL, 1 eq) were added to N,N-dimethylformamide (5 mL), and the reaction solution was stirred at 80 °C for 4 h under a carbon dioxide atmosphere of 0.1 MPa. LCMS indicated the completion of the reaction. After the reaction solution was cooled to room temperature, it was poured into a 1 M HCl aqueous solution (15 mL) and stirred at room temperature for 30 min. The filter cake was obtained by filtration and dried to obtain product 3 (440 mg, 2.13 mmol, yield 97%, purity 98%) as a yellow solid. LCMS: rt = 0.337 min, 205.1 [M+H] + , 98.972% purity. 1 1H NMR (400 MHz, DMSO-d6) δ = 11.45 (s, 1H), 11.28 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.74 - 7.63 (m, 2H), 2.61 (s, 3H).

[0146] 3) Synthesis of intermediate 4

[0147]

[0148] To the anhydrous methanol (10 mL) and dimethyl sulfoxide (3 mL), add intermediate 3 (440 mg, 2.15 mmol, 1 eq), ammonium acetate (3.55 g, 46.04 mmol, 20 eq), and sodium cyanoborohydride (677.10 mg, 10.77 mmol, 5 eq). The reaction mixture is heated at 70 °C for 12 h. LCMS shows the reaction is complete. After the reaction mixture is cooled to room temperature, it is purified by reverse-phase chromatography: mobile phase: [water (NH 4 ·H 2 O)-acetonitrile], gradient: 12%-20% B, 30 min. Lyophilization gives intermediate 4 (200 mg, 974.60 μmol, yield 45.23%, purity 100% purity) as a yellow solid. LCMS (4): rt = 0.368 min, 204.1 [M-H] - , 100% purity. 1 1H NMR (400 MHz, DMSO-d6) δ = 7.81 (d, J = 8.6 Hz, 1H), 7.32 - 7.06 (m, 2H), 4.04 (q, J = 6.6 Hz, 1H), 1.24 (d, J = 6.6 Hz, 3H).

[0149] 4) Synthesis of Compound 7

[0150]

[0151] To a microwave tube, add dioxane (2 mL), intermediate 4 (80 mg, 389.84 μmol, 1 eq), material 5 (44.12 mg, 233.90 μmol, 0.6 eq) and diisopropylethylamine (151.15 mg, 1.17 mmol, 203.71 μL, 3 eq). The reaction mixture is heated at 120 °C for 3 h. LCMS shows the reaction is complete. DMSO is added to dissolve the reaction mixture and it is purified by high-performance liquid chromatography: column: YMC-Actus Triart C18 150*30 mm*7um, mobile phase: [water (FA)-acetonitrile], gradient: 15%-45% B, 10 min to give compound 7 (5.09 mg, 13.93 μmol, yield 3.57%, purity 97.44%) as a white solid. LCMS (38): rt = 0.633 min, 358.1 [M+H] + , 97.44% purity. HPLC (38): rt = 1.734 min, 97.811% purity. 11H NMR (400 MHz, DMSO-d6) δ = 11.38 - 10.98 (m, 2H), 9.87 (br s, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.08 (s, 1H), 6.62 (br d, J = 6.2 Hz, 1H), 4.90 (td, J = 6.8, 13.8 Hz, 1H), 4.57 (br t, J = 6.8 Hz, 1H), 4.27 (s, 1H), 1.40 (d, J = 6.8 Hz, 3H), 1.27 (d, J = 6.8 Hz, 6H).

[0152] Example 8

[0153]

[0154] Synthesis route:

[0155]

[0156] Example 9

[0157]

[0158] Synthesis route:

[0159]

[0160] Examples 10, 10-1 and 10-2

[0161]

[0162] Synthesis route:

[0163]

[0164] 1) Synthesis of Intermediate 3

[0165]

[0166] Compound 1 (4.5 g, 17.93 mmol, 1 eq), compound 2 (6.47 g, 17.93 mmol, 6.06 mL, 1 eq) and tetrakis(triphenylphosphine)palladium (2.07 g, 1.79 mmol, 0.1 eq) were added to a toluene (100 mL) solution. The reaction was stirred at 120 °C under a nitrogen atmosphere for 12 hours. LCMS showed 40% of the product. After the reaction was cooled to 20 °C, potassium fluoride (2 M, 10 mL) was added, and the mixture was stirred at 20 °C for 2 hours. After filtration, it was washed with ethyl acetate (30 mL * 3). Hydrochloric acid (1 M, 20 mL) was added to the mother liquor, and the mixture was stirred at 20 °C for 1 hour, then concentrated and saturated sodium carbonate solution (30 mL) was added. It was extracted with ethyl acetate (30 mL * 3), washed with brine (30 mL), and dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure gave compound 3 (5 g, crude) as a yellow oil, which was directly used for the next step.

[0167] 2) Synthesis of Intermediate 4

[0168]

[0169] Intermediate 3 (5 g, 20.64 mmol, 1 eq) was dissolved in a dichloromethane (50 mL) solution, and then hydrochloric acid (1 M, 10 mL) was slowly added. The mixture was stirred at 25 °C for 1 hour. LCMS detected 29.3% of the product. The reaction was filtered and concentrated under reduced pressure to obtain the crude product. It was purified by column chromatography (SiO 2 , ethyl acetate / petroleum ether = 1 - 20%). Intermediate 4 (3.5 g, 16.07 mmol, 77.83% yield, 98.31% purity) was obtained as a yellow solid. LCMS: Retention time = 0.487 min, 215.2 [M + H] + .

[0170] 3) Synthesis of Intermediate 5

[0171]

[0172] Intermediate 4 (1.5 g, 7.00 mmol, 1 eq) was dissolved in methanol (20 mL), then ammonium acetate (5.40 g, 70.04 mmol, 10 eq) and 4A molecular sieve (3 g) were added, and the mixture was stirred at 20 °C for 1 hour. Then sodium cyanoborohydride (880.28 mg, 14.01 mmol, 2 eq) was added, and the mixture was stirred at 60 °C for 5 hours. LCMS detection showed 33% of the product. The reaction mixture was filtered. The filter cake was washed with MeOH (20 mL * 3), and the filtrate was concentrated to obtain the crude product. High performance liquid chromatography (C 18Column, 0.1% hydrochloric acid solution) Purification gave intermediate 5 (600 mg, 2.70 mmol, 38.51% yield, 96.74% purity) as a white oil. Prep-HPLC (column: Phenomenex luna C18 250*50mm*10um; mobile phase: [water(HCl)-ACN]; gradient: 1%-20% B over 30 min). LCMS: Retention time = 0.353 min, 199.0[M+H] + . (benzyl cation).

[0173] 4) Synthesis of intermediate 7

[0174]

[0175] Intermediate 5 (450 mg, 2.09 mmol, 1 eq) and material 6 (394.41 mg, 2.09 mmol, 1 eq) were dissolved in N-methylpyrrolidone (3 mL), then N,N-diisopropylethylamine (1.08 g, 8.36 mmol, 1.46 mL, 4 eq) was added, and the mixture was stirred in a microwave at 120 °C for 4 hours. LCMS detection showed 34.55% of the product. The reaction was filtered and concentrated under reduced pressure to obtain the crude product. Purification by high performance liquid chromatography (C 18 Column, 0.1% formic acid solution) gave intermediate 7 (160 mg, 431.07 μmol, 20.61% yield, 98.97% purity) as a white solid. Prep-HPLC (column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 28%-58% B over 15 min). LCMS: Retention time = 0.459 min, 368.1[M+H] + .

[0176] 5) Synthesis of compound 10

[0177]

[0178] Intermediate 7 (160 mg, 435.56 μmol, 1 eq) and hydrazine monohydrate (444.98 mg, 8.71 mmol, 431.18 μL, 98% purity, 20 eq) were added to a solution of N-methylpyrrolidone (16 mL) and flowed at 160 °C for 5 minutes. LCMS detection showed that 84.7% of the product was obtained. The reaction was filtered and concentrated under reduced pressure to obtain the crude product. Purification by high performance liquid chromatography (C 18 column, 0.1% formic acid solution) gave compound 10 (51.80 mg, 148.16 μmol, 34.02% yield, 99.35% purity) as an off-white solid. Prep-HPLC: column: YMC-Actus Triart C18 150*30 mm*7 μm; mobile phase: [water (FA)-ACN]; gradient: 15%-45% B over 10 min. LCMS: Retention time = 0.640 min, 348.1 [M+H] + .HPLC: retention time = 0.811 min, 99.35% purity. 1 1H NMR (400 MHz, DMSO-d6) δ = 12.00 (br s, 1H), 10.82 (br s, 1H), 9.83 (br s, 1H), 7.43 (s, 1H), 7.18 (d, J = 12.4 Hz, 1H), 6.62 - 6.49 (m, 1H), 4.90 (spt, J = 6.8 Hz, 1H), 4.57 (quin, J = 6.8 Hz, 1H), 4.40 (s, 1H), 1.43 (d, J = 6.8 Hz, 3H), 1.27 (dd, J = 2.4, 6.8 Hz, 6H).

[0179] 6) Synthesis of compounds 10-1 and 10-2

[0180]

[0181] SFC: column: DAICEL CHIRALPAK AD (250 mm*50 mm, 10 μm); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 35%, isocratic elution mode

[0182] Compound 10-1 (88.30 mg, 253.40 μmol, 30.35% yield, 99.68% purity) (ee% = 98.67) is a white solid. Prep-HPLC: column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 10 min. LCMS: Retention time = 0.656 min, 348.1 [M+H] + . HPLC: retention time = 0.810 min, 99.68% purity. 1 1H NMR (400 MHz, DMSO-d6) δ = 12.00 (br s, 1H), 10.82 (br s, 1H), 9.83 (br s, 1H), 7.43 (s, 1H), 7.18 (d, J = 12.4 Hz, 1H), 6.62 - 6.49 (m, 1H), 4.90 (spt, J = 6.8 Hz, 1H), 4.57 (quin, J = 6.8 Hz, 1H), 4.40 (s, 1H), 1.43 (d, J = 6.8 Hz, 3H), 1.27 (dd, J = 2.4, 6.8 Hz, 6H).

[0183] Compound 10-2 (97 mg, 277.92 μmol, 33.29% yield, 99.52% purity) (ee% = 86.29) is a white solid. Prep-HPLC: column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 10 min. LCMS: Retention time = 0.648 min, 348.1 [M+H] + . HPLC: retention time = 0.803 min, 99.52% purity. 11H NMR (400 MHz, DMSO-d6) δ = 12.00 (br s, 1H), 10.82 (br s, 1H), 9.83 (br s, 1H), 7.43 (s, 1H), 7.18 (d, J = 12.4 Hz, 1H), 6.62 - 6.49 (m, 1H), 4.90 (spt, J = 6.8 Hz, 1H), 4.57 (quin, J = 6.8 Hz, 1H), 4.40 (s, 1H), 1.43 (d, J = 6.8 Hz, 3H), 1.27 (dd, J = 2.4, 6.8 Hz, 6H)

[0184] Example 11

[0185]

[0186] Synthesis route:

[0187]

[0188] 1) Synthesis of Intermediate 2

[0189]

[0190] Dissolve Material 1 (3 g, 16.84 mmol, 1 eq) in anhydrous methanol (50 mL), then add ammonium acetate (12.98 g, 168.37 mmol, 10 eq) and 4A molecular sieve (6 g). Stir at 20 °C for 1 hour, then add sodium cyanoborohydride (2.12 g, 33.67 mmol, 2 eq) and heat to 60 °C and stir for 2 hours. After detecting the completion of the reaction of the raw materials by LCMS, filter the reaction solution, wash the filter cake three times with dichloromethane (200 mL), and concentrate the filtrate. After purification by reverse phase (0.1% hydrochloric acid system) and rotary evaporation concentration, Intermediate 2 (1.54 g, 7.14 mmol, 42.41% yield, HCl) is obtained as an off-white solid. LCMS: rt = 0.331 min, 163.1 [M + H - NH2] + . (benzyl cation).

[0191] 2) Synthesis of Intermediate 4

[0192]

[0193] Intermediate 2 (1.34 g, 6.21 mmol, 1 eq, HCl) and Material 3 (1.17 g, 6.21 mmol, 1 eq) were dissolved in N-methylpyrrolidone (13 mL), and then diisopropylethylamine (3.21 g, 24.85 mmol, 4.33 mL, 4 eq) was added. The mixture was heated to 120 °C under microwave conditions and reacted for 5 hours. LCMS monitoring showed that there was residual raw material and product formation. The reaction solution was prepared by reverse phase (0.1% formic acid system) and then freeze-dried to obtain Intermediate 4 (1.74 g, 4.61 mmol, 74.21% yield, FA) as a white solid. LCMS: rt = 0.439 min, 332.1 [M+H] + .。

[0194] 3) Synthesis of Compound 11

[0195]

[0196] Intermediate 4 (1.52 g, 4.03 mmol, 1 eq, FA) was dissolved in anhydrous tetrahydrofuran (12 mL) and anhydrous methanol (3 mL), and the temperature was lowered using an ice-water bath. Subsequently, 50% aqueous hydroxylamine solution (2.66 g, 40.28 mmol, 10 eq) and 1 M aqueous sodium hydroxide solution (1 M, 8.06 mL, 2 eq) were added. The reaction solution was heated to 60 °C and reacted for 0.5 hours. LCMS monitoring showed that the raw material was completely reacted and product was formed. The pH of the reaction solution was adjusted to neutral with formic acid, and then it was concentrated, prepared by reverse phase, and freeze-dried to obtain Compound 11 (924.5 mg, 2.77 mmol, 68.69% yield, 99.46% purity) as a white solid. Prep-HPLC: column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (FA)-ACN]; gradient: 5%-35% B over 21 min. LCMS: rt = 0.608 min, 333.1 [M+H] + .HPLC: rt = 0.904 min. 1 HNMR (400 MHz, DMSO-d6) δ = 11.23 (s, 1H), 9.80 (br s, 1H), 9.03 (s, 1H), 7.73 (s, 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.53-7.37 (m, 2H), 6.56 (br d, J = 6.8 Hz, 1H), 4.90 (spt, J = 6.8 Hz, 1H), 4.54 (quin, J = 6.8 Hz, 1H), 4.33 (s, 1H), 1.40 (d, J = 6.8 Hz, 3H), 1.26 (dd, J = 1.6, 6.8 Hz, 6H).

[0197] Example 12

[0198]

[0199] Synthesis route:

[0200]

[0201] 1) Synthesis of Intermediate 2

[0202]

[0203] Material 1 (3 g, 15.29 mmol, 1 eq), ammonium acetate (11.79 g, 152.92 mmol, 10 eq) and 4A molecular sieve (6 g) were added to anhydrous methanol (60 mL). The reaction solution was heated at 20 °C for 1 hour, then sodium cyanoborohydride (1.92 g, 30.58 mmol, 2 eq) was added and the mixture was heated at 60 °C for 2 hours. LCMS showed that the reaction was completed. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography: column: Phenomenex luna C18 (250*70 mm, 10 um), mobile phase: [water (FA) - acetonitrile], gradient: 2% - 32% B, 18 min. After lyophilization, Intermediate 2 (1.2 g, 6.06 mmol, yield 39.20%, purity 99.52%) was obtained as a colorless oil. Prep-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (FA) - ACN]; gradient: 2% - 32% B over 18 min). LCMS: rt = 0.338 min, 198.2 [M+H] + , 99.52% purity.

[0204] 2) Synthesis of Intermediate 4

[0205]

[0206] Add N-methylpyrrolidone (5 mL), intermediate 2 (500 mg, 2.54 mmol, 1 eq), material 3 (478.21 mg, 2.54 mmol, 1 eq) and diisopropylethylamine (1.31 g, 10.14 mmol, 4 eq) to the microwave tube. The reaction solution was heated at 120 °C for 3 hours, and LCMS showed that the reaction was complete. The reaction was cooled and filtered, and the filtrate was purified by high performance liquid chromatography: column: Phenomenex luna C18 150*40 mm*15 um, mobile phase: [water (FA)-acetonitrile], gradient: 28%-58% B, 15 min to obtain product 4 (100 mg, 286.24 μmol, yield 11.29%, purity 100%) as a white solid. Prep-HPLC (column: Phenomenex luna C18 150*40 mm*15 um; mobile phase: [water (FA)-ACN]; gradient: 28%-58% B over 15 min). LCMS: rt = 0.862 min, 350.1 [M+H] + , 100% purity.

[0207] 3) Synthesis of Compound 12

[0208]

[0209] To anhydrous methanol (1 mL) and anhydrous tetrahydrofuran (4 mL), add intermediate 4 (100 mg, 286.24 μmol, 1 eq), hydroxylamine (50% purity (aqueous solution), 189.09 mg, 2.86 mmol, 100 eq), sodium hydroxide (1 M, 572.48 μL, 2 eq). The reaction mixture was stirred at 60 °C for 0.5 h. LCMS showed that the reaction was complete. The crude product was concentrated under reduced pressure and purified by high performance liquid chromatography: column: Phenomenex luna C18 150*25 mm*10 um, mobile phase: [water (FA)-acetonitrile], gradient: 10%-40% B, 15 min. Lyophilization gave compound 12 (52.50 mg, 149.27, yield 52.15%, purity 99.61%) as a yellow colloid. Prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; gradient: 10%-40% B over 15 min). LCMS (MKL-02304): rt = 0.385 min, 351.0 [M+H]+, 100% purity. HPLC (MKL-02304): rt = 0.852 min, 99.61% purity. 1 H NMR (400 MHz, DMSO-d6) δ = 11.30 (s, 1H), 9.83 (s, 1H), 9.16 (s, 1H), 7.61 (s, 1H), 7.40 (br t, J = 11.2 Hz, 2H), 6.60 (br d, J = 6.8 Hz, 1H), 4.90 (spt, J = 6.8 Hz, 1H), 4.62 - 4.50 (m, 1H), 4.34 (s, 1H), 1.40 (d, J = 6.8 Hz, 3H), 1.27 (dd, J = 0.8, 6.8 Hz, 6H).

[0210] Example 13

[0211]

[0212] Synthetic route:

[0213] 1) Synthesis of intermediate 2

[0214]

[0215] Dissolve Material 1 (12 g, 51.49 mmol, 1 eq) in dichloromethane (120 mL) solution, and then slowly add concentrated sulfuric acid (66.24 g, 675.38 mmol, 36.00 mL, 13.12 eq). Add N-iodosuccinimide (17.38 g, 77.24 mmol, 1.5 eq) at 0 °C under a nitrogen atmosphere. Stir the mixture at 25 °C for 1 hour. Thin-layer chromatography shows that the raw material is completely consumed, forming a new spot. The reaction is filtered and concentrated under reduced pressure to obtain the crude product. Purify it by column chromatography (SiO 2 , petroleum ether). Obtain Intermediate 2 (18 g, 49.65 mmol, 96.41% yield, 99% purity) as a brown oil. LCMS: Retention time = 0.581 min, 358.6 / 360.6 [M+H] + .

[0216] 2) Synthesis of Intermediate 4

[0217]

[0218] Add Intermediate 2 (18 g, 50.15 mmol, 1 eq), Material 3 (18.11 g, 50.15 mmol, 16.94 mL, 1 eq) and tetrakis(triphenylphosphine)palladium(0) (5.80 g, 5.01 mmol, 0.1 eq) to toluene (180 mL) solution. Stir the reaction at 120 °C under a nitrogen atmosphere for 12 hours. LCMS shows that 40% of the target compound is present. After cooling the reaction to 20 °C, add potassium fluoride (2 M, 100 mL), stir at 20 °C for 2 hours, filter and wash with ethyl acetate (300 mL * 3). Add hydrochloric acid (1 M, 200 ml) to the mother liquor, stir at 20 °C for 1 hour, then concentrate and add saturated sodium carbonate solution (300 mL), extract with ethyl acetate (300 mL * 3), wash with brine (300 mL), and dry over anhydrous sodium sulfate. Filter and concentrate under reduced pressure to obtain Intermediate 4 (15 g, crude) as a yellow oil. The crude product is directly used in the next step.

[0219] 3) Synthesis of Intermediate 5

[0220]

[0221] Dissolve Intermediate 4 (15 g, 49.48 mmol, 1 eq) in dioxane (150 mL) solution, and then slowly add hydrochloric acid (1 M, 30.00 mL). Stir the mixture at 25 °C for 1 hour. LCMS detection shows that 51.45% of the target product is present. The reaction is filtered and concentrated under reduced pressure to obtain the crude product. Purify it by column chromatography (SiO 2, ethyl acetate / petroleum ether = 1 - 20%). Intermediate 5 (3 g, 10.91 mmol, 22.04% yield, 100% purity) was obtained as a white solid. LCMS: Retention time = 0.510 min, no mass signal. 1 1H NMR (400 MHz, DMSO-d6) δ = 8.54 - 8.45 (m, 1H), 8.37 - 8.34 (m, 1H), 3.91 (s, 3H), 2.64 (s, 3H).

[0222] 4) Synthesis of Intermediate 6

[0223]

[0224] Intermediate 5 (3 g, 10.91 mmol, 1 eq) was dissolved in methanol (50 mL), then ammonium acetate (8.41 g, 109.06 mmol, 10 eq) and 4A molecular sieve (6 g) were added, and the mixture was stirred at 20 °C for 1 hour. Then sodium cyanoborohydride (1.37 g, 21.81 mmol, 2 eq) was added, and the mixture was stirred at 60 °C for 5 hours. LCMS showed that there was 22% product. The reaction mixture was filtered. The filter cake was washed with MeOH (20 mL * 3), and the filtrate was concentrated to obtain the crude product. Purification by high performance liquid chromatography (C 18 column, 0.1% hydrochloric acid solution) gave Intermediate 6 (810 mg, 2.41 mmol, 22.06% yield, 92.84% purity, HCl) as a white solid. Prep-HPLC (column: Phenomenex luna C18 (250 * 70 mm, 10 um); mobile phase: [water (HCl)-ACN]; gradient: 5% - 25% B over 20 min) LCMS: Retention time = 0.415 min, 260.8 [M + H] + . (benzyl cation).

[0225] 5) Synthesis of Intermediate 8

[0226]

[0227] Intermediate 6 (430 mg, 1.38 mmol, 1 eq, HCl) and Material 7 (259.48 mg, 1.38 mmol, 1 eq) were dissolved in N-methylpyrrolidone (5 mL), and then N,N-diisopropylethylamine (711.21 mg, 5.50 mmol, 958.50 μL, 4 eq) was added. The mixture was stirred in a microwave at 120 °C for 2 hours. LCMS detection showed 24% of the target product. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. It was purified by high performance liquid chromatography (C 18 column, 0.1% formic acid solution) to obtain Intermediate 8 (90 mg, 205.95 μmol, 14.97% yield, 98% purity) as a white solid. Prep-HPLC (column: Phenomenex luna C18 150*40 mm*15 um; mobile phase: [water(FA)-ACN]; gradient: 32%-62% B over 15 min). LCMS: Retention time = 0.488 min, 428.1 / 430.1 [M+H] + .

[0228] 6) Synthesis of Intermediate 9

[0229]

[0230] Intermediate 8 (90 mg, 210.16 μmol, 1 eq) and hydrazine monohydrate (214.70 mg, 4.20 mmol, 208.05 μL, 98% purity, 20 eq) were added to a solution of N-methylpyrrolidone (9 mL) and flowed at 140 °C for 5 minutes. LCMS showed 91.97% of the target compound. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. It was purified by high performance liquid chromatography (C 18 column, 0.1% formic acid solution) to obtain Intermediate 9 (60 mg, 146.97 μmol, 69.93% yield, 100% purity) as a white solid. Prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 10 min) LCMS: Retention time = 0.403 min, 408.0 / 410.0 [M+H] + .

[0231] 7) Synthesis of Intermediate 10

[0232]

[0233] Intermediate 9 (60 mg, 146.97 μmol, 1 eq) was dissolved in methanol (5 mL), and then 1,1-bis(diphenylphosphino)ferrocene palladium chloride (10.75 mg, 14.70 μmol, 0.1 eq) and triethylamine (44.61 mg, 440.91 μmol, 61.37 μL, 3 eq) were added. The mixture was purged with carbon monoxide several times. The reaction solution was stirred at 80 °C under carbon monoxide (45 psi) for 3 hours. LCMS detection showed that 55% of the target compound was present. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. It was purified by high performance liquid chromatography (C 18 column, 0.1% formic acid solution) to obtain Intermediate 10 (21 mg, 49.33 μmol, 33.57% yield, 91% purity) as an off-white solid. Prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 10 min). LCMS: Retention time = 0.385 min, 388.2 [M+H] + .

[0234] 8) Synthesis of Compound 13

[0235]

[0236] Intermediate 10 (21 mg, 54.21 μmol, 1 eq) was dissolved in tetrahydrofuran (0.4 ml) and methanol (0.1 ml), and then an aqueous solution of hydroxylamine (35.81 mg, 542.09 μmol, 10 eq) and sodium hydroxide (1 M, 108.42 μL, 2 eq) were slowly added at 0 °C. The mixture was stirred at 60 °C for 0.5 hour. LCMS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. It was purified by high performance liquid chromatography (C 18 column, 0.1% formic acid solution) to obtain Compound 13 (1.7 mg, 4.37 μmol, 8.07% yield, 99.90% purity) as a white solid. Prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 1%-30% B over 10 min). LCMS: Retention time = 0.772 min, 389.1 [M+H] +.HPLC: retention time = 1.375 min, 99.90% purity. 1 H NMR(400 MHz, DMSO-d 6 ) δ = 11.45 (s, 1H), 11.28 (s, 1H), 10.71 (s, 1H), 9.88 - 9.76 (m, 1H), 9.10 (s, 1H), 7.74 (s, 1H), 7.66 (s, 1H), 6.56 - 6.47 (m, 1H), 4.96 - 4.84 (m, 1H), 4.58 - 4.49 (m, 1H), 4.39 (s, 1H), 1.46 (d, J = 6.8 Hz, 3H), 1.35 - 1.19 (m, 6H).

[0237] Test Example 1 Myocardial Myosin ATPase Inhibition Test

[0238] Drug preparation: Dissolve the test compound in DMSO (Sigma) at a concentration of 10 mM, aliquot 20 μL per tube and store frozen at -80 °C in a refrigerator.

[0239] Preparation of test samples: 1) Use DMSO in Echo to perform a 3-fold dilution of the compound with 8 concentration gradients, and transfer 220 nL of the compound to a 96-well plate (Corning - 3696) respectively. The drug is diluted from an initial concentration gradient of 10 μM to 4.57 nM. The positive reference is Mavacamten, and the drug is diluted from an initial concentration gradient of 50 μM to 22.86 nM. 2) Centrifuge at 1000 rpm for 15 s and seal the plate for later use.

[0240] Experimental preparation: 1) Myocardial actin buffer: 5 mM Pipes-KOH pH 7.0, 500 μM ATP, 500 μM DTT 384-well plate, Greiner; Vi-cell XR Cell Viability Analyzer, Beckman Coulter; Incubator, Thermo. 2) PM12 buffer: 12 mM Pipes-KOH, pH 7.0, 2 mM MgCl 2 .

[0241] Test method: 1) F-actin preparation: (1) Dissolve Actin in the myocardial actin buffer to a solution of 0.4 mg / mL. (2) Let it stand at room temperature for 10 min to fully dissolve the protein. (3) Add 2.0 mM MgCl 2And 2.0 mM EGTA, incubated at room temperature for 20 min to form protein polymers. 2) Preparation of thin filaments: (1) Add 200 μL of ice-cold water to dissolve 1 mg of Cardiac TT complex, with a protein concentration of 5 mg / mL. (2) Add 1000 μL of the F-actin prepared in step 1 and mix well. (3) Incubate at room temperature for 20 minutes, centrifuge at 87K xg at 4 °C for 1.5 h, discard the supernatant, and resuspend the protein with 1200 μL of PM12 buffer. 3) Prepare the reaction solution and start the experiment: (1) Add 250 μL of ice-cold PM12 buffer to 250 μg of S1 myosin, with a protein concentration of 1 mg / mL. (2) Add the reagents in the following order and mix them successively to obtain the reaction mixture: 400 μL of PM12, 400 μL of 5x MSEG (from ATPase Assay Biochem Kit), 1200 μL of actin / TT complex, 40 μL of Myosin S1, 40 μL of 100x PNP (from ATPase Assay Biochem Kit), 10.4 μL of 100 mM ATP. (3) Add 10 μL of 440 μM CaCl2 solution to a 96-well plate, preheat it in an incubator at 37 °C for 10 minutes, add 100 μL of the reaction mixture to the 96-well plate, centrifuge at 1000 rpm for 10 s, continuously read the absorbance on a SpectraMax 340PC for 10 min at intervals of 30 s, with the instrument temperature at 37 °C and the wavelength at 360 nm, and analyze the data using Prism.

[0242] Statistical method: Use the log(inhibitor) vs. response--Variable slope (four parameters) analysis method in GraphPad Prism to calculate the IC 50 .

[0243] Table 1 Inhibitory activity results of representative compounds of the present invention against cardiac myosin

[0244] Compound Number <![CDATA[IC 50 (μM)]]> Mavacamten 1.68 Aficamten 3.32 1 15.34 2 16.84 3 12.37 4 7.67 4-1 3.09 4-2 Inactive 5 15.42 6 10.52 7 17.24 10 12.34 10-1 5.04 10-2 Inactive 11 15.26 12 Inactive 13 8.84

[0245] Experimental conclusion:

[0246] The compounds of the present invention showed excellent inhibitory activity against stably expressed cardiac myosin.

[0247] The test data show that most of the compounds of the present invention exhibit inhibitory activity against cardiac myosin, such as Compounds 1-4, 4-1, 5-7, 10, 10-1, 11, 13. In particular, both Compounds 4-1 and 10-1 exhibit myosin inhibitory activity similar to that of Mavacamten / Aficamten. Therefore, compared with Mavacamten / Aficamten myosin inhibitors, the compounds of the present invention also have the prospect of preparing myosin inhibitors similar to Mavacamten / Aficamten.

[0248] Test Example 2: DPPH Free Radical Scavenging Ability Test

[0249] Test Objective: To determine the DPPH free radical scavenging ability of the compounds of the present invention.

[0250] Test Materials:

[0251] Material Manufacturer Catalog Number Dimethyl Sulfoxide Sigma D2650 D4Plus Tip Holder Tecan 30097371 T8Plus Tip Holder Tecan 30097370 96-Well Plate Corning 3599 Absolute Ethanol Sangon Biotech A500737 2,2-Diphenyl-1-picrylhydrazyl (DPPH) MCE HY-112053

[0252] Test Instruments:

[0253] Instrument Manufacturer Model DigitalDispenser Tecan HPD300 Microplate Reader FlexStation3 Molecular Devices Centrifuge Eppendorf 5425R Tissue Grinder Shanghai Jingxin JXFSTPRP-CL-48 Dry Bath Hm-Kylin GL-150B

[0254] Test Methods:

[0255] First, add 20 μL of the test compound to a 96-well plate and serially dilute it 1:2 with DMSO. Then, add 200 μL of 200 μM DPPH prepared with absolute ethanol to each well, gently shake, and incubate in the dark at room temperature for 30 minutes. Detect the absorbance value at 517 nm using a microplate reader. Calculate the DPPH scavenging rate using the following formula: DPPH scavenging rate % = (1 - Ai / A0) * 100%, where Ai is the absorbance value of the sample and A0 is the absorbance value of the DMSO control group. Process the data using XLfit 5.3.1.3 software and use a non-linear fitting formula to obtain the IC 50 value. The results are shown in Table 2.

[0256] Table 2: Test Results of DPPH Free Radical Scavenging Ability of Representative Compounds of the Present Invention

[0257]

[0258]

[0259] The results show that the compounds 1, 2, 3, 4-1, 10-2, 10-1, 11, 12, 13, etc. prepared by the present invention have DPPH free radical scavenging ability, while Mavacamten and Aficamten do not have this activity.

[0260] Test Example 3: MDA Anti-Lipid Peroxidation Ability Test

[0261] Purpose of the experiment: To test the anti-lipid peroxidation ability of the compounds of the present invention against MDA.

[0262] Experimental materials:

[0263] Material Manufacturer Catalog Number DPBS (1×) Corning 21-031-CVC Dimethyl Sulfoxide Sigma D2650 D4Plus Tip Holder Tecan 30097371 T8Plus Tip Holder Tecan 30097370 96-Well Plate Corning 3599 Ascorbic Acid ST1434 Beyotime Lipid Oxidation (MDA) Detection Kit S0131M Beyotime

[0264] Test instruments:

[0265] Instrument Manufacturer Model DigitalDispenser Tecan HPD300 Microplate Reader FlexStation3 Molecular Devices Centrifuge Eppendorf 5425R Tissue Grinder Shanghai Jingxin JXFSTPRP-CL-48 Dry Bath Hm-Kylin GL-150B

[0266] Test method:

[0267] First, extract brain tissue homogenate: Anesthetize an adult male SD rat with isoflurane and then decapitate it. Take out the whole brain, wash it twice in DPBS, peel off the meninges, transfer it to a 50 mL centrifuge tube containing 10 mL of DPBS, cut it into pieces with scissors and then divide it into 10 1.5 mL centrifuge tubes. Add three grinding beads to each tube and grind at 90 Hz for 60 min, grinding 3 times. Transfer the ground tissue homogenate to a new 50 mL centrifuge tube, add DPBS to a total volume of 30 mL, and mix well. Next, add 20 μL of the compound to be tested to a 96-well plate and serially dilute it 1:3 with DMSO. Then, add 100 μL of brain tissue homogenate, 50 μL of DPBS and 50 μL of 200 μg / ml vitamin C in sequence, and configure a series of concentration standards as the standard curve. After shaking, incubate at 37 °C for 1 hour, then add 400 μL of MDA working solution and heat at 100 °C for 15 minutes. After cooling to room temperature, centrifuge at 1000 g for 10 minutes, aspirate 200 μL of the supernatant into another new plate, and detect the absorbance value at 532 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the clearance rate of the compound against MDA through the following formula: MDA clearance rate % = [((A1 - A0) - (A2 - A0)) / ((A1 - A0) - (A3 - A0))] * 100%, where A1 refers to the absorbance value of the high control group, A2 refers to the absorbance value of the sample, A3 refers to the absorbance value of the low control group, and A0 refers to the absorbance value of the blank group. Process the data with XLfit5.3.1.3 software and use the non-linear fitting formula to obtain the IC 50 value. The results are shown in Table 3.

[0268] Table 3 Test results of the anti-lipid peroxidation ability of the compounds of the present invention against MDA

[0269]

[0270] The results show that the compounds 1, 2, 3, 4-1, 10-2, 10-1, 11, 12, 13 prepared by the present invention have anti-lipid peroxidation ability, while Mavacamten and Aficamten do not have this activity.

[0271] Experimental Example 4 Intervention Effect of Representative Compounds on Ferroptosis Process (qPCR)

[0272] 1) Experimental Purpose: By detecting the changes in the expression levels of key genes in the ferroptosis signaling pathway at the cellular level, to examine the intervention effect of a series of compounds on the ferroptosis process. The specific index is the detection of the mRNA level of the key gene PTGS2 during ferroptosis (fluorescent real-time quantitative qRT-PCR).

[0273] 2) Experimental Method:

[0274] HT-1080 cells (Catalog number: CCL-121, Shanghai Cell Bank, introduced from ATCC) were plated in a six-well plate at a density of 4×105 cells / well for 22 h; HT-1080 cells were pretreated with different concentration gradients of compounds (1.37 nM to 333 nM, a total of 6 concentration gradients) for 1 h; the classical ferroptosis inducer RSL3 (Catalog number: HY-100218A, MCE, USA) at 200 nM was used to treat HT-1080 cells for 16 h; mRNA was extracted, reverse transcribed into cDNA, and the PTGS2 mRNA level was detected by qRT-PCR (Model: ABI7500, Thermo Fisher, USA). The ferroptosis inhibitor Fer-1 (Catalog number: HY-100579, MCE, USA) was used as a positive control.

[0275] 3) Experimental Results:

[0276] Using the log(agonist) vs. response--Variable slope (four parameters) analysis method in GraphPad Prism, the IC 50 of each drug was statistically calculated, and the results are shown in Table 4.

[0277] Table 4 Intervention Effect of the Compounds of the Present Invention on the Ferroptosis Process

[0278]

[0279]

[0280] The results showed that both compound 4-1 and 10-2 prepared by the present invention could inhibit ferroptosis, while Mavacamten and Aficamten had no such activity.

[0281] The experimental data of Test Example 2-4 showed that, compared with Mavacamten / Aficamten myosin inhibitors, the compounds of the present invention, while taking into account myosin inhibitory activity, also had antioxidant / DPPH radical scavenging ability / ferroptosis resistance activity. Since studies have shown that both antioxidant and ferroptosis resistance are closely related to the pathophysiology of cardiovascular diseases, the compounds of the present invention can affect heart failure caused by systolic dysfunction through the activities tested above (antioxidant / DPPH radical scavenging ability / ferroptosis resistance), thereby overcoming the defect that Mavacamten / Aficamten myosin inhibitors need to adjust the dose according to echocardiogram.

[0282] In summary, the above pharmacological experimental data indicated that, in addition to maintaining myosin inhibitory activity similar to that of Mavacamten / Aficamten, the pyrimidine dione derivatives of the present invention improved the redox balance disorder and ferroptosis risk of patients through a new mechanism of action of increasing antioxidant / ferroptosis resistance. Therefore, the pyrimidine dione derivatives of the present invention can be used to prepare a new generation of multi-target innovative drugs for the treatment of hypertrophic cardiomyopathy, and are expected to overcome debilitating exertional dyspnea and / or adverse reactions such as those often associated with left ventricular outflow tract obstruction at this level, and to treat other heart conditions.

[0283] The above embodiments are optimized implementation schemes of the present invention, which are only used to illustrate the present invention rather than limit the present invention. Modifications or equivalent replacements made by those skilled in the art without departing from the purpose and principle of the implementation scheme of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A pyrimidinedione derivative represented by formula (I), or a pharmaceutically acceptable stereoisomer, salt or solvate thereof: in: R1 and R3: independently selected from H, D, F, Cl, Br, CN, NO2, -CO-NH(OH), -CON(OH)-R' or form a five-membered heterocyclic group, a substituted five-membered heterocyclic group, a six-membered heterocyclic group, or a substituted six-membered heterocyclic group with R2; R2: selected from a five-membered heterocyclic group, a substituted five-membered heterocyclic group, or a five-ring heterocyclic group, a substituted five-membered heterocyclic group, a six-membered heterocyclic group, or a substituted six-membered heterocyclic group formed with R1 / R3; R4: selected from H, D or CH3; R': selected from H, C1-C4 straight chain alkyl, C1-C4 branched alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; Wherein, the five-membered heterocyclic group is selected from one of: The five-membered or six-membered heterocyclic group formed by R2 and R1 / R3 includes:

2. The pyrimidinedione derivative represented by formula (I) according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or solvate thereof, characterized in that The following compounds or their pharmaceutically acceptable salts or deuterated analogs:

3. Use of the pyrimidinedione derivative represented by formula (I) as claimed in claim 1, its pharmaceutically acceptable stereoisomer, salt or solvate for preparing myosin inhibitors.

4. Use of the pyrimidinedione derivative represented by formula (I) as claimed in claim 1, its pharmaceutically acceptable stereoisomers, salts or solvates for preparing drugs for treating hypertrophic cardiomyopathy, diastolic heart failure and left ventricular hypertrophy.

5. The use according to claim 4, characterized in that The pyrimidinedione derivatives represented by formula (I), their pharmaceutically acceptable stereoisomers, salts or solvates are used in preparing drugs for inhibiting hypertrophic cardiomyopathy.

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