Ring-fused compound as well as preparation method and application thereof

Abstract

The invention relates to a ring-fused compound as shown in a formula (I), a preparation method thereof, a pharmaceutical composition containing the compound and application of the ring-fused compound in prevention or treatment of cardiovascular and cerebrovascular diseases. # imgabs0 #

Description

[0001] Cross - reference to related applications

[0002] This disclosure claims the priority and benefits of the following Chinese patent applications filed with the China National Intellectual Property Administration: Chinese Patent Application No. 202311790193.0 filed on December 22, 2023, and Chinese Patent Application No. 202410433582.6 filed on April 11, 2024. The entire texts of the above - mentioned patent applications are incorporated herein by reference in their entirety. Technical field

[0003] This disclosure relates to fused - ring compounds, methods for their preparation, pharmaceutical compositions containing such compounds, and their use in the prevention or treatment of thromboembolic diseases. Background art

[0004] The hemostatic system includes the coagulation system and the fibrinolysis system, which have important physiological functions in inhibiting bleeding and accelerating wound healing. Fibrinolysis is regulated by plasminogen and is activated by physiological plasminogen activators, tissue - type plasminogen activator (t - PA), and urokinase - type plasminogen activator (u - PA). At the same time, the activation of plasminogen can be blocked by several specific molecules to inhibit the level of activated plasmin. However, various clinical cases have shown that genetic or acquired factors can enhance or weaken the fibrinolysis system, leading to disorders between the coagulation and fibrinolysis systems, thereby causing bleeding or thrombosis.

[0005] Compared with bleeding, the formation of thrombi is much slower and less noticeable, resulting in the death of many patients. Cerebral infarction is a major cause of death in developed countries. Even if the life is saved, disabilities such as paralysis may still exist. Cerebral infarction can be treated by recanalization of the occluded site. However, the re - supply of blood flow to the damaged area of cerebral infarction may lead to hemorrhagic cerebral infarction and worsen the prognosis of life and function. As a treatment method for recanalizing occluded blood vessels in the acute phase of cerebral infarction, currently known are intravenous thrombolytics, rt - PA, and thrombolytic therapy with endovascular surgical mechanical thrombectomy. Currently, drugs for treating thrombi include aspirin, ticlopidine, warfarin, and heparin. As the first - choice drug, rt - PA can be administered within 4.5 hours after the onset. However, this drug has a strong thrombolytic effect but may cause hemorrhagic side effects, and there are also strict limitations. The proportion of patients allowed to receive the drug is less than 10% of all cerebral infarction patients. Even when rt - PA is applicable, several percent of patients experience severe intracranial hemorrhage side effects, and the risk of bleeding is a concern. Therefore, small molecules with different fibrinolysis mechanisms are ideal candidates for new anti - thrombus and thrombolytic drugs.

[0006] SMTPs (named after Stachybotrys microspora triprenyl phenols) are a new family of small molecules produced by the fungus S. microspora. SMTP-7 is one of the compounds in the SMTP family and has profound biological activities. It can enhance the proteolytic activity of plasminogen, which is the circulating zymogen of plasmin and the main protease responsible for blood clot dissolution. This activity of SMTP-7 can be attributed to the regulation of the plasminogen conformation: although plasminogen is resistant to proteolytic activation due to its helical, closed conformation, SMTP-7 relaxes its conformation into an easily activatable conformation. Therefore, SMTP-7 promotes physiological fibrinolysis and clot clearance in vivo, thus effectively treating thrombotic and embolic strokes in rodent and primate models. Notably, the action of SMTP-7 is accompanied by a reduction in hemorrhagic transformation and a wider treatment time window. SMTP compounds are expected to be used as therapeutic agents for thrombotic stroke (N.-S. Arch. Pharmacol., 382, 245 - 253 (2010)), cytoprotective agents (WO2011004620A1), etc. Given the huge unmet clinical needs, the development of SMTP derivatives has broad application prospects. Summary of the Invention

[0007] The present disclosure relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0008]

[0009] In some embodiments, the abundance of deuterium atoms is at least 4000 times the natural abundance of deuterium.

[0010] In some embodiments, the abundance of deuterium atoms is at least 5500 times the natural abundance of deuterium.

[0011] In some embodiments, the abundance of deuterium atoms is at least 6000 times the natural abundance of deuterium.

[0012] In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a sodium salt.

[0013] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:(0.01 - 3).

[0014] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:(1 - 3).

[0015] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:1, about 1:2 or about 1:3.

[0016] In some embodiments, the present disclosure provides a mixture of a compound of formula (I) or a pharmaceutically acceptable salt thereof, which comprises two or more of a compound of formula (I), a monosodium salt of the compound of formula (I), a disodium salt of the compound of formula (I), and a trisodium salt of the compound of formula (I).

[0017] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:1.

[0018] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:2.

[0019] In some embodiments, in the sodium salt of the compound of formula (I), the molar ratio of the compound of formula (I) to sodium is about 1:3.

[0020] In some embodiments, the sodium salt is selected from:

[0021]

[0022] The present disclosure also provides a solid form of the sodium salt of the compound of formula (I).

[0023] In some embodiments, the solid form of the sodium salt of the compound of formula (I) is selected from an amorphous or crystalline form.

[0024] The present disclosure also provides a method for preparing the sodium salt of the compound of formula (I), comprising the step of salifying the compound of formula (I) with a sodium-containing base.

[0025] The present disclosure also provides a solvate of the sodium salt of the compound of formula (I).

[0026] In some embodiments, the solvate of the sodium salt of the compound of formula (I) is selected from a hydrate or an ethanolate.

[0027] The present disclosure also provides a solid form of the solvate of the sodium salt of the compound of formula (I).

[0028] In some embodiments, the solid form of the solvate of the sodium salt of the compound of formula (I) is selected from an amorphous or crystalline form.

[0029] The present disclosure also provides a solid form of the compound of formula (I).

[0030] In some embodiments, the solid form of the compound of formula (I) is selected from an amorphous or crystalline form.

[0031] On the other hand, the present disclosure provides a pharmaceutical composition, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present disclosure and a pharmaceutically acceptable excipient.

[0032] On the other hand, the present disclosure provides a pharmaceutical composition comprising a solid form of a compound of formula (I) or its sodium salt according to the present disclosure and a pharmaceutically acceptable excipient.

[0033] On the other hand, the present disclosure provides a pharmaceutical composition comprising a solvate or a solid form of the sodium salt of the compound of formula (I) according to the present disclosure and a pharmaceutically acceptable excipient.

[0034] On the other hand, the present disclosure provides a method for treating mammalian cardiovascular and cerebrovascular diseases, comprising administering to a mammal in need of such treatment, preferably a human, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.

[0035] On the other hand, the present disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating cardiovascular and cerebrovascular diseases.

[0036] On the other hand, the present disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof in preventing or treating cardiovascular and cerebrovascular diseases.

[0037] On the other hand, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof for preventing or treating cardiovascular and cerebrovascular diseases.

[0038] In some embodiments, the cardiovascular and cerebrovascular diseases are selected from thromboembolic diseases.

[0039] On the other hand, the present disclosure provides the following intermediate compounds or pharmaceutically acceptable salts thereof:

[0040]

[0041]

[0042] On the other hand, the present disclosure provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof using the above intermediate compound or a pharmaceutically acceptable salt thereof.

[0043] Term Definitions and Explanations

[0044] Unless otherwise specified, the terms used in this disclosure have the following meanings. The definitions of the groups and terms described in this disclosure, including their exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. A specific term should not be considered uncertain or unclear without a special definition, but should be understood according to the ordinary meaning in the art. When a trade name appears in this text, it is intended to refer to its corresponding product or its active ingredient.

[0045] In this text represents a connection site.

[0046] Unless otherwise specified, the wedge bond and the dashed wedge bond ( and ) are used to represent the absolute configuration of a stereocenter, and the solid bond and the dashed bond ( and ) are used to represent the relative configuration of a stereocenter. For example represents that the methyl group and OH are on the same side. The wavy line is used to represent an unspecified configuration, that is, if there are chiral isomers in the chemical structure, the bond can be or or contain both and two configurations. In the chemical structure of the compounds described in this disclosure, the double bond does not specify the configuration, that is, it can be the Z configuration or the E configuration, or contain both configurations at the same time.

[0047] The term "tautomer" refers to functional group isomers produced by the rapid movement of a certain atom in a molecule between two positions. The compounds in this disclosure may exhibit tautomerism. Tautomeric compounds can exist in two or more interconvertible species. Tautomers generally exist in an equilibrium form. When attempting to isolate a single tautomer, a mixture is usually produced, and its physical and chemical properties are consistent with those of a mixture of compounds. The position of the equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; while in phenols, the enol form predominates. This disclosure includes all tautomeric forms of the compounds.

[0048] The term "stereoisomer" refers to isomers produced by different arrangements of atoms in space within a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0049] The compounds of the present disclosure may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, and thus the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis- and trans-isomers, E- and Z-geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures or other mixtures, such as enantiomer- or diastereomer-enriched mixtures. All of the above isomers and their mixtures are within the scope of the definition of the compounds of the present disclosure. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and these isomers and their mixtures involved in all substituents are also included within the scope of the definition of the compounds of the present disclosure. The compounds of the present disclosure containing asymmetric atoms may be isolated in optically pure form or in racemic form. The optically pure form may be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

[0050] The structures of the compounds of the present application can be confirmed by conventional methods well-known to those skilled in the art. If the present application relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, the single crystal X-ray diffraction method (SXRD) of the present disclosure is to collect diffraction intensity data of the cultivated single crystal with a BRUKER D8 VENTURE diffractometer, the light source is CuKα radiation, the scanning mode: ω and ф scanning. After collecting the relevant data, the crystal structure is further analyzed by the direct method (SHELXTL2018), and the absolute configuration can be confirmed. The absolute configuration of the compound can also be confirmed by the chiral structure of the starting material and the reaction mechanism of asymmetric synthesis. The absolute configuration of the compound can also be confirmed by the chiral structure of the starting material and the reaction mechanism of asymmetric synthesis.

[0051] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position is understood to have an abundance of deuterium that is at least 3000 times greater than the natural abundance of deuterium (which is 0.015%), i.e., at least 45% deuterium incorporation. In certain embodiments, the compounds of the present disclosure have an abundance of deuterium for each designated deuterium atom that is at least 3500 times the natural abundance of deuterium (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 times (60% deuterium incorporation), at least 4500 times (67.5% deuterium incorporation), at least 5000 times (75% deuterium incorporation), at least 5500 times (82.5% deuterium incorporation), at least 6000 times (90% deuterium incorporation), at least 6333.3 times (95% deuterium incorporation), at least 6466.7 times (97% deuterium incorporation), at least 6600 times (99% deuterium incorporation), or at least 6633.3 times (99.5% deuterium incorporation). The present disclosure also includes various deuterated forms of the compound of formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of the compound of formula (I) by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of the compound of formula (I), or they can be synthesized using conventional techniques with deuterated reagents, including but not limited to deuterated borane, borane-d3 tetrahydrofuran solution, lithium aluminum hydride-d, iodoethane-d and iodomethane-d, etc.

[0052] The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute and one or more molecules of a solvent. Solvates generally have a substantially fixed molar ratio of solute to solvent. This term also includes clathrate compounds, including clathrate compounds with water. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, etc. When the solvent is water, the solvate formed is a hydrate.

[0053] The term "about" is used in the present disclosure to mean approximately, around, roughly, or about. When the term "about" is used in combination with a numerical range, the range is modified by extending the upper and lower limits of the stated numerical range. Unless otherwise stated, the term "about" is used herein to modify the upper and lower limits of a stated value by a numerical value with a 10% deviation.

[0054] The term "monosodium salt of the compound of formula (I)" refers to a compound of formula (I) having a molar ratio of about 1:1 with sodium, such as 1:1. "Disodium salt of the compound of formula (I)" refers to a compound of formula (I) having a molar ratio of about 1:2 with sodium, such as 1:2. "Trisodium salt of the compound of formula (I)" refers to a compound of formula (I) having a molar ratio of about 1:3 with sodium, such as 1:3.

[0055] The term "therapeutically effective amount" means an amount of a compound of the present disclosure that (i) treats a particular disease, condition, or disorder, or (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder. The amount of the compound of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0056] The term "pharmaceutically acceptable" 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.

[0057] The term "pharmaceutically acceptable salt" refers to salts of pharmaceutically acceptable acids or bases, including salts formed by the compound with inorganic acids or organic acids, and salts formed by the compound with inorganic bases or organic bases. Salts formed by the compounds of the present disclosure with inorganic bases or organic bases include, but are not limited to, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, iron salts, or meglumine salts. The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure, or salts or solvates thereof, and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the present disclosure to an organism.

[0058] The term "pharmaceutically acceptable excipient" refers to those excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0059] The word "comprise" or "comprising" and its English variants such as "comprises" or "comprising" can be understood in an open, non-exclusive sense, i.e., "including but not limited to".

[0060] The present disclosure also includes isotopically labeled compounds of the present disclosure that are the same as those described herein, but in which one or more atoms are replaced by atoms having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N,15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0061] Certain isotopically labeled compounds of the present disclosure (e.g., labeled with 3 H and 14 C) can be used in compound and / or substrate tissue distribution analysis. Tritiation (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present disclosure can generally be prepared by substituting unlabeled reagents with isotopically labeled reagents through procedures similar to those disclosed in the following protocols and / or examples.

[0062] The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0063] Typical routes of administration of the compounds of the present disclosure, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, include but are not limited to oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

[0064] The pharmaceutical compositions of the present disclosure can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, emulsifying, freeze-drying methods, etc.

[0065] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present disclosure to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, suspensions, etc. for oral administration to a patient.

[0066] Solid oral compositions can be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of tablets or dragees. Suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants or flavoring agents, etc.

[0067] The pharmaceutical composition is also suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

[0068] In all administration methods of the compounds of general formula (I) described herein, their pharmaceutically acceptable salts or their solvates, the daily dosage in terms of the compound of formula (I) can be 0.01 mg / kg to 1000 mg / kg body weight, in the form of single or divided doses.

[0069] The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and the equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present disclosure.

[0070] The chemical reactions of the specific embodiments of the present disclosure are carried out in a suitable solvent, and the solvent must be suitable for the chemical changes of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, sometimes those skilled in the art need to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0071] An important consideration in the synthesis route planning in the art is to select suitable protecting groups for reactive functional groups (such as amino groups and hydroxyl groups in the present disclosure). For example, reference can be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are incorporated into the present disclosure as a whole.

[0072] The present disclosure uses the following abbreviations:

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 It is the absolute configuration diagram of compound P1;

[0075] Figure 2 It is the crystal cell packing diagram of compound P1;

[0076] Figure 3 Bar chart of the plasminogen activation activity of Test Example 2. Detailed implementation mode

[0077] The invention will be described in detail below through examples, but this does not mean any adverse limitation to the present disclosure. The present disclosure has been described in detail herein, and its specific implementation modes have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific implementation modes of the present disclosure without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

[0078] Unless otherwise stated, the ratio represented by the mixed solvent is the volume mixing ratio.

[0079] The compounds are named manually or by software, and commercially available compounds use the supplier's catalog names.

[0080] The structures of the compounds are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The unit of NMR shift is 10 -6 (ppm). The solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS).

[0081] The eluent below can be formed into a mixed eluent by two or more solvents, and the ratio is the volume ratio of each solvent.

[0082] "Room temperature" as described herein refers to 20 - 30 °C.

[0083] Example 1. Preparation of Compound 1f

[0084]

[0085] Step 1: Preparation of Compound 1d

[0086] At room temperature, compound 1c (4.0 g), Raney nickel (4.0 g), and deuterated methanol (45 ml) were added to a reaction flask. After D2 replacement was performed 3 times, the reaction was carried out at 50 °C in a deuterium atmosphere for 18 h. When the reaction was basically completed, Raney nickel was removed by filtration, and the reaction solution was concentrated and then purified by preparative chromatography to obtain 1.5 g of compound 1d, MS-ESI: m / z 335.3 [M+H] + .

[0087] Purification method of the preparative chromatography for compound 1d:

[0088] Instrument: Gilson GX-281

[0089] Preparation column: YMC TA-C18, 30 * 150 mm, 5 μm

[0090] Mobile phase: Mobile phase A: water (containing 7 mmol / L ammonium bicarbonate); Mobile phase B: acetonitrile

[0091] Gradient: Mobile phase B from 15% to 45%

[0092] Flow rate: 25 mL / min

[0093] Column temperature: room temperature

[0094] Detection wavelength: 254 nm / 220 nm

[0095] Separation duration: 16 min

[0096] Step 2: Preparation of compound 1e

[0097] Add compound 1d (1.2 g), DCM (15 ml), and DIPEA (0.56 g) to the reaction flask in sequence. After stirring for 10 minutes under an ice bath, slowly add Fmoc-Cl (1.11 g). After addition, stir for 10 minutes and then transfer to room temperature for reaction for 3 h. When the reaction is basically completed, add 10 ml of water to the reaction system, stir and separate the liquid. The organic phase is washed with saturated brine and then dried over anhydrous sodium sulfate. The obtained organic phase is concentrated and purified by column chromatography to obtain 1.5 g of compound 1e, MS-ESI: m / z 357.2 [M + 1 - 200] + .

[0098] Step 3: Preparation of compound 1f

[0099] Add compound 1e (1.3 g), ethyl acetate (7.0 ml), and ethyl acetate solution of 4M hydrochloric acid (5.8 ml) to the reaction flask. React at room temperature for 8 h. When the reaction is basically completed, directly filter the reaction solution. The filter cake is dried at 40 °C to obtain 0.7 g of compound 1f, which is directly used in the next step without purification. MS-ESI: m / z 357.2 [M + H] + .

[0100] Example 2: Preparation of intermediate compound 2k

[0101]

[0102] Step 1: Preparation of compound 2d

[0103] Add compound 2c (50 g) and acetonitrile (1 L) to a reaction flask under nitrogen protection. Cool the above system to -20 °C, add AlCl3 (40.0 g), and stir for 10 minutes. Then, warm the above reaction system to 10 °C, and slowly add a clear mixed solution of acetonitrile (500 ml) and NBS (47.63 g). React at room temperature for 2 h, and the reaction is basically completed. Slowly add 1N HCl aqueous solution to the above reaction system, pour the above system into ice water (2 L), add ethyl acetate (1.5 L) and stir for 10 minutes. Separate the layers, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, add 250 ml of methyl tert-butyl ether to the concentrated residue, slowly add 500 ml of n-heptane, slurry for one hour, filter, collect the filter cake and dry it under vacuum at 50 °C overnight to obtain 61 g of the title compound with a yield of 87% and a purity of 96.44%. MS-ESI: m / z 275.0 [M+H] + 。

[0104] Step 2: Preparation of compound 2e

[0105] Add compound 2d (60.5 g), tricyclohexylphosphine (12.32 g), and DMF (605 ml) to a reaction flask in sequence, stir until dissolved and clear, add compound 3c (218.0 g), displace nitrogen and add Pd(OAc)2 (4.95 g). React at 90 - 100 °C for 18 hours, and the reaction is basically completed. Cool the reaction solution to 20 - 30 °C and pour it into 3 L of ice water, stir for 20 minutes, add ethyl acetate (1 L), filter through diatomaceous earth and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and subject the concentrated residue to column chromatography to obtain 44.9 g of compound 2e with a yield of 51% and a purity of 97.6%.

[0106] MS-ESI: m / z 401.2 [M+H] + 。

[0107] Step 3: Preparation of compound 2f

[0108] Add compound 2e (69 g) and DCM (3.4 L) to a reaction flask, stir until dissolved and clear, displace nitrogen and then cool to 0 °C. Add DIPEA (33.4 g), and slowly add a mixed solution of DCM (400 ml) and MOMBr (24.8 g). Maintain the reaction at 0 °C for 1 h, and the reaction is basically completed. Add normal temperature water (1.4 L) to the reaction flask at the reaction temperature, stir for 20 minutes, separate the layers, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, concentrate, and subject the concentrated residue to column chromatography to obtain 71 g of compound 2f with a yield of 93% and a purity of 98%. MS-ESI: m / z 445.3 [M+H] + 。

[0109] Step 4: Preparation of Compound 2g

[0110] At room temperature, add Compound 2f (71.0 g) and methanol (500 ml) to a reaction flask, stir for 5 minutes under N2 protection, dropwise add the NaOCH3 / CH3OH solution (43.7 ml), react at 20 - 30 °C for 5 - 6 h. When the reaction is basically completed, drop the above reaction solution into an aqueous solution of acetic acid (28.4 g) in water (3000 ml), stir for 10 minutes, add ethyl acetate (1000 ml), separate the layers, wash the organic layer with saturated brine, dry over anhydrous sodium sulfate, concentrate, and subject the concentrate to column chromatography to obtain 60 g of Compound 2g with a yield of 85% and a purity of 96%. MS-ESI: m / z 445.3 [M+H] + 。

[0111] Step 5: Preparation of Compound 2h

[0112] Add Compound 2g (10 g) and DCM (120 ml) to a reaction flask, under argon protection, add VO(acac)2 (89.5 mg). After addition, stir for 30 minutes, add a solution of TBHP (5.85 ml) in DCM (40 ml). After addition, stir at room temperature for about 3 hours, add a solution of TFA (513 mg) in DCM (40 ml). After addition, raise the temperature to 40 °C and react for 4 - 5 hours. Add silica gel (30 g) to the reaction solution for concentration and sample mixing, and purify by column chromatography to obtain 4.58 g of Compound 2h. MS-ESI: m / z461.3 [M+H] + 。

[0113] Step 6: Preparation of Compound 2i

[0114] Add Compound 2h (4.0 g) to a reaction flask, stir with MeOH (40 ml) until clear, dropwise add an aqueous NaOH solution (4 M, 21.7 ml). After addition, raise the temperature to 50 °C and react for 0.5 h. When the reaction is basically completed, cool the reaction solution to 20 - 30 °C, add a saturated aqueous NaH2PO4 solution (250 ml) to the reaction solution, add ethyl acetate (100 ml), stir for 10 minutes, let it stand and separate the layers. Dry the organic layer over anhydrous sodium sulfate, filter, and concentrate to obtain 3.6 g of a pale yellow oil, which is Compound 2i and is directly used in the next step without purification. MS-ESI: m / z 447.2 [M+H] +

[0115] Step 7: Preparation of Compound 2j

[0116] Compound 2i (3.9 g), NaHCO3 (3.7 g), DMF (40 ml), and MeI (3.7 g) were added to a reaction flask and reacted overnight at 20 - 30 °C. When the reaction was basically completed, water (200 ml) and ethyl acetate (100 ml) were added to the reaction solution, stirred for 10 minutes, allowed to stand for liquid separation, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the concentrate was subjected to column chromatography to obtain 3.2 g of a pale yellow oil, which was compound 2j. MS-ESI: m / z 461.3 [M+H] + 。

[0117] Step 8: Preparation of compound 2k

[0118] Compound 2j was resolved by SFC to obtain 1.5 g of compound 2k (RT: 26.75 min).

[0119] SFC resolution method of compound 2j:

[0120] Instrument: YMC k-Prep LAB100G

[0121] Preparation column: ChiralPak IG, 250×40 mm I.D., 10 μm

[0122] Mobile phase: Mobile phase A: n-hexane; Mobile phase B: 93% isopropanol + 7% methanol

[0123] Gradient: 10%

[0124] Flow rate: 90 mL / min

[0125] Column temperature: Room temperature

[0126] Detection wavelength: 220 nm

[0127] MS-ESI: m / z 461.3 [M+H] +

[0128] 1 1H NMR (CDCl3, 400 MHz): 1.35 (s, 3H), 1.59 (s, 6H), 1.65 - 1.75 (m, 5H), 1.80 - 1.87 (m, 1H), 1.93 - 2.00 (m, 2H), 2.02 - 2.09 (m, 2H), 2.12 - 2.23 (m, 2H), 2.97 (dd, J = 5.2, 17.6 Hz, 2H), 3.48 (s, 3H), 3.90 (s, 3H), 3.90 - 3.96 (m, 1H), 5.01 - 5.16 (m, 2H), 5.24 (s, 2H), 6.76 (s, 1H), 10.35 (s, 1H).

[0129] Step 9: Preparation of Compound 2m

[0130] Compound 2h was resolved by SFC to obtain Compound 2l (RT: 5.68 min, 10.67 min). Compound 2m was prepared using Compound 2l with an RT of 5.68 min. The method for preparing 2m from 2l refers to the method for preparing 2i from 2h.

[0131] SFC resolution method for Compound 2h:

[0132] Instrument: Waters Prep SFC 150Mgm

[0133] Preparation column: Daicel ChiralPak AD, 40 mm I.D.×250 mm, 10 μm

[0134] Mobile phase: Mobile phase A: CO2, Mobile phase B: isopropanol

[0135] Gradient: Mobile phase B 20%

[0136] Flow rate: 120 mL / min

[0137] Column temperature: 40 °C

[0138] Detection wavelength: 210 nm / 254 nm

[0139] Step 10: Preparation of Compound 2k

[0140] The method for preparing 2k from 2m refers to the method for preparing 2j from 2i.

[0141] Example 3. Preparation of Compound 4d

[0142]

[0143]

[0144] Step 1: Preparation of Compound 4a

[0145] Compound 1f (0.7 g), DCM (37 ml), Compound 2k (1.4 g), methanol (18 ml), and acetic acid (0.4 g) were added to a reaction flask. The mixture was transferred to an ice bath, and sodium cyanoborohydride (4.0 g in total, added in portions) was added to the reaction solution in portions. After addition, the reaction was maintained at an ice-water bath for 10 h, and the reaction was basically completed. The reaction solution was filtered, water (50 ml) was added to the filtrate, and the mixture was stirred and separated. The organic phase was washed with water (50 ml) and dried over anhydrous sodium sulfate, filtered, concentrated to dryness, acetonitrile (15 ml) was added to the concentrate, and the mixture was reacted overnight at 50 - 60 °C. The reaction solution was concentrated and purified by column chromatography to obtain 0.7 g of Compound 4a.

[0146] Step 2: Preparation of Compound 4b

[0147] Add compound 4a (0.7 g), MeOH (14 ml), and Et3N (0.55 g) to a reaction flask, and react overnight at 50 - 60 °C. When the reaction is basically completed, concentrate the reaction solution to dryness. Add acetonitrile (15 ml) to the concentrate for slurrying, filter, and dry the filter cake to obtain 0.4 g of the crude product of compound 4b. Without purification, directly use it for the next step. MS-ESI: m / z 547.4 [M+H] +

[0148] Step 3: Preparation of Compound 4c

[0149] Add the crude product of compound 4b (0.4 g), DCM (10 ml), compound 2k (0.55 g), and acetic acid (0.3 g) to a reaction flask. After reacting at 0 - 10 °C for 4 hours, add sodium triacetoxyborohydride (a total of 2.0 g, added in batches) to the reaction solution. After reacting for 10 h, raise the temperature to 20 - 30 °C and react overnight. When the reaction is basically completed, add water (20 ml) to the reaction flask, stir and separate the layers. Wash the organic phase with saturated brine (20 ml) and dry it with anhydrous sodium sulfate, filter, concentrate the organic phase to dryness, and purify it by column chromatography to obtain 0.58 g of compound 4c.

[0150] Step 4: Preparation of Compound 4d

[0151] Add compound 4c (0.58 cg), PPTS (1.5 g), and t-BuOH (29 ml) to a reaction flask. Under nitrogen protection, raise the temperature to about 80 °C and react for 10 hours. When the reaction is basically completed, cool down to 20 - 30 °C. Add ethyl acetate (30 ml) and water (30 ml) to the reaction flask, stir and separate the layers. Wash the organic phase with saturated brine (30 ml), dry it with anhydrous sodium sulfate, filter, and purify the concentrated organic phase by preparative chromatography to obtain 100 mg of compound 4d.

[0152] Preparative chromatography purification method for compound 4d:

[0153] Instrument: Gilson GX-281

[0154] Preparative column: YMC TA-C18, 30*150 mm, 5 μm

[0155] Mobile phase: Mobile phase A: water (containing 7 mmol / L ammonium bicarbonate); Mobile phase B: acetonitrile

[0156] Gradient: Mobile phase B from 45% to 75%

[0157] Flow rate: 25 mL / min

[0158] Column temperature: room temperature

[0159] Detection wavelength: 254 nm / 220 nm

[0160] MS-APCI: m / z 871.6 [M+H] +

[0161] 1 HNMR (DMSO-d6, 400 MHz): 1.20–1.11 (m, 6H), 1.39 - 1.63 (m, 24H), 1.66 - 1.80 (m, 1H), 1.81 - 2.20 (m, 13H), 2.40 - 2.48 (m, 2H), 2.70 - 2.88 (m, 2H), 3.68 - 3.76 (m, 2H), 4.02 - 4.12 (d, J = 16.4 Hz, 1H), 4.12 - 4.18 (s, 2H), 4.28 - 4.40 (d, J = 16.4 Hz, 1H), 4.47 - 4.59 (m, 1H), 4.96 - 5.25 (m, 6H), 6.61 (s, 1H), 6.62 (s, 1H), 9.77 (s, 2H).

[0162] Example 4, Synthesis and Single Crystal Analysis of Intermediate Compound P1

[0163]

[0164] Compound 2m (100 mg, 0.224 mmol), MeOH (2 mL), and 4-Hydroxyphenethylamine (33.8 mg, 0.246 mmol) were added to a reaction flask and stirred at room temperature for 4 h. Then, it was placed in an ice-water bath, and sodium borohydride (40 mg, 1.12 mmol) was added. After addition, stirring was continued for 0.5 h, and glacial acetic acid (0.3 mL) was added. The temperature was raised to 60 °C (external temperature), and stirring was carried out overnight. The reaction was quenched and sent for preparative purification. After lyophilization, 55 mg of the target compound P1 was obtained. MS-ESI: m / z 550.2 [M+H] +

[0165] Preparation Chromatographic Purification Method of Compound P1

[0166] Instrument: Gilson GX-281

[0167] Preparation column: YMC TA-C18, 30 * 150 mm, 5 μm

[0168] Mobile phase: Mobile phase A: water (containing 7 mmol / L ammonium bicarbonate); Mobile phase B: acetonitrile

[0169] Gradient: Mobile phase B from 60% to 90%

[0170] Flow rate: 25 mL / min

[0171] Column temperature: Room temperature

[0172] Detection wavelength: 254 nm / 220 nm

[0173] Single crystal cultivation

[0174] Weigh 10 mg of compound P1, add 200 μl of 95% ethanol to a 1.5 ml centrifuge tube. After ultrasonic dissolution of the solid until clear, seal the tube mouth with a sealing film and make two small holes in the sealing film. After standing at room temperature for 3 days, crystals precipitate and are submitted for single crystal diffraction inspection.

[0175] Data collection was carried out on a BRUKER D8 VENTURE diffractometer. The data collection includes ω and φ scans. In the monoclinic space group C2, the structure was solved by intrinsic phasing using the SHELX software suite. Subsequently, the structure was refined by full-matrix least-squares method. All non-hydrogen atoms were located and refined using anisotropic displacement parameters.

[0176] The absolute configuration diagram of compound P1 is shown in Figure 1 . The crystallographic data are shown in Table 1 and Table 2. The structure contains two chiral centers, C53 is (S) and C54 is (R).

[0177] Table 1 Crystal data

[0178]

[0179] Table 2 Atomic coordinates (×10 4 ) and equivalent isotropic displacement parameters of P1

[0180]

[0181]

[0182]

[0183] Example 5, Preparation of Compound 4d-1

[0184]

[0185] Step 1: Preparation of Compound SM

[0186] Compound 2j was resolved by SFC to obtain 2.0 g of compound SM (RT: 17.00 min).

[0187] SFC resolution method of compound 2j:

[0188] Instrument: YMC k-Prep LAB100G

[0189] Preparation column: ChiralPak IG, 250×40mm I.D., 10μm

[0190] Mobile phase: Mobile phase A: n-hexane; Mobile phase B: 93% isopropanol + 7% methanol

[0191] Gradient: 10%

[0192] Flow rate: 90 mL / min

[0193] Column temperature: Room temperature

[0194] Detection wavelength: 220 nm

[0195] MS-ESI: m / z 461.3 [M+H] +

[0196] 1 H NMR (CDCl3, 400 MHz) δ 10.35 (s, 1H), 6.76 (s, 1H), 5.25 (s, 2H), 5.15–5.03 (m, 2H), 3.96 - 3.88 (m, 4H), 3.48 (s, 3H), 2.98 (dd, J=17.7, 5.4 Hz, 1H), 2.70 (dd, J=17.7, 6.6 Hz, 1H), 2.22–2.11 (m, 2H), 2.11–1.90 (m, 4H), 1.90–1.84 (m, 1H), 1.76–1.64 (m, 3H), 1.59 (s, 6H), 1.36 (s, 3H).

[0197] Step 2: Preparation of compound SM-1

[0198] Add compound SM (2.0 g), p-nitrobenzoic acid (1.45 g), tributylphosphine (1.76 g), and tetrahydrofuran (30 ml) to a reaction flask. Transfer it to an ice bath. Add N,N,N',N'-tetramethyldiazodicarboxamide solution (3.5 g dissolved in 10 mL of tetrahydrofuran) to the reaction solution. After addition, maintain the reaction in the ice bath for 0.5 h, then raise the temperature of the reaction solution to 60 °C and react for 48 h. The reaction is basically completed. Filter the reaction solution, add water (50 ml) to the filtrate, stir and separate the layers. Wash the organic phase with water (50 ml), dry the organic phase with anhydrous sodium sulfate, filter, concentrate to dryness, and subject the residue to column chromatography to obtain 1.8 g of compound SM-1.

[0199] 11H NMR (400 MHz, DMSO-d6) δ 10.27–10.18 (m, 1H), 8.32–8.25 (m, 2H), 8.15–8.05 (m, 2H), 6.74 (s, 1H), 5.68 (s, 2H), 5.38–5.32 (m, 1H), 5.26 (s, 2H), 5.05–4.98 (m, 1H), 4.98–4.91 (m, 1H), 3.71 (s, 3H), 3.17–3.08 (m, 3H), 2.91–2.81 (m, 1H), 2.16–2.03 (m, 2H), 1.94–1.79 (m, 4H), 1.73–1.57 (m, 2H), 1.54–1.50 (m, 3H), 1.45–1.42 (m, 5H), 1.39–1.35 (m, 3H).

[0200] Step 3: Preparation of Compound 2h-1

[0201] Add compound SM-1 (0.9 g), potassium carbonate (0.224 g), and methanol (20 ml) to a reaction flask. The reaction mixture is stirred at room temperature for 1 hour until the reaction is basically complete. Filter the reaction mixture, add water (50 ml) and ethyl acetate (50 ml) to the filtrate, stir and separate the layers. The aqueous layer is extracted twice with ethyl acetate (50 ml). The organic layer is dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and the residue is purified by column chromatography to obtain 0.49 g of compound 2h-1.

[0202] MS-ESI: m / z 461.4 [M+H] +

[0203] Step 4: Preparation of Compound 2j-1

[0204] Add compound 2h-1 (0.45 g) and methanol (5 ml) to a reaction flask, and place it in an ice bath. Add 1M aqueous sodium hydroxide solution (2.5 mL) dropwise to the reaction mixture. After addition, maintain the reaction at ice bath temperature for 0.5 h, then warm the reaction mixture to room temperature and stir for 1 hour until the reaction is basically complete. Adjust the pH of the reaction mixture to 6 with 1M dilute hydrochloric acid, stir and separate the layers. The aqueous layer is extracted twice with ethyl acetate (50 ml). The organic layer is dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and 0.4 g of compound 2j-1 is obtained.

[0205] MS-ESI: m / z 447.3 [M+H] +

[0206] Step 5: Preparation of Compound 2k-1

[0207] Compound 2j-1 (0.4 g), sodium bicarbonate (0.47 g), and N,N-dimethylformamide (5 ml) were added to a reaction flask. The mixture was transferred to an ice bath, and methyl iodide (0.477 g) was added dropwise to the reaction solution. After addition, the reaction was maintained in an ice-water bath for 0.5 h, and then the reaction solution was heated to 60 °C and reacted for 3 h. The reaction was basically completed. Water (30 ml) and ethyl acetate (30 ml) were added to the reaction solution, and the mixture was stirred and separated. The aqueous phase was extracted twice with ethyl acetate (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain 0.38 g of compound 2k-1.

[0208] MS-ESI: m / z 461.3 [M+H] +

[0209] Step 6: Preparation of compound 4a-1

[0210] Compound 1f (0.244 g), DCM (10 ml), compound 2k-1 (0.22 g), methanol (5 ml), and sodium acetate (0.078 g) were added to a reaction flask. The mixture was transferred to an ice bath, and sodium cyanoborohydride (0.2 g) was added to the reaction solution. After addition, the reaction was maintained in an ice-water bath for 1 h, and then the reaction solution was heated to 25 °C and reacted for 3 h. The reaction was basically completed. The reaction solution was filtered, and water (50 ml) was added to the filtrate. The mixture was stirred and separated. The organic phase was washed with water (50 ml) and dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. Acetonitrile (15 ml) was added to the concentrate, and the mixture was reacted overnight at 50-60 °C. The reaction solution was concentrated, and purified by column chromatography to obtain 0.2 g of compound 4a-1.

[0211] MS-ESI: m / z 769.4 [M+H] +

[0212] Step 7: Preparation of compound 4b-1

[0213] Compound 4a-1 (0.2 g), MeOH (5 ml), and Et3N (0.15 g) were added to a reaction flask and reacted overnight at 50-60 °C. The reaction was basically completed. The reaction solution was concentrated to dryness, and the concentrate was purified by a C-18 column (preparative column: spherical, 40-60 μm, 100A, 12 g, mobile phase: water (0.1% formic acid) / acetonitrile = 1:1) to obtain 0.08 g of compound 4b-1.

[0214] MS-ESI: m / z 547.3 [M+H] +

[0215] Step 8: Preparation of compound 4c-1

[0216] Compound 4b-1 (0.06 g), DCM (2 ml), compound 2k-1 (0.065 g), and sodium acetate (0.02 g) were added to a reaction flask. After reacting at 0 - 10 °C for 1 hour, sodium cyanoborohydride (0.05 g) was added to the reaction solution, and the temperature was raised to 20 - 30 °C for overnight reaction. When the reaction was basically completed, water (20 ml) was added to the reaction flask, and the mixture was stirred and separated. The organic phase was washed with saturated brine (20 ml) and dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated to dryness. After separation and purification by column chromatography, 0.1 g of compound 4c-1 was obtained.

[0217] Step 9: Preparation of compound 4d-1

[0218] Compound 4c-1 (0.1 g), PPTS (0.26 g), and t-BuOH (3 ml) were added to a reaction flask. The temperature was raised to about 80 °C under nitrogen protection and reacted for 10 hours. When the reaction was basically completed, the temperature was lowered to 20 - 30 °C. Ethyl acetate (10 ml) and water (3 ml) were added to the reaction flask, and the mixture was stirred and separated. The organic phase was washed with saturated brine (3 ml), dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated and purified by preparative chromatography to obtain 23 mg of compound 4d-1.

[0219] Purification method of compound 4d-1 by preparative chromatography:

[0220] Instrument: Gilson GX-281

[0221] Preparative column: YMC TA-C18, 30 * 150 mm, 5 μm

[0222] Mobile phase: Mobile phase A: water (containing 0.05% formic acid); Mobile phase B: acetonitrile

[0223] Gradient: Mobile phase B 65% to 95%

[0224] Flow rate: 25 mL / min

[0225] Column temperature: room temperature

[0226] Detection wavelength: 254 nm / 220 nm

[0227] MS-APCI: m / z 871.5 [M + H] +

[0228] 11H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 9.81 (s, 1H), 9.75 (s, 1H), 6.66 (s, 1H), 6.62 (s, 1H), 5.23–5.07 (m, 4H), 5.07–4.96 (m, 2H), 4.80–4.69 (m, 1H), 4.33–4.04 (m, 4H), 3.80–3.66 (m, 2H), 2.89–2.74 (m, 2H), 2.48–2.40 (m, 2H), 2.19–2.02 (m, 4H), 2.02–1.78 (m, 10H), 1.65–1.54 (m, 9H), 1.53–1.46 (m, 12H), 1.20–1.11 (m, 6H).

[0229] Example 6, Preparation of Compound 2k-2

[0230]

[0231] Add benzylamine (0.244 g), DCM (10 ml), and Compound SM-1 (0.2 g) to a reaction flask. Transfer it to an ice bath, and add sodium triacetoxyborohydride (0.14 g) to the reaction solution. After adding, maintain the reaction in an ice-water bath for 1 h, then raise the temperature of the reaction solution to 25 °C and react for 3 h. The reaction is basically completed. Filter the reaction solution, add water (50 ml) to the filtrate, stir and separate the layers. Wash the organic phase with water (50 ml), and dry the organic phase with anhydrous sodium sulfate. Filter and concentrate to dryness. Add acetonitrile (15 ml) to the concentrate and react overnight at 50 - 60 °C. Concentrate the reaction solution, and 0.15 g of Compound 2k-2 is obtained by preparation from the residue.

[0232] MS-ESI: m / z 669.19 [M+H] +

[0233] 1 1H NMR (400 MHz, DMSO-d6) δ 8.37–8.29 (m, 2H), 8.17–8.11 (m, 2H), 7.40–7.26 (m, 5H), 7.00 (s, 1H), 5.42–5.34 (m, 1H), 5.09–4.93 (m, 2H), 4.80–4.64 (m, 2H), 4.31–4.14 (m, 2H), 3.24–3.13 (m, 1H), 2.99–2.87 (m, 1H), 2.16–2.00 (m, 2H), 1.99–1.88 (m, 2H), 1.88–1.80 (m, 2H), 1.72–1.60 (m, 1H), 1.60–1.52 (m, 4H), 1.47 (s, 3H), 1.44–1.33 (m, 6H).

[0234] Preparation chromatographic purification method of Compound 2k-2:

[0235] Instrument: CAS-SH-ANA-SFC-L (Waters UPCC with PDA Detector and QDa Detector)

[0236] Preparation column: Chiralcel OJ-3 100×4.6mm I.D., 3μm

[0237] Mobile phase: A: CO2 B: Methanol (0.2% methylamine)

[0238] Gradient: Run 5% to 40% mobile phase B for 3 minutes, then run 5% mobile phase B for 1 minute.

[0239] Flow rate: 2.5 mL / min

[0240] Column temperature: 35 °C

[0241] Detection wavelength: 254 nm / 220 nm

[0242] Test examples of biological activity and related properties

[0243] The compounds in the following test examples were all prepared according to the methods of the above embodiments of the present disclosure.

[0244] Test Example 1 Activation effect of the compound on plasminogen

[0245] Prepare the mother liquor of Human Glu - Plasminogen (Enzyme research, catalog number: HPG2001) at a concentration of 150 nM (3×) using a buffer (50 mM Tris - HCl + 100 mM NaCl + 0.01% Tween - 80, pH 7.4), the mother liquor of urokinase - type plasminogen activator (uPA) (Sigma - Aldrich, catalog number: U4010) at a concentration of 15 IU / ml (3×), the mother liquor of plasmin substrate D - Val - Leu - Lys - pNA (Sigma - Aldrich, catalog number: V0882) at a concentration of 300 μM (3×), and the working solution of the test compound at 750 μM (3×) (the solvent of the compound stock solution is DMSO). Add 20 μl of 3× plasminogen (Human Glu - Plasminogen), 20 μl of the test compound (the DMSO concentration in the 3× working solution is 7.5%), and set up a control group (without adding the compound, replaced with 7.5% DMSO / buffer). After mixing, incubate at room temperature for 10 minutes. Subsequently, add 20 μl of the mixed working solution (3×) of (urokinase - type plasminogen activator (uPA) + D - Val - Leu - Lys - pNA), mix well and add it to a 96 - well plate (Corning, catalog number: 3599). Use an MD microplate reader (model: MD spectra Max i3x) for kinetic detection, with a detection wavelength of 405 nm and detect once every 1 minute. According to the absorbance value (OD 405nm ) at 405 nm wavelength and the square of time (t 2 ) to plot a curve, and use the slope of the curve to calculate the initial reaction rate, that is:

[0246] Vinitial = 10 3 ×(OD t2 - OD t1 ) / (t2 - t1) 2 , where t2 and t1 are two time points with an interval of 2 min (OD t2 is the OD 405nm value at 4 min, OD t1 is the OD 405nm value at 2 min).

[0247] The activation multiple of the test compound = the initial rate of the test compound / the initial rate of the control group, and the following results are obtained (Table 3).

[0248] Table 3. Potency of compounds in activating plasminogen

[0249] Test Compounds Concentration (μM) Activation Fold (within 5 min) Compound 4d 250 171.8

[0250] Test Example 1 shows that Compound 4d of the embodiments of the present disclosure has significant plasminogen activation activity compared to the control group. Activation of plasminogen by the compound in Test Example 2

[0251] Prepare the working solution of Human Glu-Plasminogen (Enzyme research, catalog number: HPG2001) at a concentration of 250 nM (5×) using a buffer (50 mM Tris-HCl + 100 mM NaCl + 0.01% Tween-80, pH 7.4), the working solution of urokinase-type plasminogen activator (uPA) (Sigma-Aldrich, catalog number: U4010) at a concentration of 125 IU / mL (2.5×), the working solution of plasmin substrate H-Val-Leu-Lys-pNA·2HCl (Absin, catalog number: abs45133644) at a concentration of 250 μM (2.5×), and the working solution of the compound to be tested at a 2.5× final concentration, with working solution concentrations of 750 μM and 375 μM (the solvent for the compound stock solution is DMSO, and the compound diluent is a buffer containing 6.25% DMSO). Add 40 μL / well of the mixed working solution (2.5×) of (urokinase-type plasminogen activator (uPA) + H-Val-Leu-Lys-pNA·2HCl) to a 96-well microplate (Sybio, catalog number: 100096H), add 20 μL / well of 5× plasminogen (Human Glu-Plasminogen), and then add 40 μL / well of the compound to be tested (the DMSO concentration in the 2.5× working solution is 6.25%), and set up a control group (without adding the compound, replaced with a buffer containing 6.25% DMSO). After mixing, use an MD microplate reader (model: MD spectra Max i3x) to perform kinetic detection at 37 °C, with a detection wavelength of 405 nm, detecting once every 1 minute for 30 minutes. According to the absorbance value (OD 405nm ) at the wavelength of 405 nm and time squared (t 2 ) to plot a curve, and use the slope of the curve to calculate the initial reaction rate, that is:

[0252] V initial = 10 3 ×(OD t2 - OD t1 ) / (t2 - t1) 2 , where t2 and t1 are two time points 2 minutes apart (for example: calculating the initial rate from 2 - 4 minutes, that is, OD t2 is the OD 405nm value at 4 minutes, OD t1 is the OD 405nm value at 2 minutes).

[0253] Activation multiple of the test compound = Initial rate of the test compound / Initial rate of the control group. The experimental results are shown in Table 4 and Figure 3 .

[0254] Table 4. Potency of compounds in activating plasminogen

[0255] Test Compound Number Final Concentration of Compound (μM) Activation Fold (within 5 min) 4d 300 39.5 4d 150 16.9 4d-1 300 18.5 4d-1 150 6.1

[0256] Table 4 of Test Example 2 and Figure 3 showed that at concentrations of 150 μM and 300 μM, the activation multiple of plasminogen by Compound 4d was significantly better than that of 4d-1, with statistical differences. The statistical method was Two-way ANNOVA using Graphpad Prism software, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 。 2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The abundance of deuterium atoms is at least 4000 times the natural abundance of deuterium.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claim 2, wherein: The abundance of deuterium atoms is at least 5500 times the natural abundance of deuterium.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The abundance of deuterium atoms is at least 6000 times the natural abundance of deuterium.

5. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The pharmaceutically acceptable salt is a sodium salt.

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

7. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 6, in the preparation of a medicament for preventing or treating cardiovascular and cerebrovascular diseases.

8. The use according to claim 7, wherein The cardiovascular and cerebrovascular diseases are selected from thromboembolic diseases.

9. The following compound or a pharmaceutically acceptable salt thereof:

10. A method for preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, comprising preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof by using the compound of claim 9 or a pharmaceutically acceptable salt thereof.

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