WO2024222880A1 - Pharmaceutical composition of pyrido-pyrazole derivative and use of pharmaceutical composition in medicine - Google Patents

Abstract

The present invention relates to a pharmaceutical composition of a pyrido-pyrazole derivative and a use of the pharmaceutical composition in medicine. The pharmaceutical composition comprises an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from a compound represented by general formula (I) or a stereoisomer, tautomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal thereof. The pharmaceutical composition comprises 1-1200 mg of the active ingredient A, and the content of the active ingredient A is selected from 0.5-99.0%. The present invention also relates to a use of the pharmaceutical composition in preparation of related drugs for treating cancers.

Description

A pharmaceutical composition of pyridopyrazole derivatives and its application in medicine Technical Field

The present invention belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from a compound of general formula (I) or a stereoisomer, a tautomer, a deuterated substance, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a cocrystal thereof, wherein the pharmaceutical composition comprises 1-1200 mg of the active ingredient A, and the content of the active ingredient A is selected from 0.5%-99.0%. The present invention also relates to the use of the pharmaceutical composition in preparing drugs for treating cancer.

Background Art

Protein ubiquitination in cells is a key protein modification that regulates multiple cellular processes. Protein ubiquitination is controlled by the synergistic action of E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). DUBs can cleave the isopeptide bond between ubiquitin and modified proteins, and are responsible for removing ubiquitin from target proteins and rescuing them from degradation pathways; they are also involved in the editing, maturation, and recycling of ubiquitin molecules after degradation. Currently, more than 100 deubiquitinating enzymes are known, and these proteins are subdivided into six subfamilies. The ubiquitin-specific protease (USP) subfamily is the largest of these subfamily, with 58 members currently known. USP is a cysteine protease that contains a highly conserved catalytic domain, and USP1 is a member of the USP subfamily among DUBs (Cancers, 2020, 12, 1579; Molecular Cell, 2018, 72, 925-941).

Fanconi Anemia (FA) and DNA Translesion Synthesis (TLS) pathways are the earliest discovered DNA damage tolerance and repair pathways regulated by reversible ubiquitination. USP1 can regulate the deubiquitination of specific proteins in the FA and TLS pathways to participate in the regulation of DNA damage-repair pathways (Nature Chemical Biology, 2014, 10, 298-304). USP1 plays an important role in DNA repair in tumor cells. It has been reported that the loss of USP1 leads to reduced survival and replication fork degradation in BRCA1-deficient cells (Molecular Cell, 2018, 72, 925-941). UAF1 (USP1-associated factor 1) is a cofactor of USP1, USP12, and USP46, which can enhance their deubiquitinase activity by forming a stable USP/UAF1 protein complex; the USP1/UAF1 complex deubiquitinates a variety of substrates and is associated with the regulation of DNA repair processes, tumor pathogenesis, and antiviral innate immunity (Nat Commun, 2020, 11, 6042). Currently, there are no drugs targeting USP1 protein on the market, and the research on USP1 inhibitors has broad application prospects.

Compound 1 is described in PCT/CN2023/076700, which has good USP1 inhibitory activity. When used as a therapeutic agent for treating humans, the administration of a therapeutically effective dose is a problem and may lead to toxic side effects or ineffective treatment. Therefore, it is very necessary to develop a pharmaceutical composition or pharmaceutical preparation with good safety, efficacy and stability.

Summary of the invention

The object of the present invention is to provide a pharmaceutical composition, which comprises an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from the compound described in the general formula (I) or its stereoisomer, tautomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal. The present invention also relates to the use of the pharmaceutical composition in the preparation of drugs for treating kidney disease.

The pharmaceutical composition of the invention has stable quality, good solubility, good absorption, low irritation, good safety, high bioavailability and can be absorbed orally.

The present invention relates to a pharmaceutical composition, which comprises a therapeutically effective amount of active ingredient A and a pharmaceutical excipient. The pharmaceutical composition can be in the form of a unit preparation.

The present invention relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from the compound of general formula (I) or its stereoisomer, tautomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal,

R ¹ is selected from -CH ₃ , -CHF ₂ , -CH ₂ CH ₃ ,

R ² is selected from -CH ₃ , -CHF ₂ , -CH ₂ CH ₃ ,

In some embodiments, the structure of the compound described by formula (I) is selected from one of the structures shown in Table S-1;

Table S-1 Compound Structure

In some embodiments, the compound described by general formula (I) is selected from compound 1, whose structure is:

In one aspect, the pharmaceutical composition comprises 1-1200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5-1200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5-1000 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5-900 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5-800 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 10-800 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 10-600 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 10-400 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 20-400 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 30-300 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 25-200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 20-200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 30-200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 30-100 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 50-100 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5-100 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 5 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 10 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 20 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 25 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 30 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 40 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 50 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 75 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 100 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 125 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 150 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 200 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 300 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 400 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 500 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 600 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 700 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 800 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 1000 mg of active ingredient A;

In some embodiments, the pharmaceutical composition comprises 1200 mg of active ingredient A.

In another aspect, the content of the active ingredient A in the aforementioned pharmaceutical composition is selected from 0.5%-99.0%;

In some embodiments, the content of active ingredient A is 0.5%-99.0%; in some embodiments, the content of active ingredient A is 1.0%-80.0%; in some embodiments, the content of active ingredient A is 1.0%-70.0%; in some embodiments, the content of active ingredient A is 1.0%-60.0%; in some embodiments, the content of active ingredient A is 1.0%-50.0%; in some embodiments, the content of active ingredient A is 1.0%-40.0%; in some embodiments, the content of active ingredient A is 1.0%-30.0%; in some embodiments, its content is 1.0%-20.0%; in some embodiments, the content of active ingredient A is 1.0%-10.0%; in some embodiments, the content of active ingredient A is 5.0%-90.0%; in some embodiments, the content of active ingredient A is 5.0%-85.0%; in some embodiments, the content of active ingredient A is 5.0%-70.0%; in some embodiments, the content of active ingredient A is 5.0%-60.0%; in some embodiments, the content of active ingredient A is 5.0%-50.0%; in some embodiments, the content of active ingredient A is 5.0%-40.0%; In some embodiments, the content of active ingredient A is 5.0%-30.0%; in some embodiments, the content of active ingredient A is 5.0%-20.0%; in some embodiments, the content of active ingredient A is 5.0%-10.0%; in some embodiments, the content of active ingredient A is 10.0%-90.0%; in some embodiments, the content of active ingredient A is 10.0%-80.0%; in some embodiments, the content of active ingredient A is 10.0%-75.0%; in some embodiments, the content of active ingredient A is 10.0%-70.0%; in some embodiments In some embodiments, the content of active ingredient A is 10.0%-60.0%; in some embodiments, the content of active ingredient A is 10.0%-50.0%; in some embodiments, the content of active ingredient A is 10.0%-40.0%; in some embodiments, the content of active ingredient A is 10.0%-30.0%; in some embodiments, the content of active ingredient A is 10.0%-20.0%; in some embodiments, the content of active ingredient A is 60.0%; in some embodiments, the content of active ingredient A is 30.0%; in some embodiments, the content of active ingredient A is 10.0%.

In another aspect, the pharmaceutical excipient in the aforementioned pharmaceutical composition is selected from one or more of a filler, a disintegrant, a binder, a glidant, a lubricant, a surfactant, and a pH adjuster;

In some embodiments, the filler is selected from one or more of microcrystalline cellulose, mannitol, lactose, sucrose, sorbitol, dextran, pregelatinized starch, monocalcium phosphate, starch;

In some embodiments, the disintegrant is selected from sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, One or more of cross-linked sodium carboxymethyl cellulose and calcium carboxymethyl cellulose;

In some embodiments, the binder is selected from one or more of povidone, hydroxypropyl cellulose, hypromellose, and methyl cellulose;

In some embodiments, the wetting agent is selected from one or both of water and ethanol.

In some embodiments, the glidant is selected from one or more of talc, silicon dioxide, micronized silica gel, polyethylene glycol, and magnesium dodecyl sulfate;

In some embodiments, the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, and sodium stearyl fumarate;

In some embodiments, the surfactant is selected from one or more of sodium lauryl sulfate, polysorbate, poloxamer, soluplus, polyoxyethylene castor oil.

In some embodiments, the pH adjuster is selected from one or more of citric acid, tartaric acid, and fumaric acid.

In some embodiments, the pharmaceutical excipients further comprise one or more of flavoring agents, antioxidants, preservatives, opacifiers, and film coating premixes.

In some embodiments, the aforementioned pharmaceutical compositions are in solid form.

The pharmaceutical composition according to any embodiment of the present invention comprises:

(i) Compound 1 or its succinate, in an amount of 0.5% to 99.0%; and optionally,

(ii) pharmaceutical excipients include one or more of fillers, binders, wetting agents, disintegrants, glidants, lubricants, surfactants, and surface active agents;

(iii) the filler comprises one or more of microcrystalline cellulose, mannitol, lactose, sucrose, sorbitol, dextran, pregelatinized starch, calcium dihydrogen phosphate, and starch;

(iv) the binder comprises one or more of povidone, hydroxypropyl cellulose, hypromellose, and methyl cellulose;

(v) the wetting agent comprises one or more of water and ethanol;

(vi) the disintegrant comprises one or more of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, and calcium carboxymethyl cellulose;

(vii) the glidant comprises one or more of talc, silicon dioxide, micronized silica gel, polyethylene glycol, and magnesium dodecyl sulfate;

(viii) the lubricant comprises one or more of magnesium stearate, calcium stearate, stearic acid, and sodium stearyl fumarate;

(ix) a surfactant selected from sodium lauryl sulfate, polysorbate, poloxamer, soluplus, polyoxyethylene One or more of castor oil;

(x) pH adjuster selected from one or more of citric acid, tartaric acid and fumaric acid;

(xi) The composition may further contain one or more of a flavoring agent, an antioxidant, a preservative, a sunscreen agent, and a film coating premix.

Optionally, the pharmaceutical composition described in any of the above schemes can be prepared into a preparation selected from tablets, granules, powders, oral solutions, emulsions or capsules.

Optionally, in the pharmaceutical preparation described in any of the above schemes, the binder can be added in a solution state or in a powder state; the disintegrant can be added internally, externally, or both internally and externally.

The present invention also provides the use of the pharmaceutical composition in preparing drugs related to treating cancer.

Unless otherwise stated, the terms used in the specification and claims of this application have the following meanings.

"Preparation specifications" refers to the weight of the main drug (active ingredient A) contained in each vial, tablet or other unit preparation.

The carbon, hydrogen, oxygen, sulfur, nitrogen or F, Cl, Br, I involved in the groups and compounds described in the present invention all include their isotopes, and the carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds described in the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein carbon isotopes include ¹² C, ¹³ C and ¹⁴ C, hydrogen isotopes include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super tritium), oxygen isotopes include ¹⁶ O, ¹⁷ O and ¹⁸ O, sulfur isotopes include ³² S, ³³ S, ³⁴ S and ³⁶ S, nitrogen isotopes include ¹⁴ N and ¹⁵ N, fluorine isotopes include ¹⁷ F and ¹⁹ F, chlorine isotopes include ³⁵ Cl and ³⁷ Cl, and bromine isotopes include ⁷⁹ Br and ⁸¹ Br.

"Optional" or "optionally" means that the event or circumstance described later may but need not occur, and the description includes situations where the event or circumstance occurs or does not occur. For example, "alkyl optionally substituted with F" means that alkyl may but need not be substituted with F, and the description includes situations where alkyl is substituted with F and situations where alkyl is not substituted with F.

"Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" refers to a salt of the compound of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

"Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention, or stereoisomers, tautomers, deuterated forms, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals thereof and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients and/or one or more other therapeutic agents.

A "carrier" is a substance that does not cause significant irritation to an organism and does not abrogate the biological activity and Characteristics of materials.

"Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

"Prodrug" refers to a compound of the present invention that can be converted into a biologically active compound through in vivo metabolism. The prodrug of the present invention is prepared by modifying the amino or carboxyl group in the compound of the present invention, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. When the prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free amino or carboxyl group.

"Co-crystal" refers to a crystal formed by the active pharmaceutical ingredient (API) and the co-crystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds, in which the pure state of API and CCF are solid at room temperature and there is a fixed stoichiometric ratio between the components. Co-crystal is a multi-component crystal, including binary eutectics formed between two neutral solids and multi-component eutectics formed between neutral solids and salts or solvates.

"Animal" is meant to include mammals, such as humans, companion animals, zoo animals, and livestock, preferably humans, horses, or dogs.

"Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers, and conformational isomers.

"Tautomers" refer to functional group isomers produced by the rapid movement of an atom in a molecule between two positions, such as keto-enol isomerism and amide-imino alcohol isomerism.

" _IC50 " is the concentration of a drug or inhibitor required to inhibit a specified biological process (or a component of such a process, such as an enzyme, receptor, cell, etc.) by half.

Unless otherwise specified, the "content %" of a substance in a pharmaceutical composition in this application refers to the percentage of the weight of the substance in the total weight of the pharmaceutical composition.

The "content %" in the content determination of a preparation refers to the percentage of the weight of the main drug of the preparation obtained by testing according to the determination method to the weight of the main drug in the preparation.

DETAILED DESCRIPTION

The following embodiments illustrate the technical solutions of the present invention in detail, but the protection scope of the present invention includes but is not limited to them.

The compounds used in the reactions described herein are prepared according to organic synthesis techniques known to those skilled in the art. The starting point is commercially available chemicals and/or compounds described in the chemical literature. “Commercially available chemicals” are obtained from regular commercial sources, and suppliers include: Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec, and Bailingwei Technology.

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). NMR shifts (δ) are given in units of 10 ^-6 (ppm). NMR measurements were performed using (Bruker Avance III 400 and Bruker Avance 300) NMR spectrometers, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) as the solvent, and tetramethylsilane (TMS) as the internal standard;

For MS determination (Agilent 6120B (ESI) and Agilent 6120B (APCI));

HPLC determination was performed using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6mm, 3.5μM);

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF ₂₅₄ or Qingdao GF ₂₅₄ silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.20mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm;

Column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier;

Boc: tert-butoxycarbonyl;

Ts: p-toluenesulfonyl;

Cbz: benzyloxycarbonyl;

TMS: trimethylsilyl.

Example 1: Preparation of Compound 1

Step 1: Preparation of 2A

4-(Methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid (5 g, 23.58 mmol) was dissolved in THF (60 mL), and borane tetrahydrofuran solution (47 mL, 1 M) was slowly added dropwise at 0°C, and stirred at room temperature overnight. Methanol was added at 0°C to quench the mixture, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/20-1/2) to obtain 2A (3.8 g, yield: 81%).

LCMS m/z＝199.2[M+H] ⁺

Step 2: Preparation of 2B

2A (3.8 g, 19.16 mmol) was dissolved in DCM (100 mL), and Dess-Martin periodinane (16.25 g, 38.32 mmol) was added in batches at 0°C, and the mixture was reacted at room temperature for 3 h. The reaction was completed by TLC monitoring, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/20-1/2) to obtain 2B (3.02 g, yield: 80%).

LCMS m/z＝197.1[M+H] ⁺

Step 3: Preparation of 2C

1,1-Dibromo-3,3,3-trifluoroacetone (4.98 g, 18.49 mmol, CAS: 431-67-4) was placed in a 250 mL single-mouth bottle, 30 mL of water and sodium acetate (1.89 g, 23.09 mmol) were added, and the mixture was reacted at 90 °C for 1 h. After cooling to room temperature, 60 mL of methanol, 2B (3.02 g, 15.39 mmol) and concentrated aqueous ammonia (15 mL) were added in sequence, and the mixture was reacted at 90 °C for 2 h. After cooling to room temperature, the reaction solution was poured into 200 mL of water, filtered, and the filter cake was washed with water (2 x 10 mL), and the filter cake was dried under reduced pressure to obtain 2C (2.9 g, yield: 62%).

LCMS m/z＝303.1[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ12.36-12.16(m,1H),7.68-7.55(m,1H),3.59(s,3H),1.90-1.72(m,12H).

Step 4: 2D preparation

2C (2.0 g, 6.62 mmol) was dissolved in 30 mL DMF at room temperature, and isopropyl iodide (4.50 g, 26.48 mmol) and cesium carbonate (6.47 g, 19.86 mmol) were added in sequence, and the mixture was reacted at 90°C overnight. After cooling to room temperature, water and ethyl acetate were added for extraction, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/20-1/1) to obtain 2D (1.2 g, yield: 52%).

LCMS m/z＝345.2[M+H] ⁺

Step 5: Preparation of 2E

2D (1.2 g, 3.48 mmol) was dissolved in 20 mL THF, lithium borohydride (0.38 g, 17.40 mmol) was added, and the mixture was stirred at 60 °C for 2 h. After cooling to room temperature, a saturated aqueous solution of ammonium chloride was added to quench the reaction, and water and ethyl acetate were added to extract the mixture. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2E (0.9 g, yield: 81%).

LCMS m/z＝317.2[M+H] ⁺

Step 6: Preparation of 2F

At 0°C, add methylsulfonyl chloride (64 mg, 0.56 mmol) to a solution of 2E (0.12 g, 0.37 mmol) and triethylamine (0.11 g, 1.09 mmol) in dichloromethane (15 mL), and stir at room temperature for 1 h. Add 20 mL of saturated sodium bicarbonate aqueous solution to quench, and extract with dichloromethane (20 mL×3). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purification by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/100-1/20) gave 2F (0.11 g, yield 74%).

LCMS m/z＝395.1[M+H] ⁺

Step 7: Preparation of 2G

1F (0.1 g, 0.58 mmol) and 2F (0.23 g, 0.58 mmol) were dissolved in DMF (5 mL), and cesium carbonate (0.38 g, 1.17 mmol) and sodium iodide (0.087 g, 0.58 mmol) were added, and the mixture was reacted at 140°C for 2 h. After cooling to room temperature, 20 mL of water and 20 mL of ethyl acetate were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/2) to obtain 2G (0.060 g, yield: 22%).

LCMS m/z＝470.2[M+H] ⁺

Step 8: Preparation of Compound 1

Place 2G (0.06 g, 0.13 mmol), (4-cyclopropyl-6-methoxypyrimidin-5-yl)boric acid (0.050 g, 0.26 mmol), XPhos G3 (0.011 g, 0.013 mmol, CAS: 1445085-55-1) and potassium phosphate (0.056 g, 0.27 mmol) in a 50 mL single-necked bottle, add 1,4-dioxane (4 mL) and water (1 mL), replace with nitrogen three times, heat to 90 °C and stir overnight. The mixture was cooled to room temperature, and 20 mL of water and 20 mL of ethyl acetate were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1). After concentration, the residue was purified by secondary silica gel column chromatography (dichloromethane/methanol (V/V) = 100/0-100/3) to give compound 1 (30 mg, yield: 39%).

LCMS m/z＝584.2[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD) δ8.97(d,1H),8.64(s,1H),8.38(d,1H),7.65-7.58(m,1H),4.93-4.84(m,1H) ,4.39(s,2H),3.93(s,3H),2.06-1.94(m,6H),1.74-1.59(m,7H),1.41(d,6H),1.27-1.20(m,1H),1.16 -1.09(m,1H),0.99-0.86(m,2H).

Embodiment 2:

Step 1: Preparation of 4A

Dissolve methyl 3-cyclopropyl-3-oxopropanoate (20 g, 140.66 mmol) and formamidine acetate (29.29 g, 281.32 mmol) in methanol (400 mL), add sodium methoxide (53.19 g, 984.62 mmol), and react at room temperature for 2 days. The pH was adjusted to 7, filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 0/1-1/3); (methanol/dichloromethane (V/V) = 0/1-1/10) to give 4A (9 g, yield: 47%).

LCMS m/z＝137.1[M+H] ⁺

Step 2: Preparation of 4B

4A (8.5 g, 62.43 mmol) was dissolved in acetonitrile (100 mL), and NBS (11.67 g, 65.60 mmol) was added, and the mixture was reacted at room temperature for 1 h. Water and ethyl acetate were added for extraction, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 0/1-1/1) to obtain 4B (9.1 g, yield: 67%).

LCMS m/z＝215.0[M+H] ⁺

Step 3: Preparation of 4C

4B (6 g, 27.90 mmol) was dissolved in acetonitrile (200 mL), sodium hydride (2.2 g, 54.90 mmol) was added, the mixture was stirred at room temperature for 45 min, 2-fluorosulfonyldifluoroacetic acid (8.94 g, 50.22 mmol) was added, and the mixture was reacted at room temperature for 2 h. Water and ethyl acetate were added for extraction, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 0/1-1/2) to obtain 4C (4.0 g, yield: 54%).

LCMS m/z＝265.0[M+H] ⁺

Step 4: 4D preparation

4C (1.0 g, 3.77 mmol) was dissolved in 1,4-Dioxane (15 mL), and diboronic acid pinacol ester (1.05 g, 4.16 mmol), Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (0.31 g, 0.38 mmol) and potassium acetate (0.74 g, 7.54 mmol) were added in sequence, and the mixture was reacted at 90°C overnight under nitrogen protection. After cooling to room temperature, water and ethyl acetate were added for extraction, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/1-1/20) to obtain 4D (420 mg, yield: 35%).

LCMS m/z＝313.0[M+H] ⁺

Step 5: Preparation of Compound 2

2G (100 mg, 0.21 mmol), 4D (85.21 mg, 0.27 mmol), Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (17.15 mg, 0.021 mmol) and potassium carbonate (87.07 mg, 0.63 mmol) were placed in a 25 mL single-necked bottle, 1,4-dioxane (4 mL) and water (1 mL) were added, nitrogen was replaced three times, and the mixture was stirred at 90° C. for 3 h. The mixture was cooled to room temperature, and 20 mL of water and 20 mL of ethyl acetate were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1). The crude product was further separated and purified by preparative HPLC (instrument: Waters 2767 preparative liquid phase; chromatographic column: SunFire@Prep C18 (19 mm×150 mm); mobile phase composition: mobile phase A: acetonitrile mobile phase B: water (containing 5 mM ammonium acetate)) to obtain compound 2 (20 mg, yield: 15%).

LCMS m/z＝620.3[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.10(d,1H),8.85(s,1H),8.52(d,1H),8.03-7.63(m,2H),4.88-4.74(m,1H ),4.39-4.26(m,2H),1.95-1.79(m,7H),1.61-1.46(m,6H),1.32(d,6H),1.23-1.18(m,1H),1.13-0.92(m ,3H).

Embodiment 3:

Step 1: Preparation of 5A

2C (4 g, 13.23 mmol) was dissolved in DMF (20 mL), and NaH (0.79 g, 19.84 mmol, wt% = 60%) was slowly added under ice bath, and 2-(trimethylsilyl)ethoxymethyl chloride (3.31 g, 19.85 mmol) was added after stirring under ice bath for 10 min, and the mixture was naturally warmed to room temperature and stirred for 30 min. 10 mL of saturated aqueous ammonium chloride was added to quench the reaction, and 20 mL of ethyl acetate was added for extraction. The organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/3) to obtain 5A (5.2 g, yield: 90%).

Step 2: Preparation of 5B

5A (5.2 g, 12.02 mmol) was dissolved in THF (25 mL), and lithium borohydride (1.31 g, 59.86 mmol) was added at room temperature. After the addition was complete, the mixture was stirred at 60°C for 2 h. After cooling to room temperature, 10 mL of saturated aqueous ammonium chloride solution was slowly added dropwise to quench the reaction, and 20 ml of ethyl acetate was added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 5B (5 g, yield: 100%).

LCMS m/z＝405.2[M+H] ⁺

Step 3: Preparation of 5C

5B (4.81 g, 11.89 mmol) was dissolved in DCM (30 mL), pyridine (1.41 g, 17.84 mmol) was added, trifluoromethanesulfonic anhydride (4.03 g, 14.29 mmol) was slowly added dropwise under ice bath, and the mixture was naturally heated to room temperature and stirred for 1 h. 30 mL of dichloromethane and 50 mL of water were added for extraction, and the organic layer was washed once with 30 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 5C.

Step 4: 5D preparation

1F (1.7 g, 9.91 mmol) and 5C from the previous step were dissolved in DMF (30 mL). Cesium carbonate (9.69 g, 29.73 mmol) was added under a water bath at room temperature. After the addition was completed, the mixture was stirred for 1 h. 50 mL of ethyl acetate and 50 mL of water were added for extraction. The organic layer was washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/2) to give 5D (2.3 g, yield: 41%).

LCMS m/z＝558.2[M+H] ⁺

Step 5: Preparation of 5E

5D (1 g, 1.79 mmol) and (4-cyclopropyl-6-methoxypyrimidin-5-yl)boronic acid (0.69 g, 3.56 mmol) were dissolved in a mixed solvent of 1,4-dioxane (10 mL) and water (2 mL), and XPhos G3 (0.15 g, 0.18 mmol) and potassium phosphate (0.76 g, 3.58 mmol) were added in sequence. After addition, nitrogen was replaced three times, and the temperature was raised to 90 ° C and stirred overnight. Cooled to room temperature, 20 ml of ethyl acetate and 10 ml of water were added for extraction, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1) to obtain 5E (1.1 g, yield: 91%).

Step 6: Preparation of 5F

5E (1.1 g, 1.64 mmol) was dissolved in THF (15 mL), tetrabutylammonium fluoride (4.29 g, 16.4 mmol) was added, and the temperature was raised to 70°C and stirred for 6 h. After cooling to room temperature, 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/3-1/0) to obtain 5F (0.8 g, yield 90%).

LCMS m/z＝542.2[M+H] ⁺

Step 7: 5G preparation

Dissolve isopropanol-D7 (2 g, 29.79 mmol) in DCM (20 mL), add triethylamine (6.03 g, 59.59 mmol) under ice bath, slowly add methylsulfonyl chloride (4.10 g, 35.79 mmol), and after addition, naturally warm to room temperature and stir for 20 min. Add 30 mL of water and 30 mL of dichloromethane for extraction, wash the organic layer with 20 mL of saturated sodium bicarbonate aqueous solution and 20 mL of sodium chloride aqueous solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 5G (4 g).

Step 8: Preparation of compound 3

5F (0.1 g, 0.18 mmol) and 5G (0.13 g, 0.90 mmol) were dissolved in DMF (5 mL), and cesium carbonate (0.29 g, 0.90 mmol) and sodium iodide (0.027 g, 0.18 mmol) were added in sequence, and stirred at 80°C overnight. After cooling to room temperature, 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/1) to obtain compound 3 (60 mg, yield: 56%).

LCMS m/z＝591.3[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.05(d,1H),8.72(s,1H),8.48(d,1H),7.93-7.85(m,1H),4.38-4.25(m,2H ),3.86(s,3H),1.96-1.82(m,6H),1.78-1.68(m,1H),1.62-1.45(m,6H),1.18-0.98(m,2H),0.97-0.81(m ,2H).

Embodiment 4:

5F (0.1 g, 0.18 mmol) and bromomethylcyclopropane (0.049 g, 0.36 mmol) were dissolved in DMF (5 mL), cesium carbonate (0.12 g, 0.36 mmol) was added, and the mixture was stirred at 60 °C for 3 h. After cooling to room temperature, 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/1) to give compound 4 (60 mg, yield: 56%).

LCMS m/z＝596.3[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.04(d,1H),8.72(s,1H),8.48(d,1H),7.79-7.71(m,1H),4.39-4.24(m,2H ),3.94(d,2H),3.86(s,3H),1.96-1.82(m,6H),1.79-1.68(m,1H),1.61-1.45(m,6H),1.22-1.08(m,2H ),1.05-0.98(m,1H),0.97-0.80(m,2H),0.59-0.49(m,2H),0.46-0.37(m,2H).

Embodiment 5:

Step 1: Preparation of 7A

2,2-Difluorocyclopropylmethanol (1g, 9.28mmol) was dissolved in DCM (20mL), triethylamine (1.88g, 18.54mmol) was added under ice bath, methylsulfonyl chloride (1.28g, 11.11mmol) was slowly added dropwise, and the temperature was naturally raised to room temperature and stirred for 20min. 30mL of water and 30mL of dichloromethane were added for extraction, and the organic layer was then washed with 20mL of saturated sodium bicarbonate aqueous solution and 20mL of sodium chloride aqueous solution in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 7A (1.6g).

Step 2: Preparation of compound 5

5F (0.1 g, 0.18 mmol) and 7A (0.067 g, 0.36 mmol) were dissolved in DMF (5 mL), and cesium carbonate (0.12 g, 0.36 mmol) and sodium iodide (0.027 g, 0.18 mmol) were added in sequence, and stirred at 60 °C for 3 h. The mixture was cooled to room temperature, and 20 mL of ethyl acetate and 20 mL of water were added for extraction. The organic layer was washed with saturated brine (10 mL x 3), and anhydrous sulfuric acid was added. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/1) to give compound 5 (65 mg, yield: 57%).

LCMS m/z＝632.7[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.04(d,1H),8.72(s,1H),8.48(d,1H),7.73-7.67(m,1H),4.39-4.26(m,3H ),4.19-4.08(m,1H),3.86(s,3H),2.28-2.15(m,1H),1.96-1.83(m,6H),1.77-1.62(m,3H),1.59-1.45(m ,6H),1.18-1.09(m,1H),1.06-0.97(m,1H),0.97-0.83(m,2H)

Embodiment 6:

5F (0.1 g, 0.18 mmol) and 3-bromopropyne (0.043 g, 0.36 mmol) were dissolved in DMF (5 mL), cesium carbonate (0.12 g, 0.36 mmol) was added, and the mixture was stirred at room temperature for 3 h. 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/1) to obtain compound 6 (90 mg, yield: 86%).

LCMS m/z＝580.2[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.05(d,1H),8.72(s,1H),8.48(d,1H),7.76-7.69(m,1H),5.03(d,2H), 4.41-4.24(m,2H),3.86(s,3H),3.53(t,1H),1.96-1.83(m,6H),1.79-1.69(m,1H),1.60-1.43(m,6H), 1.18-1.09(m,1H),1.06-0.98(m,1H),0.98-0.82(m,2H).

Embodiment 7:

Step 1: Preparation of 9A

4B (1 g, 4.65 mmol) was dissolved in acetonitrile (10 mL), and phosphorus oxychloride (7.13 g, 46.50 mmol) was slowly added at room temperature. After the addition, the mixture was stirred at 80°C for 4 h. After cooling to room temperature, the reaction solution was poured into ice water, and the pH was adjusted to alkaline with saturated sodium bicarbonate aqueous solution. 30 mL of ethyl acetate was added for extraction, and the organic layer was dried over anhydrous sodium sulfate and then reduced to 400 μl. The product was concentrated under reduced pressure to afford 9A.

Step 2: Preparation of 9B

2,2-Difluorocyclopropylmethanol (0.93 g, 8.60 mmol) was dissolved in THF (10 mL), and sodium hydride (0.25 g, 6.3 mmol, wt% = 60%) was slowly added under ice bath. After the addition was completed, the mixture was stirred under ice bath for 20 min, and then 9A (1 g, 4.28 mmol) was slowly added. The mixture was naturally warmed to room temperature and stirred for 20 min. 10 mL of saturated aqueous ammonium chloride solution was added to quench the reaction, and 20 mL of ethyl acetate and 20 mL of water were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 9B (1.4 g).

LCMS m/z＝305.1[M+H] ⁺

Step 3: Preparation of 9C

9B (1.3 g, 4.26 mmol) was dissolved in THF (10 mL), and isopropylmagnesium chloride tetrahydrofuran solution (3.2 mL, 6.39 mmol, 2N) was slowly added dropwise under ice bath, and stirred at room temperature for 1.5 h. 2 mL of isopropyl alcohol pinacol borate (1.19 g, 6.39 mmol) in THF was slowly added dropwise at 0°C, and stirred at room temperature for 1 h. 20 mL of water was added to quench the reaction, and 20 mL of ethyl acetate was added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 0/1-1/10) to obtain 9C (0.23 g, yield: 15%).

LCMS m/z＝353.2[M+H] ⁺

Step 4: Preparation of compound 7

2G (0.11 g, 0.23 mmol) and 9C (0.12 g, 0.34 mmol) were dissolved in a mixed solvent of 1,4-dioxane (4 mL) and water (1 mL). XPhos G3 (0.019 g, 0.023 mmol) and potassium phosphate (0.098 g, 0.46 mmol) were added in sequence. After the addition, the atmosphere was replaced with nitrogen three times and the mixture was stirred at 90 °C overnight. The mixture was cooled to room temperature, and 20 mL of ethyl acetate and 10 mL of water were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1). The crude product was further separated and purified by HPLC (instrument: waters2767 preparative liquid phase; chromatographic column: SunFire@Prep C18 (19 mm × 150 mm); mobile phase composition: mobile phase A: acetonitrile, mobile phase B: water (containing 5 mM ammonium acetate)) to obtain compound 7 (30 mg, yield: 20%).

LCMS m/z＝660.90[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.06(d,1H),8.71(d,1H),8.49(d,1H),7.91-7.84(m,1H),4.88-4.73(m,1H ),4.68-4.14(m,4H),2.17-2.00(m,1H),1.94-1.80(m,6H),1.78-1.69(m,1H),1.65-1.45(m,7H),1.43-1.28 (m,7H),1.18-1.10(m,1H),1.05-0.91(m,2H),0.90-0.80(m,1H).

Embodiment 8:

Step 1: Preparation of 10A

5D (0.7 g, 1.25 mmol) was dissolved in DCM (4 mL), trifluoroacetic acid (4 mL) was added, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, 20 mL of dichloromethane was added to the residue, 10 mL of saturated sodium bicarbonate aqueous solution was added, and the mixture was stirred for 10 min. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 10A.

Step 2: Preparation of 10B

10A (0.1 g, 0.23 mmol) was dissolved in DMF (5 mL), bromomethylcyclopropane (0.062 g, 0.46 mmol) was added, and the mixture was stirred at 60°C for 3 h. After cooling to room temperature, 20 mL of ethyl acetate and 20 mL of water were added, and the mixture was stirred to separate layers. The organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/2) to obtain 10B (0.1 g, yield: 90%).

Step 3: Preparation of Compound 8

10B (0.1 g, 0.21 mmol) and 4D (0.098 g, 0.32 mmol) were dissolved in a mixed solvent of 1,4-dioxane (4 mL) and water (1 mL), and XPhos Pd G2 (0.017 g, 0.021 mmol) and potassium phosphate (0.089 g, 0.42 mmol) were added in sequence. The atmosphere was replaced with nitrogen three times and stirred at 90 °C for 3 h. The mixture was cooled to room temperature, extracted with 20 ml of ethyl acetate and 10 ml of water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1) to obtain compound 8 (90 mg, yield: 68%).

LCMS m/z＝632.90[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.10(d,1H),8.85(s,1H),8.52(d,1H),8.05-7.62(m,2H),4.42-4.24(m,2H ),3.93(d,2H),1.94-1.81(m,7H),1.64-1.46(m,6H),1.27-1.18(m,2H),1.18-1.13(m,1H),1.06-0.93(m ,2H),0.58-0.50(m,2H),0.45-0.37(m,2H).

Embodiment 9:

Step 1: Preparation of 11A

5D (0.100 g, 0.18 mmol) and 4D (0.084 g, 0.27 mmol) were dissolved in 1,4-dioxane (4 mL) and water. Potassium phosphate (0.076 g, 0.36 mmol) and XPhos Pd G2 (0.014 g, 0.018 mmol) were added to a mixed solvent of 1 mL (1 mL), the atmosphere was replaced with nitrogen three times, and the mixture was stirred at 90°C overnight. The mixture was cooled to room temperature, 20 mL of ethyl acetate and 10 mL of water were added for extraction, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/2) to obtain 11A (0.13 g, yield: 99%).

Step 2: Preparation of 11B

11A (0.13 g, 0.18 mmol) was dissolved in DCM (4 mL), trifluoroacetic acid (4 mL) was added at room temperature, and the mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, 20 mL of dichloromethane was added to the residue, and 10 mL of saturated sodium bicarbonate aqueous solution was added, and the mixture was stirred for 10 min. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 11B.

Step 3: Preparation of compound 9

11B (0.11 g, 0.19 mmol) and 3-bromopropyne (0.045 g, 0.38 mmol) were dissolved in DMF (5 mL), cesium carbonate (0.12 g, 0.38 mmol) was added, and the mixture was stirred at room temperature for 3 h. 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/1) to obtain compound 9 (70 mg, yield: 60%).

LCMS m/z＝616.80[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.10(d,1H),8.85(s,1H),8.52(d,1H),8.03-7.62(m,2H),5.02(d,2H), 4.40-4.26(m,2H),3.51(t,1H),1.95-1.81(m,7H),1.63-1.41(m,6H),1.30-1.20(m,1H),1.12-1.01(m,2H ),1.01-0.92(m,1H).

Embodiment 10:

Step 1: Preparation of 15A

10A (0.15 g, 0.35 mmol) and 5G (0.25 g, 1.72 mmol) were dissolved in DMF (5 mL), and cesium carbonate (0.57 g, 1.75 mmol) and sodium iodide (0.052 g, 0.35 mmol) were added in sequence, and stirred at 80°C overnight. After cooling to room temperature, 20 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic layer was washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/5-1/2) to obtain compound 15A (0.1 g, yield: 60%).

Step 2: Preparation of compound 10

15A (0.1 g, 0.21 mmol) and 4D (0.098 g, 0.32 mmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL), and XPhos Pd G2 (0.017 g, 0.021 mmol) and potassium phosphate (0.089 g, 0.42 mmol) were added in sequence. The mixture was replaced with nitrogen three times and stirred at 90°C for 3 hours. The mixture was cooled to room temperature, and 20 ml of ethyl acetate and 10 ml of water were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V) = 1/10-1/1) to obtain compound 10 (70 mg, yield: 53%).

LCMS m/z＝627.80[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.10(d,1H),8.85(s,1H),8.52(d,1H),8.04-7.62(m,2H),4.40-4.25(m,2H ),1.93-1.81(m,7H),1.63-1.43(m,6H),1.24-1.16(m,1H),1.12-1.02(m,2H),1.01-0.93(m,1H).

Preparation Example:

Unless otherwise specified, the ratio (%) refers to the percentage of the weight of a component to the total weight of the formulation.

1. Preparation 1-1: Specification 25 mg/tablet:

2. Preparation 1-2; Specification: 25 mg/tablet

3. Preparation 1-3: Specification 200 mg/tablet:

4. Preparation 1-4: Specification 600 mg/tablet:

5. Preparation 1-5: Specification 1200 mg/tablet:

The above prescription preparations 1-5 adopt the following process:

1). Micro powder: weigh and mix the API and surfactant (if any)/pH adjuster (if any) according to the prescription ratio, and then micronize after mixing evenly.

2) Weighing: weigh the API-excipient micronized material, filler, binder, glidant, disintegrant according to the prescription. Lubricants;

3). Mixing: Mix the API, excipient micronized material, filler, binder, glidant, and disintegrant for 5 minutes, and add lubricant and mix for 2 minutes.

4) Tableting: Use a suitable die to press the powder in 3) into tablets with controlled tablet weight and appropriate hardness.

Biological test cases

Test Example 1: USP1/UAF1 Enzyme Inhibition Activity

The activity of deubiquitinase USP1/UAF1 after drug treatment was detected using ubiquitin rhodamine 110 as substrate. The total test system was 20 μl, and the test buffer was 50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, 0.01% Tween-20. At the beginning of the test, 0.5 nM USP1/UAF1 (Bio-Techne, E-568-050) protein was added to a 384-well plate (PerkinElmer, 6008269), and then the gradient dilution of the test compound was added, incubated at room temperature for 10 minutes, and 500 nM ubiquitin rhodamine 110 (Bio-Techne, U-555-050) was added, and incubated at room temperature for 20 minutes. Enspire ^™ Multilabel Reader (PerkinElmer) was used for detection at an excitation light of 485 nM/emission light of 535 nM. GraphPad Prism software was used to calculate the IC ₅₀ value.

Table 1 Results of the inhibitory activity of the test compounds on USP1/UAF1 enzyme

Note: A＜20nM

Conclusion: The compounds of the present invention have a good inhibitory effect on the enzymatic activity of USP1/UAF1.

Test Example 2: Beagle Dog Pharmacokinetic Test

Experimental purpose: To administer the test substance to beagle dogs by single-dose intravenous and oral gavage, determine the concentration of the test substance in beagle dog plasma, and evaluate the pharmacokinetic characteristics of the test substance in beagle dogs.

Experimental animals: Male beagle dogs, about 8-11 kg, purchased from Beijing Mas Biotechnology Co., Ltd.

Test method: On the day of the test, beagles were randomly divided into groups according to body weight. They were fasted but not watered for 14-18 hours one day before administration, and were fed 4 hours after administration. Administration was performed according to the information in the table below.

Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; intragastric administration solvent: 0.5% MC

(DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; MC: methylcellulose solution;)

Sampling: Blood was collected from the jugular vein or limb vein before and after administration and placed in an EDTAK2 centrifuge tube. The tube was centrifuged at 5000 rpm and 4°C for 10 min to collect plasma.

Blood collection time: 0, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h, 72h. All samples were stored below -60℃ before analysis. Samples were quantitatively analyzed by LC-MS/MS.

Table 2 Pharmacokinetic parameters of test compounds in beagle dog plasma

* Note: Compounds were administered ig (orally).

Conclusion: The compound synthesized by the technology of the present invention has good oral absorption performance in beagle dogs, and its oral performance is better than that of the control compound A, wherein the control compound A is Its preparation method can be found in WO2020132269.

Test Example 3: MDA-MB-436 cell clone formation test

This test uses MDA-MB-436 cells. After adding the test compound, the ability of cell clone formation is detected. At the beginning of the test, MDA-MB-436 cells (ATCC, HTB-130) were added to a 96-well plate (Corning, 3599), 500 cells per well, and adhered overnight; the next day, the compound was added with gradient dilutions, and the cells were placed in a 37°C, _CO2 -free incubator for continued culture. On the 7th day, the old culture medium was discarded, and the test compound was added with gradient dilutions again. On the 14th day, the 96-well plate was removed, the culture medium was discarded, and the cell clones were detected using the Crystal violet Assay kit. The PHERAstar FSX microplate reader (BMG LABTECH) was used for detection at OD ₅₇₀ nM. The IC ₅₀ value was calculated using GraphPad Prism software.

Table 3 Results of the inhibitory activity of the test compounds on MDA-MB-436 cell clones


Note: A＜200nM

Conclusion: The compounds of the present invention have a good inhibitory effect on the clone formation of MDA-MB-436 cells.

Test Example 4: Pharmacokinetics test in mice

Experimental animals: Male ICR mice, 25-30 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

Experimental design: On the day of the experiment, ICR mice were randomly divided into groups according to body weight. They were fasted but not watered for 12-14 hours one day before administration and fed 4 hours after administration.

(DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; MC: methylcellulose)

Blood was collected from the eye sockets at designated time points before and after administration, placed in EDTAK2 centrifuge tubes, and centrifuged at 5000rpm, 4℃ for 10min to collect plasma. The blood collection time points for the intravenous group and the gavage group were: 0, 5min, 15min, 30min, 1h, 2h, 4h, 7h, 24h. Before analysis and testing, all samples were stored below -60℃, and the samples were quantitatively analyzed by LC-MS/MS.

Table 4. Pharmacokinetic parameters of test compounds in mouse plasma

* Note: Compounds were administered ig (orally).

Conclusion: The compound synthesized by the technology of the present invention has good oral absorption performance in mice.

Claims (19)

1. A pharmaceutical composition, wherein the pharmaceutical composition comprises an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from the compound of general formula (I) or its stereoisomer, tautomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal,
   

   R ¹ is selected from -CH ₃ , -CHF ₂ , -CH ₂ CH ₃ ,

   R ² is selected from -CH ₃ , -CHF ₂ , -CH ₂ CH ₃ ,

   The pharmaceutical composition comprises 1-1200 mg of active ingredient A;

   The content of the active ingredient A is selected from 0.5%-99.0%.
2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises 5-1200 mg of active ingredient A.
3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition comprises 5-800 mg of active ingredient A.
4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition comprises 10-600 mg of active ingredient A.
5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises 25-200 mg of active ingredient A.
6. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition comprises 5 mg, 10 mg, 25 mg, 30 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 1000 mg, 1200 mg of active ingredient A.
7. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises an active ingredient A and a pharmaceutical excipient, wherein the active ingredient A is selected from a compound of the general formula (I) or a stereoisomer, a tautomer, a deuterated substance, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a cocrystal thereof, wherein the structure of the compound of the general formula (I) is selected from one of the structures shown in Table S-1.
8. The pharmaceutical composition according to claim 1, wherein the structure of the compound described in general formula (I) is selected from the following structures:
   
9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical excipient is selected from one or more of a filler, a disintegrant, a binder, a glidant, a lubricant, a surfactant, and a pH adjuster.
10. The pharmaceutical composition according to claim 9, wherein the filler is selected from one or more of microcrystalline cellulose, mannitol, lactose, sucrose, sorbitol, dextran, pregelatinized starch, calcium dihydrogen phosphate, and starch.
11. The pharmaceutical composition according to claim 9, wherein the disintegrant is selected from one or more of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, and calcium carboxymethyl cellulose.
12. The pharmaceutical composition according to claim 9, wherein the binder is selected from one or more of povidone, hydroxypropyl cellulose, hypromellose, and methyl cellulose.
13. The pharmaceutical composition according to claim 9, wherein the wetting agent is selected from one or both of water and ethanol.
14. The pharmaceutical composition according to claim 9, wherein the glidant is selected from one or more of talc, silicon dioxide, micropowdered silica gel, polyethylene glycol, and magnesium dodecyl sulfate.
15. According to the pharmaceutical composition of claim 9, the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, sodium stearyl fumarate, and glyceryl behenate.
16. The pharmaceutical composition according to claim 9, wherein the surfactant is selected from sodium lauryl sulfate, polysorbate One or more of pear esters, poloxamer, soluplus, polyoxyethylene castor oil.
17. According to the pharmaceutical composition of claim 9, the pH adjuster is selected from one or more of citric acid, tartaric acid and fumaric acid.
18. According to the pharmaceutical composition of claim 9, the pharmaceutical excipient further comprises one or more of a flavoring agent, an antioxidant, a preservative, a sunscreen, and a film coating premix.
19. Use of the pharmaceutical composition according to any one of claims 1 to 18 for preparing drugs for treating cancer.