RU2820555C2 - Fak inhibitor and drug combinations therewith - Google Patents

Abstract

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to organic and medical chemistry, namely to a combination of drugs for treating tumours mediated by FAK. Combination is characterized by that it contains a compound selected from compounds of formula 25, 41, 44, 45, 46, or a pharmaceutically acceptable salt thereof, and an immunotherapeutic drug in the same or different specified preparative forms for simultaneous or separate administration, and a pharmaceutically acceptable carrier. Immunotherapeutic agent is a PD-1 inhibitor.

EFFECT: creation of new combinations containing a compound having inhibitory activity on FAK, and an immunotherapeutic agent representing a PD-1 inhibitor, which can find application in medicine for treating FAK-mediated tumours.

4 cl, 2 dwg, 4 tbl, 5 ex

Description

Technical area

The present invention relates to the field of medicinal chemistry and, in particular, to a FAK inhibitor and combinations of drugs with it.

State of the art

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase found in cells that was first discovered in transfected V-Src chick embryonic fibroblasts. FAK has a higher expression level in most tissues, and its protein sequence has higher homology in many species (mice, toads, humans, etc.). FAK represents the intersection of several cell signaling pathways and is involved in tumor formation, proliferation, metastasis and apoptosis, cardiovascular diseases and other biological processes. Currently, it is one of the antitumor targets that has received much attention.

Recent studies have shown that FAK can be activated by a number of different factors, including integrins, G-protein coupled receptors, etc. At the same time, FAK regulates intracellular P53 and PI3K-AKT-mTOR signaling pathways through dependent and kinase-independent pathways, and also participates in the biological processes of life support, proliferation and metastasis of tumor cells. The initial attempt was to suppress tumor growth by suppressing FAK expression in tumor cells. Transfection of FAK with carboxyl terminus inactivated (FAK-CD) suppresses FAK expression, reduces cell adhesion and proliferation, and in in vivo experiments achieves suppressive effects on breast cancer cell growth. Transfection of a plasmid containing PFIK with suppressed expression of FAK (FAK-siRNA) causes suppression of cancer tumor growth in vivo. Simultaneous suppression of the expression of FAK and signaling molecules located downstream of FAK along the signal pathway (such as Src) may enhance the antitumor effect.

Due to the importance of FAK functions in tumor cells, the reliability of gene transfection, and the safety of viral vectors, small molecule inhibitors targeting the FAK signaling pathway have begun to emerge, and good results have been achieved in recent years. Currently, there are many FAK inhibitors acting as anticancer drugs that are in preclinical studies or clinical trials. According to data from the literature (a new antitumor target - focal adhesion kinase FAK and the results of studies of its inhibitors, Chen Ying et al.), TAE226, also known as NVP-226, can block the junction site of FAK and ATP, as well as phosphorylation sites Y397 and Y861 in FAK, and participate in the suppression of FAK activity. However, there is still a need in the art to develop FAK inhibitors with better inhibitory activity or better pharmacodynamic properties.

The term “deuterated drugs” means that some of the hydrogen atoms in the molecules of these drugs are replaced by deuterium. Deuterium (D) is a stable isotope of hydrogen. Because the shape and volume of deuterium in a drug are essentially the same as hydrogen, some of the hydrogen atoms in a drug molecule are replaced by deuterium, but the activity of the drug molecule remains essentially unchanged. In addition, since the mass of deuterium atoms is twice that of hydrogen, the zero-point vibrational energy of carbon-deuterium bonds (CD) is less than that of carbon-hydrogen bonds (CH), and thus the carbon-deuterium bond is more stable. Replacing some of the hydrogen atoms in drug molecules with deuterium can slow down the degradation process of the drug, prolong the action of the deuterated drug in the body, and achieve the goal of changing the metabolic rate or metabolic pathway of the drug, thereby improving the pharmacokinetics and reducing the metabolic toxicity of the drug. Due to the importance of FAK inhibitors in the field of tumor treatment and the limitations associated with them, combining them with deuterated drug technology, the discovery of new chemical compounds, reducing their effects on liver and kidney function, as well as improving the safety and effectiveness of the drug is an area of research. contributing to the further development of this type of medicine and having great practical significance.

The combined use of drugs is an effective way to improve the therapeutic effects of drugs. There are no data on the concomitant use of deuterated FAK inhibitors with other anticancer drugs or anticancer treatments.

Description of the invention

To solve the above-mentioned objects, the present invention provides a deuterated compound and its use as a FAK inhibitor, as well as a scheme for using the said deuterated compound in combination with other anti-cancer drugs.

The present invention provides a compound of formula (I) or an optical isomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof:

wherein the ring S is selected from an aromatic ring or a five-membered heterocyclic ring; A, B, X, Y and Z are each independently selected from C or N; E is absent or methylene;

and/or R ₆ selected from H or absent; R ₇ selected from H, N or absent; R ₈ is selected from haloalkyl or halogen, or R ₇ and R ₈ are linked to each other to form a ring; and/or R ₉ is selected from -NMeSO ₂ Me, -CONHOMe, -CONHMe, amide, hydrogen or absent; R ₁₀ selected from H or absent; R ₁₁ is selected from -NHSO ₂ Me, halogen, substituted piperazine or hydrogen, and the substituent on piperazine is an ethanol group; R ₁₂ is selected from -SO ₂ Me or H; R ₁₃ is selected from -CONHMe, -CONHOMe, N-alkylsulfonamide, H or absent, or R ₁₁ and R ₁₃ are linked to each other to form a ring:

Dx in formula (I) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium, and x is an integer ≥1.

In a preferred embodiment, said compound has the structure of formula (I-A):

wherein A, B, X, Y and Z are each independently selected from C or N; E is absent or represents methylene:

R ₁ and R ₅ are selected from H or methoxy group; R ₂ and R ₄ are selected from H or methoxy group; R ₃ is selected from H, -CONHMe, alkoxyamide, morpholine, methoxy group, ethylamine or sulfonamide, or R ₂ and R ₃ are linked to each other to form a ring, or R ₃ and R ₄ are linked to each other to form a ring;

and/or R ₆ selected from H or absent; R ₇ selected from H, N or absent; R ₈ is selected from haloalkyl or halogen, or R ₇ and R ₈ are linked to each other to form a ring; and/or R ₉ is selected from NMeSO ₂ Me, -CONHOMe, -CONHMe, amide, hydrogen or absent; R ₁₀ selected from H or absent; R ₁₁ is selected from -NHSO ₂ Me, halogen, substituted piperazine or hydrogen, and the substituent on piperazine is an ethanol group; R ₁₂ is selected from -SO ₂ Me or H; R ₁₃ is selected from -CONHMe, N-alkylsulfonamide, H or absent, or R ₁₁ and R ₁₃ are linked to each other to form a ring, or R ₁₃ , R ₃ and R ₄ are linked to each other to form a ring;

Dx in formula (I-A) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium, and x is an integer ≥1.

In a preferred embodiment, said compound has the structure of formula (I-B):

where A, X and Y are selected from C or N; E is missing;

R ₁₄ , R ₁₅ and R ₁₆ are each independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, methyl, ethyl or isopropyl;

and/or R ₆ represents H; R ₇ represents H; R ₈ represents halogen; R ₉ and R ₁₃ are selected from -CONHOMe, -CONHMe or H; R ₁₀ and R ₁₂ represent H;

R ₁₁ is selected from halogen, H or substituted piperazine, and the substituent on piperazine is an ethanol group;

Dx in formula (I-B) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium and x is an integer ≥ 1.

In a preferred embodiment, said compound has the structure of formula (I-C):

where A, X and Y are selected from C or N; E is methylene or absent;

R ₉ and R ₁₃ are selected from H, -NMeSO ₂ Me, -CONHOMe or -CONHMe; R ₁₀ and R ₁₂ represent H;

R ₁₁ is selected from H, substituted piperazine or halogen, and the substituent on piperazine is an ethanol group;

and/or R ₆ is selected from H; R ₇ selected from H; R ₈ is selected from haloalkyl or halogen; or R ₇ and R ₈ are linked to each other to form a ring;

and/or R ₁ , R ₄ and R ₅ are selected from H or methoxy group; R ₂ is selected from H or methoxy group; R ₃ is selected from H, -CONHMe, alkoxyamide, morpholine, methoxy group, ethylamine or sulfonamide; or R ₂ and R ₃ are linked to each other to form a ring;

Dx in formula (I-C) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium and x is an integer ≥ 1.

In a preferred embodiment, said compound has the structure of formula (I-D):

where R ₉ and R ₁₃ are selected from -CONHOMe, -CONHMe or H; R ₁₀ and R ₁₂ represent H; R ₁₁ is selected from halogen, H or substituted piperazine, and the substituent on piperazine is an ethanol group;

A, X and Y are selected from C or N; E is missing;

and/or, R ₆ is selected from H; R ₇ selected from H; R ₈ represents halogen;

and/or, R ₁₄ is selected from methyl, ethyl or isopropyl; R ₁₅ is selected from methyl or H; R ₁₆ represents H;

Dx in formula (I-D) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium, and x is an integer ≥1.

In a preferred embodiment, said compound has the structure of formula (I-E):

where B and Z are selected from C or N; E represents methylene; Y represents N; X and A represent C:

and/or, R ₆ is missing; R ₇ represents H; R ₈ represents haloalkyl:

and/or, R ₁ and R ₂ represent H; R ₃ represents -CONHMe; R ₄ represents H; or R ₃ and R ₄ are linked to each other to form a ring;

Dx in formula (1-E) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium, and x is an integer ≥1.

In a preferred embodiment, said compound has the structure of formula (I-F):

Where Dx in formula (I-F) indicates that the hydrogen on at least one carbon atom of the bracketed compound is replaced by deuterium, and x is an integer ≥1.

In a preferred embodiment, said compound has the structure of formula (I-G):

where one or more of R ₁ , R ₅ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R ₂₁ , R ₂₂ , R ₂₃ and R ₂₄ are substituted with deuterium. In a preferred embodiment, said compound is selected from, but is not limited to, the following deuterium-substituted compounds:

In a preferred embodiment, said compound is selected from, but is not limited to, one of the following compounds or one of the following deuterium-substituted compounds:

The present invention also relates to the use of the above compound or an optical isomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof for the production of a FAK inhibitor; preferably the FAK inhibitor is a drug for the treatment of cancer.

In this case, these cancers are solid tumors;

Solid tumors include mesothelioma, pancreatic cancer, soft tissue tumors, metastases, non-solid cancers, sarcomas, adenocarcinoma, lung cancer, breast cancer, lymphoma, gastrointestinal cancer, genitourinary cancer, prostate cancer and ovarian cancer ; gastrointestinal cancer includes colon cancer; Cancers of the genitourinary system include tumors of the kidneys, urothelium or testicles; and ovarian cancer includes advanced ovarian cancer:

Mesothelioma includes neurofibromas, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, KRAS-mutant non-small cell lung cancer, liver cancer, thyroid cancer, breast cancer, nervous system tumors, schwannoma, meningioma, neuroma, adenoid cystic carcinoma, ependymoma, ependymal tumors, malignant pleurisy, malignant pleural mesothelioma, triple negative breast cancer, non-hematological malignancies, melanoma, colorectal carcinoma, leukemia, adenocarcinoma, solid tumors;

Melanoma includes locally advanced melanoma, NRAS mutant melanoma, metastatic cutaneous malignant melanoma; colorectal cancer includes metastatic colorectal cancer; and leukemia includes acute myelogenous leukemia; adenocarcinoma includes adenocarcinoma; and solid tumors include locally advanced solid tumors, metastatic solid tumors and hepatocellular carcinoma.

The present invention further provides a combination of drugs for the treatment of tumors, which contains the above compound and an anti-cancer drug in the same or other specified formulations for simultaneous or separate administration, and a pharmaceutically acceptable carrier. The anticancer drug is an immunotherapy drug, a chemotherapy drug or a radiation therapy drug.

Immunotherapeutic drugs are selected from immune checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitory antibodies, TIM3 inhibitory antibodies, LAG3 inhibitory antibodies, TIGIT inhibitory antibodies, checkpoint target blocking antibodies, costimulatory antibodies or cells for CAR-T cell therapy. Said PD-1 inhibitor or PD-L1 inhibitor includes, but is not limited to: nivolumab, CT-011; AMP-224, pembrolizumab, pidilizumab, MK-3475, BMS936559, MEDI4736, MSB001071 8C, MPDL-3280A, SHR-1210, GV1308, BGB-A317, JS001, GLS-010, GB226 heptanolimab, HLX10, 03, AK104, AK105, AK112, SSI-361, JY034, KN035, SHR _1,316 , TQB2450, KL-A167, CS1001, STI-A1014, JS003, AK106, HLX-09, mPD-1 antibody;

Antibodies that block checkpoint targets include IMP321 and MGA271; The costimulatory antibody includes anti-4-1BB, anti-OX40, anti-GITR, anti-CD27, and anti-CD40.

These chemotherapeutic drugs are toxic drugs, alkylating drugs, antimetabolic drugs, antibiotics, hormone therapy drugs, natural product anticancer drugs, topoisomerase inhibitor drugs, immune drugs, platinum complex drugs, kinase inhibitors, antiproliferative drugs, antibodies, interferons, or drugs that regulate androgen signaling pathways. These toxic drugs include, but are not limited to: gemcitabine, paclitaxel and docetaxel;

These kinase inhibitors include, but are not limited to: MEK (mitogen-activated protein kinase) inhibitors, c-Met inhibitors, VEGFR2 inhibitors and EGFR inhibitors;

These drugs that regulate androgen signaling pathways include, but are not limited to: androgen synthesis inhibitors, CYP17A inhibitors, androgen receptor inhibitors, BET inhibitors, BRD4 inhibitors, RORγ inhibitors, CBP/P300 inhibitors, BMX inhibitors, PARP inhibitors; in a preferred embodiment, androgen receptor inhibitors include, but are not limited to: enzalutamide, apalutamide, bicalutamide, abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119 , procrutamide, SHR3680. The present invention further provides the use of the above combination of drugs for the production of drugs for the treatment of cancer.

Moreover, these oncological diseases are solid tumors; Solid tumors include mesothelioma, pancreatic cancer, soft tissue tumors, metastases, non-solid cancers, sarcomas, adenocarcinoma, lung cancer, breast cancer, lymphoma, gastrointestinal cancer, genitourinary cancer, prostate cancer and ovarian cancer ; gastrointestinal cancer, including colon cancer; cancer of the genitourinary system, including tumors of the kidneys, urothelium or testicles; as well as ovarian cancer, including advanced ovarian cancer;

Mesothelioma includes neurofibromas, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, KRAS-mutant non-small cell lung cancer, liver cancer, thyroid cancer, breast cancer, nervous system tumors, schwannoma, meningioma, neuroma, adenoid cystic carcinoma, ependymoma, ependymal tumors, malignant pleurisy, malignant pleural mesothelioma, triple negative breast cancer, non-hematological malignancies, melanoma, colorectal carcinoma, leukemia, adenocarcinoma, solid tumors;

Melanoma includes locally advanced melanoma, NRAS mutant melanoma, metastatic cutaneous malignant melanoma; colorectal cancer includes metastatic colorectal cancer; and leukemia includes acute myelogenous leukemia; adenocarcinoma includes adenocarcinoma; and solid tumors include locally advanced solid tumors, metastatic solid tumors and hepatocellular carcinoma.

In the present invention, the term “alkyl” includes linear or branched alkyl.

In the present invention, the term “compound of the present invention” means a compound of formula (I). The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates, optical isomers, tautomers and prodrugs of the compound of formula (I).

In the present invention, the term "pharmaceutically acceptable salt" means a salt suitable for use as a drug that is formed by the compound of the present invention and an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the alkali metal salt of the compound of the present invention. Alkali metals suitable for salt formation include, but are not limited to: lithium, sodium, potassium, calcium, magnesium, etc.

The method of administration of the compound or pharmaceutical composition of the present invention is not limited in any particular way, and standard methods of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous) and topical administration. The present invention provides a deuterated compound which, compared to the compound before deuteration, exhibits better pharmacokinetics, higher maximum plasma concentrations, higher exposure and longer half-life, and has even better metabolic properties. Moreover, the deuterated compound of the present invention can effectively inhibit the activity of FAK and has very good application prospects for the preparation of FAK inhibitors and/or drugs for the treatment of cancer. At the same time, the use of the deuterated compound of the present invention in combination with anti-cancer drugs (such as PD-1 inhibitors) can produce a synergistic effect, significantly improve the tumor suppressive effect and provide a better choice for the clinical treatment of cancer.

Naturally, based on the above content of the present invention, in accordance with generally known technical knowledge and generally accepted methods in the field, various other modifications, replacements or changes can be additionally made without deviating from the technical essence of the present invention. The content of the present invention is further illustrated by specific examples of these embodiments. However, this does not mean that the scope of the above object of the present invention is limited to the following examples. All methods carried out based on the above content of the present invention are included in the scope of the present invention.

Description of drawings

Fig. 1. Pharmacodynamic experiment to study the effect of the deuterated compound of the present invention on an animal model with an MC38 tumor.

Fig. 2. Pharmacodynamic experiment to study the effect of the deuterated compound of the present invention on an animal model with a PAN02 tumor.

Examples

The starting materials and equipment used in the present invention can be obtained through the purchase of commercially available products.

Example 1. Synthesis of N-trideuteromethyl-4-((4-(((3-(N-methanesulfonamido)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide ( connection 25)

Step 1: Synthesis of compound 25-2

25-1 (200 mg, 0.39 mmol) and DMAP (1.29 g, 10.57 mmol) were added to 10 ml of dichloromethane, to which (Boc) _2O (1.71 g, 7. 83 mmol). The system was refluxed in an oil bath for 24 hours. The next day, the reaction mixture was cooled to room temperature and dichloromethane and 0.1 N HCl solution were added to it. The mixture was extracted and then allowed to separate the layers. The organic phase was washed with brine, dried over anhydrous sodium sulfate, suction filtered and evaporated to remove the solvent. The primary product was separated by column chromatography to obtain a whitish solid 25-2 (136 mg, yield: 42.8%). MS (M+1): 811.2.

Step 2: Synthesis of compound 25-3

25-2 (136 mg, 0.17 mmol) and deuterated methylamine hydrochloride (189 mg, 2.68 mmol) were added to 5 ml of acetonitrile and the mixture was stirred at room temperature. DBU (diazabicycloundecene) (613 mg, 4.03 mmol) was then added and gradually dissolved until the reaction solution became clear. The system was then placed in an oil bath overnight to allow the reaction to proceed under reflux. The next day, the reaction mixture was cooled to room temperature and evaporated on a rotary evaporator to remove the solvent. Dichloromethane and 0.1 N HCl solution were added to the system, the reaction mixture was actively stirred and allowed to separate the layers.

The organic phase was washed first with purified water, then with saturated brine, dried over anhydrous sodium sulfate and rotary evaporated to remove the solvent. The residue was separated and purified by pre-TLC (PE/EA (petroleum ether/ethyl acetate)=2:1) to give a white solid 25-3 (42 mg, 35.3% yield). MS (M-Vos+1): 614.2.

Step 3: Synthesis of compound 25

25-3 (42 mg, 0.06 mmol) was added to 2 ml of dichloromethane and stirred at room temperature (until the solution is completely dissolved and clear), then 0.1 ml of trifluoromethanesulfonic acid was added. The reaction solution gradually became clear and pure, and the mixture was left overnight at room temperature for stirring and reaction to proceed. The next day, the solvent was removed by rotary evaporation, then ethyl acetate and saturated NaHCO ₃ solution were added to the system. The resulting mixture was vigorously stirred and then allowed to separate the layers. The pH of the aqueous phase was found to be about 7 to 8. The organic layer was washed twice with water, then with brine, and dried over anhydrous sodium sulfate.

The solvent was removed by rotary evaporation to give a white solid 25 (24 mg, yield: 80.0%).

^1H NMR (400 Hz, DMSO-d ₆ (dimethyl sulfoxide-D6): δ 9.863 (1H, s), 8.688 (1H, d, J=2.4 Hz), 8.581 (1H, d, J=2.8 Hz), 8.316 (1H, s), 8 8.180 (1H, s), 7.665-7.594 (4H, dd, J ₁ =19.6 Hz, J ₂ =8.8 Hz), 7.476-7.450 (1H, t , J=5.2 Hz), 5.001 (2H, d, J=4.8 Hz), 3.221 (3H, s), 3.199 (3H, ^s ): 514.2.

Compounds 26-40 were prepared using starting materials corresponding to the compound and a preparation method similar to the synthesis of compound 25.

Example 2. Synthesis of N-methyl-4-((4-(((3-(N-trideutermethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide ( connection 41)

Step 1: Synthesis of N-trideuteromethylmethanesulfonamide (compound 41-1) Deuterated methylamine hydrochloride (7.75 g, 109.99 mmol) was placed in a 250 mL one-neck round bottom flask while dichloromethane (120 mL) was added. This mixture was stirred at room temperature. The system was then placed in an ice water bath to cool and stirred. After 15 min, triethylamine (21.73 g, 214.75 mmol) and DMAP (128 mg, 1.05 mmol) were added sequentially. The mixture was then stirred for an additional 10 minutes in an ice water bath. Methanesulfonyl chloride (12.0 g, 104.76 mmol) was then added to the system and then removed from the ice bath. The system was left overnight at room temperature for stirring and reaction. The next day, the reaction was completed by TLC detection and the system was filtered. The filter cake was washed several times with ethyl acetate, the filtrate was combined, and the solvent was removed by rotary evaporation. Ethyl acetate (90 ml) was then added to the system and stirred vigorously for 10 minutes. After this, the system was filtered again by suction and the filter cake was washed several times with small amounts of ethyl acetate. The filtrate was combined and concentrated in vacuo to give N-trideuteromethylmethanesulfonamide (11.16 g) as a colorless, clear oily liquid, which was immediately used in the next step without further purification. Yield: 94.9%. LC/MS (ESI+): mass/charge 113.3 [M+H] ⁺ (calculated for C ₂ H ₄ D ₃ NO ₂ S, 113.1).

Step 2: Synthesis of N-(3-cyanopyrazin-2-yl)-N-trideuteromethylmethanesulfonamide (compound 41-2)

N-trideuteromethylmethanesulfonamide (6.0 g, 53.54 mmol) and 2-chloro-3-cyanopyrazine (6.23 g, 44.62 mmol) were placed in a 500 ml one-neck round bottom flask while acetonitrile (300 ml) was added. The resulting mixture was stirred at room temperature. Next, cesium carbonate (24.71 g, 75.85 mmol) was added to the system. Then the system was placed in an oil bath at 80°C and then the reaction mixture was heated and stirred. After 1.5 hours, a sample was taken and subjected to TLC. TLC showed the complete absence of starting materials. Heating was stopped and the system was left to cool naturally to room temperature, followed by filtration. The filter cake was washed several times with small amounts of acetonitrile and then the filtrate was combined. The solvent was removed by rotary evaporation. After this, ethyl acetate (150 ml) and water (150 ml) were added to the system. The resulting mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (50 ml⋅3). The organic phases were combined, washed successively with purified water (30 ml x 3) and brine (30 ml), dried over anhydrous sodium sulfate and concentrated in vacuo to give the primary product, which was separated and purified by column chromatography to give N-(3 -cyanopyrazin-2-yl)-N-deuteromethylmethanesulfonamide (5.29 g) as a pale brown-red oily liquid. Yield: 55.1%. LC/MS (ESI+): m/z 233.1 [M+H ₂ O] (calculated for C ₇ H ₅ D ₃ N ₄ O ₂ S [M+H] ⁺ , 216.1).

¹ H NMR (400 MHz, DMSO-D6) δ 8.65-8.63 (dd, J=6.0, 2.4 Hz, 2H), 3.26 (s, 3H).

Step 3: Synthesis of N-(3-(aminomethyl)pyrazin-2-yl)-N-trideuteromethylmethanesulfonamide (compound 41-3)

N-(3-cyanopyrazin-2-yl)-N-deuteromethylmethanesulfonamide (2.0 g, 9.30 mmol) was weighed into a 500 mL one-neck round bottom flask to which methanol (270 mL) was added and then the mixture stirred at room temperature until the reagents dissolved and the solution clarified. Next, wet palladized carbon (1 g) and ammonia water (20 ml) were added to the system. After this, the air was pumped out of the system and purged with argon gas 5 times to create an inert gas environment in the system. The system was again replaced with hydrogen, and after that the system continued to be stirred and kept at room temperature for the reaction to occur. After 5 hours, a sample was taken and subjected to TLC. TLC showed that the starting materials were consumed. The reaction was stopped and the hydrogenation device was disconnected. The system was filtered by suction and the filter cake was eluted several times with methanol. The filtrate was combined and the solvent was removed by rotary evaporation. The remaining water in the system was removed by rotary evaporation in several stages to obtain N-(3-(aminomethyl)pyrazin-2-yl)-N-deuteromethylmethanesulfonamide in the form of a light yellow-brown transparent oily liquid, which was immediately used in the next step without additional cleaning. LC/MS (ESI+): m/z 220.1 [M+H] ⁺ (calculated for C ₇ H ₉ D ₃ N ₄ O ₂ S, 220.1).

Step 4: Synthesis of (4-(methylcarbamyl)phenyl)carbamic acid tert-butyl ester

4-((tert-butoxycarbonyl)amino)benzoic acid (3.0 g, 12.65 mmol) was weighed out and placed in a 250 mL one-neck round bottom flask to which 50 mL of DMF was added and the mixture was stirred at room temperature. Next, EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (4.8 g, 25.29 mmol), TEA (triethanolamine) (4.5 g, 44.28 mmol), methylamine hydrochloride were added sequentially to the system (1.3 g, 18.98 mmol) and DMAP (16.0 mg, 0.13 mmol). After this, the system was stirred and left overnight at room temperature for the reaction to occur. The next day, having determined that the starting materials had been consumed, ethyl acetate (70 ml) and water (50 ml) were added to the system, and the reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (20 ml × 3). The organic layers were combined, washed first with water (20 ml x 3), then with brine (30 ml) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the primary product, which was then separated by column chromatography to give (4-(methylcarbamyl)phenyl)carbamic acid tert-butyl ester as an off-white solid (2.1 g, yield: 66.5%) . MS (IER): m/z 251.2 [M+H] ⁺ .

Step 5: Synthesis of 4-amino-N-methylbenzamide trifluoroacetate Weigh out 500.0 mg (2.00 mmol) of (4-(methylcarbamoyl)phenyl)carbamic acid tert-butyl ester into a 50 mL one-neck round bottom flask. added 10 ml of dichloromethane and stirred this mixture at room temperature. After this, trifluoroacetic acid (1 ml) was added to the system, and then the system was stirred and left overnight at room temperature for the reaction to occur. The next day, TLC showed that the reaction was complete. The reaction mixture was concentrated to remove the solvent and excess trifluoroacetic acid, and the remaining trifluoroacetic acid in the system was removed by rotary evaporation with dichloromethane in several stages until the system was completely converted into a solid, obtaining 4-amino-Nmethylbenzamide trifluoroacetate as a whitish solid (510, 0 mg), which was immediately used in the next step without further purification.

Step 6: Synthesis of the compound 4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-methylbenzamide

499.0 mg (2.30 mmol) of 2,4-dichloro-5-(trifluoromethyl)pyrimidine were weighed out and placed in a 50 mL one-neck round bottom flask to which 1,2-dichloroethane (5 mL) and tert-butanol were added ( 5 ml), and then this mixture was stirred at room temperature until the reagents dissolved and the solution clarified. The system was then placed in an ice water bath to continue cooling and mixing. After 15 minutes, zinc bromide (1.4 g, 6.00 mmol) was added to the system. The system was then further stirred for 30 minutes in an ice water bath. Then, 4-amino-N-methylbenzamide trifluoroacetate, synthesized in the previous step, and triethylamine (648.0 mg, 6.40 mmol) were added to the system. After their addition, the system was removed from the ice bath, stirred, and left overnight at room temperature to allow the reaction to proceed. The next day after the reaction was completed by detection, the solvent was removed by rotary evaporation. Ethyl acetate (30 ml) and water (20 ml) were added to the system, and then the reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (10 ml × 3). The organic layers were combined, washed successively with water (15 ml x 3) and brine (15 ml) and dried over anhydrous sodium sulfate. The reaction mixture was concentrated under reduced pressure to obtain the primary product, which was then separated by column chromatography to obtain 4-((4-chloro-5-(trifluoromethyl)shrimidin-2-yl)amino)-N-methylbenzamide as an off-white solid. (280.0 mg, yield: 42.3%). MS (IER): m/z 331.0 [M+N] ⁺ .

Step 7: Synthesis of the compound N-methyl-4-((4-(((3-(N-deuteromethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide

Add 5 mL of 1.2 -dichloroethane and 5 ml of tert-butanol and then the mixture was stirred at room temperature until the reagents dissolved and the solution became clear. Next, 4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-methylbenzamide (100.0 mg, 0.30 mmol) and diisopropylethylamine (116.3 mg, 0.30 mmol) were added to the system. 90 mmol). After their addition, the system was placed in an oil bath at 80°C and refluxed to allow the reaction to proceed. After 8 hours, the complete consumption of the starting substances was recorded using TLC. Heating was stopped and the system was then cooled to room temperature, the solvent was removed by rotary evaporation to obtain the primary product, which was then separated and purified by preliminary TLC to give N-methyl-4-((4-(((3-(N-trideuteromethylmethanesulfonamido)pyrazine -2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide in the form of a whitish solid (compound 41, 12 mg). Yield: 7.8%. MS (ESI) m/z 514.2 [M+H] ⁺ .

^1H NMR (400 MHz, DMSO- _d6 ) δ 9.83 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 8.32 (s, 1H), 8 .20 (d, J=4.0 Hz, 1H), 7.68-7.61 (dd, J=14.4, 8.4 Hz, 4GG), 7.41-7.39 (t, J =4.4 Hz, 1H), 5.01 (d, J=3.6 Hz, 2H), 3.20 (s, 3H), 2.76 (d, J=4.0 Hz, 3H).

Compounds 42 and 43 were prepared using starting materials corresponding to the compound and a preparation method similar to the synthesis of compound 41.

Example 3. Synthesis of N-trideutermethyl-4-((4-(((3-(N-trideutermethylmethanesulfonamido)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide ( compound 44) and its hydrochloride

Step 1: Synthesis of (4-(trideuteromethylcarbamoyl)phenyl)carbamic acid tert-butyl ester (compound 44-1)

N-Boc-4-aminobenzoic acid (6.0 g, 25.29 mmol) and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (7.27 g, 37.93 mmol) were respectively weighed out and placed into a 250 ml one-neck round-bottom flask with the simultaneous addition of DMF (50 ml). This mixture was stirred at room temperature. Triethylamine (6.40 g, 63.22 mmol) and deuterated methylamine hydrochloride (1.96 g, 27.82 mmol) were then added to the system. After their addition, the system was stirred and left overnight at room temperature for the reaction to proceed. The next day, a sample was taken and subjected to TLC, and when TLC indicated that the reaction was complete, ethyl acetate (50 ml) and water (50 ml) were added to the system. The reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (50 ml⋅3). The organic layers were combined, washed first with water (30 ml·3), then with brine (50 ml) and dried over anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo to obtain the primary product, which was then separated and purified by column chromatography to give (4-(deuteromethylcarbamoyl)phenyl)carbamic acid tert-butyl ester as an off-white solid (4.92 g, yield: 76. 8%). LC/MS (ESI+): m/z 254.2 [M+H] ⁺ (calculated for C ₁₃ H ₁₅ D ₃ N ₂ O ₃ , 254.2).

Step 2: Synthesis of 4-amino-N-trideuteromethylbenzamide trifluoroacetate (compound 44-2) (4-(Deuteromethijarbamoyl)phenyl)carbamic acid tert-butyl ester (3.0 g, 11.84 mmol) was weighed and placed in a one-neck round bottom flask volume of 100 ml with the simultaneous addition of dichloromethane (15 ml). This mixture was stirred at room temperature. Thereafter, trifluoroacetic acid (7 ml) was added to the system, and then the system was stirred and kept at room temperature for the reaction to proceed. After 5 hours, a sample was taken and subjected to TLC. TLC showed that the reaction was complete. The reaction mixture was rotary evaporated to remove the solvent and excess trifluoroacetic acid, and any residual trifluoroacetic acid in the system was removed by rotary evaporation with dichloromethane in batches to obtain 4-amino-N-deuteromethylbenzamide trifluoroacetate as an off-white solid, which was immediately used in next stage without additional cleaning.

Step 3: Synthesis of 4-((4-chloro-5-(trifluoromethyl)hrimidin-2-yl)amino)-N-trideuteromethylbenzamide (compound 44-3)

2,4-dichloro-5-trifluoromethylpyrimidine (2.83 g, 13.02 mmol) was weighed into a 100 mL one-neck round bottom flask to which 1,2-dichloroethane (30 mL) and tert-butanol (30 mL) were added ) and then the mixture was stirred at room temperature until the reagents dissolved and the solution clarified. The system was then placed in an ice water bath to continue cooling and mixing. When the temperature inside the system dropped to approximately 0°C, zinc bromide (8.0 g, 35.52 mmol) was added to the system. The system was then kept in an ice water bath for an additional 30 minutes to allow the reaction to proceed. 4-Amino-N-deuteromethylbenzamide trifluoroacetate, synthesized in the previous step, and triethylamine (3.83 g, 37.89 mmol) were then added to the system. After their addition, the system was removed from the ice bath, stirred, and left overnight at room temperature to allow the reaction to proceed. The next day, a sample was taken and subjected to TLC. TLC showed that the starting materials were completely consumed and the reaction was terminated. The solvent was removed by rotary evaporation. Ethyl acetate (50 ml) and water (30 ml) were added to the system, and the reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (30 ml⋅3). The organic layers were combined, washed successively with water (30 ml*3) and brine (30 ml) and dried over anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo to obtain the primary product, which was separated and purified by column chromatography to give 4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-deuteromethylbenzamide as an off-white solid (3.23 g). The yield of the two-stage reaction from the 2nd to the 3rd stage: 81.8%. LC/MS (ESI+): m/z 334.0 [M+H] ⁺ (calculated for C ₁₃ H ₇ D ₃ ClF ₃ N ₄ O, 334.0).

^1H NMR (400 MHz, DMSO- _d6 ) δ 10.89 (s, 1H), 8.87 (s, 1H), 8.31 (s, 1H), 7.84-7.77 (m, 4H).

Step 4: Synthesis of N-trideuteromethyl-4-((4-(((3-(N-trideuteromethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide ( connection 44)

4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-deuteromethylbenzamide (3.1 g, 9.29 mmol) and N-(3-cyanopyrazin-2-yl)-N β-deuteromethylmethanesulfonamide (2.0 g, 9.29 mmol) was weighed into a 250 mL one-neck round bottom flask to which 1,2-dichloroethane (80 mL) and tert-butanol (80 mL) were then added and the mixture was stirred at room temperature until the reagents dissolve and the solution becomes clear. Diisopropylethylamine (3.6 g, 27.87 mmol) was then added to the system. After this, the system was placed in an oil bath at 80°C, heated to reflux, stirred and left overnight for the reaction to occur. The next day, a sample was taken and subjected to TLC. Once TLC indicated that the reaction was complete, the solvent was removed by rotary evaporation. Ethyl acetate (100 ml) and water (50 ml) were added to the system, and the reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (50 ml⋅3). The organic layers were combined, washed successively with water (30 ml·3) and brine (50 ml) and dried over anhydrous sodium sulfate. The reaction mixture was concentrated in vacuo to obtain the primary product, which was separated and purified by column chromatography to give the title compound as a whitish solid (2.36 g). The said solid was then placed in a 250 ml single neck round bottom flask to which ethyl acetate (75 ml) was added and the mixture was stirred at room temperature until a suspension was formed. After 3 hours it was filtered by suction. The filter cake was washed several times with a small amount of ethyl acetate (45 ml) and then placed in a vacuum drying oven to dry at low temperature to obtain N-[-deuteromethyl-4-((4-(((3-(N-deuteromethylmethanesulfonamide) pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (2.11 g) as a white solid. Yield: 44.0%. LC/MS (ESI+): m/z 517.2 [M+H] ⁺ (calculated for C ₂₀ H ₁₅ D ₆ F ₃ N ₈ O ₃ S, 517.2).

¹ H NMR (400 MHz, DMSO) δ 9.83 (s, 1H), 8.69 (d, J=2.8 Hz 1H), 8.58 (d, J=2.4 Hz, 1H), 8.31 (s, 1H), 8.17 (s, 1H), 7.67-7.61 (dd, J=15.4, 8.6 Hz, 4H), 7.41 (t, J= 5.0 Hz 1H), 5.00 (d, J=4.8 Hz 2H), 3.20 (s, 3H).

Step 5: Synthesis of N-deuteromethyl-4-((4-(((3-(N-deuteromethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide hydrochloride

Compound 44 (500 mg, 0.97 mmol) was weighed into a 100 mL one-neck round bottom flask to which methanol (25 mL) was added and the mixture was stirred at room temperature. A solution of HCl in ethanol (2.25 mL, 2.0 M) was then slowly added dropwise to the system. After this, the system continued to be stirred and kept at room temperature for the reaction to occur. After 1.5 hours, the system was filtered by suction and the filter cake was washed several times with methanol (15 ml) in small quantities, and then placed in a vacuum drying oven to dry at low temperature to obtain N-deuteromethyl-4-((4-( ((3-(N-deuteromethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzoyl (517 mg) as a whitish solid. Yield: 96.6%. LC/MS (ESI+): m/z 517.2 [M+H] ⁺ (calculated for C ₂₀ H ₁₆ D ₆ ClF ₃ N ₈ O ₃ S, 517.2).

¹ H NMR (400 MHz DMSO) δ 10.02 (s, 1H), 8.68 (d, J=2.8 Hz, 1H), 8.58 (d, J=2.4 Hz, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 7.67-7.57 (m, 5H), 5.26 (br, 6H), 5.00 (d, J=4, 8 Hz, 2H), 3.19 (s, 3H).

Example 4. Synthesis of N-deuteromethyl-4-((4-(((3-(N-deuteromethanesulfonamide)pyrazin-2-yl)deuteromethyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide ( connection 45)

Step 1: Synthesis of N-(3-(aminodideuteromethyl)pyrazin-2-yl)-N-deuteromethylmethanesulfonamide (45-1)

Compound 41-2 (100.0 mg, 0.46 mmol) was weighed into a 25 mL one-neck round bottom flask, to which 5 mL of deuterated methanol (188.2 mg, 1.86 mmol) was then added and the mixture was stirred at room temperature until the reagents dissolve and the solution becomes clear. Then 20.0 mg of wet palladium carbon (treated with heavy water) and triethylamine (188.2 mg, 1.86 mmol) were added sequentially to the system. Actions were taken on the system to replace deuterium and these actions were repeated ten times. After this, the system was stirred and kept at room temperature for the reaction to occur. After 72 hours, the reaction was completed by detection. The system was then filtered by suction. The filter cake was washed several times with small amounts of deuterated methanol (10 ml). The filtrate was combined and the solvent was removed by rotary evaporation to give N-(3-(aminodeuteromethyl)pyrazin-2-yl)-N-deuteromethylmethanesulfonamide as a light tan oily liquid, which was immediately used in the next step without further purification. MS (IER): m/z 222.2 [M+H] ⁺ .

Step 2: Synthesis of the compound N-deuteromethyl-4-((4-(((3-(N-deuteromethanesulfonamide)pyrazin-2-yl)deuteromethyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (45)

To a 25 mL one-neck round bottom flask containing N-(3-(aminodeuteromethyl)pyrazin-2-yl)-N-deuteromethylmethanesulfonamide (22.1 mg, 0.10 mmol), add 2 mL of 1,2-dichloroethane and 2 mL tert-butanol and then the mixture was stirred at room temperature until the reagents dissolved and the solution clarified. Compound 44-3 (33.4 mg, 0.10 mmol) and diisopropylethylamine (30.6 mg, 0.30 mmol) were then added to the system. After their addition, the system was placed in an oil bath at 80°C and refluxed to allow the reaction to proceed. After 8 hours, the complete consumption of the starting substances was recorded using TLC. Heating was stopped and after cooling the system to room temperature, the solvent was removed by rotary evaporation to obtain the primary product, which was then separated and purified by preliminary TLC to give N-deuteromethyl-4-((4-(((3-(N-deuteromethanesulfonamide)pyrazine- 2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide as a whitish solid (8.1 mg) with a yield of 15.6%. MS (IER): m/z 519.2 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.83 (s, 1H), 8.69 (d, J=2.0 Hz, 1H), 8.58 (d, J=2.4 Hz , 1H), 8.31 (s, 1H), 8.17 (s, 1H), 7.67-7.61 (dd, J=15.2, 8.8 Hz, 4H), 7.39 ( s, 1H), 3.20 (s, 3H).

Example 5. Synthesis of 4-((4-(((3-N-trideuteromethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (compound 52)

Step 1: Synthesis of 4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (52-2)

2,4-dichloro-5-(trifluoromethyl)pyrimidine (434 mg, 2.00 mmol) was weighed out and dissolved in a 25 mL one-neck round bottom flask to which 1,2-dichloroethane (5 mL) and tert-butanol (5 ml) and then this mixture was stirred at room temperature until the reagents dissolved and the solution clarified. The system was then placed in an ice water bath to continue cooling and mixing. After 15 minutes, zinc bromide (1.2 g, 5.22 mmol) was added to the system. The system was then further stirred for 30 minutes in an ice water bath. Then 4-aminobenzamide (237 mg, 1.74 mmol), synthesized in the previous step, and triethylamine (564 mg, 5.57 mmol) were added to the system. After their addition, the system was removed from the ice bath, stirred, and left overnight at room temperature to allow the reaction to proceed. The next day after the reaction was completed by detection, the solvent was removed by rotary evaporation. Ethyl acetate (30 ml) and water (20 ml) were added to the system, and the reaction mixture was vigorously stirred and allowed to separate the layers. The aqueous phase was back-extracted with ethyl acetate (10 ml⋅3). The organic phase was combined, washed successively with water (15 ml·3) and brine (15 ml) and dried over anhydrous sodium sulfate. The reaction mixture was concentrated under reduced pressure to obtain the primary product, which was then separated by column chromatography to give 4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide as an off-white solid (294 mg , yield: 53.4%). MS (IER): m/z 317.0 [M+H] ⁺ .

Step 2: Synthesis of compound 4-((4-(((3-(N-trideuteromethylmethanesulfonamide)pyrazin-2-yl)methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (52)

To a 25 ml single-neck round-bottom flask containing compound 52-2 obtained in the previous step, 5 ml of 1,2-dichloroethane and 5 ml of tert-butanol were added and then the mixture was stirred at room temperature until the reagents dissolved and the solution cleared. Compound 44-3 (63.0 mg, 0.20 mmol) and diisopropylethylamine (78 mg, 0.60 mmol) were then added to the system. After their addition, the system was placed in an oil bath at 80°C and refluxed to allow the reaction to proceed.

The next day, the complete consumption of the starting substances was recorded using TLC. Heating was stopped and after cooling the system to room temperature, the solvent was removed by rotary evaporation to obtain the primary product, which was then separated and purified by preliminary TLC to give 4-((4-(((3-(N-deuteromethanesulfonamide)pyrazin-2-yl) methyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzamide (22 mg) as a whitish solid with a yield of 22.0%. MS (IER) m/z 500.1 [M+H] ⁺ .

^1H NMR (400 MHz, DMSO- _d6 ) δ 9.83 (s, 1H), 8.69 (s, 2H), 8.60 (s, 1H), 8.31 (s, 1H), 8 .20 (d, J=4.0 Hz, 1H), 7.68-7.60 (dd, J=15.4, 8.4 Hz, 4H), 7.41-7.39 (t, J =4.4 Hz, 1H), 5.03 (d, J=3.6 Hz, 2H), 3.20 (s, 3H).

The known compounds 4-amino-3,5-dideuterobenzoic acid and 4-amino-2,6-dideuterobenzoic acid were used as starting materials (Journal of Tabel Compounds and Radiopharmaceuticals, 53(11-12), 668-673; 2010), and compounds 48-50 and 53-55 were prepared by the methods described in the above examples.

Compounds 59-61 were prepared by the method described in the above examples using the known compound 4-amino-2,3,5,6-tetradeuterobenzoic acid (Journal of Natural Products, 79(6), 1532-1537; 2016) as a starting material. The beneficial effect of the present invention is demonstrated in the following experimental examples.

Experimental Example 1: FAK Inhibitory Activity of the Deuterated Compound of the Present Invention

(1) Experimental procedure

According to the method described in the literature (Cancer Res. 2008, 68, 1935), an experiment was carried out to study the inhibitory activity against the FAK enzyme. Details: The test compound was diluted to 1000 nM and then subjected to a serial dilution of 1:3 with DMSO (dimethyl sulfoxide). 0.1 L of the solution was placed on a 384-well plate and for each concentration, 2 wells with the same options were allocated. 5 L of FAK 2 enzyme solution was added, then centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 15 minutes. Add 5 L of substrate solution 2x and incubate at 25°C for 60 minutes. Then, 5 L of Sa-XL665 solution and 5 μL of TC (thymidine kinase) Eu3+ antibody were added and centrifuged at 1000 rpm for 1 minute, then incubated at 25°C for 60 minutes. Finally, an Envision 2104 plate reader was used to read the fluorescence signal and calculate the half-maximal inhibition concentration IC ₅₀ of each compound against the FAK enzyme. The well-known FAK inhibitor defactinib was used as a control.

(2) Experimental results

The inhibitory activity of each compound against FAK is shown in Table 1. It can be seen that the compound obtained according to the present invention can effectively inhibit the activity of FAK enzyme, and compared with the non-deuterated compound "defactinib", the deuterated compounds 41 and 45 of the present invention have higher inhibitory activity against the FAK enzyme.

Experimental Example 2: Pharmacokinetic test of the deuterated compound of the present invention in rats

(1) Experimental procedure

Accurately weigh out the appropriate amount of test drug (10 mg) and first add 0.25 ml of N,N-dimethylacetamide (DMA) to dissolve it, then slowly add 0.5% sodium carboxymethylcellulose (CMC-Na) to 5 ml. This mixture was treated with ultrasound, vortexed and further mixed well. 0.2 ml of the resulting solution was removed and stored at -20°C to determine the concentration. After an overnight fast (drinking water was available ad libitum), three healthy adult male SD rats (180–250 g, purchased from Chengdu Dossy Experimental Animal Co., Ltd.) were administered study drugs at 5 ml/kg body weight via gastric probe; before administration, as well as 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration, 0.1 ml of blood was taken from the retro-orbital venous plexus and centrifuged at 4°C for 5 minutes to separate plasma and stored at -20°C for testing. Plasma concentrations of the test compound were then determined by LC/MS/MS. The well-known FAK inhibitor defactinib was used as a control.

(2) Experimental results

Table 2. Pharmacokinetic parameters of the compounds of the present invention

As shown in Table 2, deuterated compounds 25 and 44 prepared according to the present invention showed better pharmacokinetics compared to defactinib. The compound of the present invention has a higher maximum plasma concentration _Cmax , a larger area under the pharmacokinetic exposure curve and a longer half-life. Therefore, the deuterated compound obtained according to the present invention will have better prospects for use as FAK inhibitors or drugs for the treatment of cancer.

Experimental Example 3: Pharmacodynamic experiment to study the effect of the deuterated compound of the present invention in combination with a PD-1 inhibitor in an animal tumor model

1. MC38 tumor model:

(1) Experimental procedure

Cell culture: MC-38 cells were cultured in DMEM containing 10% fetal bovine serum (FBS). MC-38 cells were collected at logarithmic growth phase and resuspended in HBSS to a concentration suitable for subcutaneous tumor inoculation in C57BL/6 mice.

Experimental Animals: 96 female C57BL/6 mice, 6-8 weeks old, weighing about 18-20 g, purchased from Beijing Vital River Experimental Animal Technology Co., Ltd. Inoculation of tumor cells: Tumor cells were collected in the logarithmic growth phase and the cell concentration was adjusted to 5x10 ⁶ /ml using HBSS, then 0.1 ml was inoculated subcutaneously into the right side of each mouse near the back, that is, 5x10 ⁵ /mouse . Growth was then monitored and tumor volume was measured. When the average tumor volume in mice increased to 50-100 mm ³ , tumor-bearing mice were randomly divided into groups according to tumor volume and injected with the drug. Details are presented in Table 3, and the day of group assignment and drug administration is designated day 0.

Calculation of tumor volume: On the 18th sushi, mice were sacrificed, tumors were removed, tumor volume was measured, and the degree of tumor suppression in each group was calculated.

Note: N: number of animals used; i.p.: intraperitoneal injection; g: intragastric administration; 2C: twice a day; 1C: once a day; 2H: twice a week.

(2) Experimental results

The pharmacodynamics of animals after 18 days of administration are shown in Fig. 1, and the calculated tumor suppression rate is shown in Table 3. It can be seen that the effectiveness of compound 44 administered alone was higher than that of mPD-1 antibody alone, indicating that the compound of the present invention had a therapeutic effect in the model MC38 tumors in mice. Moreover, compared with administration of compound 44 alone (group 4) or mPD-1 antibody alone (group 2), administration of compound 44 in combination with mPD-1 antibody (group 5) achieved significantly better tumor suppressive effects, and a synergistic effect was observed.

In addition, compared with administration of defactinib (50 mg/kg, twice daily) in combination with mPD-1 antibody (group 3), after administration of compound 44 of the present invention (50 mg/kg, once daily) in combination with mPD-1 antibody (group 5), the tumor suppressive effect was better, and an even higher level of tumor suppressive effect was achieved. That is, when used in combination with PD-1 inhibitors, the compound of the present invention at a dose half the dose of defactinib can achieve better tumor suppressive effect, indicating that the combination of the compound of the present invention and PD-1 inhibitors has significantly better antitumor effect in the MC38 tumor model than the combination of defactinib and PD-1 inhibitors.

2. Tumor model PAN02

(1) Experimental procedure

PAN-02 cells were collected at logarithmic growth phase, washed twice with PBS and then resuspended in pre-chilled PBS for inoculation. The experimental animals were female C57BL/6 mice purchased from Beijing Vital Paver Experimental Animal Technology Co., Ltd. C57BL/6 mice adapted to the laboratory environment within 3 days. PAN-02 cells were inoculated subcutaneously into the right flank ribs, and the number of inoculated cells was 1×10 ⁶ /mouse. When the tumor had grown to approximately 100 mm ³ , the mice were screened and randomly assigned to groups. Each group included 8 mice. The mice were divided into groups and administered drugs according to the dosage schedule in accordance with Table 4. The day of group division and drug administration was designated as day 1, and the administration period was 33 days.

Note: N: number of animals used; i.p.: intraperitoneal administration; g: intragastric administration; 2C: twice a day; 2H: twice a week.

(2) Experimental results

The pharmacodynamics of animals after 33 days of drug administration are shown in Fig. 2, and the calculated tumor suppression rate is shown in Table 4. It can be seen that compared with the administration of compound 44 alone (group 4) or the mPD-1 antibody alone (group 2), the administration of compound 44 in combination with the mPD-1 antibody ( group 5) achieved a significantly better suppressive effect on the tumor.

In addition, compared with the tumor suppressive effect after administration of defactinib (50 mg/kg, twice daily) in combination with mPD-1 antibody (group 3), the tumor suppressive effect after administration of compound 44 of the present invention (25 mg /kg, twice a day) in combination with the mPD-1 antibody (group 5) was significantly better. That is, when used in combination with PD-1 inhibitors, the compound of the present invention at a dose lower than that of defactinib can achieve more effective tumor suppression, indicating that administration of the compound of the present invention in combination with PD-1 inhibitors has a significantly better antitumor effect in the PAN-02 tumor model than the administration of defactinib in combination with PD-1 inhibitors.

Thus, the present invention provides a deuterated compound that exhibits better pharmacokinetics, higher maximum plasma concentrations, higher exposure and longer half-life, and even better metabolic characteristics compared to the compound before deuteration. Moreover, the deuterated compound of the present invention can effectively inhibit the activity of FAK and has very good application prospects for the preparation of FAK inhibitors and/or drugs for the treatment of cancer. At the same time, the use of the deuterated compound of the present invention in combination with anti-cancer drugs (such as PD-1 inhibitors) can produce a synergistic effect, significantly improve the tumor suppressive effect and provide a better choice for the clinical treatment of cancer.

Claims (11)

1. A combination of drugs for the treatment of FAK-mediated tumors, characterized in that it contains a compound selected from or a pharmaceutically acceptable salt thereof, and an immunotherapeutic drug in the same or different specified formulations for simultaneous or separate administration, and a pharmaceutically acceptable carrier, wherein the immunotherapeutic drug is a PD-1 inhibitor. 2. The combination of drugs according to claim 1, characterized in that the PD-1 inhibitor is an mPD-1 antibody. 3. Use of a combination of drugs according to claim 1 or 2 for the production of drugs for the treatment of cancer mediated by FAK. 4. Use according to claim 3, characterized in that said cancer is a solid tumor; solid tumors include mesothelioma, pancreatic cancer, soft tissue tumors, metastases, non-solid cancers, sarcomas, adenocarcinoma, lung cancer, breast cancer, lymphoma, gastrointestinal cancer, genitourinary cancer, prostate cancer and ovarian cancer; gastrointestinal cancer includes colon cancer; Cancers of the genitourinary system include tumors of the kidneys, urothelium or testicles; and ovarian cancer includes advanced ovarian cancer; mesothelioma includes neurofibromas, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, KRAS-mutant non-small cell lung cancer, liver cancer, thyroid cancer, breast cancer, nervous system tumors, schwannoma, meningioma, neuroma, adenoid cystic carcinoma, ependymoma, ependymal tumors, malignant pleurisy, malignant pleural mesothelioma, triple negative breast cancer, non-hematological malignancies, melanoma, colorectal carcinoma, leukemia, adenocarcinoma, solid tumors; melanoma includes locally advanced melanoma, NRAS mutant melanoma, metastatic cutaneous malignant melanoma; colorectal cancer includes metastatic colorectal cancer; and leukemia includes acute myelogenous leukemia; adenocarcinoma includes adenocarcinoma; and solid tumor includes locally advanced solid tumor, metastatic solid tumor and hepatocellular carcinoma.