Thiazole substituted pyrimidinamine compound, pharmaceutical composition thereof and use thereof

Abstract

Disclosed in the present invention are a thiazole substituted pyrimidinamine compound, a pharmaceutical composition thereof and the use thereof. Provided in the present invention is a compound as shown in formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof. The compound has relatively high CDK1 / CDK2 selectivity, and has good application prospects in the prevention and / or treatment of CDK2-related diseases.

Description

Thiazole-substituted pyrimidine amine compounds, pharmaceutical compositions thereof and uses thereof

[0001] This application claims priority to Chinese patent application 202410597688X with a filing date of 2024 / 5 / 14. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and specifically relates to thiazole-substituted pyrimidine amine compounds, pharmaceutical compositions thereof and uses thereof. BACKGROUND

[0003] Cell cycle-dependent kinases (CDKs) are a family of serine / threonine kinases whose catalytic activity is regulated by interactions with cyclins and endogenous CDK inhibitors (CKIs). The close cooperation between these three ensures the orderly progression of the cell cycle. In addition to cell cycle regulation, CDKs are also involved in the regulation of gene transcription, epigenetic regulation, metabolism, stem cell self-renewal, neuronal function, and spermatogenesis (Lim & Kaldis, 2013).

[0004] There are currently 20 members of the CDK family, designated CDK1-CDK20. With the evolutionary expansion of the CDK family, they are mainly divided into two subfamilies, the cell cycle-related subfamily (CDK1, CDK4 and CDK5) and the transcription subfamily (CDK7, CDK8, CDK9, CDK11 and CDK20) (Malumbres, 2014).

[0005] Dysregulation of the cell cycle is associated with dysregulation of CDKs, which is a defining feature of cancer, ultimately promoting abnormal proliferation, thereby promoting tumorigenesis and disease progression. In this regard, over the past few decades, several CDK inhibitors (CDKIs) have been developed (first-, second- and third-generation CDKIs) to inhibit the proliferation of cancer cells. First- and second-generation CDKIs have not benefited much in the treatment of cancer patients due to their limited specificity and high toxicity. Third-generation CDKIs have achieved the most promising results at the preclinical and clinical levels, propelling them into the late stages of clinical trials for the treatment of various malignancies (particularly breast cancer) and completely changing the traditional treatment strategy (Mughal et al., 2023).

[0006] Third-generation CDKIs specifically target CDK4 / 6 with better selectivity and low toxicity, have been clinically proven safe and effective in the treatment of ER-positive HER2-negative breast cancer patients, which form CDK4 / 6-cyclin D complex by binding with cyclin D, phosphorylate retinoblastoma protein (Rb), release and activate the previously bound transcription factor E2F, and make the cell transition from Gl phase to S phase. CDK4 / 6 inhibitors induce cell arrest in Gl phase, showing a senescent phenotype. Selective CDK4 / 6 inhibitors such as Palbociclib, Ribociclib and Abemaciclib have been approved for the treatment of ER-positive HER2-negative breast cancer patients.

[0007] CDK2 is another CDK subtype that mainly functions in the G1 to S phase of the cell cycle. In the late Gl phase, CDK2 binds with cyclin E to form a proteasome complex CDK2-cyclin E and is activated, which promotes further phosphorylation of a series of substrates including Rb, and induces the sustained expression of transcription factor E2F, thereby regulating the smooth passage of cells through Gl phase. After entering the S phase, CDK2 binds with cyclin A to form a complex CDK2-cyclin A involved in the progression of the cell cycle S phase, and completes the replication of DNA. High expression of cyclin E and activation of complex CDK2-cyclin E are a mechanism for clinical breast cancer patients to be resistant to selective CDK4 / 6 inhibitors.

[0008] Evidence suggests that CDK2 inhibitors also have application prospects as cancer targets. First, a variety of genetic changes can up-regulate cyclin E1 levels and activate CDK2. CCNE1 gene amplification is found in a significant proportion of high-grade serous ovarian cancer (HGSOC), gastroesophageal cancer and uterine serous carcinoma, and CCNE1 amplification is associated with cyclin E1 overexpression, CDK2-dependent proliferation, chemotherapy resistance and poor prognosis. Second, in some lung cancer, colorectal cancer and hematological tumors, F-box family protein FBXW7 is inactivated, thereby inhibiting the degradation of cyclin E1. In addition, the possible acquired resistance of Luminal breast cancer to CDK4 / 6 inhibitors may be due to Cyclin E amplification or overexpression. This provides a theoretical basis for studying the inhibition of CDK2 in these tumors.

[0009] First, selective CDK2 inhibitors can target CDK2 alone in cancers where CDK2 is the main driver, avoiding the toxicity and dose-limiting of CDK4 / 6 inhibition. Second, in cancers where combined inhibition of CDK4 / 6 and CDK2 is needed, more flexible dosing can improve drug efficacy and patient tolerability. Third, selective CDK2 inhibitors can provide insights into the roles of CDK2 and CDK4 / 6 in different tumors. More research has found that CDK2 inhibitors have a wider development potential, and single-agent can be used for CCNE1 amplified ovarian cancer, MYCN amplified neuroblastoma, KRAS mutant lung cancer treatment, hormone-dependent breast cancer and prostate cancer, etc. (Tadesse et al., 2020). In combination: CDK2 combined with anti-mitotic therapy can produce a synergistic effect in TNBC, combined with PI3K inhibitors to synergistically inhibit CCNE1 amplification in serous uterine cancer, combined with CDK4 / 6 inhibitors or BRAF / HSP90 inhibitors to eliminate early adaptive rebound and solve drug resistance problems (Tadesse et al., 2020).

[0010] Several selective CDK2 inhibitors have entered early clinical trials, including INX315 (Incyclix Bio), PF-07104091 (Pfizer), BLU-222 (Blueprint Medicine), INCB123667 (Incyte), and ARTS-021 (Allorion Therapeutics). However, the selectivity of these selective CDK2 inhibitors is not perfect. So far, their effectiveness and safety in clinical trials have yet to be verified. Therefore, there is still a great clinical need to develop new CDK2 inhibitors with higher selectivity. CDK2 inhibitors with higher selectivity can safely address the unmet clinical need of patients who have developed resistance to CDK4 / 6 inhibitors, as well as provide a better treatment option for new precision medicine.

[0011] Incyte discloses an imidazole-substituted pyrimidine amine compound as a CDK2 inhibitor in patent US11427567B2, the present application provides a novel chemical structure of thiazole-substituted pyrimidine amine compound, which is unexpectedly found that such novel compounds further improve the inhibitory activity of CDK2 and the selectivity of CDK1 subtype. CDK1 is the only necessary CDK in the cell cycle (Santamaría et al., 2007), and knockout of CDK1 can cause mouse embryonic death, conditional knockout of CDK1 has a greater impact on liver and brain cells, especially causing devastating damage to both male and female germ cells (Campbell et al., 2020). Most of the first and second generation CDK inhibitors in clinical trials inevitably target CDK1, which is an important factor for the lack of clinical benefits (Mughal et al., 2023). Therefore, the higher the selectivity of CDK1, the higher the expected clinical safety. High-efficiency inhibition of CDK2 activity and weak or no inhibition of CDK1 are the properties that selective CDK2 inhibitors dream of. SUMMARY

[0012] The technical problem to be solved by the present application is to provide a high-selectivity CDK2 inhibitor with a new structure. The present application aims to provide a thiazole-substituted pyrimidine amine compound, its pharmaceutical composition and application. Such compounds have strong inhibitory effect on CDK2 and no obvious inhibitory effect on CDK1, so they have higher selectivity. High selectivity brings higher safety, and has good application prospect in the prevention and / or treatment of various diseases related to CDK2.

[0013] The present application provides a compound as shown in formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

[0014] wherein:

[0015] R 1 is halogen, -CN, -NO2, -OR a , -NR b R c , C 1-6 alkyl or C 1-6 haloalkyl; R a , R b , R c independently is H, C1-C6 alkyl or C1-C6 haloalkyl;

[0016] R 2 is C1-C6 alkyl, C1-C6 haloalkyl, -NR d R e ; R d , R eindependently H, C1-C6alkyl, or C1-C6haloalkyl;

[0017] R 3 is H, D, C1-C6alkyl, or C1-C6alkoxy;

[0018] R 4 and R 5 are independently H, D, C1-C6alkyl, or C1-C6alkoxy.

[0019] In certain preferred embodiments of the present application, certain groups in the compounds of Formula (I), pharmaceutically acceptable salts thereof, or stereoisomers thereof, are defined as follows, with the unmentioned groups being as described in any of the embodiments of the present application (simply "in an embodiment of the present application").

[0020] In an embodiment of the present application, the halogen is fluorine, chlorine, bromine, or iodine; preferably fluorine or chlorine; further preferably chlorine.

[0021] In an embodiment of the present application, each of the C1-C6alkyl groups is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl; preferably methyl.

[0022] In an embodiment of the present application, each of the C1-C6haloalkyl groups is independently halomethyl, haloethyl, halonormalpropyl, haloisopropyl, halonormalbutyl, haloisobutyl, halosecondarybutyl, or halotertiarybutyl; the halo is fluoro, chloro, bromo, or iodo.

[0023] In an embodiment of the present application, each of the C1-C6alkoxy groups is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, or t-butoxy.

[0024] In an embodiment of the present application, R 1 is halogen, -OR a , or -CN; preferably halogen or -CN.

[0025] In an embodiment of the present application, R a is H.

[0026] In an embodiment of the present application, R 2 is C 1-6 alkyl or -NR d R e ; for example, -NR d R e .

[0027] In an embodiment of the present application, R d and R e are independently H.

[0028] In an embodiment of the present application, R 3 is H or D; for example, H.

[0029] In an embodiment of the present application, R 4 and R 5 are independently H or D; preferably, H.

[0030] In an embodiment of the present application, R 1 is chloro, fluoro, cyano or hydroxy.

[0031] In an embodiment of the present application, R 2 is methyl or amino.

[0032] In an embodiment of the present application, R 1 and R 2 satisfy any one of the following conditions:

[0033] (1) R 1 is -OR a or -CN; R 2 is C 1-6 alkyl;

[0034] (2) R 1 is halogen, -OR a or -CN; R 2 is -NR d R e .

[0035] In an embodiment of the present application, the compound of formula (I) is a compound of formula I-1,

[0036] wherein R 1 , R 2 , R 3 , R 4 and R 5 are as defined in any one of the present application.

[0037] In an embodiment of the present application, the compound of formula (I) is any one of the following compounds:

[0038] The present application also provides a pharmaceutical composition comprising:

[0039] (1) the compound of formula (I), pharmaceutically acceptable salt thereof or stereoisomer thereof as described above; and

[0040] (2) a pharmaceutically acceptable adjuvant.

[0041] The present application also provides the use of the above-mentioned compound represented by formula (I), pharmaceutically acceptable salt thereof or stereoisomer thereof, the above-mentioned pharmaceutical composition, which is selected from:

[0042] (1) preparing a CDK2 inhibitor;

[0043] (2) preparing a medicament for treating or preventing a disease or disorder associated with cyclin-dependent kinase 2 (CDK2);

[0044] (3) preparing a medicament for treating and / or preventing a disease or disorder, which can be cancer.

[0045] In the use, the CDK2 inhibitor can be used in vivo in a mammalian organism; can also be used in vitro, mainly as experimental use, for example: providing comparison as a standard sample or control sample, or being prepared into a kit according to the conventional method in the art, for providing rapid detection of the CDK2 inhibition effect.

[0046] In an aspect of the present application, the disease or disorder associated with cyclin-dependent kinase 2 (CDK2) is cancer associated with amplification of CCNE1 gene and / or overexpression of CCNE1.

[0047] In an aspect of the present application, the cancer is selected from ovarian cancer, gastroesophageal cancer, uterine serous carcinoma, lung cancer, colorectal cancer, breast cancer and hematological tumor.

[0048] Definitions

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Furthermore, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the application.

[0050] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0051] The term "alkyl" means a straight or branched chain alkyl group having the number of carbon atoms specified (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, n-pentyl, n-hexyl and the like.

[0052] The term "haloalkyl" means an alkyl group substituted with halogen, wherein halogen and alkyl are as defined above.

[0053] The term "alkoxy" means the group R Z -O-, wherein R Z is alkyl as defined above.

[0054] The term "pharmaceutically acceptable salt" includes "a pharmaceutically acceptable salt formed with inorganic acids or organic acids" and "a pharmaceutically acceptable salt formed with inorganic bases or organic bases".

[0055] The term "pharmaceutically acceptable excipient" means any formulation or carrier medium that does not interfere with the effectiveness of the active substance of the present application, and that is not toxic to the host or patient, and represents a representative excipient including water, oil, vegetable and mineral, paste base, lotion base, ointment base, etc. These bases include suspending agents, viscosity increasing agents, transdermal accelerators, etc. Their formulations are well known to those skilled in the art of cosmetics or topical medicine.

[0056] The term "pharmaceutical composition" means a mixture or solution of a therapeutically effective amount of an active pharmaceutical ingredient with a pharmaceutically acceptable excipient, ready for administration to a mammal, such as a human, in need thereof.

[0057] The term "treatment" relates to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein relates to the action of the verb to treat, as defined before.

[0058] The above-mentioned preferred conditions can be combined in any way, without departing from the common general knowledge of the skilled person, to give further preferred embodiments of the present application.

[0059] The reagents and materials used in the present application are commercially available.

[0060] The positive progress effect of the present application is that: the present application discloses a thiazole-substituted pyrimidine amine compound, a pharmaceutical composition thereof and application. A kind of CDK2 selective inhibitor with different structure from reported or disclosed compound is provided. It has better binding activity to CDK2, and weaker binding to CDK1;It has better catalytic inhibition activity to CDK2, and weaker catalytic inhibition activity to CDK1;And it has dose-dependent antiproliferative effect on CCNE1 high expression cell line OVCAR3. The thiazole-substituted pyrimidine amine compound has excellent CDK2 / CDK1 selectivity and high safety. DETAILED DESCRIPTION

[0061] The present application will be further described by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples, without specific conditions, are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0062] Example 1: 4-(5-(4-(azetidin-1-ylmethyl)-2-chlorophenyl)thiazol-2-yl)-N-(1-(methylsulfonyl)piperidin-4-yl)-5-(trifluoromethyl)pyrimidin-2-amine

[0063] Step 1:

[0064] 2-Chloro-4-carboxyphenylboronic acid (intermediate 1, 200 mg, 1.08 mmol) was dissolved in dichloroethane (4 mL), followed by the addition of cyclobutane (112 mg, 1.95 mmol) and acetic acid (156 mg, 2.6 mmol). The mixture was stirred at 0 °C for 1 hour. Sodium triacetoxyborohydride (1.1 g, 5.21 mmol) was then slowly added to the reaction mixture. After the addition was complete, the temperature was slowly raised to 25 °C, and the reaction was continued for 1 hour. TLC (dichloromethane / methanol = 20 / 1, product: R) was performed. f =0.4, Raw material: R f =0.8) indicates that the raw material was completely consumed. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol = 20 / 1) to give intermediate 2 (225 mg, yield: 92%), a white solid.

[0065] LCMS(ESI)m / z:226[M+H]+.

[0066] Step Two:

[0067] Intermediate 3 (8.2 g, 46.00 mmol) was dissolved in tert-butanol (80 mL), followed by the addition of intermediate 4 (10 g, 46.00 mmol) and diisopropylethylamine (17.8 g, 138.01 mmol). After the addition was complete, the mixture was heated to 80 °C and stirred for 4 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f =0.4, Raw material: R f =0.8) indicates that the raw material was completely consumed. The mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 4:1) to give a white solid intermediate 5 (10 g, yield 60%).

[0068] LCMS(ESI)m / z:359.1[M+H]+.

[0069] Step 3:

[0070] Intermediate 5 (10 g, 27.87 mmol) was dissolved in 1,4-dioxane (160 mL), followed by the addition of intermediate 6 (12.5 g, 33.45 mmol), cuprous iodide (530.85 mg, 2.79 mmol), anhydrous lithium chloride (118.16 mg, 2.79 mmol), and [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (2.04 g, 2.79 mmol). After the addition was complete, the reaction mixture was stirred at 100 °C for 2 hours under a nitrogen atmosphere. TLC (petroleum ether / ethyl acetate = 1 / 1, product: R) f =0.3, Raw material: R f= 0.4) showed that the raw material was consumed completely. The reaction solution was cooled to room temperature, filtered with diatomite, rinsed with ethyl acetate (150 mL), and the combined filtrate was concentrated under reduced pressure and purified by column chromatography (PE:EA = 1 / 1) to obtain intermediate 7 (10 g, yield 88%) as a white solid.

[0071] LCMS (ESI) m / z: 409.1 [M+H]+.

[0072] Step four:

[0073] Intermediate 7 (3 g, 1.0 eq) was dissolved in chloroform (60 mL), sodium carbonate (2.34 g, 22.09 mmol) and liquid bromine (5 mL) were added, and after addition, the reaction solution was stirred at room temperature for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: R f = 0.6, raw material: R f = 0.4) showed that the raw material was consumed completely. The reaction solution was quenched with saturated aqueous sodium sulfite solution, extracted with ethyl acetate (10.0 mL, 10.0 mL), washed with saturated sodium chloride solution (15.00 mL), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 4 / 1) to obtain intermediate 8 (3 g, yield 83%) as a yellow solid.

[0074] LCMS (ESI) m / z: 486.1 [M+H]+.

[0075] Step five:

[0076] Intermediate 8 (90 mg, 185 μmol) was dissolved in a mixed solution of 1,4-dioxane (25 mL) and water (5 mL), intermediate 2 (126 mg, 555 μmol), cesium carbonate (181 mg, 555 μmol), and [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (13.5 mg, 18.5 μmol) were added, the reaction solution was replaced with N2, and the reaction solution was stirred at 100°C for 3 hours. TLC (dichloromethane / methanol = 20 / 1, product: R f = 0.2, raw material: R f = 0.9) showed that the raw material was consumed completely. The reaction solution was cooled, water (25.0 mL) and ethyl acetate (25.0 mL, 20.0 mL) were added to the reaction solution for extraction, the organic phase was washed with saturated sodium chloride solution (25.0 mL), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain the compound of Example 1 (15 mg, purity 88%) as a white solid.

[0077] LCMS (ESI) m / z: 587 [M+H]+;

[0078] 1H NMR (400 MHz, DMSO) δ 8.84 (d, J = 13.7 Hz, 1H), 8.57-8.34 (m, 2H), 7.80 (m, 1H), 7.58 (d, J = 16.1 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 4.14-3.87 (m, 2H), 3.74-3.59 (m, 4H), 3.45 (m, 3H), 3.26 (m, 2H), 3.01-2.87 (m, 3H), 2.29-1.88 (m, 4H), 1.69 (m, 2H).

[0079] Example 2: 4-(5-(4-(azetidin-l-ylmethyl)-2-fluorophenyl)thiazol-2-yl)-N-(l- (methylsulfonyl)piperidin-4-yl)-5-(trifluoromethyl)pyrimidin-2-amine

[0080] Following the same experimental procedure as in Example 1, using 2-fluoro-4- formylphenylboronic acid instead of Intermediate 1, the title compound was obtained.

[0081] LCMS (ESI) m / z: 571.1 [M+H]+;

[0082] 1H NMR (400 MHz, DMSO) δ 8.78 (d, J = 13.2 Hz, 1H), 8.56 (d, J = 15.5 Hz, 1H), 8.39-8.32 (m, 1H), 7.93-7.85 (m, 1H), 7.53 (d, J = 21.2 Hz, 1H), 7.32-7.25 (m, 1H), 4.00-3.93 (m, 1H), 3.68-3.47 (s, 4H), 3.19-3.09 (m, 4H), 2.93-2.89 (m, 5H), 2.05-1.99 (m, 4H), 1.66-1.63 (m, 2H)

[0083] Example 3: 5-(azetidin-l-ylmethyl)-2-(2-(((l-(methylsulfonyl)piperidin-4-yl)amino)- 5-(trifluoromethyl)pyrimidin-4-yl)thiazol-5-yl)benzonitrile

[0084] Following the same experimental procedure as in Example 1, using 2-cyano-4- formylphenylboronic acid instead of Intermediate 1, the title compound was obtained.

[0085] LCMS (ESI) m / z: 578.1 [M+H]+.

[0086] 1H NMR (400MHz, DMSO) δ8.81(d,J=13.6Hz,1H),8.50(d,J=3.5Hz,1H),8.43(dd,J=24.3,7.4Hz,1H),7.91–7.83(m,2H),7.73(d,J=8.0Hz,1H),4.03 -3.90(m,2H),3.63 -3.57(m,4H),3.18(t,J=6.7Hz,4H),2.91-2.89(m,4H),2.10-2.00(m,4H),1.66-1.60(m,2H).

[0087] Example 4: 5-(azacyclobutane-1-ylmethyl)-2-(2-(((1-(methanesulfonyl)piperidin-4-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)thiazolyl)phenol

[0088] Step 1

[0089] Intermediate 8 (800 mg, 1.64 mmol) was dissolved in 1,4-dioxane (50 mL) and water (10 mL). Intermediate 9 (516 mg, 2.46 mmol), cesium carbonate (1.6 g, 4.92 mmol), and 1,1-bis(diphenylphosphine)ferrocene / palladium dichloride (120 mg, 0.16 mmol) were added. After addition, the mixture was replaced with N2, and the reaction solution was stirred at 100 °C for 3 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: R) f =0.2, Raw material: R f =0.5) indicates that the raw materials have been completely consumed. After the reaction solution was cooled, water (25.0 mL) and ethyl acetate (25.0 mL, 20.0 mL) were added to the reaction solution for extraction. The organic phase was washed with saturated sodium chloride solution (25.0 mL), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain intermediate 10 as a white solid (600 mg, yield 64%).

[0090] LCMS(ESI) m / z: 572.1 [M+H] + ;

[0091] Step Two

[0092] Intermediate 10 (600 mg, 1.05 mmol) was dissolved in tetrahydrofuran (30 mL), and the solution was slowly cooled to 0 °C. A 2.5 M solution of lithium aluminum hydride in tetrahydrofuran (1.7 mL, 4.20 mmol) was added. After the addition was complete, the solution was slowly heated to 20 °C and reacted for 1 h. TLC (dichloromethane / methanol = 20 / 1, product: R) was performed. f =0.4, Raw material: Rf = 0.8) showed that the starting material was consumed completely. Concentration under reduced pressure, purification by column chromatography (dichloromethane / methanol = 20 / 1) gave intermediate 11 white solid (300 mg, yield 52%).

[0093] LCMS (ESI) m / z: 544.1 [M+H] + .

[0094] Step three

[0095] Intermediate 11 (300 mg, 0.55 mmol) was dissolved in dichloroethane (10 mL), 1M boron tribromide (2.75 mL, 2.76 mmol) was added, after addition, slowly heated to 80°C for 12h, TLC (dichloromethane / methanol = 20 / 1, product: R f = 0.3, starting material: R f = 0.5) showed that the starting material was consumed completely. Methanol was added to quench the reaction, concentrated under reduced pressure, purified to give yellow solid intermediate 12 (300 mg, yield 92%).

[0096] LCMS (ESI) m / z: 592.1 [M+H] + .

[0097] Step four

[0098] Intermediate 12 (300 mg, 0.5 mmol) was dissolved in DMF (5 mL), azetidine (58 mg, 1.0 mmol) and potassium carbonate (105 mg, 0.75 mmol) were added, after addition, the reaction liquid was stirred at 25°C for 1h, TLC (dichloromethane / methanol = 20 / 1, product: R f = 0.4, starting material: R f = 0.8) showed that the starting material was consumed completely. Concentration under reduced pressure, purification by preparation gave Example 4 (40 mg, yield 14%).

[0099] LCMS (ESI) m / z: 569.1 [M+H]+.

[0100] 1H NMR (400 MHz, DMSO) δ 10.77 (s, 1H), 8.75 - 8.57 (m, 2H), 8.34 - 8.21 (m, 1H), 7.82 - 7.75 (m, 1H), 7.04 - 6.98 (m, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.50 - 4.44 (m, 1H), 4.01 - 3.93 (m, 2H), 3.56 - 3.50 (m, 4H), 3.41 - 3.22 (m, 3H), 3.02 - 2.78 (m, 4H), 2.14 - 1.93 (m, 4H), 1.71 - 1.53 (m, 2H).

[0101] Example 5: 4-((4-(5-(4-(azetidin-l-ylmethyl)-2-chlorophenyl)thiazol-2-yl)-5- (trifluoromethyl)pyrimidin-2-yl)amino)piperidine-l-sulfonamide

[0102] Step one

[0103] Intermediate 13 (5.0 g, 24.96 mmol) and intermediate 4 (5.42 g, 24.96 mmol) were added to tert-butanol (50 mL), DIEA (9.68 g, 74.89 mmol) was added, and the mixture was stirred at 80 °C overnight. It was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified by reverse phase purification acetonitrile: water (0.1% NH4HCO3) = 87% to give intermediate 14 as a yellow solid product (5.3 g, yield 55.7%).

[0104] LCMS (ESI) m / z: 381.1 [M+H] + .

[0105] 1 1H NMR (400 MHz, DMSO) δ 8.67 - 8.50 (m, 2H), 4.04 - 3.83 (m, 3H), 2.88 (s, 2H), 1.83 (d, J = 10.7 Hz, 2H), 1.48 - 1.33 (m, 11H).

[0106] Step two

[0107] Intermediate 14 (7.88 g, 20.69 mmol) was dissolved in 1,4-dioxane (80 mL), 2- (tri-n-butylstannyl)thiazole (9.3 g, 24.83 mmol), cuprous iodide (395 mg, 2.07 mmol), anhydrous lithium chloride (88 mg, 2.07 mmol) and [1,1-bis(diphenylphosphino) ferrocene] dichloropalladium (1.5 g, 2.07 mmol) were added, N2 was replaced, the reaction was stirred at 100 °C for 12 h. TLC (petroleum ether / ethyl acetate = 3 / 1, product: Rf = 0.5, raw material: Rf = 0.6) showed that the raw material was completely consumed. After the reaction was cooled, it was concentrated and purified by column chromatography (PE:EA = 3:1) to obtain white solid intermediate 15 (5.9 g, yield 66.2%). f f + .

[0108] LCMS (ESI) m / z: 430.1 [M+H] 1 .

[0109] + H NMR (400 MHz, DMSO) δ 8.75 (d, J = 17.6 Hz, 1H), 8.27 (dd, J = 30.9, 7.6 Hz, 1H), 8.14 (dd, J = 4.5, 3.3 Hz, 1H), 8.10 - 8.04 (m, 1H), 4.09 - 3.85 (m, 3H), 2.90 (s, 2H), 1.90 (dd, J = 29.8, 10.2 Hz, 2H), 1.51 - 1.33 (m, 11H).

[0110] Step three

[0111] Intermediate 15 (5.9 g, 13.74 mmol) was dissolved in dichloromethane (60 mL), trifluoroacetic acid (12 mL) was added, and it was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in 50 mL of water, and the ph was adjusted to 13 with 2M NaOH, and a solid was precipitated. It was filtered, the filter cake was washed with water, and dried to obtain white solid intermediate 16 (3.99 g, yield 75.1%).

[0112] LCMS (ESI) m / z: 330.1 [M+H] 1 .

[0113] Step four

[0114] Intermediate 16 (2.3 g, 6.98 mmol) was dissolved in 1,4-dioxane (50 mL), sulfonamide (intermediate 13, 6.7 g, 6.98 mmol) was added, after addition, 110 °C stirring for 12 hours. The reaction solution was concentrated, purified by column chromatography (DCM:MeOH = 20 / 1) to obtain white solid intermediate 17 (2.12 g, yield 74.3%).

[0115] LCMS (ESI) m / z: 409.1 [M+H] + .

[0116] 1 H NMR (400 MHz, DMSO) δ 8.77 (d, J = 17.8 Hz, 1H), 8.44 - 8.25 (m, 1H), 8.14 (dd, J = 6.1, 3.2 Hz, 1H), 8.08 (d, J = 3.1 Hz, 1H), 6.79 (s, 2H), 3.86 (ddd, J = 14.6, 11.0, 5.8 Hz, 1H), 3.55 - 3.45 (m, 2H), 2.76 - 2.57 (m, 2H), 2.02 (dd, J = 29.4, 10.6 Hz, 2H), 1.64 (q, J = 11.7 Hz, 2H).

[0117] Step five

[0118] Intermediate 17 (2 g, 4.90 mmol) was dissolved in chloroform (20 mL), after adding sodium carbonate (1.56 g, 14.69 mmol) at 20 °C, bromine (5 mL) was added dropwise, after dropwise addition, the reaction solution was stirred at 20 °C for 1 hour. The solvent and bromine were evaporated by rotary evaporation, and then dissolved in a mixture of methanol (20 mL) and dichloromethane (20 mL), and then poured into 200 mL of water. Filtration, oven drying, to obtain white solid 18 crude product (1.56 g, purity 80%).

[0119] LCMS (ESI) m / z: 486.8 [M+H] + .

[0120] Step six

[0121] Intermediate 18 (300 mg, 617 pmol) and intermediate 2 (417 mg, 1.85 mmol) were dissolved in a mixture of 1,4-dioxane (10 mL) and water (1 mL), [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride 90 mg, 1.85 mmol) and cesium carbonate (602 mg, 123 pmol) were added, N2 was replaced, the reaction was stirred at 100 °C for 12 h, concentrated under reduced pressure, purified by preparative TLC (DCM:MeOH = 10 / 1) to give example 8 as a white solid (20 mg, yield 5.5%).

[0122] LCMS (ESI) m / z: 588.1 [M+H] + .

[0123] 1 H NMR (400 MHz, DMSO) d 8.79 (d, J = 17.4 Hz, 1H), 8.47 - 8.29 (m, 2H), 7.75 (dd, J = 20.3, 7.9 Hz, 1H), 7.54 (s, 1H), 7.38 (d, J = 7.9 Hz, 1H), 6.79 (d, J = 10.2 Hz, 2H), 3.86 (d, J = 35.5 Hz, 1H), 3.59 (s, 2H), 3.49 (d, J = 11.6 Hz, 2H), 3.17 (t, J = 6.7 Hz, 4H), 2.66 (dd, J = 26.1, 12.2 Hz, 2H), 2.02 (dt, J = 13.9, 11.1 Hz, 4H), 1.72 - 1.55 (m, 2H).

[0124] Example 6: 4-((4-(5-(4-(azetidin-1-ylmethyl)-2-chlorophenyl)thiazol-2-yl)-5- (trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-sulfonamide-4-d

[0125] Step one

[0126] Intermediate 19 (20 g, 100 mmol) was dissolved in ammonia in methanol solution (7 M) (200 mL), cooled to 0 °C, sodium borodeuteride (4.6 g, 110 mmol) was added in portions, after addition, it was raised to room temperature and stirred for 12 h. To the reaction solution, saturated ammonium chloride solution (100 mL) was added, filtered, the filtrate was washed with dichloromethane (100 mL*3), the organic phases were combined, washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate 20 as a yellow oil (17.5 g, yield: 86%).

[0127] Step two

[0128] Following the same experimental procedure of Example 5, using intermediate 20 instead of intermediate 13 in step one of Example 5, Example 6 was synthesized to give a white solid.

[0129] LCMS (ESI) m / z: 589.1 [M+H] + .

[0130] 1H NMR (400 MHz, DMSO) δ 8.79 (d, J = 17.4 Hz, 1H), 8.38 (dd, J = 21.6, 15.4 Hz, 2H), 7.75 (dd, J = 20.2, 8.0 Hz, 1H), 7.54 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 10.0 Hz, 2H), 3.59 (s, 2H), 3.49 (d, J = 12.0 Hz, 2H), 3.17 (t, J = 6.9 Hz, 4H), 2.66 (dd, J = 25.8, 12.5 Hz, 2H), 2.10 - 1.93 (m, 4H), 1.63 (dd, J = 21.2, 9.1 Hz, 2H).

[0131] Example 7: 4-((4-(5-(4-(azetidin-1-ylmethyl)-2-cyanophenyl)thiazol-2-yl)-5- (trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-sulfonamide-4-d

[0132] Following the same experimental procedure of Example 6, using 2-cyano-4- formylphenylboronic acid instead of 2-chloro-4-formylphenylboronic acid (intermediate 1), Example 7 was synthesized to give a white solid.

[0133] LCMS (ESI) m / z: 580.1 [M+H] + .

[0134] 1H NMR (400 MHz, DMSO) δ 8.81 (d, J = 17.9 Hz, 1H), 8.49 (d, J = 4.6 Hz, 1H), 8.39 (d, J = 20.0 Hz, 1H), 8.17 (d, J = 29.1 Hz, 1H), 7.88 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 7.9, 6.2 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 6.6 Hz, 1H), 3.64 (s, 2H), 3.50 (d, J = 11.9 Hz, 2H), 3.19 (t, J = 6.5 Hz, 4H), 2.68 (td, J = 22.0, 12.0 Hz, 2H), 2.09 - 1.93 (m, 4H), 1.64 (dd, J = 20.5, 8.5 Hz, 2H).

[0135] Experimental Example 1: Testing of the affinity of the compounds to CDK2, CDK1 at the cellular level

[0136] The NanoLuc fusion protein CDK1 (NV2701, 1 pg / pL) or CDK2 (NV2781, 1 pg / pL) and CCNB1 expression vector (NV2601, 1 pg / pL) or CCNE1 expression vector (NV2641, 1 pg / pL) were transiently transfected into HEK 293 cells (ATCC, CRL-1573): 1 pg of NanoLuc fusion protein and 9 pg of expression vector were added to 1 mL of culture medium (opti-MEM) and mixed with 20 mL of cell culture medium. Plated in 384-well, incubated overnight, and different dilutions of compounds or tracers (Promega, N2640) were added. The compounds were diluted with DMSO, 4-fold dilution, 10 concentration gradients, 2.5 pL of intermediate concentration of compounds were added to each well, the final compound concentration of CDK1 ranged from 10 pM to 0.0381 nM, the final compound concentration of CDK2 ranged from 100 nM to 0.0004 nM, and the final DMSO concentration was 0.1%. The tracer DMSO stock solution was diluted with tracer dilution buffer (Promega, N2640), and 2.5 pL of tracer dilution was added to each well to make the final concentration of the tracer 0.5 pM and the final concentration of DMSO 1%. Incubated in a 37°C 5% CO2 incubator for 2 hours, and then 60 pM of 3X NanoBRET Nano-Glo substrate (Promega, N2160) was added to each well, 25 pL per well. The donor signal at 450 nm and the acceptor signal at 610 nm were measured using the Envision instrument (2105). The acceptor emission value (650 nm) of each sample was divided by the donor emission value (460 nm) to generate the original BRET ratio value. After background correction, the mBU value was calculated = [(acceptor sample / donor sample) - (acceptor no tracer control / donor no tracer control)] x 1000. The inhibition rate = (mBU_AVE of negative controls - mBU_sample) / (mBU_negative control - mBU_AVE of positive control) * 100 was calculated. IC 50 Values were obtained by fitting using PRISM software and in the model of log(inhibitor) vs response-Variable slope.

[0137] The results show that the compounds of the present application have better binding activity to CDK2 (NanoBRET_affinity test IC 50≤ 10 nM), weaker binding to CDK1 (NanoBRET_ Affinity Test IC 50 ≥ 3247 nM), thus selectively binding to CDK2, avoiding binding to CDK1. Specifically, compared with the reference PF07104091, Example 5 and 6 have stronger affinity to CDK2 / Cyclin E1 and better selectivity to CDK1 / Cyclin B1. Meanwhile, INC176 is a compound of claim 1 disclosed in patent US11427567B2, and Example 5 and 6 have similar or better CDK2 binding activity compared with INC176, but Example 5 and 6 have no detectable binding activity to CDK1, thus having higher selectivity to CDK2 / Cyclin E1 in cells.

[0138] *INC176 is Example 176 in patent US11427567B2

[0139] Experimental Example 2: Testing of compounds on CDK2 / 1 enzyme activity at the molecular level

[0140] The enzyme activity test of CDK2 / 1 uses the mobility shift assay (MSA) to test the inhibitory effect of the compound on the catalytic phosphorylation of CDK enzyme.

[0141] Preparation of compounds in the test of CDK1: the compound is diluted 10 times with DMSO (the compound is stored at a concentration of 10 mM, and at this time the concentration is 1 mM, which is the highest concentration point), and the highest concentration point is the first point, and 4-fold gradient dilution is carried out in DMSO, a total of 10 gradients.

[0142] Preparation of compounds in the test of CDK2: the compound is diluted 2000 times with DMSO (the compound is stored at a concentration of 10 mM, and at this time the concentration is 5 μM, which is the highest concentration point), and the highest concentration point is the first point, and 4-fold gradient dilution is carried out in DMSO, a total of 10 gradients.

[0143] The 6 μL of the prepared 10 gradient samples were added into 44 μL of ddH2O, 2 μL was transferred to a 384-well assay plate (the final test concentration, the test concentration of CDK1 ranged from 20 μM to 0.076 nM, the test concentration of CDK2 ranged from 100 nM to 0.0004 nM, 5 μL of 0.015 nM CDK2 / Cyclin E1 (Biortus) working solution or 5 μL of 0.01 nM CDK1 / Cyclin B1 (Biortus) working solution was added to each well, pre-incubated for 30 min, 5 μL of substrate mixture (2 μM CDK7tide and 2 mM ATP) (scilight-peptide and VWR) was added to each well, incubated at 27°C for 70 min, quenched the reaction with 4 μL of 150 mM EDTA, and the values were read on a Caliper EZ Reader II. IC 50 The values were obtained by fitting using PRISM software and in a log (inhibitor) vs response-Variable slope model.

[0144] The results show that the compounds of the present application have better catalytic inhibition activity on CDK2 (MSA_Enzyme Activity Test IC 50 ≤ 2 nM) and weaker catalytic inhibition activity on CDK1 (MSA_Enzyme Activity Test IC 50 ≥ 130 nM), thereby selectively inhibiting CDK2. Specifically, compared with the reference PF07104091 and INC176, Examples 5 and 6 have stronger catalytic inhibition ability on CDK2 / Cyclin E1. Meanwhile, the selectivity data of Examples 3, 5, 6 and 7 on CDK1 / Cyclin B1 show higher selectivity than PF07104091 and INC176.

[0145] Experimental Example 3: Anti-proliferation activity test of the compounds on tumor cells

[0146] The anti-proliferation test of the compound on human ovarian cancer cells was detected by CyQUANT method. OVCAR-3 cells were obtained from ATCC, and after the cells were recovered and stable, the cells were collected, and the viable cell count was greater than 90%. 500 cells were seeded into a 384-well plate, and the next day, an equal proportion of dilution series of the compound was added, 3-fold gradient dilution, a total of 10 gradient concentrations. 400 nL of the diluted compound was transferred to a 384-well plate containing 39.6 μL of medium and mixed well, and the final concentration range was 10000 nM to 0.51 nM. The blank control well only added medium without cells, and the solvent control well added 0.1% DMSO (total volume was 40 μL per well). After incubation for 6 days, on the 7th day, 400 rpm centrifugation for 30 seconds, remove the supernatant, add 25 μL per well of NF dye reagent (Invitrogen, C35006) diluted 501 times with HBSS buffer, incubate at 37°C for 60 minutes in the dark, and Envision reading: excitation wavelength is 485 nm, and emission detection wavelength is 530 nm. Calculate the inhibition rate (IR): IR (%) = (1-(RLU compound-RLU blank control) / (RLU solvent control-RLU blank control))*100%. IC The value was obtained by fitting using PRISM software and in the log(inhibitor)vs response-Variable slope model. 50

[0147] The results show that the compound of the present application has a dose-dependent anti-proliferation effect on the CCNE1 high-expression cell line OVCAR3. Specifically, compared with the reference PF07104091, the anti-proliferation activity of Examples 3 and 5 is slightly stronger.

[0148] In summary, the present application discloses a novel thiazole-substituted pyrimidine amine compound, which is different from the chemical structure of the reported or disclosed CDK2 inhibitors. It is unexpectedly found that the CDK2 selectivity of the present application is higher, and the excellent selectivity is expected to bring higher clinical safety and patient compliance, and has high clinical anti-tumor drug value and use.​

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, wherein: R 1 halogen, -CN, -NO2, -OR a , -NR b R c , C 1-6 1-C6alkyl or C 1-6 1-C6haloalkyl; R a , R b , R c are independently H, C1-C6alkyl or C1-C6haloalkyl; R 2 C1-C6alkyl, C1-C6haloalkyl, -NR d R e ; R d , R e are independently H, C1-C6alkyl or C1-C6haloalkyl; R 3 H, D, C1-C6 alkyl or C1-C6 alkoxy; R 4 and R 5 independently H, D, C1-C6alkyl, or C1-C6alkoxy.

2. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, one or more of the following conditions are met: (1) said halogen is fluorine, chlorine, bromine or iodine; preferably fluorine or chlorine; further preferably chlorine; (2) each said Ci-C6alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl or t-butyl; preferably methyl; (3) each said Ci-C6haloalkyl is independently halomethyl, haloethyl, halonormalpropyl, haloisopropyl, halonormalbutyl, haloisobutyl, halosecondarybutyl or halo tertiary butyl; said halo is fluoro, chloro, bromo or iodo; and (4) each said Ci-C6alkoxy is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy or t-butoxy.

3. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, one or more of the following conditions are met: (1) R 1 halogen, -OR a or -CN; preferably halogen or -CN; (2) R a is H; (3) R 2 is C 1-6 alkyl or -NR d R e ; for example, -NR d R e ; (4) R d and R e are independently H; (5) R 3 H or D; for example H; and (6) R 4 and R 5 independently H or D; preferably H.

4. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, one or two of the following conditions are met: (1) R 1 is chloro, fluoro, cyano or hydroxy; and (2) R 2 is methyl or amino.

5. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, R 1 and R 2 satisfies any one of the following conditions: (1) R 1 is -OR a or -CN; R 2 is C 1-6 alkyl; (2) R 1 halogen, -OR a or -CN; R 2 is -NR d R e .

6. The compound as shown in formula (I), pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-5, wherein, The compound as shown in formula (I) is a compound as shown in formula I-1, wherein R 1 , R 2 , R 3 , R 4 and R 5 are as defined in any one of claims 1-5.

7. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, The compound as shown in formula (I) is any one of the following compounds:

8. A pharmaceutical composition comprising: (1) a compound of Formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as claimed in any one of claims 1-7; and (2) a pharmaceutically acceptable excipient.

9. Use of a compound of Formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as claimed in any one of claims 1-7, a pharmaceutical composition of claim 8, selected from: (1) the preparation of a CDK2 inhibitor; (2) the preparation of a medicament for treating or preventing a disease or disorder associated with cyclin-dependent kinase 2 (CDK2); (3) the preparation of a medicament for treating and / or preventing a disease or disorder, which can be cancer; which can be selected from ovarian cancer, gastroesophageal cancer, uterine serous carcinoma, lung cancer, colorectal cancer, breast cancer and hematological neoplasms.

10. The use according to claim 9, characterized in that, said disease or disorder associated with cyclin-dependent kinase 2 (CDK2) is a cancer associated with amplification of the CCNE1 gene and / or overexpression of CCNE1; which can be selected from ovarian cancer, gastroesophageal cancer, uterine serous carcinoma, lung cancer, colorectal cancer, breast cancer and hematological neoplasms.

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