ITK kinase selective inhibitor and application thereof

Abstract

The invention discloses an ITK kinase selective inhibitor and application thereof, and relates to the technical field of medicinal chemistry. The structure of the ITK kinase selective inhibitor is shown in the specification, r1 is selected from or; r2 to R6 are independently selected from hydrogen, halogen, C1-5 alkyl, C1-5 alkylamino or C1-5 halogenated alkyl. The ITK kinase selective inhibitor provided by the invention has a novel structure, realizes covalent targeting of ITK Cys442, and has good selectivity. The compound disclosed by the invention has obvious in-vitro, intracellular and in-vivo activity in biological functions, not only effectively inhibits phosphorylation activity of ITK at low nanomolar concentration, but also inhibits IL-2 secretion when a TCR signal channel is activated, and prominently shows lasting pharmacodynamic characteristics obtained due to a covalent irreversible binding mechanism; covalent molecular tools and candidate drug molecules with excellent selectivity are provided for deeply exploring biological functions of ITK under physiological and pathological conditions.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a selective inhibitor of ITK kinase and its application. Background Technology

[0002] Interleukin-2 induced T-cell kinase (ITK) is a member of the Tec family and is primarily expressed on T cells, natural killer (NK) cells, and mast cells. ITK is a core regulator of T-cell receptor (TCR) signaling and is crucial for TCR-mediated immune responses. Upon TCR activation, ITK phosphorylates phospholipase Cγ1 (PLCγ1), triggering a series of events—calcium influx, diacylglycerol production, and activation of downstream effector factors such as nuclear factor activating T cells, activator protein-1, and nuclear factor κB. These effector factors regulate T-cell differentiation, proliferation, and cytokine production. ITK knockout (ITK...) - / - In mice, Th1 bias is induced, resulting in defects in Th2 and Th17 differentiation and cytokine production, while retaining Th1 differentiation and interferon-γ (IFNγ) secretion. Due to this core role, ITK has become an attractive therapeutic target for treating diseases driven by T cell signaling dysregulation, including allergies, autoimmune diseases, infections, and T cell malignancies.

[0003] Over the past few decades, several ITK inhibitors have been disclosed. Figure 1Bristol-Myers Squibb reported the first effective and selective small molecule inhibitor, BMS-509744 (compound 1). Subsequently, pharmaceutical and academic groups explored various chemical scaffolds, represented by compounds 2–8. Irreversible covalent inhibitors (compounds 5–8) were designed to covalently bind to the non-catalytic cysteine ​​residue Cys442 within the ATP-binding pocket of ITK. The covalent strategy targeting Cys442 provided a longer residence time but faced selectivity barriers against homologous kinases such as BTK (Cys481). Compound 5 exhibited potent inhibition of BTK, while compounds 6 and 7 showed slightly improved selectivity for BTK. To date, only two ITK inhibitors have entered clinical trials: JET-051 (compound 3), a non-covalent inhibitor developed by Japan Tobacco, was discontinued in a Phase II clinical trial for the treatment of rheumatoid arthritis (NCT02919475) and psoriasis (NCT03358290); CPI-818 (Soquelitinib, 8), developed by Corvus, demonstrated a 39% overall response rate (ORR) and good safety profile in an early clinical trial for patients with T-cell lymphoma (NCT03952078), and is currently in a Phase III clinical trial. Recently, CPI-818 showed significant efficacy in a Phase I clinical trial for atopic dermatitis.

[0004] Despite these advances, the successful clinical application of ITK inhibitors remains limited. The high conservation of the ATP-binding pocket of the kinase presents a significant challenge to developing highly selective ATP-competitive inhibitors. Therefore, existing technologies still require improvement and development, particularly in developing novel selective covalent inhibitors of ITK kinases based on different chemical skeletons and their applications. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a selective inhibitor of ITK kinase and its application, aiming to solve the problems of insufficient inhibitory efficacy and selectivity of existing inhibitors against ITK kinase.

[0006] The technical solution of the present invention is as follows: In a first aspect, a selective inhibitor of ITK kinases is provided, the selective inhibitor of ITK kinases having the following structure: ; Wherein, R1 is selected from , or ; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogens, and C.1-5 Alkyl, C 1-5 Alkylamino, C 1-5 One of the haloalkyl groups.

[0007] In the preferred technical solution, R1 is selected from... , , , and One of them.

[0008] A further preferred technical solution, R1 is .

[0009] In a second aspect, the use of a selective ITK kinase inhibitor as described in the first aspect in the preparation of a medicament for ITK kinase-related diseases is provided.

[0010] In a preferred embodiment, the symptoms of the ITK kinase-related disease are mediated by ITK kinase.

[0011] In a preferred embodiment, the symptoms of the ITK kinase-related disease include ITK kinase overexpression.

[0012] In a further preferred embodiment, the ITK kinase overexpression is induced by interleukin-2.

[0013] In a preferred embodiment, the ITK kinase-related diseases include at least one of allergies, autoimmune diseases, infections, and T-cell malignancies.

[0014] Beneficial Effects: Compared with existing technologies, this invention designs and synthesizes a novel selective inhibitor of ITK kinase, successfully achieving covalent targeting of ITK Cys442 while improving selectivity. In terms of biological function, the compound of this invention exhibits significant in vitro, intracellular, and in vivo activities. It not only effectively inhibits the phosphorylation activity of ITK kinase at low nanomolar concentrations but also inhibits IL-2 secretion during TCR signaling pathway activation. Furthermore, it highlights the durable pharmacodynamic properties acquired through its irreversible covalent binding mechanism, providing a highly selective covalent molecular tool and candidate drug molecule for in-depth exploration of the biological functions of ITK kinase under physiological and pathological conditions. Attached Figure Description

[0015] Figure 1 It is the structural formula of a representative ITK inhibitor in the existing technology.

[0016] Figure 2This is a diagram showing the results of the covalent binding mechanism verification experiment of the present invention; wherein, A is the chemical structure of probe 14; B is the result of the concentration-dependent labeling experiment of probe 14; and C is the result of the competitive labeling experiment of compounds 4, 9, 11 and 7 with probe 14.

[0017] Figure 3 This is a graph showing the results of LC-MS / MS analysis of compound 9 covalently binding to ITK Cys442 in this invention.

[0018] Figure 4 This is a graph showing the results of the kinase selectivity analysis of compound 9 against 25 kinases in this invention.

[0019] Figure 5 The diagram shows the results of the bioactivity evaluation of compound 9 in this invention; wherein, A is the result of compound 9 inhibiting ITK phosphorylation activity in live Jurkat cells; B and C are the results of elution experiments using irreversible inhibitors 7 and 9 and reversible inhibitor 4; and D is the result of the inhibition of IL-2 by compounds 4 and 9 in anti-CD3 / CD28 co-stimulated Jurkat cells. Detailed Implementation

[0020] This invention provides a selective inhibitor of ITK kinase and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0021] Several ITK inhibitors have been reported. Figure 1 However, due to selectivity limitations against homologous kinases (such as BTK (Cys481)), the clinical application of ITK inhibitors remains limited. Covalent targeting of specific amino acid residues is a promising strategy for achieving kinase selectivity. Furthermore, covalent ITK inhibitors may have the advantage of sustained inhibition of the TCR pathway, even under conditions of high intracellular ATP concentrations.

[0022] Based on this, embodiments of the present invention provide an ITK kinase selective inhibitor, which has the following structure: ; Wherein, R1 is selected from , or ; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogens, and C. 1-5 Alkyl, C 1-5 Alkylamino, C 1-5 One of the haloalkyl groups.

[0023] Specifically, based on the highly selective binding mode of lead compound 4, this invention designs and synthesizes a novel selective inhibitor of ITK kinase, successfully achieving covalent targeting of ITK Cys442 while maintaining or even enhancing selectivity. In terms of biological function, the compound exhibits significant in vitro, intracellular, and in vivo activities, effectively inhibiting ITK phosphorylation activity at low nanomolar concentrations and suppressing IL-2 secretion during TCR signaling pathway activation. Furthermore, it highlights the durable pharmacodynamic properties acquired through its irreversible covalent binding mechanism, providing a highly selective covalent molecular tool for in-depth exploration of the biological functions of ITK under physiological and pathological conditions.

[0024] In some implementations, R1 is selected from... , , , and One of them.

[0025] In some more specific implementations, R1 is .

[0026] This invention provides an application of the selective ITK kinase inhibitor described above in the preparation of drugs for ITK kinase-related diseases.

[0027] In some embodiments, the symptoms of the ITK kinase-related disease are mediated by ITK kinase.

[0028] In some embodiments, the symptoms of the ITK kinase-related disease include ITK kinase overexpression.

[0029] In some more specific embodiments, the ITK kinase overexpression is induced by interleukin-2 (IL-2).

[0030] In some embodiments, the ITK kinase-related diseases include at least one of allergies, autoimmune diseases, infections, and T-cell malignancies.

[0031] The ITK kinase selective inhibitors of this invention are pharmaceutically acceptable salts, including lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, iron salts, copper salts, organic ammonium salts, hydrochloride salts, phosphate salts, acetate salts, propionate salts, oxalate salts, citrate salts, etc.

[0032] The present invention will be further described below through specific embodiments.

[0033] Experimental methods not specifically described in the following examples were performed according to conventional biochemical experimental methods in the art. Unless otherwise stated, all reagents used in the examples are commercially available products.

[0034] Example 1 This embodiment provides a selective inhibitor of ITK kinase, namely compound 9, whose synthetic route is as follows:

[0035] The synthesis steps are as follows: (1) Synthesis of (R)-3-(dimethylamino)-1-phenylprop-1-ol (denoted as 1-1) The compound (R)-(+)-3-chloro-1-phenyl-1-propanol (5 g, 29.4 mmol) was dissolved in methanol (20 mL), followed by the addition of dimethylamine aqueous solution (40 wt%, 7 mL) and potassium iodide (900 mg, 5.9 mmol). The mixture was stirred overnight at 70 °C, and then concentrated under reduced pressure. The residue was diluted with water and extracted three times with a dichloromethane / methanol (10:1) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which could be used directly for the next step without further purification.

[0036] (2) Synthesis of (S)-1-(3-(dimethylamino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (denoted as 1-2) Compound 1-1 (a pale yellow oil, 2.8 g), methyl 4-nitro-1H-pyrazole-3-carboxylate (2.1 g, 12.6 mmol), and triphenylphosphine (PPh3, 4.1 g, 15.6 mmol) were dissolved in anhydrous tetrahydrofuran (30 mL), cooled to 0 °C, and stirred for 30 min. Diethyl azodicarbonate (DEAD, 1.6 mL, 15.6 mmol) was added dropwise to the mixture at 0 °C. After the addition was complete, the reaction mixture was slowly brought to room temperature and stirred for 3 h. After the reaction was complete, the mixture was concentrated under reduced pressure and extracted with a dichloromethane / methanol (20:1) mixed solvent. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 1-2 (2.1 g, 60% yield) as a pale yellow liquid. 1 H NMR(400 MHz, Chloroform-d) δ 8.16 (s, 1H), 7.41 – 7.32 (m, 5H), 5.55 (dd, J =8.2, 6.9 Hz, 1H), 3.97 (s, 3H), 2.67 – 2.52 (m, 1H), 2.30 (dq, J = 13.8, 6.8Hz, 1H), 2.23 – 2.11 (m, 9H). (3) Synthesis of (S)-1-(3-(dimethylamino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-carboxamide (denoted as 1-3) Compounds 1-2 (900 mg, 2.71 mmol) were dissolved in an ammonia solution (20 mL) and stirred overnight at room temperature. The mixture was then concentrated under reduced pressure and extracted three times with a dichloromethane / methanol (10:1) mixed solvent. The organic phases were combined, dried, concentrated, and purified by column chromatography to give compounds 1-3 (690 mg, 81% yield) as a pale yellow oil. 1 H NMR (400MHz, Chloroform-d) δ 8.92 (s, 1H), 8.10 (s, 1H), 7.45 – 7.27 (m, 5H), 7.03(s, 1H), 5.80 (dd, J = 10.9, 4.5 Hz, 1H), 2.78 (ddd, J = 12.7, 6.6, 3.1 Hz,1H), 2.42 – 2.24 (m, 2H), 2.20 (d, J = 3.9 Hz, 7H). 13 C NMR (101 MHz, CDCl3) δ159.46, 138.05, 136.28, 135.80, 132.66, 128.74, 128.57, 127.25, 61.94, 55.08,44.39, 31.27. (4) Synthesis of (S)-1-(3-(dimethylamino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-amine (denoted as 1-4) Compounds 1-3 (600 mg, 1.89 mmol) were dissolved in methanol (10 mL). N-bromosuccinimide (NBS, 500 mg, 2.8 mmol) was added under ice bath conditions, followed by dropwise addition of an aqueous solution of potassium hydroxide (630 mg, 11.3 mmol). The reaction mixture was stirred overnight, allowed to slowly rise from 0 °C to room temperature, and then heated under reflux for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was extracted with a dichloromethane / methanol (10:1) mixture. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to give compounds 1-4 (330 mg, 60% yield) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 7.47 – 7.25 (m, 5H), 6.27 (s, 2H), 5.33 (dd, J = 9.0, 6.0 Hz, 1H), 2.48 – 2.38 (m, 1H), 2.22 –2.13 (m, 1H), 2.10 (s, 8H). 13 C NMR (101 MHz, DMSO) δ 151.56, 139.93, 129.75, 129.05, 128.58, 127.59, 121.13, 63.99, 55.87, 45.67, 31.49. (5) Synthesis of tert-butyl(S)-[1-(3-dimethylamino-1-phenylpropyl)-4-nitro-1H-pyrazole-3-yl]carbamate (denoted as 1-5) Compounds 1-4 (307 mg, 1.06 mmol) were dissolved in tetrahydrofuran (10 mL). Di-tert-butyl dicarbonate (276 mg, 1.26 mmol) and triethylamine (240 mg, 1.6 mmol) were added, and the mixture was stirred at room temperature for 3 hours. The mixture was then concentrated under reduced pressure and extracted three times with a methanol / dichloromethane (1:10) mixture. The organic phases were combined, dried, concentrated, and purified by column chromatography to give compounds 1-5 (380 mg, 90% yield) as a white oil. 1 H NMR (500 MHz, DMSO-d6) δ 9.07 (s, 1H), 7.47 – 7.27 (m, 5H), 7.13 (t, J = 5.7 Hz, 1H), 5.62 (dd, J = 9.1, 5.9 Hz, 1H), 2.27 (dt, J = 13.6, 6.7 Hz, 1H), 2.18 (s, 8H), 2.05 – 1.90 (m, 1H), 1.39 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 156.02, 146.58,139.86, 132.40, 131.98, 129.15, 128.72, 127.49, 78.44, 64.33, 55.49, 37.91,31.78, 28.71. (6) Synthesis of tert-butyl(S)-[4-amino-1-(3-dimethylamino-1-phenylpropyl)-1H-pyrazol-3-yl]carbamate (denoted as 1-6) Compounds 1-5 (505 mg, 1.3 mmol) were dissolved in methanol (20 mL), and 10% Pd / C (60 mg) was added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was confirmed to be complete by TLC, the mixture was filtered through diatomaceous earth, and the filtrate was collected and concentrated under reduced pressure to obtain crude products 1-6. These crude products were used directly in subsequent reactions without further purification.

[0037] (7) Synthesis of tert-butyl(S)-[4-(6,6-dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-indazole-3-carbamate)-1-(3-dimethylamino-1-phenylpropyl)-1H-pyrazol-3-yl]carbamate (denoted as 1-7) Intermediates 6,6-dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid (388 mg, 1.2 mmol), HATU (707 mg, 1.8 mmol), and DIEA (510 mg, 4.0 mmol) were dissolved in anhydrous dichloromethane (5 mL) and stirred in an ice bath for 30 min. Compounds 1-6 (465 mg, 1.2 mmol) in anhydrous dichloromethane (3 mL) were slowly added. After stirring at room temperature for 5 h, the mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The collected organic layer was concentrated under reduced pressure and purified by column chromatography to give a pale yellow oil 1-7 (462 mg, 58% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.96 (s,1H), 8.26 (s, 1H), 7.48 (d, J = 6.4 Hz, 1H), 7.43 – 7.29 (m, 5H), 5.49 (dd, J= 8.9, 6.0 Hz, 1H), 5.36 (s, 2H), 3.56 (t, J = 7.8 Hz, 2H), 2.93 (s, 1H),2.81 – 2.56 (m, 11H), 2.47 (s, 2H), 1.43 (t, J = 6.3 Hz, 2H), 1.36 (s, 9H),0.98 (s, 6H), 0.84 (t, J = 7.8 Hz, 2H), 0.06 (s, 9H). (8) Synthesis of (S)-N-(3-amino-1-(3-(dimethylamino)-1-phenylpropyl)-1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (denoted as 1-8) Compounds 1-7 (328 mg, 0.49 mmol) were dissolved in dichloromethane (2 mL). Trifluoroacetic acid (TFA, 2 mL) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the mixture was concentrated under reduced pressure. The resulting residue was redissolved in ethanol (10 mL), and the pH was adjusted to 12 with 5 M sodium hydroxide aqueous solution. The mixture was stirred at room temperature overnight and concentrated again. The residue was extracted with a methanol / dichloromethane (1:10) mixed solvent. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The combined organic phases were concentrated and purified by column chromatography to give compounds 1-8 (115 mg, 54% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 9.31 (s, 1H), 7.88 (s,1H), 7.38 – 7.18 (m, 5H), 5.20 – 5.08 (dd, 1H), 4.78 (s, 2H), 2.65 (t, J =6.4 Hz, 2H), 2.38 (s, 3H), 2.11 (s, 9H), 1.46 (t, J = 6.4 Hz, 2H), 0.96 (s,6H). 13 C NMR (101 MHz, DMSO) δ 160.61, 147.15, 142.49, 140.50, 128.75, 127.75,127.23, 122.94, 115.07, 107.66, 62.67, 56.45, 45.76, 36.25, 34.69, 32.73,30.37, 28.10, 18.85. (9) Synthesis of (S)-N-(3-acrylamide-1-(3-(dimethylamino)-1-phenylpropyl)-1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (denoted as 9) Compounds 1-8 (70 mg, 0.16 mmol) were dissolved in anhydrous dichloromethane (2 mL). Triethylamine (21 mg, 0.2 mmol) was added under ice bath conditions, followed by dropwise addition of acrylic anhydride solution (12.6 mg, 0.1 mmol). The reaction was stirred at 0 °C until complete as monitored by TLC. The mixture was then diluted with dichloromethane and extracted with water. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to give compound 9 (45 mg, 59% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H), 10.86 (s, 1H), 10.20 (s, 1H), 8.36 (s, 1H), 7.39 – 7.26 (m, 5H), 6.53 (dd, J = 17.0, 10.2Hz, 1H), 6.31 (dd, J = 17.1, 1.9 Hz, 1H), 5.79 (dd, J = 10.2, 2.0 Hz, 1H), 5.42 (dd, J = 9.1, 5.8 Hz, 1H), 2.66 (t, J = 6.4 Hz, 2H), 2.49 – 2.41 (m,1H), 2.37 (s, 2H), 2.12 (m, 9H), 1.46 (t, J = 6.4 Hz, 2H), 0.95 (s, 6H). 13 CNMR (101 MHz, DMSO) δ 163.97, 160.15, 142.19, 141.38, 140.64, 137.27, 130.91,128.98, 128.36, 128.20, 127.34, 123.46, 115.12, 112.53, 63.49, 56.22, 45.73,36.20, 34.68, 32.55, 30.34, 28.06, 18.81. HRMS (ESI): m / z C 27 H 36 N7O2[M+H] + The calculated value is 490.2930; the measured value is 490.2928.

[0038] Example 2 This embodiment provides a selective inhibitor of ITK kinase, namely compound 10. The synthesis steps are the same as in Example 1, except that in step (9), compounds 1-8 are reacted with 4-dimethylaminobut-2-enoic acid to obtain compound 10.

[0039] Example 3 This embodiment provides a selective inhibitor of ITK kinase, namely compound 11. The synthesis steps are the same as in Example 1, except that in step (9), compounds 1-8 are reacted with 4,4,4-trifluorobutenoic acid to obtain compound 11.

[0040] Example 5 This embodiment provides a selective inhibitor of ITK kinase, namely compound 12. The synthesis steps are the same as in Example 1, except that in step (9), compounds 1-8 are reacted with propionic acid to obtain compound 12.

[0041] Example 6 This embodiment provides a selective inhibitor of ITK kinase, namely compound 13. The synthesis steps are the same as in Example 1, except that in step (9), compounds 1-8 are reacted with 2-bromo-2-chloroacetic acid to obtain compound 13.

[0042] Example 7 This embodiment provides a probe designed based on compound 9, namely compound 14, for use in subsequent competitive labeling experiments of this invention, via a click chemistry reaction between the alkynyl group and TAMRA-N3. The synthetic route of compound 14 is as follows:

[0043] The synthesis steps are as follows: (1) Synthesis of (R)-3-(methyl(prop-2-yn-1-yl)amino)-1-phenylprop-1-ol (denoted as 2-1) Compound (R)-(+)-3-chloro-1-phenyl-1-propanol (1 g, 5.88 mmol) was dissolved in methanol (10 mL), followed by the addition of N-methylpropyneamine (813 mg, 11.76 mmol) and potassium iodide (180 mg, 1.18 mmol). The mixture was stirred and refluxed overnight at 70 °C, followed by concentration under reduced pressure. The residue was diluted with water and extracted three times with a dichloromethane / methanol (10:1) mixture. The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give compound 2-1 (1015 mg, 85% yield) as a pale yellow liquid. 1 H NMR (400 MHz, DMSO- d 6) δ 7.42 – 7.04 (m, 5H), 5.31 (s, 1H), 4.58 (dd, J = 7.7, 5.1 Hz, 1H), 3.28 (d, J = 2.4 Hz, 2H), 3.08 (t, J =2.4 Hz, 1H), 2.40 (m, J = 12.1, 7.9, 6.0 Hz, 2H), 2.18 (s, 3H), 1.78 – 1.58(m, 2H). 13C NMR (101 MHz, DMSO) δ 146.70, 128.46, 127.11, 126.22, 79.50,76.10, 71.44, 52.70, 49.11, 45.59, 37.46. (2) Synthesis of (S)-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (denoted as 2-2) Compound 2-1 (1 g, 4.9 mmol), methyl 4-nitro-1H-pyrazole-3-carboxylate (1 g, 5.88 mmol), and triphenylphosphine (PPh3, 1.9 g, 7.35 mmol) were dissolved in anhydrous tetrahydrofuran (30 mL), cooled to 0 °C, and stirred for 30 min. Diethyl azodicarbonate (DEAD, 0.75 mL, 7.35 mmol) was added dropwise to the mixture under ice bath conditions. The reaction mixture was then slowly brought to room temperature and stirred for 5 h. After the reaction was complete, the mixture was concentrated under reduced pressure and extracted with a methanol / dichloromethane (1:20) mixed solvent. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography gave compound 2-2 (1.1 g, 65% yield) as a pale yellow oil. 1 H NMR (400 MHz, DMSO- d 6) δ 8.45 (s, 1H), 7.44 – 7.33 (m, 5H), 5.69 (dd, J = 9.8, 4.6 Hz, 1H), 3.99 – 3.95 (m, 5H), 3.23 (t, J = 3.0 Hz, 2H), 3.08 – 3.06 (t, 1H), 2.55 (m, J = 9.6, 6.5 Hz, 1H), 2.31 – 2.19 (m, 2H), 2.13 (s, 3H), 2.06 (m, J = 11.9, 6.6Hz, 1H). (3) Synthesis of (S)-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-carboxamide (denoted as 2-3) Compound 2-2 (1 g, 2.8 mmol) was dissolved in an ammonia solution (20 mL) and heated and stirred overnight at 50 °C. The mixture was then concentrated under reduced pressure and extracted three times with a dichloromethane / methanol (10:1) mixed solvent. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain the crude product of compound 2-3, which was used directly in subsequent reactions without further purification.

[0044] (4) Synthesis of (S)-1-[3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl]-4-nitro-1H-pyrazole-3-amine (denoted as 2-4) Compounds 2-3 (500 mg, 1.47 mmol) were dissolved in methanol (10 mL). N-bromosuccinimide (390 mg, 2.18 mmol) was added under ice bath conditions. After stirring at 0 °C for 10 min, an aqueous solution of potassium hydroxide (500 mg, 8.83 mmol) was slowly added. The reaction mixture was slowly brought to room temperature from 0 °C and stirred overnight, followed by reflux for 20 h. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was extracted with a dichloromethane / methanol (10:1) mixture. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give compound 2-4 (312 mg, 68% yield) as a yellow oil. 1 H NMR (400 MHz, DMSO- d 6) δ 8.36 (s, 1H), 7.42 – 7.24 (m, 5H), 5.56(dd, J = 8.9, 5.6 Hz, 1H), 3.63 (s, 2H), 3.07 (t, J = 2.4 Hz, 1H), 2.47 – 2.37(m, 1H), 2.32 – 2.08 (m, 6H). 13 C NMR (101 MHz, DMSO) δ 154.44, 139.10, 136.60,135.29, 129.00, 128.59, 127.94, 79.22, 76.37, 59.40, 53.38, 51.80, 45.43,32.41. (5) The synthesis of (S)-tert-butyl(1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-4-nitro-1H-pyrazole-3-yl)carbamate (denoted as 2-5) is similar to that of compounds 1-5 in Example 1.

[0045] (6) The synthesis of (S)-tert-butyl(4-amino-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-1H-pyrazole-3-yl)carbamate (denoted as 2-6) is similar to that of compounds 1-6 in Example 1.

[0046] (7) Synthesis of tert-butyl(S)-[4-(6,6-dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-indazole-3-carbamate)-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-1H-pyrazol-3-yl]carbamate (denoted as 2-7) Intermediate 6,6-dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid (300 mg, 0.93 mmol), EDCI (210 mg, 1.1 mmol), HOBT (148 mg, 1.1 mmol), and DIEA (0.32 mL, 1.85 mmol) were dissolved in anhydrous dichloromethane solution (8 mL) and stirred in an ice bath for 30 min. Compound 2-6 (356 mg, 0.93 mmol) was slowly added. After stirring at room temperature for 6 h, the mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The collected organic layer was concentrated under reduced pressure and purified by column chromatography to give a pale yellow oil 2-7 (370 mg, 57% yield). 1 H NMR (400 MHz, DMSO- d 6) δ 9.33 (s,1H), 8.82 (s, 1H), 7.85 (s, 1H), 7.28 (m, J = 22.9, 4.5 Hz, 5H), 5.41 (dd, J =8.4, 5.0 Hz, 1H), 5.33 (s, 2H), 3.48 (t, J = 7.9 Hz, 2H), 3.29 – 3.24 (t, 2H), 3.06 (t, J = 2.3 Hz, 1H), 2.69 (s, 9H), 2.45 (s, 2H), 1.53 – 1.28 (m, 11H), 0.97 (s, 6H), 0.80 (dd, J = 8.6, 7.2 Hz, 2H), -0.08 (s, 9H). (8) Synthesis of (S)-N-[3-amino-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-1H-pyrazol-4-yl]-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (denoted as 2-8) Compounds 2-7 (300 mg, 0.43 mmol) were dissolved in dichloromethane (2 mL), and TFA solution (2 mL) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by TLC, the mixture was concentrated under reduced pressure. The residue was redissolved in ethanol solution (5 mL), and then the pH was adjusted to approximately 12 with sodium hydroxide aqueous solution. The reaction mixture was stirred at room temperature overnight, concentrated again, and extracted with a dichloromethane / methanol (10:1) mixed solvent. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The combined organic layers were concentrated to obtain the crude product of compounds 2-8, which was used directly in subsequent reactions without further purification.

[0047] (9) Synthesis of (S)-N-[3-acrylamido-1-(3-(methyl(prop-2-yn-1-yl)amino)-1-phenylpropyl)-1H-pyrazol-4-yl]-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (denoted as 14) Compounds 2-8 (80 mg, 0.17 mmol) were dissolved in anhydrous dichloromethane (2 mL), and triethylamine (32 mg, 0.3 mmol) was added, followed by the slow dropwise addition of acrylic anhydride solution (12.6 mg, 0.1 mmol) under ice bath conditions. The reaction was stirred at 0 °C until complete as monitored by TLC. The mixture was then diluted with dichloromethane and extracted with water. The organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give a white solid, compound 14 (54 mg, 62% yield). 1 H NMR (400 MHz, DMSO- d 6) δ 12.85 (s, 1H), 10.28 (s, 1H), 9.01 (s,1H), 7.93 (s, 1H), 7.34 – 7.26 (m, 5H), 6.52 (dd, J = 17.0, 10.2 Hz, 1H), 6.31(dd, J = 17.0, 1.9 Hz, 1H), 5.87 (dd, J = 10.1, 1.8 Hz, 1H), 5.44 (dd, J = 8.9, 4.8 Hz, 1H), 3.29 (t, J= 2.1 Hz, 2H), 3.10 (t, J = 2.3 Hz, 1H), 2.67 (t, J =6.4 Hz, 2H), 2.47 (q, J = 7.8, 6.4 Hz, 1H), 2.39 (s, 2H), 2.21 (d, J = 16.3 Hz, 6H), 1.48 (t, J = 6.4 Hz, 2H), 0.97 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 164.10,160.66, 141.94, 141.15, 140.76, 133.02, 130.65, 128.99, 128.81, 127.97,127.48, 127.01, 115.24, 114.41, 79.29, 76.40, 58.75, 52.11, 45.43, 36.15,34.59, 33.10, 30.34, 28.04, 18.81. HRMS (ESI): m / z C 29 H 36 N7O2[M+H] + The calculated value is 514.2930; the measured value is 514.2927.

[0048] The bioactivity of the compounds of the present invention will be evaluated below.

[0049] (1) Kinase activity detection experiment With GNE-9822 ( Figure 1 Compound 4) was used as a positive control, and the results are shown in Table 1.

[0050] Table 1. Detection results of kinase activity of the compounds of the present invention.

[0051]

[0052] (2) Experiment to verify the covalent bonding mechanism Using compound 14 synthesized in Example 7 as a fluorescent probe to label recombinant ITK protein, for Figure 1 Compound 4 Figure 1 The covalent bonding mechanism of compound 7 in Example 1, compound 9 in Example 1, and compound 11 in Example 3 was verified, and the results are as follows: Figure 2 As shown.

[0053] Based on the results of the competitive labeling experiment ( Figure 2 The following conclusions can be drawn: 1. Confirmation of covalent binding: Compound 14, as a fluorescent probe, can specifically covalently bind to ITK protein in a concentration-dependent manner.

[0054] 2. Competitiveness and Mechanism Verification: Covalent inhibitors 9, 11 and 7 can block the above-mentioned markers, while reversible inhibitor 4 has no such effect, proving that the first three compete with probe 14 for the same active site of ITK and that their mechanism of action is covalent and irreversible.

[0055] Furthermore, the covalent binding sites of compound 9 were analyzed by LC-MS / MS. Figure 3 It can be seen that compound 9 is bound to ITKCys442.

[0056] (3) Selectivity analysis of compound 9 kinase Based on the kinase selectivity analysis of compound 9 (1 μM) on 25 kinases ( Figure 4 It can be seen that compound 9 has a high inhibition rate of >80% against ITK kinase, indicating good selectivity.

[0057] (4) Evaluation of the bioactivity of compound 9 The results are as follows Figure 5 As shown, the details are as follows: 1. Dose-dependent inhibition: Jurkat cells were treated with different concentrations of compound 9 and TCR was stimulated. Immunoblotting was used to detect the phosphorylation levels of downstream signaling proteins (PLCγ1 and ERK). The results showed that compound 9 effectively inhibited the phosphorylation of PLCγ1 and ERK downstream of ITK at the cellular level. 50 Reaching the nanomolar level ( Figure 5 (A)

[0058] 2. Irreversibility Verification (Elution Assay): After co-incubating cells with compound 9, the cells were thoroughly washed with PBS buffer and then stimulated again. The recovery of PLCγ1 phosphorylation was detected to determine whether the inhibition was reversible. The elution assay demonstrated that the mode of action of compound 9 is irreversible, and its inhibitory effect was maintained for a long time after the compound was removed. Figure 5 (B and C in the middle).

[0059] 3. Functional Impact Assessment: The effect of compound 9 on IL-2 (a key T cell cytokine) secretion after TCR stimulation was examined. Experimental results showed that compound 9 effectively inhibited IL-2 secretion after TCR activation, with a higher potency than reversible inhibitor 4 (…). Figure 5 (D).

[0060] In summary, this invention discloses the design, synthesis, and biological evaluation of a series of novel selective inhibitors of ITK kinases. The optimized inhibitor (compound 9) exhibits superior potency and selectivity for ITK compared to BTK and other related kinases, and significantly inhibits IL-2 secretion both in vitro and in vivo.

[0061] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A selective inhibitor of ITK kinases, characterized in that, The selective inhibitor of ITK kinase has the following structure: ； Wherein, R1 is selected from , or ; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogens, and C. 1-5 Alkyl, C 1-5 Alkylamino, C 1-5 One of the haloalkyl groups.

2. The ITK kinase selective inhibitor according to claim 1, characterized in that, R1 is selected from , , , and One of them.

3. The selective ITK kinase inhibitor according to claim 2, characterized in that, R1 is .

4. The use of a selective ITK kinase inhibitor as described in any one of claims 1-3 in the preparation of a medicament for ITK kinase-related diseases.

5. The application according to claim 4, characterized in that, The symptoms of the aforementioned ITK kinase-related diseases are mediated by ITK kinase.

6. The application according to claim 4, characterized in that, Symptoms of the aforementioned ITK kinase-related diseases include ITK kinase overexpression.

7. The application according to claim 6, characterized in that, The ITK kinase overexpression was induced by interleukin-2.

8. The application according to claim 4, characterized in that, The ITK kinase-related diseases include at least one of allergies, autoimmune diseases, infections, and T-cell malignancies.

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