Covalent inhibitor of targeting hCES2A as well as preparation method and application of covalent inhibitor

Abstract

The invention discloses a covalent inhibitor targeting hCES2A as well as a preparation method and application of the covalent inhibitor, the structural formula of the compound is as shown in the following formula I: # imgabs0 #, and R is CH3 or CH2CH3; r'is H, F, Cl, Br, OH, NH2, OCH3, CH3 or C (CH3) 3. According to the compound disclosed by the invention, N-substituted thiocarbamate is used as an electrophilic warhead to be covalently coupled with serine, so that the binding capacity is improved, a stronger inhibition effect is generated, meanwhile, a chalcone mother nucleus structure of an inhibitor molecule is not changed, a non-covalent binding part is not influenced, and relatively good selectivity is still kept. According to the invention, an electrophilic warhead structure capable of covalently modifying serine is delivered to a target amino acid residue site to form covalent coupling through targeted selectivity of drug molecules to target protein, and a new thought is provided for design and research of covalent inhibitors of other amino acid residues except cysteine.

Description

Technical Field

[0001] The present invention belongs to the technical field of covalent drug design, and in particular relates to a covalent inhibitor targeting hCES2A, a preparation method thereof and a use thereof. Background Art

[0002] Irinotecan (CPT-11) is an anticancer chemotherapy drug. After intravenous injection of irinotecan enters the human body through the blood, it can be converted into an active metabolite SN-38 (7-ethyl-l0-hydroxycamptothecin) with anti-tumor ability through hCES2A in the liver. However, due to the severe delayed diarrhea caused by the expression of hCES2A in the human intestine, it is impossible to administer itorally. Therefore, a good hCES2A inhibitor can improve the intestinal toxicity caused by oral administration of irinotecan and alleviate the pain of cancer patients.

[0003] From the perspective of drug discovery and application, an ideal hCES2A inhibitor should have the following characteristics: high specificity, potent potency, low toxicity, cell permeability (able to enter cells and block intracellular hCES2A), and acceptable metabolic stability. However, most reported hCES2A inhibitors, especially compounds with natural skeleton structures such as flavonoids, chalcones, triterpenes, and tanshinones, only show moderate inhibitory ability, poor specificity, low cell permeability, and unacceptable metabolic stability.

[0004] Covalent inhibitors, also known as irreversible inhibitors, are a class of organic small molecules that can interact with specific target proteins and form covalent bonds, leading to changes in protein conformation, thereby inhibiting protein activity. Covalent inhibitors have the advantages of higher biochemical efficiency, stronger and more persistent effects. At the same time, their dosage and frequency of administration are lower than those of traditional drugs, pharmacodynamics and pharmacokinetics are separated, free drugs are quickly cleared and can maintain efficacy for a long time, can prevent the development of drug resistance, and can target rare, non-conserved residues of specific proteins to achieve higher selectivity.

[0005] At present, there are no relevant research reports on covalent inhibitors targeting hCES2A. The present invention screened a covalent inhibitor molecular fragment targeting hCES2A, N-substituted carbamate thioester, from the covalent molecular fragment library designed and developed for targeted covalent inhibitors constructed in the early stage, and used it as an electrophilic warhead to "covalently" transform the non-covalent molecule B1 through molecular fusion technology, providing a new type of covalent inhibitor targeting hCES2A with an N-substituted carbamate thioester structure, which is used to solve the problem of delayed diarrhea caused by oral administration of itilican. Summary of the invention

[0006] The present invention is directed to the above-mentioned problems and provides a covalent inhibitor targeting hCES2A, a preparation method thereof and an application thereof in the preparation of an hCES2A inhibitor.

[0007] The technical solution of the present invention is as follows: The present invention proposes for the first time to use N-substituted carbamate thioester as an electrophilic warhead, and adopts molecular fusion technology to "covalently" transform the non-covalent lead compound B1, so that it is covalently coupled with the serine residue Ser228 in the catalytic cavity pocket of hCES2A. The modified compound does not change the parent core structure of the original molecule, and the binding mode will not be greatly affected; and it can increase the binding ability between the small molecule and the target protein through covalent action, improve the inhibitory activity, and produce an inhibitory effect that is equivalent to or better than the non-covalent lead compound B1.

[0008] The pharmaceutical preparation form of the compound described in the present invention is an oral preparation, which is used to inhibit the activity of hCES2A in the intestine and reduce delayed diarrhea during the treatment of tumors with itilican.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a covalent inhibitor targeting hCES2A, the structural formula of which is shown in the following formula I:

[0011]

[0012] Wherein, R is CH3 or CH2CH3; R' is H, F, Cl, Br, OH, NH2, OCH3, CH3 or C(CH3)3.

[0013] Preferably, in the above-mentioned covalent inhibitor targeting hCES2A, the olefinic double bond portion is in E or Z configuration.

[0014] Further preferably, the covalent inhibitor targeting hCES2A is represented by any of the following structural formulas:

[0015]

[0016]

[0017] The results of the inhibition experiment showed that the compounds with N-substituted carbamate thioesters as electrophilic warheads all showed time-dependent inhibition related to covalent interactions, and showed stronger inhibition after 33 minutes of incubation. Among them, compounds B7-4-2, B7-13-2, B7-19-1, B7-19-2 and B7-20-2 had a more prominent inhibitory effect on hCES2A protein, which was much greater than the non-covalent lead compound B1.

[0018] The results of covalent binding site determination showed that, except for compound B7-19-2 whose binding site was Ser368, the remaining compounds were able to covalently modify the serine residue Ser228 in the catalytic cavity of the hCES2A protein, thereby inhibiting the catalytic activity of the hCES2A protein.

[0019] In a second aspect, the present invention provides a method for preparing a covalent inhibitor targeting hCES2A:

[0020] 1. When R is methyl, the reaction flow of the covalent inhibitor is as follows:

[0021]

[0022] The main steps of the reaction are as follows: vanillin A is coupled with dimethylaminothiocarbonyl chloride B under alkaline conditions to obtain an intermediate C, which is then rearranged under high temperature and microwave radiation to obtain an intermediate D, which is then subjected to aldol condensation with a dihydroindanone compound E to obtain the target product F described in formula I when R is CH3.

[0023] In the above reaction process, the specific synthesis method of each step is as follows:

[0024] (1) When preparing intermediate C, the base is selected from any one of triethylamine, sodium hydroxide and potassium hydroxide, and the reaction solvent is selected from tetrahydrofuran; the preparation method is as follows: vanillin is dissolved in tetrahydrofuran, 2.5 eq. of triethylamine and 2.5 eq. of dimethylaminoformyl chloride are added at 0°C, reacted at room temperature for 4 hours, and after the reaction, an equal proportion of water is added to terminate the reaction, the mother liquor is extracted with dichloromethane 3 times, the organic phase is extracted once with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and subjected to column chromatography to obtain intermediate C;

[0025] (2) When preparing intermediate D, intermediate C is dissolved in diphenyl ether, irradiated with microwaves at 220° C., reacted for 1 to 2 hours, and intermediate D is obtained by column chromatography;

[0026] (3) When preparing the target product F, the intermediate D and the dihydroindanone compound E in a molar ratio of 8:9 are dissolved in a certain volume of alcohol solution, and a strong alkali aqueous solution with a volume of 60% of the alcohol volume and a mass volume ratio of 20% is slowly added, and the reaction is carried out at room temperature and the reaction progress is detected in real time; after the reaction is completed, an equal proportion of water is added to quench and the pH is adjusted to about 5, and then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure, and then separated by column chromatography using a dichloromethane / ethyl acetate system as the mobile phase to obtain the target product F.

[0027] 2. When R is ethyl, the reaction flow of the covalent inhibitor is as follows:

[0028]

[0029] The main steps of the reaction are as follows: Compound D is hydrolyzed under alkaline conditions and then coupled with N-methyl-N-ethylcarbamoyl chloride H to obtain intermediate I, which then undergoes aldol condensation with dihydroindanone compound E to obtain the target product J described in formula I when R is CH2CH3.

[0030] In the above reaction process, the specific synthesis method of each step is as follows:

[0031] (1) When preparing intermediate G, the base is selected from any one of triethylamine, sodium hydroxide and potassium hydroxide; the preparation process is as follows: intermediate D is dissolved in an alcohol solution, and a 5.0M alkaline aqueous solution with a volume of 20% of the alcohol solution is slowly added, and the reaction is carried out in an oil bath at 70°C and the reaction is detected; after the reaction is completed, the solvent is removed by rotary evaporation, and an equal proportion of water is added to terminate the reaction, and the pH is adjusted to 3, and the mother liquor is extracted with ethyl acetate, the organic phase is extracted with saturated brine, and the dried over anhydrous sodium sulfate and then filtered, and the dried by rotary evaporation, and the intermediate G is obtained by column chromatography;

[0032] (2) The preparation process of intermediate I is as follows: intermediate G is dissolved in tetrahydrofuran, 2.5 eq. of triethylamine and 2.5 eq. of N-ethyl-N-methylcarbamoyl chloride are added at 0°C, reacted at room temperature and the reaction is monitored in real time; after the reaction is completed, an equal proportion of water is added to terminate the reaction, the mother liquor is extracted with dichloromethane, the organic phase is extracted with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and subjected to column chromatography to obtain intermediate I;

[0033] (3) When preparing the target product J, the intermediate I and the dihydroindanone compound E in a molar ratio of 8:9 are dissolved in a certain volume of alcohol solution, and a strong alkali aqueous solution with a volume of 60% of the alcohol volume and a mass volume ratio of 20% is slowly added, and the reaction is carried out at room temperature and the reaction progress is detected in real time; after the reaction is completed, an equal proportion of water is added to quench and the pH is adjusted to about 5, and then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure, and then separated by column chromatography using a dichloromethane / ethyl acetate system as the mobile phase to obtain the target product J.

[0034] The third aspect of the present invention provides the use of the above-mentioned covalent inhibitor targeting hCES2A, its isotope-labeled compound or its pharmaceutically acceptable salt in the preparation of hCES2A inhibitor.

[0035] In a fourth aspect, the present invention provides a covalent inhibitor targeting hCES2A, comprising an active component and a pharmaceutically acceptable excipient, wherein the active component is the covalent inhibitor targeting hCES2A described above, an isotope-labeled compound thereof, or a pharmaceutically acceptable salt thereof.

[0036] Preferably, the inhibitor is in the form of an oral preparation and is used in combination with irinotecan.

[0037] Beneficial technical effects of the present invention:

[0038] The compound provided by the present invention is a hCES2A covalent inhibitor, also known as an hCES2A irreversible inhibitor. The compound can covalently bind to hCES2A, exhibits low nanomolar inhibitory activity on hCES2A, and has excellent therapeutic potential for delayed diarrhea caused by oral administration of itilican.

[0039] The compound of the present invention is covalently coupled to serine through N-substituted carbamate thioester as an electrophilic warhead. The advantages of this mode of action are: first, the compound molecule is covalently coupled to the target protein, thereby improving the binding ability and producing a stronger inhibitory effect; second, the chalcone core structure of the inhibitor molecule is not changed, and the non-covalent binding part will not be affected, and still maintains good selectivity; third, most of the current research on covalent inhibitors mainly targets cysteine ​​thiol groups, and the present invention delivers the electrophilic warhead structure that can covalently modify serine to the target amino acid residue site to form a covalent coupling through the targeted selectivity of the drug molecule to the target protein, which provides a new idea for the design and research of covalent inhibitors of amino acid residues other than cysteine.

[0040] As a hCES2A covalent inhibitor, the compound of the present invention has obvious advantages over the hCES2A reversible inhibitors in the prior art:

[0041] (1) Currently, most of the small molecule reversible inhibitors of hCES2A have the disadvantages of large dosage and short duration of action. However, the hCES2A covalent inhibitor of the present invention has a strong and long-lasting effect due to the formation of a covalent bond with the hCES2A protein, which can reduce the dosage and frequency of administration.

[0042] (2) Since the hCES2A covalent inhibitor can produce a sustained inhibitory effect on the hCES2A target protein, it can prevent the development of drug resistance. The experimental results also show that the compounds provided by the present invention can effectively inhibit the hydrolysis of oral itilican in the intestine, and still exert sustained inhibitory activity after elution. The present invention overcomes the problem that oral itilican is easily hydrolyzed by hCES2A protein in the human intestine, causing SN-38 enrichment and leading to severe delayed diarrhea. A good hCES2A inhibitor can improve the intestinal toxicity caused by oral administration of irinotecan and alleviate the suffering of cancer patients.

[0043] Therefore, the compounds provided by the present invention can be used as hCES2A covalent inhibitors to prepare drugs for preventing and treating delayed diarrhea caused by oral administration of itilican, thereby providing a new option for the development and application of clinical drugs.

[0044] In terms of preparation, the raw materials for the synthesis method of the compound of the present invention are readily available, the preparation method is unique and innovative, easy to implement, and suitable for industrial production. DETAILED DESCRIPTION

[0045] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0046] The present invention adopts molecular fusion technology and uses N-substituted carbamate thioester as an electrophilic warhead for the first time to "covalently" transform the non-covalent lead compound B1, so that the transformed molecular compound (Formula I) can covalently modify the serine residue Ser228 in the catalytic cavity of the hCES2A protein.

[0047]

[0048] Wherein, R is CH3 or CH2CH3; R' is H, F, Cl, Br, OH, NH2, OCH3, CH3 or C(CH3)3.

[0049] Specifically, the following is the preferred structural formula of the compound of the present invention:

[0050]

[0051]

[0052] Example 1: Preparation of the target product when R is CH3 in Formula I

[0053] Vanillin A is coupled with dimethylaminothiocarboxylic acid chloride B under alkaline conditions, and then rearranged under high temperature and microwave radiation to obtain an intermediate D, which is then subjected to aldol condensation with an indanone compound E to obtain the target product described in formula I when R is CH3;

[0054]

[0055] Conditions and reagents: i. 2.5 eq. of dimethylaminothiocarbonyl chloride, 2.5 eq. of triethylamine, tetrahydrofuran, room temperature, 4 hours; ii. diphenyl ether, microwave irradiation at 220°C, 1.5 hours; iii. 1-1.5 eq. of indanone compounds, 20% (w / v) aqueous solution of sodium hydroxide, ethanol, room temperature, 30 minutes.

[0056] The specific intermediate structures and reaction steps are shown below:

[0057] Synthesis of intermediate C:

[0058]

[0059] Vanillin (5 g, 32.9 mmol) was dissolved in 92 ml of tetrahydrofuran, triethylamine (8.3 g, 82.2 mmol) and dimethylaminoformyl chloride (10 g, 82.2 mmol) were added at 0°C, and the mixture was reacted for 4 hours at room temperature. The reaction was detected by thin layer chromatography (TLC). After the reaction was completed, water was added in equal proportion to terminate the reaction, the mother liquor was extracted with dichloromethane 3 times, the organic phase was extracted with saturated brine once, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. Intermediate C (5.4 g of white powder, yield 72%) was obtained by column chromatography. 1 H NMR (600MHz, DMSO-d6) δ10.00(s,1H),7.64(d,J=7.7Hz,1H),7.53(d,J=1.2,1H),7.51(dd,J=7.7,1.4Hz,1H),3.87(s,3H),3.06(s,3H),2.90(s,3H). 13 C NMR (150MHz, DMSO-d6) δ193.1,163.9,160.3,138.8,138.2,124.7,122.6,111.2,56.6,37.0.

[0060] Synthesis of intermediate D:

[0061]

[0062] Intermediate C (2.5 g, 10.5 mmol) was dissolved in 16.3 ml of diphenyl ether and reacted by microwave for 1.5 hours. Intermediate D was obtained by column chromatography as a white powder (1.9 g) with a yield of 76%. 1 H NMR (600MHz, DMSO-d6) δ9.97(s,1H),7.58(d,J=1.7Hz,1H),7.57(dd,J=8.0,1.8Hz,1H),7.28(d,J=8.0Hz,1H),3.85(s,3H),3.36(s,3H),3.32(s,3H). 13 C NMR (150MHz, DMSO-d6) δ192.5,186.0,152.5,147.6,135.3,125.4,123.8,112.5,56.5,43.4,39.0.

[0063] Synthesis of target compound B7-4-1:

[0064]

[0065] Intermediate D (620 mg, 2.4 mmol) and 1-indanone (353 mg, 2.7 mmol) were dissolved in 57 ml of ethanol, and 30 ml of 20% (w / v) sodium hydroxide aqueous solution was slowly added. The mixture was reacted at room temperature for 30 minutes, and the reaction was detected by thin layer chromatography (TLC). After the reaction was completed, water was added in equal proportion to quench the mixture, and the pH was adjusted to about 5 with 1 mol / L HCl solution. The aqueous layer was extracted with dichloromethane (3×60 ml), and the organic phases were combined, washed once with saturated brine, and then dried with anhydrous sodium sulfate. After vacuum distillation, 700 mg of yellow powder was obtained by column chromatography using dichloromethane / ethyl acetate system as the mobile phase, with a yield of 76%. 1 HNMR(600MHz, CDCl3)δ7.90(d,J=7.6Hz,1H),7.64-7.60(m,2H),7.55(d,J=8.0Hz,2H),7.44-7.4 1(m,1H),7.30(d,J=7.8Hz,1H),7.17(s,1H),4.03(s,2H),3.93(s,3H),3.14(s,3H),3.01(s,3H). 13 C NMR (150MHz, CDCl3) δ194.1,160.1,149.6,138.5,138.2,138.0,135.8,134.8 ,133.4,127.8,126.3,124.5,122.7,118.9,113.7,56.3,37.1,32.4.HRMS(ESI + ):calculated for C 20 H 19 NO3S[M+H] + 354.1158, found 354.1173.

[0066] Synthesis of target compound B7-10-1:

[0067]

[0068] The synthesis method of the target compound B7-10-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate D (300 mg, 1.2 mmol), 6-methyl-1-indanone (258 mg, 1.8 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 283 mg of a yellow powder with a yield of 74%. 1HNMR (600MHz, CDCl3) δ7.70(s,1H),7.62(s,1H),7.54(d,J=7.9Hz,1H),7.44(d,J=0.9Hz,1H),7.30(dd,J=7.9 ,1.3Hz,1H),7.17(d,J=1.0Hz,1H),3.99(s,2H),3.93(s,3H),3.14(s,3H),3.02(s,3H),2.43(s,3H).HRMS(ESI + ):calculated for C 21 H 21 NO3S[M+H] + 368.1315, found 368.1307.

[0069] Synthesis of target compound B7-11-1:

[0070]

[0071] The synthesis method of the target compound B7-11-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate D (300 mg, 1.2 mmol), 6-methoxy-1-indanone ((285 mg, 1.8 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase to obtain 336 mg of yellow powder, with a yield of 68%. 1 H NMR (600MHz, CDCl3) δ7.62-7.61(m,1H),7.54(d,J=7.9Hz,1H),7.43(d,J=8.3Hz,1H),7.34(d,J=2.5Hz,1H),7.29(dd,J=8.0,1.5Hz ,1H),7.20(dd,J=8.3,2.6Hz,1H),7.17(d,J=1.5Hz,1H),3.96(d,J=1.6Hz,2H),3.93(s,3H),3.86(s,3H),3.13(s,3H),3.02(s,3H). 13 C NMR (150MHz, CDCl3) δ194.2,165.8,160.1,159.7,142.4,139.2,138.5,138.2,136.7 ,133.3,127.0,124.2,122.7,118.9,113.7,105.9,56.3,55.7,37.1,31.7.HRMS(ESI + ):calculated for C 21 H 21 NO3S[M+H]+ 384.1264, found 384.1256.

[0072] Synthesis of target compound B7-12-1:

[0073]

[0074] The synthesis method of the target compound B7-12-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate 2 (200 mg, 0.8 mmol), 6-hydroxy-1-indanone (116 mg, 0.8 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 190 mg of a yellow powder with a yield of 70%. 1 HNMR (600MHz, DMSO-d6) δ9.87 (s, 1H), 7.52 (s, 1H), 7.50-7.48 (m, 2H), 7.43 (d, J = 0.9Hz, 1H), 7.37 (dd, J = 8.0, 1. 1Hz,1H),7.15(dd,J=8.2,2.4Hz,1H),7.09(d,J=2.4Hz,1H),4.04(s,2H),3.88(s,3H),3.06(s,3H),2.91(s,3H). 13 C NMR(150MHz,DMSO-d6)δ193.4,164.2,159.8,157.3,140.9,138.5,138.0,137.8,1 37.3,131.9,127.5,123.7,122.6,118.4,113.9,108.3,56.1,36.6,31.3.HRMS(ESI + ):calculated for C 20 H 19 NO4S[M+H]+370.1108,found 370.1103.

[0075] Synthesis of target compound B7-14-1:

[0076]

[0077] The synthesis method of the target compound B7-14-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (250 mg, 1.0 mmol), 6-bromo-1-indanone (311 mg, 1.5 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase to obtain 189 mg of yellow powder, with a yield of 70%. 1HNMR (600MHz, CDCl3) δ7.72(dd,J=8.0,1.9Hz,1H),7.65(s,1H),7.56(d,J=8.0Hz,1H),7.44(d,J=7.9Hz,1H),7.30( dd,J=8.0,1.3Hz,1H),7.16(d,J=1.1Hz,1H),3.98(d,J=1.1Hz,2H),3.93(s,3H),3.14(s,3H),3.02(s,3H).HRMS(ESI + ):calculated forC 20 H 18 BrNO3S[M+H] + 432.0264, found 434.0241.

[0078] Synthesis of target compound B7-15-1:

[0079]

[0080] The synthesis method of the target compound B7-15-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate D (200 mg, 0.8 mmol), 6-tert-butyl-1-indanone (220 mg, 1.2 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase obtains 246 mg of white powder with a yield of 72%. 1 H NMR (600MHz, CDCl3) δ7.93(s,1H),7.69(dd,J=8.0,1.5Hz,1H),7.63(s,1H),7.55(d,J=7.9Hz,1H),7.49(d,J=8 .0Hz,1H),7.31(d,J=7.9Hz,1H),7.18(s,1H),4.00(s,2H),3.94(s,3H),3.14(s,3H),3.01(s,3H),1.37(s,9H). 13 C NMR (150MHz, CDCl3) δ194.5,165.8,160.1,151.4,147.0,138.6,138.2,137.9,136.5,1 33.1,132.7,125.8,122.7,120.9,118.8,113.7,56.3,37.1,35.0,32.0,31.4.HRMS(ESI + ):calculated for C 24 H 27 NO3S[M+H] +410.1784, found 410.1774.

[0081] Synthesis of target compound B7-19-1:

[0082]

[0083] The synthesis method of the target compound B7-19-1 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate D (200 mg, 0.8 mmol), 6-fluoro-1-indanone (176 mg, 1.2 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 210 mg of a yellow powder with a yield of 68%. 1 HNMR(600MHz, CDCl3)δ7.64(s,1H),7.56-7.51(m,3H),7.35-7.29(m,1H),7.3 0(dd,J=7.9,1.2Hz,1H),7.16(s,1H),4.00(s,2H),3.14(s,3H),3.01(s,3H). 13 C NMR (150MHz, CDCl3) δ193.3,165.7,163.4,161.8,160.1,145.0,139.8,139.7,138.2,138.1,136.0 ,134.1,127.7,127.6,122.7,122.5,122.3,119.3,113.8,110.5,110.4,56.3,37.1,31.8.HRMS(ESI + ):calculated for C 20 H 18 FNO3S[M+H] + 372.1064, found 372.1055.

[0084] Synthesis of target compound B7-20-1:

[0085]

[0086] The synthesis method of the target compound B7-20-1 is the same as that of B7-4-1, and its operation method can be referred to, intermediate D (200 mg, 0.8 mmol), 6-amino-1-indanone (172 mg, 1.2 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 231 mg of a yellow powder with a yield of 75%. 1H NMR (600MHz, DMSO-D6) δ7.59(s,1H),7.54(d,J=7.9Hz,1H),7.33(d,J=8.0Hz,1H),7.30(d,J=8.0Hz,1H),7.17(s,1 H),7.14(d,J=1.9Hz,1H),6.97(dd,J=8.0,2.2Hz,1H),3.94(s,3H),3.92(s,2H),3.14(s,3H),3.02(s,3H)HRMS(ESI + ):calculated forC 20 H 20 N2O3S[M+H] + 369.1267, found 369.1249.

[0087] Example 2: Preparation of the target product when R in Formula I is CH2CH3

[0088] The compound D in Example 2 is hydrolyzed under alkaline conditions and then coupled with N-methyl-N-ethylcarbamoyl chloride to obtain intermediate I, which is then subjected to aldol condensation with an indanone compound to obtain the target product of formula I when R is CH2CH3;

[0089]

[0090] Conditions and reagents: i. 20% (w / v) potassium hydroxide aqueous solution, methanol, 70°C, 1 hour; ii. 2.5 eq. triethylamine, 2.5 eq. N-methyl-N-ethylcarbamoyl chloride, tetrahydrofuran, room temperature, 4 hours; iii. 1-1.5 eq. indanone compounds, 20% (w / v) sodium hydroxide aqueous solution, ethanol, room temperature, 1 hour.

[0091] The specific intermediate structures and reaction steps are shown below:

[0092] Synthesis of intermediate G:

[0093]

[0094] The intermediate D (33 g, 138.5 mmol) was dissolved in 298 ml of methanol, and 60.9 ml of 5.0 M potassium hydroxide aqueous solution was added dropwise, and the reaction was carried out in an oil bath at 70°C for 1 hour, and the reaction was detected by thin layer chromatography (TLC). After the reaction was completed, the ethanol solution was removed by rotary evaporation, and an equal proportion of water was added to terminate the reaction, and the pH was adjusted to 3. The mother liquor was extracted with ethyl acetate 3 times, and the organic phase was extracted with saturated brine once, dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation. Intermediate G (20 g of yellow powder, yield 78%) was obtained by column chromatography. 1HNMR (600MHz, CDCl3) δ9.89 (s, 1H), 7.57 (d, J = 7.7Hz, 1H), 7.43-7.41 (m, 2H), 5.5 (s, 1H), 3.91 (s, 3H).

[0095] Synthesis of Intermediate I:

[0096]

[0097] The intermediate G (1 g, 5.94 mmol) was dissolved in 16.6 ml of tetrahydrofuran, triethylamine (1.5 g, 14.9 mmol) and N-ethyl-N-methylcarbamoyl chloride (1.8 g, 14.9 mmol) were added at 0°C, and the mixture was reacted at room temperature for 4 hours. The reaction was detected by thin layer chromatography (TLC). After the reaction was completed, water was added in equal proportion to terminate the reaction, the mother liquor was extracted with dichloromethane 3 times, the organic phase was extracted with saturated brine once, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The intermediate I (white powder 1.1 g, yield 70%) was obtained by column chromatography. 1 H NMR (600MHz, DMSO-d6) δ10.01(s,1H),7.65(d,J=7.7Hz,1H),7.54(d,J=1.4Hz,1H),7.52(dd,J=7.7,1.6Hz,1H),3.88(s,3H),3.35(br s,2H),3.05(s,1.5H),2.89(s,1.5H). 3 C NMR (150MHz, DMSO-d6) δ193.1,163.4,138.7,138.2,124.8,111.2,56.6.

[0098] Synthesis of target compound B7-4-2:

[0099]

[0100] The synthesis method of the target compound B7-4-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (130 mg, 0.5 mmol), 1-indanone (97 mg, 0.7 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase to obtain 140 mg of yellow powder, with a yield of 74%. 1H NMR (600MHz, CDCl3) δ7.91 (d, J = 7.6 Hz, 1H), 7.65-7.61 (m, 2H), 7.57-7.55 (m, 2H), 7.43 (d, J = 7. 6Hz,1H),7.31(dd,J=8.0,1.4Hz,1H),7.17(d,J=1.4Hz,1H),4.04(s,2H),3.94(s,3H),3.47(br s,2H),3.10(br s,1.5H),2.98(br s,1.5H). 13 C NMR (150MHz, CDCl3) δ194.2,165.2,160.1,149.5,138.3,138.1,138.0,135.8, 134.8,133.4,127.8,126.3,124.5,122.7,119.1,113.7,56.2,32.4.HRMS(ESI + ):calculated for C 21 H 21 NO3S[M+H] + 368.1315, found 368.1307.

[0101] Synthesis of target compound B7-10-2:

[0102]

[0103] The synthesis method of the target compound B7-10-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (130 mg, 0.6 mmol), 6-methyl-1-indanone ((105 mg, 0.7 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase to obtain 132 mg of yellow powder, with a yield of 68%. 1 HNMR(600MHz, CDCl3)δ7.70(s,1H),7.62-7.61(m,1H),7.55(d,J=7.9Hz,1H),7.43(d,J=0.8H z,2H),7.30(dd,J=8.0,1.3Hz,1H),7.17(d,J=1.2Hz,1H),3.99(s,1H),3.93(s,3H),3.47(br s,2H),3.11(br s,1.5H),2.98(br s,1.5H),2.42(s,3H). 13C NMR (150MHz, CDCl3) δ165.2,160.1,147.0,138.5,138.1,137.8,136.3,136.1,133.2,125.9,124.6,122.7,119.0,113.7,56.3,32.1,21.2.HRMS (ESI + ):calculated for C 22 H 23 NO3S[M+H] + 382.1471, found 382.1467.

[0104] Synthesis of target compound B7-11-2:

[0105]

[0106] The synthesis method of the target compound B7-11-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (130 mg, 0.6 mmol), 6-methoxy-1-indanone (117 mg, 0.7 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase obtains 148 mg of yellow powder with a yield of 73%. 1 HNMR (600MHz, CDCl3) δ7.62-7.61(m,1H),7.55(d,J=7.9Hz,1H),7.44(d,J=8.3Hz,1H),7.34(d,J=2.5Hz,1H),7. 30(dd,J=8.0,1.4Hz,1H),7.20(dd,J=8.3,2.6Hz,1H),3.96(d,J=1.6Hz,2H),3.93(s,3H),3.86(s,3H),3.48(br s,2H),3.11(br s,1.5H),2.98(br s,1.5H). 13 CNMR (150MHz, CDCl3) δ194.2,165.2,160.1,159.7,142.4,139.2,138.4,138.1,13 6.6,133.3,127.0,124.1,122.7,119.1,113.7,105.9,56.3,55.7,31.7.HRMS(ESI + ):calculated for C 22 H 23 NO4S[M+H] + 398.1421, found 3998.1414.

[0107] Synthesis of target compound B7-12-2:

[0108]

[0109] The synthesis method of the target compound B7-12-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (190 mg, 0.7 mmol), 6-hydroxy-1-indanone (114 mg, 0.8 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase obtains 180 mg of yellow powder with a yield of 70%. 1 HNMR(600MHz,DMSO-d6)δ9.87(s,1H),7.51(s,1H),7.49(d,J=8.0Hz,1H),7.43(s,1H),7.37(d,J= 8.0Hz,1H),7.15(dd,J=8.2,2.4Hz,1H),7.09(d,J=2.3Hz,1H),4.03(s,2H),3.88(s,3H),3.45(br s,2H),3.05(br s,1.5H),2.88(br s,1.5H). 13 C NMR(150MHz,DMSO-d6)δ194.2,165.2,160.1,159.7,142.4,139.2,138.4,138.1,1 36.6,133.3,127.0,124.1,122.7,119.1,113.7,105.9,56.3,55.7,31.7.HRMS(ESI + ):calculated for C 21 H 21 NO4S[M+H] + 384.1264, found 384.1254.

[0110] Synthesis of target compound B7-13-2:

[0111]

[0112] The synthesis method of the target compound B7-13-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate D (120 mg, 0.5 mmol), 6-chloro-1-indanone (111 mg, 0.7 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase obtains 136 mg of yellow powder with a yield of 72%. 1HNMR(600MHz, CDCl3)δ7.86(d,J=1.9Hz,1H),7.64-7.63(m,1H),7.58-7.56(m,2H),7.49(d,J=8.1Hz ,1H),7.28(dd,J=8.0,1.3Hz,1H),7.15(d,J=1.2Hz,1H),3.99(d,J=1.3Hz,1H),3.93(s,3H),3.47(br s,2H),3.11(br s,1.5H),2.98(br s,1.5H).HRMS(ESI + ):calculated for C 21 H 20 ClNO3S[M+H] + 402.0925, found 402.0923.

[0113] Synthesis of target compound B7-15-2:

[0114]

[0115] The synthesis method of the target compound B7-15-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (120 mg, 0.5 mmol), 6-tert-butyl-1-indanone (126 mg, 0.7 mmol), the post-treatment method is also the same, after concentration under reduced pressure, column chromatography separation using dichloromethane / ethyl acetate system as the mobile phase to obtain 140 mg of white powder, with a yield of 70%. 1 HNMR(600MHz, CDCl3)δ7.70(dd,J=8.0,1.9Hz,1H),7.64-7.63(m,1H),7.55(d,J=7.9Hz,1H),7.49(d,J=8 .0Hz,1H),7.31(dd,J=7.9,1.4Hz,1H),7.18(d,J=1.4Hz,1H),4.00(d,J=1.5Hz,2H),3.94(s,3H),3.48(br s,2H),3.11(br s,1.5H),2.98(br s,1.5H).HRMS(ESI + ):calculated for C 25 H 29 NO3S[M+H] + 424.1941, found 424.1934.

[0116] Synthesis of target compound B7-19-2:

[0117]

[0118] The synthesis method of the target compound B7-19-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (120 mg, 0.6 mmol), 6-fluoro-1-indanone (108 mg, 0.7 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 138 mg of a yellow powder with a yield of 70%. 1 HNMR(600MHz, CDCl3)δ7.65(d,J=2.0Hz,1H),7.57-7.51(m,3H),7.35-7.32(m,1H),7 .30(dd,J=8.0,1.4Hz,1H),7.16(d,J=1.4Hz,1H),4.00(s,2H),3.94(s,3H),3.47(br s,2H),3.11(br s,1.5H),2.98(br s,1.5H) 13 C NMR (150MHz, CDCl3) δ193.3,165.2,163.5,161.8,160.1,145.0,139.8,139.8,138.2,138.1,136.0 ,134.2,127.7,127.7,122.7,122.5,122.3,119.5,113.8,110.6,110.5,56.3,31.9,0.07.HRMS(ESI + ):calculated forC 21 H 20 FNO3S[M+H] + 386.1221, found 386.1216.

[0119] Synthesis of target compound B7-20-2:

[0120]

[0121] The synthesis method of the target compound B7-20-2 is the same as that of B7-4-1, and its operation method can be referred to. Intermediate I (120 mg, 0.5 mmol), 6-fluoro-1-indanone (98 mg, 0.7 mmol), the post-treatment method is also the same. After concentration under reduced pressure, column chromatography separation is performed using a dichloromethane / ethyl acetate system as the mobile phase to obtain 137 mg of a yellow powder with a yield of 76%. 1HNMR (600MHz, CDCl3) δ7.53(s,1H),7.28(d,J=8.1Hz,1H),7.25(d,J=1.4Hz,1H),7.13(d,J=1.4Hz,1H),7. 09(d,J=2.4Hz,1H),6.92(dd,J=8.0,2.3Hz,1H),3.94(s,3H),3.93(s,3H),3.85(d,J=1.5Hz,2H),3.50(br s,1H),3.47(br s,1H),3.12(br s,1.5H),3.00(brs,1.5H).HRMS(ESI + ):calculated for C 21 H 22 N2O3S[M+H] + 383.1424, found 383.1422.

[0122] Example 3 Time-dependent inhibitory activity of compounds on hCES2A

[0123] 1. Methods and Steps

[0124] hCES2A (final concentration 0.4 μg / ml) and molecular fragment / DMSO (control group) were pre-incubated in PBS (pH 7.4) at 37°C for 3 minutes and 33 minutes respectively; then substrate FD (final concentration 4 μM) was added to the incubation mixture and reacted for 30 minutes. Fluorescence signals (excitation / emission, 480 nm / 525 nm) were continuously recorded by a microplate reader (SpectraMax iD3 Molecular Devices, Austria).

[0125] 2. Results Analysis

[0126] The time-dependent inhibitory activity results of the compounds in Example 1 on hCES2A are shown in Table 1:

[0127] Table 1 Time-dependent inhibitory effect IC of target compounds on hCES2A 50 (micromolar)

[0128]

[0129] As shown in Table 1, the compounds with N-substituted carbamate thioester as electrophilic warheads all showed time-dependent inhibition related to covalent action, and showed stronger inhibition after 33 minutes of incubation. Among them, compounds B7-4-2, B7-13-2, B7-19-1, B7-19-2 and B7-20-2 had more prominent inhibitory effects on hCES2A protein, which was much greater than the non-covalent lead compound B1.

[0130] Example 4 Detection of sites where compounds form covalent interactions with hCES2A and mass spectrometry analysis

[0131] The sites where the compounds in Example 3 form covalent interactions with hCES2A were detected and analyzed by mass spectrometry.

[0132] In order to detect the covalent binding ability of this type of covalent compounds with hCES2A protein and analyze their covalent modification sites, we selected 5 compounds with better activity for nano-liquid chromatography tandem mass spectrometry detection to determine their covalent binding sites with hCES2A protein.

[0133] 1. Methods and Steps

[0134] Purified hCES2A (final content 150 μg) was incubated with compound molecules (final concentration 100 μM) at 37°C for 4 h; the mixture was then treated with urea, DTT and IAA to complete the protein denaturation, reduction and alkylation process. The enriched protein was dissolved in NH4HCO3 solution (pH 8.0, 50 mM) containing 5% ACN and digested with chymosin and trypsin at 37°C for 16 h. After incubation overnight, the digestion was terminated with 15 μl of 10% FA. The resulting peptides were desalted on a MonoSpin C18 column (GL Sciences Inc.), and the eluate was dried and re-dissolved with 20 μl of 0.1% FA for testing.

[0135] 2. Results Analysis

[0136] The results of the detection of the binding sites of the active compounds screened in Example 3 that form covalent interactions with hCES2A are shown in Table 2.

[0137] Table 2 Binding sites of target compounds that form covalent interactions with hCES2A

[0138] Compound hCES2A binding site B7-4-2 Ser138, Ser157, Ser162, Ser228, Ser235, Ser414, Ser429 B7-13-2 Ser104, Ser108, ​​Ser228 B7-19-1 Ser228, Ser233, Ser235 B7-19-2 Ser368 B7-20-2 Ser44, Ser134, Ser228, Ser235, Ser236, Ser240

[0139] As can be seen from Table 2, except for compound B7-19-2, the other compounds can covalently modify the serine residue Ser228 in the catalytic cavity of the hCES2A protein, thereby inhibiting the catalytic activity of the hCES2A protein.

[0140] Example 5 Use of the compound in alleviating delayed diarrhea during oral administration of itilican in the treatment of tumors

[0141] In order to detect whether this type of covalent compound can relieve delayed diarrhea caused by oral administration of itilican and evaluate the efficacy of itilican after co-administration, the compound that can covalently bind to the serine residue Ser228 of the hCES2A protein obtained in Example 4 was tested, and the results showed that it can relieve delayed diarrhea caused by oral administration of itilican.

[0142] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A covalent inhibitor targeting hCES2A, characterized in that: The structural formula is shown in Formula I below: Wherein, R is CH3 or CH2CH3; R' is H, F, Cl, Br, OH, NH2, OCH3, CH3 or C(CH3)3.

2. The covalent inhibitor targeting hCES2A according to claim 1, characterized in that: The double bond portion of the alkenyl group is in the E or Z configuration.

3. The covalent inhibitor targeting hCES2A according to claim 1, characterized in that: The covalent inhibitor targeting hCES2A is shown in any of the following structural formulas:

4. The method for preparing a covalent inhibitor targeting hCES2A according to any one of claims 1 to 3, characterized in that: When R is methyl, the reaction scheme of the covalent inhibitor is as follows: The reaction steps are as follows: vanillin A is coupled with dimethylaminothiocarboxylic acid chloride B under alkaline conditions to obtain an intermediate C, which is then rearranged under high temperature and microwave radiation to obtain an intermediate D, which is then subjected to aldol condensation with a dihydroindanone compound E to obtain the target product F described in formula I when R is CH3.

5. The method for preparing the covalent inhibitor targeting hCES2A according to claim 4, Features: Wherein, when preparing intermediate C, the base is selected from any one of triethylamine, sodium hydroxide, and potassium hydroxide, and the reaction solvent is selected from tetrahydrofuran; the preparation method is as follows: vanillin is dissolved in tetrahydrofuran, 2.5 eq. of triethylamine and 2.5 eq. of dimethylaminoformyl chloride are added at 0°C, reacted at room temperature for 4 hours, and after the reaction, an equal proportion of water is added to terminate the reaction, the mother liquor is extracted with dichloromethane 3 times, the organic phase is extracted once with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and subjected to column chromatography to obtain intermediate C; When preparing intermediate D, intermediate C is dissolved in diphenyl ether, microwave irradiated at 220°C, reacted for 1 to 2 hours, and intermediate D is obtained by column chromatography; When preparing the target product F, the intermediate D and the dihydroindanone compound E with a molar ratio of 8:9 are dissolved in a certain volume of alcohol solution, and a strong alkali aqueous solution with a volume of 60% of the alcohol volume and a mass volume ratio of 20% is slowly added, and the reaction is carried out at room temperature and the reaction progress is detected in real time; after the reaction is completed, an equal proportion of water is added to quench and the pH is adjusted to about 5, and the mixture is extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure, and then separated by column chromatography using a dichloromethane / ethyl acetate system as a mobile phase to obtain the target product F.

6. The method for preparing a covalent inhibitor targeting hCES2A according to any one of claims 1 to 3, characterized in that: When R is ethyl, the reaction scheme of the covalent inhibitor is as follows: The reaction steps are as follows: Compound D is hydrolyzed under alkaline conditions and then coupled with N-methyl-N-ethylcarbamoyl chloride H to obtain intermediate I, which is then subjected to aldol condensation with dihydroindanone compound E to obtain the target product J described in formula I when R is CH2CH3.

7. The method for preparing a covalent inhibitor targeting hCES2A according to claim 6, characterized in that: in, When preparing intermediate G, the base is selected from any one of triethylamine, sodium hydroxide, and potassium hydroxide; the preparation process is as follows: dissolving intermediate D in an alcohol solution, slowly adding a 5.0M alkaline aqueous solution with a volume of 20% of the alcohol solution, reacting in an oil bath at 70°C and detecting the reaction; after the reaction, removing the solvent by rotary evaporation, adding an equal proportion of water to terminate the reaction, adjusting the pH to 3, extracting the mother liquor with ethyl acetate, extracting the organic phase with saturated brine, drying with anhydrous sodium sulfate, filtering, rotary drying, and obtaining intermediate G by column chromatography; The preparation process of intermediate I is as follows: intermediate G is dissolved in tetrahydrofuran, 2.5 eq. of triethylamine and 2.5 eq. of N-ethyl-N-methylcarbamoyl chloride are added at 0°C, reacted at room temperature and the reaction is detected in real time; after the reaction is completed, an equal proportion of water is added to terminate the reaction, the mother liquor is extracted with dichloromethane, the organic phase is extracted with saturated brine, dried with anhydrous sodium sulfate, filtered, spin-dried, and subjected to column chromatography to obtain intermediate I; When preparing the target product J, the intermediate I and the dihydroindanone compound E with a molar ratio of 8:9 are dissolved in a certain volume of alcohol solution, and a strong alkali aqueous solution with a volume of 60% of the alcohol volume and a mass volume ratio of 20% is slowly added, reacted at room temperature and the reaction progress is detected in real time; after the reaction is completed, an equal proportion of water is added to quench and the pH is adjusted to about 5, and the mixture is extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure, and then separated by column chromatography using a dichloromethane / ethyl acetate system as a mobile phase to obtain the target product J.

8. Use of the covalent inhibitor targeting hCES2A according to any one of claims 1 to 3, its isotope-labeled compound or its pharmaceutically acceptable salt in the preparation of an hCES2A inhibitor.

9. A covalent inhibitor targeting hCES2A, characterized in that It comprises an active component and a pharmaceutically acceptable excipient, wherein the active component is the covalent inhibitor targeting hCES2A according to any one of claims 1 to 3, its isotope-labeled compound or a pharmaceutically acceptable salt thereof.

10. The covalent inhibitor targeting hCES2A according to claim 9, characterized in that The inhibitor comes in an oral formulation and is used in combination with irinotecan.

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