Allosteric site covalent inhibitor of glutaminase and preparation method and application thereof

Abstract

The present invention provides a glutaminase allosteric site covalent inhibitor and its preparation method and application. Based on the existing KGA allosteric site inhibitor, a new glutaminase covalent inhibitor containing α, β-unsaturated carboxylic acid ester side chain groups is designed and synthesized as a fragment of a common covalent inhibitor, thereby improving the glutaminase inhibition and anti-tumor activity of the existing KGA allosteric site inhibitor. The covalent inhibitor of the present invention can selectively inhibit the KGA enzyme, and its activity in inhibiting tumor cell growth is more than 3 times higher than that of the lead compound CB839.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a glutaminase allosteric site covalent inhibitor and a preparation method and application thereof. Background Art

[0002] Studies have shown that abnormal glutaminase metabolism often occurs in many glutamine-dependent cancer cells, so glutaminase is a potential target that has been widely studied by researchers. Unfortunately, there are no reports on glutaminase degraders, and glutaminase inhibitors such as DON, BPTES, 968, CB839, etc. are rarely widely used due to their poor solubility, weak selectivity and low bioavailability. The only clinical drug CB839 has also recently failed in clinical trials, so we will use CB839 as a lead compound for further development and optimization.

[0003] Due to their high bonding efficiency and extended duration of action, more and more target-based covalent inhibitors are entering clinical research. α, β-unsaturated carboxylic acid ester side chain groups are common fragments of covalent inhibitors. Therefore, this study synthesized a series of α, β-KGA allosteric site inhibitors to increase biological activity. Summary of the invention

[0004] The purpose of the present invention is to provide a glutaminase allosteric site covalent inhibitor and its preparation method and application. Based on the existing KGA allosteric site inhibitor, a new glutaminase covalent inhibitor containing α, β-unsaturated carboxylic acid ester side chain groups is designed and synthesized as a fragment of a common covalent inhibitor, and to explore ways to improve the glutaminase inhibition and anti-tumor activity of the existing KGA allosteric site inhibitor.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a compound represented by formula I,

[0007]

[0008] Wherein, R1 is phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups;

[0009] R2 is H or phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms or methylenedioxy groups;

[0010] R3 is H, unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups, phenyl, pyridine, indole, or quinoline;

[0011] X is sulfur or selenium.

[0012] Preferably, the compound represented by formula I is one of the following:

[0013]

[0014] In a second aspect, the present invention provides a method for synthesizing the compound of formula I, which comprises dissolving a compound of formula II (containing an -NH2 compound), a compound of formula III (containing a -COOH compound) and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, adding N,N-diisopropylethylamine dropwise, stirring at 60°C after the addition is complete, and after the reaction is complete, post-treating the resulting reaction solution to obtain the compound of formula I; the molar ratio of the compound of formula II, the compound of formula III, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine is 1:1.2:1.6:2.5;

[0015]

[0016]

[0017] Wherein, R1 is phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups;

[0018] R2 is H or phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms or methylenedioxy groups;

[0019] R3 is H, unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups, phenyl, pyridine, indole, or quinoline;

[0020] X is sulfur or selenium.

[0021]

[0022] Preferably, the compound represented by formula I is one of the following:

[0023]

[0024] Furthermore, the volume of the N,N-dimethylformamide is 12 mL / mmol based on the amount of the compound represented by Formula II.

[0025] Further, the post-treatment is as follows: adding saturated saline (1:1 volume ratio) to the reaction solution, filtering and collecting the precipitated solid, extracting the obtained filtrate with ethyl acetate (3 times) to obtain an ethyl acetate organic phase; dissolving the solid with a mixed solution of dichloromethane and methanol in a volume ratio of 50:1 and then combining it with the ethyl acetate organic phase; the obtained mixed solution is subjected to column chromatography using a mixed solvent of dichloromethane and methanol in a volume ratio of 50:1 as an eluent, collecting the eluent containing the target product, and vacuum drying to obtain the compound shown in Formula I. The extraction step usually uses an amount equal to the volume of the filtrate; the mixed solution of dichloromethane and methanol is sufficient to dissolve the solid.

[0026] In specific implementation, the present invention first prepares the corresponding thiadiazole alkynylamine and selenadiazole alkynylamine by using thiosemicarbazide and selenoselenourea, then reacts with aromatic acetic acid, and then couples with iodopyridazinamine to obtain raw material pyridazinamine, and finally reacts with different α, β unsaturated carboxylic acids to obtain a series of compounds.

[0027] In a third aspect, the present invention provides a compound of formula I as a covalent inhibitor of glutaminase in inhibiting glutaminase activity. In particular, the compound of formula I is used in the preparation of drugs for glutaminase-related diseases, such as cytokine-induced hypertension, myeloproliferative diseases, immune diseases, neurological diseases or viral infections. The compound of formula I is one of GJ1-GJ14, and the compound of formula I is particularly preferably compound GJ2 or GJ5.

[0028] In a fourth aspect, the present invention also provides a use of the compound represented by the above formula I in the preparation of anti-tumor drugs.

[0029] Preferably, the tumor is A549 lung cancer cell, H22 liver cancer cell or HCT116 colon cancer cell.

[0030] A novel glutaminase covalent inhibitor, which uses an α, β-unsaturated carboxylate side chain group as a fragment of the covalent inhibitor, is coupled to the 320Lys residue of the KGA enzyme to completely inhibit the KGA enzyme. As shown below. Compound design and synthesis scheme:

[0031]

[0032] Reagents and conditions: (a) POCl3, reflux; (b) EDCI, DMAP, DIPEA, RT; (C) PdCl2(PPh3)2, CuI, TEA, refiux; (D) Raney-Ni, H2; (E) HBTU, DIPEA, 60℃

[0033] First, thiosemicarbazine or selenourea is used as a raw material to condense through step a to synthesize a thiadiazole or selenadiazole compound 2, then through step b, the amino group of compound 2 is condensed with different aromatic acetic acids to synthesize compound 3, and finally through steps c and d, compound 3 and iodopyridazinamine are coupled and hydrogenated to obtain compound 5. These syntheses can be referred to references [3][4]; wherein replacing thiosemicarbazine with selenourea can obtain the corresponding selenium-containing compound.

[0034] Finally, compound 5 reacts with α, β unsaturated carboxylic acid to synthesize a series of compounds 6 (GJ1-GJ14). When step a uses thiosemicarbazide and step b uses pyridineacetic acid, the corresponding pyridine covalent inhibitors GJ1 and GJ5 can be obtained. When step a uses thiosemicarbazide and step b uses pyridineacetic acid, the corresponding pyridine covalent inhibitors GJ2, GJ3, GJ4, and GJ6 can be obtained. When step a uses thiosemicarbazide and step b uses N-methylindoleacetic acid, the corresponding indole covalent inhibitors GJ7 and GJ9 can be obtained. When step a uses thiosemicarbazide and step b uses N-methylindoleacetic acid, the corresponding indole covalent inhibitors GJ8, GJ10, and GJ13 can be obtained. When step a uses thiosemicarbazide and step b uses phenylacetic acid and p-trifluoromethylphenylacetic acid, the corresponding covalent inhibitors GJ11 and GJ12 can be obtained. When step a uses thiosemicarbazide and step b uses quinolineacetic acid, the corresponding covalent inhibitor GJ14 can be obtained.

[0035] Compared with the prior art, the beneficial effect of the present invention is that the covalent inhibitor of the present invention can selectively inhibit KGA enzyme, and its activity in inhibiting tumor cell growth is more than 3 times higher than that of the lead compound CB839. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 are the structural formulas of the 14 covalent inhibitors in Example 2;

[0037] Figure 2 This is a graph showing the inhibitory activity of the covalent inhibitor on KGA and GDH enzymes in Example 3;

[0038] Figure 3 This is the enzyme activity detection diagram of the wild type hKGA and its mutants in Example 4 (OD450 value at 0.5h);

[0039] Figure 4 This is a graph showing the inhibitory effect of the covalent inhibitor on the enzyme activities of Human KGA and K320A in Example 5;

[0040] Figure 5The molecular interaction diagrams of the covalent inhibitors in Example 6 with the wild-type hKGA and mutant hKGA K320A are shown as follows: (A) The difference diagram of the molecular interaction analysis signals of the KGA protein and the CB839 compound chip with and without the inhibitors (GJ and GJ5), PBST-T20, i.e., PBST (TWEEN20), and CPD is GJ2 / GJ5; both can bind firmly with the addition of the covalent inhibitor, with almost no dose effect of the compound (B) The difference diagram of the molecular interaction analysis signals of the KGA K320A protein and the CB839 compound chip with and without the inhibitors (GJ and GJ5); the binding shows a dose effect of the compound, indicating a reversible weak binding;

[0041] Figure 6 This is a graph showing the inhibitory activity of the covalent inhibitor in Example 7 on human non-small cell lung cancer cells A549, mouse liver cancer cells H22, and human colorectal cancer HCT116 cells;

[0042] Figure 7 The drug efficacy evaluation diagram of different compounds in Example 8 on HCT116 cells based on DIP curves (endpoint DIP curves of HCT116 surviving cells after 5 days of drug treatment at different concentrations): (a) CB-839, (b) GJ2, (c) GJ5;

[0043] Figure 8 Figure 2 shows the ROS reactive oxygen species of A549 cells treated with covalent inhibitors for 12 h in Example 9: (A) DMSO control group; (B) H2O2; (CD) CB-839; (EF) GJ2; (GH) GJ5;

[0044] Fig. 9 The effect of covalent inhibitors on A549 cell clone formation in Example 10: (AB) different concentrations of compound GJ2 (CD) different concentrations of compound GJ5 (EF) DMSO control group (G) The number of cell colony formation in the drug-treated group and the non-drug-treated group was compared by unpaired t-test: *, P<0.05; **, P<0.01; ***, P<0.001;

[0045] Fig.10 , 11 , 12 are the hydrogen spectrum, carbon spectrum, and high-resolution mass spectrum of compound GJ5, respectively;

[0046] Fig.13 , 14 , 15 are the hydrogen spectrum, carbon spectrum, and high-resolution mass spectrum of compound GJ2, respectively;

[0047] Fig.16 is the hydrogen spectrum of compound GJ4;

[0048] Fig.17is the hydrogen spectrum of compound GJ6. DETAILED DESCRIPTION

[0049] Example 1. Synthesis of Intermediate 5

[0050] Synthesis of selenosemicarbazide:

[0051] 24.5 g (0.269 mol) of thiosemicarbazide, 150 ml of anhydrous ethanol and 45.8 g (0.322 mmol) of methyl iodide were added to the flask in sequence and refluxed for 2 h to obtain a light yellow clear liquid.

[0052] Into a three-necked flask, add 17.18 g (0.45 mol) of sodium borohydride, replace the air in the reaction bottle with nitrogen, inject 400 ml of anhydrous ethanol under ice bath, and then slowly inject the suspension of selenium powder and anhydrous ethanol, stirring until the solution changes from reddish brown to colorless clear liquid.

[0053] The ethanol solution of S-methylthiosemicarbazide was injected into a three-necked flask while hot, and the reaction solution was stirred overnight to obtain an orange-yellow turbid liquid. The filter cake was slurried with anhydrous ethanol, filtered and dried to obtain 20.7g of gray solid, with a yield of 65%. The synthesis route is shown in Scheme 1-2.

[0054]

[0055] Synthesis route of selenosemicarbazide of formula 1-2

[0056] Synthesis of compound 2-2a

[0057] Place 4-pentynoic acid (7.14g, 10.19mmol) and semicarbazide (10.00g, 10.19mmol) in a flask and add 90ml of phosphorus oxychloride. Under argon protection, reflux at 90℃ for 4 hours. TLC monitors the reaction to be complete, and the reaction solution is poured into 400ml of cold water while stirring to quench the reaction. Slowly add 12M sodium hydroxide solution to the reaction solution under ice bath and adjust the pH to 8-9. Extract with ethyl acetate (150ml×2) while hot. Combine the organic phases, wash with saturated brine (100ml×2), and dry over anhydrous sodium sulfate. Filter, and concentrate the filtrate under reduced pressure to obtain a crude product. Separate by column chromatography with dichloromethane: methanol = 50:1, collect the eluent containing the target compound, and rotary evaporate to obtain 7.12g of pink solid with a yield of 50%. The synthetic route is shown in Formula 1-3.

[0058]

[0059] Reaction equation of compound 2-2a of formula 1-3

[0060] Among them, 2-2b can be obtained by replacing thiosemicarbazide with selenosemicarbazide.

[0061]

[0062] 2-2a Data Characterization: 1 H NMR (500MHz, CDCl3) δ5.26 (d, 2H), 3.15–3.12 (m, 2H), 2.65–2.61 (m, 2H), 2.05 (t, J = 5.3Hz, 1H).ppm.

[0063] 2-2b Data characterization: MS (ESI): m / z calcd for C6H6N3Se: 200.0 [M+H] + ; found: 200.0. Synthesis of compound 2-3a

[0064] 2-Pyridineacetic acid hydrochloride (5.5g, 31.68mmol), carbodiimide hydrochloride (7.36g, 38.33 mmol), and p-dimethylaminopyridine (143mg, 1.17mmol) were placed in a single-mouth bottle, and 60ml of N,N dimethylformamide was added and stirred at room temperature for 15min. Thiadiazole (3.87g, 25.5mmol) was added to the reaction system and reacted at room temperature for 4h. After TLC monitoring of the reaction was complete, the reaction solution was poured into 100ml of water and extracted with ethyl acetate (100ml×2). The organic phases were combined and washed with 1N dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in sequence. The organic phase was dried over anhydrous sodium sulfate. Filtered, the filtrate was evaporated under reduced pressure to obtain a crude product. After column chromatography with dichloromethane: methanol = 50:1, the eluent containing the target compound was collected and evaporated to obtain 4.8g of yellow solid. The yield was 69%. The synthetic route is shown in Formula 1-4.

[0065]

[0066] Reaction equation of compound 2-3a of formula 1-4

[0067] Data Characterization: 1 H NMR (500MHz, CDCl3) δ12.7(s,1H),8.67–8.61(m,1H),7.72–7.69 (m,J=7.7,1.8Hz,1H),7.32(d,J=7.8Hz,1H),4.08(s,2H),3.24(t,J=7.2Hz,2H), 2.66(d,J=2.7Hz,2H),2.04(d,J=5.3Hz,1H).ppm.

[0068] Synthesis of compound 2-4a

[0069] Place 3-amino-6-chloropyridazine (15g, 0.11mol) in a two-necked bottle, add 150ml hydroiodic acid solution (55%), heat to 100°C, and reflux for 12h. After TLC monitoring of the reaction is complete, cool to room temperature, add 10mol / L sodium hydroxide solution dropwise to adjust the pH to 8-10. Filter, wash the filter cake with water, and dry to obtain 20.5g of white solid. The yield is 85%. The synthetic route is shown in Formula 1-5.

[0070]

[0071] Reaction equation of compound 2-4a of formula 1-5

[0072] Synthesis of compound 2-5a

[0073] Place 2-3a (0.85g, 3.12mmol) in a three-necked flask, add 2-4a (0.63g, 2.83mmol), cuprous iodide (0.03g, 0.28mmol), bisphenylphosphine palladium dichloride (0.1g, 0.14mmol), add 10ml of redistilled tetrahydrofuran, drop into triethylamine solution (1.15g, 11.32mmol), and replace with argon. Heat to 70°C, reflux and stir for 8h under argon protection. After TLC monitoring, the reaction solution is concentrated under reduced pressure to obtain a brown solid. After slurrying with methyl tert-butyl ether, a brown crude product is obtained. Purify by column chromatography with dichloromethane: methanol = 50: 1, collect the eluent containing the target compound and rotary evaporate to obtain 0.72g of a yellow solid. The yield is 63%.

[0074] The synthetic route is shown in Formula 1-6.

[0075]

[0076] Reaction equation of compound 2-5a of formula 1-6

[0077] Data Characterization: 1 H NMR (500MHz, DMSO-d6) δ12.71 (s, 1H), 8.52–8.46 (m, 1H), 7.77– 7.75 (m, 1H), 7.39 (d, J = 7.8Hz, 1H), 7.28 (dd, J = 7.5, 4.9Hz, 1H), 7.22 (d, J = 9.1Hz, 1H),6.69(d,J=9.2Hz,1H),6.64(s,2H),4.01(s,2H),3.26(s,2H),2.90(t,J=7.1Hz,2H).ppm.

[0078] Synthesis of Compound 2-6a

[0079] 2-5a (1g, 2.74mmol) was placed in a single-mouth bottle, and 50ml of methanol and 50ml of dichloromethane were added respectively. After stirring until dissolved, 2ml of Raney nickel was added to replace the hydrogen and stirred at room temperature for 24h. After TLC monitoring, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain 0.9g of light yellow solid. The yield was 90%. The synthesis route is shown in Formula 1-7.

[0080]

[0081] Reaction equation of compound 2-6a of formula 1-7

[0082] Data Characterization: 1 H NMR(500MHz,DMSO-d6)δ12.69(s,1H),8.52–8.46(m,1H),7.79– 7.75(m,1H),7.39(d,J=7.8Hz,1H),7.35–7.25(m,2H),6.92(d,J=9.1Hz, 1H), 6.78 (s, 2H), 4.01 (s, 2H), 3.00 (t, J = 14.1Hz, 2H), 2.70 (t, 2H), ppm.

[0083] Wherein pyridine acetic acid is replaced with 2-(1-methyl-1H-indol-2-yl)acetic acid (5.99 g, 31.68 mmol) and Example 1 is repeated to obtain

[0084]

[0085] Data Characterization:

[0086] MS(ESI):m / z calcd for C 21 H 23 N7OS:421.2[M+H] + ; found:421.2.

[0087] Example 1 was repeated by replacing pyridineacetic acid with 2-(1-methyl-1H-indol-2-yl)acetic acid (5.99 g, 31.68 mmol) and replacing 2-2a with 2-2b (5.10 g, 25.5 mmol) to obtain

[0088]

[0089] Data Characterization:

[0090] MS(ESI):m / z calcd for C 21 H 23 N7OSe:469.1[M+H] + ; found:469.1.

[0091] Wherein, pyridineacetic acid was replaced with phenylacetic acid (4.31 g, 31.68 mmol) and Example 1 was repeated to obtain

[0092]

[0093] Data Characterization:

[0094] MS(ESI):m / z calcd for C 18 H 20 N6OS:368.1[M+H] + ; found:368.1.

[0095] Wherein pyridine acetic acid is replaced by phenylacetic acid (4.31 g, 31.68 mmol) and 2-2a is replaced by 2-2b (5.10 g, 25.5 mmol) to obtain

[0096]

[0097] Data Characterization:

[0098] MS(ESI):m / z calcd for C 18 H 20 N6OSe:416.1[M+H] + ; found:416.1.

[0099] Wherein pyridine acetic acid is replaced by trifluoromethoxybenzoic acid (6.97 g, 31.68 mmol) and 2-2a is replaced by 2-2b (5.10 g, 25.5 mmol) to obtain

[0100]

[0101] Data Characterization:

[0102] MS(ESI):m / z calcd for C 19 H 19 F3N6O2Se:500.1[M+H] + ;found:500.1.

[0103] Wherein pyridineacetic acid is replaced by 2-quinolineacetic acid (5.92 g, 31.68 mmol) and 2-2a is replaced by 2-2b (5.10 g, 25.5 mmol) to obtain

[0104]

[0105] Data Characterization:

[0106] MS(ESI):m / z calcd for C 21 H 21 N7OSe:467.1[M+H] + ; found:467.1.

[0107] Example 2. The general method for the synthesis of 10 covalent inhibitors is as follows: The starting material (compound 5; synthesis reference publication [4]) reacts with a series of α, β-unsaturated carboxylic acids to obtain the above compound 6 with a yield of 18%-34%.

[0108] Take GJ2 as an example: N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazole-2-yl)-2-(pyridin-2-yl)acetamide (336 mg, 1.0 eq), 3,4-(methylenedioxy)cinnamic acid (230 mg, 1.2 eq) and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (491 mg, 1.6 eq) were added to a single-mouth bottle, N,N-dimethylformamide (10 mL) was added to dissolve, and N,N-diisopropylethylamine (261 mg, 2.5 eq) was added dropwise to the system. After the addition was complete, the mixture was stirred at 60°C overnight. After the reaction was completed as monitored by TLC, saturated brine was added to the system, and solids precipitated. The solids were collected by filtration, and the filtrate was extracted 3 times with ethyl acetate. The solid was dissolved in a mixture of dichloromethane and methanol in a volume ratio of 50:1 and then combined with the ethyl acetate organic phase. The column chromatography was performed using a mixed solvent of dichloromethane and methanol in a volume ratio of 50:1 as the eluent, and the eluent containing the target product was collected and the solvent was dried under vacuum to obtain 148 mg of a yellow solid with a yield of 31%.

[0109]

[0110] 1H NMR(600MHz,DMSO-d6)δ12.76(s,1H),11.12(s,1H),8.51–8.47(m,1H), 8.37(d,J=9.1Hz,1H),7.77(td,J=7.7,1.9Hz,1H),7.59(dd,J=12.4,3.3Hz,2H) ,7.40(d,J=7.8Hz,1H),7.29(ddd,J=7.6,4.9,1.2Hz,1H),7.16(d,J=8.2Hz,2H), 13C NMR(151MHz,DMSO)δ 165.34,160.06,155.41,154.84,149.59,149.54,148.49,141.88,137.20,129.35,12 8.60,124.65,124.40,122.66,119.88,119.06,109.20,106.90,102.10,44.65,34.83, 32.17,29.65,28.81.HRMS(ESI):m / z calcd forC 27 H 25 N7O4Se:592.1206[M+H] + ; found: 592.1203.

[0111]

[0112] Compound (GJ1) was prepared from acrylic acid (120 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide (369 mg) to obtain 127 mg of a yellow solid. The specific steps were the same as those in Example 1. Yield: 26%

[0113] MS (ESI) = 424.2 [M + H] +

[0114]

[0115] Compound (GJ3) was prepared from acrylic acid (72 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(pyridin-2-yl)acetamide (416 mg) to give a yellow solid (131 mg). The specific steps were the same as those in Example 1. Yield: 30%

[0116] 1H NMR(500MHz, DMSO-d6)δ9.21(s,1H),8.51(d,J=4.9Hz,1H),7.92(d,J=9.1Hz,1H),7.15–7.67(m,1H),7.61–7.55(m,2H ),6.91(d,J=15.6Hz,1H),6.48(s,1H),6.09(s,2H),4.00(s,2H),3.02(d,J=13.4Hz,2H),2.89(d,J=7.1Hz,2H),1.79– 1.72(m,4H).MS(ESI)=472.1[M+H] +

[0117]

[0118] Compound (GJ4) was prepared from 2-(3-(trifluoromethoxy)phenyl)acrylic acid (232 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(pyridin-2-yl)acetamide (416 mg) to give a yellow solid (113 mg). The specific steps were the same as those in Example 1. Yield: 22%

[0119] 1H NMR(600MHz,DMSO-d6)δ12.78(s,1H),8.49–8.48(m,1H),7.78–7.75(m, 1H),7.47(dd,J=32.5,15.7Hz,2H),7.39(d,J=7.8Hz,1H),7.28(dd,J=7.5,4.8Hz,2 H),7.19(d,J=9.0Hz,1H),6.78(d,J=9.1Hz,2H),6.34(s,1H),4.02(s,2H),3.00(m, 2H),2.70(t,J=7.1Hz,2H),1.79–1.72(m,4H).MS(ESI)=632.1[M+H] +

[0120]

[0121] Compound (GJ5) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide (368 mg) to obtain a yellow solid (163 mg). The specific implementation steps were the same as those in Example 1. Yield: 35%

[0122] 1H NMR(600MHz,DMSO-d6)δ12.67(s,1H),11.12(s,1H),8.52–8.48(m,1H), 8.36(d,J=9.2Hz,1H),7.77(td,J=7.6,1.9Hz,1H),7.59(dd,J=12.5,3.3Hz,2H), 7.40(d,J=7.8Hz,1H),7.31–7.27(m,1H),7.16(d,J=9.3Hz,2H),7.00(d,J=7.8Hz, 1H),6.92–6.87(m,1H),6.10(s,2H),4.01(s,2H),3.05–3.01(m,2H),2.93–2.88(m,2H),1.79–1.73(m,4H).13C NMR(151MHz,DMSO)δ168.95,165.34,164.43, 160.03,158.56,155.40,154.84,149.58,149.54,148.49,141.88,137.20,129.34,12 8.60,124.62,124.39,122.66,119.88,119.06,109.20,106.90,102.10,44.56,34.78, 29.08,29.04,28.76.HRMS(ESI):m / zcalcd for C 27 H 25 N7O4S:544.1761[M+H] + ; found: 544.1753.

[0123]

[0124] Compound (GJ6) was prepared from 2,5-difluorocinnamic acid (184 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(pyridin-2-yl)acetamide (416 mg) to give a yellow solid (157 mg). The specific steps were the same as those in Example 1. Yield: 18%

[0125] 1H NMR (600MHz, DMSO-d6) δ11.38(s,2H),8.48(d,J=4.9Hz,2H),8.36(d,J =9.1Hz,2H),7.76(t,J=7.7Hz,1H),7.68–7.54(m,3H),7.40–7.27(m,3H)7. 20(d,J=15.9Hz,1H),3.04(t,2H),2.90(t,J=7.3Hz,2H),1.79–1.72(m,4H). MS(ESI)=584.1[M+H] +

[0126]

[0127] Compound (GJ7) was prepared from acrylic acid (72 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(1-methyl-1H-indol-2-yl)acetamide (421 mg) to give a yellow solid (119 mg). The specific steps were the same as those in Example 1. Yield: 20%

[0128] 1H NMR(500MHz,DMSO-d6)δ12.64(s,1H),7.79–7.66(m,1H),7.61–7.46 (m,2H),7.40(d,J=8.2Hz,1H),7.27(s,54H),7.26–7.18(m,1H),7.15(t,J=8.2Hz, 1H),7.03(t,J=7.5Hz,1H),4.38–4.30(m,1H),3.89(s,1H),3.76(s,2H),3.58(s,1H) ,3.40(s,4H),2.99(td,J=7.2,4.7Hz,1H),2.76–2.62(m,2H),2.57(t,J=7.7Hz,1H), 1.76–1.60(m,3H).MS(ESI)=476.2[M+H]+

[0129]

[0130] Compound (GJ8) was prepared from acrylic acid (72 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(1-methyl-1H-indol-2-yl)acetamide (469 mg) to give a yellow solid (125 mg). The specific implementation steps were the same as those in Example 1. Yield: 18%.

[0131] MS (ESI) = 524.1 [M + H] +

[0132]

[0133] Compound (GJ9) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(1-methyl-1H-indol-2-yl)acetamide (421 mg) to give a yellow solid (119 mg). The specific steps were the same as those in Example 1. Yield: 32%.

[0134] MS (ESI) = 596.2 [M + H] +

[0135]

[0136] Compound (GJ10) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(1-methyl-1H-indol-2-yl)acetamide (469 mg) as raw materials to give a yellow solid (154 mg). The specific implementation steps were the same as those in Example 1. Yield: 32%.

[0137] MS (ESI) = 644.2 [M + H] +

[0138]

[0139] Compound (GJ11) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazole-2-yl)-2-phenylacetamide (416 mg) as raw materials to give a yellow solid (130 mg). The specific implementation steps were the same as those in Example 1.

[0140] MS (ESI) = 591.1 [M+H] +

[0141]

[0142] Compound (GJ12) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(4-(trifluoromethoxy)phenyl)acetamide (500 mg) as raw materials to give a yellow solid (189 mg). The specific implementation steps were the same as those in Example 1.

[0143] MS (ESI) = 675.1 [M+H] +

[0144]

[0145] Compound (GJ13) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(1-methyl-1H-indol-3-yl)acetamide (469 mg) as raw materials to give a yellow solid (150 mg). The specific implementation steps were the same as those in Example 1.

[0146] MS (ESI) = 644.1 [M + H] +

[0147]

[0148] Compound (GJ14) was prepared from 3,4-(methylenedioxy)cinnamic acid (192 mg) and N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)-2-(quinolin-2-yl)acetamide (467 mg) as raw materials to give a yellow solid (186 mg). The specific implementation steps were the same as those in Example 1.

[0149] MS (ESI) = 642.1 [M+H] +

[0150] Example 3. Evaluation of the inhibitory activity of covalent inhibitors at the enzymatic level KGA enzyme activity detection

[0151] Evaluation of the biological activity of the new covalent inhibitors KGA and GDH were tested at the enzymatic level (Table 1 and Figure 2 ) We have published the experimental method [1]. Briefly, the activity of GDH was measured by mixing the compound (0-13 μM) with 10 mM glutamate, 10 nM GDH, 400 μM NADP+ and EZMTT detection reagent for 1 hour, and then measuring the absorbance at 450 nm to calculate the inhibitory effect of GDH activity.

[0152] The activity of KGA was measured by pre-incubating the compound (0-13 μM) with 4 nM human KGA in 50 mM Tris-HCl buffer A for 0.5 h, then adding 10 mM glutamine. After 4 h of reaction, the formation of glutamate was detected for 1 h using 10 nM GDH, 400 μM NADP+ and EZMTT detection reagent. The absorbance at 450 nm was measured to calculate the inhibitory effect of KGA activity.

[0153] Table 1 and Figure 2The results of the in vitro enzyme level test of the compounds on human glutaminase (KGA) and glutamate dehydrogenase (GDH). From the data in the table, it can be seen that this series of compounds has almost no inhibitory effect on glutamate dehydrogenase (GDH), but has a good inhibitory effect on glutaminase (KGA). Among them, compounds GJ2, GJ5, GJ9 and GJ10 showed strong inhibitory activity.

[0154] Example 4. Inducible expression of wild-type human KGA and mutant proteins and detection of enzyme activity

[0155] We have already published the cloned expression of wild-type human KGA [1]. The cloned expression of mutant K320A was achieved by performing site-directed mutagenesis of wild-type human KGA, selecting human KGA 320 site and mutating lysine to alanine (K320A), tryptophan (K320W) and glutamic acid (K320E). First, 6 primers were designed:

[0156] K320A forward primer 5'-CAACGCACTATTTTTGAATGAAGATGATAAACC-3',

[0157] K320A reverse primer 5′- CAAAAATAGTGCGTTGAATCTTAGTCCACTCGGCT-3′;

[0158] K320W forward primer 5'-CAACTGGCTATTTTTGAATGAAGATGATAAACC-3',

[0159] K320W reverse primer 5′- CAAAAATAGCCAGTTGAATCTTAGTCCACTCGGCT-3′;

[0160] K320E forward primer 5'-CAACGAACTATTTTTGAATGAAGATGATAAACC-3',

[0161] K320E reverse primer 5'-CAAAAATAGTTCGTTGAATCTTAGTCCACTCGGCTC-3',

[0162] The protein was synthesized by Jierui Biotechnology. The primers synthesized were used according to the instructions to perform site-directed mutation of wild-type human KGA. After successful DNA sequencing, the protein was transformed into BL21 E. coli for expression. The operation was performed according to the expression and purification method of wild-type human KGA in the open literature [1], including inducing expression with 1mM IPTG, purifying with a nickel column, and finally storing the protein in a -80°C refrigerator. The KGA activity detection method of Example 2 was used for detection. Figure 3 It can be seen that under the same reaction conditions, the activities of the two mutant enzymes K320A and K320G are lower than those of the wild-type hKGA. However, the activity of the mutant enzyme K320A is better than that of K320G. Therefore, the wild-type human KGA and mutant K320A proteins were selected for subsequent experiments. In order to ensure the consistency of the enzyme activity of the protein, we selected the concentration corresponding to the signal values ​​of the two enzymes when they reached the same level for further compound detection.

[0163] Example 5. Inhibition experiment of covalent inhibitors on wild-type human KGA and mutant protease activity

[0164] An excess of 10 μM covalent inhibitor was reacted with 40 nM KGA or 400 nM KGA mutant K320A in 50 mM Tris-Cl buffer for 1 h, and then the mixed solution of covalent inhibitor and enzyme was diluted 3 times in a 96-well plate with 10 mM Gln buffer. After reacting for 4 hours, the KGA activity detection method of Example 2 was used for activity detection. Figure 4 As shown, when an excess of 10 μM of compound GJ2 or GJ5 interacts with wild-type KGA, the KGA activity of the non-drug group decreases with the increase of dilution multiples, but the OD value of the drug group is very small and does not change with the change of compound concentration, indicating that the compound has a strong inhibitory effect on KGA. In the case of mutant protein KGA, when an excess of 10 μM of compound GJ2 or GJ5 interacts with mutant protein KGA, the activity of the mutant protein group in the non-drug group and the drug group decreases with the increase of dilution multiples, indicating that GJ2 or GJ5 has almost no inhibitory activity on the mutant protein. These experiments show that the covalent inhibitors GJ2 or GJ5 inhibit KGA activity by forming a covalent bond with the K320 residue. KGA-K320A does not have a K320 residue site, so it cannot form a covalent bond, so it cannot inhibit the activity of the KGA-K320A mutant protein.

[0165] Example 6. Analysis of the interaction between covalent inhibitors and KGA protein

[0166] To further verify the interaction between the covalent inhibitor and KGA / KGA-K320A protein, we used BLI technology to detect the interaction between proteins. We have already made public the BLI detection method of KGA [2]. A brief description is as follows: After the compound CB-839 is fixed on the upper and lower SA sensors, the upper one is immersed in 40nM wild-type KGA (A3-A6) with the same concentration, and the lower one is immersed in 40nM wild-type KGA (B3-B6) with different concentrations of covalent inhibitor (0-3 μM). By subtracting the A3-A6 signal from the corresponding B3-B6 signal, the effect of the compound on KGA binding to CB839 is observed, thereby determining whether the covalent inhibitor is a competitive inhibitor of CB839. The detection of mutant hKGA K320A uses the same operation method as wild-type KGA.

[0167] like Figure 5 Ba, 5B-c, the results show that the signal value of KGA enzyme binding to small molecule compounds (GJ2 / 5) does not change with the concentration of the compound, which indicates that the KGA protein with a concentration of 40nM is firmly bound to the excess compound GJ2 or GJ5, and its signal value no longer changes with the concentration of the compound after reaching saturation, that is, covalent irreversible binding occurs, and the binding site is the same as CB839. When the protein mutates, when the mutant K320A enzyme binds to compounds of different concentrations, the signal value changes with the concentration, showing a concentration-dependent dose effect. This shows that the binding between the mutant protein K320A and the compound is reversible. This experiment also further shows that the series of compounds designed by us target the KGAK320 site, and there is a phenomenon of covalent irreversible binding. At the same time, when the protein mutates, K320 is lacking, and the activity of the covalent inhibitor is significantly weakened, which further shows that the compound forms a covalent bond with K320, competes with CB839, and the activity is significantly increased than CB839.

[0168] Example 7. Anti-tumor evaluation of covalent inhibitors at the cellular level

[0169] We have already made public the detection method of covalent inhibitors on tumor cells [2]. A brief description is as follows: A549 / H22 cells were seeded in a 96-well plate (1x103 / well). After 4 hours of attachment, different concentrations of compounds (0-10mM, 3-fold dilution) were added and placed in a 37° incubator. After incubation for 5 days, EZMTT detection reagent (final concentration of 1x) was added and the cells were cultured at 37°C for 1 hour. The absorbance of each well sample was measured at 450nm using an enzyme reader to calculate the cell inhibition effect.

[0170] Figure 6The results showed that this series of compounds had an inhibitory effect on the growth of three cell lines: human non-small cell lung cancer cell A549, mouse liver cancer cell H22, and human colorectal cancer cell HCT116. The results showed that compounds GJ5 and GJ6 had an IC of 1.17 and 1.12, respectively, on HCT116 cells. 50 The values ​​were 0.058μM and 0.021μM, respectively, which is more than ten times stronger than the inhibitory activity of compound CB-839. At the same time, GJ5 is also very active at the enzymatic level, which is consistent with the activity of HCT116 cells. It is worth noting that compounds GJ2 and GJ5 have very strong inhibitory activity. Compared with compound CB-839, although compounds GJ2 and GJ5 are weaker in activity against A549 cells, both compounds have strong anti-tumor activity in H22 cells. In addition, compounds GJ9 and GJ10 also have good activity (Table 1).

[0171] The IC50 of GJ2, GJ5, and GJ6 were significantly higher than that of the non-covalent inhibitor CB839.

[0172] Table 1 IC values ​​of covalent inhibitors against KGA, GDH, A549 and H22 50 (μM)

[0173]

[0174] Example 8. Evaluation of drug efficacy of HCT116 cells based on DIP curve (Drug-induced proliferation, growth rate in the presence of drugs; pharmacodynamic index)

[0175] In order to accurately detect the growth of cells after drug action, EZMTT detection reagent was added on the 5th day after drug treatment to track cell growth. The end-point DIP curve within 24 hours was drawn by reading the absorbance OD450, and the DIP rate was calculated. The end-point DIP rate was calculated as: ΔOD450 nm / ΔT (ΔOD450 nm is the absorbance difference of OD450 nm between two time points, and ΔT is the interval between two time points).

[0176] CB839 still has obvious growth at a dose of >1uM, while GJ2 and GJ5 can basically inhibit cell growth under the corresponding concentration conditions

[0177] Table 2. Comparison of inhibitors' inhibitory effects on cell growth

[0178]

[0179] DIP rate a :OD450 nm / hr;

[0180] CB-839 cannot completely inhibit cell growth. At a concentration of 1-10 μM, the growth rate is greater than 10%, indicating drug resistance. However, for GJ2 and GJ5, the inhibition rate is greater than 90% under the condition that the compound concentration is greater than 3 μM, indicating that the cells are sensitive to both compounds. Therefore, covalent inhibitors significantly increase the sensitivity of tumor cells to compounds.

[0181] Example 9. Effects of covalent inhibitors on ROS and cell clone colony assays in A549 cells

[0182] We have also published the ROS experimental method [3]. A brief description is as follows:

[0183] 7500 A549 cells / well were added with or without compounds after 4 hours of attachment. After a certain period of incubation, 100 μL DCFH-DA working solution was added to each well and the cells were incubated in a 37°C constant temperature cell culture incubator for 30 min to load the probe. The cells were washed 1-3 times with serum-free RPMI 1640 medium and photographed under a fluorescence microscope to observe the fluorescence intensity.

[0184] like Figure 8 As shown, compound GJ2 generates more ROS than CB839 at a concentration of 10 μM. It is further speculated that the inhibitory effect of this series of compounds on tumor cells is related to the increase in ROS levels.

[0185] At the same compound dose, the covalent inhibitors GJ and GJ5 generated more ROS than the non-covalent inhibitor CB839.

[0186] Example 10. Cell colony cloning experiment

[0187] It was completed according to the experimental steps of the crystal violet staining solution of Biyuntian Biotechnology Co., Ltd. A brief description is as follows: A549 cells in the exponential growth phase were digested and seeded in a six-well plate at a density of 500 cells / well, and placed in a 37° incubator overnight. Fresh culture medium was replaced, and compounds with or without different concentrations were added and incubated in a 5% CO2 constant temperature incubator for 10 days. When the clones visible to the naked eye grew, the culture was terminated, washed with PBS, and 1 ml of paraformaldehyde was added to fix for 15 minutes, followed by adding an appropriate amount of crystal violet staining for 10 minutes and then placed in the air to dry. The colonies with >50 cells were determined and counted for statistical analysis.

[0188] like Fig. 9As shown in the figure, compared with the blank group, the drug-added group significantly inhibited the proliferation of cells. At the same time, the inhibitory ability of the proliferating clones was concentration-dependent with the drug. The higher the drug concentration, the fewer the cell clones. Compound GJ2 almost completely inhibited cell proliferation at a concentration of 5 μM. Overall, it shows that both compounds effectively inhibited the proliferation of tumor cells.

[0189] Fig. 9 Effects of compound treatment on A549 cell clone formation. (AB) Compound GJ2 at different concentrations (CD) Compound GJ5 at different concentrations (EF) DMSO control group (G) Unpaired t-test was used to compare the number of cell colonies formed between the drug-treated and non-drug-treated groups: *, P<0.05; **, P<0.01; ***, P<0.001.

[0190] References

[0191] [1] Zhu M, Fang J, Zhang J, et al. Biomolecular Interaction AssaysIdentified Dual Inhibitors of Glutaminase and Glutamate Dehydrogenase ThatDisrupt Mitochondrial Func tion and Prevent Growth of Cancer Cells[J]. Anal. Chem., 2017, 89(3):1689-1696.

[0192] [2]Hou W, Fang J, Su L, et al. Design and synthesis of biotinylatedHexylselen as a pro be to identify KGA allosteric inhibitors by a convenient biomolecular interaction assay [J]. Bioorg. Med. Chem. Lett., 2019, 29(17): 2498-2502.

[0193] [3]Chen Z,Li D,Xu N,et al.Novel 1,3,4-Selenadiazole-ContainingKidney-Type Glutaminase Inhibitors Showed Improved Cellular Uptake and Antitumor Activity[J].J.Med.Chem.,2019,62(2):589-603.

[0194] [4]Xu X,Wang J,Wang M,et al.Structure-Enabled Discovery of NovelMacrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site.J MedChem.2021 Apr 22;64(8):4588-4611.

Claims

1. A compound of formula I, in, R1 is phenyl, pyridine, indole or quinoline, which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms or methylenedioxy groups; R2 is H or phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms or methylenedioxy groups; R3 is H, unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups, phenyl, pyridine, indole, or quinoline; X is sulfur or selenium.

2. Compounds represented by the following formulas GJ1-GJ14, 3. The method for preparing the compound of formula (I) as claimed in claim 1, characterized in that The method comprises: dissolving a compound represented by formula II, a compound represented by formula III and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, dropping N,N-diisopropylethylamine, stirring at 60° C. after the dropping is complete, and after the reaction is complete, post-treating the obtained reaction solution to obtain a compound represented by formula I; the molar ratio of the compound represented by formula II, the compound represented by formula III, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine is 1:1.2:1.6:2.5; Wherein, R1 is phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups; R2 is H or phenyl, pyridine, indole or quinoline which is unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms or methylenedioxy groups; R3 is H, unsubstituted or substituted by one or more of trifluoromethoxy, methoxy, halogen atoms, and methylenedioxy groups, phenyl, pyridine, indole, or quinoline; X is sulfur or selenium.

4. The method for preparing the compound of formula (I) as claimed in claim 3, characterized in that: The volume of the N,N-dimethylformamide is 12 mL / mmol based on the amount of the compound represented by Formula II.

5. The method for preparing the compound of formula I as claimed in claim 3, characterized in that The post-treatment is as follows: adding saturated brine to the reaction solution, filtering and collecting the precipitated solid, extracting the obtained filtrate with ethyl acetate to obtain an ethyl acetate organic phase; dissolving the solid with a mixed solution of dichloromethane and methanol in a volume ratio of 50:1 and then combining it with the ethyl acetate organic phase; the obtained mixed solution is subjected to column chromatography using a mixed solvent of dichloromethane and methanol in a volume ratio of 50:1 as an eluent, collecting the eluent containing the target product, and vacuum drying to obtain the compound shown in Formula I.

6. Use of the compound of formula I as claimed in claim 1 as a covalent inhibitor of glutaminase in the preparation of an agent for inhibiting glutaminase activity.

7. Use of the compound of formula I as claimed in claim 1 in the preparation of anti-tumor drugs, characterized in that The tumor is A549 lung cancer cell, H22 liver cancer cell or HCT116 colon cancer cell.

8. Use of the compound represented by formula GJ1-GJ14 as claimed in claim 2 as a covalent inhibitor of glutaminase in the preparation of an agent for inhibiting glutaminase activity, The compound represented by formula GJ1-GJ14 is one of the following:

9. The use according to claim 8, characterized in that: The compound represented by formula GJ1-GJ14 is compound GJ2 or GJ5.

10. Use of the compound represented by formula GJ1-GJ14 according to claim 8 in the preparation of anti-tumor drugs, characterized in that The tumor is A549 lung cancer cell, H22 liver cancer cell or HCT116 colon cancer cell.

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