CN114716427B - Compound serving as RIP inhibitor and preparation method and application thereof - Google Patents

Compound serving as RIP inhibitor and preparation method and application thereof Download PDF

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CN114716427B
CN114716427B CN202210015083.6A CN202210015083A CN114716427B CN 114716427 B CN114716427 B CN 114716427B CN 202210015083 A CN202210015083 A CN 202210015083A CN 114716427 B CN114716427 B CN 114716427B
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CN114716427A (en
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广兵
阳泰
董韧涵
谢建
彭坚
黄胜
刘进
覃传军
占伟
赖永新
彭向阳
许庆
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Chengdu Benuo Kecheng Biotechnology Co ltd
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Abstract

The invention provides a compound serving as a RIP inhibitor, a preparation method and application thereof, and belongs to the field of chemical medicines. The compound is a compound shown in a formula (I), or a salt, a stereoisomer, a solvate or a prodrug thereof. The compound has good inhibition activity on human RIP1 kinase, has better effect than the compound in the prior art, and can be used for preparing RIP1 inhibitors; meanwhile, the inhibition effect of the compound of the invention on TNF induced pancreatic cancer cells, colon cancer cells, lymph cancer cells and immortalized keratinocyte programmed necrosis is obviously better than that of the existing compound, and the compound of the invention can effectively inhibit the programmed necrosis of cells. Furthermore, the compound can be used for preparing medicines for treating diseases related to RIP1 and cell programmed necrosis, such as various inflammations, psoriasis, tumors and the like, and has wide application prospect.

Description

Compound serving as RIP inhibitor and preparation method and application thereof
Technical Field
The invention belongs to the field of chemical medicine, and in particular relates to a compound serving as an RIP inhibitor, and a preparation method and application thereof.
Background
Programmed necrosis (programmed necrosis), also known as necroptosis (necrosis), is a novel and controllable means of cell death with necrotic morphological features. Programmed necrosis plays a key role in embryonic development and dynamic balance of the adult organism, and is widely involved in pathophysiological processes of various diseases. Receptor interacting protein 1 (RIP 1) is located at a key position in the programmed necrosis pathway, and is a kinase which plays an upstream regulatory role as an upstream regulatory molecule of the signaling pathway, and abnormal activation of the kinase can cause a series of reactions, and RIP1 has become a central controller for determining cell fate in the death receptor signaling pathway.
Numerous studies have shown that cell death and inflammatory responses mediated by activation of RIP1 and the programmed necrotic pathway are involved in a variety of human diseases including stroke, myocardial infarction, retinal damage, lethal systemic inflammatory response syndrome and chronic enteritis, as well as malignant tumors, and the like. RIP1 kinase acts as a potential target in the programmed necrosis pathway, and inhibition of its kinase activity can inhibit the progression of the disease. The RIP1 inhibitor has important application value for treating diseases such as inflammation, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, tumors and the like. RIP inhibitors, and in particular RIP1 inhibitors, are therefore of great interest to pharmaceutical chemistry researchers.
Philip A.Harris et al report a class of RIP inhibitors with the following representative structure and found their application prospects in the treatment of inflammation or tumors. However, the therapeutic effect is still to be further improved, and RIP inhibitors with better therapeutic effects are still in urgent clinical demand.
Disclosure of Invention
The invention aims to provide a compound serving as a RIP inhibitor, and a preparation method and application thereof.
The present invention provides a compound represented by formula (I), or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof:
wherein,
R 1 is selected from hydrogen, methyl, deuterated methyl;
R 2、R3 is independently selected from hydrogen and halogen;
x is selected from O, CH 2;
R 4 is selected from S configuration-CH 3 or R configuration-CH 3;
The A ring is selected from five-membered heterocyclic ring.
Further, the method comprises the steps of,
Ring A is selected from
Further, the compound is represented by formula (II):
wherein,
R 1 is selected from hydrogen, methyl, deuterated methyl;
R 2、R3 is independently selected from hydrogen and halogen;
R 4 is selected from S configuration-CH 3 or R configuration-CH 3;
The A ring is selected from five-membered heterocycle;
preferably, the method comprises the steps of,
Ring A is selected from
Further, the compound is represented by formula (III):
wherein,
R 1 is selected from hydrogen, methyl, deuterated methyl;
R 2、R3 is independently selected from hydrogen and halogen;
The A ring is selected from five-membered heterocycle;
preferably, the method comprises the steps of,
Ring A is selected from
Further, the compound is represented by formula (IV):
wherein,
R 1 is selected from hydrogen, methyl, deuterated methyl;
The A ring is selected from five-membered heterocycle;
preferably, the method comprises the steps of,
Ring A is selected from
Further, the salt is mesylate, hydrochloride, sulfate or organic acid salt.
Further, the compound is one of the following compounds:
The invention also provides the use of the aforementioned compounds, or salts thereof, or stereoisomers thereof, or solvates thereof, or prodrugs thereof, in the preparation of a RIP inhibitor;
Preferably, the RIP inhibitor is a RIP1 inhibitor;
more preferably, the RIP inhibitor is a drug that inhibits apoptosis.
The invention also provides application of the compound, or salt, stereoisomer, solvate or prodrug thereof in preparing medicines for treating diseases related to cell programmed necrosis;
Preferably, the medicament is an anti-inflammatory, a tumor preventing and/or treating, a psoriasis preventing and/or treating medicament;
More preferably, the medicament is a medicament for preventing and/or treating pancreatic cancer, colon cancer, lung cancer, liver cancer, breast cancer, lymph cancer;
and/or the medicament is a medicament for preventing and/or treating rheumatoid arthritis and ulcerative colitis.
The invention also provides a medicine which is prepared by taking the compound, or salt, or stereoisomer, solvate or prodrug thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
In the present invention, "deuterated" refers to a compound or group in which one or more hydrogens are replaced with deuterium (D). For example, deuterated methyl is-CH 2D、-CHD2 or-CD 3.
In the present invention, halogen is fluorine, chlorine, bromine or iodine.
In the present invention, the room temperature was 25.+ -. 5 ℃ and the overnight period was 12.+ -. 2h.
The compound has good inhibition activity on human RIP1 kinase, has better effect than the compound in the prior art, and can be used for preparing RIP1 inhibitors; meanwhile, the inhibition effect of the compound of the invention on TNF induced pancreatic cancer cells, colon cancer cells, lymph cancer cells and immortalized keratinocyte programmed necrosis is obviously better than that of the existing compound, and the compound of the invention can effectively inhibit the programmed necrosis of cells. Furthermore, the compound can be used for preparing medicines for treating diseases related to RIP1 and cell programmed necrosis, such as various inflammations, psoriasis, tumors and the like, and has wide application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Synthesis of A-ring intermediates
1. Synthesis of intermediate A-1
Step 1:20 ml of ethanol is cooled to the temperature of minus 5 ℃, 10 g of acetyl chloride (0.13 mol) is added dropwise, and the ethanol is reacted for half an hour at the low temperature (0-5 ℃) after the completion of the dripping, thus obtaining freshly prepared ethanol hydrochloride gas. A mixed solution of 12.5 g of ethyl cyanoformate (0.13 mol) and 30 ml of dichloromethane is dropwise added at a low temperature (0-5 ℃) and stirred at a temperature of 0-5 ℃ for reaction overnight, solid generated by filtration reaction is imine intermediate hydrochloride, the hydrochloride is suspended in 50ml of methyl tertiary butyl ether, triethylamine (20 g) is added, stirring is carried out at room temperature for 5 hours, triethylamine hydrochloride is removed by filtration, and the methyl tertiary butyl ether is concentrated under reduced pressure to obtain 12.3 g of intermediate AM1-1.
Step 2: 9.6 g of intermediate AM1-1 (66.1 mmol) prepared in step 1 was dissolved in a mixed solution of 40 ml of ethanol and 120 ml of methyl tert-butyl ether, 10g of phenylacetyl hydrazine (66.6 mmol) was added, stirred overnight at room temperature, filtered, and the solid was washed twice with ethanol and dried to give 8.7 g of intermediate AM1-2 for the next reaction.
Step 3: 5 g of intermediate AM1-2 (20.0 mmol) was suspended in 100 ml of xylene and reacted at 170℃for 24 hours under reflux, cooled to room temperature and filtered to give 4.3 g of intermediate AM1-3.
Step 4: 4g of intermediate AM1-3 (17.3 mmol) was suspended in 40 ml of water, 1.2 g of lithium hydroxide (50.1 mmol) was added, stirred at room temperature for 3 hours, pH was adjusted to 2 with 2M dilute hydrochloric acid, solids were precipitated, filtered, the solids were washed twice with water, and dried to give 3.3 g of intermediate A-1.
2. Synthesis of intermediate A-2
Step 1: 5g of commercially available methyl 1,2, 4-triazole-3-carboxylate (39.3 mmol) were dissolved in 100 ml of acetone, 8.1 g of potassium carbonate (58.6 mmol), 6.7 g of benzyl bromide (39.1 mmol) were added, stirred overnight at room temperature, and TLC detected complete reaction of the starting material. 100 ml of water was added, extraction was performed 3 times with 150 ml of ethyl acetate, and the organic layers were combined, washed with saturated brine 1 time, and dried over 50 g of anhydrous sodium sulfate. Sodium sulfate was removed by filtration, concentrated to dryness under reduced pressure, and 50 ml of petroleum ether was added for crystallization to give 6.6 g of wet product. 6.6 g of the wet product were recrystallized from 30 ml of petroleum ether mixed solution of ethyl acetate (EA: pe=1:1), filtered and dried to give 4.1 g of intermediate AM2-1 in total.
Step 2: 2 g of intermediate AM2-1 (9.2 mmol) is taken and dissolved in 20 ml of methanol, 0.7 g of lithium hydroxide (29.2 mmol) is dissolved in 20 ml of water and added into a methanol system, the mixture is stirred for 5 hours at room temperature, TLC detection raw materials are completely reacted, pH value is regulated to 2-3 by 2M dilute hydrochloric acid, solid is separated out, the mixture is filtered, the solid is washed to be neutral by water, 1.7 g of intermediate A-2 is obtained by drying,
3. Synthesis of intermediate A-10
1-H-imidazole-4-methyl formate is taken as a raw material, and the synthesis method is similar to that of the intermediate A-2, so as to obtain an intermediate A-10.
The other intermediates A-3, A-4, A-5, A-6, A-7, A-8 and A-9 are all commercially available and have the following structures:
EXAMPLE 1 Synthesis of Compound I-1
The synthetic route is as follows:
i：NaH/DMF;ii：H2,Pd/C,MeOH;iii：TBTU,DIPEA,DMSO;iv：MeI,Cs2CO3,DMF;v：HCl,MTBE;vi：TBTU,DIPEA,DCM
the specific operation is as follows:
1. Synthesis of intermediate M1-1:
5g of BOC-L-threonine (cas: 2592-18-9,22.8 mmol) was dissolved in 50 ml of DMF, the ice-salt bath was cooled to 0℃and 1.4 g of NaH (57.0 mmol) was suspended in 10ml of DMF and dropwise added, the ice-salt bath was removed, stirring at room temperature was carried out until the bubbles disappeared, 3.2 g of 1-fluoro-2-nitrobenzene (22.7 mmol) was added dropwise, stirring at room temperature was carried out for 4 hours, and the reaction was quenched by adding ice water. 2M dilute hydrochloric acid is added into the reaction system until the pH value is 2-3, the mixture is extracted for 3 times by 50 ml of ethyl acetate respectively, the organic layers are combined, the organic layers are dried by washing, then concentrated under reduced pressure, and the residue is purified by column chromatography to obtain 6.1 g of yellow semi-solid, namely an intermediate M1-1 (molecular weight 340.13).
2. Synthesis of intermediate M2-1:
dissolving the intermediate M1-1 with 60 ml of methanol, carrying out catalytic hydrogenation reaction at room temperature by using palladium-carbon as a catalyst, filtering to remove the palladium-carbon after the reaction is completed, concentrating the filtrate under reduced pressure, crystallizing the residue by stirring with methyl tertiary butyl ether, dissolving the solid obtained by drying in 25 ml of DMSO, adding 5.7 g of TBTU (17.9 mmol) and 2.3 g of DIPEA (17.9 mmol), stirring overnight at room temperature, adding 25 ml of ice water for quenching, extracting 3 times by using 30ml of ethyl acetate respectively, merging the organic layers, washing the organic layers with water, drying under reduced pressure, crystallizing the residue by petroleum ether, filtering and drying to obtain 4.7 g of white solid, namely M2-1 intermediate (molecular weight 292.14).
3. Synthesis of intermediate M3-1:
2.0 g of intermediate M2-1 (6.8 mmol) above was dissolved in 50 ml of DMF, 3.3 g of cesium carbonate (10.2 mmol) and 1.4 g of methyl iodide (10.2 mmol) were added and stirred at room temperature until the reaction was complete. After quenching with 50 ml of ice water, extraction is performed three times with 80 ml of ethyl acetate respectively, the organic layers are combined, washed with water, dried, concentrated under reduced pressure, crystallized with petroleum ether, and filtered to obtain a solid. The above solid was added with 20 ml of methyl tert-butyl ether saturated with hydrochloric acid gas, stirred at room temperature for 6 hours, and then the solvent was concentrated under reduced pressure, and the residue was crystallized from petroleum ether to give 1.7 g of pale yellow solid, namely intermediate M3-1 (molecular weight 242.70).
4. Synthesis of target I-1:
1.7 g of intermediate M3-1 (7.0 mmol), 4.5 g of TBTU (14.0 mmol) and 1.36 g of DIPEA (10.5 mmol) were dissolved in 20 ml of DCM, 1.7 g of A-1 ring (5-benzyl-4H-1, 2, 4-triazole-3-carboxylic acid, 8.4 mmol) was added and stirred overnight at room temperature, the reaction was quenched with ice water, the organic layer was washed with water, dried and concentrated under reduced pressure, and the residue was separated by column chromatography to give 1.5 g of compound I-1.
1HNMR(400MHz,DMSO-d6)δ：14.41(s,1H),7.90(s,1H),7.52-7.43(m,1H),7.38-7.21((m,8H),4.90-4.79(m,2H),4.16(s,2H),3.36(s,3H),1.28(d,J＝5.1Hz,3H).
ESI-MS m/z:390.11[M-1]-
Example 2: synthesis of Compound I-2:
The synthetic route is as follows:
iv：CD3I,Cs2CO3，DMF；v：HCl，MTBE；vi：TBTU，DIPEA，DCM
the specific operation is as follows:
1. Synthesis of M3-2
The synthetic method is similar to the preparation method of the intermediate M3-1 in the embodiment 1 by taking M2-1 as a raw material, and deuterated iodomethane is used for replacing iodomethane to obtain the intermediate M3-2.
2. Synthesis of I-2
The procedure for the preparation of I-1 of example 1 was repeated to give the target I-2.
ESI-MS m/z:393.13[M-1]-
Example 3: synthesis of Compound I-3:
The synthetic route is as follows:
v：HCl，MTBE；vi：TBTU，DIPEA，DCM
the specific operation is as follows:
M2-1 was used as a raw material, methyl tert-butyl ether saturated with hydrochloric acid gas was added, after stirring at room temperature for 6 hours, the solvent was concentrated under reduced pressure, and the residue was crystallized from petroleum ether to give intermediate M3-3 (molecular weight 228.68).
Using the above intermediate M3-3 and intermediate A-1 as raw materials, the synthesis process was similar to step 4 of example 1, to give Compound I-3.
ESI-MS m/z:376.15[M-1]-
Example 4: synthesis of Compound I-4:
The synthetic route is as follows:
i：NaH/DMF;ii：H2,Pd/C,MeOH;iii：TBTU,DIPEA,DMSO;iv：MeI,Cs2CO3,DMF;v：HCl,MTBE;vi：TBTU,DIPEA,DCM
the specific operation is as follows:
The synthesis method is similar to example 1, using BOC-D-threonine as a raw material, to give compound I-4.
1HNMR(400MHz,CDCl3)δ：8.22(d,J＝8.9Hz,1H),7.33-7.19((m,7H),7.17-7.07(m,1H),4.84-4.74(m,1H),4.70-4.58(m,1H),4.26-4.13(m,2H),2.83(s,3H),1.38(d,J＝6.2Hz,3H).
ESI-MS m/z:390.08[M-1]-
Example 5: synthesis of Compound I-5:
The synthetic route is as follows:
iv：CD3I,Cs2CO3，DMF；v：HCl，MTBE；vi：TBTU，DIPEA，DCM
the specific operation is as follows:
The synthesis method is similar to example 2, starting with intermediate M4-2, to give compound I-5.
ESI-MS m/z:393.12[M-1]-
Example 6: synthesis of Compound I-6:
a method similar to the method of the compound I-1 adopts the A-2 ring as a raw material to prepare the compound I-6.
1H NMR(400MHz,DMSO-d6)δ：8.85(s,1H),7.95(d,J＝5.8Hz,1H),7.50-7.43(m,1H),7.42-7.23(m,8H),5.55-5.40(m,2H),4.89-4.77(m,2H),3.35(s,3H),1.30-1.21(m,3H).
ESI-MS m/z:390.08[M-1]-
Example 7: synthesis of Compound I-7:
the method is similar to the method of the compound I-2, adopts the A-2 ring as a raw material and adopts deuterated iodomethane as a raw material to prepare the compound I-7.
1H NMR(400MHz,DMSO-d6)δ:8.8(s,1H),8.00-7.91(m,1H),7.50-7.43(m,1H),7.43-7.22(m,8H),5.52-5.46(m,2H),4.89-4.80(m,2H),1.33-1.23(m,3H).
ESI-MS m/z:393.12[M-1]-
Example 8: synthesis of Compound I-8:
A method similar to the method of the compound I-3 adopts the A-2 ring as a raw material to prepare the compound I-8.
ESI-MS m/z:376.15[M-1]-
Example 9: synthesis of Compound I-9:
a method similar to the method of the compound I-1 adopts the A-3 ring as a raw material to prepare the compound I-9.
ESI-MS m/z:390.07[M-1]-
Example 10: synthesis of Compound I-10:
A method similar to the method of the compound I-1 adopts the A-4 ring as a raw material to prepare the compound I-10.
ESI-MS m/z:390.07[M-1]-
Example 11: synthesis of Compound I-11:
A method similar to the method of the compound I-1 adopts the A-5 ring as a raw material to prepare the compound I-11.
ESI-MS m/z:389.03[M-1]-
Example 12: synthesis of Compound I-12:
a method similar to the method of the compound I-1 adopts the A-6 ring as a raw material to prepare the compound I-12.
ESI-MS m/z:391.09[M-1]-
Example 13: synthesis of Compound I-13:
a method similar to the method of the compound I-1 adopts the A-7 ring as a raw material to prepare the compound I-13.
ESI-MS m/z:391.09[M-1]-
Example 14: synthesis of Compound I-14:
A method similar to the method of the compound I-1 adopts an A-8 ring as a raw material to prepare the compound I-14.
ESI-MS m/z:390.08[M-1]-
Example 15: synthesis of Compound I-15:
A method similar to the method of the compound I-1 adopts the A-9 ring as a raw material to prepare the compound I-15.
ESI-MS m/z:391.12[M-1]-
Example 16: synthesis of Compound I-16:
a method similar to the method of the compound I-1 adopts the A-10 ring as a raw material to prepare the compound I-16.
ESI-MS m/z:389.03[M-1]-
Example 17: synthesis of Compound I-17:
the method similar to the compound I-2 adopts A-10 ring and deuterated iodomethane as raw materials to prepare the compound I-17.
ESI-MS m/z:392.15[M-1]-
Example 18: synthesis of Compound I-18:
A method similar to the method of the compound I-3 adopts an A-10 ring to prepare the compound I-18.
ESI-MS m/z:375.11[M-1]-
Example 19: synthesis of Compound I-19:
the method is similar to the method of the compound I-1, and the compound I-19 is prepared by taking 2, 4-difluoronitrobenzene as a raw material.
ESI-MS m/z:408.12[M-1]-
Example 20: synthesis of Compound I-20:
The method is similar to the method of the compound I-3, and the compound I-20 is prepared by adopting 2, 4-difluoronitrobenzene as a raw material.
ESI-MS m/z:394.06[M-1]-
Example 21: synthesis of Compound I-21:
The method is similar to the method of the compound I-1, and the compound I-21 is prepared by taking 2, 6-difluoronitrobenzene as a raw material.
ESI-MS m/z:408.12[M-1]-
Example 22: synthesis of Compound I-22:
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the method is similar to the method of the compound I-3, and the compound I-22 is prepared by adopting 2, 6-difluoronitrobenzene as a raw material.
ESI-MS m/z:394.06[M-1]-
Example 23: synthesis of Compound I-23:
The method is similar to the method of the compound I-2, and the compound I-23 is prepared by taking 2, 6-difluoronitrobenzene as a raw material.
ESI-MS m/z:411.13[M-1]-
Example 24: synthesis of Compound I-24:
The method is similar to the method of the compound I-1, and 2,4, 6-trifluoro nitrobenzene is used as a raw material to prepare the compound I-24.
ESI-MS m/z:412.09[M-1]-
Example 25: synthesis of Compound I-25:
the method similar to the compound I-8 adopts 2,4, 6-trifluoro nitrobenzene as a raw material to prepare the compound I-25.
ESI-MS m/z:412.09[M-1]-
Example 26: synthesis of Compound I-26:
The method similar to the compound I-8 adopts 2, 6-difluoronitrobenzene as a raw material to prepare the compound I-26.
ESI-MS m/z:394.06[M-1]-
Example 27: synthesis of Compound I-27:
The method similar to the method of the compound I-11 adopts 2,4, 6-trifluoro nitrobenzene as a raw material to prepare the compound I-27.
ESI-MS m/z:411.09[M-1]-
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 inhibition Activity of Compounds of the invention against RIP1 kinase
The compounds of the invention and control compounds were dissolved in assay buffer (50mM Hepes pH 7.5, 50mM NaCl,30mM MgCl 2, 1mM DTT,0.02%CHAPS,0.5mg/mL BSA) and assayed in 22 spot assay at 1:1.5 (high concentration 2. Mu.M) and added to 384 well plates. 3.5. Mu.L of each concentration of inhibitor and 3.5. Mu.L of human RIP enzyme (25 nM) dissolved in assay buffer were added to the plate. After 1 hour of pre-incubation at 37 degrees celsius, 3.5 μl of ATP (15 μm to 1.5 mM) buffer was added to the plate to initiate the reaction. The reaction was carried out at room temperature for 5 hours. After completion of the reaction, 5 μl of ADP-Glo reagent containing 0.02% chaps was added to each well and incubated for 1 hour at room temperature to terminate the kinase reaction and deplete all remaining ATP. mu.L of ADP-Glo detection solution containing 0.02% CHAPS was then added to each well and incubated at room temperature for 30 minutes, and the luminosity was measured. For each ATP concentration, the luminescence data was subtracted from the control and expressed as activity data.
IC 50 at each ATP concentration was determined by calculation and the results are shown in table 1 below:
TABLE 1 inhibitory Activity of the inventive Compounds against human RIP1 kinase IC 50
Compounds of formula (I) IC50(nM)
I-1 10.19
I-2 8.68
I-4 8.97
I-6 7.36
I-7 8.18
I-16 14.40
Control Compound 1 20.35
Control Compound 2 18.29
Control Compound 3 22.64
The test results show that: the IC 50 value of the compound of the invention on the inhibition of human RIP1 kinase is obviously lower than that of the existing compound, which indicates that the compound of the invention can be used for preparing RIP inhibitor, and the effect is better than that of the existing compound.
Test example 2 inhibition of TNF-alpha induced U937 apoptosis by the inventive Compounds
Human histiocyte lymphoma cells in logarithmic growth phase U937 (purchased from ATCC) were collected, resuspended in 1640 medium, and the cell concentration was adjusted to 60 cells/. Mu.l to obtain a cell suspension, inoculated into 384 well plates, and 20. Mu.l of the cell suspension was added to each well plate. After incubation of the cells for 24 hours at 37℃in a 5% CO 2 incubator, the test and control compounds according to the invention are diluted with medium to the corresponding working concentrations set up and added to 384-well plates at 5. Mu.l/well. The final concentration of the compound was diluted from 0nM to 1000nM, 10-fold gradient. Simultaneously, a programmed necrosis inducer was added in 5. Mu.l/Kong Jiaru 384 wells, and the necrosis inducer components were TNF-. Alpha.100 ng/ml, SMAC MIMETICS AT-406,1. Mu.M, z-VAD-FMK 20. Mu.M. After the test compound and necrosis inducer were added, the cells were incubated in a 5% CO 2 incubator at 37℃for 24 hours. Then 30 mu L CELLTITER-Glo luminous solution is added to each hole, the mixture is vibrated for 2 minutes, and the absorbance is detected by an enzyme-labeled instrument after the mixture is placed for 10 minutes at room temperature. The necrosis induction rate and necrosis inhibition rate were calculated by the following formulas:
Necrosis induction rate (%) =100× (1-B/a); a is a group without necrosis inducer, B is a group with necrosis inducer
A is a necrosis inducer-free group, B is a necrosis inducer-containing group, and X is a compound-containing and necrosis inducer-containing group.
The inhibition IC 50 of the test compounds on induction of programmed necrosis was calculated by GraphPad and the results are shown in table 2 below (3 replicates per group in the experiment):
TABLE 2 inhibitory Activity of the compounds of the invention against TNF-alpha induced U937 programmed necrosis IC 50
Compounds of formula (I) IC50(nM)
I-1 2.11
I-2 0.61
I-4 0.55
I-6 0.41
I-7 0.45
I-16 4.87
Control Compound 1 7.81
Control Compound 2 10.36
Control Compound 3 6.92
The test results show that: the IC 50 value of the compound of the invention for inhibiting TNF-alpha induced human histiocyte lymphoma cell programmed necrosis is obviously lower than that of the existing compound. The compound can effectively inhibit the programmed necrosis of human histiocyte lymphoma cells.
Test example 3 inhibitory Activity of the Compounds of the invention against TNF-alpha-induced programmed necrosis of human colon cancer cells HT-29 and human pancreatic cancer cells PANC-1
Human colon cancer cells HT-29 and human pancreatic cancer cells PANC-1 (purchased from ATCC) in logarithmic growth phase were collected, resuspended in 1640 medium, and the cell concentration was adjusted to 20 cells/. Mu.l to obtain a cell suspension, which was inoculated into 384 well plates, and 20. Mu.L of the cell suspension was added to each well plate. After incubation of the cells for 24 hours at 37℃in a 5% CO 2 incubator, the test and control compounds according to the invention are diluted with medium to the corresponding working concentrations set up and added to 384-well plates at 5. Mu.l/well. The final concentration of the compound was diluted from 0nM to 1000nM, 10-fold gradient. Simultaneously, a programmed necrosis inducer was added in 5. Mu.l/Kong Jiaru 384 wells, and the necrosis inducer components were TNF-. Alpha.100 ng/ml, SMAC MIMETICS AT-406,1. Mu.M, z-VAD-FMK 20. Mu.M. After the test compound and necrosis inducer were added, the cells were incubated in a 5% CO 2 incubator at 37℃for 24 hours. Then 30 mu L CELLTITER-Glo luminous solution is added to each hole, the mixture is vibrated for 2 minutes, and the absorbance is detected by an enzyme-labeled instrument after the mixture is placed for 10 minutes at room temperature. The necrosis induction rate and necrosis inhibition rate were calculated by the following formulas:
Necrosis induction rate (%) =100× (1-B/a); a is a group without necrosis inducer, B is a group with necrosis inducer
A is a necrosis inducer-free group, B is a necrosis inducer-containing group, and X is a compound-containing and necrosis inducer-containing group.
The test compounds were calculated by GraphPad for their activity against apoptosis inducing ICs 50, results as shown in table 3 below (3 replicates per group in the experiment):
TABLE 3 inhibitory Activity of the inventive Compounds against TNF-alpha induced apoptosis
Compounds of formula (I) HT-29IC50(nM) PANC-1IC50(nM)
I-1 44.56 6.01
I-2 37.46 4.77
I-4 43.19 4.98
I-6 23.37 3.07
I-7 24.99 3.32
I-16 89.04 8.95
Control Compound 1 >100 44.10
Control Compound 2 >100 52.87
Control Compound 3 >100 55.12
The experimental results show that: the IC 50 value of the compound of the invention on TNF induced programmed necrosis of human colon cancer cells HT-29 and human pancreatic cancer cells PANC-1 is significantly lower than that of the existing compound. The compound can effectively inhibit the programmed necrosis of human colon cancer cells and human pancreatic cancer cells.
Test example 4 inhibitory Activity of the Compounds of the invention against TNF-alpha-induced apoptosis of HacaT cells
HacaT cells in logarithmic growth phase (purchased from ATCC) were collected, resuspended in 1640 medium, and cell concentrations were adjusted to 20 cells/μl to give a cell suspension, which was seeded into 384 well plates, and 20 μl of the cell suspension was added to each well plate. After incubation of the cells for 24 hours at 37℃in a 5% CO 2 incubator, the test and control compounds according to the invention are diluted with medium to the corresponding working concentrations set up and added to 384-well plates at 5. Mu.l/well. The final concentration of the compound was diluted from 0nM to 1000nM, 10-fold gradient. Simultaneously, a programmed necrosis inducer was added in 5. Mu.l/Kong Jiaru 384 wells, and the necrosis inducer components were TNF-. Alpha.100 ng/ml, SMAC MIMETICS AT-406,1. Mu.M, z-VAD-FMK 20. Mu.M. After the test compound and necrosis inducer were added, the cells were incubated in a 5% CO 2 incubator at 37℃for 24 hours. Then 30 mu L CELLTITER-Glo luminous solution is added to each hole, the mixture is vibrated for 2 minutes, and the absorbance is detected by an enzyme-labeled instrument after the mixture is placed for 10 minutes at room temperature. The necrosis induction rate and necrosis inhibition rate were calculated by the following formulas:
Necrosis induction rate (%) =100× (1-B/a); a is a group without necrosis inducer, B is a group with necrosis inducer
A is a necrosis inducer-free group, B is a necrosis inducer-containing group, and X is a compound-containing and necrosis inducer-containing group.
The inhibitory activity IC 50 of the test compounds against induction of programmed necrosis was calculated by GraphPad and the results are shown in table 4 below (3 replicates per group in the experiment):
TABLE 4 inhibitory Activity of the compounds of the invention against TNF-alpha-induced apoptosis of HacaT cells IC 50
Compounds of formula (I) IC50(nM)
I-1 6.11
I-2 4.57
I-4 4.62
I-6 3.18
I-7 3.55
I-16 8.84
Control Compound 1 10.4
Control Compound 2 16.8
Control Compound 3 9.9
The test results show that: the IC 50 value of the compound of the invention for inhibiting TNF-alpha induced HacaT cell apoptosis is obviously lower than that of the existing compound, which indicates that the compound of the invention can effectively inhibit HacaT cell apoptosis and further can be used for preventing and treating psoriasis.
In conclusion, the compound has good inhibition activity on human RIP1 kinase, has better effect than the compound in the prior art, and can be used for preparing RIP1 inhibitors; meanwhile, the inhibition effect of the compound of the invention on TNF induced pancreatic cancer cells, colon cancer cells, lymph cancer cells and immortalized keratinocyte programmed necrosis is obviously better than that of the existing compound, and the compound of the invention can effectively inhibit the programmed necrosis of cells. Furthermore, the compound can be used for preparing medicines for treating diseases related to RIP1 and cell programmed necrosis, such as various inflammations, psoriasis, tumors and the like, and has wide application prospect.

Claims (12)

1. A compound, or salt thereof, characterized by: the compound is shown in a formula (II):
wherein,
R 1 is selected from methyl, deuterated methyl;
R 2、R3 is selected from hydrogen;
R 4 is selected from S configuration-CH 3 or R configuration-CH 3;
Ring A is selected from
The saidRepresents a substitution site.
2. The compound, or salt thereof, according to claim 1, wherein: the compound is represented by the formula (III):
wherein,
R 1 is selected from methyl, deuterated methyl;
R 2、R3 is selected from hydrogen;
Ring A is selected from
The saidRepresents a substitution site.
3. The compound, or salt thereof, according to claim 2, wherein: the compound is shown in a formula (IV):
wherein,
R 1 is selected from methyl, deuterated methyl;
Ring A is selected from
The saidRepresents a substitution site.
4. A compound according to any one of claims 1 to 3, or a salt thereof, wherein: the salt is mesylate, hydrochloride, sulfate or organic acid salt.
5. A compound according to any one of claims 1 to 3, or a salt thereof, wherein: the compound is one of the following compounds:
6. Use of a compound of any one of claims 1 to 5, or a salt thereof, in the preparation of a RIP inhibitor.
7. Use according to claim 6, characterized in that: the RIP inhibitor is a RIP1 inhibitor.
8. Use according to claim 7, characterized in that: the RIP inhibitor is a medicament for inhibiting cell programmed necrosis.
9. Use of a compound according to any one of claims 1 to 5, or a salt thereof, for the manufacture of a medicament for the treatment of a disease associated with apoptosis.
10. Use according to claim 9, characterized in that: the medicine is a medicine for resisting inflammation, preventing and/or treating tumor and preventing and/or treating psoriasis.
11. Use according to claim 10, characterized in that: the medicine is used for preventing and/or treating pancreatic cancer, colon cancer, lung cancer, liver cancer, breast cancer and lymph cancer;
and/or the medicament is a medicament for preventing and/or treating rheumatoid arthritis and ulcerative colitis.
12. A medicament, characterized in that: it is prepared by taking the compound or the salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105121432A (en) * 2013-02-15 2015-12-02 葛兰素史密斯克莱知识产权发展有限公司 Heterocyclic amides as kinase inhibitors
CN109071504A (en) * 2016-02-05 2018-12-21 戴纳立制药公司 The inhibitor of receptor interacting protein kinases 1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105121432A (en) * 2013-02-15 2015-12-02 葛兰素史密斯克莱知识产权发展有限公司 Heterocyclic amides as kinase inhibitors
CN109071504A (en) * 2016-02-05 2018-12-21 戴纳立制药公司 The inhibitor of receptor interacting protein kinases 1

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