CN115057860B - ERK inhibitor and pharmaceutical application thereof - Google Patents

ERK inhibitor and pharmaceutical application thereof Download PDF

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CN115057860B
CN115057860B CN202210552373.4A CN202210552373A CN115057860B CN 115057860 B CN115057860 B CN 115057860B CN 202210552373 A CN202210552373 A CN 202210552373A CN 115057860 B CN115057860 B CN 115057860B
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CN115057860A (en
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何谷
毕志昂
蒋献
吴逢波
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides an ERK inhibitor and pharmaceutical application thereof, and belongs to the field of pharmacy. The compound shown in the formula I provided by the invention has good inhibition effect on ERK1/2 kinase, shows effective inhibition activity on colorectal cancer cell lines, can reduce the phosphorylation level of ERK1/2 and p90RSK proteins in colorectal cancer cells in cells, and can induce apoptosis of colon cancer cells. The compound can be used for preparing ERK inhibitors and medicines for preventing and/or treating diseases related to ERK activity, and has wide application prospect.

Description

ERK inhibitor and pharmaceutical application thereof
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to an ERK inhibitor and a pharmaceutical application thereof.
Background
Mitogen Activated Protein Kinase (MAPK) cascades are key signal pathways regulating various cellular processes, and play a vital role in physiological processes such as tumor cell proliferation, differentiation, survival, migration and apoptosis. Extracellular signal-regulating kinases (ERKs) are a class of serine, threonine protein kinases found at the end of the 80 s that regulate cellular signaling under both normal and pathological conditions. Expression of ERKs is critical to the developmental process of the human body and their overactivation plays a critical role in the survival and development of tumor cells. The RAS-RAF-MEK-ERK signaling cascade is the most classical pathway of MAPK, and there is a great deal of research showing that sustained deregulation or abnormal activation of protein kinases in this signaling pathway can induce various diseases such as cancer, inflammation, developmental disorders and nervous system. Thus, protein kinases associated with the ERK/MAPK pathway have become one of the important drug targets.
Over several decades of diligent efforts, small molecule inhibitors against upstream targets of the MAPK pathway BRAF, RAS and MEK have been developed successively, and some have even been FDA approved for marketing, such as vemurafenib (vemurafenib), dabrafenib (dabrafenib), trametinib (Trametinib), and the like. Although these drugs also relatively exhibit excellent antitumor activity in clinical trials, the problem of resistance caused by kinase mutation is also inevitable after several months of clinical treatment of patients, which severely limits the further development of such drugs. It was found that mutations in RAS, BRAF-related kinases upstream of the MAPK pathway lead to reactivation of the terminal kinase ERK1/2 in this pathway, a key event in the acquired resistance of BRAF and MEK inhibitors. Since ERK is located at the end of the MAPK signaling pathway, little mutation occurs, and ERK is only activated by MEK, inhibition of ERK1/2 activity can block the pathological role of this pathway. Preclinical studies indicate that ERK inhibitors can overcome acquired resistance of tumor cells to upstream kinase inhibitor induction, exhibit remarkable anti-tumor cell proliferation effects, and reverse abnormal activation of MAPK pathway caused by upstream pathway mutation.
In recent years, many ERK inhibitors have been designed and synthesized, and proved to have excellent ERK inhibitory activity and anti-tumor cell proliferation effect. Ulixertiinib (BVD-523) is the first reversible ATP-competitive small molecule ERK1/2 kinase inhibitor to enter the clinical stage, which is responsible for ERK 1/2K i The values were 0.3 and 0.04nmol/L, respectively. Studies have shown that Ulixertiinib can inhibit proliferation of BRAF mutant melanoma cells (A375) and colorectal cancer cells (Colo 205), while also exhibiting good anti-tumor activity and low toxicity in vivo K-RAS mutant pancreatic cancer xenograft tumor animal models. LY3214996 pairERK1/2 IC 50 The LY3214996 alone, with a value of 5nmol/L, not only significantly inhibited tumor cell growth in vivo, but also showed good tolerability in patients with BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic xenograft tumors. SCH772984 is an ATP-competitive ERK1/2 inhibitor which is IC to ERK1/2 50 The values are respectively 4 and 1nmol/L, and the BRAF V600E In the mutant human melanoma cell line LOX, SCH772984 not only binds inactive ERK1/2 to block kinase phosphorylation activation, but also promotes dephosphorylation of active kinases. In addition, SCH772984 also showed good anti-tumor cell proliferation in several xenograft tumor models with KRAS or BRAF mutations.
Although there have been a number of ERK inhibitors in the past into clinical studies, a significant portion of these inhibitors have terminated the clinical study due to too great toxicity, poor drug formation, or other reasons, and no ERK inhibitors have been approved for the market. Therefore, the development of more novel ERK inhibitors has important significance for promoting the early approval of ERK inhibitors to be marketed.
Disclosure of Invention
The invention aims to provide a novel ERK inhibitor and pharmaceutical application thereof.
The invention provides a compound, pharmaceutically acceptable salt thereof and stereoisomer thereof, wherein the structure of the compound is shown as a formula I:
wherein R is b Is hydrogen, R c Is unsubstituted or R d A substituted phenyl group; r is R d Selected from C 1-3 Alkyl, hydroxy, carboxyl, NR e R f ；R e 、R f Each independently selected from hydrogen, C 1-3 An alkyl group;
alternatively, R b And R is R c Connected into a ring;
Y 1 selected from CH, N, Y 2 Selected from CH, N;
m is selected from 1, 2 or 3;
R a each independently selected from C 1-5 Alkyl, halogen substituted C 1-5 Alkyl, NR g R h 3-6 membered saturated cycloalkyl which is unsubstituted or substituted with a substituent, 3-6 membered saturated heterocyclic group which is unsubstituted or substituted with a substituent; the substituents are selected from C 1-5 An alkyl group; or two adjacent R a A benzene ring is formed by connection;
R g 、R h each independently selected from hydrogen, C 1-5 An alkyl group.
Further, the structure of the compound is shown as a formula II:
wherein R is d Selected from C 1-3 Alkyl, hydroxy, carboxyl, NR e R f ；
R e 、R f Each independently selected from hydrogen, C 1-3 An alkyl group;
Y 1 selected from CH, N, Y 2 Selected from CH, N;
m is selected from 1, 2 or 3;
R a each independently selected from C 1-3 Alkyl, halogen substituted C 1-3 Alkyl, NR g R h 5-6 membered saturated cycloalkyl which is unsubstituted or substituted with a substituent, 5-6 membered saturated heterocyclic group which is unsubstituted or substituted with a substituent; the substituents are selected from C 1-3 An alkyl group;
R g 、R h each independently selected from hydrogen, C 1-3 An alkyl group.
Further, the structure of the compound is shown as a formula III-1 or a formula III-2:
wherein R is d Selected from C 1-3 Alkyl, hydroxy, carboxyl, NR e R f ；
R e 、R f Each independently selected from hydrogen, C 1-3 An alkyl group;
Y 1 is CH, Y 2 Is N; or Y 2 Is CH, Y 1 Is N;
R a1 、R a2 each independently selected from hydrogen, C 1-3 Alkyl, halogen substituted C 1-3 Alkyl, NR g R h 、And R is a1 、R a2 Are not hydrogen at the same time;
R g 、R h each independently selected from hydrogen, C 1-3 An alkyl group;
R i selected from hydrogen, C 1-3 An alkyl group.
Further, the structure of the compound is shown as a formula IV:
wherein Y is 1 Selected from CH, N, Y 2 Selected from CH, N;
m is selected from 1, 2 or 3;
R a each independently selected from C 1-3 Alkyl, halogen substituted C 1-3 Alkyl, NR g R h 5-6 membered saturated cycloalkyl which is unsubstituted or substituted with a substituent, 5-6 membered saturated heterocyclic group which is unsubstituted or substituted with a substituent; the substituents are selected from C 1-3 An alkyl group;
R g 、R h each independently selected from hydrogen, C 1-3 An alkyl group.
Further, the structure of the compound is shown as a formula V-1 or a formula V-2:
wherein Y is 1 Is CH, Y 2 Is N; or Y 2 Is CH, Y 1 Is N;
R a1 、R a2 each independently selected from hydrogen, C 1-3 Alkyl, halogen substituted C 1-3 Alkyl, NR g R h 、And R is a1 、R a2 Are not hydrogen at the same time;
R g 、R h each independently selected from hydrogen, C 1-3 An alkyl group;
R i selected from hydrogen, C 1-3 An alkyl group.
Further, the compound is selected from:
the invention also provides a medicine, which is a preparation prepared by taking the compound, the pharmaceutically acceptable salt and the stereoisomer thereof as active ingredients and adding pharmaceutically acceptable auxiliary materials.
The invention also provides application of the compound, pharmaceutically acceptable salts thereof and stereoisomers thereof in preparing ERK inhibitors.
Further, the ERK inhibitor is an ERK1 inhibitor and/or an ERK2 inhibitor.
Further, the ERK inhibitor is a medicament for preventing and/or treating diseases related to ERK activity;
preferably, the disease associated with ERK activity is cancer; the cancer is preferably colorectal cancer, lung cancer, pancreatic cancer, melanoma, acute myeloid leukemia, glioblastoma.
Experimental results show that the compound provided by the invention has good inhibition effect on ERK1/2 kinase. Wherein, the inhibition effect of the compound 13l is optimal, the inhibition rate of ERK1 and ERK2 kinase at 1 mu M is up to 62.41 percent and 65.96 percent, and the IC of the ERK1 and ERK2 kinase is realized 50 The values were 1.69. Mu.M and 0.87. Mu.M, respectively. The compounds of the invention can be used to prepare inhibitors of ERK1/2 kinase.
It is well known to those skilled in the art that various diseases including colorectal cancer, lung cancer, pancreatic cancer, melanoma, acute myeloid leukemia, glioblastoma and the like are associated with ERK1/2 kinase activity, and targeted inhibition of ERK1/2 kinase activity is an effective means for treating colorectal cancer, lung cancer, pancreatic cancer, melanoma, acute myeloid leukemia, glioblastoma and the like. Therefore, the compounds provided by the invention can be used for preparing medicines for preventing and/or treating diseases related to ERK1/2 kinase activity.
The invention also shows by experiment that compound 13l shows effective inhibitory activity against both human colorectal cancer cell lines, which has IC against SW-620 and HCT-116 cells 50 The values were 1.16. Mu.M and 0.59. Mu.M, respectively. Western blot analysis showed that compound 13l decreased the intracellular phosphorylation levels of ERK1/2 (Thr 202, tyr 204) and p90RSK (Thr 359, ser 363) proteins in HCT116 cells in a concentration-dependent manner. Finally, the invention further proves that the compound 13l can induce apoptosis of HCT116 human colon cancer cells, and the apoptosis rate of the HCT116 cells can reach 23.7 percent after the treatment of the compound 13l with 1 mu M.
In conclusion, the compound provided by the invention can be used for preparing ERK1/2 kinase inhibitors and medicines for preventing and/or treating diseases related to ERK1/2 kinase activity, and has wide application prospects.
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.
Drawings
Fig. 1: inhibition profile of ERK1/2 kinase by Compound 13 l.
Fig. 2: compound 13l and SCH772984 inhibit proliferation of colon cancer cells. * P < 0.001.
Fig. 3: effect of compound 13l on ERK1/2 and RSK phosphorylation levels in HCT116 cells.
Fig. 4: apoptosis analysis of HCT116 cells by compound 13 l.
Fig. 5: apoptosis rate of compound 13l for inducing apoptosis of HCT116 cells.
Detailed Description
Unless otherwise indicated, reagents and starting materials used in the experiments of the invention were purchased from reagent companies, all of which were analytically or chromatographically pure, and were used without further treatment.
Example 1: synthesis of target Compounds 13a-l, 14a-b, 15a-e
The synthetic route is as follows:
1. synthesis of intermediate 8
The synthesis step of intermediate 8: the N-Boc-3-cyano-4-pyrrolidone is used as a starting material to synthesize a series of compounds II. First, compound 7 (N-Boc-3-cyano-4-pyrrolidone, (21.00 g,100 mmol)) and hydrazine hydrochloride (10.30 g,150 mmol) were added to a 500mL reaction flask, and then, dehydrated alcohol (250 mL) was added thereto to dissolve the same, and then, the reaction flask was heated at 60℃under reflux for 3 hours. The reaction was monitored by TLC, after completion of the reaction, concentrated under reduced pressure, the solvent was removed, and then saturated NaHCO was slowly added with stirring 3 Adjusting pH of the aqueous solution to 7-8, extracting with EA (3X 300 mL), collecting the upper layer solution, adding appropriate amount of anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure again, recrystallizing the residue with ethyl acetate, vacuum filtering, collecting filter cake, and oven drying to obtain white solid 8, wherein intermediate 8 can be used in the next step without purification.
2. Synthesis of intermediate 9
The synthesis step of intermediate 9: in a dry 500mL round bottom flask, intermediate 8 (3-amino-5- (tert-butyl) -pyrrolo [3, 4-C) was added sequentially]Pyrazole, (11.20 g,50 mmol)), DIEA (34.80 mL,200 mmol), THF (250 mL), then the above mixture was stirred at 0 ℃ for 5 minutes, then a mixed solution of ethyl chloroformate (4.80 mL,50 mmol) diluted with anhydrous THF (120 mL) was slowly added dropwise with a dropping funnel at 0 ℃ for 2 hours, and after completion of the reaction, the mixed solution was transferred to normal temperature for stirring reaction for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, the residue was extracted with ethyl acetate and saturated aqueous NaCl solution (3X 250 mL), the upper layer solution was collected, and an appropriate amount of anhydrous Na was added 2 SO 4 Drying, concentrating under vacuum to obtain white solid 9, and purifying with silica gel chromatographic column (petroleum ether/ethyl acetate=4/1-2/1) to obtain pure intermediate 9.
3. Synthesis of intermediate 10
Synthesis of intermediates 10a, 10b, 10 d: in a 250mL reaction flask, intermediate 9 (5- (tert-butyl) -3-amino-4, 6-dihydropyrrolo [3, 4-c) was added sequentially]Pyrazole-1-carboxylic acid ethyl ester (14.80 g,50 mmol), DIEA (34.80 mL,200 mmol), THF (100 mL), followed by addition of the corresponding acid chloride (75 mmol) with stirring at 0 ℃ and stirring for 1 hour, then transferring to room temperature with stirring for 12 hours. TLC detection, after completion of the reaction, concentration under reduced pressure, and use of saturated NaHCO 3 The pH of the residue was adjusted to 7-8, extracted with ethyl acetate (3X 250 mL), the upper layer solution was collected, and an appropriate amount of anhydrous Na was added 2 SO 4 Drying, concentrating under reduced pressure, and purifying with silica gel chromatographic column (petroleum ether/ethyl acetate=1/1) to obtain pure intermediate 10.
Synthesis of intermediate 10 c: in a 250mL reaction flask, intermediate 9 (5- (tert-butyl) -3-amino-4, 6-dihydropyrrolo [3,4-c ] pyrazole-1-carboxylic acid ethyl ester, (14.80 g,50 mmol)), DIEA (34.80 mL,200 mmol), THF (100 mL) were added sequentially, followed by the corresponding acid chloride (75 mmol) with stirring at 0deg.C and stirring for 1 hour, followed by heating the reaction mixture to 45deg.C and stirring for 12 hours, during which time a precipitate formed. TLC detection is carried out, after the reaction is completed, decompression and suction filtration are carried out, THF is used for cleaning for 3 times, a filter cake is collected and dried to obtain a crude product 10c, and the crude product 10c can be used for the next step without purification.
4. Synthesis of intermediate 12
The synthesis of intermediates 11 a-d: intermediate 10 (30 mmol) was first dissolved in DCM (100 mL) in a 250mL reaction flask, then a solution of 1, 4-dioxane (30 mL) containing 4N HCl was added dropwise with stirring at 0deg.C, and the reaction was then transferred to ambient temperature with continuous stirring for 12 hours, where a precipitate formed. The reaction is monitored by TLC, after the reaction is completed, the pressure is reduced, the filtration is carried out, the reaction is washed for 3 times by methylene dichloride, a filter cake is collected and dried to obtain a crude product 11, and the crude product 11 can be used for the next step without purification.
The synthesis procedure of intermediate 12: first, intermediate 11 (5 mmol), DIEA (1.70 mL,10 mmol) and DCM (30 mL) were sequentially added to a 100mL reaction flask, and then the above mixture was slowly added to the corresponding acid chloride (7.50 mmol) with stirring at 0 ℃ for 1 hour, and then the reaction flask was transferred to a room temperature with stirring for reaction for 10 hours. TLC detection, after completion of the reaction, concentration under reduced pressure, and use of saturated NaHCO 3 Regulating pH to 7-8, extracting with ethyl acetate (3X 50 mL), collecting upper layer solution, adding proper amount of anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure, and purifying with silica gel chromatographic column (petroleum ether/ethyl acetate=1/1) to obtain pure intermediate 12.
5. Synthesis of target Compound 13a-1
The synthesis of the target compounds 13 a-l: intermediate 12 (2 mmol) was added to a dry 50mL round bottom flask, then dissolved in MeOH (20 mL), then LiOH (80 mg,2 mmol) was added with stirring, after 30min, detected by TLC, after completion of the reaction, concentrated under reduced pressure, purified by silica gel chromatography (dichloromethane/methanol=9/1) to give white solid 13, followed by recrystallisation from methanol to give pure title compound 13.
The target compounds 13a-l were synthesized as described above.
6. Synthesis of target Compounds 14a-b
The synthesis of the target compounds 14 a-b: first, intermediate 13 (2 mmol) was dissolved in a 50mL round bottom flask with 1, 4-dioxane solution (15 mL), followed by dropwise addition of 1N LiOH (1 mL) solution with stirring, and then the reaction mixture was transferred to room temperature for reaction with stirring for 3 hours. TLC detection, after the reaction is completed, regulating the pH to 4-5 by using 1N HCl solution, generating precipitate in the process, carrying out vacuum filtration, washing with water for 3 times, collecting a filter cake and drying to obtain white solid 14, then recrystallizing a crude product by using methanol, carrying out vacuum filtration, collecting the filter cake, and obtaining pure target compound 14 after drying.
7. Synthesis of target Compounds 15a-e
The synthesis of the target compounds 15 a-e: first, intermediate 13 (2 mmol) and NH 4 Cl(0.13g,2.40mmol)、EtOH(20mL)、H 2 O (5 mL), DMF (2 mL), THF (6 mL), iron powder (0.56 g,10 mmol), glacial acetic acid (3-4 drops) were added sequentially to a 100mL reaction flask. The reaction mixture was then transferred to 80 ℃ with stirring at reflux for 8 hours, during which time the solution was changed from a yellow suspension to a light brown suspension. Detecting by TLC, filtering to remove residual iron powder, concentrating under reduced pressure, and concentrating with saturated NaHCO 3 Adjusting pH to 7-8, extracting with ethyl acetate (3X 50 mL), collecting upper layer solution, adding appropriate amount of anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure, and purifying with silica gel chromatographic column (dichloromethane/methanol=9/1) to obtain pure target compound 15. The target compounds 15a-e were synthesized as described above.
Example 2: synthesis of target Compounds 19a-g
The synthetic route is as follows:
1. synthesis of intermediate 16
Synthesis of intermediate 16: intermediate 9 (5- (tert-butyl) -3-amino-4, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1-carboxylic acid ethyl ester (14.80 g,50 mmol) was added to a dry 250mL two-necked flask, then dissolved in ultra-dry DMF (100 mL) followed by N 2 Chloroethyl isocyanate (12.80 ml,150 mmol) was slowly added dropwise under protection, the mixture was stirred under ice for 5 minutes, and then the reaction mixture was transferred to room temperature and stirred for 3 hours, during which time precipitation occurred. After the reaction is finished, pouring the reactant into 500mL of ice water while the reactant is hot, performing vacuum filtration, washing 3 times with water, collecting a filter cake and drying for later use. Next, the dried crude product, potassium tert-butoxide (5.05 g,45 mmol) and THF (150 mL) were sequentially added to a 250mL two-necked flask, followed by N 2 The reaction mixture was stirred at room temperature under protection for 2 hours, and solids were precipitated during this process. After the reaction is finished, performing vacuum filtration, washing with THF for 3 times, collecting a filter cake, drying to obtain white solid 16, and finally purifying by a silica gel chromatographic column (petroleum ether/ethyl acetate=1/1) to obtain pure intermediate 16.
2. Synthesis of intermediates 18a-g
The synthesis of intermediate 17: first, intermediate 16 (5- (tert-butyl) 1-ethyl 3- (2-oxoimidazol-1-yl) -4, 6-dihydropyrrole [3,4-c ] pyrazole-1, 5-dicarboxylate, (16.43 g,45 mmol)) was dissolved in 250mL reaction flask with DCM (150 mL), then 1, 4-dioxane solution (30 mL) containing 4N HCl was added dropwise with stirring at 0 ℃, followed by transferring the reaction mass to normal temperature with continuous stirring for 12 hours, during which time precipitate formed. The reaction was monitored by TLC, after the reaction was completed, suction filtration was performed under reduced pressure, and washing with dichloromethane 3 times, and a filter cake was collected and dried to obtain a white solid 17, and the crude product 17 was used in the next step without purification.
Intermediates 18a-gIs synthesized by the steps of: first, intermediate 17 (3- (2-oxoimidazolidin-1-yl) -5, 6-dihydropyrrole [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester, (0.79 g,3 mmol), DIEA (1.02 mL,6 mmol), pyBop (1.72 g,3.3 mmol) and DCM (30 mL) were added sequentially to a 100mL round bottom flask, followed by slow addition of the corresponding acid (4.50 mmol) under ice bath, then the mixture was transferred to 0deg.C and stirred for 1 hour, then to ambient temperature and stirred for 10 hours, monitored by TLC, after completion of the reaction, concentrated under reduced pressure, reacted with saturated NaHCO 3 The residue was adjusted to pH 7-8, followed by extraction with ethyl acetate (3X 50 mL), the supernatant was collected, and an appropriate amount of anhydrous Na was added 2 SO 4 Drying, concentrating under reduced pressure, and purifying with silica gel chromatographic column (dichloromethane/methanol=30/1) to obtain pure target compound 18.
Intermediates 18a-g were synthesized as described above.
3. Synthesis of target Compounds 19a-g
The synthesis of the target compounds 19 a-g: intermediate 18 (2 mmol) was added to a dry 50mL round bottom flask, then dissolved in MeOH (20 mL), then LiOH (80 mg,2 mmol) was added with stirring, after 30min, detected by TLC, after completion of the reaction, concentrated under reduced pressure, purified by silica gel chromatography (dichloromethane/methanol=9/1) to give white solid 19, followed by recrystallisation from methanol to give pure title compound 19.
Intermediates 19a-g were synthesized as described above.
The following are the structures and characterization data for the above intermediate compounds and target compounds.
1. Structure and characterization data for intermediate compounds
TABLE 1 Structure of intermediate Compounds
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3-amino-5- (tert-butyl) -pyrrolo [3,4-C]Pyrazole (8): white powder, yield: 38.2%. 1 HNMR(400MHz,DMSO-d 6 )δ(ppm):11.19(s,1H),4.99(s,2H),4.17(d,J＝9.6Hz,2H),4.12(m,2H),1.44(s,9H).
5- (tert-butyl) -3-amino-4, 6-dihydropyrrolo [3,4-c]Pyrazole-1-carboxylic acid ethyl ester (9): white powder, yield: 88.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):5.68(s,2H),4.46(d,J＝3.2Hz,2H),4.27(q,J＝7.2Hz,2H),4.17(d,J＝3.2Hz,2H),1.44(s,9H),1.28(t,J＝7.2Hz,3H).
5- (tert-butyl) -1-ethyl-3- (4- (methoxycarbonyl) benzoylamino) -4, 6-dihydropyrrolo [3,4-c]Pyrazole-1, 5-carboxylic acid ethyl ester (10 a): white powder, yield: 72.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.86(d,J＝2.0Hz,1H),8.16(d,J＝8.0Hz,2H),8.07(d,J＝8.0Hz,2H),4.55(s,2H),4.49(s,2H),4.42(q,J＝7.6Hz,2H),3.90(s,3H),1.54(s,9H),1.35(t,J＝7.6Hz,3H).
5- (tert-butyl) -1-ethyl-3- (4-hydroxybenzoamide) -4, 6-dihydropyrrolo [3,4-c]Pyrazole-1, 5-carboxylic acid ethyl ester (10 b): white powder, yield: 76.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.86(s,1H),8.35(d,J＝8.8Hz,2H),8.26(d,J＝8.8Hz,2H),4.55(s,2H),4.49(s,2H),4.43(q,J＝7.2Hz,2H),1.49(s,9H),1.36(t,J＝7.2Hz,3H).
5- (tert-butyl) -1-ethyl-3- (4- (dimethylamino) benzoylamino) -4, 6-dihydropyrrolo [3,4-c]Pyrazole-1, 5-carboxylic acid ethyl ester (10 c): brown powder, yield: 68.7%. 1 H NMR(400MHz,Chloroform-d)δ(ppm):8.65(s,1H),7.74(d,J＝8.8Hz,2H),6.68(d,J＝8.8Hz,2H),4.73(s,2H),4.72(s,2H),4.46(q,J＝7.2Hz,2H),3.05(s,6H),1.52(s,9H),1.42(t,J＝7.2Hz,3H).
3- (4-nitrobenzamide) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (10 d): yellow powder, yield: 83.4%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.01(s,1H),8.34(d,J＝8.8Hz,2H),8.26(d,J＝8.8Hz,2H),4.55(s,2H),4.49(s,2H),4.42(q,J＝7.2Hz,2H),1.55(s,9H),1.36(t,J＝7.2Hz,3H).
3- (4-hydroxybenzoamido) -5- (4-methylbenzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 a), white powder, yield: 45.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.84(s,1H),10.23(s,1H),7.92(dd,J＝8.4Hz,2H),7.53(d,J＝7.6Hz,2H),7.34(d,J＝7.6Hz,2H),6.88(d,J＝8.4Hz,2H),4.71(s,2H),4.56(s,2H),4.44(q,J＝7.2Hz,2H),2.40(s,3H),1.38(t,J＝7.2Hz,3H).
3- (4-hydroxybenzoamido) -5- (4- (trifluoromethyl) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 b), white powder, yield: 54.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.84(s,1H),10.15(s,1H),7.91(d,J＝8.4Hz,2H),7.86(d,J＝7.6Hz,2H),7.83(d,J＝7.6Hz,2H),6.85(d,J＝8.4Hz,2H),4.74(s,2H),4.54(s,2H),4.42(q,J＝7.2Hz,2H),1.29(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzoylamino) -5- (4-methylbenzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 c): white powder, yield: 55.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.87(s,1H),7.93(d,J＝8.8Hz,2H),7.64(d,J＝8.0Hz,2H),7.36(t,J＝8.0Hz,2H),6.81(d,J＝8.8Hz,2H),4.67(s,2H),4.56(s,2H),4.43(q,J＝7.2Hz,2H),2.99(s,6H),2.35(s,3H),1.37(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzoylamino) -5- (4- (trifluoromethyl) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 d): white powder, yield: 49.0%. 1 H NMR(400MHz,Chloroform-d)δ(ppm):9.52(s,1H),8.23(d,J＝9.2Hz,2H),7.74(d,J＝3.6Hz,2H),7.71(d,J＝3.6Hz,2H),6.66(d,J＝9.2Hz,2H),5.06(s,2H),4.87(s,2H),4.55(q,J＝7.2Hz,2H),3.05(s,6H),1.49(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzeneCarboxamido) -5- (4- (dimethylamino) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 e): white powder, yield: 51.4%. 1 H NMR(400MHz,)δ(ppm):11.69(s,1H),8.28(d,J＝9.6Hz,2H),7.97(d,J＝8.8Hz,2H),7.86(m,2H),7.76(d,J＝8.8Hz,2H),5.29(s,2H),4.99(s,2H),4.42(q,J＝7.2Hz,2H),3.52(dd,J＝9.2Hz,12H),1.36(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzoyl) -5- (3- (dimethylamino) benzoyl) -5, 6-dihydropyrrole [3,4-c]Pyrazole-1 (4-hydro) -carboxylic acid ethyl ester (12 f): white powder, yield: 58.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.06(s,1H),7.88(d,J＝8.4Hz,2H),7.28(t,J＝7.4Hz,1H),6.84(s,1H),6.82(m,1H),6.81(s,1H),6.67(d,J＝8.4Hz,2H),4.80(m,2H),4.64(s,2H),4.43(q,J＝7.2Hz,2H),2.99(d,J＝12.4Hz,6H),2.93(s,6H),1.37(t,J＝7.2Hz,3H).
5- (1-naphthaloyl) -3- (4- (dimethylamino) benzamide) -5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 g), white powder, yield: 49.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.03(s,1H),8.31(s,1H),8.04(d,J＝7.6Hz,2H),7.90(s,1H),7.78(d,J＝7.8Hz,1H),7.66–7.61(m,1H),7.60(s,1H),7.59(s,1H),7.58(t,J＝2.0Hz,1H),6.68(d,J＝7.6Hz,2H),4.97(s,2H),4.41(s,2H),4.43(q,J＝7.2Hz,2H),2.98(s,6H),1.27(d,J＝7.2Hz,3H).
5- (2-naphthaloyl) -3- (4- (dimethylamino) benzamide) -5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12H.) white powder, yield: 61.8%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.07(s,1H),8.87(d,J＝4.4Hz,1H),8.32(d,J＝8.8Hz,2H),8.05(d,J＝4.4Hz,1H),8.03(d,J＝4.4Hz,1H),8.00(d,J＝4.4Hz,1H),7.83(d,J＝8.8Hz,1H),7.68(d,J＝8.4Hz,1H),7.64–7.59(m,1H),6.69(d,J＝8.8Hz,2H),4.93(s,2H),4.74(s,2H),4.45(q,J＝7.2Hz,2H),2.98(s,6H),1.38(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzamide) -5-isonicotinyl-5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 i): white powder, yield: 66.7%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.87(s,1H),8.79(d,J＝4.8Hz,2H),7.95(d,J＝5.6Hz,2H),7.63(d,J＝5.6Hz,2H),6.72(d,J＝4.8Hz,2H),4.77(s,2H),4.54(s,2H),4.43(q,J＝7.2Hz,2H),2.99(s,6H),1.36(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzamide) -5-nicotinoyl-5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 j). White powder, yield: 55.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.15(s,1H),9.08(dd,J＝6.0,2.0Hz,1H),8.93(d,J＝5.6Hz,1H),8.56(t,J＝8.8Hz,1H),8.02–7.93(m,2H),7.89(d,J＝8.8Hz,1H),6.76(d,J＝8.8Hz,2H),4.90(s,2H),4.83–4.75(m,2H),4.34(t,J＝7.2Hz,2H),3.00(s,6H),1.37(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzamido) -5- (4- (4-methylpiperazin-1-yl) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 k): white powder, yield: 46.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.70(d,J＝27.2Hz,1H),7.86(d,J＝8.8Hz,2H),7.54(d,J＝8.8Hz,2H),7.06(d,J＝8.8Hz,2H),6.81(d,J＝8.8Hz,2H),4.91(s,2H),4.68(s,2H),4.28(t,J＝7.2Hz,2H),4.04(d,J＝12.0Hz,2H),3.52(d,J＝12.0Hz,2H),3.21(t,J＝12.0Hz,2H),3.05(t,J＝12.0Hz,2H),3.02(s,6H),2.91(s,3H),1.29(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzamido) -5- (3- (4-methylpiperazin-1-yl) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 l) white powder, yield: 55.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.68(s,1H),7.93(d,J＝8.8Hz,2H),7.36(q,J＝7.6Hz,1H),7.24(d,J＝7.2Hz,2H),7.08(d,J＝7.6Hz,2H),6.98(d,J＝7.2Hz,1H),6.81(d,J＝8.8Hz,2H),4.68(m,2H),4.57(s,2H),4.44(q,J＝7.2Hz,2H),3.21(s,4H),2.99(s,6H),2.49(s,4H),2.26(s,3H),1.38(t,J＝7.2Hz,3H).
4- ((1- (ethoxycarbonyl) -5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) carbamoyl) benzoic acid (12 m). White powder, yield: 49.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.75(s,1H),8.07(d,J＝7.2Hz,2H),7.96(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.30(d,J＝7.2Hz,2H),4.84(m,2H),4.69(s,2H),4.44(q,J＝7.2Hz,2H),3.88(s,3H),2.38(s,3H),1.38(t,J＝7.2Hz,3H).
4- ((1- (ethoxycarbonyl) -5- (4- (trifluoromethyl) benzene)Formyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c]Pyrazol-3-yl) carbamoyl) benzoic acid (12 n). White powder, yield: 71.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.75(s,1H),8.09(d,J＝8.8Hz,2H),8.01(d,J＝8.0Hz,2H),7.88(d,J＝8.0Hz,2H),7.84(d,J＝8.8Hz,2H),4.86(m,2H),4.68(s,2H),4.45(q,J＝7.2Hz,2H),3.88(s,3H),1.38(t,J＝7.2Hz,3H).
5- (4-methylbenzoyl) -3- (4-nitrobenzoylamino) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 o). White powder, yield: 62.0%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.94(s,1H),7.77(d,J＝8.8Hz,2H),7.48(d,J＝8.0Hz,2H),7.30(d,J＝8.0Hz,2H),6.54(d,J＝8.8Hz,2H),5.82(s,2H),4.70(s,2H),4.42(q,J＝7.2Hz,2H),2.37(s,3H),1.36(t,J＝7.2Hz,3H).
3- (4-Nitrophenylmethylcarboxamido) -5- (4- (trifluoromethyl) benzoyl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 p). White powder, yield: 49.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.54(s,1H),7.83(d,J＝8.4Hz,2H),7.69(d,J＝8.0Hz,2H),7.61(d,J＝8.0Hz,2H),6.49(d,J＝8.4Hz,2H),4.66(m,2H),4.52(s,2H),4.38(q,J＝7.2Hz,2H),1.32(t,J＝7.2Hz,3H).
5- (3- (dimethylamino) benzoyl) -3- (4-nitrobenzoylamino) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 q.) white powder, yield: 57.7%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.43(s,1H),7.73(d,J＝8.0Hz,2H),7.28(m,1H),6.85(d,J＝7.6Hz,1H),6.80(s,1H),6.76(d,J＝7.6Hz,1H),6.52(d,J＝8.0Hz,2H),4.69(s,2H),4.48(s,2H),4.43(q,J＝7.2Hz,2H),2.91(s,6H),1.38(t,J＝7.2Hz,3H).
3- (4- (dimethylamino) benzamide) -5-nicotinoyl-5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 x). White powder, yield: 55.8%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.91(s,1H),8.72(t,J＝6.4Hz,2H),8.32(d,J＝8.4Hz,2H),8.27–8.12(m,2H),7.58(dd,J＝8.4,5.6Hz,2H),4.85(s,2H),4.72(m,2H),4.40(d,J＝7.2Hz,2H),1.37(t,J＝7.2Hz,3H).
5-nicotinoyl-3- (4-nitrobenzamide) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (12 y) white powder, yield: 49.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.87(s,1H),9.19(s,1H),8.98(dd,J＝5.6,1.6Hz,1H),8.66(d,J＝8.0Hz,1H),8.34(d,J＝8.8Hz,2H),8.27(t,J＝8.8Hz,2H),7.91(dd,J＝8.0,5.2Hz,1H),4.88(m,2H),4.55(s,2H),4.43(q,J＝7.2Hz,2H),1.36(t,J＝7.2Hz,3H).
N- (5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) -4-nitrobenzamide (13 o). White powder, yield: 57.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.28(s,1H),10.36(s,1H),7.75(d,J＝8.8Hz,2H),7.53(d,J＝8.0Hz,2H),7.32(t,J＝8.0Hz,2H),6.61(d,J＝8.8Hz,2H),4.68(s,2H),4.56(s,2H),2.37(s,3H).
4-nitro-N- (5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 p). White powder, yield: 67.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.46(s,1H),10.48(s,1H),7.91(d,J＝8.4Hz,2H),7.86(d,J＝8.4Hz,2H),7.72(d,J＝8.4Hz,2H),6.63(d,J＝8.4Hz,2H),4.71(m,2H),4.54(s,2H).
N- (5- (3- (dimethylamino) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) -4-nitrobenzamide (13 q.) white powder, yield: 71.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.39(s,1H),10.48(s,1H),7.76(d,J＝8.0Hz,2H),7.28(m,1H),6.84(d,J＝7.6Hz,1H),6.82(s,1H),6.77(d,J＝7.6Hz,1H),6.59(d,J＝8.0Hz,2H),4.67(s,2H),4.52(s,2H),2.94(s,6H).
N- (5-isonicotinyl-1, 4,5, 6-tetrahydropyrrole [3,4-c ]]Pyrazol-3-yl) -4-nitroaniline (13 x). White powder, yield: 59.8%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.61(s,1H),11.42(s,1H),8.72(d,J＝6.4Hz,2H),8.32(d,J＝8.4Hz,2H),8.27–8.12(m,2H),7.58(dd,J＝8.4,5.6Hz,2H),4.73(m,2H),4.58(s,2H).
N- (5-nicotinoyl-1, 4,5, 6-tetrahydropyrrole- [3, 4-c)]Pyrazol-3-yl) -4-nitroaniline (13 y). White powder, yield: 64.0%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.50(s,1H),11.33(s,1H),8.81(dd,J＝7.6,2.0Hz,1H),8.69(t,J＝4.4Hz,1H),8.36(d,J＝8.4Hz,1H),8.30(d,J＝8.4Hz,1H),8.25(d,J＝8.4Hz,1H),8.16(d,J＝8.4Hz,1H),8.05(t,J＝8.4Hz,1H),7.53(dd,J＝9.2,4.5Hz,1H),4.75(m,2H),4.65(s,2H).
5- (tert-butyl) 1-ethyl 3- (2-oxoimidazol-1-yl) -4, 6-dihydropyrrole [3,4-c ]]Pyrazole-1, 5-dicarboxylic acid ester (16): white powder, yield: 76.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.35(s,1H),4.43(s,2H),4.38(s,2H),4.19(t,J＝7.2Hz,2H),3.86(d,J＝8.4Hz,2H),3.79(d,J＝8.4Hz,2H),1.46(s,9H),1.25(t,J＝7.2Hz,3H).
3- (2-Oxlimidazolidin-1-yl) -5, 6-dihydropyrrole [3,4-c ]]Pyrazole-1 (4H) -carboxylic acid ethyl ester (17). White powder, yield: 85.9%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.27(s,1H),10.74(s,2H),4.35(s,2H),4.30(s,2H),4.20(q,J＝7.2Hz,2H),3.87(d,J＝8.4Hz,2H),3.80(d,J＝8.4Hz,2H),1.25(t,J＝7.2Hz,3H).
5- (4-Methylbenzoyl) -3- (2-oxoimidazolidin-1-yl) -5, 6-dihydropyrrole [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 a): white powder, yield: 44.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.36(s,1H),7.48(d,J＝7.6Hz,2H),7.27(t,J＝7.6Hz,2H),4.68(s,2H),4.56(s,2H),4.15(q,J＝7.2Hz,2H),3.83(s,2H),3.80–3.72(m,2H),2.37(s,3H),1.20(t,J＝7.2Hz,3H).
3- (2-Oximidazol-1-yl) -5- (4- (trifluoromethyl) benzoyl) -5, 6-dihydropyrrole [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 b): white powder, yield: 51.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.39(s,1H),7.90–7.83(m,2H),7.79(d,J＝8.0Hz,2H),4.71(s,2H),4.55(s,2H),4.15(q,J＝7.2Hz,2H),3.82(d,J＝4.0Hz,2H),3.81–3.72(m,2H),1.19(t,J＝7.2Hz,3H).
5- (3- (dimethylamino) benzoyl) -3- (2-oxoimidazol-1-yl) -5, 6-dihydropyrrole [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 c): white powder, yield: 49.0%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.29(s,1H),7.21(t,J＝7.6Hz,1H),6.78(d,J＝2.4Hz,1H),6.72(s,1H),6.62(d,J＝7.6Hz,1H),4.58(s,2H),4.46(s,2H),4.44(q,J＝7.2Hz,2H),3.71(d,J＝8.0Hz,2H),3.32(t,J＝8.0Hz,2H),2.88(s,6H),1.35(t,J＝7.2Hz,3H).
5-isonicotinyl-3- (2-oxoimidazole-1-)Radical) -5, 6-dihydropyrrole [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 d): white powder, yield: 42.7%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.39(s,1H),8.78(d,J＝2.4Hz,1H),8.73–8.64(m,1H),8.03(t,J＝9.6Hz,1H),7.51(t,J＝12.0Hz,1H),4.71(s,2H),4.61(s,2H),4.15(q,J＝7.2Hz,2H),3.83(d,J＝7.6Hz,2H),3.82–3.72(m,2H),1.20(t,J＝7.2Hz,3H).
5-nicotinoyl-3- (2-oxoimidazolidin-1-yl) -5, 6-dihydropyrrolo [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 e): white powder, yield: 55.4%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.28(s,1H),9.16(d,J＝4.4Hz,1H),8.97(s,1H),8.72(d,J＝7.6Hz,1H),8.06(s,1H),4.72(s,2H),4.64(s,2H),4.22(q,J＝7.2Hz,2H),3.81(d,J＝8.8Hz,2H),3.42(d,J＝8.8Hz,2H),1.24(t,J＝7.2Hz,3H).
5- (4- (4-methylpiperazin-1-yl) benzoyl) -3- (2-oxoimidazol-1-yl) -5, 6-dihydropyrrol [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 f): white powder, yield: 50.1%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.40(s,1H),7.52(d,J＝8.0Hz,2H),7.06(t,J＝8.0Hz,2H),4.70(s,2H),4.65(s,2H),4.17(d,J＝7.2Hz,2H),3.86(d,J＝6.4Hz,2H),3.84–3.71(m,4H),3.44(d,J＝6.4Hz,2H),2.84(s,4H),2.56(s,3H),1.21(t,J＝7.2Hz,3H).
5- (3- (4-methylpiperazin-1-yl) benzoyl) -3- (2-oxoimidazol-1-yl) -5, 6-dihydropyrrol [3,4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester (18 g), white powder, yield: 44.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.40(s,1H),7.35(q,J＝7.6Hz,1H),7.16(d,J＝11.6Hz,1H),7.11(d,J＝8.4Hz,1H),7.03(d,J＝7.6Hz,1H),4.68(s,2H),4.54(s,2H),4.15(q,J＝7.2Hz,2H),3.77(d,J＝8.4Hz,2H),3.40(d,J＝8.4Hz,2H),2.85(s,4H),2.54(s,4H),2.28(s,3H),1.20(t,J＝7.2Hz,3H).
2. Structure and characterization data for target compounds
TABLE 2 Structure of target Compounds
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4-hydroxy-N- (5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 a). White powder, melting point: 280.9-283.7 ℃, yield: 63.2%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.31(s,1H),10.67(s,1H),10.23(s,1H),7.87(d,J＝8.4Hz,2H),7.49(d,J＝7.6Hz,2H),7.29(d,J＝7.6Hz,2H),6.82(d,J＝8.4Hz,2H),4.67(s,2H),4.56(s,2H),2.37(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.17,164.36,161.39,139.88,134.37,130.39,130.34(2C),129.39(2C),129.27,127.45,127.27(2C),115.48,115.41(2C),50.87,45.55,21.40.HRMS(ESI) + calculated for C 20 H 18 N 4 NaO 3 ,[M+Na] + :m/z 385.1266,found:385.1271.
4-hydroxy-N- (5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 b): white powder, melting point: 241.6-243.8 ℃, yield: 68.4%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.35(s,1H),10.66(s,1H),10.12(s,1H),7.95–7.85(m,2H),7.84(d,J＝7.6Hz,2H),7.81(d,J＝7.6Hz,2H),6.83(d,J＝8.0Hz,2H),4.71(s,2H),4.54(s,2H).HRMS(ESI) + calculated for C 20 H 15 F 3 N 4 NaO 3 ,[M+Na] + :m/z 439.0986,found:439.0988.
4- (dimethylamino) -N- (5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 c). White powder, melting point: 260.8-265.1 ℃, yield: 78.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.21(s,1H),10.52(s,1H),7.87(d,J＝8.8Hz,2H),7.49(d,J＝7.6Hz,2H),7.28(d,J＝7.6Hz,2H),6.72(d,J＝8.8Hz,2H),4.67(s,2H),4.56(s,2H),2.99(s,6H),2.37(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.20,164.51,152.99,139.90,134.35,129.76,129.71(2C),129.41(2C),129.29,127.44,127.26(2C),119.75,111.16(2C),50.88,48.16(2C),45.55,21.40.HRMS(ESI) + calculated for C 22 H 24 N 5 O 2 ,[M+H] + :m/z 390.1917,found:390.1925.
4- (dimethylamino) -N- (5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 d). White powder, melting point: 303.4-305.3 ℃, yield: 69.5%. 1 HNMR(400MHz,DMSO-d 6 )δ(ppm):12.25(s,1H),10.56(s,1H),7.90(d,J＝8.4Hz,2H),7.84(d,J＝8.0Hz,2H),7.82(d,J＝8.0Hz,2H),6.73(d,J＝8.4Hz,2H),4.73(d,J＝28.4Hz,2H),4.56(s,2H),2.99(d,J＝14.0Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):168.67,168.47,164.51,152.97,141.25,130.51,129.78,129.73(2C),128.23(2C),128.10(2C),125.84,123.06,111.21,111.12(2C),50.97,48.16(2C),45.65.HRMS(ESI) + calculated for C 22 H 20 F 3 N 5 NaO 2 ,[M+Na] + :m/z 466.1454,found:466.1461.
4- (dimethylamino) -N- (5- (4- (dimethylamino) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 e). White powder, melting point: 306.8-310.2 ℃, yield: 63.9%. 1 HNMR(400MHz,TFA-d 6 )δ(ppm):11.69(s,1H),11.61(s,1H),8.21(d,J＝9.6Hz,2H),7.97(d,J＝8.8Hz,2H),7.92–7.83(m,2H),7.79(d,J＝9.6Hz,2H),5.25(s,2H),4.99(s,2H),3.48(s,12H).HRMS(ESI) + calculated for C 23 H 26 N 6 NaO 2 ,[M+Na] + :m/z 441.2014,found:441.2009.
4- (dimethylamino) -N- (5- (3- (dimethylamino) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 f). White powder, melting point: 294.6-297.8 ℃, yield: 71.3%. 1 HNMR(400MHz,DMSO-d 6 )δ(ppm):12.22(s,1H),10.49(s,1H),7.86(d,J＝8.4Hz,2H),7.26(q,J＝7.2Hz,1H),6.83(d,J＝7.6Hz,1H),6.80(s,1H),6.78(d,J＝7.6Hz,1H),6.69(d,J＝8.4Hz,2H),4.66(s,2H),4.53(s,2H),2.99(s,6H),2.93(s,6H).HRMS(ESI) + calculated for C 23 H 26 N 6 NaO 2 ,[M+Na] + :m/z 441.1999,found:441.2009.
N- (5- (1-naphthoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) -4- (dimethylamino) benzamide (13 g) white powder, melting point: 310.4-313.3 ℃, yield: 64.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.26(s,1H),10.45(s,1H),8.09–7.99(m,2H),7.93(s,1H),7.87(d,J＝3.6Hz,1H),7.72(s,1H),7.66–7.61(m,1H),7.60(s,1H),7.59(s,1H),7.58(t,J＝2.4Hz,1H),6.69(d,J＝5.6Hz,2H),4.85(s,2H),4.27(s,2H),2.98(s,6H).HRMS(ESI) + calculated for C 25 H 23 N 5 NaO 2 ,[M+Na] + :m/z448.1738,found:448.1744.
N- (5- (2-naphthoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) -4- (dimethylamino) benzamide (13 h). White powder, melting point: 313.1-315.7 ℃, yield: 52.9%. 1 H NMR(400MHz,TFA-d 6 )δ(ppm):11.86(s,1H),11.71(s,1H),8.14(d,J＝4.4Hz,1H),8.07(d,J＝8.4Hz,1H),7.98(d,J＝8.4Hz,1H),7.93(d,J＝8.8Hz,2H),7.84(d,J＝4.4Hz,1H),7.76(d,J＝8.8Hz,1H),7.68(d,J＝8.8Hz,1H),7.64–7.59(m,1H),7.41(d,J＝8.8Hz,2H),5.57–5.12(m,2H),4.97(s,2H),3.27(s,6H).HRMS(ESI) + calculated for C 25 H 23 N 5 NaO 2 ,[M+Na] + :m/z 448.1749,found:448.1744.
4- (dimethylamino) -N- (5-isonicotinyl-1, 4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 i). White powder, melting point: 303.1-305.8 ℃ and yield: 66.2%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.11(s,1H),10.52(s,1H),8.73(d,J＝4.8Hz,2H),7.86(d,J＝5.6Hz,2H),7.56(d,J＝5.6Hz,2H),6.69(d,J＝4.8Hz,2H),4.70(s,2H),4.54(s,2H),2.99(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):165.15,164.37,152.46,152.09,147.03,143.61(2C),143.45,129.87,129.76(2C),125.23,125.14(2C),112.38(2C),50.11,45.76,40.48(2C).HRMS(ESI) + calculated for C 20 H 20 N 6 NaO 2 ,[M+Na] + :m/z 399.1539,found:399.154.
4- (dimethylamino) -N- (5-nicotinoyl-1, 4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 j). White powder, melting point: 251.3-257.6 ℃ and yield: 55.8%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.25(s,1H),10.53(s,1H),8.81(dd,J＝6.0,2.4Hz,1H),8.69(t,J＝5.6Hz,1H),8.03(d,J＝8.0Hz,1H),7.96–7.77(m,2H),7.52(dd,J＝12.0,4.8Hz,1H),6.72(d,J＝8.4Hz,2H),4.70(d,J＝24.4Hz,2H),4.61(s,2H),2.99(d,J＝12.4Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):167.81,164.53,153.01,151.09,148.04,135.14,135.09,133.03,129.78,129.73(2C),124.06,123.89,111.23,111.15(2C),50.97,48.16(2C),45.55.HRMS(ESI) + calculated for C 20 H 21 N 6 O 2 ,[M+H] + :m/z 377.1715,found:377.1721.
4- (dimethylamino) -N- (5- (4- (4-methylpiperazin-1-yl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 k). White powder, melting point: 315.8-318.1 ℃, yield: 51.8%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):10.70(s,1H),9.88(s,1H),7.90(d,J＝8.8Hz,2H),7.57(d,J＝8.8Hz,2H),7.09(d,J＝8.8Hz,2H),6.80(d,J＝8.8Hz,2H),4.87(s,2H),4.48(s,2H),4.00(d,J＝12.0Hz,2H),3.56(d,J＝12.0Hz,2H),3.19(t,J＝12.0Hz,2H),3.09(t,J＝12.0Hz,2H),3.02(s,6H),2.89(s,3H).HRMS(ESI) + calculated for C 26 H 32 N 7 O 2 ,[M+H] + :m/z 474.2606,found:474.2612.
4- (dimethylamino) -N- (5- (3- (4-methylpiperazin-1-yl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (13 l) white powder, melting point: 272.3-277.8 ℃, yield: 67.4%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.15(s,1H),10.51(s,1H),7.87(d,J＝8.6Hz,2H),7.29(q,J＝7.6Hz,1H),7.08(s,1H),7.03(dd,J＝6.0,2.8Hz,1H),6.93(d,J＝7.2Hz,1H),6.72(d,J＝8.6Hz,2H),4.65(s,2H),4.53(s,2H),3.18(s,4H),2.99(s,6H),2.45(s,4H),2.22(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.33,170.06,164.42,153.00,150.26,138.36,129.77(2C),129.73,119.05,117.93,117.04,116.08,114.25,111.22,111.15(2C),53.24(2C),46.45(2C),45.51,43.49(2C),40.10,40.08.HRMS(ESI) + calculated for C 26 H 32 N 7 O 2 ,[M+H] + :m/z 474.2603,found:474.2612.
4- ((5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) carbamoyl) benzoic acid (14 a) white powder, melting point: 316.6-318.4 ℃, yield: 80.7%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.85(s,2H),11.19(s,1H),8.14(d,J＝8.0Hz,1H),8.06(d,J＝4.0Hz,1H),8.03(s,1H),8.01(s,1H),7.49(dd,J＝8.0Hz,2H),7.29(d,J＝8.0Hz,2H),4.70(s,2H),4.60(s,2H),2.37(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):167.10,163.84,139.90,137.31,134.31,134.03,129.74,129.66(2C),129.38(2C),129.27,128.57,128.51(2C),127.45,127.29(2C),50.88,45.56,21.40.HRMS(ESI) + calculated for C 21 H 18 N 4 NaO 4 ,[M+Na] + :m/z413.1216,found:413.122.
4- ((5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) carbamoyl) benzoic acid (14 b). White powder, melting point: 296.5-298.1 ℃, yield: 85.9%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):11.71(s,1H),11.95(s,1H),11.40(s,1H),8.19(d,J＝8.2Hz,1H),8.09(d,J＝5.2Hz,1H),8.07(d,J＝5.2Hz,1H),8.01(d,J＝8.2Hz,1H),7.88(d,J＝8.2Hz,2H),7.83(d,J＝8.2Hz,2H),4.75(s,2H),4.59(s,2H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):168.66,167.04,163.83,141.16,137.31,137.16,134.08,129.74,129.66(2C),129.38(2C),128.61,128.54(2C),128.25,128.12(2C),125.98,50.62,45.66.HRMS(ESI) + calculated for C 21 H 15 F 3 N 4 NaO 4 ,[M+Na] + :m/z 467.0934,found:467.0938.
4-amino-N- (5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (15 a). White powder, melting point: 180.6-188.3 ℃ and yield: 55.9%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.18(s,1H),10.32(s,1H),7.71(d,J＝8.4Hz,2H),7.49(d,J＝8.0Hz,2H),7.28(t,J＝8.0Hz,2H),6.53(s,2H),5.77(d,J＝8.4Hz,2H),4.68(s,2H),4.56(s,2H),2.37(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.15,164.59,152.88,139.85,134.41,130.01,129.96(2C),129.39(2C),129.28,127.46,127.26(2C),113.06,113.00(2C),55.35,45.55,21.41.HRMS(ESI) + calculated for C 20 H 19 N 5 NaO 2 ,[M+Na] + :m/z 384.1424,found:384.1431.
4-amino-N- (5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (15 b): white powder, melting point: 303.2-305.7 ℃ and yield: 49.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.21(s,1H),10.43(s,1H),7.87(d,J＝8.4Hz,2H),7.80(d,J＝8.4Hz,2H),7.66(d,J＝8.4Hz,2H),6.57(d,J＝8.4Hz,2H),5.80(s,2H),4.69(s,2H),4.52(s,2H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):168.67,168.47,164.60,141.28,130.02,129.97(2C),128.23,128.09(2C),125.97(2C),125.82,125.78,123.06,113.06,112.99(2C),55.35,45.66.HRMS(ESI) + calculated for C 20 H 16 F 3 N 5 NaO 2 ,[M+Na] + :m/z 438.1152,found:438.1148.
4-amino-N- (5- (3- (dimethylamino) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) benzamide (15 c). White powder, melting point: 175.6-178.3 ℃ and the yield: 43.5%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.21(s,1H),10.41(s,1H),7.71(d,J＝8.4Hz,2H),7.31–7.22(m,1H),6.82(d,J＝7.6Hz,1H),6.80(s,1H),6.77(d,J＝7.6Hz,1H),6.56(d,J＝8.4Hz,2H),5.77(s,2H),4.67(s,2H),4.51(s,2H),2.93(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.85,164.58,150.69,150.65,138.15,130.04,129.99(2C),129.52,129.40,114.28,113.68,113.08,113.02(2C),110.77,110.51,50.97,48.16(2C),45.49.HRMS(ESI) + calculated for C 21 H 22 N 6 NaO 2 ,[M+Na] + :m/z 391.1871,found:391.1877.
4-amino-N- (5 isonicotinyl-1, 4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) benzamide (15 d). White powder, melting point: 198.6-202.3 ℃ and yield: 39.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.15(s,1H),10.41(s,1H),8.72(t,J＝6.8Hz,2H),7.71(d,J＝8.4Hz,2H),7.61–7.53(m,2H),6.56(d,J＝8.4Hz,2H),5.80(s,2H),4.67(s,2H),4.51(s,2H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):165.87,164.35,150.68,145.24,144.89,130.00,129.93(2C),124.57,124.36(2C),117.41(2C),117.00,114.52(2C),50.30,45.78.HRMS(ESI) + calculated for C 18 H 17 N 6 O 2 ,[M+H] + :m/z 349.1509,found:349.1508.
4-amino-N- (5-nicotinoyl-1, 4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) benzamide (15 e). White powder, melting point: 299.8-301.7 ℃, yield: 54.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.50(s,1H),11.33(s,1H),8.81(dd,J＝7.6,2.0Hz,1H),8.69(t,J＝4.4Hz,1H),8.36(d,J＝8.4Hz,1H),8.30(d,J＝8.4Hz,1H),8.25(d,J＝8.4Hz,1H),8.16(d,J＝8.4Hz,1H),8.05(t,J＝8.8Hz,1H),7.53(dd,J＝9.2,4.4Hz,1H),6.35(s,2H),4.75(s,2H),4.65(s,2H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):165.18,164.90,163.04,149.76,149.72,145.54,143.04,142.71,142.13,134.98,129.70(2C),126.90,126.81,123.80(2C),50.60,45.84.HRMS(ESI) + calculated for C 18 H 16 N 6 NaO 2 ,[M+Na] + :m/z 371.1223,found:371.1227.
1- (5- (4-methylbenzoyl) -1,4,5, 6-tetrahydropyrrole [3,4-c ]]Pyrazol-3-yl) imidazolidin-2-one (19 a). White powder, melting point: 315.3-318.1 ℃, yield: 62.7%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.08(s,1H),7.46(d,J＝7.6Hz,2H),7.26(d,J＝7.6Hz,2H),6.85(s,1H),4.63(s,2H),4.51(s,2H),3.76(s,2H),3.38(d,J＝8.0Hz,2H),2.36(d,J＝8.0Hz,2H).HRMS(ESI) + calculated for C 16 H 17 N 5 NaO 2 ,[M+Na] + :m/z 334.1268,found:334.1274.
1- (5- (4- (trifluoromethyl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3, 4-c)]Pyrazol-3-yl) imidazolin-2-one (19 b). White powder, melting point: 313.5-317.2 ℃, yield: 80.1%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.16(s,1H),7.84(t,J＝8.0Hz,2H),7.77(d,J＝8.0Hz,2H),6.89(s,1H),4.67(s,2H),4.49(s,2H),3.77(t,J＝8.0Hz,2H),3.38(t,J＝8.0Hz,2H).HRMS(ESI) + calculated for C 16 H 14 F 3 N 5 NaO 2 ,[M+Na] + :m/z388.0985,found:388.0992.
1- (5- (3- (dimethylamino) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) imidazolin-2-one (19 c). White powder, melting point: 194.6-198.4 ℃ and yield: 77.9%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.07(s,1H),7.25(t,J＝7.6Hz,1H),6.89(m,1H),6.81(d,J＝2.4Hz,1H),6.78(s,1H),6.75(d,J＝7.6Hz,1H),4.63(s,2H),4.51(s,2H),3.76(d,J＝8.0Hz,2H),3.38(d,J＝8.0Hz,2H),2.92(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.68,170.51,150.63,138.06,137.91,129.42,114.50,114.43,113.80,110.74,110.66,45.21,43.99,40.52,40.49,37.95(2C).HRMS(ESI) + calculated for C 17 H 21 N 6 O 2 ,[M+H] + :m/z 341.1711,found:341.1721.
1- (5-Isovinyl-1, 4,5, 6-tetrahydropyrrole [3,4-c ]]Pyrazol-3-yl) imidazolin-2-one (19 d). White powder, melting point: 314.2-316.5 ℃, yield: 71.2%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.25(s,1H),9.08(d,J＝5.6Hz,2H),8.21(d,J＝5.6Hz,2H),7.01(m,1H),4.73(s,2H),4.54(s,2H),3.89–3.75(m,2H),3.41(t,J＝8.2Hz,2H).HRMS(ESI) + calculated for C 14 H 15 N 6 O 2 ,[M+H] + :m/z 299.1249,found:299.1251.
1- (5-nicotinoyl-1, 4,5, 6-tetrahydropyrrole [3, 4-c)]Pyrazol-3-yl) imidazolin-2-one (19 e). White powder, melting point: 303.2-305.8 ℃ and yield: 64.3%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.32(s,1H),9.19(t,J＝7.6Hz,1H),9.00(s,1H),8.76(d,J＝6.4Hz,1H),8.12(s,1H),6.94(s,1H),4.72(s,2H),4.64(s,2H),3.81(d,J＝8.8Hz,2H),3.42(d,J＝8.8Hz,2H).HRMS(ESI) + calculated for C 14 H 15 N 6 O 2 ,[M+H] + :m/z299.1248,found:299.1251.
1- (5- (4-methylpiperazin-1-yl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c]Pyrazol-3-yl) imidazolidin-2-one (19 f). White powder, melting point: 272.7-279.3 ℃ and the yield: 79.6%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.13(s,1H),7.48(d,J＝8.0Hz,2H),6.97(d,J＝8.0Hz,2H),6.81(s,1H),4.65(s,2H),4.60(s,2H),3.79(d,J＝8.0Hz,2H),3.44(d,J＝8.0Hz,2H),3.42–3.34(m,4H),2.56(s,4H),2.30(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):169.82,158.58,150.81,129.11,129.03(2C),127.51,118.63,115.69,115.09(2C),52.50(2C),51.11,45.43,45.17(2C),44.00,42.47,37.94.HRMS(ESI) + calculated for C 20 H 26 N 7 O 2 ,[M+H] + :m/z 396.2139,found:396.2142.
1- (5- (3- (4-methylpiperazin-1-yl) benzoyl) -1,4,5, 6-tetrahydropyrrolo [3,4-c ]]Pyrazol-3-yl) imidazolidin-2-one (19 g). White powder, melting point: 216.5-220.4 ℃ and yield: 76.1%. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm):12.07(s,1H),7.28(t,J＝7.6Hz,1H),7.05(d,J＝9.6Hz,1H),7.03(d,J＝3.6Hz,1H),6.96–6.90(m,1H),6.85(m,1H),4.62(s,2H),4.50(s,2H),3.77(d,J＝7.2Hz,2H),3.40(d,J＝7.2Hz,2H),3.20(s,4H),2.54(s,4H),2.28(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ(ppm):170.36,170.18,158.64,151.14,138.09,137.97,129.52,117.33,117.25,116.85,113.83,65.38,54.73(2C),48.01(2C),45.81,43.99,37.93,15.63.
The following experiments prove the beneficial effects of the invention.
Experimental example 1: in vitro kinase Activity evaluation
1. Experimental method
The inhibition of ERK1/2 kinase by the target compounds of the invention at a concentration of 1. Mu.M was determined using the ERK1/2 (Phospho-T202/Y204) TR-FRET cell assay kit. As a positive control, the known ERK1/2 kinase inhibitor SCH772984 was used.
ERK1/2 and ERK-TR-FRET cell detection kits were purchased from Biorbyt, and the detection method performed cell processing, cell lysis, protein detection experiments according to the detection manual of the kit, by quantifying the amount of ATP remaining in the solution after kinase reaction to measure kinase activity. The experimentally determined luminescence signal correlates with the amount of ADP present and with the amount of kinase activity.
Further, the inhibition of ERK1/2 by 13l, which is a target compound having a good inhibitory activity at a low concentration, at various concentrations was examined, and Gra was usedNon-linear regression analysis by phPad Prism software to calculate IC 50 Values. IC (integrated circuit) 50 Defined as the concentration of compound required for inhibition of 50%.
2. Experimental results
TABLE 3 inhibition of ERK1/2 kinase by target compounds at 1. Mu.M
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TABLE 4 inhibition of ERK1/2 kinase by target compounds at 1. Mu.M
It can be seen that the compounds of the present invention have good inhibitory effect on ERK1/2 kinase at lower concentrations (1. Mu.M). Among them, the compound 13l has the best inhibition effect, and the inhibition rate of ERK1 and ERK2 kinase is up to 62.41% and 65.96%. Further calculation of IC of Compound 13l for ERK1, ERK2 kinase 50 The values were 1.69. Mu.M and 0.87. Mu.M, respectively (FIG. 1).
The experimental results show that the compound can effectively inhibit ERK1/2 kinase and can be used as an ERK1/2 kinase inhibitor.
Experimental example 2: in vitro cell proliferation inhibition assay
1. Experimental method
This experiment evaluates that compound 13l carries K for both speciesAnti-proliferative activity of Ras-activated mutant SW-620 and HCT-116 tumor cell lines. First, SW620 and HCT116 human colon cancer cells in the logarithmic growth phase were formulated into cell suspensions with 10% PBS and were mixed at 1X 10 5 The density of individual cells/mL of medium was inoculated into 96-well plates, each with a volume of 100. Mu.L, followed by transfer to 37℃with 5% CO 2 Is cultured in an incubator for 12 hours. Then compound 13l was dissolved in DMSO and treated with DMEM medium, diluted to various target concentration gradient solutions (0.3. Mu.M, 0.5. Mu.M, 1.0. Mu.M, 5.0. Mu.M), and test compound at different concentrations was added to each well to treat cells, followed by transfer to 37℃with 5% CO 2 Is incubated for 24-72h in an incubator. Next, the prepared MTT solution at a concentration of 5mg/ml was added to the above-mentioned well plate (20. Mu.L/well) of the incubated cells, and transferred again to a 96-well plate containing 5% CO at 37 ℃ 2 And (2) incubating for 2-4h, then discarding the culture medium in the wells, adding 150 mu L of DMSO solution into each well, immediately oscillating the well plate by using a shaking table until the crystals are fully dissolved, finally measuring the absorbance value of each well at 570nm wavelength by using an enzyme-labeled instrument, and taking three compound wells to measure the inhibition rate. Inhibition ratio= (control group-dosing group)/control group x 100%. Calculation of IC using GraphPad Prism6 to analyze data 50 Values.
As a positive control, the known ERK1/2 kinase inhibitor SCH772984 was used.
2. Experimental results
TABLE 5 IC of Compound 13l against human colorectal cancer cells 50 Value of
The data in Table 5 and FIG. 2 show that Compound 13l exhibits potent inhibitory activity against both human colorectal cancer cell lines, IC against SW-620 and HCT-116 cells 50 The values were 1.16. Mu.M and 0.59. Mu.M, respectively.
Experimental example 3: protein immunofluorescence imprinting assay
1. Experimental method
(1) Sample preparation
SW620 and HCT116 human colon cancer cells in logarithmic growth phase were cultured at 1X 10, respectively 5 The density of cells/well was distributed on a 6-well plate, and after adding an appropriate amount of DMEM medium, it was left at 37 ℃ and contained 5% co 2 Is incubated for 12h in an incubator. After the cell density reached 80% -90%, both cells were treated with 13l of medium containing different concentrations (0.5. Mu.M, 1.0. Mu.M) of the compound, respectively, for 24h. The cells were then collected with a clean spatula, then washed with 3ml of 4℃pre-chilled PBS buffer, centrifuged at 1500rpm for 5 minutes, the supernatant discarded, and the washing operation repeated twice to wash out the culture. The washed cells are then lysed on ice with an appropriate amount of lysis buffer containing protease inhibitor and phosphatase inhibitor, which requires frequent shaking back and forth and should be performed on ice for at least 30 minutes in order to allow the cells to be fully lysed. After lysis, the cell debris and lysate were transferred with a gun to a 1.5ml centrifuge tube, followed by centrifugation at 12000rpm for 10 minutes in a centrifuge pre-chilled at 4 ℃. And collecting supernatant fluid, and then carrying out the next protein content measurement experiment.
(2) Protein content determination
Firstly, preparing BCA working solution 2mL for later use according to the volume ratio of 50:1 of the solution A and the solution B, then diluting the protein sample concentration to 0.5mg/mL by using PBS, and then carrying out gradient dilution according to the following table 6 for drawing a standard concentration curve.
TABLE 6 Standard concentration Curve gradient dilution Table
Numbering device 1 2 3 4 5 6 7 8
Protein standard diluent (mu L) 0 2 4 6 8 12 16 20
PBS Diluent(μL) 20 18 16 14 12 8 4 0
Final protein concentration (mg/mL) 0 0.05 0.10 0.15 0.20 0.30 0.40 0.50
Next, 20. Mu.L of each sample in the table was carefully added to the 96-well plate, then 200. Mu.L of BCA working solution was added to each well, and after shaking uniformly, the wells were allowed to develop at 37℃for 15-30min. And finally, measuring the absorbance value of each hole by using an enzyme-labeled instrument at 570nm wavelength, drawing a standard curve by using a blank control to obtain a regression equation, and substituting the absorbance value of the measuring tube into the equation to obtain the protein concentration of each group.
(3) SDS-PAGE electrophoresis
And clamping the cleaned glass plate by using a clamp and vertically clamping the glass plate on a glue preparation frame, preparing 10% of separation glue according to detection proteins, then adding TEMED and uniformly mixing the separation glue, then slowly pouring the glue along a glass crack by using a pipetting gun, and then adding isopropanol into the upper layer of the glue to seal the glue so as to enable the glue to solidify faster. Standing at room temperature for 30min, pouring out isopropanol for sealing after the glue is fully solidified, cleaning with distilled water, and wiping off water for later use. Adding 4% concentrated gel according to the same method, adding TEMED, mixing thoroughly, pouring gel again, horizontally inserting the comb into the concentrated gel, standing at room temperature for 30min again, slowly pulling out the comb after the gel is completely solidified, washing the gel, placing the gel into an electrophoresis tank completely, and adding samples with measured protein content into sample adding holes respectively. Finally, electrophoresis is carried out for 3-4 hours under 80V until the electrophoresis is finished, and then the next experiment can be carried out.
(4) Transfer film
First, 6 pieces of filter paper and 1 piece of PVDF membrane were cut according to the size of the gel for use, and then the activated PVDF membrane was immersed in methanol. Then slowly taking the gel out of the glass plate for standby, placing the membrane into a transfer liquid, placing the black surface of the membrane clamp upwards, sequentially laying a layer of foam cushion, three layers of filter paper, gel, PVDF membrane, three layers of filter paper and a layer of foam cushion, and finally closing the red surface of the membrane clamp (air bubbles should be avoided in the process). Finally, the prepared transfer film clamp is placed into a transfer film groove, and after the transfer liquid is filled up, the transfer film is transferred for 2h under 80V (in the process, a little ice block is added on the other side of the groove to dissipate heat generated during electric transfer).
(5) Immune response
First, the above PVDF membrane was rinsed three times with PBST buffer, and then transferred to PBST blocking solution containing 5% nonfat milk powder for 1h at room temperature. And then rinsing the sealed PVDF membrane with PBST buffer solution for three times, transferring the PVDF membrane into a hybridization bag containing a proper amount of primary antibody diluent solution, incubating the PVDF membrane on a shaking table for 1-2 hours at room temperature, rinsing the PVDF membrane with PBST buffer solution for three times again, and incubating the PVDF membrane with secondary antibody diluent solution for 1-2 hours at room temperature again by the same method.
(6) Color development
And rinsing the incubated PVDF membrane with PBST buffer solution for three times, then uniformly dripping developing solution on the membrane protein surface, and immediately detecting the experimental result after 1 minute.
2. Experimental results
The expression level of the apoptosis-related protein of HCT116 cell after the treatment of compound 13l was examined. As shown in the results of FIG. 3, compound 13l decreased the intracellular phosphorylation levels of ERK1/2 (Thr 202, tyr 204) and p90RSK (Thr 359, ser 363) proteins in HCT116 cells in a concentration-dependent manner.
Experimental example 4: apoptosis experiments
1. Experimental method
The experiment adopts an Annexin V-FITC/PI staining method and a Hoechst 33258 staining method to determine the apoptosis rate of the cells. First, HCT116 human colon cancer cells in the logarithmic growth phase were cultured at 1X 10 5 The density of cells/well was distributed on a 6-well plate, and after adding an appropriate amount of DMEM medium, it was left at 37 ℃ and contained 5% co 2 Is incubated for 12h in an incubator. After the cell density reached 80% -90%, HCT116 cells were treated with 13l of medium containing different concentrations (0, 0.5. Mu.M, 1.0. Mu.M) of the compound, respectively. After 24h 13l of treated and untreated cells were harvested, whereupon the treated cells were digested with EDTA-free trypsin, centrifuged at 3000rpm for 5 min, the cells were washed twice with pre-chilled PBS, the supernatant was subsequently discarded, and 1-5X 10 was selected for collection 5 Individual cells. Next, 100. Mu.L of the binding buffer was added at room temperature for air-blown mixing, followed by further addition of 5Mu L of Annexin-VFITC and 5 mu L of PI dye solution are uniformly mixed by shaking, incubated for 15 minutes at room temperature in a dark place, and finally the cells are observed and detected by a flow cytometer, and the results are analyzed by using Flowjo software.
2. Experimental results
The experiment searches the death cause of the cells by detecting the apoptosis rate of the cells through a flow cytometer. First, concentrated bright apoptotic nuclei were readily observed after staining HCT116 cells treated with Compound 13l with Hoechst 33258 (as shown in FIG. 4). Next, after treating HCT116 cells with compound 13l at concentrations of 0, 0.5 and 1. Mu.M, respectively, for 24 hours, the cells were stained by Annexin V-FITC/PI, followed by observation and detection on the cells by a flow cytometer. The results of apoptosis are shown in FIG. 5, which shows that compound 13l can induce apoptosis of HCT116 human colon cancer cells in a concentration-dependent manner. At a concentration of 1. Mu.M for compound 13l, the total apoptosis rate of HCT116 cells can reach 23.7%. It was further confirmed that compound 13l induced apoptosis in human colon cancer cells.
In summary, the invention provides an ERK inhibitor and pharmaceutical application thereof. The compound provided by the invention has good inhibition effect on ERK1/2 kinase, shows effective inhibition activity on colorectal cancer cell lines, can reduce the phosphorylation level of ERK1/2 (Thr 202, tyr 204) and p90RSK (Thr 359, ser 363) proteins in colorectal cancer cells in cells, and can induce apoptosis of colon cancer cells. The compound can be used for preparing ERK1/2 kinase inhibitors and medicines for preventing and/or treating diseases related to ERK1/2 kinase activity, and has wide application prospect.

Claims (7)

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: the compound is as follows:
。
2. a medicament, characterized in that: the preparation is prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
3. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of an ERK inhibitor.
4. Use according to claim 3, characterized in that: the ERK inhibitor is ERK1 inhibitor and/or ERK2 inhibitor.
5. Use according to claim 3 or 4, characterized in that: the ERK inhibitor is a medicament for preventing and/or treating diseases related to ERK activity.
6. Use according to claim 5, characterized in that: the disease associated with ERK activity is cancer.
7. Use according to claim 6, characterized in that: the cancer is colorectal cancer, lung cancer, pancreatic cancer, melanoma, acute myeloid leukemia, glioblastoma.
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