CN115057860B - An ERK inhibitor and its pharmaceutical use - Google Patents

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

An ERK inhibitor and its pharmaceutical use

Technical Field

The invention belongs to the field of pharmaceuticals, and in particular relates to an ERK inhibitor and pharmaceutical use thereof.

Background Art

The mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway that regulates a variety of cellular processes and plays a vital role in physiological processes such as tumor cell proliferation, differentiation, survival, migration and apoptosis. Extracellular signal-regulated kinase (ERK) is a class of serine and threonine protein kinases discovered in the late 1980s that regulate cell signaling under both normal and pathological conditions. The expression of ERK is essential for the development of the human body, and their overactivation plays a vital role in the survival and development of tumor cells. The RAS-RAF-MEK-ERK signaling cascade is the most classic MAPK pathway, and a large number of studies have shown that persistent dysregulation or abnormal activation of protein kinases in this signaling pathway can induce a variety of diseases such as cancer, inflammation, developmental disorders and the nervous system. Therefore, protein kinases related to the ERK/MAPK pathway have become one of the important drug targets.

After decades of unremitting efforts, small molecule inhibitors targeting upstream targets of the MAPK pathway, such as BRAF, RAS, and MEK, have been developed one after another, and some have even been approved by the FDA for marketing, such as vemurafenib, dabrafenib, and trametinib. Although these drugs have also shown relatively excellent anti-tumor activity in clinical trials, they inevitably develop drug resistance caused by kinase mutations after a few months of clinical treatment, which seriously limits the further development of such drugs. Studies have found that mutations in RAS and BRAF-related kinases upstream of the MAPK pathway, leading to the reactivation of the terminal kinase ERK1/2 in the pathway, are key events in the acquired resistance of BRAF and MEK inhibitors. Since ERK is located at the end of the MAPK signaling pathway, it rarely mutates, and ERK can only be activated by MEK, so inhibiting the activity of ERK1/2 can block the pathological effects of this pathway. Preclinical studies have shown that ERK inhibitors can overcome the acquired resistance of tumor cells to upstream kinase inhibitors, exhibit significant anti-tumor cell proliferation effects, and reverse the abnormal activation of the MAPK pathway caused by upstream pathway mutations.

In recent years, many ERK inhibitors have been designed and synthesized, and have been shown to have excellent ERK inhibitory activity and anti-tumor cell proliferation effects. Ulixertinib (BVD-523) is the first reversible ATP-competitive small molecule ERK1/2 kinase inhibitor to enter the clinical stage, with _Ki values for ERK1/2 of 0.3 and 0.04 nmol/L, respectively. Studies have shown that Ulixertinib can inhibit the proliferation of BRAF-mutated melanoma cells (A375) and colorectal cancer cells (Colo205), and also shows good anti-tumor activity and low toxicity in an in vivo K-RAS-mutated pancreatic cancer xenograft tumor animal model. LY3214996 has an IC ₅₀ value of 5nmol/L for ERK1/2. Oral administration of LY3214996 alone not only significantly inhibits the growth of tumor cells in vivo, but also shows good tolerability in patients with BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal cancer, lung cancer and pancreatic cancer xenografts. SCH772984 is an ATP-competitive ERK1/2 inhibitor with IC ₅₀ values of 4 and 1nmol/L for ERK1/2, respectively. In the BRAF ^V600E mutant human melanoma cell line LOX, SCH772984 not only binds to inactive ERK1/2 to block the phosphorylation activation of the kinase, but also promotes the dephosphorylation of the active kinase. In addition, SCH772984 also showed good anti-tumor cell proliferation effects in several xenograft tumor models with KRAS or BRAF mutations.

Although several ERK inhibitors have entered clinical research in the early stage, a considerable number of these inhibitors have terminated clinical research due to excessive toxicity, poor drugability or other reasons, and no ERK inhibitor has been approved for marketing. Therefore, the development of more new ERK inhibitors is of great significance to promote the early approval of ERK inhibitors for marketing.

Summary of the invention

The purpose of the present invention is to provide a new ERK inhibitor and its pharmaceutical use.

The present invention provides a compound, a pharmaceutically acceptable salt thereof, and a stereoisomer thereof, wherein the structure of the compound is shown in Formula I:

Wherein, R _b is hydrogen, R _c is phenyl which is unsubstituted or substituted by R _d ; R _d is selected from C _1-3 alkyl, hydroxyl, carboxyl, NR _e R _f ; R _e and R _f are each independently selected from hydrogen and C _1-3 alkyl;

Alternatively, R _b and R _c are connected to form a ring;

_Y1 is selected from CH, N, _Y2 is selected from CH, N;

m is selected from 1, 2 or 3;

_Ra is independently selected from _C1-5 alkyl, halogen-substituted _C1-5 alkyl, _{NRgRh ,} unsubstituted or substituted 3-6 membered saturated cycloalkyl, unsubstituted or substituted 3-6 membered saturated heterocyclic group; the substituent is selected from _C1-5 alkyl; or two adjacent _Ra are connected to form a benzene ring;

R _g and R _h are each independently selected from hydrogen and C _1-5 alkyl.

Furthermore, the structure of the compound is shown in Formula II:

Wherein, R _d is selected from C _1-3 alkyl, hydroxyl, carboxyl, NR _e R _f ;

R _e and R _f are each independently selected from hydrogen and C _1-3 alkyl;

_Y1 is selected from CH, N, _Y2 is selected from CH, N;

m is selected from 1, 2 or 3;

_Ras are each independently selected from _C1-3 alkyl, halogen-substituted _C1-3 alkyl, _NRgRh , unsubstituted or substituted 5-6 membered saturated cycloalkyl, unsubstituted or substituted 5-6 membered _saturated heterocyclic group; the substituent is selected from _C1-3 alkyl;

R _g and R _h are each independently selected from hydrogen and C _1-3 alkyl.

Furthermore, the structure of the compound is as shown in Formula III-1 or Formula III-2:

Wherein, R _d is selected from C _1-3 alkyl, hydroxyl, carboxyl, NR _e R _f ;

R _e and R _f are each independently selected from hydrogen and C _1-3 alkyl;

_Y1 is CH, _Y2 is N; or _Y2 is CH, _Y1 is N;

R _a1 and R _a2 are each independently selected from hydrogen, C _1-3 alkyl, halogen-substituted C _1-3 alkyl, NR _g R _h , and _Ra1 and _Ra2 are not hydrogen at the same time;

R _g and R _h are each independently selected from hydrogen and C _1-3 alkyl;

R _i is selected from hydrogen, C _1-3 alkyl.

Furthermore, the structure of the compound is shown in Formula IV:

Wherein, Y ₁ is selected from CH, N, and Y ₂ is selected from CH, N;

m is selected from 1, 2 or 3;

_Ras are each independently selected from _C1-3 alkyl, halogen-substituted _C1-3 alkyl, _NRgRh , unsubstituted or substituted 5-6 membered saturated cycloalkyl, unsubstituted or substituted 5-6 membered _saturated heterocyclic group; the substituent is selected from _C1-3 alkyl;

R _g and R _h are each independently selected from hydrogen and C _1-3 alkyl.

Furthermore, the structure of the compound is shown in Formula V-1 or Formula V-2:

wherein _Y1 is CH and _Y2 is N; or _Y2 is CH and _Y1 is N;

R _a1 and R _a2 are each independently selected from hydrogen, C _1-3 alkyl, halogen-substituted C _1-3 alkyl, NR _g R _h , and _Ra1 and _Ra2 are not hydrogen at the same time;

R _g and R _h are each independently selected from hydrogen and C _1-3 alkyl;

R _i is selected from hydrogen, C _1-3 alkyl.

Furthermore, the compound is selected from:

The present invention also provides a medicine, which is a preparation prepared by using the above compound, its pharmaceutically acceptable salt, or its stereoisomer as active ingredients and adding pharmaceutically acceptable auxiliary materials.

The present invention also provides the use of the above compound, its pharmaceutically acceptable salt, and its stereoisomer in the preparation of ERK inhibitors.

Furthermore, the ERK inhibitor is an ERK1 inhibitor and/or an ERK2 inhibitor.

Furthermore, the ERK inhibitor is a drug 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.

The experimental results show that the compounds provided by the present invention have a good inhibitory effect on ERK1/2 kinases. Among them, compound 131 has the best inhibitory effect, with an inhibition rate of 62.41% and 65.96% for ERK1 and ERK2 kinases at 1 μM, and IC ₅₀ values for ERK1 and ERK2 kinases of 1.69 μM and 0.87 μM, respectively. The compounds of the present invention can be used to prepare ERK1/2 kinase inhibitors.

It is well known to those skilled in the art that a variety of diseases including colorectal cancer, lung cancer, pancreatic cancer, melanoma, acute myeloid leukemia, glioblastoma, etc. are related to 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, etc. Therefore, the compounds provided by the present invention can be used to prepare drugs for preventing and/or treating diseases related to ERK1/2 kinase activity.

The present invention also proves through experiments that compound 131 exhibits effective inhibitory activity against two human colorectal cancer cell lines, and its IC ₅₀ values for SW-620 and HCT-116 cells are 1.16 μM and 0.59 μM, respectively. Western blot analysis shows that compound 131 reduces the intracellular phosphorylation levels of ERK1/2 (Thr202, Tyr204) and p90RSK (Thr359, Ser363) proteins in HCT116 cells in a concentration-dependent manner. Finally, the present invention further confirms that compound 131 can induce apoptosis of HCT116 human colon cancer cells. After treatment with 1 μM of compound 131, the apoptosis rate of HCT116 cells can reach 23.7%.

In summary, the compounds provided by the present invention can be used to prepare ERK1/2 kinase inhibitors, as well as drugs for preventing and/or treating diseases related to ERK1/2 kinase activity, and have broad application prospects.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Inhibition curve of compound 131 on ERK1/2 kinase.

Figure 2: Inhibitory effects of compound 131 and SCH772984 on the proliferation of colon cancer cells. *** indicates P < 0.001.

Figure 3: Effect of compound 131 on the phosphorylation levels of ERK1/2 and RSK in HCT116 cells.

Figure 4: Apoptosis analysis of HCT116 cells by compound 131.

Figure 5: Apoptosis rate of HCT116 cells induced by compound 131.

DETAILED DESCRIPTION

Unless otherwise specified herein, all reagents and raw materials used in the experiments of the present invention were purchased from reagent companies, all reagents were of analytical grade or chromatographic grade, and were used directly without further treatment.

Example 1: Synthesis of target compounds 13a-1, 14a-b, 15a-e

Synthesis route:

1. Synthesis of Intermediate 8

Synthesis steps of intermediate 8: N-Boc-3-cyano-4-pyrrolidone was used as the starting material to synthesize the compounds of series 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 500 mL reaction bottle, and then anhydrous ethanol (250 mL) was added to dissolve it. Then, the reaction bottle was heated to reflux at 60 ° C for 3 hours. The reaction was monitored by TLC. After the reaction was completely completed, it was concentrated under reduced pressure to remove the solvent. Then, saturated NaHCO ₃ aqueous solution was slowly added under stirring, and the pH was adjusted to 7-8. EA was extracted (3×300 mL), the upper layer solution was collected, and an appropriate amount of anhydrous Na ₂ SO ₄ was added to dry, and it was concentrated under reduced pressure again. The residue was recrystallized with ethyl acetate, and filtered under reduced pressure. The filter cake was collected and dried to obtain a white solid 8. Intermediate 8 can be used in the next step without purification.

2. Synthesis of Intermediate 9

Synthesis steps of intermediate 9: In a dry 500mL round-bottom flask, add intermediate 8 (3-amino-5-(tert-butyl)-pyrrolo[3,4-C]pyrazole, (11.20g, 50mmol)), DIEA (34.80mL, 200mmol), THF (250mL) in sequence, then stir the above mixture at 0℃ for 5 minutes, then slowly add a mixed solution of ethyl chloroformate (4.80mL, 50mmol) diluted with anhydrous THF (120mL) at 0℃ using a dropping funnel. This process takes 2 hours. After the reaction is completed, the mixed solution is transferred to room temperature and stirred for 12 hours. Monitor by TLC. After the reaction is complete, concentrate under reduced pressure. Extract the residue with ethyl acetate and saturated aqueous NaCl solution (3×250 mL). Collect the upper layer solution, add an appropriate amount of anhydrous Na ₂ SO ₄ for drying, and concentrate under reduced pressure to obtain a white solid 9. Purify it using a silica gel column (petroleum ether/ethyl acetate=4/1 to 2/1) to obtain pure intermediate 9.

3. Synthesis of Intermediate 10

Synthesis steps of intermediates 10a, 10b and 10d: In a 250 mL reaction bottle, add 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) and THF (100 mL) in sequence, then add the corresponding acid chloride (75 mmol) under stirring at 0°C, and continue stirring for 1 hour, then transfer to room temperature and continue stirring for 12 hours. TLC detection, wait until the reaction is completely completed, concentrate under reduced pressure, adjust the pH of the residue to 7-8 with saturated NaHCO ₃ , and then extract with ethyl acetate (3×250 mL), collect the upper layer solution, add an appropriate amount of anhydrous Na ₂ SO ₄ to dry, concentrate under reduced pressure, and purify using a silica gel column (petroleum ether/ethyl acetate=1/1) to obtain pure intermediate 10.

Synthesis steps of intermediate 10c: In a 250mL reaction bottle, add intermediate 9 (5-(tert-butyl)-3-amino-4,6-dihydropyrrolo[3,4-c]pyrazole-1-carboxylic acid ethyl ester, (14.80g, 50mmol)), DIEA (34.80mL, 200mmol), THF (100mL) in sequence, then add the corresponding acid chloride (75mmol) under stirring at 0°C, and continue stirring for 1 hour, then heat the reaction mixture to 45°C and stir for 12 hours, during which a precipitate will form. TLC detection, after the reaction is complete, reduce the pressure and filter, and wash with THF 3 times, collect the filter cake and dry it to obtain the crude product 10c, which can be used in the next step without purification.

4. Synthesis of Intermediate 12

The synthesis steps of intermediates 11a-d are as follows: first, intermediate 10 (30 mmol) is dissolved in a 250 mL reaction bottle with DCM (100 mL), and then a 1,4-dioxane solution (30 mL) containing 4N HCl is added dropwise under stirring at 0°C, and then the reactants are transferred to room temperature and stirred for 12 hours. During this process, precipitates are continuously generated. The reaction is monitored by TLC. After the reaction is completely completed, the mixture is filtered under reduced pressure and washed with dichloromethane three times. The filter cake is collected and dried to obtain the crude product 11, which can be used in the next step without purification.

Synthesis steps of intermediate 12: First, intermediate 11 (5 mmol), DIEA (1.70 mL, 10 mmol), and DCM (30 mL) were added to a 100 mL reaction bottle in sequence, and then the corresponding acyl chloride (7.50 mmol) was slowly added to the mixture under stirring at 0°C and continued to stir for 1 hour, and then the reaction bottle was transferred to room temperature for stirring and reacted for 10 hours. TLC detection, after the reaction was completely completed, it was concentrated under reduced pressure, the pH was adjusted to 7-8 with saturated NaHCO ₃ , and then extracted with ethyl acetate (3×50 mL), the upper layer solution was collected, and an appropriate amount of anhydrous Na ₂ SO ₄ was added for drying, and vacuum concentrated, and purified using a silica gel column (petroleum ether/ethyl acetate=1/1) to obtain pure intermediate 12.

5. Synthesis of target compound 13a-1

The synthesis steps of the target compound 13a-1 are as follows: the intermediate 12 (2 mmol) was added into a dry 50 mL round-bottom flask, and then MeOH (20 mL) was added to dissolve it, and then LiOH (80 mg, 2 mmol) was added under stirring. After 30 minutes, the mixture was detected by TLC. After the reaction was complete, it was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane/methanol = 9/1) to obtain a white solid 13. Next, it was recrystallized from methanol to obtain the pure target compound 13.

The target compounds 13a-1 were synthesized according to the above method.

6. Synthesis of target compounds 14a-b

The synthesis steps of the target compound 14a-b are as follows: First, the intermediate 13 (2 mmol) is dissolved in a 50 mL round-bottom flask with a 1,4-dioxane solution (15 mL), and then a 1 N LiOH (1 mL) solution is added dropwise under stirring. Next, the reaction mixture is transferred to room temperature and stirred for 3 hours. After the reaction is completely completed, the pH is adjusted to 4-5 with a 1 N HCl solution. During this process, a precipitate is generated. The mixture is filtered under reduced pressure and washed with water 3 times. The filter cake is collected and dried to obtain a white solid 14. Subsequently, the crude product is recrystallized with methanol, filtered under reduced pressure, and the filter cake is collected. After drying, the pure target compound 14 is obtained.

7. Synthesis of target compounds 15a-e

The synthesis steps of the target compounds 15a-e are as follows: First, the intermediate 13 (2mmol), NH ₄ Cl (0.13g, 2.40mmol), EtOH (20mL), H ₂ O (5mL), DMF (2mL), THF (6mL), iron powder (0.56g, 10mmol), and glacial acetic acid (3-4 drops) are added to a 100mL reaction bottle in sequence. The reaction mixture is then transferred to 80°C and stirred and refluxed for 8 hours. During the process, the solution changes from a yellow suspension to a light brown suspension. After the reaction is completed, the remaining iron powder is removed by hot filtration by TLC detection, and the solution is concentrated under reduced pressure. The pH is adjusted to 7-8 with saturated NaHCO ₃ , and then extracted with ethyl acetate (3×50mL). The upper layer solution is collected, dried with an appropriate amount of anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and finally purified using a silica gel column (dichloromethane/methanol=9/1) to obtain the pure target compound 15. The target compounds 15a-e are synthesized according to the above method.

Example 2: Synthesis of target compounds 19a-g

Synthesis route:

1. Synthesis of Intermediate 16

Synthesis steps of intermediate 16: Add intermediate 9 (5-(tert-butyl)-3-amino-4,6-dihydropyrrolo[3,4-c]pyrazole-1-carboxylic acid ethyl ester, (14.80g, 50mmol)) to a dry 250mL two-necked flask, then add ultra-dry DMF (100mL) to dissolve it, then slowly add chloroethyl isocyanate (12.80mL, 150mmol) under _N2 protection, then stir the above mixture in an ice bath for 5 minutes, then transfer the reaction mixture to room temperature and stir for 3 hours, during which precipitation will be generated. TLC detection, after the reaction is completed, pour the above reactant into 500mL ice water while hot, then reduce pressure and filter, wash with water 3 times, collect the filter cake and dry it for use. Next, the dried crude product, potassium tert-butoxide (5.05 g, 45 mmol) and THF (150 mL) were added to a 250 mL two-necked bottle in sequence, and then the reaction mixture was stirred at room temperature for 2 hours under _N2 protection. During this process, solids were precipitated. TLC detection, after the reaction was completed, vacuum filtration was performed, and the mixture was washed with THF 3 times. The filter cake was collected and dried to obtain a white solid 16. Finally, it was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1) to obtain pure intermediate 16.

2. Synthesis of Intermediates 18a-g

Synthesis steps of intermediate 17: First, dissolve 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)) in a 250 mL reaction bottle with DCM (150 mL), then add 1,4-dioxane solution (30 mL) containing 4N HCl dropwise at 0°C with stirring, then transfer the reactants to room temperature and continue stirring for 12 hours. Precipitation will continue to form during this process. Monitor the reaction by TLC. After the reaction is completely completed, filter under reduced pressure and wash with dichloromethane 3 times. Collect the filter cake and dry it to obtain white solid 17. The crude product 17 can be used in the next step without purification.

Synthesis steps of intermediates 18a-g: First, intermediate 17 (ethyl 3-(2-oxoimidazolidin-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate, (0.79 g, 3 mmol)), DIEA (1.02 mL, 6 mmol), PyBop (1.72 g, 3.3 mmol) and DCM (30 mL) were added to a 100 mL round-bottom flask in sequence, and then the corresponding acid (4.50 mmol) was slowly added under an ice bath. Then, the mixture was transferred to 0°C and stirred for 1 hour, and then transferred to room temperature and stirred for 10 hours. The reaction was monitored by TLC. After the reaction was completely completed, it was concentrated under reduced pressure, and the pH of the residue was adjusted to 7-8 with saturated NaHCO ₃ , and then extracted with ethyl acetate (3×50 mL). The upper layer solution was collected and an appropriate amount of anhydrous Na ₂ SO ₄ was dried, concentrated under reduced pressure again, and finally purified by silica gel column chromatography (dichloromethane/methanol=30/1) to obtain pure target compound 18.

Intermediates 18a-g were synthesized according to the above method.

3. Synthesis of target compounds 19a-g

The synthesis steps of the target compound 19a-g are as follows: the intermediate 18 (2 mmol) was added into a dry 50 mL round-bottom flask, and then MeOH (20 mL) was added to dissolve it, and then LiOH (80 mg, 2 mmol) was added under stirring. After 30 minutes, the mixture was detected by TLC. After the reaction was complete, it was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane/methanol = 9/1) to obtain a white solid 19. Next, it was recrystallized from methanol to obtain the pure target compound 19.

Intermediates 19a-g were synthesized according to the above method.

The following are the structures and characterization data of the above intermediate compounds and target compounds.

1. Structure and characterization data of intermediate compounds

Table 1. Structures of intermediate compounds

3-Amino-5-(tert-butyl)-pyrrolo[3,4-c]pyrazole (8). White powder, yield: 38.2%. ¹ HNMR (400 MHz, 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 (400 MHz, 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)benzamido)-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-carboxylic acid ethyl ester (10a). White powder, yield: 72.3%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.86 (d, J=2.0 Hz, 1H), 8.16 (d, J=8.0 Hz, 2H), 8.07 (d, J=8.0 Hz, 2H), 4.55 (s, 2H), 4.49 (s, 2H), 4.42 (q, J=7.6 Hz, 2H), 3.90 (s, 3H), 1.54 (s, 9H), 1.35 (t, J=7.6 Hz, 3H).

5-(tert-Butyl)-1-ethyl-3-(4-hydroxybenzamide)-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-carboxylic acid ethyl ester (10b). White powder, yield: 76.5%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.86 (s, 1H), 8.35 (d, J=8.8 Hz, 2H), 8.26 (d, J=8.8 Hz, 2H), 4.55 (s, 2H), 4.49 (s, 2H), 4.43 (q, J=7.2 Hz, 2H), 1.49 (s, 9H), 1.36 (t, J=7.2 Hz, 3H).

5-(tert-Butyl)-1-ethyl-3-(4-(dimethylamino)benzamido)-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-carboxylic acid ethyl ester (10c). Brown powder, yield: 68.7%. ¹ H NMR (400 MHz, Chloroform-d) δ (ppm): 8.65 (s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.73 (s, 2H), 4.72 (s, 2H), 4.46 (q, J = 7.2 Hz, 2H), 3.05 (s, 6H), 1.52 (s, 9H), 1.42 (t, J = 7.2 Hz, 3H).

Ethyl 3-(4-nitrobenzamide)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (10d). Yellow powder, yield: 83.4%. ¹ H NMR (400 MHz, 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-Hydroxybenzamido)-5-(4-methylbenzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12a). White powder, yield: 45.3%. ¹ H NMR (400 MHz, 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,2 H), 4.44 (q, J = 7.2Hz, 2H), 2.40 (s, 3H), 1.38 (t, J = 7.2Hz, 3H).

Ethyl 3-(4-hydroxybenzamido)-5-(4-(trifluoromethyl)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (12b). White powder, yield: 54.3%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.84 (s, 1H), 10.15 (s, 1H), 7.91 (d, J=8.4 Hz, 2H), 7.86 (d, J=7.6 Hz, 2H), 7.83 (d, J=7.6 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 4.74 (s, 2H), 4.54 (s, 2H), 4.42 (q, J=7.2 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H).

3-(4-(Dimethylamino)benzamido)-5-(4-methylbenzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12c). White powder, yield: 55.6%. ¹ H NMR (400 MHz, 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).

Ethyl 3-(4-(dimethylamino)benzamido)-5-(4-(trifluoromethyl)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (12d). White powder, yield: 49.0%. ¹ H NMR (400 MHz, Chloroform-d) δ (ppm): 9.52 (s, 1H), 8.23 (d, J = 9.2 Hz, 2H), 7.74 (d, J = 3.6 Hz, 2H), 7.71 (d, J = 3.6 Hz, 2H), 6.66 (d, J = 9.2 Hz, 2H), 5.06 (s, 2H), 4.87 (s, 2H), 4.55 (q, J = 7.2 Hz, 2H), 3.05 (s, 6H), 1.49 (t, J = 7.2 Hz, 3H).

Ethyl 3-(4-(dimethylamino)benzamido)-5-(4-(dimethylamino)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (12e). White powder, yield: 51.4%. ¹ H NMR (400 MHz,) δ (ppm): 11.69 (s, 1H), 8.28 (d, J = 9.6 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H), 7.86 (m, 2H), 7.76 (d, J = 8.8 Hz, 2H), 5.29 (s, 2H), 4.99 (s, 2H), 4.42 (q, J = 7.2 Hz, 2H), 3.52 (dd, J = 9.2 Hz, 12H), 1.36 (t, J = 7.2 Hz, 3H).

3-(4-(dimethylamino)benzoyl)-5-(3-(dimethylamino)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12f). White powder, yield: 58.6%. ¹ H NMR (400 MHz, 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-naphthoyl)-3-(4-(dimethylamino)benzamide)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12 g). White powder, yield: 49.3%. ¹ H NMR (400 MHz, 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-Naphthoyl)-3-(4-(dimethylamino)benzamide)-5,6-dihydropyrrolo[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-isonicotinoyl-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12i). White powder, yield: 66.7%. ¹ H NMR (400 MHz, 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-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12j). White powder, yield: 55.3%. ¹ H NMR (400 MHz, 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 (12k). White powder, yield: 46.5%. ¹ H NMR (400 MHz, 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 (121). White powder, yield: 55.3%. ¹ H NMR (400 MHz, 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 (12m). White powder, yield: 49.6%. ¹ H NMR (400 MHz, 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)benzoyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)carbamoyl)benzoic acid (12n). White powder, yield: 71.5%. ¹ H NMR (400 MHz, 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).

Ethyl 5-(4-methylbenzoyl)-3-(4-nitrobenzamido)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (12o). White powder, yield: 62.0%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.94 (s, 1H), 7.77 (d, J=8.8 Hz, 2H), 7.48 (d, J=8.0 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 6.54 (d, J=8.8 Hz, 2H), 5.82 (s, 2H), 4.70 (s, 2H), 4.42 (q, J=7.2 Hz, 2H), 2.37 (s, 3H), 1.36 (t, J=7.2 Hz, 3H).

Ethyl 3-(4-nitrobenzamido)-5-(4-(trifluoromethyl)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (12p). White powder, yield: 49.6%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.54 (s, 1H), 7.83 (d, J=8.4 Hz, 2H), 7.69 (d, J=8.0 Hz, 2H), 7.61 (d, J=8.0 Hz, 2H), 6.49 (d, J=8.4 Hz, 2H), 4.66 (m, 2H), 4.52 (s, 2H), 4.38 (q, J=7.2 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H).

5-(3-(Dimethylamino)benzoyl)-3-(4-nitrobenzamido)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12q). White powder, yield: 57.7%. ¹ H NMR (400 MHz, 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-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12x). White powder, yield: 55.8%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.91 (s, 1H), 8.72 (t, J=6.4 Hz, 2H), 8.32 (d, J=8.4 Hz, 2H), 8.27–8.12 (m, 2H), 7.58 (dd, J=8.4, 5.6 Hz, 2H), 4.85 (s, 2H), 4.72 (m, 2H), 4.40 (d, J=7.2 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

5-Nicotinyl-3-(4-nitrobenzamide)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (12y). White powder, yield: 49.6%. ¹ H NMR (400 MHz, 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＝8.0,5.2 Hz, 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 (13o). White powder, yield: 57.6%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.28 (s, 1H), 10.36 (s, 1H), 7.75 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.0 Hz, 2H), 7.32 (t, J=8.0 Hz, 2H), 6.61 (d, J=8.8 Hz, 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 (13p). White powder, yield: 67.3%. ¹ H NMR (400 MHz, 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 (13q). White powder, yield: 71.5%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.39 (s, 1H), 10.48 (s, 1H), 7.76 (d, J=8.0 Hz, 2H), 7.28 (m, 1H), 6.84 (d, J=7.6 Hz, 1H), 6.82 (s, 1H), 6.77 (d, J=7.6 Hz, 1H), 6.59 (d, J=8.0 Hz, 2H), 4.67 (s, 2H), 4.52 (s, 2H), 2.94 (s, 6H).

N-(5-isonicotinyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-nitrobenzamide (13x). White powder, yield: 59.8%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.61 (s, 1H), 11.42 (s, 1H), 8.72 (d, J=6.4 Hz, 2H), 8.32 (d, J=8.4 Hz, 2H), 8.27–8.12 (m, 2H), 7.58 (dd, J=8.4, 5.6 Hz, 2H), 4.73 (m, 2H), 4.58 (s, 2H).

N-(5-nicotinoyl-1,4,5,6-tetrahydropyrrole-[3,4-c]pyrazol-3-yl)-4-nitrobenzamide (13y). White powder, yield: 64.0%. ¹ H NMR (400 MHz, 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,1 H),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-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate (16). White powder, yield: 76.5%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.35 (s, 1H), 4.43 (s, 2H), 4.38 (s, 2H), 4.19 (t, J=7.2 Hz, 2H), 3.86 (d, J=8.4 Hz, 2H), 3.79 (d, J=8.4 Hz, 2H), 1.46 (s, 9H), 1.25 (t, J=7.2 Hz, 3H).

3-(2-Oxoimidazolidin-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (17). White powder, yield: 85.9%. ¹ H NMR (400 MHz, 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=7.2 Hz, 2H), 3.87 (d, J=8.4 Hz, 2H), 3.80 (d, J=8.4 Hz, 2H), 1.25 (t, J=7.2 Hz, 3H).

Ethyl 5-(4-methylbenzoyl)-3-(2-oxoimidazolidin-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (18a). White powder, yield: 44.3%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.36 (s, 1H), 7.48 (d, J=7.6 Hz, 2H), 7.27 (t, J=7.6 Hz, 2H), 4.68 (s, 2H), 4.56 (s, 2H), 4.15 (q, J=7.2 Hz, 2H), 3.83 (s, 2H), 3.80–3.72 (m, 2H), 2.37 (s, 3H), 1.20 (t, J=7.2 Hz, 3H).

3-(2-Oxoimidazol-1-yl)-5-(4-(trifluoromethyl)benzoyl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18b). White powder, yield: 51.6%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.39 (s, 1H), 7.90–7.83 (m, 2H), 7.79 (d, J=8.0 Hz, 2H), 4.71 (s, 2H), 4.55 (s, 2H), 4.15 (q, J=7.2 Hz, 2H), 3.82 (d, J=4.0 Hz, 2H), 3.81–3.72 (m, 2H), 1.19 (t, J=7.2 Hz, 3H).

5-(3-(Dimethylamino)benzoyl)-3-(2-oxoimidazol-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18c). White powder, yield: 49.0%. ¹ H NMR (400 MHz, 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-oxyimidazol-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18d). White powder, yield: 42.7%. ¹ H NMR (400 MHz, 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-Nicotinyl-3-(2-oxoimidazolidin-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18e). White powder, yield: 55.4%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.28 (s, 1H), 9.16 (d, J=4.4 Hz, 1H), 8.97 (s, 1H), 8.72 (d, J=7.6 Hz, 1H), 8.06 (s, 1H), 4.72 (s, 2H), 4.64 (s, 2H), 4.22 (q, J=7.2 Hz, 2H), 3.81 (d, J=8.8 Hz, 2H), 3.42 (d, J=8.8 Hz, 2H), 1.24 (t, J=7.2 Hz, 3H).

5-(4-(4-methylpiperazin-1-yl)benzoyl)-3-(2-oxoimidazol-1-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18f). White powder, yield: 50.1%. ¹ H NMR (400 MHz, 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-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylic acid ethyl ester (18 g). White powder, yield: 44.6%. ¹ H NMR (400 MHz, 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 of target compounds

Table 2. Structures of target compounds

4-Hydroxy-N-(5-(4-methylbenzoyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide (13a). White powder, melting point: 280.9-283.7°C, yield: 63.2%. ¹ H NMR (400 MHz, 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 (13b). White powder, melting point: 241.6-243.8°C, yield: 68.4%. ¹ H NMR (400 MHz, 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 (13c). White powder, melting point: 260.8-265.1°C, yield: 78.3%. ¹ H NMR (400 MHz, 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/z390.1917,found:390.1925.

4-(Dimethylamino)-N-(5-(4-(trifluoromethyl)benzoyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide (13d). White powder, melting point: 303.4-305.3°C, 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 (13e). White powder, melting point: 306.8-310.2°C, 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 (13f). 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 forC 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°C, yield: 64.3%. ¹ H NMR (400 MHz, 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 forC ₂₅ H ₂₃ N ₅ NaO ₂ ,[M+Na] ⁺ :m/z448.1738, found:448.17 44.

N-(5-(2-naphthoyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(dimethylamino)benzamide (13h). White powder, melting point: 313.1-315.7°C, yield: 52.9%. ¹ H NMR (400 MHz, 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 (13i). White powder, melting point: 303.1-305.8°C, yield: 66.2%. ¹ H NMR (400 MHz, 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 (100 MHz, 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 (13j). White powder, melting point: 251.3-257.6°C, 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＝12.0,4.8 Hz, 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 (13k). 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/z474.2606,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 (13l). White powder, melting point: 272.3-277.8°C, 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 (14a). 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,2 H),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 (14b). White powder, melting point: 296.5-298.1°C, 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),1 25.98,50.62,45.66.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 (15a). White powder, melting point: 180.6-188.3°C, yield: 55.9%. ¹ H NMR (400 MHz, 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 (15b). White powder, melting point: 303.2-305.7°C, yield: 49.6%. ¹ H NMR (400 MHz, 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 CNMR (100 MHz, 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). 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 (15c). White powder, melting point: 175.6～178.3℃, 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 forC 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 (15d). White powder, melting point: 198.6-202.3°C, yield: 39.6%. ¹ H NMR (400 MHz, 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 (100 MHz, 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 (15e). 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,1 H), 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-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)imidazolidin-2-one (19a). White powder, melting point: 315.3-318.1°C, yield: 62.7%. ¹ H NMR (400 MHz, 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 (19b). White powder, melting point: 313.5-317.2°C, yield: 80.1%. ¹ H NMR (400 MHz, 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) ⁺ calculatedfor 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 (19c). White powder, melting point: 194.6～198.4℃, 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(ES) I) ⁺ calculated for C ₁₇ H ₂₁ N ₆ O ₂ ,[M+H] ⁺ :m/z 341.1711,found:341.1721.

1-(5-isonicotinyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)imidazolin-2-one (19d). White powder, melting point: 314.2-316.5°C, yield: 71.2%. ¹ H NMR (400 MHz, 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-Nicotinyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)imidazolin-2-one (19e). White powder, melting point: 303.2-305.8°C, yield: 64.3%. ¹ H NMR (400 MHz, 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-tetrahydropyrrol[3,4-c]pyrazol-3-yl)imidazolidin-2-one (19f). White powder, melting point: 272.7～279.3℃, 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-tetrahydropyrrol[3,4-c]pyrazol-3-yl)imidazolidin-2-one (19 g). White powder, melting point: 216.5-220.4°C, yield: 76.1%. ¹ H NMR (400 MHz, 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 beneficial effects of the present invention are demonstrated by experimental examples below.

Experimental Example 1: In vitro kinase activity evaluation

1. Experimental Methods

The inhibition rate of the target compound of the present invention on ERK1/2 kinase at a concentration of 1 μM was determined using the ERK1/2 (Phospho-T202/Y204) TR-FRET cell detection kit, and the known ERK1/2 kinase inhibitor SCH772984 was used as a positive control.

ERK1/2 and ERK-TR-FRET cell detection kits were purchased from Biorbyt. The detection method was carried out according to the detection manual of the kit for cell treatment, cell lysis, and protein detection experiments. It measures kinase activity by quantifying the amount of ATP remaining in the solution after the kinase reaction. The luminescent signal measured in the experiment is related to the amount of ADP present and the amount of kinase activity.

The inhibitory effect of the target compound 131, which has good inhibitory activity at low concentrations, on ERK1/2 at different concentrations was further determined, and the IC ₅₀ value was calculated by nonlinear regression analysis using GraphPad Prism software. IC ₅₀ is defined as the concentration of the compound required to achieve 50% inhibition.

2. Experimental results

Table 3. Inhibition rate of target compounds on ERK1/2 kinase at 1 μM

Table 4. Inhibition rate of target compounds on ERK1/2 kinase at 1 μM

It can be seen that the compounds of the present invention have a good inhibitory effect on ERK1/2 kinase at a relatively low concentration (1 μM). Among them, compound 131 has the best inhibitory effect, with inhibition rates of 62.41% and 65.96% on ERK1 and ERK2 kinases. Further calculations show that the IC ₅₀ values of compound 131 on ERK1 and ERK2 kinases are 1.69 μM and 0.87 μM, respectively (Figure 1).

The above experimental results show that the compounds of the present invention can effectively inhibit ERK1/2 kinase and can be used as ERK1/2 kinase inhibitors.

Experimental Example 2: In vitro cell proliferation inhibition experiment

1. Experimental Methods

This experiment evaluated the antiproliferative activity of compound 13l against two tumor cell lines carrying K-Ras activating mutations, SW-620 and HCT-116. First, SW620 and HCT116 human colon cancer cells in the logarithmic growth phase were prepared into cell suspensions with 10% PBS and seeded into 96-well plates at a density of 1×10 ⁵ cells/mL culture medium, with a volume of 100 μL per well, and then transferred to an incubator containing 5% CO ₂ at 37°C for 12 h. Next, compound 13l was dissolved in DMSO and treated with DMEM medium, diluted into various target concentration gradient solutions (0.3 μM, 0.5 μM, 1.0 μM, 5.0 μM), and then different concentrations of the test compound were added to each well to treat the cells, and then transferred to an incubator containing 5% CO ₂ at 37°C for incubation for 24-72 h. Next, the prepared 5 mg/ml MTT solution was added to the 96-well plate with the incubated cells (20 μL/well), and then transferred to an incubator containing 5% CO ₂ at 37°C for incubation for 2-4 hours. Then, the culture medium in the wells was discarded, and 150 μL of DMSO solution was added to each well. Then, the well plate was shaken on a shaker until the crystals were fully dissolved. Finally, the absorbance value of each well was measured at a wavelength of 570 nm using an ELISA reader, and three replicate wells were taken to calculate the inhibition rate. Inhibition rate = (control group-drug group)/control group × 100%. GraphPad Prism6 was used to analyze the data and calculate the IC ₅₀ value.

SCH772984, a known ERK1/2 kinase inhibitor, was used as a positive control.

2. Experimental results

Table 5. IC ₅₀ values of compound 131 against human colorectal cancer cells

The data in Table 5 and Figure 2 show that compound 131 exhibits potent inhibitory activity against both human colorectal cancer cell lines, with IC ₅₀ values of 1.16 μM and 0.59 μM for SW-620 and HCT-116 cells, respectively.

Experimental Example 3: Protein Immunofluorescence Immunoblotting Analysis

1. Experimental Methods

(1) Sample preparation

SW620 and HCT116 human colon cancer cells in the logarithmic growth phase were distributed on 6-well plates at a density of 1×10 ⁵ cells/well, and after adding an appropriate amount of DMEM culture medium, they were placed in an incubator at 37°C and containing 5% CO ₂ and incubated for 12 hours. After the cell density reached 80% to 90%, the two cells were treated with culture medium containing different concentrations (0.5 μM, 1.0 μM) of compound 131 for 24 hours. Next, the cells were collected with a clean scraper, and then washed with 3 ml of 4°C pre-cooled PBS buffer. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, and the washing operation was repeated twice to wash away the culture medium. Then, the washed cells were lysed on ice with an appropriate amount of lysis buffer containing protease inhibitors and phosphatase inhibitors. In order to fully lyse the cells, this process requires frequent shaking back and forth and should be carried out on ice for at least 30 minutes. After lysis, the cell fragments and lysate need to be transferred to a 1.5 ml centrifuge tube with a gun, and then centrifuged at 12000 rpm for 10 minutes in a pre-cooled centrifuge at 4°C. After collecting the supernatant, the next step of the protein content determination experiment can be carried out.

(2) Protein content determination

First, prepare 2 mL of BCA working solution with a volume ratio of 50:1 between solution A and solution B, and then use PBS to dilute the protein sample concentration to 0.5 mg/mL. Next, perform gradient dilution according to Table 6 below to draw the standard concentration curve.

Table 6. Gradient dilution table of standard concentration curve

serial number 1 2 3 4 5 6 7 8 Protein standard diluent (μL) 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, carefully add 20 μL of each sample in the table to a 96-well plate, and then add 200 μL of BCA working solution to each well. After shaking evenly, place at 37°C to allow color to develop for 15-30 minutes. Finally, use an ELISA reader to measure the absorbance value of each well at a wavelength of 570 nm, draw a standard curve using the blank control, and obtain a regression equation. Substitute the absorbance value of the measurement tube into the formula to obtain the protein concentration of each group.

(3) SDS-PAGE electrophoresis

Clamp the cleaned glass plate with a clip and clamp it vertically on the glue rack. Prepare 10% separation gel according to the detection protein, then add TEMED and mix it evenly, then use a pipette to slowly pour the gel along the glass slit, and then add isopropanol to the upper layer of the gel to seal it to make it solidify faster. Let it stand at room temperature for 30 minutes. After the gel is fully solidified, pour out the liquid-sealed isopropanol, wash it with distilled water and wipe it dry for later use. Next, prepare 4% concentrated gel in the same way, then add TEMED and mix it thoroughly. After pouring the gel again, insert the comb horizontally into the concentrated gel, let it stand at room temperature for another 30 minutes, wait until the gel is completely solidified, slowly pull out the comb, wash the gel, put it in the electrophoresis tank intact, and add the samples after the protein content is determined to the sample wells. Finally, electrophoresis at 80V for 3 to 4 hours until the electrophoresis is over and the next experiment can be carried out.

(4) Transfer

First, cut 6 filter papers and 1 PVDF membrane according to the size of the gel for later use, and then soak the PVDF membrane in methanol for activation. Next, slowly remove the gel from the glass plate for later use. After the membrane is placed in the transfer solution, place the black side of the transfer clip upwards, and lay a layer of sponge pad, three layers of filter paper, gel, PVDF membrane, three layers of filter paper and a layer of sponge pad in sequence, and finally close the red side of the transfer clip (avoid bubbles during this process). Finally, put the above transfer clip into the transfer tank, and after it is filled with transfer solution, transfer the membrane at 80V for 2h (during this process, a little ice should be added to the other side of the tank to dissipate the heat generated during the electrotransfer).

(5) Immune response

First, the PVDF membrane was rinsed three times with PBST buffer, and then transferred to PBST blocking solution containing 5% skim milk powder for decolorization at room temperature for 1 hour. The blocked PVDF membrane was then rinsed three times again with PBST buffer, and then transferred to a hybridization bag containing an appropriate amount of primary antibody diluent, and then incubated on a shaker at room temperature for 1 to 2 hours, and then rinsed three times again with PBST buffer, and incubated again at room temperature for 1 to 2 hours with secondary antibody diluent in the same way.

(6) Color rendering

After the incubated PVDF membrane was rinsed three times with PBST buffer, the developer was evenly added to the membrane protein surface, and the experimental results were immediately detected after 1 minute.

2. Experimental results

The expression levels of apoptosis-related proteins in HCT116 cells were detected after treatment with compound 131. As shown in Figure 3, compound 131 reduced the intracellular phosphorylation levels of ERK1/2 (Thr202, Tyr204) and p90RSK (Thr359, Ser363) proteins in HCT116 cells in a concentration-dependent manner.

Experimental Example 4: Cell apoptosis experiment

1. Experimental Methods

In this experiment, Annexin V-FITC/PI staining and Hoechst 33258 staining were used to determine the apoptosis rate of cells. First, HCT116 human colon cancer cells in the logarithmic growth phase were distributed on a 6-well plate at a density of 1×10 ⁵ cells/well. After adding an appropriate amount of DMEM culture medium, they were placed in an incubator at 37°C and containing 5% CO ₂ and incubated for 12 hours. After the cell density reached 80% to 90%, HCT116 cells were treated with culture medium containing different concentrations (0, 0.5 μM, 1.0 μM) of compound 13l. After 24 hours, 13l-treated and untreated cells were harvested, and then the cells were digested with EDTA-free trypsin. After centrifugation at 3000 rpm for 5 minutes, the cells were washed twice with pre-cooled PBS, and then the supernatant was discarded, and 1 to 5×10 ⁵ cells were selected and collected. Next, add 100 μL of binding buffer at room temperature and mix by pipetting, then add 5 μL of Annexin-VFITC and 5 μL of PI dye solution. After shaking and mixing, incubate at room temperature in the dark for 15 minutes. Finally, observe and detect the cells using a flow cytometer, and use Flowjo software to analyze the results.

2. Experimental results

This experiment explored the cause of cell death by detecting the apoptosis rate of cells using flow cytometry. First, after HCT116 cells treated with compound 13l were stained with Hoechst 33258, concentrated and bright apoptotic nuclei were easily observed (as shown in Figure 4). Next, HCT116 cells were treated with compound 13l at concentrations of 0, 0.5, and 1 μM for 24 hours, and then the cells were stained with Annexin V-FITC/PI, and then the cells were observed and detected by flow cytometry. The results of cell apoptosis are shown in Figure 5, which show that compound 13l can induce apoptosis of HCT116 human colon cancer cells in a concentration-dependent manner. At a concentration of 1 μM of compound 13l, the total apoptosis rate of HCT116 cells can reach 23.7%. It was further confirmed that compound 13l can induce apoptosis of human colon cancer cells.

In summary, the present invention provides an ERK inhibitor and its pharmaceutical use. The compound provided by the present invention has a good inhibitory effect on ERK1/2 kinase, shows effective inhibitory activity on colorectal cancer cell lines, can reduce the intracellular phosphorylation level of ERK1/2 (Thr202, Tyr204) and p90RSK (Thr359, Ser363) proteins in colorectal cancer cell cells, and can induce apoptosis of colon cancer cells. The compound of the present invention can be used to prepare ERK1/2 kinase inhibitors, as well as drugs for preventing and/or treating diseases related to ERK1/2 kinase activity, and has broad application prospects.

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.