Analog of plasminogen activator and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an analogue of a cysteine zymogen activator (a PAC-1 analogue containing a carvone structure) and application of the analogue in cancer treatment. The invention relates to an analogue of a cysteaspase-3 activator, which is a PAC-1 analogue containing a carvone structure as shown in a formula I. The PAC-1 analogue disclosed by the invention is obtained by retaining structures such as piperazine ring and hydrazide, introducing a monoterpenoid carvone and carrying out structural modification on benzene rings on two sides, and a novel procaspase-3 activator is developed. The derivatives have stronger physiological activity. Pharmacological tests prove that the PAC-1 analogue can inhibit proliferation of various tumor cells and has good anti-cancer activity, and the preparation method of the PAC-1 analogue is simple, feasible and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to an analogue of procaspase activator (PAC-1 analogue containing carvone structure) and its application in the treatment of cancer. Background Art

[0002] Cancer has always been regarded as one of the diseases seriously endangering human health. Drug treatment is one of the important means for clinical treatment of tumors today, and the tumor field has always been a key area for new drug research and development. With the continuous in-depth research on anti-tumor drugs by scientists, achieving anti-tumor effects by inducing cell death has become a hot topic in current drug research (Duan Jingshi, Yu Yaqin, He Xinya, et al. Research progress on the mechanism of anti-tumor drugs inducing paraptosis in tumor cells [J]. Life Sciences, 2021, 33(07): 888-895.).

[0003] During the process of apoptosis, procaspase-3 plays an important role. Compared with normal cells, procaspase-3 often has a higher expression in various tumor cells. Procaspase-Activating Compound 1 (PAC-1) can chelate with zinc ions to affect the expression of procaspase-3, thereby promoting apoptosis of tumor cells and showing good anti-tumor prospects (West D C, Qin Y, Peterson Q P, et al. Differential Effects of Procaspase-3Activating Compounds in the Induction of Cancer Cell Death[J]. MolecularPharmaceutics, 2012, 9(5):1425-1434.). Currently, PAC-1 has been in a phase I clinical trial in cancer patients. The research results show that the PAC-1 drug has inhibitory activity against various tumors such as ovarian cancer, hemangioendothelioma, and hepatocellular carcinoma. (Danciu O C, Nicholas M K, Emmadi R, et al. Phase I Study of Procaspa se ActivatingCompound-1(PAC-1)in the Treatment of Advanced Malign ancies[J]. Molecular&Cellular Proteomics, 2018, 11(12):E217-E222.). Targeted drugs are highly specific, but mutations in the target are likely to lead to the failure of drug treatment. Therefore, the multi-target cooperative design of PAC-1 is a current research hotspot. Some scholars have integrated other anti-tumor pharmacophores into its skeleton and synthesized PAC-1 diarylurea conjugates, benzothiazole PAC-1 derivatives, etc. Some of these derivatives show better inhibitory effects on tumor cells than PAC-1. (Hergenrother PJ, Roth HS. Derivatives of Procaspase-Activating Compound 1(PAC-1)and their AnticancerActivities[J]. Current Medicinal Chemistry, 2016, 23(3):)

[0004] In the aspect of brain tumors, the combined treatment of PAC-1 and temozolomide has shown good efficacy (Tonogai E J, Shan H, Botham R C, et al. Evaluation of a procaspase-3 activator with hydroxyurea or temozolomide against high-grade meningioma in cell culture and canine cancer patients[J]. Neuro-Oncology, 2021(10):10.). However, there is currently a lack of research on the treatment of brain tumors with PAC-1 alone. The poor penetration ability of PAC-1 itself and its certain neurotoxicity also limit its application in brain tumors. Therefore, it is necessary to develop verified derivatives to solve the corresponding problems and achieve better effects. Summary of the Invention

[0005] The object of the present invention is to provide a procaspase-3 activator PAC-1 as a lead compound to obtain PAC-1 analogs (containing carvone structure) and their applications in the treatment of cancer.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] An analog of procaspase activator, characterized in that: the analog is a PAC-1 analog containing a carvone structure shown in Formula I:

[0008]

[0009] In the formula,

[0010] R1, R2, and R3 can be the same or different and are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, hydroxyl, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form a C3-C8 ring or aromatic ring;

[0011] n, m, and x are the same or different and are independently selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

[0012] Preferably, in the formula of the analog, R1, R2, and R3 can be the same or different and are independently selected from C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form a C3-C8 ring or aromatic ring;

[0013] n, m, and x are the same or different and are independently selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

[0014] Further preferably, in the formula of the analog, R1, R2, and R3 may be the same or different and are independently selected from C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, nitro or halogen; or, any two adjacent substituents among R1, R2, and R3 form an aromatic ring;

[0015] n, m, and x are the same or different and are each selected from 1, 2 or 3, and n + m + x is less than or equal to 5.

[0016] Even further preferably, in the formula of the analog, R1, R2, and R3 may be the same or different and are independently selected from C1-C4 alkyl, C1-C2 alkoxy, C2-C4 alkenyl, nitro or halogen; or, any two adjacent substituents among R1, R2, and R3 form an aromatic ring;

[0017] n, m, and x are the same or different and are each selected from 1, 2 or 3, and n + m + x is less than or equal to 5.

[0018] More preferably, the analog is

[0019]

[0020]

[0021] The present invention also provides a preparation method of the PAC-1 analog:

[0022]

[0023] In the above preparation process:

[0024] The solvent used is a conventional reaction solvent and there are no special requirements.

[0025] A pharmaceutical composition, wherein the composition contains an analog of the procaspase activator shown in formula I.

[0026] An application of the analog of the procaspase activator or the pharmaceutical composition as described above, an application of the analog of the procaspase activator shown in formula I or the composition in the preparation of an anti-tumor drug.

[0027] The tumor is melanoma, breast cancer, glioblastoma, glioma, cervical cancer, liver cancer, fibrosarcoma, colon cancer, lymphoma, chronic myelogenous leukemia or promyelocytic leukemia.

[0028] Advantages of the present invention:

[0029] The PAC-1 analogues described in the present invention are obtained by retaining structures such as piperazine rings and hydrazides, introducing the monoterpene compound carvone, and modifying the structures of the benzene rings on both sides, thereby developing novel procaspase-3 activators, and these derivatives have stronger physiological activities.

[0030] The PAC-1 analogues of the present invention have been proven by pharmacological experiments to inhibit the proliferation of various tumor cells, have good anti-cancer activities, and their preparation methods are simple, feasible, and easy to operate. Detailed implementation manners

[0031] The following examples are used to illustrate the feasibility of the present invention. Those skilled in the art should understand that according to the teachings of the prior art, modifying or replacing the corresponding technical features still falls within the scope protected by the present invention.

[0032] Example 1

[0033] Preparation of (R)-2-methyl-5-(1-chloromethyl)vinylcyclohex-2-enone

[0034]

[0035] 5 g (33.33 mmol) of (-)-carvone and 100 mL of n-hexane were added to a 250 mL eggplant-shaped flask. While stirring in an ice bath, 4.15 mL (36.67 mmol) of tert-butyl hypochlorite was added dropwise. After the addition, the mixture was warmed to room temperature and stirred for another 3 h. After the reaction was completed, the reaction solution was washed successively with saturated aqueous sodium sulfite solution and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography to obtain 1.58 g of a pale yellow oil, with a yield of 55.2%. 1H NMR (600 MHz, Chloroform-d) δ 6.72 - 6.66 (m, 1H), 5.20 (s, 1H), 4.99 (s, 1H), 4.06 - 4.00 (m, 2H), 2.96 - 2.77 (m, 1H), 2.61 - 2.23 (m, 4H), 1.73 (s, 3H).

[0036] Example 2

[0037] Preparation of tert-butyl (R)-4-[2-(4-methyl-5-oxocyclohex-3-en-1-yl)allyl]piperazine-1-carboxylate

[0038]

[0039] Add 2 g (10.87 mmol) of (R)-2-methyl-5-(1-chloromethyl)vinylcyclohex-2-enone, 6.03 g (32.61 mmol) of N-Boc piperazine, 2 mg (2.17 mmol) of KI and 60 ml of absolute ethanol to a 250 ml single-necked flask. Reflux the reaction for 7 hours until the reaction is complete. Evaporate and concentrate to obtain a yellow viscous liquid, extract with water and EA, and purify by column chromatography to obtain 3.36 g of a white solid with a low melting point, with a yield of 92.5%. 1 H NMR (600 MHz, Chloroform-d) δ 6.75 - 6.60 (m, 1H), 5.06 - 4.74 (m, 2H), 3.38 - 3.27 (m, 4H), 2.92 - 2.83 (m, 2H), 2.82 - 2.76 (m, 1H), 2.57 - 2.51 (m, 1H), 2.46 - 2.39 (m, 1H), 2.37 - 2.22 (m, 6H), 1.75 - 1.66 (m, 3H), 1.38 (s, 9H).

[0040] Example 3

[0041] Preparation of (R)-2-methyl-5-[3-(piperazin-1-yl)prop-1-en-2-yl]cyclohex-2-en-1-one

[0042]

[0043] Dissolve 1 g of tert-butyl (R)-4-[2-(4-methyl-5-oxocyclohex-3-en-1-yl)allyl]piperazine-1-carboxylate in 30 ml of absolute methanol, add 10 ml of saturated hydrochloric acid methanol solution, and stir at room temperature for 3 hours. After the reaction is complete, adjust the pH to 8, evaporate and concentrate to remove the solvent, extract with water and DCM, dry over anhydrous sodium sulfate, evaporate and concentrate to obtain 0.49 g of a pale yellow oil, with a yield of 64%. 1 H NMR (400 MHz, Chloroform-d) δ 6.69 - 6.58 (m, 1H), 5.04 - 4.80 (m, 2H), 3.07 - 1.85 (m, 15H), 1.72 - 1.65 (m, 3H).

[0044] Example 4

[0045] Preparation of N'-benzylidene-2-chloroacetohydrazide

[0046]

[0047] Dissolve 0.5 g of benzaldehyde (4.70 mmol) in 20 mL of absolute ethanol, add 252 μL of hydrazine hydrate (5.19 mmol), and reflux at 80 °C for half an hour. After the reaction, remove the solvent by distillation under reduced pressure. 0.55 g of pale yellow oily benzaldehyde hydrazone is obtained, with a yield of 98%. Dissolve 0.2 g (1.67 mmol) of benzaldehyde hydrazone in dichloromethane, add 0.7 g (5.01 mmol) of potassium carbonate as an acid-binding agent, add 141 μL (1.84 mmol) of chloroacetyl chloride, and react at room temperature for 30 min. After the reaction, filter by suction to collect the filtrate, and remove the solvent under reduced pressure. After purification by thin-layer chromatography (DCM:MeOH = 20:1), 38 mg of white solid is obtained, with a yield of 12%. MS(ESI) m / z: 219.0 [M+Na] +

[0048] Example 5

[0049] Synthesis of Compound A1

[0050]

[0051] Add 50 mg (0.24 mmol) of (R)-2-methyl-5-[3-(piperazin-1-yl)prop-1-en-2-yl]cyclohex-2-en-1-one, 47 mg (0.24 mmol) of N'-benzylidene-2-chloroacetylhydrazine, 3 mg (0.02 mmol) of potassium iodide, and 99 mg (0.72 mmol) of potassium carbonate to a 50 mL single-necked flask. Add 15 mL of acetonitrile, heat to 82 °C and react for 2 hours. Remove the solvent under reduced pressure, and purify by thin-layer chromatography (DCM:MeOH = 20:1) to obtain 25 mg of pale yellow solid A1, with a yield of 26% and a melting point of 59 - 60 °C. 1 H NMR(400MHz,Chloroform-d)δ10.02(s,1H),8.14(s,1H),7.76 - 7.60(m,2H),7.35 - 7.31(m,3H),6.71 - 6.66(m,1H),4.96(s,1H),4.88(s,1H),3.12(s,2H),2.94 - 2.86(m,2H),2.81 - 2.74(m,1H),2.58 - 2.22(m,12H),1.72(s,3H). 13 C NMR(101MHz,CDCl3)δ198.86,165.33,147.47,146.43,143.78,134.41,132.53,129.55,127.67,126.71,112.26,62.04,59.99,52.61,52.10,42.34,37.79,30.58,14.70.HRMS(ESI)calcd for C23 H 31 N4O2[M+H] + 395.2447, found 395.2451 HRMS(ESI) calcd for C 23 H 31 N4O2[M+H] + 395.2447, found 395.2451.

[0052] Example 6

[0053] According to the methods of Examples 1 to 5 above, benzaldehyde and N'-benzylidene-2-chloroacetohydrazide in Examples 4 and 5 were respectively replaced with benzaldehyde and chloroacetohydrazide containing the above substituents to further synthesize Compounds A2 to A29.

[0054] A2: Pale yellow solid, yield 24%, mp 114 °C. 1 H NMR(600 MHz, Chloroform-d) δ 10.96(s, 1H), 10.10(s, 1H), 8.45(s, 1H), 7.32 - 7.28(m, 1H), 7.21 - 7.18(m, 1H), 7.00 - 6.96(m, 1H), 6.94 - 6.86(m, 1H), 6.77 - 6.73(m, 1H), 5.03(s, 1H), 4.95(s, 1H), 3.19(s, 2H), 3.01 - 2.93(m, 2H), 2.90 - 2.23(m, 13H), 1.80 - 1.76(m, 3H). 13 C NMR(151 MHz, CDCl3) δ 198.86, 164.77, 157.59, 150.39, 146.28, 143.79, 134.41, 130.94, 129.95, 118.32, 116.34, 116.23, 112.42, 61.97, 59.85, 52.58, 51.97, 42.33, 37.76, 30.56, 14.71. HRMS(ESI) calcd for C 23 H 31 N4O3[M+H] + 411.2399, found 411.2396.

[0055] A3: Pale yellow solid, yield 32%, mp 124 - 126 °C. 11H NMR (400 MHz, Chloroform-d) δ 11.17 (s, 1H), 10.00 (s, 1H), 8.35 (s, 1H), 7.14 - 7.09 (m, 1H), 7.05 - 6.97 (m, 1H), 6.80 - 6.74 (m, 1H), 6.71 - 6.66 (m, 1H), 6.02 - 5.90 (m, 1H), 5.04 - 4.87 (m, 4H), 3.42 - 3.37 (m, 2H), 3.13 (s, 2H), 2.94 - 2.84 (m, 2H), 2.82 - 2.75 (m, 1H), 2.57 - 2.22 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 198.85, 164.74, 155.40, 150.36, 146.34, 143.78, 135.50, 134.41, 131.30, 128.16, 127.23, 118.05, 115.85, 114.64, 112.37, 62.00, 59.85, 52.61, 52.02, 42.33, 37.78, 32.82, 30.57, 14.70. HRMS (ESI) calcd for C 26 H 35 N4O3 [M + H] + 451.2710, found 451.2709.

[0056] A4: Pale yellow oil, yield 22%. 1 1H NMR (600 MHz, Chloroform-d) δ 11.14 (s, 1H), 10.01 (s, 1H), 8.30 (s, 1H), 7.11 - 7.08 (m, 1H), 6.99 - 6.96 (m, 1H), 6.74 - 6.71 (m, 1H), 6.70 - 6.66 (m, 1H), 4.96 (s, 1H), 4.87 (s, 1H), 3.15 - 3.08 (m, 2H), 2.93 - 2.87 (m, 2H), 2.80 - 2.75 (m, 1H), 2.54 - 2.24 (m, 12H), 2.21 (s, 3H), 1.72 (s, 3H). 1313C NMR (151 MHz, Chloroform-d) δ 195.17, 161.04, 152.07, 146.55, 142.60, 140.13, 130.63, 128.32, 123.92, 121.49, 114.10, 111.78, 108.60, 58.27, 56.12, 48.88, 48.29, 38.59, 34.04, 26.84, 10.98. HRMS (ESI) calcd for C 24 H 33 N4O3 [M+H] + 425.2557, found 425.2553.

[0057] A5: Pale yellow oil, yield 26%. 1 1H NMR (600 MHz, Chloroform-d) δ 11.47 (s, 1H), 9.99 (s, 1H), 8.29 (s, 1H), 7.29 - 7.21 (m, 1H), 7.01 - 6.92 (m, 1H), 6.78 - 6.72 (m, 1H), 6.70 - 6.65 (m, 1H), 4.96 (s, 1H), 4.87 (s, 1H), 3.11 (s, 2H), 2.93 - 2.84 (m, 2H), 2.80 - 2.73 (m, 1H), 2.56 - 2.21 (m, 12H), 1.72 (s, 3H), 1.35 (s, 9H). 13 13C NMR (151 MHz, DMSO-d6) δ 195.18, 161.05, 153.11, 147.13, 142.65, 140.15, 132.89, 130.64, 124.58, 113.90, 112.43, 108.58, 58.30, 56.13, 48.90, 48.32, 38.61, 34.05, 30.19, 26.84, 24.60, 10.99. HRMS (ESI) calcd for C27H38N4O3Na [M+Na] + 489.2842, found 489.2842.

[0058] A6: Pale yellow oil, yield 31%. 11H NMR (400 MHz, Chloroform-d) δ 8.62 (s, 1H), 7.17 - 7.11 (m, 1H), 6.71 - 6.66 (m, 1H), 6.55 - 6.49 (m, 1H), 6.33 - 6.27 (m, 1H), 4.96 (s, 1H), 4.87 (s, 1H), 3.77 (s, 3H), 3.10 (s, 2H), 2.95 - 2.85 (m, 2H), 2.81 - 2.74 (m, 1H), 2.57 - 2.27 (m, 12H), 1.72 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 198.92, 164.57, 159.07, 157.83, 146.44, 145.58, 143.88, 134.36, 131.60, 112.24, 109.10, 105.77, 99.86, 62.02, 59.85, 54.72, 52.66, 52.04, 42.32, 37.78, 30.57, 14.70. HRMS (ESI) calcd for C 24 H 33 N4O4 [M + H] + 441.2502, found 4412507.

[0059] A7: Pale yellow oil, yield 29%. 1 1H NMR (600 MHz, Chloroform-d) δ 8.50 (s, 1H), 7.12 - 7.01 (m, 1H), 6.97 - 6.86 (m, 1H), 6.82 - 6.72 (m, 1H), 6.72 - 6.60 (m, 1H), 4.97 (s, 1H), 4.89 (s, 1H), 3.14 (s, 2H), 3.00 - 2.85 (m, 2H), 2.83 - 2.22 (m, 13H), 1.84 - 1.62 (m, 3H). 13 13C NMR (151 MHz, Chloroform-d) δ 198.88, 165.01, 151.22, 149.86, 149.60, 145.82 (d, J = 12.4 Hz), 143.80, 134.42, 124.85 (d, J = 3.6 Hz), 118.51 (d, J = 3.5 Hz), 117.88 (d, J = 7.2 Hz), 117.24, 112.49, 61.94, 59.84, 52.57, 51.92, 42.32, 37.76, 30.56, 14.71. HRMS (ESI) calcd for C 23 H 30 N4O3F [M + H] +429.2305, found 429.2302.

[0060] A8: Pale yellow oil, yield 26%. 1 H NMR(600MHz, Chloroform-d) δ8.48(s, 1H), 7.39 - 7.24(m, 1H), 7.14 - 7.01(m, 1H), 6.88 - 6.74(m, 1H), 6.74 - 6.62(m, 1H), 4.97(s, 1H), 4.88(s, 1H), 3.12(s, 2H), 3.01 - 2.83(m, 2H), 2.82 - 1.96(m, 13H), 1.84 - 1.60(m, 3H). 13 C NMR(151MHz, Chloroform-d) δ198.90, 165.09, 153.18, 149.54, 146.34, 143.81, 134.41, 131.08, 128.31, 120.82, 118.61, 117.63, 112.35, 62.00, 59.91, 52.64, 52.00, 42.34, 37.77, 30.57, 14.71. HRMS(ESI) calcd for C 23 H 30 N4O3Cl [M + H] + 429.2006, found 429.2009.

[0061] A9: Pale yellow oil, yield 28%. 1 H NMR(600MHz, Chloroform-d) δ8.47(s, 1H), 7.48(dd, J=7.9, 1.5Hz, 1H), 7.11(dd, J=7.7, 1.5Hz, 1H), 6.73(t, J=7.8Hz, 1H), 6.71 - 6.64(m, 1H), 4.98(s, 1H), 4.89(s, 1H), 3.14(s, 2H), 2.99 - 2.86(m, 2H), 2.84 - 2.19(m, 13H), 1.76 - 1.68(m, 3H). 13 C NMR(151MHz, Chloroform-d) δ198.86, 165.00, 154.05, 149.49, 146.21, 143.78, 134.42, 134.12, 129.12, 119.17, 117.53, 112.50, 110.03, 61.94, 59.83, 52.57, 51.95, 42.32, 37.76, 30.56, 14.71.. HRMS(ESI) calcd for C23 H 30 N4O3Br[M+H] + 489.1501, found 489.1504.

[0062] A10: Pale yellow solid, yield 28%, mp 70 - 72 °C. 1 H NMR (400 MHz, Chloroform-d) δ 10.83 (s, 1H), 10.04 (s, 1H), 8.34 (s, 1H), 7.05 - 6.98 (m, 1H), 6.75 - 6.57 (m, 3H), 4.98 (s, 1H), 4.89 (s, 1H), 3.14 (s, 2H), 2.98 - 2.86 (m, 2H), 2.84 - 2.22 (m, 16H), 1.72 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 198.95, 164.75, 157.49, 150.35, 146.31, 143.88, 141.96, 134.38, 129.81, 119.49, 116.63, 113.83, 112.37, 61.98, 59.85, 52.58, 51.99, 42.32, 37.77, 30.57, 20.67, 14.70. HRMS (ESI) calcd for C 24 H 33 N4O3[M+H] + 425.2555, found 425.2553.

[0063] A11: Pale yellow solid, yield 28% yield 32%: mp 78 - 80 °C. 1 H NMR (400 MHz, Chloroform-d) δ 10.79 (s, 1H), 10.00 (s, 1H), 8.33 (s, 1H), 7.08 - 7.03 (m, 1H), 6.96 - 6.92 (m, 1H), 6.88 - 6.80 (m, 1H), 6.70 - 6.66 (m, 1H), 4.96 (s, 1H), 4.88 (s, 1H), 3.11 (s, 2H), 2.94 - 2.86 (m, 2H), 2.81 - 2.74 (m, 1H), 2.55 - 2.21 (m, 13H), 1.71 (s, 3H), 1.22 (s, 9H). 1313C NMR (101 MHz, Chloroform-d) δ 198.88, 164.77, 157.31, 155.11, 150.19, 146.33, 143.85, 134.37, 129.57, 115.81, 113.78, 113.23, 112.35, 61.99, 59.88, 52.60, 52.00, 42.32, 37.78, 33.98, 30.56, 30.02, 14.70. HRMS(ESI) calcd for C 27 H 39 N4O3 [M+H] + 467.3023, found 467.3022.

[0064] A12: Pale yellow solid, yield 28%, mp 64 °C. 1 1H NMR (400 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.05 - 6.99 (m, 1H), 6.71 - 6.65 (m, 1H), 6.46 - 6.37 (m, 2H), 4.96 (s, 1H), 4.88 (s, 1H), 3.74 (s, 3H), 3.10 (s, 2H), 2.94 - 2.86 (m, 2H), 2.82 - 2.73 (m, 1H), 2.57 - 2.22 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 198.91, 164.63, 161.91, 159.56, 150.37, 146.35, 143.84, 134.39, 131.08, 112.33, 109.81, 106.02, 100.45, 62.00, 59.87, 54.40, 52.60, 52.02, 42.33, 37.78, 30.58, 14.70. HRMS(ESI) calcd for C 24 H 33 N4O4 [M+H] + 441.2504, found 441.2502

[0065] A13: Pale yellow solid, yield 24%, mp 134 °C. 11H NMR (400 MHz, Chloroform-d) δ 8.37 (s, 1H), 7.12 - 7.08 (m, 1H), 7.01 - 6.91 (m, 2H), 6.70 - 6.67 (m, 1H), 4.96 (s, 1H), 4.88 (s, 1H), 3.11 (s, 2H), 2.94 - 2.86 (m, 2H), 2.82 - 2.74 (m, 1H), 2.54 - 2.22 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 198.89, 165.00, 158.12, 149.40, 146.32, 143.82, 134.39, 130.76, 124.72, 121.70, 119.52, 115.49, 112.36, 62.00, 59.87, 52.62, 52.00, 42.33, 37.77, 30.56, 14.71. HRMS (ESI) calcd for C 23 H 30 N4O3F [M + H] + 429.2296, found 429.2302.

[0066] A14: Pale yellow oil, yield 25%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.41 (s, 1H), 7.10 - 7.03 (m, 1H), 6.97 - 6.90 (m, 1H), 6.85 - 6.77 (m, 1H), 6.73 - 6.66 (m, 1H), 4.97 (s, 1H), 4.89 (s, 1H), 3.13 (s, 2H), 2.91 (m, 2H), 2.82 - 2.74 (m, 1H), 2.68 - 1.87 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 198.84, 165.04, 156.13, 148.87, 146.31, 143.75, 134.46, 130.62, 128.92, 122.94, 117.74, 117.44, 112.40, 61.98, 59.85, 52.62, 52.03, 42.37, 37.81, 30.60, 14.72. HRMS (ESI) calcd for C 23 H 30 N4O3Cl [M + H] + 445.2011, found 445.2006.

[0067] A15: Pale yellow oily substance, yield 21%. 1 H NMR (400 MHz, Chloroform-d) δ8.39 (s, 1H), 7.13 - 7.09 (m, 1H), 7.01 - 6.93 (m, 2H), 6.72 - 6.66 (m, 1H), 4.97 (s, 1H), 4.89 (s, 1H), 3.12 (s, 2H), 2.97 - 2.87 (m, 2H), 2.82 - 2.75 (m, 1H), 2.56 - 2.24 (m, 12H), 1.72 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ198.84, 164.96, 158.15, 149.54, 146.30, 143.76, 134.43, 130.76, 124.75, 121.71, 119.55, 115.49, 112.40, 61.99, 59.86, 52.62, 51.99, 42.33, 37.78, 30.56, 14.70. HRMS (ESI) calcd for C 23 H 30 N4O3Br [M + H] + 489.1494, found 489.1501.

[0068] A16: Pale yellow solid, yield 27%, mp 134 °C. 1 H NMR (400 MHz, Chloroform-d) δ10.73 (s, 1H), 10.04 (s, 1H), 8.39 (s, 1H), 7.09 (dd, J = 8.4, 2.2 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.78 - 6.72 (m, 1H), 5.12 - 4.86 (m, 2H), 3.17 (s, 2H), 3.03 - 2.91 (m, 2H), 2.90 - 2.80 (m, 1H), 2.76 - 2.29 (m, 12H), 2.26 (s, 3H), 1.78 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ198.85, 164.83, 155.40, 150.47, 146.37, 143.80, 134.40, 131.77, 130.01, 127.37, 115.99, 112.33, 62.00, 59.89, 52.61, 52.03, 42.34, 37.80, 30.57, 19.28, 14.70. HRMS (ESI) calcd for C 24 H 33N4O3[M+H] + 425.2557, found 425.2553.

[0069] A17: Pale yellow oil, yield 25%. 1 H NMR(400MHz, Chloroform-d) δ8.40(s,1H), 7.26(dd, J=8.7, 2.4Hz,1H), 7.12(d, J=2.4Hz,1H), 6.86(d, J=8.6Hz,1H), 6.71 - 6.66(m,1H), 4.96(s,1H), 4.88(s,1H), 3.11(s,2H), 2.94 - 2.86(m,2H), 2.81 - 2.74(m,1H), 2.55 - 2.23(m,12H), 1.72(s,3H), 1.21(s,9H). 13 C NMR(101MHz, Chloroform-d) δ198.89, 164.83, 155.26, 150.96, 146.34, 143.85, 140.99, 134.36, 128.32, 126.48, 115.73, 115.56, 112.31, 62.01, 59.89, 52.61, 52.03, 42.32, 37.77, 32.95, 30.57, 30.38, 14.70. HRMS(ESI) calcd for C 27 H 39 N4O3[M+H] + 467.3025, found 467.3022.

[0070] A18: Pale yellow oil, yield 28%. 1 H NMR(400MHz, Chloroform-d) δ10.45(s,1H), 10.05(s,1H), 8.39(s,1H), 6.88 - 6.81(m,2H), 6.71 - 6.63(m,2H), 4.97(s,1H), 4.89(s,1H), 3.69(s,3H), 3.13(s,2H), 2.96 - 2.87(m,2H), 2.82 - 2.74(m,1H), 2.58 - 2.24(m,12H), 1.72(s,3H). 1313C NMR(101MHz,CDCl3)δ198.81,164.79,151.77,151.42,150.26,146.28,143.74,134.43,117.69,116.96,116.18,113.65,112.44,61.97,59.85,54.87,52.58,51.96,42.34,37.80,30.57,14.69.HRMS(ESI)calcd for C 24 H 33 N4O4[M+H] + 441.2502,found 441.2502.

[0071] A19: Pale yellow solid, yield 22%, mp 144 °C. 1 1H NMR(400MHz,Chloroform-d)δ8.34(s,1H),6.96 - 6.90(m,1H),6.88 - 6.81(m,2H),6.71 - 6.66(m,1H),4.96(s,1H),4.88(s,1H),3.12(s,2H),2.94 - 2.86(m,2H),2.81 - 2.74(m,1H),2.57 - 2.24(m,12H),1.72(s,3H). 13 13C NMR(101MHz,Chloroform-d)δ198.86,165.06,155.91,153.68,148.93(d,J = 2.9Hz),146.32,143.81,134.40,117.76(d,J = 23.4Hz),117.26(d,J = 7.7Hz),116.44(d,J = 7.7Hz),114.98(d,J = 23.9Hz),112.38,61.99,59.88,52.62,51.99,42.34,37.80,30.57,14.69.HRMS(ESI)calcd for C 23 H 30 N4O3F[M+H] + 429.2301,found 429.2302.

[0072] A20: Pale yellow oil, yield 25%. 11H NMR (400 MHz, Chloroform-d) δ 8.35 (s, 1H), 7.18 - 7.10 (m, 2H), 6.89 - 6.84 (m, 1H), 6.71 - 6.67 (m, 1H), 4.97 (s, 1H), 4.89 (s, 1H), 3.13 (s, 2H), 2.95 - 2.86 (m, 2H), 2.82 - 2.74 (m, 1H), 2.57 - 2.23 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 198.82, 165.02, 156.11, 148.85, 146.30, 143.74, 134.43, 130.62, 128.92, 122.94, 117.74, 117.42, 112.39, 61.99, 59.86, 52.63, 52.00, 42.35, 37.80, 30.58, 14.69. HRMS (ESI) calcd for C 23 H 30 N4O3Cl [M + H] + 445.2008, found 445.2006.

[0073] A21: Pale yellow oil, yield 28%. 1 1H NMR (600 MHz, Chloroform-d) δ 8.32 (s, 1H), 7.29 (dd, J = 8.8, 2.4 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.71 - 6.67 (m, 1H), 4.97 (s, 1H), 4.89 (s, 1H), 3.13 (s, 2H), 2.94 - 2.87 (m, 2H), 2.81 - 2.75 (m, 1H), 2.57 - 2.25 (m, 12H), 1.72 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 198.89, 164.99, 156.56, 148.61, 146.26, 143.82, 134.39, 133.41, 131.88, 118.16, 118.05, 112.43, 109.82, 61.96, 59.82, 52.58, 51.94, 42.32, 37.76, 30.56, 14.71. HRMS (ESI) calcd for C 23 H 30 N4O3Br [M + H] + 489.1501, found 489.1505.

[0074] A22: Pale yellow oil, yield 21%. 1 H NMR(400MHz,Chloroform-d)δ11.22(s,1H),10.12(s,1H),8.51(s,1H),7.20-7.11(m,1H),6.74-6.65(m,2H),6.56-6.47(m,1H),4.97(s,1H),4.88(s,1H),3.13(s,2H),2.96-2.86(m,2H),2.82-2.73(m,1H),2.55-2.22(m,12H),1.72(s,3H). 13 C NMR(101MHz,Chloroform-d)δ198.90,164.83,160.68(d,J=251.9Hz),158.73(d,J=4.0Hz),146.35,143.83,142.91(d,J=8.1Hz),134.40,131.46(d,J=11.3Hz),112.35,112.12(d,J=3.3Hz),105.76(d,J=13.0Hz),104.42(d,J=21.1Hz),61.99,59.80,52.65,52.00,42.33,37.77,30.58,14.70.HRMS(ESI)calcd for C 23 H 30 N4O3F[M+H] + 429.2302,found429.2304.

[0075] A23: Pale yellow oil, yield 24%. 1 H NMR(400MHz,Chloroform-d)δ8.74(s,1H),7.16-7.09(m,1H),6.88-6.81(m,2H),6.72-6.67(m,1H),4.97(s,1H),4.89(s,1H),3.15(s,2H),2.95-2.89(m,2H),2.82-2.75(m,1H),2.56-2.23(m,12H),1.72(s,3H). 1313C NMR (101 MHz, Chloroform-d) δ 198.89, 164.82, 159.16, 146.37, 146.30, 143.81, 134.40, 133.40, 131.12, 119.36, 115.44, 113.80, 112.41, 61.96, 59.81, 52.64, 51.94, 42.33, 37.77, 30.57, 14.70. HRMS (ESI) calcd for C 23 H 30 N4O3cCl [M+H] + 445.2006, found 445.2010.

[0076] A24: Pale yellow oil, yield 22%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 7.08 - 7.02 (m, 2H), 6.91 - 6.85 (m, 1H), 6.71 - 6.67 (m, 1H), 4.97 (s, 1H), 4.88 (s, 1H), 3.15 (s, 2H), 2.95 - 2.88 (m, 2H), 2.83 - 2.75 (m, 1H), 2.56 - 2.22 (m, 12H), 1.72 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 198.90, 164.88, 159.16, 148.96, 146.31, 143.84, 134.39, 131.39, 123.49, 122.81, 116.17, 114.99, 112.39, 61.96, 59.84, 52.65, 51.94, 42.33, 37.77, 30.57, 14.71. HRMS (ESI) calcd for C 23 H 30 N4O3Br [M+H] + 489.1501, found 489.1504

[0077] A25: Pale yellow solid, yield 30%, mp 60 - 62 °C. 11H NMR (400 MHz, Chloroform-d) δ 11.25 (s, 1H), 9.96 (s, 1H), 8.35 (s, 1H), 7.33 - 7.28 (m, 1H), 6.99 - 6.94 (m, 1H), 6.71 - 6.66 (m, 1H), 4.96 (s, 1H), 4.88 (s, 1H), 3.12 (s, 2H), 2.96 - 2.84 (m, 2H), 2.83 - 2.73 (m, 1H), 2.65 - 2.17 (m, 12H), 1.72 (s, 3H), 1.36 (s, 9H), 1.22 (s, 9H). 13 13C NMR (101 MHz, Chloroform-d) δ 198.85, 164.64, 154.58, 151.62, 146.37, 143.79, 139.80, 135.91, 134.40, 125.91, 124.67, 115.33, 112.32, 62.02, 59.86, 52.61, 52.06, 42.33, 37.78, 34.10, 33.12, 30.45, 28.40, 14.69. HRMS (ESI) calcd for C 31 H 47 N4O3 [M + H] + 523.3648, found 523.3652.

[0078] A26: Pale yellow oil, yield 27%. 1 1H NMR (400 MHz, Chloroform-d) δ 11.66 (s, 1H), 9.84 (s, 1H), 8.50 (s, 1H), 6.72 - 6.63 (m, 1H), 6.10 - 6.04 (m, 1H), 5.92 - 5.87 (m, 1H), 4.96 (s, 1H), 4.88 (s, 1H), 3.77 - 3.70 (m, 6H), 3.10 (s, 2H), 2.95 - 2.86 (m, 2H), 2.81 - 2.30 (m, 13H), 1.72 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 198.87, 164.21, 162.76, 160.81, 158.74, 146.40, 145.85, 143.81, 134.39, 112.29, 99.59, 92.70, 89.48, 62.00, 59.83, 54.65, 54.44, 52.63, 52.02, 42.33, 37.79, 30.58, 14.69. HRMS (ESI) calcd for C 25 H 35N4O5[M+H] + 471.2607, found 471.2609.

[0079] A27: Pale yellow oil, yield 26%. 1 H NMR(400MHz, Chloroform-d) δ 12.25(s, 1H), 10.13(s, 1H), 9.28(s, 1H), 7.94 - 7.88(m, 1H), 7.74 - 7.67(m, 2H), 7.45 - 7.38(m, 1H), 7.30 - 7.24(m, 1H), 7.16 - 7.10(m, 1H), 6.70 - 6.65(m, 1H), 4.98(s, 1H), 4.88(s, 1H), 3.17(s, 2H), 2.96 - 2.87(m, 2H), 2.82 - 2.75(m, 1H), 2.59 - 2.24(m, 12H), 1.72(s, 3H). 13 C NMR(101MHz, CDCl3) δ 198.84, 164.74, 158.18, 146.90, 146.32, 143.76, 134.42, 132.23, 131.08, 128.14, 127.13, 126.47, 122.47, 118.85, 118.42, 112.40, 106.83, 61.99, 59.93, 52.65, 51.99, 42.34, 37.81, 30.58, 14.69. HRMS(ESI) calcd for C 27 H 33 N4O3[M+H] + 461.2553, found 461.2558.

[0080] A28: Yellow oil, yield 19%. 1 H NMR(600MHz, Chloroform-d) δ 8.49(s, 1H), 8.22 - 7.96(m, 2H), 7.07 - 6.90(m, 1H), 6.73 - 6.65(m, 1H), 4.97(s, 1H), 4.89(s, 1H), 3.16(s, 2H), 2.97 - 2.85(m, 2H), 2.83 - 2.23(m, 13H), 1.79 - 1.65(m, 3H). 1313C NMR (151 MHz, Chloroform-d) δ 198.93, 165.31, 162.75, 147.68, 146.27, 143.85, 139.34, 134.42, 126.13, 125.81, 117.06, 116.26, 112.44, 61.99, 59.82, 52.65, 51.97, 42.35, 37.77, 30.56, 14.71. HRMS (ESI) calcd for C 23 H 30 N5O5 [M+H] + 456.2247, found 456.2251.

[0081] Example 7

[0082] The MTT method was used to detect the inhibitory effects of the compounds obtained in the above examples on melanoma cells, breast cancer cells, glioblastoma, glioma, and human glioma cells.

[0083] Cells were seeded in DMEM medium containing 10% (v / v) fetal bovine serum, 100 mg / L penicillin, and 100 mg / L streptomycin and incubated in an incubator at 37 °C and 5% CO2. Cells in the logarithmic growth phase were digested with trypsin and made into cell suspensions. 100 μL (cell number 5×103) of the cell suspension was seeded in a 96-well plate (the edges were sealed with PBS) and cultured overnight in an incubator at 37 °C and 5% CO2. The drug was diluted with DMEM medium containing 1% (v / v) serum, and then the diluted drug was added to the 96-well cell culture plate, 100 μL per well, so that the final concentrations of the drug were 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L respectively. The control group was replaced with a culture medium containing the same volume of solvent. Three replicates were set for each concentration, and the cells were cultured for another 48 h. After culturing for 48 h, 100 μL of freshly prepared medium containing 0.5 mg / ml MTT was added to each well. After 3 h, the culture medium in the wells was discarded, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was mixed evenly with a micro oscillator for 8 min. After the formed formazan was completely dissolved, the OD492 nm value was measured with an enzyme-linked immunosorbent assay detector.

[0084] The inhibition rate of the drug on the growth of tumor cells was calculated according to the following formula: Tumor cell growth inhibition rate (%) = [A492 (negative control) - A492 (drug-treated group)] / A492 (negative control) × 100%. The half-maximal inhibitory concentration (IC50) of the sample was calculated using Graphpad Prism 6.02 software. The experimental results are shown in the following table.

[0085] Table 1 IC50 values of the example compounds and PAC-1 against A375 cells

[0086]

[0087] The cells were cultured with different samples at different concentrations for 48 hours

[0088] Table 2 IC50 values of the example compounds and PAC-1 against U251 cells

[0089]

[0090] The cells were cultured with different samples at different concentrations for 48 hours

[0091] Table 3 IC50 values of the example compounds and PAC-1 against LN229 cells

[0092]

[0093]

[0094] The cells were cultured with different samples at different concentrations for 48 hours

[0095] Table 4 IC50 values of the example compounds and PAC-1 against MDA-MB-231 cells

[0096]

[0097] The cells were cultured with different samples at different concentrations for 48 hours

[0098] Table 5 IC50 values of the example compounds and PAC-1 against MCF-7 cells

[0099]

[0100] The cells were cultured with different samples at different concentrations for 48 hours

[0101] In summary, the modification of PAC-1 in the present invention increases its anti-tumor action targets, reduces the off-target risk and drug resistance, enhances its anti-tumor activity, and significantly enhances the inhibitory effect on U251 cells of human brain glioma.

[0102] The above examples describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above examples. What is described in the above examples and the specification only illustrates the principle of the present invention. Without departing from the principle of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. An analogue of caspase activator, characterized in that: The analog is a PAC-1 analog containing a carvone structure represented by Formula I: In the formula, R1, R2, and R3 may be the same or different and are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, hydroxy, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form a C3-C8 ring or an aromatic ring; n, m, and x are the same or different and are each selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

2. The analog of caspase activator according to claim 1, characterized in that: In the formula of the said analog, R1, R2, and R3 may be the same or different and are independently selected from C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form a C3-C8 ring or an aromatic ring; n, m, and x are the same or different and are each selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

3. The zymogen activator analogue according to claim 2, characterized in that: In the formula of the said analog, R1, R2, and R3 may be the same or different and are independently selected from C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form an aromatic ring; n, m, and x are the same or different and are each selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

4. The analogue of caspase activator according to claim 3, characterized in that: In the formula of the said analog, R1, R2, and R3 may be the same or different and are independently selected from C1-C4 alkyl, C1-C2 alkoxy, C2-C4 alkenyl, nitro, or halogen; or, any two adjacent substituents among R1, R2, and R3 form an aromatic ring; n, m, and x are the same or different and are each selected from 1, 2, or 3, and n + m + x is less than or equal to 5.

5. The analogue of caspase activator according to claim 4, wherein: The said analog is 6. A pharmaceutical composition, characterized in that: The composition contains an analog of the procaspase activator represented by Formula I as claimed in claim 1.

7. Use of an analogue of the procaspase activator according to claim 1 or the pharmaceutical composition according to claim 6, characterized in that: Use of the analog of the procaspase activator represented by Formula I as claimed in claim 1 or the composition as claimed in claim 6 in the preparation of an anti-tumor drug.

8. The application according to claim 7, wherein: The said tumor is melanoma, breast cancer, glioblastoma, glioma, cervical cancer, liver cancer, fibrosarcoma, colon cancer, lymphoma, chronic myelogenous leukemia, or promyelocytic leukemia.

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