Analogs of procaspase activators and uses thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to an analog of pro-caspase activator (PAC-1 analog containing carvone structure) and application thereof in treating cancer. The analog of pro-caspase activator is a PAC-1 analog containing carvone structure shown in formula I: The PAC-1 analog in the application retains structures such as piperazine ring and hydrazide, introduces monoterpene carvone, and modifies structures of two benzene rings, thereby obtaining a new procasepase-3 activator. The derivatives have stronger physiological activity. The PAC-1 analog in the application is proved by pharmacological tests to be capable of inhibiting proliferation of various tumor cells, has good anticancer activity, and has a simple and feasible preparation method and easy operation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an analog of procysteine ​​activator (PAC-1 analog containing carvone structure) and its application in the treatment of cancer. Background Technology

[0002] Cancer has always been considered one of the diseases that seriously endanger human health. Drug therapy is one of the important means of treating tumors in clinical practice today, and the field of oncology has always been a key area for new drug research and development. With the continuous deepening of scientists' research on anti-tumor drugs, the induction of cell death to achieve anti-tumor effects 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 tumor cell paraapoptosis [J]. Life Sciences, 2021, 33(07): 888-895.).

[0003] Procaspase-3 plays a crucial role in apoptosis, and its expression is often higher in various tumor cells compared to normal cells. Procaspase-Activating Compound 1 (PAC-1) can chelate with zinc ions, affecting the expression of procaspase-3 and thus promoting tumor cell apoptosis, showing promising anti-tumor potential (West DC, Qin Y, Peterson QP, et al. Differential Effects of Procaspase-3 Activating Compounds in the Induction of Cancer Cell Death[J]. Molecular Pharmaceutics, 2012, 9(5):1425-1434.). Currently, PAC-1 is undergoing phase I clinical trials in cancer patients, and the results show that PAC-1 has inhibitory activity against various tumors such as ovarian cancer, hemangioendothelioma, and hepatocellular carcinoma. (Danciu OC, Nicholas MK, Emmadi R, et al. Phase I Study of Procaspase Activating Compound-1 (PAC-1) in the Treatment of Advanced Malignancies[J]. Molecular & Cellular Proteomics, 2018, 11(12):E217–E222.). Targeted drugs are highly specific, but mutations at the target site can easily lead to the failure of drug treatment. Therefore, multi-target synergistic design of PAC-1 is currently a research hotspot. Some researchers have integrated other anti-tumor pharmacophores into its scaffold to synthesize PAC-1 diarylurea conjugates, benzothiazole PAC-1 derivatives, etc., some of which have shown better tumor cell inhibition effects than PAC-1. (HergenrotherP J, Roth HS.Derivatives of Procaspase-Activating Compound 1 (PAC-1) and their Anticancer Activities[J]. Current Medicinal Chemistry, 2016, 23(3): 201-241)

[0004] In the treatment of brain tumors, PAC-1 combined with temozolomide has shown good efficacy (Tonogai EJ, Shan H, Botham RC, et al. Evaluation of a procaspase-3 activator with hydroxyurea ortemozolomide against high-grade meningioma in cell culture and canine cancer patients[J]. Neuro-Oncology, 2021(10):10.). However, research on PAC-1 alone for the treatment of brain tumors is currently lacking. PAC-1 itself has poor penetration ability and certain neurotoxicity, which limits its application in brain tumors. Therefore, it is necessary to develop validated derivatives to address these issues and achieve better results. Summary of the Invention

[0005] The purpose of this invention is to provide a PAC-1 analog (containing carvone structure) obtained by using procaspase-3 activator PAC-1 as a lead compound and its application in cancer treatment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An analogue of procysteine ​​activator, characterized in that: the analogue is a PAC-1 analogue containing a carvone structure as shown in Formula I:

[0008] Formula I

[0009] In the formula,

[0010] R1, R2, and R3 may be selected independently from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, hydroxyl, nitro, or halogen, respectively; or, any two adjacent substituents in R1, R2, and R3 may form a C3-C8 ring or aromatic ring.

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

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

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

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

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

[0016] In a further preferred embodiment, in the analogue formula, R1, R2, and R3 may be the same or different and independently selected from C1-C4 alkyl, C1-C2 alkoxy, C2-C4 alkenyl, nitro, or halogen; or, any two adjacent substituents in R1, R2, and R3 may form an aromatic ring.

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

[0018] More preferably, the analogue is

[0019]

[0020]

[0021]

[0022] This invention also provides a method for preparing the PAC-1 analogue:

[0023]

[0024] In the above preparation process:

[0025] The solvent used is a conventional reaction solvent, with no special requirements.

[0026] A pharmaceutical composition comprising an analogue of the activator of procysteine ​​as shown in Formula I.

[0027] The use of an analogue of the activator of caspase I or the pharmaceutical composition thereof, and the use of the analogue of the activator of caspase I or the composition thereof in the preparation of an antitumor drug.

[0028] The tumors mentioned are melanoma, breast cancer, glioblastoma, glioma, cervical cancer, liver cancer, fibrosarcoma, colon cancer, lymphoma, chronic myeloid leukemia, or promyelocytic leukemia.

[0029] Beneficial effects of this invention:

[0030] The PAC-1 analogues described in this invention retain the piperazine ring, hydrazide, and other structures, and introduce the monoterpenoid compound carvone to modify the structure of the two benzene rings, thereby obtaining novel procasepase-3 activators. These derivatives have stronger physiological activity.

[0031] The PAC-1 analogue of the present invention has been shown by pharmacological tests to inhibit the proliferation of various tumor cells and has good anti-cancer activity. Its preparation method is simple, feasible and easy to operate. Detailed Implementation

[0032] The feasibility of the present invention will be illustrated below through examples. Those skilled in the art should understand that modifications or substitutions to the corresponding technical features based on the teachings of the prior art still fall within the scope of protection claimed by the present invention.

[0033] Example 1

[0034] ( R Preparation of 2-methyl-5-(1-chloromethyl)vinylcyclohexyl-2-enone

[0035] 5 g (33.33 mmol) of L-carvone and 100 mL of n-hexane were added to a 250 mL round-bottom flask. 4.15 mL (36.67 mmol) of tert-butanol hypochlorite was added dropwise with stirring in an ice bath. After the mixture was brought to room temperature, stirring was continued for 3 h. Once the reaction was complete, the reaction solution was washed successively with saturated sodium sulfite aqueous 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] ( R Preparation of tert-butyl piperazine-1-carboxylate

[0038] Add 2g (10.87mmol) to a 250ml single-necked flask. R2g (10.87mmol) of 2-methyl-5-(1-chloromethyl)vinylcyclohexyl-2-enone N -6.03 g (32.61 mmol) of tert-butyloxycarbonylpiperazine, 2 mg (2.17 mmol) of KI, and 60 mL of anhydrous ethanol. The reaction was refluxed for 7 hours until complete. The mixture was evaporated and concentrated to obtain a yellow viscous liquid, which was extracted with water and EA, and purified by column chromatography to give 3.36 g of a low-melting-point white solid, 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).

[0039] Example 3

[0040] Preparation of (R)-2-methyl-5-[3-(piperazin-1-yl)prop-1-en-2-yl]cyclohex-2-en-1-one

[0041]

[0042] 1 g of (R)-4-[2-(4-methyl-5-oxocyclohexyl-3-en-1-yl)allyl]piperazine-1-carboxylic acid tert-butyl ester was dissolved in 30 ml of anhydrous methanol, and 10 ml of a saturated solution of hydrochloric acid in methanol was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the pH was adjusted to 8, the solvent was removed by evaporation and concentration, and the mixture was extracted with water and DCM. After drying with anhydrous sodium sulfate, the mixture was evaporated and concentrated to give 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).

[0043] Example 4

[0044] Preparation of N'-benzylmethyl-2-chloroacetylhydrazine

[0045]

[0046] 0.5 g of benzaldehyde (4.70 mmol) was dissolved in 20 mL of anhydrous ethanol, and 252 μL of hydrazine hydrate (5.19 mmol) was added. The mixture was refluxed at 80 °C for half an hour. After the reaction was complete, the solvent was removed by distillation under reduced pressure. 0.55 g of a pale yellow oily substance, benzaldehyde hydrazone, was obtained, with a yield of 98%. 0.2 g (1.67 mmol) of benzaldehyde hydrazone was dissolved in dichloromethane, and 0.7 g (5.01 mmol) of potassium carbonate was added as an acid-binding agent. 141 μL (1.84 mmol) of chloroacetyl chloride was added, and the mixture was reacted at room temperature for 30 min. After the reaction was complete, the filtrate was collected by filtration, and the solvent was removed under reduced pressure. After purification by thin-layer chromatography (DCM:MeOH = 20:1), 38 mg of a white solid was obtained, with a yield of 12%. MS (ESI) m / z: 219.0 [M+Na] +

[0047] Example 5

[0048] Synthesis of compound A1

[0049] Add 50 mg (0.24 mmol) (R)-2-methyl-5-[3-(piperazin-1-yl)prop-1-en-2-yl]cyclohex-2-en-1-one, 47 mg (0.24 mmol) N'-benzylmethyl-2-chloroacetylhydrazine, 3 mg (0.02 mmol) potassium iodide, and 99 mg (0.72 mmol) potassium carbonate to a 50 mL single-necked flask. Add 15 mL of acetonitrile and react at 82 °C for 2 hours. Remove the solvent under reduced pressure and purify by thin-layer chromatography (DCM:MeOH = 20:1) to give 25 mg of pale yellow solid Al, yield 26%, melting point 59-60 °C. 1 H NMR (400 MHz, Chloroform-d) δ 10.02 (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). 13CNMR (101 MHz, CDCl3) δ 198.86, 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 C 23 H 31 N4O2[M+H] ＋ 395.2447, found395.2451HRMS (ESI) calcd for C 23 H 31 N4O2[M+H] ＋ 395.2447, found 395.2451.

[0050] Example 6

[0051] Following the methods described in Examples 1 to 5 above, benzaldehyde and N'-benzylmethyl-2-chloroacetylhydrazine in Examples 4 and 5 were replaced with benzaldehyde and chloroacetylhydrazine containing the above-mentioned substituents, respectively, to synthesize compounds A2 to A28.

[0052] A2: Pale yellow solid, yield 24%, mp 114℃. 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). 13C 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 forC 23 H 31 N4O3[M+H] ＋ 411.2399, found 411.2396.

[0053] A3: Pale yellow solid, yield 32%, mp 124-126℃. 1 H NMR (400 MHz, Chloroform-d) δ11.17 (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 C 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 forC 26 H 35 N4O3[M+H] ＋ 451.2710, found 451.2709.

[0054] A4: Pale yellow oily substance, yield 22%. 1 H 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). 13 C 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 was found; 425.2553 was also found.

[0055] A5: Pale yellow oily substance, yield 26%. 1 H 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). 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 was found.

[0056] A6: Pale yellow oily substance, yield 31%. 1 H 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 441.2507.

[0057] A7: Pale yellow oily substance, yield 29%. 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 C NMR (151 MHz, Chloroform-d) δ198.88, 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), HRMS (ESI) calcd for C 23 H 30 N4O3F [M+H] ＋ 429.2305, found 429.2302.

[0058] A8: Pale yellow oily substance, yield 26%. 1 H NMR (600 MHz, Chloroform-d) δ 8.48 (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 (151 MHz, 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 was found as 429.2009.

[0059] A9: Pale yellow oily substance, yield 28%. 1 H NMR (600 MHz, Chloroform- d ) δ 8.47 (s, 1H), 7.48 (dd, J = 7.9, 1.5 Hz, 1H), 7.11 (dd, J = 7.7, 1.5 Hz, 1H), 6.73 (t, J =7.8 Hz, 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 ) δ 198.86, 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 C 23 H 30 N4O3Br [M+H] ＋ 489.1501, found 489.1504.

[0060] A10: Pale yellow solid, yield 28%, mp 70-72℃. 1H NMR (400 MHz, Chloroform- d ) δ10.83 (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.

[0061] A11: Pale yellow solid, yield 28% 32%; mp 78-80℃. 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). 13 C 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.

[0062] A12: Pale yellow solid, yield 28%, mp 64℃. 1 H 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 C NMR (101 MHz, CDCl3) δ198.91, 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, found441.2502

[0063] A13: Pale yellow solid, yield 24%, mp 134℃. 1 H 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 C 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.

[0064] A14: Pale yellow oily substance, yield 25%. 1 H 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 C 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, found445.2006.

[0065] A15: Pale yellow oily substance, yield 21%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.39 (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) δ 198.84, 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.

[0066] A16: Pale yellow solid, yield 27%, mp 134℃. 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) δ 198.85,164.83, 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 33 N4O3[M+H] ＋ 425.2557 was found; 425.2553 was also found.

[0067] A17: Pale yellow oily substance, yield 25%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.40 (s, 1H), 7.26 (dd, J = 8.7, 2.4 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 8.6 Hz,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). 13C NMR (101 MHz, Chloroform- d ) δ 198.89, 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.

[0068] A18: Pale yellow oily substance, yield 28%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.45 (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). 13 C NMR (101 MHz, CDCl3) δ 198.81, 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.

[0069] A19: Pale yellow solid, yield 22%, mp 144℃. 1H NMR (400 MHz, Chloroform- d ) δ 8.34(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 C NMR (101 MHz, Chloroform- d ) δ198.86, 165.06, 155.91, 153.68, 148.93 (d, J = 2.9 Hz), 146.32, 143.81, 134.40,117.76 (d, J = 23.4 Hz), 117.26 (d, J = 7.7 Hz), 116.44 (d, J = 7.7 Hz), 114.98 (d, J = 23.9 Hz), 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.

[0070] A20: Pale yellow oily substance, yield 25%. 1 H 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). 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.

[0071] A21: Pale yellow oily substance, yield 28%. 1 H 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 C 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.

[0072] A22: Pale yellow oily substance, yield 21%.1 H NMR (400 MHz, Chloroform-d) δ 11.22 (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) δ 198.90, 164.83, 160.68 (d, J = 251.9 Hz), 158.73 (d, J = 4.0Hz), 146.35, 143.83, 142.91 (d, J = 8.1 Hz), 134.40, 131.46 (d, J = 11.3 Hz), 112.35, 112.12 (d, J = 3.3 Hz), 105.76 (d, J = 13.0 Hz), 104.42 (d, J = 21.1 Hz),61.99, 59.80, 52.65, 52.00, 42.33, 37.77, 30.58, 14.70.HRMS (ESI) calcd forC 23 H 30 N4O3F [M+H] ＋ 429.2302, found 429.2304.

[0073] A23: Pale yellow oily substance, yield 24%. 1H NMR (400 MHz, Chloroform-d) δ 8.74 (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). 13 C 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 was found to be 445.2010.

[0074] A24: Pale yellow oily substance, yield 22%. 1 H 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). 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

[0075] A25: Pale yellow solid, yield 30%, mp 60-62℃. 1 H NMR (400 MHz, Chloroform-d) δ11.25 (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 C 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.

[0076] A26: Pale yellow oily substance, yield 27%. 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 C NMR (101MHz, 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 35 N4O5[M+H] ＋ 471.2607, found 471.2609.

[0077] A27: Pale yellow oily substance, yield 26%. 1 H NMR (400 MHz, 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). 13C NMR (101 MHz, CDCl3) δ198.84, 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.

[0078] A28: Yellow oily substance, yield 19%. 1 H NMR (600 MHz, 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). 13 C 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 forC 23 H 30 N5O5[M+H] ＋ 456.2247, found 456.2251.

[0079] Example 7

[0080] The inhibitory effects of the compounds obtained in the above examples on melanoma cells, breast cancer cells, glioblastoma, glioma, and human glioma cells were detected using the MTT assay.

[0081] Cells were seeded in DMEM medium containing 10% (v / v) fetal bovine serum, 100 mg / L penicillin, and 100 mg / L streptomycin, and incubated at 37°C in a 5% CO2 incubator. Logarithmically growing cells were harvested, digested with trypsin to prepare a cell suspension, and 100 μL (5 × 10³ cells) per well was seeded into 96-well plates (bordered with PBS) and cultured overnight at 37°C in a 5% CO2 incubator. The drug was diluted with DMEM medium containing 1% (v / v) serum, and then 100 μL of the diluted drug was added to each well of a 96-well cell culture plate to achieve final drug concentrations of 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L. The control group was treated with a medium containing an equal volume of solvent. Each concentration was tested in triplicate, and cells were cultured for another 48 h. After 48 h of culture, 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 well was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The mixture was stirred with a micro-shaker for 8 min. After the formazan was completely dissolved, the OD492 nm value was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0082] The inhibition rate of the drug on tumor cell growth was calculated using 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 determined using Graphpad Prism 6.02 software. The experimental results are shown in the table below.

[0083] Table 1. IC50 values ​​of the compounds in the examples and PAC-1 against A375 cells.

[0084]

[0085] Cells were cultured together with different samples of varying concentrations for 48 hours.

[0086] Table 2. IC50 values ​​of the compounds in the examples and PAC-1 against U251 cells.

[0087]

[0088] Cells were cultured together with different samples of varying concentrations for 48 hours.

[0089] Table 3. IC50 values ​​of the compounds in the examples and PAC-1 against LN229 cells.

[0090]

[0091] Cells were cultured together with different samples of varying concentrations for 48 hours.

[0092] Table 4. IC50 values ​​of the compounds in the examples and PAC-1 against MDA-MB-231 cells.

[0093]

[0094] Cells were cultured together with different samples of varying concentrations for 48 hours.

[0095] Table 5. IC50 values ​​of the compounds in the examples and PAC-1 against MCF-7 cells.

[0096]

[0097] Cells were cultured together with different samples of varying concentrations for 48 hours.

[0098] In summary, the modification of PAC-1 in this invention increases its anti-tumor targets, reduces off-target risks and drug resistance, enhances its anti-tumor activity, and significantly strengthens its inhibitory effect on human glioma U251 cells.

[0099] The above embodiments 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 to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. An analogue of procysteine ​​activator, characterized in that: The analogue is 。 2. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains an analogue of the procystein activator of claim 1.

Images