Celastrol derivatives, preparation method thereof and medical use

Abstract

The present invention discloses a celastrol derivative with a structure as shown in Formula IV, where X = O or NH; L is selected from -(CH2) n -, n is an integer from 2 to 5; R is selected from. The celastrol derivative of the present invention has a significant inhibitory effect on the proliferation of various tumor cells, and is significantly superior to the parent compound celastrol. The present invention also discloses the pharmaceutical use of the celastrol derivative in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, relates to celastrol derivatives, and particularly relates to celastrol pyrimidine / purine derivatives, a preparation method thereof, and a medical use thereof. Background Art

[0002] Cancer has become the leading cause of death globally. According to statistics from the World Health Organization (WHO), the most common cancers are breast cancer, lung cancer, colon cancer, rectal cancer, and prostate cancer. Cancer has gradually become a world problem endangering human life and health, and scientists urgently need to find effective solutions.

[0003] Celastrol (CEL) is an effective active ingredient derived from the root of the traditional Chinese medicine Tripterygium wilfordii, belonging to pentacyclic triterpenoid compounds. It has a wide range of pharmacological activities, such as anti-tumor, anti-inflammatory, antioxidant, immunosuppressive, etc.

[0004]

[0005] Although celastrol has relatively clear anti-tumor activity, its activity and drug-likeness need to be improved. Currently, there have been a large number of structural modification studies on celastrol, but no candidate compounds that can enter clinical trials have been obtained. Therefore, it is of great significance to find celastrol derivatives with stronger anti-tumor activity and better drug-likeness. Summary of the Invention

[0006] Pyrimidine and its fused pyrimidine (purine) derivatives are a class of heterocyclic aromatic scaffolds with a wide range of biological and pharmacological activities. Due to their good anti-tumor activity, they often appear as basic structural units in commercially available anti-tumor drugs. It has been found that pyrimidine and purine compounds show strong inhibitory effects on cyclin-dependent kinases (CDKs) and histone deacetylases (HDACs), etc. Based on this, the inventors introduced pyrimidine / purine analogs at the C-20 carboxyl position of celastrol to synthesize celastrol pyrimidine / purine derivatives, hoping to improve the anti-tumor activity of the derivatives.

[0007] The present invention is achieved by the following technical solutions:

[0008] A celastrol derivative having a structure as shown in formula IV:

[0009]

[0010] wherein, X = O or NH;

[0011] L is selected from -(CH2) n -, and n is an integer from 2 to 5;

[0012] R is selected from

[0013] Preferably, X = O, and L is selected from -(CH2) n -, n is an integer from 2 to 5, and R is selected from or X = NH, and L is selected from -(CH2) n -, n is an integer from 2 to 5, and R is selected from but does not include X = O, and L is selected from -(CH2)3-

[0014] More preferably, X = O, and L is selected from -(CH2) n -, n is 4 or 5, and R is selected from or X = O, and L is selected from -(CH2) n -, n is an integer from 3 to 5, and R is selected from or X = NH, and L is selected from -(CH2) n -, n is an integer from 2 to 4, and R is selected from but does not include X = O, and L is selected from -(CH2)3-

[0015]

[0016] As one of the technical solutions of the celastrol derivative of the present invention, the structure of the celastrol derivative is shown in Formula I:

[0017]

[0018] Among them, R1 is selected from

[0019] n is an integer from 2 to 5.

[0020] Preferably, n = 4 or 5.

[0021] As one of the technical solutions of the celastrol derivative of the present invention, the structure of the celastrol derivative is shown in Formula II:

[0022]

[0023] Among them, n is an integer from 2 to 5.

[0024] Preferably, n is an integer from 2 to 4.

[0025] As one of the technical solutions of the celastrol derivative of the present invention, the structure of the celastrol derivative is shown in Formula III:

[0026]

[0027] Among them, R2 = Cl, Br; R3 = NH2, H; n is an integer from 2 to 5.

[0028] Preferably, R2 = Cl, Br; R3 = NH2, H; n is an integer from 3 to 5, excluding the case where R2 = Br, R3 = H, and n = 3.

[0029] Specifically, the celastrol derivatives of the present invention are selected from the following compounds:

[0030]

[0031]

[0032] Most preferably, the structure of the celastrol derivative of the present invention is shown by the following formula:

[0033]

[0034] Another object of the present invention is to provide a preparation method of the celastrol derivative. When R is selected from the synthetic route is as follows:

[0035]

[0036] Among them, R1 is selected from H or n is an integer from 2 to 5. Preferably, n = 4 or 5.

[0037] It includes the following steps:

[0038] Step (1): Using pyridine as an acid-binding agent and anhydrous dichloromethane as a reaction solvent, the 2-aminopyrimidine analogue shown by the formula reacts with the acyl chloride shown by the formula to obtain intermediate a;

[0039] Step (2): Using NaHCO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent, intermediate a reacts with celastrol to obtain the target compound.

[0040] In step (1), the molar ratio of the 2-aminopyrimidine analogue to the acyl chloride is 1:1.2 to 1:1.5; the molar ratio of the acyl chloride to pyridine is 1:1 to 1:1.25.

[0041] Specifically, first dissolve the 2-aminopyrimidine analogue in anhydrous dichloromethane, then add pyridine, add the acyl chloride under ice bath conditions, and react at room temperature for 4 to 6 hours. After the reaction is completed, spin-dry the solvent, redissolve with a small amount of methanol, and purify through silica gel column chromatography with dichloromethane:methanol = 100:1 to 80:1 V / V as the eluent to obtain intermediate a.

[0042] In step (2), the molar ratio of celastrol to intermediate a is 1:1.5 to 1:2; the molar ratio of celastrol to NaHCO3 is 1:4 to 1:5, the reaction temperature is 60°C to 70°C, and the reaction time is 12 to 18 hours.

[0043] When R is selected from the synthesis route is as follows:

[0044]

[0045] wherein n is an integer from 2 to 5, preferably, n is an integer from 2 to 4.

[0046] It includes the following steps:

[0047] Step (1): Using triethylamine as a base catalyst and anhydrous ethanol as a reaction solvent, 2-chloropyrimidine reacts with the diamine shown in formula to obtain intermediate b;

[0048] Step (2): Using anhydrous dichloromethane as a reaction solvent, intermediate b and the carboxyl group of celastrol undergo an amidation reaction in the presence of EDCI, HOBT, and DIPEA to obtain the target compound;

[0049] In step (1), the molar ratio of 2-chloropyrimidine to the diamine is 1:1.5 to 1:3; the reaction temperature is 70 to 80°C, and the reaction time is 12 to 18 hours.

[0050] In step (2), the molar ratio of celastrol to intermediate b is 1:2 to 1:3; the molar ratio of celastrol to EDCI, HOBT, and DIPEA is 1:1.5 to 3:1.5 to 3:2 to 5; the reaction temperature is 25°C to 40°C, and the reaction time is 8 to 12 hours.

[0051] When R is selected from the synthesis route is as follows:

[0052]

[0053] wherein, R2 = Cl, Br; R3 = NH2, H; n is an integer from 2 to 5;

[0054] It includes the following steps:

[0055] Step (1): Using potassium carbonate as an acid-binding agent and acetone as a reaction solvent, the purine analogue shown in formula reacts with the dibromoalkane shown in formula to obtain intermediate c;

[0056] Step (2): Using NaHCO3 as the base catalyst and N,N-dimethylformamide as the reaction solvent, intermediate c reacts with celastrol to obtain the target compound;

[0057] In step (1), the molar ratio of the purine analogue to the dibromoalkane is 1:3 to 1:4; the molar ratio of the purine analogue to potassium carbonate is 1:3 to 1:4; the reaction temperature is 50°C to 60°C, and the reaction time is 7 to 12 hours.

[0058] In step (2), the molar ratio of celastrol to intermediate c is 1:1.5 to 1:2; the molar ratio of celastrol to NaHCO3 is 1:4 to 1:5; the reaction temperature is 60°C to 70°C, and the reaction time is 12 to 18 hours.

[0059] Another object of the present invention is to provide the pharmaceutical use of the celastrol derivative in the preparation of anti-tumor drugs.

[0060] The tumors are breast cancer, prostate cancer, lung cancer, and liver cancer.

[0061] An anti-tumor drug composition, which uses the celastrol derivative of the present invention as the active ingredient and is supplemented with a pharmaceutically acceptable carrier to form any pharmaceutically acceptable dosage form.

[0062] The dosage forms are tablets, capsules, dripping pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, and nano-formulations.

[0063] Advantages of the present invention:

[0064] The structure of the celastrol pyrimidine / purine derivatives of the present invention is novel, the preparation method is simple, the reaction conditions are mild, the reagents used are low-toxic, the raw materials are cheap and easily available, the post-treatment is convenient, no harmful substances are produced, which conforms to "green science", and the yield of the target product is relatively high.

[0065] Pharmacological experiments show that the celastrol pyrimidine / purine derivatives of the present invention have obvious inhibitory effects on the proliferation of various tumor cells, and are significantly superior to the parent compound celastrol. The compounds of the present invention are expected to become new anti-tumor candidate drugs and are worthy of in-depth study. Specific embodiments

[0066] The following further introduces the substantial content of the present invention in combination with examples. These examples are illustrative and do not limit the protection scope of the present invention accordingly.

[0067] Example 1: Preparation of Compound I1

[0068]

[0069] 2-Aminopyrimidine (190.2 mg, 2 mmol) was dissolved in 10 mL of anhydrous dichloromethane. Pyridine (242 μL, 3 mmol) was added, and 5-bromovaleryl chloride (322 μL, 2.4 mmol) was added under ice bath conditions. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC (the developing solvent was dichloromethane:methanol = 40:1 V / V). After reacting for 6 h, the reaction was completed. The solvent was evaporated, and a small amount of methanol was added for re-dissolution. It was purified by silica gel column chromatography (the eluent was dichloromethane:methanol = 100:1 V / V) to obtain intermediate a1 (180.6 mg, yield about 35.0%).

[0070] Celastrol (90 mg, 0.2 mmol), intermediate a1 (103 mg, 0.4 mmol) and NaHCO3 (67 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Stir at 60 °C for 16 h, add 60 mL of water for dilution, extract with ethyl acetate (70 mL × 3), combine the organic layers, wash with saturated NaCl solution (200 mL × 1), dry over anhydrous Na2SO4 overnight, filter by suction, concentrate the organic solution, and purify by silica gel column chromatography (the eluent was dichloromethane:methanol = 70:1 - 50:1 V / V), and dry under vacuum to obtain compound Ⅰ1 (94 mg, yield 74.9%), an orange-red powder.

[0071] Structure identification data of compound Ⅰ1: 1 H-NMR (400 MHz, CDCl3, TMS) δ 8.57 (d, J = 4.7 Hz, 2H), 7.04–6.99 (m, 2H), 6.54 (d, J = 1.4 Hz, 1H), 6.34 (d, J = 7.2 Hz, 1H), 3.96 (qt, J = 11.2, 6.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 1.44 (s, 3H), 1.26 (s, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.55 (s, 3H). 13C-NMR(500MHz,CDCl3)δ178.39,178.36,173.71,170.28,164.80,158.31,157.57,146.10(C×2),134.19,127.43,119.66,118.17,117.30,116.20,64.16,45.10,44.30,42.97,40.44,39.45,38.25,36.85,36.38,34.78,33.56,32.84,31.61,30.80,30.59,29.84,29.63,28.68,28.05,21.64,21.42,18.51,10.28.LC-TOF-MS(ESI)calculated for C 38 H 49 N3O5[M+H] + 628.37,found 628.7.

[0072] Example 2: Preparation of Compound Ⅰ2

[0073]

[0074] Referring to the preparation method of Compound I1 in Example 1, 6-bromohexanoyl chloride in an equimolar amount was used instead of 5-bromovaleryl chloride to obtain Compound Ⅰ2 (113.8 mg, yield 88.6%), an orange powder.

[0075] Structure identification data of Compound Ⅰ2: 1 H-NMR(400MHz,CDCl3,TMS)δ8.60(d,J = 4.8Hz,2H),7.03(dd,J = 7.1,1.4Hz,1H),6.99(t,J = 4.8Hz,1H),6.55(d,J = 1.5Hz,1H),6.34(d,J = 7.2Hz,1H),4.00–3.80(m,2H),2.68(p,J = 7.6Hz,2H),2.21(s,3H),1.44(s,3H),1.26(s,4H),1.17(s,3H),1.09(s,3H),0.54(s,3H). 1313C-NMR (500 MHz, CDCl3) δ 178.38, 178.36, 172.76, 170.57, 164.95, 158.35, 157.68, 146.19 (C×2), 134.47, 127.40, 119.62, 118.25, 117.61, 116.23, 64.65, 45.09, 44.32, 42.95, 40.45, 39.48, 38.26, 37.26, 36.39, 34.74, 33.84, 32.79, 31.62, 30.76, 30.58, 29.90, 29.62, 28.66, 28.38, 26.11, 24.61, 21.54, 18.38, 10.30. LC-TOF-MS (ESI) calculated for C 39 H 51 N3O5 [M+H] + 642.38, found 642.7.

[0076] Example 3: Preparation of Compound Ⅰ3

[0077]

[0078] 4-(3-Pyridyl)-2-aminopyrimidine (344.4 mg, 2 mmol) was dissolved in 10 mL of anhydrous dichloromethane. Pyridine (242 μL, 3 mmol) was added, and 5-bromovaleryl chloride (296.5 μL, 2.2 mmol) was added under ice bath conditions. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC (the developing solvent was dichloromethane:methanol = 40:1 V / V). After 6 h, the reaction was completed. The solvent was evaporated under reduced pressure, and a small amount of methanol was added to redissolve. The product was purified by silica gel column chromatography (the eluent was dichloromethane:methanol = 100:1 - 80:1 V / V) to obtain intermediate a3 (258.6 mg, yield about 38.6%).

[0079] Celastrol (90 mg, 0.2 mmol), intermediate a1 (134.1 mg, 0.4 mmol) and NaHCO3 (67 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide. After stirring at 60 °C for 16 h, 60 mL of water was added for dilution, and the mixture was extracted with ethyl acetate (70 mL × 3). The organic layers were combined, washed with saturated NaCl solution (200 mL × 1), dried over anhydrous Na2SO4 overnight, filtered, and the organic solution was concentrated. The product was purified by silica gel column chromatography (the eluent was dichloromethane:methanol = 70:1 - 50:1 V / V) and dried under vacuum to obtain Compound Ⅰ3 (80.9 mg, yield 57.4%), an orange-red powder.

[0080] Structure identification data of Compound Ⅰ3: 1H-NMR(400MHz, CDCl3, TMS) δ 9.25 (s, 1H), 8.78–8.72 (m, 1H), 8.66 (d, J=5.2 Hz, 1H), 8.37 (d, J=8.0 Hz, 1H), 7.46 (dd, J=8.0, 4.8 Hz, 1H), 7.42 (d, J=5.2 Hz, 1H), 7.01–6.98 (m, 1H), 6.53 (d, J=1.5 Hz, 1H), 6.33 (d, J=7.1 Hz, 1H), 4.04–3.90 (m, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.19 (s, 3H), 1.43 (s, 3H), 1.25 (s, 4H), 1.17 (s, 3H), 1.09 (s, 3H), 0.55 (s, 3H). 13 C-NMR(500MHz, CDCl3) δ 178.36, 170.26, 164.79, 162.99, 159.36, 157.89, 151.98, 148.51, 146.07, 134.70, 134.19, 131.96, 127.41, 123.88, 119.63, 118.16, 117.29, 111.65, 64.14, 45.09, 44.29, 42.96, 40.45, 39.44, 38.24, 36.95, 36.38, 34.78, 33.54 (C×2), 32.85, 31.61, 30.79, 30.58 (C×2), 29.85, 29.62, 28.67, 28.08, 21.63, 21.34, 18.53, 10.28. LC-TOF-MS(ESI) calculated for C 43 H 52 N4O5 [M+H] + 705.39, found 705.7.

[0081] Example 4: Preparation of Compound Ⅰ4

[0082]

[0083] Referring to the preparation method of Compound I3 in Example 3, 6-bromohexanoyl chloride in an equimolar amount was used instead of 5-bromovaleryl chloride to obtain Compound Ⅰ4 (62.8 mg, yield 43.7%), an orange powder.

[0084] Structure identification data of Compound Ⅰ4: 1H-NMR(400MHz,CDCl3,TMS)δ9.26(s,1H),8.74(d,J=4.9Hz,1H),8.69(d,J=5.3Hz,1H),8.40(d,J=8.0Hz,1H),7.44(t,J=5.0Hz,2H),7.02(d,J=7.1Hz,1H),6.55(s,1H),6.34(d,J=7.1Hz,1H),3.91(ddt,J=39.9,11.1,6.3Hz,2H),2.81(d,J=6.8Hz,2H),2.20(s,3H),1.43(s,3H),1.25(s,3H),1.17(s,3H),1.09(s,3H),0.54(s,3H). 13 C-NMR(500MHz,CDCl3)δ178.34,170.49,164.94,162.99,159.39,158.00,151.96,148.52,146.14,134.75,134.42,132.02,127.39,123.86,119.57,118.23,117.51,111.68,64.62,45.09,44.32,42.95,40.44,39.47,38.25,37.35,36.38,34.75,33.81,32.78,31.61,30.76,30.57,29.90,29.63,28.66,28.41,26.94,26.11,25.30,24.55,21.55,18.40,10.29.LC-TOF-MS(ESI)calculated for C 44 H 54 N4O5[M+H] + 719.41,found719.7.

[0085] Example 5: Preparation of Compound Ⅱ1

[0086]

[0087] Dissolve 2-chloropyrimidine (229 mg, 2 mmol) in 8 mL of ethanol, and successively add ethylenediamine (267 μL, 4 mmol) and triethylamine (340 μL, 4 mmol). Reflux at 80 °C overnight, and monitor the reaction progress by TLC (the developing solvent is PE:EA = 3:1). After the reaction is completed, remove the solvent by rotary evaporation to obtain intermediate b1.

[0088] Celastrol (90 mg, 0.2 mmol), EDCI (57.5 mg, 0.3 mmol), and HOBt (40.5 mg, 0.3 mmol) were dissolved in 10 mL of dichloromethane. Then, N,N - diisopropylethylamine (DIPEA, 142 μL, 0.8 mmol) was added, and the reaction was activated at room temperature (25 °C) for 3 h. After TLC detection showed that celastrol was completely activated, intermediate b1 (83 mg, 0.6 mmol) was added, and the reaction was continued at room temperature for 12 h. After the reaction was completed, an appropriate amount of dichloromethane was added for dilution, and the mixture was washed with 5% HCl solution (30 mL × 3). The organic layer was taken, washed with saturated NaCl solution (80 mL × 1), dried over anhydrous Na2SO4 overnight, filtered by suction, the filtrate was concentrated, and purified by silica gel column chromatography (the eluent was dichloromethane:methanol = 70:1 V / V) to obtain compound Ⅱ1 (84.0 mg, yield 73.6%), an orange - red powder.

[0089] Structural identification data of compound Ⅱ1: 1 1H - NMR (400 MHz, CDCl3, TMS) δ 8.26 (d, J = 4.7 Hz, 2H), 6.99 (dd, J = 7.1, 1.4 Hz, 1H), 6.57 (t, J = 4.8 Hz, 1H), 6.52 (d, J = 1.7 Hz, 1H), 6.31 (d, J = 7.1 Hz, 1H), 3.53 (p, J = 5.8 Hz, 2H), 3.31 (p, J = 5.4 Hz, 2H), 2.20 (s, 3H), 1.41 (s, 3H), 1.22 (s, 3H), 1.10 (d, J = 8.6 Hz, 6H), 0.58 (s, 3H). 13 13C - NMR (500 MHz, CDCl3) δ 178.43, 170.38, 164.76, 162.99, 162.53, 158.05, 146.11, 134.08, 127.39, 119.66, 118.03, 117.19, 111.10, 110.74, 45.07, 44.43, 43.00, 42.50, 40.13, 39.38, 38.21, 36.43, 34.88, 33.81, 33.54, 31.59, 31.20, 30.78, 29.90, 29.72, 29.27, 28.64, 21.69, 18.20, 10.29. LC - TOF - MS (ESI) calculated for C 35 H 46 4N4O3 [M + H] + 571.36, found 571.7.

[0090] Example 6: Preparation of compound Ⅱ2

[0091]

[0092] Referring to the preparation method of Compound Ⅱ1 in Example 5, 1,3-propanediamine in an equimolar amount was used instead of ethylenediamine to obtain Compound Ⅱ2 (77.6 mg, yield 63.2%), an orange-red powder.

[0093] Structure identification data of Compound Ⅱ2: 1 H-NMR(400 MHz, CDCl3, TMS) δ 8.25 (d, J = 4.8 Hz, 2H), 6.99 (dd, J = 7.1, 1.4 Hz, 1H), 6.54 (t, J = 4.8 Hz, 1H), 6.51 (d, J = 1.5 Hz, 1H), 6.32 (d, J = 7.2 Hz, 1H), 3.46 (q, J = 6.3 Hz, 2H), 3.19 (qt, J = 13.1, 6.5 Hz, 2H), 2.20 (s, 3H), 1.43 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 1.13 (s, 3H), 0.65 (s, 3H). 13 C-NMR(500 MHz, CDCl3) δ 178.41, 177.78, 170.25, 164.75, 162.81, 158.08 (C×2), 146.08, 133.99, 127.44, 119.60, 118.01, 117.08, 110.64, 45.07, 44.44, 43.02, 40.39, 39.41, 38.18, 38.15, 36.43, 35.91, 35.15, 33.99, 33.48, 31.70, 30.98, 30.88, 30.15, 29.79, 29.41, 28.72, 21.80, 18.39, 10.28. LC-TOF-MS(ESI) calculated for C 36 H 48 N4O3 [M + H] + 585.37, found 585.7.

[0094] Example 7: Preparation of Compound Ⅱ3

[0095]

[0096] Referring to the preparation method of Compound Ⅱ1 in Example 5, 1,4-butanediamine in an equimolar amount was used instead of ethylenediamine to obtain Compound Ⅱ3 (68.7 mg, yield 57.4%), an orange-red powder.

[0097] Structure identification data of Compound Ⅱ3: 1H-NMR (400 MHz, CDCl3, TMS) δ 8.24 (d, J = 4.8 Hz, 2H), 7.00 (dd, J = 7.1, 1.4 Hz, 2H), 6.55–6.48 (m, 2H), 6.33 (d, J = 7.2 Hz, 1H), 3.38 (q, J = 6.6 Hz, 2H), 3.16 (q, J = 6.5 Hz, 2H), 2.20 (s, 3H), 1.43 (s, 3H), 1.25 (s, 4H), 1.12 (d, J = 8.6 Hz, 6H), 0.62 (s, 3H). 13 C-NMR (500 MHz, CDCl3) δ 178.42, 177.73, 170.16, 164.73, 162.32, 158.06, 158.03, 146.08, 134.00, 127.46, 119.62, 118.05, 117.10, 110.50, 45.06, 44.38, 43.01, 40.96, 40.34, 39.39, 39.35, 38.17, 36.39, 35.08, 33.84, 33.48, 31.63, 31.03, 30.87, 30.21, 29.46, 28.70, 26.97, 26.58, 21.81, 18.34, 10.29. LC-TOF-MS (ESI) calculated for C 37 H 50 N4O3 [M+H] + 599.39, found 599.7.

[0098] Example 8: Preparation of Compound Ⅲ1

[0099]

[0100] 2-Amino-6-chloropurine (149 mg, 1 mmol) was dissolved in 10 mL of acetone. Potassium carbonate (552 mg, 4 mmol) and 1,3-dibromopropane (406 μL, 4 mmol) were added successively. The reaction was refluxed at 50 °C. The reaction progress was monitored by TLC (developing solvent: dichloromethane:methanol = 40:1 V / V). The reaction was carried out for 7 h. At this time, the reaction was completed. The solvent was evaporated to dryness, and a small amount of methanol was added to redissolve. The product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1 V / V) to obtain intermediate c1 (191.8 mg, yield about 66.0%).

[0101] Celastrol (90 mg, 0.2 mmol), intermediate c1 (116.2 mg, 0.4 mmol) and NaHCO3 (67 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide and stirred at 60 °C for 16 h. Then 60 mL of water was added for dilution, and the mixture was extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with saturated NaCl solution (200 mL × 1), dried over anhydrous Na2SO4 overnight, filtered by suction, and the organic solution was concentrated. After silica gel column chromatography (the eluent was dichloromethane:methanol = 70:1 - 50:1 V / V), vacuum drying gave compound Ⅲ1 (71.3 mg, yield 54.0%), an orange-red powder.

[0102] Structure identification data of compound Ⅲ1: 1 1H-NMR (400 MHz, CDCl3, TMS) δ 7.75 (s, 1H), 7.01 (d, J = 7.1 Hz, 1H), 6.55 (s, 1H), 6.34 (d, J = 7.1 Hz, 1H), 4.17 (t, J = 7.0 Hz, 2H), 4.10–3.91 (m, 2H), 2.21 (s, 3H), 1.44 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.09 (s, 3H), 0.51 (s, 3H). 13 13C-NMR (500 MHz, CDCl3) δ 178.37, 178.02, 169.92, 164.74, 159.23, 153.84, 151.49, 146.08, 141.97, 134.17, 127.48, 125.33, 119.59, 118.27, 117.36, 61.41, 45.04, 44.18, 42.91, 41.01, 40.46, 39.35, 38.30, 36.31, 34.64, 33.51, 32.80, 31.55, 30.78, 30.58, 29.83, 29.65, 28.74, 28.65, 21.61, 18.68, 10.30. LC-TOF-MS (ESI) calculated for C 37 H 46 ClN5O4 [M + H] + 661.33, found 661.6.

[0103] Example 9: Preparation of compound Ⅲ2

[0104]

[0105] Referring to the preparation method of Compound Ⅲ1 in Example 8, 1,4-dibromobutane in an equimolar amount was used instead of 1,3-dibromopropane to obtain Compound Ⅲ2 (80.5 mg, yield 59.7%), an orange-red powder.

[0106] Structural identification data of Compound Ⅲ2: 1 H-NMR (400 MHz, CDCl3, TMS) δ 7.75 (s, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.54 (s, 1H), 6.34 (d, J = 7.2 Hz, 1H), 4.12 (t, J = 7.0 Hz, 2H), 3.93 (ddt, J = 44.5, 11.5, 5.4 Hz, 2H), 2.21 (s, 3H), 1.45 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 0.50 (s, 3H). 13 C-NMR (500 MHz, CDCl3) δ 178.39, 178.13, 170.02, 164.77, 159.21, 153.89, 151.42, 146.08, 142.09, 134.25, 127.44, 125.30, 119.54, 118.24, 117.34, 63.36, 45.06, 44.22, 43.42, 42.93, 40.42, 39.39, 38.26, 36.32, 34.69, 33.47, 32.78, 31.57, 30.77, 30.56, 29.81, 29.65, 28.64, 26.50, 25.70, 21.62, 18.56, 10.29. LC-TOF-MS (ESI) calculated for C 38 H 48 ClN5O4 [M + H] + 674.34, found 674.7.

[0107] Example 10: Preparation of Compound Ⅲ3

[0108]

[0109] Referring to the preparation method of Compound Ⅲ1 in Example 8, 1,5-dibromopentane in an equimolar amount was used instead of 1,3-dibromopropane to obtain Compound Ⅲ3 (91.3 mg, yield 66.3%), an orange-red powder.

[0110] Structural identification data of Compound Ⅲ3: 11H-NMR (400 MHz, CDCl3, TMS) δ 7.74 (s, 1H), 7.02 (dd, J = 7.2, 1.3 Hz, 1H), 6.52 (d, J = 1.5 Hz, 1H), 6.34 (d, J = 7.2 Hz, 1H), 4.08 (t, J = 7.2 Hz, 2H), 3.90 (ddt, J = 37.0, 11.1, 6.5 Hz, 2H), 2.20 (s, 3H), 1.44 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 0.51 (s, 3H). 13 13C-NMR (500 MHz, CDCl3) δ 178.35, 178.21, 170.08, 164.82, 159.19, 153.88, 151.32, 146.06, 142.16, 134.29, 127.41, 125.28, 119.47, 118.23, 117.34, 63.93, 45.06, 44.26, 43.52, 42.94, 40.40, 39.43, 38.27, 36.34, 34.72, 33.59, 32.73, 31.58, 30.75, 30.56, 29.84, 29.63, 29.26, 28.64, 27.78, 23.16, 21.60, 18.49, 10.30. LC-TOF-MS (ESI) calculated for C 39 H 50 ClN5O4 [M + H] + 688.36, found 688.7.

[0111] Example 11: Preparation of Compound Ⅲ4

[0112]

[0113] 6-Bromopurine (199 mg, 1 mmol) was dissolved in 10 mL of acetone. Potassium carbonate (552 mg, 4 mmol) and 1,3-dibromopropane (406 μL, 4 mmol) were added successively. The reaction was refluxed at 50 °C. The reaction progress was monitored by TLC (developing solvent: dichloromethane:methanol = 40:1 V / V). The reaction was carried out for 7 h. At this time, the reaction was completed. The solvent was evaporated to dryness, and a small amount of methanol was added to redissolve. The product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 180:1 V / V) to obtain intermediate c4 (139.8 mg, yield about 43.7%).

[0114] Celastrol (90 mg, 0.2 mmol), intermediate c4 (128 mg, 0.4 mmol) and NaHCO3 (67 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide and stirred at 60 °C for 16 h. Then 60 mL of water was added for dilution, and the mixture was extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with saturated NaCl solution (200 mL × 1), dried over anhydrous Na2SO4 overnight, filtered by suction, and the organic solution was concentrated. After silica gel column chromatography (the eluent was dichloromethane:methanol = 60:1 - 30:1 V / V), vacuum drying gave compound Ⅲ4 (103.5 mg, yield 75.0%), an orange-red powder.

[0115] Structure identification data of compound Ⅲ4: 1 1H-NMR (400 MHz, CDCl3, TMS) δ 8.70 (s, 1H), 8.13 (s, 1H), 7.01 (dd, J = 7.1, 1.4 Hz, 1H), 6.51 (d, J = 1.5 Hz, 1H), 6.34 (d, J = 7.2 Hz, 1H), 4.37 (td, J = 7.0, 1.6 Hz, 2H), 4.00 (ddt, J = 36.8, 11.5, 5.9 Hz, 2H), 2.20 (s, 3H), 1.45 (s, 3H), 1.26 (s, 3H), 1.15 (s, 3H), 1.09 (s, 3H), 0.50 (s, 3H). 13 13C-NMR (500 MHz, CDCl3) δ 178.35, 178.06, 169.65, 164.62, 152.03, 150.63, 146.06, 144.82, 143.38, 134.33, 134.02, 127.50, 119.62, 118.25, 117.17, 61.04, 45.02, 44.19, 42.89, 41.84, 40.49, 39.39, 38.28, 36.31, 34.71, 33.56, 32.80, 31.56, 30.80, 30.56, 29.78, 29.75, 28.90, 28.64, 21.64, 18.67, 10.29. LC-TOF-MS (ESI) calculated for C 37 H 45 BrN4O4 [M + H] + 689.26, found 689.6.

[0116] Example 12: Preparation of compound Ⅲ5

[0117]

[0118] Referring to the preparation method of Compound Ⅲ4 in Example 11, 1,4-dibromobutane was used in an equimolar amount to replace 1,3-dibromopropane, and Compound Ⅲ5 (63.0 mg, yield 44.8%) was obtained, which was an orange-red powder.

[0119] Structural identification data of Compound Ⅲ5: 1 H-NMR(400MHz,CDCl3,TMS)δ8.70(s,1H),8.14(s,1H),7.01(dd,J=7.1,1.4Hz,1H),6.51(d,J=1.4Hz,1H),6.33(d,J=7.2Hz,1H),4.32(t,J=7.2Hz,2H),3.95(ddt,J=62.8,11.6,5.4Hz,2H),2.21(s,3H),1.45(s,3H),1.25(s,3H),1.14(s,3H),1.09(s,3H),0.51(s,3H). 13 C-NMR(500MHz,CDCl3)δ178.35,178.20,169.86,164.71,151.99,150.63,146.06,144.90,143.32,134.31,134.11,127.46,119.57,118.22,117.22,63.17,45.04,44.22,42.91,40.44,39.39,38.28,36.33,34.72,33.53,32.81,31.58,30.81,30.56(C×2),29.78,29.70,28.64,26.60,25.69,21.63,18.58,10.29.LC-TOF-MS(ESI)calculated for C 38 H 47 BrN4O4[M+H] + 705.28,found 705.6.

[0120] Example 13: Preparation of Compound Ⅲ6

[0121]

[0122] Referring to the preparation method of Compound Ⅲ4 in Example 11, 1,5-dibromopentane was used in an equimolar amount to replace 1,3-dibromopropane, and Compound Ⅲ6 (73.5 mg, yield 51.2%) was obtained, which was an orange-red powder.

[0123] Structural identification data of Compound Ⅲ6: 1H-NMR(400MHz, CDCl3, TMS) δ 8.67(s, 1H), 8.13(s, 1H), 7.00(dd, J = 7.1, 1.4Hz, 1H), 6.48(d, J = 1.5Hz, 1H), 6.33(d, J = 7.1Hz, 1H), 4.29(t, J = 7.1Hz, 2H), 3.88(ddt, J = 34.5, 10.9, 6.4Hz, 2H), 2.19(s, 3H), 1.43(s, 3H), 1.25(s, 4H), 1.09(d, J = 6.5Hz, 6H), 0.51(s, 3H). 13 C-NMR(500MHz, CDCl3) δ 178.28, 178.17, 170.00, 164.74, 151.90, 150.66, 146.03, 144.98, 143.23, 134.22, 134.16, 127.41, 119.48, 118.21, 117.24, 63.80, 45.05, 44.30, 44.24, 42.90, 40.40, 39.40, 38.26, 36.33, 34.70, 33.62, 32.72, 31.58, 30.75, 30.55, 29.80, 29.67, 29.57, 28.65, 27.84, 23.20, 21.60, 18.49, 10.28. LC-TOF-MS(ESI) calculated for C 39 H 49 BrN4O4[M + H] + 717.29, found 717.6.

[0124] Example 14: In vitro anti-tumor activity study

[0125] The MTT colorimetric method was used to test the in vitro anti-tumor activity of celastrol derivatives, and CEL and CDDO-Me (Bardoxolone methyl) were selected as positive control drugs.

[0126] Instruments: Laminar flow hood (SW-CJ-1FD, AIRTECH, Sujing Antai), CO2 incubator (Heracell VIOS 160i, Thermo, USA), biological microscope (IX71, Olympus, Japan), multi-functional microplate reader (POLARstar, Omega, USA).

[0127] Reagents: DMEM medium (Gibco), fetal bovine serum (BI), trypsin (Sigma), DMSO (Sigma).

[0128] Cell lines: human triple-negative breast cancer cell line MDA-MB-231, human breast cancer cell line MCF-7, human prostate cancer cell line PC-3, human liver cancer cell line HepG2, human non-small cell lung cancer cell line A549 (all provided by Jiangsu Kaygee Biotechnology Co., Ltd.).

[0129] Methods:

[0130] Preparation of complete medium: Add 50 mL of fetal bovine serum to 450 mL of DMEM medium to prepare complete medium, and store it in a refrigerator at 4 °C for later use. All media mentioned below are this complete medium.

[0131] Preparation of compound solution: Weigh a certain amount of celastrol derivative or positive control drug and dissolve it in DMSO to prepare a compound stock solution with a concentration of 10 mM, and then dilute it with complete medium to a test compound solution with a concentration of 100 μM. All compound solutions mentioned below are this 100 μM compound solution.

[0132] Preparation of blank control group solution: Similar to the preparation of 100 μM compound solution, replace the compound stock solution with an equal volume of DMSO.

[0133] Resuscitate the cryopreserved human cell lines (MDA-MB-231, MCF-7, PC-3, HepG2, A549) in a cell culture incubator at 37 °C and 5% CO2, and change the medium once a day. When the cell growth state is good and the cell density increases to about 80%, discard the old medium, wash with PBS, add 1 mL of 0.25% trypsin to digest until the cells shrink and become round (except for HepG2 which requires 4 min, the digestion time for the other cells is 1 - 2 min), add 1 mL of new complete medium to terminate the digestion, transfer the cells to a centrifuge tube, centrifuge at 1000 r / min for 5 min; add new complete medium to the cell pellet, gently pipette to mix evenly, count, and inoculate the cell suspension (4.5 - 5.0×10 4 cell / ml) into a 96-well plate, 100 μL / well, and continue to culture in a CO2 incubator for 24 h.

[0134] Dilute the compound solution and the blank control group solution with medium to the test concentration and add them to the 96-well plate (a total of 5 concentrations, in triplicate), continue to culture for 48 h, add 10 μL of freshly prepared 5 mg / ml MTT solution to each well, incubate in the dark for 4 h, then discard the supernatant, and add 100 μL of DMSO to each well under dark conditions, shake on a plate shaker for 10 min to dissolve the crystals. Immediately use a multi-functional microplate reader to measure the absorbance value of each well at a wavelength of 570 nm, and calculate the inhibition rate of the compound on the cells according to the following formula. The average value of the 3 primary screening results is its final inhibition rate. Further concentration gradient screening of the test compound, calculate its IC50 Values (calculated by Graphpad Prism 8 software), and the results of three repeated experiments were the final IC 50 values of the tested compounds.

[0135] Cell inhibition rate (Inhibition rate, %) = [(OD value of blank control - OD value of drug administration group) / OD value of blank control group] × 100%

[0136] Using the MTT method, with CEL and CDDO-Me as positive controls, the inhibition rates of the target compounds on 5 human tumor cells MDA-MB-231, MCF-7, PC-3, HepG2, and A549 at a drug administration concentration of 1.0 μM were measured (Table 1). Further, the IC 50 values of all compounds on MDA-MB-231, MCF-7, and PC-3 were measured, as well as the IC

[0137] values of Compounds I1, I2, 50 II1-II3 on HepG2 and A549 (selection criteria: compounds with activity superior to CEL at 1.0 μM, inhibition rate on HepG2 > 50%, and inhibition rate on A549 > 75%). The results are shown in Table 2.

[0138] Table 1 Inhibitory effects of all compounds on five different human cancer cell lines (1.0 μM)

[0139]

[0140]

[0141] Table 2 In vitro anti-proliferative effects of selected compounds on five different human cancer cell lines

[0142]

[0143] Note: ND indicates not measured.

[0144] The following conclusions can be drawn from Table 1 and Table 2:

[0145] (1) Generally speaking, compared with CEL, the anti-proliferative activities of most celastrol derivatives on 5 tumor cells have been improved, and the anti-proliferative activities of some compounds are significantly superior to those of CEL and CDDO-ME. Except for Compound III6, the cytotoxic effects of pyrimidine (I and II) derivatives are generally superior to those of purine (III) derivatives.

[0146] (2) Compounds modified only with pyrimidine rings (types I1, I2, and II) showed a significant increase in the anti-proliferative activity against 5 types of tumor cells; moreover, the in vitro anti-proliferative activity of amide derivatives (type II) was generally superior to that of ester derivatives (I1, I2). When the linking fragment was pyrimidine, compound II2 with a linker arm length of 3C had the strongest anti-proliferative activity and was the most sensitive to MDA-MB-231 cells (II2: IC 50 = 0.25 μM; CEL: IC 50 = 0.82 μM; CDDO-Me: IC 50 = 1.19 μM). When the linking chain was the same, the addition of a pyridine fragment (pyrimidine-fused pyridine) weakened the anti-proliferative activity of compounds (I3, I4).

[0147] (3) Compounds conjugated with purine (type III) had strong anti-proliferative activity against 3 types of human tumor cells, MDA-MB-231, MCF-7, and PC-3, while their anti-proliferative activity against HepG2 and A549 was significantly weakened, indicating that celastrol purine derivatives were more sensitive to MDA-MB-231, MCF-7, and PC-3, and had the strongest anti-proliferative activity against MDA-MB-231 cells. Among the purine derivatives, compound III6 had the most prominent anti-proliferative activity.

[0148] In summary, celastrol derivatives showed better anti-proliferative effects than CEL against 5 human tumor cell lines, and the anti-proliferative activity of the derivatives in triple-negative breast cancer cells was significantly better than that in other human tumor cell lines. Among them, compound II2 had the strongest inhibitory effect on MDA-MB-231, with an IC 50 value of 0.25 ± 0.02 μM, which was 3.3 times more active than CEL. Therefore, compound II2 can be used as a preferred compound, worthy of further study and expected to become a new anti-tumor candidate.

Claims

1. Celastrol derivative with the structure shown in Formula Ⅳ: Among them, X = O, L is selected from -(CH2) n -, n is an integer from 2 to 5, R is selected from Or X = NH, L is selected from -(CH2) n -, n is an integer from 2 to 4, R is selected from Provided that it does not include X = O, L is selected from -(CH2)3-, R is selected from 2. The celastrol derivative according to claim 1, wherein: X = O, L is selected from -(CH2) n -, n is 4 or 5, R is selected from or X = O, L is selected from -(CH2) n -, n is an integer from 3 to 5, R is selected from or X = NH, L is selected from -(CH2) n -, n is an integer from 2 to 4, R is selected from provided that X = O, L is selected from -(CH2)3-, R is selected from 3. Celastrol derivative with the structure shown in the following formula:

4. Celastrol derivative with the structure shown in the following formula:

5. A preparation method of the celastrol derivative according to claim 1, characterized in that: When R is selected from the synthetic route is as follows: wherein, R1 is selected from H or n is an integer from 2 to 5; It includes the following steps: Step (1): Using pyridine as an acid-binding agent and anhydrous dichloromethane as a reaction solvent, reacting the 2-aminopyrimidine analogue shown in formula with the acyl chloride shown in formula to obtain intermediate a; Step (2): Using NaHCO3 as the base catalyst and N,N-dimethylformamide as the reaction solvent, intermediate a reacts with celastrol to obtain the target compound; When R is selected from the synthetic route is as follows: Wherein, n is an integer from 2 to 5; It includes the following steps: Step (1), using triethylamine as the base catalyst and absolute ethanol as the reaction solvent, reacting 2-chloropyrimidine with the diamine shown in the formula to obtain intermediate b; Step (2): Using anhydrous dichloromethane as the reaction solvent, intermediate b undergoes an amidation reaction with the carboxyl group of celastrol in the presence of EDCI, HOBT and DIPEA to obtain the target compound; When R is selected from the synthetic route is as follows: wherein, R2 = Cl, Br; R3 = NH2, H; n is an integer from 2 to 5; provided that L is not selected from -(CH2)3-, and R is selected from It includes the following steps: Step (1), using potassium carbonate as an acid-binding agent and acetone as a reaction solvent, reacting the purine analogue represented by formula with the dibromoalkane represented by formula to obtain intermediate c; Step (2): Using NaHCO3 as the base catalyst and N,N-dimethylformamide as the reaction solvent, intermediate c reacts with celastrol to obtain the target compound.

6. Use of the celastrol derivative according to any one of claims 1-4 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The tumors are breast cancer, prostate cancer, lung cancer, liver cancer.

8. A pharmaceutical composition, characterized in that: Using the celastrol derivative according to any one of claims 1-4 as the active ingredient, supplemented with a pharmaceutically acceptable carrier, to make any pharmaceutically acceptable dosage form.

9. The pharmaceutical composition according to claim 8, wherein: The dosage forms are tablets, capsules, dripping pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, nano-formulations.

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