A targeted tripterine derivative and a preparation method and use thereof

Abstract

The application discloses a tripterine derivative with a structure as shown in formula I, wherein R1 is selected from H, and R2 is an integer from 3 to 6. Compared with tripterine, the tripterine derivative has enhanced inhibition effect on tumor cells, significantly reduced toxicity, can target mitochondria, and can kill cells by increasing the ROS level in tumor cells. The application further discloses a use of the tripterine derivative in preparation of an anti-tumor drug. The application further discloses a use of the tripterine derivative in preparation of a mitochondria-targeting anti-tumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry and pharmacology, and relates to a triptolide derivative, a preparation method thereof and use of the triptolide derivative in preparation of an anticancer drug. BACKGROUND

[0002] Malignant tumor is one of the biggest health problems in the world today, and its causes and processes are complex and changeable. At present, although a variety of cancer treatment drugs have been marketed, due to problems such as toxicity, side effects, long-term drug resistance, and the like, the therapeutic effect of anticancer drugs is still not ideal.

[0003] Natural active products are an important source for developing anticancer drugs. Triptolide is a pentacyclic triterpenoid compound derived from traditional Chinese medicine Tripterygium wilfordii. Studies have shown that it has multiple biological activities such as anti-tumor, anti-inflammatory, and anti-obesity, among which the anti-tumor effect has attracted widespread attention. Although triptolide has good pharmacological activity, it is difficult to be directly applied to clinical use due to problems such as poor targeting, high toxicity, poor water solubility, and low bioavailability.

[0004] SUMMARY

[0005] The purpose of the present application is to modify the structure of triptolide as a lead compound, and to provide a triptolide derivative. Pharmacological experiments show that the anti-tumor activity of the triptolide derivative of the present application is significantly improved, and the toxicity is significantly reduced compared with triptolide.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The triptolide derivative has the structure shown in formula I:

[0008]

[0009] wherein R1 is selected from H, R2 is selected from n is an integer of 3-6.

[0010] Preferably, R1 is selected from H, and R2 is selected from n is an integer of 3-5; R1 is selected from H, R2 is selected from n is an integer of 3-6; R1 is selected from H, and R2 is selected from n is an integer of 3-6.

[0011] More preferably, R1 is selected from H, and R2 is selected from n is 3, 4 or 5; R1 is selected from H, and R2 is selected from n is an integer of 3-6; R1 is selected from H, and R2 is selected from n is 3, 5, 6; R1 is selected from R2 is selected from n is 4.

[0012] In particular, the tripterine derivative of the structure shown in formula I is selected from the following compounds:

[0013]

[0014]

[0015] Another object of the present application is to provide a preparation method of the tripterine derivative, when R1=H, the synthetic route is as follows:

[0016]

[0017] wherein n is as described above;

[0018] comprising the following steps:

[0019] Step (1), intermediate III is obtained by reacting biotin shown in formula II with dibromoalkane shown in formula at room temperature, using K2CO3 as base catalyst and N,N-dimethylformamide (DMF) as reaction solvent; wherein the molar ratio of biotin to dibromoalkane is 1:3-1:5; the molar ratio of biotin to K2CO3 is 1:3-1:4;

[0020] Step (2), tripterine biotin derivative shown in formula Ia is obtained by reacting intermediate III with tripterine under reflux, using NaHCO3 as base catalyst and N,N-dimethylformamide (DMF) as reaction solvent; wherein the molar ratio of tripterine to intermediate III is 1:1.1-1:1.3; the molar ratio of tripterine to NaHCO3 is 1:4-1:6;

[0021] when R1=H, the synthetic route is as follows:

[0022]

[0023] wherein n is as described above;

[0024] comprising the following steps:

[0025] Step (1), 7-(diethylamino)coumarin-3-carboxylic acid shown in formula VI is reacted with dibromoalkane shown in formula The dibromoalkane shown was reacted under reflux to give intermediate VII; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane was 1:3 to 1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 was 1:3 to 1:4; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI was 1:0.5.

[0026] Step (2): Using NaHCO3 as an alkaline catalyst and N,N-dimethylformamide (DMF) as a reaction solvent, intermediate VII reacts with triptolide under reflux to obtain the triptolide coumarin derivative shown in Formula Ib; wherein, the molar ratio of triptolide to intermediate VII is 1:1.1 to 1:1.3; and the molar ratio of triptolide to NaHCO3 is 1:4 to 1:6;

[0027] When R1 = H, The synthesis route is as follows:

[0028]

[0029]

[0030] Wherein, n is as described above;

[0031] Includes the following steps:

[0032] Step (1): Using K2CO3 as the alkaline catalyst and acetone as the reaction solvent, triptolide and formula... The dibromoalkane shown was reacted under reflux to give intermediate IV; wherein the molar ratio of triptolide to dibromoalkane was 1:3 to 1:5; and the molar ratio of triptolide to K2CO3 was 1:3 to 1:4.

[0033] Step (2): Using N,N-dimethylformamide (DMF) as the reaction solvent, in the presence of HOBT, EDCI, and DIPEA, 7-(diethylamino)coumarin-3-carboxylic acid of formula VI reacts with... The reaction at room temperature yields intermediate VIII; wherein the 7-(diethylamino)coumarin-3-carboxylic acid reacts with... The molar ratios are 1:2 to 1:3; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to HOBT is 1:1.5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to EDCI is 1:1.5; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to DIPEA is 1:4.

[0034] Step (3), the intermediate VIII is subjected to acid hydrolysis to remove the Boc protecting group at room temperature in the presence of trifluoroacetic acid and in anhydrous dichloromethane as a reaction solvent; wherein the volume ratio of trifluoroacetic acid to anhydrous dichloromethane is 1:5-1:10;

[0035] Step (4), the intermediate IX is reacted with the intermediate IV in refluxing in the presence of K2CO3 as a base catalyst and anhydrous acetonitrile as a reaction solvent to obtain the emodin coumarin derivative shown in formula Ic; wherein the molar ratio of the intermediate IV to the intermediate IX is 1:1.1; the molar ratio of the intermediate IV to K2CO3 is 1:2-1:3;

[0036] When , the synthetic route is as follows:

[0037]

[0038]

[0039] wherein n is as described above;

[0040] comprising the following steps:

[0041] Step (1), the 7-(diethylamino)coumarin-3-carboxylic acid shown in formula VI is reacted with the dibromoalkane shown in formula in refluxing in the presence of K2CO3 as a base catalyst, KI as a catalyst and N,N-dimethylformamide (DMF) as a reaction solvent to obtain the intermediate VII; wherein the molar ratio of the 7-(diethylamino)coumarin-3-carboxylic acid to the dibromoalkane is 1:3-1:5; the molar ratio of the 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3-1:4; the molar ratio of the 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5;

[0042] Step (2), the intermediate VII is reacted with emodin in refluxing in the presence of NaHCO3 as a base catalyst and N,N-dimethylformamide (DMF) as a reaction solvent to obtain the emodin coumarin derivative shown in formula Ib; wherein the molar ratio of emodin to the intermediate VII is 1:1.1-1:1.3; the molar ratio of emodin to NaHCO3 is 1:4-1:6;

[0043] Step (3), the reaction of the tripterine coumarin derivative shown in formula Ib and the biotin shown in formula II in the presence of DMAP and EDCI to obtain the tripterine-coumarin-biotin derivative shown in formula Id, wherein the molar ratio of the tripterine coumarin derivative to biotin is 1:3, the molar ratio of the tripterine coumarin derivative to DMAP is 1:0.5, and the molar ratio of the tripterine coumarin derivative to EDCI is 1:1.5.

[0044] Compared with tripterine, the tripterine derivative has enhanced inhibitory effect on tumor cells and significantly reduced toxicity.

[0045] Another object of the present application is to provide the use of the tripterine derivative in the preparation of a mitochondria-targeted anti-tumor drug.

[0046] The tumor is breast cancer or lung cancer, preferably breast cancer.

[0047] Another object of the present application is to provide a pharmaceutical composition comprising the tripterine derivative as an effective component and a pharmaceutically acceptable carrier in different dosage forms.

[0048] The dosage form is selected from tablets, capsules, dripping pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, and nano-preparations for oral, injection or topical administration in clinic. In these different preparations, the content of the tripterine derivative of the present application can be 0.1%-99.9%.

[0049] Advantages of the present application:

[0050] Compared with tripterine, the tripterine derivative has enhanced inhibitory effect on tumor cells and significantly reduced toxicity.

[0051] The preparation method of the tripterine derivative has mild reaction conditions, low-toxicity reagents, easily available raw materials, convenient post-treatment, and high yield. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Fluorescence colocalization effect of compound I-17 on mitochondria of MDA-MB-231 cells, wherein A is an image taken under a 20-fold microscope, and B is an image taken under a 20-fold microscope.

[0053] Figure 2The effect of compound I-17 on ROS level in MDA-MB-231 cells. Specific embodiments

[0054] To further illustrate the technical solutions of the present application, a series of examples are listed below. These examples are illustrative and should not be construed as limiting the present application.

[0055] Example 1

[0056] Synthesis of compound A2

[0057]

[0058] In a single-neck flask, biotin (0.98 mmol, 0.24 g), anhydrous potassium carbonate (2.94 mmol, 0.41 g) were added, dissolved in 5 mL of N,N-dimethylformamide, and then 1,3-dibromopropane (0.30 mL, 2.94 mmol) was added. The reaction was carried out at room temperature. After TLC monitoring showed that the reaction was complete, 50 mL of dichloromethane was added to dilute the reaction solution, which was washed with water 3 times, and the organic phase was combined. The organic phase was washed with saturated sodium bicarbonate solution 3 times, saturated brine 3 times, and anhydrous sodium sulfate overnight. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (DCM:MeOH = 60:1, V:V) to obtain compound A2 (white solid) 0.26 g, yield 72.6%.

[0059] ESI-MS: Bromine isotope characteristic peaks 365.3, 367.3 [M+H] + .

[0060] Synthesis of compound I-2

[0061]

[0062] Compound A2 (0.11 mmol, 0.04 g) was added after stirring, and the reaction was heated to reflux at 50°C until TLC detection showed that the reaction was complete. 20 mL of ethyl acetate was added to dilute the reaction solution, which was washed with water 3 times, and the organic phase was combined. The organic phase was washed with saturated sodium bicarbonate solution 3 times, saturated brine 3 times, and anhydrous sodium sulfate overnight. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (DCM:MeOH = 60:1, V:V) to obtain compound I-2 (orange-red solid) 30.5 mg, yield 41.5%.

[0063] ESI-MS: 735.4 [M+H] + .

[0064] 1 H-NMR (300 MHz, CDC13, TMS), δ ppm: 7.00 (dd, J = 7.1, 1.4 Hz, 1H), 6.51 (d, J = 1.4 Hz, 1H), 6.34 (d, J = 7.1 Hz, 1H), 4.50 (dd, J = 7.8, 4.8 Hz, 1H), 4.36 (m, 1H), 3.85 (m, 4H), 3.13 (td, J = 7.4, 4.4 Hz, 1H), 2.91 (dd, J = 12.8, 4.9 Hz, 1H), 2.73 (d, J = 12.8 Hz, 1H), 2.20 (s, 3H), 1.43 (s, 3H), 1.25 (s, 4H), 1.17 (s, 3H), 1.09 (s, 3H), 0.53 (s, 3H).

[0065] Example 2

[0066] Synthesis of compound A3

[0067]

[0068] With 1,4-dibromobutane 0.35 mL (2.94 mmol) instead of 1,3-dibromopropane in Example 1, and other conditions unchanged, compound A3 (white solid) 0.26 g, yield 70.0% was obtained.

[0069] ESI-MS: bromine isotope characteristic peak 379.1, 381.1 [M+H] + .

[0070] Synthesis of compound I-3

[0071]

[0072] Referring to the preparation method of compound I-2 in Example 1, with equimolar amount of compound A3 instead of compound A2, and other conditions unchanged, the target compound I-3 (orange red solid) 31.6 mg, yield 42.2% was obtained.

[0073] ESI-MS: 749.4 [M+H] + .

[0074] 1H-NMR (300 MHz, CDC13, TMS), δ ppm: 7.03 (dd, J = 7.1, 1.4 Hz, 1H), 6.52 (d, J = 1.4 Hz, 1H), 6.37 (d, J = 7.2 Hz, 1H), 4.53 (s, 1H), 4.40 (m, 1H), 3.96 (m, 3H), 3.96 (m, 1H), 3.18 (q, J = 6.8 Hz, 1H), 2.94 (dd, J = 12.8, 4.7 Hz, 1H), 2.75 (d, J = 12.9 Hz, 1H), 2.23 (s, 3H), 1.47 (s, 3H), 1.28 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 0.56 (s, 3H).

[0075] Example 3

[0076] Synthesis of compound A4

[0077]

[0078] With 1,5-dibromopentane 0.40 mL (2.94 mmol) instead of 1,3-dibromopropane in Example 1, and other conditions unchanged, compound A4 (white solid) 0.31 g, yield 80.5% was obtained.

[0079] ESI-MS: bromine isotope characteristic peaks 393.1, 395.1 [M+H] + .

[0080] Synthesis of compound I-4

[0081]

[0082] Referring to the preparation method of compound I-2 in Example 1, with equimolar amount of compound A4 instead of compound A2, and other conditions unchanged, the target compound I-4 (orange red solid) 41.3 mg, yield 54.1% was obtained.

[0083] ESI-MS: 763.4 [M+H] + .

[0084] 1H-NMR (300 MHz, CDC13, TMS), δ ppm: 7.04 (dd, J = 7.1, 1.4 Hz, 1H), 6.54 (d, J = 1.4 Hz, 1H), 6.37 (d, J = 7.1 Hz, 1H), 4.48 (m, 1H), 4.30 (m, 1H), 4.08 (t, J = 6.6 Hz, 2H), 4.00 (dt, J = 12.1, 6.4 Hz, 1H), 3.86 (dt, J = 10.9, 6.5 Hz, 1H), 3.18 (q, J = 7.1 Hz, 1H), 2.94 (dd, J = 12.8, 4.9 Hz, 1H), 2.76 (d, J = 12.8 Hz, 1H), 2.23 (s, 3H), 1.47 (s, 3H), 1.39 (s, 3H), 1.19 (s, 3H), 1.12 (s, 3H), 0.56 (s, 3H).

[0085] Example 4

[0086] Synthesis of compound A10

[0087]

[0088] In a single-neck flask, 7-(diethylamino)coumarin-3-carboxylic acid (1.0 mmol, 0.26 g), anhydrous potassium carbonate (3.0 mmol, 0.42 g), potassium iodide (0.5 mmol, 0.083 g) were added, 5 mL of N,N-dimethylformamide was added to stir and dissolve, 1,3-dibromopropane 0.30 mL (3.0 mmol) was added, and the reaction was heated to reflux at 50°C until the reaction was completed by TLC monitoring. After the reaction was completed, 50 mL of dichloromethane was added to dilute the reaction solution, washed with water 3 times, the organic layer was combined, the organic layer was washed with saturated brine 3 times, and anhydrous sodium sulfate was dried overnight. The organic phase was rotary evaporated, and separated by normal phase silica gel column chromatography (PE:EA = 20:1, V:V) to obtain compound A10 (yellow solid) 0.17 g, yield 44.5%.

[0089] ESI-MS: Bromine isotope characteristic peak 382.1, 384.1 [M+H] + .

[0090] Synthesis of compound I-10

[0091]

[0092] Compound I-10 was synthesized according to the procedure described in Example 4, using compound A10 instead of compound A9. Compound I-10 (orange red solid) 43.1 mg, yield 52.1%.

[0093] ESI-MS: 752.4 [M+H] + .

[0094] 1 H-NMR (400 MHz, DMSO, TMS), δ ppm: 8.59 (s, 1H), 8.42 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.96 (dd, J = 7.1, 1.4 Hz, 1H), 6.73 (dd, J = 9.1, 2.4 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 6.33-6.26 (m, 1H), 4.38-4.28 (m, 1H), 4.16 (dt, J = 11.5, 6.1 Hz, 1H), 4.08-3.93 (m, 2H), 3.48 (q, J = 7.1 Hz, 4H), 2.00 (s, 3H), 1.33 (s, 3H), 1.18 (s, 3H), 1.14 (s, 3H), 1.05 (s, 3H), 0.49 (s, 3H).

[0095] Example 5

[0096] Synthesis of compound A11

[0097]

[0098] Compound A11 (yellow solid) 0.20 g, yield 50.7% was obtained by replacing 1,3-dibromopropane in Example 4 with 1,4-dibromobutane 0.35 mL (3.0 mmol) and other conditions unchanged.

[0099] ESI-MS: Br isotope characteristic peaks 396.1, 398.1 [M+H] + .

[0100] Synthesis of compound I-11

[0101]

[0102] Referring to the preparation method of compound I-10 in Example 4, compound A11 was used to replace compound A10 in an equimolar amount, and other conditions were unchanged to obtain the target compound I-11 (orange red solid) 43.6 mg, yield 56.9%.

[0103] ESI-MS: 766.4 [M+H] + .

[0104] 1 H-NMR (300 MHz, DMSO, TMS), δ ppm: 8.74 (s, 1H), 8.56 (s, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.03 (d, J = 6.9 Hz, 1H), 6.78 (dd, J = 9.1, 2.4 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.40-6.26 (m, 1H), 4.22 (t, J = 5.8 Hz, 2H), 4.06-3.82 (m, 2H), 3.48 (q, J = 6.8 Hz, 4H), 2.07 (s, 3H), 1.40 (s, 3H), 1.31 (s, 3H), 1.17 (s, 3H), 1.06 (s, 3H), 0.45 (s, 3H).

[0105] Example 6

[0106] Synthesis of compound A12

[0107]

[0108] Referring to the preparation method of compound I-10 in Example 4, compound A11 was used to replace compound A10 in an equimolar amount, and other conditions were unchanged to obtain the target compound I-11 (orange red solid) 43.6 mg, yield 56.9%.

[0109] ESI-MS: 766.4 [M+H] + .

[0110] Synthesis of compound I-12

[0111]

[0112] Referring to the preparation method of compound I-10 in Example 4, compound A11 was used to replace compound A10 in an equimolar amount, and other conditions were unchanged to obtain the target compound I-11 (orange red solid) 43.6 mg, yield 56.9%.

[0113] ESI-MS: 766.4 [M+H] + .

[0114] 1 H-NMR (400 MHz, DMSO, TMS), δ ppm: 8.72 (s, 1H), 8.55 (s, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.03 (d, J = 6.9 Hz, 1H), 6.78 (dd, J = 9.0, 2.4 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.30 (d, J = 7.1 Hz, 1H), 4.21 (tt, J = 6.3, 3.2 Hz, 2H), 3.85 (ddt, J = 32.0, 11.3, 5.9 Hz, 2H), 3.48 (q, J = 7.0 Hz, 4H), 2.07 (s, 3H), 1.33 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 0.98 (s, 3H), 0.42 (s, 3H).

[0115] Example 7

[0116] Synthesis of compound A13

[0117]

[0118] With 1,6-dibromopentane 0.49 mL (3.0 mmol) instead of 1,3-dibromopropane in Example 4, and other conditions unchanged, compound A13 (yellow solid) 0.18 g, yield 42.9% was obtained.

[0119] ESI-MS: Br isotope characteristic peak 424.1, 426.1 [M+H] + .

[0120] Synthesis of compound I-13

[0121]

[0122] Referring to the preparation method of compound I-10 in Example 4, with compound A13 instead of compound A10 in equimolar amount, and other conditions unchanged, the target compound I-13 (orange red solid) 21.6 mg, yield 27.2% was obtained.

[0123] ESI-MS: 794.5 [M+H] + .

[0124] 1H-NMR (400 MHz, DMSO, TMS), δ ppm: 8.71 (s, 1H), 8.52 (s, 1H), 7.61 (s, 1H), 7.00 (s, 0H), 6.73 (s, 1H), 6.50 (s, 1H), 6.34 (s, 1H), 4.16 (s, 2H), 3.82 (d, J = 25.4 Hz, 2H), 3.45 (s, 4H), 2.07 (s, 3H), 1.54 (s, 3H), 1.36 (s, 3H), 1.19 (s, 3H), 0.97 (s, 3H), 0.40 (s, 3H).

[0125] Example 8

[0126] Synthesis of compound A14

[0127]

[0128] In a single-neck flask, 7-(diethylamino)coumarin-3-carboxylic acid (1.0 mmol, 0.26 g), N-Boc-piperazine (2.0 mmol, 0.37 g), HOBT (1.5 mmol, 0.20 g), EDCI (1.5 mmol, 0.29 g) were added and dissolved in 5 mL of N,N-dimethylformamide, 0.70 mL (4.0 mmol) of DIPEA was added dropwise, and the reaction was stirred at room temperature until TLC detection showed that the reaction of 7-(diethylamino)coumarin-3-carboxylic acid was complete. The reaction solution was diluted with 50 mL of ethyl acetate, washed with water 3 times, and the organic phase was combined. The organic phase was washed with saturated brine 3 times and dried over anhydrous sodium sulfate overnight. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (PE:EA = 6:1, V:V) to obtain compound A14-1 (yellow-green solid) 0.25 g. 0.25 g of compound A14-1 was dissolved in 4 mL of anhydrous dichloromethane, and 0.4 mL of trifluoroacetic acid was added dropwise. The reaction was carried out at room temperature, and the reaction was monitored by TLC. After the reaction was completed, it was concentrated to obtain compound A14 (yellow sticky substance) 0.22 g, yield 67.7%.

[0129] ESI-MS: 330.2 [M+H] + .

[0130]

[0131] In a single-neck flask, compound A6 (0.3 mmol, 0.135 g) was dissolved in 5 mL of acetone, anhydrous potassium carbonate (0.9 mmol, 0.12 g) was added, 1,3-dibromopropane 0.091 mL (0.9 mmol) was added, and the reaction was heated to reflux at 50 °C until TLC detection showed that the reaction was complete. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (PE:EA = 20:1, V:V) to obtain compound A7 (orange red solid) 0.12 g, yield 70.0%.

[0132] ESI-MS: Isotope characteristic peaks 571.2, 573.2 [M+H] + .

[0133] Synthesis of compound I-14

[0134]

[0135] Compound A14 (0.22 mmol, 0.073 g) was dissolved in 5 mL of anhydrous acetonitrile, anhydrous potassium carbonate (0.44 mmol, 0.061 g) was added, and the reaction was heated to reflux at 50 °C for 30 min, then compound A6 (0.2 mmol, 0.11 g) was added, and the reaction was heated to reflux until TLC detection showed that the reaction was complete. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (DCM:MeOH = 60:1, V:V) to obtain compound I-14 (orange red solid) 23.4 mg, yield 14.3%.

[0136] ESI-MS: 820.5 [M+H] + .

[0137] 1 H-NMR (300 MHz, CDC13, TMS), δ ppm: 7.85 (s, 1H), 7.32 (d, J = 8.9 Hz, 1H), 7.04 (dd, J = 7.1, 1.4 Hz, 1H), 6.61 (dd, J = 8.9, 2.4 Hz, 1H), 6.52 (d, J = 1.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.36 (d, J = 7.2 Hz, 1H), 4.05 (dt, J = 11.0, 6.4 Hz, 1H), 3.90 (dt, J = 11.4, 6.2 Hz, 1H), 3.77 (s, 1H), 3.45 (q, J = 7.2 Hz, 7H), 2.59 - 2.37 (m, 8H), 2.22 (s, 3H), 1.46 (s, 3H), 1.24 (s, 3H), 1.19 (s, 3H), 1.11 (s, 3H), 0.56 (s, 3H).

[0138] Example 9

[0139] Synthesis of compound A8

[0140]

[0141] With 1,5-dibromopentane 0.12 mL (0.9 mmol) instead of 1,3-dibromopropane in the preparation of compound A6 of Example 8, the target compound A8 (orange red solid) 0.12 g was obtained in 66.9% yield.

[0142] ESI-MS: Isotope characteristic peaks 599.3, 601.3 [M+H] + .

[0143] Synthesis of compound I-15

[0144]

[0145] With equimolar amount of compound A8 instead of compound A6, the target compound I-15 (orange red solid) 19.1 mg was obtained in 11.3% yield according to the preparation method of compound I-14 of Example 8.

[0146] ESI-MS: 848.5 [M+H] + .

[0147] 1 H-NMR (300 MHz, CDCl3, TMS), δ ppm: 7.84 (s, 1H), 7.30 (s, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.59 (d, J = 8.9 Hz, 1H), 6.49 (d, J = 11.6 Hz, 2H), 6.34 (d, J = 7.0 Hz, 1H), 3.88 (m, 4H), 3.42 (m, 6H), 2.69-2.33 (m, 6H), 2.20 (s, 3H), 1.44 (s, 3H), 1.23 (s, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.54 (s, 3H).

[0148] Example 10

[0149] Synthesis of compound A9

[0150]

[0151] With 1,6-dibromopentane 0.15 mL (0.9 mmol) instead of 1,3-dibromopropane in the preparation of compound A6 of Example 8, the target compound A9 (orange red solid) 0.12 g was obtained in 65.3% yield.

[0152] ESI-MS: Isotope characteristic peaks 613.3, 615.3 [M+H] + .

[0153] Synthesis of compound I-16

[0154]

[0155] The target compound I-16 (orange red solid) 17.9 mg, yield 10.4% was prepared according to the preparation method of compound I-14 in Example 8, replacing compound A6 with equimolar compound A9, and other conditions were unchanged.

[0156] ESI-MS: 862.5 [M+H] + .

[0157] 1 H-NMR (300 MHz, CDC13, TMS), δ ppm: 7.85 (s, 1H), 7.33 (s, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.61 (d, J = 9.0 Hz, 2H), 6.54 (s, 1H), 6.49 (s, 1H), 6.37 (d, J = 7.2 Hz, 1H), 3.95 (m, 4H), 3.84 (m, 4H), 3.45 (d, J = 7.6 Hz, 4H), 2.48 (m, 4H), 2.22 (s, 3H), 1.47 (s, 3H), 1.35 (s, 3H), 1.19 (s, 3H), 1.11 (s, 3H), 0.56 (s, 3H).

[0158] Example 11

[0159] Synthesis of compound I-17

[0160]

[0161] In a single-neck flask, compound I-11 (0.13 mmol, 0.10 g), DMAP (0.07 mmol, 0.0086 g), EDCI (0.20 mmol, 0.038 g), biotin (0.39 mmol, 0.095 g) were added and dissolved in 3 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature until TLC detection showed that the reaction was complete. 30 mL of dichloromethane was added to dilute the reaction solution, washed with water 3 times, and the organic phase was combined. The organic phase was dried with saturated brine 3 times and dried overnight with anhydrous sodium sulfate. The organic phase was rotary evaporated and separated by normal phase silica gel column chromatography (DCM:MeOH = 10:1, V:V) to obtain compound I-17 (yellow solid) 33.6 mg, yield 26.1%.

[0162] ESI-MS: 992.5 [M+H]+ .

[0163] 1 H-NMR (400 MHz, CDC13, TMS), δ ppm: 8.42 (s, 1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H), 7.06 (dd, J = 7.1, 1.4 Hz, 1H), 6.60 (dd, J = 9.0, 2.5 Hz, 1H), 6.44 (dd, J = 3.7, 1.9 Hz, 1H), 6.31 (d, J = 7.2 Hz, 2H), 4.50 (dt, J = 8.1, 4.2 Hz, 1H), 4.32 (t, J = 6.3 Hz, 3H), 4.06 (dt, J = 11.9, 6.1 Hz, 1H), 3.90 (dt, J = 11.5, 5.8 Hz, 1H), 3.43 (q, J = 7.3 Hz, 4H), 3.14 (dq, J = 11.2, 6.5 Hz, 1H), 2.91 - 2.79 (m, 1H), 2.75 (dd, J = 12.9, 5.5 Hz, 1H), 2.12 (s, 3H), 1.27 (s, 3H), 1.21 (s, 3H), 1.17 (s, 3H), 1.08 (s, 3H), 0.55 (s, 3H).

[0164] Example 12

[0165] (I) In vitro anti-tumor cell proliferation experiment of the compound

[0166] The anti-tumor activity of the tripterine derivatives (compound I-2 to compound I-4, compound I-10 to compound I-17) of the application was tested by tetrazolium blue colorimetry (MTT method), and tripterine (CEL) and CDDO-Me (Bardoxolone methyl) were selected as positive control drugs. An appropriate amount of the test compound, CEL and CDDO-Me were weighed, dissolved in DMSO to prepare a stock solution with a concentration of 10 mmol / L, and then diluted with culture medium to prepare a series of solutions with gradient concentrations.

[0167] Instruments: super-clean workbench (SW-CJ-1FD, AIRTECH, Suqing Antai), constant-temperature CO2 incubator (3111, Thermo, USA), high-pressure sterilization pot (YXO.SG41.280, Shanghai Huaxian), centrifuge (SIGMA), multi-biology microscope (IX71, Olympus, Japan), multifunctional enzyme marker (POLARstar, Omega, USA).

[0168] Reagents: DMEM culture medium (KeyGEN), fetal bovine serum (GIBCO), trypsin (SIGMA), DMSO (SIGMA).

[0169] Cell lines: human breast cancer cell line MDA-MB-231, human breast cancer cell line MCF-7, human lung cancer cell line A549, human normal breast cell line MCF-10A, and human normal liver cell line L02 (all purchased from Jiangsu Kaiji Biotechnology Co., Ltd.), all cultured in DMEM (high glucose) medium.

[0170] Methods: Frozen cell lines were thawed and cultured in a 37℃, 5% CO2 incubator, with the medium changed daily. Once the cells were in the exponential growth phase and in good condition, they were plated. 1 mL of 0.25% trypsin digestion solution was added, and digestion was performed for 1-2 min. Cell status was observed under a microscope. When adherent cells became rounded and shrunken, the digestion solution was removed. 1-2 mL of culture medium containing 10% fetal bovine serum was added to prepare a cell suspension. Cell counting was performed at a density of 5 × 10⁶ cells per well. 3 Calculate the required cell suspension volume based on the number of cells and the total number of wells. Seed the cell suspension into 96-well plates at 100 μL / well, seal with PBS, and incubate at 37°C with 5% CO2 for 24 h. Discard the old culture medium and add a series of solutions of the test compound, CEL, or CDDO-Me at gradient concentrations, and set up a blank control (an equal volume of DMSO solution at the highest concentration of the test drug). Each drug concentration is used in triplicate, and the plates are incubated for 48 h. Add MTT reagent (5 mg / mL) to the 96-well plates at 10 μL / well and incubate for another 4 h. Aspirate the culture medium from the plate, add 150 μL of DMSO to each well, and measure the absorbance of each well at 570 nm using a multi-mode microplate reader. Calculate the cell inhibition rate using the following formula.

[0171] Cell inhibition rate % = [(OD value of blank control group - OD value of drug treatment group) / OD value of blank control group] × 100%.

[0172] The average of the three initial screening results is the final inhibition rate. The IC50 of the test drug is then calculated. 50 The IC50 value (using GraphPad software) is the final IC50 value of the compound obtained from three repeated experiments. 50 value.

[0173] Selectivity SI=IC 50 (MCF-10A) / IC 50 (MDA-MB-231) or IC 50 (L02) / IC 50 (MDA-MB-231).

[0174] Table 1. Inhibition of the tested compounds on MDA-MB-231, MCF-7 and A549 cell lines

[0175]

[0176]

[0177] Note: NA means no detection.

[0178] As can be seen from Table 1, compared with celastin, the breast cancer cell inhibition effect of the celastin derivative of the present application is significantly enhanced, and the normal cell selectivity is improved. Among them, the selectivity of compound I-17 is the best, which is about 7.34 times higher than that of celastin, and about 14.11 times higher than that of human normal liver cells.

[0179] (ii) Detection of compound I-17 targeting cell mitochondria

[0180] Instrument: Laser confocal microscope (Zeiss LSM800)

[0181] Reagent: Mito RedCMXRos mitochondrial red fluorescence probe (Yixing Biological).

[0182] Method: Referring to the cell digestion counting method in the "in vitro anti-tumor cell proliferation experiment of the compound", collect the cell suspension (1x10 5 / mL), and inoculate the MDA-MB-231 cells uniformly in the laser confocal small dish; after 24h of culture, discard the original culture medium, add the culture medium containing 20μM compound I-17 (refer to "in vitro anti-tumor cell proliferation experiment of the compound"), and continue to culture for 2h. Then, according to the instructions of the kit, perform the fluorescence co-localization experiment.

[0183] Add 100nM mitochondrial probe in the laser confocal small dish, incubate for 40min, and then observe the mitochondrial localization of the cells and the compound under the appropriate excitation light source by laser confocal microscope. As shown in Figure 1 , wherein A is the image taken under 20x lens, B is the image taken under 20x lens, the red image is the fluorescence of the mitochondrial probe, and the green image is the fluorescence of compound I-17, which highly coincides to present orange color. The experimental results show that compound I-17 effectively accumulates in the mitochondria of MDA-MB-231 cells, can further induce the generation of ROS in mitochondria, affect oxidative stress, and play an anti-tumor role. Generally, tumor cells have higher mitochondrial membrane potential than normal cells, and drugs with the ability to target mitochondria can more easily accumulate in the mitochondria of tumor cells; and the lead compound celastin does not have the ability to target mitochondria, so compared with the former, the celastin derivatives including compound I-17 of the present application have better selectivity in anti-tumor.

[0184] (III) Effect of compound I-17 on the level of ROS in MDA-MB-231 cells

[0185] Instrument: inverted fluorescence microscope (Olympus).

[0186] Reagent: reactive oxygen species (ROS) assay kit (KeyGen).

[0187] Method: According to the cell digestion counting method in the "in vitro anti-tumor cell proliferation experiment of the compound", collect the cell suspension (1x10 5 / mL), and inoculate the MDA-MB-231 cells uniformly in a 6-well plate and culture for 24 h. Divide into an NAC (N-acetyl-L-cysteine) group, different dose compound I-17 treatment group, an emodin treatment group, and a blank control (DMSO) group. The NAC (N-acetyl-L-cysteine) group is pretreated with NAC (5 mM) for 2 h before administration, and the rest of the treatment group and the blank control group are incubated for 2 h without NAC. Discard the original culture medium, and add the culture medium containing the set concentration (1, 2, 4 μM) of compound I-17 (refer to "in vitro anti-tumor cell proliferation experiment of the compound") to the different dose compound I-17 treatment group, add the culture medium containing 1 μM emodin to the emodin treatment group, add the culture medium containing 4 μM compound I-17 to the NAC group, and add the same volume of DMSO as the highest concentration of the tested drug I-17 to the blank control, and continue to culture for 24 h. Then, detect the intracellular ROS level according to the instructions of the kit.

[0188] Load DCH-DA probe in a 6-well plate, and observe the ROS level of the cells treated with the compound under the appropriate excitation light source by inverted fluorescence microscope. As shown in Figure 2 , with the increase of the dose of compound I-17, the number of green spots is observed to increase, indicating that the intracellular ROS level increases with the increase of the drug concentration. After the cells are pretreated with NAC to remove the intracellular ROS, the ROS level in the cells treated with 4 μM concentration of compound I-17 is significantly lower than that of the cells without pretreatment, indicating that the emodin derivatives including compound I-17 in the present application kill tumor cells by increasing the intracellular ROS level.

[0189] (IV) Acute toxicity test of compound I-17

[0190] Instrument: AOT425 StatPgm software (Organization for Economic Cooperation and Development, OECD).

[0191] Reagent: DMSO (Shanghai Huishi), Tween 80 (Shanghai Huishi), PBS (KeyGEN).

[0192] Method: The acute toxicity of compound I-17 and tripterine was tested by up and down method. Appropriate amount of tripterine was weighed, and mixed solvent (DMSO: Tween 80: PBS = 1:1:8, and sonicated if necessary) was used as solvent to prepare series of solutions (injection dose 5.5, 17.5 mg / kg, 200 μL per mouse); appropriate amount of compound I-17 was weighed, and mixed solvent (DMSO: Tween 80: PBS = 1:1:8) was used as solvent to prepare series of solutions (injection dose 17.5, 55, 175 mg / kg, 200 μL per mouse). One mouse was randomly selected to be injected with one dose, and then observed. The injection dose of the next mouse was selected according to the death or survival of the mouse within 2 days after injection. The observation lasted for 7 days, and the short-term (2 days) and long-term (7 days) survival (marked as O) or death (marked as X) of all mice were recorded. The results were input into AOT425 program to calculate the LD 50 value.

[0193] Table 2. Acute toxicity test results of tripterine

[0194]

[0195] Table 3. Acute toxicity test results of compound I-17

[0196]

[0197] The results are shown in Tables 3 and 4. The LD 50 of tripterine was about 9.811 mg / kg, and the LD 50 of compound I-17 was about 103 mg / kg, which was about 10 times less toxic than tripterine.

Claims

1. A tripterine derivative having a structure as shown in formula I: wherein R1is selected from H, R2is selected from n is an integer from 3 to 6.

2. The tripterine derivative according to claim 1, characterized in that: R1is selected from H, R2is selected from n is an integer from 3 to 5; R1is selected from H, R2is selected from n is an integer from 3 to 6; R1is selected from H, R2is selected from n is an integer from 3 to 6.

3. The tripterine derivative according to claim 2, characterized in that: R1is selected from H, R2is selected from n is 3, 4 or 5; R1is selected from H, R2is selected from n is an integer from 3 to 6; R1is selected from H, R2is selected from n is 3, 5, 6; R1is selected from R2is selected from n is 4.

4. A tripterine derivative having a structure as shown in formula:

5. A preparation method of the tripterine derivative according to claim 1, characterized in that: When R1= H, the synthesis route is as follows: wherein n is as defined in claim 1; comprising the following steps: Step (1) : intermediate III is obtained by reacting biotin with dibromoalkane in the presence of K2CO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of biotin to dibromoalkane is 1:3-1:5; the molar ratio of biotin to K2CO3 is 1:3-1:4; wherein the formula of dibromoalkane is shown in the following: Step (1) : intermediate III is obtained by reacting biotin with dibromoalkane in the presence of K2CO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of biotin to dibromoalkane is 1:3-1:5; the molar ratio of biotin to K2CO3 is 1:3-1:4; wherein the formula of dibromoalkane is shown in the following: Step (2), intermediate III and tripterine are reacted under reflux to obtain a tripterine-biotin derivative shown in formula Ia with NaHCO3 as an alkali catalyst and N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of tripterine to intermediate III is 1:1.1-1:1.3; and the molar ratio of tripterine to NaHCO3 is 1:4-1:6; When R1= H, the synthesis route is as follows: wherein n is as defined in claim 1; comprising the following steps: Step (1) : intermediate VII is obtained by refluxing 7-(diethylamino)coumarin-3-carboxylic acid with dibromoalkane in the presence of K2CO3 as base catalyst, KI as catalyst and N,N-dimethylformamide as reaction solvent; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3-1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3-1:4; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5; Step (1) : intermediate VII is obtained by refluxing 7-(diethylamino)coumarin-3-carboxylic acid with dibromoalkane in the presence of K2CO3 as base catalyst, KI as catalyst and N,N-dimethylformamide as reaction solvent; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3-1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3-1:4; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5; Step (2), intermediate VII and tripterine are reacted under reflux to obtain a tripterine-coumarin derivative shown in formula Ib with NaHCO3 as an alkali catalyst and N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of tripterine to intermediate VII is 1:1.1-1:1.3; and the molar ratio of tripterine to NaHCO3 is 1:4-1:6; When R1= H, the synthesis route is as follows: wherein n is as defined in claim 1; comprising the following steps: Step (1) : intermediate IV was obtained by refluxing tripterine with dibromoalkane in the presence of K2CO3 as base catalyst and acetone as reaction solvent; wherein, the molar ratio of tripterine to dibromoalkane was 1:3-1:5; the molar ratio of tripterine to K2CO3 was 1:3-1:

4. Step (1) : intermediate IV was obtained by refluxing tripterine with dibromoalkane in the presence of K2CO3 as base catalyst and acetone as reaction solvent; wherein, the molar ratio of tripterine to dibromoalkane was 1:3-1:5; the molar ratio of tripterine to K2CO3 was 1:3-1:

4. Step (2) is carried out in the presence of HOBT, EDCI and DIPEA in N,N- dimethylformamide as the reaction solvent, and 7-(diethylamino)coumarin-3-carboxylic acid is reacted with formula to obtain the intermediate VIII at room temperature; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to is 1:2-1:3; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to HOBT is 1:1.5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to EDCI is 1:1.5; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to DIPEA is 1:

4. Step (3), intermediate VIII is subjected to acid hydrolysis to remove a Boc protecting group to obtain intermediate IX in the presence of trifluoroacetic acid with anhydrous dichloromethane as a reaction solvent; wherein the volume ratio of trifluoroacetic acid to anhydrous dichloromethane is 1:5-1:10; Step (4), intermediate IX and intermediate IV are reacted under reflux to obtain a tripterine-coumarin derivative shown in formula Ic with K2CO3 as an alkali catalyst and anhydrous acetonitrile as a reaction solvent; wherein the molar ratio of intermediate IV to intermediate IX is 1:1.1; and the molar ratio of intermediate IV to K2CO3 is 1:2-1:3; When the synthetic route is as follows: wherein n is as defined in claim 1; comprising the following steps: Step (1) : intermediate VII is obtained by refluxing 7-(diethylamino)coumarin-3-carboxylic acid with dibromoalkane in the presence of K2CO3 as base catalyst, KI as catalyst and N,N-dimethylformamide as reaction solvent; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3-1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3-1:4; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.

5. Step (1) : intermediate VII is obtained by refluxing 7-(diethylamino)coumarin-3-carboxylic acid with dibromoalkane in the presence of K2CO3 as base catalyst, KI as catalyst and N,N-dimethylformamide as reaction solvent; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3-1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3-1:4; and the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.

5. Step (2), intermediate VII and tripterine are reacted under reflux to obtain a tripterine-coumarin derivative shown in formula Ib with NaHCO3 as an alkali catalyst and N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of tripterine to intermediate VII is 1:1.1-1:1.3; and the molar ratio of tripterine to NaHCO3 is 1:4-1:6; Step (3), a tripterine-coumarin-biotin derivative shown in formula Id is obtained by reacting a tripterine-coumarin derivative shown in formula Ib with a biotin shown in formula II at room temperature in the presence of DMAP and EDCI with N,N-dimethylformamide as a reaction solvent; wherein the molar ratio of the tripterine-coumarin derivative to the biotin is 1:3; the molar ratio of the tripterine-coumarin derivative to DMAP is 1:0.5; and the molar ratio of the tripterine-coumarin derivative to EDCI is 1:1.

5.

6. Use of the tripterine derivative according to any one of claims 1-4 in the preparation of an anti-tumor drug, wherein the tumor is breast cancer or lung cancer.

7. Use of the derivative of tripterine according to any one of claims 1-4 in the preparation of a mitochondria-targeted anti-tumor drug, wherein the tumor is breast cancer, lung cancer.

8. A pharmaceutical composition, characterized by: It is prepared into different dosage forms with a pharmaceutically acceptable carrier, wherein the derivative of tripterine according to any one of claims 1-4 is an effective component.

9. The pharmaceutical composition of claim 8, wherein: The dosage form is selected from the group consisting of tablets, capsules, dripping pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, nano-preparations.

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