Targeting tripterine derivative as well as preparation method and application thereof

Abstract

The invention discloses a tripterine derivative with a structure shown as a formula I. In the tripterine derivative, R1 is selected from H, # imgabs 0 # R2 is selected from # imgabs 1 # and is an integer from 3 to 6. Compared with tripterine, the tripterine derivative disclosed by the invention has the advantages that the inhibition effect on tumor cells is enhanced, the toxicity is obviously reduced compared with that of tripterine, mitochondria can be targeted, and meanwhile, the cells can be killed by increasing the ROS level in the tumor cells. The invention also discloses an application of the tripterine derivative in preparation of antitumor drugs. The invention also discloses an application of the tripterine derivative in preparation of mitochondrial targeting antitumor drugs. # imgabs2 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and pharmacotherapy, and relates to a tripterygium wilfordii derivative and a preparation method thereof and use thereof in the preparation of anticancer drugs. Technical Background

[0002] Malignant tumors are one of the biggest health problems in the world today. Their causes and progression are complex and changeable. Although a variety of cancer treatment drugs are currently on the market, the therapeutic effects of anticancer drugs are still unsatisfactory due to toxic side effects, long-term drug resistance and other issues.

[0003] Natural active products are an important source for the development of anticancer drugs. Tripterygium wilfordii is a pentacyclic triterpenoid compound derived from the 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 received widespread attention. Although Tripterygium wilfordii has good pharmacological activity, its poor targeting, high toxicity, poor water solubility, and low bioavailability make it difficult to be directly applied in clinical practice.

[0004] Summary of the invention

[0005] The purpose of the present invention is to use tripterygium wilfordii as a lead compound for structural modification and transformation, and to provide a tripterygium wilfordii derivative. Pharmacological experiments show that compared with tripterygium wilfordii, the tripterygium wilfordii derivative of the present invention has significantly improved anti-tumor activity and significantly reduced toxicity.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The structure of the tripterygium wilfordii derivative is shown in Formula I:

[0008]

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

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

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

[0012] Specifically, the tripterygium wilfordii derivative having a structure as shown in Formula I is selected from the following compounds:

[0013]

[0014]

[0015] Another object of the present invention is to provide a method for preparing the tripterygium wilfordii derivative. The synthetic route is as follows:

[0016]

[0017] Where n is as mentioned above;

[0018] The following steps are involved:

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

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

[0021] When R1=H, The synthetic route is as follows:

[0022]

[0023] Where n is as mentioned above;

[0024] The following steps are involved:

[0025] Step (1), using K2CO3 as a base catalyst, KI as a catalyst, and N,N-dimethylformamide (DMF) as a reaction solvent, 7-(diethylamino)coumarin-3-carboxylic acid represented by formula VI and formula The dibromoalkane shown in the reaction was refluxed to obtain intermediate VII; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3 to 1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3 to 1:4; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5;

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

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

[0028]

[0029]

[0030] Where n is as mentioned above;

[0031] The following steps are involved:

[0032] Step (1), using K2CO3 as a base catalyst and acetone as a reaction solvent, celastrol and formula The dibromoalkane shown is reacted under reflux to obtain intermediate IV; wherein the molar ratio of tripterygium wilfordii to dibromoalkane is 1:3 to 1:5; the molar ratio of tripterygium wilfordii to K2CO3 is 1:3 to 1:4;

[0033] Step (2), using N,N-dimethylformamide (DMF) as a reaction solvent, in the presence of HOBT, EDCI and DIPEA, 7-(diethylamino)coumarin-3-carboxylic acid represented by formula VI is reacted with At room temperature, the intermediate VIII is obtained; wherein the 7-(diethylamino)coumarin-3-carboxylic acid and 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; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to DIPEA is 1:4;

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

[0035] Step (4), using K2CO3 as a base catalyst and anhydrous acetonitrile as a reaction solvent, reacting intermediate IX with intermediate IV under reflux to obtain a tripterygium wilfordii coumarin derivative shown in formula Ic; 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 to 1:3;

[0036] when The synthetic route is as follows:

[0037]

[0038]

[0039] Where n is as mentioned above;

[0040] The following steps are involved:

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

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

[0043] Step (3), using N,N-dimethylformamide (DMF) as a reaction solvent, in the presence of DMAP and EDCI, the tripterygium wilfordii coumarin derivatives shown in formula Ib react with biotin shown in formula II at room temperature to obtain the tripterygium wilfordii-coumarin-biotin derivatives shown in formula Id; the molar ratio of the tripterygium wilfordii coumarin derivatives to biotin is 1:3; the molar ratio of the tripterygium wilfordii coumarin derivatives to DMAP is 1:0.5; the molar ratio of the tripterygium wilfordii coumarin derivatives to EDCI is 1:1.5.

[0044] Compared with tripterygium wilfordii, the tripterygium wilfordii derivatives of the present invention have enhanced inhibitory effects on tumor cells, significantly reduced toxicity compared with tripterygium wilfordii, and can target mitochondria, while killing cells by increasing the level of ROS in tumor cells. Another object of the present invention is to provide the use of the tripterygium wilfordii derivatives in the preparation of anti-tumor drugs.

[0045] Another object of the present invention is to provide the use of the tripterygium wilfordii derivative in the preparation of mitochondrial targeted anti-tumor drugs.

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

[0047] Another object of the present invention is to provide a pharmaceutical composition, which uses a tripterygium wilfordii derivative as an active ingredient and a pharmaceutical carrier to form different dosage forms.

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

[0049] Beneficial effects of the present invention:

[0050] Compared with tripterygium wilfordii, the inhibitory effect of the tripterygium wilfordii derivatives of the present invention on tumor cells is enhanced, and the toxicity is significantly reduced compared with tripterygium wilfordii.

[0051] The preparation method of the tripterygium wilfordii derivative of the present invention has mild reaction conditions, low-toxic reagents, easy-to-obtain raw materials, convenient post-processing and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The fluorescence co-localization effect of compound I-17 on mitochondria of MDA-MB-231 cells, wherein A is an image taken under a 20x microscope, and B is an image taken under a 20x magnification microscope.

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

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

[0055] Example 1

[0056] Synthesis of Compound A2

[0057]

[0058] In a single-necked bottle, add biotin (0.98mmol, 0.24g), anhydrous potassium carbonate (2.94mmol, 0.41g), add 5mL N,N-dimethylformamide to dissolve, then add 1,3-dibromopropane (0.30mL, 2.94mmol), and react at room temperature. After TLC monitoring, add 50mL dichloromethane to the reaction solution to dilute, wash with water 3 times, combine the organic phase, wash the organic phase with saturated sodium bicarbonate solution 3 times, wash with saturated brine 3 times, and dry over anhydrous sodium sulfate overnight. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (DCM: MeOH = 60: 1, V: V) to obtain 0.26g of compound A2 (white solid), with a yield of 72.6%.

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

[0060] Synthesis of Compound I-2

[0061]

[0062] Dissolve tripterygium wilfordii (0.1mmol, 0.045g) in 2mL N,N-dimethylformamide, add NaHCO3 (0.4mmol, 0.034g), stir, add compound A2 (0.11mmol, 0.04g), heat under reflux at 50°C, until TLC detection shows that the reaction is complete, add 20mL ethyl acetate to the reaction solution for dilution, wash with water 3 times, combine the organic phase, wash the organic phase with saturated sodium bicarbonate solution 3 times, wash with saturated brine 3 times, and dry over anhydrous sodium sulfate overnight. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (DCM: MeOH = 60: 1, V: V) to obtain 30.5mg of compound I-2 (orange-red solid), with a yield of 41.5%.

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

[0064] 1 H-NMR (300MHz, CDCl3, TMS), δppm: 7.00 (dd, J=7.1, 1.4Hz, 1H), 6.51 (d, J=1.4Hz, 1H),6.34(d,J=7.1Hz,1H),4.50(dd,J=7.8,4.8Hz,1H),4.36(m,1H),3.85(m,4H) ,3.13(td,J=7.4,4.4Hz,1H),2.91(dd,J=12.8,4.9Hz,1H),2.73(d,J=12.8Hz,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] 0.35 mL (2.94 mmol) of 1,4-dibromobutane was used to replace the 1,3-dibromopropane in Example 1. The other conditions remained unchanged to obtain 0.26 g of compound A3 (white solid) with a yield of 70.0%.

[0069] ESI-MS: Bromine isotope characteristic peaks 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, compound A2 was replaced by compound A3 in an equal molar amount, and other conditions remained unchanged to obtain 31.6 mg of target compound I-3 (orange-red solid) with a yield of 42.2%.

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

[0074] 1H-NMR (300MHz, CDCl3, TMS), δppm: 7.03 (dd, J=7.1, 1.4Hz, 1H), 6.52 (d, J=1.4H z,1H),6.37(d,J=7.2Hz,1H),4.53(s,1H),4.40(m,1H),3.96(m,3H),3.96(m,1 H),3.18(q,J=6.8Hz,1H),2.94(dd,J=12.8,4.7Hz,1H),2.75(d,J=12.9Hz,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] 0.40 mL (2.94 mmol) of 1,5-dibromopentane was used to replace the 1,3-dibromopropane in Example 1. The other conditions remained unchanged to obtain 0.31 g of compound A4 (white solid) with a yield of 80.5%.

[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, compound A4 was used to replace compound A2 in an equal molar amount, and other conditions remained unchanged to obtain 41.3 mg of target compound I-4 (orange-red solid) with a yield of 54.1%.

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

[0084] 1H-NMR (300MHz, CDCl3, TMS), δppm: 7.04 (dd, J=7.1, 1.4Hz, 1H), 6.54 (d, J=1.4Hz, 1H), 6.37 (d ,J=7.1Hz,1H),4.48(m,1H),4.30(m,1H),4.08(t,J=6.6Hz,2H),4.00(dt,J=12.1,6.4Hz,1H) ,3.86(dt,J=10.9,6.5Hz,1H),3.18(q,J=7.1Hz,1H),2.94(dd,J=12.8,4.9Hz,1H),2.76(d,J =12.8Hz,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-necked bottle, add 7-(diethylamino)coumarin-3-carboxylic acid (1.0mmol, 0.26g), anhydrous potassium carbonate (3.0mmol, 0.42g), potassium iodide (0.5mmol, 0.083g), add 5mL N,N-dimethylformamide and stir to dissolve, add 0.30mL (3.0mmol) of 1,3-dibromopropane, and heat under reflux at 50°C until the reaction is completed as monitored by TLC. After the reaction is completed, add 50mL of dichloromethane to the reaction solution to dilute, wash with water 3 times, combine the organic layers, wash the organic layers with saturated brine 3 times, and dry over anhydrous sodium sulfate overnight. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (PE:EA=20:1, V:V) to obtain 0.17g of compound A10 (yellow solid), with a yield of 44.5%.

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

[0090] Synthesis of Compound I-10

[0091]

[0092] Dissolve tripterygium wilfordii (0.1mmol, 0.045g) in 2mL N,N-dimethylformamide, add NaHCO3 (0.4mmol, 0.034g), stir, add 0.039g (0.11mmol) compound A10, heat under reflux at 50°C until TLC detection shows that the reaction is complete, add 20mL ethyl acetate to the reaction solution for dilution, wash with water 3 times, combine the organic phases, wash the organic phases with saturated brine 3 times, and dry over anhydrous sodium sulfate overnight. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (DCM: MeOH = 100: 1, V: V) to obtain 43.1mg of compound I-10 (orange-red solid), with a yield of 52.1%.

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

[0094] 1 H-NMR (400MHz, DMSO, TMS), δppm: 8.59 (s, 1H), 8.42 (s, 1H), 7.48 (d, J = 9.0Hz, 1H), 6.96 (dd,J=7.1,1.4Hz,1H),6.73(dd,J=9.1,2.4Hz,1H),6.49(d,J=2.3Hz,1H),6.33-6.26(m ,1H),4.38-4.28(m,1H),4.16(dt,J=11.5,6.1Hz,1H),4.08-3.93(m,2H),3.48(q,J=7. 1Hz,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] 0.35 mL (3.0 mmol) of 1,4-dibromobutane was used to replace the 1,3-dibromopropane in Example 4. The other conditions remained unchanged to obtain 0.20 g of compound A11 (yellow solid) with a yield of 50.7%.

[0099] ESI-MS: Bromine 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 equal molar amount, and other conditions remained unchanged to obtain 43.6 mg of target compound I-11 (orange-red solid) with a yield of 56.9%.

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

[0104] 1 H-NMR (300MHz, DMSO, TMS), δppm: 8.74 (s, 1H), 8.56 (s, 1H), 7.64 (d, J = 9.0Hz, 1 H),7.03(d,J=6.9Hz,1H),6.78(dd,J=9.1,2.4Hz,1H),6.55(d,J=2.4Hz,1H),6. 40-6.26(m,1H),4.22(t,J=5.8Hz,2H),4.06-3.82(m,2H),3.48(q,J=6.8Hz,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] 0.40 mL (3.0 mmol) of 1,5-dibromopentane was used to replace the 1,3-dibromopropane in Example 4. The other conditions remained unchanged to obtain 0.21 g of compound A12 (yellow solid) with a yield of 51.5%.

[0109] ESI-MS: Bromine isotope characteristic peaks 410.1, 412.1 [M+H] + .

[0110] Synthesis of Compound I-12

[0111]

[0112] Referring to the preparation method of compound I-10 in Example 4, compound A10 was replaced with an equal molar amount of compound A12, and other conditions remained unchanged to obtain 39.1 mg of target compound I-11 (orange-red solid) with a yield of 50.1%.

[0113] ESI-MS: 780.4[M+H] + .

[0114] 1 H-NMR (400MHz, DMSO, TMS), δppm: 8.72 (s, 1H), 8.55 (s, 1H), 7.63 (d, J = 9.0Hz, 1H), 7.0 3(d,J=6.9Hz,1H),6.78(dd,J=9.0,2.4Hz,1H),6.55(d,J=2.4Hz,1H),6.30(d,J=7.1Hz ,1H),4.21(tt,J=6.3,3.2Hz,2H),3.85(ddt,J=32.0,11.3,5.9Hz,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] 0.49 mL (3.0 mmol) of 1,6-dibromopentane was used to replace the 1,3-dibromopropane in Example 4. The other conditions remained unchanged to obtain 0.18 g of compound A13 (yellow solid) with a yield of 42.9%.

[0119] ESI-MS: Bromine isotope characteristic peaks 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, compound A10 was replaced with an equimolar amount of compound A13, and other conditions remained unchanged to obtain 21.6 mg of target compound I-13 (orange-red solid) with a yield of 27.2%.

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

[0124] 1H-NMR (400MHz, DMSO, TMS), δppm: 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=25.4Hz,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-necked bottle, add 7-(diethylamino)coumarin-3-carboxylic acid (1.0mmol, 0.26g), N-Boc-piperazine (2.0mmol, 0.37g), HOBT (1.5mmol, 0.20g), EDCI (1.5mmol, 0.29g), add 5mL N,N-dimethylformamide to dissolve, drop 0.70mL (4.0mmol) DIPEA, stir at room temperature to react until TLC detection shows that the reaction of 7-(diethylamino)coumarin-3-carboxylic acid is complete, add 50mL ethyl acetate to the reaction solution to dilute, wash with water 3 times, combine the organic phases, wash the organic phases with saturated brine 3 times, and dry over anhydrous sodium sulfate overnight. The organic phase was spin-dried and separated by normal phase silica gel column chromatography (PE:EA=6:1, V:V) to obtain 0.25 g of compound A14-1 (yellow-green solid); 0.25 g of compound A14-1 was dissolved in 4 mL of anhydrous dichloromethane, 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, the reaction was concentrated to obtain 0.22 g of compound A14 (yellow viscous substance), with a yield of 67.7%.

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

[0130]

[0131] In a single-necked bottle, add tripterygium wilfordii (0.3mmol, 0.135g), add 5mL acetone and stir to dissolve, add anhydrous potassium carbonate (0.9mmol, 0.12g), 1,3-dibromopropane 0.091mL (0.9mmol), heat under reflux at 50°C until TLC detection shows that the reaction is complete. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (PE:EA=20:1, V:V) to obtain 0.12g of compound A6 (orange-red solid), with a yield of 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 mixture was heated to reflux at 50°C for 30 min. Compound A6 (0.2 mmol, 0.11 g) was then added, and the mixture was heated to reflux until TLC detection showed that the reaction was complete. The organic phase was spin-dried and separated by normal phase silica gel column chromatography (DCM:MeOH=60:1, V:V) to obtain 23.4 mg of compound I-14 (orange-red solid) with a yield of 14.3%.

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

[0137] 1 H-NMR (300MHz, CDCl3, TMS), δppm: 7.85 (s, 1H), 7.32 (d, J = 8.9Hz, 1H), 7.04 (dd, J = 7.1, 1.4Hz, 1H ),6.61(dd,J=8.9,2.4Hz,1H),6.52(d,J=1.4Hz,1H),6.49(d,J=2.4Hz,1H),6.36(d,J=7.2Hz,1H) ,4.05(dt,J=11.0,6.4Hz,1H),3.90(dt,J=11.4,6.2Hz,1H),3.77(s,1H),3.45(q,J=7.2Hz,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] 0.12 mL (0.9 mmol) of 1,5-dibromopentane was used to replace 1,3-dibromopropane in the preparation of Compound A6 in Example 8. Other conditions remained unchanged to obtain 0.12 g of Compound A8 (orange-red solid) with a yield of 66.9%.

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

[0143] Synthesis of Compound I-15

[0144]

[0145] Referring to the preparation method of compound I-14 in Example 8, compound A6 was replaced with an equal molar amount of compound A8, and other conditions remained unchanged to obtain 19.1 mg of target compound I-15 (orange-red solid) with a yield of 11.3%.

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

[0147] 1 H-NMR (300MHz, CDCl3, TMS), δppm: 7.84 (s, 1H), 7.30 (s, 1H), 7.02 (d, J = 7.0Hz, 1H), 6.59 (d, J = 8.9Hz, 1H), 6.49 (d, J = 11.6Hz, 2H), 6.34 (d, J=7.0Hz,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] 0.15 mL (0.9 mmol) of 1,6-dibromopentane was used to replace 1,3-dibromopropane in the preparation of Compound A6 in Example 8. Other conditions remained unchanged to obtain 0.12 g of Compound A9 (orange-red solid) with a yield of 65.3%.

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

[0153] Synthesis of Compound I-16

[0154]

[0155] Referring to the preparation method of compound I-14 in Example 8, compound A6 was replaced by an equal mole of compound A9, and other conditions remained unchanged to obtain 17.9 mg of target compound I-16 (orange-red solid) with a yield of 10.4%.

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

[0157] 1 H-NMR (300MHz, CDCl3, TMS), δppm: 7.85 (s, 1H), 7.33 (s, 1H), 7.04 (d, J = 7.9Hz, 1H), 6.61 (d, J = 9.0Hz, 2H), 6.54 (s, 1H), 6.49 (s, 1H), 6.37 (d, J = 7.2H) z,1H),3.95(m,4H),3.84(m,4H),3.45(d,J=7.6Hz,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] Embodiment 11

[0159] Synthesis of Compound I-17

[0160]

[0161] In a single-necked bottle, add compound I-11 (0.13mmol, 0.10g), DMAP (0.07mmol, 0.0086g), EDCI (0.20mmol, 0.038g), biotin (0.39mmol, 0.095g), add 3mL N,N-dimethylformamide to dissolve, react at room temperature until TLC detection shows that the reaction is complete, add 30mL dichloromethane to the reaction solution to dilute, wash with water 3 times, combine the organic phase, wash the organic phase with saturated brine 3 times, dry over anhydrous sodium sulfate overnight. The organic phase is spin-dried and separated by normal phase silica gel column chromatography (DCM: MeOH = 10: 1, V: V) to obtain 33.6mg of compound I-17 (yellow solid), with a yield of 26.1%.

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

[0163] 1 H-NMR (400MHz, CDCl3, TMS), δppm: 8.42 (s, 1H), 7.37 (dd, J = 9.0, 2.4Hz, 1H), 7.06 (dd, J = 7.1, 1.4Hz, 1H), 6.60 (dd, J = 9 .0,2.5Hz,1H),6.44(dd,J=3.7,1.9Hz,1H),6.31(d,J=7.2Hz,2H),4.50(dt,J=8.1,4.2Hz,1H),4.32(t,J=6.3Hz,3H),4 .06(dt,J=11.9,6.1Hz,1H),3.90(dt,J=11.5,5.8Hz,1H),3.43(q,J=7.3Hz,4H),3.14(dq,J=11.2,6.5Hz,1H),2.91-2. 79(m,1H),2.75(dd,J=12.9,5.5Hz,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 assay of compounds

[0166] The anti-tumor activity of the tripterygium wilfordii derivatives (Compound I-2-Compound I-4, Compound I-10-Compound I-17) of the present invention was tested by MTT method, and tripterygium wilfordii (CEL) and CDDO-Me (Bardoxolone methyl) were selected as positive control drugs. Appropriate amounts of the test compound, CEL and CDDO-Me were weighed, dissolved in DMSO to prepare a mother solution with a concentration of 10 mmol / L, and then diluted with culture medium to form a series of solutions with gradient concentrations.

[0167] Instruments: clean bench (SW-CJ-1FD, AIRTECH, Sujing Antai), constant temperature CO2 incubator (3111, Thermo, USA), high pressure sterilizer (YXO.SG41.280, Shanghai Huaxian), centrifuge (SIGMA), multi-biological microscope (IX71, Olympus, Japan), multi-function microplate reader (POLARstar, Omega, USA).

[0168] Reagents: DMEM 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, human normal liver cell line L02 (all purchased from Jiangsu KeyGen Biotech Co., Ltd.), all cultured in DMEM (high glucose) medium.

[0170] Methods: The frozen cell lines were revived and cultured in a constant temperature 37°C, 5% CO2 incubator. The medium was changed once a day. When the cells were in the exponential growth phase and in good condition, they could be plated. 1 mL of 0.25% trypsin digestion solution was added and digested for 1-2 min. The cell state was observed under a microscope. When the adherent cells became round and shrunk, the digestion solution was removed and 1-2 mL of culture medium containing 10% fetal bovine serum was added to make a cell suspension. The cells were counted and 5×10 cells were added to each well. 3 The amount of cell suspension required was calculated based on the number of cells and the total number of wells. The cell suspension was inoculated on a 96-well plate at 100 μL / well, sealed with PBS, and placed in a constant temperature 37°C, 5% CO2 incubator for 24 hours. The old culture medium was discarded, and a series of solutions with gradient concentrations of the test compound, CEL or CDDO-Me were added, and a blank control (an equal volume of DMSO solution equal to the highest concentration of the test drug) was set up. Three replicate wells were set up for each drug concentration and cultured for 48 hours. MTT reagent (5 mg / mL) was added to the 96-well plate at 10 μL / well and continued to incubate for 4 hours. The culture medium in the plate was removed, 150 μL DMSO was added to each well, and the absorbance of each well was detected at a wavelength of 570 nm using a multifunctional microplate reader, and the cell inhibition rate was calculated according to the following formula.

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

[0172] The average of the three initial screening results is the final inhibition rate, and the IC of the tested drug is calculated 50 The final IC value of the compound was obtained by 3 repeated experiments (graphpad software). 50 value.

[0173] Selective 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 test compounds on MDA-MB-231, MCF-7 and A549 cell lines

[0175]

[0176]

[0177] Note: NA means not tested.

[0178] As shown in Table 1, compared with celastrol, the inhibitory effect of the celastrol derivatives of the present invention on breast cancer cells is significantly enhanced, and the selectivity for normal cells is improved. Among them, compound I-17 has the best selectivity, and compared with celastrol, the selectivity for normal human breast cells is increased by about 7.34 times, and the selectivity for normal human liver cells is increased by about 14.11 times.

[0179] (II) Compound I-17 targeting cell mitochondria detection

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

[0181] Reagent: Mito RedCMXRos mitochondrial red fluorescent probe (Yisheng Biotechnology).

[0182] Methods: Referring to the cell digestion and counting method in the “in vitro anti-tumor cell proliferation experiment of compounds”, the cell suspension (1×10 5 / mL), MDA-MB-231 cells were uniformly inoculated in a laser confocal microplate; after culturing for 24 hours, the original culture medium was discarded, and a culture medium containing 20 μM compound I-17 (refer to "In vitro anti-tumor cell proliferation experiment of compounds") was added, and the culture was continued for 2 hours. Subsequently, the fluorescence co-localization experiment was performed according to the instructions of the kit.

[0183] 100 nM mitochondrial probe was added to the laser confocal microscopy dish, and after incubation for 40 min, the mitochondrial localization of cells and compounds was observed under a laser confocal microscope under a suitable excitation light source. Compound I-17 has a significant co-localization effect with mitochondria. Figure 1 As shown, A is an image taken under a 20x microscope, and B is an image taken under a 20x microscope; the red image is the fluorescence of the mitochondrial probe, and the green image is the fluorescence of compound I-17, and the two are highly overlapped and appear orange. The experimental results show that compound I-17 effectively accumulates in the mitochondria of MDA-MB-231 cells, and can further induce mitochondria to generate ROS, affect oxidative stress, and exert anti-tumor effects. Tumor cells usually have a higher mitochondrial membrane potential than normal cells, and drugs with the ability to target mitochondria will be more easily enriched in the mitochondria of tumor cells; and the lead compound tripterygium wilfordii does not have the ability to target mitochondria, so compared with the former, the tripterygium wilfordii derivatives including compound I-17 of the present invention have better selectivity in anti-tumor.

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

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

[0186] Reagents: Reactive oxygen species (ROS) assay kit (KeyGen).

[0187] Methods: Referring to the cell digestion and counting method in the “in vitro anti-tumor cell proliferation experiment of compounds”, the cell suspension (1×10 5 / mL), MDA-MB-231 cells were evenly inoculated in 6-well plates and cultured for 24h. They were divided into NAC (N-acetyl-L-cysteine) group, different doses of compound I-17 treatment group, tripterygium wilfordii treatment group, and blank control (DMSO) group. NAC (5mM) was added to the NAC (N-acetyl-L-cysteine) group for pretreatment for 2h before administration, and the remaining treatment groups and blank control group were incubated without NAC for 2h. The original culture medium was discarded, and different doses of compound I-17 treatment groups were added with culture medium containing set concentrations (1, 2, 4μM) of compound I-17 (refer to "In vitro anti-tumor cell proliferation experiment of compounds"), tripterygium wilfordii treatment group was added with culture medium containing 1μM tripterygium wilfordii, NAC group was added with culture medium containing 4μM compound I-17, and blank control was added with an equal volume of DMSO equal to the highest concentration of the test drug I-17, and continued to be cultured for 24h. Subsequently, the intracellular ROS level was detected according to the instructions of the kit.

[0188] The DCDH-DA probe was loaded in a 6-well plate, and the ROS level of cells treated with the compound was observed under an inverted fluorescence microscope under a suitable excitation light source. Figure 2 As shown, with the increase of the dose of compound I-17, the number of green spots was observed to increase, indicating that the intracellular ROS level increased with the increase of drug concentration. After the cells were pretreated with NAC to remove intracellular ROS, the ROS level in the cells treated with 4 μM concentration of compound I-17 was significantly reduced compared with the cells without pretreatment, indicating that the tripterygium wilfordii derivatives including compound I-17 of the present invention 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 Co-operation and Development, OECD).

[0191] Reagents: DMSO (Shanghai Hushi), Tween 80 (Shanghai Hushi), PBS (KeyGEN).

[0192] Methods: The acute toxicity of compound I-17 and tripterygium wilfordii was tested by the up-down method. An appropriate amount of tripterygium wilfordii was weighed and a mixed solvent of DMSO: Tween 80: PBS with a volume ratio of 1:1:8 (if necessary, increase the ratio of Tween 80 and ultrasonicate, the same below) was used as a solvent to prepare a series of solutions (injection dose 5.5, 17.5 mg / kg, 200 μL per mouse); an appropriate amount of compound I-17 was weighed and a mixed solvent of DMSO: Tween 80: PBS with a volume ratio of 1:1:8 was used as a solvent to prepare a series of solutions (injection dose 17.5, 55, 175 mg / kg, 200 μL per mouse). A mouse was randomly selected for injection and then observed. The next mouse's injection dose was selected based on its death or survival within 2 days after administration. The observation was conducted for a total of 7 days. The short-term (2 days) and long-term (7 days) survival (marked as O) or death (marked as X) results of all mice were recorded. The results were input into the AOT425 program to calculate the LD 50 value.

[0193] Table 2. Results of acute toxicity test of tripterygium wilfordii

[0194]

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

[0196]

[0197] The results are shown in Table 3 and Table 4. The LD of tripterygium wilfordii was calculated. 50 The LD of compound I-17 is about 9.811 mg / kg. 50 It is about 103 mg / kg, and its toxicity is about 10 times lower than that of celastrol.

Claims

1. A tripterygium wilfordii derivative having a structure as shown in Formula I: in, R1 is selected from H, R2 is selected from n is an integer of 3 to 6.

2. The tripterygium wilfordii derivative according to claim 1, characterized in that: R1 is selected from H, R2 is selected from n is an integer of 3 to 5; R1 is selected from H, R2 is selected from n is an integer of 3 to 6; R1 is selected from H, R2 is selected from n is an integer of 3 to 6.

3. The tripterygium wilfordii derivative according to claim 2, characterized in that: R1 is selected from H, R2 is selected from n is 3, 4 or 5; R1 is selected from H, R2 is selected from n is an integer of 3 to 6; R1 is selected from H, R2 is selected from n is 3, 5, or 6; R1 is selected from R2 is selected from n is 4.

4. The structure of the tripterygium wilfordii derivative is as follows:

5. A method for preparing the tripterygium wilfordii derivative according to claim 1, characterized in that: When R1=H, The synthetic route is as follows: Wherein, n is as described in claim 1; The following steps are involved: Step (1), using K2CO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent, biotin and formula The dibromoalkane shown is reacted at room temperature to obtain intermediate III; wherein the molar ratio of biotin to dibromoalkane is 1:3 to 1:5; the molar ratio of biotin to K2CO3 is 1:3 to 1:4; Step (2), using NaHCO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent, reacting the intermediate III with tripterygium wilfordii under reflux to obtain a tripterygium wilfordii biotin derivative shown in formula Ia; wherein the molar ratio of the tripterygium wilfordii to the intermediate III is 1:1.1 to 1:1.3; and the molar ratio of the tripterygium wilfordii to NaHCO3 is 1:4 to 1:6; When R1=H, The synthetic route is as follows: Wherein, n is as described in claim 1; The following steps are involved: Step (1), using K2CO3 as a base catalyst, KI as a catalyst, N,N-dimethylformamide as a reaction solvent, 7-(diethylamino)coumarin-3-carboxylic acid and formula The dibromoalkane shown in the figure is refluxed to obtain intermediate VII; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3 to 1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3 to 1:4; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5; Step (2), using NaHCO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent, reacting the intermediate VII with triptolide under reflux to obtain a triptolide coumarin derivative shown in formula Ib; wherein the molar ratio of the triptolide to the intermediate VII is 1:1.1 to 1:1.3; and the molar ratio of the triptolide to NaHCO3 is 1:4 to 1:6; When R1=H, The synthetic route is as follows: Wherein, n is as described in claim 1; The following steps are involved: Step (1), using K2CO3 as a base catalyst and acetone as a reaction solvent, celastrol and formula The dibromoalkane shown is reacted under reflux to obtain intermediate IV; wherein the molar ratio of tripterygium wilfordii to dibromoalkane is 1:3 to 1:5; the molar ratio of tripterygium wilfordii to K2CO3 is 1:3 to 1:4; Step (2), using N,N-dimethylformamide as the reaction solvent, in the presence of HOBT, EDCI and DIPEA, 7-(diethylamino)coumarin-3-carboxylic acid and formula At room temperature, the intermediate VIII is obtained; wherein the 7-(diethylamino)coumarin-3-carboxylic acid and 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; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to DIPEA is 1:4; Step (3), using anhydrous dichloromethane as the reaction solvent, in the presence of trifluoroacetic acid, the intermediate VIII is subjected to acid hydrolysis to remove the Boc protecting group to obtain the intermediate IX; wherein the volume ratio of trifluoroacetic acid to anhydrous dichloromethane is 1:5 to 1:10; Step (4), using K2CO3 as a base catalyst and anhydrous acetonitrile as a reaction solvent, reacting intermediate IX with intermediate IV under reflux to obtain a tripterygium wilfordii coumarin derivative shown in formula Ic; 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 to 1:3; when The synthetic route is as follows: Wherein, n is as described in claim 1; The following steps are involved: Step (1), using K2CO3 as a base catalyst, KI as a catalyst, N,N-dimethylformamide as a reaction solvent, 7-(diethylamino)coumarin-3-carboxylic acid and formula The dibromoalkane shown in the figure is refluxed to obtain intermediate VII; wherein the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to dibromoalkane is 1:3 to 1:5; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to K2CO3 is 1:3 to 1:4; the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to KI is 1:0.5; Step (2), using NaHCO3 as a base catalyst and N,N-dimethylformamide as a reaction solvent, reacting the intermediate VII with triptolide under reflux to obtain a triptolide coumarin derivative shown in formula Ib; wherein the molar ratio of the triptolide to the intermediate VII is 1:1.1 to 1:1.3; and the molar ratio of the triptolide to NaHCO3 is 1:4 to 1:6; Step (3), using N,N-dimethylformamide as reaction solvent, in the presence of DMAP and EDCI, the tripterygium wilfordii coumarin derivatives shown in formula Ib react with biotin shown in formula II at room temperature to obtain the tripterygium wilfordii-coumarin-biotin derivatives shown in formula Id; the molar ratio of the tripterygium wilfordii coumarin derivatives to biotin is 1:3; the molar ratio of the tripterygium wilfordii coumarin derivatives to DMAP is 1:0.5; the molar ratio of the tripterygium wilfordii coumarin derivatives to EDCI is 1:1.

5.

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

7. Use of the tripterygium wilfordii derivative according to any one of claims 1 to 4 in the preparation of mitochondrial targeted anti-tumor drugs.

8. The use according to claim 6 or 7, characterized in that: The tumors are breast cancer and lung cancer.

9. A pharmaceutical composition, characterized in that: The invention uses the tripterygium wilfordii derivative described in any one of claims 1 to 4 as an effective component and is prepared into different dosage forms with a pharmaceutical carrier.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage form is selected from tablets, capsules, pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, and nano preparations.

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