CN111518157B

CN111518157B - Triptolide derivative and preparation method and application thereof

Info

Publication number: CN111518157B
Application number: CN202010529052.3A
Authority: CN
Inventors: 范培红; 宋慧娜
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2021-02-23
Anticipated expiration: 2040-06-11
Also published as: CN111518157A

Abstract

The invention provides a triptolide derivative, which has a structure shown in a formula I:

Description

Triptolide derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a triptolide derivative and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The great problem of tumor treatment is poor selectivity and high toxicity of chemotherapeutic drugs. Designing a drug based on the difference between tumor cells and normal cells is expected to achieve a certain selectivity of the antitumor drug. Research shows that the mitochondrial membrane potential of tumor cells is far greater than that of normal cells, and with the understanding of mitochondrial function and structure change in tumor cells, the specific targeting of compounds to mitochondria becomes a new field of drug development.

The natural product is a treasure house for finding the compound with the anti-tumor activity. In 1972, Kupchan isolated and identified a cyclic diterpenoid compound, Triptolide, with significant anti-leukemia activity from an ethanol extract of Tripterygium wilfordii for the first time. Research shows that triptolide is an effective antitumor agent and can inhibit the growth of cancer cells in vitro and in vivo, but because triptolide has poor water solubility and a narrow treatment window, serious adverse reaction can be caused, and the cytotoxicity is high, so that the clinical application of the triptolide is severely limited.

Disclosure of Invention

Therefore, the invention aims to provide the triptolide derivative, the preparation method and the application thereof, compared with the triptolide, the triptolide derivative has better selectivity on tumor cells while keeping better antitumor activity, reduces the toxicity on normal cells, has better water solubility, is beneficial to the in-vivo absorption and the drug effect of the drug, and reduces the adverse reaction, and has the potential of being used as an antitumor drug.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a triptolide derivative, which has a structure represented by formula I:

wherein n is an integer of 4 to 10, preferably 5 to 10.

The compounds of the invention are cationic compounds with oleophilic and hydrophilic amphiphilicity, have improved water solubility compared with triptolide, and can be gathered at the cell mitochondrial site under the push of mitochondrial transmembrane potential. Because the mitochondrial membrane potential of tumor cells is higher than that of normal cells, it can provide a driving force for selective accumulation in the mitochondria of tumor cells, thus having mitochondrial targeting and the compounds of the invention exhibit mitochondrial toxicity which can lead to tumor cell death.

The inventor of the present invention studied the effect of structural change on the antitumor activity and the attenuation of normal cells in the research process of the present invention, and found that, as shown in the following structure, the A, B, C three parts are necessary structures for achieving the antitumor effect and the attenuation of normal cells of the present invention, and n in the B part is an integer of 4 to 10, and particularly, as shown in the following structure, the C part can not only bring about the targeting effect, but also exhibit a very desirable attenuation of normal cells while maintaining a good antitumor effect when it is linked to the A, B (n is an integer of 4 to 10) two parts in the following manner. However, if the value of n in part B is too high, for example, n is 11 or more, the inhibitory effect on breast cancer cells is significantly reduced and the inhibitory activity (in terms of IC) is significantly lowered₅₀Value counting) canThe reduction is about 40 times, the inhibition effect on liver cancer cells is poor, and the liver cancer cells hardly have attenuation effect on normal cells.

In addition, the invention also synthesizes

The results show that the compounds with two structures can show certain activity of inhibiting liver cancer cells, wherein,

when the value of n is an integer of 4-10, the compound can show a cancer inhibition effect equivalent to triptolide, but cannot reduce the toxic effect on normal cells;

in the formula, when n is an integer of 3-10, the compound has a certain inhibitory effect on breast cancer cells, namely IC₅₀The value was about 0.6 to 0.8. mu.M, but the attenuation effect on normal cells was not exhibited.

In a more preferred embodiment of the invention, n is preferably 4, 5, 6, 7, 10, said compound being selected from the following structures:

in a second aspect of the present invention, there is provided a method for preparing the triptolide derivative described in the first aspect, comprising: taking triptolide TP as a starting raw material, carrying out esterification reaction on C-14 beta-OH of triptolide to obtain an intermediate compound, and then carrying out salt forming reaction on the intermediate compound and triphenylphosphine to generate the triptolide derivative shown in the formula I;

wherein n is an integer of 4 to 10.

In some embodiments provided herein, the method is carried out via the following reaction scheme:

wherein n is an integer of 4 to 10;

in some embodiments of the invention, the condensation agent of the esterification reaction is selected from one or more of N, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), preferably EDCI; the catalyst for the esterification reaction is selected from one or more of 4-Dimethylaminopyridine (DMAP), Triethylamine (TEA) and N, N-Diisopropylethylamine (DIPEA), and is preferably DMAP.

In a further embodiment of the present invention, the esterification reaction is performed by the EDCI/DMAP method, the solvent for the esterification reaction is one or more selected from the group consisting of chloroform, N-dimethylformamide, and dichloromethane, preferably dichloromethane, and the reaction temperature is room temperature.

In some embodiments of the invention, the solvent for the salt-forming reaction is selected from one or more of toluene, N-dimethylformamide, N-butanol and acetonitrile, preferably acetonitrile.

In some embodiments of the invention, the salt formation reaction is carried out under heating reflux conditions at 80-85 ℃.

In some preferred embodiments, the method comprises: taking triptolide as a starting material, and carrying out the reaction in dichloromethane at room temperature by an EDCI/DMAP method

Performing esterification reaction, detecting to obtain yellow clear solution, and separating to obtain intermediate compound

And then carrying out salt-forming reaction with triphenylphosphine in acetonitrile as a solvent under the condition of heating reflux at the temperature of 80-85 ℃, and separating to obtain the compound of the formula X after the detection reaction is finished.

Wherein in the method, triptolide, EDCI, DMAP,

The adding amount ratio of dichloromethane is 1mol:4mol:2mol:3mol:55 mL; the adding amount ratio of the intermediate compound, triphenylphosphine and acetonitrile is 1mol: (3-3.1) mol: 120 mL.

It will be appreciated that in the light of the present disclosure, one skilled in the art can further optimize the reaction conditions based on the present disclosure, such as selecting alternative solvents among the more commonly used reagents in the art, such as further scaling up or scaling down the reaction in routine adjustments to achieve higher yields or reduce the production of impurities based on the amounts of materials disclosed herein, and of course, it will be understood that after each reaction, one skilled in the art can select operations to purify the product to further refine the next reaction, and that routine purification methods can be selected either as an option or after routine experimentation.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the triptolide derivative as described in the first aspect above.

In a fourth aspect of the present invention, there is provided a pharmaceutical preparation comprising the triptolide derivative of the first aspect and at least one pharmaceutically acceptable carrier or adjuvant.

The "composition" as described herein refers to a pharmaceutical product comprising a therapeutically effective amount of the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The triptolide derivative or the pharmaceutical composition or the pharmaceutical preparation containing the triptolide derivative can be administered in a unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, etc.

The pharmaceutical composition or pharmaceutical preparation of the present invention may further comprise conventional carriers, including but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances (e.g. phosphates, glycerol, sorbitan esters, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts) or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The amount of carrier in the pharmaceutical composition or formulation may be from 1% to 98% by weight, usually about 80% by weight. Preservatives, buffers and the like may be present in the carrier as is conventional.

The pharmaceutically acceptable excipients include, but are not limited to, excipients which may be binders, fillers, lubricants, disintegrants, buffers, stabilizers, preservatives, and the like. The auxiliary material refers to a component except for an effective component in the pharmaceutical composition or the pharmaceutical preparation, is nontoxic to a subject, and can stably coexist with a pharmaceutical active component or stably coexist after adopting a proper means.

Oral tablets and capsules may contain binders such as syrup, acacia, sorbitol, tragacanth or polyvinylpyrrolidone; fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine; lubricants such as magnesium stearate, talc, polyethylene glycol, silica; a disintegrant such as potato starch, or an acceptable humectant such as sodium lauryl sulfate may be present. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, hydrogenated edible fats and oils, emulsifying agents, such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

In a fifth aspect of the invention, there is provided a mitochondrially targeted pharmaceutical carrier comprising a triptolide derivative as described in the first aspect above.

In the embodiment of the invention, the triptolide derivative can be selectively accumulated in the mitochondria of the tumor cells by utilizing the membrane potential difference of the tumor cells, so that the triptolide derivative has good mitochondrial targeting of the tumor cells.

In a sixth aspect of the invention, there is provided a delivery system comprising a triptolide derivative as described in the first aspect above or a mitochondrially targeted pharmaceutical carrier as described in the fifth aspect above. The triptolide derivative can be used as a drug delivery system or a main component in the drug delivery system due to good targeting property on tumor cell mitochondria, and can selectively deliver more active drugs into the tumor cell mitochondria to realize good tumor treatment effect.

In a seventh aspect of the present invention, there is provided an application of the triptolide derivative of the first aspect, or the pharmaceutical composition of the third aspect, or the pharmaceutical preparation of the fourth aspect, or the mitochondrion-targeted pharmaceutical carrier of the fifth aspect, or the delivery system of the sixth aspect, in preparing an anti-tumor drug.

In an embodiment of the present invention, the tumor is liver cancer or breast cancer.

In the embodiment of the invention, the compound of the invention has better selectivity on tumor cells and lower toxicity on normal cells compared with triptolide while retaining better antitumor activity (the antitumor activity on liver cancer and breast cancer is equivalent to that of triptolide, but is obviously better than that of doxorubicin hydrochloride), wherein the toxicity of the triptolide derivative on normal liver cells is reduced by up to 827 times (by IC) compared with the triptolide₅₀Value calculation), can be reduced by at least 6-20 times, has better selectivity with tumor cells and is safer for normal cells.

Meanwhile, compared with triptolide, the triptolide derivative disclosed by the invention has better water solubility, is beneficial to in vivo absorption and drug effect exertion of a medicament, and reduces adverse reactions.

Also, the present invention provides a method for treating tumors, especially liver cancer and breast cancer, especially liver cancer, comprising administering to a subject a therapeutically effective amount of the triptolide derivative of the first aspect or the pharmaceutical composition of the third aspect or the pharmaceutical formulation of the fourth aspect or the mitochondrially targeted pharmaceutical carrier of the fifth aspect or the delivery system of the sixth aspect; the subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

The invention has the following beneficial effects: the triptolide derivative can target tumor cell mitochondria, has equivalent inhibitory activity to tumor cells compared with triptolide, but has better water solubility and selectivity to tumor cells, greatly reduces the toxic effect to normal cells, can effectively inhibit liver cancer cells and breast cancer cells, can be beneficial to the in vivo absorption and the drug effect of drugs due to the improvement of the water solubility, can greatly reduce the adverse reaction of the drugs due to the lower toxic effect to the normal cells, and has the potential of being developed into a new antitumor drug.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1Preparation of Compound Mito-TP-1

Triptolide (0.055mmol, 20mg), 5-bromovaleric acid (0.165mmol, 29.9mg), EDCI (0.22mmol, 42.2mg), DMAP (0.11mmol, 13.4mg) were added to 2ml of anhydrous dichloromethane, reacted at room temperature for 8 hours under nitrogen protection, and the reaction progress was checked by TLC to give a yellow clear solution. Extracting with saturated sodium bicarbonate solution for three times, collecting the dichloromethane layer, adding appropriate amount of anhydrous magnesium sulfate, drying for 20 min, vacuum filtering, rotary evaporating, and concentrating. Separating by silica gel preparative plate method (petroleum ether: ethyl acetate 1: 1) to obtain white solid powder, dissolving intermediate 1(0.0382mmol, 20.0mg) in 3mL acetonitrile, adding triphenylphosphine (0.116mmol, 29.3mg), heating and refluxing at 80 ℃ in oil bath for 48h, detecting that the reaction is basically completed by TLC, and concentrating by rotary evaporation. Separation was carried out by silica gel preparative plate method (dichloromethane: methanol ═ 15: 1) to obtain 13.1mg of white solid, i.e., Mito-TP-1.

The synthetic route is as follows:

spectral data:

intermediate 1:¹H NMR(600MHz,CD₃OD)δ5.09(s,1H,H-14),4.79(m,2H,H-19),3.97(d,J＝3.2Hz,1H,H-11),3.64(d,J＝3.0Hz,1H,H-12),3.49(m,2H,H-5'),3.49(m,1H,H-7),2.79(m,1H,H-5),2.41(m,2H,H-2'),2.27(m,1H,H-2a),2.27(m,1H,H-6a),2.07(m,1H,H-2b),1.90(m,1H,H-15),1.90(m,1H,H-6b),1.90(m,4H,H-3',H-4'),1.52(m,1H,H-1a),1.35(m,1H,H-1b),1.05(s,3H,H-20),0.96(d,J＝7.0Hz,3H,H-17),0.86(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.24(C-18),174.29(CH ₂CO-,C-1'),164.02(C-4),125.69(C-3),72.89(C-14),72.13(C-19),65.04(C-13),64.33(C-9),62.92(C-8),61.28(C-7),56.90(C-11),56.39(C-12),41.60(C-5),36.97(C-10),34.32(C-2'),33.95(C-5'),33.08(C-4'),30.97(C-1),29.79(C-15),24.89(C-6),24.33(C-3'),18.06(C-17),18.06(C-2),17.26(C-16),14.34(C-20).

compound Mito-TP-1:¹H NMR(600MHz,CD₃OD)δ7.78(m,3H,H-4”),7.65(m,6H,H-2”,H-6”),7.61(m,6H,H-3”,H-5”),4.99(s,1H,H-14),4.77(m,2H,H-19),4.01(d,J＝3.3Hz,1H,H-11),3.68(d,J＝3.1Hz,1H,H-12),3.45(d,J＝5.7Hz,1H,H-7),3.23(m,2H,H-5'),2.70(m,1H,H-5),2.45(m,2H),2.14(m,2H),1.97(m,1H),1.75(m,5H),1.51(m,1H),1.32(m,1H,H-1a),1.20(m,1H,H-1b),0.78(d,J＝7.0Hz,3H,H-20),0.68(d,J＝6.9Hz,3H,H-16),0.62(s,3H,H-20)；¹³C NMR(150MHz,CD₃OD)δ176.15(C-18),174.07(C-1'),163.83(C-4),136.49(d,J＝3.0Hz,C-4”),135.02(d,J＝10.1Hz,C-2”,C-6”),131.72(d,J＝12.9Hz,C-3”,C-5”),125.69(C-3),120.02(d,J＝86.5Hz,C-1”),72.91(C-14),72.11(C-19),65.00(C-13),64.24(C-9),63.10(C-8),61.35(C-7),57.04(C-11),56.25(C-12),41.49(C-5),36.89(C-10),34.18(C-2'),30.93(C-1),29.77(C-15),26.77(d,J＝17.6Hz,C-4'),24.30(C-6),22.98(d,J＝4.2Hz,C-3'),22.58(d,J＝51.8Hz,C-5'),18.07(C-17),18.00(C-2),17.26(C-16),14.46(C-20).HPLC purity:99％.HR-ESI-MS m/z 705.2981(C₄₃H₄₆O₇P⁺[M-Br]⁺calculations 705.2976)

Example 2Preparation of Compound Mito-TP-2

Intermediate 2 and compound Mito-TP-2 were prepared according to the procedure of example 1, starting from the following synthetic route.

The synthetic route is as follows:

spectral data:

intermediate 2:¹H NMR(600MHz,CD₃OD)δ5.08(s,1H,H-14),4.80(m,2H,H-19),3.96(d,J＝3.2Hz,1H,H-11),3.63(d,J＝3.1Hz,1H,H-12),3.48(m,2H,H-6'),3.48(m,1H,H-7),2.78(m,1H,H-5),2.48(m,2H,H-2'),2.27(m,1H,H-2a),2.27(m,1H,H-6a),2.06(m,1H,H-2b),1.85(m,1H,H-15),1.85(m,1H,H-6b),1.85(m,2H,H-5'),1.67(m,2H,H-3'),1.50(m,2H,H-4'),1.50(m,1H,H-1a),1.33(m,1H,H-1b),1.04(s,3H,H-20),0.95(d,J＝7.0Hz,3H,H-17),0.85(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.18(C-18),174.53(C-1'),163.98(C-4),125.66(C-3),72.77(C-14),72.12(C-19),65.03(C-13),64.35(C-9),62.85(C-8),61.24(C-7),56.86(C-11),56.35(C-12),41.59(C-5),36.96(C-10),35.10(C-2'),34.29(C-6'),33.83(C-5'),30.96(C-1),29.73(C-15),28.66(C-4'),25.40(C-3'),24.32(C-6),18.09(C-17),18.05(C-2),17.28(C-16),14.37(C-20).

compound Mito-TP-2:¹H NMR(600MHz,CD₃OD)δ7.90(m,3H,H-4”),7.86(m,6H,H-2”,H-6”),7.77(m,6H,H-3”,H-5”),5.06(s,1H,H-14),4.79(m,2H,H-19),3.95(d,J＝3.2Hz,1H,H-11),3.61(d,J＝2.5Hz,1H,H-12),3.46(m,1H,H-7),3.42(m,2H,H-6'),2.76(d,J＝13.2Hz,1H,H-5),2.37(m,2H,H-2'),2.25(m,1H,H-2a),2.25(m,1H,H-6a),2.00(s,1H,H-2b),1.82(m,1H,H-15),1.82(m,1H,H-6b),1.74(m,6H,H-3',H-4',H-5'),1.30(m,2H,H-1),0.93(d,J＝7.0Hz,3H,H-17),0.90(s,3H,H-20),0.84(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.19(C-18),174.26(C-1'),163.95(C-4),136.48(d,J＝3.6Hz,C-4”),135.02(d,J＝10.5Hz,C-2”,C-6”),131.72(d,J＝12.9Hz,C-3”,C-5”),125.63(C-3),120.08(d,J＝86.6Hz,C-1”),72.84(C-14),72.14(C-19),65.12(C-13),64.30(C-9),62.98(C-8),61.29(C-7),57.05(C-11),56.25(C-12),41.50(C-5),36.91(C-10),34.64(C-2'),30.91(C-1),30.53(d,J＝16.7Hz,C-5'),29.85(C-15),25.16(C-3'),24.29(C-6),23.12(d,J＝5.1Hz,C-4'),22.81(d,J＝51.3Hz,C-6'),18.11(C-17),18.02(C-2),17.29(C-16),14.50(C-20).HPLC purity:98％.HR-ESI-MS m/z 719.3122(C₄₄H₄₈O₇P⁺[M-Br]⁺calculations 719.3132)

Example 3Preparation of Compound Mito-TP-3

Intermediate 3 and compound Mito-TP-3 were prepared according to the procedure of example 1, starting from the following synthetic route.

The synthetic route is as follows:

spectral data:

intermediate 3:¹H NMR(600MHz,CD₃OD)δ5.08(s,1H,H-14),4.80(m,2H,H-19),3.96(d,J＝3.1Hz,1H,H-11),3.63(d,J＝2.5Hz,1H,H-12),3.46(m,2H,H-7'),3.46(m,1H,H-7),2.78(m,1H,H-5),2.45(m,2H,H-2'),2.27(m,1H,H-2a),2.27(m,1H,H-6a),2.06(m,1H,H-2b),1.85(m,1H,H-15),1.85(m,1H,H-6b),1.85(m,2H,H-6'),1.67(m,2H,H-3'),1.50(m,2H,H-5'),1.50(m,2H,H-4'),1.50(m,1H,H-1a),1.33(m,1H,H-1b),1.04(s,3H,H-20),0.95(d,J＝7.0Hz,3H,H-17),0.85(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.24(C-18),174.73(C-1'),164.03(C-4),125.68(C-3),72.78(C-14),72.13(C-19),65.04(C-13),64.36(C-9),62.83(C-8),61.26(C-7),56.85(C-11),56.39(C-12),41.61(C-5),36.98(C-10),35.22(C-2'),34.42(C-7'),34.00(C-6'),30.97(C-1),29.78(C-15),29.21(C-4'),29.02(C-5'),26.08(C-3'),24.35(C-6),18.07(C-17),18.07(C-2),17.26(C-16),14.36(C-20).

compound Mito-TP-3:¹H NMR(600MHz,CD₃OD)δ7.89(m,3H,H-4”),7.85(m,6H,H-2”,H-6”),7.76(m,6H,H-3”,H-5”),5.06(m,1H,H-14),4.78(m,2H,H-19),3.93(d,J＝3.2Hz,1H,H-11),3.60(d,J＝2.4Hz,1H,H-12),3.45(d,J＝5.6Hz,1H,H-7),3.40(m,2H,H-7'),2.76(m,1H,H-5),2.39(m,2H,H-2'),2.24(m,1H,H-2a),2.24(m,1H,H-6a),2.00(m,1H,H-2b),1.81(m,1H,H-15),1.81(m,1H,H-6b),1.69(m,6H,H-3',H-4',H-6'),1.45(m,2H,H-5'),1.45(m,1H,H-1a),1.31(m,1H,H-1b),0.93(d,J＝7.0Hz,3H,H-17),0.88(s,3H,H-20),0.83(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.18(C-18),174.60(C-1'),163.93(C-4),136.47(d,J＝3.6Hz,C-4”),135.00(d,J＝10.2Hz,C-2”,C-6”),131.71(d,J＝13.2Hz,C-3”,C-5”),125.68(C-3),120.11(d,J＝86.2Hz,C-1”),72.77(C-14),72.11(C-19),65.01(C-13),64.29(C-9),62.84(C-8),61.36(C-7),56.91(C-11),56.32(C-12),41.48(C-5),36.91(C-10),35.10(C-2'),31.33(d,J＝16.4Hz,C-6'),30.85(C-1),29.83(C-15),29.05(C-4'),25.83(C-3'),24.33(C-6),23.41(d,J＝4.8Hz,C-5'),22.74(d,J＝50.8Hz,C-7'),18.09(C-17),18.05(C-2),17.26(C-16),14.40(C-20).HPLC purity:94％.HR-ESI-MS m/z733.3292(C₄₅H₅₀O₇P⁺[M-Br]⁺calculations 733.3289)

Example 4Preparation of Compound Mito-TP-4

Intermediate 4 and compound Mito-TP-4 were prepared according to the procedure of example 1, starting from the following synthetic route.

The synthetic route is as follows:

spectral data:

intermediate 4:¹H NMR(600MHz,CD₃OD)δ5.07(s,1H,H-14),4.79(m,2H,H-19),3.96(d,J＝3.2Hz,1H,H-11),3.62(m,1H,H-12),3.45(m,2H,H-8'),3.45(m,1H,H-7),2.78(m,1H,H-5),2.39(m,2H,H-2'),2.27(m,1H,H-2a),2.27(m,1H,H-6a),2.08(m,1H,H-2b),1.87(m,1H,H-15),1.87(m,1H,H-6b),1.87(m,2H,H-7'),1.67(m,2H,H-3'),1.47(m,2H,H-6'),1.47(m,2H,H-4'),1.47(m,1H,H-1a),1.36(m,2H,H-5'),1.36(m,1H,H-1b),1.04(s,3H,H-20),0.95(d,J＝7.0Hz,3H,H-17),0.85(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.22(C-18),174.79(C-1'),164.04(C-4),125.68(C-3),72.75(C-14),72.13(C-19),65.04(C-13),64.36(C-9),62.80(C-8),61.24(C-7),56.83(C-11),56.39(C-12),41.61(C-5),36.98(C-10),35.28(C-2'),34.56(C-8'),34.09(C-7'),30.97(C-1),29.92(C-4'),29.77(C-5'),29.64(C-6'),29.18(C-15),26.17(C-3'),24.36(C-6),18.07(C-17),18.06(C-2),17.27(C-16),14.37(C-20).

compound Mito-TP-4:¹H NMR(600MHz,CD₃OD)δ7.89(m,3H,H-4”),7.80(m,6H,H-2”,H-6”),7.75(m,6H,H-3”,H-5”),5.06(s,1H,H-14),4.78(m,2H,H-19),3.94(d,J＝3.2Hz,1H,H-11),3.61(d,J＝2.3Hz,1H,H-12),3.45(d,J＝5.7Hz,1H,H-7),3.39(m,2H,H-8'),2.77(m,1H,H-5),2.37(m,2H,H-2'),2.25(m,1H,H-2a),2.25(m,1H,H-6a),2.03(m,1H,H-2b),1.82(m,1H,H-15),1.82(m,1H,H-6b),1.64(m,6H,H-3',H-4',H-7'),1.39(m,4H,H-5',H-6'),1.39(m,2H,H-1),0.96(s,3H,H-20),0.93(d,J＝7.0Hz,3H,H-17),0.83(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.20(C-18),174.67(C-1'),163.99(C-4),136.46(d,J＝3.6Hz,C-4”),134.98(d,J＝10.4Hz,C-2”,C-6”),131.69(d,J＝12.4Hz,C-3”,C-5”),125.68(C-3),120.15(d,J＝86.2Hz,C-1”),72.76(C-14),72.12(C-19),65.03(C-13),64.32(C-9),62.78(C-8),61.30(C-7),56.87(C-11),56.33(C-12),41.53(C-5),36.95(C-10),35.21(C-2'),31.52(d,J＝16.5Hz,C-7'),30.89(C-1),29.79(C-15),29.62(C-5'),29.56(C-4'),26.01(C-3'),24.35(C-6),23.54(d,J＝5.0Hz,C-6'),22.76(d,J＝50.8Hz,C-8'),18.09(C-17),18.06(C-2),17.26(C-16),14.40(C-20).HPLC purity:96％.HR-ESI-MS m/z747.3457(C₄₆H₅₂O₇P⁺[M-Br]⁺calculations 747.3445)

Example 5Preparation of Compound Mito-TP-5

Intermediate 5 and compound Mito-TP-5 were prepared according to the procedure of example 1, starting from the following synthetic route.

The synthetic route is as follows:

spectral data:

intermediate 5:¹H NMR(600MHz,CD₃OD)δ5.07(s,1H,H-14),4.80(m,2H,H-19),3.96(d,J＝3.2Hz,1H,H-11),3.63(d,J＝3.0Hz,1H,H-12),3.45(m,2H,H-11'),3.45(m,1H,H-7),2.78(m,1H,H-5),2.39(m,2H,H-2'),2.26(m,1H,H-2a),2.26(m,1H,H-6a),2.08(m,1H,H-2b),1.87(m,1H,H-15),1.87(m,1H,H-6b),1.87(m,2H,H-10'),1.66(m,2H,H-3'),1.51(m,1H,H-1a),1.43(m,2H,H-4'),1.35(m,1H,H-1b),1.35(m,10H,H-5',H-6',H-7',H-8',H-9'),1.04(s,3H,H-20),0.95(d,J＝7.0Hz,3H,H-17),0.85(d,J＝6.9Hz,3H,H-16)；¹³CNMR(150MHz,CD₃OD)δ176.23(C-18),174.86(C-1'),164.04(C-4),125.69(C-3),72.74(C-14),72.14(C-19),65.03(C-13),64.35(C-9),62.77(C-8),61.24(C-7),56.82(C-11),56.39(C-12),41.61(C-5),36.98(C-10),35.36(C-2'),34.63(C-11'),34.15(C-10'),30.97(C-1),30.66(C-6'),30.63(C-7'),30.52(C-5'),30.11(C-4'),29.98(C-8'),29.78(C-9'),29.32(C-15),26.30(C-3'),24.38(C-6),18.08(C-17),18.07(C-2),17.27(C-16),14.38(C-20).

compound Mito-TP-5:¹H NMR(600MHz,CD₃OD)δ7.80(m,3H,H-4”),7.70(m,6H,H-2”,H-6”),7.65(m,6H,H-3”,H-5”),4.97(s,1H,H-14),4.68(m,2H,H-19),3.84(d,J＝3.2Hz,1H,H-11),3.52(m,1H,H-12),3.35(d,J＝5.7Hz,1H,H-7),3.29(m,2H,H-11'),2.68(m,1H,H-5),2.27(m,2H,H-2'),2.15(m,1H,H-2a),2.15(m,1H,H-6a),1.94(m,1H,H-2b),1.75(m,1H,H-15),1.75(m,1H,H-6b),1.55(m,4H,H-3',H-4'),1.44(m,2H,H-10'),1.37(m,1H,H-1a),1.22(m,10H,H-5',H-6',H-7',H-8',H-9'),1.22(m,1H,H-1b),0.91(s,3H,H-20),0.84(d,J＝7.0Hz,3H,H-17),0.73(d,J＝6.9Hz,3H,H-16)；¹³C NMR(150MHz,CD₃OD)δ176.20(C-18),174.81(C-1'),164.04(C-4),136.46(d,J＝3.6Hz,C-4”),134.96(d,J＝10.3Hz,C-2”,C-6”),131.69(d,J＝13.1Hz,C-3”,C-5”),125.65(C-3),120.16(d,J＝86.3Hz,C-1”),72.72(C-14),72.13(C-19),65.03(C-13),64.33(C-9),62.79(C-8),61.25(C-7),56.83(C-11),56.36(C-12),41.57(C-5),36.96(C-10),35.33(C-2'),31.69(d,J＝16.5Hz,C-10'),30.94(C-1),30.47(C-7'),30.42(C-6'),30.07(C-8'),29.94(C-5'),29.77(C-15),26.25(C-4'),24.35(C-3'),23.63(d,J＝5.0Hz,C-9'),22.74(d,J＝51.1Hz,C-11'),18.09(C-17),18.07(C-2),17.26(C-16),14.36(C-20).HPLC purity:99％.HR-ESI-MS m/z 789.3919(C₄₉H₅₈O₇P⁺[M-Br]⁺calculations 789.3915)

Example 6Control research on antitumor activity of triptolide and triptolide derivatives

1. Experimental Material

Control drugs: triptolide (TP for short) and doxorubicin hydrochloride (DO for short);

experimental drugs: the intermediates 1, 2, 3, 4, 5, and the compounds Mito-TP-1, Mito-TP-2, Mito-TP-3, Mito-TP-4, and Mito-TP-5, which were prepared in examples 1-5 of the present invention.

Cell culture: l-02 normal liver cell lines, Shanghai Qiaoxin Biotechnology, Inc.; HepG-2 human hepatoma cell line, college of medicine, Shandong university; MCF-7 human Thymus cell line, Shandong university college of medicine; RPMI-1640 culture solution, DMEM culture solution, fetal calf serum, trypsin containing 0.25% EDTA, MTT (tetramethyl azone), DMSO (dimethyl sulfoxide), penicillin-streptomycin solution (100x), 0.5% ciprofloxacin lactate sodium chloride injection and PBS (phosphate buffered saline).

The instrument comprises the following steps: an enzyme-labeling instrument: bio Ted, USA; a liquid transfer device: eppendorf, Germany; ultra-clean biological safety cabinet: thermo Fisher Scientific, usa; a carbon dioxide incubator: thermo Fisher Scientific, usa; and (3) sterilizing the pan: sanyo, japan; and (3) inverting the microscope: olympus, japan; minispin centrifuge: eppendorf, germany.

2. Experimental methods

For human liver cancer cells (HepG-2): placing human liver cancer cell (HepG-2) in DMEM culture solution containing 10% fetal calf serum, 1% streptomycin mixture (100X), 0.5% ciprofloxacin lactate and sodium chloride injection, and placing the cell in DMEM culture solution containing 5% CO₂Culturing in an incubator at 37 ℃ until the culture medium is paved on the bottom of a dish (d-60mm culture dish), sucking out the culture medium, washing with 1mL of PBS once, adding 500 mu L of DMEM containing EDTA and pancreatin for digestion for 3min, stopping digestion with 1mL of 10% FBS, transferring to a 1.5mL centrifuge tube, centrifuging at 1000rpm for 4min, discarding the supernatant, and taking 1mL of the culture medium for re-suspension to prepare cell suspension. 10 μ L of the cell suspension was taken and counted using a hemocytometer. Calculating the formula: cell count/ml primary cell suspension (total number of cells in four quadrants/4) dilution of cell suspension multiple 10⁴. Adding 300 μ L of cell suspension into 11mL of DMEM medium containing 10% FBS, and inoculating 100 μ L of cells into 96-well plate at a concentration of (8-10) × 10³/ml，5％CO₂And cultured overnight in an incubator at 37 ℃. After the cells are attached and grow to the logarithmic growth phase, the drugs are diluted by 2% FBS DMEM medium, the cells are treated by the drugs with different concentrations, meanwhile, a control group (blank control group: 2% FBS DMEM medium added with 0.1% DMSO) is set up, each well is 100 mu L, and the drug action time is 48 h. After the drug action time is over, 1mL of MTT is added into 9mL of serum-free DMEM medium, the DMEM medium containing 2% FBS of the drug is aspirated, 100 mu L of the drug is added into each well, and 5% CO is added into each well₂And culturing for 4 hours in an incubator at 37 ℃. After the MTT incubation is finished, the DMEM medium containing MTT is absorbed and removed, 100 mu L DMSO is added into each well, the plate is shaken for 1min, and the absorbance (OD value) is measured at 570nm of an enzyme-labeling instrument.

Human hepatic normal cell L-02: placing normal human liver cells (L-02) in a mixture containing 10% fetal calf serum, 1% streptomycin (100X), and 0.5% lactic acidIn RPMI-1640 culture solution of ciprofloxacin sodium chloride injection, cells are placed in a medium containing 5% CO₂Culturing in an incubator at 37 ℃ until the culture medium is paved on the bottom of a dish (d-60mm culture dish), sucking out the culture medium, washing with 1mL PBS once, adding 500 mu L of RPMI-1640 culture medium containing EDTA and pancreatin for digestion for 2min and 1mL of 10% FBS to stop digestion, transferring to a 1.5mL centrifuge tube, centrifuging at 1000rpm for 4min, discarding the supernatant, and taking 1mL of culture medium to resuspend to prepare cell suspension. 10 μ L of the cell suspension was taken and counted using a hemocytometer. Calculating the formula: cell count/ml primary cell suspension (total number of cells in four quadrants/4) dilution of cell suspension multiple 10⁴. Adding 300 μ L of cell suspension into 11mL of RPMI-1640 medium containing 10% FBS, and inoculating cells into 96-well plate at a concentration of (8-10) × 10 (100 μ L/well)³/ml，5％CO₂And cultured overnight in an incubator at 37 ℃. After the cells are attached and grow to the logarithmic growth phase, the drug is diluted by 2% FBS RPMI-1640 culture medium, the cells are treated by the drug with different concentrations, and a control group (blank control group: 2% FBS RPMI-1640 culture medium added with 0.1% DMSO) is set up at the same time, each well is 100 muL, and the drug action time is 48 h. After the drug action time is over, 1mL of MTT is added into 9mL of serum-free RPMI-1640 medium, and the RPMI-1640 medium containing 2% FBS is aspirated, wherein each well contains 100 mu L of 5% CO₂And culturing for 4 hours in an incubator at 37 ℃. After the incubation of MTT, the medium containing MTT was aspirated, 100. mu.L of DMSO was added to each well, the plate was shaken for 1min, and the absorbance (OD value) was measured at 570nm using a microplate reader.

For human breast tumor cells (MCF-7): the operation steps are the same as those of normal human liver cells (L-02).

All results are in accordance with the formula: lgIC₅₀＝Xm-I*[sigP-(3-Pm-Pn)/4]Calculate the IC₅₀Wherein Xm is the logarithm of the highest concentration of the drug; sigP is the sum of all inhibition rates; lg (highest concentration of drug/concentration of drug adjacent to the highest concentration); pm is the maximum killing rate; pn is the minimum kill rate.

3. Results of the experiment

IC of compound Mito-TP-1, compound Mito-TP-2, compound Mito-TP-3, compound Mito-TP-4, compound Mito-TP-5 and triptolide₅₀(μ M) results of the comparison are shown in Table 1.

TABLE 1 triptolide derivatives and triptolide IC₅₀(μ M) comparison

As can be seen from table 1: compared with triptolide, the triptolide derivative prepared by the invention has equivalent inhibitory activity on liver cancer cells and breast cancer cells, and has better inhibitory activity compared with doxorubicin hydrochloride; the intermediate compounds 1-5 of the triptolide derivative also show inhibitory activity on liver cancer cells and breast cancer cells, but the toxicity of Mito-TP-1-Mito-TP-5 on normal cells is generally reduced compared with triptolide, wherein the toxicity of the compounds Mito-TP-1, Mito-TP-2, Mito-TP-3, Mito-TP-4 and Mito-TP-5 on normal liver cells L-02 is reduced by about 827 times, 16 times, 15 times, 6 times and 20 times respectively, while the intermediate compounds 1-5 do not have obvious attenuation activity and have equivalent toxic effect with the triptolide.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A triptolide derivative has a structure shown in formula I:

wherein n is an integer of 4 to 10.

2. The triptolide derivative according to claim 1, wherein the compound is selected from the following structures:

3. a method for preparing the triptolide derivative of claim 1 or 2, comprising: taking triptolide TP as a starting raw material, carrying out esterification reaction on C-14 beta-OH of triptolide to obtain an intermediate compound, and then carrying out salt forming reaction on the intermediate compound and triphenylphosphine to generate the triptolide derivative shown in the formula I;

wherein n is an integer of 4 to 10.

4. The process according to claim 3, characterized in that it is carried out by the following reaction scheme:

wherein n is an integer of 4 to 10.

5. The process of claim 3, wherein the condensation agent for the esterification reaction is selected from one or more of DCC, DIC and EDCI; the catalyst for the esterification reaction is one or more selected from DMAP, TEA and DIPEA.

6. The method according to claim 3, wherein the esterification reaction is performed by EDCI/DMAP method, the solvent of the esterification reaction is one or more selected from chloroform, N-dimethylformamide and dichloromethane, and the reaction temperature is room temperature.

7. The method according to claim 3, wherein the solvent for salt forming reaction is selected from one or more of toluene, N-dimethylformamide, N-butanol and acetonitrile.

8. The process of claim 3, wherein the salt formation reaction is carried out under heating reflux conditions at 80-85 ℃.

9. A pharmaceutical composition comprising the triptolide derivative of claim 1 or 2.

10. A pharmaceutical formulation comprising the triptolide derivative of claim 1 or 2 and at least one pharmaceutically acceptable carrier or adjuvant.

11. A mitochondrially targeted drug carrier comprising the triptolide derivative of claim 1 or 2.

12. A delivery system comprising the triptolide derivative of claim 1 or 2 or the mitochondrially-targeted pharmaceutical carrier of claim 11.

13. Use of the triptolide derivative of claim 1 or 2, or the pharmaceutical composition of claim 9, or the pharmaceutical formulation of claim 10, or the mitochondrially-targeted pharmaceutical carrier of claim 11, or the delivery system of claim 12 for the preparation of an anti-tumor medicament.

14. The use of claim 13, wherein the tumor is liver cancer or breast cancer.