CN114478684A

CN114478684A - Triptolide prodrug, preparation method and medical application thereof

Info

Publication number: CN114478684A
Application number: CN202210072229.0A
Authority: CN
Inventors: 胡立宏; 王均伟; 康迪; 朱学军; 潘祥; 宋祎; 刘琰
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-05-13
Anticipated expiration: 2042-01-21
Also published as: CN114478684B; WO2023138147A1

Abstract

The invention discloses triptolide prodrugs, a preparation method and medical application thereof. Belongs to the field of pharmaceutical chemistry; the preparation steps are as follows: 1. triptolide reacts with acyl chloride under the action of DMAP to generate an intermediate II; 2. the intermediate II reacts with a carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate a compound III; 3. salifying the compound III with acid to obtain a target compound I. The invention has better water solubility and pharmacokinetic properties, has higher transformation speed in vivo blood environment and higher efficient synthesis method compared with Minnelide, and has better treatment effect on malignant tumor, inflammatory disease, autoimmune disease and the like; in addition; because of its good water solubility, it is not only effective for oral administration, but also can be made into injection for use.

Description

Triptolide prodrug, preparation method and medical application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and relates to triptolide water-soluble prodrugs or pharmaceutically acceptable salts thereof, a pharmaceutical composition containing the compounds, a preparation method and medical application thereof.

Background

Tripterygium wilfordii hook.f. is a plant of Tripterygium genus of Celastraceae family, has the effects of dispelling wind and removing dampness, promoting blood circulation and removing obstruction in collaterals, relieving swelling and pain, killing insects and removing toxicity and the like, and is a preferred traditional Chinese medicine for clinically treating autoimmune diseases. At present, various tripterygium wilfordii extractive drugs such as tripterygium wilfordii polyglycoside tablets, tripterygium wilfordii tablets (999), tripterygium wilfordii double-layer tablets, tripterygium wilfordii total terpene tablets and the like are marketed and applied to the treatment of various immune and inflammatory diseases such as rheumatoid arthritis, autoimmune hepatitis, nephritis, nephrotic syndrome and the like. The main active ingredients of the radix Tripterygii Wilfordii extract comprise diterpenes, triterpenes and alkaloids, wherein triptolide in diterpenes is one of the main active ingredients of radix Tripterygii Wilfordii, and is also the main active ingredient of preparations such as radix Tripterygii Wilfordii polyglycoside tablet and radix Tripterygii Wilfordii tablet.

Triptolide, one of the highly active components of tripterygium wilfordii, has strong pharmacological actions such as immunosuppression, anti-inflammation, and anti-tumor [ J Am Chem Soc,1972,94(20): 7194-; drugs R D,2003,4(1): 1-18; trends Pharmacol Sci,2019,40(5):327-341 ]. Since 1969, the traditional Chinese medicine tripterygium wilfordii has been widely used for treating rheumatoid arthritis, and a double-blind clinical study of the american college of rheumatology also shows that tripterygium wilfordii can obviously improve the symptoms of rheumatoid arthritis, and the effective rate reaches 58% [ J Rheumatol 2003, 30 (3): 465-. Tripterygium wilfordii shows satisfactory effects for other autoimmune and inflammatory diseases, such as systemic lupus erythematosus, psoriasis, ankylosing spondylitis, asthma, nephritis, ulcerative colitis, pulmonary fibrosis, etc. [ chi J Mod Appl Pharm, 1999,16 (2): 10-13; br J Clin Pharmacol 2012 Sep; 74(3) 424-36; rheum Dis Clin North Am 2000; am J Pathol 2001; 158(3) 997-; 26(1):29-50 ]. In view of the immunosuppressive effects of tripterygium wilfordii, many researchers find that triptolide can effectively prevent rejection caused by transplanted organs after heart, kidney, liver or bone marrow Transplantation, and significantly prolong the survival time of animals [ Transplantation 2000; 70, (3) 447-55; transplant Proc 1999; 31(7): 2719-23). In the cancer field, triptolide has broad-spectrum antitumor activity by controlling the overall transcription level of cells through covalent binding and inhibition of XPB activity in TFHII transcription complex [ Angew Chem Int Ed Engl,2015,54(6): 1859) -1863; mol Cancer Ther,2003,2: 65-72 ]. In addition, triptolide exhibits potent anti-HIV effects in vitro [ J Nat Prod 2000; 357-61, and several clinical realizations are ongoing [ NCT 03403569; NCT 01817283; NCT 02002286. However, their structure has poor water solubility, which limits their clinical applications. Therefore, the water-soluble prodrug of triptolide can be designed and synthesized by introducing the water-soluble group, so that the water solubility of the triptolide is improved and the drug property of the triptolide is improved under the condition of not influencing the drug effect of the triptolide.

The design strategy of the water-soluble prodrug is mainly to connect some water-soluble groups at the C-14 position of the triptolide through ester bonds or acetals. For example, PG490-88 is a prodrug of triptolide, which is the first triptolide prodrug to be clinically studied, by introducing a water-soluble carboxylic acid directly through an ester bond at the C-14 position of triptolide [ US5663335 ]. Although the toxic side effects of most patients can be controlled in clinical trials, two patients have fatal side effects, one of which dies at a dose of 12mg/kg from neutropenic sepsis and the other of which dies at a dose of 18mg/kg from complex clinical syndromes. Subsequent pharmacokinetic experiments show that PG490-88 cannot be rapidly transformed into triptolide in vivo, and the triptolide has great difference in different species (including mice, monkeys and humans), and also shows 2-3 times of transformation difference in the same 18 volunteers. The reason for this is probably that PG490-88 has a large steric hindrance at C-14 position, which prevents esterase from hydrolyzing ester bonds, resulting in slow and incomplete conversion of PG490-88 in vivo. Furthermore, the safe dosage of the drug is difficult to control due to differences in esterase activity between individuals. Therefore, the phase I clinical trial of PG490-88 was also forced to end in 2009.

In order to overcome the problem of incomplete conversion of PG490-88 in vivo, researchers have attempted to optimize the C-14 linkage of PG490-88 to avoid steric hindrance effects on bond scission. In 2010, Georg et al used methylal as a linking arm to introduce a phosphate group to synthesize a water-soluble triptolide prodrug Minnelide [ WO2010/129918 ]. Because the steric hindrance beside the phosphoester bond is small, Minnelide is easily hydrolyzed by phosphatase to generate a hydroxymethyl intermediate, and the structure of the intermediate hydroxymethyl is unstable, so that the hydroxyl at the C14 position can be released by automatic hydrolysis. Therefore, Minnelide showed better pharmacokinetic properties in phase I clinical trial and entered phase II clinical trial.

Although the structure of methylal is easy to be broken by hydrolysis, in vitro human plasma conversion experiments, the Minnelide is found to be completely converted into triptolide for more than 24 hours, and the Minnelide can not be related to high content of alkaline phosphatase in plasma. In addition, because the triptolide raw material is expensive, the synthesis difficulty of Minnelide is higher, and the total yield is lower (39%), so that the development cost is higher; and the reaction conditions are harsh, so that the method is not beneficial to large-scale preparation. Therefore, the triptolide prodrug which has good water solubility, can be quickly and completely converted and has simple synthesis process has important market prospect.

The carboxylesterase is a hydrolase which is most abundant in human and animal bodies and participates in the metabolic processes of various endogenous and exogenous compounds and medicines, and a water-soluble aliphatic nitrogenous heterocycle is introduced at the C-14 position of triptolide in a connection mode of hydroxy acid ester (a structure which is easy to be hydrolyzed by esterase), so that a triptolide water-soluble prodrug which is good in water solubility, rapid in-vivo conversion and easy to synthesize (the total yield reaches 61%) is discovered. The prodrug has good water solubility and effective oral administration, can be completely converted into triptolide in human plasma within 1 hour, and is suitable for effectively treating various inflammatory diseases, immune diseases, hematological malignant tumors and solid tumors of triptolide.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a triptolide prodrug or pharmaceutically acceptable salt thereof, which is shown in a general formula (I) and has remarkably improved water solubility and can be quickly converted in blood plasma.

The technical scheme is as follows: the triptolide prodrug of the invention, or pharmaceutically acceptable salt, polymorph or solvate thereof, is introduced into a fatty nitrogen-containing heterocycle through a hydroxy acid ester connecting arm which is easy to self-degrade; the chemical structural formula of the derivative is shown as the formula (I):

wherein:

n1 is 1 to 6;

n2 is 1 to 6;

n3 is 0 or 1;

x is a carbon, nitrogen or oxygen atom;

HA is selected from hydrochloric acid, sulphuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulphonic acid, benzenesulphonic acid, p-toluenesulphonic acid or ferulic acid;

typical preferred compounds of the invention are as follows (the triptolide prodrug is selected from the following compounds), but are not limited to:

the invention also aims to provide a preparation method for preparing the triptolide prodrug or pharmaceutically acceptable salt, polymorph or solvate thereof; the preparation method comprises the following specific steps:

the first step is as follows: triptolide reacts with acyl chloride under the action of DMAP to generate an intermediate II;

dissolving triptolide and DMAP in anhydrous dichloromethane, adding acyl chloride at the low temperature of-10-5 ℃, and then placing the mixture at the temperature of 0-30 ℃ for reaction for 6-12 hours; washing the obtained reaction solution with dilute hydrochloric acid, saturated sodium bicarbonate and brine in sequence, and drying with anhydrous sodium sulfate; carrying out suction filtration, concentrating the filtrate, and carrying out column chromatography purification to obtain an intermediate II;

the second step is that: the intermediate II reacts with a corresponding carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate a compound III;

dissolving the purified intermediate II in anhydrous DMF, adding sodium iodide and carboxylic acid, reacting for 0.5-1 hour, adding potassium carbonate, heating to 40-70 ℃, and reacting for 3-12 hours to obtain a reaction solution; then pouring the reaction liquid into water, extracting with ethyl acetate, washing with sodium bicarbonate water solution and saline solution, drying, carrying out suction filtration, concentrating the filtrate, and carrying out column chromatography purification to obtain a compound III;

the third step: salifying the compound III with corresponding acid to obtain a target compound I;

and dissolving the purified compound III in ethyl acetate, adding an inorganic acid or an organic acid, reacting at the temperature of 0-30 ℃ for 6-12 hours, carrying out suction filtration, and drying to obtain the target compound I.

The invention aims to provide a pharmaceutical composition, which comprises the triptolide prodrug, pharmaceutically acceptable salt, polymorph or solvate thereof, and at least one pharmaceutically acceptable carrier, additive, auxiliary agent or excipient.

The invention also aims to provide the triptolide prodrug or pharmaceutically acceptable salt and application of a pharmaceutical composition thereof in preparing antitumor drugs.

The triptolide prodrug provided by the invention can be completely converted into triptolide in human plasma, so that the triptolide prodrug or pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof can be used as a single therapeutic agent or used in combination with other antitumor drugs, and are used for treating various malignant tumors in which the triptolide is effective, specifically various malignant tumors such as acute myeloid leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma, neuroblastoma and the like.

The other purpose of the invention is to provide the triptolide prodrug or pharmaceutically acceptable salt and the application of the pharmaceutical composition thereof in preparing drugs for treating acute myeloid leukemia; in-vitro anti-tumor activity experiments show that the compound can obviously inhibit the proliferation of acute myeloid leukemia cells; the whole animal experiment shows that the compound has good curative effect on acute myeloid leukemia, the effective dose is only 25 mug/kg, and the compound has synergistic effect when combined with FLT3 inhibitor; thus, the compounds of the present invention, or pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, may be used as monotherapies or in combination with FLT3 inhibitors for the treatment of acute myeloid leukemia.

The other purpose of the invention is to provide the triptolide prodrug or the pharmaceutically acceptable salt and the application of the pharmaceutical composition thereof in preparing anti-inflammatory, immunosuppressive and virus (such as anti-HIV) drugs; the triptolide prodrug can be completely converted into triptolide in human plasma; therefore, the triptolide prodrug or the pharmaceutically acceptable salt thereof and the pharmaceutical composition thereof provided by the invention can be used for treating various indications with triptolide, including rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, systemic vasculitis, psoriasis, idiopathic dermatitis, inflammatory bowel disease, asthma, pulmonary fibrosis, nephritis, nephrotic syndrome, immune rejection, and cytokine release syndrome caused by LPS induction, CAR-T therapy, bacterial infection, viral infection and the like.

Has the advantages that: compared with the prior art, the invention has the characteristics that: the triptolide prodrug disclosed by the invention has obvious in-vivo antitumor and immunosuppressive activities, has better water solubility and pharmacokinetic properties compared with triptolide, and has the advantages of rapider in-vivo conversion, simpler synthesis, higher yield (61%), lower cost and good industrial prospect compared with Minnelide.

Drawings

FIG. 1 is a flow chart of the preparation of the present invention;

FIG. 2 is a schematic diagram of experiments of the in vitro transformation of TP-P1, TP-P2 and TP-P5 in rat plasma in example 11 of the present invention;

FIG. 3 is a schematic diagram of experiments of in vitro transformation of TP-P1 and TP-P5 in human plasma in example 12 of the present invention;

FIG. 4 is a schematic diagram of the experiment of in vitro transformation of TP-P1, PG490-88Na, Minnelide in rat plasma in example 13 of the present invention;

FIG. 5 is a schematic diagram of the experiment of in vitro transformation of TP-P1, PG490-88Na, Minnelide in human plasma in example 14 of the present invention;

FIG. 6 is a schematic diagram of the in vitro transformation experiment of different concentrations of TP-P1 in human plasma in example 15 of the present invention.

Detailed Description

In order to more clearly illustrate the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings:

example 1

Synthesis of Compound TP-P1

Synthesis of intermediate II-1: dissolving triptolide TP (1.8g,5.0mmol) in 50mL of anhydrous dichloromethane, adding DMAP (3.05g,25.0mmol) at 0 ℃, then dropwise adding chloroacetyl chloride (4.0mL,50.00mmol), and reacting at 25 ℃ for 12 hours after dropwise adding; after the reaction is finished, washing the reaction solution by using 5% of dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride solution in sequence, and then drying by using anhydrous sodium sulfate; the filtrate was filtered under suction, concentrated, and purified by column chromatography (dichloromethane: methanol: 200:1 to 100:1) to obtain 1.77g of a white solid with a yield of 88.8%.¹H NMR(500MHz,MeOD)δ(ppm): 5.14(1H,s),4.78-4.85(2H,m),4.31(2H,d,J＝1.0Hz),3.99(1H,d,J＝3.2Hz),3.67(1H,d,J＝ 2.6Hz),3.53(1H,d,J＝5.7Hz),2.79-2.81(1H,m),2.24-2.32(2H,m),2.07-2.12(1H,m), 1.88-1.98(2H,m),1.50-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.98(3H,d,J＝7.0Hz), 0.86(3H,d,J＝6.9Hz).

Synthesis of intermediate III-1: compound II-1(1.0g,2.3mmol) was dissolved in 50mL anhydrous DMF and sodium iodide (860mg,4.6mmol) and morpholin-4-ylacetic acid (670mg,4.6mmol) were added; after 40 minutes reaction at room temperature, potassium carbonate (320mg,2.3mmol) was added, followed by heating to 50 ℃ for 4 hours reaction; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-50: 1) to obtain 1.01g of white solid with the yield of 80.4%;¹H NMR(500MHz,MeOD)δ(ppm):5.13(1H,s),4.82-4.85(2H,m),4.77-4.79(2H,m), 3.99(1H,d,J＝3.2Hz),3.72-3.75(4H,m),3.65(1H,d,J＝2.5Hz),3.51(1H,d,J＝2.5Hz), 3.43(2H,s),2.79-2.81(1H,m),2.63-2.67(4H,m),2.23-2.32(2H,m),2.04-2.10(1H,m), 1.88-1.98(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.98(3H,d,J＝10.7Hz), 0.86(3H,d,J＝6.9Hz).

synthesis of Compound TP-P1: compound III-1(1.0g,1.83mmol) was dissolved in 20mL of ethyl acetate, and 20mL of a saturated ethyl acetate solution of hydrogen chloride was added to the solution to react at room temperature for 6 hours; after the reaction was completed, suction filtration was performed, and the filter cake was washed with ethyl acetate and then vacuum-dried to obtain 0.91g of a white solid with a yield of 85.3%.¹H NMR(500MHz,DMSO-d₆)δ (ppm):10.93(1H,s),5.46(1H,s),4.88-4.92(2H,m),4.77-4.84(2H,m),4.59(1H,m),4.37(2H, s),3.88(1H,d,J＝4.9Hz),3.72-3.85(4H,m),3.56(1H,d,J＝5.5Hz),3.05-3.30(4H,m), 2.64-2.74(1H,m),2.15-2.24(2H,m),1.95-1.98(2H,m),1.80-1.88(1H,m),1.36-1.41(1H,m), 1.25-1.32(1H,m),0.93(3H,d,J＝6.5Hz),0.90(3H,s),0.78(3H,d,J＝6.6Hz).¹³C NMR(126 MHz,DMSO-d₆)δ(ppm):173.6,166.6,166.5,162.5,123.7,75.5,75.4(2C),70.8,66.8,62.5, 61.8,59.5(2C),58.1,57.4,52.3(2C),35.4,34.6,30.4,29.2,22.5,17.1,16.3,15.9(2C),14.6. LCMS(ESI):m/z[M+H]⁺calcd for C₂₈H₃₇ClNO₁₀ ⁺,582.2；found,582.0.

Example 2

Synthesis of Compound TP-P2

Synthesis of intermediate III-2: compound II-1(100mg,0.23mmol) was dissolved in 10mL anhydrous DMF and sodium iodide (86mg,0.46mmol) and 4-methyl-1-piperazineacetic acid (73mg,0.46mmol) were added; after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-40: 1) to obtain 93mg of white solid with the yield of 72.3%;¹H NMR(500MHz,MeOD-d₆)δ(ppm):5.12(1H,s),4.81-4.85(2H,m), 4.77-4.80(2H,m),3.98(1H,d,J＝3.1Hz),3.65(1H,d,J＝2.8Hz),3.50(1H,d,J＝5.7Hz), 3.47(2H,s),2.79-2.83(1H,m),2.66-2.78(8H,m),2.40(3H,s),2.26-2.32(2H,m),2.04-2.10(1H, m),1.89-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.97(3H,d,J＝7.0 Hz),0.86(3H,d,J＝6.9Hz).

synthesis of Compound TP-P2: dissolving a compound III-2(100mg,0.18mmol) in 5mL of ethyl acetate, adding 3mL of saturated ethyl acetate solution of hydrogen chloride, reacting at room temperature for 6 hours, performing suction filtration after the reaction is finished, washing a filter cake with ethyl acetate, and then performing vacuum drying to obtain a white solid of 75mg with a yield of 70.4%;¹H NMR(500MHz,MeOD)δ(ppm): 4.83-4.86(2H,m),4.82(2H,s),4.74(1H,s),4.32(1H,d,J＝5.3Hz),3.95(1H,d,J＝5.4Hz), 3.84-3.93(2H,m),3.59-3.71(2H,m),3.53(1H,d,J＝6.1Hz),3.37-3.47(2H,m),3.12-3.28(4H, m),2.96(3H,s),2.82-2.85(1H,m),2.27-2.33(2H,m),2.08-2.14(2H,m),1.93-1.99(1H,m), 1.56-1.59(1H,m),1.35-1.41(1H,m),1.05(3H,s),1.01(3H,d,J＝6.8Hz),0.88(3H,d,J＝6.9 Hz).¹³C NMR(126MHz,MeOD)δ(ppm):174.6,168.4,166.9,162.3,124.3,75.4,75.2,70.7, 67.0,62.2,60.5,59.3,58.0,56.8,56.0,52.6(2C),48.9(2C),42.1,39.6,35.3,30.3,28.9,22.4, 16.5,14.8,14.4,13.2.LCMS(ESI):m/z[M+H]⁺calcd for C₂₉H₄₀ClN₂O₉ ⁺,595.2；found,595.2.

example 3

Synthesis of Compound TP-P3

Synthesis of Compound TP-P3: compound II-1(100mg,0.23mmol) was dissolved in 10mL anhydrous DMF and sodium iodide (86mg,0.46mmol) and 2-piperidinylacetic acid (66mg,0.46mmol) were added; after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-40: 1) to obtain 67mg of a white solid with a yield of 53.4%;¹H NMR(500MHz,CDCl₃)δ(ppm):5.11(1H,s),4.81-4.85(2H,m),4.65-4.73(2H, m),3.84(1H,d,J＝3.1Hz),3.64(2H,d,J＝4.2Hz),3.56(1H,d,J＝2.8Hz),3.48(1H,d,J＝ 5.7Hz),2.87-2.98(4H,m),2.32-2.36(1H,m),2.16-2.26(2H,m),1.89-1.95(2H,m),1.76-1.80 (4H,m),1.52-1.59(2H,m),1.32-1.38(2H,m),1.06(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d, J＝6.9Hz).¹³C NMR(126MHz,CDCl₃)δ(ppm):173.2,168.0,167.0,159.9,125.7,72.4,69.9, 63.5,63.1,61.3,60.8,59.4,57.9,55.4,55.0,53.4(2C),40.3,35.7,29.9,28.0,24.6(2C),23.4, 23.0,17.5,17.1,16.7,13.8.LCMS(ESI):m/z[M+H]⁺calcd for C₂₉H₃₈NO₉ ⁺,544.2；found,544.3.

example 4

Synthesis of Compound TP-P4

Synthesis of Compound TP-P4: compound II-1(100mg,0.23mmol) was dissolved in anhydrous 10mL DMF and sodium iodide (86mg,0.46mmol) and 2- (pyrrolidin-1-yl) acetic acid (59mg,0.46mmol) were added; after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, adding the reaction solution into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-40: 1) to obtain 64mg of white solid with the yield of 52.3%;¹H NMR(500MHz,MeOD)δ(ppm):5.14(1H,m),4.81-4.85(2H,m), 3.98-4.03(2H,m),3.73(1H,m),3.66(1H,d,J＝7.3Hz),3.51(1H,d,J＝5.7Hz),3.27(2H,s), 3.14-3.18(1H,m),2.79-2.81(1H,m),2.59-2.62(1H,m)2.22-2.32(2H,m),2.02-2.09(4H,m), 1.85-1.92(2H,m),1.49-1.53(1H,m),1.35-1.42(4H,m),1.05(3H,s),0.97(3H,d,J＝7.0Hz), 0.86(3H,d,J＝6.9Hz).¹³C NMR(126MHz,MeOD)δ(ppm):174.6,172.4,162.4,161.9,124.1, 71.7,70.6,65.9,63.5,62.7,61.4,59.6,59.5,55.4,54.8,54.3,52.9,40.0,35.4,29.4,28.2,22.8 (2C),22.7,16.5(2C),15.7,12.7.LCMS(ESI):m/z[M+H]⁺calcd for C₂₈H₃₆NO₉ ⁺,530.2；found, 530.2.

example 5

Synthesis of Compound TP-P5

Synthesis of intermediate II-5: dissolving triptolide (180mg,0.50mmol) in 20mL anhydrous dichloromethaneDMAP (305mg,2.50mmol) was added at 0 ℃ and 4-bromobutyryl chloride (0.56mL,5.00 mmol) was added dropwise, after which the reaction was carried out at 25 ℃ for 12 hours; after the reaction is finished, washing the reaction solution by using 5% of dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride solution in sequence, and then drying by using anhydrous sodium sulfate; the filtrate was filtered under suction, concentrated, and purified by column chromatography (dichloromethane: methanol: 200:1 to 100:1) to obtain 193mg of a white solid with a yield of 75.6%.¹H NMR(500MHz, CDCl₃)δ(ppm):5.11(1H,s),4.65-4.73(2H,m),4.33(1H,t,J＝7.1Hz),3.85(1H,d,J＝2.9Hz), 3.52-3.55(2H,m),3.49(1H,d,J＝5.6Hz),2.60-2.72(4H,m),2.51(1H,t,J＝8.1Hz),2.24-2.30 (3H,m),1.89-1.98(2H,m),1.57-1.62(1H,m),1.26-1.35(1H,m),1.07(3H,s),0.98(3H,d,J＝ 6.9Hz),0.87(3H,d,J＝6.9Hz).

Synthesis of intermediate III-5: compound II-5(117mg,0.23mmol) was dissolved in 10mL anhydrous DMF and sodium iodide (86mg,0.46mmol) and morpholin-4-ylacetic acid (67mg,0.46mmol) were added; after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-50: 1) to obtain 83mg of white solid with the yield of 63.2%;¹H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.24(2H,t,J ＝6.4Hz),3.98(1H,d,J＝3.2Hz),3.73(4H,t,J＝4.7Hz),3.65(1H,d,J＝2.8Hz),3.50(1H,d, J＝5.7Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J＝4.6Hz),2.46-2.57(2H,m),2.24-2.32 (2H,m),2.09-2.12(1H,m),2.01-2.06(2H,m),1.86-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39 (1H,m),1.05(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).

synthesis of Compound TP-P5: dissolving compound III-5(100mg,0.17mmol) in 5mL of ethyl acetate, adding 3mL of saturated ethyl acetate solution of hydrogen chloride, and reacting at 25 ℃ for 6 hours; after the reaction was completed, suction filtration was performed, and the filter cake was washed with ethyl acetate and then dried in vacuo to obtain 53mg of a white solid with a yield of 51.3%. mp: 239-.¹H NMR (500MHz,MeOD)δ(ppm):4.81-4.85(2H,m),4.73(1H,s),4.37-4.39(2H,m),4.33(1H,m), 4.14(2H,s),3.89-4.03(4H,m),3.55(1H,d,J＝4.9Hz),3.34(1H,d,J＝5.5Hz),2.83-2.86(1H, m),2.48-2.58(2H,m),2.28-2.32(2H,m),2.06-2.11(4H,m),1.94-2.04(2H,m),1.53-1.62(1H, m),1.32-1.40(3H,m),1.25-1.28(1H,m),1.05(3H,s),1.01(3H,d,J＝6.0Hz),0.90(3H,d,J＝ 6.0Hz).¹³C NMR(126MHz,MeOD)δ(ppm):174.7,171.9,165.9,162.4,124.2,75.4,74.1,70.7, 66.9,65.1,63.7(2C),62.3,59.7,57.9,56.9,55.9,52.6(2C),39.6,35.3,30.3,30.2,29.1,23.5, 22.4,16.6,14.8,14.4,13.1.LCMS(ESI):m/z[M+H]+calcd for C₃₀H₄₁ClNO₁₀ ⁺,610.2；found, 610.2.

Example 6

Synthesis of Compound TP-P6

Synthesis of intermediate II-6: dissolving triptolide (180mg,0.50mmol) in 20mL of anhydrous dichloromethane, adding DMAP (305mg,2.50mmol) at 0 ℃, then dropwise adding 5-bromovaleryl chloride (0.70mL,5.00mmol), and reacting at 25 ℃ for 12 hours after dropwise adding; after the reaction is finished, washing the reaction solution by using 5% of dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride solution in sequence, and then drying by using anhydrous sodium sulfate; the filtrate was filtered with suction, concentrated, and purified by column chromatography (dichloromethane: methanol: 200:1 to 100:1) to obtain 205mg of a white solid in a yield of 78.4%.¹H NMR(500MHz,CDCl₃)δ (ppm):5.10(1H,s),4.65-4.73(2H,m),3.84(1H,d,J＝3.2Hz),3.55(1H,d,J＝3.0Hz), 3.47-3.49(1H,m),3.43(2H,t,J＝6.6Hz),2.69-2.72(1H,m),2.50-2.58(1H,m),2.42(2H,t,J＝ 7,4Hz),2.31-2.36(1H,m),2.11-2.21(2H,m),1.98-2.01(1H,m),1.90-1.96(2H,m),1.78-1.84 (2H,m),1.57-1.60(1H,m),1.22-1.29(1H,m),1.07(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d, J＝6.9Hz).

Synthesis of Compound TP-P6: compound II-6(120mg,0.23mmol) was dissolved in 10mL anhydrous DMF and sodium iodide (86mg,0.46mmol) and morpholin-4-ylacetic acid (67mg,0.46mmol) were added; after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-50: 1) to obtain 88mg of white solid with the yield of 65.1%;¹H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.19 (2H,t,J＝5.5Hz),3.98(1H,d,J＝3.0Hz),3.73(4H,t,J＝4.5Hz),3.65(1H,d,J＝3.0Hz), 3.50(1H,d,J＝5.6Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J＝4.6Hz),2.48-2.54(1H, m),2.39-2.45(1H,m),2.26-2.31(2H,m),2.05-2.11(1H,m),1.85-1.99(2H,m),1.76-1.79(2H, m),1.50-1.54(1H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J＝7.0Hz),0.86(3H,d,J＝ 6.9Hz).¹³C NMR(126MHz,MeOD)δ(ppm):174.6,172.7,170.2,162.4,124.1,71.3,70.6,66.2 (2C),64.0,63.5,62.8,61.3,59.7,58.6,55.3,54.8,52.9(2C),40.1,35.4,33.2,29.4,28.3,27.5, 22.8,21.3,16.6,16.5,15.7,12.8.LCMS(ESI):m/z[M+H]+calcd for C₃₁H₄₂NO₁₀ ⁺,588.3；found, 588.3.

example 7

Synthesis of Compound TP-P7

Synthesis of intermediate II-7: triptolide (180mg,0.50mmol) was dissolved in 20mL anhydrous dichloromethane inDMAP (305mg,2.50mmol) was added at 0 ℃ followed by dropwise addition of 6-bromohexanoyl chloride (0.77mL,5.00mmol) and reaction at 25 ℃ for 12 hours after completion of the dropwise addition; after the reaction is finished, washing the reaction solution by using 5% of dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride solution in sequence, and then drying by using anhydrous sodium sulfate; the filtrate was filtered, concentrated, and purified by column chromatography (dichloromethane: methanol: 200:1 to 100:1) to obtain 191mg of a white solid in a yield of 71.2%.¹H NMR(500MHz,CDCl₃)δ (ppm):5.07(1H,s),4.63-4.71(2H,m),3.82(1H,d,J＝3.2Hz),3.53(1H,d,J＝2.5Hz),3.46 (1H,d,J＝6.1Hz),3.40(2H,t,J＝6.8Hz),2.66-2.69(1H,m),2.43-2.49(1H,m),2.36(2H,t,J＝ 7.4Hz),2.22-2.32(1H,m),2.12-2.19(1H,m),1.84-1.89(2H,m),1.68-1.73(2H,m),1.60-1.65 (2H,m),1.55-1.57(1H,m),1.45-1.51(2H,m),1.18-1.24(1H,m),1.04(3H,s),0.94(3H,d,J＝ 7.0Hz),0.82(3H,d,J＝6.9Hz).

Synthesis of Compound TP-P7: compound II-7(124mg,0.23mmol) was dissolved in 10mL anhydrous DMF and sodium iodide (86mg,0.46mmol) and morpholin-4-ylacetic acid (67mg,0.46mmol) were added. After reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-50: 1) to obtain 83mg of white solid with the yield of 60.1%;¹H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.16(2H,t,J ＝6.6Hz),3.98(1H,d,J＝3.0Hz),3.72(4H,t,J＝4.4Hz),3.64(1H,d,J＝3.0Hz),3.48(1H,d, J＝5.6Hz),3.28(2H,s),2.78-2.81(1H,m),2.62(4H,t,J＝4.4Hz),2.45-2.51(1H,m),2.35-2.42 (1H,m),2.24-2.32(2H,m),2.05-2.11(1H,m),1.82-1.95(2H,m),1.64-1.79(2H,m),1.45-1.57 (2H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).¹³C NMR(126MHz,MeOD)δ(ppm):174.6,172.9,170.1,162.4,124.1,71.3,70.6,66.1(2C),64.3, 63.5,62.8,61.3,59.7,58.6,55.3,54.8,52.9(2C),40.1,35.4,33.6,29.4,28.3,28.0,24.9,24.3, 22.8,16.6,16.5,15.8,12.9.LCMS(ESI):m/z[M+H]+calcd for C₃₂H₄₄NO₁₀ ⁺,602.3；found, 602.3.

example 8

Synthesis of Compound TP-P8

Synthesis of Compound TP-P8: dissolving compound II-1(100mg,0.23mmol) in 10mL anhydrous DMF, adding sodium iodide (86mg,0.46mmol) and 3- (4-morpholinyl) propionic acid (73mg,0.46 mmol); after reacting at 25 ℃ for 40 minutes, potassium carbonate (32mg,0.23mmol) was added, followed by heating to 50 ℃ for 4 hours; after the reaction is finished, pouring the reaction liquid into water, extracting by ethyl acetate, combining organic layers, washing by sodium bicarbonate aqueous solution and saturated sodium chloride solution in sequence, and drying by anhydrous sodium sulfate; performing suction filtration, concentrating the filtrate, and purifying by column chromatography (dichloromethane: methanol: 100: 1-50: 1) to obtain 84mg of white solid with the yield of 65.4%;¹H NMR(500MHz,CDCl₃)δ(ppm):5.10(1H,s),4.75-4.82(2H,m), 4.65-4.72(2H,m),3.87(4H,t,J＝4.7Hz),3.85(1H,d,J＝3.1Hz),3.56(1H,d,J＝2.7Hz),3.48 (1H,d,J＝5.7Hz),3.14(2H,t,J＝7.3Hz),2.90-2.92(4H,m),2.85-2.89(2H,m),2.69-2.72(1H, m),2.32-2.36(1H,m),2.16-2.21(2H,m),1.88-1.95(2H,m),1.55-1.59(1H,m),1.32-1.36(1H, m),1.07(3H,s),0.98(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).¹³C NMR(126MHz,CDCl₃)δ (ppm):170.5,167.2,165.0,159.8,125.7,72.3,70.0,65.1(2C),63.6,63.1,61.3,60.9,59.4,55.4, 55.0,52.6,52.3(2C),40.3,35.7,29.9,29.5,28.1,23.4,17.5,17.1,16.7,13.8.LCMS(ESI):m/z [M+H]+calcd for C₂₉H₃₈NO₁₀ ⁺,560.2；found,560.3.

example 9

Long term stability test of Compounds

The experimental method comprises the following steps: the compounds were left open at room temperature for more than 90 days and the purity of the compounds was determined by HPLC normalization (table 1).

Table 1 long-term stability test results of the compounds of the examples

The results show that: the series of compounds have good chemical stability, and can be placed in an open air at room temperature for 90 days and subjected to HPLC-UV¹The purity of the compound is not obviously reduced by H-NMR spectrum analysis.

Example 10

Stability test of Compound TP-P1 in pure Water and acidic aqueous solution

The experimental method comprises the following steps: compound TP-P1 was dissolved in pure water at pH7 and an acidic aqueous solution at pH4 and pH2, samples were taken at different time points, and the purity of the compound was determined by HPLC normalization (table 2).

TABLE 2 stability test results of TP-P1 Compound in pure Water and acidic aqueous solution

The results show that: the compound TP-P1 showed no significant change in purity in 6 hours in aqueous solution at pH7 and pH2, pH 4.

Example 11

Experiments on the in vitro transformation of TP-P1, TP-P2, TP-P5 in rat plasma

In consideration of the water solubility of the compounds, salt-forming compounds TP-P1, TP-P2 and TP-P5 with better water solubility are selected for carrying out in vitro plasma transformation experiments of rats.

The experimental method comprises the following steps: adding equal volume of 100 μ g/mL TP-P1, TP-P2 and TP-P5 aqueous solution into 250 μ L rat blank plasma, incubating at 37 ℃ for 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12 and 24h in a constant temperature oscillator, taking 20 μ L drug-containing plasma in precooled 60 μ L methanol, vortexing for 3min, centrifuging at 14000rpm/min for 10min at 4 ℃, and taking supernatant for HPLC analysis;

the liquid phase analysis method comprises the following steps: ACN:0.1％TFA-H₂O35: 65 isocratic elution, flow rate: 0.6mL/min

The liquid phase analysis method comprises the following steps: ACN 0.1% TFA-H₂O35: 65 isocratic elution, flow rate: 0.6mL/min

The experimental results are shown in figure 2;

the results show that: three prodrugs TP-P1, TP-P2 and TP-P5 were all converted to TP completely within 30min in rat plasma, but at the same time, TP-P1 had been converted 52.3% at 15min, while TP-P2 and TP-P5 were less than 50%, so the efficiency of TP-P1 prodrug conversion to TP was slightly higher than TP-P2 and TP-P5.

Example 12

In vitro transformation experiments of TP-P1 and TP-P5 in human plasma

Since compounds TP-P1 and TP-P2 both use a glycolic acid linkage, and the conversion rate of TP-P1 is better than that of TP-P5 in the same time, TP-P1 and TP-P5 are selected for human in vitro plasma conversion experiments.

The experimental method comprises the following steps: adding equal volume of TP-P1 or TP-P5 aqueous solution (100 mu g/mL or 2 mg/mL) into 250 mu L of human blank plasma, incubating at 37 ℃ for 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10 and 24h in a constant temperature oscillator, taking 20 mu L of drug-containing plasma in precooled 60 mu L of methanol, whirling for 3min, 4 ℃, 14000rpm/min, centrifuging for 10min, and taking supernatant for HPLC analysis.

The experimental results are shown in fig. 3;

the results show that: TP-P1 takes 45min to convert completely to TP at low concentration, and 60min to convert completely to TP at high concentration; the TP-P5 needs 45min to be completely converted into TP at low concentration, and the TP is completely converted into TP at high concentration for 90 min; the influence of the drug concentration on the plasma conversion is shown, and the conversion rate is higher at low concentration. TP-P5 compared with TP-P1, it took 45min to convert completely to TP at low concentration, but TP-P1 had already converted 90% at 30min, but TP-P5 was less than 80%, so TP-P1 converted to TP faster than TP-P5 in human plasma at equivalent concentration.

Example 13

In vitro transformation experiment of TP-P1, PG490-88Na and Minnelide in rat plasma

The experimental method comprises the following steps: mu.L of rat blank plasma was taken, added with equal volume of 1. mu.g/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubated at 37 ℃ and 60r/min in a constant temperature oscillator, 40. mu.L of drug-containing plasma was taken in pre-cooled 120. mu.L methanol (IS 1ng/mL) for 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h, vortexed for 3min, 4 ℃, 14000rpm/min for 10min, and the supernatant was taken for UPLC-MS/MS analysis.

The liquid phase analysis method comprises the following steps: mobile phase 0.1% FA-H2O (a) and acn (b); flow rate: 0.3 mL/min; gradient elution degree: 0-2 min, 15-80% of B; 2-3 min, 80% B-80% B; for 3-4 min, 80-15% of B; 4-5 min, 15% B-15% B; sample introduction amount: 5 mu L of the solution;

the experimental results are shown in fig. 4;

the results show that: TP-P1 can be converted into TP in the plasma of rats quickly, and complete conversion of TP can be realized within 30 min; PG490-88Na can also be completely converted to TP within 90min in the plasma of rat; the conversion of Minnelide is relatively slow, and the complete conversion of TP can be realized only after 6 hours, probably because the relative content of phosphatase in the plasma of the rat is low; thus, under rat plasma conditions, the prodrug TP-P1 converted to TP at a much higher rate than PG490-88Na and Minnelide.

Example 14

In vitro transformation experiment of TP-P1, PG490-88Na and Minnelide in human plasma

The experimental method comprises the following steps: mu.L of human blank plasma was taken, added with equal volume of 1. mu.g/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubated at 37 ℃ and 60r/min in a constant temperature oscillator, 40. mu.L of drug-containing plasma was taken in pre-cooled 120. mu.L methanol (IS 1ng/mL) for 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h, vortexed for 3min, 4 ℃, 14000rpm/min for 10min, and the supernatant was taken for UPLC-MS/MS analysis.

The liquid phase analysis method comprises the following steps: mobile phase 0.1% FA-H2O (a) and acn (b); flow rate: 0.3 mL/min; gradient elution degree: 0-2 min, 15% B-80% B; 2-3 min, 80% B-80% B; for 3-4 min, 80-15% of B; 4-5 min, 15% B-15% B; sample introduction amount: 5 mu L of the solution;

the experimental results are shown in fig. 5;

the results show that: TP-P1 can be converted into TP in human plasma quickly, and can be completely converted within 1 h; PG490-88Na converts in human plasma very slowly, 24h conversion is less than 15%, far lower than conversion rate in rat plasma; the Minnelide conversion was also relatively slow, approaching 80% for 24h conversion, probably also due to the relatively low levels of phosphatase in human plasma. Thus, under human plasma conditions, the prodrug TP-P1 converted to TP at a much higher rate than PG490-88Na and Minnelide.

Example 15

Experiment for in vitro transformation of different concentrations of TP-P1 in human plasma

The experimental method comprises the following steps: adding equal volume of TP-P1 aqueous solution (10 mu g/mL, 1 mu g/mL and 100ng/mL) into 400 mu L of human blank plasma, incubating at 37 ℃ for 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12 and 24h in a constant temperature oscillator for 60r/min, taking 40 mu L of drug-containing plasma in precooled 120 mu L of methanol (IS ═ 1ng/mL), vortexing for 3min, 4 ℃, 14000rpm/min for 10min, taking supernatant, and carrying out UPLC-MS/MS analysis;

the experimental results are shown in fig. 6;

the results show that: TP-P1 was able to convert faster to TP at various low concentrations (50ng/mL, 500ng/mL, 5000ng/mL) in human plasma, and essentially complete conversion was achieved within 1 h.

Example 16

Proliferation inhibitory Activity of TP-P1 and TP against various tumor cell lines

TABLE 3 in vitro proliferation inhibitory Activity of TP-P1 on various tumor cells

The experimental method comprises the following steps: MV-4-11, THP-1, KG-1 and HL-60 are human acute myeloid leukemia cells, PANC-1 is human pancreatic cancer cells, U937 is human histiocyte lymphoma cells, CAG, ARP-1 and H929 are human myeloma cells, HepG2 and Hep3B are human liver cancer cells, HT-29 and HCT-116 are human colon cancer cells, MDA-MB-231 is human breast cancer cells, Hela is human cervical cancer cells, A549 is human lung cancer cells; suspension growth cells the in vitro antiproliferative activity of compounds on tumor cells was determined by the CCK-8 method: diluting the tested compound with 3-fold gradient of culture medium to twice the final concentration, and taking 200 mu L to 2mL of EP tube for later use; taking a proper amount of cells in a logarithmic growth phase, re-suspending the cells in a culture medium, adding the cells into the culture medium containing the tested compound in an equal volume, reversing the cells up and down for 10 times, uniformly mixing, and sequentially adding the cells into a 96-well plate, wherein each well is 100 mu L; at 37 ℃ with 5% CO₂After 48h of incubator culture, adding 10 mu L of CCK-8 into each hole, and continuing to incubate for 2 h; reading the OD450 absorbance value by an enzyme-labeling instrument, and repeating the experiment twice; the data were analyzed and processed using Graphpad Prism 8 software to determine IC₅₀(ii) a The adherent growth cells are used for measuring the in vitro antiproliferative activity of the compound on solid tumor cells by adopting an MTT method: trypsinizing the cells in logarithmic growth phase, counting, resuspending the appropriate amount of cells in culture medium, adding 100. mu.L/well to a 96-well plate, overnight culturing, adding 100. mu.L/well of 3-fold gradient diluted test compound or control medium at 37 deg.C and 5% CO₂After 48h of incubator culture, adding 20 mu L of MTT into each hole, continuing to incubate for 4h at 37 ℃, reading OD490 absorbance value by an enzyme-labeling instrument, and repeating the experiment twice; the data were analyzed and processed using Graphpad Prism 8 software to determine IC₅₀The value is obtained.

The results show that: TP-P1 and TP have strong proliferation inhibitory activity to most tumor cells, wherein the inhibitory activity to human acute myelocytic leukemia cell lines THP-1, KG-1, MV-4-11 and HL-60 is strongest, and the inhibitory activity to myeloma, lymphoma and other solid tumor cells is weaker than that of human acute myelocytic leukemia cell lines (Table 3).

Example 17

Antitumor effect of TP-P1 on MV-4-11 nude mouse transplantation tumor model

The experimental method comprises the following steps: MV-4-11 cells are cultured and expanded in vitro, a proper amount of cells in logarithmic growth phase are taken to be resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and the suspension is prepared into 5 multiplied by 10 under aseptic condition⁶A 100 mu L cell suspension, and inoculating the 100 mu L cell suspension under the axilla of the anterior left limb of the male Balb/c nude mouse by using a syringe; when the tumor volume grows to 100-200mm³Selecting animals with moderate tumor sizes, and randomly grouping, wherein each group comprises 5 animals; blank vehicle (PBS), low dose (25 μ g/kg/d) of test compound TP-P1, medium dose (50 μ g/kg/d) of TP-P1, and high dose (100 μ g/kg/d) of TP-P1 were administered separately, i.p. once a day for 4 weeks; during the administration period, nude mice body weight and tumor size were measured daily; after the experiment, the cervical vertebrae were dislocated and sacrificed, and the tumor was weighed.

The formula for Tumor Volume (TV) is: TV 1/2 × a × b²And a represents the tumor major axis; b represents the tumor minor axis.

TABLE 4 antitumor Effect of TP-P1 on the model of MV-4-11 nude mouse transplantable tumor

P < 0.05; p < 0.01; p <0.001 (compared to solvent control).

The results show that: compound TP-P1 can inhibit tumor growth in a dose-dependent manner on a MV-4-11 nude mouse transplantation tumor model by continuous intraperitoneal injection for 4 weeks, and has no influence on mouse body weight; wherein the tumor inhibition rate under the dosage of 25 mu g/kg/d reaches 54.31 percent, the dosage of 100 mu g/kg/d can completely eliminate the transplanted tumor, and the tumor inhibition rate reaches 100 percent (Table 4).

Example 18

Antitumor effect of TP-P1 on THP-1 nude mouse transplantation tumor model

The experimental method comprises the following steps: THP-1 cell in vitro culture and amplification, taking a proper amount of cells in logarithmic growth phase to be resuspended in serum-free 1640 cultureThe nutrient and Matrigel (1:1) suspension is prepared into 5X 10 under the aseptic condition⁶A 100 mu L cell suspension, and inoculating the 100 mu L cell suspension under the axilla of the anterior left limb of the male Balb/c nude mouse by using a syringe; when the tumor volume grows to 100-200mm³Selecting animals with moderate tumor sizes, and randomly grouping, wherein each group comprises 5 animals; blank vehicle (PBS), positive control TP (180. mu.g/kg/d), test compound TP-P1 (100. mu.g/kg/d), TP-P1 (300. mu.g/kg/d), TP-P1 (600. mu.g/kg/d) and TP-P1 (1200. mu.g/kg/d) were administered by intraperitoneal injection once a day for 4 weeks; during the administration period, body weight and tumor size of nude mice were measured daily. After the experiment, the cervical vertebrae were dislocated and sacrificed, and the tumor was weighed.

TABLE 5 antitumor Effect of TP-P1 on THP-1 nude mouse transplanted tumor model

P < 0.05; p < 0.01; p <0.001 (compared to solvent control).

The results show that: in the THP-1 nude mouse transplantation tumor model, compound TP-P1 can inhibit tumor growth in a dose-dependent manner by continuous intraperitoneal injection for 4 weeks, and has no influence on mouse body weight. Wherein the tumor inhibition rate under the dosage of 100 mu g/kg/d reaches 93.87 percent, the dosage of 300 mu g/kg/d and above can completely eliminate the transplanted tumor, and the tumor inhibition rate reaches 100 percent (Table 5).

Example 19

Antitumor effect of TP-P1 and Giritinib co-administered in MV-4-11 nude mouse transplantation tumor model

The experimental method comprises the following steps: MV-4-11 cells are cultured and expanded in vitro, a proper amount of cells in logarithmic growth phase are taken to be resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and the suspension is prepared into 5 multiplied by 10 under aseptic condition⁶A 100 mu L cell suspension, and inoculating the 100 mu L cell suspension under the axilla of the anterior left limb of the male Balb/c nude mouse by using a syringe; when the tumor volume grows to 100-200mm³Selecting animals with moderate tumor sizes, and randomly grouping into 6 animals in each group; blank vehicle (PBS) and sodium carboxymethylcellulose (CMC-Na), test compound TP-P1 dose group (50 μ g/kg/d), Giletinib low dose (0.5mg/kg/d), Giletinib high dose (1mg/kg/d), TP-P1 combination Giletinib low dose group; TP-P1 is injected into the abdominal cavity every day, and the administration is carried out once by the gavage of the Girritinib for 3 weeks; during the administration period, nude mice body weight and tumor size were measured daily; after the experiment, the cervical vertebrae were dislocated and sacrificed, and the tumor was weighed.

TABLE 6 anti-tumor Effect of TP-P1 in combination with Girardinib on MV-4-11 nude mouse transplant tumor model

P < 0.05; p < 0.01; p <0.001 (compared to solvent control). ^ p < 0.05; ^ p < 0.01; and (c) p <0.001 (compared to the Girardinib low dose group).

The results show that: on the MV-4-11 nude mouse transplantation tumor model, with continuous administration for 3 weeks, the tumor inhibition rate of the combination group was 78.12% higher than that of the corresponding dose single-drug giritinib group (48.27%), and higher than that of the two-dose single-drug giritinib group (71.96%) (table 6); therefore, the compound TP-P1 and the Giritinib are combined to have synergistic effect in the treatment of acute myeloid leukemia.

Example 20

TP-P1 relieving lung inflammation of sepsis mouse induced by LPS

The experimental method comprises the following steps: dividing female Balb/c mice into 5 groups by adopting a random grouping method, wherein each group comprises 6 mice, namely a solvent control group, a model group, a TP-P1 low dose group (500 mu g/kg/d), a TP-P1 medium dose group (1000 mu g/kg/d) and a TP-P1 high dose group (1500 mu g/kg/d), performing preventive administration on each group two days before LPS injection, performing intraperitoneal injection once a day, continuously administering for 2 days, and ensuring that the administration volume is 10 ml/kg; on the 3 rd day, 10mg/kg LPS is injected into the abdominal cavity once respectively for the model group and the TP-P1 low, medium and high dose groups, and the administration volume is 5 ml/kg; on the 4 th day, mouse lung tissues are picked up aseptically, the expression conditions of inflammatory factors such as IL-1 beta, IL-6, TNF-alpha, IFN-gamma and the like are detected by RT-qPCR technology, and each group of data is normalized with a solvent control group respectively.

TABLE 7 effects of TP-P1 in relieving LPS-induced pulmonary inflammation in septic mice

P < 0.05; p < 0.01; p <0.001 (compared to solvent control). ^ p < 0.05; ^ p < 0.01; and (c) a, p <0.001 (drug-administered versus model).

The results show that: TP-P1 can inhibit the release of inflammatory factors such as IL-1 beta, IL-6, TNF-alpha, IFN-gamma and the like, and is dose-dependent and relieves the lung inflammation of sepsis mice induced by LPS (Table 7).

Example 21

Pharmacokinetic Properties of TP-P1

The experimental method comprises the following steps: dividing 8 male SD rats into 2 groups by a random grouping method, wherein the randomly grouped SD rats are fasted overnight but can freely drink water; after 12h, the first group was administered an aqueous solution of compound TP-P1 at a dose of 1.6 mg/kg for intragastric administration; a second group is administered with olive oil suspension of TP, and is administered by intragastric administration, and the administration dose is 1.0 mg/kg; adopting orbital hemospasia, taking blood respectively at 2min, 5min, 10min, 15min, 30min, 45min, 60min, 90min, 2h, 4h and 6h, transferring into a centrifuge tube of 1.5mL pretreated with heparin sodium, centrifuging (8000rpm/min, 5min, 4 ℃) to obtain plasma, and storing in a refrigerator at-80 ℃.

The treatment method comprises the following steps: treating a plasma sample by a 1:3 methanol protein precipitation method, carrying out vortex centrifugation, taking supernatant, analyzing TP blood concentration in the plasma by UPLC-MS/MS, and processing data by Phoenix 64 software to obtain pharmacokinetic parameters.

TABLE 8 pharmacokinetic Properties of TP-P1

The results show that: AUC of TP-P1_(0-t)Pharmacokinetic parameters such as Cmax were all better than TP, and TP-P1 absorption was significantly higher than TP for equimolar oral dosing (Table 8).

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.

Claims

1. A triptolide prodrug or a pharmaceutically acceptable salt, a polymorph or a solvate thereof, wherein the chemical structural formula of the compound is shown as the formula (I):

wherein:

n1 is 1 to 6;

n2 is 1 to 6;

n3 is 0 or 1;

x is a carbon, nitrogen or oxygen atom;

HA is selected from hydrochloric acid, sulphuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulphonic acid, benzenesulphonic acid, p-toluenesulphonic acid or ferulic acid.

2. The class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs, or solvates thereof according to claim 1,

the triptolide prodrug is selected from the following compounds:

3. the method for preparing the triptolide prodrugs or the pharmaceutically acceptable salts, polymorphs, or solvates thereof according to claims 1 and 2, comprising the following steps:

the first step is as follows: triptolide reacts with acyl chloride under the action of DMAP to generate an intermediate II:

the second step is that: and reacting the intermediate II with a carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate a compound III:

dissolving the purified intermediate II in anhydrous DMF, adding sodium iodide and carboxylic acid, reacting for 0.5-1 hour, adding potassium carbonate, heating to 40-70 ℃, and reacting for 3-12 hours to obtain a reaction solution; pouring the reaction solution into water, extracting with ethyl acetate, washing with sodium bicarbonate water solution and saline solution, drying, performing suction filtration, concentrating the filtrate, and performing column chromatography purification to obtain a compound III;

the third step: salifying the compound III with an acid to obtain a target compound I:

dissolving the purified compound III in ethyl acetate, adding an inorganic acid or an organic acid, reacting at the temperature of 0-30 ℃ for 6-12 hours, carrying out suction filtration, and drying; finally obtaining the target compound I.

4. A pharmaceutical composition comprising a class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs, or solvates thereof as claimed in claim 1.

5. A method for preventing/treating malignant tumor, inflammatory disease, immune disease and virus infectious disease by using the pharmaceutical composition of claim 4.

6. The malignant tumor according to claim 5, wherein the malignant tumor comprises acute myelogenous leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, bile duct cancer, stomach cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma, and neuroblastoma.

7. Immune and inflammatory diseases as claimed in claim 5, characterized in that said immune and inflammatory diseases comprise rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, systemic vasculitis, psoriasis, idiopathic dermatitis, inflammatory bowel disease, asthma, pulmonary fibrosis, nephritis, nephrotic syndrome, immune rejection and LPS induction, CAR-T therapy, bacterial infections, cytokine release syndrome due to viral infections.

8. Use of a pharmaceutical composition according to claim 4 in combination with an FLT3 inhibitor for the treatment of acute myeloid leukemia.