WO2023138147A1 - Triptolide prodrug, preparation method therefor and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to the field of medicinal chemistry. Disclosed are a triptolide prodrug, a preparation method therefor and a pharmaceutical use thereof. The preparation steps are: 1, making triptolide react with acyl chloride under the action of DMAP to generate an intermediate II; 2, making the intermediate II react with a carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate a compound III; and 3, carrying out salt formation on the compound III and an acid to obtain a target compound I. The present invention has better water solubility and pharmacokinetic properties, and has a faster transformation speed in an in vivo blood environment compared with Minnelide, a more efficient synthesis method, and a better therapeutic effect on malignant tumors, inflammatory diseases, autoimmune diseases, etc. In addition, the triptolide prodrug has good water solubility, and therefore is effective in oral administration, and can be prepared into an injection for use.

Description

A kind of triptolide prodrug, its preparation method and its medical use technical field

The invention belongs to the field of medicinal chemistry, and relates to a class of triptolide water-soluble prodrugs or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing these compounds, their preparation methods and medical applications.

Background technique

Tripterygium wilfordii Hook.f. is a plant of the genus Tripterygium wilfordii Hook.f. in the family Celastraceae. It has the functions of expelling wind and dampness, promoting blood circulation and dredging collaterals, reducing swelling and pain, killing insects and detoxifying, etc. It is the first choice of traditional Chinese medicine for clinical treatment of autoimmune diseases. At present, there are a variety of tripterygium extract drugs such as Tripterygium wilfordii Polyglycoside Tablets, Tripterygium wilfordii Tablets (999), Tripterygium wilfordii Double-layer Tablets, Tripterygium Total Terpene Tablets, etc., which are used in the treatment of various immune and inflammatory diseases such as rheumatoid arthritis, autoimmune hepatitis, nephritis and nephrotic syndrome. The main active components of Tripterygium wilfordii extract are diterpenoids, triterpenoids and alkaloids, among which triptolide, a diterpenoid, is one of the main active ingredients of Tripterygium wilfordii, and it is also the main active ingredient of preparations such as tripterygium glycosides and tripterygium.

As one of the highly active components of Tripterygium wilfordii, triptolide has strong immunosuppressive, anti-inflammatory, anti-tumor and other pharmacological effects [J Am Chem Soc, 1972, 94(20): 7194-7195; 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 to treat rheumatoid arthritis. A double-blind clinical study by the American College of Rheumatology also showed that Tripterygium wilfordii can significantly improve the symptoms of rheumatoid arthritis, with an effective rate of 58% [J Rheumatol 2003, 30(3): 465-467]. For other autoimmune and inflammatory diseases, such as systemic lupus erythematosus, psoriasis, ankylosing spondylitis, asthma, nephritis, ulcerative colitis, pulmonary fibrosis, etc., Tripterygium wilfordii has shown satisfactory effects [Chin J Mod Appl Pharm, 1999,16(2): 10-13; Br J Clin Pharmacol 2012Sep; 74(3): 424-36; Rheum Dis Clin North Am 2000; Am J Pathol 2001; 158(3):997-1004; 26(1):29-50]. In view of the immunosuppressive effect of tripterygium wilfordii, many scholars have found that triptolide can effectively prevent the rejection caused by transplanted organs after heart, kidney, liver or bone marrow transplantation, and significantly prolong the survival period of animals [Transplantation 2000; 70(3):447-55; Transplant Proc 1999; 31(7):2719-23]. In the field of cancer, triptolide regulates the overall transcription level of cells by covalently binding and inhibiting XPB activity in the TFHII transcription complex, so that triptolide has broad-spectrum anti-tumor activity [Angew Chem Int Ed Engl, 2015, 54(6): 1859-1863; Mol Cancer Ther, 2003, 2: 65-72]. In addition, triptolide has shown effective anti-HIV effects in vitro [J Nat Prod 2000; 63(3):357-61], and multiple clinical implementations are in progress [NCT03403569; NCT01817283; NCT02002286]. However, due to the poor water solubility of its diterpene lactone structure, its clinical application is limited. Therefore, the water-soluble prodrug of triptolide can be designed and synthesized by introducing a water-soluble group, so as to improve its water solubility and improve its druggability without affecting its drug efficacy.

The design strategy of water-soluble prodrugs is mainly to connect some water-soluble groups at the C-14 position of triptolide through ester bonds or acetals. For example, PG490-88 introduces a water-soluble carboxylic acid directly through an ester bond at the C-14 position of triptolide, and is the first triptolide prodrug to enter clinical research [US5663335]. In clinical trials, although the toxic side effects of most patients were controllable, two patients had fatal side effects, one of whom died of neutropenic sepsis at a dose of 12 mg/kg, and the other died of complex clinical syndrome at a dose of 18 mg/kg. Subsequent pharmacokinetic experiments found that PG490-88 could not be rapidly converted into triptolide in vivo, which not only showed great differences in different species (including mice, monkeys and humans), but also showed a 2-3 times conversion difference in the same batch of 18 volunteers. The reason may be that the C-14 steric hindrance of PG490-88 hinders the hydrolysis of ester bonds by esterase, resulting in slow and incomplete transformation of PG490-88 in vivo. In addition, due to differences in esterase activity among individuals, it is also difficult to control the safe dosage of drugs. Therefore, the phase I clinical trial of PG490-88 was also forced to terminate in 2009.

In order to overcome the problem of incomplete transformation of PG490-88 in vivo, researchers have devoted themselves to optimizing the connection method of the C-14 position of PG490-88 to avoid the influence of steric hindrance on bond breaking. In 2010, Georg et al. designed and synthesized the water-soluble triptolide prodrug Minnelide [WO2010/129918] by using methylal as a linker to introduce a phosphate group. Due to the small steric hindrance next to the phosphate bond, Minnelide is easily hydrolyzed by phosphatase to generate a hydroxymethyl intermediate. The structure of the intermediate hydroxymethyl ether is very unstable, and it will be automatically hydrolyzed to release the hydroxyl group at the C14 position. Therefore, Minnelide showed good pharmacokinetic properties in Phase I clinical trials and has entered Phase II clinical trials.

Although the structure of methylal is easily broken by hydrolysis, we found that it still takes more than 24 hours for Minnelide to be completely converted into triptolide in the in vitro human plasma conversion experiment, which may be related to the low content of alkaline phosphatase in plasma. In addition, due to the expensive raw materials of triptolide, the synthesis of Minnelide is difficult and the total yield is low (39%), making its development cost high; and due to the harsh reaction conditions, it is not conducive to large-scale preparation. Therefore, the development of a triptolide prodrug with good water solubility, rapid and complete conversion, and simple synthesis process has an important market prospect.

Carboxylesterase is the most abundant hydrolase in humans and animals, and participates in the metabolic process of various endogenous and exogenous compounds and drugs. The present invention introduces a water-soluble aliphatic nitrogen-containing heterocyclic ring at the C-14 position of triptolide through the connection of a hydroxyester (a structure that is easily hydrolyzed by esterases), and discovers a class of triptolide water-soluble prodrugs that have good water solubility, rapid in vivo transformation, and are easy to synthesize (total yield reaches 61%). The prodrug has good water solubility, is effective when taken orally, and can be completely converted into triptolide in human plasma within 1 hour. Therefore, it is suitable for the treatment of various inflammatory diseases, immune diseases, hematological malignancies and solid tumors that triptolide can effectively treat.

Contents of the invention

Object of the invention: The object of the present invention is to provide a triptolide prodrug or a pharmaceutically acceptable salt thereof whose water solubility is significantly improved and can be rapidly transformed in plasma as represented by general formula (I).

Technical solution: a class of triptolide prodrugs or their pharmaceutically acceptable salts, polymorphs or solvates that introduce an aliphatic nitrogen-containing heterocycle through a hydroxyester linking arm that is easy to self-degrade according to the present invention; the chemical structural formula of the derivative is shown in formula (I):

in:

n1 is 1~6;

n2 is 1~6;

n3 is 0 or 1;

X is a carbon, nitrogen or oxygen atom;

HA is selected from hydrochloric acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid;

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

Another object of the present invention is to provide a method for preparing the triptolide-like prodrug or its pharmaceutically acceptable salt, polymorph or solvate; its specific preparation steps are as follows:

The first step: triptolide reacts with acid chloride under the action of DMAP to generate intermediate II;

Dissolving triptolide and DMAP in anhydrous dichloromethane, adding acid chloride at a low temperature of -10 to 5°C, and reacting at a temperature of 0 to 30°C for 6 to 12 hours; then washing the obtained reaction solution with dilute hydrochloric acid, saturated sodium bicarbonate and brine in sequence, and drying with anhydrous sodium sulfate; suction filtration, concentration of the filtrate, and purification by column chromatography to obtain intermediate II;

Second step: Intermediate II reacts with the corresponding carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate compound III;

Dissolve the purified intermediate II in anhydrous DMF, add sodium iodide and carboxylic acid, react for 0.5 to 1 hour, add potassium carbonate, then heat it to 40 to 70°C for 3 to 12 hours to obtain a reaction solution; then pour the reaction solution into water, extract with ethyl acetate, wash with aqueous sodium bicarbonate and saline, dry, filter with suction, concentrate the filtrate, and purify by column chromatography to obtain compound III;

The third step: Salt formation of compound III with the corresponding acid to obtain the target compound I;

Dissolve the purified compound III in ethyl acetate, add inorganic acid or organic acid, react at a temperature of 0-30° C. for 6-12 hours, filter with suction, and dry to obtain the target compound I.

The object of the present invention is to provide a pharmaceutical composition, including the triptolide prodrug, its pharmaceutically acceptable salt, polymorph or solvate, and at least one pharmaceutically acceptable carrier, additive, auxiliary agent or vehicle.

Another object of the present invention is to provide the use of the triptolide prodrug or pharmaceutically acceptable salt, and its pharmaceutical composition in the preparation of antitumor drugs.

The triptolide prodrug of the present invention can be completely converted into triptolide in human plasma. Therefore, the triptolide prodrug or its pharmaceutically acceptable salt and pharmaceutical composition thereof provided by the present invention can be used as a single therapeutic agent, or used in combination with other antineoplastic drugs, for the treatment of various malignant tumors for which triptolide is effective, specifically including acute myeloid leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, gallbladder cancer, etc. Tube cancer, gastric cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma, neuroblastoma and other malignant tumors and other malignant tumors.

Another object of the present invention is to provide the triptolide prodrug or pharmaceutically acceptable salt and its pharmaceutical composition in the preparation of drugs for the treatment of acute myeloid leukemia; in vitro anti-tumor activity experiments show that the compound of the present invention can significantly inhibit the proliferation of acute myeloid leukemia cells; whole animal experiments show that the compound of the present invention has a good curative effect on acute myeloid leukemia, the effective dose is only 25 μg/kg, and it has a synergistic effect when used in combination with FLT3 inhibitors; therefore, the compound of the present invention or its pharmaceutically acceptable The salts of and pharmaceutical compositions thereof can be used as a single therapeutic agent or used in combination with FLT3 inhibitors for the treatment of acute myeloid leukemia.

Another object of the present invention is to provide the purposes of the triptolide prodrug or pharmaceutically acceptable salt and its pharmaceutical composition in the preparation of anti-inflammatory, immunosuppressive and viral (for example: anti-HIV) drugs; the triptolide prodrug of the present invention can be completely converted into triptolide in human plasma; therefore, the triptolide prodrug or its pharmaceutically acceptable salt provided by the present invention, and its pharmaceutical composition can be used for the treatment of various indications that triptolide is effective, including rheumatoid arthritis, acute 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, etc.

Beneficial effects: Compared with the prior art, the present invention is characterized in that the triptolide prodrug of the present invention has significant anti-tumor and immunosuppressive activity in vivo, and has better water solubility and pharmacokinetic properties than triptolide, and compared with Minnelide, not only the in vivo transformation is faster, but also the synthesis is simpler, the yield is higher (61%), the cost is lower, and it has good industrialization prospects.

Description of drawings

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

Figure 2 is a schematic diagram of the in vitro transformation experiment of TP-P1, TP-P2 and TP-P5 in rat plasma in Example 11 of the present invention;

3 is a schematic diagram of the in vitro transformation experiment of TP-P1 and TP-P5 in human plasma in Example 12 of the present invention;

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

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

Fig. 6 is a schematic diagram of in vitro conversion experiments of different concentrations of TP-P1 in human plasma in Example 15 of the present invention.

Detailed ways

In order to illustrate the technical solution of the present invention more clearly, the technical solution of the present invention is described in further detail below in conjunction with the accompanying drawings:

Example 1

Synthesis of compound TP-P1

Synthesis of Intermediate II-1: Dissolve triptolide TP (1.8g, 5.0mmol) in 50mL of anhydrous dichloromethane, add DMAP (3.05g, 25.0mmol) at 0°C, and then add chloroacetyl chloride (4.0mL, 50.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with sodium chloride solution, and then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 1.77g of white solid with a yield of 88.8%. ¹ H 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 of anhydrous DMF, and sodium iodide (860mg, 4.6mmol) and morpholin-4-ylacetic acid (670mg, 4.6mmol) were added; after 40 minutes of reaction at room temperature, potassium carbonate (320mg, 2.3mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, ethyl acetate Extracted, combined organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 1.01 g of a white solid with a 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, 20mL of saturated hydrogen chloride in ethyl acetate was added, and reacted at room temperature for 6 hours; after the reaction was completed, suction filtered, 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%. ¹ H NMR(500MHz,DMSO-d ₆ )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). ¹³ C NMR(126MHz,DMSO-d ₆ )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 C ₂₈ H ₃₇ ClNO ₁₀ ⁺ ,582.2；found,582.0.

Example 2

Synthesis of compound TP-P2

Synthesis of Intermediate III-2: Dissolve compound II-1 (100mg, 0.23mmol) in 10mL of anhydrous DMF, add sodium iodide (86mg, 0.46mmol) and 4-methyl-1-piperazineacetic acid (73mg, 0.46mmol); after reacting at 25°C for 40 minutes, add potassium carbonate (32mg, 0.23mmol), then heat to 50°C for 4 hours; after the reaction, pour the reaction solution into In water, extracted with ethyl acetate, combined the organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 93 mg of white solid, yield 72.3%; ¹H NMR (500MHz, MeOD-d ₆)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(3 H,s),0.97(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).

Synthesis of compound TP-P2: Dissolve compound III-2 (100 mg, 0.18 mmol) in 5 mL of ethyl acetate, add 3 mL of saturated hydrogen chloride in ethyl acetate, react at room temperature for 6 hours, after the reaction is completed, filter with suction, wash the filter cake with ethyl acetate, and dry in vacuo to obtain 75 mg of a white solid, 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(1 H,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＝6.9Hz). 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 C ₂₉h ₄₀ClN ₂o ₉ ⁺, 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 of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and 2-piperidinylacetic acid (66mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; Extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 67 mg of white solid, yield 53.4%; ¹H NMR (500MHz, CDCl ₃)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, CDCl ₃( 2C), 23.4, 23.0, 17.5, 17.1, 16.7, 13.8.LCMS(ESI):m/z[M+H] ⁺calcd for C ₂₉h ₃₈NO ₉ ⁺, 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, sodium iodide (86mg, 0.46mmol) and 2-(pyrrolidin-1-yl)acetic acid (59mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; The reaction solution was added to water, extracted with ethyl acetate, the organic layers were combined, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 64 mg of a white solid, with a 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 C ₂₈h ₃₆NO ₉ ⁺, 530.2; found, 530.2.

Example 5

Synthesis of compound TP-P5

Synthesis of Intermediate II-5: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 4-bromobutyryl chloride (0.56mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 193 mg of white solid, yield 75.6%. ¹ H NMR(500MHz, CDCl ₃ )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 of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and morpholin-4-ylacetic acid (67mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, Extracted with ethyl acetate, combined the organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 83 mg of white solid, yield 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: Compound III-5 (100 mg, 0.17 mmol) was dissolved in 5 mL of ethyl acetate, 3 mL of saturated hydrogen chloride in ethyl acetate was added, and reacted at 25° C. for 6 hours; after the reaction was completed, suction filtered, the filter cake was washed with ethyl acetate, and then vacuum-dried to obtain 53 mg of a white solid, with a yield of 51.3%. mp:239-241℃. ¹ H 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). ¹³ C 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 C ₃₀ H ₄₁ ClNO ₁₀ ⁺ ,610.2；found,610.2.

Example 6

Synthesis of compound TP-P6

Synthesis of Intermediate II-6: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 5-bromovaleryl chloride (0.70mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, and then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 205 mg of white solid with a yield of 78.4%. ¹ H NMR(500MHz,CDCl ₃ )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 of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and morpholin-4-ylacetic acid (67mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, Extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 88 mg of white solid, yield 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 C ₃₁h ₄₂NO ₁₀ ⁺,588.3; found, 588.3.

Example 7

Synthesis of compound TP-P7

Synthesis of Intermediate II-7: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 6-bromohexanoyl chloride (0.77mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 191 mg of white solid, with a yield of 71.2%. ¹ H NMR(500MHz,CDCl ₃ )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 (124 mg, 0.23 mmol) was dissolved in 10 mL of anhydrous DMF, and sodium iodide (86 mg, 0.46 mmol) and morpholin-4-ylacetic acid (67 mg, 0.46 mmol) were added.于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体83mg，收率60.1％； ¹ H 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). ¹³ C 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 C ₃₂ H ₄₄ NO ₁₀ ⁺ ,602.3；found,602.3.

Example 8

Synthesis of compound TP-P8

Synthesis of compound TP-P8: Dissolve compound II-1 (100 mg, 0.23 mmol) in 10 mL of anhydrous DMF, add sodium iodide (86 mg, 0.46 mmol) and 3-(4-morpholinyl) propionic acid (73 mg, 0.46 mmol); after reacting at 25°C for 40 minutes, add potassium carbonate (32 mg, 0.23mmol), then heat to 50°C for 4 hours; Pour into water, extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 84 mg of white solid, yield 65.4%; ¹H NMR (500MHz, CDCl ₃)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),1.55-1.5 9(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, CDCl ₃)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, 2 9.5, 28.1, 23.4, 17.5, 17.1, 16.7, 13.8. LCMS (ESI): m/z[M+H]+calcd for C ₂₉h ₃₈NO ₁₀ ⁺, 560.2; found, 560.3.

Example 9

Compound long-term stability test

Experimental method: the compound was left open at room temperature for more than 90 days, and the purity of the compound was determined by the HPLC normalization method (Table 1).

The long-term stability test result of table 1 embodiment compound

The results showed that the chemical stability of this series of compounds was very good, and the purity of the compounds did not decrease significantly after being left open at room temperature for 90 days by HPLC-UV and ¹ H-NMR spectrum analysis.

Example 10

Stability experiment of compound TP-P1 in pure water and acidic aqueous solution

Experimental method: Compound TP-P1 was dissolved in pure water with pH=7 and acidic aqueous solution with pH=4 and pH=2, samples at different time points were collected, and the purity of the compound was determined by HPLC normalization method (Table 2).

Table 2 Stability test results of compound TP-P1 in pure water and acidic aqueous solution

The results showed that the purity of the compound TP-P1 did not change significantly within 6 hours in the aqueous solution of pH7, pH2, and pH4.

Example 11

In Vitro Transformation Experiment of TP-P1, TP-P2, TP-P5 in Rat Plasma

Considering the water solubility of the compounds, we selected salt-forming compounds TP-P1, TP-P2, and TP-P5 with better water solubility for in vitro plasma conversion experiments in rats.

Experimental method: Take 250 μL rat blank plasma, add an equal volume of 100 μg/mL TP-P1, TP-P2, TP-P5 aqueous solution, incubate in a constant temperature oscillator at 60 r/min, 37 °C, take 20 μL of drug-containing plasma in pre-cooled 60 μL methanol, and vortex 3 min, centrifuge at 4°C, 14000rpm/min for 10min, and take the supernatant for HPLC analysis;

Liquid phase analysis method: ACN: 0.1% TFA-H ₂ O = 35:65 isocratic elution, flow rate: 0.6mL/min

Liquid phase analysis method: ACN: 0.1% TFA-H ₂ O = 35:65 isocratic elution, flow rate: 0.6mL/min

The experimental results are as shown in Figure 2;

The results showed that the three prodrugs of TP-P1, TP-P2 and TP-P5 could be completely converted into TP within 30 minutes in rat plasma, but at the same time, 52.3% of TP-P1 had been converted in 15 minutes, while TP-P2 and TP-P5 were less than 50%. Therefore, the conversion efficiency of TP-P1 prodrug into TP was slightly higher than that of TP-P2 and TP-P5.

Example 12

In Vitro Transformation Experiment of TP-P1 and TP-P5 in Human Plasma

In view of the fact that both compounds TP-P1 and TP-P2 use glycolic acid linkages, and the conversion rate of TP-P1 is better than that of TP-P5 within the same time period, we chose TP-P1 and TP-P5 for human in vitro plasma conversion experiments.

Experimental method: Take 250 μL of human blank plasma, add an equal volume of TP-P1 or TP-P5 aqueous solution (100 μg/mL or 2 mg/mL), incubate in a constant temperature shaker at 60 r/min, 37 °C, take 20 μL of drug-containing plasma in pre-cooled 60 μL methanol, vortex for 3 min, 4 °C , Centrifuge at 14000rpm/min for 10min, and take the supernatant for HPLC analysis.

Liquid phase analysis method: ACN: 0.1% TFA-H ₂ O = 35:65 isocratic elution, flow rate: 0.6mL/min

The experimental results are as shown in Figure 3;

The results showed that it took 45 minutes for TP-P1 to be completely converted into TP at low concentrations, and 60 minutes at high concentrations; 45 minutes for TP-P5 to be completely converted to TP at low concentrations, and 90 minutes at high concentrations to completely convert to TP; it indicated that drug concentration had an impact on plasma conversion, and the conversion rate was faster at low concentrations. Compared with TP-P1, it takes 45 minutes for TP-P5 to be completely converted into TP at a low concentration, but 90% of TP-P1 has been converted in 30 minutes, while TP-P5 is less than 80%. Therefore, at the same concentration, the conversion rate of TP-P1 into TP in human plasma is faster than that of TP-P5.

Example 13

In Vitro Transformation Experiment of TP-P1, PG490-88Na and Minnelide in Rat Plasma

Experimental method: Take 400μL rat blank plasma, add an equal volume of 1μg/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubate in a constant temperature oscillator at 60r/min, 37℃, take 40μL drug-containing plasma at 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h Vortex for 3 min in pre-cooled 120 μL methanol (IS=1 ng/mL), centrifuge at 14000 rpm/min at 4°C for 10 min, and take the supernatant for UPLC-MS/MS analysis.

Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;

The experimental results are as shown in Figure 4;

The results show that: TP-P1 can be converted into TP quickly in the plasma of rats, and the complete conversion of TP can be realized within 30 minutes; PG490-88Na can also be completely converted into TP within 90 minutes in the plasma of rats; and the conversion of Minnelide is relatively slow, and it takes 6 hours to realize the complete conversion of TP, which may be due to the relatively low content of phosphatase in rat plasma; therefore, under the condition of rat plasma, the conversion rate of prodrug TP-P1 into TP is much higher than that of PG490-88 Na and Minnelide.

Example 14

In Vitro Transformation Experiment of TP-P1, PG490-88Na and Minnelide in Human Plasma

Experimental method: Take 400μL human blank plasma, add an equal volume of 1μg/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubate in a constant temperature oscillator at 60r/min, 37°C, take 40μL of drug-containing plasma at 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h Vortex for 3 min in pre-cooled 120 μL methanol (IS=1 ng/mL), centrifuge at 14000 rpm/min at 4°C for 10 min, and take the supernatant for UPLC-MS/MS analysis.

Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;

The experimental results are as shown in Figure 5;

The results show that: TP-P1 can also be converted into TP quickly in human plasma, and can be completely converted within 1 hour; while PG490-88Na is very slow in human plasma, and the conversion rate is less than 15% in 24 hours, which is far lower than the conversion rate in rat plasma; the conversion of Minnelide is also relatively slow, and the conversion in 24 hours is close to 80%, which may also be due to the relative low content of phosphatase in human plasma. Therefore, under the condition of human plasma, the conversion rate of prodrug TP-P1 to TP is much higher than that of PG490-88Na and Minnelide.

Example 15

In Vitro Transformation Experiment of Different Concentrations of TP-P1 in Human Plasma

Experimental method: Take 400 μL of human blank plasma, add equal volume of TP-P1 aqueous solution (10 μg/mL, 1 μg/mL, 100 ng/ml), incubate in a constant temperature oscillator at 60 r/min, 37 ° C, take 40 μL of drug-containing plasma in pre-cooled 120 μL methanol ( IS=1ng/mL), vortex for 3min, centrifuge at 14000rpm/min for 10min at 4°C, and take the supernatant for UPLC-MS/MS analysis;

Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;

The experimental results are as shown in Figure 6;

The results show that: TP-P1 can be converted into TP relatively quickly at different low concentrations (50ng/mL, 500ng/mL, 5000ng/mL) in human plasma, and complete conversion can be basically achieved within 1h.

Example 16

Proliferation inhibitory activity of TP-P1 and TP on various tumor cell lines

Table 3 In vitro proliferation inhibitory activity of TP-P1 on various tumor cells

Experimental method: 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, H929 are human myeloma cells; HepG2 and Hep3B are human liver cancer cells; HT-29 and HCT-116 are human colon cancer cells; Human breast cancer cells, Hela is human cervical cancer cells, A549 is human lung cancer cells; CCK-8 method was used to determine the in vitro anti-proliferation activity of the compound on tumor cells: dilute the test compound with a 3-fold gradient in the medium to twice the final concentration, and take 200 μL to 2 mL EP tube for later use; take an appropriate amount of cells in the logarithmic growth phase and resuspend in the medium, add an equal volume to the medium containing the test compound, mix up and down 10 times, and add to a 96-well plate in turn, 100 per well μL; at 37°C, 5% CO ₂After 48 hours of incubation in the incubator, add 10 μL of CCK-8 to each well and continue to incubate for 2 hours; read the OD450 absorbance value with a microplate reader, and repeat the experiment twice; use Graphpad Prism 8 software to analyze and process the data to obtain the IC ₅₀; For adherent growth cells, the in vitro anti-proliferation activity of the compound on solid tumor cells was determined by the MTT method: cells in the logarithmic growth phase were digested with trypsin, counted, and an appropriate amount of cells were resuspended in culture medium, and 100 μL per well was added to a 96-well plate. ₂After culturing in the incubator for 48 hours, add 20 μL MTT to each well, continue to incubate at 37°C for 4 hours, read the OD490 absorbance value with a microplate reader, and repeat the experiment twice; use Graphpad Prism 8 software to analyze and process the data to obtain the IC ₅₀value.

The results showed that TP-P1 and TP had strong proliferation inhibitory activity on most tumor cells, among which the inhibitory activity on human acute myeloid leukemia cell lines THP-1, KG-1, MV-4-11 and HL-60 was the strongest, and the inhibitory activity on myeloma, lymphoma and other solid tumor cells was weaker than that of human acute myeloid leukemia cell lines (Table 3).

Example 17

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

Experimental method: MV-4-11 cells were cultured and expanded in vitro, and an appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and prepared under sterile conditions into 5×10 ⁶/100μL cell suspension, use a syringe to inoculate 100μL cell suspension subcutaneously in the armpit of the anterior left limb of male Balb/c nude mice; wait until the tumor volume grows to 100-200mm ³At the same time, animals with moderate tumor size were randomly divided into 5 groups. They were given blank vehicle (PBS), low dose of TP-P1 (25 μg/kg/d), medium dose of TP-P1 (50 μg/kg/d), and high dose of TP-P1 (100 μg/kg/d) respectively, and injected intraperitoneally once a day for 4 weeks. During the administration period, the body weight and tumor diameter of nude mice were measured every day;

The calculation formula of tumor volume (TV) is: TV=1/2×a×b ² , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.

Table 4 Anti-tumor effect of TP-P1 on MV-4-11 nude mouse xenograft model

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

The results showed that: on the MV-4-11 nude mouse xenograft tumor model, administered intraperitoneally for 4 weeks, the compound TP-P1 could dose-dependently inhibit the growth of the tumor, and had no effect on the body weight of the mice; the tumor inhibition rate at a dose of 25 μg/kg/d reached 54.31%, and the dose of 100 μg/kg/d could completely regress the xenograft tumor, and the tumor inhibition rate reached 100% (Table 4).

Example 18

Anti-tumor effect of TP-P1 on THP-1 nude mouse xenograft tumor model

实验方法：THP-1细胞体外培养扩增，取适量处于对数生长期的细胞重悬于无血清1640培养基与Matrigel(1:1)混悬液中，无菌条件下制备成5×10 ⁶ /100μL细胞悬液，用注射器将100μL细胞悬液接种于雄性Balb/c裸小鼠前左肢腋窝皮下；待肿瘤体积生长至100-200mm ³时，选取肿瘤大小适中的动物随机分组，每组5只；分别给予空白媒介(PBS)、阳性对照药TP(180μg/kg/d)、受试化合物TP-P1(100μg/kg/d)、TP-P1(300μg/kg/d)、TP-P1(600μg/kg/d)，TP-P1(1200μg/kg/d)，每天腹腔注射一次，给药4周；给药期间，每天测量裸小鼠体重和瘤径。 After the experiment, the animals were killed by cervical dislocation, and the tumors were weighed.

The calculation formula of tumor volume (TV) is: TV=1/2×a×b ² , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.

Table 5 Antitumor effect of TP-P1 on THP-1 nude mouse xenograft model

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

The results showed that: on the THP-1 nude mouse xenograft tumor model, the compound TP-P1 could dose-dependently inhibit the growth of the tumor after continuous intraperitoneal injection for 4 weeks, and had no effect on the body weight of the mice. Among them, the tumor inhibition rate at a dose of 100 μg/kg/d reached 93.87%, and the dose of 300 μg/kg/d and above could completely regress the transplanted tumor, and the tumor inhibition rate reached 100% (Table 5).

Example 19

Anti-tumor effect of TP-P1 combined with geritinib on MV-4-11 nude mouse xenograft tumor model

Experimental method: MV-4-11 cells were cultured and expanded in vitro, and an appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and prepared under sterile conditions into 5×10 ⁶/100μL cell suspension, use a syringe to inoculate 100μL cell suspension subcutaneously in the armpit of the anterior left limb of male Balb/c nude mice; wait until the tumor volume grows to 100-200mm ³At the same time, the animals with moderate tumor size were randomly divided into groups, 6 animals in each group; they were given blank medium (PBS) and carboxymethylcellulose sodium (CMC-Na), the test compound TP-P1 dose group (50 μg/kg/d), geritinib low dose (0.5 mg/kg/d), geritinib high dose (1 mg/kg/d), TP-P1 combined with geritinib low dose group; TP-P1 intraperitoneal injection every day, geritinib intragastric administration once, The administration was administered for 3 weeks; during the administration period, the body weight and tumor diameter of the nude mice were measured every day; after the experiment, the mice were sacrificed by cervical dislocation, and the tumors were weighed.

The formula for calculating tumor volume (TV) is: TV=1/2×a×b ² , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.

Table 6 Anti-tumor effect of TP-P1 combined with geritinib on MV-4-11 nude mouse xenograft model

*, p<0.05; **, p<0.01; ***, p<0.001 (compared to solvent control). ^, p<0.05; ^^, p<0.01; ^^^, p<0.001 (compared with low-dose geritinib group).

The results showed that: on the MV-4-11 nude mouse transplanted tumor model, after continuous administration for 3 weeks, the tumor inhibition rate of the combination group was 78.12% higher than that of the corresponding dose of single drug geritinib group (48.27%), and higher than that of the double dose of single drug geritinib group (71.96%) (Table 6); therefore, the combination of compound TP-P1 and geritinib for the treatment of acute myeloid leukemia has a synergistic effect.

Example 20

The role of TP-P1 in alleviating LPS-induced lung inflammation in septic mice

Experimental method: Female Balb/c mice were randomly divided into 5 groups, 6 in each group, respectively solvent control group, model group, TP-P1 low-dose group (500 μg/kg/d), TP-P1 medium-dose group (1000 μg/kg/d), TP-P1 high-dose group (1500 μg/kg/d), each group was given preventive administration two days before LPS injection, intraperitoneal injection once a day, continuous administration for 2 days, and the administration volume was 1 0ml/kg; on the third day, the model group, TP-P1 low, medium, and high dose groups were given an intraperitoneal injection of 10 mg/kg LPS, and the administration volume was 5ml/kg; on the fourth day, the lung tissue of the mice was aseptically removed, and the expressions of inflammatory factors such as IL-1β, IL-6, TNF-α, IFN-γ were detected by RT-qPCR technology, and the data of each group were normalized with the solvent control group.

Table 7 TP-P1 alleviates the effect of LPS-induced sepsis mice lung inflammation

*, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control group). ^, p<0.05; ^^, p<0.01; ^^^, p<0.001 (comparison between administration group and model group).

The results showed that: TP-P1 can inhibit the release of inflammatory factors such as IL-1β, IL-6, TNF-α, IFN-γ, and in a dose-dependent manner, relieve LPS-induced lung inflammation in septic mice (Table 7).

Example 21

Pharmacokinetic properties of TP-P1

Experimental method: 8 male SD rats were divided into 2 groups by random grouping. The randomly grouped SD rats were fasted overnight but allowed to drink water freely; 12 hours later, the first group was given the aqueous solution of compound TP-P1, and the dosage was 1.6 mg/kg; the second group was given the olive oil suspension of TP, and the dosage was 1.0 mg/kg; Blood was collected at min, 90min, 2h, 4h, and 6h, transferred to a 1.5mL centrifuge tube pretreated with sodium heparin, and centrifuged (8000rpm/min, 5min, 4°C) to obtain plasma, which was stored in a -80°C refrigerator.

Processing method: Plasma samples were processed by 1:3 methanol protein precipitation method, and after vortex centrifugation, the supernatant was taken to analyze the concentration of TP in plasma by UPLC-MS/MS, and the data were processed by Phoenix 64 software to obtain pharmacokinetic parameters.

The pharmacokinetic property of table 8 TP-P1

The results showed that the pharmacokinetic parameters such as AUC _(0-t) and Cmax of TP-P1 were superior to those of TP, and the absorption of TP-P1 was significantly higher than that of TP in the case of equimolar oral administration (Table 8).

The above are only preferred implementations of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims (8)

A class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof, characterized in that the chemical structural formula of the compound is as shown in formula (I):

in: n1 is 1~6; n2 is 1~6; n3 is 0 or 1; X is a carbon, nitrogen or oxygen atom; HA is selected from hydrochloric acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid. A class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof according to claim 1, characterized in that, Described triptolide prodrug is selected from following compounds:

The preparation method of a class of triptolide prodrug or its pharmaceutically acceptable salt, polymorph or solvate as claimed in claim 1 and 2, is characterized in that, its specific preparation steps are as follows:

The first step: triptolide reacts with acid chloride under the action of DMAP to generate intermediate II: Dissolving triptolide and DMAP in anhydrous dichloromethane, adding acid chloride at a low temperature of -10 to 5°C, and reacting at a temperature of 0 to 30°C for 6 to 12 hours; then washing the obtained reaction solution with dilute hydrochloric acid, saturated sodium bicarbonate and brine in sequence, and drying with anhydrous sodium sulfate; suction filtration, concentration of the filtrate, and purification by column chromatography to obtain intermediate II; Second step: intermediate II reacts with carboxylic acid compound under the effect of sodium iodide and potassium carbonate to generate compound III: Dissolve the purified intermediate II in anhydrous DMF, then add sodium iodide and carboxylic acid, react for 0.5 to 1 hour, add potassium carbonate, then heat it to 40 to 70°C and react for 3 to 12 hours to obtain a reaction solution; then pour the reaction solution into water, extract with ethyl acetate, wash with aqueous sodium bicarbonate and saline, dry, filter with suction, concentrate the filtrate, and purify by column chromatography to obtain compound III; The third step: Salt formation of compound III with acid to obtain target compound I: Dissolve the purified compound III in ethyl acetate, add inorganic acid or organic acid, react at a temperature of 0-30° C. for 6-12 hours, filter with suction, and dry; the target compound I is finally obtained. A pharmaceutical composition, comprising a class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof as described in claim 1. A pharmaceutical composition as claimed in claim 4 is used in the preparation of medicines for preventing/treating malignant tumors, inflammatory diseases, immune diseases and viral infectious diseases. The malignant tumor according to claim 5, wherein the malignant tumor comprises acute myeloid leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, bile duct cancer, gastric cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma and neuroblastoma. According to the immune disease and inflammatory disease described in claim 5, it is characterized in that, the immune disease and inflammatory disease include 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 infection, cytokine release syndrome caused by viral infection. A use of the pharmaceutical composition as claimed in claim 4 in combination with an FLT3 inhibitor for the treatment of acute myeloid leukemia.

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