A triterpene saponin and its use in the treatment of cancer

Abstract

The application discloses a trptolide derivative targeted to ROS activation and a preparation method and application thereof. The ROS-sensitive prodrug is prepared by introducing a phenylboronic acid group into a free hydroxyl group of trptolide (TP), and has remarkable beneficial effects. First, by using the characteristic that the ROS level in IPF lung tissue is significantly higher than that in normal lung tissue, after the prodrug enters the body, it can be specifically activated by the high-concentration ROS in the IPF lung tissue, and accurately release TP to play an anti-pulmonary fibrosis role. In normal tissues, due to the low concentration of ROS, the amount of prodrug converted to TP is very small, greatly reducing the toxicity of the drug to normal tissues, effectively solving the problems of narrow treatment window and large toxic side effects of TP, and improving the drug safety.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a triptolide derivative targeting ROS activation, its preparation method, and its uses. Background Technology

[0002] Idiopathic pulmonary fibrosis (PF) is a progressive lower respiratory tract disease of unknown etiology, characterized by diffuse alveolitis and alveolar structural disorder that ultimately leads to pulmonary fibrosis. It is prevalent in people over 65 years of age, with smoking, dust inhalation, and genetic factors being significant risk factors. IPF is fatal and irreversible. Clinical manifestations include progressive dyspnea, dry cough, and often accompanied by weight loss, malaise, fatigue, and fever. The patient's condition gradually deteriorates over months or years, ultimately leading to death from respiratory failure. Most patients experience a rapid decline to the end stage of the disease within 2-5 years of symptom onset, with an average survival of only 2.8 years. Therefore, it is often referred to as a "tumor-like disease" and was included in the first batch of rare disease catalogs in 2018.

[0003] Currently, treatment options for IPF are very limited. Pirfenidone and nintedanib are FDA-approved drugs for IPF, but these two drugs can only alleviate symptoms to a certain extent and are unlikely to significantly prolong patient survival. Although lung transplantation is considered a relatively effective treatment in clinical practice, the 5-year survival rate after treatment is less than 42%. Due to the extremely high mortality rate of IPF and the fact that its pathogenesis is not fully understood, there are still no truly effective treatments. Therefore, finding and developing new drugs to treat IPF is of great significance for improving patient survival rates and reducing mortality.

[0004] Triptolide (TP), a natural product extracted from the root of *Tripterygium wilfordii*, a plant in the Celastraceae family, has shown therapeutic potential in delaying and inhibiting pulmonary fibrosis. However, TP suffers from poor water solubility and a narrow therapeutic window, which greatly limits its clinical application and development. How to reduce its toxic side effects while preserving TP's anti-IPF activity, making it safe and effective for clinical treatment, has become a crucial issue that urgently needs to be addressed. Summary of the Invention

[0005] The main objective of this invention is to propose a triptolide derivative targeting ROS activation, its preparation method, and its uses. The aim is to provide a triptolide derivative that reduces the toxic side effects of TP while maintaining the anti-IPF activity of TP. This compound can be used in the preparation of drugs that delay and inhibit pulmonary fibrosis, thus solving the toxicity problem in clinical applications.

[0006] To achieve the above objectives, this invention proposes a triptolide derivative targeting ROS activation, the chemical structural formula of which is shown in Formula I or Formula II below:

[0007]

[0008] Formula I

[0009]

[0010] Formula II

[0011] Where Q is Q1 or Q2, and T is any one of T1, T2, T2, T3, T4, T5, and T6;

[0012] The structure of Q1 is shown below:

[0013] ;

[0014] The structure of Q2 is shown below:

[0015] ;

[0016] The structure of T1 is shown below:

[0017]

[0018] The structure of T2 is shown below:

[0019]

[0020] The structure of T3 is shown below:

[0021]

[0022] The structure of T4 is shown below:

[0023]

[0024] The structure of T5 is shown below:

[0025]

[0026] The structure of T6 is shown below:

[0027]

[0028] Y = O or NH;

[0029] R1, R2 = H or CH3.

[0030] Preferably, the triptolide derivative is selected from the following structures:

[0031] ; ;

[0032] ; ;

[0033] ; ;

[0034] ;

[0035] .

[0036] This invention also proposes a method for preparing the ROS-targeting triptolide derivative as described above, comprising the following steps:

[0037] (1) Add anhydrous DMF, TP and NPC to the reactor, pass in an inert gas, stir under an ice-water bath, dissolve anhydrous TEA and DMAP in anhydrous DMF to obtain a mixture, slowly add the mixture dropwise to the reactor, stir, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, dry, concentrate to obtain an oily crude product, namely compound TP-NPC.

[0038] (2) Add the compound TP-NPC, anhydrous triethylamine, and 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF. Under inert gas protection, stir until the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate to obtain a yellow oily crude product, and obtain a white solid by reverse column chromatography, which is the ROS-targeted triptolide derivative.

[0039] Preferably, in step (1), the molar ratio of TP, NPC, TEA and DMAP is 1:(4~6):(4~6):(4~6); the inert gas includes nitrogen; the washing is performed using saturated Na2CO3 and NaCl; and the drying is performed using Na2SO4.

[0040] In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, and 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4~6):(4~6).

[0041] This invention also proposes a method for preparing the ROS-targeting triptolide derivative as described above, comprising the following steps:

[0042] (1) Add anhydrous DMF, TP and NPC to the reactor, pass in an inert gas, stir under an ice-water bath, dissolve anhydrous TEA and DMAP in anhydrous DMF to obtain a mixture, slowly add the mixture dropwise to the reactor, stir, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, dry, concentrate to obtain an oily crude product, namely compound TP-NPC.

[0043] (2) Add the compound TP-NPC, anhydrous triethylamine, and 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF. Under inert gas protection, stir until the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate to obtain a yellow oily crude product, and obtain a white solid by reverse column chromatography.

[0044] (3) Add acetone and water to the reactor, add the white solid, sodium periodate and ammonium acetate, stir under inert gas protection, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate under reduced pressure to obtain white solid, that is, the ROS-targeted triptolide derivative.

[0045] Preferably, in step (1), the molar ratio of TP, NPC, TEA and DMAP is 1:(4~6):(4~6):(4~6); the inert gas includes nitrogen; the washing is performed using saturated Na2CO3 and NaCl; and the drying is performed using Na2SO4.

[0046] In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, and 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4~6):(4~6);

[0047] In step (3), the molar ratio of the white solid, sodium periodate and ammonium acetate is 1:(1~3):(1~3).

[0048] This invention also proposes a method for preparing the ROS-targeting triptolide derivative as described above, comprising the following steps:

[0049] (1) Add anhydrous THF, 4-(hydroxymethyl)phenylboronic acid pinacol ester, NPC and TEA in a molar ratio of 1:(1~3):(1~3) to the reactor, stir under inert gas protection, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, add DCM, wash and concentrate to obtain a yellow oily crude product, and purify to obtain a white solid.

[0050] (2) Add anhydrous THF to the reactor, add the white solid, tert-butyl (pyrrolidine-2-ylmethyl)carbamate and TEA in a molar ratio of 1:(1~3):(1~3), stir under inert gas protection, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add DCM, wash and concentrate to obtain a yellow oily crude product, and purify to obtain a colorless oily product;

[0051] (3) Add the colorless oily product to the reactor, add anhydrous DCM and TFA, stir, and concentrate under reduced pressure to obtain a pink oily product;

[0052] (4) Add TP-NPC, anhydrous triethylamine and the pink oily product in a molar ratio of 1:(4~6):(4~6) to the reactor. Stir under inert gas protection. After the reaction is complete, concentrate under reduced pressure to remove the solvent. Add DCM, wash and concentrate to obtain a yellow oily crude product. Purify to obtain a white solid, which is the ROS-targeted triptolide derivative.

[0053] This invention also proposes a method for preparing the ROS-targeting triptolide derivative as described above, comprising the following steps:

[0054] Anhydrous DMF was added to the reactor, along with TP, 4-(bromomethyl)phenylboronic acid pinacol ester, and Ag2O in a molar ratio of 1:(4~6):(4~6). The mixture was slowly heated to room temperature at 0~2℃. Under inert gas protection, the mixture was stirred, concentrated under reduced pressure to remove the solvent, DCM was added, and the mixture was washed, dried, concentrated, purified, and evaporated to obtain a white solid, which is the ROS-targeted triptolide derivative.

[0055] The present invention also proposes the use of a triptolide derivative targeting ROS activation as described above in the preparation of a medicament for delaying or inhibiting pulmonary fibrosis.

[0056] Preferably, the triptolide derivative targeting ROS activation can reduce the toxic side effects of TP.

[0057] Reactive oxygen species (ROS) are important signaling molecules in the body, playing a crucial role in metabolic processes. Normal lung tissue maintains a low ROS concentration, but in IPF lung tissue, ROS levels are significantly increased. Therefore, they can be used to develop ROS-sensitive prodrugs. Introducing phenylboronic acid groups as carriers into the original drug to design ROS-sensitive prodrugs has become an effective approach for ROS-based drug development. This invention introduces phenylboronic acid groups into the free hydroxyl group of TP (potassium phosphate). After the compound enters the body, it is activated by the high concentration of ROS in IPF lung tissue to generate TP. Because the ROS concentration in normal tissues is low, the amount of drug converted to TP in normal tissues is less, thereby reducing the drug's toxicity to normal tissues. The reaction mechanism is as follows:

[0058]

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] (1) The technical solution provided by this invention prepares a ROS-sensitive prodrug by introducing phenylboronic acid groups into the free hydroxyl groups of triptolide (TP), which has significant beneficial effects. First, taking advantage of the fact that the ROS level in IPF lung tissue is significantly higher than that in normal lung tissue, the prodrug can be specifically activated by the high concentration of ROS in IPF lung tissue after entering the body, and accurately release TP to exert its anti-pulmonary fibrosis effect. In normal tissue, due to the low concentration of ROS, the amount of TP converted from the prodrug is extremely small, which greatly reduces the toxicity of the drug to normal tissue, effectively solves the problem of narrow therapeutic window and large toxic side effects of TP, and improves the safety of medication.

[0061] (2) The activation mechanism based on ROS concentration differences in this invention enables targeted drug release at the lesion site. Compared with traditional drug delivery methods, it allows TP to act more concentratedly on the IPF lesion area, increasing the drug concentration at the lesion site, enhancing the therapeutic effect on IPF, and helping to delay and inhibit the progression of pulmonary fibrosis, providing a safer and more effective treatment option for IPF patients. Meanwhile, the approach of designing ROS-sensitive prodrugs using phenylboronic acid groups as carriers provides new ideas and methods for developing new drugs based on ROS to treat diseases such as IPF, which has significant theoretical implications and broad application prospects. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0063] Figure 1 The diagram shows the metabolic transformation of compound 1 in plasma (A) and plasma with 200 µM H2O2 added (B) in Example 10 of the present invention.

[0064] Figure 2 The images show the serological indicators of mice after two weeks of continuous intraperitoneal injection of compound 1 in Example 11 of this invention. A is a quantitative detection graph of liver function indicators (ALT and AST) after administration to mice, and B is a quantitative detection graph of kidney function indicators (BUN and Creatine).

[0065] Figure 3This is a stained section of lung tissue from mice after two weeks of continuous intraperitoneal injection of compound 1 in Example 11 of the present invention.

[0066] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0068] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0069] Example 1 Synthesis of ROS-targeted triptolide derivative (compound 1)

[0070]

[0071] The steps are as follows:

[0072] (1) Add 2 mL of anhydrous DMF to a 10 mL double-necked round-bottom flask, then add TP (0.056 mmol) and NPC (0.28 mmol). Purge with nitrogen and start stirring in an ice-water bath. Dissolve anhydrous TEA (0.28 mmol) and DMAP (0.28 mmol) in 1 mL of anhydrous DMF and slowly add the solution dropwise to the flask. Stir for 24 hours and check the reaction is complete by TLC. Remove the solvent by vacuum concentration, add 5 mL of DCM, wash with saturated Na2CO3 and NaCl, dry with Na2SO4, and concentrate to obtain an oily crude product, namely compound TP-NPC.

[0073]

[0074] (2) Add 2 mL of anhydrous THF to a 15 mL round-bottom flask, then add TP-NPC (0.056 mmol), anhydrous triethylamine (0.28 mmol), and 4-(aminomethyl)phenylboronic acid pinacol ester (0.28 mmol) from the above steps. Stir for 1 hour under nitrogen protection, and the reaction is confirmed to be complete by TLC. The solvent is removed by concentration under reduced pressure, 5 mL of DCM is added, and the mixture is washed with saturated Na2CO3 and NaCl. The crude product is concentrated to obtain a yellow oily substance, which is then subjected to reverse column chromatography to obtain a white solid. The structure is confirmed to be compound 1 (16.62 mg, yield 47%). 1 H NMR (400 MHz, Chloroform-d) δ 7.78 (s, 1H), 7.76(s, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 5.38 (t, J = 6.1 Hz, 1H), 4.97 (s, 1H), 4.67 (s, 2H), 4.42 (qd, J = 15.3, 6.0 Hz, 2H), 3.81 (d, J = 3.2 Hz, 1H), 3.55– 3.43 (m, 2H), 2.68 (d, J = 12.9 Hz, 1H), 2.39 – 2.24 (m, 1H), 2.23 – 2.10(m, 2H), 2.01 – 1.85 (m, 2H), 1.55 (dd, J = 12.6, 5.2 Hz, 1H), 1.33 (s, 12H), 1.06 (s, 3H), 0.99 (d, J = 7.0 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H). ESI [M+H]+:620.2.

[0075] Example 2 Synthesis of ROS-targeted triptolide derivative (compound 2)

[0076]

[0077] The steps are as follows:

[0078] In a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, followed by compound 1 (0.026 mmol) obtained in Example 1, sodium periodate (0.052 mmol), and ammonium acetate (0.052 mmol). The mixture was stirred at room temperature for 2 hours under nitrogen protection, and the reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the mixture was washed with saturated NaCl. The solution was then concentrated under reduced pressure to obtain a white solid, which was identified as compound 2 (12.49 mg, 90% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.67 (d, J = 7.5 Hz, 2H), 7.28 (d, J = 7.5 Hz, 2H), 5.55 (d, J = 41.8Hz, 1H), 4.99 (t, J = 4.8 Hz, 1H), 4.68 (s, 2H), 4.45 (dt, J = 28.1, 5.2 Hz, 2H), 3.86 (dd, J = 15.5, 3.2 Hz, 1H), 3.52 (t, J = 4.6 Hz, 2H), 2.70 (d, J =13.0 Hz, 1H), 2.32 (d, J = 16.6 Hz, 1H), 2.18 – 2.10 ESI [M+H]+: 538.1.

[0079] Example 3 Synthesis of ROS-targeted triptolide derivative (compound 3)

[0080]

[0081] The steps are as follows:

[0082] (1) Add 2 mL of anhydrous DMF to a 10 mL double-necked round-bottom flask, then add TP (0.056 mmol) and NPC (0.28 mmol). Purge with nitrogen and start stirring in an ice-water bath. Dissolve anhydrous TEA (0.28 mmol) and DMAP (0.28 mmol) in 1 mL of anhydrous DMF and slowly add the solution dropwise to the flask. Stir for 24 hours and check the reaction is complete by TLC. Remove the solvent by vacuum concentration, add 5 mL of DCM, wash with saturated Na2CO3 and NaCl, dry with Na2SO4, and concentrate to obtain an oily crude product, namely compound TP-NPC.

[0083] (2) 2 mL of anhydrous THF was added to a 15 mL round-bottom flask, followed by TP-NPC (0.056 mmol), anhydrous triethylamine (0.28 mmol), and pinacol 4-(hydroxymethyl)phenylboronic acid (0.28 mmol). The mixture was stirred for 2 hours under nitrogen protection, and the reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated Na2CO3 and NaCl, and concentrated to obtain a yellow oily crude product. The crude product was purified by reverse column chromatography to obtain a white solid, which was identified as compound 3 (9 mg, yield 26.42%). 1 H NMR (400 MHz, Chloroform-d) δ 7.69 – 7.62 (m, 2H), 7.28 – 7.19 (m,2H), 5.13 (d, J = 12.6 Hz, 1H), 5.05 (d, J = 12.6 Hz, 1H), 4.70 (d, J = 0.9Hz, 1H), 4.61 – 4.45 (m, 3H), 3.66 (d, J = 3.1 Hz, 1H), 3.38 (dd, J = 3.1,1.0 Hz, 1H), 3.32 (d, J = 5.5 Hz, 1H), 2.21 – 2.10 (m, 1H), 2.02 (dq, J =16.0, 5.1, 4.4 Hz, 3H), 1.82 – 1.74 (m, 2H), 1.47 – 1.38 (m, 1H), 1.20 (s,12H), 0.92 (d, J = 3.3 Hz, 3H), 0.81 (dd, J = 7.0, 2.0 Hz, 3H), 0.69 (dd, J =6.9, 1.9 Hz, 3H). ESI [M+H]+: 621.3.

[0084] Example 4 Synthesis of ROS-targeted triptolide derivative (compound 4)

[0085]

[0086] The steps are as follows:

[0087] In a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, along with compound 3 (0.014 mmol) obtained in Example 3, sodium periodate (0.028 mmol), and ammonium acetate (0.028 mmol). The mixture was stirred under nitrogen protection at room temperature for 2 hours, and the reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the mixture was washed with saturated NaCl. The solution was then concentrated under reduced pressure to obtain a white solid, which was identified as compound 4 (7.3 mg, 90% yield). 1 H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J = 7.6 Hz, 2H), 7.45 (d, J = 7.7 Hz, 2H), 5.30 – 5.12 (m, 2H), 4.77 (d, J = 10.7 Hz, 1H), 4.59 (d, J = 3.6 Hz, 2H), 3.74 (d, J = 3.1 Hz, 1H), 3.45 (dd, J = 7.5, 3.0 Hz, 1H), 3.43 – 3.34 (m, 1H), 2.61 (dt, J = 14.0,5.0 Hz, 1H), 2.28 – 2.18 (m, 1H), 2.10 – 2.03 (m, 1H), 1.86 (dq, J = 13.8,6.4 Hz, 2H), 1.50 (dd, J = 12.5, 5.1 Hz, 1H), 1.27 (s, 1H), 1.19 – 1.11 (m,1H), 1.01 (s, 3H), 0.88 (dd, J = 9.2, 6.8 Hz, 3H), 0.77 (d, J = 7.0 Hz, 3H).ESI [M+H]+: 539.2.

[0088] Example 5 Synthesis of ROS-targeted triptolide derivative (compound 5)

[0089]

[0090] The steps are as follows:

[0091] (1) Add 5 mL of anhydrous THF to a 50 mL round-bottom flask, then add 1.28 mmol of 4-(hydroxymethyl)phenylboronic acid pinacol ester, 2.56 mmol of NPC, and 2.56 mmol of TEA. Stir under nitrogen protection at room temperature for 10 hours, and the reaction is confirmed to be complete by TLC. Remove the solvent by vacuum concentration, add 5 mL of DCM, wash with saturated Na2CO3 and NaCl, and concentrate to obtain a yellow oily crude product. Purify by silica gel column chromatography (PE:EA = 10:1) to obtain a white solid, which is identified as compound A1 (365.78 mg, yield 71.5%). 1 H NMR (400 MHz, Chloroform-d) δ 8.32 – 8.24 (m, 2H),7.85 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 7.7 Hz, 2H), 7.41 – 7.34 (m, 2H), 5.31(s, 2H), 1.35 (s, 12H). ESI [M+H]+: 400.1.

[0092]

[0093] (2) In a 50 mL round-bottom flask, add 5 mL of anhydrous THF, compound A1 (0.64 mmol), tert-butyl (pyrrolidone-2-ylmethyl)carbamate (1.28 mmol), and TEA (1.28 mmol). Stir under nitrogen protection at room temperature for 3 hours, and the reaction is confirmed to be complete by TLC. The solvent is removed by concentration under reduced pressure, 5 mL of DCM is added, and the mixture is washed with saturated Na2CO3 and NaCl. The crude product is concentrated to obtain a yellow oily product, which is purified by silica gel column chromatography (PE:EA = 4:1) to obtain a colorless oily product. The structure is confirmed to be compound A2. (244.82 mg, yield 85.6%). 1 H NMR (400 MHz, Chloroform-d) δ 7.73 (d, J = 7.8Hz, 2H), 7.29 (d, J = 7.6 Hz, 2H), 5.07 (d, J = 4.8 Hz, 2H), 3.87 (s, 1H),3.35 (dd, J = 6.9, 4.1 Hz, 2H), 3.28 – 3.04 (m, 2H), 1.87 (s, 2H), 1.78 (s,2H), 1.36 (d, J = 2.4 Hz, 9H), 1.27 (s, 12H). ESI [M+H]+: 461.3.

[0094]

[0095] (3) In a 25 mL round-bottom flask, compound A2 (0.55 mmol) was added, along with anhydrous DCM and TFA. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain a pink oily product. The structure was identified as compound A3 (232.58, yield 95%). 1 H NMR(400 MHz, Methanol-d4) δ 7.74 (d, J = 7.7 Hz, 2H), 7.38 (d, J = 7.6 Hz, 2H), 5.24 – 5.11 (m, 2H), 4.10 (dtt, J = 11.0, 7.2, 3.7 Hz, 1H), 3.56 (dt, J =10.8, 7.4 Hz, 1H), 3.48 (q, J = 5.3, 4.8 Hz, 1H), 3.14 – 2.97 (m, 2H), 2.13 (dt, J = 12.7, 7.8 Hz, 1H), 1.99 – 1.89 (m, 2H), 1.77 (dq, J = 11.4, 5.2 Hz,1H), 1.34 (s, 12H). ESI [M+H]+: 361.2.

[0096]

[0097] (4) The synthesis method was the same as that of compound 1. TP-NPC (0.056 mmol), anhydrous triethylamine (0.28 mmol), and compound A3 (0.28 mmol) were added and stirred at room temperature for 2 hours under nitrogen protection. The reaction was detected by TLC to be complete. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the mixture was washed with saturated Na2CO3 and NaCl. The crude product was concentrated to obtain a yellow oily product. The product was purified by reverse column chromatography to obtain a white solid. The structure was identified as compound 5 (12.60 mg, yield 26.42%). 1H NMR (400 MHz, Chloroform-d) δ 7.73 (d, J = 7.6 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 5.11 –5.01 (m, 2H), 4.85 (d, J = 3.8 Hz, 1H), 4.60 (d, J = 3.7 Hz, 2H), 3.91 (s,1H), 3.74 (d, J = 3.4 Hz, 1H), 3.44 (d, J = 3.4 Hz, 2H), 3.37 (dd, J = 13.1,4.7 Hz, 3H), 3.26 (s, 1H), 2.61 (d, J = 13.0 Hz, 1H), 2.24 (d, J = 17.7 Hz,1H), 2.14 – 1.98 (m, 2H), 1.86 (td, J = 13.7, 6.3 Hz, 4H), 1.50 (dd, J =12.4, 5.3 Hz, 2H), 1.27 (s, 12H), 1.20 – 1.09 (m, 2H), 0.98 (d, J = 8.2 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.78 (dd, J = 6.8, 3.5 Hz, 3H). ESI [M+H]+:747.4.

[0098] Example 6 Synthesis of ROS-targeted triptolide derivative (compound 6)

[0099]

[0100] The steps are as follows:

[0101] In a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, followed by compound 5 (0.016 mmol) obtained in Example 5, sodium periodate (0.032 mmol), and ammonium acetate (0.032 mmol). The mixture was stirred under nitrogen protection at room temperature for 2 hours, and the reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the mixture was washed with saturated NaCl. The solution was then concentrated under reduced pressure to give a white solid, which was identified as compound 6. (9.61 mg, 90% yield) 1H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J = 7.6 Hz, 2H), 7.31 (s, 2H), 5.09 (s, 2H), 4.85 (s, 1H), 4.59(s, 2H), 3.90 (s, 1H), 3.75 (s, 1H), 3.66 (q, J = 7.0 Hz, 1H), 3.49 – 3.39(m, 4H), 3.39 (s, 2H), 2.60 (s, 1H), 2.24 (d, J = 17.4 Hz, 1H), 2.06 (s, 2H), 1.88 (s, 2H), 1.76 (s, 3H), 1.48 (s, 2H), 1.01 – 0.96 (m, 3H), 0.93 – 0.89 (m, 3H), 0.78 (d, J = 6.9 Hz, 3H). ESI [M+H]+: 665.1.

[0102] Example 7 Synthesis of ROS-targeted triptolide derivative (compound 7)

[0103]

[0104] The steps are as follows:

[0105] 2 mL of anhydrous DMF was added to a 10 mL round-bottom flask, followed by TP (0.056 mmol), 4-(bromomethyl)phenylboronic acid pinacol ester (0.28 mmol), and Ag₂O (0.28 mmol). The mixture was slowly heated to room temperature from 0°C and stirred for 12 hours under nitrogen protection. The solvent was removed by vacuum concentration, and 5 mL of DCM was added. The mixture was washed with saturated Na₂CO₃ and NaCl, dried over Na₂SO₄, and concentrated to obtain an oily crude product. This product was purified by liquid chromatography, and the samples were combined and evaporated to dryness to obtain a white solid. The structure was identified as compound 7 (7.5 mg, yield 23%). 1H NMR (400 MHz, Chloroform-d) δ 7.82 – 7.76 (m, 2H), 7.26 (dt, J = 8.6, 1.0 Hz, 2H), 4.85 (dt, J = 12.5, 1.1 Hz, 1H), 4.85 – 4.78 (m,1H), 4.67 – 4.57 (m, 3H), 3.86 (d, J = 3.7 Hz, 1H), 3.62 (t, J = 3.8 Hz, 1H), 3.37 (dd, J = 5.1, 2.4 Hz, 1H), 3.00 (tq, J = 6.3, 1.3 Hz, 1H), 2.40 – 2.31(m, 1H), 2.28 – 2.19 (m, 1H), 2.09 (ddd, J = 12.5, 6.1, 2.5 Hz, 1H), 1.97 (ddd, J = 12.5, 6.2, 5.2 Hz, 1H), 1.86 (dtd, J = 11.0, 5.5, 1.7 Hz, 1H), 1.43(ddd, J = 12.1, 7.3, 4.9 Hz, 1H), 1.36 (ddd, J = 12.4, 7.5, 4.9 Hz, 1H), 1.26(s, 6H), 1.21 (s, 6H), 1.01 – 0.96 (m, 9H). [M+H]+: 577.3.

[0106] Example 8 Synthesis of ROS-targeted triptolide derivative (compound 8)

[0107]

[0108] The steps are as follows:

[0109] In a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, along with compound 7 (0.013 mmol) obtained in Example 7, sodium periodate (0.026 mmol), and ammonium acetate (0.026 mmol). The mixture was stirred under nitrogen protection at room temperature for 2 hours, and the reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the mixture was washed with saturated NaCl. The solution was then concentrated under reduced pressure to give a white solid, which was identified as compound 8 (5.79 mg, 90% yield). 1H NMR (500 MHz, Chloroform-d) δ 7.67 – 7.61 (m, 2H), 7.16 (dt, J = 8.3, 1.1 Hz, 2H), 4.87 (dt, J = 12.4, 1.1 Hz, 1H), 4.82 (dt, J = 12.4, 1.0 Hz, 1H), 4.67 – 4.57 (m,3H), 3.86 (d, J = 3.7 Hz, 1H), 3.62 (t, J = 3.8 Hz, 1H), 3.37 (dd, J = 5.1,2.4 Hz, 1H), 3.00 (tq, J = 6.2, 1.2 Hz, 1H), 2.36 (dddq, J = 12.3, 7.1, 4.9,1.1 Hz, 1H), 2.28 – 2.19 (m, 1H), 2.09 (ddd, J = 12.5, 6.1, 2.5 Hz, 1H), 1.97(ddd, J = 12.4, 6.2, 5.2 Hz, 1H), 1.87 (pd, J = 5.5, 3.7 Hz, 1H), 1.43 (ddd,J = 12.1, 7.3, 4.8 Hz, 1H), 1.36 (ddd, J = 12.4, 7.5, 4.9 Hz, 1H), 1.01 –0.96 (m, 9H). ESI [M+H]+: 495.2.

[0110] Example 9: Effects of ROS-targeting triptolide derivatives on TGF-β1-induced transformation of lung fibroblasts into lung myofibroblasts

[0111] Lung fibroblasts were seeded in DMEM mixed medium containing 10% fetal bovine serum and cultured in a 5% CO2 incubator. After treatment with 20 ng / mL TGF-β1, cells were divided into a control group (treated with only 20 ng / mL TGF-β1) and a TP derivative treatment group. After cell administration (0.1 µM), cells were cultured for another 24 h, the supernatant was discarded, cells were washed three times with PBS, and cells were collected. Sample processing and hydroxyproline quantification were performed according to the method described in the literature (Eur J Med Chem. 279 (2024) 116839), as shown in Table 1. The inhibition rate was calculated as follows: Inhibition rate (%) = (Control group - amount of hydroxyproline after administration) / Control group * 100%. The results showed that the TP derivative could inhibit the TGF-β1-induced biosynthesis of proline.

[0112] Table 1. Inhibition of TGF-β1-induced hydroxyproline production by TP derivatives

[0113] compound Inhibition rate (%) compound Inhibition rate (%) control group - 5 59.5 1 80.5 6 63.3 2 74.9 7 54.8 3 65.7 8 50.2 4 68.2

[0114] Example 10: Study on ROS-dependent activation of compound 1

[0115] TP derivatives were co-incubated with mouse plasma and with mouse plasma supplemented with 200 µM H2O2 for different times. The incubation solutions were then analyzed by liquid chromatography to determine the concentration of the TP derivatives. The experimental results are as follows: Figure 1 As shown. Experimental results indicate that compound 1 is metabolically stable in mouse plasma, and its metabolic half-life t0 is [not specified]. 1 / 2 > 1h ( Figure 1 (A). In mouse plasma containing 200 µM H2O2, compound 1 can be activated by H2O2 to generate TP, whose t 1 / 2 = 37.2 min ( Figure 1 (B)

[0116] Example 11: In vivo toxicity and anti-idiopathic pulmonary fibrosis study of compound 1

[0117] A mouse model of pulmonary fibrosis (IPF) was established by direct intratracheal injection of bleomycin (3 mg / kg) into 6-8 week old C57BL / 6 mice (20-25 g). The mice were divided into five groups: a blank control group, a model group, a nibunida (BIBF1120) group (60 mg / kg, po), a low-dose compound 1 group (0.33 mg / kg), and a high-dose compound 1 group (0.66 mg / kg). Administration began on day 1 of bleomycin induction or day 5 after bleomycin induction. The positive control group received oral BIBF1120 (dissolved in 0.5% sodium carboxymethyl cellulose (MC) + 0.2% Tween 80 + 99.3% H2O) once daily, while the compound 1 group received intraperitoneal injection of the corresponding concentration of compound 1 (dissolved in H2O) once daily. After 14 days of continuous administration, the following procedures were performed: (1) Blood was collected and serum was separated. The concentrations of alanine aminotransferase (ALT) (Wuhan Huamei Biotechnology Co., Ltd.; CAS: CSB-E16539m), aspartate aminotransferase (AST) (Wuhan Huamei Biotechnology Co., Ltd.; CAS: CSB-E12649m), blood urea nitrogen (BUN) (Qingdao Jieshikang Biotechnology Co., Ltd.; CAS: SJH-033595) and creatine (Cream) (Shanghai Huabang Biotechnology Co., Ltd.; CAS: HB-P9S3251X) in the serum were detected. Figure 2 (2) Animals were weighed and euthanized. After taking photographs of the tissues, the liver, kidney, and lung tissues were immersed in 10% formalin for pathological staining and observation. Figure 3 .

[0118] Figure 2 The serological parameters of mice after continuous intraperitoneal injection of compound 1 for two weeks are shown in Figure A, where quantitative detection of liver function parameters (ALT and AST) and renal function parameters (BUN and Creatine) are shown in Figure B. The control group represents the untreated group. The results show that after continuous intraperitoneal injection of compound 1 for two weeks, there was no significant difference in serological parameters between the compound 1-treated group and the normal control group, indicating that compound 1 has no significant toxic side effects on the liver and kidneys.

[0119] Figure 3 This image shows stained sections of mouse lung tissue after two weeks of continuous intraperitoneal injection of compound 1. In a bleomycin-induced IPF model in mice, compound 1, after two weeks of continuous administration, significantly reduced bleomycin-induced lung inflammation and inhibited bleomycin-induced pulmonary fibrosis, and significantly improved bleomycin-induced lung damage in mice. The high-dose group of compound 1 showed better improvement in pathological fibrosis of mouse lung tissue than the positive control drug nibunida.

[0120] In summary, the ROS-targeting triptolide prodrug compound of this invention can inhibit or delay pulmonary fibrosis and reduce the toxicity of the drug to normal tissues, effectively solving the problems of narrow therapeutic window and large toxic side effects of TP. It has great research value in anti-IPF.

[0121] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A triptolide derivative targeting ROS activation, characterized in that, The triptolide derivative is selected from the following structures: ； ； ； ； ； ； ； 。 2. A method for preparing a ROS-targeting triptolide derivative as described in claim 1, characterized in that, Includes the following steps: (1) Add anhydrous DMF, TP and NPC to the reactor, pass in an inert gas, stir under an ice-water bath, dissolve anhydrous TEA and DMAP in anhydrous DMF to obtain a mixture, slowly add the mixture dropwise to the reactor, stir, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, dry, concentrate to obtain an oily crude product, namely compound TP-NPC. (2) Add the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester or 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stir under inert gas protection, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate to obtain a yellow oily crude product, and obtain a white solid by reverse column chromatography, which is the ROS-targeted triptolide derivative; The structure of the NPC is as follows: ； The structural formula of the TP-NPC is as follows: 。 3. The method for preparing the ROS-targeted triptolide derivative as described in claim 2, characterized in that, In step (1), the molar ratio of TP, NPC, TEA and DMAP is 1:(4~6):(4~6):(4~6); the inert gas includes nitrogen; the washing is performed using saturated Na2CO3 and NaCl; the drying is performed using Na2SO4. In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester or 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4~6):(4~6).

4. A method for preparing a ROS-targeting triptolide derivative as described in claim 1, characterized in that, Includes the following steps: (1) Add anhydrous DMF, TP and NPC to the reactor, pass in an inert gas, stir under an ice-water bath, dissolve anhydrous TEA and DMAP in anhydrous DMF to obtain a mixture, slowly add the mixture dropwise to the reactor, stir, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, dry, concentrate to obtain an oily crude product, namely compound TP-NPC. (2) Add the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester or 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stir under inert gas protection, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate to obtain a yellow oily crude product, and obtain a white solid by reverse column chromatography. (3) Add acetone and water to the reactor, add the white solid, sodium periodate and ammonium acetate, stir under inert gas protection, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, concentrate under reduced pressure to obtain white solid, i.e. ROS-targeted triptolide derivative. The structure of the NPC is as follows: ； The structural formula of the TP-NPC is as follows: 。 5. The method for preparing the ROS-targeted triptolide derivative as described in claim 4, characterized in that, In step (1), the molar ratio of TP, NPC, TEA and DMAP is 1:(4~6):(4~6):(4~6); the inert gas includes nitrogen; the washing is performed using saturated Na2CO3 and NaCl; the drying is performed using Na2SO4. In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester or 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4~6):(4~6); In step (3), the molar ratio of the white solid, sodium periodate and ammonium acetate is 1:(1~3):(1~3).

6. A method for preparing a ROS-targeting triptolide derivative as described in claim 1, characterized in that, Includes the following steps: (1) Add anhydrous THF and 4-(hydroxymethyl)phenylboronic acid pinacol ester, NPC and TEA in a molar ratio of 1:(1~3):(1~3) to the reactor. Stir under inert gas protection. After the reaction is complete, concentrate under reduced pressure to remove the solvent. Add DCM, wash and concentrate to obtain a yellow oily crude product. Purify to obtain a white solid. (2) Add anhydrous THF to the reactor, add the white solid, tert-butyl (pyrrolidine-2-ylmethyl)carbamate and TEA in a molar ratio of 1:(1~3):(1~3), stir under inert gas protection, after the reaction is complete, concentrate under reduced pressure to remove the solvent, add DCM, wash and concentrate to obtain a yellow oily crude product, and purify to obtain a colorless oily product. (3) Add the colorless oily product to the reactor, add anhydrous DCM and TFA, stir, and concentrate under reduced pressure to obtain a pink oily product; (4) Add TP-NPC, anhydrous triethylamine and the pink oily product in a molar ratio of 1:(4~6):(4~6) to the reactor. Stir under inert gas protection. After the reaction is complete, concentrate under reduced pressure to remove the solvent. Add DCM, wash and concentrate to obtain a yellow oily crude product. Purify to obtain a white solid, which is the ROS-targeted triptolide derivative. The structure of the NPC is as follows: ； The structural formula of the TP-NPC is as follows: 。 7. A method for preparing a ROS-targeting triptolide derivative as described in claim 1, characterized in that, Includes the following steps: Anhydrous DMF was added to the reactor, along with TP, 4-(bromomethyl)phenylboronic acid pinacol ester, and Ag2O in a molar ratio of 1:(4~6):(4~6). The mixture was slowly heated to room temperature at 0~2℃. Under inert gas protection, the mixture was stirred, concentrated under reduced pressure to remove the solvent, DCM was added, and the mixture was washed, dried, concentrated, purified, and evaporated to obtain a white solid, which is the ROS-targeted triptolide derivative.

8. Use of a triptolide derivative targeting ROS activation as described in claim 1 in the preparation of a medicament for delaying or inhibiting pulmonary fibrosis.

9. The use as described in claim 8, characterized in that, The triptolide derivative targeting ROS activation can reduce the toxic side effects of TP.

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