Targeted ROS (reactive oxygen species) activated triptolide derivative as well as preparation method and application thereof

Abstract

The invention discloses a targeted ROS (reactive oxygen species) activated triptolide derivative as well as a preparation method and application thereof, ROS sensitive prodrugs are prepared by introducing phenylboronic acid groups into free hydroxyl groups of triptolide (TP), and the targeted ROS activated triptolide derivative has remarkable beneficial effects. Firstly, by means of the characteristic that the ROS level in IPF lung tissue is remarkably higher than that in normal lung tissue, after entering the body, the prodrug can be specifically activated by high-concentration ROS in the IPF lung tissue, TP is accurately released, and the anti-pulmonary fibrosis effect is achieved. And in normal tissues, because the ROS concentration is relatively low and the amount of TP generated by prodrug conversion is extremely small, the toxicity of the drug to the normal tissues is greatly reduced, the problems of narrow TP therapeutic window and large toxic and side effects are effectively solved, and the medication safety is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a triptolide derivative targeting ROS activation, a preparation method thereof, and uses thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (PF) is a progressive lower respiratory tract disease of unknown etiology, characterized by diffuse alveolitis and alveolar structural disorder, ultimately leading to pulmonary fibrosis. Approximately 5 million people worldwide suffer from this disease. With the aging population in my country, the number of IPF patients has been rising annually, projected to reach 320,000 by 2032. The disease is most common in people over 65 years old, with smoking, dust inhalation, and genetic factors as significant risk factors. IPF is fatal and irreversible, with clinical manifestations including progressive dyspnea and a dry cough, often accompanied by weight loss, malaise, fatigue, and fever. Patients' condition gradually worsens over months or years, ultimately leading to death from respiratory failure. Most patients experience rapid deterioration to the terminal stage within 2-5 years of symptom onset, with an average survival of only 2.8 years. It has been dubbed a "tumor-like disease" and was added to the First List of Rare Diseases in 2018.

[0003] Currently, treatment options for IPF are very limited. Pirfenidone and nintedanib are FDA-approved IPF treatments, but these two drugs can only alleviate patients' symptoms to a certain extent and are unlikely to significantly prolong their survival. Although lung transplantation is currently considered a relatively effective treatment option, the 5-year survival rate after recovery is less than 42%. Due to the extremely high mortality rate of IPF and the incomplete understanding of its pathogenesis, there is still no effective treatment available. Therefore, the search and development of new drugs to treat IPF is of great significance for improving patient survival and reducing mortality.

[0004] Triptolide (TP), a natural product extracted from the root of the Celastraceae plant Tripterygium wilfordii, has shown therapeutic potential in delaying and inhibiting pulmonary fibrosis. However, TP suffers from poor water solubility and a narrow therapeutic window, which significantly limits its clinical application and development. Reducing TP's toxic side effects while preserving its anti-IPF activity remains a key challenge, ensuring its safe and effective clinical application. Summary of the Invention

[0005] The main purpose of the present invention is to propose a triptolide derivative that targets ROS activation, its preparation method, and use. The purpose is to provide a triptolide derivative that reduces the toxic and side effects of TP while maintaining the anti-IPF activity of TP. The compound can be used to prepare drugs for delaying and inhibiting pulmonary tissue fibrosis, which can solve the toxicity problem in clinical application.

[0006] To achieve the above objectives, the present invention provides a triptolide derivative targeting ROS activation, wherein the chemical structure of the triptolide derivative is shown in Formula I or Formula II below:

[0007]

[0008] Wherein, Q is Q1 or Q2, and T is any one of T1, T2, T2, T3, T4, T5, and T6;

[0009] The structure of Q1 is as follows:

[0010]

[0011] The structure of Q2 is as follows:

[0012]

[0013] The structure of T1 is as follows:

[0014]

[0015] The structure of T2 is as follows:

[0016]

[0017] The structure of T3 is as follows:

[0018]

[0019] The structure of T4 is as follows:

[0020]

[0021] The structure of T5 is as follows:

[0022]

[0023] The structure of T6 is as follows:

[0024]

[0025] Y=O or NH;

[0026] R1, R2 = H or CH3.

[0027] Preferably, the triptolide derivative is selected from the following structures:

[0028]

[0029]

[0030] The present invention also provides a method for preparing the triptolide derivative targeting ROS activation as described above, comprising the following steps:

[0031] (1) Anhydrous DMF, TP, and NPC were added to a reactor, inert gas was introduced, and the mixture was stirred under an ice-water bath. Anhydrous TEA and DMAP were dissolved in anhydrous DMF to obtain a mixed solution, which was slowly added dropwise to the reactor and stirred. After the reaction was complete, the solvent was removed by concentration under reduced pressure, triethylamine was added, the mixture was washed, dried, and concentrated to obtain an oily crude product, namely, compound TP-NPC;

[0032] (2) Add the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stir under the protection of inert gas, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, and concentrate to obtain a yellow oily crude product, which is subjected to reverse column chromatography to obtain a white solid, i.e., a triptolide derivative targeting ROS activation.

[0033] 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 method uses saturated Na2CO3 and NaCl; the drying method uses Na2SO4;

[0034] In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4-6):(4-6).

[0035] The present invention also provides a method for preparing the triptolide derivative targeting ROS activation as described above, comprising the following steps:

[0036] (1) Anhydrous DMF, TP, and NPC were added to a reactor, inert gas was introduced, and the mixture was stirred under an ice-water bath. Anhydrous TEA and DMAP were dissolved in anhydrous DMF to obtain a mixed solution, which was slowly added dropwise to the reactor and stirred. After the reaction was complete, the solvent was removed by concentration under reduced pressure, triethylamine was added, the mixture was washed, dried, and concentrated to obtain an oily crude product, namely, compound TP-NPC;

[0037] (2) adding the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stirring under the protection of inert gas, and after the reaction is complete, concentrating under reduced pressure to remove the solvent, adding triethylamine, washing, and concentrating to obtain a yellow oily crude product, which is then subjected to reverse phase column chromatography to obtain a white solid;

[0038] (3) Add acetone and water to the reactor, add the white solid, sodium periodate and ammonium acetate, and stir under the protection of inert gas. After the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, and concentrate under reduced pressure to obtain a white solid, which is a triptolide derivative targeting ROS activation.

[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 method uses saturated Na2CO3 and NaCl; the drying method uses Na2SO4;

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

[0041] In step (3), the molar ratio of the white solid, sodium periodate and ammonium acetate is 1:(1-3):(1-3).

[0042] The present invention also provides a method for preparing the triptolide derivative targeting ROS activation as described above, comprising the following steps:

[0043] (1) Anhydrous THF, 4-(hydroxymethyl)phenylboronic acid pinacol ester, NPC, and TEA in a molar ratio of 1:(1-3):(1-3) were added to a reactor and stirred under the protection of an inert gas. After the reaction was complete, the solvent was removed by concentration under reduced pressure, and DCM was added. The product was washed and concentrated to obtain a yellow oily crude product, which was purified to obtain a white solid.

[0044] (2) adding anhydrous THF to a reactor, adding the white solid, tert-butyl (pyrrolidin-2-ylmethyl)carbamate, and TEA in a molar ratio of 1:(1-3):(1-3), stirring under the protection of inert gas, and after the reaction is complete, concentrating under reduced pressure to remove the solvent, adding DCM, washing, and concentrating to obtain a yellow oily crude product, which is purified to obtain a colorless oily product;

[0045] (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;

[0046] (4) TP-NPC, anhydrous triethylamine, and the pink oily product in a molar ratio of 1:(4-6):(4-6) are added to the reactor, and stirred under the protection of inert gas. After the reaction is complete, the solvent is removed by concentration under reduced pressure, DCM is added, washed, and concentrated to obtain a yellow oily crude product, which is purified to obtain a white solid, i.e., a triptolide derivative targeting ROS activation.

[0047] The present invention also provides a method for preparing the triptolide derivative targeting ROS activation as described above, comprising the following steps:

[0048] Anhydrous DMF was added to the reactor, and TP, 4-(bromomethyl)phenylboronic acid pinacol ester and Ag2O in a molar ratio of 1:(4-6):(4-6) were added. The temperature was slowly raised to room temperature at 0-2°C. Under the protection of inert gas, the mixture was stirred and concentrated under reduced pressure to remove the solvent. DCM was added, the mixture was washed, dried, concentrated, purified, and spin-dried to obtain a white solid, which was a triptolide derivative targeting ROS activation.

[0049] The present invention also proposes a use of the triptolide derivative targeting ROS activation as described above in preparing a drug for delaying or inhibiting pulmonary tissue fibrosis.

[0050] Preferably, the triptolide derivative targeting ROS activation can reduce the toxic side effects of TP.

[0051] Reactive oxygen species (ROS) are important signaling molecules in the body and play a vital role in the body's metabolic processes. The ROS concentration in normal lung tissue is maintained at a low level, but in IPF lung tissue, the ROS level is significantly increased. Therefore, it can be used to develop ROS-sensitive prodrugs. The introduction of phenylboronic acid groups as carriers into the original drug to design ROS-sensitive prodrugs has become an effective way to develop new drugs based on ROS. The present invention introduces phenylboronic acid groups into the free hydroxyl group of TP. After the compound enters the body, it is activated by the high concentration of ROS in IPF lung tissue to generate TP. Since the ROS concentration in normal tissue is low, the amount of drug converted to TP in normal tissue is small, thereby reducing the toxicity of the drug to normal tissue. The reaction mechanism is as follows:

[0052]

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) In the technical solution provided by the present invention, a ROS-sensitive prodrug is prepared by introducing a phenylboronic acid group into the free hydroxyl group of triptolide (TP), which has significant beneficial effects. First, by utilizing 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, accurately releasing TP to exert its anti-pulmonary fibrosis effect. In normal tissue, due to the low ROS concentration, the amount of TP converted by the prodrug is extremely small, which greatly reduces the toxicity of the drug to normal tissue, effectively solving the problem of TP's narrow therapeutic window and large toxic side effects, and improving the safety of the drug.

[0055] (2) This activation mechanism based on ROS concentration differences in the present invention achieves targeted drug release at the lesion site. Compared with traditional drug delivery methods, it can enable TP to act more concentratedly on the IPF lesion area, increase the concentration of the drug at the lesion site, enhance the therapeutic effect on IPF, help delay and inhibit the progression of lung tissue fibrosis, and provide a safer and more effective treatment option for IPF patients. At the same time, the approach of designing ROS-sensitive prodrugs using phenylboronic acid groups as carriers provides new ideas and methods for the development of new ROS-based drugs for the treatment of diseases such as IPF, which has important theoretical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0057] Figure 1 Graph showing the metabolic transformation of compound 1 in Example 10 of the present invention in plasma (A) and in plasma with 200 μM H 2 O 2 added (B);

[0058] Figure 2 These are the serum indicators of mice after continuous intraperitoneal injection of Compound 1 for two weeks in Example 11 of the present invention. Panel A shows the quantitative detection of liver function indicators (ALT and AST) in mice after administration, and Panel B shows the quantitative detection of renal function indicators (BUN and Creatine).

[0059] Figure 3 This is a staining image of a section of mouse lung tissue after continuous intraperitoneal injection of Compound 1 for two weeks in Example 11 of the present invention.

[0060] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0061] In order to make the purpose, 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. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0062] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0063] Example 1 Synthesis of a triptolide derivative (Compound 1) targeting ROS activation

[0064]

[0065] Here are the steps:

[0066] (1) In a 10 mL two-necked round-bottom flask, 2 mL of anhydrous DMF was added, followed by TP (0.056 mmol) and NPC (0.28 mmol). Nitrogen was introduced and stirring was started in an ice-water bath. Anhydrous TEA (0.28 mmol) and DMAP (0.28 mmol) were dissolved in 1 mL of anhydrous DMF and slowly added dropwise to the flask. The mixture was stirred for 24 hours. TLC confirmed the reaction was 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, dried over Na2SO4, and concentrated to obtain an oily crude product, namely compound TP-NPC.

[0067]

[0068] (2) A 15 mL round-bottom flask was charged with 2 mL of anhydrous THF, and TP-NPC (0.056 mmol), anhydrous triethylamine (0.28 mmol), and 4-(aminomethyl)phenylboronic acid pinacol ester (0.28 mmol) from the above step were added. The mixture was stirred under nitrogen for 1 hour. TLC confirmed the reaction was 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 mixture was concentrated to obtain a yellow oily crude product. Reverse phase column chromatography gave a white solid, which was identified as compound 1 (16.62 mg, 47% yield).1 H NMR(400MHz,Chloroform-d)δ7.78(s,1H),7.76(s,1H),7.30(s,1H),7.28(s,1H),5.38(t,J=6.1Hz,1 H),4.97(s,1H),4.67(s,2H),4.42(qd,J=15.3,6.0Hz,2H),3.81(d,J=3.2Hz,1H),3.55–3.43(m,2H), 2.68(d,J=12.9Hz,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. 2Hz,1H),1.33(s,12H),1.06(s,3H),0.99(d,J=7.0Hz,3H),0.86(d,J=6.8Hz,3H).ESI[M+H]+:620.2.

[0069] Example 2 Synthesis of a triptolide derivative (Compound 2) targeting ROS activation

[0070]

[0071] Here are the steps:

[0072] To a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, along with compound 1 (0.026 mmol) obtained in Example 1, sodium periodate (0.052 mmol), and ammonium acetate (0.052 mmol). Under nitrogen, the mixture was stirred at room temperature for 2 hours. TLC confirmed the reaction was complete. The solvent was removed by concentration under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated NaCl and concentrated under reduced pressure to yield a white solid, which was identified as compound 2 (12.49 mg, 90% yield). 1H NMR(400MHz,Chloroform-d)δ7.67(d,J=7.5Hz,2H),7.28(d,J=7.5Hz,2H),5.55(d,J=41.8Hz,1H),4.9 9(t,J=4.8Hz,1H),4.68(s,2H),4.45(dt,J=28.1,5.2Hz,2H),3.86(dd,J=15.5,3.2Hz,1H),3.52(t,J=4 .6Hz,2H),2.70(d,J=13.0Hz,1H),2.32(d,J=16.6Hz,1H),2.18–2.10(m,2H),1.95(dt,J=13.3,6.2Hz, 2H),1.58–1.54(m,1H),1.08(d,J=5.8Hz,3H),1.02–0.98(m,3H),0.89–0.85(m,3H).ESI[M+H]+:538.1.

[0073] Example 3 Synthesis of a triptolide derivative (Compound 3) targeting ROS activation

[0074]

[0075] Here are the steps:

[0076] (1) In a 10 mL two-necked round-bottom flask, 2 mL of anhydrous DMF was added, followed by TP (0.056 mmol) and NPC (0.28 mmol). Nitrogen was introduced and stirring was started in an ice-water bath. Anhydrous TEA (0.28 mmol) and DMAP (0.28 mmol) were dissolved in 1 mL of anhydrous DMF and slowly added dropwise to the flask. The mixture was stirred for 24 hours. TLC confirmed the reaction was 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, dried over Na2SO4, and concentrated to obtain an oily crude product, namely compound TP-NPC.

[0077] (2) In a 15 mL round-bottom flask, 2 mL of anhydrous THF was added, followed by the addition of compound TP-NPC (0.056 mmol), anhydrous triethylamine (0.28 mmol), and 4-(hydroxymethyl)phenylboronic acid pinacol ester (0.28 mmol). The mixture was stirred under nitrogen for 2 hours. TLC confirmed the reaction was 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 mixture was concentrated to obtain a yellow oily crude product, which was purified by reverse phase column chromatography to obtain a white solid. The structure was confirmed to be compound 3 (9 mg, yield 26.42%). 1H NMR(400MHz,Chloroform-d)δ7.69–7.62(m,2H),7.28–7.19(m,2H),5.13(d,J=12.6Hz,1H),5.05(d,J=12.6H z,1H),4.70(d,J=0.9Hz,1H),4.61–4.45(m,3H),3.66(d,J=3.1Hz,1H),3.38(dd,J=3.1,1.0Hz,1H),3.32(d,J =5.5Hz,1H),2.21–2.10(m,1H),2.02(dq,J=16.0,5.1,4.4Hz,3H),1.82–1.74(m,2H),1.47–1.38(m,1H),1.2 0(s,12H),0.92(d,J=3.3Hz,3H),0.81(dd,J=7.0,2.0Hz,3H),0.69(dd,J=6.9,1.9Hz,3H).ESI[M+H]+:621.3.

[0078] Example 4 Synthesis of a triptolide derivative (Compound 4) targeting ROS activation

[0079]

[0080] Here are the steps:

[0081] To 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). Stirring was continued under nitrogen at room temperature for 2 hours. TLC confirmed the reaction was complete. The solvent was removed by concentration under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated NaCl and concentrated under reduced pressure to yield a white solid. The structure was confirmed to be compound 4 (7.3 mg, 90% yield). 1H NMR(400MHz,Chloroform-d)δ8.13(d,J=7.6Hz,2H),7.45(d,J=7.7Hz,2H),5.30–5.12(m,2H),4.77(d,J=10.7Hz, 1H),4.59(d,J=3.6Hz,2H),3.74(d,J=3.1Hz,1H),3.45(dd,J=7.5,3.0Hz,1H),3.43–3.34(m,1H),2.61(dt,J=14. 0,5.0Hz,1H),2.28–2.18(m,1H),2.10–2.03(m,1H),1.86(dq,J=13.8,6.4Hz,2H),1.50(dd,J=12.5,5.1Hz,1H),1 .27(s,1H),1.19–1.11(m,1H),1.01(s,3H),0.88(dd,J=9.2,6.8Hz,3H),0.77(d,J=7.0Hz,3H).ESI[M+H]+:539.2.

[0082] Example 5 Synthesis of a triptolide derivative (Compound 5) targeting ROS activation

[0083]

[0084] Here are the steps:

[0085] (1) In a 50 mL round-bottom flask, 5 mL of anhydrous THF was added, followed by 4-(hydroxymethyl)phenylboronic acid pinacol ester (1.28 mmol), NPC (2.56 mmol), and TEA (2.56 mmol). The mixture was stirred under nitrogen and stirred at room temperature for 10 hours. TLC confirmed the reaction was 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 oil. The crude product was purified by silica gel column chromatography (PE:EA = 10:1) to obtain a white solid. The structure was confirmed to be Compound A1 (365.78 mg, yield 71.5%). 1 H NMR(400MHz,Chloroform-d)δ8.32–8.24(m,2H),7.85(d,J=7.6Hz,2H),7.44(d, J=7.7Hz,2H),7.41–7.34(m,2H),5.31(s,2H),1.35(s,12H).ESI[M+H]+:400.1.

[0086]

[0087] (2) In a 50 mL round-bottom flask, 5 mL of anhydrous THF was added, followed by Compound A1 (0.64 mmol), tert-butyl (pyrrolidin-2-ylmethyl)carbamate (1.28 mmol), and TEA (1.28 mmol). The mixture was stirred under nitrogen and stirred at room temperature for 3 hours. TLC confirmed the reaction was 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 oil. The crude product was purified by silica gel column chromatography (PE:EA = 4:1) to obtain a colorless oil. The structure was confirmed to be Compound A2 (244.82 mg, yield 85.6%). 1 HNMR(400MHz,Chloroform-d)δ7.73(d,J=7.8Hz,2H),7.29(d,J=7.6Hz,2H),5.07(d,J=4.8Hz,2H),3.87(s,1H),3.35(dd ,J=6.9,4.1Hz,2H),3.28–3.04(m,2H),1.87(s,2H),1.78(s,2H),1.36(d,J=2.4Hz,9H),1.27(s,12H).ESI[M+H]+:461.3.

[0088]

[0089] (3) Compound A2 (0.55 mmol) was added to a 25 mL round-bottom flask, followed by anhydrous DCM and TFA. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain a pink oily product, which was identified as Compound A3 (232.5 g, 95% yield). 1 H NMR(400MHz,Methanol-d4)δ7.74(d,J=7.7Hz,2H),7.38(d,J=7.6Hz,2H),5.24 –5.11(m,2H),4.10(dtt,J=11.0,7.2,3.7Hz,1H),3.56(dt,J=10.8,7.4Hz,1H), 3.48(q,J=5.3,4.8Hz,1H),3.14–2.97(m,2H),2.13(dt,J=12.7,7.8Hz,1H),1. 99–1.89(m,2H),1.77(dq,J=11.4,5.2Hz,1H),1.34(s,12H).ESI[M+H]+:361.2.

[0090]

[0091] (4) The synthesis method was the same as 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 complete by TLC. The solvent was removed by concentration under reduced pressure, 5 mL of DCM was added, and the product was washed with saturated Na2CO3 and NaCl. The product was concentrated to obtain a yellow oily crude product. The product was purified by reverse phase column chromatography to obtain a white solid. The structure was confirmed to be compound 5 (12.60 mg, yield 26.42%). 1 H NMR(400MHz,Chloroform-d)δ7.73(d,J=7.6Hz,2H),7.30(d,J=7.8Hz,2H),5.11–5.01(m,2H),4.85(d,J=3.8Hz,1H),4.60( d,J=3.7Hz,2H),3.91(s,1H),3.74(d,J=3.4Hz,1H),3.44(d,J=3.4Hz,2H),3.37(dd,J=13.1,4.7Hz,3H),3.26(s,1H),2.61( d,J=13.0Hz,1H),2.24(d,J=17.7Hz,1H),2.14–1.98(m,2H),1.86(td,J=13.7,6.3Hz,4H),1.50(dd,J=12.4,5.3Hz,2H),1. 27(s,12H),1.20–1.09(m,2H),0.98(d,J=8.2Hz,3H),0.91(d,J=6.7Hz,3H),0.78(dd,J=6.8,3.5Hz,3H).ESI[M+H]+:747.4.

[0092] Example 6 Synthesis of a triptolide derivative (Compound 6) targeting ROS activation

[0093]

[0094] Here are the steps:

[0095] To a 15 mL round-bottom flask, 2 mL of acetone and 1 mL of water were added, along with 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 and allowed to stir at room temperature for 2 hours. TLC confirmed the reaction was complete. The solvent was removed by concentrating under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated NaCl and concentrated under reduced pressure to yield a white solid, which was identified as compound 6 (9.61 mg, 90% yield). 1H NMR(400MHz,Chloroform-d)δ7.72(d,J=7.6Hz,2H),7.31(s,2H),5.09(s,2H),4.85(s,1 H),4.59(s,2H),3.90(s,1H),3.75(s,1H),3.66(q,J=7.0Hz,1H),3.49–3.39(m,4H),3.39 (s,2H),2.60(s,1H),2.24(d,J=17.4Hz,1H),2.06(s,2H),1.88(s,2H),1.76(s,3H),1.4 8(s,2H),1.01–0.96(m,3H),0.93–0.89(m,3H),0.78(d,J=6.9Hz,3H).ESI[M+H]+:665.1.

[0096] Example 7 Synthesis of a triptolide derivative (Compound 7) targeting ROS activation

[0097]

[0098] Here are the steps:

[0099] A 10 mL round-bottom flask was charged with 2 mL of anhydrous DMF, TP (0.056 mmol), 4-(bromomethyl)phenylboronic acid pinacol ester (0.28 mmol), and AgO (0.28 mmol). The mixture was slowly warmed from 0°C to room temperature and stirred under nitrogen for 12 hours. The solvent was removed by concentration under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated NaCO and NaCl, dried over NaSO, and concentrated to obtain an oily crude product. The product was purified by liquid chromatography, and the collected samples were combined and dried to obtain a white solid. The structure was confirmed to be compound 7 (7.5 mg, 23% yield). 1H NMR(400MHz,Chloroform-d)δ7.82–7.76(m,2H),7.26(dt,J=8.6,1.0Hz,2H),4.85(dt,J=12.5,1.1Hz,1H),4.85–4.78(m,1H),4.67– 4.57(m,3H),3.86(d,J=3.7Hz,1H),3.62(t,J=3.8Hz,1H),3.37(dd,J=5.1,2.4Hz,1H),3.00(tq,J=6.3,1.3Hz,1H),2.40–2.31(m,1H ),2.28–2.19(m,1H),2.09(ddd,J=12.5,6.1,2.5Hz,1H),1.97(ddd,J=12.5,6.2,5.2Hz,1H),1.86(dtd,J=11.0,5.5,1.7Hz,1H),1.4 3(ddd,J=12.1,7.3,4.9Hz,1H),1.36(ddd,J=12.4,7.5,4.9Hz,1H),1.26(s,6H),1.21(s,6H),1.01–0.96(m,9H).ESI[M+H]+:577.3.

[0100] Example 8 Synthesis of a triptolide derivative (Compound 8) targeting ROS activation

[0101]

[0102] Here are the steps:

[0103] To a 15-mL round-bottom flask, 2 mL of acetone and 1 mL of water were added compound 7 (0.013 mmol) obtained in Example 7, sodium periodate (0.026 mmol), and ammonium acetate (0.026 mmol). Stirring was continued under nitrogen at room temperature for 2 hours. TLC confirmed the reaction was complete. The solvent was removed by concentrating under reduced pressure, and 5 mL of DCM was added. The mixture was washed with saturated NaCl and concentrated under reduced pressure to yield a white solid, which was identified as compound 8 (5.79 mg, 90% yield). 1H NMR(500MHz,Chloroform-d)δ7.67–7.61(m,2H),7.16(dt,J=8.3,1.1Hz,2H),4.87(dt,J=12.4,1.1Hz,1H),4.82(dt,J=12.4,1.0Hz, 1H),4.67–4.57(m,3H),3.86(d,J=3.7Hz,1H),3.62(t,J=3.8Hz,1H),3.37(dd,J=5.1,2.4Hz,1H),3.00(tq,J=6.2,1.2Hz,1H),2.36( dddq,J=12.3,7.1,4.9,1.1Hz,1H),2.28–2.19(m,1H),2.09(ddd,J=12.5,6.1,2.5Hz,1H),1.97(ddd,J=12.4,6.2,5.2Hz,1H),1.87( pd,J=5.5,3.7Hz,1H),1.43(ddd,J=12.1,7.3,4.8Hz,1H),1.36(ddd,J=12.4,7.5,4.9Hz,1H),1.01–0.96(m,9H).ESI[M+H]+:495.2.

[0104] Example 9 Effects of triptolide derivatives targeting ROS activation on TGF-β1-induced transformation of pulmonary fibroblasts into pulmonary myofibroblasts

[0105] Lung fibroblasts were seeded in a DMEM mixed culture medium supplemented with 10% fetal bovine serum and cultured in a 5% CO2 incubator. After treatment with 20 ng / mL of TGF-β1, the cells were divided into a control group (treated with 20 ng / mL TGF-β1 alone) and a TP derivative-treated group. After 24 hours of treatment with 0.1 μM TGF-β1, the cells were cultured, the supernatant discarded, and the cells were washed three times with PBS. Sample processing and hydroxyproline quantification were performed according to the literature (Eur J Med Chem. 279(2024)116839), resulting in Table 1. The inhibition rate was calculated as follows: Inhibition rate (%) = (control group - amount of hydroxyproline after administration) / control group * 100%. The results show that TP derivatives can inhibit TGF-β1-induced proline biosynthesis.

[0106] Table 1. TP derivatives inhibit TGF-β1-induced hydroxyproline production

[0107] 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

[0108] Example 10 Study on Compound 1's Dependence on ROS Activation

[0109] TP derivatives were incubated with mouse plasma and mouse plasma added with 200 μM H2O2 for different time periods, and the incubation solution was taken for liquid phase analysis to detect the concentration of TP derivatives. Figure 1 The experimental results showed that compound 1 was metabolically stable in mouse plasma, and its metabolic half-life was t 1 / 2 >1h( Figure 1 A). In 200 μM H2O2 mouse plasma, compound 1 can be activated by H2O2 to generate TP. 1 / 2 =37.2min( Figure 1 B).

[0110] Example 11 In vivo toxicity and anti-idiopathic pulmonary fibrosis study of compound 1

[0111] Six- to eight-week-old C57BL / 6 mice (20-25g) were injected directly into the trachea with bleomycin (3 mg / kg) to establish a mouse pulmonary fibrosis (IPF) model. Five groups were divided into the following groups: a blank control group, a model group, a nibendazim (BIBF1120) group (60 mg / kg, po), a low-dose TP-DEA2 group (0.33 mg / kg), and a high-dose TP-DEA2 group (0.66 mg / kg). Dosing began on the first or fifth day after bleomycin induction. The positive control group received BIBF1120 (dissolved in 0.5% sodium carboxymethylcellulose (MC) + 0.2% Tween 80 + 99.3% H2O) orally once daily. The TP-DEA2 group received a once-daily intraperitoneal injection of the corresponding concentration of TP-DEA2 (dissolved in H2O). After 14 days of continuous administration, the following procedures were performed: (1) blood was collected and serum was separated, and serum ALT, AST, urea nitrogen (BUN) and creatine (Creatine) concentrations were detected using ALT (Wuhan Huamei Biotechnology Co., Ltd.; CAS: CSB-E16539m), AST (Wuhan Huamei Biotechnology Co., Ltd.; CAS: CSB-E12649m), BUN (Qingdao Jieshikang Biotechnology Co., Ltd.; CAS: SJH-033595) and creatine (Shanghai Huabang Biotechnology Co., Ltd.; CAS: HB-P9S3251X) kits to obtain serum ALT, AST, urea nitrogen and creatine concentrations. Figure 2 (2) The animals were weighed and killed, and the tissues were taken and photographed. The liver, kidney, and lung tissues were immersed in 10% formalin for pathological staining observation. Figure 3 .

[0112] Figure 2Figure 1 shows serological parameters in mice after two weeks of continuous intraperitoneal injection of Compound 1. Panel A shows the quantitative measurement of liver function indicators (ALT and AST), while Panel B shows the quantitative measurement of renal function indicators (BUN and Creatine). The control group represents a group that received no treatment. The experimental results show that after two weeks of continuous intraperitoneal injection of Compound 1, there were no significant differences in serological parameters between the Compound 1-treated group and the control group, indicating that Compound 1 has no significant toxic side effects on the liver and kidneys.

[0113] Figure 3 This image shows a stained section of mouse lung tissue following two weeks of continuous intraperitoneal injection of Compound 1. In a bleomycin-induced IPF mouse model, Compound 1 significantly reduced bleomycin-induced lung inflammation and inhibited bleomycin-induced lung fibrosis after two weeks of continuous administration. Compound 1 significantly improved bleomycin-induced lung damage in mice. The high-dose group showed superior improvement in lung pathological fibrosis compared to the active drug, nibendazim.

[0114] In summary, the triptolide prodrug compound targeting ROS activation of the present invention can inhibit or delay lung tissue fibrosis and reduce the toxicity of the drug to normal tissues, effectively solving the problems of TP's narrow therapeutic window and large toxic side effects, and has great in-depth research value in the fight against IPF.

[0115] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A triptolide derivative targeting ROS activation, characterized in that: The chemical structural formula of the triptolide derivative is shown in Formula I or Formula II below: Wherein, Q is Q1 or Q2, and T is any one of T1, T2, T2, T3, T4, T5, and T6; The structure of Q1 is as follows: The structure of Q2 is as follows: The structure of T1 is as follows: The structure of T2 is as follows: The structure of T3 is as follows: The structure of T4 is as follows: The structure of T5 is as follows: The structure of T6 is as follows: Y=O or NH; R1, R2 = H or CH3.

2. The triptolide derivative targeting ROS activation according to claim 1, characterized in that The triptolide derivative is selected from the following structures:

3. A method for preparing a triptolide derivative targeting ROS activation according to claim 1, characterized in that: The following steps are involved: (1) Anhydrous DMF, TP, and NPC were added to a reactor, inert gas was introduced, and the mixture was stirred under an ice-water bath. Anhydrous TEA and DMAP were dissolved in anhydrous DMF to obtain a mixed solution, which was slowly added dropwise to the reactor and stirred. After the reaction was complete, the solvent was removed by concentration under reduced pressure, triethylamine was added, the mixture was washed, dried, and concentrated to obtain an oily crude product, namely, compound TP-NPC; (2) Add the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stir under the protection of inert gas, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, and concentrate to obtain a yellow oily crude product, which is subjected to reverse column chromatography to obtain a white solid, i.e., a triptolide derivative targeting ROS activation.

4. The method for preparing a triptolide derivative targeting ROS activation according to claim 3, wherein: 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 method uses saturated Na2CO3 and NaCl; the drying method uses Na2SO4; In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:(4-6):(4-6).

5. A method for preparing a triptolide derivative targeting ROS activation according to claim 1, characterized in that: The following steps are involved: (1) Anhydrous DMF, TP, and NPC were added to a reactor, inert gas was introduced, and the mixture was stirred under an ice-water bath. Anhydrous TEA and DMAP were dissolved in anhydrous DMF to obtain a mixed solution, which was slowly added dropwise to the reactor and stirred. After the reaction was complete, the solvent was removed by concentration under reduced pressure, triethylamine was added, the mixture was washed, dried, and concentrated to obtain an oily crude product, namely, compound TP-NPC; (2) adding the compound TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 4-(hydroxymethyl)phenylboronic acid pinacol ester to anhydrous THF, stirring under the protection of inert gas, and after the reaction is complete, concentrating under reduced pressure to remove the solvent, adding triethylamine, washing, and concentrating to obtain a yellow oily crude product, which is then subjected to reverse phase column chromatography to obtain a white solid; (3) Add acetone and water to the reactor, add the white solid, sodium periodate and ammonium acetate, and stir under the protection of inert gas. After the reaction is complete, concentrate under reduced pressure to remove the solvent, add triethylamine, wash, and concentrate under reduced pressure to obtain a white solid, which is a triptolide derivative targeting ROS activation.

6. The method for preparing a triptolide derivative targeting ROS activation according to claim 5, wherein: 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 method uses saturated Na2CO3 and NaCl; the drying method uses Na2SO4; In step (2), the molar ratio of TP-NPC, anhydrous triethylamine, 4-(aminomethyl)phenylboronic acid pinacol ester / 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).

7. A method for preparing a triptolide derivative targeting ROS activation according to claim 1, characterized in that: The following steps are involved: (1) Anhydrous THF, 4-(hydroxymethyl)phenylboronic acid pinacol ester, NPC, and TEA in a molar ratio of 1:(1-3):(1-3) were added to a reactor and stirred under the protection of an inert gas. After the reaction was complete, the solvent was removed by concentration under reduced pressure, and DCM was added. The product was washed and concentrated to obtain a yellow oily crude product, which was purified to obtain a white solid. (2) adding anhydrous THF to a reactor, adding the white solid, tert-butyl (pyrrolidin-2-ylmethyl)carbamate, and TEA in a molar ratio of 1:(1-3):(1-3), stirring under the protection of inert gas, and after the reaction is complete, concentrating under reduced pressure to remove the solvent, adding DCM, washing, and concentrating to obtain a yellow oily crude product, which is purified 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) TP-NPC, anhydrous triethylamine, and the pink oily product in a molar ratio of 1:(4-6):(4-6) are added to the reactor, and stirred under the protection of inert gas. After the reaction is complete, the solvent is removed by concentration under reduced pressure, DCM is added, washed, and concentrated to obtain a yellow oily crude product, which is purified to obtain a white solid, i.e., a triptolide derivative targeting ROS activation.

8. A method for preparing a triptolide derivative targeting ROS activation according to claim 1, characterized in that: The following steps are involved: Anhydrous DMF was added to the reactor, and TP, 4-(bromomethyl)phenylboronic acid pinacol ester and Ag2O in a molar ratio of 1:(4-6):(4-6) were added. The temperature was slowly raised to room temperature at 0-2°C. Under the protection of inert gas, the mixture was stirred and concentrated under reduced pressure to remove the solvent. DCM was added, the mixture was washed, dried, concentrated, purified, and spin-dried to obtain a white solid, which was a triptolide derivative targeting ROS activation.

9. Use of the triptolide derivative targeting ROS activation as claimed in claim 1 in preparing a drug for delaying or inhibiting pulmonary tissue fibrosis.

10. The use according to claim 9, characterized in that The triptolide derivative targeting ROS activation can reduce the toxic and side effects of TP.

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