Preparation method and application of camptothecin prodrugs CPT-40 and CPT-38

Abstract

The invention discloses a preparation method and application of prodrugs CPT-40 and CPT-38 based on camptothecin, and belongs to the technical field of medicines. The invention aims to solve the technical problems of poor water solubility, low in-vivo stability and large toxic and side effects of the existing camptothecin drugs. The key point of the technical scheme of the invention is to provide a CPT-40 compound as shown in a formula (I) and a CPT-38 compound as shown in a formula (II), a preparation method of the CPT-40 compound and the CPT-38 compound, and application of the CPT-40 compound and the CPT-38 compound in preparation of antitumor drugs. The prodrug can be specifically activated by thioredoxin reductase highly expressed in tumor tissues to release active camptothecin so as to kill tumor cells. The compound is mainly used for preparing drugs for targeted therapy of tumors, and has the advantages of high selectivity, good plasma stability and high oral bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel class of camptothecin prodrugs CPT-40 and CPT-38, their preparation methods, pharmaceutical compositions comprising the compounds, and their application in the preparation of antitumor drugs. Background Technology

[0002] Cancer is a major disease threatening human health. Chemotherapy is one of the most commonly used treatments in clinical practice, but traditional chemotherapy drugs such as camptothecin (CPT) have drawbacks such as poor water solubility, unsatisfactory distribution in the body, and significant toxic side effects, which severely limit their clinical application. Camptothecin is an alkaloid extracted from the camptotheca tree and is effective against various cancers, but its lactone ring is easily hydrolyzed and deactivated under physiological conditions, and its bioavailability is low.

[0003] The thioredoxin system (including thioredoxin Trx and its reductase TrxR) plays a central role in maintaining intracellular redox homeostasis. Studies have shown that this system is overexpressed and activated in various tumor cells and is closely associated with tumor proliferation, metastasis, and drug resistance. This provides a novel strategy for developing anti-tumor drugs targeting TrxR.

[0004] Therefore, developing a drug that can improve the solubility, stability and bioavailability of CPT and can target the specific release of CPT into the tumor microenvironment has important clinical significance and application value. Summary of the Invention

[0005] The technical problem to be solved:

[0006] This invention aims to address the technical problems of poor water solubility, low in vivo stability, and significant toxic side effects of existing camptothecin-based drugs. It provides a novel camptothecin prodrug that can be specifically activated by thioredoxin reductase (TrxR), which is highly expressed in tumor tissue, thereby achieving targeted tumor therapy and reducing toxic side effects.

[0007] Technical solution:

[0008] This invention designs and synthesizes two novel camptothecin derivatives, namely CPT-40 and CPT-38, by linking the 20-hydroxyl group of camptothecin to a specific linker containing a disulfide bond.

[0009] In a first aspect, the present invention provides a camptothecin derivative CPT-40 as shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0010]

[0011] (I)

[0012] In a second aspect, the present invention provides a camptothecin derivative CPT-38 as shown in formula (II), or a pharmaceutically acceptable salt thereof:

[0013]

[0014] (II)

[0015] In a third aspect, the present invention provides a method for preparing compound CPT-40 of formula (I), characterized in that camptothecin is used as a raw material, reacted with triphosgene to generate a chloroformate intermediate, and then reacted with a bicyclic piperazine compound as shown in formula (7C) to obtain the target product CPT-40.

[0016] The compound of formula (7C) is prepared by a multi-step reaction involving sulfonamide, methanesulfonylation, thioacetylation, and deprotection of cis-2-butene-1,4-diol.

[0017] In a fourth aspect, the present invention provides a method for preparing compound CPT-38 of formula (II), characterized in that camptothecin is used as a raw material, reacted with triphosgene to generate a chloroformate intermediate, and then reacted with a bicyclic piperazine compound as shown in formula (7T) to obtain the target product CPT-38.

[0018] The compound of formula (7T) is prepared by a multi-step reaction of trans-2-butene-1,4-diol, including sulfonation, methanesulfonation, thioacetylation, and deprotection.

[0019] Beneficial effects:

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Targeted activation with high selectivity: The prodrugs CPT-40 and CPT-38 described in this invention are resistant to glutathione (GSH) and can be specifically reduced by thioredoxin reductase (TrxR) highly expressed in tumor tissue, thereby releasing the active drug CPT and killing tumor cells. Experiments have shown that under the action of the TrxR / Trx system, CPT-40 and CPT-38 released 70.6% and 80.3% of CPT, respectively, within 8 h, while the amount of CPT released after GSH treatment was extremely low. In TrxR knockdown cells, the cytotoxicity of CPT-40 was significantly reduced, further confirming its activation specificity.

[0022] (2) High plasma stability and long half-life: The prodrugs described in this invention greatly improve the instability of CPT under physiological conditions. After incubation in mouse plasma for 24 h, CPT-40 and CPT-38 still maintain more than 80% stability, and their half-life exceeds 24 h, which is far superior to CPT itself.

[0023] (3) Significantly improved oral bioavailability: Pharmacokinetic studies have shown that the oral absorption rate of CPT-40 in rat models reached 59.8%, which solved the problem of poor oral absorption of CPT and made it possible to administer it orally.

[0024] (4) Reduced toxicity: Cytotoxicity experiments showed that the prodrug molecules CPT-40 and CPT-38 had reduced toxicity to normal cells compared to the parent drug CPT, and had better safety potential.

[0025] In summary, CPT-40 and CPT-38 provided by this invention, as novel TrxR-activated camptothecin prodrugs, possess good targeting, stability, and pharmacokinetic properties, and are expected to be developed into highly effective and low-toxicity antitumor drugs. Attached Figure Description

[0026] Figure 1 Cell viability curves of HeLa, HepG2, A549 and L02 cells after 24 h of treatment with CPT-40, CPT-38 and CPT as determined by MTT assay.

[0027] Figure 2 A is a schematic diagram of the TrxR activation and release mechanism of the prodrug described in this invention; B is a bar chart of CPT release rates of CPT-40 and CPT-38 after incubation under different conditions (GSH, TrxR / Trx, etc.) detected by LC-MS.

[0028] Figure 3 A and C represent the cytotoxicity curves of CPT, CPT-40, and CPT-38 in HeLa-shNT and HeLa-shTrxR cells, respectively; D is the result of Western Blot verification of the TrxR1 knockdown efficiency in HeLa-shTrxR1 cells.

[0029] Figure 4 Stability bars of CPT-40 and CPT-38 after incubation in PBS buffer and mouse plasma for different times.

[0030] Figure 5 Plasma drug concentration-time curves of CPT-40 in SD rats after intraperitoneal injection (5 mg / kg) and oral administration (10 mg / kg), respectively. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Example 1: Synthesis of compounds CPT-40 and CPT-38

[0033] 1. Synthesis of intermediates and CPT-40

[0034]

[0035] Compound 2C ((E)-bis(2-nitrophenyl)but-2-ene-1,4-diylbis(sulfonamide)): trans-2-buten-1,4-diol (0.19 mL, 2.3 mmol) was dissolved in acetonitrile (12 mL), and 2-nitrobenzenesulfonamide (1.0 g, 5.0 mmol), iodine (58 mg, 0.23 mmol), and sodium hypochlorite pentahydrate (822 mg, 5.0 mmol) were added at room temperature. The resulting mixture was stirred at room temperature for 3 h, then heated to 70 °C and stirred for another 15 h. After the reaction was complete, an aqueous solution of sodium thiosulfate and ethyl acetate were added, the mixture was separated, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a colorless solid 2C (562 mg, 50% yield). Structural confirmation data:¹H-NMR (400 MHz, DMSO-d6) δ 8.08–7.51 (m, 10H), 4.40 (s, 2H), 3.55 (p, J=5.3 Hz, 2H), 3.41 (h, J=5.6 Hz, 4H).

[0036] Compound 3C (((E)-but-2-ene-1,4-diylbis(sulfonyl))bis(azadiyl))bis(2,1-phenylene)dimethanesulfonate): 2C (722 mg, 1.5 mmol) was dissolved in pyridine (10 mL), and methanesulfonyl chloride (356 µL, 4.6 mmol) was added. After the reaction was complete, the mixture was precipitated in aqueous solution and washed with diethyl ether to give a colorless solid 3C (694 mg, 73% yield). Confirmatory structural data: ¹H-NMR (400 MHz, DMSO-d6) δ 8.50–7.78 (m, 8H), 4.19 (d, J=8.4 Hz, 2H), 4.05 (dd, J=10.2, 6.2 Hz, 2H), 3.89 (s, 2H), 2.87 (s, 6H).

[0037] Compound 4C (S,S'-((E)-but-2-ene-1,4-dimethylbis(thio))diethyl ester): 3C (694 mg, 1.1 mmol) was dissolved in acetone (21 mL), and potassium thioacetate (366 mg, 3.2 mmol) was added. After stirring at room temperature for 3 h, the solution was poured into water, the solid was collected, and dried to give a colorless solid 4C (579 mg, 90% yield). Confirmatory structural data: ¹H-NMR (400 MHz, DMSO-d6) δ 8.17–7.84 (m, 8H), 3.54 (t, J=9.0 Hz, 2H), 3.20 (dd, J=13.9, 3.6 Hz, 2H), 2.77 (dd, J=13.9, 9.4 Hz, 2H), 2.03 (s, 6H).

[0038] Compound 5C (((E)-but-2-ene-1,4-diylbis(thio))diyl)diphenylsulfonamide): 4C (572 mg, 0.95 mmol) was dissolved in methanol (35 mL), and hydrochloric acid (4.0 M dioxane solution, 11.9 mL) was added. The mixture was heated to 70 °C and stirred for 15 h. After cooling, the mixture was poured into water, and the solid was collected and dried to give a colorless solid 5C (484 mg, 98% yield). Confirmatory structural data: ¹H-NMR (400 MHz, DMSO-d6) δ 8.15–7.79 (m, 8H), 3.76 (br-s, 2H), 3.46 (br-s, 4H).

[0039] Compound 6C: 5C (545 mg, 1.05 mmol) was dissolved in anhydrous dichloromethane (20 mL), and DIPEA (623 µL, 3.66 mmol) and VTT (383 mg, 1.15 mmol) were added. The reaction was purified by solid-phase extraction to give a colorless solid 6C (498 mg, 87% yield). Structural confirmation data: ¹H-NMR (400 MHz, DMSO-d6) δ 8.05–7.76 (m, 8H), 4.18 (d, J=7.0 Hz, 2H), 3.74–3.46 (m, 6H), 3.07 (d, J=13.9 Hz, 2H).

[0040] Compound 7C ((4aR,8aS,9aR)-decahydro-1H-pyrazino[2,1-c][1,2,4]thiadiazole-1-onium 2,2-dioxide chloride): 6C (1,192 mg, 2.18 mmol) was dissolved in THF (45 mL), and reacted with a methanolic solution of sodium methoxide. After deprotection, precipitation with a dioxane solution of hydrochloric acid gave 7C (851 mg, 99% yield) as a pale yellow solid. Structural confirmation data:¹H-NMR (400 MHz, DMSO-d6) δ 10.01 (s, 4H), 4.02 (d, J=7.0 Hz, 4H), 3.93 (d, J=12.7 Hz, 2H), 3.60 (d, J=15.0 Hz, 2H), 3.00 (d, J=11.2 Hz, 2H).

[0041] CPT-40: Camptothecin (1.0 equivalent) was suspended in dry dichloromethane, and solutions of triphosgene (1.2 equivalent) and DMAP (2.0 equivalent) in dry dichloromethane were added sequentially. After stirring at room temperature for 3 h, a solution of 7C bicyclopiperazine (1.4 equivalent) and DMAP (2.8 equivalent) in dichloromethane was added in situ. After stirring at room temperature for 1 h, the mixture was evaporated to dryness and purified by column chromatography to obtain colorless solid CPT-40 in 25% yield. Structural confirmation data:¹H-NMR (600 MHz, CDCl₃) δ 8.41 (d, J=5.8 Hz, 1H), 8.22 (dd, J=8.6, 2.9 Hz, 1H), 7.96 (dd, J=8.1, 4.7 Hz, 1H), 7.85 (ddt, J=8.7, 6.7, 1.7 Hz, 1H), 7.69 (ddt, J=9.6, 7.9, 1.9 Hz, 1H), 5.69 (dd, J=17.3, 8.6 Hz, 1H), 5.42 (dd, J=17.3, 9.1 Hz, 1H), 5.35–5.23 (m, 2H), 4.52–2.82 (m, 10H), 2.60 (ddd, J=72.1, 13.4, 3.9 Hz, 1H), 2.37-2.06 (m, 2H), 0.99 (t, J=7.5 Hz,3H). HRMS (m / z): [M+H]⁺ calcd for C 28 H 28 N4O6S2, 551.1345; found, 551.1426. HPLCpurity: 96.6%.

[0042] 2. Synthesis of CPT-38

[0043]

[0044] The synthesis method of CPT-38 is the same as that of CPT-40, except that the starting material trans-2-buten-1,4-diol is replaced with cis-2-buten-1,4-diol to obtain the corresponding cis-configured product. Structural confirmation data:¹H-NMR (600 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.14 (dd, J=17.3, 8.4 Hz, 2H), 7.86 (t, J=7.7 Hz, 1H), 7.71 (t, J=7.5 Hz, 1H), 7.11 (s, 1H), 5.51–2.78 (m, 16H), 2.39 (s, 1H), 2.18 (q, J=7.4 Hz, 2H), 0.92 (t, J=7.4 Hz, 3H). HRMS (m / z): [M+H]⁺ calcd for C 28 H 28 N4O6S2, 551.1345; found, 551.1432. HPLC purity: 96.0%.

[0045] Example 2: In vitro cytotoxicity experiment

[0046] The cytotoxicity of CPT-40, CPT-38, and CPT against human cervical cancer cells (HeLa), human hepatocellular carcinoma cells (HepG2), human non-small cell lung cancer cells (A549), and normal human hepatocytes (L02) was evaluated using the MTT assay. Logarithmic growth phase cells were seeded at 5000 cells per well in 96-well plates and cultured for 24 h. Different concentrations (0-100 μM) of the test compounds were then added, and the cells were cultured for another 24 h. MTT solution was added to each well, and after incubation for 4 h, formazan was dissolved in DMSO. The absorbance was measured at 570 nm, and cell viability was calculated.

[0047] The results are as follows Figure 1 As shown, the toxicity of CPT-40 and CPT-38 to the aforementioned tumor cells was lower than that of the parent drug CPT, indicating that as prodrugs, their toxicity was released in a sustained manner, and they have better safety potential.

[0048] Example 3: Study on the selective activation and CPT release characteristics of TrxR

[0049] CPT-40 and CPT-38 (50 μM) were incubated with different systems in TE buffer (pH 7.4) at 37°C, and the release of CPT was detected by LC-MS. The systems included: NADPH; TrxR+NADPH; Trx+NADPH; TrxR / Trx; TrxR / Trx+NADPH; mutant TrxR (U498C)+NADPH; GSH+GR+NADPH.

[0050] The results are as follows Figure 2 As shown in Figure B, after incubation for 24 h in a GSH (5 mM) system simulating physiological concentrations, CPT-40 and CPT-38 released almost no CPT (<3%). However, after incubation for 8 h in a TrxR (66 nM) / Trx (10 μM) / NADPH system, CPT-40 and CPT-38 released 70.6% and 80.3% of CPT, respectively. Even in a system containing only TrxR and NADPH, approximately 30% or more of CPT was released after 8 h. This indicates that the prodrug of this invention has a highly selective activation capacity for TrxR.

[0051] Example 4: Targeting validation based on TrxR1 knockdown cell model

[0052] A HeLa cell line with stable TrxR1 knockdown (HeLa-shTrxR1) and a control cell line (HeLa-shNT) were constructed via lentiviral transfection. Western blotting was used to verify the knockdown efficiency. Figure 3 D). Then, CPT-40, CPT-38, and CPT were incubated with the two cell types respectively, and cytotoxicity was determined using the MTT assay.

[0053] The results are as follows Figure 3 As shown in AC, there was no significant difference in toxicity between the parent drugs CPT and CPT-38 to the two cell types. However, CPT-40 was significantly less toxic to HeLa-shTrxR1 cells than to HeLa-shNT cells. Figure 3 B). This result strongly demonstrates that the activation and cytotoxicity of CPT-40 within cells are highly dependent on TrxR, reflecting its excellent targeting ability.

[0054] Example 5: Plasma stability experiment

[0055] CPT-38 and CPT-40 (final concentration 50 μM) were incubated with PBS buffer and mouse plasma, respectively, at 37°C. Samples were taken at preset time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24 h), methanol was added to terminate the reaction, and the supernatant was collected after centrifugation for LC-MS analysis to determine the remaining prodrug content.

[0056] The results are as follows Figure 4 As shown, after 24 hours of incubation in mouse plasma, CPT-40 and CPT-38 maintained more than 80% of their original drug concentrations, with half-lives exceeding 24 hours. In contrast, CPT itself is extremely unstable in plasma, with a very short half-life. These results demonstrate that the prodrug of this invention significantly improves the plasma instability problem of CPT.

[0057] Example 6: Pharmacokinetic Study in Rats

[0058] Eight-week-old male SD rats (weighing 250±30 g) were divided into two groups. One group received a single intraperitoneal injection (5 mg / kg), while the other received a single oral gavage administration (10 mg / kg). Blood samples were collected from the orbital sinus at different time points after administration (0, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 h). Plasma was separated, and after protein precipitation with acetonitrile, the concentration of CPT-40 in the plasma was determined by LC-MS.

[0059] The results are as follows Figure 5 As shown, peak plasma concentrations were reached approximately 1 hour after intraperitoneal injection and approximately 2 hours after oral administration. The area under the curve (AUC) yielded an absolute oral bioavailability of 59.8% for CPT-40. These results indicate that CPT-40 not only exhibits good in vivo stability but also excellent oral absorption characteristics.

[0060] In summary, this invention has successfully prepared a novel class of camptothecin prodrugs, CPT-40 and CPT-38. In vitro and in vivo experiments have confirmed that these prodrugs can be specifically activated by the tumor-associated enzyme TrxR, exhibiting advantages such as high plasma stability, high oral bioavailability, and low toxicity, demonstrating great potential for development into novel antitumor drugs.

Claims

1. A class of camptothecin derivatives CPT-40 as shown in formula (Ⅰ), or a pharmaceutically acceptable salt thereof; Equation (Ⅰ).

2. A class of camptothecin derivatives CPT-38 as shown in formula (II), or a pharmaceutically acceptable salt thereof, Formula (II).

3. A method for preparing compound CPT-40 of formula (I) as described in claim 1, characterized in that, Includes the following steps: Using camptothecin as a raw material, it reacts with triphosgene to generate a chloroformate intermediate, which is then reacted with a bicyclic piperazine compound as shown in formula (7C) to obtain the target product CPT-40; The structure of the compound of formula (7C) is as follows:

4. A method for preparing compound CPT-38 of formula (II) as described in claim 2, characterized in that, Includes the following steps: Using camptothecin as a raw material, it reacts with triphosgene to generate a chloroformate intermediate, which is then reacted with a bicyclic piperazine compound as shown in formula (7T) to obtain the target product CPT-38. The structure of the compound of formula (7T) is as follows:

5. The preparation method according to claim 3, characterized in that, The compound of formula (7C) was prepared by a multi-step reaction of cis-2-butene-1,4-diol.

6. The preparation method according to claim 4, characterized in that, The compound of formula (7T) was prepared by a multi-step reaction of trans-2-butene-1,4-diol.

7. Use of the compound of claim 1 or 2 in the preparation of a medicament for treating and / or preventing tumors.

8. The use as described in claim 7, characterized in that, The tumor is cervical cancer, liver cancer, or non-small cell lung cancer.

9. Use of the compound of claim 1 or 2 in the preparation of an antitumor drug specifically activated by thioredoxin reductase (TrxR).

10. Use of the compound of claim 1 or 2 in the preparation of an antitumor drug having good plasma stability and oral bioavailability.

Images