Bis (2-chloroethyl) carbamate modified SN-38 derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of modernization of traditional Chinese medicines, and mainly relates to a novel camptothecin derivative as well as a preparation method and medical application thereof. Specifically, on the basis of structural optimization of a traditional Chinese medicine active ingredient camptothecin, 7-ethyl-10-hydroxycamptothecin (SN-38) is chemically modified through a bis (2-chloroethyl) carbamate group, and a novel derivative containing a nitrogen mustard pharmacophore and a camptothecin mother nucleus at the same time is designed and synthesized. And further introducing a phosphate group into the alcoholic hydroxyl group through phosphorylation modification or a salt forming reaction or forming inorganic acid salt to finally obtain the target compound as shown in the formula I. In-vivo and in-vitro experiments prove that the SN-38 derivative disclosed by the invention has the advantages of improving the anti-tumor effect, increasing the drug stability, reducing the toxic and side effects of the drug and the like.

Description

Technical Field

[0001] This invention belongs to the field of modern Chinese medicine technology, and mainly relates to a novel camptothecin derivative, its preparation method and pharmaceutical application, especially to a technical solution that improves the water solubility, stability and antitumor activity of the drug by introducing bis(2-chloroethyl)carbamate and salt groups. Background Technology

[0002] Camptothecin is a natural antitumor active ingredient extracted from the traditional Chinese medicine plant *Camptotheca acuminata*. While its derivative SN-38, as a topoisomerase I inhibitor, possesses significant antitumor potential, its application in clinical formulation development is severely limited by bottlenecks such as extremely low water solubility, rapid in vivo metabolism, and significant toxic side effects. This invention optimizes the structure of the traditional Chinese medicine active ingredient camptothecin by chemically modifying 7-ethyl-10-hydroxycamptothecin (SN-38) with a bis(2-chloroethyl)carbamate group, designing and synthesizing a novel derivative containing both a nitrogen mustard pharmacophore group and a camptothecin core. Further, phosphorylation modification or salt formation reactions introduce phosphate ester groups into the alcohol hydroxyl groups or form acid salts, ultimately yielding the target compound as shown in Formula I. In vitro and in vivo experiments demonstrate that the novel camptothecin derivative involved in this invention has numerous advantages, including improved antitumor efficacy, increased drug stability, and reduced drug toxicity.

[0003] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0004] This invention aims to provide a novel class of bis(2-chloroethyl)carbamate-modified SN-38 derivatives and their preparation method, to address the limitations in clinical application of existing SN-38 products due to low water solubility, insufficient bioavailability, and toxicity. Furthermore, by combining SN-38 with a nitrogen mustard pharmacophore and introducing acid / salt modification, a synergistic effect of enhanced antitumor activity, optimized physicochemical properties, and reduced toxic side effects is achieved, providing more efficient and safer candidate drugs for the treatment of malignant tumors such as cervical cancer, liver cancer, and colon cancer.

[0005] This invention discloses a novel prodrug molecule based on SN-38, the general structural formula of which is shown in Formula I:

[0006]

[0007] Wherein, R is selected from H, phosphoric acid (PO3H2), sodium phosphate (PO3Na2), etc.; XH is a pharmaceutically acceptable acid such as hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), hydrobromic acid (HBr), nitric acid (HNO3), citric acid (C6H8O7), maleic acid (C4H4O4), tartaric acid (C4H6O6), methanesulfonic acid (CH3SO3H), acetic acid (CH3COOH), fumaric acid (C4H4O4), succinic acid (C4H6O4), malic acid (C4H6O5), benzoic acid (C7H6O2), lactic acid (C3H6O3), glutamic acid (C5H9NO4), aspartic acid (C4H7NO4).

[0008] The synthetic route of the SN-38 derivative described above is as follows:

[0009]

[0010] The product is characterized by the following steps: (1) Step 1: In the presence of a base (such as triethylamine, pyridine or its analogues) and a catalyst (such as DMAP or its derivatives), SN-38 and bis(2-chloroethyl)carbamoyl chloride (BCH, or its corresponding carbamic acid, etc. as raw materials) are reacted at room temperature in a halocarbon solvent (such as dichloromethane, chloroform) for 12-24 hours, and then extracted and precipitated by column chromatography to obtain compound 1; (2) Step 2: Compound 1 is reacted with concentrated hydrochloric acid or other inorganic acids (such as sulfuric acid, hydrobromic acid) or organic acids (such as citric acid, maleic acid, tartaric acid, methanesulfonic acid, acetic acid, fumaric acid, succinic acid, malic acid, benzoic acid, lactic acid, glutamic acid, aspartic acid, etc.) in an alcohol-ether mixed solvent (such as methanol / diethyl ether, ethanol / isopropyl ether) for 0.5-2 hours at room temperature, and the acid salt is precipitated to obtain compound 2-HX; (3) Step 3: Compound 1 is reacted stepwise with a phosphorylating agent (such as P2O5, POCl3 or tetraethyl pyrophosphate) in an inert solvent (such as dioxane, toluene) at 60-100℃ for 12-48 hours, and purified by precipitation to obtain phosphate ester compound 3; (4) Step 4: Compound 3 is reacted with a strong base (such as NaOH) in a polar solvent (such as ethanol, methanol) at room temperature for 8-12 hours, and dried to obtain water-soluble sodium phosphate compound 4. This method can efficiently prepare a series of SN-38 derivatives by optimizing reaction conditions (including but not limited to catalyst type, solvent selection, temperature control and purification method), and its protection scope covers reasonable alternatives to the reaction parameters in the above steps.

[0011] The use of the SN-38 derivatives as described above in the preparation of drugs for treating cervical cancer and / or liver cancer and / or colon cancer.

[0012] Furthermore, the SN-38 derivative is any one of the compounds and / or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the cervical cancer is human cervical cancer cell line HeLa, the liver cancer is human liver cancer cell line HepG2, and the colon cancer is mouse colon cancer cell line CT26. Attached Figure Description

[0014] Figure 1 The study aimed to investigate the effects of different compounds on CT26 cell migration, quantitative analysis of migration, and flow cytometry analysis and quantitative analysis of the effects of each compound on the CT26 cell cycle.

[0015] Figure 2 Annexin V-FITC / PI staining analysis and apoptosis analysis of CT26 cells induced by various compounds.

[0016] Figure 3 This document presents representative photographs of animal model establishment and drug administration regimens, tumor volume growth curves, body weight changes, tumor weight, tumor inhibition rate, and tumor ablation in mice of each treatment group. Detailed Implementation

[0017] To better understand the present invention, the following detailed description is provided in conjunction with embodiments. However, the scope of protection of the present invention is not limited to the scope represented by the embodiments.

[0018] Unless otherwise specified, all raw materials used in this invention are commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the art. The quantities of all substances used in this invention are conventionally used quantities.

[0019] Experimental Example 1: Synthesis of Compounds

[0020] The final product of the synthesis is 1: (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-1,3,4,12,14,14a-hexahydro-13λ 4 -Pyran[3',4':6,7]indole[1,2-b]quinoline-9-ylbis(2-chloroethyl)carbamate (1,MN33-45)

[0021]

[0022] Step 1: Take a 50 mL pear-shaped flask and add SN-38 (100.0 mg, 0.26 mmol) and bis(2-chloroethyl)carbamoyl chloride (BCH, 37.8 μL, 0.26 mmol) to the flask equipped with a magnetic stirrer. Then add triethylamine (43.0 μL, 0.31 mmol) and 4-dimethylaminopyridine (62.0 mg, 0.51 mmol) to the flask, along with 6 mL of dichloromethane as the reaction solvent. Under argon protection, stir at room temperature for 18 h. TLC detection showed that the reaction was complete, and the reaction solution changed from a pale yellow mixture to a colorless solution. The reaction solution was poured into 100 mL of water, transferred to a 250 mL separatory funnel, and extracted three times with 50 mL × 3 ethyl acetate. The organic layer was collected, dried over anhydrous Na2SO4, concentrated, and then separated and purified by 200-300 mesh silica gel column chromatography (DCM:MeOH = 60:1) to give 163.0 mg of white solid target compound 1 (MN33-45), with a yield of 100.0%. 1 H NMR (400 MHz, DMSO-d6): δ 8.12 (d, J = 9.1 Hz, 1H), 7.98 (d, J = 2.3Hz, 1H), 7.62 (dd, J = 9.1, 2.4 Hz, 1H), 7.26 (s, 1H), 6.49 (s, 1H), 3.12 (dd, J = 14.6, 7.0 Hz, 2H), 1.85 - 1.90 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ 172.41, 156.78, 153.78, 151.78, 149.98, 149.60,146.34, 145.88, 145.21, 131.04, 128.45, 126.99, 125.70, 118.92, 114.96,96.56, 72.34, 65.25, 61.81, 45.35, 42.22, 41.35, 30.88, 22.21, 13.77, 7.71.HRMS (+ESI) (m / z) [M+H] + Calculated for C 27 H 27 Cl2N3O6, 559.1277, found 560.1348.

[0023] The final product of the synthesis is 2: (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-1,3,4,12,14,14a-hexahydro-13λ 4 -Pyran[3',4':6,7]indole[1,2-b]quinoline-9-ylbis(2-chloroethyl)carbamate hydrochloride (2,MN33-45)

[0024]

[0025] Step 1: Weigh 35.0 mg (0.06 mmol) of compound 1 into a 50 mL flask, add it to a mixture of ether and methanol = 1.2 mL: 2 mL, and then slowly add 0.1 mL of concentrated hydrochloric acid dropwise to the system. Stir at room temperature for 1 h. After the reaction is complete, evaporate the solvent, add ether, and a pale yellow solid precipitates. Filter the solid, wash the filter cake with ether, and collect the filter cake to obtain 37.2 mg of the pale yellow solid target compound 2 (MN33-47), with a yield of 100.0%. 1H NMR (400 MHz, DMSO-d6): δ 8.19(dd, J = 9.0, 1.8 Hz, 1H), 8.05 (s, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.32 (s,1H), 5.44 (s, 2H), 5.33 (s, 2H), 3.98 (t, J = 6.1 Hz, 2H), 3.90 (t, J = 5.9Hz, 2H), 3.85 (t, J = 6.4 Hz, 2H), 3.73 (t, J = 6.2 Hz, 2H), 3.19 (d, J = 7.5Hz, 2H), 1.79-1.92 (m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 172.46, 156.83, 153.84, 151.76, 150.06,149.603, 146.34, 145.88, 145.27, 131.06, 128.43, 127.02, HRMS (+ESI) (m / z) [M-Cl] + Calculated for C 27 H 28 Cl3N3O6, 595.1044, found 560.1409

[0026] The final product of the synthesis is 3: (4S)-4,11-diethyl-3,14-dioxo-4-(phosphono)-1,3,4,12,14,14-hexahydro-13λ 4 -Pyran[3',4':6,7]indole[1,2-b]quinoline-9-ylbis(2-chloroethyl)carbamate (3,MN33-53)

[0027]

[0028] Step 1: Weigh compound 1 (50.0 mg, 0.09 mmol) and P2O5 (16.5 mg, 0.12 mmol) into a 50 mL eggplant-shaped reaction flask, add 6 mL of 1,4-dioxane, and protect with argon gas. Heat to 80 °C and react for 12 h, then lower to 60 °C. Add 0.2 mL of water to the system, stir for 30 min, then raise to 90 °C and react for 24 h. After the reaction is complete, evaporate the solvent, filter, wash the residue with diethyl ether, and collect the filter cake to obtain 70.1 mg of the yellow solid target compound 3 (MN33-53), with a yield of 75%. 1 H-NMR (400 MHz, DMSO-d6): δ 8.51 (s, 1H), 8.20 (d, J = 9.1 Hz, 1H), 8.01 (s, 1H), 7.70 (s, 1H), 6.14 (s, 1H), 5.60 (s, 1H), 5.24 (s, 2H), 4.65 (d, J =16.4 Hz, 2H), 3.96 (d, J = 5.1 Hz, 2H), 3.90 (d, J = 5.1 Hz, 2H), 3.83 - 8.60(m, 2H), 3.72 - 3.75 (m, 2H), 3.56 (s, 2H), 3.16 (d, J = 7.3 Hz, 2H), 1.29(t, J = 7.1 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H).HRMS (+ESI) (m / z) [M+Na] + Calculated for C 27 H 28 C l2 N3O9P, 639.0490, found 663.0660.

[0029] Synthesized to produce sodium phosphate prodrug 4 (MN33-63), a final product of SN-38 derivative.

[0030] Step 1: Weigh compound 3 (30.0 mg, 0.05 mmol) and NaOH (4.0 mg, 0.1 mmol) into a reaction flask containing 3 mL of anhydrous ethanol. Stir the reaction at room temperature for 10 h. After the reaction is complete, evaporate the solvent and dry to obtain 34.0 mg of the pale yellow solid target compound 4 (MN33-63), with a yield of 99.4%. 1H NMR (400 MHz, DMSO-d6): δ 8.02 (d, J = 9.7 Hz, 1H), 7.42(d, J = 6.7 Hz, 2H), 7.24 (s, 1H), 5.42 (s, 2H), 5.28 (s, 2H), 4.25 – 4.29(m, 2H), 3.77 (t, J = 5.9 Hz, 2H), 3.57 – 3.61 (m, 2H), 3.48 – 3.50 (m, 2H), 3.06 – 3.11 (m, 2H), 1.80 – 1.91 (m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 0.87 (t,J = 7.2 Hz, 3H).HRMS (+ESI) (m / z) [M+H] + Calculated for C 27 H 26 Cl2N3O9PNa2,684.0657, found 685.0739.

[0031] Table 2

[0032]

[0033] Example 2: Cell anti-proliferative activity assay (CCK-8 assay)

[0034] Experimental steps:

[0035] (1) Cell Culture: Six different tumor cell lines (A549, HGC-27, CT26, HCT-15, HeLa, and HepG2) were used in the experiment. Based on cell state and characteristics, cells were seeded at different densities in 96-well plates: 8000 cells / well for HepG2 and HCT-15, 10000 cells / well for HeLa, CT26, and HGC-27, and 12000 cells / well for A549. After trypsin digestion, cells were counted, mixed, and 100 μL of cell suspension was added to each 96-well plate using a multipipe. The cells were then cultured at 37°C and 5% CO2 for 24 hours.

[0036] (2) Drug treatment: One blank group, one control group, and seven drug treatment groups (CH, SN-38, CH+SN-38, MN33-45, MN33-53, MN33-47 and MN33-63) were set up. The positive control and four prodrugs were first prepared into 20 mM stock solutions with DMSO, and then serially diluted to different concentrations with the corresponding complete cell culture medium. The culture medium of different concentrations of the compounds was replaced into each well, 100 μL per well. The 96-well plates after drug addition were placed in an incubator and incubated for 72 hours.

[0037] (3) Detection: After incubation, discard the culture medium. Prepare a solution of blank culture medium and CCK-8 reagent at a volume ratio of 9:1. Add 100 µL to each well and incubate for 2 hours. Then, use a microplate reader to detect the absorbance at 450 nm. Calculate the survival rate and inhibition rate based on the OD value.

[0038] (4) Experimental results (Table 3)

[0039] In various tumor cell lines, the half-maximal inhibitory concentration (IC50) of compounds such as MN33-45, MN33-47, MN33-53, and MN33-63 was significantly reduced. 50 The antiproliferative activity of MN33-53 was significantly lower than that of SN-38, CH monotherapy, and the combination therapy of CH and SN-38, demonstrating stronger tumor cell proliferation inhibitory activity. Taking the A549 cell model as an example, the antiproliferative activity of MN33-53 was more than 60 times that of SN-38, more than 4100 times that of CH monotherapy, and more than 19 times that of the CH / SN-38 combination therapy group. In HGC-27 cells, the antiproliferative capacity of MN33-63 was more than 23 times that of SN-38, approximately 13,000 times that of CH monotherapy, and more than 25 times that of the combination therapy group. These data fully highlight the significant advantages of the MN series compounds in inhibiting tumor cell proliferation.

[0040] Table 3. Antiproliferative activity of MN33-45, MN33-47, MN33-53 and MN33-63 against six cancer cell types after 72 hours (n=3).

[0041]

[0042] Example 3: Cell scratch assay and cell cycle assay

[0043] Experimental steps:

[0044] (1) Cell scratch test

[0045] The inhibitory effect of the test compound on cancer cell migration was evaluated using a cell scratch assay. CT26 cells were cultured at 10 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates. After full adhesion, a straight incision was made in the confluent cell monolayer using a 200 μL pipette tip, and the cells were washed with PBS to remove any detached cells. Subsequently, the cells were treated with 0.1 μM of the test compound for 24 and 48 hours, respectively. Cell migration was observed and images were acquired at 0, 24, and 48 hours using an inverted microscope (DXS-5).

[0046] (2) Cell cycle analysis experiment

[0047] CT26 cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours to allow for complete adhesion. After adhesion, 1 μM of the test compound was added, and the cells were cultured for another 48 hours. After treatment, cells were collected, washed twice with PBS, and fixed at -20°C with pre-chilled 68% (v / v) ethanol for 24 hours. The fixed cells were collected by centrifugation, washed again with PBS, and finally resuspended in 250 μL of PBS. The treated samples were incubated at 37°C in the dark for 30 minutes, filtered, and then analyzed for cell cycle using a flow cytometer (FC500-MPL). Data analysis was performed using FlowJo software.

[0048] (3) Results Analysis and Discussion:

[0049] Cancer metastasis is a key factor leading to poor patient prognosis; therefore, studying the inhibitory effects of prodrugs on tumor invasion and metastasis is of significant research value for improving cancer treatment strategies. This study used a wound healing assay (WHMA). Figure 1 (A–1B) This study systematically evaluated the inhibitory effects of the prodrugs MN33-47 and MN33-63 on tumor cell migration. Six groups were set up: a control group, a CH group, a SN-38 group, a CH+SN-38 combination group, a MN33-47 group, and a MN33-63 group. Cell migration was dynamically observed at 0 h, 24 h, and 48 h. The results showed that compared with the CH and SN-38 groups, the MN33-47, MN33-63, and CH+SN-38 combination groups significantly enhanced the inhibitory effect on cell migration area, and were significantly better than the single-drug treatments. Notably, the prodrug MN33-63 showed the most significant inhibition of cancer cell migration at 24 h and 48 h, with migration rates of only 2.51% and 4.42%, respectively, significantly better than all other experimental groups.

[0050] Elucidating the mechanism of cell cycle arrest is a crucial step in evaluating the antitumor efficacy of drugs. In this study, CT26 cells were treated with 100 nM CH, SN-38, CH+SN-38, MN33-47, and MN33-63 for 48 hours. After perforation, fixation, and propidium iodide (PI) staining, cell cycle distribution was analyzed by flow cytometry (Figures 4C–4I). The results showed that the proportion of G2 / M phase cells in the untreated control group was 8.98% (…). Figure 1 D); the G2 / M phase ratios in the CH-only treatment group, the SN-38-only treatment group, and the CH+SN-38 combined treatment group were 16.7% ( Figure 1 E), 19.6% Figure 1 F) and 22.7% ( Figure 1 G); while the prodrugs MN33-47 and MN33-63 caused significant G2 / M phase blockade, with proportions reaching 46.3% (G); Figure 1 H) and 59.8% Figure 1 (I) Simultaneously, the proportion of cells in the G1 phase decreased accordingly, with MN33-63 causing the most significant G2 / M phase arrest. These results clearly demonstrate that, at the same concentration, the cell cycle arrest induced by the prodrugs MN33-47 and MN33-63 (especially MN33-63) was significantly stronger than that SN-38 alone or in combination with CH, highlighting the design advantage of prodrugs in enhancing antitumor activity by effectively arresting the cancer cell cycle.

[0051] Figure 1 (A) Effects of different compounds (CH, SN-38, CH+SN-38, MN33-47, and MN33-63, 100 nM) on CT26 cell migration. (B) Quantitative analysis of CT26 cell migration after treatment with each compound (n=3). ***P < 0.005. (C) Quantitative flow cytometry analysis of the effects of compounds CH, SN-38, CH+SN-38, and prodrugs MN33-47 and MN33-63 (100 nM) on the CT26 cell cycle (n=3). (D–I) Flow cytometry analysis of CT26 cell cycle distribution in each treatment group (D: control group; E: CH group; F: SN-38 group; G: CH+SN-38 combined group; H: MN33-47 group; I: MN33-63 group).

[0052] Example 4: Apoptosis Experiment

[0053] (1) Experimental steps:

[0054] CT26 cells were spaced at 1 × 10⁶ cells per well. 6Cells were seeded at a density of 1000 mcg / well in 6-well plates and incubated at 37°C for 24 hours to allow for complete adhesion. After adhesion, the cells were treated with 0.1 μM of the assay compound for 48 hours. After treatment, the culture medium was removed, and the cells were digested with EDTA-free trypsin, centrifuged, and the cell pellet was collected. The cell pellet was washed 2-3 times with PBS, gently resuspended in 1× Binding Buffer, and the cell density was adjusted to 1 × 10⁶ cells / well. 6 Cells were prepared at a density of [number] cells / mL to form a homogeneous cell suspension. 100 μL of the cell suspension was transferred to a pre-chilled flow cytometry tube, and 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added sequentially. The mixture was vortexed for 10 seconds and incubated at 25 °C in the dark for 15 minutes to allow dye binding. After incubation, 400 μL of 1× Binding Buffer was added to each tube and gently mixed. The prepared samples were analyzed within 1 hour using a flow cytometer (FC500-MPL), detecting the fluorescence signal at an excitation wavelength of 488 nm. Data analysis was performed using FlowJo software.

[0055] Figure 2 (A) Analysis of apoptosis induced in CT26 cells by prodrugs MN33-47 and MN33-63 at a concentration of 100 nM (detected by Annexin V-FITC / PI staining). The groups were: a, control group; b, CH group; c, SN-38 group; d, CH+SN-38 combined group; e, MN33-47 group; f, MN33-63 group. (B) Quantitative analysis of apoptosis in CT26 cells after treatment with each compound at 100 nM (control group, CH, SN-38, CH+SN-38, MN33-47, and MN33-63) by Annexin V-FITC / PI staining. All experiments were repeated three times.

[0056] (2) Results Analysis and Discussion:

[0057] To evaluate the pro-apoptotic effects of the prodrugs MN33-47 and MN33-63 on CT26 cells, we used Annexin V-FITC / PI double staining combined with flow cytometry for analysis. Figure 2(A–2B). All treatment groups (a. control group; b. CH single drug; c. SN-38 single drug; d. CH+SN-38 physical mixture; e. MN33-47; f. MN33-63) were administered at a concentration of 100 nM. Results showed that compared with the blank control group, the apoptosis rates of each control group (CH, SN-38, and CH+SN-38 mixture) were significantly increased: 15.91% in the CH group, 25.00% in the SN-38 group, and 38.20% in the CH+SN-38 mixture group. At the same concentration (100 nM), the apoptosis rates induced by the prodrugs MN33-47 and MN33-63 were significantly higher, at 50.63% and 61.10%, respectively, indicating that their pro-apoptotic effects were significantly superior to those of CH, SN-38 single drugs, and their physical mixtures. This superior performance is attributed to its innovative molecular design: as a covalent conjugate of SN-38 (a topoisomerase I inhibitor) and chlorambucil (CH, a DNA alkylating agent), these two prodrugs release two active ingredients simultaneously upon intracellular activation; SN-38 induces DNA damage by stabilizing the topoisomerase I–DNA complex, while CH induces DNA cross-linking and strand breaks through alkylation. The synergistic effect of these two different DNA damage mechanisms results in significantly enhanced cytotoxicity compared to either the single drug or its physical mixture (CH+SN-38), thereby effectively inducing apoptosis. Crucially, this synergistic effect effectively overcomes the inherent resistance of CT26 cells to SN-38. Compared to the moderate antiproliferative activity (IC50) exhibited by SN-38 monotherapy, this effect significantly enhances the cytotoxicity. 50 With a concentration of 112.6 nM and limited apoptosis-inducing ability (25.00%), MN33-47 and MN33-63 exhibited potent inhibitory activity, with an IC50 concentration of 112.6 nM and 25.00% respectively. 50 The values ​​were 4.77 nM and 9.78 nM, respectively, and the apoptosis-inducing effect was significantly enhanced. These results confirm that the covalent dual-drug strategy successfully bypassed the inherent resistance barrier of CT26 cells to SN-38 through a synergistic mechanism.

[0058] Example 5: Pharmacodynamic evaluation of tumor-bearing mice

[0059] Experimental steps

[0060] (1) Model building

[0061] Cell preparation: CT26 cells in the exponential growth phase were collected by trypsin digestion, washed twice with PBS and centrifuged, then diluted with PBS and counted to adjust the cell concentration to 1×10⁻⁶. 7 Cells / mL, take 100μL (containing 1×10⁻⁶ cells / mL) 6 CT26 cell suspension (1 cell) was subcutaneously injected into the axilla of 4-week-old female BALB / c mice.

[0062] Animal feeding and grouping: Mice were purchased from Beijing Spefol Biotechnology Co., Ltd. After purchase, they were acclimatized for 2-3 days in a standard laboratory environment, and their weight was measured and recorded. When the tumor volume reached 50 mm³, the experimental mice were randomly divided into several groups (n=4), including a blank control group, a positive control group (SN-38 2 mg / kg, CH 1 mg / kg), and a test compound group (MN33-47: 1.56, 3.12, 6.24 mg / kg; MN33-63: 1.79, 3.58, 7.16 mg / kg). All groups were fed standardized feed and maintained under the same environmental conditions.

[0063] (2) Dosing regimen

[0064] Drug preparation: The positive control and test compound groups were diluted with physiological saline, and each sample was treated with 5% Tween 80 solubilizer and sonicated.

[0065] Administration method and cycle: Mice were administered intraperitoneal injections daily. The model group was injected with physiological saline containing 5% Tween 80, while other groups were administered the prescribed doses. The experiment lasted for 10 days. The dosage settings were as follows: for II-1, the three gradient doses were 1.56, 3.12, and 6.24 mg / kg, respectively; for II-3, the three gradient doses were 1.79, 3.58, and 7.16 mg / kg, respectively; the positive controls SN-38 were 2 mg / kg, and CH was 1 mg / kg. All doses were converted to body weight (unit: mg / kg).

[0066] (3) Evaluation indicators

[0067] Basic indicator monitoring: During the experiment, the mice were weighed daily using an electronic balance and the tumor volume was measured using a vernier caliper. The calculation formula was: tumor volume (mm³) = 0.5 × length × width². At the same time, the physiological status of the mice was systematically assessed, including mental state, behavioral patterns, and coat condition.

[0068] Tissue sample collection and processing: After the administration was completed, blood was collected from the eyeballs of each mouse to collect serum samples for assessing liver and kidney function; after the mice were sacrificed, heart, liver, spleen, lung, kidney and tumor tissues were collected. The tumor tissues were weighed and photographed using an electronic balance. The collected samples were fixed in 4% paraformaldehyde solution at room temperature.

[0069] H&E staining and pathological analysis: The fixed tissue was subjected to gradient dehydration, clearing, paraffin embedding, and preparation of 4 μm thick paraffin sections. After dewaxing and hydration, the sections were stained with Harris hematoxylin and eosin in sequence. After dehydration and clearing, the sections were mounted with neutral resin and observed under a microscope to acquire images and assess the pathological damage of tumors and organ tissues.

[0070] Figure 3(A) Animal model establishment and administration regimen. BALB / c female mice carrying tumors were subcutaneously injected with 1×10⁻⁶ saturates in the right ventral region. 6 Mice were randomly divided into 6 groups (n=5) when the tumor volume reached approximately 50 mm³ on day 9. From day 10, mice received intraperitoneal injections daily for 10 consecutive days. The groups and dosages were as follows: control group (physiological saline containing 5% Tween 80), CH (1 mg / kg), SN-38 (2 mg / kg), CH+SN-38 combination therapy (1 mg / kg + 2 mg / kg), MN33-47 (1.56, 3.12, 6.24 mg / kg), and MN33-63 (1.79, 3.58, 7.16 mg / kg). Animals were sacrificed and samples were collected after the last administration. (B) Tumor volume growth curves of mice in different treatment groups. (C) Weight changes of mice in different treatment groups over time. (D) Actual tumor weight of each treatment group. (E) Tumor weight inhibition rate of each treatment group. (F) Representative photographs of tumors removed from each group after 10 days of treatment. Treatment conditions for all groups were as follows: Solvent control: physiological saline containing 5% Tween 80 (a); CH: 1 mg / kg (b); SN-38: 2 mg / kg (c); CH+SN-38: 1 mg / kg + 2 mg / kg (d); MN33-47 low, medium, and high doses: 1.56 (e), 3.12 (f), and 6.24 mg / kg (g); MN33-63 low, medium, and high doses: 1.79 (h), 3.58 (i), and 7.16 mg / kg (j). ***P < 0.005.

[0071] (4) Results and Discussion:

[0072] We further evaluated its in vivo efficacy in a CT26 tumor-bearing BALB / c mouse model. The experimental protocol is as follows: Figure 3 As shown in Figure A, tumor-bearing mice were randomly assigned to the following groups: solvent control group, CH single-drug group (1 mg / kg), SN-38 single-drug group (2 mg / kg), CH+SN-38 physical mixture group (1 mg / kg + 2 mg / kg), and gradient dose groups of MN33-47 (1.56, 3.12, 6.24 mg / kg) and MN33-63 (1.79, 3.58, 7.16 mg / kg). Treatment continued for 10 consecutive days, and mice were sacrificed and tumors removed on day 12.

[0073] During days 1 to 10 of treatment, both prodrugs MN33-47 and MN33-63 exhibited significant tumor growth inhibition. Figure 3(B) The tumor volume in these groups was significantly smaller than that in the blank control group. The medium-dose prodrug showed better tumor-suppressive effects than the SN-38 monotherapy group, while high-dose treatment further reduced tumor volume, achieving an inhibition level comparable to the CH control group and the CH+SN-38 physical mixture group. The tumor growth curve in the prodrug group was flatter, indicating that it has stronger and more durable antitumor activity in vivo.

[0074] However, weight monitoring during treatment showed that mice in both the CH control group and the CH+SN-38 physical mixture group experienced a significant decrease in body weight. Figure 3 C), indicating that these regimens have systemic toxicity. In contrast, no weight loss was observed in any of the prodrug groups (dose of MN33-47 and MN33-63) and the SN-38 group, indicating better safety.

[0075] Analysis of the weight of the tumor stripped at the endpoint showed that both MN33-47 and MN33-63 exhibited a clear dose-dependent inhibitory effect. Figure 3 D). The high-dose prodrug group showed significantly stronger inhibitory effects than CH, SN-38 monotherapy, and the CH+SN-38 physical mixture group, and also exhibited the lowest tumor mass. Further analysis of the inhibition rate ( Figure 3 E) It was found that the tumor inhibition rates of the high-dose MN33-47 and MN33-63 groups reached 75.73% and 77.10%, respectively, which were significantly higher than those of the CH monotherapy group (60.44%), the SN-38 monotherapy group (33.85%), and the CH+SN-38 physical mixture group (61.85%). Consistent with the above results, the dissected tumor photographs ( Figure 3 F) clearly shows that the tumor volume in the high-dose prodrug groups (MN33-47: 6.24 mg / kg, g group; MN33-63: 7.16 mg / kg, j group) was significantly smaller than that in the blank control group (a group), the SN-38 monotherapy group (b group), and the CH monotherapy group (c group), and even smaller than the corresponding physical mixture groups (d group). In summary, these results demonstrate that MN33-47 and MN33-63 have significantly better antitumor effects in vivo than any single drug or their physical mixture, suggesting a synergistic antitumor effect.

Claims

1. A bis(2-chloroethyl)carbamate-modified SN-38 derivative, the general structural formula of which is shown in formula (I): in, R is selected from H, phosphoric acid (PO3H2), sodium phosphate (PO3Na2), etc.; XH is a pharmaceutically acceptable acid such as hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), hydrobromic acid (HBr), nitric acid (HNO3), citric acid (C6H8O7), maleic acid (C4H4O4), tartaric acid (C4H6O6), methanesulfonic acid (CH3SO3H), acetic acid (CH3COOH), fumaric acid (C4H4O4), succinic acid (C4H6O4), malic acid (C4H6O5), benzoic acid (C7H6O2), lactic acid (C3H6O3), glutamic acid (C5H9NO4), aspartic acid (C4H7NO4).

2. A bis(2-chloroethyl)carbamate-modified SN-38 derivative of formula (I), its enantiomer, racemate, or mixture thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, characterized in that the compound is selected from the group consisting of: Table 1:

3. The method for preparing the SN-38 derivative as described in claim 1 or 2, characterized in that: The synthetic route of the method is as follows:

4. The method for preparing the SN-38 derivative according to claim 1 or 2, characterized in that: Starting with SN-38, the target compounds 1, 2-HX, 3, and 4 were synthesized sequentially through carbamate esterification, hydrochlorination, phosphorylation, and neutralization salt formation reactions. The specific steps are as follows: (1) Step 1: In the presence of a base (such as triethylamine, pyridine, or their analogues) and a catalyst (such as DMAP or its derivatives), SN-38 and bis(2-chloroethyl)carbamoyl chloride (BCCC, or its corresponding carbamic acid, etc. as raw materials) were reacted at room temperature for 12-24 hours in a halocarbon solvent (such as dichloromethane, chloroform). Compound 1 was obtained by extraction and column chromatography. (2) Step 2: Compound 1 reacts with concentrated hydrochloric acid or other inorganic acids (such as sulfuric acid, hydrobromic acid) or organic acids (such as citric acid, maleic acid, tartaric acid, methanesulfonic acid, acetic acid, fumaric acid, succinic acid, malic acid, benzoic acid, lactic acid, glutamic acid, aspartic acid, etc.) in an alcohol-ether mixed solvent (such as methanol / diethyl ether, ethanol / isopropyl ether) at room temperature for 0.5-2 hours, and the acid salt is precipitated to obtain compound 2-HX; (3) Step 3: Compound 1 reacts with phosphorylation reagent (such as P2O5, POCl3 or tetraethyl pyrophosphate) in an inert solvent (such as dioxane, toluene) at 60-100℃ in steps for 12-48 hours, and is purified by precipitation to obtain phosphate ester compound 3; (4) Step 4: Compound 3 reacts with strong base (such as NaOH) in a polar solvent (such as ethanol, methanol) at room temperature for 8-12 hours, and is dried to obtain water-soluble sodium phosphate salt compound 4. This method can efficiently prepare a series of SN-38 derivatives by optimizing reaction conditions (including but not limited to catalyst type, solvent selection, temperature control and purification method), and its protection scope covers reasonable alternatives to the reaction parameters in the above steps.

5. A bis(2-chloroethyl)carbamate-modified SN-38 derivative as described in claim 1 or 2, characterized in that, The inhibitors include compounds as described in any one of claims 1-2, their enantiomers, diastereomers, racemates and mixtures thereof, as well as their pharmaceutically acceptable salts, hydrates and solvates.

6. A pharmaceutical composition, characterized in that, include: (A) A therapeutically effective amount of the compound of claim 1, including its enantiomers, diastereomers, racemates and mixtures thereof, and one or more of its pharmaceutically acceptable salts, hydrates and solvates; and (B) a pharmaceutically acceptable carrier of the compound of claim 1. In another preferred embodiment, the pharmaceutical composition optionally further comprises pharmaceutically acceptable excipients selected from the group consisting of: binders, fillers, diluents, disintegrants, suspending agents, suspending agents, sustained-release agents, lyophilization protectants, coating agents, enteric materials, lubricants, flow aids, anti-adhesives, sweeteners, flavoring agents, plasticizers, light-blocking agents, solubilizers, humectants, solvents, osmotic pressure regulators, colorants, pigments, surfactants, emulsifiers, water-soluble matrices, fat-soluble matrices, lipophilic matrices, pore-forming agents, gelling agents, preservatives, buffers, chelating agents, antioxidants, or combinations thereof. The pharmaceutical composition is characterized by its use in the preparation and treatment of cancer-related diseases. In another preferred embodiment, the cancer-related disease is selected from the group consisting of: liver cancer, lung cancer, breast cancer, stomach cancer, colorectal cancer, esophageal cancer, cervical cancer, ovarian cancer, bladder cancer, pancreatic cancer, acute and chronic myeloid leukemia, choriocarcinoma, etc.

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