Camptothecin derivatives, their preparation methods and applications

Abstract

This invention belongs to the field of pharmaceutical compound technology, and discloses a camptothecin derivative, its preparation method, and its application. The preparation method of the camptothecin derivative includes the following reaction steps: adding camptothecin, isovaleraldehyde / n-butyraldehyde, and Mes-Acr to dichloromethane. + TPA and TFA are mixed under inert gas protection and undergo a Minisci reaction. The product after the reaction is separated and purified to obtain a camptothecin derivative. In summary, a novel camptothecin derivative is provided, and the preparation method is simple. In addition, the novel camptothecin derivative based on this invention can inhibit tumor cell growth and serves as a potential anticancer drug. Specifically, its inhibitory activity against HepG2 liver cancer cells is superior to CPT and SN-38, and its inhibitory activity against MCF-7 breast cancer cells is superior to SN-38.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to camptothecin derivatives, their preparation methods, and applications. Background Technology

[0002] Camptothecin is a natural alkaloid isolated from the camptotheca tree. It exhibits significant antitumor activity by inhibiting DNA topoisomerase I and has good cytotoxic activity against various cancers such as liver cancer, breast cancer, colon cancer, lung cancer, and leukemia.

[0003] Although camptothecin exhibits broad and significant antitumor activity, it still has many problems, such as poor solubility, dose-limited toxicity, rapid diffusion of the Topo I-DNA complex, and plasma instability caused by lactone ring hydrolysis and binding to human serum proteins. Therefore, there is an urgent need to develop camptothecin derivatives with better solubility, higher stability, and better activity. Summary of the Invention

[0004] In view of this, in order to solve the problems mentioned in the background art, the purpose of the present invention is to provide camptothecin derivatives, their preparation methods and applications.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Option 1

[0007] Camptothecin derivatives, including compounds of formula (I) or (II), or pharmaceutically acceptable salts thereof, or stereoisomers thereof;

[0008]

[0009] Option 2

[0010] The preparation method of camptothecin derivatives involves preparing compounds of formula (I) or formula (II), or pharmaceutically acceptable salts thereof, or stereoisomers thereof, according to the following reaction route:

[0011]

[0012] Option 3

[0013] The preparation method of camptothecin derivatives includes the following reaction steps:

[0014] Add camptothecin, isovaleraldehyde / n-butyraldehyde, and Mes-Acr to dichloromethane + TPA and TFA are mixed under inert gas protection to undergo a Minisci reaction. The product after the reaction is separated and purified to obtain camptothecin derivatives as shown in formula (I) or formula (II).

[0015]

[0016] Preferably, the inert gas is argon.

[0017] Preferably, the concentration of the dichloromethane is 0.05M.

[0018] Preferably, the camptothecin, isovaleraldehyde / n-butyraldehyde, and Mes-Acr + The molar ratio of TPA to TFA is 1:2:0.05:0.1:2.

[0019] Preferably, the Minisci reaction is carried out at a temperature of 25–35°C for 8–10 hours.

[0020] Preferably, the separation and purification includes:

[0021] An aqueous solution of sodium bicarbonate was added to the product after the reaction, and the first aqueous phase and the first organic phase were separated.

[0022] The first aqueous phase was extracted with dichloromethane to obtain the second organic phase;

[0023] The first organic phase and the second organic phase were combined, dried, and separated by thin-layer chromatography to obtain camptothecin derivatives as shown in formula (I) or formula (II);

[0024] The first aqueous phase was filtered, and the resulting solid was dissolved in an organic solvent and then separated by thin-layer chromatography to obtain camptothecin derivatives as shown in formula (I) or formula (II).

[0025] Preferably, the organic solvent comprises dichloromethane and methanol in a volume ratio of 1 to 2:1.

[0026] Option 4

[0027] The use of camptothecin derivatives as shown in formula (I) or formula (II) in the preparation of drugs for treating cancer, wherein said cancers include liver cancer and breast cancer;

[0028]

[0029] Option 5

[0030] A tumor cell growth inhibitor prepared from a camptothecin derivative as shown in formula (I) or formula (II), wherein the inhibitor is used to inhibit the growth activity of HepG2 liver cancer cells and MCF-7 breast cancer cells;

[0031]

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1) This invention provides a novel camptothecin derivative, and the preparation method is simple;

[0034] 2) This invention is based on the novel camptothecin derivatives that can be used to prepare tumor cell growth inhibitors and anticancer drugs. Specifically, the inhibitory activity against HepG2 liver cancer cells is superior to that against CPT and SN-38, and the inhibitory activity against MCF-7 breast cancer cells is superior to that against SN-38. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The following is a list of abbreviations used in the examples.

[0037] CPT: Camptothecin

[0038] DCM: Dichloromethane

[0039] MeOH: Methanol

[0040] TFA: Trifluoroacetic acid

[0041] TPA: Its CAS number is 1445903-27-4

[0042] Mes-Acr + : 10-Methyl-9-trimethylmethylacridinium perchlorate, CAS number 674783-97-2

[0043] Na2SO4: Sodium sulfate

[0044] Example 1

[0045] Synthesis of camptothecin derivatives

[0046] The reaction route is as follows:

[0047]

[0048] Based on the above reaction route, the specific reaction steps include:

[0049] S1. Take 4 mL of 0.05 M dichloromethane as the reaction solvent and add the reaction solvent to a 10 mL test tube containing a magnetic particle;

[0050] S2. Add camptothecin (0.10 mmol, 1.0 equiv.), isovaleraldehyde (0.20 mmol, 2.0 equiv.), and Mes-Acr to the test tube. + (0.005mmol, 0.05equiv.), TPA (0.01mmol, 0.1equiv.), TFA (0.40mmol, 2.0equiv.);

[0051] S3. Place the test tube in ice water and degas the mixture in the test tube with argon gas for 15 minutes to achieve inert gas protection;

[0052] S4. Place the test tube on a magnetic stirrer and mix. Then, irradiate the mixture with a 36W blue light strip (about 2cm away from the light source) at a temperature of 25-35℃ for 8-10 hours.

[0053] S5. Add a 1M sodium bicarbonate aqueous solution to the product after the reaction until the pH of the mixed solution is 7.

[0054] S6. Separate the mixed solution to obtain the first aqueous phase and the first organic phase;

[0055] S7. Extract the first aqueous phase with dichloromethane to obtain the second organic phase;

[0056] S8. Combine the first organic phase and the second organic phase, dry with Na2SO4 and separate by thin-layer chromatography (DCM:MeOH = 20:1) to obtain the camptothecin derivative as shown in formula (Ⅰ);

[0057] S9. The first aqueous phase is filtered, and the resulting solid is treated with an organic solvent (V). 二氯甲烷 :V 甲醇 After dissolving (1-2:1), the mixture was separated by thin-layer chromatography (DCM:MeOH = 20:1) to obtain the camptothecin derivative as shown in formula (Ⅰ).

[0058] The structure of the camptothecin derivative shown in formula (Ⅰ) was identified by nuclear magnetic resonance spectroscopy:

[0059] R f =0.30(DCM / MeOH 20:1(v / v)).

[0060] Nuclear magnetic resonance spectroscopy: 1 H NMR (500MHz, DMSO-d6)δ

[0061] 8.16 (dd, J = 8.0, 5.1 Hz, 2H),

[0062] 7.83(t,J=7.6Hz,1H),7.71(t,J=7.7Hz,1H),7.33(s,1H),6.53(s,1H),5.92(s,1H),5.67(d,J=10.3Hz,1H),5.51-5.28(m,4H) ,2.06-2.03(m,2H),1.90-1.76(m,2H),1.43-1.33(m,1H),1.10(d,J=6.5Hz,3H),0.93(d,J=6.7Hz,3H),0.89(t,J=7.3Hz,3H); 13 C NMR(126MHz,DMSO-d6)δ172.97,157.28,

[0063] 153.22,150.45,148.81,148.26,145.74,130.40,130.33,128.04,126.54,125.27,124.31,119. 40,96.91,72.87,67.33,65.75,51.41,46.30,30.77,24.90,24.07,21.79,8.25; HRMS(APCI)m / z calcd for C 25 H 27 N2O5

[0064] [M+H] + :435.1914,found:435.1912.

[0065] Example 2

[0066] Synthesis of camptothecin derivatives

[0067] The reaction route is as follows:

[0068]

[0069] Based on the above reaction route, the specific reaction steps include:

[0070] S1. Take 4 mL of 0.05 M dichloromethane as the reaction solvent and add the reaction solvent to a 10 mL test tube containing a magnetic particle;

[0071] S2. Add camptothecin (0.10 mmol, 1.0 equiv.), n-butyraldehyde (0.20 mmol, 2.0 equiv.), and Mes-Acr to the test tube. + (0.005mmol, 0.05equiv.), TPA (0.01mmol, 0.1equiv.), TFA (0.40mmol, 2.0equiv.);

[0072] S3. Place the test tube in ice water and degas the mixture in the test tube with argon gas for 15 minutes to achieve inert gas protection;

[0073] S4. Place the test tube on a magnetic stirrer and mix. Then, irradiate the mixture with a 36W blue light strip (about 2cm away from the light source) at a temperature of 25-35℃ for 8-10 hours.

[0074] S5. Add a 1M sodium bicarbonate aqueous solution to the product after the reaction until the pH of the mixed solution is 7.

[0075] S6. Separate the mixed solution to obtain the first aqueous phase and the first organic phase;

[0076] S7. Extract the first aqueous phase with dichloromethane to obtain the second organic phase;

[0077] S8. Combine the first organic phase and the second organic phase, dry with Na2SO4 and separate by thin-layer chromatography (DCM:MeOH = 20:1) to obtain the camptothecin derivative as shown in formula (Ⅱ);

[0078] S9. The first aqueous phase is filtered, and the resulting solid is treated with an organic solvent (V). 二氯甲烷 :V 甲醇 After dissolving (1-2:1), the solution was separated by thin-layer chromatography (DCM:MeOH = 20:1) to obtain the camptothecin derivative as shown in formula (Ⅱ).

[0079] The structure of the camptothecin derivative shown in formula (II) was identified by nuclear magnetic resonance spectroscopy:

[0080]

[0081] R f =0.30(DCM / MeOH 20:1(v / v))

[0082] Nuclear magnetic resonance spectroscopy: 1 H NMR (500MHz, DMSO-d6) δ8.27(d,J=8.4Hz,1H),8.17(d,J=8.4Hz,1H),7.85(t,J=7.6Hz,1H),7.71(t,J=7.6Hz,1H),7.34(s,1H),6.53(s ,1H),5.92(d,J=3.3Hz,1H),5.69–5.60(m,1H),5.51–5.40(m,3H),5.35(d,J=19.4Hz,1H),1.98–1.71(m,4H),1.51(m,2H),0.90(m,6H); 13C NMR(126MHz,DMSO-d6)δ172.98,157.30,153.17,150.47,148.83,147.93,145.78,130.37,130.34,127.98,126.70, 125.53,124.78,119.41,96.90,72.88,68.82,65.76,51.56,30.77,29.44,19.01,14.28,8.25; HRMS(APCI)m / zcalcd for C 24 H 25 N₂O₅[M+H] + :421.1758,found:421.1755.

[0083] Example 3

[0084] Cytotoxicity test of camptothecin derivatives

[0085] To investigate the antitumor activity of the obtained camptothecin derivatives, cell proliferation inhibition experiments were conducted on HepG2 liver cancer cells and MCF-7 breast cancer cells.

[0086] Culture of human hepatocellular carcinoma cell line HepG2 and human breast cancer cell line MCF-7:

[0087] (1) Cell resuscitation: Take out the cryopreservation tubes containing HepG2 and MCF-7 cells from the liquid nitrogen tank, place them in a 37°C constant temperature water bath to thaw, and then centrifuge them in a centrifuge at 1000r / min for 3min to remove DMSO from the cryopreservation solution.

[0088] (2) Cell culture—Add DMEM medium to the cryovials and repeatedly pipette to disperse the cell clusters into individual cells. In a clean bench, transfer the thawed cells to a culture dish using a pipette, mix the cells using the cross-shaped mixing method, and incubate at 37°C in a 5% CO2 incubator.

[0089] (3) Cell passage – When the cells reach 80-90% confluence, remove the old culture medium, wash with PBS solution, remove the PBS solution, add trypsin to digest the cells to change them from an adherent state to a floating state, and then add fresh culture medium to stop the digestion. Transfer the cell suspension to a sterile centrifuge tube using a pipette, centrifuge, discard the supernatant, add DMEM culture medium and repeatedly pipette, and transfer to a new culture dish for culture.

[0090] MTT assay of camptothecin derivatives:

[0091] HepG2 and MCF-7 cells in logarithmic growth phase were used at a concentration of 8 × 10⁻⁶. 4 and 5×104 The cells were seeded at a density of 10 cells / mL in 96-well plates and incubated in a cell culture incubator for 24 hours. Then, different concentrations (0.1, 1, 5, 10, 50, 100 μM) of camptothecin derivatives were added and incubated for 48 hours.

[0092] Then, add 100 μL of 5 mg / mL MTT solution, incubate in a cell culture incubator for 2.5 h, then aspirate the MTT solution, add 150 μL of DMSO to dissolve the cells, shake for 10 min, measure the absorbance at 490 nm, and calculate the cell viability using the following formula:

[0093] Cell viability (%) = [(A 490,Sample -A 490,Blank ) / (A 490,Control -A 490,Blank )]×100.

[0094] Controlled experiment:

[0095] SN-38 (7-ethyl-10-hydroxycamptothecin) and CPT were used as control compounds. The experimental procedures and cell viability calculation methods were the same as those for the MTT assay of the above-mentioned camptothecin derivatives.

[0096] In summary, the cell proliferation inhibition activities were calculated and shown in the table below.

[0097]

[0098] The data results shown in the table above indicate that the camptothecin derivative of the present invention has strong inhibitory activity against the proliferation of HepG2 cells and MCF-7 cells, and its inhibitory activity against HepG2 liver cancer cells is superior to that against CPT and SN-38, and its inhibitory activity against MCF-7 breast cancer cells is superior to that against SN-38.

[0099] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A camptothecin derivative, characterized in that: Selected from compounds represented by formula (I) or formula (II); 2. The method for preparing the camptothecin derivative as described in claim 1, characterized in that, The compound shown in formula (Ⅰ) was prepared according to the following reaction route: Alternatively, the compound shown in formula (II) can be prepared by following the reaction route:

3. The preparation method according to claim 2, characterized in that, The reaction steps include the following: Add camptothecin, isovaleraldehyde, and Mes-Acr to dichloromethane + TPA and TFA are mixed under an inert gas atmosphere to undergo a Minisci reaction. The product is then separated and purified to obtain a camptothecin derivative as shown in formula (I). Alternatively, camptothecin, n-butyraldehyde, and Mes-Acr are added to dichloromethane. + TPA and TFA were mixed under inert gas protection to undergo a Minisci reaction. The product after the reaction was separated and purified to obtain the camptothecin derivative as shown in formula (II).

4. The preparation method according to claim 3, characterized in that: The inert gas is argon.

5. The preparation method according to claim 3, characterized in that: The concentration of dichloromethane is 0.05M; the camptothecin, isovaleraldehyde or n-butyraldehyde, and Mes-Acr... + The molar ratio of TPA to TFA is 1:2:0.05:0.1:

2.

6. The preparation method according to claim 3, characterized in that: The Minisci reaction is carried out at a temperature of 25–35°C for 8–10 hours.

7. The preparation method according to claim 3, characterized in that, The separation and purification process includes: An aqueous solution of sodium bicarbonate was added to the product after the reaction, and the first aqueous phase and the first organic phase were separated. The first aqueous phase was extracted with dichloromethane to obtain the second organic phase; The first organic phase and the second organic phase were combined, dried, and separated by thin-layer chromatography to obtain camptothecin derivatives as shown in formula (I) or (II); The first aqueous phase was filtered, and the resulting solid was dissolved in an organic solvent and then separated by thin-layer chromatography to obtain camptothecin derivatives as shown in formula (I) or (II).

8. The preparation method according to claim 7, characterized in that: The organic solvent is dichloromethane and methanol in a volume ratio of 1 to 2:

1.

9. The use of the camptothecin derivative as described in claim 1 in the preparation of a medicament for treating cancer, wherein the cancer is selected from liver cancer or breast cancer.

10. A tumor cell growth inhibitor containing a camptothecin derivative as described in claim 1, wherein the inhibitor is used to inhibit the growth activity of HepG2 liver cancer cells and MCF-7 breast cancer cells.

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