Camptothecin derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical compounds, and discloses a camptothecin derivative and a preparation method and application thereof.The preparation method of the camptothecin derivative comprises the following reaction steps that camptothecin, isovaleraldehyde / n-butyraldehyde, Mes-Acr < + >, TPA and TFA are added into dichloromethane and mixed under protection of inert gas for a Minisci reaction, and the camptothecin derivative is obtained. And separating and purifying the product after the reaction to obtain the camptothecin derivative. In conclusion, the novel camptothecin derivative is provided, and the preparation method is simple. Besides, based on the novel camptothecin derivative disclosed by the invention, the growth of tumor cells can be inhibited, and the novel camptothecin derivative can be used as a potential anti-cancer drug, particularly, the inhibitory activity on liver cancer cells HepG2 is superior to that of CPT and SN-38, and the inhibitory activity on breast cancer cells MCF-7 is superior to that of SN-38.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and in particular relates to camptothecin derivatives and preparation methods and applications thereof. Background Art

[0002] Camptothecin is a natural alkaloid isolated from Camptotheca acuminata. It produces significant anti-tumor 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 anti-tumor activity, it still has many problems, such as poor solubility, dose-limiting toxicity, rapid diffusion of Topo I-DNA complex, plasma instability caused by hydrolysis of the lactone ring and binding to human serum proteins, etc. Therefore, it is urgent 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 raised in the above background technology, the purpose of the present invention is to provide camptothecin derivatives and preparation methods and applications thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Solution 1

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

[0008]

[0009] Solution 2

[0010] The preparation method of camptothecin derivatives is to prepare the compound shown in formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to the following reaction route:

[0011]

[0012] Option 3

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

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

[0015]

[0016] Preferably, the inert gas is argon.

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

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

[0019] Preferably, the reaction temperature of the Minisci reaction is 25-35° C., and the reaction time is 8-10 h.

[0020] Preferably, the separation and purification comprises:

[0021] Adding a sodium bicarbonate aqueous solution to the product after the reaction to separate into a first aqueous phase and a first organic phase;

[0022] Extracting the first aqueous phase with dichloromethane to obtain a second organic phase;

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

[0024] The first aqueous phase is filtered, and the obtained solid is dissolved in an organic solvent and then separated by thin layer chromatography to obtain a camptothecin derivative represented by 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] Use of a camptothecin derivative as shown in formula (I) or (II) in the preparation of a drug for treating cancer, wherein the cancer includes liver cancer and breast cancer;

[0028]

[0029] Option 5

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

[0031]

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

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

[0034] 2) The present invention is based on novel camptothecin derivatives that can be used to prepare tumor cell growth inhibitors and anticancer drugs. Specifically, the inhibitory activity against liver cancer cells HepG2 is better than that of CPT and SN-38, and the inhibitory activity against breast cancer cells MCF-7 is better than that of SN-38. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 is 1445903-27-4

[0042] Mes-Acr + : 10-Methyl-9-mesityl acridine perchlorate, CAS number is 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.), Mes-Acr + (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 by bubbling argon for 15 min to achieve inert gas protection;

[0052] S4. Place the test tube on a magnetic stirrer for mixing and stirring, and irradiate the reaction with a 36W blue light strip (about 2 cm from the light source) at a temperature of 25 to 35°C for 8 to 10 hours;

[0053] S5. A 1M aqueous sodium bicarbonate solution was added to the reaction product until the pH value of the mixed solution reached 7;

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

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

[0056] S8. The first organic phase and the second organic phase were combined, dried with Na2SO4, and separated by thin layer chromatography (DCM: MeOH = 20: 1) to obtain a camptothecin derivative as shown in formula (I);

[0057] S9. The first aqueous phase is filtered and the obtained solid is treated with an organic solvent (V 二氯甲烷 :V 甲醇 =1-2:1) and then separated by thin layer chromatography (DCM:MeOH=20:1) to obtain a 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] NMR Spectroscopy: 1 H NMR (500 MHz, 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.), Mes-Acr + (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 by bubbling argon for 15 min to achieve inert gas protection;

[0073] S4. Place the test tube on a magnetic stirrer for mixing and stirring, and irradiate the reaction with a 36W blue light strip (about 2 cm from the light source) at a temperature of 25 to 35°C for 8 to 10 hours;

[0074] S5. A 1M aqueous sodium bicarbonate solution was added to the reaction product until the pH value of the mixed solution reached 7;

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

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

[0077] S8. The first organic phase and the second organic phase were combined, dried with Na2SO4, and separated by thin layer chromatography (DCM: MeOH = 20: 1) to obtain a camptothecin derivative as shown in formula (II);

[0078] S9. The first aqueous phase is filtered and the obtained solid is treated with an organic solvent (V 二氯甲烷 :V 甲醇 =1-2:1) and then separated by thin layer chromatography (DCM:MeOH=20:1) to obtain a 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] NMR 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] In order to investigate the anti-tumor activity of the obtained camptothecin derivatives, liver cancer cells HepG2 and breast cancer cells MCF-7 were selected for cell proliferation inhibition activity experiments.

[0086] Human liver cancer cell line HepG2 and human breast cancer cell line MCF-7 cell culture:

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

[0088] (2) Cell culture - Add DMEM culture medium to the cryopreservation tube, and use a pipette to blow several times to disperse the cell clusters into single cells. In the clean bench, transfer the thawed cells to the culture dish with a pipette, shake the cells evenly with the cross-shaking method, and place them in a 37°C, 5% CO2 incubator for culture;

[0089] (3) Cell passaging: When the cell growth reaches 80-90%, remove the old culture medium, add PBS solution for washing, remove the PBS solution, add trypsin to digest the cells so that the cells change from an adherent state to a floating state, and then add new culture medium to terminate the digestion. Use a pipette to transfer the cell suspension to a sterile centrifuge tube and centrifuge, discard the supernatant, add DMEM culture medium and pipette several times, and transfer to a new culture dish for culture.

[0090] MTT assay of camptothecin derivatives:

[0091] HepG2 and MCF-7 cells in the logarithmic growth phase were cultured at 8×10 4 and 5×104 Cells were seeded at a density of 100 μg / mL in a 96-well plate and incubated in a cell culture incubator for 24 h. Then, camptothecin derivatives of different concentrations (0.1, 1, 5, 10, 50, 100 μM) were added and allowed to act for 48 h.

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

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

[0095] Controlled trials:

[0096] SN-38 (7-ethyl-10-hydroxycamptothecin) and CPT were used as control compounds, and the test procedures and the method for calculating cell viability were the same as those of the MTT test of camptothecin derivatives.

[0097] In summary, the cell proliferation inhibitory activity was calculated as shown in the following table

[0098]

[0099] From the data results shown in the above table, it can be seen that the camptothecin derivatives of the present invention have strong proliferation inhibitory activity on both HepG2 cells and MCF-7 cells, and the inhibitory activity on liver cancer cells HepG2 is better than that of CPT and SN-38, and the inhibitory activity on breast cancer cells MCF-7 is better than that of SN-38.

[0100] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A camptothecin derivative, characterized in that: Including compounds represented by formula (I) or (II), or pharmaceutically acceptable salts thereof, or stereoisomers thereof; 2. The method for preparing a camptothecin derivative according to claim 1, characterized in that: The compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof can be prepared according to the following reaction scheme: Alternatively, the compound represented by formula (II), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof can be prepared according to the following reaction route:

3. The preparation method according to claim 2, characterized in that: The process comprises the following reaction steps: Add camptothecin, isovaleraldehyde, Mes-Acr + , TPA and TFA, mixed under the protection of inert gas to produce Minisci reaction, and the product after the reaction is separated and purified to obtain a camptothecin derivative as shown in formula (I) or camptothecin, n-butyraldehyde, Mes-Acr are added to dichloromethane + , TPA and TFA are mixed under the protection of an inert gas to undergo a Minisci reaction, and the product after the reaction is separated and purified to obtain a 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 the dichloromethane is 0.05M; the camptothecin, isovaleraldehyde or n-butyraldehyde, Mes-Acr + , the mixing molar ratio of TPA and TFA is 1:2:0.05:0.1:

2.

6. The preparation method according to claim 3, characterized in that: The reaction temperature of the Minisci reaction is 25-35° C., and the reaction time is 8-10 h.

7. The preparation method according to claim 3, characterized in that: The separation and purification comprises: Adding a sodium bicarbonate aqueous solution to the product after the reaction to separate into a first aqueous phase and a first organic phase; Extracting the first aqueous phase with dichloromethane to obtain a second organic phase; The first organic phase and the second organic phase are combined, dried, and separated by thin layer chromatography to obtain a camptothecin derivative as shown in formula (I) or (II); The first aqueous phase is filtered, and the obtained solid is dissolved in an organic solvent and then separated by thin layer chromatography to obtain a camptothecin derivative as shown in formula (I) or (II).

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

1.

9. Use of the camptothecin derivative according to claim 1 in the preparation of a drug for treating cancer, wherein the cancer includes liver cancer and breast cancer.

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

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