CN110423259B - Camptothecin glycoconjugate and preparation method and application thereof - Google Patents
Camptothecin glycoconjugate and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a camptothecin glycoconjugate as well as a preparation method and application thereof. The invention provides a camptothecin glycoconjugate which is synthesized by connecting camptothecin and hexose by using adipic acid as a linker. Experimental results show that the camptothecin glycoconjugate has an anti-tumor effect, and has high-efficiency and low-toxicity effects compared with camptothecin. GLUT1 is widely distributed on blood brain barrier and erythrocyte, the experimental result shows that the camptothecin glycoconjugate can be identified and transported by GLUT1, and enters tumor cells through GLUT1 to achieve the anti-tumor effect, and the camptothecin glycoconjugate can inhibit DNA topoisomerase (TOPO I) as a cytotoxic quinoline alkaloid.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a camptothecin glycoconjugate as well as a preparation method and application thereof.
Background
Malignant tumors, namely cancers, are the second leading cause of death in the world, and in 2018, new cases of cancers exceed 170 thousands of people, and by 2030, the number of new cancer cases is expected to increase to 2360 thousands every year.
Camptothecin is an alkaloid isolated from Camptotheca acuminata fruit. Camptothecin has strong cytotoxic activity, and can be used for treating various malignant tumors such as colon cancer, ovarian cancer, bone cancer, bladder cancer and leukemia. Has great modification value. However, camptothecin is highly cytotoxic to normal cells.
Disclosure of Invention
In view of the above, the present invention provides a camptothecin glycoconjugate, and a preparation method and an application thereof, which are used for solving the problem of high cytotoxicity of camptothecin on normal cells.
The specific technical scheme of the invention is as follows:
a camptothecin glycoconjugate is synthesized by connecting camptothecin and hexose by using adipic acid as a linker.
Experimental results show that the camptothecin glycoconjugate has an anti-tumor effect, and has high-efficiency and low-toxicity effects compared with camptothecin.
In the present invention, hexose is a Glucose transporter 1 (GLUT 1) ligand.
Preferably, the hexose is selected from D-glucose, D-galactose, D-mannose or 2-deoxy-D-glucose.
In the invention, D-glucose, D-galactose, D-mannose and 2-deoxy-D-glucose are GLUT1 ligands, and experimental results show that the camptothecin glycoconjugate can be identified and transported by GLUT1, is transported through a GLUT1 channel, penetrates through a tumor cell membrane, enters a tumor cell to achieve an anti-tumor effect, and solves the problem in the transportation of anti-tumor drugs. In addition, the camptothecin glycoconjugate can inhibit DNA topoisomerase (TOPO I) as a cytotoxic quinoline alkaloid.
Preferably, the camptothecin glycoconjugate is selected from
The invention also provides a preparation method of the camptothecin glycoconjugate in the technical scheme, which comprises the following steps:
a) carrying out a first reaction on hexose and trimethylchlorosilane under an alkaline condition to obtain a first product;
b) carrying out a second reaction on the first product and ammonium acetate to obtain a second product;
c) carrying out a third reaction on camptothecin and adipic acid under the catalysis of EDC & HCl (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) to obtain a third product;
d) carrying out a fourth reaction on the second product and the third product under the catalysis of DCC (dicyclohexylcarbodiimide) and DMAP to obtain a fourth product;
e) and carrying out a fifth reaction on the fourth product and trifluoroacetic acid to obtain the camptothecin glycoconjugate.
The preparation method of the invention uses trimethylsilyl as a hydroxyl protecting group, takes camptothecin and hexose as a linker with adipic acid, then carries out deprotection to obtain camptothecin glycoconjugate, esterifies 20-hydroxyl of camptothecin, eliminates hydrogen bond in camptothecin molecule, increases steric hindrance of adjacent carbonyl, reduces activity of carbonyl, and leads nucleophilic reagent not to attack carbonyl, thereby leading lactone ring to slowly open in vivo to play the role of anti-tumor.
Preferably, the temperature of the first reaction is 20-30 ℃, preferably 25 ℃, and the time is 2.5-4 h, preferably 4 h;
the temperature of the second reaction is 20-30 ℃, preferably 25 ℃, and the time is 8-12 h, preferably 9 h;
the temperature of the third reaction is 30-40 ℃, preferably 40 ℃, and the time is 12-24 h, preferably 16 h;
the temperature of the fourth reaction is 30-40 ℃, preferably 30 ℃, and the time is 12-36 h, preferably 24 h;
the temperature of the fifth reaction is-5 ℃ to 0 ℃, preferably 0 ℃, and the time is 2.5h to 4h, preferably 4 h.
Preferably, the basic reagent for the basic conditions of step a) is selected from triethylamine, N-diisopropylethylamine or pyridine, preferably triethylamine;
the molar ratio of the hexose, the trimethylchlorosilane and the alkaline reagent is 1: (5.5-6): (5.5-6), preferably 1:5.5: 5.5.
In the present invention, the first reaction of step a) is preferably carried out in a solvent selected from the group consisting of N, N-Dimethylformamide (DMF) and/or N, N-dimethylacetamide, preferably N, N-dimethylformamide.
The hexose is selected from D-glucose, D-galactose, D-mannose or 2-deoxy-D-glucose, and the first product is a compound 1 to a compound 4 in sequence:
step a) is particularly preferably: the hexose was dissolved in a mixed solution of triethylamine and DMF (25ml, 61.05mmol), and then the solution system was cooled to 0 ℃ with a low-temperature constant-temperature reaction bath. At 0 ℃, slowly dropwise adding trimethylchlorosilane into the solution system, and then stirring at room temperature to perform a first reaction. The progress of the reaction was checked by TLC, and after completion of the reaction, hexane and crushed ice were added to the reaction mixture in this order. Extracting the aqueous solution with hexane, combining the organic phases, washing twice with water and brine, respectively, anhydrous Na2SO4Drying, concentrating under reduced pressure, and purifying by column chromatography (petroleum ether: ethyl acetate 20: 1) to obtain the first product.
Preferably, step b) the molar ratio of said first product to said ammonium acetate is 1: (1.5-2), more preferably 1: 2;
the second reaction is preferably carried out in a solvent selected from one or more of dichloromethane, methanol, acetonitrile, tetrahydrofuran and acetone, preferably dichloromethane and methanol, preferably in a volume ratio of 1: 1.
The first product is a compound 1 to a compound 4, and the second product is a compound 5 to a compound 8:
step b) is particularly preferably: and (3) carrying out a second reaction on the first product and ammonium acetate under the conditions of dichloromethane and methanol (v: v ═ 1:1) as solvents, wherein the reaction temperature is 25 ℃, the reaction time is 9 hours, the reduced pressure rotary evaporation is carried out, and column chromatography purification (petroleum ether: ethyl acetate ═ 20: 1) is carried out to obtain a second product.
Step c) the molar ratio of camptothecin, adipic acid, EDC & HCl and DMAP is 1: (3-5): (4-8): (3-5), preferably 1: 4: 8: 4;
the third reaction is preferably carried out in a solvent selected from dichloromethane, tetrahydrofuran or acetone, preferably dichloromethane.
The third product is compound 9:
step c) is particularly preferably: dissolving camptothecin in dry CH2Cl2Adding EDC & HCl, DMAP and adipic acid respectively under magnetic stirring, performing magnetic stirring, refluxing for 16h for a third reaction, cooling to room temperature, washing with 10% citric acid, distilled water and saturated brine respectively (200 mL. times.2), drying the organic layer with anhydrous sodium sulfate, filtering, concentrating, and performing column Chromatography (CH)2Cl2: MeOH, 40: 1) a yellow solid, the third product, was obtained.
The molar ratio of the second product, the third product, DCC and DMAP in the step d) is (1.5-3): 1: (2-3): (0.2 to 0.5), preferably 2: 1: 2.4: 0.2.
the fourth reaction is preferably carried out in a solvent selected from dichloromethane, tetrahydrofuran or acetone, preferably dichloromethane.
The second product is a compound 5 to a compound 8, and the fourth product is a compound 10 to a compound 13 in sequence:
the step d) is particularly preferably: dissolving the second product in dry CH2Cl2Adding DCC and DMAP at-5 deg.C, mixing and stirring, and drippingAdding dichloromethane solution of the third product, stirring at 30 deg.C for 24 hr for fourth reaction, filtering, concentrating the filtrate under reduced pressure, and purifying by column Chromatography (CH)2Cl2：CH3OH 80:1) to yield a light yellow solid, the fourth product.
Preferably, the fifth reaction of step e) is carried out in a solvent selected from dichloromethane and/or acetonitrile, preferably dichloromethane;
the volume content of the trifluoroacetic acid in the reaction system is 2.5-10%, preferably 2.5%.
The fourth product is compound 10-compound 13, and the camptothecin glycoconjugates are compound a-compound d in sequence.
Step e) is particularly preferably: dissolving the fourth product in dichloromethane, cooling to 0 deg.C, adding trifluoroacetic acid for fifth reaction, detecting by TLC, concentrating under reduced pressure, and purifying by column Chromatography (CH)2Cl2：CH3OH ═ 12:1) to give a yellow solid, the camptothecin glycoconjugate.
In the preparation method, the synthetic route is as follows:
the invention also provides the application of the camptothecin glycoconjugate in the technical scheme and/or the camptothecin glycoconjugate prepared by the preparation method in the technical scheme in the preparation of antitumor drugs.
The camptothecin glycoconjugate is a water-soluble glycoconjugate, and experimental results show that the camptothecin glycoconjugate has an anti-tumor effect and has a low-toxicity effect compared with camptothecin.
Tumor cell growth is dependent on a constant supply of energy, while glucose is the major source of energy for tumor cells. The GLUT1 is most important among many transporters. GLUT1 is widely distributed on the blood-brain barrier and erythrocytes, and is characterized by high expression in a variety of tumors. Therefore, the delivery effect of targeting tumor cells can be achieved by adding the carrier capable of being recognized by GLUT1 to the anti-tumor medicament. Experimental results show that the camptothecin glycoconjugate can be identified and transported by GLUT1, and enters tumor cells through a GLUT1 channel to achieve the anti-tumor effect. In addition, the camptothecin glycoconjugate can inhibit DNA topoisomerase (TOPO I) as a cytotoxic quinoline alkaloid.
The intake of the camptothecin glycoconjugate is regulated and controlled by GLUT1, GLUT1 is highly expressed in breast cancer and glioma, and the camptothecin glycoconjugate is preferably applied to the preparation of anti-breast cancer and anti-glioma drugs.
The invention also provides a medicament, which comprises the camptothecin glycoconjugate in the technical scheme and/or the camptothecin glycoconjugate prepared by the preparation method in the technical scheme.
The camptothecin glycoconjugate in the drug can be identified and transported by GLUT1, and is transported through a GLUT1 channel, penetrates through a tumor cell membrane and enters a tumor cell to achieve the anti-tumor effect, so that the problem in the transportation of anti-tumor drugs is solved.
In summary, the invention provides a camptothecin glycoconjugate, which is synthesized by connecting camptothecin and hexose by using adipic acid as a linker. Experimental results show that the camptothecin glycoconjugate has an anti-tumor effect and has a low-toxicity effect compared with camptothecin. GLUT1 is widely distributed on blood brain barrier and erythrocyte, the experimental result shows that the camptothecin glycoconjugate can be identified and transported by GLUT1, and enters tumor cells through GLUT1 to achieve the anti-tumor effect, and the camptothecin glycoconjugate can inhibit DNA topoisomerase (TOPO I) as a cytotoxic quinoline alkaloid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph of the effect of camptothecin glycoconjugates on brain microvascular endothelial cell survival (24h) (P < 0.0001 compared to camptothecin group) in an example of the present invention, wherein Control represents negative Control group, CPT represents camptothecin group, and a, b, c represent compound a, compound b and compound c, respectively;
fig. 2 is a graph of the growth inhibition of glioma cells U87 by camptothecin and camptothecin glycoconjugates in example 2 of the present invention (24h) (P < 0.01, P < 0.0001 compared to the camptothecin group), wherein CPT represents the camptothecin group, a, b, c represent compound a, compound b and compound c, respectively;
FIG. 3 is a graph showing the effect of D-glucose on the survival rate of glioma cells U87 (24h) in example 2 of the present invention, wherein Control represents a negative Control group and 200 represents a D-glucose Control group;
fig. 4 is a graph of growth inhibition of glioma cells U87 in different groups of example 2 of the present invention (24h) (P < 0.0001 compared to the group without D-glucose), wherein CPT represents camptothecin group, and a, b, c represent compound a, compound b and compound c, respectively.
Detailed Description
The invention provides a camptothecin glycoconjugate and a preparation method and application thereof, which are used for solving the problem of high cytotoxicity of camptothecin on normal cells.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The culture solution adopted in the embodiment of the invention is a mixture of 10% Fetal Bovine Serum (FBS) supplemented by DEME culture medium and 1% penicillin and streptomycin.
Example 1
This example carried out the preparation of a camptothecin glycoconjugate (compound a) comprising the following steps:
1) d-glucose (2g, 11.1mmol) was dissolved in a mixed solution of triethylamine (8.5ml, 61.05mmol) and DMF (25ml, 61.05mmol), followed by a low-temperature constant-temperature reaction bathThe solution system was cooled to 0 ℃. Trimethylchlorosilane (7.75ml, 61.05mmol) was slowly added dropwise to the solution system at 0 ℃ and then stirred at room temperature for 4 hours to carry out the first reaction. The progress of the reaction was checked by TLC, and after completion of the reaction, hexane and crushed ice were added to the reaction mixture in this order. Extracting the aqueous solution with hexane, combining the organic phases, washing twice with water and brine, respectively, anhydrous Na2SO4Drying, concentration under reduced pressure, and purification by column chromatography (petroleum ether: ethyl acetate 20: 1) gave 1.632g of compound 1 as a clear oil in 27.2% yield. The nuclear magnetic data for compound 1 is:1H NMR(400MHz,CDCl3)δ＝4.86(d,J＝3.1,1H),3.66–3.50(m,4H),3.28–3.17(m,2H),0.03–-0.04(m,45H)。
2) compound 1(1g, 1.848mmol) was purified in dichloromethane and methanol (v: v ═ 1:1) as a solvent, and ammonium acetate (0.285g, 3.696mmol) at 25 ℃ for 9 hours, followed by rotary evaporation under reduced pressure, column chromatography purification (petroleum ether: ethyl acetate ═ 20: 1) to give 509mg of Compound 5 as a clear oil in 58.74% yield. The nuclear magnetic data for compound 5 is:1H NMR(400MHz,CDCl3)δ＝5.00(d,J＝3.0,1H),3.90–3.58(m,4H),3.46(d,J＝8.9,1H),3.34(dd,J＝9.1,3.0,1H),1.70(s,1H),0.20–0.10(m,36H)。
3) camptothecin (0.20g, 0.574mmol) was dissolved in 200mL dry CH2Cl2EDC. HCl (0.88g, 4.59mmol), DMAP (0.14g, 1.15mmol) and adipic acid (0.336g, 2.04mmol) were added under magnetic stirring, respectively, the mixture was stirred magnetically, refluxed for 16 hours for the third reaction, cooled to room temperature, washed with 10% citric acid, distilled water and saturated brine (200 mL. times.2), respectively, the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (CH. times.2)2Cl2: MeOH, 40: 1) 184mg of a yellow solid, Compound 9, are obtained in 67.3% yield. The nuclear magnetic data for compound 9 is:1H NMR(400MHz,DMSO)δ＝11.98(s,1H),8.69(s,1H),8.15(dd,J＝17.1,8.1,2H),7.87(ddd,J＝8.4,6.9,1.4,1H),7.79–7.58(m,1H),7.06(s,1H),5.49(s,2H),5.30(s,2H),2.51(s,2H),2.24(s,4H),1.65–1.43(m,4H),0.92(t,J＝7.4,3H)。
4) compound 5(531mg, 1.133mmol) was dissolved in 50mL dry CH2Cl2Adding DCC (280.4mg, 1.36mmol) and DMAP (13.8mg, 0.133mmol) at-5 deg.C, mixing and stirring for 30min, adding 100ml of compound 9(270mg, 0.567mmol) in dichloromethane dropwise, stirring at 30 deg.C for 24 hr to perform fourth reaction, filtering the reaction product, concentrating the filtrate under reduced pressure, and purifying by column Chromatography (CH)2Cl2：CH3OH 80:1) to yield 110mg of compound 10 as a pale yellow solid in 20.9% yield. The nuclear magnetic data for compound 10 is:1H NMR(400MHz,CDCl3)δ＝8.25(s,1H),8.09(d,J＝8.5,1H),7.80(d,J＝8.0,1H),7.73–7.64(m,1H),7.57–7.47(m,1H),7.07(s,1H),5.51(s,1H),5.29(s,1H),5.15(s,2H),4.86(d,J＝3.0,1H),4.18(dd,J＝11.8,2.2,1H),3.85(dd,J＝11.8,5.4,1H),3.74(ddd,J＝9.5,5.4,2.1,1H),3.63(t,J＝8.8,1H),3.30–3.16(m,2H),2.46–2.19(m,4H),2.18–2.11(m,1H),2.02(dq,J＝14.9,7.5,1H),1.56(dd,J＝6.0,3.0,4H),0.84(t,J＝7.5,3H),0.16–-0.11(m,36H)。
5) dissolving compound 10(100mg, 0.1078mmol) in 30ml dichloromethane, cooling the solution to 0 deg.C, adding trifluoroacetic acid (750 μ L) for fifth reaction, detecting by TLC after 2.5h, concentrating under reduced pressure, and purifying by column Chromatography (CH)2Cl2：CH3OH ═ 12:1) to give 48mg of a yellow solid, compound a, in 69.7% yield. The nuclear magnetic data for compound a is:1H NMR(400MHz,DMSO)δ＝8.70(s,1H),8.15(dd,J＝16.1,8.3,2H),7.88(t,J＝7.7,1H),7.72(t,J＝7.5,1H),7.07(s,1H),6.64(d,J＝6.6,<1H),6.32(d,J＝4.6,<1H),5.49(s,2H),5.36–5.24(m,2H),5.04(dd,J＝23.5,5.5,1H),4.91(d,J＝4.4,0H),4.90–4.86(m,1H),4.72(d,J＝3.9,<1H),4.50(d,J＝6.6,<1H),4.25(td,J＝13.6,4.4,m,>1H),4.09(d,J＝4.8,<1H),3.94(td,J＝11.4,6.5,1H),3.79–3.72(m,<1H),3.48–3.38(m,>1H),3.17(d,J＝4.2,1H),3.15–3.07(m,1H),3.07–2.95(m,1H),2.89(t,J＝8.4,<1H) 2.55(dd, J ═ 18.3,12.5,2H),2.33(d, J ═ 4.0,2H), 2.23-2.07 (m,2H),1.58(s,4H),0.92(s, 3H); the mass spectrum result of compound a is: HRMS (ESI) Calcd for C32H34N2O12 638.5,found661.5[M+Na]+。
Example 2
This example carried out the preparation of a camptothecin glycoconjugate (compound b) comprising the following steps:
1) 9.26g of Compound 2 was prepared in the same manner as in step 1) of example 1, in 62.8% yield, starting from D-galactose. The nuclear magnetic data for compound 2 is:1H NMR(400MHz,CDCl3)δ＝4.91(d,J＝2.0,1H),3.76(s,2H),3.68(d,J＝1.8,2H),3.52–3.45(m,1H),3.39(dd,J＝9.6,5.6,1H),0.03–-0.05(m,45H)。
2) using compound 2 as a starting material, 1.663g of compound 6 was prepared in the same manner as in step 2) of example 1 in 74.67% yield as a transparent oil. The nuclear magnetic data for compound 6 is:1H NMR(400MHz,CDCl3)δ＝4.96(d,J＝1.7,1H),3.84–3.79(m,1H),3.72–3.67(m,3H),3.67–3.61(m,1H),1.70(d,J＝147.6,1H),0.02–-0.05(m,36H)。
3) starting from compound 6 and compound 9 obtained in example 1, 50mg of a pale yellow solid, compound 11, was prepared in the same manner as in example 1) step 4), with a yield of 8.92%. The nuclear magnetic data for compound 11 is:1H NMR(400MHz,CDCl3)δ＝8.27(s,1H),8.10(d,J＝8.5,1H),7.81(s,1H),7.71(s,1H),7.55(s,1H),7.09(s,1H),6.83–6.81(m,0H),5.55(d,J＝17.2,1H),5.29(d,J＝17.2,1H),5.17(d,J＝3.0,2H),4.92(s,1H),3.95(dd,J＝8.5,4.6,4H),3.68(d,J＝0.8,1H),3.37(s,1H),2.44–2.32(m,2H),2.20(d,J＝4.3,2H),2.15(dd,J＝14.4,6.9,1H),2.08–1.98(m,1H),1.60–1.56(m,4H),0.85(t,J＝7.5,3H),0.03–-0.05(m,36H)。
4) starting from compound 11, prepared in the same manner as in example 1) step 5) was 48mg of a yellow solid, compound b, in 69.7% yield. The nuclear magnetic data for compound b is:1H NMR(400MHz,DMSO)δ＝8.59(s,1H),8.04(dd,J＝13.9,8.1,2H),7.76(td,J＝6.9,1.7,1H),7.61(dd,J＝11.0,4.0,1H),6.95(s,1H),6.47(d,J＝6.7,<1H),6.13–6.08(m,<1H),5.42–5.32(m,2H),5.19(s,2H),4.83–4.78(m,<1H),4.62(d,J＝4.2,<1H),4.56(d,J＝5.0,<1H),4.44(d,J＝4.4,<1H),4.39(dd,J＝4.9,3.2,<1H),4.34(t,J＝5.5,<1H),4.20(d,J＝6.7,<1H),4.14–4.05(m,<1H),3.84(ddd,J＝17.6,11.2,3.7,2H),3.61–3.53(m,<1H),3.50(d,J＝3.6,<1H),3.42(dd,J＝5.5,30,1H), 3.40-3.34 (m,1H), 3.16-3.08 (m,1H), 2.50-2.40 (m,2H),2.21(d, J ═ 3.8,2H), 2.08-1.99 (m,2H),1.47(s,4H), 0.83-0.78 (m, 3H); the mass spectrum result of compound b is: HRMS (ESI) Calcd for C32H34N2O12638.6,found 661.5[M+Na]+。
Example 3
This example carried out the preparation of a camptothecin glycoconjugate (compound c) comprising the following steps:
1) 9.7g of Compound 3 was prepared in the same manner as in step 1) of example 1, using D-mannose as a starting material, in a yield of 65.8%. The nuclear magnetic data for compound 3 is:1H NMR(400MHz,CDCl3)δ＝4.75(d,J＝2.0,1H),3.70–3.65(m,2H),3.60–3.55(m,2H),3.49(s,1H),3.43(tdd,J＝8.7,5.7,2.5,1H),0.01–-0.03(m,45H)。
2) using compound 2 as a starting material, 4.756g of compound 7 was prepared in the same manner as in step 2) of example 1 in 70.45% yield as a transparent oil. The nuclear magnetic data for compound 7 is:1H NMR(400MHz,CDCl3)δ＝4.76(d,J＝2.1,1H),3.72(t,J＝5.4,2H),3.55–3.47(m,3H),1.79(s,1H),0.04–-0.04(m,36H)。
3) starting from compound 7 and compound 9 obtained in example 1, 48mg of a pale yellow solid, compound 12, was prepared in the same manner as in example 1) step 4), with a yield of 6%. The nuclear magnetic data for compound 12 is:1H NMR(400MHz,CDCl3)δ＝8.39(s,1H),8.22(d,J＝8.6,1H),7.94(d,J＝8.2,1H),7.83(s,1H),7.67(s,1H),7.21(s,1H),5.67(d,J＝17.2,1H),5.41(d,J＝17.2,1H),5.29(d,J＝3.1,2H),4.90(d,J＝2.1,1H),4.31(s,1H),3.97(dd,J＝11.6,5.7,1H),3.85–3.82(m,1H),3.63(t,J＝1.9,1H),3.48(d,J＝4.0,2H),2.56–2.42(m,2H),2.34(s,2H),2.27(dd,J＝14.4,6.9,1H),2.15(dq,J＝15.0,7.5,1H),1.70(dd,J＝8.1,4.5,4H),0.97(t,J＝7.5,3H),0.14(ddd,J＝14.0,6.9,3.9,36H)。
4) starting from compound 12, prepared in the same manner as in example 1) step 5) was 18mg of a yellow solid, compound b, in 54.4% yield. Nuclear magnetic data for compound c is: 1H NMR (400MHz, DMSO) δ 8.70(s,1H),8.16(dd, J16.0, 8.3,2H), 7.92-7.84 (m,1H),7.72(t, J7.5, 1H),7.07(s,1H),6.35(s,1H),5.50(d, J1.0, 2H), 5.30H(s,2H),4.85(s,1H),4.26(dd, J ═ 11.5,1.7,1H),3.95(dd, J ═ 11.7,6.9,1H), 3.73-3.65 (m,1H),3.53(ddd, J ═ 12.3,6.1,2.4,3H),3.42(d, J ═ 16.5,4H), 3.29-3.26 (m,1H), 2.62-2.52 (m,2H),2.33(d, J ═ 4.9,2H),2.16(q, J ═ 7.1,2H),1.59(d, J ═ 3.2,4H),0.92(t, J ═ 7.4, 3H); the mass spectrum result of compound b is: HRMS (ESI) Calcd for C32H34N2O12 638.4,found661.4[M+Na]+。
Example 4
This example determines the toxicity of camptothecin glycoconjugates on cells and tests the cell viability of brain microvascular endothelial cells bend.3 using MTT staining.
Culturing brain microvascular endothelial cell Bend.3 in culture solution, taking logarithmically growing cell, and preparing 5 × 10 with the culture solution4cells/ml single cell suspension were seeded at 100. mu.l/well density in 96-well plates. At 37 ℃ with 5% CO2After standing culture in an incubator for 24h, the culture solution is aspirated, each group of concentration gradient is provided with 6 multiple holes, and 200 mu L of culture solution containing the to-be-detected product is added in each hole. The concentration of the sample in the culture medium was 50. mu.M and 25. mu.M. And (3) reserving six holes, adding only culture solution without cells to serve as a blank control, simultaneously taking camptothecin original drug molecules with the same concentration as a positive control group, and taking culture solution holes without drugs to serve as negative controls. And after 24 hours of culture, removing the supernatant, adding 100 mu l of 5mg/ml MTT solution, continuing to culture for 2 hours, centrifuging for 12min, discarding the supernatant again, adding 100 mu l of DMSO, and shaking for 5-10 min to fully dissolve the bluish purple crystals. The OD (absorbance) value of the compound was measured at 570nm absorbance wavelength using an enzyme linked immunosorbent assay, the optical density value was measured at 490nm wavelength using an enzyme linked immunosorbent assay, all optical density values were first subtracted from the blank control, and the relative cell viability of the experimental group drug was obtained by comparison with the control value, and the cell activity was ═ OD experiment-OD blank)/(OD negative-OD blank) × 100%.
As a result, referring to fig. 1, which is a graph (24h) (P < 0.0001 compared to camptothecin group) of the effect of camptothecin glycoconjugate on the survival rate of brain microvascular endothelial cell bend.3 in the present example, wherein Control represents negative Control group, CPT represents camptothecin group, and a, b, and c represent compound a, compound b, and compound c, respectively, the result shows that compound a, compound b, and compound c of camptothecin glycoconjugate have higher survival rate and significant difference on brain microvascular endothelial cell bend.3 compared to camptothecin, and the camptothecin glycoconjugate of the present invention has lower toxicity than camptothecin.
Example 5
This example performs the determination of the antitumor activity of camptothecin glycoconjugates and studies their antitumor mechanisms.
Taking logarithmic growth glioma cell U87, preparing 5 × 10 with culture solution4cells/ml single cell suspension were seeded at 100. mu.l/well density in 96-well plates. At 37 ℃ with 5% CO2After the culture medium is statically cultured in an incubator for 24 hours, the culture medium is sucked, 10 groups of experiments are respectively carried out, each group is provided with 6 compound holes, and four groups are medicine groups: adding 100 μ L of culture solution containing drug into each well, wherein the concentration of drug in the culture solution is 50 μ M; four groups were D-glucose + drug group: adding 100 μ L of culture solution containing D-glucose and drug into each well, wherein the concentration of D-glucose in the culture solution is 200mM, and the concentration of drug in the culture solution is 50 μ M; one group is a D-glucose control group, 100 mu L of culture solution containing only D-glucose and no medicine is added into each well, and the concentration of the D-glucose in the culture solution is 200 mM; one group was a negative control group, and 100. mu.L of a culture medium containing no D-glucose and no drug was added to each well. Six wells were left with culture medium without cells as a blank and the supernatant removed after 24h incubation. Adding 100 mu l of 5mg/ml MTT solution, continuously culturing for 4h, centrifuging, removing the supernatant again, adding 100 mu l of DMSO, and shaking for 5-10 min to fully dissolve the blue-violet crystals. Measuring optical density value at 490nm with enzyme-linked immunosorbent assay detector, subtracting blank control group from all optical density values, and comparing with negative control to obtain relative cell inhibition rate of experimental group drug, wherein cell survival rate is (OD experiment-OD blank)/(OD negative control-OD blank) x 100%, and drug cell inhibition rate is [1- (OD experiment-OD blank)/(OD negative control-OD blank)]×100％
Please refer to fig. 2, which is a graph illustrating the growth inhibition of U87 on glioma cells by camptothecin and camptothecin glycoconjugates in example 2 of the present invention, wherein CPT represents camptothecin group, and a, b, and c represent compound a, compound b, and compound c, respectively. The results show that, compared with camptothecin, the camptothecin glycoconjugate has higher effect of inhibiting the growth of glioma cells after the glioma cells U8724 h are incubated, and the significant difference exists.
FIG. 3 is a graph showing the effect of D-glucose on the survival rate of glioma cells U87 in example 2 of the present invention, wherein Control represents the negative Control group and 200 represents the D-glucose Control group. The result shows that the survival rate of the glioma cell U87 is more than 85% under the condition of culturing the D-glucose at the concentration of 200mM, and the cytotoxicity test of competitive inhibition of D-glucose on glycoconjugate through GLUT1 pathway can be carried out.
Please refer to fig. 4, which is a graph illustrating the growth inhibition of U87 glioma cells of different groups in example 2, wherein CPT represents camptothecin group, and a, b, and c represent compound a, compound b, and compound c, respectively. The results showed that the inhibitory effect of compound a at a concentration of 50 μ M on tumor cells was weakened by about 20.48% (52.92-42.09)/52.92 x 100% ═ 20.48%) when the cells were incubated for 24h with the drug in the presence of D-glucose at a concentration of 200 mM; compound b at a concentration of 50 μ M attenuated about 36.68% (53.21-33.69)/53.21 × 100% ═ 36.68%) of the inhibitory effect on tumor cells; the inhibitory effect of compound c at a concentration of 50 μ M on tumor cells was reduced by about 36.68% (58.52-38.39)/58.52 × 100% ═ 34.4%). The antitumor effect of camptothecin in the presence and absence of 200mM is not obviously different.
The results show that the taking of the camptothecin glycoconjugate by the glioma cells U87 can be obviously inhibited in the presence of 200mM glucose. Therefore, it can be preliminarily predicted that the camptothecin glycoconjugate mainly plays an anti-tumor role through GLUT1 recognition via GLUT1 pathway and transportation into glioma cell U87 cell, indicating that the uptake of the camptothecin glycoconjugate is regulated by GLUT 1.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A camptothecin glycoconjugate, wherein the camptothecin glycoconjugate is selected from the group consisting of
2. A process for the preparation of the camptothecin glycoconjugate of claim 1 comprising the steps of:
a) carrying out a first reaction on hexose and trimethylchlorosilane under an alkaline condition to obtain a first product;
b) carrying out a second reaction on the first product and ammonium acetate to obtain a second product;
c) carrying out a third reaction on camptothecin and adipic acid under the catalysis of EDC & HCl and DMAP to obtain a third product;
d) carrying out a fourth reaction on the second product and the third product under the catalysis of DCC and DMAP to obtain a fourth product;
e) and carrying out a fifth reaction on the fourth product and trifluoroacetic acid to obtain the camptothecin glycoconjugate.
3. The preparation method according to claim 2, wherein the temperature of the first reaction is 20-30 ℃ and the time is 2.5-4 h;
the temperature of the second reaction is 20-30 ℃, and the time is 8-12 h;
the temperature of the third reaction is 30-40 ℃, and the time is 12-24 h;
the temperature of the fourth reaction is 30-40 ℃, and the time is 12-36 h;
the temperature of the fifth reaction is-5 ℃ to 0 ℃, and the time is 2.5h to 4 h.
4. The process according to claim 2, wherein the basic reagent of the basic conditions of step a) is selected from triethylamine, N-diisopropylethylamine or pyridine;
the molar ratio of the hexose, the trimethylchlorosilane and the alkaline reagent is 1: (5.5-6): (5.5-6).
5. The method of claim 2, wherein the molar ratio of the first product of step b) to the ammonium acetate is 1: (1.5-2);
step c) the molar ratio of camptothecin, adipic acid, EDC & HCl and DMAP is 1: (3-5): (4-8): (3-5);
the molar ratio of the second product, the third product, DCC and DMAP in the step d) is (1.5-3): 1: (2-3): (0.2-0.5).
6. The method of claim 2, wherein the fifth reaction of step e) is carried out in a solvent;
the volume content of the trifluoroacetic acid in the reaction system is 2.5-10%.
7. Use of the camptothecin glycoconjugate of claim 1 and/or the camptothecin glycoconjugate prepared by the preparation method of any one of claims 2 to 5 in the preparation of an anti-tumor medicament.
8. A medicament comprising the camptothecin glycoconjugate of claim 1 and/or the camptothecin glycoconjugate prepared by the preparation method of any one of claims 2 to 5.
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