CN108434457B - Adriamycin polyethylene glycol epothilone B conjugate and preparation method thereof - Google Patents
Adriamycin polyethylene glycol epothilone B conjugate and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an adriamycin pegylated epothilone B and a preparation method thereof, and the method mainly comprises the following steps: 1) reacting adriamycin with hydrazide polyethylene glycol carboxyl under the action of phosphoric acid to obtain an adriamycin polyethylene glycol carboxyl intermediate, wherein the molar ratio of adriamycin to phosphoric acid is 1: 0.01-0.02; 2) under the action of a second coupling agent and a catalyst, the epothilone B reacts with the adriamycin polyethylene glycol carboxyl intermediate to obtain the adriamycin pegylated epothilone B conjugate.
Description
Technical Field
The invention relates to the field of medicine synthesis, and in particular relates to an adriamycin pegylated epothilone B conjugate and a preparation method thereof.
Background
Doxorubicin is one of the most widely used small molecule chemotherapeutic drugs, but due to the defects of poor water solubility, non-selectivity of drug distribution and the like, doxorubicin generates serious toxic and side effects on normal tissues after entering the body. At the same time, if adriamycin is used for a long time, the adriamycin also induces the tumor cells to generate strong drug resistance, namely, the sensitivity of the cancer cells to the same dose of the adriamycin is reduced. The structural formula of the adriamycin is shown as a formula 1.
How to improve the specificity of the distribution of the adriamycin in vivo, enhance the chemotherapy curative effect of the adriamycin and simultaneously reduce the toxic and side effect of the adriamycin becomes an urgent problem to be solved. PEG is used as a medical material approved by FDA, and is widely used for delivery of anticancer drugs due to its advantages of low toxicity, high water solubility, low immunogenicity, low protein binding property, and the like. In addition, after the drug is delivered into the body, whether the drug can be specifically responded and released at the tumor site is also an important factor influencing the curative effect of the drug.
Chinese patent publication No. CN103768612B discloses a PEG peptide dendrimer targeting drug delivery system loaded with adriamycin.
The secondary metabolite epothilone, which was first found by microbiologists at the national center for biotechnology research (GBF) in the end of the twentieth eighties in soil by Sorangium cellulosum (So ce), was the first metabolite. Reichenbach and
in 1987 it was established that the myxobacteria sorangium cellulosum So ce90 was able to produce an antifungal and cytotoxic active substance, two new macrolide compounds were isolated and designated epothilone a and B. The structural formula of the epothilone is shown in formula 2.
They were found experimentally to have inhibitory activity against cultured fungal pathogens. The study of activity was discontinued in 1994 due to side effects and clinical problems. Fortunately, in the same year, the national cancer society of america found that epothilone B had inhibitory activity against tumor cells. Soon thereafter, epothilones were again discovered by the Merck laboratory in experiments screening 7000 natural extracts for paclitaxel conjugates. It was found to have at least the same effect as paclitaxel in tubulin polymerization experiments. The fact that epothilone has an inhibitory effect on tumor cells is demonstrated. As the epothilone is an anti-tumor chemotherapeutic drug, the epothilone has certain cytotoxicity and can cause gastrointestinal reaction, bone marrow suppression and other toxic and side effects.
Disclosure of Invention
Based on the above research background, the present invention proposes a scheme to bond hydrophilic PEG to hydrophobic doxorubicin molecules, using PEG as the hydrophilic end and doxorubicin and Epothilone B as the hydrophobic end of the whole prodrug, and the prepared prodrug doxorubicin polyethylene glycol Epothilone B (DOX-PEG-Epothilone B) has multiple advantages: greatly increases the water solubility of the adriamycin and the epothilone B; the prodrug DOX-PEG-Epothilone B can be effectively self-assembled in a physiological environment to form stable nanoparticles, so that targeted drug delivery can be performed by utilizing the EPR effect of the nano-drug, and the enrichment of adriamycin and Epothilone B at tumor sites is increased. The invention aims to provide an adriamycin pegylated epothilone B conjugate and a preparation method thereof, wherein hydrazone bonds are connected through an acid-sensitive connecting bond with a pH response function, the conjugate is stable under a weak alkaline condition, and the hydrazone bonds can be rapidly broken under a weak acidic condition (such as tumor tissues and intracellular lysosome environment), so that a drug is released.
The invention is realized by the following modes:
an doxorubicin pegylated epothilone B conjugate having the structural formula:
wherein n is an integer from 22 to 410.
A preparation method of an adriamycin pegylated epothilone B conjugate mainly comprises the following steps: 1) reacting adriamycin with carboxyl polyethylene glycol carboxyl in DMF under the action of phosphoric acid to prepare an adriamycin polyethylene glycol carboxyl intermediate, wherein the molar ratio of the adriamycin to the phosphoric acid is 1: 0.01-0.02; 2) and under the action of a second coupling agent and a catalyst, reacting the epothilone B with the adriamycin polyethylene glycol carboxyl intermediate to obtain the adriamycin pegylated epothilone B conjugate.
Preferably, the peptide coupling agent in the step 2) is 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, and the N-heterocyclic catalyst is N-methylmorpholine.
Preferably, the preparation method of the doxorubicin pegylated epothilone B conjugate is characterized in that the molar ratio of the doxorubicin polyethylene glycol carboxyl, the 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, the N-methylmorpholine and the epothilone B is 1:1-2:1-2: 2-3.
Preferably, the preparation method of the doxorubicin pegylated epothilone B conjugate is characterized in that the organic solvent in step 1) is dimethyl sulfoxide, and the solvent in step two is N, N-dimethylformamide.
The reaction formula is shown as formula 3:
the invention has the following beneficial effects:
1, the synthesized prodrug DOX-PEGn-Epothilone B shows good water solubility, and compared with small molecular drugs of adriamycin and Epothilone B, the solubility of the drugs is effectively improved.
2 to synthesize the new prodrug DOX-PEGn-Epothilone B. The nano-drug can be self-assembled in a physiological environment to form the nano-drug, so that passive targeted drug delivery can be performed by the EPR effect. Meanwhile, as hydrazide polyethylene glycol carboxyl (Hz-PEGn-COOH) is used for reacting with the carbonyl of the adriamycin, the synthesized DOX-PEGn-Epothilone B is an acid-sensitive hydrazone bond connector, and has the characteristics of keeping stability in blood (weak alkaline) circulation and quickly releasing adriamycin raw drug once reaching tumor tissues (weak acidic), thereby realizing tumor targeted drug delivery and reducing toxic and side effects while enhancing the curative effect of chemotherapy.
3, the invention utilizes cheap and easily available phosphoric acid, DCC and DMAP to carry out catalytic coupling reaction, thereby saving cost and ensuring that the reaction keeps high selectivity and yield.
The conjugates of the present invention and their preparation are described below with reference to examples, which are not intended to limit the present invention, the scope of which is defined by the claims.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
An doxorubicin pegylated epothilone B conjugate having the structural formula:
wherein n is an integer from 22 to 410.
Reacting adriamycin with hydrazide polyethylene glycol carboxyl under the action of phosphoric acid to obtain an adriamycin polyethylene glycol carboxyl intermediate, wherein the molar ratio of adriamycin to phosphoric acid is 1: 0.01-0.02; 2) and reacting the epothilone B with an doxorubicin polyethylene glycol carboxyl intermediate under the action of a second coupling agent (N' -dicyclohexylcarbodiimide) and a catalyst (N-methylmorpholine) to obtain the doxorubicin pegylated epothilone B conjugate. In the reaction, the molar ratio of the adriamycin polyethylene glycol carboxyl, the 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, the N-methylmorpholine and the epothilone B is 1:1-2:1-2: 2-3. The reaction of step 1) and step 2) is carried out in the presence of an organic solvent. The organic solvent in the step 1) is dimethyl sulfoxide, and the solvent in the step 2) is N, N-dimethylformamide.
Example 1:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively22-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Epothilone B (6mmol) and DOX-PEG22-COOH (6mmol) were weighed into a 25mL round bottom flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (6mmol) as a coupling agent and DMAP (12mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 22-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 2:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively72-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution of pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (88% yield). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Epothilone B (12mmol) and DOX-PEG72-COOH (6mmol) were weighed into a 25mL round bottom flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (6mmol) as a coupling agent and DMAP (6mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 72-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 3:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively120-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Approximately 12mmol of Epothilone B and 6mmol of DOX-PEG120-COOH were weighed into a 25mL round-bottomed flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (12mmol) as a coupling agent and DMAP (12mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 120-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 4:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively160-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Approximately 12mmol of Epothilone B and DOX-PEG160-COOH (6mmol) were weighed into a 25mL round-bottomed flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (12mmol) as a coupling agent and DMAP (6mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 160-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 5:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively220-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Epothilone B (18mmol) and DOX-PEG220-COOH (6mmol) were weighed into a 25mL round bottom flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (12mmol) as a coupling agent and DMAP (6mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 220-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 6:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively280-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Approximately 18mmol of Epothilone B and 6mmol of DOX-PEG280-COOH were weighed into a 25mL round-bottomed flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (12mmol) as a coupling agent and DMAP (12mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 280-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H), 2.70 (ddH-13H-13, 2H-1H), 2H-21-7 (m,1H), 2H-1H-15, ddd, 2H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 7:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively360-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG22-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Epothilone B (18mmol) and DOX-PEG360-COOH (6mmol) were weighed into a 25mL round bottom flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (6mmol) as a coupling agent and DMAP (6mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 360-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H-70), 2.70 (ddH, 1H-7H), 2H-7 (1H-1H), 5.42(1H, ddd, H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Example 8:
Hz-PEG was added to a 50ml three-necked flask equipped with a mechanical stirrer, a thermometer, and a nitrogen gas bag, respectively410-COOH (10mmol), phosphoric acid (1mmol) and 30ml DMF. The system was stirred at room temperature 25 ℃ for 48 hours under nitrogen protection in the dark. Adding 100 microliter Triethylamine (TEA), dialyzing with dialysis membrane in phosphate buffer solution with pH 8.0 until the dialyzate is no longer red, taking out the dialyzate, and freeze-drying to obtain intermediate compound DOX-PEG410-COOH (yield 90%). The synthesis of this product was confirmed by both nuclear magnetic and high performance liquid chromatography.
Approximately 12mmol of Epothilone B and DOX-PEG410-COOH (6mmol) were weighed into a 25mL round-bottomed flask and dissolved with stirring. Under the protection of nitrogen, 5mL of a dimethylsulfoxide solution containing DCC (6mmol) as a coupling agent and DMAP (12mmol) as a catalyst was slowly added. The reaction was carried out in an ice bath for 2h and then removed, and the reaction was stirred at room temperature for further 24h as monitored by Thin Layer Chromatography (TLC) using chloroform/methanol (10/1, V/V) as a developing solvent. After the reaction, the reaction solution was filtered and repeated three times to obtain a solution, which was poured into iced ether in an excess amount of about 10 times, and a large amount of white precipitate was immediately formed, which was left to stand at-20 ℃ overnight and recrystallized. Filtering, evaporating the filtrate at 40 deg.C under reduced pressure to obtain bright yellow solid, subjecting to Sephadex LH-20 column chromatography and silica gel column chromatography to obtain target product, spin drying, and vacuum drying to obtain off-white powder, i.e. DOX-PEG 410-Epothilone B. The assignments of specific peaks of nuclear magnetic HNMR are summarized as 9.05(s,1H), 8.89-8.86 (m,1H), 8.55-8.52 (m,2H), 7.87-7.85 (m,1H), 7.76-7.73 (m,1H),7.40(s,1H),6.98(1H, s, H-19), 6.60(1H, s, H-17), 6.51(s,1H),5.73(1H, dd, H-7), 5.42(1H, ddd, H-15), 4.75-4.74(m,2H),4.25(1H, m, H-3), 4.22-4.19(t,2H),3.56-3.82(-OCH2CH2), 3.38(3H, CH3O-), 2.81(1H, ddH-13H), 2.70 (ddH-13H-13, 2H-1H), 2H-21-7 (m,1H), 2H-1H-15, ddd, 2H-15), 2.36(1H), 2.12(1H, dd, 14a), 2.09(3H, d, H-27),2.04-1.98 (m,2H),1.92(1H, dd, 14b), 1.73(1H, m, H-8), 1.38(3H, s, H-28), 1.27(3H, s, H-26), 1.18(3H, d, H-24), 1.09(3H, s, H-22), 0.94-0.90 (m, 6H).
Claims (5)
1. An doxorubicin pegylated epothilone B conjugate, wherein the conjugate has the structural formula:
wherein n is an integer of 22 to 410, and the doxorubicin and the polyethylene glycol are linked in the form of a hydrazone bond.
2. The method of claim 1, wherein the method comprises the steps of:
1) reacting adriamycin with hydrazide polyethylene glycol carboxyl under the action of a first coupling agent and phosphoric acid to prepare an adriamycin polyethylene glycol carboxyl intermediate, wherein the molar ratio of the adriamycin to the phosphoric acid is 1: 0.01-0.02;
2) and under the action of a second coupling agent and a catalyst, reacting the epothilone B with the adriamycin polyethylene glycol carboxyl intermediate to obtain the adriamycin pegylated epothilone B conjugate.
3. The method for preparing an doxorubicin pegylated epothilone B conjugate according to claim 2, wherein the second coupling agent in step 2) is mainly a peptide coupling agent and the catalyst is a N-heterocyclic catalyst.
4. The process of claim 2, wherein said doxorubicin pegylated epothilone B is formed as a carboxylic intermediate having the formula:
5. the method for preparing an doxorubicin pegylated epothilone B conjugate according to claim 2, wherein the organic solvent of the reaction of step 1) is dimethyl sulfoxide and the organic solvent of the reaction of step 2) is N, N-dimethylformamide.
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