Design,Synthesis And The Multy Drug Resistance Reversal Mechenism Of A Novel Anthracyclines Drug, Azide Doxorubicin (Adox)

Anthracyclines, one kind of the most commonly used anti-tumor antibiotics in clinical, was thought to be one kind of the most effective anti-tumor drugs, either when it was used alone and in combination. Daunorubicin (DNR) and idarubicin (IDA, Zavedos(?)) are commonly used to treat leukemia and lymphoma, while adriamycin (doxorubicin, DOX) and epirubicin (EPI, Farmorubicin (?)) are widely used in the treatment of leukemia, lymphoma and various solid tumors, including breast cancer, non-small cell lung cancer, cervical cancer and head and neck cancer. Although have efficient anticancer activities, the toxic effects on cardiac cells greatly affect the survival and life quality of cancer patients, which limit its wide clinical applications. Besides, anthracyclines drugs is easy to produce drug resistance in its long term application, including cross-resistance among this class of drugs as well as cross-resistance to vincristine.In order to overcome the cardiac toxicity and multidrug resistance (MDR) of anthracyclines, the development of safer, more effective and lower toxic derivertives is imperative. In this study, based on the molecular docking experiments, a novel analogue of DOX,3'-azide doxorubicin (ADOX), was designed and synthesized. Its anti-tumor activity and avoidance of MDR protein recognition to overcome the P-gp mediated drug resistance were evaluated both in cellular and tumor-bearing animals' level. The research contents and results were summarized as follows.1Design of a novel anthracycline and molecular dockingThe3'-NH2group of anthracyclines, which has been reported to be critical for P-gp binding, was designed to be replaced by a3'-azido group to produce a novel anthracycline, ADOX. The crystal structure of P-gp (PDB entry:3G60) was chosen for molecular docking with DOX and ADOX using Autodock3.0, Sybyl7.1and UCSF Chimera DOCK6softwares.DOX was easily docked to P-gp. It could form H-bonds with Asp133A, Gln129A, Gln130A(x2), Asn126B and Gln130B of P-gp and the binding energy was-10.0kcal/mol. Compared with DOX, ADOX was a poor substrate for P-gp and it could only formed H-bonds with Gln130B(x2), Gln130A, with the binding energy2.5kcal/mol. This result may be caused by the bulky azido substitution that would push ADOX away from the active cavity.2Synthesis of ADOXIn this study, a novel DOX derivative with unique azido group, ADOX, was synthesized by two methods. The first method is very complicated, while ADOX could be conveniently prepared from DOX via one pot diazo transfer reaction in moderate yield of80%by the second method.3Effect and mechanism of ADOX on Drug-resistant tumor cells and MDR3.1Drug-resistant cell killing and MDR reversal activities of ADOXTetrazolium[3-(4,5-dimethythiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulf ophenyl)-2H-tetrazolium, inner salt (MTS) assays were employed to study the effect of DOX and ADOX on both drug sensitive cells (MCF-7and K562) and drug resistant cells (MCF-7/DNR and K562/DOX). Both DOX and ADOX exhibited potent lethal activity in drug-sensitive tumor cells in a dose-dependent manner, while only ADOX showed lethal activity in drug-resistant cells, its IC50in MCF-7/ADR was3.5mM and IC50in K562/DOX was0.87μM, which were lower than that of DOX (20μM and27μM, separately). The drug resistance index value (DRI, ratio of IC50in drug-resistant cells over IC50in drug-sensitive cells) was calculated according the formula:DRI=IC50(drug resistant cells)/IC50(drug sensitive cells). The DRIs of ADOX were1.6and1.4, respectively, which were much smaller than those of DOX (182and338). The MTS assay results indicated that, unlike DOX, ADOX could efficiently overcome drug resistance in cellular level.3.2MDR reversal mechanim of ADOXRT-PCR was used to analize the expression of MDR1in drug-resistant cells, Western blot was used to detect the expression of P-gp, and flow cytometry (FACS) was employed to study the drug accumulation of DOX or ADOX in drug resistant cell, K562/DOX. The results showed that the expression of MDR1in drug-resistant cells was high, and the results of FACS showed that the concentration of ADOX in this kind of cells was also high. This indicated that, the anti-MDR mechanism of ADOX was the avoidance of P-gp recognization.4Biological activity evaluation of ADOX on reversing drug resistance of tumor cells4.1Anticancer activity of ADOX against drug-resistant cancer xenograft model in nude miceAfter the leucemia nude mice model was established, DOX and ADOX were i.p. twice per week for three weeks. The body weight, tumor size and survival were detected. One week after administration, tumor in blank control grows fast, while the grow speeds in ADOX and DOX group (5mg/kg) were much slower, and ADOX showed a better effect than DOX. The body weight decrease of DOX group was more than82%, and after3weeks treatment, all mice of DOX group died. Body weight decrease in ADOX and blank group were not obvious. All mice of ADOX group survived in50d after treatment, while50%of DOX group died for tumor and drug toxicity in20d.50%animal of blank group died in25d, and all died in33d.These results showed that ADOX have better anti-tumor activity and lower toxicity.4.2Acute toxicity of ADOX determined by mouse LD50test Balb/c mice (half male and half female, weighing between18-22grams) were randomly divided into7groups. The mice were observed for14days after one-dose-administration of ADOX, i.p. Numbers of dead mice in each group were recorded, and the LD50value was calculated by using software Origin7.5. The LD50(35mg/kg) of ADOX was higher than that (10mg/kg) of DOX.5Chronic cardiac muscle toxicity of ADOX and DOX in ratsRat cardiac muscle injury model was established according to the previous reference with little modification. After five weeks of continuous administration, the heart rate, QRS duration and Q-T interval were detected at2d,4w and8w after the last adminisition.(1) Heart rate:compared with control group, the heart rate of DOX group slow down obviously, and with the increase of administration times, the slow of heart rate was more obvious (P<0.05); While, the heart rate of ADOX group had no obvious difference with that of blank group (P>0.05)(2) QRS duration:compared with control group, QRS durations of both DOX group and ADOX group prolonged obviously (P<0.05), but the prolonged time of ADOX group is shorter than that of DOX(P<0.05).(3) Q-T interval:compared with control group, Q-T intervals of both DOX group and ADOX group prolonged obviously (P<0.05), but the prolonged time of ADOX group is shorter than that of DOX (P<0.05).Compared with DOX, ADOX showed decreased toxic effect on heart rate, QRS duration and Q-T interval.ELISA was employed to test the concentration of cardiac troponin T (cTnT) in rats before and after administration. When the cumulative doses of DOX were3,6,9,12and15mg/kg respectively, the concentrations of cTnT were0.68,1.17,2.42,5.11,12.76and18.8ng/ml respectively. The oncentration of cTnT rose with the increase of cumulative dose of DOX in a dose-dependent manner. The results indicated that the injury on cardiac cells enhanced with the increase of cumulative dose. When the cumulative doses of ADOX were3,6,9,12and15mg/kg respectively, the concentrations of cTnT were1.13,1.29,1.75,1.99,2.03and2.42ng/ml respectively. The injury on cardiac cells of ADOX enhanced in a much smaller degree compared with that of DOX, which indicated that ADOX showed lower cardiac toxicity than DOX.The main results and conclusions were as follows:(1) Molecular docking experiment showed that DOX was a proper substrate for P-gp and ADOX was a poor substrate, which was helpful for ADOX to overcome the disadvantage of MDR of DOX.(2) A novel DOX analogue with unique azido group, ADOX, was synthesized.(3) The high expression of MDR1in drug-resistant cells (MCF-7/ADR and K562/DOX) and the high ADOX concentration in this kind of cells indicated that the mechanism of anti-MDR activity of ADOX was because of the avoidance of P-gp recognization.(4FACS proved the molecular docking result:ADOX is a poor substrate for P-gp. This was helpful to explain the mechanism of MDR reversal activity of ADOX.(5) Compared with DOX group, the tumor size of ADOX group was smaller, the survival rate was higher and the body weight showed less decrease. This result indicated that ADOX showed better anti-tumor activity and less toxicity in drug-resistant cancer model.(6) Compared with DOX, ADOX showed decreased toxic effect on heart rate, QRS duration and Q-T interval.