Design, Synthesis And Anti-tumor Activity Of Two Mutual Prodrugs Of Doxorubicin

AIM: According to the principle of prodrug, two conjugates of doxorubicin with celecoxib or with - linolenic acid were designed and synthesized in order to increase the therapeutic effects of doxorubicin and decrease its side effects.METHODS: 1. Sulfonyl amino of celecoxib and 3'-amino of doxorubicin were chosen for structure modification. Succinic anhydride was used as the cross-linking agent. This mutual prodrug of doxorubicin and celecoxib was synthesized through amide bond. 2. -linolenic acid was reacted with BocNHNH2 to get hydrazide of -linolenic acid with the protection of Boc. After removed the Boc-protected group and reacted with doxorubicin, the mutual prodrug through a hydrazone bond was synthesized. 3. Doxorubicin was designed as a positive control. Culture medium without drugs was designed as blank control. After HepG2 cells were incubated with different concentrations of two mutual prodrugs of doxorubicin for 48 h, MTT assay was used to measure the antitumor activity of the two mutual prodrugs, and IC50 was used to identify the activity of the two mutual prodrugs in vitro. 4. The mutual prodrug of celecoxib-doxorubicin was injected to rats by caudal vein. Blood was collected from the orbit of rats at points of the set time and was anticoagulated by heparin. Plasma was separated by centrifugation. The drugs in plasma were extracted twice by ethyl acetate. Ethyl acetate was then evaporated to dryness at 45℃under a gentle stream of nitrogen by the Zymark TurboVap LV Evaporator. The residue was dissolved in 50μL of methanol. 20μL solution was drawn and then detected by LC/MS/MS. Mobile phase constituted with acetonitrile and water (1‰formic acid) (70:30, V/V). An electrospray ionization-mass spectrometry (ESI-) source was used as detector and was operated in selected multiple reaction monitoring (MRM) mode.RESULTS: 1. Two mutual prodrugs were synthesized, and their structures were characterized. 2. In HepG2 cells, IC50 of DOX, C-DOX and L-DOX were 5μM, 32μM and 9μM, respectively. 3. LC/MS/MS was used to determine drug concentration in plasma. Regression equation for C-DOX was y=0.01x-0.1134. The linear range was 1-2000 ng/mL. Regression equation for DOX, CELA and CEL were y=0.0006x-0.0002, y=0.0018x+0.0076, y=0.0016x+0.0006, respectively. The linear range was 1-200 ng/mL. Within-day and between-day precision expressed by relative standard derivation(RSD) were less than 15%. The recovery rates of analytical method for the four drugs were from 90% to 110%. The AUC were 1133 ng·h·mL-1, 272 ng·h·mL-1, 243 ng·h·mL-1 and 313 ng·h·mL-1 for H-DOX, DOX, CELA and CEL, respectively.CONCLUSION: 1. After being identified by spectrum, the two mutual prodrugs of doxorubicin are the target compounds. 2. Both of the two mutual prodrugs of doxorubicin inhibited the growth of HepG2 cells. The mutual prodrug of doxorubicin with -linolenic acid had greater antitumor activity in HepG2 cells than C-DOX. 3. Pharmacokinetics study of C-DOX in rats showed that little amount of doxorubicin and celecoxib could be released in blood. This suggested that the prodrug in rats was ralatively stable.