Design, Synthesis And Structure-Bioactivity Relationships Of Otobain Derivatives

Etoposide, a semisynthetic derivative of podophyllotoxin, is clinically useful medicines against various types of cancers, including:small-cell lung cancer, testicular cancer, acute leukemin, lymphoma. However, their high toxicity, low water solubility and drug resistance have limited their application in cancer chemotherapy. On the other hand, the natural supply of podophyllotoxin becomes scarcer and the economical synthesis of podophyllotoxin is not feasible. Therefore, searching for safer and more effective anticancer agents by expanding the study scope of podophyllotoxin on structural modification, stereo configuration and structure-activity relationship is an urgent need. In this respect, this paper carried out the following research:1) According to the structure-activity relationship of podophyllotoxin and structure combination strategy, lead compound 7a was designed by introducing trans-fused y-lactone and C-4 hydroxy to otobain. Further according to drug combination principle and bioisosterism, novel otobain C-4 ester, amide and amine derivatives were designed.2) Starting from commercially available piperonal, lead compound 7a was synthesized by aldehyde protection, michael addition to furan-2(5H)-one, nucleophilic addition to another molecule piperonal, intramolecular Friedel-Crafts cyclization, deprotection and reduction in six steps with 21% overall yield. By esterification, azide substitution, reduction, acylation and substitution reaction,47 novel target compounds were obtained. The assignment of relative stereochemistry for target compounds was based on’H NMR and single-crystal X-ray diffraction. The relative configuration of otobain ester derivatives was assigned as r-1, trans-2, trans-3, trans-4. The relative configuration of otobain amide and amine derivatives were assigned as r-1, trans-2, trans-3, cis-4.3) All the synthesized compounds were consequently evaluated for their inhibitory activity against A549 and HL60 cancer cell lines in vitro. The results showed that ester derivative 8at, amide derivatives 11ak, 11al, 11ap, 11aq and amine derivatives 12aa,12ab and 12ae showed good inhibitory activity that was comparable with etoposide. Compounds with good inhibitory activity in preliminary screening were further evaluated in vitro anti-tumor activity against five human tumor cell lines (A549, HL60, SGC7901, MCF7 and HCT116) and one human normal cell line (HaCat). Notably, compound 12ae showed potent actions against HL60 (IC50= 1.06 μM), SGC7901 (IC50= 4.16μM), MCF7 (IC50= 2.25 μM), HCT116 (IC50= 1.65 μM) and A549 (IC50= 2.32 μM) cancer cell lines that was comparable with the positive control drug etoposide. Moreover, compound 12ae (i.e., IC50= 1.16 μM) showed less toxicity to human normal cell HaCat than that of etoposide (i.e., IC50= 0.58 μM). In vivo anti-tumor assay also showed compound 12ae exhibited certain inhibitory activity in A549 cancer cell line. The body weights in the etoposide -treated group were reduced by 16.75% at the end of the experiments. However, both in the low dosage compound 12ae-treated group (20 mg/kg) and high dosage compound 12ae-treated group (40 mg/kg), the body weights of mice were not obviously affected. Colony formation assay showed that compound 12ae almost blocked the anchor growth of A549 cells at 3 μM. Cell cycle assay, apoptosis assay and western blot assay showed that compound 12ae caused G2/M phase arrest in A549 cells, but it did not induce apoptosis even at 10 μM. These finding indicated that the anti-tumor mechanisms of otobain derivatives may be different from that of podophyllotxin and its representative semi-synthetic derivatives such as etoposide. These compounds inhibited cancer cells proliferation by arresting cell cycle at G2/M phase.4) Taking compound 12ae as a representative, its racemic cis isomer 12be and four chiral isomers 12ae1,12ae2,12be1 and 12be2 were synthesized. The enantionmeric excess of target enantiomerically pure compounds were higher than 97%. The absolute configuration of compound 4bel was assigned as R, R, S, R by successfully obtained X-ray crystal structure of the sulfur-containing compound 4bel. Then the absolute configuration of each chiral intermediates and four target enantiomerically pure compounds were assigned based on the stereochemical process of the reaction and NMR methods.Target compounds rac-12ae, rac-12be and their pure enantiomers were evaluated for in vitro cytotoxicity against four human tumor cell lines (A549, HCT116, MCF7 and SGC7901) and one human normal cell line (HaCat). The results showed that r-1, trans-2, trans-3, cis-4 configuration racemic compound 12ae and its two enantiomers 12ael and 12ae2, possessing a different relative configuration from that of etoposide, exhibited potent cytotoxicity. Among them, the cytotoxicity of the enantiomer(1R,2S,3R,4R)-12ae2 against tumor cell lines A549, HCT116, MCF7 and SGC7901 was about 10.74 times,4.28 times,4 times and 8 times more active than that of etoposide, respectively. The cytotoxicity of enantiomer 12ae2 against HaCat was about 1/3 that of etoposide. These results revealed that stereochemistry of otobain derivatives, especially 1,2-trans relative configuration, had a great influence on their activities.5) In our investigation on the synthesis of 6-bromo piperonal, an important intermediate for synthesis of otobain derivatives, a selective and efficient method of oxidizing benzyl alcohols to benzaldehydes and methyl benzoates by using 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) as oxidant and by using dichloromethane and methanol as solvent is developed. One-step oxidation of benzyl alcohols to corresponding methyl benzoates was achieved in good yields (86%-94%) by reacting benzyl alcohol with DBDMH at a ratio of 1:4 in methanol at room temperature for 12 h. The selective oxidation of benzyl alcohols to benzaldehydes was achieved in excellent yields (89～99%) by reacting benzyl alcohol with DBDMH at a ratio of 1:1 in dichloromethane at room temperature for 0.5 h. Subsequently, the proposed method was used to synthesize 6-bromo piperonal. In summary, a mild, efficient and low cost method of chemoselectively converting benzyl alcohols to benzaldehyde and methyl benzoate using DBDMH was developed. Moreover, the reaction was metal free and acid-base free, and can thus be a valuable alternative to existing approaches for use at laboratory and industrial scales.The screened compound 12ae2 in this article has significant potential for further development as new anti-tumor candidate compound. Moreover, it can be obtained by total synthesized starting from easy getting piperonal. Thus, the work is worthy of further studied.