Desigh, Synthesis And Evaluation Of Novel Sophoridinic Acid Derivatives As Anticancer Agents

Sophoridine (figure 1), an alkaloid monomer extracted from Chinese traditional medicine Sophora alopecuroides L, has a wide range of biological activities. Sophoridine Hydrochloride Injection was approved by SFDA in 2005 as an anticancer drug against malignant trophoblastic tumors with considerable efficacy and safety. Because of its unique chemical scaffold and biological activity, sophoridine was applied as the lead compound in our earlier study and its preliminary anti-tumor structure -activity relationship (SAR) was elucidated. It was found that sophoridinic acid 6B (figure 1) bearing a novel structure skeleton demonstrated a significantly higher activity in human HepG2 cell line than sophoridine (Bioorg Med Chem Lett.,2012). Compared with sophoridine, the 3-ring scaffold sophoridinic acid has several advantages. First, the 4-ring scaffold was derived to a simpler 3-ring scaffold. Second, more modification sites (11-side and 12-substitution) appeared, which can be transformed into various derivatives. Last, sophoridinic acid had a strong anti-tumor activity; the antiproliferative activity of 6B exceeded that of sophoridine by more than an order of magnitude.Figurel. The structure and activity of sophoridine,6B and modifiable sites (right)Based on these works, sophoridinic acid 6B was taken as the lead compound, systematic structure modifications and optimizations were carried out focusing on the 11-side chain,12-substitution as well as the chiral configuration of the 5-C atom. By the following in vitro anticancer assay against HepG2 hepatoma cells, the anti-tumor SAR of sophoridinic acid derivatives were summarized as follows.1) 5R configuration was essential for the activity. The activity decreased dramatically or even disappeared when the 5R configuration was converted to 5S.2) Introduction of an aryl methylene (methylnaphthalene, methylanthracene and halobenzyl) group on the 12-N atom could obviously increase the activity. Excessive lipophicity (produced by methylnaphthalene or methylanthracene) may lead to increasement of toxicity. So halobenzyl was fixed/retained on the 12-N atom in later study.3) When p-chlorobenzyl on the 12-N atom was turned into p-chlorobenzoyl, the activity decreased, while the p-chlorobenzenesulfonyl substitution may remain the activity.4) The carboxyl group on the 11-side chain was not crucial for the activity. The analogues derived to ester, primary alcohol, hemiacetal, hydroxamic, ketone, tertiary alcohol or alkene displayed improved activity, while the ether, amide and oxime ether conversion led to a decrease or disappereance of activity. However, ester, alcohol, hemiacetal and hydroxyl oxime groups were vulnerable to metabolism in vivo.5) Target compounds with good druggablity including considerable PK. properties and toxicity features could be gained by by the regulation of 11-side chain on the basis of retaining the pharmacorephore on the 12-N atom.9 target compounds with diverse structure types (Z08, C08, C34, C35, T08A, X08C, A08C, A08D, A08F) with an IC50 lower than 10 μM were screened out for further investigations, including antitumor spectra analysis, activity against drug-resistant tumor, mechanism study, druggability evaluation and in vivo pharmacodynamic evaluation.The antitumor spectra analysis showed that compounds Z08 and C08 showed high cytotoxicities against five tumor cell lines (HCT116, H1299, U87, MCF-7, KB) with IC50 values ranging from 4 μM to20 μM, indicating a broad antiproliferative spectra of these compounds. C45, C35, T08A, X08C exhibited potent anti-tumor effect against MCF-7/ADrR cell lines which were resistant to adriamycin with IC50 values ranging from 5.7 μM to 9.5 μM. There was no cross drug-resistance between sophoridinic acid derivatives and adriamycin, indicating a different anticancer mechanism from adriamycin.It was indicated in the mechanism study that C08 and C34 were able to cause the G0/G1 arrest in the cell cycle in HepG2 cells. Besides, C08 also displayed a significant inhibitory activity against DNA Top I at the concentration of 0.6 mg/ml, in consistence with sophoridine. And it was found by molecular docking model LigandFit that the C08 had a very good affinity to DNA Top I-DNA complex.The in vivo pharmacokinetic experiment showed that C08, C34, A08C had ideal pharmacokinetic profiles (Cmax:3.4～5.9μM, Tmax:0.6～4.6 h, T1/2:3.8～13.0 h). It was demonstrated by the acute toxicity assay that the Oral LD50 of C08 is higher than 500mg/kg, the LD50 of A08C in mice by intraperitoneal injection was between 25 and 50 mg/kg, and the body weight of the administration mice group by a LD50 dose did not change significantly compared to the control, indicating good druggabilities of C08 and A08C.Based on these studies, A08C was finally chosen as the candidate compound to carry on the in vivo pharmacodynamic evaluation. It was showed that A08C could effectively inhibit the growth of transplantable tumor in nude mouse in a dose-dependent manner, and had no impact on the body weight of mice compared with the positive control, and thus was worthy further inverstigation.Totally 127 novel compounds were synthesized,117 of which were target compounds, whose structures were confirmed by 1H NMR,13C NMR and HRMS.33 derivatives gave IC50 values less than 10 μM against HepG2 cells. Some candidates showed broad antiproliferative spectra as well as potent inhibition to drug-resistant tumor. Besides, candidate compounds were equipped with good PK profiles and safety in vivo.These results hinted that an anti-tumor compound reservoir bearing a novel structure skeleton was constructed by sophoridinic acid derivatives with a 3-ring scaffold. These derivatives had strong anti-tumor activity, broad antiproliferative spectra and good druggability, and they were effective against resistant tumors with a mechanism different from that of adriamycin, and were thus very promising. Intensive mechanism of action of related candidates is in progress.Relevant research has applied for a Chinese patent (Appl. No.:CN201310268482.4), which is in substantive examination stage now. Some work has been published in European Journal of Medicinal Chemistry (2014) and ACS Medicinal Chemistry Letters (2014).