br Fig Summary of MDR
Fig. 11. Summary of MDR reversal profile of different derivatives of 5-oxo-hexahydroquinoline scaffold.
S. Ranjbar et al.
agents, also leads to a higher collateral sensitivity, as described for flavonoid compounds (Lorendeau et al., 2014), D series compounds are good leads in order to develop better CS agents.
3.6. Expression of ABC transporters in cells
Protein expression levels in MRP1-transfected Flp-In and BCRP-transfected HEK293 ML-210 were determined by western blot. The results showed high expression of ABC transporters in transfected cells and non-detectable levels in parental cells (Supplementary Fig. 1).
4. Discussion and conclusions
In this study, 5-oxo-hexahydroquinoline derivatives bearing 3-(pyridin-2-yl)ethyl carboxylate (C series) or 3-(pyridin-2-yl)propyl carboxylate (D series) at C3 position were synthesized and evaluated for their MDR potency. A brief MDR reversal profile assessment of different derivatives of designed scaffold is presented in Fig. 11.
Flow cytometric detection of rhodamine 123 in P-gp–overexpressing MES-SA/DX5 cells revealed that most of the compounds including D6, C6, D2, D3, C3, C4 and D5 showed significant inhibition of P-gp transporter while, the first 4 compounds including D6, C6, D2 and D3 were even more potent than verapamil as the positive control at 25 μM concentration. According to our previous studies and the above-men-tioned results for the 5-oxo-hexahydroquinoline derivatives, it can be deduced that the length of the linker between pyridine ring and car-boxylate moiety plays a crucial role in P-gp inhibitory activity. Therefore, considering Log P values in Table 1, it is shown that more lipophilic 2-pyridyl ethyl carboxylate and 2-pyridyl propyl carboxylate substituents (groups C and D compounds) were better P-gp modulators than 2-pyridyl methyl carboxylate substituents (group A compounds in Ranjbar et al. report (Ranjbar et al., 2017)). In the current study, we also examined the effect of the synthesized compounds on 2 other im-portant ABC transporters, MRP-1 and BCRP. Flow cytometric determi-nation of calcein-AM accumulation in MRP1-overexpressing Flp-In HEK293 cells proved that compounds D5, D6, C5, D1, C4, C1 and D3 were the best MRP1 inhibitors at 10 μM as compared with the standard verapamil. It was deduced from flow cytometric detection of mitoxan-trone accumulation in BCRP-overexpressing HEK293 cells that com-pound D3 as the most potent derivative, inhibited BCRP effectively at 10 and 20 μM. Moreover, compounds C3, C4, C5, D6 and D2 efficiently inhibited the BCRP pump at 20 μM. r> The modulatory effect of 5-oxo-hexahydroquinoline derivatives on the efflux pumps is obviously affected by the nature of the substitutions at the 4-phenyl residue. It is concluded from flow cytometry analysis that ana-logs containing chlorine moiety on the phenyl ring, including D6 (bearing 3-chloro), C4 (bearing 2,4-dichloro) and D3 (bearing 4-chloro) showed modulatory activity on all three efflux pumps (Table 2 and Fig. 11). On the other hand, introducing a nitro substitution at the para po-sition of 4-phenyl moiety caused D2 to be a semi-selective P-gp in-hibitor. D4 derivative, containing 3-flourophenyl moiety at C4 position of hexahydroquinoline core, could be introduced as a selective MRP1 modulator as the compound did not show inhibitory activity against P-gp and BCRP transporters (Table 2 and Fig. 11). Finally, compound C2, with 4-methoxyphenyl function at C4 position of hexahydroquinoline nucleus, showed no considerable activity against any of the three transporters.
Determination of the cytotoxicity and the amount of intracellular GSH depletion in BHK-21 and MRP1-transfected BHK-21 cell lines de-monstrated that D series compounds including D4, D5 and D6 were good leads in order to develop better CS agents. Compound D4, bearing flour substitution at the meta position of phenyl ring, demonstrated remarkable specific inhibitory effect and CS activity on the MRP1 transporter.