Figure 1.
Structure of representative camptothecins and luotonin A.
Camptothecin is a natural topoisomerase 1 inhibitor that has been used as a lead for the development of a family of anticancer agents in clinical use, exemplified by topotecan. Nevertheless, the camptothecins suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring that leads to an inactive carboxylate form. Luotonin A is a plant alkaloid whose structure strongly resembles that of camptothecin but lacks the lactone moiety. The discovery that luotonin A is also a topoisomerase 1 inhibitor, although less potent than the camptothecins, provided a unique opportunity for drug discovery in the anticancer area.
Figure 2.
Preparation of synthetic precursors.
Compounds 1a–d are commercially available. Compound 1e was prepared by bromination of 1c, using a literature method. Compounds 1f and 1g were prepared by addition of 3,5-dimethylphenylmagnesium bromide to the corresponding 2-aminobenzonitrile derivative to generate an imine that was hydrolyzed in situ by addition of acid.
Figure 3.
Synthesis of luotonin A and its analogues by CAN-catalyzed Friedländer reactions.
The reaction between the o-aminocarbonyl compounds 1 and the dehydrovasicinone 2 was performed in refluxing ethanol, in the presence of Ce(IV) ammonium nitrate (CAN) as a Lewis acid.
Table 1.
Scope and yields of the CAN-promoted Friedländer synthesis of luotonins.
Figure 4.
Isolation of open luotonin analogues that behave as intermediates of the Friedländer reaction.
By interrupting the reaction at mid-course, it was possible to isolate enamines 4 and prove their role as intermediates of the Friedländer reaction by verifying that they afforded the final products under our standard conditions.
Figure 5.
DNA relaxation inhibition assay.
Drug concentration was 10 µM. Activities are expressed relative to that of camptothecin (CPT): (+) 1–25%, (++) 26–50%, (+++) 51–75%, (++++) 76–100% of the CPT inhibition. All compounds were at least as potent as luotonin A and compound 3g showed a remarkable activity, comparable to that of camptothecin.
Figure 6.
Docking of luotonin A, topotecan and compound 3c.
A. A full view of the topoisomerase 1-DNA complex with luotonin A docked onto the active site. B. Expansion of the binding site. Luotonin A (navy blue) is shown compared with the position found for topotecan (brown) by crystallography, showing an excellent concordance between both poses. The base pairs are coloured in red (−1) and green (+1). The topoisomerase Arg364 residue responsible for hydrogen bonding with the compounds is displayed in blue. C. Docking of compound 3c, showing the hydrogen bonding interaction of Arg364 with both nitrogens N5 and N6.
Table 2.
Quantification of the affinities of compounds 3a–g for their binding sites and its correlation with their inhibitory activity.
Figure 7.
B-ring substitution improves hydrogen bonding to Arg364.
A. Overlay of luotonin A (3a) and compounds 3b–e at their binding sites, showing that the presence of the substituent at the C-14 position of ring B induces a rotation of the docking pose that leads to improved hydrogen bonding to Arg364 and hence to a better fit to the binding site. B. Docking of luotonin A showing the position of Asn352, which would interfere with any ring-B substituent. C. Docking of the B-phenyl substituted compound 3c, also showing the position of Asn352.
Figure 8.
The two binding orientations of compounds 3.
Docking studies revealed two different modes of binding: A. In the case of compounds 3a–e, the docking pose was similar to the one found for topotecan by X-Ray crystallography. B. Compounds 3f and 3g, on the other hand, changed their mode of binding in order to shield the highly hydrophobic dimethylphenyl moiety from the aqueous environment by burying it between the base pairs of the scissile strand and the surface of the protein. C Detail of the newly described binding feature of the human topoisomerase 1-DNA complex uncovered by our study.
Table 3.
Main interactions located for compounds 3a–g at their binding sites.
Figure 9.
Results of the cytotoxicity assays.
The cytotoxicities of compounds 3a–g are expressed as percentage of cell growth relative to the negative control. Error bars account for the standard error. Asterisks indicate statistically significant differences between a drug and the model compound 3a. Camptothecin was employed as a positive control (0% growth) at 25 and 2.5 µM concentrations.