Design, synthesis and biological evaluation of di-substituted noscapine analogs as potent and microtubule-targeted anticancer agents

Abstract

Noscapine is an opium-derived kinder-gentler microtubule-modulating drug, currently in Phase I/II clinical trials for cancer chemotherapy. Here, we report the synthesis of four more potent di-substituted brominated derivatives of noscapine, 9-Br-7-OH-NOS (2), 9-Br-7-OCONHEt-NOS (3), 9-Br-7-OCONHBn-NOS (4), and 9-Br-7-OAc-NOS (5) and their chemotherapeutic efficacy on PC-3 and MDA-MB-231 cells. The four derivatives were observed to have higher tubulin binding activity than noscapine and significantly affect tubulin polymerization. The equilibrium dissociation constant (KD) for the interaction between tubulin and 2, 3, 4, 5 was found to be, 55±6μM, 44±6μM, 26±3μM, and 21±1μM respectively, which is comparable to parent analog. The effects of these di-substituted noscapine analogs on cell cycle parameters indicate that the cells enter a quiescent phase without undergoing further cell division. The varying biological activity of these analogs and bulk of substituent at position-7 of the benzofuranone ring system of the parent molecule was rationalized utilizing predictive in silico molecular modeling. Furthermore, the immunoblot analysis of protein lysates from cells treated with 4 and 5, revealed the induction of apoptosis and down-regulation of survivin levels. This result was further supported by the enhanced activity of caspase-3/7 enzymes in treated samples compared to the controls. Hence, these compounds showed a great potential for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.

Keywords: Anticancer activity; Noscapine; Tubulin polymerization.

Figure 1. Molecular docking analysis of di-substituted noscapine analogs

Conformations of compounds in tubulin active site, hydrogen bond interactions (left) and compound orientation in binding pocket (right) for compounds 2, 3, 4 and 5 (A, B, C, and D) respectively. Compounds are shown in ball and stick model with green color.

Figure 2. Binding free energy calculation of ligand-tubulin complex and docked pose of compound 2 showing interaction with tubulin

Binding free energies (kcal/mol) of the compounds (2, 3, 4 and 5) calculated from the MD trajectory using MMGBSA method (left) and model representation of compound 2 in tubulin heterodimer (α- subunit in cyan and β-subunit in light blue) (right). Compound 2 was shown in space filling model (green), GTP in ball and stick model, Mg²⁺ (pink) in VDW model and Br-in brown color.

Figure 3. Fluorescence quenching of tubulin by derivatives of noscapine

Double reciprocal plots showing a dissociation constant (K_D) of 55 ± 6 µM for compound 2 binding to tubulin, 44 ± 6 µM for compound 3, 26 ± 3 µM for compound 4 and 21 ± 1 µM for compound 5. Values are mean ± S.D. for three independent experiments performed in triplicate (p < 0.05).

Figure 4. Tubulin polymerization determined via turbidity (at 350 nm)

Relative absorbance of compounds 2–5 is measured with increasing concentrations (0–25µM) indicated compounds for 30 min with 12 µM tubulin.

Figure 5. Percentage of cell survival measured using MTT assay

A and B represent the sensitivity profile of PC-3 and MDA-MB 231 cells to the four di-substituted noscapine analogs. C is a bar-graphical representation of IC₅₀ values of noscapine analogs in PC-3 and MDA-MB 231 cells. The values and error bars shown in all the graphs represent average and standard deviations, respectively, of three independent experiments (p <0.05).

Figure 6. Cell cycle profile in a 3 dimensional disposition for compounds 2–5 at different time points (0–48h)

The bar-graphs represent the percent G2/M and sub-G1populations at different time points for compounds 2–5. The distribution of cell population between G2/M and sub-G1 has been represented as bar graphs in the panels for each compound.

Figure 7. Immunoblot analysis of surviving and cleaved PARP protein and caspase-3/7 activity with compounds 2, 3, 4, and 5

A. Protein samples from treated and control cells probed for survivin and cleaved PARP. β-actin was the loading control. B. Bar graphical representation of relative fluorescence of caspase-3/7 activity in control and treated samples.

Scheme 1

Synthesis of di-substituted noscapine analogs Reagents & Conditions a) NaN₃Nal, DMF, 140 °C, 3h. b) Ac₂O, Dimethylamino pyridine, THF, rt, 4h. c) Dimethylamino pyridine, CH₂Cl₂, RNCO, rt, 6–8 h.