Original article
Studies on indolizines. Evaluation of their biological properties as microtubule-interacting agents and as melanoma targeting compounds

https://doi.org/10.1016/j.ejmech.2014.10.041Get rights and content

Highlights

  • Synthesis and biological screening of indolizine-containing inhibitors of tubulin polymerization.

  • Evaluation of new indolizines for cytotoxicity on an NCI-60 human cancer cell lines panel.

  • Discovery of indolizine derivatives with GI50 values in the nanomolar range on melanoma MDA-MB-435 cell lines.

  • SAR study on indolizin-3-yl(3,4,5-trimethoxyphenyl) methanones as microtubule-interacting agents.

Abstract

With the aim of investigating new analogues of phenstatin with an indolizin-3-yl unit, in particular as the B-ring, three new series of compounds (68, 934 and 54) were synthesized and tested for interactions with tubulin polymerization and evaluated for cytotoxicity on an NCI-60 human cancer cell lines panel. The replacement of the 3′-hydroxy-4′-methoxyphenyl B-ring of phenstatin with substituted indolizine unit results in the conservation of both antitubulin and cytotoxic effect. Indolizines 9 and 17 were the most effective in the present study and showed the highest antiproliferative effect on melanoma cell lines MDA-MB-435 (GI50 = 30 nM) and could serve as new lead compounds for the development of anti-cancer therapeutics.

Introduction

Tubulin is a heterodimer of closely related and tightly linked globular α- and β-tubulin proteins. α- and β-Tubulin heterodimers are polymerized in hollow tubes called microtubules, forming the mitotic spindle. Their importance in cell division makes microtubules an important target for anti-cancer drugs [1]. Tubulin binding agents interfere with the dynamic instability of microtubules, and thereby arrest mitotic cells in the G2/M-phases of the cell division cycle, leading to induction of apoptosis [2]. Colchicine and its analogues predominantly bind to a high affinity site, called the colchicine binding site, located at the interface of homodimers α/β [3].

A known tubulin polymerization inhibitor that binds to the colchicine site of the protein is phenstatin which resulted from a SAR-study on combretastatin A-4 (CA-4) and exhibited potent inhibition of tubulin polymerization and of cancer cell growth [4].

The literature abounds of pharmacomodulations performed on known inhibitors of tubulin polymerization. The majority of structural modifications of the phenstatin have concerned either the carbonyl bridge or the B-ring; the A-ring (3,4,5-trimethoxyphenyl unit) was generally kept intact and it has long been considered essential to the biological activity [5]. However, some modulations on the A-ring are permitted without reducing the biological potency [6].

We were puzzled by the recent discovery of phenstatin analogs with 5,6-fused bicyclic heteroaromatic scaffolds as B-ring, and we have pointed out that only four indolizin-3-yl-(3,4,5-trimethoxyphenyl)methanones were described [7], [8], [9], [10], [11] (compounds 14, Fig. 1). Indolizine 1 showed potent anti-mitotic and anti-proliferative activity, and authors concluded that this compound could serve as important tool for the development of anti-cancer therapeutics [7]. Concerning compound 2, only synthesis details and physico-chemical properties are available in the literature [8]. The biological evaluation of this compound could provide useful information for the development of new chemical entities in this family of microtubule-interacting agents. Indolizine 3 is described as a tubulin binding anti-cancer agent [9]. However, its potential on tubulin and on cancer cell growth is not reported. Finally, the indolizin-3-yl-(3,4,5-trimethoxyphenyl)methanone 4 showed a growth inhibitory activity in vitro on gastric carcinoma MKN-45 cells [10], [11]. On the other hand, some closely related ketones such as compound 5 (Fig. 1) acts as apoptosis inducer [12]; that enhances the biological potential of products with this type of structure.

Our recent identification of phenstatin analogues bearing an indolizin-3-yl scaffold as B-ring [6c], and the absence of a complete SAR study in these series encouraged us to explore some structure–activity relationships in this new family of tubulin polymerization inhibitors. Therefore, we present here our synthetic strategies for the replacement of the classical 3′-hydroxy-4′-methoxyphenyl B-ring with substituted indolizines and the biological evaluation of target compounds (compounds 634 and 54, Fig. 1).

Section snippets

Chemistry

The objective of the present study was to highlight the substituents on the indolizine ring that may play an important role in the biological activity. In order to obtain diverse substituents on the indolizine B-ring, a [3 + 2] cycloaddition synthesis was privileged for the target compounds.

The bromoacetyl-4-methoxybenzene 35A was commercially available, and the other starting bromoacetyl derivatives 35BD were synthesized by reacting the corresponding di- or tri-methoxyacetophenone with

Conclusions

Three new series of phenstatin analogues with a substituted indolizin-3-yl B-ring have been designed and synthesized. The lack of RSA in the literature concerning indolizin-3-yl-(3,4,5-trimethoxyphenyl)methanones encouraged us to explore some structure–activity relationships in this new family of compounds. Newly synthesized indolizines were tested for interactions with tubulin polymerization and evaluated for cytotoxicity on an NCI-60 human cancer cell lines panel. The replacement of the

Materials and methods

Starting materials are commercially available and were used without further purification. Melting points were measured on a Buchi 510 apparatus and are raw values. NMR spectra were acquired at 400 MHz for 1H NMR and 100 MHz for 13C NMR on a Bruker DRX 400 spectrometer or on a Varian 400 MHz Premium Shielded® spectrometer with tetramethylsilane as internal standard, at room temperature. Chemical shifts (δ) are expressed in ppm relative to TMS as internal standard. Thin layer chromatography (TLC)

Acknowledgments

This work was supported by a grant of the Romanian Ministry of Education, CNCS-UEFISCDI, project number PN-II-RU-PD-2012-3-0426. The authors gratefully acknowledge the National Cancer Institute (NCI) for biological evaluation of compounds on their 60-cell panel: the testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (the URL to the Program's website: http://dtp.cancer.gov).

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