Indolizine-phenothiazine hybrids as the first dual inhibitors of tubulin polymerization and farnesyltransferase with synergistic antitumor activity

Highlights

• Design, synthesis and biological evaluation of indolizine-phenothiazine hybrids.

• Molecular docking on studied targets.

• Eaton’s reagent-mediated acylation of 10H-phenothiane and 10-methyl-10H-phenothiazine.

• Identification of dual inhibitors of tubulin polymerization and farnesyltransferase.

• High cytotoxicity effect on NCI-60 cancer cell line panel (up to GI₅₀ = 1.8 nM).

Abstract

In the incessant search for innovative cancer control strategies, this study was devoted to the design, synthesis and pharmacological evaluation of dual inhibitors of farnesyltransferase and tubulin polymerization (FTI/MTIs). A series of indolizine-phenothiazine hybrids 16 (amides) and 17 (ketones) has been obtained in a 4-step procedure. The combination of the two heterocycles provided potent tubulin polymerization inhibitors with similar efficiency as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones 17 were also able to inhibit human farnesyltransferase (FTase) in vitro. Interestingly, three molecules 17c, 17d and 17f were very effective against both considered biological targets. Next, nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b were evaluated for their cell growth inhibition potential on the NCI-60 cancer cell lines panel. Ketones 17a-f were the most active and displayed promising cellular activities. Not only they arrested the cell growth of almost all tested cancer cells, but they displayed cytotoxicity potential with GI₅₀ values in the low nanomolar range. The most sensitive cell lines upon treatment with indolizine-phenothiazine hybrids were NCI-H522 (lung cancer), COLO-205 and HT29 (colon cancer), SF-539 (human glioblastoma), OVCAR-3 (ovarian cancer), A498 (renal cancer) and especially MDA-MB-435 (melanoma). Demonstrating the preclinical effectiveness of these dual inhibitors can be crucial. A single dual molecule could induce a synergy of antitumor activity, while increasing the effectiveness and reducing the toxicity of the classical combo treatments currently used in chemotherapy.

Introduction

Indolizine 1 (Fig. 1), formerly named pyrroline, pyrindole, 8-pyrrolopyridine and also pyrrolo[1,2-a]pyridine, is an aromatic heterocycle consisting of a pyridine ring condensed with a pyrrole cycle. Indolizines were discovered by Angeli in 1890 and first synthesized by Scholtz in 1912 [1]. So far, the aromatic form has not been found in the structure of natural products, while the hydrogenated form of indolizine seems to be common, many such derivatives being isolated from plants, microbes, marine organisms, insects or fungi [2]. For example, (-)-swainsonine (compound 2, Fig. 1) is a natural compound present in some plants and fungi and exploited for the antitumor potential [3]. This compound has shown promising potential for the treatment of glioma and gastric carcinoma [4], [5]. (-)-Swainsonine also enhances the in vivo efficacy of chemotherapeutic agents such as cisplatin and acts against tumors by stimulating macrophages, protecting the hematopoietic system of the toxicity of chemotherapeutic agents [6], [7]. (+)-Castanospermine (compound 3, Fig. 1) is extracted from Castanospermum austral seeds and inhibits the multiplication of human immunodeficiency virus (HIV) [8]. (-)-Ipalbidine (compound 4, Fig. 1) is another natural compound of interest, being an analgesic preventing addiction, as well as a leukocyte respiratory burst inhibitor [9]. (R)-Antofine 5 is also a natural indolizinic alkaloid with antitumor potential [9], [10], [11]. (R)-Tylophorine 6 extracted from Tylophora exhibits potential anticancer and anti-inflammatory activity [12]. Camptothecin 7 (Fig. 1), an important representative of alkaloids inhibiting topoisomerase I, contains the partially hydrogenated indolizine nucleus in the structure [13]. (20S)-Camptothecin is extracted from the stem and bark of the decorative tree Camptotheca acuminata, used in Chinese traditional medicine for the treatment of cancer [14]. Moreover, many analogues of this alkaloid have been synthesized in the laboratory and are used in chemotherapy [15].

Due to the pronounced similarity both structurally and chemically with the indole nucleus, the biological potential of indolizine was envisaged. Analogues of biologically active products in which indolizine took the place of the indole unit were rapidly synthesized. Thus, Carbon and Brehm [16] proposed 1-indolizinealanine as a possible antimetabolite of tryptophan. The literature highlights a variety of indolizine derivatives with biological activity such as antimicrobial, antioxidant, anti-inflammatory and anticancer [17]. In particular, many indolizines have been reported for their anticancer potential. For instance, such structures containing a cyclopropylketone in position 3 displayed in vitro antiproliferative activity on Hep-G2 cell lines [18], the most active in the study being compound 8 (IC₅₀ (Hep-G2) = 0.2 µg/mL, Fig. 2). Other products decorated with indolizine ring are involved in the migration and proliferation of tumor cells by preventing protein–protein interaction in which the endothelial vascular growth factor (VEGF) is involved (compound 9, Fig. 2) [19].

The first tubulin polymerization inhibitors containing an indolizine moiety were published as phenstatin analogues, known antimitotic agent (Fig. 2) [20]. Among these compounds, promising activity was reported for derivatives with an indolizine ring linked to a trimethoxyphenyl nucleus through a carbonyl group. Their cytotoxic effect was especially registered on MDA-MB-435 melanoma cells [21]. When indolizine was combined with triazine and p-bromobenzoyl (compound 10, Fig. 2), an inhibition activity of the growth of SNB-75 SNC cancer cells and MDA-MB-231/ATCC breast cancer cells was observed [22]. As for indolizine-glyoxylamide 11, it displayed cytotoxic activity against cancer cell lines which have developed resistance to chemotherapy such as Taxol-resistant HL60/TX1000 cell lines (Fig. 2) [23].

The use of indolizine ring in the conception and synthesis of some analogs of phenstatin led to promising results by providing compounds with good biological activity [21], [22]. Thus, we were able to confirm that indolizine may replace successfully the cycle B of model phenstatin. In the previously reported studies, however, the active molecules were those presenting the classic 3,4,5-trimethoxyphenyl ring A (e.g. compound 13, Fig. 2) [21]. In addition, our research group discovered that other indolizines inhibited the human farnesyltransferase (FTase) (e.g. compound 12: IC₅₀ (FTase) = 1.3 µM [24], Fig. 2).

Tubulin is a well-documented target in oncology due to its role in chromosome segregation and cell division. This requires extremely fast dynamics of microtubules polymerization and depolymerization, both mediated by the tubulin. These dynamics can be blocked by different classes of inhibitors of tubulin that, by interfering with the dynamics of the microtubules, stop the cancer cell in mitosis (arrest of cells in G2/M phase of the cell cycle), eventually leading to cell death, both through apoptosis and necrosis [25]. The FTase is another studied target in oncology. It catalyzes the addition of a lipid group to the terminal carboxyl of several proteins, including Ras proteins having a proto-oncogenic role. The substrates of this enzyme undergo a process of maturation consisting of: fixation of a prenyl chain, loss of the three terminal amino acids and methylation of the C-terminal carboxyl group. Sometimes a palmitoylation phase ends the process. The prenylation step is a key step, as its inhibition prevents proteins to attach to the target membrane [26]. Inhibitors of FTase are also capable of inhibiting bipolar spindle formation and chromosomal alignment during metaphase [27].

In the incessant search for molecules with improved antitumor activity, a new series of compounds has been conceived in this study (target compounds, Fig. 2) where the cycle B is an indolizine unit and the cycle A is a phenothiazine. Chemistry of the latter azaheterocycle is part of the laboratory's expertise and proved to be a successful substitute for the classical trimethoxyphenyl group in the structure of antimitotic agents (e.g. phenothiazine derivative 14: IC₅₀ (tubulin) = 9.48 µM) [28]. Phenothiazine derivative 15 was not active on tubulin but displayed activity against human FTase (IC₅₀ = 0.6 µM [29], Fig. 2).

The ambition of this work was to obtain a synergy of antitumor effects using a unique compound bearing the two heterocycles that are each present in the structure of previously identified antitumor agents as phenstatin analogues as well as FTase inhibitors. These targeted indolizine-phenothiazine hybrids may constitute dual inhibitors of tubulin polymerization and farnesyltransferase. Substituents privileged on each heterocycle are those previously identified to be beneficial for the pursued biological activity: methyl, methoxy, acetyl or bromo (target compounds, Fig. 2).

The use of specific FTase inhibitors to treat different cancers has been disappointing in clinical trials due to the ability of the cancer cells to circumvent the problem of the FTase inhibition and go through FTase inhibition by using a second prenylation route mediated by structurally related geranylgeranyltransferase. Demonstrating the effectiveness of dual inhibitors of tubulin and FTase (MTI-FTI hybrids) can be crucial, leading to an innovative strategy for the design of new anticancer compounds. A single molecule may induce a synergy of antitumor action increasing the effectiveness and reducing the toxicity of the classical combo treatments.