Design, synthesis and biological evaluation of novel 5-bromo derivatives of indole phytoalexins

The increasing diversity of small molecule libraries is a major source for the discovery of new drug candidates. In term of this trend, we report the synthesis five series 5-bromosubstituted derivatives of indole phytoalexins Type A-E using straightforward synthetic approach. Novel compounds were screened in vitro for antiproliferative/cytotoxic activity against seven human cancer cell lines by MTT assay. Evaluation of their antiproliferative potency showed that the activity of some analogues was better or comparable to that of cisplatin and at the same time the toxicity of these compounds on 3T3 cells was lower than that of cisplatin. We found that all 5-bromosubstituted analogues of indole phytoalexins Type A-E exhibited lower or approximately the same activities as a previously studied corrensponding non-brominated compounds.


Introduction
Cruciferous vegetables (such as broccoli, mustard greens, cabbage, cauliflower and turnip) are prolific producers of indole-sulfur substances when are exposed to physical, chemical (heavy metals, UV radiation) or biological stress (pathogen infection). These compounds, termed 2 indole phytoalexins, are a hallmark of the family Brassicaceae and serve as an important defense mechanism for the plants. The majority of indole phytoalexins are rather simple compounds. The basic structure of these compounds is an indole, oxindole or indoline nucleus with a linear chain or annexed heterocycle (1,2,6, Fig. 1.) Some of the indole phytoalexins carry unique structural features with spiro attached thiazoline ring (3-5, Fig. 1) [1].
In addition, a naturally occurring brassinin (1), is one of the most biologically active indole phytoalexins and exhibits various pharmacological effects. Its activities is partly a result of its dithiocarbamate group. Brassinin (1) acts as a mitochondrial inhibitor and antioxidant [17].
Brassinin (1) induces cell cycle arrest in G1 phase through inhibition of the PI3K signaling pathway in HT-29 human colon cancer cells [18]. Another study showed that brassinin (1) is bioavailable indoleamine 2,3-dioxygenase inhibitor (IDO -enzyme that promotes tumor escape via mechnisms of immune tolerance) [19]. In several studies, brassinin has been the subject of combination therapy. Brassinin (1) in combination with capsaicin has synergistic anticancer effect on PC-3 human prostate cancer cells [20]. Lee et al. have revealed that a combination of brassinin (1) and paclitaxel synergistically inhibited A549 lung cancer cell growth [21]. It has also recently been found that the combination of brassinin-imatinib synergistically induces cytotoxicity and apoptosis in SW480 colon cancer cells. The combined treatment of brassinin (1) and imatinib have also revealed the anti-metastatic potencial of treatment [22]. Three novel biological activities of brassinin (1) were recently described. Brassinin (1) inhibits TNF-induced vascular inflammation in human umbilical vein endothelial cells (HUVECs) and may serve as a potencial therapeutic agent for atherosclerosis [23]. Brassinin (1) also effectively suppresses lipid accumulation in 3T3-L1 adipocytes and obesity-induced inflammatory responses through the Nrf2-HO-1 signaling pathway in an adipocyte-macrophage co-culture [24]. 3 The indole phytoalexins are class of natural products displaying unique promising properties for the development of new drugs leads, and they are a wonderful challenge to synthetic chemists. Various synthetic structural and positional modifications of indole phytoalexins have been made to evaluate their antiproliferative activities for development of novel anticancer agents. 1-Boc substituted derivative of brassinin (Type I, Fig. 1) and thiourea derivatives of brassinins (Type III) display higher potencies of antiproliferative activity than the lead compounds 1 and 2 [25][26][27]. Homobrassinin (Type II) is more active than brassinin (1) and has been shown to cause ROS dependent apoptosis in Caco-2 colorectal cancer cells [28]. Likewise, the introduction of a substituted phenyl amino group to the compounds 3 and 4 (Type V) resulted in enhanced antiproliferative effect against human cancer cell lines [27,29]. Structural modification of phytoalexin (2R,3R)-(-)-1-methoxyspirobrassinol methyl ether [(2R,3R)-(-)-6a] -synthetic 2-aminoanalogues (Type VII), 2'-aminoanalogues (Type VIII) or 2,2'diaminoanalogues (Type IX) exhibited remarkable anticancer activity [25,26,[29][30][31][32]. Synthetic analogues of cyclobrassinin (Type XI, Fig. 1) with phenyl amino group instead of methylthio group have shown extraordinary anticancer properties [25,33].
According to the literature reports, the halogenation of natural products is a one of the most popular modification that allows optimalization of the biological activity of molecules. The majority of halogenated metabolites contain bromine. An interesting fact was observed in all types of indole scaffolds that halogenations generally occur at C-5, sometimes at C-6 or at both C-5 and C-6 of the indole ring. The bromination of many natural compounds is associated with increased biological activity [34].
From the group of synthetic derivatives of indole phytoalexins, 5-bromobrassinin (Type IV, Fig. 1) contains another bromine atom in the indole nucleus C-5. 5-Bromobrassinin has a better pharmacologic profile than brassinin with slower clearance [35]. 5-Bromobrassinin induced tumour regressions of mammary gland tumors in MMTV-Neu mice in combination with paclitaxel [36]. 5-Bromobrassinin belongs to the class of compounds having IDO inhibitor activity. 5-Bromobrassinin suppressed growth of highly aggressive B16-F10 melanoma isograft tumor [19]. The presence of bromine at the C-5 position of the indole nucleus of spiroindoline phytoalexins (Type VI, Type X) led to a partial increase of antiproliferative activities on leukaemic cells compared to natural non-brominated indole phytoalexins [37].     into Types B-E target compounds

Synthesis
The synthetic ease and diversity of derivatives of indole phytoalexins as well as the reported potency of phytoalexins prompt us to investigated the effect of 5-bromo substitution on antiproliferative activity.
The synthesis of the Type A target compounds 9-11 starts from oxime 7. Oxime 7 was reduced to a labile amine 8 by sodium cyanoborohydride and titanium trichloride catalysis using the methodology optimised in our group´s previuos work [37]. The key step of the preparation of thioureas 9-11 was the reaction of crude amine 8 with the appropriate isothiocyanate and triethylamine in methanol. Target thioureas 9-11 were prepared in 59-66% yield after two reaction steps starting from oxime 7 (Scheme 1). Synthesis of target compounds 14-17 was achieved in a similar fashion than for molecules 9-11. In this manner, reduction of the oxime 12 with NaBH4 using NiCl2.6H2O as a catalyst produced unstable amine 13 [37]. Amine 13 was reacted with appropriate isothiocyanate and triethylamine in methanol to give the consequent thioureas 14-17 in 46-50% after two reaction steps (Scheme 2). Aminoanalogues of 5-bromobrassinin 9-11 were the basic substrates on which we tested oxidative spirocyclization reactions. Following a procedure that mimics the process used to produce 1-methoxyspirobrassinin [(±)-(4)], Type B target compounds (±)-18-20 were prepared by cyclization with CrO3 (5 eq.) in acetic acid and dioxane in reasonable yields (52-61%, Scheme 3). It should be noted that numerous attempts to synthesize spirocompounds (±)-18-20 using pyridinium chlorochromate (according to published report 38) were unsuccessful.
Then we tested the substrate scope 9-11, 14-17 with bromine as a cyclization reagent.
Obtained IC50 values of prepared aminoanalogues of 5-bromo-1-methoxybrasinin 9-11 and 5bromo-1-Boc-brasinin 14-17 are shown in Table 1. Table 1   ntnot tested The potency of compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72 h incubation of cells and presented as IC50 (concentration of a given compound that decreased amount of viable cells to 50% relative to untreated control cells).

Conclusion
We have prepared a library of 5-bromosubstituted analogues of indole phytoalexins Type A-E.
Type A target compounds -thioureas served as key intermediates for synthesis other interesting series. Synthesis of 2´-aminoanalogues of 1-methoxyspirobrassinin (Type B target compounds) was achieved via oxidative cyclization of thioureas. The spirocyclization reaction of thioureas with bromine has been employed to prepare the 2´-aminoanalogues and 2,2´-diaminoanalogues