Antioxidant, α-glucosidase inhibitory and in vitro antitumor activities of coumarin-benzothiazole hybrids

Glycosidases are a class of carbohydrate-hydrolase enzymes that catalyze hydrolysis of glycosidic bonds in oligosaccharides [1], [[2]. The development of α-glycosidase inhibitors with potential therapeutic applications has received considerable attention recently 3], [[4]. The emergence of drug resistance to cancer chemotherapeutic agents has directed significant research efforts toward development of new agents for cancer treatment utilizing molecular hybridization (MH) strategy of different pharmacophores with the aim of obtaining superior anticancer activity compared to the parent molecules [5], [6], [7]. Limited examples of lead compounds from natural sources display promising antitumor activity based on their potent glycosidase inhibition activity 8].

The imbalance between overproduction of reactive oxygen species (ROS) and cellular detoxification machinery in favor of ROS production, known as oxidative stress, leads to cellular damage and malfunction [[9], [10]. Oxidative stress is directly associated with cancer progression, and there is a pressing need for development of potent antioxidant agents that can protect cellular organelles from ROS 11]. In this context, coumarin derivatives demonstrate intriguing antioxidant activity owing to scavenging of the initial radicals and propagating peroxyl radicals [12], [[13]. Moreover, coumarin-based compounds show promising antitumor activity 14] and are potent inhibitors of α-glycosidase [15], [[16]. Benzothiazole is a versatile synthetic scaffold with a wide spectrum of biological effects including potential antioxidant 17] and antitumor [18] activities. In addition, benzothiazole-containing agents show α-glycosidase inhibitory activity [19], [20].

Bromophenols (BPs), isolated from marine algae, demonstrate promising α-glycosidase inhibitory activity which has been attributed to the presence of bromo and hydroxy substituents [21], [[22]. Therefore, BPs are promising lead compounds for the design of potential α-glycosidase inhibitors. Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE, Figure 1) is a potent α-glycosidase inhibitor with a half maximal inhibitory concentration (IC₅₀) value of 0.098 μm21] and a potential antitumor agent [23]. Investigation of the binding interactions between BDDE and α-glycosidase has identified a charged-hydrophobic-polar (C-H-P) binding pocket in α-glycosidase that fits BDDE [24]. The hydroxy groups of BDDE are involved in multiple hydrogen bonds with residues in the polar areas of the binding pocket, while the rest of the molecule is stabilized by hydrophobic interactions with nearby residues. The binding mode of BDDE to α-glycosidase is consistent with the structure-activity relationship established for hydroxycoumarin derivative (Figure 1) as potent α-glycosidase inhibitor with IC₅₀ values in the nanomolar range [25]. It is proposed that hydrogen bonding and extensive hydrophobic interactions in a cooperative fashion are involved in the α-glycosidase inhibitory activity of the hydroxycoumarin derivatives. The basis of the cooperative hydrogen bonding and hydrophobic interactions has been derived from X-ray crystallographic analysis of maltose in a complex with Thermotoga maritima α-glucosidase AglA [26]. In the crystal structure, one of the glucose rings of maltose is bound by multiple hydrogen bonds to charged residues in the binding pocket, while the rest of the molecule is hydrophobically stacked to stabilize the interactions.

Figure 1

α-Glycosidase inhibitors reported in the literature and coumarin-benzothiazole hybrid compounds 5a–c.

In this investigation, the design strategy of the coumarin-benzothiazole hybrids as α-glycosidase inhibitors (Figure 1) interrogates structural features of both the marine natural BDDE and coumarins. It was anticipated that the benzothiazole core with bromo and hydroxy substituents would be implicated in hydrophobic and hydrogen-bonding interactions with α-glycosidase similar to BPs. The coumarin moiety in the new hybrid compounds was speculated to be involved in additional hydrophobic and hydrogen bonding interactions in the hydrophobic and polar areas of the binding pocket. The synthesized coumarin-benzothiazole hybrids (Figure 1) were evaluated for their antitumor and antioxidant activities.

The target compounds 5a–c of this study were synthesized according to the general approach outlined in Scheme 1. As can be seen, the starting aminothiophenol 2 was synthesized by hydrolysis of the benzothiazole 1 with aqueous potassium hydroxide. Treatment of methyl substituted coumarin derivatives 3a–c with selenium dioxide in xylene proceeded smoothly to furnish formyl derivatives 4a–c in serviceable yields. Subsequent condensation of 2 and 4a–c in glacial acetic acid yielded coumarin-benzothiazole hybrids 5a–c (Scheme 1).

Scheme 1

Synthesis of compounds 5a–c.

Compounds 5a–c were evaluated by the National Cancer Institute (NCI) in vitro for their antitumor activity [27]. A single dose (10 μm) of the tested compounds was used in the full NCI-60 cell lines panel assay. Compounds 5a,b exhibited weak antitumor activity against all tested cell lines except for moderate activity of 5a against central nervous system (CNS) and breast cancer cell lines. Compound 5c displayed lethal effects (>100% inhibition) against non-small-cell lung cancer (HOP-62), CNS cancer (SNB-75) and ovarian cancer (SK-OV-3) cell lines. Subsequently, compound 5c after passing this primary antitumor assay was carried over to the NCI five-dose screening. It can be suggested that the presence of a polar ionizable group on the coumarin moiety of these coumarin-benzothiazole hybrids is essential for antitumor activity. Potent antitumor activity of 5c was evident against CNS cancer (SNB-75) and ovarian cancer (OVCAR-4) cell lines with half maximal growth inhibition values of 0.24 and 0.33 μm, respectively.

Compounds 5a–c were also evaluated for their in vitro α-glucosidase inhibitory activity [28]. The results showed that compound 5c exhibits promising inhibitory activity with an IC₅₀ value of 6.32±0.51 μm in comparison to miglitol as a reference compound (IC₅₀=0.39±0.02 μm). Compounds 5a,b display moderate α-glucosidase inhibitory activity with IC₅₀ values of 38.9±1.43 and 21.47±0.91 μm, respectively.

Antioxidant activities of compounds 5a–c were determined using diphenylpicrylhydrazyl (DPPH) radical scavenging method [29]. In this test, compound 5c displayed moderate antioxidant activity with an IC₅₀ value of 35.17±1.34 μm which is comparable to the activity of the standard reference ascorbic acid (IC₅₀=22.8±0.71 μm). Compounds 5a,b displayed similar antioxidant capacity with IC₅₀ values of 44.5±2.66 and 41.36±2.12 μm, respectively. The correlation between antitumor activity of 5c to its α-glucosidase inhibitory and antioxidant activities in comparison to 5a,b suggests that these activities represent the basis of its antitumor profile.

Saccharomyces cerevisiae isomaltase crystal structure (PDB ID: 3AJ7) shows high sequence similarity (72.4%) with α-glucosidase and was utilized in this investigation for molecular docking studies. Compound 5c displays significantly preferential binding to the target enzyme with an estimated binding energy of −21.59 kcal mol⁻¹, which is in good agreement with the result of the in vitro α-glucosidase inhibition assay. The detailed analysis of the binding interaction is displayed in the two-dimensional (2D) binding mode of 5c in Figure 2. As can be seen, the coumarin moiety of 5c is stretched into a hydrophobic pocket of the target enzyme revealing hydrophobic interactions with Phe303, Phe178 and Tyr158. The carbonyl group of the coumarin scaffold interacts by hydrogen bonding with Gln353. It is noteworthy to mention that hydrogen bonding of the amino group in 5c with Glu277 further stabilizes embedding of the coumarin moiety in the hydrophobic pocket, compared to 5a, b that lack this interaction. The benzothiazole moiety of 5c is involved in π-π interactions with Phe314 and Lys156, as well as hydrogen bonding with Arg315 (Figure 2).

Figure 2

Interaction of compound 5c with the binding site of the target enzyme. Dashed lines represent hydrogen bonds. Hydrophobic interactions are shown by green solid lines.

Three-dimensional (3D) and 2D pharmacophoric maps for the structural features of compound 5c (the most active member of this study) were created by LigandScout software and are presented in Figure 3A and B, respectively. The investigated pharmacophoric features include hydrogen bond donors and acceptors as directed vectors, positive and negative ionizable regions as well as lipophilic areas that are represented by spheres. These pharmacophoric maps of 5c may help design more potent antitumor coumarin-benzothiazole hybrids.

Figure 3

The 3D and 2D pharmacophoric maps of compound 5c. (A) The 3D pharmacophoric map; the pharmacophore color coding is red for hydrogen acceptor, yellow for hydrophobic regions and green for hydrogen donors. (B) The 2D pharmacophoric map; HBA is hydrogen bond acceptor, H is hydrophobic center, HBD is hydrogen bond donor and AR is aryl.

In conclusion, coumarin-benzothiazole hybrids 5a–c were introduced in this investigation as a novel scaffold of potential antitumor agents. The substitution pattern of the coumarin moiety in the new hybrid molecules greatly affects their biological activity. Compound 5c is the most active member of this study according to NCI’s single- and five-dose assays. Intriguing antioxidant and α-glucosidase inhibitory activities of 5c are in good agreement with the antitumor screening results. The preliminary results reported in this study may help design new coumarin-benzothiazole hybrids as antitumor agents based on the nature and number of polar substituents on the coumarin nucleus.