Antioxidant Activity and Cytotoxicity of Selenium Incorporated Biologically Inspired N-Heteroaryl Compounds

Abstract

Selenium is one of the essential trace elements in humans and animals. It is incorporated in the redox-active selenocysteine (Sec), the 21st proteinogenic amino acid, which is found in a limited number of proteins/enzymes. The reactivity of the Sec residue in these proteins allows them to perform diverse functions such as reducing oxidative stress, maintaining redox homeostasis, and thyroid hormone metabolism. An important family of biomolecules is derived from pyridyl-based systems (e.g., 2-pyridinol, 2-nicotinamide). Several of these molecules are also responsible for a wide range of biological activities like redox reactions (e.g., NAD ⇌ NADH; NADP⁺ ⇌ NADPH). Due to their presence in natural systems, these scaffolds are widely used as pharmaceutical motifs in drug design and development. This is supported by the FDA database which reveals that over 60% of small molecule drugs contain N-heterocycles. Thus, a variety of selenium-incorporated pyridyl derivatives with reference to their antioxidant activity have been synthesized and studied. Antioxidant activity and cytotoxicity of these compounds have also been investigated in cellular models. The nature of the substituent at the C-3 position of the pyridine ring significantly influences the structure of the molecule as well as its antioxidant activity and cytotoxic properties. In this article, the work published by us and other groups on pyridyl selenium compounds is reviewed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12011-025-04694-y.

Introduction

Approximately 0.1% of our body mass is made up of essential trace elements—usually known as micronutrients [1]. These elements, except iodine and molybdenum, are those with atomic numbers ranging between 23 and 34 of the periodic table. These elements, in general, exhibit one-electron redox processes. In some cases, two-electron redox process is also observed. These elements are involved in numerous structural, physiological, catalytic, and regulatory functions. For the sustenance of various biological activities, the functional forms of these elements must be maintained within a narrow limit. Their excess as well as deficiency results in numerous ill effects on human health. The requirement of each trace element differs significantly and varies in different age groups (infants, children, adults, during pregnancy), gender, and region. Trace elements are usually expressed in parts per million (ppm) or µg/g while some are expressed in parts per billion (ppb) or ng/g (Table 1). The amount of trace elements may appear insignificant, but they have an incredible effect on the sustenance of life.

Table 1.

Essential trace elements (micronutrients)

Element	Quantity in average healthyhuman adult (70 kg body weight)	Oxidation states
Vanadium	~10 mg	+3, +4, +5
Chromium	~6 mg	+2 to +6
Manganese	12–20 mg	+2, +3
Iron	3–5 g	+2, +3, +4
Cobalt	~1 mg	+1, +2, +3
Nickel	~6–8 mg	+2, +3, +4
Copper	80–120 mg	+1, +2
Zinc	2–3 g	+2 (redox inactive)
Selenium	13–20 mg	0, +2, +4
Molybdenum	~5 mg	+2 to +6
Iodine	10–25 mg	−1
Fluorine	~3 mg	−1

The element with atomic number 34 is selenium. A daily intake of 50–80 µg is essential for human development and normal growth. Its deficiency weakens the immune system, leading to health problems manifested in several associated diseases, while excess intake (> 200 µg/day) could cause health hazards.

Unlike sulfur, selenium in humans is found in much smaller quantities, primarily as the redox-active amino acid selenocysteine (Sec) (1) in about 25 selenoproteins, which are involved in various redox processes. Within the selenoprotein family, glutathione peroxidase (GPx), consisting of five Sec-containing enzymes, plays a crucial role in antioxidant defense. It protects the organism against oxidative damage by neutralizing intracellular hydrogen peroxide and other hydroperoxides generated during oxidative stress.

Synthetic chemists have made significant progress in the past few years to design and synthesize new selenium compounds and have examined them for a variety of biological activities. Organic (as organoselenium compounds) and inorganic (as selenite) selenium compounds showed different types of biological activity, as they are metabolized differently in the body. Studies reveal that the efficacy and toxicity of selenium compounds are modulated by a variety of factors, including concentration, chemical form, hetero atom substitution, and the selenium redox state [2].

Pyridyl based systems (e.g., 2-pyridinol, 2-nicotinamide) (Scheme 1) constitute an important family of biomolecules. Several of these molecules are also responsible for a wide range of biological activities like redox reactions (e.g., NAD ⇌ NADH; NADP⁺ ⇌ NADPH). Owing to the structural similarity with the endogenous systems, pyridyl systems can interact easily with bio-components in the cell. Therefore, medicinal chemists have extensively studied similar scaffolds such as pyridine, quinolone, pyridazine, pyrimidine, and pyrazine and found potent pharmaceutical activity [3]. The importance of these scaffolds in small molecule drug design and discovery is evident from the FDA database, which reveals that over 60% of approved drugs utilize these structural scaffolds. The first FDA-approved antiviral drug, idoxuridine, used in 1963 for the treatment of herpes, was developed using these motifs [4].

Scheme 1.

Pyridyl based systems (e.g., 2-pyridinol, 2-nicotinamide)

Inspired by the biological activity of GPx, selenium-incorporated endogenous N-heteroaryl systems have been conceived as potential antioxidants as well as promising candidates for other biological activities. Accordingly, we have designed and developed several small molecules incorporating selenium in N-heteroaryl scaffolds (Scheme 2). In this article, we review the work published by us and other researchers on pyridyl-based selenium compounds.

Scheme 2.

Small molecules incorporating selenium in N-heteroaryl scaffolds

Synthesis of N-Heteroaryl Diselenides

The most commonly employed strategy to prepare N-heteroaryl diselenides involves the reaction between N-heterocyclic halo compounds and diselenide dianion (Se₂²⁻) which is usually generated in situ by reduction of selenium with a variety of reducing agents (Scheme 3). Another frequently employed method is the insertion of selenium across the M-C bond in N-heteroaryl lithium and Grignard reagents (Scheme 3) [5]. Several selenium compounds derived from the nicotinic acid motif have been synthesized using Na₂Se₂ in appropriate solvents (Scheme 4) [6].

Scheme 3.

The reaction between N-heterocyclic halo compounds and diselenide dianion

Scheme 4.

Selenium compounds derived from the nicotinic acid motif synthesized using Na₂Se₂

Structures of Di N-Heteroaryl Diselenides

Di-N-heteroaryl diselenides are pale-yellow to light orange in color. All the compounds have been invariably characterized by NMR spectroscopy. The ⁷⁷Se NMR chemical shifts appear in a narrow region δ 300–525 ppm with reference to Me₂Se. Attempts to correlate the observed ⁷⁷Se NMR chemical shifts of N-heteroaryl diselenides either with the nature of substituents in the heterocyclic ring or with the number of substituents met with little success (Scheme 5) [6–12], as the ⁷⁷Se NMR data on such compounds are limited. The ⁷⁷Se NMR chemical shift may show a solvent effect (Scheme 5).

Scheme 5.

⁷⁷Se NMR chemical shifts (reference Me₂Se) of some N-heteroaryl diselenides [6–12]

Molecular structures of several N-heteroaryl diselenides have been determined by single crystal X-ray diffraction analyses (Table S1, supplementary material) [6–40]. The following general features can readily be identified.

• The Se–C distances in di N-heteroaryl diselenides lie at ∼1.92 Ǻ while the Se–Se distances vary in the range 2.28–2.40 Ǻ and are influenced by the nature of electronic substitutes in the N-heteroaryl ring. The electron-withdrawing groups at the C-3 position lead to slight elongation of the Se-Se bond, while electron-releasing group results in shortening of the Se-Se bond (e.g., 3-Mepy₂Se₂, py₂Se₂, and 3-CF₃py₂Se₂). The bent structures have shorter Se-Se distances than the planar structures.

• The lone pair of electrons of nitrogen of the N-heteroaryl ring can adopt different orientations with respect to the Se-Se bond, resulting in cis-cis, cis–trans, and trans–trans conformations (Scheme 6). In general, molecules with planar geometry prefer cis-cis conformation, while non-planar compounds exist in trans–trans or cis–trans geometry.

Scheme 6.

Different conformations of di N-heteroaryl diselenides

• The di N-heteroaryl diselenides adopt either a bent or planar structure. The C-Se-Se-C torsion (dihedral) angles (bent (74–93°) or planar (~ 180°)) are highly influenced by the electronic properties of the substituents and their position in the pyridyl ring (Scheme 7).

• Unsubstituted pyridyl ring and ring substituted by electron releasing group (e.g., 3-Me) at C-3 position results in an angular structure, whereas ring substituted by electron withdrawing group (e.g., Br, CF₃, COOH, COOBu^t, CONH₂, CONHPh, CONMe₂) at C-3 position leads to planar molecules. However, the nature of secondary interactions, such as π-π stacking and hydrogen bonding, can defy such a generalization. For instance, bis(3-hydroxy-2-pyridyl) diselenide, despite having hydroxyl group at C-3 position, adopts a bent structure with the C-Se-Se-C angles of 93.04 and 94.21°, although the compound di{6-bromo-4,5-(carboethoxy)−3-hydroxyl-2-pyridyl} diselenide acquires the planar geometry. The compound [2-NC₅H₃(3-CONH₂)Se]₂, despite substituting by electron withdrawing group at C-3 position, can be isolated in both planar as well as bent geometries, each showing hydrogen bonding (Fig. 1) [11]. The π-π stacking also results in planarity. Bis(3-bromo-2-pyridyl) diselenide forms a dimer. This dimer formation is driven by π-π stacking interactions with a stacking distance of 3.569 Å between the two pyridyl ring centroids [18]. Similar stacking can be noted in bis(3,5-dimethyl-2-pyridyl) diselenide [28].

• Substituents at other positions in the pyridyl ring invariably produce angular structures, regardless of their electron-donating or electron-withdrawing properties.

Scheme 7.

Dihedral angle in diorgano diselenides

Fig. 1.

Molecular structure of [2-NC₅H₃(3-CONH₂)Se]₂ (redrawn from Phadnis PP, et al. (2019) Indian J Chem Section A, 58A: 18–28) [11]

Polymorphism in Di N-Heteroaryl Diselenides

Polymorphism plays a crucial role in drug development. Polymorphism can dramatically influence the efficacy of pharmaceutical drugs. The existence of multiple polymorphs of active pharmaceutical ingredients (APIs) is quite common and can have significant implications for drug development and formulation. A substantial proportion, exceeding 50%, of active pharmaceutical ingredients (APIs) exhibit polymorphism, which accounts for variations in their properties. Some dipyridyl diselenides exist in different polymorphs.

The prototype of di-N-heteroaryl diselenide, viz., 2,2-dipyridyl diselenide, can be isolated in monoclinic and orthorhombic forms on recrystallization from a diethyl ether–petroleum ether mixture and petroleum ether, respectively [13–15]. The main structural variation lies in the position of the pyridyl rings around the Se-Se bond. In the monoclinic form, the Se-Se bond is coplanar with both the pyridyl rings, whereas in the orthorhombic form, it is coplanar with only one.

Three polymorphs of 2,2′-diseleno bis(3-nicotinamide), viz., monoclinic, orthorhombic, and triclinic, could be isolated upon recrystallization from DMSO and DMF [11, 22]. The C-Se-Se-C torsion angles are 180° and ~ 93° for monoclinic and triclinic forms, respectively. Each polymorph exhibits secondary hydrogen bonding interactions with the lattice solvent molecules (Fig. 1). Hydrogen bonding in orthorhombic and triclinic forms takes place between amide hydrogen and carbonyl oxygen of DMF, the only difference being in the way the amide groups are H-bonded. In the orthorhombic form, amide groups exhibit identical hydrogen bonding with two DMF molecules. Conversely, in the triclinic form, amide groups are engaged in hydrogen bonding interactions differently. In both cases, a sinusoidal pattern is formed [11, 22]. In the monoclinic form, amide hydrogen atoms from individual diselenide molecules are hydrogen bonded to DMSO molecules, leading to the formation of one-dimensional strands (Fig. 1) [11].

2,2′-Diselenobis(3-pyridinol) is another diselenide which shows polymorphism. Upon recrystallization from methanol–water and methanol-chloroform mixtures, the compound crystallized in the monoclinic space group with varying cell parameters and distinct crystal morphologies [10]. Both forms are linked by robust, but distinctly directed hydrogen bonds. Crystals obtained from methanol–water showed that the molecules are interlocked through intermolecular O–H⋯N hydrogen bonds producing 3-D sheets. In contrast, molecules in the crystals obtained from methanol-chloroform mixtures form helical chains of two distinct orientations. These chains are formed by utilizing both intra- and inter-molecular O–H⋯N hydrogen bonds.

Several di-N-heteroaryl diselenides have been examined for different types of biological activities. The pyridyl-based diselenides exhibit multifunctional biological activities such as GPx mimic, free radical scavenger, and cytotoxicity. These are briefly described below.

Antioxidant Activity of Di N-Heteroaryl Diselenides

In vitro, GPx-mimicking activity of pyridyl and pyrimidyl organoselenium compounds was assessed by ¹H NMR and HPLC methods [7]. Structure–activity relationships reveal that compounds with stronger electron-withdrawing groups at the C-3 position exhibit enhanced GPx-like antioxidant activity.

The NMR technique was used to measure the time (t₅₀) required for 50% of the DTT^red to undergo oxidation to DTT^ox. This t₅₀ value served as an indicator of the 50% reduction of H₂O₂ by the thiol cofactor (DTT^red). Using the HPLC method, the relative decay of GSH over time (in minutes) was plotted, and the t₅₀ value was determined as the time required for a 50% decay of GSH. The GPx activity in both the assays of these compounds followed an order as py₂Se₂ > pym₂Se₂ > Ph₂Se₂ (Table 2). The significance of the Se-Se bond for GPx activity is evident from the fact that the diselenides show much higher activity than that of the corresponding mono selenides (selenoethers) [7].

Table 2.

GPx activity of some diaryl diselenides

Compound	t50 in min (by NMR)	t50 in min (by HPLC)
py2Se2	21	52
pym2Se2	>350	>300
Ph2Se2	>350	>300

A similar study was carried out by Nogueira and coworkers around the same time [41]. This group reported that py₂Se₂ exhibits better in vitro antioxidant activity than other diaryl diselenides in the lipid peroxidation assay by measuring TBARS (thiobarbituric acid reactive substances). The py₂Se₂ is more potent than other aryl compounds in reducing lipid peroxidation in rat liver homogenate models. The IC₅₀ value for lipid peroxidation in rat liver homogenates is shown below:

$$\begin{array}{cc}& {\mathrm{py}}_2{\mathrm{Se}}_2<{\left(4,-,{\mathrm{FC}}_6,{\mathrm{H}}_4,\mathrm{Se}\right)}_2<{\left(3,-,{\mathrm{CF}}_3,{\mathrm{C}}_6,{\mathrm{H}}_4,\mathrm{Se}\right)}_2<{\left(4,-,{\mathrm{MeOC}}_6,{\mathrm{H}}_4,\mathrm{Se}\right)}_2<{\left(4,-,{\mathrm{ClC}}_6,{\mathrm{H}}_4,\mathrm{Se}\right)}_2\hfill \\ \hfill {\mathrm{IC}}_{50}\left(\mu ,\mathrm{M}\right):& 16.982.886.3142.2147.7\hfill \end{array}$$

Collins et al. reported that 2,2′-dipyridyl diselenide exhibits antioxidant activity in a skin cell model of UVA (320–400 nm) induced stress. In contrast, bis(quinolin-8-yl) diselenide displayed slight antioxidant activity at a lower concentration (1 µM) but exhibited pro-oxidant effects at higher concentrations (5 µM) [42].

Singh and coworkers [8, 9] synthesized pyridoxine-based diselenides (2 and 3) and investigated their GPx-like activities by the coupled reductase assay. These compounds showed better GPx activity than ebselen (61 µm/min). Compounds substituted at the 6-position by bromide are more active (217 µm/min for 2 (X = Br) and 143 µm/min for 3 (X = Br) than the unsubstituted derivatives (72 µm/min for 2 (X = H) and 100 µm/min for 3 (X = H)) [8, 9].

The GPx mimicking activity of several nicotinamide derivatives (Scheme 4) has been evaluated using ¹H NMR spectroscopy and HPLC methods [6, 43]. The compound [2-NC₅H₃(3-CONH₂)Se]₂ is the most active among all the derivatives. At a concentration of 0.015 mmol (10 mol %), the reaction was instantaneous, resulting in complete conversion of DTT^red to DTT^ox. Therefore, a lower concentration (0.003 mmol; 2 mol %) was used, where the t₅₀ was ~ 4 min. Interestingly, the compound [2-NC₅H₃(3-CONH₂)Se]₂ exhibits six times higher activity than the well-studied selenium derivative, ebselen (Fig. 2) [43].

Fig. 2.

Formation of GSSG as a function of reaction time during oxidation of GSH in the presence of H₂O₂, catalyzed by organoselenium compound (10 µM). (Reproduced with permission from Jain and co-workers, Indian J. Chem., 53A (2014) 781) [43]

Enzyme kinetics were employed to investigate the GPx-like catalytic activity of [2-NC₅H₃(3-CONH₂)Se]₂, and the resulting intermediates were characterized by ⁷⁷Se NMR, HPLC, mass spectrometry, and absorption spectroscopy. The stopped-flow kinetic studies indicated that the reaction of GSH with the diselenide to form selone occurs with a rate constant of 4.8 × 10³ M⁻¹ s⁻¹ [44]. The activity was initiated by the reduction of diselenide by GSH to RSeSG (R = NC₅H₃CONH₂) and a stable selone. The latter is believed to be responsible for efficient GPx-like activity. The quantum chemical modelling demonstrates that selone is the reduced form rather than selenol. The selenol is less stable than selone by more than 10 kcal mol⁻¹ in water. Selone reacts with H₂O₂ much slower, with a rate constant of 18 M⁻¹ s⁻¹. In the ⁷⁷Se NMR spectra, characteristic resonances attributable to selone, selenenyl sulfide, selenenic acid, and seleninic acid were identified. From the ⁷⁷Se NMR data, various steps involved in the catalytic cycle can be identified as follows. The catalytic cycle is initiated by the reaction of diseleno dinicotinamide with GSH to form RSeSG (IV) and selenol (I) which undergoes keto-enol tautomerization to give selone (II). The selone can react with H₂O₂ to form RSeO₂H (V) through the intermediary of RSeOH (III). The RSeO₂H (V) formed can react with GSH to form RSeSG (IV) which can either react with GSH or H₂O₂ to form RSeH (I) or RSeOH (III), respectively, thus completing the catalytic cycle. The path followed by diseleno dinicotinamide during its GPx mimicking activity can be summarized as shown in Scheme 8 [44].

Scheme 8.

The path followed by diseleno dinicotinamide during its GPx mimicking activity

Free Radical Scavenging Activity of Di N-Heteroaryl Diselenides

Reactive oxygen species (ROS) are oxygen-containing molecules that can exist as both free radicals and non-radical molecules. To assess free radical scavenging activity, some of these compounds were evaluated using the DPPH assay and deoxyribose assay. The radical scavenging (H^• transfer) activity of organoselenium compounds was evaluated by scavenging 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical and was measured spectrophotometrically, whereas the OH^• radical scavenging capacity was assessed using the deoxyribose assay [6, 43].

The antioxidant activity of nicotinamide derivatives was assessed by measuring their ability to scavenge DPPH free radicals at 517 nm, and this scavenging capacity was correlated with the concentration of the selenium compounds. The IC₅₀ values, representing the concentration of the selenium compound needed to scavenge 50% of DPPH free radicals, were determined. The results show that [2-NC₅H₃(3-CONH₂)Se]₂ significantly outperformed other compounds of Scheme 2 in free radical scavenging ability.

Lipid peroxidation and DNA damage were assessed via gel electrophoresis, using AAPH (2,2′-azobis (2-methylpropionamidine) dihydrochloride) and γ-radiation as damage-inducing sources. 2,2′-Diseleno bis(3-nicotinamide) at micromolar concentrations provides protection against lipid and DNA damage caused by AAPH and γ-radiation-generated radicals (Fig. 3) [43].

Fig. 3.

Inhibition of TBARS in lipid against AAPH (20 mM) and γ–radiation (50 Gy) induced lipid peroxidation in the presence of 2-selenonicotinamide (reproduced with permission from Jain and co-workers, Indian J. Chem., 53A (2014) 781–786) [43]

2-Deoxyribose degradation in the presence of a Fenton reagent provides a measure of hydroxyl radical (^•OH) scavenging ability of antioxidants. The IC₅₀ values, which indicate the concentration of the selenium compound needed to inhibit degradation by 50%, were calculated and compared to those of mannitol (positive control), a standard reference compound. The scavenging potential was the highest for D-mannitol, followed by [2-NC₅H₃(3-CONH₂)Se]₂ with IC₅₀ values of 49.80 and 76.84 µM, respectively.

Bis(3-amino-2-pyridyl)diselenide shows antioxidant capabilities by scavenging ABTS (2,2′-azino bis-3-ethylbenzothiazoline-6 sulfonic acid) radicals and also exhibits significant inhibition of acetylcholinesterase (AChE) activity [19].

Radioprotection Activity of Di N-Heteroaryl Diselenides

A few selenopyridyl derivatives have been examined for their potential radioprotective activity. Ionizing radiation is used in cancer radiation therapy. While it is useful for killing cancer cells, exposure to normal cells is unwanted. Depending on the amount of absorbed radiation dose, it can cause many irreversible changes in cells that can lead to mutations or even cell death [45]. To protect normal cells from unwanted radiation damage, radioprotectors are used. Among various radioprotectors, amifostine, a thiol-containing compound, is commonly used. Being a chalcogen, belonging to the same group, several selenium compounds show efficient radioprotection; sodium selenite formulation is being marketed as selenase®, a potential drug. Diselenodipropionic acid, a compound developed in our group, is being explored in the clinic as a radioprotector to reduce lung fibrosis, a side effect of thoracic radiotherapy [46].

Being promising GPx mimics, nicotinamide derivatives have been evaluated for their radioprotective effects. Their antioxidant ability could provide protection from ionizing radiation-induced cellular damage. Among all the compounds, diseleno dinocotinamide ([2-NC₅H₃(3-CONH₂)Se]₂) showed promising radioprotective activity in Chinese Hamster Ovary (CHO) cells, which are the most commonly used models for radiation experiments. Diseleno dinocotinamide showed radioprotection at low concentrations (up to 25 µM); however, at higher concentrations, it showed a pro-oxidant effect. Up to a 25 µM concentration, the compound did not show any toxicity. At this concentration, the dose modification factor (DMF) was 1.26 at a radiation exposure of 1–12 Gy. The advantage of this compound over other selenium derivatives is the stability of both diselenide as well as its reduced species, mono selenide, a selone moiety. The compound acted as a substrate for thioredoxin reductase (TrxR) during which the diselenide is converted into selone. TrxR is an important enzyme maintaining redox balance in cells. Selone has also been reported to exhibit effective GPx activity. The mechanism for this radioprotection has been attributed to the GPx enzyme-like activity, increased levels of GSH in the cells, acting as a substrate to TrxR, and also the ability of selone to remove ROS. In CHO cells, the compound provides protection even when added after radiation exposure, indicating its ability to promote DNA repair. Interestingly, 2,2′-diseleno bis(3-nicotinamide) also protects lymphocytes from radiation-induced cell death and apoptosis [47].

Anti-cancer Activity of Di N-Heteroaryl Diselenides

Di N-heteroaryl diselenides have been examined by several groups for anti-cancer effects in many cellular models. Rizvi et al. evaluated the cytotoxicity of several aromatic diselenides including dipyridyl diselenide in a number of human cancer cell lines such as HL-60 (leukemia), OVCAR-5 (epithelial), 786-O (Renal), HT-29 (colorectal), and PC-3 (prostate) by MTT assay. The IC₅₀ values (concentration of drug necessary to inhibit 50% growth of cells) for dipyridyl diselenide varied in the range 26–30 μM in different cell lines [48]. Dhau et al. reported the anti-carcinogenic activity of methyl substituted dipyridyl selenides and dipyridyl diselenides [49]. The preliminary studies were performed in acute lymphoid leukemia cells by MTT assay using curcumin as a reference standard. The IC₅₀ values (concentration of drug necessary to inhibit 50% growth of cells) indicated that the anti-cancer activity of pyridyl selenides decreased with increasing number of methyl substitutions in the pyridyl ring. The diselenides are 2–5 times more active than the corresponding mono selenides [49].

Gandhi et al. evaluated the anti-cancer effects of 2,2′-dipyridyl diselenide in human lung carcinoma (A549) cells and compared them with normal lung fibroblast (WI38) cells [50]. The IC₅₀ values are slightly higher in WI38 cells as compared to A549 cells. A new concept for cytotoxicity for these diselenides has been attributed to the induction of reductive stress. As observed in radioprotection studies, the compound is reduced by TrxR to a selone that acts initially as a ROS scavenger. Later, it causes significant DNA damage, G1 phase arrest, and apoptosis in A549 cells. The reductive stress is inferred through the inhibition of redox enzymes and its effect on GSH biosynthesis [50].

Anti-cancer activity of hydroxy substituted dipyridyl diselenide, viz., 2,2′-diselenobis (3-pyridinol) has also been investigated [10, 51]. Cytotoxicity of [2-C₅H₃N(3-OH)Se]₂, both in bulk and polymorphic forms, was assessed in A549 human lung carcinoma cells. A similar toxicity pattern was observed across all the samples. The effective concentration needed to reduce the cellular viability by 50% or the IC₅₀ values of all the samples has been found to be ~ 10 µM. Both bulk and its polymorphs have been shown to exhibit similar cytotoxicity (Fig. 4) [10]. Gandhi et al. further showed that [2-C₅H₃N(3-OH)Se]₂ also gets reduced to form a stable mono selenide, selone. As observed with the unsubstituted compound, this hydroxy derivative caused cytotoxicity through reductive stress. The cells after treatment with the compound caused DNA damage, G1 phase arrest, and apoptosis in cells. The compound also increased the GSH levels and inhibited redox enzymes. The IC₅₀ value for this compound is also comparable to that observed for the unsubstituted derivatives [51].

Fig. 4.

Cytotoxic effect of [2-C₅H₃N(3-OH)Se]₂ and its polymorphs in A549 human lung carcinoma cells after 48 h of incubation by MTT assay. Data presented as percentage toxicity with respect to control cells (DMSO, 0.25%). The results are presented as mean ± SEM (n = 3).

(Reproduced with permission from Elsevier (Phadnis PP et al. (2017) J Organomet Chem, 852: 1–7) [10]

Kim et al. examined the anti-proliferative ability of diaryl diselenides and substituted dipyridazinyl diselenides (Scheme 9) in human breast cancer (MCF-7) cells using the CCK-8 assay [52]. 1,2-Bis(3-chloropyridazinyl) diselenide showed the highest potency among all the compounds. It inhibited the growth of MCF-7 cells (IC₅₀ = 10.34 µM) in a dose-dependent manner [52].

Scheme 9.

The anti-proliferative ability of diaryl diselenides and substituted dipyridazinyl diselenides

Conclusions and Future Directions

A variety of N-heteroaryl diselenides have been developed with reference to their biological activities. They show diverse structural features, which are strongly dependent on the substituents in the N-heteroaryl ring. The C-Se-Se-C torsion angle (bent (74–93°) or planar (~ 180°)) is highly influenced by the electronic properties of the substituents and their position in the pyridyl ring. Electron-withdrawing substituents at the C-3 position lead to a planar structure. Polymorphism is quite common in these compounds.

These systems show multifunctional biological activities, like excellent GPx mimic, free radical scavenger, and radio protector. These compounds are emerging as new anti-cancer agents. So far, only a few compounds (unsubstituted and hydroxy/methyl/amide substituted derivatives) have been examined. Among them, diseleno dinicotinamide is found to be non-toxic to normal cells but slightly toxic to cancer cells. More interesting effects can be observed if the substituents can interact with the selenium moiety through non-bonding interactions. This can influence the reduction of the diselenide moiety and therefore can modulate the cellular redox environment in a more subtle form and consequently influence the cytotoxicity.

Based on the promising results reported under in vitro and cellular systems, it is necessary to study them in detail in animal models. Especially interesting are diseleno dinicotinamide and pyridinol derivatives. The studies so far propose activity in micromolar concentrations. However, this can vary significantly with suitable substitutions in the aromatic ring. Substitutions having groups containing heterocyclic atoms like N and O can significantly alter the selenium redox status and also its bio-activity. Another interesting area is the bio-activity of purine and pyrimidine containing diselenides, which are not fully explored for biological studies. Synthetic chemists can consider designing and synthesizing more such compounds that are stable and easily accessible for biological studies. With their similarity to DNA bases, purine and pyrimidine compounds can be promising agents for anti-cancer studies. Further, future studies should also be focused on identifying the important molecular biological pathways sensitized by the administration of such compounds, as this would provide an understanding of the sites of interaction and metabolism.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

The work reported in the MS is our joint project work executed over several years. VKJ wrote the MS, KIP worked on biological section. Both the authors have read and reviewed the MS several times.

Funding

Open access funding provided by Department of Atomic Energy.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.