Cytoprotective effects of imidazole-based [S₁] and [S₂]-donor ligands against mercury toxicity: a bioinorganic approach

Abstract

Here we report the coordination behaviour of an imidazole-based [S₁]-donor ligand, 1,3-dimethyl-imidazole-2(3H)-thione (L1), and [S₂]-donor ligand, 3,3′-methylenebis(1-methyl-imidazole-2(3H)-thione) (L2) or 4,4′-(3,3′-methylenebis-(2-thioxo-2,3-dihydro-imidazole-3,1-diyl))dibutanoic acid (L3), with HgX₂ (X = Cl, Br or I) in solution and the solid state. NMR, UV-Vis spectroscopic, and single crystal X-ray studies demonstrated that L1 or L2 coordinated rapidly and reversibly to the mercury center of HgX₂ through the thione moiety. Treatment of L2 with HgCl₂ or HgBr₂ afforded 16-membered metallacycle k¹-(L2)₂Hg₂Cl₄ or k¹-(L2)₂Hg₂Br₄ where two Cl or Br atoms are located inside the ring. In contrast, treatment of L2 with HgI₂ afforded a chain-like structure of k¹-[L2Hgl₂]_n, possibly due to the large size of the iodine atom. Interestingly, [S₁] and [S₂]-donor ligands (L1, L2, and L3) showed an excellent efficacy to protect liver cells against HgCl₂ induced toxicity and the strength of their efficacy is in the order of L3 > L2 > L1. 30% decrease of ROS production was observed when liver cells were co-treated with HgCl₂ and L1 in comparison to those cells treated with HgCl₂ only. In contrast, 45% and 60% decrease of ROS production was observed in the case of cells co-treated with HgCl₂ and thiones L2 and L3, respectively, indicating that [S₂]-donor ligands L2 and L3 have better cytoprotective effects against oxidative stress induced by HgCl₂ than [S₁]-donor ligand L1. Water-soluble ligand L3 with N-(CH₂)₃CO₂H substituents showed a better cytoprotective effect against HgCl₂ toxicity than L2 in liver cells.

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Significance to metallomics

Thiols, dithiols and other sulfhydryl-containing ligand interactions play a crucial role in the detoxification of mercury in cellular systems. Clinically used bidentate chelating agents DMPS and DMSA and the endogenous thiol glutathione are known to play significant roles in detoxification of Hg(II) and methylmercury. In this work, we have demonstrated the interaction of thione based [S₁] and [S₂]-donor ligands, inspired by bidentate chelating agents, with Hg(II) in solution and the solid state and also studied their efficacy to protect hepatocytes against Hg(II) toxicity. Our results showed that [S₂]-donor ligands are, indeed, effective in reducing Hg(II) toxicity in liver cells.

1. Introduction

Heavy metals, especially mercury (Hg), cadmium (Cd), arsenic (As) and lead (Pb) are extremely toxic to the environment. Mercury and mercury related compounds including inorganic mercury like HgX₂ (X = Cl, Br, and I) and organomercurial compound methylmercury (MeHg⁺), in particular, are highly toxic to humans and animals.¹ Although all forms of mercury are toxic, the toxicity profile of mercury compounds depends mostly on the chemical or molecular form, the level of exposure, the duration of exposure, and the route of exposure.² Mercury ions (Hg²⁺ or MeHg⁺) have a high affinity towards thiol or selenol groups of proteins including various essential antioxidant enzymes, thioredoxine reductase (TrxR), glutathione peroxidase (GPx), selenoprotein-P and glutathione reductase (GR).^3–7 Most of the mercury, inorganic or organic form, within the various tissues and fluid compartments of mammals is bound to low molecular weight endogenous thiol molecules like L-cysteine and glutathione (GSH), which are present in large concentrations and facilitate the transport of mercury in various organs.^8,9 The methylmercury–cysteine complex (MeHgCys) can easily cross the cellular membranes including placental and blood–brain barriers with the help of L-type large neutral amino acid transporter, LAT1.¹⁰ On the other hand, GSH is known to play a crucial role in removing inorganic mercury from hepatocytes into bile by forming a mercury–glutathione complex in the hepatocytes followed by the biliary secretion of this complex. Detailed investigation of interrelation between the biliary transport of GSH and of inorganic mercury by Clarkson et al. revealed that the increase in the rate of GSH secretion into bile after GSH administration is accompanied by an increase in the rate of mercury secretion into bile.¹¹ As mercury toxicity is mostly associated with the inhibition of antioxidant enzymes and the reduced GSH concentration in the cellular system, it leads to the production of reactive oxygen species (ROS), causing oxidative damage of DNA, proteins and lipids, and ultimate cell death.^12,13

Several bacteria are resistant to both inorganic and organomercury compounds due to the presence of the mercury resistance mer operon that codes for many Mer proteins including MerP, MerG, MerT, MerB, and MerA. The gene merB encodes an organomercurial lyase (MerB) that catalyses the protonolytic cleavage of carbon–mercury bonds. The cytosolic mercuric reductase MerA reduces inorganic mercury, Hg²⁺, to less toxic elemental Hg(0).^14,15

Several synthetic molecules with vicinal thiols such as British antilewiste (BAL), sodium 2,3-dimercaptopropanesulfate (DMPS), and meso-2,3-dimercaptosuccinic acid (DMSA) have shown promising effects in removing mercury and other heavy metals like lead and arsenic and have been clinically used for mercury chelation (Fig. 1).^16,17 This is probably due to the fact that Hg²⁺ might have strong preference for bis-thiolate coordination in addition to the chelate effect provided by these bidentate ligands, which is supported by the presence of the crystal structures of two cysteine coordinated mercury bound MerB, MerP, MerT, and the N-terminal domain of MerA.^16,18 Nevertheless, the presence of more Hg²⁺–thiolate bonds (tri- or tetra-coordinated Hg–S bond) is also known in the literature.^19,20 Notably, BAL has been discontinued due to the presence of side effects and low therapeutic index.^21a Although DMPS and DMSA are not effective in chelating mercury in the brain, they have shown promising results in enhancing urinary excretion of mercury and thus, currently they are the best choice for treatments for mercury toxicity.²¹

Fig. 1 Chemical structures of known Hg²⁺ chelating agents BAL, DMPS, and DMSA, and imidazole-based [S₁] and [S₂]-donor ligands L1, L2, and L3.

Recently, we have reported that imidazole-based [S₂]-donor ligand 3,3′-methylenebis(1-methyl-1H-imidazole-2(3H)-thione) (L2) is more effective in cleaving the, otherwise inert, Hg–C bond of MeHg⁺ at high temperature but, less effective in cleaving the Hg–C bond of different MeHg⁺ species at room temperature (21 °C). We observed 50% Hg–C bond cleavage of MeHgI whereas only 15% Hg–C bond cleavage in the case of [MeHg]BF₄, by L2 at 21 °C.²² Although imidazole based thiones with different sulfur donor groups, like [S₂] and [S₃]-donor, have recently been broadly studied to investigate their effect on Hg–C bond cleavage of organomercurials, RHg⁺, the coordination behaviour of [S₂]-donor ligand L2 with inorganic mercury compounds HgX₂ (X = Cl, Br, or I) in solution and the solid state has not been studied so far.²³ Since the liver is the main mercury detoxification organ^11d,24 we have employed HepG2 cells in our study to investigate the cytoprotective effects of imidazole-based thiones, 1,3-dimethyl-1H-imidazole-2(3H)-thione (L1), L2 and newly designed water soluble 4,4′-(3,3′-methylenebis(2-thioxo-2,3-dihydro-1H-imidazole-3,1-diyl))-dibutanoic acid (L3), against HgCl₂-induced toxicity in cells. Herein, we report the coordination behaviour of [S₁] and [S₂]-donor ligands L1, L2, or L3 with mercury(II) halides in solution and the solid state. In addition, we have investigated the efficacy of these [S₁] and [S₂]-donor ligands to protect liver cells against HgCl₂-induced toxicity.

2. Results and discussion

2.1 Nature of coordination of [S₁] and [S₂]-donor ligands

NMR experiment. In order to understand the coordination behaviour of N,N-disubstituted imidazole-based [S₁] and [S₂] donor-ligands like L1 and L2 or L3 toward mercury(II) salts both in solution and the solid state we have employed HgX₂ (X = Cl, Br, I) in our study and performed a series of experiments such as NMR, UV-Vis and single crystal X-ray studies as mentioned below. Here, it is pertinent to note that although the crystal structures of analogues of 1-methylimidazoline-2(3H)-thione, and 3,4,5,6-tetrahydropyrimidine-2-thione with mercury(II) halides have been reported in the literature,²⁵ the coordination properties of [S₁] and [S₂]-donor ligands L1, L2, or L3 in solution and the solid state with HgX₂ (X = Br or I), except HgCl₂ for L1,^25e and the effects of anion (X⁻; X = Cl, Br or l) on the coordination properties of these ligands toward HgX₂ have not been studied. ¹H NMR titration experiments between L1 or L2 and HgI₂ at room temperature (21 °C) showed a significant downfield shift of proton resonance of both –NCH₃ and olefinic protons of L1 or L2, indicating an interaction between the thione group of ligands to the mercury centre of HgI₂, Fig. 2 and Fig. S1–S6 (ESI†). The resulting solution of L1 and HgI₂, in a 1 : 1 molar ratio, showed resonances for protons of –NCH₃ and olefin at 3.649 and 7.452 ppm, respectively, which are 0.20 ppm and 0.35 ppm downfield chemical shifts in comparison to the resonances of those protons of free ligand L1 (3.447 ppm for –NCH₃ and 7.103 ppm for olefinic proton), Fig. 2a. Interestingly, the gradual addition of excess L1 (1–11 equivalents) into the solution of HgI₂ always resulted in a single set of resonances, with gradual shift of the resonances towards free ligand L1, suggesting that the interaction between L1 and Hg²⁺ is reversible in nature and rapid, faster than the NMR time scale at room temperature.^23,26 The variation of proton resonances of –NCH₃ proton and olefinic protons upon gradual addition of L1 (1–11 equivalents) into the solution of HgX₂ (X = I, Br, or Cl) is summarized in Fig. 2b and Fig. S7 (ESI†). Interestingly, we found that the difference of chemical shift in the presence of 1 equivalent of HgX₂ with respect to the free ligand (i.e. Δδ) increased with increasing electronegativity of the X atom, in the order of HgI₂ < HgBr₂ < HgCl₂, as shown in Fig. 3 for Δδ of –NCH₃ proton.²⁷ Conversely, unlike proton resonance, in the case of ¹³C NMR we observed significantly large upfield shift (10.1 ppm) for C2 resonance of L1 upon treatment with HgI₂ as illustrated in Fig. 2c and Fig. S8 (ESI†), indicating the decrease in double bond character of C=S of L1 upon coordination to the mercury center.^28,29 In contrast, the C4 (3.5 ppm) and C5 (1.4 ppm) resonances shifted slightly downfield because of the increase in the double bond character of the C=N groups in a 5-membered heterocycle ring, Fig. 2c and Table S1 (ESI†).

Fig. 2 (a) Stack spectra of ¹H NMR titration of HgI₂ (0.05 M) with various equivalents of L1. (b) Variation of the ¹H NMR chemical shift of the methyl group (–NCH₃) of L1 in the presence of different molar ratios of [L1]/[HgX₂]. The concentration of HgX₂ was 0.05 M and L1 was varied from 1 to 15 equiv. and ∞ (ligand only). (c) The difference of the ¹³C NMR chemical shift (Δδ) of the carbon resonances of L1 in the presence of HgI₂ (0.05 M) against free Ligand L1. The [L1]/[HgI₂] molar ratio was varied from 1 to 4, [L1]/[HgI₂] = ∞ (free ligand only). (d) Variation of the ¹H NMR chemical shift of the methyl group (–NCH₃) of L2 in the presence of HgX₂ (0.05 M) as a function of the molar ratio, as mentioned in (b). (e) The difference of ¹H NMR chemical shift (Δδ) of –NCH₃, –NCH₂–N–, and olefinic protons of L2 in the presence of HgI₂ (0.05 M) with respect to free Ligand L2. The [L2]/[HgI₂] molar ratio was varied from 1 to 11, [L2]/[HgI₂] = ∞ (free ligand only). All NMR experiments were performed in DMSO-d₆ at 21 °C (* = DMSO-d₆, # = water in DMSO-d₆). ¹H NMR for L1 only: 3.447 ppm for –NCH₃ and 7.103 ppm for olefinic proton; ¹H NMR for L2 only: 3.463 ppm for –NCH₃, 7.118 and 7.395 ppm for olefinic protons, and 6.129 ppm bridged methylene (–NCH₂N–) proton.

Fig. 3 ¹H NMR chemical shift of –NCH₃vs. electronegativity of X (X = Cl, Br and I) from (1 : 1) complexes of L1 and L2 with HgX₂.

Likewise, similar fluxional behaviour was also noticed in the case of [S₂]-donor ligand L2 when it was treated with HgX₂ (Fig. 2d). The titration profile between L2 and HgX₂ showed significant amounts of downfield shift of resonances of both bridged methylene (–NCH₂N–, H_b) and olefinic protons (H_a and H_c), and slight downfield shift of resonance of –NCH₃ protons (H_d), Fig. 2e. In a 1 : 1 molar ratio ([L2] : [HgX₂] = 1), the Δδ values for the bridged methylene protons (H_b) were 0.454, 0.454, and 0.447 ppm for HgI₂, HgBr₂, and HgCl₂, respectively. Similarly, for olefinic proton H_a, Δδ values were 0.472, 0.494, and 0.519 ppm and the same values for the H_c proton were 0.418, 0.445, and 0.475 ppm for HgI₂, HgBr₂, and HgCl₂, respectively. In contrast, the Δδ values for –NCH₃ (H_d) were 0.129, 0.171, and 0.205 ppm for HgI₂, HgBr₂, and HgCl₂, respectively, indicating a small chemical shift of –NCH₃ proton resonance as compared to the bridged methylene and olefinic protons.

Significantly, on treatment of one equivalent of HgCl₂, the C2 resonance of [S₁]-donor ligand L1 was shifted significantly more in comparison to the C2 or C2′ (both C=S moieties of L2 are equivalent) resonance of [S₂]-donor ligand L2, Table 1 and Fig. 4a. 13.5 ppm upfield shift of the C2 resonance of L1 was observed against 7.33 ppm upfield shift of the C2 and C2′ resonances of L2, showing that the two C=S groups of L2 are coordinating to the mercury centre of HgCl₂ to an equal extent but to a lesser extent than the single C=S group of L1. Furthermore, we have performed ¹⁹⁹Hg NMR experiments to understand the nature of coordination between the sulphur atom of L1 or L2 to the mercury centre of HgCl₂ as ¹⁹⁹Hg NMR chemical shifts provide a sensitive probe for the complexation between them. Treatment of one equivalent of L2 into the solution of HgCl₂ has shifted the ¹⁹⁹Hg mercury NMR peak position drastically from −1501 ppm to −878 ppm (Δ¹⁹⁹Hg = 632 ppm), showing the possibility of immediate formation of k²-fashioned di-sulfur coordinated 1 : 1 complex k²-L2HgCl₂ which further converted into a thermodynamically more stable k¹-fashioned di-sulfur coordinated 2 : 2 complex (k¹-L2)₂Hg₂Cl₄ (Table 1, Fig. 4b and Fig. S3, ESI,†Scheme 1), confirmed by single crystal X-ray experiment (vide infra). DFT calculations also suggest that k¹-(L2)₂Hg₂Cl₄ is almost 28.5 kcal mol⁻¹ more stable than k²-L2HgCl₂. In contrast, the treatment of one equivalent of L1 into the solution of HgCl₂ shifted the ¹⁹⁹Hg mercury NMR peak to 360 ppm only (from −1501 ppm to −1141 ppm), almost half of the Δ¹⁹⁹Hg value for L2. However, addition of one more equivalent of L1 shifted the ¹⁹⁹Hg mercury NMR position further to −902 ppm, indicating the formation of a di-sulfur coordinated 2 : 1 complex of (L1)₂HgCl₂ (Fig. S9, ESI†).²⁸

Table 1 Variation of ¹³C NMR of the C2-carbon and ¹⁹⁹Hg chemical shift values for titration^a of HgCl₂ with L1 and L2 in DMSO-d₆

([L]/[M])	L1 and HgCl2 complex (δ ppm)			L2 and HgCl2 complex (δ ppm)
	^13C of C2	^199Hg^b	Δ^199Hg	^13C of C2	^199Hg	Δ^199Hg
a All NMR experiments were carried out at room temperature (21 °C) in DMSO-d6, [HgCl2] = 0.1 M and ligand concentrations were varied from 0.1 M to 0.4 M. b ^199Hg NMR of HgCl2 appeared at −1501 ppm. ∞ = ligand only.
∞	161.8	—		162.73	—
1	148.3	−1141	360	155.40	−878	623
2	151.6	−902	599	154.45	−900	601
3	153.8	−874	627	159.9	−869	632
4	156.1	−860	641	161.13	−867	634

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Fig. 4 (a) ¹³C NMR chemical shift variations of L2 (1 to 4 equiv.) in the presence of HgCl₂ (1 equiv.). All ¹³C NMR experiments were performed in DMSO-d₆ at 21 °C. (b) Scheme for an energetically stable complex of L2 with HgCl₂.

Scheme 1 Synthetic routes for the formation of (L1HgI₂)₂, (L1)₃Hg₂I₄, (L1)₂HgCl₂, (L1)₂HgBr₂, k¹-(L2)₂Hg₂Cl₄, k¹-(L2)₂Hg₂Br₄ and k¹-[L2HgI₂]_n.

UV-Vis experiment. The nature of interaction between L1 or L2 with HgX₂ was monitored by UV-Vis spectrophotometer by following the ligand to metal charge transfer transition bands, S → Hg, in the solution state. In solution both the thiones L1 and L2 showed an absorption band at 269 nm, which was attributed to n → π* electronic transition of C=S and a shoulder absorption band at 235 nm corresponding to the π → π* electronic transition of the ligands (Fig. 5).³⁰ Treatment of HgX₂ into the solution of L1 or L2 (50 μM) showed a lower energy band that appeared in the range of 290 to 330 nm, which could be attributed to the ligand-to-metal charge-transfer (LMCT) band (Fig. S10, ESI†). For instance, upon gradual addition of HgI₂ (0.1–2.0 equiv.) to the solution of L1 (50 μM), a new LMCT band at 323 nm gradually increased whereas the band at 269 nm of L1 decreased substantially (Fig. 5a and Fig. S10, ESI†). The Job's plot, obtained by varying the concentrations of L1 and HgI₂, suggested the possible formation of 1 : 1 adduct L1HgI₂ in solution with an inflection point at 0.5 with respect to 323 nm (Fig. 5b), which converted into a thermodynamically more stable product (L1HgI₂)₂, a dimer of L1HgI₂, confirmed by single crystal X-ray study (vide infra). Likewise, gradual addition of HgCl₂ (0.1–1.0 equiv.) into the solution of L2 showed the increase of an LMCT peak at 290 nm and the decrease of the peak at 269 nm of L2 (Fig. 5c and Fig. S12e, ESI†). Titration spectra of L2 and HgCl₂ showed the generation of a new peak at 290 nm, which saturated with one equivalent of HgCl₂ with the standard isobestic point, as shown in Fig. 5c. Formation of a 1 : 1 complex in solution was observed from the Job's plot of L2 and HgCl₂, which showed an inflection point at 0.5 with respect to 290 nm (Fig. 5d), indicating the possible formation of a 1 : 1 complex of k²-L2HgCl₂ in solution which later converted into a thermodynamically stable product of a 2 : 2 complex of k¹-(L2)₂Hg₂Cl₄ (vide infra). Interestingly, from the Job's plot, the initial formation of a 1 : 1 complex in solution was also noticed when L1 was treated with HgCl₂, showing possible formation of a 1 : 1 adduct of L1HgCl₂ (Fig. S11, ESI†), like L1HgI₂, which slowly converted into a thermodynamically stable product of a 2 : 1 complex of (L1)₂HgCl₂ as confirmed by single crystal X-ray experiment.

Fig. 5 (a) UV titration of L1 (5 × 10⁻⁵ M) with HgI₂ up to 1 equiv. in acetonitrile. (b) Job's plot for the L1 (5 × 10⁻⁵ M) and HgI₂ complex system. (c) UV titration of L2 (5 × 10⁻⁵ M) with HgCl₂ (0.1–1 equiv.) in ACN. (d) Job's plot for the L2 (5 × 10⁻⁵ M) and HgCl₂ complex system.

Single crystal X-ray study. Considering the solution state coordination behaviour we intended to study the structural properties of the complexes between L1 and L2 with HgX₂ in the solid state. Slow evaporation of the acetone solution of L1 and HgI₂, 1 : 1 molar ratio, at room temperature afforded the formation of 1 : 1 complex [L1HgI₂]₂ (CCDC number: 1857557†), whereas excess L1 (4 equiv.) led to the formation of an unusual crystal of (L1)₃Hg₂I₄, as illustrated in Scheme 1 and Fig. 6. The single crystal X-ray structure of [L1HgI₂]₂ showed the formation of a diamond shaped bridge between two asymmetric units of L1HgI₂ through a weak covalent interaction between Hg and I atoms resulting in the formation of two different types of Hg–I bonds (d_(Hg1–I1) = d_{(Hg1′–I1′)}: 2.694 Å; and for bridged Hg–I bonds d_(Hg1–I2) = d_{(Hg1′–I2′)}: 2.844 Å and d_{(Hg1–I2′)} = d_{(Hg1′–I2)}: 2.973 Å). The S atom of L1 strongly coordinated to the Hg centre with Hg–S and C–S bond distances of 2.471 Å and 1.705 Å, respectively, in [L1HgI₂]₂, indicating a slight elongation of the C–S bond upon coordination to the Hg centre in comparison to that in free L1 (d_(C=S): 1.689 Å). The formation of a binuclear complex might be due to the weak mercurophilic interaction between the two Hg centres (d_{(Hg1–Hg1′)}: 4.235 Å) of [L1HgI₂]₂.³¹ The crystal packing showed strong intermolecular hydrogen bonding (H-bonding) between the H atom of –NCH₃ and the bridged iodine atom (d_(H⋯l2): 3.137 Å), Fig. S13 (ESI†). The unusual crystal of (L1)₃Hg₂I₄ formed due to a weak covalent interaction between the two unsymmetrical units, (L1)₂HgI₂ and L1HgI₂, through the iodine atom of (L1)₂HgI₂ to the Hg atom of L1HgI₂, leading to the distorted tetrahedral geometry at the Hg centres, with τ₄ values of 0.81 (Hg1), and 0.87 (Hg2), Fig. 6 and Table 2. As a result, the bridged I atom formed longer Hg–I bonds (d_(Hg1–I2): 3.222 Å; d_(Hg2–I2): 2.811 Å) in comparison to the other Hg–I bonds in the complex (d_(Hg1–I1): 2.689 Å; d_(Hg2–I3): 2.745 Å; d_(Hg2–I4): 2.765 Å). Three L1 units are coordinated unsymmetrically to the Hg centres with three different Hg–S bond lengths (d_(Hg1–S1): 2.465 Å; d_(Hg1–S2): 2.651 Å, d_(Hg2–S3): 2.458 Å). The crystal packing of (L1)₃Hg₂I₄ showed a chain like structure with intermolecular S⋯I interactions (Fig. S14, ESI†). On the other hand, mononuclear (L1)₂HgX₂, X = Cl^25e or Br, types of crystals were obtained when L1 was treated with 0.5 or 1 equivalent of HgX₂ in an ACN/DCM solvent mixture (1 : 1) at room temperature, as shown in Fig. 6c. (L1)₂HgBr₂ formed strong intermolecular H-bonding between the Br-atom of one molecular unit with the H-atom of –NCH₃ and olefinic H atom of another molecular unit (d_(Br1⋯Hb): 2.807 Å; d_(Br⋯Ha): 2.904 Å) leading to the formation of a dimeric structure, as illustrated in Fig. 6d.

Fig. 6 Molecular structure of (a) [L1HgI₂]₂, (b) (L1)₃Hg₂I₄, and (c) (L1)₂HgBr₂. (d) Image showing intermolecular H-bonding interaction between two units of (L1)₂HgBr₂.

Table 2 The Hg–S and Hg–X bond lengths and τ₄ values in the complexes

Compounds	Bond length		τ 4
	Hg–S (Å)	Hg–X (Å)
[L1HgI2]2	2.471	2.694, 2.844, 2.973	0.89
(L1)3Hg2I4	2.465, 2.651, 2.458	2.689, 3.222, 2.811, 2.745, 2.765	0.81, 0.87
(L1)2HgBr2	2.511, 2.576	2.637, 2.597	0.89
k ^1-(L2)2Hg2Cl4	2.465	2.458, 2.651	0.90
k ^1-(L2)2Hg2Br4	2.505	2.622, 2.751	0.88
k ^1-[L2Hg2I2]n	2.503, 2.521	2.637, 2.683, 2.693, 2.779	0.91

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Unlike L1, [S₂]-donor ligand L2 afforded a chain or ring-like structure in the solid state on treatment with one equiv. of HgX₂. X-ray structure analysis of the yellow colour diamond shaped single crystal of the complex between L2 and HgI₂ confirmed the formation of a polymeric structure of k¹-[L2HgI₂]_n, where two S atoms of L2 interact with the two geometrically different mercury centers, as shown in Fig. 7d. On the other hand, in the case of HgX₂ where X = Cl or Br, we observed the formation of 16-membered metallacycle k¹-(L2)₂Hg₂X₄, with two mercury centres that are geometrically equivalent, in a tetrahedral geometry with τ₄ values of 0.90, and 0.88 for k¹-(L2)₂Hg₂Cl₄ and k¹-(L2)₂Hg₂Br₄, respectively, Fig. 7a and b. In k¹-(L2)₂Hg₂Cl₄, the S atoms of two L2 ligands coordinated symmetrically (d_(Hg–S1): 2.465 Å) with Hg centres of two HgCl₂ units leading to the formation of a 16-membered ring-like structure, in which two Cl atoms are located inside of the metallocycle (sandwiched between the two 5-membered imidazole rings) with significantly elongated Hg–Cl bond lengths (d_(Hg1–Cl2) = d_(Hg1–Cl4) = 2.651 Å) and the other two Cl atoms are located outside of the ring with shorter Hg–Cl bond lengths (d_(Hg1–Cl1) = d_(Hg1–Cl3) = 2.458 Å) (Fig. S15, ESI†). Similar to k¹-(L2)₂Hg₂Cl₄, we also observed two different types of Hg–Br bonds in k¹-(L2)₂Hg₂Br₄, d_(Hg1–Br1) = d_(Hg1–Br3) = 2.622 Å; d_(Hg1–Br2) = d_(Hg1–Br4) = 2.751 Å. Br1 and Br3, oriented at the outside of the 16-membered metallacycle, formed a shorter Hg–Br bond, whereas Br2 and Br4 are sandwiched between two 5-membered imidazole rings of the metallacycle, as shown in Fig. 7c, and formed longer Hg–Br bonds. Crystal packing arrangement of k¹-(L2)₂Hg₂Br₄ showed that Br2 or Br4 are involved in strong H-bonding interaction with H-atoms of –NCH₃ & olefin-H of another unit of the metallacycle, whereas the outer Br atom (Br1 or Br3) participated in halogen bonding (Br⋯S interaction) interaction with a S atom of another unit of the metallacycle, as shown in Fig. S16 (ESI†).

Fig. 7 ORTEP images of 16-membered metallacycles k¹-(L2)₂Hg₂Cl₄ (a) (solvent DMSO is omitted for clarity), and k¹-(L2)₂Hg₂Br₄ (b). (c) Mercury image of k¹-(L2)₂Hg₂Br₄ showing that two Br atoms are sandwiched between two imidazole rings. ORTEP images of the polymeric structure of k¹-[L2HgI₂]_n (d) and L3 (e).

2.2 Cytoprotective effects of [S₁] and [S₂]-donor ligands against Hg(II) induced toxicity

Cytotoxicity. After detailed studies to understand the nature of coordination of [S₁] and [S₂]-donor ligands to the mercury center of various Hg(II) salts in both solution and solid states, we investigated the protective effect of these imidazole-based thiones against Hg(II) induced toxicity in a cellular system. To examine the protective effect of the imidazole-based thiones against Hg(II)-induced toxicity in hepatocytes we have also employed another new N,N-disubstituted imidazole-based [S₂]-donor ligand L3 with –N(CH₂)₃CO₂H substituents. The synthetic procedure of L3 is mentioned in the supporting information and the crystal structure is shown in Fig. 6e. First, we determined the cytotoxicity of these imidazole-based thiones in human HepG2 cells. To investigate the cytotoxic effect, cells (1.0 × 10⁴) were seeded in 96 well-plates and incubated with various concentrations of ligands (0–100 μM) for 24 h and their toxic effects were analysed using standard MTT assays.^32a To our delight, we found that these thione based ligands are not cytotoxic to HepG2 cells even up to 100 μM concentrations, and more than 90% of cell viability was observed in the presence of 100 μM ligands, as illustrated in Fig. 8a, suggesting that these thiones can be used safely even up to 100 μM concentration to study their protective effect against Hg(II) toxicity. Next we investigated the cytotoxic effect of HgCl₂ in a dose dependent manner in HepG2 cells. As mentioned above, cells (1.0 × 10⁴) were seeded into 96 well-plates and incubated with various concentrations of HgCl₂ (0–50 μM) for 24 h to analyse its cytotoxic effect.^32b Almost 90% cell death was observed at 50 μM and ∼50% cell survival was observed at 25 μM concentration of HgCl₂ (IC₅₀ = 25 μM) and, thus, 25 μM concentration of HgCl₂ was used to study the protective effect of thiones (L1, L2 or L3) against HgCl₂ toxicity (Fig. S28, ESI†). Cells were co-treated with HgCl₂ (25 μM) and various concentrations of thiones (0–100 μM) and incubated for 24 h. In the case of [S₁]-donor ligand L1, we have observed only 10% and 15% protection of HepG2 cells at 50 μM and 100 μM concentrations of L1, respectively, in comparison to the cells treated with 25 μM HgCl₂ only. Whereas in the case of [S₂]-donor ligands, we have observed up to 25% and 35% protection at 100 μM concentration of L2 and L3, respectively, as illustrated in Fig. 8b. These observations clearly suggest that [S₂]-donor ligands L2 and L3 have greater protective effects in comparison to the [S₁]-donor ligand L1. Again, the among [S₂]-donor ligands, L3 has more protective effect than L2 against HgCl₂ toxicity in liver cells, indicating that L3 possibly coordinates effectively with Hg²⁺ ions in a cellular system and protects enzymes, proteins and GSH from Hg²⁺.

Fig. 8 (a) Effect of L1, L2 and L3 (5–100 μM) on cell viability in HepG2 cells. (b) Percentage of cell viability of HepG2 cells treated with HgCl₂ (25 μM) and co-treated with HgCl₂ (25 μM) and various amounts of ligands (5–100 μM).

Intracellular ROS estimation. It is well-known that the inorganic mercury toxicity is mainly due to its strong affinity toward thiol and selenol containing proteins including many vital antioxidant enzymes like thioredoxin (Trx), thioredoxin reductase (TrxR) and glutathione peroxidase (GPx), glutathione reductase (GR), and endogenous thiols including GSH and L-CysH and thereby it reduces the concentration of GSH, an important antioxidant, thiol containing tripeptide present in living cells, leading to the production of reactive oxygen species (ROS) in various tissues which causes DNA damage, protein oxidation, and lipid peroxidation. Overproduction of ROS within the cells leads to a situation called oxidative stress, which ultimately leads to cell death.^6,11,12 When cells were treated with 20 μM HgCl₂ for 2 h followed by the treatment with DCFH-DA for 0.5 h, we observed remarkably strong fluorescence signal of DCF due to the production of huge amounts of ROS in HgCl₂-treated HepG2 cells in comparison to that observed in untreated cells (UT), as shown in Fig. 9a and b. In accordance, the bright field image of HgCl₂-treated HepG2 cells confirmed that the cells were completely stressed in the presence of 20 μM HgCl₂. However, co-treatment of cells with 20 μM of HgCl₂ and 100 μM of ligand (HgCl₂/Ln hereafter) decreased the production of ROS in the cells and as a result we observed a significant amount of decrease of fluorescence intensity in co-treated cells, Fig. 9c–e and 10a. The mean intensity profile showed ∼30% decrease of ROS production in cells co-treated with HgCl₂/L1 in comparison to the cells treated with HgCl₂ only, as indicated by weaker fluorescence signal of DCF. However, to our delight, in the case of [S₂]-donor ligands, we observed ∼45% and ∼60% decrease of ROS production in cells co-treated with HgCl₂/L2 and HgCl₂/L3, respectively, in comparison to the cells treated with HgCl₂ only, as indicated by much weaker fluorescence signal of DCF. These observations strongly suggest that [S₂]-donor ligands L2 and L3 have more cytoprotective effect against HgCl₂ induced toxicity, 1.5 and 2 times, respectively, in comparison to L1. Interestingly, in accordance with the ROS results, the bright field images confirmed that the cells are not in stress and, moreover, the shapes of the HepG2 cells remained intact when they were co-treated with HgCl₂ and 100 μM of L2 or L3.

Fig. 9 Bright field (top) and the corresponding fluorescence images (bottom) of untreated (a), 20 μM HgCl₂ (b), 20 μM HgCl₂ + 100 μM L1 (c), 20 μM HgCl₂ + 100 μM L2 (d), and 20 μM HgCl₂ + 100 μM L3 (e) treated HepG2 cells.

Fig. 10 (a) The relative mean intensity profile of ROS production in HeG2 cells treated with HgCl₂ and co-treated with HgCl₂ plus ligands (L1, L2 or L3). (b) HRMS of L3 detected in cell lysates.

In order to investigate the internalization of the active compound L3 into the HepG2 cells we have incubated cells with L3 (100 μM) for 4 h and performed HRMS-QTOF analysis with cell lysates after thorough washing of cells with PBS buffer. A detailed experimental procedure is mentioned in the ESI.† Identification of L3 in cell lysates by mass spectrometry [L3: m/z for (M + Na) = 407.0813], Fig. 10b, confirmed internalization of L3 into the HepG2 cell. Next we investigated the protecting effect of L3 due to the intracellular complexation with mercury. For this, at first, cells were incubated in the absence (control experiment) and presence of L3 (100 μM) for various times (4 h, 12 h, and 24 h) in 6-well plates. After incubation of cells with L3 at various times, the medium was completely removed, the cells were washed with PBS buffer and then fresh medium was added in 6-well plates to treat the cells further with HgCl₂. Cells were incubated with HgCl₂ (20 μM) for 2 h and then the production of ROS in the cells was measured using DCFH-DA as mentioned and compared with that observed in untreated cells, as shown in Fig. S29 (ESI†). The mean fluorescence intensity of the DCF profile, Fig. S30 (ESI†), showed 15%, 28%, and 36% reduction of ROS level in cells pre-treated with L3 for 4 h, 12 h and 24 h, respectively, in comparison to the untreated cells.

3. Conclusions

In summary, we demonstrated the coordination behaviour of [S₁] and [S₂]-donor ligands such as L1, L2 or L3 with mercury(II) halides in solution and the solid state by NMR, UV-Vis, and single crystal X-ray diffraction studies. NMR studies revealed that L1 or L2 ligand coordinated rapidly and reversibly to the mercury center of HgX₂ (X = Cl, Br, or I) at room temperature. A significant chemical shift of the ¹H and ¹³C resonance of the ligands was observed upon coordination to the mercury center of HgX₂ in comparison to the free ligand, indicating strong interaction between [S₁] or [S₂]-donor ligands with Hg²⁺ in solution. The UV-Vis titration and Job's plot confirmed that [S₁]-donor ligand L1 formed a 1 : 1 complex with HgI₂ in solution when both were mixed in a 1 : 1 molar ratio at room temperature. Single crystal X-ray studies also confirmed the formation of a 1 : 1 bimetallic complex of [L1HgI₂]₂ in the solid state when L1 and HgI₂ reacted in a 1 : 1 molar ratio. However, addition of excess L1 into the solution of HgI₂ afforded (L1)₃Hg₂I₄, a 3 : 2 bimetallic complex. In contrast, treatment of [S₂]-donor ligand L2 to the solution of HgI₂, in a 1 : 1 molar ratio, afforded a polymeric structure of k¹-[L2HgI₂]_n. Interestingly, treatment of L2 with HgX₂ (X = Cl or Br) yielded stable sixteen-membered neutral binuclear metallacycle k¹-(L2)₂Hg₂Cl₄ or k¹-(L2)₂Hg₂Br₄ where two Cl or Br atoms are sandwiched in between the two five-membered imidazole rings of the metallacycle. However, possibly due to the large size of iodine, the mixture of L2 and HgI₂ afforded a stable polymeric structure of k¹-[L2HgI₂]_n. In vitro investigation of the cellular toxicity of ligands L1, L2 and L3 in HepG2 cells suggests that these ligands are not cytotoxic to human liver cells. Water-soluble [S₂]-donor ligand L3 with N-(CH₂)₃CO₂H substituents showed excellent cytoprotective effects against HgCl₂ induced toxicity in hepatocytes. In MTT assay, 35% more cell survival was observed when HepG2 cells were co-treated with HgCl₂ (25 μM) and L3 (100 μM) in comparison to that in the case of cells treated with HgCl₂ (25 μM) alone. Fluorescence imaging study, for the estimation of ROS production induced by HgCl₂, in HepG2 cells demonstrated that L3 has an excellent property to reduce the oxidative stress in a cellular system, mostly through binding to HgCl₂.

4. Experimental section

4.1 General experimental

Methyl imidazole, diiodomethane, sulfur powder, 4-bromo butyrate MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], DCFH-DA (2′,7′-dichlorofluorescin diacetate) and DMSO-d₆ were purchased from Sigma-Aldrich. Mercuric chloride, mercuric bromide, and mercuric iodide were purchased from CDH chemicals and other chemicals were purchased from local companies. The L1 and L2 ligands were prepared by following the literature procedure,^33–35 and L3 was synthesized in a new synthetic method (ESI†). All the synthetic experiments were carried out under anhydrous and anaerobic conditions using standard Schlenk techniques for the synthesis. Mass spectrometric analysis was carried out using an Agilent 6540 accurate mass Q-TOF HRLC/MS equipped with an electrospray ionisation source (ESI). ¹H (400 MHz), ¹³C (100 MHz), and ¹⁹⁹Hg (71.6 MHz) NMR spectra were obtained on a Bruker Advance 400 MHz NMR spectrometer using the solvent as an internal standard for ¹H and ¹³C. Chemical shifts (¹H, ¹³C) are cited with respect to tetramethylsilane (TMS). The ¹⁹⁹Hg NMR spectra are reported in ppm relative to neat Me₂Hg (δ = 0 ppm) and HgCl₂ (δ = −1501 ppm for 1 M solution in DMSO-d₆ at 21 °C) was used as an external standard. Electronic spectra were recorded on a UV-1800 Shimadzu UV-Vis spectrophotometer at 298 K in acetonitrile.

4.2 Synthesis

Synthesis of [L1HgI₂]₂. To a stirred solution of HgI₂ (100 mg, 0.21 mmol) in acetone a solution of L1 (27 mg, 0.21 mmol) in acetone was added and the reaction mixture was stirred at room temperature for 0.5 h. Upon slow evaporation of the reaction solution, a pale yellow coloured crystalline product was obtained. Yield: 89.2 g (73%). ¹H NMR (DMSO-d₆) δ = 3.68 (s, 12H), 7.51 (s, 4H), ¹³C NMR (DMSO-d₆) δ = 35.8, 121.5, 151.7. HR-ESIMS (m/z): calcd for [M]⁺ C₁₀H₁₆N₄S₂Hg₂I₄ = 1165.638, observed value: [M − HgI₃]⁺ = 584.9590.

Synthesis of (L1)₃Hg₂I₄. To a stirred solution of HgI₂ (100 mg, 0.21 mmol) in acetone a solution of excess L1 (108 mg, 0.84 mmol) in methanol was added and reaction mixture was stirred at room temperature for 0.5 h. The reaction solution was removed and washed with dichloromethane to get a pale yellow coloured crude product. The complex was crystallized from hot DMSO solvent as cube shaped crystals. Yield: 84.2 g (66%). ¹H NMR (DMSO-d₆) δ = 3.57 (s, 18H), 7.31 (s, 4H), ¹³C NMR (DMSO-d₆) δ = 35.2, 119.9, 156.5.

Synthesis of (L1)₂HgBr₂. To a solution of L1 (50 mg, 0.39 mmol) in dichloromethane one equivalent of HgBr₂ (140.4 mg, 0.39 mmol) dissolved in acetonitrile was added and immediate formation of a white precipitate was observed. The suspension was stirred for another 0.5 h at room temperature. The white precipitate was filtered and washed with dichloromethane and dried under high vacuum. The obtained product was crystallized from hot DMF solvent as colourless needle-shaped crystals. Yield: 103.2 g (86%). ¹H NMR (DMSO-d₆) δ = 3.68 (s, 12H), 7.50 (s, 4H), ¹³C NMR (DMSO-d₆) δ = 35.5, 121.1, 151.6. HR-ESIMS (m/z): calcd for [M]⁺ C₁₀H₁₆N₄S₂HgBr₂ = 617.8854, observed value: [M − Br]⁺ = 536.9713.

Synthesis of k¹-(L2)₂Hg₂Cl₄. To a 5 mL solution of L2 (50 mg, 0.21 mmol) in dichloromethane one equivalent of mercury(II) chloride (56.47 mg, 0.21 mmol) dissolved in acetonitrile was added. The immediate formation of white precipitates occurred and the suspension was stirred for another 0.5 h at 30 °C. A white solid of (L2)₂Hg₂Cl₄ was filtered from the reaction mixture and suitable single crystals were obtained from a DMSO solution of the complex. Yield: 89.2 g (83%). ¹H NMR (DMSO-d₆) δ = 3.66 (s, 12H), 6.57 (s, 4H), 7.59–7.59 (d, J = 1.6 Hz, 4H), 7.86–7.87 (d, J = 1.6 Hz, 4H), ¹³C NMR (DMSO-d₆) δ = 35.5, 57.3, 120.3, 122.1, 154.1. HR-ESIMS (m/z): calcd for [M]⁺ C₁₈H₂₄N₈S₄Hg₂Cl₄ = 1023.95, observed value: [M − HgCl₃]⁺ = 717.0430, [M-C₉H₁₂N₄S₂HgCl₃]⁺ = 476.9911.

Synthesis of k¹-(L2)₂Hg₂Br₄. k ¹-(L2)₂Hg₂Br₄ was synthesized following a procedure similar to that for k¹-(L2)₂Hg₂Cl₄, except mercury(II) bromide (75 mg, 0.21 mmol) was added in place of mercury(II) chloride. Over time, block shaped single crystals of the complex were settled from the saturated solution of the complex in DMSO at room temperature. Yield: 100 mg (76%). ¹H NMR (DMSO-d₆) δ = 3.60 (s, 12H), 6.57 (s, 4H), 7.53–7.54 (d, J = 1.6 Hz, 4H), 7.89–7.890 (d, J = 1.6 Hz, 4H), ¹³C NMR (DMSO-d₆) δ = 36.0, 57.7, 121.2, 122.4, 152.3. HR-ESIMS (m/z): calcd for [M]⁺ C₁₈H₂₄N₈S₄Hg₂Br₄ = 1201.7082, observed value: [M − HgBr₃]⁺ = 760.9913, [M − C₉H₁₂N₄S₂HgBr₃]⁺ = 520.9402.

Synthesis of k¹-[L2HgI₂]_n. To 10 mL of L2 (50 mg, 0.21 mmol) in MeOH, one equivalent of mercuric iodide (98.7 mg, 0.21 mmol) was added and the reaction mixture was stirred for another 30 minutes at 30 °C. After completion, the reaction solution was concentrated and dried under vacuum to obtain a white solid. Diamond shaped single crystals of k¹-[L2HgI₂]_n were obtained from the solution of acetone/DMSO solution. Yield: 84 mg. ¹H NMR (DMSO-d₆) δ = 3.59 (s, 6H), 6.58 (s, 2H), 7.53–7.54 (d, J = 2 Hz, 2H), 7.91–7.92 (d, J = 2 Hz, 2H), ¹³C NMR (DMSO-d₆) δ = 36.1, 57.4, 120.6, 122.1, 154.7.

4.3 NMR titration experiments of L1 and L2 with HgX₂ (X = Cl, Br or I)

A solution of HgX₂ (0.05 mmol) in DMSO-d₆ (0.6 mL) was titrated with various equivalents (0–11 equiv.) of L1 (or L2) in DMSO-d₆ and the ¹H NMR spectra were recorded at room temperature.²⁶ The variation of the chemical shifts of the proton (¹H) and carbon (¹³C) resonances of the N-CH₃ group, olefinic-C and olefinic-H of L1, and bridged-methylene (N-CH₂-N), N-CH₃ group, olefinic-H or olefinic-C of L2 were reported with respect to solvent the residual peak (DMSO-d₆, ¹H δ = 2.5 ppm), as shown in Fig. S1–S8 (ESI†). Full titration spectra are presented in the ESI.†

4.4 UV-Visible spectroscopic analysis

UV-Visible titration studies were performed using a UV-1800 Shimadzu UV-Vis spectrophotometer at 298 K in acetonitrile. 50 μM of L1 (or L2) solution in 1 mL acetonitrile was transferred into a 1 cm path length UV cuvette and various amounts of HgX₂ were added (0.1–2 or 3 equiv.) to get a titration profile. For determining the complex composition in solution by Job's method we used 5 × 10⁻⁵ M of HgX₂ and 5 × 10⁻⁵ M of ligands (L1 or L2 or L3) in acetonitrile. In total, nine mixtures of HgX₂ and ligands were prepared. The volumes of ligand solution varied from 9 to 1 mL and those of HgX₂ solution from 1 to 9 mL to obtain 1 to 9 mole fractions. The total volume was always kept at 10 mL. The complex inflection point in the Job's plot was calculated with respect to the LMCT wavelength of the corresponding complex.

4.5 Protection of HepG2 cells

Cell viability. Cell culture experiment and cell viability assay were performed following the general procedure. HepG2 cells were continuously grown in a C25-mL cultural flask in RPMI 1640 supplemented with 5% FBS, 100 U mL⁻¹ penicillin and 100 U mL⁻¹ streptomycin in a CO₂ incubator (5%) at 37 °C. MTT assay was performed to quantify the viability of the cells treated with ligands L1, L2, L3 and HgCl₂ as mentioned below. In brief, 1 × 10⁴ HepG2 cells were seeded into each well of a 96-well cell culture plate. After being grown for another 24 h, the cells were treated with various concentrations of ligands L1, L2 and L3 (1 to 100 μM) to determine the cytotoxicity of each ligand. After incubation of the cells with different concentrations of ligand for 24 h, the medium was removed and the cells were treated with 100 μL of 500 μg mL⁻¹ of MTT (thiazolyl blue tetrazolium bromide) and incubated for another 4 h. Finally, MTT solution was removed and the formazan product was dissolved by adding 100 μL DMSO, and allowing for vibration for 10 min. Absorbance was read on an ELISA plate reader (Thermo Scientific, USA) at 590 nm. Likewise, the cytotoxicity of HgCl₂ was also determined using the MTT assay, as mentioned above. To determine the IC₅₀ value, the cells were treated with various concentrations of HgCl₂ (5–50 μM). To investigate the protective effect of the ligands against HgCl₂ toxicity, the cells were co-treated with ligand of various concentrations (5–100 μM) and HgCl₂ (25 μM) and a similar procedure was followed, as mentioned above.

4.5 ROS estimation

The intracellular oxidative stress, induced by HgCl₂, was detected by fluorescent imaging study. DCFH-DA was used as a fluorescent dye to measure the intracellular ROS level following the literature procedure.³⁶ In brief, HepG2 cells (5 × 10⁵) were seeded in 12-well plates and grown in a complete medium in a CO₂ incubator to 80% confluence. Cells were exposed to 20 μM HgCl₂ (alone) and co-treated with 100 μM concentration of the ligands (L1, L2, or L3) for 2 h. After 2 h incubation, the cells were washed with PBS and labelled with 10 μM DFCH–DA and incubated for 0.5 h at 37 °C. After being washed with PBS (3 times), the intracellular ROS level was determined using a Nikon eclipse Ti-u fluorescence microscope with the excitation wavelength set at 488 nm and emission wavelength at 530 nm.

4.6 Single crystal X-ray analysis

Single crystal X-ray diffraction data were collected on a D8 Venture Bruker AXS single crystal X-ray diffractometer equipped with a CMOS PHOTON 100 detector having monochromatized microfocus sources (Mo-Kα = 0.71073 Å). Single crystals of L3 (CCDC 1857559), (L1)₂HgBr₂ (CCDC 1486402), [L1HgI₂]₂ (CCDC 1857557), (L1)₃Hg₂I₄ (CCDC 1857558), k¹-(L2)₂Hg₂Cl₄·DMSO (CCDC 1534013), k¹-(L2)₂Hg₂Br₄ (CCDC 1857556) and k¹-[L2Hg₂I₂]_n (CCDC 1857560) suitable for X-ray diffraction study were obtained from a slow evaporation process as described in the Experimental section.† Crystallographic parameters of L3 and the complexes are mentioned in the ESI† (Tables S1 and S2).³⁷ All crystal data were collected at room temperature and solved using the SHELX program implemented in APEX3.^38–41 The non-H atoms were located in successive difference fourier syntheses and refined with anisotropic thermal parameters. All the hydrogen atoms were placed at the calculated positions and refined using a riding model with appropriate HFIX commands. The program “Mercury” was used for molecular packing analysis.⁴² The crystal structure of [L2HgI₂]_n was disordered at the Hg2 atom, and the disordered mercury atom was treated using the PART command with occupancy (74 : 26)% (Hg2A : Hg2B). Similarly, k¹-(L2)₂Hg₂Cl₄·DMSO having solvent molecule disorder at the O1 atom was split by the PART command with occupancy (63 : 37)% (O1A : O1B).⁴³ The crystal structure of k¹-(L2)₂Hg₂Br₄ has disorder in the solvent molecule which was removed by SQUEEZE option using PLATON software.⁴⁴

Funding source

This work was supported by Shiv Nadar University (SNU), Shiv Nadar Foundation (SNF), and SERB (SB/S1/IC-44/2013), Govt. of India.

Caution

Mercuric salts are highly toxic to humans, and thus appropriate safety precautions must be taken in handling these toxic chemicals.

Conflicts of interest

The authors declare no competing financial interest and have no conflicts to declare.

Acknowledgements

We thank Shiv Nadar University (SNU), Shiv Nadar Foundation (SNF) and SERB (SB/S1/IC-44/2013), Govt. of India for funding. RK, and AC thank the SNU for fellowships.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1486402, 1534013, 1857556–1857560. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8mt00237a

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