A rationally designed molecule for removal of cyanide from human blood serum and cytochrome c oxidase

Abstract

Compound 3 having three dimethoxyphenolic units exhibited excellent selectivity and competitive binding with CN⁻. Various experiments revealed the capability of this compound to act through all the three dimethoxyphenolic units and efficiently remove cyanide from human blood serum – more precisely from cytochrome c oxidase.

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1. Introduction

Besides the accidental cases of NaCN and KCN poisoning; gold mining, electroplating and metallurgical processes¹ are some of the common sources of exposure to cyanide² and once the permissible level of 1.9 μM (ref. 3) is exceeded, it proves lethal. The extreme toxicity of cyanide is attributed to its binding to Fe²⁺ of cytochrome c oxidase (CcOX) and consequent inhibition of mitochondrial electron transport chain. This results in decreased oxidative metabolism and diminishing oxygen utilization.⁴

Chemical capturing of CN⁻ and disposal of the product as safe metabolite/s is desirable to get rid of cyanide poisoning. Irrespective of a number of reports,^5–12 only a few of the compounds including hydroxocobalamin, dicyano-cobalt(III)-porphyrins, vitamin B12 analogues, metallophthalocyanines and hexahydrated dichloride of cobalt(II) and nickel(II)^13–18 find compatibility to the living system and hence safely used in scavenging CN⁻ from the biological medium. Moreover, the limited number of available kits mainly use NO for removal of cyanide from cytochrome c oxidase.^19,20 Therefore, the dire consequences of cyanide poisoning make the detection and removal of this poison from the environmental as well as biological systems a key challenge to contemporary chemical/medicinal science. Here, we demonstrate the working of a new molecule for removal of cyanide from human blood serum and also its capability to restore the cyanide inhibited CcOX activity.

After the recent disclosure of dimethoxyphenolic substituted pyrazolone (1, Chart 1)²¹ for removal of cyanide from the human blood serum, it was envisaged that by increasing the phenolic sites in the molecule, its sensitivity for CN⁻ may get enhanced. As a result, less quantity of the compound will be required per unit of cyanide and consequently the detection limit will improve. Replacement of pyrazole moiety of compound 1 with oxindole in compound 2 did not change its behaviour towards CN⁻. Mode of action as well as the sensitivity of compound 2 for CN⁻ was similar to that observed for compound 1 (Fig. S1–S4†). Advantageously compound 2 was further derivatized and as per our plan of increasing the number of dimethoxyphenolic units in the molecule, compound 3 was prepared and it was checked for detection/removal of CN⁻ from the biological medium.

Chart 1

2. Experimental section

Materials

Assay buffer, enzyme dilution buffer, dithiothretol solution, cytochrome c, cytochrome c oxidase, dimethyl sulphoxide, mesitylene, oxindole, AlCl₃, formaldehyde, HBr–AcOH, NaCN, TBACN.

Synthesis of 3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (2)

A finely ground mixture of syringaldehyde (182 mg, 1 mmol) and oxindole (160 mg, 1.2 mmol) was heated at 150 °C for 30 min. The reaction mixture was washed with diethyl ether (4 × 50 ml) to get pure product, 80%, yellow solid, mp 203–204 °C, ν_max/cm⁻¹: 3349 (NH), 3162 (OH), 1679 (C=O); δ_H (500 MHz; CDCl₃; Me₄Si) 3.95 (6H, s, OCH_3(major)), 3.98 (6H, s, OCH_3(minor)), 6.85–6.96 (2H, m, ArH_(major)), 6.99 (1H, br, OH), 7.01 (2H, m, ArH_(minor)), 7.16–7.18 (1H, m, ArH_(major)), 7.19–7.21 (1H, m, ArH_(minor)), 7.45–7.50 (1H, m, ArH_(major)), 7.52–7.53 (1H, m, ArH_(minor)), 7.63 (1H, s, bridged H_(major)), 7.66 (1H, s, bridged H_(minor)), 7.80–7.84 (1H, m, ArH_(major)), 7.96–7.99 (1H, m, ArH_(minor)), 8.28 (1H, s, CH_(major)), 8.34 (1H, s, CH_(minor)), 10.04 (1H, br, NH_(minor)), 10.05 (1H, br, NH_(major)). δ_C (normal/DEPT-135; CDCl₃ + DMSO-d₆) 61.1 (+ve, OCH₃), 61.1 (+ve, OCH₃), 112.0 (+ve, CH), 114.2 (+ve, CH), 115.0 (+ve, CH), 115.1 (+ve, CH), 125.6 (+ve, CH), 125.7 (+ve, CH), 126.5 (C), 127.2 (+ve, CH), 127.3 (+ve, CH), 128.8 (C), 129.8 (C), 129.9 (C), 130.5 (C), 130.6 (C), 132.7 (+ve, CH), 134.1 (+ve, CH), 141.8 (+ve, CH), 141.9 (+ve, CH), 142.5 (+ve, CH), 143.5 (C), 144.9 (C), 147.4 (C), 152.0 (C), 152.5 (C), 174.8 (C=O).174.8 (C=O). HRMS (ESI) m/z for C₁₇H₁₅NO₄ (M + H)⁺ calcd 298.1074, found 298.1071 (Fig. S5–S9†).

Synthesis of 3,4,5-trimethoxybenzaldehyde²²

To the stirred solution of syringaldehyde (3 g, 16.5 mmol) in DMF (40 ml); K₂CO₃ (3.41 g, 1.5 equiv.), CH₃I (2.8 g, 19.71 mmol) and KI (catalytic amount) were added. The reaction was allowed to stir overnight at room temperature. After completion of the reaction, it was quenched by adding water and extracted with ethyl acetate (4 × 25 ml). The organic layer was separated, dried over Na₂SO₄ and concentrated under vacuum to procure pure product, creamish white solid (90%), mp 75–76 °C, δ_H (500 MHz; CDCl₃; Me₄Si) 3.93 (6H, s, 2× OCH₃), 3.94 (3H, s, OCH₃), 7.13 (2H, s, ArH), 9.87 (1H, s, CHO); δ_C (normal/DEPT-135) (CDCl₃) 56.2 (+ve, OCH₃), 60.9 (+ve, OCH₃), 131.7 (C), 143.6 (C), 153.6 (C), 191.0 (C=O).

Synthesis of compound 4

3,4,5-Trimethoxybenzaldehyde (1.5 g, 7.6 mmol) and oxindole (1 g, 7.6 mmol) were heated at 145 °C for 1 h. The reaction mass was purified by column chromatography to obtain pure compound 4, yellow solid (80%), 145 °C, δ_H (500 MHz; CDCl₃; Me₄Si) 3.90 (6H, s, OCH_3(major)), 3.964 (3H, s, OCH_3(major)) 3.968 (3H, s, OCH_3(minor)), 3.99 (6H, s, OCH_3(minor)), 6.89–6.93 (3H, m, ArH_(major)), 6.94–6.95 (3H, m, ArH_(minor)), 7.23–7.28 (2H, m, ArH_(major)), 7.50–7.55 (2H, m, ArH_(minor)), 7.78 (1H, s, bridged H_(major)), 7.81 (1H, s, bridged H_(minor)), 7.82 (1H, s, CH_(minor)), 7.84 (1H, s, CH_(major)), 8.65 (1H, br, NH_(minor)), 8.78 (1H, br, NH_(major)). Integration of the signals corresponding to major and minor isomers is in the ratio 3 : 1 (Fig. S10a†). HPLC of compound 4 (Fig. S10b†) also showed the presence of two isomers in the ratio 3 : 1.

Synthesis of tris-(bromomethyl)mesitylene) (5)

To the mixture of mesitylene (2.4 g, 20 mmol), paraformaldehyde (2 g, 70 mmol) and glacial acetic acid (10 ml); 14 ml of 31 wt% HBr–acetic acid solution was added rapidly. The reaction mixture was kept for 12 h at 95–110 °C and then poured into 100 ml of water. The solid was filtered, washed with water and dried in vacuum to obtain a white solid (91%), mp 185–186 °C, δ_H (300 MHz; CDCl₃; Me₄Si) 4.57 (6H, s, CH₂Br), 2.46 (9H, s, CH₃).

Synthesis of compound 6

Solution of compound 4 (100 mg, 0.32 mmol) and KOH (144 mg, 2.5 mmol) in DMSO (1 ml) was stirred for 5–10 min. Then compound 5 (43 mg, 0.1 mmol) was added and stirred the reaction mixture for 1 h (TLC). After completion of the reaction, it was quenched with water, solid separated out which was filtered and washed with water. The crude product was purified through column chromatography to get compound 6 as yellow solid, 60%, mp 172–173 °C, δ_H (500 MHz; CDCl₃; Me₄Si) 2.39–2.41 (3H, m, CH₃), 2.44–2.46 (6H, m, 2× CH₃), 3.85–3.86 (12H, m, 4× OCH₃), 3.92–3.93 (9H, m, 3× OCH₃), 3.97–3.98 (6H, m, 2× OCH₃), 5.14–5.18 (6H, m, CH₂Ph), 5.90–5.94 (1H, m, ArH), 6.02–6.07 (2H, m, ArH),6.54–6.78 (5H, m, ArH), 6.88–6.90 (5H, m, ArH), 7.46–7.48 (2H, m, ArH), 7.74–7.75 (2H, m, ArH), 7.79–7.81 (2H, m, ArH), 7.84–7.85 (2H, m, ArH). ¹H NMR spectrum of compound 6 showed two sets of signals in their integration ratio 2 : 1; δ_C (normal/DEPT-135) (CDCl₃) 16.4 (+ve, CH₃), 17.3 (+ve, CH₃), 40.3 (−ve, CH₂), 40.5 (−ve, CH₂), 56.2 (+ve, OCH₃), 60.9 (+ve, OCH₃), 61.0 (+ve, OCH₃), 106.7 (+ve, CH), 109.2 (+ve, CH), 110.0 (+ve, CH), 118.5 (+ve, CH), 121.2 (+ve, CH), 121.3 (+ve, CH), 122.6 (+ve, CH), 124.1 (C), 124.5 (C), 125.8 (C), 128.6 (+ve, CH), 129.2 (+ve, CH), 129.6 (+ve, CH), 129.7 (+ve, CH), 130.0 (C), 131.6 (C), 131.8 (C), 137.1 (C), 137.2 (C), 137.6 (+ve, CH), 137.7 (+ve, CH), 137.8 (+ve, CH), 139.3 (C), 140.6 (C), 140.9 (C), 141.0 (C), 143.2 (C), 143.3 (C), 143.4 (C), 152.6 (C), 153.2 (C), 165.9 (C=O), 168.2 (C=O). Because of the presence of compound 4 in two isomeric forms (E- and Z-), its coupling with compound 5 may form different isomers of 6 in which all the three fragments, coming from compound 4, exist either in E-configuration (6-i) or Z-configuration (6-ii). Another possibility is the presence of two fragments in the E-form, one in the Z-form (6-iii) and vice versa. Although compound 6 gave single spot on TLC, further confirmation about the isomeric form/s of this compound was made after its conversion to compound 3, the target compound (Fig. S11–S13†).

Synthesis of compound 3

The reaction mixture obtained by the slow addition of anhydrous AlCl₃ (62 mg, 0.45 mmol) to solution of compound 6 (100 mg, 0.09 mmol) in dry DCM (10 ml)²³ was stirred for 2 h. The reaction was quenched with water (10 ml) and extracted with DCM (4 × 25 ml). The organic layer was washed with brine, dried over Na₂SO₄ and evaporated under vacuum. The crude product was purified through column chromatography to get compound 3 as a yellowish brown solid, 31%, mp 187–188 °C, ν_max/cm⁻¹: 3430 (OH), 1682 (C=O), δ_H (500 MHz; CDCl₃; Me₄Si) 2.38–2.41 (3H, m, CH₃), 2.42–2.44 (6H, m, 2× CH₃), 3.85–3.92 (12H, m, 4× OCH₃), 3.93–4.01 (6H, m, 2× OCH₃), 5.12–5.18 (6H, m, CH₂Ph), 5.90–5.93 (1H, m, ArH), 5.83 (1H, br, OH), 5.99–6.06 (3H, m, ArH),6.51–6.59 (3H, m, ArH), 6.74–6.78 (3H, m, ArH), 6.88–6.93 (5H, m, ArH), 7.34–7.47 (2H, m, ArH), 7.75–7.80 (4H, m, ArH), 7.94–7.95 (1H, m, ArH). Similar to compound 6, ¹H NMR spectrum of compound 3 also showed two sets of signals in their integral ratio 2 : 1; δ_C (normal/DEPT-135) (CDCl₃) 17.3 (+ve, CH₃), 40.5 (−ve, CH₂), 56.4 (+ve, OCH₃), 56.5 (+ve, OCH₃), 106.7 (+ve, CH), 109.2 (+ve, CH), 110.0 (+ve, CH), 118.2 (+ve, CH), 121.4 (+ve, CH), 122.3 (+ve, CH), 124.8 (C), 125.8 (C), 129.4 (+ve, CH), 131.7 (C), 136.5 (C), 137.2 (C), 138.3 (+ve, CH), 143.1 (C), 146.6 (C), 147.0 (C), 166.1 (C=O), 168.4 (C=O). HRMS (ESI) m/z for C₆₃H₅₇N₃O₁₂ (M + Na)⁺ calcd 1070.3798, found 1070.3810 (Fig. S14†).

3. Results and disscusion

Characterization of compound 3

NMR spectral data of compound 3 was investigated in detail so that its exact structure is defined. ¹H NMR spectrum of compound 3 showed two signals for OCH₃ protons with their intensity ratio 2 : 1 (Fig. S14b†). Characteristically, the two signals due to H-10 were observed at δ 7.78 and 7.35 and their integration ratio was also 2 : 1. To check if it is the presence of two isomeric forms of compound 3 (coming from 6-i and 6-ii) or the existence of compound 3 in the isomeric form corresponding to the precursor 6-iii which is responsible for its characteristic ¹H NMR spectrum, ROESY, HSQC and HMBC NMR spectra of compound 3 were recorded. ROESY spectrum of compound 3 clearly showed the presence of ROE between H-11 and H-17 as well as ROE between H-11 and H-24 (Fig. S14g†). These observations rule out the possibility of isomeric form 6-ii as the precursor of compound 3 and point towards the possibility of existence of this compound in one isomeric form only. Presence of two sets of H-10 signals in intensity ratio 2 : 1 (downfield H-10 signal corresponds to two protons) is probably due to the E-configuration at two olefinic groups (say A and B) and Z-configuration at olefinic group of fragment C. Therefore, two types of signals in the NMR spectra of compound 3 are due to the difference in the local environment of fragments A/B and C (Scheme 1). In contrast to two peaks in the HPLC of compound 4, compound 3 has a single peak (Fig. S14a†). Moreover, the HSQC and HMBC NMR spectra of compound 3 also showed one set of signals for the mesitylene unit (resonances corresponding to C1–C7) and two sets of signals for the remaining C/H resonances (Fig. S14k†). Detailed investigations of the HSQC and HMBC spectra of compound 3 indicated the correlations of H-20 with C-19, C-10, C-21, and C-17 in HMBC spectrum, and with C-20 in HSQC spectrum. It was observed that the C17–H20 cross-peak in HMBC and C20–H20 cross-peak in HSQC spectra are completely overlapping (encircled in dotted green circle) (Fig. S14l†), i.e., have same chemical shift. Since the proton is common in both cases, this is a clear indication that the ¹³C chemical shift of C-17 and C-20 are identical. In other words, these carbons experience same chemical environment. This is possible only if the hydroxyl group is located in a symmetrical fashion, i.e., in between the methoxy groups. The possibilities of putting this hydroxyl group in a non-symmetrical fashion will lead to significant differences in chemical shifts of C-17 and C-20 and hence, no overlap between C17–H20 and C20–H20 cross-peaks in HMBC and HSQC, respectively. Therefore, unambiguously the structure of compound 3 was established (Fig. S14j†).

Scheme 1 Synthesis of compound 3.

UV-vis spectral studies of compound 3: selective and competitive binding with CN⁻

Addition of 1 ml tap water (Table S1†) to 1 ml 200 nM solution of compound 3 in ultrapure water turned the color of the solution dark red. In order to check which salt of tap water is responsible for color change, a calculated amount of various salts (Table S2†) was added to the solution of compound 3. Interestingly, only the addition of 1 ml of 1.0 nM NaCN/CuCN/TBACN resulted into the appearance of orange color (Fig. S15†).

UV-vis spectrum of solution of compound 3 (1 μM, H₂O–DMSO (2 : 1), pH 7.0) showed decrease in absorbance at 269 and 359 nm with concomitant increase at 501 nm when 0.01 μM solution of NaCN was added stepwise (1–17 equiv.) (Fig. 1). Same observations were recorded when the titrations of compound 3 against CN⁻ were performed in dry CHCl₃ taking tetrabutylammonium cyanide (TBACN) as the source of CN⁻ (Fig. S16†). It was noticed that the color change and UV-vis spectral change in the solution of compound 3 during the presence of CN⁻ takes place at pH 5–8 indicating the suitability of working of compound 3 in the desirable physiological pH (upper digestive part in mammals exhibit slightly acidic pH). The selectivity of compound 3 for CN⁻ as well as its competitive binding with CN⁻ was evident from Fig. 2. Adduct of compound 3 with CN⁻ (3·CN⁻) exhibited 1 : 3 stoichiometry (Fig. S17†) and binding constant (K_a) was 1.5 × 10⁶ M⁻¹. The detection limit of this dendrimer for CN⁻ was found to be 5 nM (Fig. S18†). Therefore, in consistent with the design, compound 3 showed significantly efficient, competitive and selective response to CN⁻.

Fig. 1 Change in the UV-vis spectra of compound 3 (red trace) on incremental addition of CN⁻ (up to 17 equiv.). Inset: plot of absorption intensity at 501 nm vs. concentration of CN⁻.

Fig. 2 Monitoring of UV-vis absorbance of solution of compound 3 at 501 nm in the presence of anions other than CN⁻ (front bars) and in the presence of CN⁻ in combination with other anions (rear bars) showing respectively selective and competitive binding of CN⁻ with compound 3.

Removal of CN⁻ from blood serum

A sample of human blood serum containing NaCN was diluted in ultrapure water (10 ml, 1 nM CN⁻). The presence of 200 nM compound 3 (H₂O–DMSO, 2 : 1) turned the serum solution dark red. After extraction with ethyl acetate, the aqueous part did not respond to compound 3 indicating that there is no CN⁻ left in the aqueous part. Apparently, the CN⁻ was removed with ethyl acetate. However, extraction of the solution of blood serum and NaCN (without adding compound 3) with ethyl acetate and treatment of the aqueous part with compound 3 resulted into the color change of the solution to dark red. It means ethyl acetate did not remove NaCN alone from the blood serum. A comparison of IR spectra of compound 3 and the chemical species obtained after concentrating ethyl acetate extract showed the disappearance of OH peak and emergence of a new peak at 2087 cm⁻¹ in case of ethyl acetate extract (Fig. 3A and B). Apparently, CN get incorporated into compound 3. High resolution mass spectrum of the ethyl acetate extract gave characteristic mass peak at m/z 1192.3547 which corresponds to m/z of compound 7 (Fig. 3C, Scheme 2). It is proposed that CN⁻ might have reacted at all the phenolic groups of compound 3 to form compound 7 (Scheme 2). ¹H NMR spectra of compound 3 were recorded after stepwise addition of TBACN. It was observed that signal due to OH at δ 5.83 (D₂O exchange, Fig. S19 and S20†) got vanished on addition of 0.06 equiv. of CN⁻. The reaction of CN⁻ at the phenolic part of compound 3 and hence its mode of action was further confirmed by the treatment of compound 6 with CN⁻. No color and UV-vis spectral change was observed when NaCN was added to the H₂O–DMSO solution of compound 6. Comparison of scanning electron microscope images of compound 3 and compound 7 showed loss of aggregation of particles in compound 7 (Fig. S21†). This might be due to the replacement of OH groups with ONa in compound 7 and thereby disappearance of intermolecular H-bonding (Scheme S1†). Same observations were recorded when above experiments were performed by using CuCN in place of NaCN.

Fig. 3 (A) IR spectrum of compound 3; (B) IR spectrum of compound 7; (C) mass spectrum of ethyl acetate extract of solution of compound 3 with 6 equiv. NaCN showing formation of compound 7 with m/z 1192.3547 (calcd m/z 1192.3546 [M + H]⁺) (Scheme 1); (D) mass spectrum of EtOH–H₂O solution of compound 7 after stirring for 30 min.

Scheme 2 Mode of action of compound 3 with CN⁻, formation of compound 7 and subsequent formation of compound 8 at pH 9.5–10.0.

Ethanol–water (1 : 1) solution of compound 7 at pH 9.5–10 was stirred and the changes were monitored by high resolution mass spectrometry. After 30 min of stirring, a mass peak at m/z 1318.3057 was detected which indicated that the cyanide units were hydrolyzed to corresponding carboxyl groups and compound 8 was formed (calcd m/z 1318.3055 [M + H]⁺) (Fig. 3D). Continuous stirring of the solution for another 30 min and recording of mass spectra showed the breakdown of compound 7/8 to smaller components (Fig. S22, Chart S1†). Therefore, capturing of cyanide by compound 3, consequent formation of compound 7 and conversion of 7 to 8 under physiological pH (pH of lower intestine 9–10) resulted into the safe disposal of cyanide.

Removal of CN⁻ bound to cyt c oxidase

Since the blood serum contains Fe²⁺ in the porphyrin system, and the cyanide was probably bound to CcOX, it is apparent from the results of foregoing experiments that compound 3 has more affinity for cyanide in comparison to Fe²⁺. This was further checked by the enzyme immunoassay which was based on UV-vis monitoring of CcOX activity for oxidation of cyt c Fe²⁺ to Fe³⁺. Addition of 50 nM CcOX (diluted in enzyme buffer) to solution of cyt c in assay buffer made the UV-vis absorption band at 550 nm (due to Fe²⁺) to disappear (Fig. 4A). Michaelis–Menten constant (K_m) was 39 M⁻¹ (Fig. 4B). The presence of 2000 nM cyanide (NaCN) in the CcOX and cyt c assay buffer left the 550 nm band unchanged indicating the inhibition of CcOX activity (Fig. 4C and D). In consistent with the literature report,²⁴ K_i (inhibition constant) of cyanide for CcOX was 1.1 μM (Fig. S23†). The binding affinity (K_a) of cyanide for CcOX was found to be 3.8 × 10⁵ M⁻¹ which was relatively less than the binding affinity of cyanide with compound 3. However, the presence of compound 3 in the solution of CcOX did not affect its activity for cyt c indicating no interaction of compound 3 with the enzyme. Interestingly, when compound 3 was added to the assay buffer containing cyt c and cyanide bound CcOX, an increase in the enzymatic activity was attained as evident from the decrease in intensity of 550 nm absorption band (Fig. 4F). The activity of CcOX was fully recovered in the presence of 13 μM compound 3 (Fig. 4G). However, presence of compound 4 and 6 in the assay buffer did not recover the activity of cyanide bound CcOX whereas 50 μM of compound 2 was required for recovering the CcOX activity of 50 nM enzyme, inhibited by 2000 nM cyanide (Fig. 4G and S24†). UV-vis spectra of the assay buffer containing cty c, cyanide bound CcOX and compound 3 was recorded after every 2 min until there was no change in the 550 nm absorption band. It was noticed that the activity of CcOX started recovering after 2 min of addition of compound 3 and completely restored in 15 min (Fig. 4H). Earlier, the experiments on mice²⁰ showed that it takes 20–25 min for restoration of activity after the administration of cyanide antidote (NaNO₂/isoamyl nitrite). Found over human tumor cell lines, the LC₅₀ (concentration of the compound causing 50% death of the cells) of compound 3 was >10⁻⁴ M. It is worth to mention that as per the results of our experiments, ∼13 μM of compound 3 is sufficient to reverse the effect of 2000 nM of cyanide. This concentration of the compound 3 is much lower than its lethal dose.

Fig. 4 (A) UV-vis spectra of cyt c alone (red trace) and in presence of CcOX (green trace); (B) Lineweaver–Burk (double reciprocal) plot for enzymatic activity of CcOX; (C) UV-vis spectra showing inhibition of CcOX activity by CN⁻; (D) CcOX activity as a function of CN⁻ conc.; (E) time dependent change in the inhibition of CcOX activity in the presence of CN⁻; (F) stepwise addition of compound 3 to the solution of CN⁻ bound CcOX and cyt c. The absorption band at 550 nm corresponding to the oxidation of Fe²⁺ to Fe³⁺ of cyt c is undergoing stepwise change, (G) recovery of cyanide bound CcOX activity in presence of compound 2, 3, 4 and 6; (H) graph showing the time taken in recovering the CcOX activity in the presence of 13 μM compound 3. All the experiments were repeated and deviation of ±0.02 was noticed. Data shown here is average of the two experiments.

4. Conclusions

In accordance with the design; compound 3, containing three dimethoxyphenolic units exhibited better response to CN⁻ than its precursor 2 which has only one dimethoxyphenolic unit. Compound 3, exhibiting significant selectivity and competitive binding, removed CN⁻ from the blood serum. The activity of CN⁻ bound CcOX was also recovered in the presence of compound 3. Further studies of compound 3 for examining its effect on the cyanide poisoned animals are under investigation.

Acknowledgements

Financial assistance by DST, and CSIR, New Delhi is gratefully acknowledged. SK and AS thank CSIR and DST, New Delhi, respectively for fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra09658d

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