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

Article abstract of DOI:10.1039/c4ra09658d

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.

Full text of DOI:10.1039/c4ra09658d

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A rationally designed molecule for removal of
cyanide from human blood serum and cytochrome
c oxidase†
Cite this: RSC Adv., 2014, 4, 61884
Sukhmeet Kaur, Amrinder Singh, Venus Singh Mithu and Palwinder Singh*
Received 2nd September 2014
Accepted 3rd November 2014
Compound 3 having three dimethoxyphenolic units exhibited excellent selectivity and competitive binding
À
with CN . Various experiments revealed the capability of this compound to act through all the three
DOI: 10.1039/c4ra09658d
dimethoxyphenolic units and efficiently remove cyanide from human blood serum – more precisely
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from cytochrome c oxidase.
increasing the phenolic sites in the molecule, its sensitivity
for CN may get enhanced. As a result, less quantity of the
1
. Introduction
À
Besides the accidental cases of NaCN and KCN poisoning; compound will be required per unit of cyanide and conse-
1
gold mining, electroplating and metallurgical processes are quently the detection limit will improve. Replacement of
2
some of the common sources of exposure to cyanide and pyrazole moiety of compound 1 with oxindole in compound 2
À
once the permissible level of 1.9 mM (ref. 3) is exceeded, it did not change its behaviour towards CN . Mode of action as
À
proves lethal. The extreme toxicity of cyanide is attributed to well as the sensitivity of compound 2 for CN was similar to
2
+
its binding to Fe of cytochrome c oxidase (CcOX) and that observed for compound 1 (Fig. S1–S4†). Advantageously
consequent inhibition of mitochondrial electron transport compound 2 was further derivatized and as per our plan of
chain. This results in decreased oxidative metabolism and increasing the number of dimethoxyphenolic units in the
4
diminishing oxygen utilization.
molecule, compound 3 was prepared and it was checked for
À
À
Chemical capturing of CN and disposal of the product as detection/removal of CN from the biological medium.
safe metabolite/s is desirable to get rid of cyanide poisoning.
5
–12
Irrespective of a number of reports,
only a few of the
compounds including hydroxocobalamin, dicyano-cobalt(III)-
porphyrins, vitamin B12 analogues, metallophthalocyanines
1
3–18
and hexahydrated dichloride of cobalt(II) and nickel(II)

nd compatibility to the living system and hence safely used
À
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.
1
9,20
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.
Aer the recent disclosure of dimethoxyphenolic
2
1
substituted pyrazolone (1, Chart 1) for removal of cyanide
from the human blood serum, it was envisaged that by
Department of Chemistry, UGC Sponsored Centre for Advanced Studies, Guru Nanak
Dev University, Amritsar-143005, India. E-mail: palwinder_singh_2000@yahoo.com;
Fax: +91 183 2258819; Tel: +91 183 2258802 ext. 3495
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra09658d
Chart 1
1
61884 | RSC Adv., 2014, 4, 61884–61890
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H
(major)), 7.81 (1H, s, bridged H(minor)), 7.82 (1H, s, CH(minor)),
2
. Experimental section
7.84 (1H, s, CH(major)), 8.65 (1H, br, NH(minor)), 8.78 (1H, br,
Materials
NH(major)). Integration of the signals corresponding to major
Assay buffer, enzyme dilution buffer, dithiothretol solution, and minor isomers is in the ratio 3 : 1 (Fig. S10a†). HPLC of
cytochrome c, cytochrome c oxidase, dimethyl sulphoxide, compound 4 (Fig. S10b†) also showed the presence of two
mesitylene, oxindole, AlCl
TBACN.
3
, formaldehyde, HBr–AcOH, NaCN, isomers in the ratio 3 : 1.
Synthesis of tris-(bromomethyl)mesitylene) (5)
Synthesis of 3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-
-one (2)
To the mixture of mesitylene (2.4 g, 20 mmol), para-
formaldehyde (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 ltered, washed with water
2
A nely 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 Â
ꢀ
and dried in vacuum to obtain a white solid (91%), mp 185–
5
n
0 ml) to get pure product, 80%, yellow solid, mp 203–204 C,
ꢀ
À1
186 C, d
(
H
(300 MHz; CDCl
).
; Me Si) 4.57 (6H, s, CH Br), 2.46
3 4 2
max/cm : 3349 (NH), 3162 (OH), 1679 (C]O); dH (500 MHz;
9H, s, CH
3
CDCl ; Me Si) 3.95 (6H, s, OCH3(major)), 3.98 (6H, s, OCH3(minor)),
3
4
6
.85–6.96 (2H, m, ArH(major)), 6.99 (1H, br, OH), 7.01 (2H, m,
Synthesis of compound 6
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, Solution of compound 4 (100 mg, 0.32 mmol) and KOH (144 mg,
ArH(minor)), 7.63 (1H, s, bridged H(major)), 7.66 (1H, s, bridged 2.5 mmol) in DMSO (1 ml) was stirred for 5–10 min. Then
H
(minor)), 7.80–7.84 (1H, m, ArH(major)), 7.96–7.99 (1H, m, compound 5 (43 mg, 0.1 mmol) was added and stirred the
ArH(minor)), 8.28 (1H, s, CH(major)), 8.34 (1H, s, CH(minor)), 10.04 reaction mixture for 1 h (TLC). Aer completion of the reaction,
1H, br, NH(minor)), 10.05 (1H, br, NH(major)). d (normal/DEPT- it was quenched with water, solid separated out which was
(
C
1
1
1
(
1
1
35; CDCl3 + DMSO-d ) 61.1 (+ve, OCH ), 61.1 (+ve, OCH ), ltered and washed with water. The crude product was puried
6 3 3
12.0 (+ve, CH), 114.2 (+ve, CH), 115.0 (+ve, CH), 115.1 (+ve, CH), through column chromatography to get compound 6 as yellow
ꢀ
25.6 (+ve, CH), 125.7 (+ve, CH), 126.5 (C), 127.2 (+ve, CH), 127.3 solid, 60%, mp 172–173 C, d
(500 MHz; CDCl
), 3.85–3.86 (12H,
), 3.97–3.98 (6H, m,
Ph), 5.90–5.94 (1H, m, ArH),
; Me Si) 2.39–
H
3 4
+ve, CH), 128.8 (C), 129.8 (C), 129.9 (C), 130.5 (C), 130.6 (C), 2.41 (3H, m, CH
), 2.44–2.46 (6H, m, 2Â CH
), 3.92–3.93 (9H, m, 3Â OCH
), 5.14–5.18 (6H, m, CH
3
3
32.7 (+ve, CH), 134.1 (+ve, CH), 141.8 (+ve, CH), 141.9 (+ve, CH), m, 4Â OCH
3
3
42.5 (+ve, CH), 143.5 (C), 144.9 (C), 147.4 (C), 152.0 (C), 152.5 2Â OCH
3
2
(
(
C), 174.8 (C]O).174.8 (C]O). HRMS (ESI) m/z for C17
H
15NO
4
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–
.81 (2H, m, ArH), 7.84–7.85 (2H, m, ArH). H NMR spectrum of
+
M + H) calcd 298.1074, found 298.1071 (Fig. S5–S9†).
1
7
2
2
compound 6 showed two sets of signals in their integration
ratio 2 : 1; dC (normal/DEPT-135) (CDCl ) 16.4 (+ve, CH ), 17.3
Synthesis of 3,4,5-trimethoxybenzaldehyde
3
3
To the stirred solution of syringaldehyde (3 g, 16.5 mmol) in
DMF (40 ml); K CO (3.41 g, 1.5 equiv.), CH I (2.8 g, 19.71
(
+ve, CH ), 40.3 (Àve, CH ), 40.5 (Àve, CH ), 56.2 (+ve, OCH ),
3
2
2
3
2
3
3
60.9 (+ve, OCH
3
3
), 61.0 (+ve, OCH ), 106.7 (+ve, CH), 109.2 (+ve,
mmol) and KI (catalytic amount) were added. The reaction was
allowed to stir overnight at room temperature. Aer completion
of the reaction, it was quenched by adding water and extracted
with ethyl acetate (4 Â 25 ml). The organic layer was separated,
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
dried over Na
2
SO
4
and concentrated under vacuum to procure
(
(
+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]
ꢀ
pure product, creamish white solid (90%), mp 75–76 C, d
H
(500
),
(normal/DEPT-135)
), 131.7 (C), 143.6
MHz; CDCl
.13 (2H, s, ArH), 9.87 (1H, s, CHO); d
CDCl ) 56.2 (+ve, OCH ), 60.9 (+ve, OCH
C), 153.6 (C), 191.0 (C]O).
3
; Me
4
Si) 3.93 (6H, s, 2Â OCH
3 3
), 3.94 (3H, s, OCH
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-conguration (6-i) or
Z-conguration (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 conr-
mation about the isomeric form/s of this compound was made
aer its conversion to compound 3, the target compound
(Fig. S11–S13†).
7
C
(
(
3
3
3
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 puried by column chromatography to obtain pure
ꢀ
compound 4, yellow solid (80%), 145 C, d
H
(500 MHz; CDCl
3
;
Me Si) 3.90 (6H, s, OCH3(major)), 3.964 (3H, s, OCH3(major)) 3.968
4
Synthesis of compound 3
(3H, s, OCH3(minor)), 3.99 (6H, s, OCH3(minor)), 6.89–6.93 (3H, m,
ArH(major)), 6.94–6.95 (3H, m, ArH(minor)), 7.23–7.28 (2H, m, The reaction mixture obtained by the slow addition of anhy-
ArH(major)), 7.50–7.55 (2H, m, ArH(minor)), 7.78 (1H, s, bridged drous AlCl
3
(62 mg, 0.45 mmol) to solution of compound 6
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23
(
100 mg, 0.09 mmol) in dry DCM (10 ml) was stirred for 2 h. that the C17–H20 cross-peak in HMBC and C20–H20 cross-peak
The reaction was quenched with water (10 ml) and extracted in HSQC spectra are completely overlapping (encircled in dotted
with DCM (4 Â 25 ml). The organic layer was washed with brine, green circle) (Fig. S14l†), i.e., have same chemical shi. Since
dried over Na SO and evaporated under vacuum. The crude the proton is common in both cases, this is a clear indication
2
4
1
3
product was puried through column chromatography to get that the C chemical shi of C-17 and C-20 are identical. In
compound 3 as a yellowish brown solid, 31%, mp 187–188 C, other words, these carbons experience same chemical envi-
ꢀ
À1
n
max/cm : 3430 (OH), 1682 (C]O), d
H
(500 MHz; CDCl
), 2.42–2.44 (6H, m, 2Â CH ), 3.85–3.92 a symmetrical fashion, i.e., in between the methoxy groups. The
), 3.93–4.01 (6H, m, 2Â OCH ), 5.12–5.18 (6H, possibilities of putting this hydroxyl group in non-
m, CH Ph), 5.90–5.93 (1H, m, ArH), 5.83 (1H, br, OH), 5.99–6.06 symmetrical fashion will lead to signicant differences in
3 4
; Me Si) ronment. This is possible only if the hydroxyl group is located in
2
(
.38–2.41 (3H, m, CH
3
3
12H, m, 4Â OCH
3
3
a
2
(3H, m, ArH),6.51–6.59 (3H, m, ArH), 6.74–6.78 (3H, m, ArH), chemical shis of C-17 and C-20 and hence, no overlap between
6
.88–6.93 (5H, m, ArH), 7.34–7.47 (2H, m, ArH), 7.75–7.80 (4H, C17–H20 and C20–H20 cross-peaks in HMBC and HSQC,
1
m, ArH), 7.94–7.95 (1H, m, ArH). Similar to compound 6, H respectively. Therefore, unambiguously the structure of
NMR spectrum of compound 3 also showed two sets of signals compound 3 was established (Fig. S14j†).
in their integral ratio 2 : 1; d
+ve, CH ), 56.4 (+ve, OCH
06.7 (+ve, CH), 109.2 (+ve, CH), 110.0 (+ve, CH), 118.2 (+ve, CH),
21.4 (+ve, CH), 122.3 (+ve, CH), 124.8 (C), 125.8 (C), 129.4 (+ve,
C
(normal/DEPT-135) (CDCl
3
) 17.3
(
1
1
3
), 40.5 (Àve, CH
2
3
), 56.5 (+ve, OCH
3
),
UV-vis spectral studies of compound 3: selective and
competitive binding with CN
À
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 resul-
ted into the appearance of orange color (Fig. S15†).
CH), 131.7 (C), 136.5 (C), 137.2 (C), 138.3 (+ve, CH), 143.1 (C),
46.6 (C), 147.0 (C), 166.1 (C]O), 168.4 (C]O). HRMS (ESI) m/z
1
+
57 3
for C63H N O12 (M + Na) calcd 1070.3798, found 1070.3810
(Fig. S14†).
3. Results and disscusion
UV-vis spectrum of solution of compound 3 (1 mM, H O–
Characterization of compound 3
2
DMSO (2 : 1), pH 7.0) showed decrease in absorbance at 269 and
NMR spectral data of compound 3 was investigated in detail so
that its exact structure is dened. H NMR spectrum of
compound 3 showed two signals for OCH protons with their
3
359 nm with concomitant increase at 501 nm when 0.01 mM
1
solution of NaCN was added stepwise (1–17 equiv.) (Fig. 1).
Same observations were recorded when the titrations of
intensity ratio 2 : 1 (Fig. S14b†). Characteristically, the two
signals due to H-10 were observed at d 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 corre-
sponding to the precursor 6-iii which is responsible for its
À
compound 3 against CN were performed in dry CHCl
3
taking
À
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
À
presence of CN takes place at pH 5–8 indicating the suitability
of working of compound 3 in the desirable physiological pH
1
characteristic H NMR spectrum, ROESY, HSQC and HMBC
(
upper digestive part in mammals exhibit slightly acidic pH).
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 possi-
bility of existence of this compound in one isomeric form only.
Presence of two sets of H-10 signals in intensity ratio 2 : 1
À
The selectivity of compound 3 for CN as well as its competitive
binding with CN was evident from Fig. 2. Adduct of compound
3
À
À
À
with CN (3$CN ) exhibited 1 : 3 stoichiometry (Fig. S17†) and
6
À1
binding constant (K ) was 1.5 Â 10 M . The detection limit of
a
À
this dendrimer for CN was found to be 5 nM (Fig. S18†).
Therefore, in consistent with the design, compound 3 showed
signicantly efficient, competitive and selective response to
(downeld H-10 signal corresponds to two protons) is probably
À
CN .
due to the E-conguration at two olenic groups (say A and B)
and Z-conguration at olenic group of fragment C. Therefore,
two types of signals in the NMR spectra of compound 3 are due
À
Removal of CN from blood serum
to the difference in the local environment of fragments A/B and A sample of human blood serum containing NaCN was
À
C (Scheme 1). In contrast to two peaks in the HPLC of diluted in ultrapure water (10 ml, 1 nM CN ). The presence of
2
compound 4, compound 3 has a single peak (Fig. S14a†). 200 nM compound 3 (H O–DMSO, 2 : 1) turned the serum
Moreover, the HSQC and HMBC NMR spectra of compound 3 solution dark red. Aer extraction with ethyl acetate, the
also showed one set of signals for the mesitylene unit (reso- aqueous part did not respond to compound 3 indicating that
À
À
nances corresponding to C1–C7) and two sets of signals for the there is no CN le in the aqueous part. Apparently, the CN
remaining C/H resonances (Fig. S14k†). Detailed investigations was removed with ethyl acetate. However, extraction of the
of the HSQC and HMBC spectra of compound 3 indicated the solution of blood serum and NaCN (without adding
correlations of H-20 with C-19, C-10, C-21, and C-17 in HMBC compound 3) with ethyl acetate and treatment of the aqueous
spectrum, and with C-20 in HSQC spectrum. It was observed part with compound 3 resulted into the color change of the
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Scheme 1 Synthesis of compound 3.
solution to dark red. It means ethyl acetate did not remove compound 3 were recorded aer stepwise addition of TBACN.
NaCN alone from the blood serum. A comparison of IR It was observed that signal due to OH at d 5.83 (D O exchange,
2
spectra of compound 3 and the chemical species obtained Fig. S19 and S20†) got vanished on addition of 0.06 equiv. of
À
À
aer concentrating ethyl acetate extract showed the disap- CN . The reaction of CN at the phenolic part of compound 3
pearance of OH peak and emergence of a new peak at 2087 and hence its mode of action was further conrmed by the
À1
À
cm in case of ethyl acetate extract (Fig. 3A and B). Appar- treatment of compound 6 with CN . No color and UV-vis
ently, CN get incorporated into compound 3. High resolution spectral change was observed when NaCN was added to the
mass spectrum of the ethyl acetate extract gave characteristic H O–DMSO solution of compound 6. Comparison of scan-
2
mass peak at m/z 1192.3547 which corresponds to m/z of ning electron microscope images of compound 3 and
À
compound 7 (Fig. 3C, Scheme 2). It is proposed that CN
compound 7 showed loss of aggregation of particles in
might have reacted at all the phenolic groups of compound 3 compound 7 (Fig. S21†). This might be due to the replace-
1
to form compound 7 (Scheme 2). H NMR spectra of ment of OH groups with ONa in compound 7 and thereby
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Fig. 1 Change in the UV-vis spectra of compound 3 (red trace) on
À
incremental addition of CN (up to 17 equiv.). Inset: plot of absorption
À
intensity at 501 nm vs. concentration of CN .
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
+
1
192.3547 (calcd m/z 1192.3546 [M + H] ) (Scheme 1); (D) mass spec-
2
trum of EtOH–H O solution of compound 7 after stirring for 30 min.
Fig. 2 Monitoring of UV-vis absorbance of solution of compound 3 at
À
5
01 nm in the presence of anions other than CN (front bars) and in the
À
presence of CN in combination with other anions (rear bars) showing
respectively selective and competitive binding of CN with compound 3.
À
disappearance of intermolecular H-bonding (Scheme S1†).
Same observations were recorded when above experiments
were performed by using CuCN in place of NaCN.
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. Aer 30 min of stirring, a mass peak at m/z
À
Scheme 2 Mode of action of compound 3 with CN , formation of
compound 7 and subsequent formation of compound 8 at pH 9.5–
1318.3057 was detected which indicated that the cyanide units
were hydrolyzed to corresponding carboxyl groups and
1
0.0.
+
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 from the results of foregoing experiments that compound 3 has
2
+
compound 7/8 to smaller components (Fig. S22, Chart S1†). more affinity for cyanide in comparison to Fe . This was further
Therefore, capturing of cyanide by compound 3, consequent checked by the enzyme immunoassay which was based on
2
+
formation of compound 7 and conversion of 7 to 8 under UV-vis monitoring of CcOX activity for oxidation of cyt c Fe to
3
+
physiological pH (pH of lower intestine 9–10) resulted into the Fe . Addition of 50 nM CcOX (diluted in enzyme buffer) to
safe disposal of cyanide.
solution of cyt c in assay buffer made the UV-vis absorption
2
+
band at 550 nm (due to Fe ) to disappear (Fig. 4A). Michaelis–
À1
Menten constant (K ) was 39 M (Fig. 4B). The presence of
m
À
Removal of CN bound to cyt c oxidase
2000 nM cyanide (NaCN) in the CcOX and cyt c assay buffer le
2
+
Since the blood serum contains Fe in the porphyrin system, the 550 nm band unchanged indicating the inhibition of CcOX
24
and the cyanide was probably bound to CcOX, it is apparent activity (Fig. 4C and D). In consistent with the literature report,
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550 nm absorption band. It was noticed that the activity of CcOX
started recovering aer 2 min of addition of compound 3 and
completely restored in 15 min (Fig. 4H). Earlier, the experi-
20
ments on mice showed that it takes 20–25 min for restoration
of activity aer the administration of cyanide antidote (NaNO
isoamyl nitrite). Found over human tumor cell lines, the LC50
2
/
(
concentration of the compound causing 50% death of the cells)
À4
of compound 3 was >10 M. It is worth to mention that as per
the results of our experiments, ꢁ13 mM 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.
4. Conclusions
In accordance with the design; compound 3, containing three
À
dimethoxyphenolic units exhibited better response to CN than
its precursor 2 which has only one dimethoxyphenolic unit.
Compound 3, exhibiting signicant selectivity and competitive
À
binding, removed CN from the blood serum. The activity of
À
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
Fig. 4 (A) UV-vis spectra of cyt c alone (red trace) and in presence of Financial assistance by DST, and CSIR, New Delhi is gratefully
CcOX (green trace); (B) Lineweaver–Burk (double reciprocal) plot for
enzymatic activity of CcOX; (C) UV-vis spectra showing inhibition of
acknowledged. SK and AS thank CSIR and DST, New Delhi,
respectively for fellowship.
À
À
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
À
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 cor-
Notes and references
À
2+
3+
responding to the oxidation of Fe to Fe 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 mM compound 3.
All the experiments were repeated and deviation of Æ0.02 was noticed.
Data shown here is average of the two experiments.
1
C. A. Young, L. G. Twidwell and C. G. Anderson, Cyanide:
Social, Industrial, and Economic Aspects, TMS (The Minerals,
Metals & Materials Society), 2001.
2
R. Koeing, Science, 2000, 287, 1737–1738.
3 Guidelines for Drinking Water Quality, World Health
Organization, Geneva, 1996.
4
(a) S. I. Baskin, J. B. Kelly, B. I. Maliner, G. A. Rockwood and
C. K. Zoltani, Medical Aspects of Chemical Warfare, ed. S. D.
Tuorinsky, TMM publications, Washington, DC, 2008, ch.
Ki (inhibition constant) of cyanide for CcOX was 1.1 mM
(
Fig. S23†). The binding affinity (K ) of cyanide for CcOX was
a
5
À1
found to be 3.8 Â 10 M 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
mM 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 mM 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 aer every 2 min until there was no change in the
11, pp. 371–410; (b) J. L. Way, Annu. Rev. Pharmacol.
Toxicol., 1984, 24, 451–481.
M. R. Ajayakumar, P. Mukhopadhyay, S. Yadav and S. Ghosh,
Org. Lett., 2010, 12, 2646–2649.
5
6
D.-S. Kim, Y.-M. Chung, M. Jun and K. H. Ahn, J. Org. Chem.,
2009, 74, 4849–4854.
7
8
Y. Sun, G. Wang and W. Guo, Tetrahedron, 2009, 65, 3480.
K. Watanabe, H. Kitagishi and K. Kano, ACS Med. Chem. Lett.,
2011, 2, 943–947.
9
S. H. Kim, S. J. Hong, J. Yoo, S. K. Kim, J. L. Sessler and
C.-H. Lee, Org. Lett., 2009, 11, 3626–3629.
1
1
0 C. M ¨a nnel-Crois ´e and F. Zelder, Inorg. Chem., 2009, 48,
272–1274.
1 S. E. Patterson, A. R. Monteil, J. F. Cohen, D. L. Crankshaw,
1
R. Vince and H. T. Nagasawa, J. Med. Chem., 2013, 56, 1346–
1349.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 61884–61890 | 61889

View Article Online
RSC Advances
Paper
1
1
1
1
2 R. Bhattacharya, N. Gopalan, A. N. Singh, P. Singh and 19 L. L. Pearce, E. L. Bominaar, B. C. Hill and J. Peterson, J. Biol.
S. K. Yadav, Pharm. Biol., 2014, 52, 137–143. Chem., 2003, 278, 52139–52145.
3 P. Hambright and R. Langley, J. Inorg. Biochem., 1988, 32, 20 L. K. Cambal, A. C. Weitz, H. H. Li, Y. Zhang, X. Zheng,
1
97–205.
L. L. Pearce and J. Peterson, Chem. Res. Toxicol., 2013, 26,
828–836.
4 O. S. Benz, Q. Yuan, A. A. Amoscato, L. L. Pearce and
J. Peterson, Chem. Res. Toxicol., 2012, 25, 2678–2686.
21 P. Singh and M. Kaur, Chem. Commun., 2011, 47, 9122–9124.
5 J. L. Fortin, S. Waroux, J. P. Giocanti, G. Capellier, 22 A. M. Deveau, N. E. Costa, E. M. Joshi and T. L. Macdonald,
M. Ruttimann and J. J. Kowalski, J. Emerg. Med., 2010, 39,
20–324.
6 W. W. Reenstra and W. P. Jencks, J. Am. Chem. Soc., 1979,
01, 5780–5791.
7 Y. H. Kim and J. I. Hong, Chem. Commun., 2002, 512–513.
Bioorg. Med. Chem. Lett., 2008, 18, 3522–3525.
3
23 A. Shirali, M. Sriram, J. J. Hall, B. L. Nguyen,
R. Guddneppanavar, J. F. Ackley, C. J. Jelinek, R. C. Brown,
V. L. Murrell, A. McMordie, S. Sharma, D. J. Chaplin and
K. G. Pinney, J. Nat. Prod., 2009, 72, 414–421.
1
1
1
1
8 P. Anzenbacher, D. S. Tyson, K. Jurs ´ı kov ´a and 24 E. Antonini, M. Brunori, C. Greenwood, B. G. Malmstrom
F. N. Castellano, J. Am. Chem. Soc., 2002, 124, 6232–6233.
and G. C. Rotilio, Eur. J. Biochem., 1971, 23, 396–400.
61890 | RSC Adv., 2014, 4, 61884–61890
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