Kinetics of the Reaction between Cobinamide and Isoniazid in Aqueous Solutions

Article abstract of DOI:10.1134/S0036024419020274

Abstract: The kinetics and mechanism of the reaction of diaquacobinamide (Cbi(III)) with isoniazid (iso-nicotinoyl hydrazide (INH)) are studied. It is determined that the composition of the products depends on the ratio between the concentrations of the reactants. Adding excess INH to cobinamide results in the rapid formation of a stable complex of Cbi(III) with two isoniazid molecules. If the concentrations of isoniazid and cobinamide are close, or cobinamide is in excess, then a complex of Cbi(III) with one isoniazid molecule initially forms. There is then a fast inner-sphere electron transfer to yield an unstable complex of reduced Cbi(II) with hydrazyl radical (RN₂H₂)(Cbi(II)) that decomposes to form reduced cobinamide and the products of the oxidation of isoniazid: isonicotinamide, pyridine-4-carboxaldehyde, and isonicotinic acid (INA). It is concluded that with a 1000% excess of cobinamide, the main product of the oxidation of INH is INA.

Full text of DOI:10.1134/S0036024419020274

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2019, Vol. 93, No. 2, pp. 265–270. © Pleiades Publishing, Ltd., 2019.
Russian Text © S.O. Tumakov, I.A. Dereven’kov, D.S. Sal’nikov, S.V. Makarov, 2019, published in Zhurnal Fizicheskoi Khimii, 2019, Vol. 93, No. 2, pp. 230–236.
PHYSICAL CHEMISTRY
OF SOLUTIONS
Kinetics of the Reaction between Cobinamide and Isoniazid
in Aqueous Solutions
S. O. Tumakova, I. A. Dereven’kova, D. S. Sal’nikova,*, and S. V. Makarova
aIvanovo State University of Chemistry and Technology, Ivanovo, 153000 Russia
*е-mail: densal@isuct.ru
Received April 18, 2018
Abstract—The kinetics and mechanism of the reaction of diaquacobinamide (Cbi(III)) with isoniazid (iso-
nicotinoyl hydrazide (INH)) are studied. It is determined that the composition of the products depends on
the ratio between the concentrations of the reactants. Adding excess INH to cobinamide results in the rapid
formation of a stable complex of Cbi(III) with two isoniazid molecules. If the concentrations of isoniazid and
cobinamide are close, or cobinamide is in excess, then a complex of Cbi(III) with one isoniazid molecule ini-
tially forms. There is then a fast inner-sphere electron transfer to yield an unstable complex of reduced Cbi(II)
with hydrazyl radical (RN2H2)(Cbi(II)) that decomposes to form reduced cobinamide and the products of
the oxidation of isoniazid: isonicotinamide, pyridine-4-carboxaldehyde, and isonicotinic acid (INA). It is
concluded that with a 1000% excess of cobinamide, the main product of the oxidation of INH is INA.
Keywords: kinetics, reaction mechanism, antidote, cobinamide, isoniazid
DOI: 10.1134/S0036024419020274
INTRODUCTION
quacobinamide is a more efficient antidote for cyanide
[10, 11] and hydrogen sulfide [12–15] poisoning than
aquacobalamin, and it is an efficient NO scavenger [16].
In [17], we studied the interaction between vitamin
B12 and isoniazid. It was shown that during the reac-
tion cobalamin reversibly binds with the neutral INH
molecule. However, we failed to find information on
Poisoning with toxic alcohols, cyanide, hydrogen
sulfide, and carbon monoxide, along with intentional
or unintentional overdoses of medications, are the
second leading cause of injury-related morbidity and
mortality [1, 2]. One way of preventing poisoning is to
use special substances (antidotes) that can reduce or
eliminate the effects of poisons on the human body. the interaction between isoniazid and cobinamide.
There are several mechanisms behind the action of
antidotes: (1) interacting with poisons to form non-
toxic substances, (2) accelerating the removal of poi-
sons from the body, (3) reducing the rate of the trans-
formation of poison into more toxic substances, (4)
competing with the poison for essential receptor sites,
and (5) blocking essential receptor sites that enhance
the toxic effect of the poison [1, 3].
Isoniazid (isonicotinoyl hydrazide (INH))
(Fig. 1a) is a highly efficient antituberculosis drug [4,
5] on the list of vital and essential medicines. Among
clinical issues in using INH are its neuro- and hepato-
toxicity [4, 5]. An overdose of isoniazid adversely
affects the human body [6]. It is recommended that
pyridoxal (vitamin B6) be used to reduce its toxic effect
[7]. However, there are literature data that testify to the
ineffectiveness of using pyridoxal to treat isoniazid
overdoses [8].
The aim of this work was to find the kinetic param-
eters and mechanism behind the reaction between iso-
niazid and cobinamide.
EXPERIMENTAL
Isoniazid (>98%), isonicotinic acid (99%), and
isonicotinamide (99%) (Alfa Aesar) were used in our
experiments. Diaquacobinamide was synthesized
according to the procedure in [18]. The other reagents
were of chemically pure grade.
The products of the reaction of isoniazid with
cobinamide (Cbi(III)) in a neutral medium were
determined as follows. An aqueous isoniazid solution
was added to an aqueous cobinamide solution under
anaerobic conditions. The Cbi(III)-to-INH ratio in
the obtained solution was 1 : 2 and 4 : 1. This solution
was kept for several hours until the completion of the
reaction. The solution was then passed through a silica
gel (1 g) column and eluted with 100 mL of distilled
water. Cobinamide was adsorbed and retained by silica
Cobinamide (Fig. 1b) is a precursor of vitamin B12
(cobalamin (Cbl)) during its biosynthesis [9]. Cobin-
amide differs from vitamin B12 by the absence of a ribo-
nucleotide fragment. It has been determined that dia- gel, while isoniazid and the products of its oxidation
265

266
TUMAKOV et al.
(a)
O
(b)
O
NH2
O
NH2
O
H2N
H2N
NH2
OH2
N
N
N
N
O
O
NH2
N
H
Co
H
N
H2O
NH2
O
O
NH
OH
Fig. 1. Structures of (a) isoniazid and (b) diaquacobinamide.
were eluted from the column. The aqueous solution using a 1-cm sealed quartz cell. Fast reactions were stud-
passed through the chromatographic column and was ied using an RX2000 rapid mixing stopped-flow unit.
evaporated in vacuum at 40°C until a dry residue
formed. The residue was dissolved in ethanol (95 vol %)
and analyzed via gas chromatography–mass spec-
trometry on a Shimadzu GCMS-QP2010 Ultra gas
chromatograph–mass spectrometer using a Zebron
ZB-5ms capillary column. The mobile phase was
helium (30 mL/min). The column temperature
(120°C) was held constant using a thermostat.
The anaerobic conditions were created using
argon.
The rate of the reaction between cobinamide and
isoniazid was determined from the results of measur-
ing the absorbance at wavelengths of 412, 460, 520,
and 537 nm.
RESULTS AND DISCUSSION
Kinetic studies were performed under anaerobic
conditions with a Varian Cary 50 spectrophotometer
It was found that adding excess isoniazid to an
aqueous cobinamide solution changes the color of the
solution from red to yellow in the pH range of 4–13.
Analysis of the UV–Vis spectra recorded during the
reaction of cobinamide with isoniazid at pH ≥7.5
revealed two successive events (Fig. 2).
A
1.2
The first event was very fast and depended on the
isoniazid concentration. At INH concentrations of
<1 mM, the peaks of the initial cobinamide at 348,
495, and 517 nm vanished (Fig. 2, spectrum 1), and
new peaks emerged at 350 and 497 nm (Fig. 2, spec-
trum 2). At high (>5 mM) isoniazid concentrations,
peaks at 355, 507, and 532 nm emerge (Fig. 2, spec-
trum 3). Such changes are characteristic of the reac-
tions of complexation of cobinamide with different
ligands (L): thiocyanate [19], cyanamide [20], hydro-
gen sulfide [13], and cyanide [21]. In these reactions,
coordinated water molecules are successively substi-
tuted to form LCbi(III) and (L)2Cbi(III).
0.8
4
2
3
0.4
0
1
300
400
500
600
λ, nm
Fig. 2. UV–Vis spectra recorded during the reaction
between cobinamide and isoniazid at pH 7.5: (1) initial
The second event was accompanied by the vanish-
ing of the peaks of the first intermediate and the emer-
gence of new peaks at 315 and 469 nm (Fig. 2, spec-
trum 4). The final spectrum of the reaction product
cobinamide, (2, 3) intermediates, and (4) final product;
−5
[Cbi(III)] = 5 × 10 M; [INH] = (2) 1 and (3) 50 mM
0
0
at 25°C under anaerobic conditions.
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KINETICS OF THE REACTION BETWEEN COBINAMIDE AND ISONIAZID
267
Overall Stoichiometry of the Reaction
A470
0.9
Figure 3 presents the results from spectrophoto-
metric titration of cobinamide with isoniazid under
anaerobic conditions.
It was found that the full reduction of Cbi(III) was
attained at [IHN] : [Cbi(III)] = 1 : 4.
Note that in redox reactions, isoniazid acts as a
one- or four-electron reducer [23, 24].
0.7
0.5
Determining Products of the Reaction between Isoniazid
and Cobinamide in a Neutral Medium
Gas chromatography–mass spectrometry was used
to determine the products of the reaction of isoniazid
with cobinamide in a neutral medium: isonicotinic
acid, isonicotinamide, and pyridine-4-carboxalde-
hyde (Table 1). The initial isoniazid was not found in
the studied sample, indicating the isoniazid was fully
oxidized.
0
0.4
0.8
1.2
[INH]0/[Cbi(III)]0
Fig. 3. Spectrophotometric titration of cobinamide with
isoniazid at pH 7.5.
From the areas under the peaks, it was found that at
[Cbi(III)] : [IHN] = 1 : 2, the main product is isonic-
otinamide (68%, Table 1); at [Cbi(III)] : [IHN] = 4 :
1, it is isonicotinic acid (90%, Table 1).
Similar changes were observed in the oxidation of
isoniazid by catalase peroxidase from Mycobacterium
tuberculosis [25].
corresponded to the reduced form of cobinamide Co2+
(Cbi(II)) [13, 22].
When pH is reduced to 4.5, the reaction between iso-
niazid and cobinamide proceeds in one step and is
accompanied by the vanishing of the peaks of the initial
cobinamide (349 and 520 nm) and the emergence of new
peaks (315 and 469 nm) corresponding to Cbi(II).
The overall scheme of the oxidation of isoniazid by
This shows that cobinamide acts as an oxidant of cobinamide in aqueous solutions under anaerobic
isoniazid over a wide range of pH.
conditions at pH 7.5 can thus be presented as
O
O
O
O
NH2
N
(1)
OH
NH2
H
+
+
H
Cbi, H2O
N
N
N
N
Kinetic Studies
It is seen from Fig. 4 that the initial absorbance
grows along with the isoniazid concentration. This is
explained by the formation of a complex of cobin-
amide with two INH molecules (Fig. 2). It was also
found that the drop in the absorbance (which corre-
sponds to the accumulation of the reduced form of
cobinamide (Cbi(II))) slows as the excess of isoniazid
with respect to cobinamide grows.
We failed to study the kinetics of the reaction of com-
plexation of cobinamide with isoniazid because this reac-
tion is very fast and finishes in the time it takes to mix the
reagents throughout the investigated pH range.
Figure 4 shows typical kinetic curves of cobinamide
reduction.
Table 1. Characteristics of the products of the reaction
Yield, %
Rt, min
Substance
m/z
[Cbi(III)] : [INH] = 1 : 2 [Cbi(III)] : [INH] = 4 : 1
Pyridine-4-carboxaldehyde
Isonicotinamide
Isonicotinic acid
Isoniazid
2.170
3.537
107
122
123
137
16
68
16
—
–
10
90
—
4.346
16.257
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268
A535
TUMAKOV et al.
A535
1.20
0.4
1.15
4
1.10
0.3
1.05
0.2
0.1
3
1.00
0.95
2
1
0
500
1000
1500
τ, s
0
200
400
600
800
τ, s
Fig. 5. Typical kinetic curve of the reduction of cobin-
Fig. 4. Kinetic curves of the reduction of cobinamide with
–5
amide by isoniazid with a deficiency of the latter (second
an excess of isoniazid: [Cbi(III)] = 5 × 10 M; [INH] =
0
0
−4
−5
step): [Cbi(III)] = 1.5 × 10 M; [INH] = 1.25 × 10
M
(1) 0.25, (2) 1, (3) 5, and (4) 50 mM at 25°C and pH 7.5
0
0
at 25°C and pH 7.5 under anaerobic conditions.
under anaerobic conditions.
Co(III) and prevents reduction. Note that similar
The kinetic curves were linearized in coordinates of
(A0 – At)/(Af – At) versus time and are described by the results were obtained for the interaction between
Cbi(III) and hydrogen sulfide [13].
equation
Figure 5 shows a typical kinetic curve of the oxida-
tion of INH by an excess of Cbi(III). Processing of the
kinetic data in semilog coordinates showed that the
A0 + ktAf [Cbi(III)]0
1 + kt[Cbi(III)]0
where A0, At, and Af are the initial, current, and final
absorbances, respectively; [Cbi(III)]0 is the total
cobinamide concentration in the reaction, M; k is the
second-order reaction rate constant; and t is time, sug-
gesting that the order of the reaction with respect to
cobinamide is 2 (kobs2, M−1 s−1) [26].
The dependence of kobs2 on the isoniazid concen-
tration at pH 7.5 is nonlinear; kobs2 falls as the isoniazid
concentration grows (Table 2).
The drop in the rate of the cobinamide reduction
reactions upon a simultaneous increase in the concen-
(2)
A =
,
t
partial order of the reaction was 1 ( ' , s−1).
kobs2
The dependence of the observed rate constant for
the oxidation of INH on the Cbi(III) concentration is
nonlinear, but it can be linearized in coordinates of
'
versus [Cbi(III)]2. This indicates the order of the
kobs2
reaction with respect to cobinamide was 2. Linear fit-
ting of this dependence allowed us to calculate third-
order constant k' = (4.55 0.2) × 105 M−2 s−1 at pH 7.5
and 25°C under anaerobic conditions.
Our kinetic studies thus demonstrated that two
tration of the complex of cobinamide(III) with two Cbi(III) molecules are required for the formation of
isoniazid molecules shows that adding two INH mol- reduced cobinamide, and the reaction is described by
ecules to Cbi(III) stabilizes the oxidation state of the kinetic equation
kobs2 = k'[Cbi(III)]2,
(3)
H2O
H2O
Co(III)
OH
OH
pKa1
pKa2
,
(4)
(5)
Co(III)
H2O
Co(III)
OH
+H+
+H+
O
O
O
O−
NH+3
NH+3
NH2
N
N−
pKa3
pKa4
pKa5
N
H
N
H
,
H
+H+
+H+
+H+
HN
N
N
N
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KINETICS OF THE REACTION BETWEEN COBINAMIDE AND ISONIAZID
269
(6)
H2O
Co(III)
OH
RN2H3
Co(III)
OH
RN2H3
Co(III)
H2O
k1
pKa6
−H+
,
+ RN2H3
+ H2O
RN2H3
Co(III)
H2O
RN2H3
Co(III)
RN2H3
k2
+ RN2H3
+ H2O,
(7)
(8)
RN2H3
Co(III)
H2O
RN•2H3
Co(II)
RN•2H2
Co(II)
k3
pKa7
+ H2O
Co(II)
+ H+,
•
RN2H2
(9)
k4
Co(II)
2
2
+ 2RNH2 + N2,
RN•2H3
H2O
k5
+ 2RN2H2 + H+ + 2H2O,
+ ROH + H+ + 4H2O + N2.
(10)
Co(II)
Co(II)
Co(III)
+
2
H2O
H2O
k6
Co(II)
Co(III)
H2O
+ RN2H2
2
2
(11)
In an aqueous solution, cobinamide exists as dia-
quacobinamide (H2O)2Cbi(III), aquahydroxocobin-
Based on the data on the products and stoichiome-
try of the reaction and the results from kinetic studies,
we can propose the following mechanism of the infor-
mation of isoniazid with cobinamide in a neutral
medium:
amide (H2O)(OH–)Cbi(III), and the dihydroxo form
(HO–)2Cbi(III) with pKa1 = 5.9 and pKa2 = 10.2 (reac-
tion (4)) [13]. Note that only diaquacobinamide and
aquahydroxocobinamide participate in the complex-
ation reactions, since the hydroxide ion is inert to sub-
stitution in the inner sphere of the complex.
This mechanism includes reversible steps of
sequential binding of two molecules of isoniazid
(RN2H3) by cobinamide (reactions (6) and (7)). The
complex with one isoniazid molecule is unstable;
there is an electron transfer to form a complex of
reduced Cbi(II) with hydrazyl radical (reaction (8)).
The bonding of the second isoniazid molecule (reac-
tion (7)) prevents reaction (8) and stabilizes the 3+
oxidation state of cobalt in cobinamide. The forma-
tion of the complex of cobinamide with two isoniazid
molecules shows that the main form produced after
INH also has acid–base properties and exists in the
solution in four forms (reaction (5)) with pKa3 = 1.99,
pKa4 = 3.67, and pKa5 = 10.89 [23]. At physiological
pH values, isoniazid is primarily in the neutral form
(RN2H3, where R is acyl of isonicotinic acid).
adding
the
first
INH
molecule
is
Table 2. Dependence of kobs2 on the isoniazid concentra-
tion (pH 7.5, 25°C; anaerobic conditions)
(RN2H3)(H2O)Cbi(III), since OH– is inert to substi-
tution.
kobs2, M–1 s–1
[INH], mM
The complex (RN2H2)(Cbi(II)) of reduced Cbi(II)
with the hydrazyl radical is unstable and decomposes.
The difference between the reaction products suggests
this process proceeds through two mechanisms. The
first is the disproportionation of this complex to form
two Cbi(II) molecules, isonicotinamide (RNH2), and
0.25
1
5
659
546
119
7.8
50
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TUMAKOV et al.
2. J. M. Marraffa, V. Cohen, and M. A. Howland, Am. J.
nitrogen (reaction (9)). The second is the oxidation of
complex (RN2H2)(Cbi(II)) by a second Cbi(III) mole-
cule to form two Cbi(II) molecules and the product of
the two-electron oxidation of isoniazid (reaction (10)).
In both cases, the reaction order with respect to cobin-
amide is two because the rate-determining step
involves two cobinamide molecules.
Health Syst. Pharm. 69, 199 (2012).
3. S. Bradberry and A. Vale, Medicine 35, 562 (2007).
4. P. Preziosi, Curr. Drug. Metab. 8, 839 (2007).
5. P. Wang, Acta Pharm Sin. B 6, 384 (2016).
6. R. C. Watkins, E. L. Hambrick, G. Benjamin, and
S. N. Chavda, J. Natl. Med. Assoc. 82, 57 (1990).
The product of the two-electron oxidation of isoni-
azid (RN2H2) is unstable and rapidly oxidized to ison-
icotinic acid (ROH) and nitrogen by an excess of
cobinamide (reaction (11)).
7. M. L. Sievers and R. N. Herrier, Am. J. Hosp. Pharm
32, 202 (1975).
8. W. P. Tai, H. Yue, and P. J. Hu, Adv. Ther. 25, 1085
(2008).
9. A. I. Scott and C. A. Roessner, Biochem. Soc. Trans.
CONCLUSIONS
30, 613 (2002).
10. K. E. Broderick et al., Exp. Biol. Med. 231, 641 (2006).
11. A. Chan et al., Clin. Toxicol. 48, 709 (2010).
12. D. S. Salnikov, P. N. Kucherenko, I. A. Dereven’kov,
et al., Eur. J. Inorg. Chem., 852 (2014).
13. D. S. Salnikov, S. V. Makarov, R. van Eldik, et al., Eur.
J. Inorg. Chem., 4123 (2014).
It was shown in this work that at near-physiological
pH values, cobinamide can rapidly form a complex
with one or two isoniazid molecules. The binding of
isoniazid to cobinamide under the same conditions
(pH 7.5, 25°C) is approximately 300 times faster than
the binding by aquacobalamin (vitamin B12) [17]. The
complex of Cbi(III) with one isoniazid molecule
(which is formed primarily when the isoniazid and
cobinamide concentrations are close, or when cobin-
amide is in excess) is unstable and forms reduced
cobinamide and products of the oxidation of isonia-
zid: isonicotinic acid, isonicotinamide, and pyridine-
4-carboxaldehyde. The main product of the oxidation
of INH by an excess of cobinamide is isonicotinic
acid, which is less toxic than INH. The formation of
INA also testifies to the ability of cobinamide to oxi-
dize active metabolites (radical forms) of INH, which
form via the oxidation of INH and inactivate biologi-
cal substances [27–29].
14. M. Brenner, S. Benavides, S. B. Mahon, et al., Clin.
Toxicol. 52, 490 (2014).
15. J. Jiang, A. Chan, S. Ali, et al., Sci. Rep. 6, 20831
(2016).
16. K. E. Broderick, V. Singh, S. Zhuang, et al., J. Biol.
Chem. 280, 8678 (2005).
17. S. O. Tumakov, I. A. Dereven’kov, D. S. Salnikov, and
S. V. Makarov, Russ. J. Phys. Chem. A 91, 1839 (2017).
18. W. C. Blackledge, A. Griesel, S. B. Mahon, et al., Anal.
Chem. 82, 4216 (2010).
19. I. A. Dereven’kov, D. S. Salnikov, S. V. Makarov, et al.,
J. Inorg. Biochem. 125, 32 (2013).
Unlike cobinamide, aquacobalamin cannot oxi-
dize INH [17].
20. P. N. Kucherenko, D. S. Salnikov, Thu Thuy Bui, et al.,
Macroheterocycles 6, 262 (2013).
The complex of cobinamide with two INH mole-
cules (which forms in a large excess of isoniazid) is sta-
ble at pH 7.5 and 25°C; there is no oxidation of isoni-
azid.
21. W. C. Blackledge, C. W. Blackledge, A. Griesel, et al.,
Anal. Chem. 82, 4216 (2010).
22. I. A. Dereven’kov, D. S. Salnikov, R. Silaghi-Dumi-
trescu, et al., Coord. Chem. Rev. 309, 68 (2016).
23. J. Dong, Y. Ren, S. Sun, et al., Dalton Trans. 46, 8377
(2017).
ACKNOWLEDGMENTS
24. V. Bogdándi, G. Lente, and I. Fábián, RSC Adv. 5,
This work was supported by the Russian Science
Foundation, project no. 14-23-00204 P. The authors
thank researcher N. Pechnikova at the Laboratory of
Gas Chromatography, Mass Spectrometry, and EPR
Spectrometry, Shared Scientific Resource Center,
Ivanovo State University of Chemistry and Technol-
ogy, Ivanovo, Russia, for her help in analyzing the
products of the oxidation of isoniazid via gas chroma-
tography–mass spectrometry.
67500 (2015).
25. K. Johnsson and P. G. Schultz, J. Am. Chem. Soc. 116,
7425 (1994).
26. M. J. Hynes, Proc. R. Irish Acad., Sect. B: Biol., Geol.
Chem. Sci. 89, 435 (1989).
27. B. K. Sinha and R. P. Mason, J. Drug. Metab. Toxicol.
5, 168 (2014).
28. I. Metushi, J. Uetrecht, and E. Phillips, Br. J. Clin.
Pharmacol. 81, 1030 (2016).
29. L. V. Forbes et al., Biochem. Pharmacol. 84, 949
REFERENCES
(2012).
1. Reference Guide for Emergency Doctor, Ed. by A. L. Vertkin
Translated by V. Glyanchenko
(GEOTAR-MED, Moscow, 2001) [in Russian].
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