J Biol Inorg Chem (2012) 17:275–283
DOI 10.1007/s00775-011-0848-x
ORIGINAL PAPER
Isoniazid metal complex reactivity and insights
for a novel anti-tuberculosis drug design
Eduardo Henrique Silva Sousa • Luiz Augusto Basso • Diógenes S. Santos
Izaura Cirino Nogueira Diógenes • Elisane Longhinotti •
Luiz Gonzaga de França Lopes • Ícaro de Sousa Moreira
Received: 27 May 2011 / Accepted: 10 September 2011 / Published online: 28 September 2011
Ó SBIC 2011
Abstract For over a decade, tuberculosis (TB) has been the
leading cause of death among infectious diseases. Since
the 1950s, isoniazid has been used as a front-line drug in the
treatment of TB; however, resistant TB strains have limited
its use. The major route of isoniazid resistance relies on KatG
enzyme disruption, which does not promote an electron
transfer reaction. Here, we investigated the reactivity of
isoniazid metal complexes as prototypes for novel self-
activating metallodrugs against TB with the aim to overcome
resistance. Reactivity studies were conducted with hydrogen
peroxide, hexacyanoferrate(III), and aquopentacyanofer-
rate(III). The latter species showed a preference for the inner-
sphere electron transfer reaction pathway. Additionally,
electron transfer reaction performed with either free isonia-
zid or (isoniazid)pentacyanoferrate(II) complex resulted in
similar oxidized isoniazid derivatives as observed when the
In memory of Ícaro de Sousa Moreira.
E. H. S. Sousa (&)  I. C. N. Diógenes  E. Longhinotti 
L. G. de França Lopes  Í. de Sousa Moreira
Departamento de Quı́mica Orgânica e Inorgânica,
Laboratório de Bioinorgânica,
Universidade Federal do Ceará,
P.O. Box 6021,
Fortaleza 60455-970, Brazil
e-mail: eduardohss@dqoi.ufc.br
L. A. Basso  D. S. Santos
Centro de Pesquisas em Biologia Molecular e Funcional,
Pontifı́cia Universidade Católica do Rio Grande do Sul, Av.
Ipiranga, 6681, Tecnopuc, Prédio 92A Partenon,
Porto Alegre, RS 90619-900, Brazil
L. A. Basso  D. S. Santos  L. G. de França Lopes
Instituto Nacional de Ciência e Tecnologia em Tuberculose,
Pontifı́cia Universidade Católica do Rio Grande do Sul,
Porto Alegre, Brazil
•
KatG enzyme was used. However, upon metal coordination,
a significant enhancement in the formation of isonicotinic
acid was observed compared with that of isonicotinamide.
These results suggest that the pathway of a carbonyl-cen-
tered radical might be favored upon coordination to the
Fe(II) owing to the p-back-bonding effect promoted by this
metal center; therefore, the isoniazid metal complex could
serve as a potential metallodrug. Enzymatic inhibition assays
conducted with InhA showed that the cyanoferrate moiety is
not the major player involved in this inhibition but the pres-
ence of isoniazid is required in this process. Other isoniazid
metal complexes, [Ru(CN)5(izd)]3- and [Ru(NH3)5(izd)]2?
(where izd is isoniazid), were also unable to inhibit InhA,
supporting our proposed self-activating mechanism of
action. We propose that isoniazid reactivity can be rationally
modulated by metal coordination chemistry, leading to the
development of novel anti-TB metallodrugs.
Keywords Tuberculosis  Isoniazid  Metallodrugs 
Enoyl reductase  Cyanoferrate
Abbreviations
DMSO Dimethyl sulfoxide
HPLC High-performance liquid chromatography
IQG607 Sodium (isoniazid)pentacyanoferrate(II)
TB Tuberculosis
Introduction
Mycobacterium tuberculosis infection has affected mankind
for centuries, leading to the death of billions of people.
Extremely efficient therapeutic compounds were discovered
in the mid-1900, leading to great expectation of eradicating
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tuberculosis (TB) [1]. Unfortunately, this goal has not been
attained and a worse-case scenario has emerged in which TB
has become the leading cause of death worldwide compared
with other known infectious diseases. It latently infects about
two billion people, one third of the world’s population,
creating a huge reservoir of this bacterium [2]. Each year,
there are about eight million new TB cases and two million
deaths [3]. The growing number of multiple-drug-resistant
M. tuberculosis strains and the recently discovered extre-
mely drug resistant strains are a cause for alarm among health
agencies around the world. According to the WHO, multiple-
drug-resistant M. tuberculosis strains are found in at least 72
countries, accounting for 3–41% of TB cases [4]. In 2005, it
was reported that there were 27,000 cases of extremely drug-
resistant TB in developed and underdeveloped countries [5].
Despite the fact that TB can be cured using therapy
involving multiple drugs, the minimum treatment time of
6 months is too long and the drugs are highly toxic. These
factors make patient compliance with therapy very
difficult, and as a result, this noncompliance leads to drug-
resistant M. tuberculosis. Since the most recently devel-
oped TB drug is about 40 years old, there is a strong
requirement for the development of new drugs that would
shorten the treatment time, lower the toxicity levels, and
yet be effective against the various resistant strains [3, 6].
Isoniazid is the front-line and most effective anti-TB drug
discovered so far, along with rifampin. The discovery of
isoniazid was a major milestone in chemotherapy for TB
because it was highly active, inexpensive, and relatively less
toxic [7]. Its molecular mechanism of action was unknown
for decades long after its first clinical use, but a more detailed
picture has emerged during recent decades [8]. At least five
different genes (katG, inhA, ahpC, kasA, and ndh) have been
found to correlate with isoniazid resistance [6]. However, the
primary target of isoniazid has been shown to be the product
of the inhA structural gene [9]. A proteomic approach using
isoniazid adducts was conducted, which led to the identifi-
cation of a panel of other promising potential isoniazid tar-
gets in M. tuberculosis [10]; hence, this suggests that there
may be a broader spectrum of isoniazid anti-TB action. In
addition, there is a search for novel molecular anti-TB targets
for a generation of new drugs, including those potentially
involved in M. tuberculosis latency [11–13].
It has been shown that isoniazid is a prodrug that is
activated by an electron transfer reaction catalyzed by the
catalase–peroxidase KatG [14]. This reaction can generate
a range of reactive oxygen species and reactive organic
radicals, which can attack multiple targets in M. tubercu-
losis [3]. Several reports have suggested that the main
mechanism for isoniazid is based on the formation of a
radical intermediate species [15, 16]. This isonicotinic acyl
radical would promptly react with NAD(H) and produced a
NAD–isoniazid adduct [15]. It has been shown this adduct
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J Biol Inorg Chem (2012) 17:275–283
works as an efficient inhibitor for InhA enzyme
(Kd = 0.4 nM) in a slow-onset inhibition step [17]. This
protein is an enoyl reductase enzyme involved in one of the
steps for mycolic acid biosynthesis and it is a very efficient
therapeutic target.
Approximately 50% of isolated isoniazid-resistant
strains have either a deletion or mutations in the katG gene
[18, 19]. It appears that the enzymatic activation of isoni-
azid through an electron transfer reaction is a major step in
TB resistance, as described elsewhere [20]. These reports
along with our previous chemical and biochemical data
prompted us to design novel ‘‘self-activating metallodrugs’’
for TB treatment, which would potentially overcome iso-
niazid resistance. Isoniazid is an acylhydrazine pyridine
derivative and, from an inorganic chemistry view point, has
appealing properties. For decades, pyridine derivatives
have been shown to form stable and well-characterized
complexes with cyanoferrates [21–24]. Moreover, there
have been a number of reports on electron transfer reac-
tions involving cyanoferrate complexes [21, 25–27]. Oxi-
dation of many organic ligands exhibits large kinetic
barriers, which are usually dramatically lowered by
metalloenzymes. Intramolecular electron transfer reactions
promoted by metals could be used as a strategy to mimic
biocatalysts. This strategy has just recently gained appeal
in metallodrug design. Isoniazid requires enzymatic catal-
ysis to promote an electron transfer reaction leading to drug
activation; a coordinated metal complex could produce a
faster alternative route for intramolecular-assisted ligand
oxidation. On the basis of this, we have asked the question
whether the cyanoferrate moiety could indeed favor a faster
intramolecular electron transfer reaction of isoniazid in
support of this strategy. Additionally, we evaluated how
similar these oxidized species were to enzymatically and
nonenzymatically generated active species and what their
implications are for the design of a novel anti-TB drug. It
was shown that sodium (isoniazid)pentacyanoferrate(II)
(IQG607) could efficiently inhibit the TB target enzyme
(InhA) in vitro and kill M. tuberculosis, including isoni-
azid-resistant strains [28–30]. A mechanism of action has
been proposed that is based on the chemical reactivity of
this complex along with biochemical and biological data
supporting this compound as a prototype for metallodrugs
against M. tuberculosis. However, there was no clear evi-
dence that supported the mechanism mentioned above.
Materials and methods
Materials
All experiments were performed under an inert atmosphere
(N2 or Ar) using conventional techniques. Milli-Q grade or
J Biol Inorg Chem (2012) 17:275–283
doubly distilled water was used throughout. Pyridine
derivatives (isonicotinamide, isonicotinic acid, 4-pyri-
dinemethanol, 4-pyridinecarboxaldehyde) used as stan-
dards in high-performance liquid chromatography (HPLC)
along with NAD? and NADH were purchased from Sigma.
Isoniazid, from Aldrich, was recrystallized in methanol
solution. Potassium hexacyanoferrate(III) was purchased
from Mallinckrodt. Na3[FeII(CN)5(NH3)]3H2O and
Na2[FeIII(CN)5(NH3)]H2O complexes were synthesized as
previously described [29, 30] with minor modifications
[20]. All other chemicals were of analytical reagent grade.
Synthesis of Na3[Fe(CN)5(izd)]4H2O (IQG607)
A 100.0 mg sample of Na3[Fe(CN)5(NH3)]3H2O was
dissolved in 5 mL of water under argon flow. The isoniazid
ligand (90.0 mg), dissolved in 2 mL of water, was added to
the complex solution. Upon mixing, the resulting solution
immediately developed an orange color and was allowed to
react for 2 h under argon, with stirring, and in the
absence of light. The reaction vessel was then cooled in an
ice bath, and cold NaI-saturated solution in ethanol was
added dropwise until precipitation was complete.
Na3[Fe(CN)5(izd)]4H2O (IQG607; where izd is isoniazid)
as a red solid was collected by filtration, washed with
ethanol and diethyl ether, dried, and stored under a vacuum
in the absence of light. Anal. Calc.: C: 28.4; H: 3.2; N: 24;
Found: C: 28.0; H: 3.5; N: 23.7. 1H NMR: d(D2O, 500
MHz) 9.11 (d, 2H, H-2,20 ), 7.41 (d, 2H, H-3,30 ). The yield
was greater than 85%.
Reaction of isoniazid and Na2[FeIII(CN)5(NH3)]H2O
A 100.0-mg sample of Na2[Fe(CN)5(NH3)]H2O was dissolved
in 5 mL of water. The isoniazid ligand (100.0 mg), dissolved in
2 mL of water, was added to the complex solution. Upon
mixing, bubbling of gas and a color change occurred immedi-
ately. The reaction was allowed to proceed for 2 h under argon,
with stirring, and in the absence of light. The reaction vessel
was then cooled in an ice bath, and ice-cold 1:2 diethyl ether/
ethanol was added dropwise until precipitation was complete.
The orange solid was collected by filtration, washed with eth-
anol and diethyl ether, dried, and stored under a vacuum in the
absence of light. The yield was better than 85%. Formula
proposed: Na3[FeII(CN)5(isn)]3H2O (where isn is isonicoti-
namide) Anal. Calc.: C: 30.6; H: 2.8; N: 22.7; Found: C: 30.0;
H: 2.6; N: 23.0. 1H NMR: d(D2O, 500 MHz) 9.18 (d, 2H,
H-2,20 ), 7.57 (d, 2H, H-3,30 ).
Apparatus
The electronic absorption spectra were acquired with a
Hewlett-Packard 8453 diode-array scanning spectrophoto-
277
meter in the UV and visible range. A 1.0-cm quartz cell
was used and sample concentrations were kept between
1.0 9 10-3 and 1.0 9 10-2 M for most of the experiments
unless otherwise stated. IR spectra were taken with a Shi-
madzu FTIR-8300 spectrophotometer, using solid samples
dispersed in KBr pellets. Electrochemical experiments
were conducted with a BAS 100BW electrochemical ana-
lyzer (Bioanalytical Systems, West Lafayette, IN, USA).
A conventional three-electrode glass configuration cell with
a glassy carbon electrode of 0.126-cm2 geometrical area, and
a platinum foil were used as working and auxiliary elec-
trodes, respectively. A 0.1 M sodium trifluoroacetate solu-
tion, pH 5.0, was used as the electrolyte, at 25 °C. Potential
values were measured versus Ag/AgCl (3.5 M KCl, Bioan-
alytical Systems) as a reference electrode. All potentials
were converted to the normal hydrogen electrode and are
cited accordingly throughout the text. The Mössbauer
spectra were obtained with a conventional constant accel-
eration spectrometer operating in a triangular wave mode
with a 57Co source in a rhodium matrix. All spectra were
taken with the source and absorber at room temperature, and
sodium nitroprusside was used as a reference.
Chromatography measurements were conducted with a
Shimadzu liquid chromatograph (HPLC) equipped with a
model LC-10AD pump and an SPD-M10A UV–vis pho-
todiode-array detector with a CBM-10AD interface. An
octadecyl silica column (250 mm 9 4.6-mm inner diame-
ter, 5-lm particles, from Alltech) was employed and
injection volumes of 5 lL were used for most of the
analyses unless otherwise stated.
Kinetic measurements
Outer-sphere electron transfer reaction rates were deter-
mined using a Hewlett-Packard 8453 spectrophotometer at
25 °C. Solutions of the reactants were freshly prepared
under an argon atmosphere for each run. Buffer solutions
consisting of a mixture of K2HPO4 and KH2PO4 were used.
The ionic strength of the solutions (0.5 M) was adjusted by
the addition of KCl. The pH measurements were performed
with a Corning 440 pH meter equipped with a Cole-Parmer
glass electrode. Values of kobs were calculated from a least-
squares fitting to the equation ln(At - A?) = ln(Ao -
A?) - kobst, where At, Ao, and A? are absorbances at time
t, at time zero (first point), and at longer times (four or
more half lives), respectively. The calculations were done
using the Hewlett-Packard 8453 spectrophotometer
kinetic software. The reaction progress was followed at
420 nm, the absorbance maximum of [Fe(CN)6]3-, under
pseudo-first order conditions. Kinetic measurements for
the intramolecular reaction were performed with an
Aminco-Morrow 126550-3 stopped-flow spectrophoto-
meter.
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Reactions with hexacyanoferrate(III)
The reactions of isoniazid with hexacyanoferrate(III) in
excess were performed in buffered solution, pH 7.0, fol-
lowed by electronic spectroscopy (k = 420 nm) until
completion. The products of this reaction were also char-
acterized using HPLC. We injected 5 lL of the product
reaction mixture into the HPLC system and separated the
mixture by isocratic elution. Standards were injected to
identify the products formed.
The reaction of 2.0 mM NAD? and isoniazid, in buf-
fered phosphate solution pH 7.0, was started by addition of
4 mM hexacyanoferrate(III) and the reaction was allowed
to proceed for 1 h. This mixture was loaded on a C18
reverse-phase column and separated by a gradient elution
of 5–60% acetonitrile/water over a period of 40 min. The
adduct products were identified by the appearance of two
new peaks with typical electronic spectra. Standard mix-
tures of only two of the three components were prepared to
characterize and validate the appearance of the peak.
After mixing, we loaded three control mixtures: (1)
NAD? and hexacyanoferrate(III), (2) NAD? and isoniazid,
and (3) isoniazid and hexacyanoferrate(III), and their peaks
were identified and excluded from the reaction conducted
with the three components [NAD?, isoniazid, and hexa-
cyanoferrate(III)].
Reactions with hydrogen peroxide
Free and coordinated isoniazid (1.0 mM) were oxidized
using 66.0 mM hydrogen peroxide solution, in phosphate
buffer pH 7.0, at 25 °C and the reaction was allowed to
proceed for 6 h. Their oxidized products were identified on
the basis of HPLC injections of standards. The H2O2-oxi-
dized isoniazid iron complex was subjected to a 100-fold
concentration excess of dimethyl sulfoxide (DMSO) to
ensure complete ligand displacement, and to prevent any
further oxidation reaction, ascorbic acid was added to the
mixture. This mixture was loaded on the HPLC column and
the organic products released from the complex were
promptly characterized by the injection of standards.
Enzyme inhibition assays
Wild-type InhA and I21V InhA were expressed, purified,
and characterized as described in the literature [28]. Inhi-
bition assays were performed as described elsewhere
[28, 31]. Briefly, experiments on time-dependent inacti-
vation of the enoyl reductases were conducted in
100.0 mM NaH2PO4/Na2HPO4, pH 7.5 at 25 °C, with
either 3.0 lM wild-type InhA or 3.0 lM I21V, in the presence
of 100.0 lM complexes. Aliquots were taken at specified
times, and steady-state enzyme activities were determined
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J Biol Inorg Chem (2012) 17:275–283
from the rates of decrease in absorbance at 340 nm using
2-trans-dodecenoyl-CoA (100.0 lM) and NADH (200 lM).
The substrate 2-trans-dodecenoyl-CoA was synthesized
from 2-trans-dodecenoic acid and CoA by the mixed anhy-
dride method [28, 31] and was purified by reverse-phase
HPLC using a 19 mm 9 300 mm C18 lBondapak column
(Waters Associates, Milford, MA, USA) as previously
described [31]. The ratio of the absorbance of purified
2-trans-dodecenoyl-CoA at 232 and 260 nm was 0.62, a
value that meets the established criterion for pure thioesters
(A232 nm/A260 nm C 0.52). Wild-type InhA and I21V mutant
enzymes were expressed and purified to homogeneity as
described elsewhere [28, 29]. The specific activities of wild-
type InhA and I21V mutant with 2-trans-dodecenoyl-CoA
(100 lM) and NADH (200.0 lM) were 13.5 ± 0.4 and
8.1 ± 0.3 U mg-1, respectively. Activity measurements for
both wild-type and I21V InhA proteins were performed for
up to 1 h in 100 mM Na2HPO4, pH 7.5 at 25 °C, but no loss
of enzyme activity was observed in the time range studied.
Results and discussion
A product of the reaction of isoniazid
with [FeIII(CN)5(NH3)]2-
On the basis of previous observations of electron transfer
reactions involving isoniazid-like compounds, we conducted
a direct reaction of isoniazid with [FeIII(CN)5(NH3)]2-. The
final product was isolated and further analyzed. This isolated
metal complex showed the presence of a peak profile com-
patible with diamagnetic iron(II) with predominant signals at
d 9.18 and d 7.57, which, respectively, correspond to the
H-2,20 and H-3,30 of a pyridine derivative such as isonico-
tinamide. Additionally, this result indicates that the coordi-
nation occurs through the nitrogen atom of the pyridine ring.
The IR spectroscopy measurements were also consistent
with a reduced pentacyanoferrate(II) complex as assigned by
mCN and mFe–C at 2,046 and 569 cm-1, respectively [20, 21,
32]. The electronic spectrum showed an intense solvent-
dependent band with a maximum at 438 nm (e = 4.8 9
103 M-1 cm-1) assigned to a metal-to-ligand charge trans-
fer transition, which matches a band found in the (isonico-
tinamide)pentacyanoferrate(II) complex [22]. Furthermore,
the cyclic voltammogram with E1/2 at 578 mV was consis-
tent with an isonicotinamide ligand bound to a pentacyano-
ferrate(II) complex.
HPLC was used to further identify and quantify the
production of pyridine derivatives. It is well known that
pentacyanoferrate complexes are very hydrophilic and their
separation on reverse-phase columns is poor and close to
the void time; hence, a new strategy was developed to take
full advantage of this technique. Since organic ligands
J Biol Inorg Chem (2012) 17:275–283
usually interact well with C18 reverse-phase columns, the
strategy was to dislodge these ligands from the pentacy-
anoferrate moiety for this study. Taking into account the
well-known use of DMSO in substitution reactions of
pentacyanoferrate(II) complexes [33, 34], we made the
isolated complex react with DMSO in a pseudo-first-order
condition and investigated the products by HPLC. The
DMSO molecule displaces the sixth ligand, thus forming
[Fe(CN)5(dmso)]3- in a reaction in which the aquation is
the rate-determining step. Two peaks assigned to isonico-
tinamide (major, over 90%) and isonicotinic acid (minor)
were detected, meaning that the reaction of pentacyano-
ferrate(III) with isoniazid resulted in the formation of
mainly [Fe(CN)5(isn)]3- (where isn is isonicotinamide)
and impurities of [Fe(CN)5(isa)]3- (where isa is isonicot-
inic acid). These results are consistent with the mechanism
suggested for acylhydrazine decomposition [35] and are
in agreement with isoniazid oxidation investigated using
mycobacterial enzymes and manganese compounds
[35, 36].
To further provide solid evidence for the iron redox state
in this isolated complex, Mössbauer spectroscopy was
employed. The Mössbauer spectra of iron pentacyano
complexes are shown as doublets owing to the presence of
an electronic field gradient with noncubic symmetry in the
57
Fe nucleus [37]. We observed a doublet with an isomer
shift (d) and quadrupolar splitting (D) of 0.229 ± 0.003
and 0.893 ± 0.004 mm/s, respectively, which is a typical
profile for low-spin iron(II) cyano complexes. Our results
are consistent with NMR profiles, which indicate charac-
teristics of diamagnetic species.
Kinetics of the electron transfer reaction
As mentioned earlier, an electron transfer process is
involved in the reaction of isoniazid with
[FeIII(CN)5(H2O)]2- (note ammonia is quickly released
upon dissolution in water, resulting in the aquo compound).
This reaction can occur through an inner-sphere or an
outer-sphere mechanism, although a mixture of both is also
possible. To study this mechanism, stopped-flow mea-
surements were performed under pseudo-first-order reac-
tion conditions for isoniazid at 438 nm (pH 7.0). kobs of
12 s-1 was determined and was independent of the con-
centration, indicating an inner-sphere mechanism. To fur-
ther validate the data, the contribution of an outer-sphere
mechanism using hexacyanoferrate(III) ([FeIII(CN)6]3-)
was examined. This compound, with E1/2 = 450 mV,
should react with isoniazid through the outer-sphere path-
way faster than [FeIII(CN)5(H2O)]2- (E1/2 = 390 mV) on
the basis of Marcus theory. The kinetic data for the hexa-
cyanoferrate reaction showed a second-order rate with kobs
dependent on the pH (k = 0.5 and 41 M-1 s-1 for pH 7.0
279
and 8.0, respectively). Therefore, the reaction with
[FeIII(CN)5(H2O)]2- is at least 1,000 times faster than the
outer-sphere electron transfer reaction using [FeIII(CN)6]3-
under identical conditions. Thus, it supports an inner-
sphere mechanism where isoniazid is oxidized by
[FeIII(CN)5(H2O)]2- producing [FeII(CN)5(izd*)]3-, where
izd* are products of oxidation, such as isonicotinamide and
isonicotinic acid. Another relevant point in this study is a
significant pH dependence for outer-sphere oxidation. As
far as we know, no connection between mycobacterial
enzymatic oxidation and acid–base assistance has been
reported. Previously, we reported that pyridine thioamide
compounds showed a similar pH-dependent behavior, but
at higher pH [20]. Thus, it is evident that a base assistance
can accelerate catalytic oxidation of these compounds,
which might also take place in the active sites of the
activating enzymes KatG and EthA. Isoniazid-activating
enzyme KatG, a heme catalase–peroxidase, has a con-
served amino acid triad of arginine–tryptophan–histidine in
the active site, which could work well in acid–base catal-
ysis. The X-ray structures available for three KatG
enzymes from M. tuberculosis [Protein Data Bank (PDB)
ID 1SJ2)], Haloarcula marismortui (PDB ID 1ITK), and
Burkholderia pseudomallei (PDB ID 1MWV) showed that
these residues are close to the heme group. In addition,
Wengenack et al. [38] showed by NMR spectroscopy that
the acylhydrazine group of isoniazid is close to the heme
center of KatG, which would support a hypothesis of
acid–base assistance during catalysis.
Isoniazid and metal complex oxidations
The reaction of isoniazid with an excess of [FeIII(CN)6]3-
was monitored by HPLC using a UV–vis diode array.
Figure 1 shows the chromatogram of the final products,
where four peaks with retention times of 2.3, 3.2, 5.2, and
10 min were observed and assigned to [FeIII(CN)6]3-,
isonicotinic acid, isonicotinamide, and 4-pyridinecarbo-
xaldehyde, respectively, on the basis of the electronic
spectra and injection of standards. These pyridine deriva-
tives matched those observed for in vivo and in vitro
enzymatic oxidation of isoniazid [35, 36, 39, 40]. Inter-
estingly, the relative proportion of isonicotinamide and
isonicotinic acid switched on altering the isoniazid
concentration and this indicates that isonicotinic acid pro-
duction strongly decreased at a concentration above
20 mM, whereas isonicotinamide production increased.
This behavior has also been observed for enzymatic reac-
tion of KatG, which highlights a suitable use of this
oxidizing reactant for modeling and reactivity comparison
[35, 40].
Manganese complexes have been used as a model
reactant for the isoniazid activation through oxidation
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Fig. 1 Chromatogram for the reaction of isoniazid (2 mM) with
hexacyanoferrate(II), in molar excess, pH 7.0. Isocratic elution at
10% acetonitrile pH 3.5, flow rate of 1.0 mL min-1, monitored at
260 nm, after 1 h of reaction at 25 °C
[35, 36]. This reaction conducted in the presence of NAD?
leads to the formation of NAD–isoniazid adducts. To
evaluate the potential use of [FeIII(CN)6]3- instead of
manganese complexes, isoniazid was mixed with NAD?,
which triggered a reaction upon addition of [FeIII(CN)6]3-
at pH 7.0. The chromatogram showed two new peaks at 7.1
and 8.2 min, which were not seen in the standards or
control mixtures (isoniazid plus [FeIII(CN)6]3-, NAD? plus
isoniazid, or NAD? plus [FeIII(CN)6]3-) examined for
identical incubation time and medium. Those peaks were
found in the electronic spectra, which showed typical bands
at 260 and 330 nm consistent with dihydropyridine com-
pounds. In addition, A260 nm/A330 nm absorbance ratios of
about 3.2 were obtained, which is consistent with NAD–
isoniazid adducts described in the literature [15, 41].
Optimization of product formation and the conditions to
obtain better yields is clearly necessary and is under way.
However, this result suggests the possibility of employing a
very common and clean outer-sphere oxidizing compound,
[FeIII(CN)6]3-, to prepare these important adducts.
Oxidative reaction of isoniazid (1 mM) promoted by
hydrogen peroxide (66 mM) exhibited kinetics slower than
initially expected in comparison with [FeIII(CN)6]3- oxi-
dation (Fig. 2). This was also found in previous reports,
where it was reported that a slow reaction with hydrogen
peroxide was indeed accelerated by the catalytic effect of
the catalase–peroxidase KatG enzyme [14]. In the work
reported in the literature, 200 lM isoniazid and 25 mM
H2O2 in the presence of KatG was used and yet still
resulted in incomplete reaction even after 6 h [14]. The
reaction performed in our studies produced isonicotin-
amide as the major product despite it being used at rela-
tively low isoniazid concentration. To evaluate and
compare the reactivity of free isoniazid and isoniazid
coordinated to pentacyanoferrate(II), the oxidation of this
metal complex with hydrogen peroxide was conducted.
The peaks assigned to isonicotinic acid, isonicotinamide,
and 4-pyridinecarboxaldehyde were observed in the
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J Biol Inorg Chem (2012) 17:275–283
Fig. 2 Chromatogram for the reaction of hydrogen peroxide
(66 mM) with isoniazid (1 mM) (a) and (isoniazid)pentacyanofer-
rate(II) (b). Isocratic elution at 10% acetonitrile pH 3.5, flow rate of
1.0 mL min-1, monitored at 260 nm, after 6 h of reaction at 25 °C
chromatograms obtained for both reactions, indicating
general similarities (Fig. 2). However, remarkable changes
in the ratio of isonicotinic acid to isonicotinamide might
reflect a preferential reaction pathway, suggesting that the
metal bonding could be influencing the isoniazid reactivity.
For free isoniazid, the relative area of the peak assigned to
isonicotinamide is more intense than that of isonicotinic
acid. However, the opposite profile was seen for the reaction
containing isoniazid coordinated to pentacyanoferrate(II)
([FeII(CN)5(izd)]3-). Also, after 5 h of reaction more iso-
niazid was oxidized in the reaction with the complex than in
the reaction with the free ligand (Fig. 2). Two pathways
have been proposed for the formation of isonicotinamide
and isonicotinic acid and are based on the origin of the
radical produced as well as the initial concentration of
isoniazid [35]. Isonicotinamide is produced by a nitrogen-
based radical that decomposes by a bimolecular reaction,
whereas isonicotinic acid is produced by an acyl-based
radical in a reaction with water. When this latter radical is
engaged in resonance, the p-back-bonding effect is a rea-
sonable explanation for the observed result. This effect may
favor the stabilization of p-resonant radicals, thus enhanc-
ing the formation of the acyl radical. Hence, the p-back-
bonding phenomenon manifested in pentacyanoferrate(II)
complexes can be used as a way to adjust isoniazid
reactivity. One interesting example is 4-pyridine-
carboxaldehyde, which in solution is found to react with
water and reaches an equilibrium between the hydrated
J Biol Inorg Chem (2012) 17:275–283
form (alcohol) and the aldehyde. The free ligand produces
an equilibrium constant of 0.84, but once the ligand is
coordinated to pentacyanoferrate(II), equilibrium constant
shifts to of 0.48. What is particularly interesting is that
aldehyde formation is favored. This new equilibrium is due
to the extended electronic density delocalization of the p
orbitals in the back-bonding, which favors the aldehyde
instead of the hydrated form (alcohol). In our studies, the
favored product formed was isonicotinic acid and upon
oxidation it might have important functional implications
since it has been suggested that the acyl radical is the
‘‘preactive’’ form of isoniazid in vivo [3]. Therefore having
a higher production level or a longer lifetime for this radical
could improve the action and performance of isoniazid as a
reactivity strategy. Further investigations of radical forma-
tion employing EPR spectroscopy and suitable spin traps
are under way.
InhA inhibition assays
As previously reported [28] [FeII(CN)5(izd)]3- (IQG607)
showed very efficient enzymatic inhibition of wild-type
InhA and its isoniazid-resistant mutant InhA I21V. Its
ability to inhibit the enzyme even in the absence of KatG
and NAD(H) was also remarkable and suggested its
potential use as a metallodrug against KatG- and InhA-
resistant strains. More recently, it was shown that this
compound is indeed effective against isoniazid-sensitive
and isoniazid-resistant strains [30]. A comparative inhibi-
tion study was done with [FeII(CN)5(izd)]3- and
[FeIII(CN)6]3- to evaluate the role of the inorganic moiety.
It was observed that [FeIII(CN)6]3- had no ability to inhibit
100
% Activity Remaining
80
60
40
20
0 routes to generate activated drug are shown. In the first route, as
0 20 40 60
TIME (minutes)
Fig. 3 Assay of inactivation of wild-type InhA (3 lM) by 100 lM
[Fe(CN)6]3- (circles), 100 lM [Ru(NH3)5(izd)]2? (where izd is
isoniazid) (triangles), 100 lM [Ru(CN)5(izd)]3- (squares), and
100 lM [Fe(CN)5(izd)]3- (IQG607) (diamonds), at 25 °C, as
described in ‘‘Materials and methods’’
281
the InhA enzyme either in the presence or in the absence of
NAD(H) or even in its reduced form, as illustrated in
Fig. 3. The data suggest the isoniazid moiety is still a major
and relevant pharmacophore in this compound. However, it
is evident that cyanoferrate has altered the mechanism of
action of isoniazid. This new metal compound inhibits
InhA enzyme without KatG, bypassing the enzymatic
activation. This behavior could explain the action of
IQG607 against an isoniazid-resistant strain as was recently
observed [30]. The mechanism we predicted was based on
an electron transfer reaction scheme (Fig. 4) in which an
inner-sphere reaction would be used to promote faster
isoniazid oxidation, bypassing the enzymatic activation as
discussed elsewhere [20]. The metal atom, which is
kinetically easier to oxidize, triggers the oxidation of the
coordinated isoniazid, thus forming activated species. To
investigate our mechanistic hypothesis, we prepared two
other isoniazid metal complexes, [Ru(NH3)5(izd)]2? and
[Ru(CN)5(izd)]3-. The former has a lower electrochemical
potential (E1/2 = 356 mV) than IQG607, whereas the latter
has a much higher value (E1/2 * 1,000 mV). Remarkably,
both compounds were unable to inhibit InhA (see Fig. 4)
even though [Ru(CN)5(izd)]3- exhibited high similarity to
[FeII(CN)5(izd)]3- (IQG607), including its overall size and
charge. The electrochemical potentials of both compounds,
however, are very different, thus reinforcing the potential
role of an intramolecular electron transfer reaction in InhA
inhibition. Furthermore, evaluation of toxicity has shown
Fig. 4 Self-activating anti-tuberculosis metallodrug scheme. Two
proposed in the literature, free isoniazid is enzymatically converted to
an active species, but this has suffered from resistance, particularly
deletion or mutation in key activating enzymes, which has blocked
this pathway. The alternative route proposed here relies on the
generation of the active species using an intramolecular electron
transfer reaction pathway promoted by a metal complex which would
bypass the enzymatic system of activation, thus leading to isoniazid
function even against isoniazid-resistant strains
123
282
that IQG607 shows promise as a viable candidate for fur-
ther development as an anti-TB agent on the basis of tox-
icological profile assays and the ability to mass produce it
on a large scale [28].
Conclusions
As reported in the literature, isoniazid must be activated by
electron transfer reactions in vivo, which depends on KatG
enzyme. It is also known that the highest percentage of
isoniazid-resistant strains is based on katG gene deletion or
mutation in approximately 50% of strains. These data
indicate that there is an obvious need for the development
of drugs that could overcome TB resistance and yet are still
keep the isoniazid efficacy against M. tuberculosis. To
accomplish this goal we proposed an isoniazid metal
complex that could be self-activated in vivo by endogenous
oxidizing species found in macrophages. We were able to
find the mechanism of oxidation of isoniazid with
[FeIII(CN)5(H2O)]2-, followed by a preferential inner-
sphere electron transfer process. This concept supports our
earlier proposal to design ‘‘self-activating’’ metallodrugs
against TB [20]. On the basis of our data, isoniazid is
intramolecularly oxidized by pentacyanoferrate(III), which
gives us hope to generate an activated drug. Another
interesting observation was that the coordinated pentacy-
anoferrate(II) has likely favored a pathway for isonicotinic
acyl radical formation resulting in the major production of
isonicotinic acid. Despite current debate regarding the
isoniazid activation, several reports suggest that an acyl
radical is the actual active isoniazid intermediate [35, 42].
This species would be directly involved in the reaction with
NAD(H) to produce NAD–isoniazid adducts in vivo as
well as isonicotinic acid in in vitro experiments.
Our proposal for how this complex could work in vivo as
an anti-TB metallodrug is quite straightforward. It is known
that M. tuberculosis faces a strong oxidizing environment
inside the macrophage, where it is able to supply endogenous
activating agents such as hydrogen peroxide and superoxide.
Therefore, we came up with the idea to have a metal complex
of isoniazid where the metal unit can be quickly oxidized
then subsequently trigger isoniazid activation intramolecu-
larly (Fig. 4); hence, it would no longer require the presence
of activating enzymes such as KatG. Now instead, this metal
compound would serve as a way to kill isoniazid-resistant
strains. Our data obtained using the pentacyanoferrate
complex support this hypothesis. Other pyridine thioamide
and isoniazid drugs have also been reported to support this
concept [20, 28]. Our proposed mechanism, tested with these
metallodrugs, as shown in Fig. 4 indicates that an
inner-sphere electron transfer reaction occurs between the
metal and isoniazid, thereby activating this prodrug and
123
J Biol Inorg Chem (2012) 17:275–283
overcoming the necessity for KatG. The inhibition data for
InhA show KatG is not required, thus supporting our pro-
posal. The InhA inhibition experiment conducted with
ruthenium(II) (isoniazid)pentamine shows that it does not
inhibit InhA (Fig. 3); however, it has a much lower elec-
trochemical potential than the cyanoferrate complex. The
(isoniazid)pentacyanoruthenate(II) complex was also unable
to inhibit InhA, likely because of its very high electro-
chemical potential which was harder to reach. Further studies
are under way to provide evidence for this mechanism as well
as to examine other analogous metal complexes to support
this hypothesis. Our data reinforce the idea that electro-
chemical potential might be a key to design these novel
inhibitors. Interestingly, a somehow related process was
reported that was based on the enhanced activation of iso-
niazid activation by KatG in the presence of Mn2?. It was
proposed that transient Mn3? would be produced by KatG,
which would oxidize isoniazid and reduce the metal back to
Mn2?, which is reminiscent of our proposal [43]. However,
this proposed mechanism is still unclear and it is unknown
whether it is fully viable in vivo. We are currently trying to
answer this question with the complexes used in our own
studies.
Another important point in our anti-TB metallodrug
design is the use of an iron compound as a deadly bait.
Following this similar strategy, we believe it might facili-
tate drug uptake by taking advantage of the iron trans-
porters, which could serve as a shuttle for this metallodrug.
This could overcome one of the drawbacks in anti-TB drug
design and therapy, namely, improving the bioavailability
of the drug in M. tuberculosis, but clearly this idea remains
to be proven. However, this kind of strategy can potentially
serve as a platform for other pathogenic systems such as
malaria.
Acknowledgments The authors thank the Brazilian agencies CNPq
and CAPES for financial support. This work was also supported by
the National Institute of Science and Technology on Tuberculosis
(Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES) and the Millennium
Initiative Program (CNPq), Brazil, to D.S.S. and L.A.B.. D.S.S. also
acknowledges a grant awarded by BNDES. L.G.F.L. (301478/2008-2),
I.C.N.D. (303530/2008-1), D.S.S. (304051/1975-06), and L.A.B.
(520182/99-5) are research career awardees of the National Council for
Scientific and Technological Development of Brazil (CNPq). We are
also thankful to CENAUREMN/UFC and Douglas W. Franco for the
use of NMR and stopped-flow equipment.
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