Pentacyanoferrate(II) complex of pyridine-4- and pyrazine-2-hydroxamic acid as source of HNO: investigation of anti-tubercular and vasodilation activities

Article abstract of DOI:10.1007/s00775-020-01805-z

A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO^.)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na₃[Fe^II(CN)₅] moiety. The corresponding pentacyanoferrate(II) complex Na₄[Fe^II(CN)₅(PyzCONHO^?)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with H₂O₂ was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H₂O₂- and N-coordinated pyrazine Fe^II species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.

Full text of DOI:10.1007/s00775-020-01805-z

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-020-01805-z
ORIGINAL PAPER
Pentacyanoferrate(II) complex of pyridine‑4‑
and pyrazine‑2‑hydroxamic acid as source of HNO: investigation
of anti‑tubercular and vasodilation activities
Edinilton Muniz Carvalho1,2,3 · Tercio de Freitas Paulo1,2,3 · Alix Sournia Saquet1 · Bruno Lopes Abbadi4,5
·
Fernanda Souza Macchi4,5 · Cristiano Valim Bizarro4,5 · Rafael de Morais Campos6 · Talles Luann Abrantes Ferreira6 ·
Nilberto Robson Falcão do Nascimento6 · Luiz Gonzaga França Lopes3,5 · Remi Chauvin1,2
Eduardo Henrique Silva Sousa3,5 · Vania Bernardes‑Génisson1,2
·
Received: 4 May 2020 / Accepted: 5 July 2020
© Society for Biological Inorganic Chemistry (SBIC) 2020
Abstract
A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabo-
lites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplifed by
combining two anti-mycobacterial drugs: pyrazinamide (frst line) and delamanid (third line) whose active metabolites are
pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO.)), respectively. Aiming to generate those active species, a
hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na3[FeII(CN)5] moi-
ety. The corresponding pentacyanoferrate(II) complex Na4[FeII(CN)5(PyzCONHO−)] was synthesized and characterized
by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with
H2O2 was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis
(Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H2O2- and N-coordinated pyrazine FeII species
are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of
a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin
trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively grow-
ing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly
lesser toxic efects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic
cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as
a possible pharmacophore model for an alternative vasodilator drug.
Keywords Blood vessel vasodilation · Hybrid pro-drug activation · Metallodrug · Pyrazinamide · Sodium nitroprusside
derivative · Tuberculosis
Introduction
Tuberculosis, a millennial disease due to the infection by
Mycobacterium tuberculosis (Mtb), remains today a world-
wide health problem causing more than 1 million deaths
Electronic supplementary material The online version of this
annually and being responsible for latent infections in about
article (https://doi.org/10.1007/s00775-020-01805-z) contains
one third of the global population [1].
supplementary material, which is available to authorized users.
Although the cure of tuberculosis can be achieved by a
therapeutic regimen involving four frontline drugs, isonia-
zid (INH), rifampicin, ethambutol and pyrazinamide (PYZ)
(Scheme 1), without treatment, tuberculosis becomes lethal.
Moreover, the frst-line therapy is inefcient against multi
* Eduardo Henrique Silva Sousa
eduardohss@dqoi.ufc.br
* Vania Bernardes-Génisson
vania.bernardes-genisson@lcc-toulouse.fr
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

JBIC Journal of Biological Inorganic Chemistry
O
and extensively drug-resistant Mtb strains, which are of
increasing importance.
F
F
F
N
Second- and third-line drugs should thus be used against
these virulent Mtb strains. However, these drugs display
numerous toxic side efects, and even for them, resistant Mtb
strains are emerging.
O
O
O
N
N
O-
N+
DELAMANID = PRO-DRUG
Ddn activation
HNO / NO = ACTIVE METABOLITE
Recently, a novel antitubercular drug, delamanid [2]
(Scheme 2), was approved as a third-line drug of the anti-
tuberculosis therapy. This molecule has two particular
interests: on the one hand, only a few cases of resistance
have been yet reported, and, on the other, it acts upon both
dormant and replicating Mtb strains [3, 4]. Delamanid, a
nitro-dihydro-imidazole derivative, is reported as a pro-drug
and thus requires an enzymatic activation step. The active
metabolite is described as being nitroxyl, i. e. HNO (proto-
nated one-electron reduced form of nitric oxide, NO.), result-
ing from a reductive decomposition of the nitroimidazole
group by a mycobacterial nitroreductase enzyme (Ddn) [5,
6]. However, a possible action of the NO. radical cannot be
entirely ruled out. On the other hand, no organic metabolite
arisen from this bio-reductive activation step was until now
identifed as a bacterial growth inhibitor.
O
Required parameters are missing or incorrect.
Scheme 2 Structure of the delamanid anti-tubercular pro-drug and its
active metabolite(s)
agent [9]. Indeed, cardiovascular diseases are among the
major causes of deaths around the world, for which there
is an urgent need for new therapeutic agents, including
HNO and nitric oxide (NO.) donors. Sodium nitroprusside,
a nitrosylpentacyanoferrate(II) complex, is the only clini-
cally approved metal-based nitric oxide donor, which has
been used in cardiovascular emergency procedures for over
50 years. In contrast, there is no HNO donor agent clinically
available so far. Together these examples show that HNO
It is interesting to note that while HNO/NO. may exhibit
anti-tuberculosis activities, it is also known to play a very
important role on other biological processes as an anti-
oxidant [7], anti-angiogenic [8], and also as a vasorelaxant
HO
NHNH2
O
O
H
N
N
N
N
H
NH2
N
OH
ETHAMBUTOL
ISONIAZID (INH)
PYRAZINAMIDE (PYZ)
PRO-DRUGS
HO
O
H
O
O
O
OH
O
OH OH
N
OH
NH
O
N
N
N
N
O
OH
O
N
isonicotinoyl radical
pyrazinoic acid
O
RIFAMPICIN
ACTIVE METABOLITES
Scheme 1 First-line anti-tubercular (pro-)drugs and reactive species generated from the isoniazid and pyrazinamide pro-drugs.
1 3

JBIC Journal of Biological Inorganic Chemistry
donor molecules can be of great interest in the medical feld
as potential therapeutic agents.
generate, in an opportunistic way, two antitubercular agents,
HNO and pyrazinoic acid. Both metabolites could be ideally
released without the need of mycobacterial enzymes (over-
coming resistance phenomena) and could act upon multiple
targets simultaneously, reducing the chances of selection of
resistant bacterial cells.
Lately, some of the present authors have studied
and developed a biomimetic system, as an alternative
non enzyme-dependent way to activate the INH anti-
tuberculosis pro-drug (KatG=activation enzyme of INH),
based on the redox reactivity of a metal complex of INH
(called IQG607) in the presence of oxidizing agents such
as H2O2 [10–12]. The anti-mycobacterial activity (in vitro
MICMtb = 1.56 μg/mL ~ 3.5 μM) [13], chemical stabil-
ity, and absence of toxicity (selectivity index SI > 4000,
LD50 > 2000 mg/kg) [14] of the [FeII(CN)5(INH)]3− com-
plex motivated us to extend this activation approach to heter-
oaryl hydroxamic acids used as ligands for the development
of new HNO donor complexes. Thereby, to prepare HNO
donors from [FeII(CN)5(heteroaryl hydroxamic acid)] com-
plexes with wide spectra of biological activities, including
antituberculosis, the pyrazine-type nitrogen aryl ring was
naturally frst selected and used in this study. The pyra-
zine moiety is already present in the structure of a frst-line
antitubercular drug, pyrazinamide (PYZ, see Scheme 1).
Pyrazinamide, like isoniazid and delamanid, is also a pro-
drug and is activated by pyrazinamidase (PncA), an enzyme
of Mtb [15, 16]. This reaction converts the pro-drug into
the corresponding carboxylic acid (pyrazinoic acid =Pyz-
COOH) that is assigned to be the efective toxic agent for
Mtb (Scheme 1). The sensitivity of Mtb to this drug is partly
due to an inefcient efux of PyzCOOH, that is found accu-
mulated in the interior of the cell [17, 18].
This paper mainly describes the synthesis, characteri-
zation, and chemical–biological studies of pyrazine-2-hy-
droxamic acid (2) and its pentacyanoferrate(II) complex 3
(Scheme 3). As proposed above, the ligand 2 is actually a
functional hybrid of two diferent pharmacophores corre-
sponding to the active metabolites of PYZ (PyzCOOH) and
delamanid (HNO). After chemical activation of the proto-
type complex 3 (Fe(II) converted to Fe(III) upon oxidation),
and independently of the functional state of the activating
enzyme of Mtb (mutated or not), the released pharmacoph-
ores are expected to recognize diferent targets and act by
diferent mechanisms, killing growing and, more particu-
larly, non-growing Mtb strains (Scheme 3).
Isonicotinohydroxamic acid has also been synthesized in
view of the fact that putative isonicotinic acid, formed after
the activation step, can be considered as a bio-isostere of
pyrazinoic acid.
Material and methods
Chemicals
Considering that the major route for PYZ resistances
relies on point mutations on the PncA enzyme that is respon-
sible for the conversion of PYZ into pyrazinoic acid [17–19],
the synthesis of the Na3[FeII(CN)5(PyzCONHOH)] complex,
could be judicious because the proposed complex can be
considered as a hybrid source of the active metabolites of
PYZ and delamanid that, under oxidative conditions, could
Available reagents and solvents were purchased from
commercial suppliers and used without further purifca-
tion: methyl isonicotinate, methyl pyrazine-2-carboxylate
(Alfa Aesar); hydroxylamine solution (NH2OH, 50% in
water) from Sigma-Aldrich, pyrazinoic acid, isonicotinic
acid and potassium ferricyanide (III) (K3[Fe(CN)6]·3H2O)
from Sigma-Aldrich; isoniazid (INH) from ACROS
3-
O
O
N
N
N
non-replicating
OH
Mtb strains
N
NH3
FeII
CN
Fe(CN)5
PYZ active metabolite
3-
NC
CN
CN
O
O
Na
3 NC
MULTI-
TARGETS
N
N
N
NHOH H2O2
NHOH
N
Fe(CN)5
non-replicating and
replicating Mtb strains
HNO
Delamanid
active metabolite
2
3
Scheme 3 Possible outcome route for oxidation of the Na3[FeII(CN)5(PyzCONHOH)] complex by H2O2
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JBIC Journal of Biological Inorganic Chemistry
Organics; sodium dihydrogen phosphate (NaH2PO4),
disodium hydrogen phosphate (Na2HPO4) and sodium
trifuoroacetate (NaC2F3O2) from PROLABO and Fluka;
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-
1-oxyl-3-oxide potassium salt (c-PTIO) and N-tert-
butyl-α-phenylnitrone (PBN), used as radical trapping
agents, from Thermo Fisher Scientifc and Alfa Aesar,
respectively.
modulation amplitude of 0.5 G, a microwave power of 5.15
mW, with an attenuation of 16 dB with repeated number
of 2 scans. The samples were transferred into a capillary
(Hirschmann, Duran, Ringcaps, 50 μL), which was then
placed inside a larger quartz tube enabling the sample to
be accurately positioned inside the resonator. All samples
were prepared as 40 mM solutions in phosphate bufer pH
7.4, at 25 °C, by mixing suitable amounts of the studied
compound and spin trap (cPTIO or PBN). The reactions
were initiated by the addition of the H2O2 and the spectra
immediately recorded.
Na3[FeII(CN)5(NH3)]·3H2O was prepared as previously
described [20]. Methanol, ethanol, dichloromethane, and
acetone were of chromatographic grade, and water used in
the experiments was ultra-purifed using a Milli-Q system
(Millipore).
Synthetic procedures
General procedure for hydroxamic synthesis
Instruments
To a solution of methyl heteroaryl-carboxylate derivative
(3.62 mmol) in methanol (10 mL) at room temperature and
under stirring, 3.9 mL of hydroxylamine 50% (65.2 mmol)
was slowly added. The solution was stirred for 72 h. The
solvent was evaporated under vacuum and the solid was
resuspended in dichloromethane, fltered, and thus washed
from traces of pyrazine-2-carboxylate. Then, the solid was
resuspended in water (4 mL) to remove traces of hydroxy-
lamine: the pyrazine-2-hydroxamic acid being poorly solu-
ble in water, the mixture was cooled down in an ice bath for
3 h to assist the product precipitation. Then, the solid was
fltered, washed with cold acetone, and dried under vacuum.
1H NMR spectra were recorded on a Bruker spectrom-
eter at 300 and 400 MHz using D2O and DMSO-d6 as
solvents, with increasing chemical shifts at low feld vs
1
the tetramethylsilane H nucleus reference derived from
the deuterium lock signal of the solvent. 13C NMR spec-
tra were recorded on a Bruker spectrometer at 101 MHz
using D2O and DMSO-d6 as solvents, with increasing
chemical shifts at low feld vs the tetramethylsilane 13C
nucleus reference derived from the deuterium lock sig-
nal of the solvent. 15N NMR spectra were recorded on
a Bruker spectrometer at 50.7 MHz and external sample
of CH3NO2 was used as the reference. Electrospray mass
spectra (ESI) were obtained on a Perkin–Elmer SCIEX
API 365 instrument high resolution and desorption chem-
ical ionization (DCI) mass spectra were acquired on a
Finnigan TQS 7000 spectrometer. Infrared spectra were
obtained using a Perkin–Elmer Spectrum One spectrom-
eter. UV–Vis spectra were recorded on a Perkin–Elmer
UV/Vis/NIR Spectrometer—Lambda 950. Normal Raman
(NR) spectra of the samples were acquired by means of an
Xplora (Horiba) microspectrometer: the excitation radia-
tion was the 785 nm line and the laser beam was focused
on the sample by a×50 long distance objective. Elemental
analyses were obtained using a PERKIN ELMER 2400
series II instrument. Electrochemical measurements were
performed using a BAS Epsilon E2 818 potentiostat/gal-
vanostat system within a conventional three-electrode
cell. Vitreous carbon, platinum, and calomel electrodes
were used as working, auxiliary and reference electrodes,
respectively. Voltammograms were recorded in phosphate
bufer solution (PBS) 0.1 M, pH 7.4. Electron paramag-
netic resonance (EPR, or electron spin resonance = ESR)
spectra were recorded at room temperature (ca. 291 K) on
a Bruker Elexsys-II E500 (X-Band) spectrometer using a
Pyrazine‑2‑hydroxamic acid (2)
1
Yield = 85% (0.43 g), white solid. H NMR (400 MHz,
DMSO-d6) δ (ppm): 11.62 (s, 1H), 9.28 (s, 1H), 9.12
(d, J = 1.5 Hz, 1H), 8.84 (d, J = 2.5 Hz, 1H), 8.68 (dd,
J = 2.5, 1.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
(ppm): 160.65 (C=O), 147.75 (CH), 145.52 (Cq), 143.85
(CH), 143.67 (CH). 15N NMR (50.7 MHz, D2O) δ (ppm):
319.30 (N1), 323.36 (N4). IR symmetric stretching (ꢀs),
anti-symmetric stretching (ꢀas), symmetric bending (δs)
and twisting (τ) (cm−1): 3212 (ꢀs O–H), 3061 (ꢀs N–H),
2823 (ꢀs C–H), 1662 (ꢀs C=O), 1585 – 1524 ꢀ C=N, ꢀ
C=C), 1417 (δs N–H), 1386 (δs C–H), 1023 (ꢀas C=N),
s
916 (τ C–H). UV–Vis (H2O) ꢀmax/nm (ꢀ/M−1 cm−1) = 211
(8169), 272 (7248), 312 (876). MS (DCI/CH4) m/z: 140.04
[M + H+], 124.05 [(M + H+)-16]. HRMS (DCI/CH4) m/z:
for {(C5H5N3O2)+H+} calcd.: 140.0460 found: 140.0464.
Elemental analysis calcd. for C5H5N3O2: C, 43.17; H, 3.62;
N, 30.21. Found: C, 42.91; H, 3.51; N, 29.81. TLC (dichlo-
romethane/methanol 90:10) Rf =0.42. M.p.=165 °C. Elec-
trochemistry in 0.1 M phosphate bufer, pH 7.4, Epa =0.684
and 1.119 V vs NHE.
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JBIC Journal of Biological Inorganic Chemistry
Pyridine‑4‑hydroxamic acid (5)
and 753 (π C–H), 652 (ꢀs C=N). UV–Vis (H2O) ꢀmax/nm (ε/
M−1 cm−1)=216 (18,820), 270 (6228), 486 (3638). Elemen-
tal analysis calcd. for C10H4FeN8O2Na4·3H2O: C, 25.55; H,
2.14; N, 23.84. Found: C, 25.93; H, 2.16; N, 23.94. Electro-
chemistry in phosphate bufer (0.1 M, pH 7.4): Epa =0.722
and 1.091 V vs NHE.
1
Yield = 98% (0.49 g), white solid. H NMR (300 MHz,
DMSO-d6) δ (ppm): 11.50 (s, 1H), 9.32 (s, 1H), 8.70
(d, J = 6.1 Hz, 2H), 7.67 (d, J = 6.1 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 162.48 (Cq), 150.68 (CH),
140.31 (Cq), 121.40 (CH). IR symmetric stretching (ꢀs),
anti-symmetric stretching (ꢀas), symmetric bending (δs) and
twisting (τ) (cm−1) ꢀmax (cm−1): 3324 (ꢀs O–H), 3132 (ꢀs
N–H), 1640 (ꢀs C=O), 1540–1520 (ꢀs C=N, ꢀs C=C), 1410
(δs N–H), 1316 (δs C–H), 1025 (ꢀas C=N), 908 (τ C–H).
UV–Vis (H2O) ꢀmax/nm (ε/M−1 cm− 1) = 235 (3760), 260
(3710). HRMS (DCI/CH4): for {(C6H6N2O2)+H+} calcd.:
139.0508 found: 139.0515. Elemental analysis calcd. for
C6H6N2O2: C, 52.17; H, 4.38; N, 20.28. Found: C, 52.12;
H, 4.21; N, 20.25. TLC (dichloromethane/methanol 90:10)
Rf = 0.24. M.p = 154 °C. Electrochemistry in 0.1 M phos-
phate bufer, pH 7.4, Epa =0.767 and 1.207 V vs NHE.
Na4[FeII(CN)5(PyCONHO−)] (Na46)
(PyCONHO− = pyridine‑4‑hydroxamate)
1
Yield= 79% (0.173 g), yellow solid. H NMR (400 MHz,
D2O) δ (ppm): 9.05 (d, J=4.96, 2H), 7.39 (d, J=4.76, 2H).
13C NMR (101 MHz, D2O) δ (ppm): 180.64 (CNeq), 176.39
(CNax), 164.02 (Cq), 157.34 (CH), 141.63 (Cq), 120.52
(CH). IR symmetric stretching (ꢀs), antisymmetric stretching
(ꢀas), symmetric bending (δs), antisymmetric bending (δas),
wagging (π), rocking (ρ) and twisting (τ) (cm−1): 3483 (ꢀs
N–H), 3382–3047 (ꢀs C–H), 2052 (ꢀs C≡N), 1647 (ꢀs C=O),
1546 (ꢀs C=N, C=C), 1492—1414 (δs C–H), 1313 (π C–H),
1227 (τ C–H), 1165 (ꢀs C–C). 908 (δas C–H), 846 (π C–H),
698 (π N–H). UV–vis (H2O) ꢀmax/nm (ε/M−1 cm−1) = 233
(14,400), 266 (5123), 439 (4052). Elemental analysis calcd.
for C11H5FeN7O2Na4·3.5H2O: C, 27.64; H, 2.53; N, 20.51.
Found: C, 27.66; H, 2.21; N, 20.76. Electrochemistry in
0.1 M phosphate bufer (pH 7.4): Epa =0.551 and 1.099 V
vs NHE.
General procedure for preparation
of Na4[FeII(CN)5(HeteroarylCONHO−)]compounds
To a solution of Na3[FeII(CN)5(NH3)]·3H2O (0.150 g,
0.46 mmol, 1 equiv.) in water (3 mL), it was slowly added a
solution of the ligand (pyrazine-2-hydroxamic acid, 0.077 g,
0.55 mmol, 1.2 equiv. or pyridine-4-hydroxamic acid,
0.076 g, 0.55 mmol, 1.2 equiv.) in water (2 mL). The mixture
was stirred at room temperature, under argon atmosphere
and protected from light for 3 h. Then 150 mL of a cold solu-
tion of ethanol containing an excess of sodium iodide (30
equiv.) was added dropwise. The resulting precipitate was
kept overnight at −20 °C before it was collected by fltration,
washed with cold ethanol, and dried in a vacuum desiccator.
CAUTION: since these complexes are moderately light and
oxygen sensitive, they must be stored in a vacuum desiccator
protected from light to extend their lifetime.
Computational details
All calculations were carried out using the density functional
theory (DFT) with the B3LYP [21–23] functional as imple-
mented in the Gaussian 09 program package, Revision D.01
(Gaussian Inc., Wallingford, CT) [24]. The 6-311++G(d,p)
basis set was used for non-metal atoms, while the LAN-
L2DZ relativistic efective core potential basis set was used
for Fe atoms. Vibrational frequency analyses were carried
out to confrm convergence to a minimum energy geometry
by the absence of imaginary frequencies. A scaling factor of
0.9679 for the calculated harmonic vibrational wavenumbers
considering the 6-311++G(d,p) basis set and the B3LYP
functional. The vertical excitation energies were determined
by the time-dependent density functional theory protocol
(TD-DFT) using the B3LYP functional and mixed basis sets
mentioned above. The polarizable continuum model (PCM)
[25] was used in the case of vertical excitation energies to
take into account the solvent efect, where the dielectric con-
stant of water was considered. The FTIR, UV–Vis spectra
were extracted from output fles, while Raman spectra were
calculated with the GaussSum 3.0 program [26]. This pro-
gram was used to calculate the Raman intensity considering
an exciting radiation of 785 nm.
Na4[FeII(CN)5(PyzCONHO−)] (Na43)
(PyzCONHO− = pyrazine‑2‑hydroxamate)
Yield = 87% (0.180 g), orange solid. 1H NMR (400 MHz,
D2O) δ (ppm): 9.46 (s, 1H), 9.18 (d, J=3.3 Hz, 1H), 8.27
(d, J = 3.2 Hz, 1H). 13C NMR (101 MHz, D2O) δ (ppm):
178.69 (CNeq), 173.75 (CNax), 160.69 (C=O), 153.77
(CH), 150.66 (CH), 145.50 (Cq), 142.33 (CH). 15N NMR
(50.7 MHz, D2O) δ (ppm): 301.10 (N4), 308.56 (N1). IR
symmetric stretching (ꢀs), antisymmetric stretching (ꢀas),
symmetric bending (δs), antisymmetric bending (δas) and
wagging (π) (cm−1): 3418 (ꢀs N–H), 3452–3250 (ꢀs C–H),
2053 (ꢀs C≡N), 1609 (ꢀs C=O), 1577 (ꢀs C=N, C=C),
1391—1173 (δs C–H), 1033 (τ C–H). 908 (δas C–H), 854
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JBIC Journal of Biological Inorganic Chemistry
control. This assay was performed using seven independent
measurements prepared with diferent animals for statisti-
cal analysis. All procedures were performed according to
the ethics committee of State University of Ceará number
2897836/15.
Antimycobacterial activity
In vitro activity of the compounds 2, 3, 5 and 6 was evalu-
ated against the Mycobacterium tuberculosis H37Rv ATCC
27294 reference strain (American Type Culture Collection,
Rockville, Md.), by performing minimum inhibitory con-
centration assays (MIC), as described elsewhere [27]. This
strain was grown up to mid-log phase (OD600 0.8–1.0) in
Middlebrook 7H9 broth (Difco™), supplemented with 10%
ADC (albumin, dextrose and catalase—BD BBL™) and
0.05% Tween 80 (Sigma-Aldrich), with agitation (100 rpm),
at 37 °C. Following that, a mycobacterial suspension was
prepared using sterile glass beads (4 mm), which was ali-
quoted and stored at -80 °C until use. Compounds were frst
solubilized in ultrapure water (4 mg/mL) and diluted in 7H9
broth (200 µg/mL). Serial twofold dilutions (100 µL) of each
compound were performed directly in 96-well plates in 7H9
broth, giving a concentration range of 100–0.2 µg/mL. An
aliquot of mycobacterial suspension was thawed and diluted
in 7H9 broth to a theoretical OD of 0.006, which was added
to each well (100 µL). Compound and culture-free wells
were used as positive and negative growth controls, respec-
tively. Isoniazid (INH, ACROS Organics) was used as an
anti-TB drug reference. Plates were sealed with Paraflm M®
and incubated inside plastic bags in a bacteriological incuba-
tor (37 °C, 5% CO2) for 7 days, before the addition of 60 µL
of 0.01% (w/v) resazurin (Sigma-Aldrich) solution to each
well. Minimum inhibitory concentration (MIC) was consid-
ered as the lowest compound concentration that prevented
a color conversion from blue (no growth) to pink (growth).
MIC values reported for each compound were the most fre-
quently occurring value observed among three independent
assays. INH was used as a positive control of MIC assays.
Systemic hemodynamics
Male WistarKyoto (WKY) or spontaneously hypertesive
rats (SHR) (250–300 g) were anesthetized with sodium
pentobarbital (50 mg/kg) and a polyethylene catheter (PE
240) was inserted into the trachea to help with spontaneous
breathing. The left femoral artery was isolated and canulated
with a polyethylene catheter (PE 10) and this catheter was
coupled to a pressure transducer (MLT844, ADInstruments,
Sydney, Australia) for continuous recording of blood pres-
sure by means of a data acquisition system (Powerlab, ADIn-
struments) and a Labchart 7.0 software. The right carotid
artery was canulated with a microtip pressure–volume cath-
eter (SPR-869, Millar Instruments; Houston, TX) and this
catheter was inserted into the left ventricle for continuous
measurement of cardiac parameters such as cardiac output.
Results and discussion
Synthesis
The ligand moiety, pyrazine-2-hydroxamic acid (2) (Pyz-
CONHOH) [28, 29], of the targeted hybrid complex 3, was
directly prepared from methyl pyrazinoate (1) in NH2OH
solution with 85% yield (Scheme 4) and fully character-
ized. Then, the coordination of 2 with Na3[Fe(CN)5NH3]
was carried out in aqueous medium to aford Na43 in
87% yield (Scheme 4). This compound was analyzed by
1H/13C/15N NMR, IR, UV–Vis and Raman spectroscopy,
along with cyclic voltammetry, elemental analysis and
DFT calculations. Attempts to obtain crystals of the com-
plex Na43 for X-ray difraction analysis were unsuccessful.
Additionally, procedures to exchange the sodium cations
by other more crystallogenic/solubilizing cations (e.g.
Vasodilation assay
Rats were sacrificed by overdose of sodium thiopental
and the thoracic aorta was carefully removed and cut into
rings of about 5 mm in length. The aortic rings were mounted
in a 5 mL organ bath containing Krebs–Henseleit medium
with the following composition: 120 mM NaCl, 4.7 mM
KCl, 1.8 mM CaCl2, 1.43 mM MgCl2, 25.0 mM NaHCO3,
1.17 mM KH2PO4, glucose and maintained at 37 °C. The
rings were attached to a force transducer (TRI202P, Panlab,
Barcelona, Spain) coupled to a Powerlab data acquisition
systema (ADInstruments, Sydney, Australia) and data were
recorded and analyzed using the Labchart 7.0 software.
After equilibration, the rings were pre-contracted with 1 μM
phenylephrine (PE), and once a stable plateau was achieved,
cumulative concentration–response curves were constructed
using the ligand 2 and 5 and the metal complexes 3 and 6 in
a concentration range from 0.1 nM to 100 μM. Sodium nitro-
prusside (SNP, Na2[Fe(CN)5(NO)]) was used as positive
+
PPN+, NEt4 ) also failed. (Protocol a: to a solution of salt
+
(PPN+ or NEt4 ) in ethyl acetate, the iron complex was
added. The mixture was stirred overnight in the shadow.
The solution was fltered to remove the unsolubilized
complex. The fltered solution was rotoevaporated and
the crude product was characterized by 1H-NMR. Protocol
b: an aqueous solution containing the iron complex was
+
added to a solution of salt cation (PPN+ or NEt4 ) in ethyl
acetate. This mixture was stirred overnight in the shadow.
Then the organic and aqueous phases were separated. The
organic phase was rotoevaporated and the solid obtained
1
was characterized by H-NMR. No signal corresponding
1 3

JBIC Journal of Biological Inorganic Chemistry
4-
O
O
O
1
N
N
Na3[Fe(CN)5NH3]/H2O
NaI / EtOH
N
N
N
NH2OH 50%
MeOH / H2O
2
NHO-
OCH3
NHOH
6
5
3
N
4
85%
87%
Fe(CN)5
1
2
3
Scheme 4 Synthesis of the pentacyanoferrate(II) complex of pyrazine-2-hydroxamate from methyl pyrazine-2-carboxylate
to the complex framework was observed, just signals of
Characterizations
+
the cation PPN+ or NEt4 . The aqueous solution was also
analyzed by 1H-NMR; however, no characteristic signal of
these salts was identifed).
NMR spectroscopy
The 1H NMR spectrum of 3 shows the aromatic protons in
The pentacyanoferrate(II) complexes of pyridine-
4-hydroxamic acid (PyCONHOH) and sodium pyrazine-
2-carboxylate (PyzCOONa) were both synthesized for
the purpose of comparison and for control experiments,
respectively. Treatment of methyl isonicotinate 4 with
hydroxylamine originated the corresponding hydroxamic
acid 5 [30], which was converted to the metal complex
Na46, using the same protocol employed for the prepara-
tion of 3 (Scheme 5). Likewise, deprotonation of pyrazi-
noic acid 7 led to sodium pyrazinoate 8, which was then
coordinated to the FeII(CN)5 moiety with 64% yield in the
complex 9 (see Supporting Information).
3–
the vicinal positions from the N-Fe(CN)5 fragment (H3
s, 9.46 and H5 d, 9.18 ppm) are both shifted to low feld as
compared to those of the free protonated ligand 2 (PyzCON-
HOH: s, 9.11 and d, 8.80 ppm). On the other hand, the distal
aromatic proton H6 of 3 is shifted up-feld (8.27 ppm) with
respect to the corresponding proton in the protonated free
ligand 2 (8.68 ppm). The same trend was observed in the 13C
NMR spectra for the corresponding aromatic 13CH nuclei.
The 2D 1H-15N HMQC NMR spectrum of 3 unambiguously
shows that coordination of 2 occurs selectively at the N-4
position of the pyrazine ring (Figure S1). As observed for
the [Fe(CN)5(INH)]3− complex, [31] the 15N1 and 15N4 sig-
nals of 2 at 319.30 and 323.36 ppm are, respectively, both
shifted up-feld to 308.56 and 301.11 ppm, for the complex.
The more pronounced shifting undergone by N4 indicates
In all cases, the coordination to the diamagnetic Fe(II)
center was shown to take place only at the less hindered N
atom of the heteroaromatic ring as evidenced by spectro-
scopic analysis (see Sect. 3.2).
Scheme 5 Synthesis of pentacyanoferrate(II) complexes of pyridine-4-hydroxamic acid and sodium pyrazine-2-carboxylate
1 3

JBIC Journal of Biological Inorganic Chemistry
that the coordination of Fe(CN)5 moiety occurs at this center.
The high resolution of the NMR spectra of 3, without signal
broadening vs the spectra of 2, provides a strong evidence
that the iron atom of 3 is indeed in a low-spin+2 oxidation
state (Figure S1).
The pKa value of 3 in water solution, determined by a titri-
metric method using 0.025 M HCl, is indeed as low as 8.5.
Cyclic voltammetry
The cyanide ligands of 3 are revealed by two 13C
NMR signals at 178.7 and 173.8 ppm, characteristic of
sp 13CN nuclei in equatorial and axial positions vs the
Fe–N axis. These results are consistent with those pre-
viously obtained for the pyrazine hydrazide complex
Na3[Fe(CN)5(PyzCONHNH2)] [32].
Cyclic voltammograms of the ligand 2 and complex 3 in a
phosphate bufer solution (0.1 M, pH 7.4) were recorded
using a glassy carbon working electrode (Figure S2). For
the free ligand 2, two oxidative waves are observed at
0.68 V and 1.12 V vs NHE as irreversible electrochemical
processes. In the cyclic voltammogram of the complex 3H+
(Figure S2 red line), the oxidation wave is observed in the
potential range of 0.50–0.90 V vs NHE. This wave can be
attributed to two processes corresponding to the local oxida-
tion of the FeII –>FeIII and coordinated ligand. Additionally,
a second irreversible electrochemical process is observed
at 1.091 V vs NHE and assigned to the second ligand oxi-
dation. The oxidation of the organic moiety likely initiates
via an intramolecular process involving the formation of a
Fe(III) center, although an intermolecular reaction cannot be
strictly ruled out. Furthermore, there are kinetic evidences
of an intramolecular electron transfer during the chemical
oxidation of the [Fe(CN)5(INH)]3− complex (IQG607), a
process which might be similar in the chemical oxidation
of 3H+ [10a]. In the cathodic scanning, only one wave at
0.54 V vs NHE is observed and attributed to the FeIII –>FeII
reduction process.
Vibrational spectroscopy
The cyanide ligands were also characterized by infrared
(IR) and Raman spectroscopy, where two bands in the
range 2050–2100 cm−1 are assigned to the FeC≡N stretch-
ing frequencies ν(C≡N) [33]. The most relevant infrared and
Raman vibration modes and wavenumbers can be found in
the Supporting information (Table S1).
Unexpectedly, the experimental normal Raman spectrum
of 3 in the solid state (Fig. 1) was indeed much more com-
patible with the corresponding DFT-simulated spectrum,
than with the DFT-simulated spectrum of the O-protonated
form (3H+, Scheme 4). The O-deprotonated form of the
ligand 2 in the tetraanionic complex 3 was also supported
by elemental analysis (see experimental section). IR data
reveal that the C=O band at 1662 cm−1 in the protonated
free ligand 2 is shifted to ca. 1580 cm−1 in the complex 3,
in agreement with the anionic hydroxamate form of 2 in 3
(Scheme 4). The formation of 3 is a consequence of the basic
medium (pH>>9) used for the preparation of this complex.
UV–Vis absorption spectroscopy
The electronic spectra of the protonated free ligand 2 and
complex 3H+ were recorded in slightly acidic aqueous
Fig. 1 Experimental normal Raman (a) and IR (b) spectra of the complex 3 under tetraanionic form, Na4[Fe(CN)5L], L=PyzCONHO– (plain
line), in the solid state, and DFT-simulated spectra for [Fe(CN)5L]4− (dotted line)
1 3

JBIC Journal of Biological Inorganic Chemistry
solution (pH = 6.8). The free ligand 2 exhibits two main
bands at 211 and 272 nm, and a shoulder at 312 nm, while
the complex 3H+ exhibits three main electronic transitions
with absorption maxima at 216, 270, and 486 nm (Figure
S3). The two frst ones are consistent with bands relative
to the ligand moiety, while the last band is characteristic of
a metal-to-ligand charge transfer transition (MLCT). This
type of transition occurs from an electron-rich Fe center to
π-accepting ligands, which is in agreement with the+2 oxi-
dation state of the iron center. TD-DFT calculations further
validate the character of these transitions (see Supporting
information, Table S2 and Figure S4).
enzyme, which is often found mutated in PYZ-resistant
Mtb strains. In these conditions, oxidation of 3 under
its major hydroxamic acid form 3H+ and the release of
PyzCOO−, without assistance of PncA, was thus investi-
gated by UV–vis and 1H NMR spectroscopy.
Before addition of H2O2, the trianionic complex 3H+
(bufer solution of 3 at pH = 7.4) showed an absorption
band with a maximum at 489 nm, characteristic of the
MLCT transition as previously discussed. This band dis-
appeared almost completely within 2 h (Fig. 2, red line)
after addition of H2O2, indicating modifcation of the
metal moiety. This result supports the hypothesis that this
complex is activated by H2O2 via FeII – > FeIII oxidation
(Fig. 2).
Chemical oxidation of the Fe(II) complex
under physiological pH conditions
and characterization of drug metabolites active
against Mtb
1H NMR analysis of 3 in deuterated bufer solution
and in the presence of 2.5 equivalents of H2O2, after 52 h,
revealed that the signals relative to the aromatic protons
of the trianionic complex 3H+ had completely disap-
peared, while new signals corresponding to free pyrazine
carboxylate (8) were observed. This was evidenced by a
As argued earlier, the complex Na43 has been devised as a
precursor of the anti-Mtb metabolites of pyrazinamide and
delamanid, i.e. pyrazinoic acid (PyzCOOH) and nitroxyl
(HNO/NO.), respectively. The search for evidences of the
formation of the later organic and inorganic products upon
chemical oxidation with H2O2 is addressed below:
1
perfect matching of the new aromatic H NMR signals
(Fig. 3, spectrum C) with those of an authentic sample of
sodium pyrazinoate (8) (Fig. 3, spectrum E). Remarkably,
after oxidative activation, the organic metabolite 8 was
found to be spontaneously released from the metal center,
while only very little ligand remains coordinated to the
FeII(CN)53– unit (Fig. 3, spectrum C and D)
Formation of PyzCOOH upon oxidation of 3
Chemical oxidation of 3 or its protonated form 3H+ by
action of H2O2 in a phosphate bufer solution (40 mM,
pH = 7.4) can be considered as a mimic of the biologi-
cal activation of PYZ by the pyrazinamidase (PncA)
It is worth noting that the free hydroxamic acid 2 in the
presence of H2O2 is unable to produce pyrazinoic acid even
after 52 h. In addition, in the presence of Na3[FeIII(CN)6]
(2.5 equiv.), the hydroxamic group of 2 is only very slowly
Fig. 2 UV–Vis monitoring
of the reaction of 3 under the
protonated main form 3H+
(171 µM) with H2O2 (427.5 µM)
in phosphate bufer solution,
40 mM, pH 7.4, 22 °C. Reaction
at t=0 min, before addition of
H2O2, (blue line), and after 2 h
(red line). The black curve cor-
responds to a solution of sodium
2-pyrazinoate (110 µM). Inset
shows the kinetic curve based
on changes at 489 nm
1 3

JBIC Journal of Biological Inorganic Chemistry
Fig. 3. 1H-NMR spectra at
400 MHz: of the complex 3
under its protonated form 3H+
(20 mM) at 0 h (a) and at 52 h
(b) without addition of H2O2,
at 52 h after reaction of 3H+
with H2O2 (50 mM) (c), and
of authentic samples of the
complex 9 (20 mM) (d) and
pyrazinoate salt (8) (20 mM)
(e). Solutions in 40 mM phos-
phate bufer, pH 7.4, at 25 °C
and partially oxidized, and converted to the corresponding
carboxylic acid PyzCOOH (even after 64 h), after hydroly-
sis of the putative dimer N,O-dipyrazinoylhydroxylamine
(PyzC(O)NHOC(O)Pyz) formed as intermediate [34]. The
latter intermediate has never been observed for the ligand
of 3 in oxidative conditions. All together, these results show
that both H2O2 and the N-coordinated FeII unit are needed
for oxidative activation of the hydroxamic moiety, ruling out
a random mechanism passing through a direct hydrolysis of
the hydroxamic acid function (PyzCONHOH+H2O→Pyz-
COOH+NH2OH) or an alternative activation route where
the hydroxamic acid group would coordinate metal ions
occasionally present in bufer solutions.
signatures diferent from the quintuplet signal of cPTIO (a
septet for cPTI) [34]. cPTIO also reacts with HNO, by which
it is reduced to the EPR-silent nitronyl-hydroxylamine, while
releasing the NO radical (Scheme S1) [35, 36].
The frst control of cPTIO with the free hydroxamic acid
2 or complex 3 showed that the EPR signal of cPTIO was
not altered, even after 15 min (Fig. 4b, d). The same sta-
bility of cPTIO was observed in the presence of H2O2 and
with the mixture of FeCl2 +H2O2, a Fenton-reaction system
generating hydroxyl radicals (Fig. 4c, e). Moreover, EPR
monitoring did not show any evidence of reaction of cPTIO
with Na3[FeIII(CN)6] (Fig. 4f). However, a net decrease of
the cPTIO EPR signal was observed upon treatment of the
FeII complex 3 with H2O2, indicating neutralization of the
nitroxide radical, which would be a priori due to the release
of HNO (Fig. 4a).
Formation of HNO/NO upon oxidation of 3
The inorganic metabolite of delamanid, i.e. the weak acid
nitroxyl (HNO) or radical nitric oxide (NO), was tar-
geted. Discrimination between these possible products
was envisaged by EPR spectroscopy in the presence of the
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-
3-oxide potassium salt (cPTIO). This compound is a water-
soluble nitronyl-nitroxide known to be readily reduced by
NO, of which it is a specifc scavenging agent, to give the
corresponding cPTI nitroxide and the NO2 radical (Scheme
S1). This species (cPTI) presents characteristic EPR
Our previous studies on the activation of
Na3[Fe(CN)5(INH)] (IQG607) complex in the presence
of H2O2 gave evidences (by EPR spectroscopy) for the
formation of the isonicotinoyl radical as an intermediate
towards isonicotinoic acid [31, 37]. On this basis, it was
decided to check whether the hydroxamate complex 3 or
its acidic form 3H+ could also generate pyrazinoic acid via
a transient radical intermediate what could be an indirect
evidence for the formation of NO.. By employing the same
methodology used before for the investigation of the INH
1 3

JBIC Journal of Biological Inorganic Chemistry
Fig. 4 a EPR signal of the cPTIO radical in the presence of the
complex 3 under its protonated form 3H+ and H2O2 (t0 =black line
and t10min =red line) in phosphate bufer solution, 40 mM, pH 7.4.
b Control of the stability of the cPTIO EPR signal in the presence
of Na3Fe(CN)5-PyzCONHOH] (t0 =black line and t15min =red line). c
Control of the stability of the cPTIO EPR signal in the presence of
H2O2 (t0 =black line and t15min =red line). d Control of the stability
of the cPTIO EPR signal in the presence of the ligand PyzCONHOH
and H2O2 (t0 =black line and t20min =red line). e Control of the sta-
bility of the cPTIO EPR signal in Fenton reaction conditions [FeCl2
(5 mM) and H2O2 (200 mM)] (t0 =black line and t15min =red line). f
Control of the stability of the cPTIO EPR signal in the presence of
[Fe(CN)6]3− and H2O2 (t0 =black line and t20min =red line)
complex, EPR spectra were recorded using N-tert-butyl-α-
phenylnitrone (PBN) as the spin trapping agent (Scheme
S1B). In contrast to the parent iron complexes of isoniazid
and of pyrazinoic acid hydrazide [30], Na3[Fe(CN)5(INH)]
and Na3[Fe(CN)5(PyzCONHNH2)], respectively, no radical
could be trapped upon treatment of the pyrazine and even of
the pyridine hydroxamic acid complexes (3 and 6, respec-
tively), with H2O2 (Schemes 4 and 5).
HNO + ArC•=O → NO• + PyzCHO, would yield an alde-
hyde, which was not evidenced by 1H NMR spectroscopy.
Moreover, the benzile-like product of the radical coupling
process 2 ArCO• →ArC(O)–C(O)Ar also failed to be iso-
lated and even detected.
Another possibility is thus that, in the hydroxamic series,
the carboxylic acid does not arise from an aroyl radical,
but from another intermediate instead. This intermediate
could be the undissociated aroyl-nitroso compound, gener-
ated upon an oxidation step, which could undergo a rapid
heterolytic cleavage by the surrounding water molecules
(Scheme 6), releasing HNO without any radical species, as
previously suggested to occur from other hydroxamic acids
[38, 39].
To explain the latter results, two hypotheses can
be proposed. In the case of the hydroxamic acid com-
plexes, possibly formed HNO along with the aroyl radi-
cal (by analogy with the INH complex, Scheme 3) might
quench the latter thus shortening its half-life time and
preventing its detection by EPR. However, the process
1 3

JBIC Journal of Biological Inorganic Chemistry
complex 6 exhibits lower EC50 (64 nM) than 3 and SNP is
only a fvefold more potent than 6. The benefcial role of
the iron complex moiety for vasodilation properties can be
promptly noted by comparing the EC50 values of 2 and 3 and
also 5 and 6 (Fig. 5). The concentration–response curve for
the vasodilation experiments showed that the complex 3 as
compared to SNP has the same efcacy and similar potency,
which is a priori promising for the design of an alternative
drug. Although SNP has several indications in cardiology
such as heart failure, angina, after cardiac surgeries, hyper-
tensive crisis, hypertensive encephalopathy, cardiogenic
pulmonary oedema, it also has several contraindications
and limitations such as liver toxicity, refex tachycardia, and
electrocardiographic alterations.
Inhibitory activity against Mtb
The antimycobacterial activity of the ligand 2 and complex
3 was investigated against the Mtb H37Rv reference strain,
using INH as positive reference compound. The ligand 5
and the complex 6 were also introduced in these biological
assays for comparison purposes. In this anti-mycobacterial
assay, resazurin dye was employed to measure the lowest
concentration of the tested compounds that could prevent
any color change, which indicates a disruption of the growth.
Those values were reported as MIC (minimum inhibitory
concentration), which, for compounds 2 and 3, 5 and 6
were, respectively, 100 and>100 μg/mL, and for INH was
0.39 μg/mL. These results indicate that even at considerably
high concentrations, there is no measurable inhibition of
Mtb growth under these experimental conditions. However,
putative activity against of Mtb strains within activated mac-
rophage, or even in the non-replicating Mtb state, deserves
further investigations.
Although the water-soluble complexes 3 and 6, able to
release HNO/NO, are less potent than SNP, this can be an
advantage because SNP, releasing NO rapidly, can cause
major drops in blood pressure, thus requiring a careful con-
trol through titration.
Vasodilatory and antihypertensive activity
Aiming to test the hypothesis that metal complex 3 or 3H+
can release HNO/NO, vasodilation assays were performed.
Again, the ligand 2 and 5 and the complex 6 were also tested
for comparison. The NO species is indeed known to induce
vasodilation of blood vessels, and NO carrier molecules,
such as sodium nitroprusside (SNP, Na2[FeII(CN)5(NO)),
have an important therapeutic efect for hypertensive crises
[40]. At the same time, HNO, the reduced and protonated
form of NO., has also been reported to exhibit vasodila-
tion activity, and shares many similar biological features
of NO.[38, 39]. The structural analogy of the complexes 3
and 6 with SNP was thus a natural argument for such inves-
tigations, along with our further experimental evidences
for HNO production (Fig. 4). Indeed, an assay carried out
side-by-side with SNP and complexes 3 and 6 showed an
interesting profle of vasodilation. While the free ligands 2
and 5 exhibited an expressively higher EC50 value for vaso-
dilation activity at 19 μM, the metal complex 3 had an EC50
value of 250 nM only, a remarkable enhancement of 76-fold.
Additionally, EC50 values for the complex 3 is 19-fold higher
than SNP as measured side-by-side (EC50 = 13 nM). The
Fig. 5 Relaxation efects on aortic rings pre-contracted with 0.1 µM
phenylephrine: [Fe(CN)5(pyrazine-2-hydroxamic acid)]3− (3) (blue),
Fe(CN)5(pyridine-4-hydroxamic acid)]3− (6) (red), pyrazine-2-hy-
droxamic acid (2) (green), isonicotinoic hydroxamic acid (5) (purple)
and SNP (black). *p<0.05 vs. pyrazine-2-hydroxamic acid and isoni-
cotinoic hydroxamic acid. The IC50 and respective 95% confdence
interval were calculated only for 3 (250 nM, 91–690 nM) and 6 (64
nM, 25–160 nM)
3-
2-
3-
O
O
O
O
H2O
N
N
pH = 7.4
N
N
N
N
N
N
O-
O
NHOH
NHOH
N
H2O2
HNO
OH + -OH
2H+ + 1e-
FeII(CN)5
FeIII(CN)5
FeII(CN)5
pyrazinamide
delamanid active
metabolite
active metabolite
Scheme 6 Proposed outcome of the chemical oxidation of complex 3 under its trianionic form 3H+ with H2O2
1 3

JBIC Journal of Biological Inorganic Chemistry
The complexes 3 and 6 can be all the more interesting
since their efcacy (maximum relaxation efect) is quite
similar to that of SNP. We should also point out that, in
contrast to SNP, whose release of NO also promotes cyanide
dissociation, the process is greatly reduced for new pentacy-
anoferrate complexes. Actually, a quick comparison of the
lethal dose of SNP and the pentacyanoferrate(II)-INH com-
plex (IQG607) illustrates this topic (mouse orally adminis-
tered with SNP and IQG607 LD50=61 mg/kg and 2970 mg/
kg, respectively), where SNP is over 48-fold more lethal
[14, 41]. It is also noteworthy that those values correspond
to oral dosages, but considering that pentacyanoferrate(II)
complexes are particularly instable in acidic conditions—
occurring in the rats’ stomach— leading to decomposition
and cyanide release—the real activity level might be even
higher with other administration routes.
a
120
100
80
60
40
20
0
WKY
SHR
*
*
*
*
*
*
*
Control 0.1
0.5
1
5
10
Compound 6 (mg/Kg)
b
150
100
50
*
All these results indicate that the complex 3 or 6 might be
a suitable model for designing alternative anti-hypertensive
drugs. Modulation of the electronic efects on the metal
center and changes on the aromatic hydroxamic ligand moi-
ety might lead to more efcient tuning of their properties
to achieve adjustable HNO/NO release. For instance, the
compound 6, which exhibited the lower EC50 (64 nM), was
demonstrated to decrease blood pressure more efciently in
hypertensive rats (SHR) than its normotensive matched con-
trols (Fig. 6). The doses of 1, 5, and 10 mg/kg promoted a
drop in blood pressure equivalent to 45.6 1.9%, 62.1 2.5%
and 65.1 3.1% in SHR rats and 30.8 2.3, 50.9 4.2 and
53.3 3.3 in normotensive rats, respectively. This compound
did also increase cardiac output signifcantly, probably sec-
ondary to a decreased afterload and increased coronary fow.
*
WKY
SHR
*
*
* *
0
Control 0.1
0.5
1
5
10
Compound 6 (mg/kg)
Fig. 6 Antihypertensive (a) and increased cardiac output (b) induced
by compound 6 in normotensive (WKY) rats or spontaneously hyper-
tensive rats (SHR). *p<0.05 vs. control (administration of the vehi-
cle)
Conclusion
promising advances have been established for antihyper-
tensive therapy, where the hydroxamic complexes 3 and 6
have been shown to be globally as active as the SNP drug,
with a more regular action and signifcantly lower toxicity.
Additionally, comparison of vasodilation properties of 3
and 6 with those of 2 and 5 respectively, clearly show that
the pentacyanoferrate moiety plays a benefcial role in the
efciency of the release of HNO from hydroxamic acids.
Investigation of the expected longer duration of vasodila-
tory action of 3 and 6 will be performed in due course by
systematic pharmacodynamic (PD) studies.
The proposed strategy for metal-mediated oxidative acti-
vation of hybrid prodrugs, has been investigated for the
anti-Mtb metabolites of pyrazinamide and delamanid in
the hydroxamic hybrid series. The results unveil biological
prospects of Fe(II) coordination complexes in pharmaco-
phore design, while spanning a bridge between far-related
medicinal challenges, from anti-bacterial to vasodilation
efects. The complex 3 in the presence of H2O2 gave direct
and indirect evidences for the release of pyrazinoic acid
and HNO, respective active metabolites of pyrazinamide
and delamanid. Although this complex did not exhibit
anti-Mtb activity on actively growing non-resistant
strains, further investigations may be warranted employ-
ing either/both single-condition whole-cell models (e.g.,
fatty acid carbon sources, hypoxia, low pH, bioflm, and
carbon starvation) or/and multistress condition whole-cell
models that try to recapitulate nonreplicating persistent
bacilli, upon which pyrazinamide is active. Furthermore,
Acknowledgements The authors would also like to acknowledge
fnancial support given by CNPq/FAPERGS/CAPES/BNDES to the
National Institute of Science and Technology on Tuberculosis (INCT-
TB), Brazil [grant numbers: 421703-2017-2/17-1265-8/14.2.0914.1;
LGFLopes: CNPq 303355/2018-2; EHSS: CNPq 308383/2018-4, Uni-
versal 403866/2016-2) and COFECUB Project n° Ph-C 883/17. This
study was fnanced in part by the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.
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JBIC Journal of Biological Inorganic Chemistry
14. Abbadi BL, Rodrigues-Junior VS, Dadda AS, Pissinate K, Villela
AD, Campos MM, Lopes LGF, Bizarro CV, Machado P, Sousa
EHS, Basso LA (2018) Is IQG-607 a potential metallodrug or
metallopro-drug with a defned molecular target in Mycobacte-
rium tuberculosis? Front Microbiol 9:880
Additionally, we are also in debt with Iury A. Paz, Renata Oliveira
Santiago and Ariana Gomes da Silva for their assistance on the pre-
liminary biological studies.
Compliance with ethical standards
15. Zhang Y, Mitchison D (2003) The curious characteristics of
pyrazinamide: a review. Int J Tuberc Lung Dis 7:6–21
Conflict of interest The authors declare have no confict of interest.
16. Vandal OH, Nathan CF, Ehrt S (2009) Acid resistance in Myco-
bacterium tuberculosis. J Bacteriol 191:4714–4721
Ethical approval All procedures were performed according to the ethics
17. Zhang Y, Mitchison D, Shi W, Zhang W (2014) Mechanisms
of pyrazinamide action and resistance. Microbiol. Spectr. 2:
MGM2-0023-2013
committee of State University of Ceará number 2897836/15.
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Zhang T (2016) Pyrazinamide resistance in Mycobacterium tuber-
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Publisher’s Note Springer Nature remains neutral with regard to
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Afliations
Edinilton Muniz Carvalho1,2,3 · Tercio de Freitas Paulo1,2,3 · Alix Sournia Saquet1 · Bruno Lopes Abbadi4,5
·
Fernanda Souza Macchi4,5 · Cristiano Valim Bizarro4,5 · Rafael de Morais Campos6 · Talles Luann Abrantes Ferreira6 ·
Nilberto Robson Falcão do Nascimento6 · Luiz Gonzaga França Lopes3,5 · Remi Chauvin1,2
Eduardo Henrique Silva Sousa3,5 · Vania Bernardes‑Génisson1,2
·
1
4
CNRS, Laboratoire de Chimie de Coordination,
LCC, UPR 8241, 205 Route de Narbonne, BP 44099,
31077 Cedex 4 Toulouse, France
Centro de Pesquisas Em Biologia Molecular E Funcional
(CPBMF), Pontifícia Universidade Católica Do Rio Grande
Do Sul (PUCRS), Porto Alegre, Brazil
2
3
5
6
Université de Toulouse, Université Paul Sabatier, UPS, 118
Route de Narbonne, 31062 Cedex 9, Toulouse, France
Instituto Nacional de Ciência E Tecnologia Em Tuberculose
(INCT-TB), Porto Alegre, Brazil
Grupo de Bioinorgânica, Departamento de Química Orgânica
E Inorgânica, Universidade Federal Do Ceará, Campus Pici,
Fortaleza, CE 60455-760, Brazil
Laboratório de Farmacologia Cardiovascular E Renal,
Universidade Estadual Do Ceará, Campus do Itaperi,
Fortaleza, CEP 60714-903, Brazil
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