Synthesis, properties, and antibacterial activity of a new nitric oxide donor — a nitrosyl iron complex with 5-phenyl-1H-1,2,4-triazole-3-thiol

Article abstract of DOI:10.1007/s11172-019-2691-0

A new binuclear μ-NSC-type nitrosyl iron complex [Fe₂(SR)₂(NO)₄] (R is 5-phenyl-1H-1,2,4-triazole-3-thiolyl) was synthesized. The composition and structure of the newly synthesized complex were determined by atomic absorption, IR, EPR, and M?ssbauer spectroscopy and SQUID magnetometry. Amperometric analysis showed that the new complex is an effective nitric oxide (NO) donor in a 1% aqueous DMSO solution. The amounts of NO generated by the complex [Fe₂(SR)₂(NO)₄] in aqueous solutions at physiological pH values were determined. The new complex was shown to have higher antibacterial activity against the gram-positive bacteria Micrococcus luteus compared to kanamycin and streptomycin. According to assays, sulfur-containing ligands of the 1,2,4-triazole-3-thiol series are promising for the design of new antibacterial agents containing the {Fe(NO)₂} structural moiety.

Full text of DOI:10.1007/s11172-019-2691-0

Russian Chemical Bulletin, International Edition, Vol. 68, No. 12, pp. 2225—2231, December, 2019
2225
Synthesis, properties, and antibacterial activity of a new nitric oxide donor —
a nitrosyl iron complex with 5-phenyl-1H-1,2,4-triazole-3-thiol*
N. A. Sanina,a,b,c V. A. Mumyatova,a A. A. Terent´ev,a,b,c R. B. Morgunov,a N. S. Ovanesyan,a and A. V. Kulikova
aInstitute of Problems of Chemical Physics, Russian Academy of Sciences,
1 prosp. Akad. Semenova, 142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 522 3507. E-mail: sanina@icp.ac.ru
bDepartment of Fundamental Physical and Chemical Engineering, Lomonosov Moscow State University,
1 Leninskie Gory, 119991 Moscow, Russian Federation.
Fax: +7 (495) 939 0175
cScientific and Educational Center "Medicinal Chemistry", Moscow State Regional University,
24 ul. Very Voloshinoi, 141014 Mytishchi, Moscow region, Russian Federation.
Fax: +7 (499) 261 4377
A new binuclear -NSC-type nitrosyl iron complex [Fe2(SR)2(NO)4] (R is 5-phenyl-1H-
1,2,4-triazole-3-thiolyl) was synthesized. The composition and structure of the newly synthe-
sized complex were determined by atomic absorption, IR, EPR, and Mössbauer spectroscopy
and SQUID magnetometry. Amperometric analysis showed that the new complex is an effective
nitric oxide (NO) donor in a 1% aqueous DMSO solution. The amounts of NO generated by
the complex [Fe2(SR)2(NO)4] in aqueous solutions at physiological pH values were determined.
The new complex was shown to have higher antibacterial activity against the gram-positive
bacteria Micrococcus luteus compared to kanamycin and streptomycin. According to assays,
sulfur-containing ligands of the 1,2,4-triazole-3-thiol series are promising for the design of new
antibacterial agents containing the {Fe(NO)2} structural moiety.
Key words: nitric oxide, nitric oxide donors, binuclear tetranitrosyl iron complexes,
Mössbauer spectroscopy, EPR spectroscopy, antibacterial activity.
According to the World Health Organization (WHO),1
infectious diseases are among the leading causes of death
worldwide. An estimated 700 000 people around the world
die each year from drug-resistant strains of common
pathogens.2 In Russia, tuberculosis, viral hepatitis, and
HIV infection are the leading causes of death from infec-
tious and parasitic diseases.3 The development of new
chemotherapeutic approaches to search for more effective
antibacterial agents and drugs is a way to address this big-
gest challenge of humanity.
The goal of our studies is to discover effective chemi-
cal compounds and drugs based on these compounds with
the structures and properties similar to cellular intermedi-
ates that are present in the human body that would not
only show high efficacy but also would not contribute to
the development of new antimicrobial resistance. Within
the framework of the NO-therapy method (nitric oxide
therapy), earlier we have designed compounds exhibiting
antibacterial activity. Thus, we synthesized hybrid systems,
which bear aliphatic (thioamines, thioures) and aromatic
(thiophenols, mercaptopyridine derivatives) sulfur-con-
taining moieties as inhibitors of DNA synthesis and con-
tain NO groups as a source of nitric oxide capable of
inhibiting bacterial growth. Our studies showed4—6 that
antibacterial activity of these hybrid molecules is compa-
rable with that of the currently used antibiotics.
Mercapto-1,2,4-triazoles are also promising sulfur-
containing ligands (inhibitors of DNA synthesis). Apart
from fungicidal5—12 and antiviral13 activities, mercapto-
1,2,4-triazoles and their derivatives have antibacterial
properties. For example, 1,2,4-triazole sulfone derivatives
displayed activity comparable with that of norfloxacin.14
Quinoline derivatives containing the 1,2,4-triazole-3-thiol
moiety have moderate or high antibacterial activity against
Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, and Klebsiella pneumoniae (Friedländer bacil-
lus) with a minimum inhibitory concentration (MIC) of
6.25—50 g mL–1 comparable with that of ciprofloxacin
(MIC is 6.25 g mL–1).15 3-Aryloxymethyl-4-[2-(benz-
imidazolylthio)acetamido]-5-mercapto-1,2,4-triazole
derivatives were reported16 to have activity against S. aureus
and E. coli comparable with that of penicillin and strep-
* Based on the materials of the 4th Russian Conference on
Medicinal Chemistry with International participation (June 9—14,
2019, Ekaterinburg, Russia).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2225—2231, December, 2019.
1066-5285/19/6812-2225 © 2019 Springer Science+Business Media, Inc.

2226 Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Sanina et al.
tomycin. 4-Alkylidene(arylidene)-4-ethyl-5-(2-furyl)-
1,2,4-triazole-3-mercaptoacetic acid hydrazides are active
against strains of S. aureus, Staphylococcus epidermidis,
Friedländer bacillus, and E. coli: the MIC of these com-
pounds against S. aureus is 2.4 g mL–1, whereas MIC of
moiety) displayed the highest activity. Various triazolyl-
thiazole derivatives were synthesized, and their antimy-
cobacterial activity was evaluated against M. tuberculo-
sis.24,27,28 Antituberculosis activity of these compounds
(MIC is about 8 g mL–1) is much higher than that of
their precursors, which do not contain the triazole ring.
This is an evidence that the 1,2,4-triazole moiety can
enhance antituberculosis activity under certain conditions.
Hence, the drug design for NO-therapy of infectious
diseases based on nitrosyl iron complexes bearing, apart
from NO groups, sulfur-containing ligands of the mer-
capto-1,2,4-triazole series, holds considerable promise.
In the present work, we report the synthesis of the new
nitrosyl iron complex with 5-phenyl-1H-1,2,4-triazole-
3-thiol. The properties of the newly synthesized complex
were studied in the solid state and solution, and its cyto-
toxicity and antibacterial properties were evaluated in
models of gram-positive and gram-negative bacteria.
cefuroxime is 1.2 g mL–1 17
3-(3-Aryl-1H-pyrazol-5-
.
yl)-4-substituted 5-mercapto-1,2,4-triazoles are much
more active against S. aureus, E. coli, Shigella dysenteriae,
and Salmonella typhi (the causative agent of typhoid fever)
than chloramphenicol.18 Fused heterocyclic derivatives,
4,6-disubstituded 1,2,4-triazole-1,3,4-thiadiazoles, were
synthesized and were shown to be active against E. coli,
Pseudomonas fluorescens, Bacillus subtilis, and Xanthomonas
campestris (the causative agent of a variety of plant diseases
including black rot in cruciferous plants).19 Substituted
4-arylideneamino-3-mercapto-5-[(1H-indol-3-yl)meth-
yl]-4H-1,2,4-triazoles and 6-aryl-3-[(1H-indol-3-yl)-
methyl]-7H-1,2,4-triazole-1,3,4-thiadiazines are active
against S. aureus.20 5-(3,5-Dibromo-2-hydroxyphenyl)-
2,3,4-trihydro-1,2,4-triazole-3-thione and a series of
5-[3-(amino-5-mercapto-4H-1,2,4-triazol-3-yl)phenyl]-
3-phenylcyclohex-2-en-1-ones proved to be more active
against E. coli, P. aeruginosa, S. aureus, S. epidermidis,
and B. subtilis than ciprofloxacin.21 The investigation
of new nalidixic acid analogs, 3-(4-arylideneamino-5-
mercapto-4H-1,2,4-triazol-3-yl)-1-ethyl-7-methyl-1,8-
naphthyridin-4(1H)-ones, exhibiting activity against
P. aeruginosa, B. subtilis, E. coli, K. pneumoniae, Streptococcus
pneumoniae, and P. aeruginosa showed that compounds
bearing electron-donating substituents at the phenyl ring
are more active.22 3-N-Substituted carboxamidomethyl-
thio-4H-1,2,4-triazoles23 are active against S. aureus,
B. subtilis, E. coli, K. pneumoniae, S. pneumoniae, and
P. aeruginosa.23 Compounds containing electron-with-
drawing substituents (a chlorine atom, methoxy or nitro
groups) at the phenyl ring are more active than strepto-
mycin and ciprofloxacin.
Experimental
The following commercially available chemical reagents
and solvents were used: 5-phenyl-1H-1,2,4-triazole-3-thiol
(95%), diethylenetriamine NO adduct (97%), FeSO4•7H2O,
Na2S2O3•5H2O, and KOH (Sigma-Aldrich). Complex
Na2[Fe2(S2O3)2(NO)4]•4H2O was synthesized by a procedure
described prevuously.29 Nitrogen(II) oxide (NO) was prepared
by the reaction of a solution of FeSO4•7H2O with a solution of
sodium nitrite NaNO2 (reagent grade) in concentrated HCl
(reagent grade) by a known procedure.30 All solutions were
prepared, mixed, and stored under an argon atmosphere.
Elemental analysis was performed and the IR spectra were
measured in the Multiple-user Analytical Center of the Institute
of Problems of Chemical Physics of the Russian Academy of
Sciences. Elemental analysis was carried out on a Vario Micro
cube CHNS/O elemental analyzer. The IR spectra were re-
corded on a Perkin-Elmer Spectrum 100X spectrophotometer at
room temperature.
The evaluation of antituberculosis activity of mercapto-
1,2,4-triazole derivatives occupies a special place in studies
of these compounds. For example, a series of 4-alkyl(aryl)-
2,4-dihydro-5-{(6-(4-bromophenyl)imidazolo[2,1-b]thi-
azol-3-yl)methyl}-3H-1,2,4-triazole-3-thiones,24 2-sub-
stituted 5-isopropylthiazoles and 1,2,4-triazoles,25 and
4-amino-5-(4-pyrrol-1-yl-phenyl)-2,4-dihydro-1,2,4-
triazole-3-thione derivatives26 are active against Myco-
bacterium tuberculosis H37Rv. These compounds exhibited
antituberculosis activity with MIC comparable to that of
isoniazid (MIC 0.25 g mL–1). 3-Benzylsulfonyl deriva-
tives of 1,2,4-triazole and 4-methyl-1,2,4-triazole are
active against various mycobacterial species.13 The syn-
thesized triazole derivatives have moderate or low anti-
mycobacterial activity and are less active against M. tuber-
culosis and M. kansasii compared to isoniazid. 1,2,4-Tri-
azole derivatives containing electron-withdrawing sub-
stituents (a nitro group, a trifluoromethyl group, a thioamide
Complex [Fe2(SR)2(NO)4] (1) was synthesized from 5-phenyl-
1H-1,2,4-triazole-3-thiol by a known procedure.31 5-Phenyl-
1H-1,2,4-triazole-2-thiol (0.3322 g, 1.88 mmol) was added with
vigorous stirring to a solution of KOH (0.1050 g, 1.88 mmol) in
methanol (10 mL) and water (20 mL). The reaction mixture was
filtered into a flask under an argon atmosphere. Then a solution
of Na2[Fe2(S2O3)2(NO)4]•4H2O (0.0718 g, 0.188 mmol) and
Na2S2O3•5H2O (0.0620 g, 0.250 mmol) in water (10 mL) was
filtered into the flask with stirring under argon. The solution
turned intensely dark brown. The mixture was stirred for 20 min
that resulted in the formation of small dark red-brown polycrystals.
The reaction mixture was kept at ~4 C for 16 h, and the result-
ing crystals were filtered off and dried in air. The yield was 0.061 g
(84%). Found (%): C, 34.81; H, 2.19; N, 18.03; Fe, 19.90;
S, 11.78. Fe2C16H12N10S2O4. Calculated (%): C, 35.29; H, 2.21;
N, 18.38; Fe, 20.00; S, 11.76. IR, /cm–1: 3486 (m), 3264 (m),
1809 (NO, v.s), 1760 (NO, v.s), 1611 (w), 1565 (w), 1552 (w),
1510 (w), 1489 (m), 1466 (m), 1427 (w), 1350 (w), 1227 (w),
1121 (w), 1074 (w), 1028 (w), 980 (w), 968 (w), 863 (w), 772 (m),
688 (v.s).

Novel antibacterial nitric oxide donor
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2227
Mössbauer absorption spectra were recorded on a WissEl
Mössbauer spectrometer (Germany) in constant acceleration
mode using 57Co isotope in the Rh matrix as the -radiation
source. The low-temperature spectra were measured using
a CF-506 temperature-controlled helium flow cryostat (Oxford
Instruments). The Mössbauer spectra were fitted by the least-
squares procedure assuming the Lorentzian line shape of com-
posite absorption peaks.
Magnetic moment of complex 1 was measured on a Quantum
Design MPMS magnetometer at 6—300 К in a magnetic field of
1 kOe. A powdered sample with a weight of ~10 mg was placed
in a cell with the compensated intrinsic magnetic moment
~100 times lower than the magnetic moment of the sample.
EPR spectra of powders and solutions of complex 1 in DMSO
were recorded on a Radiopan SE/X 2544 spectrometer at room
temperature. Powders (~10 mg) were placed in 5 mm tubes;
solutions (~2 mmol L–1), in 1 mm tubes. The number of para-
magnetic centers in the sample was estimated by comparing the
second integrals of the EPR spectra of the sample and the stan-
dard. A CuSO4•5H2O powder or a nitroxide radical solution in
water was used as the standard. The g-factor of complex 1 was
determined using 2,2-diphenyl-1-picrylhydrazyl with the g-factor
of 2.0036 as the standard. The percentage of paramagnetic com-
plexes (N) was evaluated as the ratio of the number of paramag-
netic centers determined by EPR to the number of the com-
plexes estimated using the weighted sample.
Amperometric analysis of NO. The NO-donor ability of
complex 1 was studied by amperometry with an inNO-T Nitric
Oxide Measuring System using an amiNO-700 selective sensor
electrode (Innovative Instruments, USA). The method is based
on the measurement of the diffusion current caused by changes
in the electrode potential due to the NO release by the compound
under study. The concentration of NO, which was generated by
complex 1 (at a concentration of 4•10–6 mol L–1) in a 1% aque-
ous DMSO (reagent grade) solution (25 C), was recorded for
500 s every 0.2 s. The amperometric sensor was calibrated with
a standard 1 M NaNO2 aqueous solution, which was added to
the mixture containing H2O (18 mL), KI (20 mg), and 1 M H2SO4
(reagent grade, GOST-4204-77; 2 mL). The reaction is described
by the equation
Cytotoxicity of complex 1 was evaluated against the Vero cell
line (African green monkey kidney epithelium) used as the stan-
dard in toxicology assays.32 The cells were seeded in 96-well
plates and incubated overnight. Then complex 1 was added at
different concentrations to the incubation medium. The incuba-
tion was performed overnight, and then the cells were stained
with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) (the final MTT concentration in the cells was
0.5 mg mL–1). After the incubation with the dye for 2 h, the
culture medium was removed, and the formazan crystals that
formed were dissolved by the addition of DMSO (100 L per
each well). The absorbance of the solutions was measured by
spectrophotometry using a Spark 10M plate reader (Tecan,
Switzerland) at a wavelength of 570 nm. The nonspecific absor-
bance was measured at a wavelength of 650 nm. The intensity of
MTT staining was calculated from the difference between the
absorbance at 570 and 650 nm. The MTT staining of the cells in
the control sample was taken as 100%.33
Evaluation of antibacterial activity of complex 1 was per-
formed by determining the minimum inhibitory concentration,
which prevents visible growth of the microorganism in the broth
culture, by means of serial dilutions.34 The double-series dilu-
tion method was employed. A stock solution of complex 1
was prepared in DMSO with the maximum concentration of
20 mmol L–1 and was added to 1 mL of sterile incubation LB
medium (1% peptone, 0.5% yeast extract, 1% NaCl, 0.1% glu-
cose, pH 6.8—7.0). The final concentrations of the complex in
the samples varied from 3.9 mol L–1 to 2 mmol L–1. The final
maximum concentration of DMSO in the samples was 10%. This
DMSO concentration has no effect on the viability of the bacte-
rial agent. The inoculated broth culture (1 mL) with a density
0.5 McFarland standard was added to the test tubes containing
complex 1 (the final concentration of the microorganisms in the
samples was 5•105 CFU mL–1). The test tubes were incubated
in a thermostat at 37 C; the control samples were incubated for
the same time at 4 C. The results were recorded after 24 h. The
concentration of complex 1, at which no visible growth of mi-
croorganisms was observed in the medium (compared to the
control samples), was taken as MIC. Laboratory cultures of the
gram-negative bacteria E. coli (strain BB) and the gram-positive
bacteria Micrococcus luteus (strain 21/26) were used as bacterial
agents. Streptomycin, ceftriaxone, and kanamycin dissolved in
water served as the standard samples. The concentration range
2 NaNO2 + 2 KI + 2 H2SO4
=
= 2 NO + K2SO4 + 2 H2O + Na2S2O4+ I2.
of the antibiotics was from 0.49 to 250 mol L–1
.
According to the reaction equation, the components react in
a ratio of 1 : 1 : 1. Therefore, the concentration of the released NO
is equivalent to the concentration of the added sodium nitrite. The
addition of the standard sodium nitrite solution (10 L) resulted
in the release of NO (50 nmol L–1) in the working solution (20 mL).
The calculation was performed by the following equation:
Results and Discussion
Complex 1 was synthesized in three steps: 1) the
preparation of gaseous NO, 2) the preparation of the ni-
trosyl iron complex with a thiosulfate ligand, and 3) the
preparation of the complex with 5-phenyl-1H-1,2,4-tri-
azole-3-thiol (Scheme 1, Fig. 1).
M1•V1 = M2•V2,
where M1 is the molarity of the standard sodium nitrite solution,
V1 is the added volume of the standard sodium nitrite solution, M2
is the molarity of the sodium nitrite solution after the addition of
the standard to the sample, V2 is the total volume of the sample.
Then the weighted sample of nitrosyl complex 1 was dissolved
in 10 mL of DMSO (reagent grade), an aliquot of 0.5 mL of the
sample was taken and added to a Hydrion buffer solution (Aldrich,
49.5 mL), pH 6.0, 7.0, or 8.0, and the NO release was measured.
The diethylenetriamine NO adduct was used as the standard.
Scheme 1
RSH is 5-phenyl-1H-1,2,4-triazole-3-thiol.

2228 Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Sanina et al.
length between the iron atom and the nitrogen atom of
the heterocyclic ligand is, on average, 2 Å; the bond
length between the iron and sulfur atoms is 2.3 Å. The
bond length and angle distribution in the aromatic ligands
of all -SCN-type complexes corresponds to the thiol
form of the ligand. The bond lengths in the {Fe(NO)2}
structural fragment of all complexes of this type vary
in a narrow range. The partial oxidation/reduction in
the Fe—NO moiety can occur due to the weak direct
(dFe*NO) and back donation accompanied by shorten-
ing/elongation of Fe—N bonds and elongation/shortening
of N—O bonds. A decrease in the bond length between
the iron atom and the nitrogen atom of the heterocycle is
generally indicative of good stability of the complex in
protic media.
According to SQUID magnetometry, complex 1 is
paramagnetic (Fig. 2). The effective magnetic moment of
the paramagnetic center {Fe(NO)2} at 150—300 К is
1.8 B, which is similar to the calculated effective magnetic
moment eff = g(S(S + 1))1/2 = 1.73 B for the spin S = 1/2
(see Fig. 2, a). At T < 100 К, the effective magnetic mo-
ment per magnetic center decreases with decreasing
Fig. 1. Proposed structure of the complex [Fe2(SR)2(NO4)] (1).
Complex 1 is stable during storage in light in the ab-
sence of moisture, readily soluble in alcohols, acetonitrile,
and DMSO, and insoluble in nonpolar solvents. The de-
composition temperature of complex 1 is about 177 C.
The IR spectrum of complex 1 shows synphase vib-
rations of the NO group (NO(sym)) at ~1809 cm–1
and antiphase vibrations (NO(asym)) at ~1760 cm–1
.
The differences between the frequencies NO(sym) and
NO(asym) is 49 cm–1. The stretching bands (doublets) of
NO groups, the intensity of which is an order of magnitude
higher than that of the other lines, are the major lines in
the IR spectrum of complex 1. This effect is attributed not
only to the enhancement of polarization of the N—O bond
in the complex but also to the fact that the donor-accep-
tor electron transfer is sensitive to the N—O distance in
the {Fe—NO} moiety. This binding feature accounts for
not only a considerable intensity of the stretching bands
of NO groups in the spectra of the complexes but also
eff/B
a
1.6
1.2
0.8
noticeable differences in the splitting of the doublet NO
,
varying from 13 cm–1 for equivalent NO groups to 82 cm–1
for nonequivalent NO groups, with the difference in the
N—O and Fe—N distances of 0.01 Å.35
The Mössbauer spectral parameters of complex 1 at 85 К
are as follows: the isomeric shift Fe = 0.301(1) mm s–1 and
the quadrupole splitting EQ = 0.925(4) mm s–1 with the
linewidth Г = 0.287(3) mm s–1. These parameters are
similar to those of the known neutral binuclear iron com-
plexes of the -NCS structural type, the structure and the
physicochemical properties of which were studied in our
previous work.36 These complexes are used as the basis for
the development of polymer composites, namely, nitric
oxide delivery systems37 promising for medical applica-
tions. In neutral binuclear complexes of this structural
type, in particular, in the binuclear complex with 3-amino-
5-mercapto-1,2,4-triazoles,38 the ligands are bound to the
iron atoms in a bridging mode through the sulfur and
nitrogen atoms39—41 (see Fig. 1). The iron atoms also have
a tetrahedral configuration and are at a distance, on aver-
age, of 4 Å, from each other, as opposed to complexes
of the -S structural type, Roussin's red esters42 (Fe…Fe,
2.7 Å; these complexes are diamagnetic). In these para-
magnetic binuclear sulfur nitrosyl complexes, the bond
100
200
T/К
mol/cm3 mol–1
b
4
2
0
0
100
200
T/К
Fig. 2. Temperature dependence of the effective magnetic mo-
ment (a) and the molar magnetic susceptibility (b) of complex 1
in a magnetic field of 1 kOe. In Fig. 2 a, the calculated effective
magnetic moment for the spin 1/2 is indicated by a horizontal line.

Novel antibacterial nitric oxide donor
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2229
[NO]/nmol L–1
a
a
1
2
30
25
20
15
10
5
g = 2.033
g = 2.014
315
320
325
330
B/mT
b
g = 2.028
3
323
Fig. 3. EPR spectra of complex 1: a, powder; b, a solution in DMSO
at a concentration of ~2 mmol L–1
324
325
326
B/mT
100
[NO]/nmol L–1
28
200
300
400
500 t/s
.
b
temperature, reaching 1.0 B at T = 6 K. The decrease in
the effective magnetic moment at T < 100 K can be attri-
buted to the crystal field splitting and the spin—orbit
coupling. The distance between the complexes is too large
for the exchange interaction to cause a noticeable decrease
in the effective magnetic moment with decreasing tem-
perature. The calculated spin of the paramagnetic center
is in agreement with the results of the study of binuclear
complexes of the -NCS structural type with two [Fe(NO)2]
groups containing nine outer-shell electrons each, which
were synthesized and structurally characterized previ-
ously.43,44 In the case of the nearly linear Fe—N—O group,
* electrons of NO form a strong exchange-coupled pair
(covalent bond) with two t2g electrons of the 3d7 shell of
the iron with the temperature-independent resulting spin
S = 1/2 of the [Fe(NO)2] group. However, the effective
magnetic moment decreases with decreasing temperature
due to mixing of the orbital moment of the iron ion.
Figure 3 presents the EPR spectra of complex 1 in the
solid state and in a DMSO solution. The spectrum of
crystalline complex 1 shows anisotropy of the g-factor
(g = 2.014 and g = 2.033). It should be noted that the
fraction of paramagnetic complexes is very low N = 0.048.
Besides, the spectrum has a weak broad Lorentzian line
with a width of ~50 mT and the g-factor of ~2 (in Fig. 3,
a, this line is not shown), for which N  0.3.
The EPR spectrum of complex 1 in a DMSO solution
(see Fig. 3, b) displays splitting at two equivalent nitrogen
nuclei with the constant of 0.19 mT. The fraction of the
paramagnetic complexes N = 0.56.
Complex 1 was found to generate NO upon hydrolysis
without additional activation. The release of NO (Fig. 4)
was observed already within the first seconds after dissolu-
tion. The kinetic curves reach a plateau ~150 s after the
beginning of the experiment, and then the amount of
generated NO does not decrease due apparently to the
establishment of the equilibrium. The experiments at 25 C
were performed in solutions at different pH values using
the diethylenetriamine NO adduct (DETA-NONOate) as
2
24
20
16
12
8
3
1
4
100
200
300
400
500
t/s
Fig. 4. Time dependence of the concentration of NO generated
by complex 1 (0.4•10–5 mol L–1) in a 1% aqueous DMSO solu-
tion at pH 6 (1), 7 (2), and 8 (3) at 25 C under anaerobic (a)
and aerobic (b) conditions.
the standard. The latter is a representative of NONOates,
the efficient NO donors used in the clinical practice.
Complex 1 was found to be a more effective NO donor
than DETA-NONOate. Within 100 s after the dissolution
of complex 1, the amount of NO in the solution was
~10—30 nmol per 1 mole of the compound, which cor-
responds to the two times higher NO-donor activity
compared to DETA-NONOate (~15 nmol per 1 mole of
the compound at pH 7.0). The presence of dissolved oxy-
gen has almost no effect on the generation of NO by
complex 1 in neutral and alkaline media. In an acidic
medium, the NO generation by complex 1 is almost halved,
from 30.0 nmol L–1 at pH 7 to 14.5 nmol L–1 at pH 6
(see Fig. 4, b).
The cytotoxicity evaluation against Vero cells showed
that complex 1 taken at concentrations lower than
50 mol L–1 exhibits low toxicity, whereas at concentra-
tions of 100 mol L–1 it almost completely suppresses the
cell viability. Therefore, the concentration IC50 for com-
plex 1 is about 70 mol L–1. Hence, this complex can be
considered as moderately toxic.45 5-Phenyl-1H-1,2,4-

2230
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Sanina et al.
Table 1. Minimum inhibitory concentrations (MIC)
of complex 1 and standard compounds against gram-
negative and gram-positive bacteria
kanamycin and streptomycin. Based on the results of in-
vestigation, it can be concluded that sulfur-containing
ligands of the mercapto-1,2,4-triazole series hold prom-
ise for the design of new nitrosyl iron complexes with
antibacterial activity.
Compound
MIC/mol L–1
E. coli
M. luteus
This study was financially supported by the Russian
Foundation for Basic Research (Project No. 17-03-00837).
1
500
125
250
115
>250
>500
>500
0.98
Kanamycin
Streptomycin
Ceftriaxone
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Received September16, 2019;
in revised form November 1, 2019;
accepted November 8, 2019