Synthesis, structure, and PDE inhibiting activity of the anionic DNIC with 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyl, the nitric oxide donor

Article abstract of DOI:10.1016/j.ica.2021.120559

A new inhibitor of phosphodiesterase (PDE), i.e., nitric oxide (NO) donor, a water soluble DNIC Na[Fe(C₇H₅N₄S)₂(NO)₂]?2·.5H₂O (1), has been synthesized. The structure of the new complex, its physical–chemical properties in the solid state and in solutions have been studied experimentally (X-ray analysis, IR, UV–Vis, Mossbauer spectroscopy, mass-spectrometry, and amperometry) and theoretically (quantum-chemical modeling by DFT method).

Full text of DOI:10.1016/j.ica.2021.120559

Inorganica Chimica Acta 527 (2021) 120559
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, structure, and PDE inhibiting activity of the anionic DNIC with
5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyl, the nitric oxide donor
,b,c,
Natalia A. Saninaa
*, Yuliya A. Isaevaa, Andrey N. Utenysheva, Pavel V. Dorovatovskiid,
Nickolai S. Ovanesyana, Nina S. Emel’yanovaa b, Olesya V. Pokidovaa, Liliya V. Tat’yanenkoa,
,
Il’ya V. Sulimenkove, Alexandr I. Kotel’nikova b, Sergey M. Aldoshina
,
,b
a Institute of Problems of Chemical Physics, RAS, 1, Acad. Semenov Av., 142432 Chernogolovka, Moscow Region, Russian Federation
b Faculty of Fundamental Physical-Chemical Engineering of M.V. Lomonosov MSU, Leninskie Gory, 119991, Moscow, GSP-1, Russian Federation
c Medical-Chemical Scientific-Educational Center of Moscow Region State University, 24, Vera Voloshina Street, Mytischi, Moscow Region, 141014, Russia
d National Research Centre“Kurchatov Institute”, 1 Acad. Kurchatov Square, 123182 Moscow, Russian Federation
e Chernogolovka Branch of N.N. Semenov Federal Research Center of Chemical Physics of RAS, 1, Acad. Semenov Av., 142432 Chernogolovka, Moscow Region, Russian
Federation
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new inhibitor of phosphodiesterase (PDE), i.e., nitric oxide (NO) donor, a water soluble DNIC Na[Fe
(C7H5N4S)2(NO)2]⋅2⋅.5H2O (1), has been synthesized. The structure of the new complex, its physical–chemical
properties in the solid state and in solutions have been studied experimentally (X-ray analysis, IR, UV–Vis,
Mossbauer spectroscopy, mass-spectrometry, and amperometry) and theoretically (quantum-chemical modeling
by DFT method).
Nitric oxide donors
Dinitrosyl iron complex
5-(3-pyridyl)-4H-1,2, 4-triazole-3-thiol
X-ray diffraction
Mossbauer spectroscopy
IR spectroscopy
UV–Vis spectroscopy
Amperometry
Electrospray mass-spectrometry
TDDFT
Fermentative activity
Phosphodiesterase
1. Introduction
types of aliphatic and azaheterocyclic ligands containing S-[12–24], N-
[25–40], and O-[41–43] donor groups. However, there are few works on
The therapy by nitric oxide (NO) is one of the modern strategies for
treatment of socially relevant diseases, which needs new medicines
characterized by a wide range of activity and no resistance. NO is known
to be a multifunctional molecule able to interact with a lot of cell targets
[1–4]. In vivo, NO can be stabilized and stored as dinitrosyl iron com-
plexes (DNICs) bound with protein, and released from low-molecular
DNICs (LMW-DNICs) existing in the cells [5]. Mimetics of dinitrosyl
non-heme proteins, a class of chemical compounds easily generating
nitric oxide, have a unique chemical structure and a typical EPR signal at
g = 2.03 [6–9]. Design of new DNICs containing the [Fe(NO)2] fragment
with biologically active ligands becomes more and more important due
to the discovery of their interesting biological properties [10,11].
Synthetic analogues of LMW-DNICs were obtained mainly with three
synthesis of LMW-DNICs with ligands having mercapto- and/or thion-
substituents in the heterocyclic ring of 1,2,4-triazole [44]. Such LMW-
DNICs can be of practical application because of the presence of two
pharmacophores, i.e., the NO groups and mercapto-1,2,4-triazolyls. The
latter ones are characterized by a wide range of biological activity
(antibacterial, antifungal, anti-tuberculosis, anti-tumor, etc.) [45–50].
Mercapto-triazoles are effective in inhibiting phosphodiesterases (PDE),
which are known to be responsible for the signal control at different
stages of phosphorylation and protein–protein interaction, and hence, to
play an important role in immune reactions and in control of inflam-
matory processes [51]. The amount of cyclic nucleotides in the cell is
known to be also controlled through activation of enzyme guanylate
cyclase by another signal molecule, nitric oxide NO, which can be
* Corresponding author at: Institute of Problems of Chemical Physics, RAS, 1, Acad. Semenov Av., 142432 Chernogolovka, Moscow Region, Russian Federation.
E-mail address: sanina@icp.ac.ru (N.A. Sanina).
https://doi.org/10.1016/j.ica.2021.120559
Received 28 June 2021; Received in revised form 4 August 2021; Accepted 4 August 2021
Available online 11 August 2021
0020-1693/© 2021 Elsevier B.V. All rights reserved.

N.A. Sanina et al.
Inorganica Chimica Acta 527 (2021) 120559
regulated by introduction of exogenous LMW-DNICs. Purposeful devel-
opment of new compounds which are capable of site-specific effect on
therapeutic targets through two mechanisms simultaneously (as a spe-
cific PDE inhibitor and NO donor) might enhance the regulation of cyclic
nucleotides amount in vivo. As follows from the data we have obtained
earlier, binuclear tetranitrosyl iron complex with 3-amino-5-mercapto-
1,2,4-triazole has a high PDE inhibiting activity [52], but its applica-
tion is limited by its insolubility in aqueous solutions. Therefore, a tar-
geted design of non-toxic and water soluble (i.e., bioavailable) LMW-
DNICs containing mercapto-1,2,4-triazolyls as effective PDE inhibitors
and simultaneously possessing NO donor ability is of current impor-
tance. The study of such DNICs influence on the activity of PDE iso-
enzymes related to various pathologies, which prolong or enhance the
impacts of physiological processes on cAMP and cGMP, could result in
the creation of new agents for NO therapy, as well as in the development
of unique therapeutic strategies for prophylaxis of various socially
relevant diseases.
by the least-squares refinement of Lorentzian shape peaks.
2.2. X-ray analysis for complex 1
X-ray analysis of 1 was performed at 100 K at a ‘Belok’ beamline of
the National Research Center “Kurchatov Institute” (Moscow, Russia)
using a Rayonix SX165 detector at λ = 0.96990 Å. The data were
indexed and integrated by means of iMOSFLM utility in CCP4 program
[55], and then scaled within the Scala program taking into account
absorption [56]. Table 1 shows crystallographic data and main refine-
ment parameters. The structure was solved by the direct method [57].
Positions and temperature parameters of non-hydrogen atoms were
refined in anisotropic approximation by full matrix least-squares
method [57]. Positions of all hydrogen atoms were calculated geomet-
rically and then refined with the “riding” model [57]. All calculations
were performed by means of SHELXTL program complex [57]. Because
of a low quality of the crystals, the hydrogen atoms of the crystallization
water were not localized.
In this work, we report on the synthesis of a new PDE inhibitor, a
water soluble sodium salt with DNIC anion of Na[Fe(SR)2(NO)2]⋅2,5
H2O composition, where R = 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyl
(1). Properties of complex 1 were studied by the methods of X-ray
diffraction, Mossbauer spectroscopy, absorption spectroscopy in UV–vis
region, electro-mass spectrometry, amperometry, and quantum-
chemical calculations.
Mass-spectral studies of complex 1 solutions were performed at an
Exactive Orbitrap mass-spectrometer (ThermoFisher Scientific, Ger-
many) using ionic source with electrospraying (ESI-MS). Working res-
olution of the mass-spectrometer was 10,000 (FWHM). Relative error for
measuring the ion mass to its charge ratio (m/z) was better than 5⋅10-6.
The sample concentration in the solution was about 100 μM.
2. Materials and methods
2.3. The procedure of operation in the inert gas atmosphere
The procedure of operation in argon atmosphere was described in
The following chemicals were used in the work: tris (hydroxymethyl)
aminomethane “Serva”, Germany), sulfanilamide (SA) (“Sigma”, USA),
dihydrochloride N-(1-naphthyl) ethylenediamine (NEDA) (Germany).
Water was distilled in a Bi / Duplex distiller (Germany).
[58].
2.4. Amperometric determination of NO release activity
2.1. Synthesis of complex 1
Quantity of NO generated by complex 1 in solutions was measured
using an amiNO-700 sensor electrode of “inNO Nitric Oxide Measuring
System” (Innovative Insruments, Inc., Tampa, Florida, USA). All exper-
iments were performed in anaerobic and aerobic 1% aqueous DMSO
solutions at 25 ◦C and pH 7.0. NO concentration was recorded during ~
500 s (with 0.2 s pace) in aqueous solution, with NO donor concentra-
Complex 1 was synthesized using Na2[Fe2(S2O3)2(NO)4]⋅4H2O [53],
5- (3-pyridyl)-4H-1,2,4-triazole-3-thiol (Sigma-Aldrich), NaOH (Sigma-
Aldrich), and Na2S2O3⋅5H2O (Sigma-Aldrich). Methanol was purified
using procedure in [54].
1.67 g (9.38 mmol) of 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiol and
0.71 g (12.63 mmol) of the alkali were dissolved in 8 ml of methanol.
This solution was dropwise added to the reaction flask containing so-
lution of Na2[Fe2(S2O3)2(NO)4]⋅4H2O (0.36 g, 0.63 mmol) and 0.31 g
(1.25 mmol) of Na2S2O3⋅5H2O in 8 ml of water at room temperature and
with intense mixing. After 7 days of storing the reaction mixture at
T=+8◦C, black fine crystalline product was filtered through the porous
filter and dried on air. Yield: 87%.
tion being 0.1 μM. DMSO was purified according to procedure in [54].
Commercial buffers Hydrion (Sigma-Aldrich, N 239089) were used (pH
of the solutions were measured using a membrane “HI 8314” pH-meter
(HANNA instruments, Germany)). To calibrate the electrochemical
sensor, a standard aqueous solution of NaNO2 (100 μM) was used, which
was added to the mixture containing 20 mg of KI (“Aldrich”), 2 ml of 1 M
Table 1
Analysis of C, H, N, S, O elements in complex 1 was performed at a
“Vario El cube” CHNS/O element analyzer; Fe and Na were determined
at an “AAS-3” atom-absorption spectrometer in Analytical Center of
IPCP RAS. For C14H15N10O4,5S2FeNa. Found, % C- 31,25, H ꢀ 2,80, N ꢀ
26,00, O – 13,40, S ꢀ 11,87; Fe ꢀ 10,40; Na – 4,30. Calculated, % C ꢀ
31,23, H ꢀ 2,79, N ꢀ 26,02, O – 13,38, S ꢀ 11,90, Fe ꢀ 10,41; Na – 4,28.
IR spectra for 1 (cmꢀ 1) were registered at a Fourier spectrometer
(Brucker ALPHA in the frequency range of в 400–4000 cmꢀ 1 in the mode
of attenuated total reflectance (ATR) at room temperature. IR spectrum
(cmꢀ 1): 3480 (w), 2670 (w), 1773 (vs), 1735 (vs), 1665 (s), 1467 (w),
1412 (w), 1327 (w), 1308 (m), 1114 (m), 1029 (m), 992 (vw), 809 (m),
Main crystallographic data and parameters of the experiment for 1 at 100 K.
Empirical formula
[C14H10FeN10O2S2]Na⋅2⋅.5H2O
Formula weight
547.29
Temperature
100(2) K
0.79313 A
P2(1)/n
Wavelength
Crystal system, space group
Unit cell dimensions
a = 7.5200(15) Å
α
= 90◦
b = 18.480(4) Å β = 98.10(3)◦
c = 14.990(3) Å γ = 90◦
2062.3(7) A3
Volume
Z, Calculated density
Absorption coefficient
F(000)
4, 1.730 mg/cm3
1.354 mmꢀ 1
741 (m), 729 (cpeдняя), 636 (m), 538 (m), 473 (m), 435 (m), νNO =
1084
1773 cmꢀ 1, 1735 cmꢀ 1
.
Theta range for data collection
Limiting indices
1.96◦ to 30.98◦
57Fe Mossbauer spectra of 1 at ambient temperature were recorded
at a Wissel GMBH spectrometer operating in the mode of constant ac-
celeration using a ca. 5 mCi 57Co/Rh source. Low-temperature spectra
were measured using a continuous-flow helium cryostat (CF-506, Ox-
ford Instr. Ltd) operating in a temperature range of 4.2 to 305 K. Velocity
ꢀ 9≤h≤9, –23≤k≤23, ꢀ 19≤l≤19
25,758 / 4475 [R(int) = 0.0781]
Full-matrix least-squares on F2
4475/0/325
Reflections collected / unique
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
1.035
R1 = 0.0765, wR2 = 0.2143
R1 = 0.1207, wR2 = 0.2602
2.8 d ꢀ 1.027 e.A3
calibration was performed with enriched 57Fe 1
μ iron foil. The
Largest diff. peak and hole
¨
Mossbauer spectra comprised single quadrupole doublet that was solved
2

N.A. Sanina et al.
Inorganica Chimica Acta 527 (2021) 120559
H2SO4 solution (pure grade, Khimmed), and 18 ml of water.
2.5. Determination of NO2– concentration from Griess reaction
Scheme 1. Fermentative hydrolysis of cGMP.
8.8 ⋅ 10-5 M solutions of complex 1 were prepared on air in various
solvents: 50% DMSO, 50% ethanol, and Tris-HCl Buffer, pH 7.0. Aliquots
of the reaction mixture (0.5 ml) were taken and introduced in the vessels
containing 1.5 ml of 0.5% sulfanilamide solution in 0.25 M HCl. After 5-
minute incubation, 1 ml of 0.02% solution of N-(1-naphthyl)ethylene
diamine dihydrochloride (NEDA) was added to 0.5 M HCl. In 10 min,
optical density at 540 nm was determined. Kinetics of NO2– accumula-
tion in the system was registered during 40 or 250 min. NO2– concen-
tration was calculated from the calibration curve plotted with respect to
sodium nitrite.
(Reachim, Russia) after additional purification, Tris-HCl buffer (Serva,
Germany) were used. cGMP-PDE enzyme was isolated from the cerebral
cortex of the Wistar line rats [62]. The cerebral cortex tissue was ho-
mogenized in Potter homogenizer with 10-fold amount of cooled 0.2 M
Tris-HCl buffer, pH 7.55. The homogenate was spun in the centrifuge for
1 h at 40,000 g. The supernatant containing cGMP-PDE was frozen in
liquid nitrogen.
To determine cGMP-PDE activity, an aliquot of cGMP-PDE solution
containing 0.1 mg of protein was added to 1 ml of 0.2 M Tris-HCl buffer
(pH 7.6). The compounds under study (complex 1, 5- (3-pyridyl)-4H-
1,2,4-triazole-3-thiol or nitrite) were added as 0.2 ml of DMSO solutions
(complex 1, 5- (3-pyridyl)-4H-1,2,4-triazole-3-thiol) or buffer solutions
(nitrite). The final concentration was 0.1 mmol, 0.01 mmol, or 0.001
mmol. The total volume of the sample was 2.0 ml. After 20 min of
preincubation at room temperature, 0.1 mmol of cGMP was added to the
sample. The samples were kept for 20 min at 30 ◦C; then they were
2.6. Complex 1 decomposition in Tris-HCl buffer (pH 7.0), DMSO, and
ethanol under anaerobic conditions
Anaerobic solution (0.05 M tris-HCl buffer (pH 7.0), DMSO, and
ethanol) was added to the vessel filled with argon and containing a
sample of complex 1, so that concentration of the complex in solution
was 2∙10-3 M. Dissolution was performed using a magnetic stirrer during
3–5 min at 23 ◦C. Then, the aliquot of the solution was taken under
argon flow and introduced in an anaerobic experimental cuvette of 4-ml
volume with the optical path length 1 cm, which contained the corre-
sponding solvent. The final concentration of complex 1 solution was
4∙10-5 M. The reference cuvette contained the same solvent as the
experimental one.
placed in the boiling water bath for 3 min. Then 50 μg of nucleotidase (as
cobra poison) was added to the samples cooled to ambient temperature
and kept at 30 ◦C for 10 min. The reaction was terminated by adding 0.2
ml of 55% TCA to each sample. The reaction mixture was spun in the
centrifuge at 10,000 g during 5 min. To determine cGMP-PDE activity,
concentration of inorganic phosphate accumulating in the fermentative
reaction was determined from the reaction with ammonium molibdate
by a spectroscopic method at a Specord Mꢀ 40 spectrophotometer at λ =
735 nm. The relative activity of the enzyme was calculated from
formula:
2.7. Absorption spectra in UV–Vis region
Absorption spectra in UV–Vis region were registered at an Agilent
Cary 60 UV–Vis at 23 ◦C in the range of 200–750 nm at regular intervals.
I = 100(A0 ꢀ A)/A0
where I – relative activity; A0 – specific concentration of inorganic
phosphate in the reference sample (without complex 1); A – specific
concentration of inorganic phosphate in the experimental sample (in the
presence of 1).
2.8. Interaction of 1 with Hb
The homogenous solution of HbO2 was isolated from fresh blood of
experimental animals using the available technique [59]. To transform
HbO2 into Hb, the protein solution in 10-ml vessels was blown with
argon during 10 min. 2.8⋅10ꢀ 3 M solutions of 1 prepared in DMSO (see
above) were used. The reaction was started by introduction of 50 µl of 1
solution in the experimental cuvette containing Hb solution in 0.05 M
Tris-HCl buffer pH 7.0, and in the reference cuvette containing 0.05 M
Tris-HCl buffer pH 7.0. The complex concentration in both cuvettes was
4.7⋅10-5 M. Hb concentration was 5.6⋅10-6 M. Kinetics of HbNO accu-
mulation was registered for 21 h.
To determine the effect of NO on enzyme activity, anaerobic solution
of PDE was prepared as described above (Section 2.7). The buffer so-
lution was saturated with gaseous NO. Then, aliquots of the saturated
NO buffer solution were injected into anaerobic PDE solutions. After
incubation with PDE and cGMP, NO was removed from the solution by
blowing the solution with argon, and the phosphate content was
determined according to the standard method.
3. Results and discussion
3.1. Synthesis of complex 1
2.9. Quantum-chemical calculations
Complex 1 was obtained by substitution of the thiosulfate ligands in
the binuclear tetranitrosyl thiosulfate complex by 5-(3-pyridyl)-4H-
1,2,4-triazole-3-thiolyls (Scheme 2). The reaction occurs through the
formation of a water soluble dinitrosylthiosulfate complex in the pres-
ence of Na2S2O3, a reducing agent.
Quantum-chemical calculations were performed with full geometry
optimization of the initial, final and intermediate complexes using
Gaussian 09 Program (D version) [60] by means of meta-hybrid TPSSh
functional [61] and a basis set 6-311++G**/6-31G*, taking into ac-
count solvation in aqueous solution in the frame of polarized continuum
model (PCM). The electronic spectra were calculated by the time-
dependent density functional theory (TDDFT).
3.2. Description of 1 structure
2.10. Effect of complex 1 and the products of its decomposition on
phosphodiesterase (PDE) activity
In the known anionic mononuclear DNICs containing S-donor li-
gands, which have been characterized by X-ray analysis [12–24], the
iron atom has a distorted tetrahedral geometry with two slightly curved
– –
Fe-NO fragments. An average value for the Fe N O angles in these
Activity of cGMP phosphodiesterase (PDE) was determined from the
quantity of GMP forming in the fermentative reaction, which corre-
sponds to the quantity of inorganic phosphate yielding from GMP upon
addition of nucleotidase [62] (Scheme 1):
complexes is ~ 165◦, and in each complex these angles differ incon-
◦
–
siderably (0.1-10 ), together with the N O and Fe-N(O) bond lengths,
for which the range is 1.144–1.199 Å and 1.642–1.721 Å, respectively.
The following cations: Et4N+ [12–14], N2H5 [15], bis(triphenylphos-
+
In the work, cGMP, nucleotidase (as cobra poison), ATF (Sigma-
Aldrich, USA), DMSO, trichloroacetic acid (TCA), ammonium molibdate
phoranylidene)ammonium
(PPN+),
bis(mercapto-ethane-
3

N.A. Sanina et al.
Inorganica Chimica Acta 527 (2021) 120559
Scheme 2. Synthesis of 1.
diazacyclooctane) (H + bme-daco) [16,19–21], and K-18-crown-ether
[17] are counter ions in these DNICs.
Table 2
Bond lengths [Å] and valence angles [degrees] for 1.
Complex 1 is a crystal hydrate of the sodium salt with DNIC anion
(Fig. 1). The sodium cation and crystallization water molecules are
disordered. The anion is a mononuclear DNIC with 5-(3-pyridyl)-4H-
1,2,4-triazole-3-thiol. The Fe atom has a slightly distorted tetrahedral
coordination. Distribution of bond lengths and valence angles values in
the Fe coordination unit is as follows: Fe-N(1) = 1.708(6), Fe-N(2) =
1.687(6), Fe-S(1) = 2.293(2), Fe-S(2) = 2.322(2) Å; N(2)-Fe-N(1) =
111.1(3), N(2)-Fe-S(1) = 103.4(2), N(1)-Fe-S(1) = 106.8(2), N(2)-Fe-S
(2) = 111.0(2), N(1)-Fe-S(2) = 109.8(2), S(1)-Fe-S(2) = 109.3(1)◦. The
Bond
Length [Å]
Angle
[degrees]
Fe-N(1)
1.708(6)
1.687(6)
2.293(2)
2.322(2)
1.753(6)
1.741(6)
1.327(7)
1.349(7)
1.323(6)
1.356(6)
1.331(7)
1.362(6)
1.326(7)
1.333(7)
1.345(7)
1.355(6)
1.338(7)
1.345(6)
1.348(7)
1.351(7)
1.387(7)
1.396(7)
1.476(7)
1.394(7)
1.401(7)
1.463(7)
1.151(7)
2.159(8)
1.129(7)
1.387(7)
1.380(7)
1.400(7)
1.395(7)
N(2)-Fe-N(1)
116.1(3)
103.4(2)
106.8(2)
114.8(4)
111.0(2)
109.8(2)
109.3(1)
97.38(19)
102.71(19)
118.7(5)
103.4(4)
103.1(4)
118.4(5)
103.1(4)
109.8(4)
103.9(5)
109.7(4))
114.1(4)
121.0(5)
124.8(5)
114.2(5)
121.1(5)
124.7(5)
123.3(5)
122.9(5)
109.0(5)
128.6(4)
122.4(4)
109.6(5)
122.9(4)
127.6(4)
118.3(5)
122.3(5)
123.1(5)
166.8(6)
177.7(6)
Fe-N(2)
N(2)-Fe-S(1)
Fe-S(1)
N(1)-Fe-S(1)
Fe-S(2)
Fe(1)-Fe-S(2)
S(1)-C(1)
N(2)-Fe-S(2)
S(2)-C(1A)
N(6)-C(4)
N(6)-C(5)
N(4)-C(2)
N(4)-N(5)
N(4A)-C(2A)
N(4A)-N(5A)
N(6A)-C(5A)
N(6A)-C(4A)
N(3)-C(1)
N(3)-C(2)
N(5)-C(1)
N(3A)-C(1A)
N(3A)-C(2A)
N(5A)-C(1A)
C(3A)-C(4A)
C(3A)-C(7A)
(3A)-C(2A)
C(3)-C(7)
N(1)-Fe-S(2)
S(1)-Fe-S(2)
C(1)-S(1)-Fe
C(1A)-S(2)-Fe
–
N
O bond lengths are: O(1)-N(1) = 1.151(7) Å and O(2)-N(2) = 1.129
C(4)-N(6)-C(5)
C(2)-N(4)-N(5)
C(2A)-N(4A)-N(5A)
C(5A)-N(6A)-C(4A)
C(1)-N(3)-C(2)
C(1)-N(5)-N(4)
C(1A)-N(3A)-C(2A)
C(1A)-N(5A)-N(4A)
N(4)-C(2)-N(3)
N(4)-C(2)-C(3)
N(3)-C(2)-C(3)
N(4A)-C(2A)-N(3A)
N(4A)-C(2A)-C(3A)
N(3A)-C(2A)-C(3A)
N(6)-C(4)-C(3)
N(6A)-C(4A)-C(3A)
N(3A)-C(1A)-N(5A)
N(3A)-C(1A)-S(2)
N(5A)-C(1A)-S(2)
N(5)-C(1)-N(3)
N(4A)-C(1)-S(1)
N(3)-C(1)-S(1)
C(7A)-C(6A)-C(5A)
N(6)-C(5)-C(6)
N(6A)-C(5A)-C(6A)
O(1)-N(1)-Fe
(7) Å. The minimum distance between the iron atoms linked by the
inversion center {1-x, 1-y, 1-z} is 5.291(3) Å. The other bond lengths and
valence angles are shown in Table 2.
The ligands are essentially planar: the angle between the planes
formed by the triazole cycle C(1), C(2), N(3)-N(5) and the pyridine cycle
C(3)-C(7), N(6) is 2.9◦, while the similar angle formed by the triazole
cycle C(1A), C(2A), N(3A)-N(5A) and the pyridine cycle C(3A)-C(7A), N
(6A) is 3.7◦. The ligands themselves are practically parallel (the angle
between the averaged planes of the ligands is 0.5◦), but are displaced
with respect to each other (Fig. 1). The shortest intramolecular contact is
N(6)… N(6A) = 3.694 Å , and the largest intramolecular contact N(3)…
N(3A) = 3.847 Å. Values of other intramolecular contacts lie in this
range.
C(3)-C(4)
C(3)-C(2)
In the crystal, the complex anions form stacks along a short a crys-
tallographic direction (Fig. 2).
O(1)-N(1)
O(1)-Fe(1)
O(2)-N(2)
C(7)-C(6)
To determine possible intermolecular contacts in the stack and be-
tween the stacks in the crystalline structure of 1, quantum-chemical
calculations were performed for two anionic mononuclear complexes
1 participating in the formation of the stacked structure and bound by
(1 + x, y, z) translation, and two anionic mononuclear complexes from
the neighboring stacks (1-x, 1-y, 1-z), within the GAUSSIAN-09 program
[63] in the frame of density functional theory in TPPSH/TZVP approx-
imation. Geometry of mononuclear complexes was taken from X-ray
C(7A)-C(6A)
C(6)-C(5)
C(6A)-C(5A)
O(2)-N(2)-Fe
Fig. 1. General view of 1, spatial arrangement of the ligands in the complex.
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N.A. Sanina et al.
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Fig. 2. Fragment of the crystalline structure of 1.
data, and scf calculation was performed for them. The corresponding
wave function was obtained. Based on the wave function, topological
analysis of the electron density distribution was performed in the frame
of Bader’s QTAIM theory [64–66]. Fig. 3 presents molecular graph of
two mononuclear complexes 1, and pairs of atoms are shown between
which critical points (CP) of the electron density (3, ꢀ 1) are found,
which correspond to the chemical bond [64–66].
… N(11), N(5)… C(19), C(15)… C(26), and (28)… C(36) contacts (Fig. 3)
are ꢀ 0.83, ꢀ 0.68, ꢀ 0.73, ꢀ 0.72 kcal/mol, respectively. The same
values of energy correspond to N(49)… N(50), N(44)… C(58), C(54)… C
(65), and (67)… C(75) contacts. Energies of intermolecular interactions
in critical points corresponding to S(3)… S(41), N(13)… N(47), N(6)… C
(57), C(14)… C(63), and (32)… C(73) contacts (Fig. 3) are equal to
ꢀ 0.71, ꢀ 0.92, ꢀ 0.71, ꢀ 0.71, ꢀ 1.62 kcal/mol, respectively.
As follows from Fig. 3, critical points of CP(3, ꢀ 1) type are both
intramolecular (CP1 – CP4 and CP10 – CP13), and intermolecular (CP5 –
CP9); they provide stacked crystalline packing of the complex anions.
Energies of intermolecular bonds were calculated from Espinosa-
Lecomte formula Ea-b≈ 1/2νe(r), where Ea-b – energy of A-B bond, and
νe(r) – potential energy density in the critical point of A-B bond. Energies
of intramolecular interactions in critical points corresponding to N(10)
The nitrosyl groups from the neighboring stacks (1-x, 1-y, 1-z) are
directed to each other in such a way that distances between the oxygen
atoms are 3.305(3) Å, and distances between the oxygen and iron atom
are 3.488 (3) Å (Fig. 4). As can be seen from Fig. 4, in complex 1, both a
parallel arrangement of the NO groups (stacking interaction), and a
perpendicular one (T like interaction) are available [67]. There is a
critical point CP(3, ꢀ 1) with energy 0.92 kcal/mol between the iron
Fig. 3. System of critical points of CP (3, -1) type in the stacks formed by the anion of compound 1.
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Fig. 4. Mutual arrangement of Fe-coordination units (1-x, 1-y, 1-z) and corresponding interatomic distances in complex 1, Å.
atom and oxygen atom of the NO groups, but there are no critical points
(3, ꢀ 1) between the oxygen atoms and nitrogen atoms of the nitrosyl
groups, as it was in earlier studied complexes [68]. Similar to earlier
studied complexes [68], in complex 1 there are CP(3, ꢀ 1) between Fe…
S(1) [Fe-S(1) = 2.293(2) Å] and Fe… S(2) [Fe-S(2) = 2.322(2) Å], which
are equivalent in energy (-27.9 kcal/mol and ꢀ 26.2 kcal/mol, respec-
atom. Its value depends on the electron shielding effects and bond
lengths change. The isomeric shift is proportional to s-electron density
on the nucleus, which in its turn depends on shielding by d-electrons.
Quadrupole splitting, ΔEQ, informs on the interaction of the 57Fe nucleus
quadrupole moment with the gradient of the electrical field surrounding
the nucleus. δFe values for 1 and salt with N2H5þ (DNIC with 5-nitropyr-
idine-2-thiolyl) are almost twice higher than for the salt with thiophenol
DNIC [12], which points to the decrease of the charge density on the iron
atom in these salts and is consistent with the increase of the Fe-S bond
length in 1 (2.328 Å) (see Section 3.2). An average Fe-S bond length in
DNIC with thiophenolyls is 2.280 Å.
tively), and are characterized by a considerable degree of π-component:
ellipticity values are in the range of 0.09–0.08, which suggests that there
are no different types of chemical bonds between the iron and sulfur
atoms.
3.3. Spectroscopy of complex 1 in the solid state
3.4. Investigation of complex 1 decomposition in solutions
3.3.1. IR spectroscopy of 1
In the IR spectra of DNICs, there are usually two absorption bands of
the NO group valence vibrations. The difference between these bands
(ΔνNO) depends on the ligands coordinated to the iron atom [69], and it
can be due to non-equivalence of the nitrosyl groups in the {Fe(NO)2}
fragments. For anionic DNICs with two sulfur-containing ligands ΔνNO
is ~ 45 cmꢀ 1 on the average [12–15,16,18–23 ], for neutral ones it is ~
50 cmꢀ 1 [17,44], and for cationic ~ 67 cmꢀ 1 [23].
Fig. 5 shows ESI-MS spectrum of complex 1 solution in the negative
mode. By analyzing the isotopic structure of the ionic peaks and precise
measuring the m/z values of the ions, all main registered ions have been
identified. The ionic peak with the maximal intensity and m/z =
469.9774 corresponds to [Fe(R-H)2(NO)2]- ions (R = C7H6N4S). Relative
intensities of main ions in the mass-spectrum are shown in Table 3.
Variations of intensities for [Fe(R-H)2(NO)-H]-, [Fe(R-H)2(NO)]-, and
[Fe(R-H)2(NO)2]- ions within the period of the mass-spectrum recording
(1 min) did not exceed 5%.
Complex 1 is characterized by a typical NO group absorption (1773
cmꢀ 1, 1735 cМ-1) and ΔνNO = 38 cmꢀ 1. A shift of the NO vibrational
frequencies to the region of higher values as compared to those in
available DNICs [12–14,16,18–23] is observed, which points to the in-
crease of NO positive charge, a lower nucleophilic activity and a higher
nitrosating ability of complex 1 as compared to anionic DNICs
[12–14,16,18–23]. We have observed a similar behavior for anionic
DNIC (N2H5)[Fe(SC5H3N2O2)2(NO)2] with 5-nitropyridine-2-thiolyl
Earlier, various types of DNICs were shown to decompose upon
dissolving, and their stability was dependent on the structure of thio
ligand, solvent, oxygen presence, etc. [70–72]. In this work, to analyze
complex 1 transformation in various solvents (Tris-HCl buffer, pH 7.0,
DMSO and ethanol) method of kinetic spectrophotometry was used, too.
Fig. 6 presents time dependence of the absorption spectra of complex
1 in anaerobic Tris-HCl buffer, pH 7.0. The spectrum has one intense
absorption band at 236 nm which increases with DNIC decomposition.
The rate constant of the reaction is (1.5 ± 0.2)⋅10-4 sꢀ 1. In addition,
there is a shoulder at 267–288 nm in the initial spectrum whose optical
density also increases inconsiderably during the day. Interestingly, that
the anion does not have a band at 390 nm typical for mononuclear
complexes with aliphatic thio ligands [73], which is also true for DNIC
with thiourea ligands [71].
– –
[15]. In that case, contribution of π-binding to the Fe N O fragment
decreases, which leads to weakening of the Fe-N bond and strengthening
–
of the N O bond. Consequently, position of the NO absorption bands in
the IR spectrum of 1 correlates with the decrease of the NO bond lengths
(see Section 3.2).
57
¨
3.3.2. Fe Mossbauer spectroscopy of complex 1
¨
Mossbauer spectrum of 1 polycrystals is a single quadrupole doublet.
Values of the isomeric shift, δFe = 0.149(1) mm⋅sꢀ 1, quadrupole splitting
0.937(2) mm⋅sꢀ 1, and the line width, 0.314(5) mm⋅sꢀ 1 of 1 at 294 K do
not differ significantly from those of the anionic DNIc with 5-nitropyri-
dine-2-thiolyl (δ =158(2) mm/s, ΔEQ,=0.956(4)mm/s with the line
width of 0.246(3F)emm/s) [15]. Isomeric shift δFe shows the covalence of
the iron-ligand bond, as well as the oxidation and spin state of the iron
NO release by the anion of complex 1 was recorded using a selective
electrode (Fig. 7), as well as from HbNO formation in the reaction with
deoxyhemoglobin (Hb) (Fig. 8). Hb is favorable for spectrophotometric
registration of NO accumulation kinetics because it is a trap for NO (the
rate of NO binding with Hb heme is close to diffusion rate, with the
ꢀ
1
equilibrium constant 3 ∙ 1010
M
[74]). In addition, the yielding
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Fig. 5. ESI mass-spectra of 1 in MeOH (the sample concentration is 100
μ
M). The insert shows the comparison of the experimental mass-spectra of [Fe(R-H)2(NO)2]-
ions (R=C7H6N4S) and the calculated isotopic distribution for these ions (red lines).
region of the initial shoulder at 210–240 nm; in the buffer only the band
at 236 nm increases. The effective rate constants for decomposition
processes suggest that in ethanol the process occurs 2.4 times slower (k
= (1.5 ± 0.2)⋅10-4 sꢀ 1 for buffer and (6.2 ± 0.2)⋅10-5 sꢀ 1 for ethanol).
The band at 280 nm in the buffer changes little, while in ethanol and
DMSO, a strong increase of the optical density is observed in this region
(the inserts in Figs. 9 and 10). Analysis of kinetics at this wavelength
shows that decomposition in ethanol solution is more prolonged than in
Table 3
m/z values (experimental and calculated) and relative intensities of main ions in
ESI mass-spectra of complex 1 solution.
Ion
m/z
m/z
Average relative intensity within 1
min
(exp)
(calc)
[R-H]-
177.02
261.95
177.02
261.95
0.15
0.45
[Fe(R-H)2(NO)-
H]-
DMSO (k = (7.2 ± 0.7)⋅10-5
s
ꢀ 1 in ethanol and (10.6 ± 0.9)⋅10-4
s
ꢀ 1 in
[Fe(R-H)2(NO)]-
[Fe(R-H)2(NO)2]-
439.98
469.98
439.98
469.98
0.83
1
DMSO). In addition, in both solvents new bands appear as a result of
decomposition: an expressed shoulder at 300–360 nm in ethanol and
broad bands at 360 nm and 440 nm in DMSO.
nitrosyl hemoglobin (HbNO) has a specific absorption spectrum: one
absorption maximum of Hb (556 nm) splits into two (545 nm and 575
nm) in the process of HbNO formation, which is convenient for deter-
mination of the reaction extent. Thus, NO release from the anion of 1 is
accompanied by HbNO formation, which is registered by the spectro-
photometer. Decomposition of the absorption spectra of the reaction
system into the spectra of the reaction components (Hb and HbNO) to
determine their concentration was performed using the MathCAD
program.
For the further analysis of the mechanism of the complex decom-
position in these solvents, NO donor activity of the anion with respect to
nitrite ions formation was estimated using Griess reaction. Nitrite ions
are known to be main products of NO transformation in aqueous aerobic
solutions [76]. Therefore, by analyzing kinetics of nitrite ions accumu-
lation, ability of 1 to release NO can be estimated. For nitrites formation
from NO, the presence of water and oxygen is necessary, therefore the
experiments were performed on air, and the solvents were mixed with
the buffer.
As follows from the experiment with the sensor electrode (Fig. 7),
concentration of the released NO is considerably less than the initial
concentration of the complex, and the curve is striving for reaching the
plateau. So, we can assume that the equilibrium is reached between the
initial complex 1 and the product obtained after NO release (1 ↔
product + NO), this being observed earlier for other DNICs [71,72,75].
Interestingly, in contrast to other complexes, in this case the kinetic
curve does not reach the plateau within 500 s, which testifies to a more
prolonged character of NO release by complex 1.
We can see from Fig. 11 that in the buffer solution the formation
curve for the nitrite ions reaches the plateau in 10 min after the start of
dissolution. Similarly, for 50% ethanol solution it reaches the maximal
value in 9–10 min. The rate constants for these processes are close ((3.2
± 0.3)⋅10-3 sꢀ 1 for the buffer and (2.6 ± 0.3)⋅10-3 sꢀ 1 for 50% ethanol).
In contrast to the buffer and ethanol, for 50% DMSO solution the curve
does not reach the plateau even within 4 h. Obviously, DMSO stabilizes
the complex, and it becomes a more prolonged NO donor.
Thus we can assume that if NO release occurs in 100% DMSO, the
process will be slower than in ethanol and the buffer.
However, introduction of Hb as a trap into the solution shifts the
equilibrium and results in a more effective NO donation by the complex
(Fig. 8). In this case, the plateau is reached in 5 h from the reaction start,
and this corresponds to the complete saturation of the protein by NO.
From the analysis of the kinetic curve, the effective rate constant of the
process, k=(1.9 ± 0.2)⋅10-4 sꢀ 1, can be determined.
3.5. Quantum-chemical modeling of TDDFT spectra of decomposition
products of complex 1 anion in various solvents
To further analyze the influence of solvents on the mechanism of
complex 1 decomposition, quantum-chemical modeling of probable
decomposition products of 1 anion and their TDDFT spectra was
We can see that decomposition processes of 1 in these solvents vary
considerably. In ethanol, the absorption increase is observed in the
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Fig. 6. Time dependence of the absorption spectra of complex 1 (4⋅10ꢀ 5 M) in anaerobic Tris-HCl buffer, pH 7.0 (2% alcohol solution). In the insert: Kinetics of this
process, effective constant of the reaction rate is (1.5±0.2)⋅10-4 s-1 (at 236 nm).
Fig. 7. Time dependence of concentration of NO released by complex 1
Fig. 8. Kinetics of HbNO formation in the reaction of the anion of complex 1
(0.4⋅10ꢀ 5 M) in 1 % anaerobic DMSO aqueous solution, pH 7.0, 25 ◦C.
(4.7⋅10ꢀ 5 M) with 5.6⋅10-6 M Hb. Solvent - Tris-HCl buffer, pH 7.0, 23 ◦C.
performed for three solvents used in the experiment.
transition, correspond.
At the first stage, optimization of complex 1 geometry was performed
in PCM framework in water, ethanol, and DMSO, taking into account the
complex solvation, and TDDFT spectra of the initial anion were plotted
in these solvents (Fig. 12).
As follows from the experiment, intensity of the band at ~ 240 nm
increases in aqueous and ethanol solutions with time. In ethanol and
DMSO intensity of the band at 280 nm increases considerably. Addi-
tionally, in ethanol solution shoulders at ~ 260 and 300–325 nm appear,
while in DMSO solution at ~ 360 nm. Based on these data, we can as-
sume that either the complex changes differently in different solvents, or
the solvent affects the spectrum of the same compound upon
coordination.
The Figure shows that the spectra for the initial anion in three sol-
vents coincide, and the bands available are similar to those in the
experimental absorption spectra. Like in the experimental spectra, the
intense band at 240 nm, which corresponds to
π → π* transition of the
conjugated system of the triazole ligands, is the main one for all three
The next stage involved quantum-chemical modeling of several
possible transformation processes for the anion of 1 (structures 2–4 in
Table 5), which occur with solvent molecules participation in anaerobic
solution; spectra of probable products were plotted. Solvation of the
theoretical spectra; however, π(Fe-NO) → π*(Fe-NO) transitions also
contribute to the spectra, as distinct from the cationic complex with thio
urea [70]. The shoulder at ~ 280 nm is the second intense band, to
which the same electron transitions, as well as
π
(Fe-S) → p(S) + dz2(Fe)
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Fig. 9. Time dependence of the absorption spectra of 1 (4⋅10-5 M) in anaerobic ethanol solution. In the insert: kinetics of the process; effective constants of the
reaction rate are (6.2±0.6)⋅10-5 s-1 (at 236 nm) and (7.2±0.7)⋅10-5 s-1 (at 280 nm).
Fig. 10. Time dependence of the absorption spectra of 1 (4⋅10-5 M) in anaerobic DMSO solution. In the insert: kinetics of the process; effective constant of the
reaction rate is (10.6±0.9)⋅10-4 (at 280 nm).
anion with hydrogen bonds formation at the expense of the hydrogen
atoms of water molecules or ethanol, and the nitrogen atoms of the li-
gands, and at the expense of the oxygen atoms of DMSO and the
hydrogen atoms of the ligands was considered (fig. 13a).
of complexes 2–4 participate in the formation of the hydrogen bond. For
the structure with DMSO, only one stable hydrogen bond forms, there-
fore there is no such change of the initial spectrum as for ethanol and
water. On the whole, the spectrum variation does not correspond to the
experiment because intensity of the bands at 240 nm and 280 nm de-
creases. However, this does not mean that in the experiment no solva-
tion of the ligands occurs, this process being rather energetically
favorable (21.0 kcal/mol for water, 20.9 kcal/mol for ethanol and 9.5
kcal/mol for DMSO). We can conclude that the initial experimental
As follows from Fig. 13b the ligands solvation in the anion of 1 leads
to intensity decrease for main spectrum bands, ~240 nm and ~ 280 nm,
in water and ethanol, which is reasonable because main electron tran-
sitions for these bands are
π
→
π
* transitions of the conjugated system of
-system in the anions
triazole ligands, while the nitrogen atoms of this
π
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in aqueous solution the band at 240 nm is more intense than in the initial
anion, because upon coordination of two water molecules, two rather
intense electron transitions appear at 238.16 and 229.57 nm in the
theoretical spectrum, which contribute to the band at 240 nm (Fig. 14).
π
-orbitals of the Fe(NO)2 fragment and π-systems of the ligands are
involved in these transitions. Decomposition of the initial complex 1 in
water can be presented as follows (scheme 3):
The first stage is an energetically favorable process of the ligands
solvation to yield complex 2. Then six-coordinated complex 3 forms,
which easily dissociates to release either NO (complex 4) or the triazole
ligand (complex 5). This is accompanied by the formation of five-
coordinated products, for which intensity increase for the band at
240 nm is also observed. All these processes are not very energetically
costly, and are most likely equilibrium. Fig. 15 shows theoretical spectra
of the products presented in scheme 2. We can see that transformation of
complex 2 to any of complexes 3–5 is accompanied by the absorption
increase - at 240 nm, while if it occurs due to NO ligand removal – at 280
nm. In the experiment, we see both the absorption increase at 240 nm,
and the appearance of the band at 280 nm, so products 3 and 4 are most
likely in the equilibrium. However, we realize that the process is
complicated, and anionic complex 4 and neutral complex 5 could be not
final products, and could transform further, and other six-coordinated
iron complexes could contribute to the band at 240 nm.
Fig. 11. Kinetic curves for nitrite ions accumulation obtained upon decompo-
sition of 1 (8.8⋅10-5 M) in various solvents (50% DMSO, 50% ethanol and Tris-
HCl buffer) in aerobic conditions. Rate constants of the reaction are: (5.7±0.6)⋅
10-4 s-1 for 50% DMSO in Tris-HCl buffer, pH 7.0, (2.6±0.3)⋅10-3 s-1 for 50%
ethanol in Tris-HCl buffer, pH 7.0, and (3.2±0.3)⋅10-3 s-1 for Tris-HCl buffer,
pH 7.0.
In addition, we are dubious about product 5 (removal of the triazole
ligand). In its theoretical spectrum, there is a band at 333.12 nm which
corresponds to the electron transitions in the removed triazole ligand.
Consequently, the absence of the band at ~ 330 nm in the experimental
spectrum (Fig. 6) suggests that no dissociation of the initial anion over
the Fe-S bond occurs in aqueous solution.
spectra correspond to the solvated products.
Interestingly, in the experimental spectrum of 1 anion in water the
band at 240 nm changes rather slowly. As far as this band corresponds to
the electron transformations in the ligands and the Fe(NO)2 fragment, in
case of a simple dissociation over Fe-O and Fe-S bonds we could expect a
decrease of its intensity. Consequently, there is another process,
apparently related to the water molecules. The assumption can be made
that the iron atom coordination number increases to six due to two
solvent molecules addition.
In contrast to aqueous solution, the final experimental spectrum in
ethanol solution is more complicated (Fig. 9). The intensity of the band
at 240 nm also increases, which can be explained by a higher coordi-
nation number of the iron atom. Indeed, modeling the spectrum of the
anion of the six-coordinated complex analogous to structure of 2 in the
presence of ethanol molecules is characterized by the intensity increase
of the band at 240 nm while transforming from four-coordinated to six-
In the modeled spectrum of the anion of the six-coordinated complex
Fig. 12. TDDFT spectra of 1 in water (blue), ethanol (red), and DMSO (green).
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Fig. 13a. Optimized geometries of the solvated complex 1.
Fig. 13b. Calculated TDDFT spectra of structures 1-4 (taking into account the ligands solvation): water (blue), ethanol (red), DMSO (green), initial anion of 1
without solvation (black).
-
coordinated complex (Fig. 16). In addition, the band at 280 nm in-
creases, too, and this is observed for UV-spectrum of the anionic com-
plex 1 in ethanol solution. This increase in the theoretical spectrum is
explained by 1.5 times growth of oscillation force for the transition at
271.72 nm in six-coordinated structure as compared to the transition at
272.25 nm in four-coordinated one. It can be seen in Fig. 16 that these
transitions mainly contribute to the band at 280 nm. Depending on the
complex coordination number, not only the intensity of these transitions
changes, but also their character: for the anion of the four-coordinated
Theoretical TDDFT spectrum of Fe(SN3C2HC5H4N)2(NO)(C2H5OH)2
anion is presented in Fig. 17. It has, taking into account Lorentzian
broadening, a band at 310 nm and a shoulder at 263 nm. In Fig. 17, the
orbitals responsible for electron transitions contributing to these bands
are shown. They are mainly π(Fe-NO) → π*(Fe-NO) in the structure of
the mononitrosyl fragment. Hence, appearance of these bands in the UV
spectrum can be attributed to the formation of this complex, which is
accompanied by NO ligand removal. Thus, we can conclude that in
ethanol solution, like in aqueous solution, the formation of six-
coordinated iron complex occurs due to coordination of two solvent
molecules at the iron atom, and NO ligand removal. The concurrent
presence of the bands responsible for these two processes suggests that
NO ligand removal is a reversible process [71,72,75], and both a six-
coordinated product and a five-coordinated complex without NO
ligand are present in solution in equilibrium. The difference between the
spectra in water and ethanol is due to the solvent nature, because
structurally similar complexes with different coordinating solvent have
different electron transitions in UV spectra. This is true for dissociation
over the Fe-S bond, too. The band responsible for electron transition LP
complex,
six-coordinated anion, p-orbitals of the sulfur atom and the oxygen atom
of ethanol, and (NO) orbitals are involved.
π → π* transitions in the triazole ligands occur, while for the
π
Thus, in ethanol solution the intensity increase for the bands at 240
and 280 nm indicates the growth of the iron atom coordination number
to 6, because of coordination of two ethanol molecules. This suggests the
same scheme of the initial anion decomposition in ethanol as in water
(Scheme 3). It should be noted that in ethanol NO donor properties are
kept, as follows from the experiment. Assuming that other changes in the
spectrum, i.e., arising of two shoulders at ~ 250–260 nm and 300–325
nm are related to NO donation, the structure similar to that of complex 4
(five-coordinated without NO ligands) but with ethanol as solvent was
modeled, and its theoretical spectrum was plotted.
(S) + π(N3C2H) → π*(C5H4N) in the removed ligand is at ~ 360 nm,
while in aqueous solution the similar transition corresponds to 333.12
nm.
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Fig. 14. Calculated TDDFT spectra of the anion of the six-coordinated complex (red) as compared to the initial one (black).
Scheme 3. Decomposition of the anion of 1 in water.
The absence of the intensity growth in this region in the experimental
spectrum suggests that there is no dissociation over the Fe-S bond in the
anion of 1 in ethanol. The scheme of the reaction is likely as follows
Scheme 4.
to the increase of the coordination number, and the second one to the
thiotriazole ligand removal. As follows from the experiment (Fig. 11),
complex 1 in DMSO is a weak NO donor. Therefore, first, modeling of
-
spectra for six-coordinated Fe(SN3C2HC5H4N)2(NO)2(DMSO)2 and Fe
The experimental UV spectrum of the initial anion in DMSO shows
the growth of the band intensity at 280 nm and appearance of a shoulder
at 360 nm (Fig. 10). Based on the conclusions made for aqueous and
ethanol solutions, assumption can be made that the first band is related
(SN3C2HC5H4N)(NO)2(DMSO)2- - (SN3C2HC5H4N)- was made. They are
shown in Fig. 18 in comparison with the initial spectrum of 1 anion in
DMSO. In the theoretical spectra, the intensity increase for the band at
280 nm is observed, which is due to the growth of the iron atom
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Fig. 15. Calculated TDDFT spectra. Red – anion of complex 2 with hydrated ligands; black – anion of six-coordinated complex 3; green – 4 (NO removal); blue – 5
(removal of the triazole ligand).
Fig. 16. Calculated TDDFT spectra of the anion of the six-coordinated complex (black) as compared to the initial complex (red).
coordination number; there is electron transition LP(S) +
π
(N3C2H) →
π
*
coordinated complex. Transformation of 1 in DMSO occurs ten times
faster than in ethanol, and in 50% aerobic DMSO mixtures 1 releases NO
in a more prolonged way, which should be taken into account in bio-
logical experiments. So, in the studies of PDE inhibiting ability of 1,
DMSO aqueous solutions were used.
(C5H4N) at 363.27 nm in the removed ligand, which induces the cor-
responding band at ~ 360 nm. This is in a good agreement with the
experiment (Fig. 10). Consequently, decomposition of the anion of 1 in
DMSO differs from that in water and ethanol, i.e., main dissociation
occurs not over the Fe-NO bond, but over the Fe-S bond (Scheme 5).
Thus, complex 1 was shown to decompose in water and ethanol to
yield a six-coordinated complex with the corresponding solvent, and to
release NO, while in DMSO the triazole ligand separates from the six-
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Fig. 17. TDDFT spectrum of Fe(SN3C2HC5H4N)2(NO)(C2H5OH)2- anion.
Scheme 4. Decomposition of the anion of 1 in ethanol.
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triazole derivatives of Shiff bases exhibits a stronger inhibiting effect on
the enzyme functions as compared to EDTA which is used for treatment
of rheumatoid arthritis, diabetes, multiple sclerosis, and some skin dis-
eases [77]. A similar inhibiting effect was observed for a series of tri-
azole derivatives with 5-phenyl-2-furane residues, triazole-pyridine
dicarbonitriles, etc. [78–80]. However, no systematic study has been
performed on the influence of properties of DNICs based on mercapto-
triazoles on PDE fermentative function. Therefore, analysis of inhibiting
activity of 1 and its thioligand seemed to be interesting.
The experimental data (Table 4) show that complex 1 in the con-
centration range of 0.1 to 0.001 mmol has a modulating effect on cGMP-
PDE activity, i.e., it inhibits the enzyme activity by 50–60% in concen-
tration of 0.1 mmol, having approximately the same activity as a classic
inhibitor, theophylline [81]. Half-maximal inhibitory concentration
(IC50) of complex 1 is 0.07 mM.
Inhibiting activity of 1 was compared to a similar activity of the
ligand (Table 4). For 0.1 mmol concentration, a considerable difference
in the effect on cGMP-PDE activity is observed: 5-(3-pyridyl)-4H-1,2,4-
triazole-3-thiol ligand inhibits the fermentative function three times
weaker (18.6 ± 2 % vs 59 ± 6 %). This difference reduces with the
decrease of the initial concentration of complex 1 and its ligand.
In addition to the thioligand, the effect of NO (and a mixture of NO
with the thioligand) on the enzyme activity was analyzed. NO was
blown out after incubation with PDE and cGMP to prevent acidification
of the medium and its interaction with other system components. It was
found that NO in all studied systems did not affect PDE activity.
It is necessary to emphasize that NO is a short-lived molecule that is
rapidly oxidized to nitrites under these conditions [76]. According to
our experiments, nitrites are accumulated in the system within 10 min
(Fig. 11). The study of their effect on PDE activity showed that they did
not inhibit enzyme functions.
Fig. 18. Calculated TDDFT spectra. Red – structure 4 with solvated ligands,
-
black – six-coordinated Fe(SN3C2HC5H4N)2(NO)2(DMSO)2 , green – removal of
the triazole ligand Fe(SN3C2HC5H4N)(NO)2(DMSO)2- - (SN3C2HC5H4N)-.
3.6. Inhibitory effect of complex 1 and the products of its decomposition
on the enzyme activity of cGMP-PDE
As mentioned above, investigations of pharmacological activity of
mercaptotriazoles involve development of related PDE inhibitors and
design of anti-inflammatory drugs for treatment of asthma, chronic
obstructive lung disease, Parkinson disease, etc. At present, vast data
have been obtained on synthesis of such promising inhibitors and
analysis of their pharmacological activity. For example, a series of
Scheme 5. Decomposition of the anion of 1 in DMSO.
15

N.A. Sanina et al.
Inorganica Chimica Acta 527 (2021) 120559
CRediT authorship contribution statement
Table 4
Inhibition of cGMP-PDE (% of the reference) by complex 1 and its ligand 5-(3-
pyridyl)-4H-1,2,4-triazole-3-thiol.
Compound
Natalia A. Sanina: Conceptualization, Methodology, Investigation,
Writing – original draft, Visualization. Yuliya A. Isaeva: Investigation.
Andrey N. Utenyshev: Investigation. Pavel V. Dorovatovskii: Inves-
tigation. Nickolai S. Ovanesyan: Investigation. Nina S. Emel’yanova:
Investigation. Olesya V. Pokidova: Investigation. Liliya V. Tat’ya-
nenko: Investigation. Il’ya V. Sulimenkov: Investigation. Alexandr I.
Kotel’nikov: Writing - review & editing, Supervision. Sergey M.
Aldoshin: Investigation, Writing - review & editing, Supervision, Proj-
ect administration.
Concentration, mmol
0.1
0.01
0.001
Complex 1
59 ± 6
18.6 ± 2
63 ± 8
29 ± 3
17.9 ± 2
45 ± 5
20.2 ± 2
15.5 ± 2
30 ± 3
5- (3-pyridyl) ꢀ 4H-1,2,4-triazole-3-thiol
Theophylline
*The data are presented as an average value and mean squared deviation, from
at least three experiments.
It should be noted that the experiments on inhibiting ability of 1 and
its ligand have been performed on the homogenate of mice cerebral
cortex, which contained PDE mixture. At present, PDE isoforms are
classified in 11 families that differ in substrate specificity, affinity,
unique tissue distribution, and regulator mechanisms [82–83]. From
literature data, two isoforms (PDE1A and PDE1B) present above 90% of
total activity of the brain enzyme [84]. In addition, in brain PDE4, PDE7,
and other types of PDE family are expressed [85,86]. Thus, we can as-
sume that further investigation of pharmacological activity of complex 1
should focus on these isoforms as main targets.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors acknowledge the assistance of M.Z. Aldoshina.
The work has been performed with financial support of the Ministry
of Science and Higher Education of the Russian Federation (Agreement
on the provision of grants from the federal budget in the form of sub-
sidies in accordance with paragraph 4 of Article 78.1 of the Budget Code
of the Russian Federation, Moscow, October 1, 2020, No. 075-15-2020-
777).
4. Conclusions
In this work, a new water soluble DNIC, a sodium salt with the anion
in which tetrahedral iron atom is coordinated by two NO groups and two
5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyls (complex 1) has been synthe-
sized. Properties of 1 have been studied in solid and in solutions by X-ray
analysis, IR, Mossbauer, UV–Vis spectroscopy, mass-spectrometry, and
amperometry. Complex 1 was shown to form long-lived nitrosyl in-
termediates, this being responsible for its prolonged NO donor activity.
By comparing theoretical and experimental spectra in various solvents,
conclusion can be made that the intensity increase of the bands at 240
and 280 nm is due to the increase of the iron atom coordination number
to six because of coordination of two solvent molecules. The bands at
260 and 320 nm indicate formation of a mononitrosyl fragment instead
of a dinitrosyl one. The band at 360 nm suggests the appearance of a free
thiotriazole ligand. The assumption can be made that in water and
ethanol the initial anion decomposes to release NO through the forma-
tion of a six-coordinated complex with the solvent, and total decom-
position reaction in the buffer (at 236 nm) occurs 2.4 times faster than in
ethanol (k=(1.5 ± 0.2)⋅10-4 sꢀ 1 for the buffer and k=(6.2 ± 0.6)⋅10-5 sꢀ 1
for ethanol). The solvents affect the spectra shape. In DMSO the triazole
ligand rives from the six-coordinated complex. Interestingly, the anion
transformation in DMSO occurs ten times faster than in ethanol: k=
(10.6 ± 0.9)⋅10-4 sꢀ 1 for DMSO and k=(7.2 ± 0.7)⋅10-5 sꢀ 1 for ethanol
(at 280 nm). Consequently, the solvent influences the mechanism of the
anion decomposition, which can be seen in the spectrum shape.
Analysis of NO donor activity of 1 using the sensor electrode and
HbNO formation shows that in aqueous anaerobic solutions the complex
releases NO reversibly, and in the presence of Hb as a trap the complex
becomes a more efficient NO donor. In addition, comparison of complex
1 ability to release NO on air in the buffer and in 50% mixtures with
DMSO and ethanol has shown that DMSO causes the complex stabili-
zation, which makes it a more prolonged NO donor. In this case, a weak
difference is observed between the rate of NO donation in the buffer and
50% ethanol solution: (2.6 ± 0.3)⋅10-3 sꢀ 1 for 50% ethanol and (3.2 ±
0.3)⋅10-3 sꢀ 1 for Tris-HCl buffer, pH 7.0.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ica.2021.120559.
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