Synthesis, structure, NO donor activity of iron-sulfur nitrosyl complex with 2-aminophenol-2-yl and its antiproliferative activity against human cancer cells

Article abstract of DOI:10.1080/00958972.2013.848980

A new tetranitrosyl binuclear iron complex, [Fe₂(SC ₆H₆N)₂(NO)4] (1), has been synthesized by two methods. Molecular and crystalline structure of 1 were determined by X-ray analysis; the complex is binuclear of -S type with ~2.7052(4) A between the irons. The compound crystallizes in monoclinic, space group P21/n, Z = 2; parameters of the unit cell: a = 6.6257(2) A, b = 7.9337(2) A, c = 16.7858(4) A, β = 96.742(2)°, V = 876.26(4) A³. Parameters of Moessbauer spectrum for 1 are: isomer shift δ_Fe = 0.096(1) mm/s, quadrupole splitting ΔEQ = 1.122(1) mm/s, line width 0.264(1) mm/s at 293 K. As follows from the electrochemical analysis of aqueous solutions of 1, it generates NO in protonic media without additional activation. NO amount and the rate of its activation are much higher in acidic solutions than in neutral and alkali ones. The constants of hydrolytic decomposition of 1 were calculated. The geometry and electronic structure of isolated 1 were studied using the density functional theory. Differential sensitivity of four lines of human tumor cells of various genesis to 1 has been determined (ovarian carcinoma (SCOV3), large intestine cancer (LS174T), mammary gland carcinoma (MCF7), and non-small cell carcinoma of lung (A549)); dependence of tumor cells amount on the complex concentration has been studied in order to use the complex as a promising antitumor agent for trials in vivo.

Full text of DOI:10.1080/00958972.2013.848980

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Synthesis, structure, NO donor
activity of iron–sulfur nitrosyl complex
with 2-aminophenol-2-yl and its
antiproliferative activity against human
cancer cells
N.A. Saninaa, G.I. Kozuba, O.S. Zhukovab, N.S. Emel’yanovaa, T.A.
Kondrat’evaa, D.V. Korchagina, G.V. Shilova, N.S. Ovanesyana &
S.M. Aldoshina
a Russian Academy of Sciences Institute of Problems of Chemical
Physics, Chernogolovka, Russian Federation
b Russian Academy of Medical Sciences N.N. Blokhin Russian
Cancer Research Center, Moscow, Russian Federation
Accepted author version posted online: 27 Sep 2013.Published
online: 30 Oct 2013.
To cite this article: N.A. Sanina, G.I. Kozub, O.S. Zhukova, N.S. Emel’yanova, T.A. Kondrat’eva,
D.V. Korchagin, G.V. Shilov, N.S. Ovanesyan & S.M. Aldoshin (2013) Synthesis, structure, NO
donor activity of iron–sulfur nitrosyl complex with 2-aminophenol-2-yl and its antiproliferative
activity against human cancer cells, Journal of Coordination Chemistry, 66:20, 3602-3618, DOI:
10.1080/00958972.2013.848980
To link to this article: http://dx.doi.org/10.1080/00958972.2013.848980
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Journal of Coordination Chemistry, 2013
Vol. 66, No. 20, 3602–3618, http://dx.doi.org/10.1080/00958972.2013.848980
Synthesis, structure, NO donor activity of iron–sulfur nitrosyl
complex with 2-aminophenol-2-yl and its antiproliferative
activity against human cancer cells
N.A. SANINA*†, G.I. KOZUB†, O.S. ZHUKOVA‡, N.S. EMEL’YANOVA†,
T.A. KONDRAT’EVA†, D.V. KORCHAGIN†, G.V. SHILOV†, N.S. OVANESYAN† and
S.M. ALDOSHIN†
†Russian Academy of Sciences Institute of Problems of Chemical Physics, Chernogolovka,
Russian Federation
‡Russian Academy of Medical Sciences N.N. Blokhin Russian Cancer Research Center,
Moscow, Russian Federation
(Received 30 May 2013; accepted 28 August 2013)
A new tetranitrosyl binuclear iron complex, [Fe2(SC6H6N)2(NO)4] (1), has been synthesized by two
methods. Molecular and crystalline structure of 1 were determined by X-ray analysis; the complex is
binuclear of “μ-S” type with ~2.7052(4) Å between the irons. The compound crystallizes in mono-
clinic, space group P21/n, Z = 2; parameters of the unit cell: a = 6.6257(2) Å, b = 7.9337(2) Å,
c = 16.7858(4) Å, β = 96.742(2)°, V = 876.26(4) Å3. Parameters of Mössbauer spectrum for 1
are: isomer shift δFe = 0.096(1) mm/s, quadrupole splitting ΔEQ = 1.122(1) mm/s, line width 0.264
(1) mm/s at 293 K. As follows from the electrochemical analysis of aqueous solutions of 1, it gener-
ates NO in protonic media without additional activation. NO amount and the rate of its activation
are much higher in acidic solutions than in neutral and alkali ones. The constants of hydrolytic
decomposition of 1 were calculated. The geometry and electronic structure of isolated 1 were
studied using the density functional theory. Differential sensitivity of four lines of human tumor cells
of various genesis to 1 has been determined (ovarian carcinoma (SCOV3), large intestine cancer
(LS174T), mammary gland carcinoma (MCF7), and non-small cell carcinoma of lung (A549));
dependence of tumor cells amount on the complex concentration has been studied in order to use
the complex as a promising antitumor agent for trials in vivo.
Keywords: Sulfur-nitrosyl iron complexes; X-ray analysis; MTT assay
1. Introduction
The search for new approaches to the development of effective and non-toxic
“non-platinum” chemotherapeutic preparations based on metal coordination compounds has
been intense [1]. Nitrosyl derivatives of biogenic iron are of a particular interest [2]. During
the latest decade, nitric oxide (NO) was discovered to participate in carcinogenesis [3], in
addition to its wide range of biological actions and ability to affect the organism systems,
*Corresponding author. Email: sanina@icp.ac.ru
© 2013 Taylor & Francis

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3603
fermentation activity, processes of cell fission, and death. Depending on NO local
concentration in vivo, it can affect cell targets in different ways: the products of its reactions
can either stimulate or, on the contrary, inhibit cancer growth. NO changes apoptosis level
of tumor cells, activity of p53 gene, and neoplasm of vessels that nourish a tumor [4] and
suppress activity of the key reparation protein of mammals O6-methyl-guanine-DNA-
methyl-transferase [5]. Due to these properties, NO donors can be considered as a new class
of anticancer agents [6]. However, available synthetic NO donors of different classes are
not used as mono-therapeutic drugs for the treatment of oncological diseases, but for the
activity boost of the existing chemotherapeutic preparations or radiotherapy [7–11]. That is
why the search for new compounds, i.e. exogenous NO-donating drugs (NODD) that have
direct antitumor effect, is of current importance [12–21].
Nitrosyl iron complexes (NIC) are intermediates in the decomposition of proteins and
formation of S-nitrosothiols, which are catalyzed by the iron, and are reservoirs and trans-
porters of NO in vivo [22, 23]. Based on these data, methods for synthesis and investigation
of physical–chemical properties of synthetic analogs of NIC have been developed, with the
aim to use them as NO donors in the monotherapy of oncological diseases. Recently, high
anticancer activity has been shown for a series of iron nitrosyl complexes – synthetic
models of the active sites of nitrosyl iron-sulfur proteins [24–26]. Functional sulfur-
containing ligands in such NIC are reversible inhibitors for synthesis of cellular DNA, and
they suppress the growth of tumors of various genesis, while the NO group, being the
second component of the hybrid complex, is a key signal molecule that controls tumor
growth.
The present work deals with synthesis, study of structure, and cytotoxic activity of a
new NO donor,
a
representative of synthetic NIC of general composition
[Fe2(μ-SR)2(NO)4] (1), with R-2-aminothiophenol-2-yl, a structural analog of DNA
pyridine bases belonging to the class of antimetabolites that inhibit the growth of
malignant tumors of various genesis. The latter ones provide enhancement of the NO
effect in the nitrosyl iron complex.
2. Experimental
2.1. Materials and general methods
All materials and solvents were purchased commercially and used as supplied, unless
otherwise mentioned. Analytical grade solvents used for physical–chemical and in vivo
studies were further purified by literature methods [27] before use.
2.2. Instrumentation
IR-spectrum (cm−1) was recorded on Perkin–Elmer Spectrum 100X at room temperature.
57Fe Mössbauer absorption spectra of polycrystals of 1 were recorded on WissEl operating
in constant acceleration mode. 57Co in Rh matrix was used as the source. Spectra at low
temperatures were measured using continuous flow helium cryostat CF-506 (Oxford
Instruments) with controllable temperature. Mössbauer spectra were processed by the least
square method assuming the Lorentzian form of the individual spectral components.

3604
N.A. Sanina et al.
2.3. Preparation and characterization of the complex
Complex 1 has been obtained by two methods.
Method 1. Polycrystals of 1 were obtained by the reported method [28]. 20 mL aqueous
mixture of 0.3 g (7.5 mM) of KOH and 0.62 g (5 mM) of 2-aminothiophenol were added
to 20 mL of aqueous solution of 0.496 g (2 mM) of Na2S2O3 ⋅ 5H2O and 0.57 g (1 mM) of
Na2[Fe2(S2O3)2(NO)4] ⋅ 4H2O obtained by the reported method [29] in an argon atmosphere
within 20 min. The precipitate was separated by filtration and desiccated in air for 24 h.
Fine-dispersed bright black crystals-bars were obtained by re-crystallization from acetoni-
trile. Yield: 75.5%.
Elemental analysis of polycrystals of 1 has been performed at the Analytical Center of
IPCP RAS. Calcd for Fe2S2C12H12N6O4 (%): Fe – 23.30; S – 13.40; N – 17.50; C – 30.00;
H – 2.50. Found (%): Fe – 23.29; S – 13.38; N – 17.48; C – 29.83; H – 2.46.
IR-spectrum (cm−1) of 1: 3523, 3456 (vw); 3367 (w); 3187, 3058, 3020, 2621, 2444,
1949, 1920 (vw); 1780, 1725 (vs); 1608 (s); 1562 (vw); 1512 (vw); 1474, 1448 (s); 1304,
1249 (m); 1160, 1146 (w); 1077, 1050 (vw); 1016 (w); 976, 946 (vw); 853 (w); 832,
754 (s); νNO 1780, 1725 cm−1.
Method 2. Single crystals of 1 have been obtained in two steps under inert atmosphere [30].
Step 1. 1 g (3.2 mm) of bis-(2-nitrophenol-2-yl) disulfide in 50 mL of ethanol was
reduced by 5 g (86.2 mm) of hydrazine hydrate. After complete dissolution of the initial
disulfide in alcohol (30 min), the solution became red. Then the alcohol was removed by
evaporation in vacuum, and hydrazinium salt of 2-nitrophenol-2-yl was isolated as
water-soluble red oil.
Step 2. Mixture of solutions: 0.6 g (1 mM) of Na2[Fe2(S2O3)2(NO)4] ⋅ 4H2O [29] in 20 mL
of H2O and 0.6 g (2.3 mM) of Na2S2O3 ⋅ 5H2O were placed in a three-necked flask in an
argon atmosphere. 1 g (5.3 mM) of hydrazinium salt in 20 mL of H2O was dripped under
intense mixing. The reaction mixture was left in the refrigerator at temperature + 3 °C for
24 h. The precipitate was filtered through a porous glass filter N4, and washed thoroughly
with several batches of absolute ether to separate a fraction containing an admixture phase.
The deposit was dissolved in methylene chloride. Then methylene chloride was evaporated
to the minimal volume and the solution was left in the freezer at –18 °C. 24 h later, black-
red crystals of 1 were filtered from the solution. Yield of 1, 20%. The ether fraction was
evaporated in vacuum. Yield of the admixture powder, 35%.
Elemental analysis of the admixture powder was performed at the Analytical Center of
IPCP RAS. Calcd for Fe2S2C12H8N6O8 (%): Fe – 20.68; S – 11.87; N – 15.55; C – 26.69;
H – 1.49. Found (%): Fe – 20.36; S – 11.97; N – 15.11; C – 26.76; H – 1.68.
IR spectrum (cm−1) of the admixture powder was recorded on Perkin–Elmer Spectrum
100X at room temperature: 3459, 3372 (vw); 3094, 3018, 2964, 2615, 2442 (vw); 1777,
1725 (vs); 1610 (w); 1590, 1566 (w); 1508 (s), 1474 (w), 1449 (w); 1337, 1306 (m); 1256 (w);
1161, 1148 (vw); 1106, 1040 (w); 959 (vw); 854 (w); 832, 804, 783, 752 (w); 731 (s);
νNO 1777, 1725 cm−1.
2.4. X-ray analysis
X-ray diffraction analysis of 1 was carried out on a CCD diffractometer Agilent XCalibur
with EOS detector (Agilent Technologies UK Ltd., Yarnton, Oxfordshire, England). Data

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3605
collection, determination and refinement of the unit cell parameters were carried out using
CrysAlis PRO program suite [31]. X-ray diffraction data were collected at 100(2) K using
MoKα (λ = 0.71073 Å) radiation. Coverage of unique data was 95.2% complete to 75.6°
(2θ). The structure was solved by direct methods. The positions and thermal parameters of
non-hydrogen atoms were refined isotropically and then anisotropically by full-matrix
least-squares. Selected crystallographic parameters and the data collection and refinement
statistics are given in table 1. The bond lengths and angles in the structure of 1 are listed in
table 2. All the hydrogen atom positions were located from the difference map. All
calculations were performed with the SHELXTL program package [32].
The X-ray crystal structure data have been deposited with the Cambridge
Crystallographic Data Center with reference code CCDC 932314. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Center, 12
Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033.
2.5. Investigation
Investigation of the polycrystalline powder of 1 obtained by method 1 was performed on
powder X-ray difractometer ARLX’TRA (Thermo Electron Corporation).
Table 1. Crystal data and structure refinement for 1.
Formula (fw)
Fe2S2C12H12N6O4 (480.1)
Temperature (K)
Crystal system
Space group
100(2)
Monoclinic
P21/n
Unit cell dimensions
a (Å)
6.6257(2)
b (Å)
7.9337(2)
c (Å)
16.7858(4)
β (°)
96.742(2)
Volume (Å3), Z
Crystal size (mm)
Reflections collected/unique
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Largest diff. peak and hole (eÅ−3
876.26(4), 2
0.1 × 0.2 × 0.25
15,330/4483 [R(int) = 0.0401]
4483/0/142
1.016
R1 = 0.0352, wR2 = 0.0733
R1 = 0.0522, wR2 = 0.0783
0.936 and −0.704
)
Table 2. Selected bond lengths [Å] and angles [°] for 1.
Bond lengths (Å)
Fe(1)–N(1)
N(1)–O(1)
Fe(1)–S(1)
Fe(1)–Fe(1A)
1.671(1)
1.176(2)
2.2696(4)
2.7052(4)
Fe(1)–N(2)
N(2)–O(2)
Fe(1)–S(1A)
S(1)–C(1)
1.678(1)
1.161(2)
2.2748(4)
1.783(1)
Bond angles (°)
O(1)–N(1)–Fe(1)
N(1)–Fe(1)–S(1)
N(1)–Fe(1)–N(2)
C(1)–S(1)–Fe(1)
170.6(1)
O(2)–N(2)–Fe(1)
N(2)–Fe(1)–S(1)
S(1)–Fe(1)–S(1A)
C(1)–S(1)–Fe(1A)
171.9(1)
109.60(4)
118.37(6)
110.92(5)
107.36(4)
106.93(1)
109.25(5)

3606
N.A. Sanina et al.
2.6. Electrochemical determination of NO
Sensor electrode “amiNO-700” of “inNO Nitric Oxide Measuring System” (Innovative
Instruments, Inc., Tampa, FL, USA) was used for determination of the NO amount gener-
ated by 1 in solution. The NO concentration was recorded during ~500 s (with pace 0.2 s)
in 1% DMSO aqueous solution with the NO donor concentration 0.1 μM. DMSO was puri-
fied according to reported procedure [27]. For calibration of the electrochemical sensor, a
standard NaNO2 aqueous solution (100 μM) was used, which was added to the mixture of
0.12 M of KI and 2 mL of 1 M H2SO4 in 18 mL of water. All experiments were performed
in aerobic solutions at 250 °C and pH 6.5 (pH of solutions was measured by membrane
pH-meter “HI 8314” (HANNA instruments, Germany).
2.7. Quantum-chemical calculations
Theoretical study of 1 was performed by density functional method in three approxima-
tions, i.e. using local functional BP86, hybrid functional B3LYP in the basis set
6–311++G**//6–31G* in the frame of program GAUSSIAN03 [33], and local functional
PBE with SBK basis in the frame of program PRIRODA [34]. The absence of imaginary
frequencies confirms stability of the structures. While comparing the energies of the
optimized structures, the contribution of zero frequency energies was taken into account. To
take into account solvation energies, polarizable continuum model (PCM) and extended
basis set 6–311++G** were used (GAUSSIAN03 program).
2.8. MTT Assay
Cytotoxic activity of the complex was studied on lines of human tumor cells of various
genes. Tumor cells were grown in RPMI 1640 medium containing 10% of calf embryonal
serum and 5% of CO2 at 37 °C. The cells were disseminated in 96-cavity plates and grown
under similar conditions. Before the experiment, the compound was dissolved in 100 μL of
dimethylsulfoxide (DMSO), then nutrient medium RPMI 1640 was used to get the desired
concentration. The final concentration of DMSO in the samples was below 0.1% and did
not affect the cell growth. Cytotoxic activity was determined from survival value of the
cells in the samples. Survival test was performed using MTT test based on the ability of
dehydrogenases of living cells to reduce achromatic MTT into blue crystals of formazan
soluble in DMSO [35]. The complex (20 μL) was placed in the cavities with final
concentrations of 100, 50, and 25 μM. The total incubation volume was 200 μL. The cells
with the preparation were incubated under the above conditions for 72 h. After completion
of the incubation, MTT reagent was added to the cells and under the same conditions
incubation was performed for 2 h. Then, the formazan crystals were dissolved in 100 μL of
DMSO during 20 min at 37 °C. Optical absorption of DMSO solutions was measured at an
optical counter for multi-cell trays at wavelength 540 nm. The results (average values for 4
parallel measurements) were expressed as survival value in % (ex/control) × 100. A
compound was considered active if cells survival value was equal to or less than 50% on
one of three lines at concentration 100 μM (IC50 ≤ 100 μM). The experimental error was
below 5%.

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3. Results and discussion
3.1. Synthesis of the complex
3607
Polycrystals of 1 were obtained from replacement of thiosulfate ligands of the sodium salt
of tetranitrosyl binuclear anion by the corresponding ligand – 2-aminophenol-2-yl (see
method 1 in section 2), according to scheme 1.
1 is insoluble in water, poorly soluble in alcohols, and soluble in such solvents as
CH2Cl2, acetonitrile, DMSO, acetone, CHCl3, and tetrahydrofuran. Figure 1 shows poly-
crystals obtained by re-crystallization of the reaction product from acetonitrile.
Single crystals of 1 appropriate for X-ray analysis were obtained by method 2 (see
section 2) according to scheme 2. In this method, distinct from method 1, hydrazinium
salt of 2-nitrophenyl-2-thiolate, which was obtained as water soluble oil from the
corresponding functional disulfide, is used for the replacement of thiosulfate ligands in
Na2[Fe2(S2O3)2(NO)4] ⋅ 4H2O by aromatic thiol.
In this method, conversion of the functional thiol to the salt is performed without using
concentrated alkali solution. Alkalis are responsible for the formation of Fe(OH)3 as a side
product, thus leading to decrease of the target product yield. While synthesizing binuclear
nitrosyl iron complex from hydrazinium salt of 2-nitropheno-2-yl, we intended to synthesize
a new tetranitrosyl iron complex with NO2 in the ortho-position of the aromatic ligand. How-
ever, the mixture of two tetranitrosyl iron complexes yields 1 (with 2-aminophenol-2-yl)
and complex with 2-nitrophenol-2-yl. We assume that during the synthesis, the NO2 group in
the ortho-position is reduced by the hydrazinium salt (which is present in a 5-fold excess) in
the presence of the iron salt. In organic compounds, the nitro group can be reduced to amine
by hydrazine in the presence of metals, mainly Raney nickel [36, 37] and iron [38].
In the powder obtained by method 1, in addition to single crystals of 1, binuclear nitrosyl
iron complex with 2-nitrophenol as ligand was detected (its content can reach 50% in differ-
ent experiments). The mixture of the complexes was separated by extraction with absolute
ether (which easily dissolves the complex with 2-nitrophenol). Both fractions were identi-
fied by elemental analysis and IR-spectroscopy. IR-spectra of single crystals of 1 obtained
by method 2 are similar to those of polycrystals obtained by method 1, and have a charac-
teristic absorption of NH2, 1608 cm−1, and valence vibrations of NO, 1780, 1725 cm−1.
From elemental analysis and IR spectra of the admixture powder, we can assume that it is
nitrosyl iron complex with 2-nitrophenol-2-yl (see section 2). The complex has characteris-
tic absorptions for NO2 (1508 cm−1) and valence vibrations of NO, 1777, 1725 cm−1 (see
figure 1 of Supplementary material).
To identify 1 obtained by method 1, diffraction patterns of polycrystal samples were
recorded, which coincide with theoretical pattern for a single crystal of 1 synthesized by
method 2. Polycrystals of 1 from method 1 were used for the physical–chemical and biolog-
ical experiments.
; n OH
SH
NH2
[Fe2(S2O3)2(NO)4]2
+
S2O32
[Fe(S2O3)2(NO)2]3
[Fe2(SC6H6N)2(NO)4]
2S2O23
Scheme 1.
'

3608
N.A. Sanina et al.
Figure 1. Polycrystals of 1 obtained by re-crystallization of the reaction product (scheme 1) from acetonitrile.
3.2. IR studies
In experimental spectra of polycrystals of 1 intense absorption of the NO (νNO = 1780,
1725 cm−1) and at 1608 cm−1 typical for NH2 are observed. However, in the spectrum of a
single crystal of 1, there is no absorption at 1500–1570 cm−1 typical for NO2 of the initial
disulfide.
3.3. Mössbauer effect studies
The Mössbauer spectrum of polycrystals of 1 is a doublet. Parameters of the Mössbauer
spectrum at 293 K (quadrupole splitting ΔEQ = 1.122(1) mm/s, isomer shift δFe = 0.096
(1) mm/s and width of the absorption lines 0.264(1) mm/s) are close to those for neutral
complexes with thiophenol, pyridil-2-yl [24], pyrimidil-2-yl [28], and 5-nitropyridine-2-yl
[3]. This suggests structural similarity of two irons in 1 and the structure of nitrosyl com-
plex with 2-aminophenol-2-yl with iron coordinated by two NO groups being linked with
two bridging sulfurs. This is confirmed by X-ray data.
3.4. Single-crystal X-ray diffraction
From X-ray data, 1 has a centrosymmetric binuclear structure (figure 2) similar to that of
anionic thiosulfate NIC Q2[Fe(S2O3)2(NO)4] with Q = Na, Me, Et, n-Pr, n-Bu [29, 39-41]
and of neutral NIC [Fe2(μ-SR)2(NO)4] with R = Me, Et, n-C5H11, CMe3 [40–43], Py [44],
Pym [28], and 5-nitroPy [30]. Interatomic Fe-Fe distance is 2.7052(4) Å, consistent with
the presence of a chemical bond. Bond length C(1)-S(1) is 1.783(1) Å, equal, in the limits
of accuracy, to similar bonds in μ-S binuclear complexes (see [24]), and exceeding the
length of the double S = C bond (1.684 Å) [45]. This allows us to state that the thiol
form of the ligand participates in the formation of this complex. The amino-group has a

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3609
N2H4 H2O ; C2H5OH
SN2H5
S
S
NO2
NO2
NH2
+
Na2Fe2(S2O3)2(NO)4
+
Na2S2O3
Fe2(SC6H6N)2(NO)4 + impurity
Scheme 2.
non-planar, pyramidal structure. N(3) deviates from the plane H(3A)C(2)H(3B) by 0.23 Å;
the angle between the planes of the phenyl ring and NH2 is 38.8°. Similar to the structure
with pH substituent [46], the plane of the phenyl ring is almost perpendicular (89.8°) to the
plane S(1)Fe(1)S(1A)Fe(1A), thus making impossible the presence of shortened intramolec-
ular contacts of the NO group with 2-aminophenylthiol, as previously observed [30].
Similar to the compounds studied earlier [28, 30, 40–44], in 1 there are small differences
in the structure of the NO groups, which leads to different Fe–N and N–O bond lengths
and valence angles Fe–N–O (table 2).
Figure 3 shows the fragment of the crystalline structure projection on the crystallographic
plane (YZ). Intermolecular contacts S(1)···H(3A) 2.81 Å, O(1)···H(6) 2.52 Å are shown by
the dotted line. Intermolecular contacts of O(2) of the nitrosyl group with C(1) and C(2)
atoms of the phenyl group of the adjacent complex (figure 4) point to the interaction of the
lone pair of O(2) with the aromatic π-system. Consequently, C(1)–C(2) 1.410(2) and C(2)–
C(3) 1.414(2) Å, which are the nearest to O(2), are elongated a little as compared to the
other C–C bonds of phenyl (1.378(3)–1.399(2) Å). Interatomic interactions of O···π type
were observed earlier in NIC and identified from the precision X-ray analysis [47].
3.5. Study of NO donor activity of the complex in solutions
Figure 5 shows experimental and theoretical time dependence of NO amount in solution
upon complex 1 decomposition. In the protic media, the compound generates NO without
thermo-, photo-, or fermentative activation. The kinetic dependences have maxima, which
can be due to further NO conversion. We have used the simplest two-step scheme [28] for
description of the process:
k1
LFeðNOÞ ꢀ! LFe þ NO; where L ¼ ½Feðl2 ꢀ SRÞ2ðNOÞ3ꢁ

3610
N.A. Sanina et al.
k2
NO ꢀ! products;
which produces the following time dependence of NO concentration:
Ck1
ðeꢀk t ꢀ eꢀk t
Þ
1
2
½NOꢁ ¼
k2 ꢀ k1
Constant C is the maximum NO concentration in the absence of its further transforma-
tion. Values C, k1, and k2 were obtained by least squares from the experimental data
(figure 6); for 1 k1 and k2 are 34.9 × 10−4 s−1 and 352.3 × 10−4 s−1, respectively.
As follows from the experiments with pH increase from 6.5 to 8, the amount of NO gen-
erated by 1 decreases by a factor of four (table 3). Probably, the complex properties change
under the action of hydroxide, though these processes should be examined additionally.
From comparative analysis of the NO activity of 1 and the complex with phenolyl [48] in
solutions at pH = 6.5 in anaerobic medium, 1 generates twice as much NO (~40 nM) as the
latter (~20 nM). In aerobic solutions, the NO amount generated by phenolyl complex
increases by five times (109 nM), while in 1 it almost does not change (~37 nM). 1 keeps
rather high NO activity in DMSO solutions while storing in air. Figure 6 shows the experi-
mental time dependence of NO generated upon decomposition of 1 in 1% aqueous DMSO
solution. The measurements were performed during five hours, and each sample was taken
from the basic solution every 30 min. For 1, the maximum evolution of NO was observed
at the very beginning and was constant during 30 min (~40 nM). Then NO amount
decreased to 5 nM. For the phenolyl complex, NO donor activity upon storing reduces to
Figure 2. The general view of 1 with representation of atoms by thermal motion ellipsoids ( p = 50%).

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3611
this value within an hour. Probably, in solutions of 1 stable nitrosyl intermediates exist,
which are responsible for prolonged NO generation.
3.6. Quantum-chemical calculations
In table 4, the key parameters of the optimized geometry of 1 obtained by using various
functionals are presented to compare them with the experimental data (table 2). All selected
methods describe the structure of the complex satisfactorily. The complex is diamagnetic
and its basic spin state should be singlet. However, for solutions obtained with B3LYP, the
triplet state is lower by 16.6 kcal/M. On one hand, this can be a general overestimate of a
high-spin state by hybrid functional [49]. It has been shown earlier [50] that the electronic
structure of diamagnetic binuclear complexes of [Fe2(SR)2(NO)4] type corresponds to the
opposite orientation of the spins of the unpaired electrons on each Fe(NO)2 unit. Such state
can be realized upon binding the spin 3/2 of Fe with the opposite-oriented spins S = 1/2 of
two NO groups, and thus the triplet state has the correct spin structure (figure 7). Value
of <S2> = 5.47 is also close to the theoretical value 6 for the electron configuration corre-
sponding to three unpaired electrons on iron and one electron on each NO. With such
approach, the singlet state could be realized provided the presence of several open shells,
and it cannot be described in the framework of singlet-determinant approximation. A sin-
gle-determinant state with distorted symmetry, with the spin density arising on each Fe and
NO, can be an approximation to the accurate multi-determinant solution (figure 7).
Figure 3. Fragment of the projection of the crystalline structure on the crystallographic plane (YZ).

3612
N.A. Sanina et al.
Figure 4. Intermolecular contacts of one of the nitrosyl groups with the π-system O(2)···C(2B) 3.00 Å, O(2)···C
(1B) 3.14 Å.
Figure 5. Experimental (1) and theoretical (2) time dependences of the NO amount generated in solution upon 1
decomposition in anaerobic 1% aqueous solution of DMSO at pH 7.0 and 25 °C.

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3613
Figure 6. Time dependence of the NO amount generated by 1 (4 × 10−6M l−1) in anaerobic 1% aqueous solution
of DMSO at 25 °C in distilled water at pH 6.5.
Distribution of the spin density (B3LYP/6–31G*) almost equally between Fe and two
NO in the Fe(NO)2 fragment points to the fact that a d orbital engaged by the unpaired
electron does not have preferential localization either on the metal or on the ligand. Such
molecular orbital can form from the orbitals of the metal and the NO ligands with close
energetic characteristics. Residual two electrons on Fe and the electrons on the NO groups,
which had opposite-oriented spins in the ground state, now engage two binding molecular
orbitals Fe–NO, also of the delocalized type. Due to this delocalization, there is consider-
able contribution of ionic states Fe3+(NO–)2 and Fe–(NO+)2 in the wave function, responsi-
ble for increase of energy in the singlet state with filled shells.
Using quantum-chemical modeling, we examined the energy aspects of dissociation of
the Fe–NO bond in 1. The estimate was based on the difference of the energies of the initial
complex and products upon escape of one NO in gaseous phase, as well as in water and
DMSO. The effect of solvation in the polarized continuum model was taken into account
[50, 51].
All approaches we have used lead, upon optimization of the geometry, to the structure
presented in figure 8. Upon dissociation of the Fe–NO bond, a free coordination site arises
Table 3. Amount of NO generated by 1 at various pH in
anaerobic conditions at T = 25 °C.
NO amount, nM
Complex
pH 6.5
40.8
pH 7.0
15.6
pH 8.0
9.4
1

3614
N.A. Sanina et al.
Table 4. Selected optimized geometry parameters (bond lengths (Å), angles (°)) for 1.
B3LYP/6–31G* (triplet)
BP86/6–31G*
PBE/SBK
Fe1–N1
Fe1–N2
Fe1–S1
Fe1–S1A
N1–O1
N2–O2
S1–C1
Fe–Fe
1.627 (1.727)
1.630 (1.731)
2.219 (2.397)
2.226 (2.415)
1.171 (1.180)
1.169 (1.177)
1.782 (1.800)
2.606 (3.367)
114.5 (115.5)
166.0 (163.6)
167.0 (166.0)
1.641
1.642
2.231
2.242
1.187
1.191
1.793
2.608
113.2
166.2
166.7
1.661
1.661
2.252
2.252
1.172
1.172
1.779
2.664
115.5
168.1
168.4
N1–Fe1–N2
O1–N1–Fe1
O2–N2–Fe1
Figure 7. Distribution of the spin density in 1 (B3LYP/6–31G*). Bold type – triplet state, italics – singlet state
with broken symmetry.

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3615
Figure 8. Theoretical structure forming upon NO separation from 1.
in 1. This results in the formation of intramolecular coordination bond Fe–N (2.10–2.13 Å),
with nitrogen of NH2 of the phenyl. This leads to stabilization of the forming structure and
to decrease of its energy. The energy of NO separation from this complex was almost twice
less than that for the complex with phenyl ligand [50] (table 5). Consequently, it should
generate more NO in aqueous DMSO solution. Table 5 shows that in the condensed phase,
the energy of separation decreases considerably for both complexes, thus pointing to the
facility of this reaction in solutions and sufficient stability in air. All these data are in
agreement with the experiment in solution, in which 1 generates twice as much NO as the
complex with the phenyl ligand. On the other hand, formation of the stable structure upon
separation of one NO should prevent further dissociation of the complex, consistent with

3616
N.A. Sanina et al.
Table 5. Energy of NO separation.
Energy of NO separation
Condensed phase
Complex
Gaseous phase
H2O
DMSO
1
−12.6
−24.1
1.9
−8.7
2.2
−10.4
[Fe2(NO)4(SPh)2]
prolonged NO generation in DMSO solution under the conditions of the experiment (see
section 3.5). Such NO generation is significant in anticancer therapy.
3.7. Investigation of anti-proliferative activity of the complex
Cytotoxic activity of 1 has been studied on four lines of human tumor cells: ovarian
carcinoma (SCOV3), large intestine cancer (LS174T), mammary gland carcinoma (MCF7),
and non-small cell carcinoma of lung (A549). 1 has different cytotoxic activity on the above
cell lines (table 6) with the highest activity shown for A549 and SCOV3. Dependence of
activity on concentration of 1 has been determined (table 7). The complex with phenolyl
[25] exhibits maximum activity on the cells of myeloleukemia K562 (IC50 = 20 μM), and
its activity is close to that of the platinum complex cis-DDP (IC50 = 20 μM). Lung carci-
noma (A549) and ovarian carcinoma (SCOV3) are stable to the action of the phenolyl com-
plex. Investigations of the phenolyl complex on transplantable tumors of mice Ca-755 and
melanoma B-16 (mice are the first generation hybrids BDF1 (C57Bl/6 x DBA/2) and DBA/2,
weight 18–25 g, obtained in the animal laboratory of N.N. Blokhin Russian Cancer
Research Center of RAMS) showed a moderate statistically important anticancer effect
when administered intra-abdominally during 5 days in 10–75 mg/kg dose as compared to
the control experiments. Toxicity of the complex was much lower (LD100 = 60 mg/kg) than
that of cisplatin (LD100 = 16 mg/kg).
Table 6. IC50 values (μM, for 1 on the lines of human tumor cells).
Complex
SKOV3
27
LS174T
37
MCF7
74
A549
25
1
Table 7. Dependence of survival of tumor cells on the concentration of 1.
Survival (%)
Concentration (μM)
Cells
25
50
100
IC50 (μM)
MCF7
SCOV3
LS174T
100
52
100
24.3
6.3
83.6
12.5
6.9
13.9
37
27
74

Synthesis, structure, NO donor activity of iron–sulfur nitrosyl complex
3617
From the above data on cytotoxic activity, we suggest that the complex with
2-aminophenolyl is a more promising anticancer agent for testing in vivo as a prolonged-
release NO donor compared to the complex with phenolyl.
4. Conclusion
Single crystals of nitrosyl iron complex with sulfur-containing ligand 2-aminophenol-2-yl,
which is a structural analog of DNA thiopyridine base, have been synthesized; their
molecular and crystalline structure have been studied by X-ray method, and the electron
structure has been determined by quantum chemistry and Mössbauer spectroscopy. The use
of a sulfur-containing ligand with the amino-group in the ortho-position of the phenyl ring
was shown to yield the nitrosyl iron complex with prolonged NO generation in aqueous
solutions and with a wider range of cytotoxic activities compared to the nitrosyl iron com-
plex with thiophenol studied earlier.
Supplementary material
Supplemental data for this article can be accessed http://dx.doi.org/10.1080/00958972.
2013.848980.
Acknowledgments
The work has been supported by the Program of the Presidium of RAS “Fundamental sciences
for medicine.”
References
[1] J.C. Dabrowiak. Metals in Medicine, p. 342 John Wiley & Sons, (2009).
[2] R.L. Elliott, J.F. Head. J. Cancer Therapy, 3, 278 (2012).
[3] S. Moccelin. Curr. Cancer Drug Targets, 9, 214 (2009).
[4] B. Brune, N. Scheneiderhan. Toxicol. Lett., 193, 19 (2003).
[5] L. Liu, M. Xu-Welliver, S. Kanugula, A.E. Pegg. Cancer Res., 62, 3037 (2002).
[6] D. Wink., J. Vodovoz, J. Cook. Biochemistry, 63, 948 (1998).
[7] N.P. Konovalova, S.A. Goncharova, L.M. Volkova, T.A. Raevskaya, L.T. Eremenko, A.M. Korolev. Nitric
Oxide, 8, 59 (2003).
[8] W. Yang, P.A. Rogers, H. Ding. J. Biol. Chem., 277, 12868 (2002).
[9] O. Siri, A. Tabard, P. Pullumbi, R. Guilard. Inorg. Chim. Acta, 350, 633 (2003).
[10] J.L. Burgaud, E. Jngini, P. Del Soldato, N.Y. Ann. Acad. Sci., 962, 360 (2002).
[11] T.I. Karu, L.V. Pyatibrat, G.S. Kalendo. Toxicol. Lett., 121, 57 (2001).
[12] A. Millet, A. Bettaieb, F. Renaud, L. Prevotat, A. Hammann, E. Solary, B. Mignotte, J.F. Jeannin.
Gastroenterology, 123, 235 (2002).
[13] V.J. Findlay, D.M. Townsend, J.E. Saavedra, G.S. Buzard, M.L. Citro, L.K. Keefer, X. Ji, K.D. Tew. Mol.
Pharmacol., 65, 1070 (2004).
[14] S.L. Pan, J.H. Guh, C.Y. Peng, S.W. Wang, Y.L. Chang, F.C. Cheng, J.H. Chang, S.C. Kuo, F.Y. Lee, C.M.
Teng. J. Pharmacol. Exp. Ther., 314, 35 (2005).
[15] E.J. Yeo, Y.S. Chun, Y.S. Cho, J. Kim, J.C. Lee, M.S. Kim, J.W. Park. J. Natl. Cancer Inst., 95, 516 (2003).
[16] B. Rigas, K. Kashfi. Trends Mol. Med., 10, 324 (2004).
[17] J.L. Williams, S. Borgo, I. Hasan, E. Castillo, F. Traganos, B. Rigas. Cancer Res., 61, 3285 (2001).

3618
N.A. Sanina et al.
[18] C. Levagna, P. Del Soldato, J.L. Burgaud, P. Rampal. Curr. Cancer Drug Targets, 3, 407 (2003).
[19] J.L. Williams, N. Nath, J. Chen, T.R. Hundley, J. Gao, L. Kopelovich, K. Kashfi, B. Rigas. Cancer Res.,
63, 7613 (2003).
[20] Y.J. Lee, K.H. Lee, J.M. Jessup, D.W. Seol, T.H. Kim, T.R. Billar, Y.K. Song. Oncogene, 20, 1476 (2001).
[21] M. Chawla-Sarkar, J.A. Bauer, J.A. Lupica, B.H. Morrison, Z. Tang, R.K. Oates, A. Almasan, J.A. DiDonato,
E.C. Borden, D.J. Lindner. J. Biol. Chem., 278, 39461 (2003).
[22] A.R. Butler, I.I. Megson. Chem. Rev., 102, 1155 (2002).
[23] H. Lewandowska, M. Kalinowska, K. Brzoska, K. Wojciuk, G. Wojciuk, M. Kruszewski. Dalton Trans.,
40, 8273 (2011).
[24] N.A. Sanina, S.M. Aldoshin Russ. Chem. Bull. (Engl. Transl.), 60, 1223 (2011).
[25] N.A. Sanina, O.S. Zhukova, S.M. Aldoshin, N.S. Emel’yanova, G.К. Gerasimova. Patent RU 2429242,
(2011).
[26] N.A. Sanina, K.A. Lysenko, O.S. Zhukova, T.N. Roudneva, N.S. Emelyanova, S.M. Aldoshin, Patent US
8,067,628 B2 (2011).
[27] A. Weissberger, E. Proskauer, J.A. Riddick, E.E. Toops. Organic Solvents: Physical Properties and Methods
of Purification, Interscience Publishers Inc., New York (1955).
[28] N.A. Sanina, G.V. Shilov, S.M. Aldoshin, A.F. Shestakov, L.A. Syrtsova, N.S. Ovanesyan, E.S. Chudinova,
N.I. Shkondina, N.S. Emel’yanova, A.I. Kotel’nikov. Russ. Chem. Bull. (Engl. Transl.), 58, 572 (2009).
[29] N.A. Sanina, S.M. Aldoshin, T.N. Rudneva, N.I. Golovina, G.V. Shilov, Yu. M. Shul’ga, V.M. Martynenko,
N.S. Ovanesyan, Russ. J. Coord. Chem., 31, 301 (2005).
[30] G.I. Kozub, N.A. Sanina, T.A. Kondrat’eva, G.V. Shilov, D.V. Korchagin, N.S. Ovanesyan, S.M. Aldoshin,
Russ. J. Coord. Chem., 38, 671 (2012).
[31] Agilent. CrysAlis PRO (Version 171.35.19), Agilent Technologies UK Ltd, Yarnton, Oxfordshire, England
(2011).
[32] G.M. Sheldrick. SHELXTL (Version 6.14), Structure Determination Software Suite, Bruker AXS, Madison,
Wisconsin, USA (2000).
[33] M.J. Frisch, G.W. Trucks, H.B., Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr.,
T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M.L.X. Klene, J.E. Knox, H.P. Hratchian, J.B. Cross,
C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich,
A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz,
Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi,
R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill,
B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople. Gaussian 03 Revision D.01, Gaussian Inc.,
Wallingford, CT (2004).
[34] D.N. Laikov. Chem. Phys. Lett., 81, 151 (1997).
[35] O.S. Zhukova. Russ. Biother. J., 3, 12 (2004).
[36] Organicum. Practical work on organic chemistry, Vol. 2, p. 224 Mir, Moscow (1979).
[37] V.A. Reznikov. Chemistry of nitrogen-containing compounds, p. 75, NSU, Novosibirsk (2006). К. Veigand,
Experimental methods in organic chemistry, Vol. 1, p. 346, Khimiya, Moscow (1968).
[38] A.Kh. Shakhnes, S.S., Vorob’ev, S.A. Shevelev. Russ. Chem. Bull., 55, 938 (2006)
[39] N.A. Sanina, O.A. Rakova, S.M. Aldoshin, I.I. Chuev, E.G. Atovmyan, N.S. Ovanesyan. Russ. J. Coord.
Chem., 27, 179 (2001).
[40] C. Glidewell, R.J. Lambert, N.B. Hursthouse, M. Motevalli. J. Chem. Soc., Dalton Trans., 1989 (2061).
[41] J.T. Thomas, J.H. Robertson, E.G. Cox. Acta Crystallogr., 11, 599 (1958).
[42] C. Glidewell, M.E. Harman, M.B. Hursthouse, I.L. Johnson, M. Motevalli. J. Chem. Res. (S), 212, 1676 (1988).
[43] O.A. Rakova, N.A. Sanina, G.V. Shilov, Yu. M. Shul’ga, V.M. Martynenko, N.S. Ovanesyan, S.M. Aldoshin.
Russ. J. Coord. Chem., 28, 341 (2002).
[44] N.A. Sanina, L.A. Syrtsova, N.I. Shkondina, E.S. Malkova, A.I. Kotelnikov, S.M. Aldoshin. Russ. Chem.
Bull. (Engl. Transl.), 56, 761 (2007).
[45] J. Jolley, W.I. Cross, R.G. Pritchard, C.A. McAuliffe, K.B. Nolan. Inorg. Chim. Acta, 315 (2001).
N.A. Sanina, N.S. Emel’yanova, A.N. Chekhlov, A.F. Shestakov, I.V. Sulimenkov, S.M. Aldoshin. Russ. Chem.
Bull. (Engl. Transl.), 59, 1126 (2010).
[47] S.M. Aldoshin, K.A. Lysenko, MYu Antipin, N.A. Sanina, V.V. Gritsenko. J. Mol. Struct., 875, 309 (2008).
[48] T.S. Stupina, I.I. Parkhomenko, I.V. Balalaeva, G.V. Kostyuk, N.A. Sanina. A.A. Terent’ev, Russ. Chem. Bull.
(Engl. Transl.), 60, 1488 (2011).
[49] M. Radon, M. Srebro, E. Broclawik. J. Chem. Theory Comput., 5, 1237 (2009).
[50] A.F. Shestakov, Yu.N. Shul’ga, N.S. Emel’yanova, N.A. Sanina, S.M. Aldoshin. Russ. Chem. Bull., 55, 2133
(2006).
[51] N.S. Emel’yanova, A.F. Shestakov, I.V. Sulimenkov, T.N. Rudneva, N.A. Sanina, S.M. Aldoshin. Russ. Chem.
Bull. (Engl. Transl.), 61, 1 (2012).