Nitro-imidazoles in ferrocenyl alkylation reaction. Synthesis, enantiomeric resolution and in vitro and in vivo bioeffects

Article abstract of DOI:10.1016/j.jorganchem.2018.06.019

Ferrocenylalkyl nitro-imidazoles (4a-h, 5a-h) were prepared via the regiospecific reaction of the α-(hydroxy)alkyl ferrocenes, FcCHR (OH) (1a–h; Fc = ferrocenyl; R = H, Me, Et, Pr, i-Pr, Ph, ortho-Cl-Ph, ortho-I-Ph), with nitro-imidazoles in aqueous organic medium (H₂O-CH₂Cl₂) at room temperature in the presence of HBF₄, within several minutes in good yields. X-ray structural data for racemic (R,S)-1-N-(benzyl ferrocenyl)-2-methyl-4-nitroimidazole (5f) were determined. The resulting enantiomers were resolved into enantiomers by analytical HPLC on modified amylose or cellulose chiral stationary phases. The viabilities of 4b, 4d, 5b, 5c in vitro, and in experiments in vivo antitumor effects of 1-N-ferrocenylethyl-4-nitroimidazole (4b) against murine solid tumor system Ca755 carcinoma were evaluated.

Full text of DOI:10.1016/j.jorganchem.2018.06.019

Accepted Manuscript
Nitro-imidazoles in ferrocenyl alkylation reaction. Synthesis, enantiomeric resolution
and in vitro and in vivo bioeffects
Lubov V. Snegur, Maria V. Lyapunova, Daria D. Verina, Vadim V. Kachala, Alexander
A. Korlyukov, Mikhail M. Ilyin, Jr., Vadim A. Davankov, Larissa A. Ostrovskaya,
Natalia V. Bluchterova, Margarita M. Fomina, Victor S. Malkov, Kseniya V. Nevskaya,
Alexandra G. Pershina, Alexander A. Simenel
PII:
S0022-328X(18)30515-1
10.1016/j.jorganchem.2018.06.019
JOM 20478
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 19 February 2018
Revised Date: 18 June 2018
Accepted Date: 24 June 2018
Please cite this article as: L.V. Snegur, M.V. Lyapunova, D.D. Verina, V.V. Kachala, A.A. Korlyukov,
M.M. Ilyin Jr., V.A. Davankov, L.A. Ostrovskaya, N.V. Bluchterova, M.M. Fomina, V.S. Malkov, K.V.
Nevskaya, A.G. Pershina, A.A. Simenel, Nitro-imidazoles in ferrocenyl alkylation reaction. Synthesis,
enantiomeric resolution and in vitro and in vivo bioeffects, Journal of Organometallic Chemistry (2018),
doi: 10.1016/j.jorganchem.2018.06.019.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Nitro-imidazoles in Ferrocenyl Alkylation Reaction. Synthesis, Enantiomeric
Resolution and in Vitro and in Vivo Bioeffects
1
∗
2
3
4
Lubov V. Snegur , Maria V. Lyapunova , Daria D. Verina , Vadim V. Kachala , Alexander
1
,5
1
1
6
A. Korlyukov , Mikhail M. Ilyin, Jr. , Vadim A. Davankov , Larissa A. Ostrovskaya , Natalia
V. Bluchterova , Margarita M. Fomina , Victor S. Malkov , Kseniya V. Nevskaya ,
Alexandra G. Pershina , and Alexander A. Simenel
6
6
2
7
7
1,8
1
A.N. Nesmeyanov Institute of OrganoElement Compounds, Russian Academy of Sciences, 28
Vavilov St, 119991 Moscow, Russian Federation
National Research Tomsk State University, 36 Lenin Ave., 634050 Tomsk, Russian Federation
2
3
D.I. Mendeleev University of Chemical Technology of Russia, 9 Miusskaya sq., 125047
Moscow, Russian Federation
4
N.D. Zelynski Institute of Organic Chemistry RAS, 47 Leninsky Ave., 119991 Moscow, Russian
Federation
5
N.I. Pirogov Russian National Research Medical University, 1 Ostrovityanov St., 117997
Moscow, Russian Federation
6
N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosigin St,
1
19991 Moscow, Russian Federation
7
Siberian State Medical University, 2 Moskovsky trakt, 634055 Tomsk, Russian Federation
National University of Science and Technology "MISIS", Chemistry Department, 4 Leninskiy
8
Ave., 119049 Moscow, Russian Federation
Ferrocenylalkyl nitro-imidazoles (4a-h, 5a-h) were prepared via the regiospecific reaction of the α-
(
hydroxy)alkyl ferrocenes, FcCHR(OH) (1a–h; Fc=ferrocenyl; R=H, Me, Et, Pr, i-Pr, Ph, ortho-Cl-Ph,
ortho-I-Ph), with nitro-imidazoles in aqueous organic medium (H O-CH Cl ) at room temperature in the
2
2
2
presence of HBF , within several minutes in good yields. X-ray structural data for racemic (R,S)-1-N-
4
(
benzyl ferrocenyl)-2-methyl-4-nitroimidazole (5f) were determined. The resulting enantiomers were
resolved into enantiomers by analytical HPLC on modified amylose or cellulose chiral stationary phases.
The viabilities of 4b, 4d, 5b, 5c in vitro, and in experiments in vivo antitumor effects of 1-N-
ferrocenylethyl-4-nitroimidazole (4b) against murine solid tumor system Ca755 carcinoma were
evaluated.
Keywords: ferrocene compounds; nitro-imidazoles; enantiomeric resolution; X-ray crystal structure;
toxicity in vitro; bioactivity in vivo
1
. Introduction
The conjugation of nitrogen-containing hetero cycles with ferrocene represents perspective
directions for design and synthesis of bioactive compounds with different types of activities
including antianemic, tuberculostatic, antimalarial, antimicrobic and antiproliferative [1−10].
The antiproliferative activity has been especially investigated by numerous research groups
[
10−15].
Among heterocycles, azoles and especially imidazole cause a high interest because represent
essential components of DNA, RNA, histidine amino acid or drugs [10−14,16-20]. On the other
∗
Corresponding author
E-mail address: snegur@ineos.ac.ru (L.V. Snegur)

ACCEPTED MANUSCRIPT
hand, such nitrogen- heterocycle−ferrocene ensembles possess positive enthalpies of formation
to give highly energetic structures [21]. Well known, that both ferrocenes and nitro-compounds
were applied for solid rocket propellant in the early 1970s [10]. Moreover, nitro groups being in
the structures of nitro-glycerin and trinitro-toluene make these compounds high explosive. To
our knowledge, there are no systematic studies on synthesis of compounds combining both nitro-
imidazole and ferrocene fragments excluding the data in the review article of Tverdoxlebov with
co-authors [21].
Ferrocene-modification of organic compounds was widely represented in reviews [17,22,23]
and special recent issue [24] The ferrocenylalkylation method for the introduction of
ferrocenylalkyl groups into various nucleophilic substrates was based on the substitution
reactions of ferrocenylalkyl amines or α-(hydroxy)alkyl ferrocenes with nucleophiles [22]. The
later one is extensively explored approach to ferrocene-based compounds [23,25,26]. Recently,
for example, Moiseev and co-workers elaborated new synthetic route to ferrocenes of such type
through unstable α-ferrocenylalkyl carbonates under neutral conditions [27]. Some more original
pathway for modificated ferrocenes through the direct C−H functionalization of aromatics by the
C−C coupling of halogen-free (hetero)arenes with lithium ferrocenes allows the preparing of
planar chiral ferrocenes [28].
Other well-known pathway to ferrocene heterocyclic molecules is consists in formation of
carbonyl-connected ferrocene-C(O)-heterocycle through a dehydrohalogenation reaction
between ferrocenoyl chloride and NH-heterocycle [29]. Redox amination of ferrocenyl ketones
and formylferrocene with pyrroline allows preparing ferrocene-substituted pyrroles [30].
In this paper, the development of our ferrocene-devoted works is reported [11–13,31-34].
Early we proposed a simple route to ferrocene-based compounds [35]. Moreover, using this
protocol, enantiomeric-enriched ferrocene-based compounds were prepared recently [36].
Herein, according to this synthetic approach a series of unknown ferrocene-containing nitro-
1
imidazoles was prepared in good yields. All synthesized compounds were characterized by H,
1
3
C NMR, EI-MS and microanalytical data. For compound 5f X-ray structural data were
obtained. A series of racemic (S,R)-ferrocene nitro-imidazoles (14 pairs of ferrocene-based
compounds) was separated into enantiomers by analytical HLPC on chiral modified amylose.
The antitumor effects were evaluated for compound 4b against Ca755 carcinoma experimental
tumor model. The index of tumor growth inhibition (TGI) amounting to 22% (in comparison
−1
with controls) was determined for compound 4b in daily dose equal to 10 mg kg . At the dose
−1
of 20 mg kg the tested compound 4b was not effective against Ca755 carcinoma. On the
contrary, a stimulation effect equal to 46% was observed in this case. Cytotoxicities were

ACCEPTED MANUSCRIPT
evaluated by MTT test for compounds 4b, 4d, 5b, 5c, and for compound 4b maximal tolerated
dose was found in in vivo experiments.
2
. Experimental
2
.1. Starting Materials and Analytical Instrumentations
The starting ferrocenyl methanol (1a) was obtained from trimethylferrocenyl
methylammonium iodide according to a well-known procedure [37]. Others ferrocenyl alcohols
1b-h) were synthesized from ferrocene by acylation with the corresponding acid chlorides
(
according to the Friedel–Crafts procedure and subsequent reduction either by sodium
borohydride in water [38] or by lithium aluminum hydride in diethyl ether or THF [39,12]. 4(5)-
Nitroimidazole (2a) and 2-methyl-4(5)-nitroimidazole (2b) were prepared according to patents
[
40,41]. Syntheses were carried out in a 100 mL flask with fluoroplastic propeller mixer and
thermometer. Sulfuric acid 92 % (66.3 g) and 10.4 g (0.15 mol) imidazole or 2-methyl-4(5)-
nitroimidzole (2b) were added into the flask with stirring. The temperature was raised to 145°C,
then, 29.8 g (0.35 mol) sodium nitrate was added in portions to the mixture. After stirring at
1
45°C during 6 hours cold water (100 mL) was added to the flask and the mixture was cooled to
the room temperature. The crud products were isolated by 25 % ammonia solution to pH 10,
filtered and dried by lyoplilization at −52°C and 0.27 mbar for 16 h. Both imidazoles represented
white powders, yield 2a 75%, m.p. 306-310°C; yield 2b 95%, m.p. 253-256°C.
1
13
All solvents were purified and dried by standards techniques. H and C NMR spectra were
obtained on a Bruker Avance instrument at 400 MHz for protons and 100 MHz for carbon
respectively. Chemical shifts are given in ppm relative to solvent residual protons. EI mass
spectra were taken on a Finnigan Polaris Q spectrometer at 70 eV and the temperature of the ion
chamber 250°C. IR spectra were recorded on a UR-20 (Karl Zeiss) spectrophotometer.
2
.2. General Procedure
To a mixture of 1.0 mmol of ferrocene alcohol and 1.0 mmol of the corresponding
nitroimidazole in 1.0 ml of methylene dichloride, 0.18 ml of 45 % aqueous solution of
fluoroboric acid was added under vigorous stirring. The agitation was continued for 5 min then
diethyl ether (15 ml), the same amount of cold water, and 5-10 mg of ascorbic acid were added
to the reaction flask. After vigorous shaking of the mixture, the organic solution was separated,
washed with cold water (3×15 ml), the solvents were removed in vacuo, and the residue was
dried over CaCl in a desiccator.
2
4
-Nitro-1-(ferrocenylmethyl)-1H-imidazole (4a). Yield 64%. Orange crystals, m.p. 206 °С. EI-
+
1
MS, m/z (RI, %) 311 [M ] (67). Н NMR (CDCl , δ, ppm): 4.21 (s, 5H, Fc); 4.25 (s, 2Н, Fc);
3
1
3
4
.28 (s, 2Н, Fc); 4.95 (s, 2Н, CH ); 7.41 (s, 1Н, Im(C-2)); 7.70 (s, 1Н, Im(C-5)). С NMR
2
(
CDCl , δ, ppm): 48.28 (CH ); 68.89 (C H ); 68.99 (C H ); 69.64 (C H ); 79.80 (C H -ipso);
3 2 5 4 5 5 5 4 5 4
1
18.76 (Im(C-5)); 135.22 (Im(C-2)); 148.37 (Im(C-NO ). Anal.: C 53.99; H 4.17; N 13.48%.
2
Calc. for C H FeN O : C, 54.05; H, 4.21; N, 13.51%.
1
4
13
3
2

ACCEPTED MANUSCRIPT
4
-Nitro-1-(1-ferrocenylethyl)-1H-imidazole (4b). Yield 65 %. Yellow crystals, m.p. 106 °С. EI-
+
1
MS, m/z (RI, %) 325 [M ] (25). Н NMR (CDCl , δ, ppm): 1.89 (d, J = 6.9 Hz, 3H, CH ); 4.14
3
3
(s, 1H, Fc); 4.19 (s, 5H, Fc); 4.26 (s, 1H, Fc); 4.28 (s, 2H, Fc); 5.25 (q, J = 6.9 Hz, 1H, CH); 7.42
1
3
(
s, 1Н, Im(C-2)); 7.69 (s, 1Н, Im(C-5)). С NMR (CDCl , δ, ppm): 21.95 (CH ); 54.82 (CH);
3 3
6
5.79 (C H ); 67.95 (C H ); 68.78 (C H ); 69.12 (C H ); 69.40 (C H ); 86.35 (C H -ipso);
5 4 5 4 5 4 5 5 5 4 5 4
1
17.76 (Im(C-5)); 134.50 (Im(C-2)); 147.45 (Im(C-NO )). Anal.: C 55.43; H 4.54; N 12.94.
2
Calc. for C H FeN O : C, 55.41; H, 4.65; N, 12.92%.
1
5
15
3
2
4
-Nitro-1-(1-ferrocenylpropyl)-1H-imidazole (4c). Yield 50%. Red-brown crystals, m.p. 87 °С.
+
1
EI-MS, m/z (RI, %) 339 [M ] (34). Н NMR (CDCl , δ, ppm): 0.95 (t, J = 7.4 Hz, 3H, CH );
3
3
2
.04 (m, 1H, CH ); 2.36 (m, 1H, CH ); 4.07 (s, 1H, Fc); 4.15 (s, 5H, Fc); 4.23 (s, 1H, Fc); 4.26
2 2
1
3
(
s, 2H, Fc); 4.88 (dd, J = 10.8 Hz, 3.8, 1H, CH); 7.47 (s, 1Н, Im(C-2)); 7.72 (s, 1Н, Im(C-5)). С
NMR (CDCl , δ, ppm): 11.19 (CH ); 29.04 (CH ); 61.61 (CH); 66.30 (C H ); 67.14 (C H );
3
3
2
5
4
5
4
6
8.53 (C H ); 69.07 (C H ); 69.15 (C H ); 86.96 (C H -ipso); 117.73 (Im (C-5)); 135.10 (Im (C-
5 4 5 5 5 4 5 4
2
)); 147.98 (Im (C- NO )). Anal.: C 56.68; H 5.05; N 12.41. Calc. for C H FeN O : C, 56.66;
2 16 17 3 2
H, 5.05; N, 12.39%.
4
-Nitro-1-(1-ferrocenylbutyl)-1H-imidazole (4d). Yield 48 %. Red-brown crystals, m.p. 111 °С.
+
1
EI-MS, m/z (RI, %) 353 [M ] (29). Н NMR (CDCl , δ, ppm): 0.98 (t, J = 7.2 Hz, 3H, CH );
3
3
1
4
7
.28 (m, 2Н, CH ); 2.03 (m, 1Н, CH ); 2.21 (m, 1Н, CH ); 4.05 (s, 1Н, Fc); 4.13 (s, 5Н, Fc);
2 2 2
.20 (s, 1Н, Fc); 4.24 (s, 2Н, Fc); 4.99 (dd, J = 10.8 Hz, 3.5, 1H, CH); 7.47 (s, 1Н, Im(C-2));
1
3
.73 (s, 1Н, Im(C-5)). С NMR (CDCl , δ, ppm): 13.55 (CH ); 19.58 (СH ); 37.74 (CH ); 59.53
3
3
2
2
(
CH); 66.25 (C H ); 67.14 (C H ); 68.50 (C H ); 69.07 (C H ); 69.14 (C H ); 87.14 (C H -ipso);
5 4 5 4 5 4 5 5 5 4 5 4
1
17.83 (Im(C-2)); 135.20 (Im (C-5)); 147.93 (Im (C-NO ). Anal.: C 58.86; H 5.73; N 11.45.
2
Calc. for C H FeN O : C, 58.87; H, 5.76; N, 11.44%.
1
7
19
3
2
4
-Nitro-1-(1-ferrocenyl-2-methylpropyl)-1H-imidazole (4e). Yield 51%. Red-brown crystals,
+
1
m.p. 138 °С. EI-MS, m/z (RI, %) 353 [M ] (36). Н NMR (CDCl , δ, ppm): 0.80 (d, J = 6.7 Hz,
3
3
4
1
6
1
H, CH ); 0.90 (d, J = 6.7 Hz, 3Н, CH ); 2.15 (м, 1Н, CH); 3.94 (s, 5Н, Fc); 4.03 (s, 1Н, Fc);
3
3
.22 (s, 1Н, Fc); 4.25 (s, 2Н, Fc); 4.53(d, J = 8.4 Hz, 1H, CH); 7.59 (s, 1Н, Im(C-2)); 7.89 (s,
1
3
Н, Im(C-5)). С NMR (CDCl , δ, ppm): 19.63 (CH ); 20.51 (CH ); 35.35 (CH); 65.65 (CH);
3 3
3
6.60 (C H ); 67.67 (C H ); 68.88 (C H ); 69.19 (C H ); 69.50 (C H ); 86.88 (C H -ipso);
5
4
5
4
5
5
5
4
5
4
5
4
18.58 (Im(C-2)); 135.91 (Im (C-5)); 147.78 (Im (C-NO ). Anal.: C 58.84; H 5.74; N 11.46.
2
Calc. for C H FeN O : C, 58.87; H, 5.76; N, 11.44%.
1
7
19
3
2
4
-Nitro-1-(ferrocenyl(phenyl)methyl)-1H-imidazole (4f). Yield 47%. Yellow-brown crystals,
+
1
m.p. 68 °С. EI-MS, m/z (RI, %) 387 [M ] (50). Н NMR (CDCl , δ, ppm): 4.04 (s, 1H, Fc); 4.06
3
(s, 5Н, Fc); 4.21 (s, 1Н, Fc); 4.32 (s, 1Н, Fc); 4.36 (s, 1Н, Fc); 6.22 (s, 1Н, CH); 7.25 (m, 2Н,

ACCEPTED MANUSCRIPT
1
3
Ph); 7.34 (s, 1Н, Im(C-2)); 7.43 (m, 3Н, Ph); 7.59 (s, 1Н, Im (C-5)). С NMR (CDCl , δ, ppm):
3
6
3.51 (CH); 68.23 (C H ); 68.51 (C H ); 69.29 (C H ); 69.52 (C H ); 69.54 (C H ); 84.59
5 4 5 4 5 5 5 4 5 4
(
C H -ipso); 119.16 (Im (C-5)); 127.41 (C H ); 129.07 (C H ); 129.12 (C H ); 135.62 (C H );
5 4 6 5 6 5 6 5 6 5
1
38.15 (Im(C-2)); 147.60 (Im (C-NO )). Anal.: C 62.05; H 4.44; N 10.89. Calc. for
2
C H FeN O : C, 62.04; H, 4.43; N, 10.85%.
20
17
3
2
4
-Nitro-1-((2-chlorophenyl)ferrocenylmethyl)-1H-imidazole (4g). Yield 52%. Red-brown
+
1
crystals, m.p. 149 °С. EI-MS, m/z (RI, %) 421 [M ] (36). Н NMR (CDCl , δ, ppm): 4.10 (s, 1H,
3
Fc); 4.14 (s, 5H, Fc); 4.19 (s, 1H, Fc); 4.38 (s, 2H, Fc); 6.77 (s, 1H, CH); 7.17(d, J = 9.2 Hz, 1H,
Ph); 7.31 (m, 1H, Ph); 7.37 (m, 1H, Ph); 7,49 (s, 1H, Im(C-2)); 7.52 (m, 1H, Ph); 7.65 (s, 1H,
1
3
Im(C-5)). С NMR (CDCl , δ, ppm): 59.38 (CH); 67.53 (C H ); 68.37 (C H ); 69.04 (C H );
3
5
4
5
4
5
4
6
1
1
3
4
9.36 (C H ); 69.75 (C H ); 84.6 (C H -ipso); 118.98 (Im(C-5)); 127.62 (C H ); 129.49 (C H );
5 5 5 4 5 4 6 4 6 4
30.29 (C H ); 130.63 (C H ); 133.46 (C H (C-ipso)); 135.91 (C H (C-Cl)); 135.98 (Im(C-2));
6
4
6
4
6
4
6
4
47.60 (Im(C-NO ). Anal.: C 56.92; H 3.80; N 9.96. Calc. for C H ClFeN O : C, 56.97; H,
2
20 16
3
2
.82; N, 9.97%.
-Nitro-1-((2-iodophenyl)ferrocenylmethyl)-1H-imidazole (4h). Yield 52%. Yellow crystals,
+
1
m.p. 85 °С. EI-MS, m/z (RI, %) 513 [M ] (45). Н NMR (CDCl , δ, ppm): 4.02 (s, 1H, Fc); 4.15
3
(s, 5H, Fc ); 4.19 (s, 1H, Fc); 4.33 (s, 1H, Fc); 4.36 (s, 1H, Fc); 6.59 (s, 1H, CH); 7.11 (m, 2H,
13
Ph); 7.36 (m, 1H, Ph); 7.52 (s, 1H, Im(C-2)); 7.62 (s, 1H, Im(C-5)); 7.93 (m, 1H, Ph). С NMR
(
(
(
CDCl , δ, ppm): 67.02 (СН); 67.27 (C H ); 67.95 (C H ); 68.77 (C H ); 69.43 (C H ); 69.79
3 5 4 5 4 5 4 5 5
C H ); 85.47 (C H -ipso); 100.37 (C H (C-I)); 119.05 (Im(C-5)); 129.06 (C H ); 129.45
5
4
5
4
6
4
6
4
C H ); 131.04 (C H ); 136.25 (Im(C-2)); 140.39 (C H ); 140.51 (C H ); 147.62 (Im(C-NO ).
6
4
6
4
6
4
6
4
2
Anal.: C 46.76; H 3.20; N 8.19. Calc. for C H FeIN O : C, 46.82; H, 3.14; N, 8.19%.
2
0
16
3
2
2
-Methyl-4-nitro-1-(ferrocenylmethyl)-1H-imidazole (5a). Yield 43%. Yellow-orange crystals,
+
1
m.p. 89 °С. EI-MS, m/z (RI, %) 325 [M ] (71). Н NMR (CDCl , δ, ppm): 2.42 (s, 3Н, CH );
3
3
4
.18 (s, 5Н, Fc); 4.20 (s, 2Н, Fc); 4.24 (s, 2Н, Fc); 4.79 (s, 2Н, CH ); 7.58 (s, 1Н, Im(C-5)).
2
1
3
NMR (CDCl , δ, ppm): 13.17 (CH ); 46.96 (CH ); 68.31 (C H ); 68.77 (C H ); 68.84 (C H );
3
3
2
5
4
5
4
5
5
6
9.33 (C H ); 69.35 (C H ); 79.88 (C H -ipso); 119.33 (Im(C-5)); 144.01 (Im(C-2)); 145.84
5 4 5 4 5 4
(
Im(C-NO ). Anal.: C 54.80; H 4.72; N 12.78. Calc. for C H FeN O : C, 55.41; H, 4.65; N,
2 15 15 3 2
1
2
2.92%.
-Methyl-4-nitro-1-(1-ferrocenylethyl)-1H-imidazole (5b). Yield 52%. Yellow-orange crystals,
+
1
m.p. 83 °С. EI-MS, m/z (RI, %) 339 [M ] (87). Н NMR (CDCl , δ, ppm): 1,78 (d, J = 6.9 Hz,
3
3
5
2
Н, CH ); 2.47 (s, 3Н, CH ); 4.08 (s, 1Н, Fc); 4.17 (s, 5Н, Fc); 4.21 (s, 1Н, Fc); 4.25 (s, 2Н, Fc);
3 3
1
3
.15 (q, J = 6.9 Hz, 1Н, CH); 7.84 (s, 1Н, Im(C-5)). NMR (CDCl , δ, ppm): 13.31 (CH (Im));
3
3
1.18 (CH ); 53.03 (CH); 65.84 (C H ); 67.77 (C H ); 68.53 (C H ); 69.01 (C H ); 69.35
3
5
4
5
4
5
4
5
5
(
C H ); 86.63 (C H -ipso); 117.51 (Im(C-5)); 143.52 (Im(C-2)); 146.10 (Im(C-NO ). IR (ν,
5 4 5 4 2

ACCEPTED MANUSCRIPT
−1
cm ): 3150, 3094, 2984, 1537 (s, NO ), 1498 (s, NO ), 1447, 1392 (s, NO ), 1380 (s, NO ),
2
2
2
2
1
263, 1144, 1122, 1106, 829, 752, 508, 483. Anal.: C 58.69; H 5.79; Fe 15.32 N 11.37. Calc. for
C H FeN O : C, 58.87; H, 5.76; N, 11.44%.
16
17
3
2
2
-Methyl-4-nitro-1-(1-ferrocenylpropyl)-1H-imidazole (5c). Yield 54%. Orange powder, m.p.
+
1
1
41 °С. EI-MS, m/z (RI, %) 353 [M ] (57). Н NMR (CDCl , δ, ppm): 0.92 (t, J = 7.6 Hz, 3H,
3
CH ); 2.05 (m, 1H, CH ); 2.37 (m, 1H, CH ); 2.49 (s, 3H, CH ); 4.05 (s, 1H, Fc); 4.16 (s, 5H,
3
2
2
3
Fc); 4.19 (s, 1H, Fc); 4.21 (s, 1H, Fc); 4.23 (s, 1Н, Fc); 4.86 (dd, J = 10.9 Hz, 3.6, 1H, CH); 7.62
1
3
(
s, 1Н, Im(C-5)). С NMR (CDCl , δ, ppm): 11.09 (CH ); 13.72, (CH (Im)); 28.91 (CH );
3 3 3 2
5
9.26 (CH); 66.23 (C H ); 67.07 (C H ); 68.31 (C H ); 69.04 (C H ); 69.10 (C H ); 87.46
5 4 5 4 5 4 5 5 5 4
(
C H -ipso); 117.34 (Im (C-5)); 144.39 (Im (C-2)); 146.81 (Im (C- NO )). Anal.: C 58.85; 5
5
4
2
4
.74; N 11.49. Calc. for C H FeN O : C, 58.87; H, 5.76; N, 11.44%.
17 19 3 2
2
-Methyl-4-nitro-1-(1-ferrocenylbutyl)-1H-imidazole (5d). Yield 87%. Yellow crystals, m.p. 168
+
1
°
С. EI-MS, m/z (RI, %) 367 [M ] (53). Н NMR (CDCl , δ, ppm): 0.98 (t, J = 7.2 Hz, 3H,
3
CH ); 1.25 (m, 2Н, CH ); 2.04 (m, 1Н, CH ); 2.25 (m, 1H, CH ); 4.05 (s, 1Н, Fc); 4.15 (s, 5Н,
3
2
2
2
Fc); 4.18 (s, 1Н, Fc); 4.23 (s, 1H, Fc); 4.95 (dd, J = 10.7 Hz, 3.6, 1H, CH); 7.64 (s, 1Н, Im(C-
1
3
5
)). С NMR (CDCl , δ, ppm): 13.71 (CH (Im)); 19.57 (CH ); 37.75 (CH ); 57.39 (CH ); 66.25
3 3 3 2 2
(
СН); 67.00 (C H ); 68.28 (C H ); 69.04 (C H ); 87.67 (C H -ipso); 117.36 (Im (C-5)); 144.25
5 4 5 4 5 5 5 4
(
Im(C-2)); 146.59 (Im (C-NO ). Anal.: C 58.92; H 5.71; N 11.47. Calc. for C H FeN O : C,
2 18 21 3 2
5
2
8.87; H, 5.76; N, 11.44%.
-Methyl-4-nitro-1-(1-ferrocenyl-2-methylpropyl)-1H-imidazole (5e). Yield 47%. Yellow
+
1
crystals, m.p. 124 °С. EI-MS, m/z (RI, %) 367 [M ] (38). Н NMR (CDCl , δ, ppm): 0.79 (d, J =
3
6
5
1
.4 Hz, 3H, CH ); 0.87 (d, J = 6.8 Hz, 3Н, CH ); 2.01 (m, 1Н, СН); 2.59 (s, 3Н, CH ); 3.89 (s,
3 3 3
Н, Fc); 4.05 (s, 1Н, Fc); 4.20 (s, 2Н, Fc); 4.27(s, 1H, Fc); 4.38 (d, J = 9.6 Hz, 1H, CH); 7.84 (s,
1
3
Н, Im(C-5)). С NMR (CDCl , δ, ppm): 13.99 (CH (Im)); 20.08 (CH ); 20.42 (CH ); 36.14
3
3
3
3
(
CH); 64.13 (CH); 67.19 (C H ); 68.69 (C H ); 69.25 (C H ); 69.91 (C H ); 87.84 (C H -ipso);
5 4 5 5 5 4 5 4 5 4
1
17.36 (Im(C-5)); 144.60 (Im(C-2)); 153.14 (Im (C-NO ). Anal.: C 58.79; H 5.74; N 11.41.
2
Calc. for C H FeN O : C, 58.87; H, 5.76; N, 11.44%.
1
8
21
3
2
2
-Methyl-4-nitro-1-(ferrocenyl(phenyl)methyl)-1H-imidazole (5f). Yield 55%. Yellow crystals,
+
1
m.p. 132 °С. EI-MS, m/z (RI, %) 401 [M ] (72). Н NMR (CDCl , δ, ppm): 2.24 (s, 3H, CH );
3
3
3
.87 (s, 1Н, Fc); 4.08 (s, 5Н, Fc); 4.23 (s, 1Н, Fc); 4.30 (s, 1Н, Fc); 4.38 (s, 1Н, Fc); 6.14 (s, 1Н,
1
3
CH); 7.22 (m, 2H, Ph); 7.39 (m, 1H, Ph); 7.41 (m, 2Н, Ph); 7.45 (s,1H, Im(C-5)). С NMR
(
CDCl , δ, ppm): 13.63 (CH ); 62.04 (CH); 68.49 (C H ); 68.56 (C H ); 69.10 (C H ); 69.18
3 3 5 4 5 4 5 4
(
C H ); 69.93 (C H ); 84.84 (C H -ipso); 119.15 (Im(C-5)); 127.07 (C H ); 128.59 (C H );
5 5 5 4 5 4 6 5 6 5
−1
1
28.78 (C H ); 137.17 (C H ); 144.45 (Im(C-2)); 145.47 (Im (C-NO ). IR (ν, cm ): 3168, 3096,
6 5 6 5 2

ACCEPTED MANUSCRIPT
3
8
6
2
030, 2923, 1532 (s, NO ), 1487 (s, NO ), 1455, 1380 (s, NO ), 1263, 1151, 1107, 1003, 993,
2 2 2
20, 750, 730, 703, 506, 488, 479. Anal.: C 62.85; H 4.70; N 10.52. Calc. for C H FeN O : C,
2
1
19
3
2
2.86; H, 4.77; N, 10.47%.
-Methyl-4-nitro-1-((2-chlorophenyl)ferrocenylmethyl)-1H-imidazole (5g). Yield 55 %. Orange
+
1
crystals, m.p. 92 °С. EI-MS, m/z (RI, %) 435 [M ] (63). Н NMR (CDCl , δ, ppm): 2.34 (s, 3H,
3
CH ); 4.03 (s, 1H, Fc); 4.12 (s, 5H, Fc); 4.19 (s, 1H, Fc); 4.36 (s, 1H, Fc); 4.37 (s, 1H, Fc); 6.61
3
(s, 1H, CH); 7.12 (m, 1H, Ph); 7.29-7,38 (m, 2H, Ph); 7.47 (s, 1H, Im(C-5)); 7.52 (m, 1H, Ph).
1
3
С NMR (CDCl , δ, ppm): 13.81 (CH (Im)); 57.46 (CH); 67.51 (C H ); 68.69 (C H ); 69.39
3
3
5
4
5
4
(
C H ); 69.88 (C H ); 84.19 (C H -ipso); 119.18 (Im(C-5)); 127.73 (C H ); 129.08 (C H );
5 5 5 4 5 4 6 4 6 4
1
30.14 (C H ); 132.86 (C H (C-ipso)); 135.41 (C H (C-Cl)); 144.60 (Im(C-2)); 145.63 (Im(C-
6 4 6 4 6 4
NO ). Anal.: C 57.79; H 4.14; N 9.69. Calc. for C H ClFeN O : C, 57.89; H, 4.16; N, 9.64%.
2
21 18
3
2
2
-Methyl-4-nitro-1-((2-iodophenyl)ferrocenylmethyl)-1H-imidazole (5h). Yield 43%. Orange
+
1
crystals, m.p. 84 °С. EI-MS, m/z (RI, %) 527 [M ] (83). Н NMR (CDCl , δ, ppm): 2.40 (s, 3H,
3
CH ); 4.09 (s, 2H, Fc ); 4.15 (s, 5H, Fc); 4.38 (s, 2H, Fc); 6.39 (s, 1H, CH); 7.08 (m, 1H, Ph);
3
1
3
7
1
.37-7.44 (m, 2H, Ph); 7.56 (s, 1H, Im(C-5)); 7.98 (m, 1H, Ph). С NMR (CDCl , δ, ppm):
3
4.87 (CH ); 65.55 (СН); 67.12 (C H ); 68.95 (C H ); 69.29 (C H ); 69.52 (C H ); 69.59
3
5
4
5
4
5
4
5
4
(
C H ); 85.03 (C H -ipso); 100.12 (C H (C-I)); 119.39 (Im(C-5)); 128.79(C H ); 129.13 (C H );
5 5 5 4 6 4 6 4 6 4
1
30.64 (C H ); 140.02 (C H (C- ipso)); 140.35 (C H ); 145.12 (Im(C-2)); 146.57 (Im(C-NO ).
6 4 6 4 6 4 2
Anal.: C 47.81; H 3.45; N 7.99. Calc. for C H FeIN O : C, 47.85; H, 3.44; N, 7.97%.
2
1
18
3
2
2
.3. X-ray crystallography
Single crystals of C H FeN O (5f) were orange prisms crystallized from acetone. A
2
1
19
3
2
suitable crystal was selected from crud product, and intensities of reflections were measured on a
Bruker SMART CCD 1K diffractometer in Centre for molecular composition studies of INEOS
RAS. The crystal was kept at room temperature during data collection. The structure was solved
with the XS structure solution program [42] using Direct Methods and refined with the ShelXL
[
43] refinement package using Least Squares minimisation. Molecular graphics were drawn
using OLEX2 program [44] (Fig. 1). All crystallographic data were submitted to Cambridge
Crystallographic Data Centre (CCDC deposition number 1816071). Selected crystallographic
parameters were summarized in Table 1.
Table 1. Crystallographic data for 5f.
Empirical formula
Formula weight
T, K
C H FeN O
2
1
19
3
2
401.24
293
Space group, Z
P2 /n, 4
1

ACCEPTED MANUSCRIPT
a, Å
b, Å
c, Å
α, °
β, °
12.6743(7)
10.2407(6)
14.6595(9)
90
98.4280(10)
90
γ, °
3
V, Å
1882.16(19)
1.416
−3
Densitycalc, g·cm
−1
µ, cm
F(000)
θmax, °
8.22
832
2
57.99
Reflections collected
Independent reflections
Reflections with I>2σ(I)
Parameters
14116
4972
3690
245
R1 [I>2σ(I)]
0.0387
0.1062
1.028
wR2 [all reflections]
GOF
−3
Residual electron density, e·Å (ρmin/ρmax
)
0.544/-0.215
2
.4.Racemic resolution by HPLC
The following chiral columns (250 × 4.6 mm, 5 µm) were used: 3-AmyCoat, Chiracel OD, Chiracel
OJ. Chromatographic resolution was carried out on an HPLC system (Advanced Separation
Thechnologies, Inc., Whippany, NJ, USA), with a Bruker LC 31 instrument equipped with a UV detector
−
1
(
254 nm); the flow rate was 1.0 mL min at an ambient temperature.
2
.5. Cytotoxicity
Rat hepatoma HTC cells were cultured in the DMEM/F12 medium with addition of 10% fetal calf
serum (HyClone, USA), L-glutamine (PanEco, Russia) and gentamicin as antibiotic (PanEco, Russia).
5
The cells were seated on 96-well plates in concentration of 2×10 per well and cultured in a CO2
incubator at 37°C for 24 h. Then the solutions of 1N-(ferrocenylethyl)-4-nitro-imidazole (4b), 1N-
(
(
ferrocenylbutyl)-4-nitro-imidazole (4d), 1N-(ferrocenylethyl)-2-methyl-4-nitroimidazole (5b) and 1N-
ferrocenylpropyl)-2-methyl-4-nitroimiazole (5c) in increasing concentrations of 5.0, 10.0, 15.0, 20.0, and
2
5.0 µM were added to the cells. As a control was the solution of 0.68 M DMSO in water. After 2, 24 and
4
8 hours the viability was assessed by the MTT assay. All experiments were performed in triplicate at
each concentration level and repeated three times. The data have been given in terms of percent growth
inhibition relative to untreated controls (see Supplementary Materials).

ACCEPTED MANUSCRIPT
2
.6. Antitumor test
Carcinoma 755 (Ca755) was transplanted subcutaneously to the inbred mice BDF1
f1(C57Bl6×DBA2)/ males, a hybrid line of C57Bl6 females and DBA2 males, with weight 18–20 g, in
accordance with the standard procedure. The agents 4b and 1N-FcCH(CH )-Im were administered on the
/
3
−1
next day after tumor inoculation. The tested daily doses were 10.0 and 20.0 mg kg (total doses were
−1
5
0.0 and 100.0 mg kg ). Ethanol-water solutions (10% by volume) of compounds 4b and FcCH(CH )-Im
3
were administered intraperitoneally in daily doses (Table 3) five times every day starting from the next
day after tumor inoculation. Each group comprised five to seven animals, including the control group of
animals.
The kinetics of tumor growth was studied by measurement of tumor size during the whole period of
tumor development. Two cross-coupling tumor sizes were measured and the volume of the tumor was
2
calculated as V = ab /2, where a is the length and b is the width and the height of the tumor. As estimated
−
3
previously, the density of tumor tissue is equal to 1.0 g cm . So it is assumed that the weight of tumor in
3
grams is equal to the volume of tumor in cm . The index of tumor growth inhibition (TGI) was calculated
as (C − T)/C, %, where C and T are the mean tumor weight in groups of control and treated animals,
respectively.
3
.
Results and Discussion
3
.1.Synthesis
Imidazoles and benzimidazoles, being the central ingredients in many drugs, are often used
for chemical modifications by ferrocene for medicinal investigations [12,16,45,46].
Scheme 1. Synthesis of ferrocenylalkyl-4-nitro-imidazoles (4a–h, 5a–h) from ferrocenyl alcohols (1a–h)
and 4-nitro-imidazoles (2, 3).
A high effective approach to ferrocenyalkylated imidazole and benzimidazoles was realized
previously in neutral media using N,N'-carbonyldiimidazole or N,N'-thiobenzimidazoles as
nucleophilic agents [47,48]. Imidazole has the highest basicity among five-membered nitrogen-
containing heterocycles in living organisms with basic pK 6.98. Nitro-imidazoles have
a
significantly lower basicity than imidazole due to electron-withdrawing effect of the nitro-group.
So, we carried out the Fc-alkylation reaction of nitro-imidazoles through in situ generation of
+
thermodynamically stable ferrocenylcarbenium ions FcC H(R) by adding of strong fluoroboric

ACCEPTED MANUSCRIPT
acid and equimolar ratio of reagents avoiding protonation of heterocycles. An advantage of this
method is the simplicity, the accessibility of initial reagents and the possibility to vary the
lipophility of final products by changing ferrocenyl alcohols. Ferrocenylalkyl nitro-imidazoles
(4a–f, 5 a–f, Scheme 1) were synthesized via the reaction of 4-nitro-benzimidazole (2) or 2-
methyl-4-nitro-benzimidazole (3) with eight different ferrocenyl alcohols, FcCHR(OH) (1a-h,),
in methylene dichloride at room temperature in the presence of 45% aqueous fluoroboric acid
(with an equimolar ratio of ferrocenyl alcohol, nitro-imidazole and the acid). The products of the
reactions were isolated in satisfactory to good yields (43−87%) without column chromatography.
To prevent the oxidation of the final product during the work-up, ascorbic acid was added. Fc-
Alkylation appeared to be a regiospecific process and 1-N-ferrocenylalkyl nitro-imidazoles were
the only products formed, which were proved basing on NMR spectral data.
1
5
14
16
2
1
1
3
1
17 CH
18
3
1
0
2
9
8
4
N
N
1
1
7
6
1
Fe
20 19
5
NO
2
2
3
Figure 1. Fragment of HMBC spectrum of 1N-(ferrocenylbenzyl)-2-methyl-4-nitro-imidazole (5f).
1
The H NMR spectra of compounds 4a–f and 5 a–f, in CDCl shows several sets of signals
3
assigned, respectively, to the protons of the substituted and unsubstituted cyclopentadienyl rings,
to the CH(R)-bridge protons, their substituents R, and to the protons of nitro-imidazoles and their
1
3
methyl substituent. The assignments of the signals of the C NMR spectra of Fc-compounds
with 4-nitro-imidazole and 2-methyl-4-nitro-imidazole were based on HSQC spectra. The
1
13
1
13
structures of compounds were assigned on the basis of H and C NMR spectra and H/ C
heteronuclear correlations. Particularly in the HMBC spectrum of 1N-(ferrocenylbenzyl)-2-

ACCEPTED MANUSCRIPT
methyl-4-nitro-imidazole (5f) (Fig. 1), there are essential correlations between singlet at 6.14
ppm attributed to the CH linked to the ferrocene moiety (on Fig. 1 C-11), and unsubstituted C-
atom in imidazole (C-20, 119.15 ppm) and carbon atom at Me-group (C-18, 144.45 ppm) of 2-
methyl-4-nitro-imidazole. There are no correlations between CH-linked proton and carbon atom
bearing NO -group imidazole (C-19). Thus, ferrocenylalkylation proceeded in 1N-position of
2
nitro-imidazole ring and appeared to be regiospecific process. Regiospecifity of alkylation of the
others derivatives was proved in the same way.
3
.2.Crystal Structure
According to single crystal X-ray diffraction study the bond lengths in ferrocene and imidazole
moieties are very close to those in previously studied derivatives of α-(hydroxy)alkyl ferrocenes (see
caption to Fig. 2 and X-ray data for Fc-imidazoles [12]). Moreover, the nitro group in the heterocycle
does not cause any change in the bond length N1-C5 connecting imidazole and α-carbon atom. For 5f this
length is equaled 1.486(2) Å, for Fc-CH(Ph)Im − 1.487 Å [12].The most noticeable intermolecular
interactions in the crystal packing of 5f are weak the C-H…π interactions between ferrocene and
imidazole fragments. Nitro groups participated in weak C-H…O bonds with ferrocene groups and methyl
groups.
Figure 2. Molecular structure of 5f presented in thermal ellipsoids at 50% probability. Selected lengths,
(
Å) and angles (°), C5-C17 1.508(3); N1-C5 1.486(2); C5-C6 1.519(3); Fe1-C17 2.0310(17); N1-C1

ACCEPTED MANUSCRIPT
1
1
.312(3); N1-C3 1.354(2); N3-C2 1.356(3); N1-C5-C17 109.55(14); N1-C5-C6 111.04(15); C5-C17-C21
24.45(16); C6-C5-C17 113.46(15).
3
.3.Chiral resolutions by HPLC
In this section, racemic ferrocenylalkyl nitro-imidazoles were considered. Chiral centers in the
investigated compounds (4b–h and 5b–h) are represented by the carbon atom connecting a ferrocene
moiety with a heterocycle. These racemic mixtures were successfully separated into enantiomers on silica
packings modified by amylose as chiral selector, using the HPLC analytical method.
Earlier, this method of separation was initially applied for racemic ferrocene compounds carrying
various simple substituents [49]. The chiral sorbents based upon β- and γ-cyclodextrins turned out to be
effective in this case, as well as for the separation of ferrocene pyrazoles [36]. To separate mixtures of
racemic ferrocene derivatives having bulky substituents such as ferrocenylalkyl azoles [35,48] or
ferrocenylalkyl thiopyrimidines [32], modified cellulose was used as the chiral stationary phase. Columns
with amylose derivatives were used to separate into enantiomers ferrocene derivatives of mercaptoazoles
[
50].
The enantiomeric resolution analytical data are summarized in Table 2. We successfully separated all
4 pairs of investigated compounds. Some interesting conformities should be noted. First, retention
factors k' and k' for compounds 4b-4d and 5b-5d with aliphatic substituents R decrease with the
1
1
2
growing chain length of R. For example, for 4b and 4d from k' 10.47 and k' 13.52 to 6.16 and 10.31,
1
2
respectively; the same for derivatives with methyl group in the imidazole ring − from 4.90 and 6.34 for
b, and 3.73 and 4.07 for 5d. It should be marked that methyl group in the heterocycle makes retention
5
factors low. On the other hand, for phenyl containing compounds, vice versa, retention factors increase
monotonously on introduction of chlorine and iodine substituents into the phenyl ring. For example, for 5f
k' equals 3.33 and k' equals 4.93; for 5h 5.04 and 9.52, correspondingly. The most efficient HPLC
1
2
separation was achieved in the case of compounds 5j and 5h, with halogen-containing phenyl linkers, α =
2
.07 for 5j and α = 1.89 for 5h (Table 2).
The recognition mechanism on amylose is apparently connected, with the formation of specific
hydrogen bonds between the strongly basic nitrogen atom of the corresponding heterocyclic fragments
or/and nitro-groups and carbamate units of the modified amylose.
Table 2. Enantiomeric resolution of 4b-h and 5b-h racemic mixtures on column 3-Amy Coat.
(b)
HPLC data
(a)
Molecule and compound number
Retention
Retention Separation
factor, k'1
factor, k'2
factor, α
10.47
13.52
1.29
4b

ACCEPTED MANUSCRIPT
9.32
6.16
5.20
11.17
10.13
7.35
1.20
4
c
1.64
4d
1.41
4e
11.41
13.28
1.16
(c)
4f
7.15
8.03
1.12
4g
4
8.93
61.03
1.25
1.29
(d)
4h
4
.90
6.34
5b

ACCEPTED MANUSCRIPT
4.77
3.73
5.59
5.63
1.18
1.09
1.87
5
c
4.07
5d
10.46
5e
3.33
4.27
5.04
4.93
8.86
9.52
1.48
2.07
1.89
5
f
5g
5h
(
a)
(b)
(c)
∗
in the structures means the stereogenic center;
Mobile phase, hexane–isopropanol 9:1 (v/v);
For
For
(d)
compound 4f, experimental data were obtained on the modified cellulose, column Chiralcel OD;
compound 4h, experimental data were obtained on the modified cellulose, column Chiralcel OJ.
3
.4.Biological tests. Cytotoxicity and antitumor activity
Since the end of the 1990s, increased interest in the biochemistry of ferrocenes has emerged,
especially in the therapeutic areas of oncology [1,51]. At present, several significant review papers
devoted to antitumor activities of ferrocene compounds including ferrocene-modified nucleic bases,

ACCEPTED MANUSCRIPT
nitrogen heterocycles, aromatic substances, and drugs were published [3−6,9,10,15]. This problem was
carefully treated including mechanistic aspects [4,10,13]. According to recently published analysis, the
most probable mechanism of the anticancer action of ferrocene compounds is the initiation of tumor cell
apoptosis by protecting a telomere from the effect of telomerase and/or decreasing the telomerase activity
[
4]. Most likely, the development of therapeutic schemes for potential ferrocene-based drugs will
continue in the near future.
Herein, in sequel of our works [12,35,50,52] ferrocene-modified nitro-imidazoles were investigated.
Nitro-imidazole-based drugs are widely used in clinical and medicinal practices as anaerobic
antimicrobial agents, namely, tinidazole, metronidazole, dimetridazole, ornidazole, nimorazole,
secnidazole, satranidazol [53-55].
The exceptional role of imidazole derivatives in living systems is well known. They take part in the
essential biochemical processes, since the imidazole ring is an indispensable structural fragment of
nucleic acids, histidine and its decarboxylation product histamine.
At the same time, introduction of the ferrocene moiety in a biomolecule may change the normal route
of their interactions with biological targets, pharmacokinetics and pharmacodynamics or the direction of
binding [4,15]. Therefore, such approach in construction of new biologically active ferrocene-modified
molecules is in progress now [29,56-59]. However, in contrast to the rather well developed ferrocene-
based area, only limited representatives of ferrocene-containing nitro-imidazoles are known [46]. Thus,
the synthesis of new ferrocene-based nitro-imidazoles as potential drug candidates is of current interest.
3
.4.1. MTT assay. Viability of the tested compounds
The viability of four ferrocene compounds, namely, 1N-(ferrocenylethyl)-4-nitro-imidazole (4b), 1N-
(
(
ferrocenylbutyl)-4-nitro-imidazole (4d), 1N-(ferrocenylethyl)-2-methyl-4-nitroimidazole (5b) and 1N-
ferrocenylpropyl)-2-methyl-4-nitroimiazole (5c) was evaluated by the MTT assay in increasing
concentrations of 5.0, 10.0, 15.0, 20.0, and 25.0 µM. Rat hepatoma cell line (HTC) was used to study the
sensitivity to ferrocene nitro-imidazoles with different alkyl substituents R (Fig 1, from methyl for 4b and
5
b to ethyl for 5c and propyl for 4d).
Cytotoxicity (%) = As/Amc ×100%, where As − optical density of tested sample, Amc − mean optical
density of the control sample.
As shown in Table 4 (Supplementary materials), a distinct proliferative effect was observed for
compound 4b during the incubation of the rat hepatoma cells for 2 hours. However, this effect was not
observed for incubation times approaching 24 hours. Moreover, when the solution with a concentration of
2
5 µM was used, the cell viability decreased. Also it is noteworthy that the viability of rat hepatoma cells
decreased with the increasing of the incubation time, when compound 4b was used in concentrations of
2
0 µM and 25 µM.
Compound 4d in a concentration of 5 µM leads to a decrease in the viability of cells by 17.6% in the
rat hepatoma cell line HTC after a 2-hour cultivation. Increasing in the duration of culturing to 24 hours is

ACCEPTED MANUSCRIPT
accompanied by a decrease of the cell viability, when this compound 4d is added in an amount of 10−25
µM. A 48-hour incubation was not accompanied by a cytotoxic effect.
Compound 5b did not have any cytotoxic effect on the cells during the HTC culture incubation for 2,
2
4 and 48 hours at the investigated concentrations. In contrast, some stimulation of the cell proliferation
was observed during incubation with the compound in a concentration of 5 µM for 48 hours.
Incubation of the compound 5c with the rat hepatoma cells was not accompanied by cytotoxic effects
at the concentrations and time periods investigated. It should be noted that the increasing of proliferation
activity of the cells during the incubating of the compounds with the HTC cells for 2 and 48 hours and
concentration of 25 µM.
As a result, no cytotoxic effects of 1N-(ferrocenyl)nitro-imidazoles 5b and 5c on the culture of rat
hepatoma were found with the exception of compounds 4b and 4d at relatively high concentrations and
long incubation periods (a day or more). This opens perspectives for further detailed study of the specific
pharmacological properties of the compounds obtained.
3
.4.2. Antitumor activity
The antitumor effects of ferrocene-based thymine [31], benzimidazole [12], thiopyrimidine [32], and
thiobenzimidazole [50] against some solid tumor models such as Ca755 carcinoma (Ca755) and Lewis
lung carcinoma (LLC) transplanted in mice have been studied earlier. The index of tumor growth
inhibition as high as 95%, in comparison with control was achieved in some cases [32]. This effectiveness
was comparable with that of cisplatin. It was marked that solid tumor models, namely, Ca755 carcinoma
and Lewis lung carcinoma were considerably more sensitive to ferrocene compounds than ascite ones,
such as L1210 and P388 leukemia.
For the assessment of antitumor activity Ca755 carcinoma was used. The tested doses were 10.0 and
−
1
2
0.0 mg kg . The solutions were administered intraperitoneally. Tumor sizes were measured during the
whole period of tumor growth. The index of tumor growth inhibition was calculated at the time point
where the antitumor activity of the drug was maximal. This was after 16 days for 4b and after 12 days for
its analog without nitro-group, FcCH(CH )Im. The results of activity against the above-mentioned murine
3
tumor are summarized in Table 3. As seen from Table 3, carcinoma 755 is sensitive to both compounds.
−
1
An insignificant antitumor effect of compound 4b was shown on Ca755 at the dose of 10.0 mg kg .
Moreover, 4b and FcCH(CH )Im, as was proved experimentally, stimulated the growth of solid tumor
3
Ca755. Such an activity reversal phenomenon has not been observed before. This tumor model showed
that some imidazole‐based organometallics can exhibit stimulation effects.
To understand these unexpected facts we analyzed some biological results. Earlier, on solid tumor
models an inverse dose–effect response was found for ferrocenylmethyl benzimidazoles [12] (Lewis lung
−
1
carcinoma, dose 5.0 mg kg , tumor growth inhibition 70%), ortho-carboxybenzoyl ferrocene sodium salt
−
1
[
60] (Ca755, dose 2.5 mg kg , tumor growth inhibition 70%), 1N-ferrocenylmethyl thymine against
−
1
Ca755, dose 2.5 mg kg , tumor growth inhibition 70% [31], as well as for some other ferrocene
derivatives [11], that is, a decrease of the dose gave an increase of the effects. Then, it was suggested that

ACCEPTED MANUSCRIPT
the found dose–efficiency dependence – the achievement of the maximum antitumor effect after
application of the agent in a rather low doses – is typical for ferrocene compounds of this kind [31]. A
preliminary conclusion was being drawn: the anomalies in dose–effect response may be connected to the
increased immunogenicity of the ferrocene derivatives. A large dose causes enhanced immune response
and, as a consequence, the earlier destruction of the compound [31].
Table 3. Antitumor activity of 1N-(ferrocenylethyl)-4-nitroimidazole (4b) against Ca755 carcinoma in
vivo.
Adenocarcinoma 755
Daily
Compound
dose,
mg
−1
Mean
tumor
Tumor growth
kg
(a)
inhibition, %
weight, g
(
b)
FcCH(CH )Im
10.0
20.0
4.5±0.7
4.8±0.7
+50 (stimulation)
+60 (stimulation)
3
FcCH(CH )Im
3
Control
−
3.0±0.6
−
4
4
-NO FcCH(CH )Im (4b)
10.0
20.0
3.2±0.4
6.0±0.7
22
2
3
-NO FcCH(CH )Im (4b)
+46 (stimulation)
2
3
Control
−
4.1±0.6
−
Initial solvent, physiological solution-ethanol 90:10, percentage by volume;
drug administration, intraperitoneal; during five days after tumor inoculation.
(
a)
Evaluation of the index of tumor growth inhibition (%), day 14 after tumor Ca755 inoculation for
FcCH(CH )Im; day 16 after tumor Ca755 inoculation for 4b.
This compound was prepared according Reference [47].
3
(b)
Conclusion
A series of nontoxic ferrocene nitro-imidazoles have been easily obtained in good yields by reaction
of commercially or synthetically available ferrocene alcohols with nitro-imidazoles. X-ray structural data
for racemic (R,S)-1-N-(benzyl ferrocenyl)-2-methyl-4-nitroimidazole (5f) were determined. The resulting
enantiomers were resolved into enantiomers by analytical HPLC method. Cytotoxicity studies of (R,S)-
ferrocenyl(alkyl) nitro-imidazoles were realized. Antitumor activity tests were made for compound 4b.
DFT calculations and investigations into the unusual reactivity and biological properties of these
novel ferrocene derivatives are in progress now in our laboratories.
Supplementary data
Table 4. Cytotoxicity studies of ferrocenyl(alkyl) nitro-imidazoles 4b, 4d, 5b, and 5c.
Concentration, 4b
Time
5
µM
10 µM
15 µM
20 µM
25 µM
Rat hepatoma cells viability, %

ACCEPTED MANUSCRIPT
2
h
113.5
109.7
116.3
118.2
113.4
(
107.2; 117.8)*
(101.8; 117.9)*
(110.0; 125.3)*
(115.5; 122.7)*
(101.6; 117.8)
1
16.0
99.5
(84.9; 116.6)
85.7
(78.7; 96.5)#
85.7
(78.7; 96.5)#
79.2
(75.0; 96.8)*,#
2
4
4 h
8 h
(
102.2; 127.5)
102.8
94.6
87.4
57,2
61,9
(
89.9; 109.3)
(90.9; 99.3)
(71.4; 107.9)
(53.4; 72.0)*,##
(53.5; 74.1)*,##
Concentration, 4d
Time
5 µM
10 µM
15 µM
20 µM
25 µM
2
h
82.4
79.6; 101.1)*
94.8
99.9
(90.7; 104.6)
72.2
100.1
(90.8; 106.3)
83.5
108.9
(100.1; 116.4)
92.1
106.0
(86.0; 116.5)
71.1
(
2
4
4 h
8 h
(
72.2; 113.8)
(65.8; 83.9)*,#
(59.3; 92.1)*,#
(65.5; 94.0)*,#
(67.8; 83.5)*,#
94.6
100.9
94.9
105.6
76.7
(
84.5; 103.0)
(83.1; 108.9),## (84.4; 112.5)##
(80.1; 123.3)##
(68.9; 109.6)
Concentration, 5b
Time
5 µM
10 µM
15 µM
20 µM
25 µM
2
h
94.8
97.1
99.0
101.2
107.5
(
86.0; 98.3)
(87.5; 100.4)
(94.8; 101.1)
(95.5; 104.8)
(99.3; 113.1)
2
4
4 h
8 h
83.8
65.3; 99.5)
94.3
(91.7; 112.7)
95.1
(92.6; 111.0)
90.1
(71.5; 96.8)#
75.5
(66.0; 99.1)#
(
1
16.1
104.8
89.8; 112.6)
120.2
(103.0; 127.5)
104.5
(96.9; 139.9)##
109.7
(102.3; 114.6)##
(
103.7;
32.6)*,##
(
1
Concentration, 5c
Time
5 µM
10 µM
15 µM
20 µM
25 µM
2
h
103.8
102.0
103.9
102.2
118.6
(
(
93.7; 111.9)
(98.2; 111.0)
(89.6; 109.4)
(98.7; 113.4)
(116.4; 121.9)*
2
4
4 h
8 h
79.6
56.6; 113.9)
119.7
90.1
(79.1; 125.0)
95.7
97.3
(84.2; 117.2)
118.0
92.9
(69.3; 111.8)
110.3
85.4
(77.6; 93.1)#
121.9
(
99.4; 133.7)##
(80.0; 125.9)
(105.2; 125.2)
(99.7; 119.7)
(101.3; 131.1)*, ##
*
#
#
statistically valid differences in comparison with intact cells (р<0.05)
statistically valid differences in comparison with cells cultured for 2 hours (р<0.05)
# statistically valid differences in comparison with cells cultured for 24 hours (р<0.05)

ACCEPTED MANUSCRIPT
Tumor weight, g
7
6
5
4
3
2
1
0
3
1
2
7
9
11
13
15
17
Days postimplant
Figure 3. Antitumor activity of compound 4b against Са755 carcinoma
−1
−1
1
control, 2 10 mg kg per day , 3 20 mg kg per day
Intraperitoneal drug administration, days 1-5 postimplant

ACCEPTED MANUSCRIPT
Т/С,%
1
1
1
1
1
80
В
60
40
20
00
Са755
3
1
8
6
4
2
0
0
0
0
0
2
9
14
Days postimplant
Figure 4. Antitumor activity of compound FcCH(CH )Im against Ca755 carcinoma (B)
19
24
3
−1
−1
1
control, 2 10 mg kg per day , 3 20 mg kg per day
Oral drug administration, days 1−5 postimplant.
Acknowledgments: This work was supported by the Russian Academy of Sciences (Presidium Program
“
“
Fundamental Sciences for Medicine”), by the Department of Chemistry and Materials Science (Program
Medicinal Chemistry”) and by the Tomsk State University competitiveness improvement Program.
References
1
2
3
. G. Jaouen (Ed.). Bioorganometallics: Biomolecules, Labeling, Medicine; Wiley-VCH:
Weinheim, Germany, 2006.
. E.G. Perevalova, M.D. Reshetova, K.I. Grandberg, Ferrocene and Related Compounds; Nauka:
Moscow, Russia, 1983. (In Russian)
. L.V. Snegur, V.N. Babin, A.A. Simenel, Yu.S. Nekrasov, L.A. Ostrovskaya, N.S. Sergeeva,
Antitumor activities of ferrocene compounds. Russ. Chem. Bull., Int. Ed. 2010, 59(12), 2167-
2
178.
4
. V.N. Babin, Y.A. Belousov, V.I. Borisov, V.V. Gumenyuk, Y.S. Nekrasov, L.A. Ostrovskaya,
I.K. Sviridova, N.S. Sergeeva, A.A. Simenel, L.V. Snegur. Ferrocenes as potential anticancer
drugs. Facts and hypotheses. Russ. Chem. Bull. 2014, 63, 2405–2422.
5
. C. Ornelas, Application of ferrocene and its derivatives in cancer research. New J. Chem. 2011,
3
5, 1973–1985.
. G. Gasser, I. Ott, N. Metzler-Nolte. Organometallic anticancer compounds. J. Med. Chem. 2011,
4, 3–25.
6
5

ACCEPTED MANUSCRIPT
7
8
. G.M. Maguene, J. Jakhlal, M. Ladyman, A. Vallin, D.A. Ralambomanana, T. Bousquet,
J. Maugein, J. Lebibi, L. Pélinski, Synthesis and antimycobacterial activity of a series of
ferrocenyl derivatives. Eur. J. Med. Chem. 2011, 46, 31–38.
. V.N. Kulikov, R.S. Nikulin, A.N. Rodionov, E.S. Babusenko, V.N. Babin, L.V. Kovalenko,
Yu.A. Belousov. Synthesis and antimycobacterial activity of N-isonicotinoyl-N´-
alkylideneferrocenecarbohydrazides. Russ. Chem. Bull. 2017, 66(6), 1122–1125. DOI:
1
0.1007/s11172-017-1864-y
9
. F.A. Larik, A. Saeed, T.A. Fattah, U. Muqadar, P.A. Channar. Recent advances in the synthesis,
biological activities and various applications of ferrocene derivatives. Appl. Organomet. Chem.
2
017, 31(7), e3664. DOI: 10.1002/aoc.3664
1
0. E.W. Neuse. Macromolecular ferrocene compounds as cancer drug models. J. Inorg. Organomet.
Polym. Mater. 2005, 15, 3–31.
1
1. L.V. Popova, V.N. Babin, Y.A. Belousov, Y.S. Nekrasov, A.E. Snegireva, N.P. Borodina,
G.M. Shaposhnikova, O.B. Bychenko, P.M. Raevskii, N.B. Morozova, A.I. Ilyina, K.G. Shitkov,
Antitumor effects of binuclear ferrocene derivatives. Appl. Organomet. Chem. 1993, 7, 85–94.
2. L.V. Snegur, A.A. Simenel, Y.S. Nekrasov, E.A. Morozova, Z.A. Starikova, S.M. Peregudova,
Y.V. Kuzmenko, V.N. Babin, L.A. Ostrovskaya, N.V. Bluchterova, M.M. Fomina. Synthesis,
structure and redox potentials of biologically active ferrocenylalkyl azoles. J. Organomet. Chem.
1
2
004, 689, 2473–2479. https://doi.org/10.1016/j.jorganchem.2004.05.001
1
3. L.V. Snegur,; Y.S. Nekrasov, N.S. Sergeeva, Z.V. Zhilina, V.V. Gumenyuk, Z.A. Starikova,
A.A. Simenel, N.B. Morozova, I.K. Sviridova, V.N. Babin. Ferrocenylalkyl azoles: Bioactivity,
synthesis, structure. Appl. Organomet. Chem. 2008, 22, 139–147.
1
4. M.D. Joksovic, V. Markovic, Z.D. Juranic, T. Stanojkovic, L.S. Jovanovic, I.S. Damljanovic,
K.M. Szécsényi, N. Todorovic, K.M. Trifunovic, R.D. Vukicevi´c. Synthesis, characterization
and antitumor activity of novel N-substituted a-amino acids containing ferrocenyl pyrazole-
moiety. J. Organomet. Chem. 2009, 694, 3935–3942.
1
5. G. Jaouen, A. Vessières, S. Top. Ferrocifen type anticancer drugs. Chem. Soc. Rev. 2015, 44,
8
802–8817.
1
6. B. Narasimhan, D. Sharma, P. Kumar. Biological importance of imidazole nucleus in the new
millennium. Med. Chem. Res. 2011, 20(8), 1119−1140. DOI 10.1007/s00044-010-9472-5.
7. K. Kowalski. Ferrocenyl-nucleobase complexes: Synthesis, chemistry and applications. Coord.
Chem. Rev. 2016, 317, 132-156. http://dx.doi.org/10.1016/j.ccr.2016.02.008
1
1
8. J.L. Kedge, H.V. Nguyen, Z. Khan, L. Male, M.K. Ismail, H.V. Roberts, N.J. Hodges,
S.L. Horswell, Y. Mehellou, J.H.R. Tucker. Organometallic Nucleoside Analogues: Effect of
Hydroxyalkyl Linker Length on Cancer Cell Line Toxicity. Eur. J. Inorg. Chem. 2017, 466-476.
DOI: 10.1002/ejic.201600853
1
9. M. Hocek, P. Stepnicka, J. Ludvik, I. Cisarova, I. Votruba, D. Reha, P. Hobza. Chem. Eur. J.
Ferrocene-Modified Purines as Potential Electrochemical Markers: Synthesis, Crystal Structures,
Electrochemistry and Cytostatic Activity of (Ferrocenylethynyl)- and (Ferrocenylethyl)purines.
2
004, 10, 2058-2066. https://doi.org/10.1002/chem.200305621
2
0. J. Skiba, Q. Yuan, A. Hildebrandt, H. Lang, D. Trzybiński, K. Woźniak, R.K. Balogh,
B. Gyurcsik, V. Vrčekб K. Kowalski. Ferrocenyl GNA nucleosides: a bridge between organic
and organometallic xeno-nucleic acids. ChemPlusChem 2018, 83, 77-86.
http://dx.doi.org/10.1002/cplu.201700551
2
2
2
1. A.V. Sachivko, V.P. Tverdohlebov, I.V. Celinskii. Polynitrogen ferrocene derivatives. Russ.
Chem. J. 1997, XLI (2), 119-129.
2. V.I. Boev, L.V. Snegur, V.N. Babin, Y.S. Nekrasov. α-Metallocenylalkylation. Russ. Chem. Rev.
1
997, 66, 613–636.
3. L.V. Snegur, A.A. Simenel, A.N. Rodionov, V.I. Boev. Ferrocene modification of organic
compounds for medicinal applications. Russ. Chem. Bull. 2014, 63, 26-36.

ACCEPTED MANUSCRIPT
2
2
4. The Multifaced Chemistry of Ferrocene (Ed. P. Štepnička) Eur. J. Inorg. Chem. 2017, 212-526.
) K. Kowalski. Ferrocenyl-nucleobase complexes: Synthesis, chemistry and applications. Coord.
2
Chem. Rev. 2016, 317, 132-156.
5. D. Onyancha, V. Nyamori, C. W. McCleland, C. Imrie, T.I.A. Gerber. Solvent-free reactions of
N,N`-thiocarbonyldiimidazole with ferrocenylcarbinols. J. Organomet. Chem. 2009, 694, 207–
2
12.
2
2
6. B. Gharib, A. Hirsch. Synthesis and Characterization of New Ferrocene-Containing Ionic
Liquids. Eur. J. Org. Chem. 2014, 4123-4136. DOI: 10.1002/ejoc.201400061
7. E.V. Shevaldina, A.D. Shagina, V.N. Kalinin, A.B. Ponomaryov, A.F. Smol’yakov,
S.K. Moiseev. α-Ferrocenylalkyl carbonates: Reagents for ferrocenylalkylation reactions under
mild neutral conditions. J. Organomet. Chem. 2017, 836-837, 1-7. DOI:
1
0.1016/j.jorganchem.2017.02
2
8. I.A. Utepova, O.N. Chupakhin, P.O. Serebrennikova, A.A. Musikhina, V.N. Charushin. Two
Approaches in the Synthesis of Planar Chiral Azinylferrocenes. J. Org. Chem. 2014, 79, 8659-
8
667.
2
9. S. Realista, S. Quintal, P.N. Martinho, A.I. Melato, A. Gil, T. Esteves, M.de Deus Carvalho,
L.P. Ferreira, P.D. Vaz, M.J. Calhorda. Electrochemical studies and potential anticancer activity
in ferrocene
derivatives.
J.
Coord. Chem.
2017,
70(2), 314–327.
DOI:
org/10.1080/00958972.2016.1257125
3
0. K.Y. Zherebker, A.N. Rodionov, M.M. Il´in, A.A. Korlyukov, D.E. Arkhipov, Y.A. Belousov,
A.A. Simenel. Synthesis and properties of N-ferrocenylalkylated pyrroles. Russ. Chem. Bull., In.
Ed. 2014, 63(10), 2281—2284. https://doi.org/10.1007/s11172-014-0735-z
3
1. A.A. Simenel, E.A. Morozova, L.V. Snegur, S.I. Zykova, V.V. Kachala, L.A. Ostrovskaya,
N.V. Bluchterova, M.M. Fomina. Simple route to ferrocenylalkyl nucleobases. Antitumor activity
in vivo. Appl. Organomet. Chem. 2009, 23, 219–224.
3
3
2. A.A. Simenel, G.A. Dokuchaeva, L.V. Snegur,; A.N. Rodionov, M.M. Ilyin, S.I. Zykova,
L.A. Ostrovskaya, N.V. Bluchterova, M.M. Fomina, V.A. Rikova. Ferrocene-modified
thiopyrimidines: Synthesis, enantiomeric resolution, antitumor activity. Appl. Organomet. Chem.
2
011, 25, 70–75.
3. L.V. Snegur, S.I. Zykova, A.A. Simenel, Y.S. Nekrasov, Z.A. Starikova, S.M. Peregudova,
M.M. Ilyin, V.V. Kachala, I.K. Sviridova, N.S. Sergeeva, Redox-active ferrocene-modified
pyrimidines and adenine as antitumor agents: Structure, separation of enantiomers, and inhibition
of DNA synthesis in tumor cells. Russ. Chem. Bull. 2013, 62, 2056–2064.
3
3
3
4. A.N. Rodionov, L.V. Snegur, A.A. Simenel, Yu.V. Dobryakova, V.A. Markevich. Ferrocene-
modification of Amino Acids: Synthesis and in vivo Bioeffects on Hippocampus. Russ. Chem.
Bull. 2017, 66(1), 136-142.
5. L.V. Snegur, V.I. Boev, Y.S. Nekrasov, M.M. Ilyin, V.A. Davankov, Z.A.Starikova,
A.I. Yanovsky, A.F. Kolomiets, V.N. Babin, Synthesis and structure of biologically active
ferrocenylalkyl polyfluoro benzimidazoles. J. Organomet. Chem. 1999, 580, 26–35.
6. L.V. Snegur, Y.A. Borisov, Y.V. Kuzmenko, V.A. Davankov, M.M. Ilyin, M.M. Ilyin, Jr.,
D.E. Arhipov, A.A. Korlyukov, S.S. Kiselev, A.A. Simenel. Enantiomeric-Enriched Ferrocenes:
Synthesis, Chiral Resolution, and Mathematic Evaluation of CD-chiral Selector Energies with
Ferrocene-Conjugates. Molecules 2017, 22, 1410.
3
7. J.K. Lindsay, C.R. Hauser. Aminomethylation of ferrocene to form N,N-
dimethylaminomethylferrocene and its conversion to the corresponding alcohol and aldehyde. J.
Org. Chem. 1957, 22, 355-357.
3
3
4
4
8. M. Rausch, M. Vogel, H. Rosenberg. Derivatives of Ferrocene. II. Some Reduction Products of
Benzoylferrocene and 1,1`-Dibenzoylferrocene. J. Org. Chem. 1957, 22, 903-906.
9. F.S. Arimoto, A.C. Haven, Jr. Derivatives of Dicyclopentadienyliron. J. Am. Chem. Soc. 1955,
7
7, 6295–6297. DOI: 10.1021/ja01628a068
0. М.V. Lyapunova, S.I. Belikh, V.S. Mal´kov. Method of obtaining of 4(5)-nitroimidazole. RU
Patent 2610267, 16 February 2016. http://www.findpatent.ru/patent/261/2610267.html
1. М.V. Lyapunova, V.S. Malkov, N.Y. Selikhova.The method of nitration of 2-methylimidazole.
RU Patent 2526125. 29 March 2013. http://www.findpatent.ru/patent/252/2523125.html

ACCEPTED MANUSCRIPT
4
2. G.M. Sheldrick, A short history of SHELX. Acta Crystallogr. Sect. A 2008, A64, 112-122.
https://doi.org/10.1107/S0108767307043930
4
3. G.M. Sheldrick. Acta Crystallogr. 2015, C71, 3-8.
4
4. O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H.J. Puschmann. OLEX2: a
complete structure solution, refinement and analysis program. J. Appl. Cryst. 2009, 42, 339-341.
https://doi.org/10.1107/S0021889808042726
4
4
5. S. Quintal, M.C. Gimeno, A. Laguna, M.J. Calhorda. Silver(I) and copper(I) complexes with
ferrocenyl ligands bearing imidazole or pyridyl substituents. J. Organomet. Chem. 2010, 695(4),
5
58-566. https://DOI.10.1016/j.jorganchem.2009.11.013.
6. P. Toro, A.H. Klahn, B. Pradines, F. Lahoz, A. Pascual, C. Biot, R. Arancibia. Organometallic
benzimidazoles: Synthesis, characterization and antimalarial activity. Inorg. Chem. Commun.
2
013, 35, 126–129.
4
4
4
7. A.A. Simenel, E.A. Morozova, Yu.V. Kuzmenko, L.V. Snegur. Simple route to
ferrocenyl(alkyl)imidazoles. J. Organomet. Chem. 2003, 665, 13-14.
8. A.A. Simenel, Yu.V. Kuzmenko, E.A. Morozova, M.M. Ilyin, I.F. Gúnko, L.V. Snegur. J.
Organomet. Chem. 2003, 688, 138-143.
9. D.W. Armstrong, W. DeMond, B.P. Czech. Separation of metallocene enentiomers by liquid
chromatography: Chiral recognition via cyclodextrin bonded phases. Anal. Chem. 1985, 57, 481–
4
84. DOI: 10.1021/ac50001a037
5
0. A.N. Rodionov, K.Ya. Zherebker, L.V. Snegur, A.A. Korlyukov, D.E. Arhipov, A.S. Peregudov,
M.M. Ilyin, M.M. Ilyin, Jr., O.M. Nikitin, N.B. Morozova, A.A. Simenel. Synthesis, structure and
enantiomeric resolution of ferrocenylalkyl mercaptoazoles. Antitumor activity in vivo. J.
Organomet. Chem. 2015, 783, 83-91. https://doi.org/10.1016/j.jorganchem.2015.01.031
2
6
5
5
1. X. Wu, M.L. Go. Fe The Use of Iron-Based Drugs in Medicine, in the book, in: M. Gielen,
E.R.T. Tiekink (Eds.). Metallotherapeutic Drugs and Metal-based Diagnostic Agents: the Use of
Metals in Medicine. John Wiley & Sons, Ltd, 2005.
2. М.V. Lyapunova, V.S. Malkov. Experimental Comparison of 2-methylimidazole Nitration by
Nitric Acid and Nitrate Salts of Alkali Metals. Adv. Mater. Res. 2015, 1085, 143-147. DOI:
1
0.4028/www.scientific.net/AMR.1085.143.
5
3. https://www.vidal.ru/drugs
5
4. A. Mital. Synthetic Nitroimidazoles: Biologacal Activities and Mutagenicity. Sci. Pharm. 2009,
7
7, 497−520. http://dx.doi.org/10.3797/scipharm.0907-14
5
5
5
5
5
6
5. K.C. Lamp, C.D. Freeman, N.E. Klutman, M.K. Lacy, Pharmacokinetics and pharmacodynamics
of the nitroimidazole antimicrobials. Clin Pharmacokinetic. 1999, 36(5), 353-73.
DOI:10.2165/00003088-199936050-00004
6. H. Parveen, M.A. Alsharif, M.I. Alahmdi, S. Mukhtar, A. Azam, Novel Pyrimidine‐based
Ferrocenyl substituted Organometallic Compounds: Synthesis, Characterization and Biological
Evaluation. Appl. Organomet. Chem. 2018, e4261. https://doi.org/10.1002/aoc.4261
7. F. Asghar, S. Fatima, S. Rana, A. Badshah, I. Butler, N. Tahir. Synthesis, spectroscopic
investigation, and DFT study of N,N′-disubstituted ferrocene-based thiourea complexes as potent
anticancer agents. Dalton Transactions, 2017, DOI: 10.1039/C7DT04090C
8. A.N. Rodionov, M.D. Gerasimova, E.Yu. Osipova, A.A. Korlyukov; A.S. Peregudov;
A.A. Simenel. Synthesis of bis-ferrocenylpyrazoles via ferrocenylalkylation reaction. Monatsh.
Chem. 2017, 148, 925-932. https://doi.org/10.1007/s00706-016-1895-3
9. E.Y. Osipova, A. S. Ivanova, A. N. Rodionov, A. A. Korlyukov, D. E. Arkhipov, A. A. Simenel,
Ferrocenylalkylation of 2-mercaptobenzoxazoles. Russ. Chem. Bull. 2016, 65, 2868-2872.
https://doi.org/10.1007/s11172-016-1670-y
0. A.A. Simenel, S.V. Samarina, L.V. Snegur, Z.A. Starikova, L.A. Ostrovskaya, N.V. Bluchterova,
M.M. Fomina, o-Carboxybenzoylferrocene. Bioactivity and chemical Modifications. Appl.
Organomet. Chem. 2008, 22, 276–280.

ACCEPTED MANUSCRIPT
Highlights (for review)
Synthesis of ferrocenylalkyl nitro-imidazoles, racemic resolution, X-ray structure and bio effects in vitro
and in vivo