Synthesis and pharmacological activity of N-hetaryl-3(5)-nitropyridines

Article abstract of DOI:10.1134/S1068162015030048

Abstract Previously undescribed 2-, 4or 6-substituted hetaryl-3(5)-nitropyridines were synthesized by the interaction of a number of chlorosubstituted 3(5)-nitropyridines with some diazoles or 3-chloropyridazin-6one. In addition, pyrazolyl-3-nitropyridines were prepared by both the above method and cyclization of hydrazinopyridines, which, in turn, were synthesized by the treatment of chlorosubstituted 3-nitropyridines with hydrazine. It has been shown that these compounds have a moderate antibacterial activity against some pathogenic Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli) and a strong protistocidal effect on protozoa species Colpoda steinii surpassing in this respect clinically used reference drugs.

Full text of DOI:10.1134/S1068162015030048

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 4, pp. 402–408. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © A.I. Klimenko, L.N. Divaeva, A.A. Zubenko, A.S. Morkovnik, L.N. Fetisov, A.N. Bodryakov, 2015, published in Bioorganicheskaya Khimiya, 2015,
Vol. 41, No. 4, pp. 454–461.
Synthesis and Pharmacological Activity
of NꢀHetarylꢀ3(5)ꢀNitropyridines
, 1
A. I. Klimenkoc, L. N. Divaevaa , A. A. Zubenkob, A. S. Morkovnika,
L. N. Fetisovb, and A. N. Bodryakovb
aInstitute of Organic and Physical Chemistry, South Federal University,
pr. Stachky 194/2, RostovꢀonꢀDon, 344090 Russia
bNorthꢀCaucasian Zonal Scientific Research Veterinary Institute, Novocherkassk, Russia
cDon State Agrarian University, Novocherkassk, Russia
Received October 31, 2014; in final form, December 9, 2014
Abstract—Previously undescribed 2ꢀ, 4ꢀ or 6ꢀsubstituted hetarylꢀ3(5)ꢀnitropyridines were synthesized by the
interaction of a number of chlorosubstituted 3(5)ꢀnitropyridines with some diazoles or 3ꢀchloropyridazinꢀ6ꢀ
one. In addition, pyrazolylꢀ3ꢀnitropyridines were prepared by both the above method and cyclization of
hydrazinopyridines, which, in turn, were synthesized by the treatment of chlorosubstituted 3ꢀnitropyridines
with hydrazine. It has been shown that these compounds have a moderate antibacterial activity against some
pathogenic Gramꢀpositive and Gramꢀnegative bacteria (Staphylococcus aureus and Escherichia coli) and a
strong protistocidal effect on protozoa species Colpoda steinii surpassing in this respect clinically used referꢀ
ence drugs.
Keywords: Nꢀhetarylꢀ3(5)ꢀnitropyridines, synthesis, hetarylation, nucleophilic substitution, cyclization, reduction,
antimicrobial activity, Staphylococcus aureus, Escherichia coli, protistocidal activity, Colpoda steinii
DOI: 10.1134/S1068162015030048
1
INTRODUCTION
affect significant pathogens, such as Eimeria tenella. It
was shown over the last years that some complex subꢀ
stituted derivatives of imidazo[1,2ꢀa]pyridine, which
The creation of new natural and synthetic antiꢀ
infectious drugs is known to be one of the urgent probꢀ
lems of modern science because of, first of all, the wide
spread of soꢀcalled resistant infections (bacterial, proꢀ
tozoal, viral, etc.) insensitive to drugs, which is one of
the greatest threats to human health [1–5]. Multiresisꢀ
tant bacterial infection is of particular concern
because it is the reason for an annual death of about
25000 patients, even in the European Union. The
analogous problems are also typical for veterinary
medicine due in particular to the spread of resistant
forms of coccidia (parasitic unicellular protozoa speꢀ
cies of the Eimeria class), which cause Coccidiosis
epidemics and mass death of animals, birds, and fish
[6–8].
combine annelated imidazole and pyridine cycles in
their structure, were especially strong coccidiostatics.
These compounds were demonstrated to inhibit
cGMPꢀdependent protein kinase (PKG) vital to the
micropathogen [20, 21]. The most active among them
are, however, potentially genotoxic [22, 23] and have
relatively narrow range of actions [24]. Imidazo[1,2ꢀ
a]pyridines without these disadvantages are not very
active in vivo; they are only a few times more potent
than coccidiostatic salinomycin [25].
In this work, we continued the search of antiꢀinfecꢀ
tious preparations among nitrogen heterocycles and
synthesized different previously unknown
3)5ꢀnitro(amino)pyridines (II)–(VIII), (
ing the imidazoleꢀ1ꢀyl, benzimidazolꢀ1ꢀyl, pyrazolꢀ1ꢀyl,
or 3ꢀchloropyridazinꢀ6ꢀonꢀ2ꢀyl group as the ꢀhetaryl
substituent, and studied the antibacterial and antiꢀ
coccidal activities of these ꢀbihetaryls.
Nꢀhetarylꢀ
X) containꢀ
Different heterocyclic compounds, particularly,
imidazole, benzimidazole, and pyridine are of great
interest in the search for new antimicrobial and antiꢀ
protozoal drugs. For example, compounds containing
ureide [9] or aryloxyethyl [10–13] substituents, active
against Gramꢀpositive bacteria, have been recently
found among benzimidazoles. It is also known that
some derivatives of imidazole [14] and nitropyridines
[15–19] have a pronounced coccidiostatic activity and
N
N,C
RESULTS AND DISCUSSION
The initial compounds for the synthesis of
hetarylpyridines were isomeric chloroꢀ3(5)ꢀnitropyꢀ
ridines. Their chlorine atom was subjected to the
1
Corresponding author: phone: +7 (863) 2975196; eꢀmail:
divaevaln@mail.ru.
nucleophilic substitution by the
Nꢀhetaryl group
402

SYNTHESIS AND PHARMACOLOGICAL ACTIVITY
403
under the action of
Nꢀanions, which were generated the twoꢀstep method. The first step was the substituꢀ
from corresponding 1,2ꢀ and 1,3ꢀdiazoles or 3ꢀchloꢀ
ropyridazinꢀ6ꢀone in the presence of bases, such as
K2CO3 (method A) or NaH (method B). The reaction
occurs in DMF with yields ranging from 65 to 90%
under mild conditions.
tion of the chlorine atom in 2ꢀchloroꢀ or 4ꢀchloroꢀ3ꢀ
nitropyridine (Ia or Ib, respectively) by the hydrazine
group and the second step was cyclization of hydrazine
derivatives (IXa, b) with acetylacetone, with pyrazole
ring closure being occurred in the presence of catalytic
amounts of acetic acid. Both steps proceed very easily
in ethanol with high yields (Scheme 1).
Thus, 2ꢀhetarylꢀ3ꢀnitropyridines
were obtained from 3ꢀnitroꢀ2ꢀchloropyridine (Ia
and 4ꢀhetarylꢀ3ꢀnitroꢀ and 2ꢀhetarylꢀ5ꢀnitropyꢀ
(
IIa)–(IVa
)
)
5ꢀAminoꢀ2ꢀ(4,5ꢀdichloroimidazolꢀ1ꢀyl)ꢀpyridine
ridines (IIb, c), (IIIc)–(VIIIc), from 4ꢀchloroꢀ3ꢀ
nitroꢀ and 2ꢀchloroꢀ5ꢀnitropyridines
(Scheme 1).
(
Ib
,
c
)
(
XI) was synthesized by reduction of the nitro group in
2ꢀ(4,5ꢀdichloroimidazolꢀ1ꢀyl)ꢀnitropyridine IIc
with metallic iron in an ethanolꢀHCl mixture
thesis of pyrazolyl nitropyridines was carried out by (Scheme 1).
(
)
In addition to the above oneꢀstep method, the synꢀ
H2N
NO2
NO2
Het
+
HetH
Fe/H
for (IIc
)
Cl
N
N
N
N
N
Cl
(
(
IIа–c), (IIIа, c
IVа, c), (Vc)–(VIIIc
)
(
Iа–c
)
(XI)
Cl
)
NH NH
2
2
NO2
NHNH2
NO2
N
CH COCH COCH
3
2
3
N
N
N
CH3
H3C
(
IXа
,
b
)
(Xа
,
b
)
(
Ia): 2ꢀCl; (Ib): 4ꢀCl; (Ic): 6ꢀCl
Het: (IIа ): 4,5ꢀdichloroimidazolꢀ1ꢀyl; (IIIа
imidazolꢀ1ꢀyl; (VIc 3,5ꢀdimethylꢀ4ꢀchloropyrazolꢀ1ꢀyl; (VIIc
VIIIc 3ꢀchloropyridzinꢀ6ꢀonꢀ1ꢀyl; (Xа, b 3,5ꢀdimethylpyrazolꢀ1ꢀyl
–
c
,
c
)
4ꢀchloropyrazolꢀ1ꢀyl; (IVа
,
c
)
4ꢀbromopyrazolꢀ1ꢀyl;
(
(
Vc)
)
) benzimidazolꢀ1ꢀyl;
)
)
Scheme 1. Synthesis of 2ꢀ, 4ꢀ, and 6ꢀhetarylꢀsubstituted derivatives of 3(5)ꢀnitro(amino)ꢀpyridine.
The structure of the resultant hetarylpyridines was hetaryl fragments with the nitro group, if it is in an adjaꢀ
confirmed by 1H NMR and IR spectroscopy and eleꢀ cent position. These factors can significantly impede
the conjugation of these groups with the pyridine sysꢀ
tem. The reduction of nitrocompound (IIc) to aminoꢀ
derivative (XI) due to the strong influence of the
amino group leads to a significant enhancement of the
shielding of protons and the pronounced shift of NMR
ment analysis. Taking into account these data and the
synthetic methods used, the structure does not give
rise to any doubt. Chemical shifts of 2ꢀ, 4ꢀ, and 6ꢀproꢀ
tons in the pyridine ring of these compounds strongly
depends on the electronꢀacceptor properties of the
hetaryl substituent and its position and vary in ranges
of 8.69–9.43, 7.62–8.80, and 8.01–8.85 ppm, respecꢀ
tively. As can be expected, the compounds containing
especially strong electronꢀaccepting hetaryls, i.e.,
benzimidazoleꢀ2ꢀyl and 3ꢀchloropyridazinꢀ6ꢀonꢀ1ꢀ
yl, have the maximal shift values. Chemical shifts of
protons of the hetaryl groups often significantly
exceed those observed for similar protons in correꢀ
signals in a strong field, i.e., in the region of
δ ≤
7.85 ppm. In this case, the NMR spectrum contains
the additional signal of the primary amino group in the
form of a twoꢀproton singlet at 5.57 ppm, and the IR
spectrum contains the corresponding two strongly
broadened and significantly overlapping bands of symꢀ
metric and asymmetric bands of stretching vibrations
of the NH bonds at 3295 and 3410 cm–1.
sponding
Nꢀunsubstituted diazoles and 3ꢀchloropyꢀ
The biological properties of synthesized
Nꢀhetarylꢀ
ridazinꢀ6ꢀone. This is, obviously, due to the cumulaꢀ
tive and overall deshielding influence of some factors,
such as high electronegativity of the present 3(5)ꢀnitroꢀ
3(5)ꢀnitropyridines (II)–(VIII), and ( ) were studied
with on example of their antimicrobial and protisꢀ
tocidal activity. The antimicrobial activity was studied
X
pyridyl groups, the anisotropic effect of the lone pair of in test cultures of two standard bacterial strains Staꢀ
their ring nitrogen atom, and steric interactions of the phylococcus aureus Pꢀ209 and Escherichia coli 078
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015

404
KLIMENKO et al.
ꢀhetaryl)ꢀ3(5)ꢀnitropyridines
Table 1. Antibacterial and protistocidal activity of (
N
Minimal inhibiting concentration (MIC), µg/mL
Compound
E. coli 078
(field strain)
S. aureus Pꢀ209
C. steinii
(
(
(
(
(
(
(
(
(
(
(
(
(
(
IIa
IIb
)
)
100
100
100
500
500
500
500
500
500
100
100
500
100
100
–
100
100
100
500
500
500
500
500
500
100
500
500
100
100
–
31.25
62.5
15.62
125
500
500
500
500
500
500
500
500
500
62.5
–
IIc
IIIa
IIIc
IVa
IVc
Vc
VIc
VIIc
VIIIc
)
)
)
)
)
)
)
)
)
Xa
Xb
)
)
XI)
Bicocs
Ciprofloxacin
1
10
–
(field strain), which were usually used for testing the
Thus, the results demonstrate that among a numꢀ
antimicrobial activity of hetarylnitropyridines. Ciproꢀ ber of studied hetarylꢀ3(5)ꢀnitro(amino)pyridines, the
floxacin was used as the reference antibacterial agent.
The cultures were grown for a day at 37 on a Luriaꢀ
Bertani (LB) agar nutrient medium by the method of
compounds containing the imidazolyl (as the hetaryl)
group and having the protistocidal activity may be of
practical interest. Their structure should be optiꢀ
mized, and after achieving the required activity,
advanced toxicological studies should be performed.
°
C
twoꢀfold serial dilutions.
The protistocidal activity was studied in vitro
against ciliates Colpoda steinii (field isolate) by the
serial dilution method.
EXPERIMENTAL
The results presented in Table 1 show that 2ꢀ, 4ꢀ,
and 6ꢀhetarylꢀsubstituted nitropyridines exhibit rather
weak, a little variable, and usually almost equal antiꢀ
bacterial activity against Staphylococcus aureus and
Escherichia coli. At the same time, the considered
compounds possess the protistocidal activity, which
varies much more widely depending on their structure.
This activity is quite strong for pyridines containing
the 4,5ꢀdichloroimidazolꢀ1ꢀyl group, i.e., nitroderivꢀ
We used commercial reactants from Fluka (Gerꢀ
many) and AldrichꢀSigma (United States), and solꢀ
vents purified by standard methods.
1H NMR spectra of the synthesized bihetaryls were
recorded on a Varian Unityꢀ300 spectrometer
(300 MHz) (United States) in DMSOꢀd6; in case of
compound (IIа), the spectrum was recorded in
atives (IIa
pound (IIc) having the strongest effect (MIC =
15.6 g/mL).
Imidazolyl nitropyridines (IIa–c) were compared
–c) and aminoderivative (XI), with comꢀ
CDCl3. Chemical shifts of protons ( , ppm) are meaꢀ
δ
sured relative to the residual proton signals of deuterꢀ
ated dimethylsulfoxide and chloroform (2.49 and
7.25 ppm, respectively). IR spectrum was recorded on
the spectrometer “Varian Excalibur 3100 FTꢀIR” in
powder by the method of frustrated total internal
reflection. All melting points were determined on a
FisherꢀJohns melting point apparatus. (Fisher Scienꢀ
tific, United States). The element analysis was perꢀ
formed by the classical microanalytical method [26].
The reactions and the purity of the synthesized comꢀ
pounds were monitored by TLC (Al2O3 plates,
III degree of activity; eluent, CHCl3; visualization of
spots with iodine vapor in a humid chamber).
μ
with the best out of currently used coccidiostatics
(Baycox, Bayer AG, and amprolium), as well as with
furazolidon, sulfadimethoxine, trichopol, and ciprofꢀ
loxacin (Table 2), which were inactive in the studied
range of concentrations (<1000
μ
g/mL). The comparꢀ
ison of compounds (IIa ) with the two aboveꢀmenꢀ
–c
tioned specialized and equally effective coccidiostatic
drugs allows one to conclude that bihetaryl (IIa) is not
inferior to them in activity, whereas its isomers (IIb
)
and (IIc) show 2–4 times higher activity.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015

SYNTHESIS AND PHARMACOLOGICAL ACTIVITY
405
Table 2. Comparison of protistocidal activity of most active hetarylꢀ3(5)ꢀnitropyridines (IIa–c) and clinically used drugs
Accounted concentration of preparation, µg/mL
Preparation
1000
500
250
125
62.5
31.25
15.62
0.78
0.39
(
(
(
IIa
)
)
+
+
+
–
–
–
–
+
+
–
+
+
+
–
–
–
–
+
+
–
+
+
+
–
–
–
–
+
+
–
+
+
+
–
–
–
–
+
+
–
+
+
+
–
–
–
–
+
+
–
–
+
+
–
–
–
–
–
–
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
IIb
IIc)
Furazolidon
Sulfadimethoxine
Trihopol
Ciprofloxacin
Bicocs (toltrazuril)
Amprolium
Water
Designations: (+), all protozoa died; (–) all were alive.
General procedure for the synthesis of hetarylꢀ3(5)ꢀ a yield of 50%. M.p., 106–108
nitropyridines (IIa–c), (IIIa,c), (IVa,c), (Vc)–(VIIIc). spectrum: 8.01 (1 H, d, 1 5.1, H5), 8.22 (1 H, s, H2'),
Method A. A mixture of corresponding chloronitropyꢀ 9.19 (1 H, d, 5.1, H6), 9.51 (1 H, s, H2). Found, %:
ridine (Ia ) (1.59 g, 10 mmol), diazole or 3ꢀchloroꢀ C 36.63; H 1.40; Cl 27.05; N 21.33. С8H4Cl2N4O2
pyridazinꢀ6ꢀone (10 mmol), and powdered K2CO3 Calculated, %: C 37.09; H 1.56; Cl 27.37; N 21.63.
°
C
(from CCl4). 1H NMR
J
J
–c
.
(4.14 g) was thoroughly stirred for several hours at a
temperature from 45 to 65 (the exact time and temꢀ
perature values are shown for the individual comꢀ
pound). The mixture was then cooled, poured into
100 mL of H2O, and the formed precipitate was filꢀ
tered off and washed with water.
2ꢀ(4,5ꢀDichloroimidazolꢀ1ꢀyl)ꢀ3ꢀnitropyridine (IIc)
°C
was synthesized from 2ꢀchloroꢀ5ꢀnitropyridin and
4,5ꢀdichloroimidazole by method A at 50–55 C for
°
4 h in a yield of 85%. M.p., 116–118
°
C
(from AcOEt).
9.0, H3), 8.34
1H NMR spectrum: 8.06 (1 H, d,
J
(1 H, s, H2'), 8.85 (1 H, dd, J1 9.0, J2 3.0, H4), 9.27
(1 H, d, 2.7, H6). Found, %: C 36.71; H 1.31;
J
Method B. Solution of NHꢀheterocycle (10 mmol)
Cl 27.03; N 21.40. С8H4Cl2N4O2. Calculated, %:
C 37.09; H 1.56; Cl 27.37; N 21.63.
3ꢀNitroꢀ2ꢀ(4ꢀchloropyrazolꢀ1ꢀyl)pyridine (IIIa)
was synthesized by method B from 2ꢀchloroꢀ3ꢀnitroꢀ
in dry DMF (12 mL) was cooled to
0°C, followed by
the portionwise addition of NaH (2.9 mg, 12 mmol)
under stirring. The mixture was stirred for 15 min at
0°C, and corresponding chloronitropyridine (1.59 g,
10 mmol) was portionwise added to the mixture. After
30 min of stirring, the mixture was brought to room
temperature, treated with water (100 mL), and the
formed precipitate of hetarylꢀ3(5)ꢀnitropyridine was
filtered off.
pyridine and 4ꢀchloropyrazol in a yield of 65%. M.p.,
1
116–118
°
C
(from EtOH). H NMR spectrum: 7.62
(1 H, dd, J1 8.1, J2 4.8, H5), 7.76 (1 H, s, H5'), 8.38
(1 H, dd, J1 8.1, J2 1.5, H4), 8.55 (1 H, s, H3'), 8.67
(1 H, dd, J1 4.8, J2 1.5, H6). Found, %: 42.55; H 2.01;
Cl 15.50; N 24.65. С8H5ClN4O2. Calculated, %:
C 42.78; H 2.24; Cl 15.78; N 24.94.
5ꢀNitroꢀ2ꢀ(4ꢀchloropyrazolꢀ1ꢀyl)pyridine (IIIc)
was synthesized by method B from 2ꢀchloroꢀ5ꢀnitroꢀ
2ꢀ(4,5ꢀDichloroimidazolꢀ1ꢀyl)ꢀ3ꢀnitropyridine (IIa)
was synthesized from 2ꢀchloroꢀ3ꢀnitropyridine and
4,5ꢀdichloroimidazole by method A. The reaction was
carried out at 50–55
°
C for 5 h. Yield, 75%; M.p.,
1
pyridine and 4ꢀchloropyrazol in a yield of 70%. M.p.,
114–116 (from AcOEt). H NMR spectrum: 7.75
°
C
1
147–149
(1 H, s, H5'), 8.14 (1H, d,
H3'), 8.74 (1 H, dd, J1 9.0, J2 2.7, H4) 9.23 (1 H, d,
2.4, H6). Found, %: C 42.48; H 2.12; Cl 15.49;
24.71. С8H5ClN4O2. Calculated, %: C 42.78;
°
C
(from AcOEt). H NMR spectrum: 7.85
2
(1 H, dd, J1 8.1, J2 4.8, H5), 7.79 (1 H, s, H2' ), 8.58
J
9.0, H3), 8.71 (1 H, s,
(1 H, dd, J1 8.4, J2 1.5, H4), 8.88 (1 H, dd, J1 4.8,
J2 1.5, H6). Found, %: C 36.88; H 1.49; Cl 27.00;
J
N
N
21.40. С8H4Cl2N4O2. Calculated, %: 37.09; H 1.56;
H 2.24; Cl 15.78; N 24.94.
Cl 27.37; N 21.63.
2ꢀ(4ꢀBromopyrazolꢀ1ꢀyl)ꢀ3ꢀnitropyridine (IVa) was
4ꢀ(4,5ꢀDichloroimidazolꢀ1ꢀyl)ꢀ3ꢀnitropyridin (IIb)
synthesized by method B from 2ꢀchloroꢀ3ꢀnitropyriꢀ
was synthesized from 4ꢀchloroꢀ3ꢀnitropyridin and
dine and 4ꢀbromopyrazole in a yield of 70%. M.p.,
4,5ꢀdichloroimidazole by method A at 45 C for 3 h in
°
1
129–130
°
C
(from EtOH). H NMR spectrum: 7.63
2
(1 H, dd, J1 7.8, J2 4.8, H5), 7.72 (1 H, s, H5'), 8.41
(1 H, dd, J1 8.1, J2 1.5, H4), 8.57 (1 H, s, H3'), 8.69
Here and below, the numbers with a prime indicate position of
protons of nonpyridine hetaryl substitient.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015

406
KLIMENKO et al.
(1 H, dd, J1 4.8, J2 1.5, H6). Found, %: C 35.60; filtered and thoroughly dried. The mixture of resultant
) (1.54 g, 10 mmol), acetylacetone
H 1.50; Br 29.46; N 20.65. С8H5BrN4O2. Calculated, %: compound (IXa, b
C 35.71; H 1.87; Br 29.70; N 20.85.
2ꢀ(4ꢀBromopyrazolꢀ1ꢀyl)ꢀ5ꢀnitropyridine (IVc) was
synthesized by method B from 2ꢀchloroꢀ5ꢀnitropyriꢀ
(1.51 mg, 15 mmol), and acetic acid (0.1 mL) in
20 mL of EtOH was boiled for 3 h and cooled, followed
by the addition of water (100 mL). The formed precipiꢀ
tate of pyrazolylꢀ3ꢀnitropyridine was filtered off.
dine and 4ꢀbromopyrazole in a yield of 90%. M.p.,
1
152–154
(1 H, d,
J1 9.0,
2.4, N6). Found, %: C 35.52; H 1.36; Br 29.61;
20.61. С8H5BrN4O2. Calculated, %: C 35.71;
°C
(from EtOH). H NMR spectrum: 8.09
2ꢀ(3,5ꢀDimethylpyrazolꢀ1ꢀyl)ꢀ3ꢀnitropyridine (Xa)
J
9.3, H3), 8.11 (1 H, s, H5'), 8.76 (1 H, dd,
was synthesized from 3ꢀnitroꢀ2ꢀchloropyridine by the
J
2 2.7, H4), 8.91 (1 H, s, H3'), 9.27 (1 H, d,
above method in a yield of 90%. M.p., 107–109 C
°
1
J
N
(from AcOEt). H NMR spectrum: 2.10 (3 H, s,
5'СН3), 2.50 (3 H, s, 3'СН3), 6.16 (1 H, s, H4'), 7.67
(1 H, dd, J1 7.8, J2 4.8, H5), 8.50 (1 H, dd, J1 8.1,
H 1.87; Br 29.70; N 20.82.
J
2 1.5, H4), 8.77 (1 H, dd, J1 4.8, J2 1.5, H6). Found, %:
2ꢀ(Imidazolꢀ1ꢀyl)ꢀ5ꢀnitropyridine (Vc) was syntheꢀ
C 54.66; H 4.28; N 25.41. С10H10N4O2. Calculated, %:
C 55.04; H 4.62; N 25.67.
sized by method B from 2ꢀchloroꢀ5ꢀnitropyridine and
imidazole in a yield of 80%. M.p., 217–219
°
C
(from
EtOH). H NMR spectrum: 7.20 (1H, s, H4'), 8.08
(1 H, s, H2'), 8.09 (1 H, d, 9.3, H3), 8.69 (1 H, s,
H5'), 8.78 (1 H, dd, 1 9.0, 2 2.7, H4), 9.29 (1 H, d,
2.4, H6). Found, %: C 50.18; H 2.95; N 29.10.
1
4ꢀ(3,5ꢀDimethylpyrazolꢀ1ꢀyl)ꢀ3ꢀnitropyridine (Xb)
was synthesized from 4ꢀchloroꢀ3ꢀnitropyridine simiꢀ
larly to compound (Xa) in a yield of 88%. M.p., 155–
158
s, 5'СН3), 2.34 (3 H, s, 3'СН3), 6.21 (1 H, s, H4'), 7.86
(1 H, d, 5.4, H5), 8.95 (1 H, d, 5.4, H6), 9.19 (1 H,
J
J
J
C
(from AcOEt). 1H NMR spectrum: 2.12 (3 H,
J
°
С8H6N4O2. Calculated, %: C 50.53; H 3.18; N 29.46.
J
J
2ꢀ(3,5ꢀDimethylꢀ4ꢀchloropyrazolꢀ1ꢀyl)ꢀ5ꢀnitropyꢀ
ridine (VIc) was synthesized by method B from
2ꢀchloroꢀ5ꢀnitropyridine and 3,5ꢀdimethylꢀ4ꢀchloroꢀ
s, H2). Found, %: C 54.70; H 4.00; N 25.21.
С10H10N4O2. Calculated, %: C 55.04; H 4.62; N 25.67.
pyrazole in a yield of 65%. M.p., 140–142
°
C
(from
AcOEt). H NMR spectrum: 2.26 (3 H, s, 5'СН3),
2.65 (3 H, s, 3'СН3), 8.05 (1 H, dd, 1 9.0, 2 6.0, H3),
8.70 (1 H, d, J1 9.0, J2 3.0, H4), 9.26 (1 H, d, 2.7
5ꢀAminoꢀ2ꢀ(4,5ꢀdichloroimidazolꢀ1ꢀyl)ꢀpyridine (XI).
The mixture of iron (33.6 g, 0.6 gꢀatom), EtOH
(150 mL), and conc. HCl (2 mL) was boiled under
stirring for two hours. After the portionwise addition of
nitropyridine (IIc) (25.9 g, 0.1 mol), the mixture was
boiled for another three hours. К2СО3 (5 g) was then
added by portions of 0.2–0.3 g, and the reaction mixꢀ
ture was boiled for 30 min. The mixture was filtered
1
J
J
J
,
H6). Found, %: C 47.40; H 3.25; Cl 13.70, N 22.00.
С10H9ClN4O2. Calculated, %: C 47.54; H 3.59;
Cl 14.03, N 22.17.
2ꢀ(Benzimidazolꢀ1ꢀyl)ꢀ5ꢀnitropyridine (VIIc) was
synthesized by method A from 2ꢀchloroꢀ5ꢀnitropyriꢀ
hot and washed with hot ethanol (
(100 mL) was evaporated, water (100 mL) was added,
3 20 mL). Ethanol
×
dine and benzimidazole at 50–60
°
C for 2 h in a yield
1
and the precipitate of amine (XI) was filtered off. The
of 90%. M.p., 206–208 (from EtOH). H NMR
°
C
1
yield, 20.6 g (90%). M.p., 179–181 C (from EtOH).
°
spectrum: H NMR spectrum: 7.37–7.47 (2 H, m,
1H NMR spectrum: 5.57 (2 H, s, NH2), 7.06–7.17
(2 H, m, H3,4), 7.77 (1 H, s, H2'), 7.85 (1 H, d, 1 4.8
H5', 6'), 7.79–7.81 [1 H, m, H7' (or H4')], 8.25 [1 H,
J
,
d,
H3), 8.82 (1 H, dd, J1 9.0, J2 3.0, H4), 9.17 (1 H, s,
H2'), 9.43 (1H, d, 3.0, H6). Found, %: C 59.68;
J 9.0, H4' (or H7')], 8.48 (1 H, dd, J1 7.8, J2 1.6,
H6). Found, %: C 41.66; H 2.25; Cl 30.69; N 24.02.
С8H6Cl2N4. Calculated, %: C 41.95; H 2.64; Cl 30.95;
N 24.46.
J
H 2.98; N 23.00. С12H8N4O2. Calculated, %: C 60.00;
H 3.36; N 23.32.
Study of antimicrobial properties of prepared comꢀ
pounds. Solutions of the studied compounds (2 mL) of
different concentrations obtained by twoꢀfold serial
dilutions in a liquid medium [27–30] were added to
the prepared suspensions of bacteria (2 mL). The
accounted microbial load was 250000 microbial bodꢀ
ies in 1 mL. The resultant solutions were incubated for
5ꢀNitroꢀ2ꢀ[(3ꢀchloropyridazinꢀ6ꢀon)ꢀ1ꢀyl]ꢀpyridine
(VIIIc) was synthesized by method A from 2ꢀchloroꢀ
5ꢀnitropyridine and 3ꢀchloropyridinꢀ6ꢀone at 60–
65
(from AcOEt). 1H NMR spectrum: 7.25 (1 H, d,
H4'), 7.71 (1 H, d, 9.9, H5'), 8.01 (1 H, d, 8.7, H3),
8.80 (1 H, dd, 1 9.0, 2 2.7, H4), 9.40 (1 H, d, 2.4,
°
C
for 1.5 h in a yield of 70%. M.p., 147–149
°
C
,
J
9.9
J
J
18 h at 37 C. The nutrient medium containing bacteꢀ
°
J
J
J
ria at the concentration of 250000 microbial bodies in
1 mL was used as the positive control. The nutrient
medium without bacteria was used as the negative
control. The experiments were carried out using the
standard bacterial strains, i.e., S. aureus Pꢀ209, E. coli
078 (field strain).
H6). Found, %: C 42.55; H 1.68; Cl 13.69; N 21.79.
С9H5ClN4O3. Calculated, %: C 42.79; H 2.00;
Cl 14.03; N 22.18.
General procedure of twoꢀstep synthesis of pyraꢀ
zolylꢀ3ꢀnitropyridines (Xa, b). Hydrazine hydrate
(0.65 mL, 20 mmol) was dropwise added to 2ꢀ or
4ꢀchloroꢀ3ꢀnitropyridine (1.4 g, 10 mmol) in 15 mL of
Study of protistocidal activity was performed by the
EtOH and boiled for 30 min. The reaction mixture was method [31, 32] on protozoa of the C. stenii species
cooled, followed by the addition of water (20 mL). The (field isolate, collection of the Laboratory of Parasitolꢀ
formed precipitate of hydrazine pyridine (IXa, b) was ogy of North Caucasian Zonal Scientific Research
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015

SYNTHESIS AND PHARMACOLOGICAL ACTIVITY
407
4. Bald, D. and Koul, A., Drug Discovery Today, 2013,
Veterinary Institute, Russia). Experiments were carꢀ
ried out in the enzyme immunoassay microplates. A
mixture of boiled tap water and sterile distilled water in
equal volumes was used as the medium for the expoꢀ
sure of protozoa. The substances were initially diluted
by distilled water. The serial dilutions of the substances
were performed as follows.
vol. 18, pp. 250–255.
5. Bacterial Resistance to Antimicrobials, 2nd ed.,
Wax, R.G., Lewis, K., Salyers, A.A., and Taber, H.,
Eds., New York: Taylor and Francis Group, CRC Press,
2008.
6. Scribner, A., Dennis, R., Lee, S., Ouvry, G., Perrey, D.,
Fisher, M., Wyvratt, M., Leavitt, P., Liberator, P., Gurꢀ
nett, A., Brown, C., Mathew, J., Thompson, D.,
Schmatz, D., and Biftu, T., Eur. J. Med. Chem., 2008,
vol. 43, pp. 1123–1151.
7. Kolabskii, N.A. and Pashkin, P.I., Koktsidiozy sel’skoꢀ
khozyaistvennykh zhivotnykh (Coccidioses of Farm
Animals), Leningrad: Kolos, 1974.
8. Khovanskikh, A.E., Ilyushekkin, Yu.P., and Kirillov, A.I.,
Koktsidioz sel’skokhozyaistvennoi ptitsy (Coccidiosis of
Poultry), Leningrad: Agropromizdat, 1990.
9. Charifson, P.S., Grillot, A.L., Grossman, T.H., Parꢀ
sons, J.D., Badia, M., Bellon, S., Deininger, D.D.,
Drumm, J.E., Gross, C.H., LeTiran, A., Liao, Y.,
Mani, N., Nicolau, D.P., Perola, E., Ronkin, S., Shanꢀ
non, D., Swenson, L.L., Tang, Q., Tessier, P.R.,
Tian, S.K., Trudeau, M., Wang, T., Wei, Y., Zhang, H.,
and Stamos, D., J. Med. Chem., 2008, vol. 51,
pp. 5243–5263.
10. Morkovnik, A.S., Divaeva, L.N., Zubenko, A.A., Podꢀ
ladchikova, O.N., Fetisov, L.N., and Akopova, A.R.,
RF Patent no. 2423335, Byull. Izobret., 2011, no. 19.
11. Zubenko, A.A., Klimenko, A.I., Morkovnik, A.S.,
Divaeva, L,N., Fetisov, L.N., and Bodryakov, A.N., RF
Patent no. 2, Byull. Izobret., 2014, no. 12.
Solution 1. The compound (5 mg) was added to
70% aqueous DMSO (50
tion of distilled water (5 mL). The final concentration
was 1000 g/mL.
μL), followed by the addiꢀ
μ
Solutions 2–12. A mixture of boiled tap water and
sterile distilled water (1 : 1) was added to wells 2–12
(150
150
ring, 150
well 3, and so on until the end of the row; 150
well 12 was removed after stirring. Threeꢀday culture
of C. steinii (30 L) was added to each well. Well 1 was
filled with solution 1 (150 L) and of protozoa suspenꢀ
sion (30 L). The protozoa suspension was prepared
μ
L/well) with an automatic 8ꢀchannel pipette;
L of solution 1 was added to well 2 and after stirꢀ
L of the resultant solution was added to
L from
μ
μ
μ
μ
μ
μ
so that 10–15 active species were observed in each
field of view at low magnification. After addition of
protozoa, the plate was covered with a lid and allowed
to stay at room temperature (20–22
Results were accounted for as follows. The mixture
of well 12 (30 L) was applied onto a clean glass slide
and examined under a microscope at low magnificaꢀ
tion (10 15). The presence or absence of living proꢀ
°C) for 18–20 h.
μ
×
12. Divaeva, L.N., Morkovnik, A.S., Klimenko, A.I.,
Zubenko, A.A., Fetisov, L.N., and Bodryakov, A.N.,
RF Patent no. 2514196, Byull. Izobret., 2014, no. 12.
13. Morkovnik, A.S., Divaeva, L.N., Kuz’menko, T.A.,
Podladchikova, O.N., Zubenko, A.A., Akopova, A.R.,
and Fetisov, L.N., in Materialy VII Vserossiiskoi nauchꢀ
noi konferentsii “Khimiya i meditsina, Orkhimedꢀ2009”
(Proc. VII AllꢀRussia Sci. Conf. “Chemistry and Medꢀ
icine, Orkhimedꢀ2009”), Ufa, 2009, pp. 57–58.
14. Cuckler, A.C., Chapin, L.R., Malanga, C.M.,
Rogers, E.F., Becker, H.J., Clark, R.L., Leanza, W.J.,
Pessolano, A.A., Shen, T.Y., and Sarett, L.H., Proc.
Soc. Exp. Biol. Med., 1958, vol. 98, pp. 167–170.
tozoa was noted. The view was performed from right to
left. Well 1 without living species was considered as
that containing minimal protistocidal concentration
of the studied compound.
The following solutions were used as controls:
⎯
control of the medium (boiled tap water + sterile
distilled water), 5 wells;
control of the solvent (50
L of distilled water and all serial dilutions as in the
case of the studied compounds), 12 wells;
reference drug, Baycox.
⎯
μL of 70% DMSO +
5
μ
⎯
15. Morisawa, Y., Kataoka, M., Kitano, N., and Matꢀ
suzawa, T., J. Med. Chem., 1977, vol. 20, no. 1,
pp. 129–133.
ACKNOWLEDGMENTS
16. Morisawa, Y., Kataoka, M., and Kitano, N., J. Med.
Chem., 1977, vol. 20, no. 4, pp. 483–487.
17. Morisawa, Y., Kataoka, M., Sakamoto, T., Hitoshi, N.,
Kitano, N., and Kusano, K., J. Med. Chem., 1978,
vol. 21, no. 2, pp. 194–199.
The work was supported as part of the State project
in the field of scientific activity (project
no. 4.196.2014/K) and performed using the equipꢀ
ment of the Center of Collective Use “Molecular
spectroscopy” of South Federal University.
18. Morisawa, Y., Kataoka, M., Nagahori, H., Sakamoto, T.,
Kitano, N., Kusano, K., and Sato, K., J. Med Chem.
1980, vol. 23, no. 12, pp. 1376–1380.
,
REFERENCES
19. Bodryakov, A.N., Zubenko, A.A., and Fetisov, L.N., in
Materialy Vserossiiskoi Nauchnoꢀprakticheskoi konfeꢀ
rentsii “Nauchnoe obespechenie ustoichivogo razvitiya
zhivotnovodstva Rossiiskoi federatsii” (Proc. AllꢀRussia
Sci.ꢀPract. Conf. “Scientific Support for Sustainable
Livestock Development in the Russian Federation”),
Novocherkassk, 2012, pp. 116–119.
1. Urgently needed: New antibiotics (Editorial), Lancet,
2009, vol. 374, p. 1868.
2. Vento, S. and Cainelli, F., The Lancet, 2010, vol. 375,
p. 637.
3. Panasevich, L.C., Nation’s Health, 2004, vol. 34, no. 7,
p. 8.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015

408
KLIMENKO et al.
20. Gurnett, A.M., Liberator, P.A., Dulski, P.M., Salowe, S.P.,
Donald, R.G., Anderson, J.W., Wiltsie, J., Diaz, C.A.,
Harris, G., Chang, B., DarkinꢀRattray, S.J., Nare, B.,
Crumley, T., Blum, P.S., Misura, A.S., Tamas, T., Sarꢀ
dana, M.K., Yuan, J., Biftu, T., and Schmatz, D.M.,
J. Biol. Chem., 2002, vol. 277, pp. 15913–15922.
21. Donald, R.G.K. and Liberator, P.A., Mol. Biochem.
Parasitol., 2002, vol. 180, p. 136.
22. Scribner, A., Dennis, R., Hong, J., Lee, S., McIntyre, D.,
Perrey, D., Feng, D., Fisher, M., Wyvratt, M., Leavitt, P.,
Liberator, P., Gurnett, A., Brown, C., Mathew, J.,
Thompson, D., Schmatz, D., and Biftu, T., Eur.
J. Med. Chem., 2007, vol. 42, pp. 1334–1357.
23. Biftu, T., Feng, D., Fisher, M., Liang, G.B., Qian, X.,
Scribner, A., Dennis, R., Lee, S., Liberator, P.A.,
Brown, C., Gurnett, A., Leavitt, P.S., Thompson, D.,
Mathew, J., Misura, A., Samaras, S., Tamas, T.,
Sina, J.F., McNulty, K.A., McKnight, C.G.,
Schmatz, D.M., and Wyvratt, M., Bioorg. Med. Chem.
Lett., 2006, vol. 16, pp. 2479–2483.
24. Liang, G.ꢀB., Qian, X., Feng, D., Fisher, M.,
Brown, C.M., Gurnett, A., Leavitt, P.S., Liberator, P.A.,
Misura, A.S., Tamas, T., Schmatz, D.M., Wyvratt, M.,
and Biftu, T., Bioorg. Med. Chem. Lett., 2007, vol. 17,
pp. 3558–3561.
New Pharmaceuticals), Khabriev, R.U., Ed., Moscow:
Meditsina, 2005.
28. Rukovodstvo po provedeniyu doklinicheskikh issledovanii
lekarstvennykh sredstv (Guidelines for Preclinical Trials
of Medicinal Products), Mironov, A.N., Ed., Moscow:
Grif i K, 2012, part 1.
29. Opredelenie chuvstvitel’nosti mikroorganizmov k antiꢀ
bakterial’nym preparatam. Metodicheskie ukazaniya.
MUK 4.2.1890ꢀ04 (Determination of the Sensitivity of
Microorganisms to Antibiotics: Guidelines MUK
4.2.1890ꢀ04), Moscow: Meditsina, 2004.
30. Pershin, G.N., Metody eksperimental’noi khimioterapii
(Methods of Experimental Chemotherapy), Moscow:
Meditsina, 1971.
31. Fetisov, L.N., Zubenko, A.A., Bodryakov, A.N., and
Bodryakova, M.A., The search for new protistocidal
agents: problems of normative regulation in veterinary,
in Materialy mezhdunarodnogo parazitologicheskogo
simpoziuma “Sovremennye problemy obshchei i chastnoi
parazitologii” (Proc. Int. Parasitol. Symp. “Modern
Problems of General and Special Parasitology”), 2012,
pp. 70–73.
32. Zubenko, A.A., Fetisov, L.N., and Bodryakov, A.N.,
Opredelenie protistotsidnoi aktivnosti novykh soedineꢀ
nii v ryadu azotsoderzhashchikh geterotsiklov (Deterꢀ
mination of protistocidal activity of new compounds in
the range of nitrogenꢀcontaining heterocycles), in
Materialy Vserossiiskoi nauchno prakticheskoi konferenꢀ
tsii GNU SKZNIVI “Nauchnoe obespechenie innovatꢀ
sionnogo razvitiya otechestvennogo zhivotnovodstva”
(Proc. AllꢀRussia Sci.ꢀPract. Conf. GNU SKZNIVI
“Scientific Support of Innovative Development of
Domestic Livestock”), Novocherkassk, 2011, pp. 162–
165.
25. Feng, D., Fisher, M., Liang, G.B., Qian, X., Brown, C.,
Gurnett, A., Leavitt, P.S., Liberator, P.A., Mathew, J.,
Misura, A., Samaras, S., Tamas, T., Schmatz, D.M.,
Wyvratt, M., and Biftu, T., Bioorg. Med. Chem. Lett.
2006, vol. 16, pp. 5978–5981.
,
26. Gel’man, N.E., Terent’eva, E.A., Shanina, T.M., and
Kiparenko, L.M., Metody kolichestvennogo organiꢀ
cheskogo elementnogo analiza (Methods of Quantitative
Organic Elemental Analysis), Moscow: Khimiya, 1987.
27. Rukovodstvo po eksperimental’nomu (doklinicheskomu)
izucheniyu novykh farmakologicheskikh veshchestv
(Guidelines for Experimental (Preclinical) Studies of
Translated by A. Levina
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015