6-Nitrotriazolo[1,5-a]pyrimidines as promising structures for pharmacotherapy of septic conditions

Article abstract of DOI:10.1134/S1068162017040094

Promising 6-nitro-1,2,4-triazolo[1,5-a]pyrimidine analogues, structural analogues of synthetic inhibitors of adenosine receptors, were sorted out on the basis of quantum-chemical calculations. The compounds were synthesized by nitration and chlorodeoxygenation reactions. The in vivo activity of 6-nitroheterocycles was studied and the affinity to adenosine receptor A_2A was demonstrated in regard to septic conditions.

Full text of DOI:10.1134/S1068162017040094

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 4, pp. 421–428. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © K.V. Savateev, E.N. Ulomsky, V.V. Fedotov, V.L. Rusinov, K.V. Sivak, M.M. Lyubishin, N.N. Kuzmich, A.G. Aleksandrov, 2017, published in Bioorganicheskaya
Khimiya, 2017, Vol. 43, No. 4, pp. 402–410.
6
-Nitrotriazolo[1,5-a]pyrimidines as Promising Structures
for Pharmacotherapy of Septic Conditions
a
a, 1
a
a
b
b
K. V. Savateev , E. N. Ulomsky , V. V. Fedotov , V. L. Rusinov , K. V. Sivak , M. M. Lyubishin ,
b
b
N. N. Kuzmich , and A. G. Aleksandrov
a
Boris Yeltsin Federal University, Department of Organic Chemistry, Yekaterinburg, 620002 Russia
b
Research Institute of Influenza, Ministry of Health of the Russian Federation, St. Petersburg, 197376 Russia
Received September 2, 2016; in final form, December 5, 2016
Abstract⎯ Promising 6-nitro-1,2,4-triazolo[1,5-a]pyrimidine analogues, structural analogues of syn-
thetic inhibitors of adenosine receptors, were sorted out on the basis of quantum-chemical calculations.
The compounds were synthesized by nitration and chlorodeoxygenation reactions. The in vivo activity
of 6-nitroheterocycles was studied and the affinity to adenosine receptor A was demonstrated in regard
2
A
to septic conditions.
Keywords: adenosine receptors, chlorodeoxygenation, nitroderivatives, sepsis, triazolopyrimidines
DOI: 10.1134/S1068162017040094
INTRODUCTION
attack on A AR could result in the reduction of the
2A
inflammatory reaction via mediated signal cascades.
In most countries, sepsis is among ten major fac-
tors causing patient death in departments of resuscita-
tion and intensive care [1]. The frequency of incidents
of severe sepsis among these patients is 2–18% and
that of septic shock, about 4% [2]. The mortality due
The therapeutic potential of the A AR interaction
2A
with small molecules is generally accepted [12]. A
series of selective A AR agonists and antagonists are
2A
to sepsis can achieve 30%, and due to septic shock, being tested in clinical trials as potential drugs for the
more than 50%. Since microorganisms have been treatment of Parkinson’s disease, inflammation, can-
considered major causative agents of sepsis so far,
antibiotics still play the leading role in its therapy in
spite of the development of new drugs [3]. However,
with all positive results of antibiotics therapy, it cannot
affect the advanced inflammatory response, which
plays one of the key roles in sepsis pathogenesis [4, 5].
It is necessary to develop a complex approach for sep-
cer, ischemia, diabetic nephropathy, infections, and
disorders of the central nervous system [12]. At the
same time, a similar arsenal of drugs for the therapy
of sepsis is practically absent. Condensed azo-
loazines, structural purine analogues, are among
antiseptic agents involved in the regulation of ade-
sis treatment including the search for new targets for nosine receptor functioning [13, 14]. Azoloazines
antiseptic drugs.
containing a bridge nitrogen atom, such as 1,2,4-
azolo[1,3,5]triazines of the ZM-241385 type [16],
SCH 442416 analogues, 1,2,4-triazolo[1,5-a]pyrim-
Recently, the adenosine receptor A2A (A AR) is
2A
regarded as a promising target. It was shown previ-
ously that the activation of this receptor reduced idines, [16], and others occupy a specific place
inflammation and the use of synthetic adenosine ana- among effectors of A2A receptors.
logues for sepsis therapy increased viability [6, 7]. The
In this work, we proposed 2,5-substituted-6-nitro-
A AR activation in animal models in vivo resulted in
2A
the reduction of the effect associated with chronic 1,2,4-triazolo[1,5-a]pyrimidine-7(4H)-ones of type
inflammation, probably, due to the intensification of (A) and 2,5-substituted-6-nitro-7-amino-1,2,4-tri-
cAMP synthesis [6, 8–11]. Moreover, mice defective azolo[1,5-a]pyrimidines of type (B), structural ana-
in the A AR gene were sensitive to minimal effects
stimulating inflammation [5]. It suggested that the
2A
logues of purines and ZM-241385, as new antiseptic
agents taking into account that the electron effect of
the nitro group in the azine cycle was equivalent to that
of the pyridine nitrogen (Scheme 1).
1
Corresponding author: phone: +7 (909) 005-3245; e-mail:
ulomsky@yandex.ru.
421

4
22
SAVATEEV et al.
R3
OH
O
NH2
N
NH
NO2
N
N
N
N
N
R1
NO2
N
N
N
N
R1
N
H
R2
O
N
N
H
N
R2
(A)
(B)
ZM-241385
Scheme 1. The structure of the known ZM-241385 and related 5-substituted 6-nitro-1,2,4-triazolo[1,5-a]pyrimidine-
(4H)-ones of type (A) and 2,5-substituted-6-nitro-7-amino-1,2,4-triazolo[1,5-a]pyrimidines of type (B).
7
In this work, we reported the results of the struc- cycle conformational flexibility (when relevant), stan-
tural analysis of the affinity of a series of triazolo[1,5- dard settings were used by default.
a]pyrimidines (A, B) to the A receptor, the selection
2
A
The pK values were calculated by the equation
i
of promising molecules, their synthesis, and biological
activity in regard to septic conditions.
pK = –log[K ], where K was the equilibrium constant
i
i
i
of the (L*R) complex formation of the ligand (L) with
the receptor (R) (K = [R*L]/[R]*[L] (Table 1)).
i
RESULTS AND DISCUSSION
The results of docking clearly demonstrated that
most of the compounds under study were involved in
the complex formation with the receptors by means of
hydrogen bonds with a moderate affinity to the recep-
The choice of molecular structures relevant for
adenosine receptors
At the first stage, we estimated the affinity of target
compounds toward the A receptor using computer
2A
tor. The exception was compound (Ia) having pK > 7
i
modelling. As a comparative model we used ZM-
and compound (V), 7-N-[2'-(4''-chlorophenyl)]ethyl-
amino-6-nitro[1,2,4]triazolo[1,5-a]pyrimidine, which
showed the most effective binding within the triazol-
opyrimidine series. The three-dimensional pattern of
compound (V) interaction with the A receptor is
2
41385.
The process of preparation of the receptor to dock-
ing involved the removal of different cofactors, inor-
ganic ions, small inorganic molecules, and all water
molecules outside the active site. Three-dimension
protonation with consideration of pH (7.4) and char-
acteristic ionization states of amino acid side chains as
well as geometric optimization using molecular
mechanics (the AMBER99 force field specially
parametrized for macromolecules) were performed.
The protein molecule was validated using a Ramach-
andran diagram [17] and estimation of permissible
bond lengths and valence angles.
2A
2
given in Fig. 1. The sp -hybrid nitrogen atom in the
heterocycle position 3 formed a hydrogen bond with
the amide proton of asparagine 253 side chain,
whereas oxygen atoms of the nitro group served accep-
tors of the hydrogen bond with the hydroxyl proton of
threonine 88. An essential structural feature of triazo-
lopyrimidine (V) is an advantageous position of a
stretched hydrophobic 2-(4'-chlorophenyl)ethyl
group in the receptor active site (the receptor structure
was taken from the PDB, Protein Data Bank,
www.rcsb.org, PDB code: 3EML).
All the structures were minimized by the DFT
Density Functional Theory) method using a M06-2X
meta hybrid functional [18] and a 31G(d) basic set of
functions. The correctness of stationary points was
confirmed by the lack of imaginary frequencies.
Quantum chemical calculations were carried out using
the Gaussian 09 program [19].
The docking procedure was performed using a Sur-
flex-Dock flexible algorithm implemented in the
Sybyl-X 1.1 software [20]. The method uses the
empiric Hammerhead scoring function [21, 22] and
employs an idealized active site ligand (called a Proto-
mol) for the generation of ligand positions by the step-
wise cross-sectional design, which can link fragments
from different positions. Being one of the most effec-
tive algorithms for docking [23], Surflex-Dock is
especially effective for sorting out false positive dock-
(
However, despite rather high pKi values, all the
designed triazolopyrimidines were inferior in their
affinity to the reference ZM-241385. It is known that
both polar (hydrogen bonds, etc.) and hydrophobic
interactions are important for optimal binding. A
visual analysis of two-dimensional ligand formulas
and three-dimensional receptor–ligand complexes
demonstrated that the shape of the referent antagonist
was better adjusted to the active site pocket. In addi-
tion, it bears bulk hydrophobic groups providing effec-
tive nonpolar interactions and contributing a consid-
erable energetic effect to the complex formation.
To summarize, the 1,2,4-triazolo[1,5-a]pyrimi-
ing algorithms upon screening. With the exception of dines under study are promising molecules for the
the options of the active site conformation adaptation development of their synthesis and evaluation of their
to the ligand conformation and consideration of the antiseptic activities.
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6-NITROTRIAZOLO[1,5-a]PYRIMIDINES AS PROMISING STRUCTURES
423
Table 1. The results of the docking test of compounds (I)–(VI) and ZM-241385 in respect to the A A receptor
2A
R3
NH2
N
O
NH
NO2
R2
OH
N
N
N
N
N
N
R1
NO2
R2
N
N
N
O
N
H
N
N
H
N
(
Ia, c), (IIb, c, d)
(III)−(VI)
(IId)
ZM-241385
No.
R1
(Ia)
(Ic)
(IIb)
(IIc)
H
(III)
H
(IV)
H
(V)
(VI)
H
ZM-241385
H
Me
Me
H
–
–
N
R2
H
–
H
Me
Me
OH
H
H
Me
Cl
O
R3
–
–
–
–
–
n-Bu
O
–
pKi
7.14
5.23
5.99
5.27
5.11
5.34
6.53
7.54
6.48
10.00
1
Synthetic Part
The first stage of the synthetic part was dedicated to
A general procedure for the synthesis of 2-R -5-
2
R -6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-ones
consisted in the coupling of 5-R -3-amino-1,2,4-tri-
1
1
the development of methods of synthesis of 2-R -5-
2
R -6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-ones (I) azoles (VII) with β-ketocarboxylic acids or their deriv-
and (II) (Scheme 2).
atives. Particularly, heating of aminotriazole (VII)
Fig. 1. The A2A receptor complex with triazolopyrimidine (V).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 4 2017

4
24
SAVATEEV et al.
Table 2. The impact of triazolopyrimidine derivatives (I)–(VI) on the viability of mice with septic shock induced by an LPS
injection
Group/Injected
compound
Effect,
died/totally
Injected
compound
Effect,
died/totally
Viability, %
Viability , %
Intact mice
Placebo (LPS only)
Hydrocortisone, 15 mg/kg
0/5
5/5
2/5*
4/5
5/5
5/5
100
0
60
20
0
(V)
2/5*
5/5
1/5*
4/5
2/5*
2/5*
60
0
80
20
60
60
(IIb)
(Ia)
(IV)
(III)
(VI)
(
(
(
Ic)
IIc)
IId)
0
*
р < 0.05.
with ethyl 2-nitro-3-ethoxyacrylate (VIII) resulted in acetate [24, 25], an alternative synthetic scheme was
ethyl 2-nitro-3-(5'-(furyl-2'')-1,2,4-triazol-3'-yl)amino-
1
used [26]. At the first stage, 5-R -3-amino-1,2,4-
2
-nitroacrylate (IX), which was successively treated
triazole (VII) was coupled with ethyl acetoacetate
with 2M sodium carbonate and concentrated hydro-
chloric acid to give 2-(fur-2'-yl)-6-nitro-1,2,4-tri-
azolo[1,5-a]pyrimidin-7-one (Iа) (Scheme 2). The
structure of compound (Iа) was ascribed according to
1
(
X) in acetic acid to give 2-R -5-methyl-1,2,4-tri-
azolo[1,5-a]pyrim7idin-7-ones (XI) (Scheme 2).
Nitration, most commonly with a mixture of nitric
1
1
IR and H NMR spectroscopy and element analysis.
and sulfuric acids [26], resulted in 2-R -5-methyl-6-
If α-nitro-β-ketoester was unavailable due to its nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-ones
(II)
instability, as it took place in the case of 2-nitroaceto- (Scheme 2).
O
NO2
O
EtO
NO2
N
N
N
N
EtO (VIII)
N
NH
NH
O
Na CO
2
3
O
N
70°C
O
N
O
RT
N
H
NH2
N
H
OEt
R1
NO2
(
VIIa)
O
(IX)
(Ia)
O
N
O
EtO
(X)
NO2
N
N
N
O
N
NH
N
NHO3
R1
R1
H2SO4
N
N
N
H
N
H
NH2
VIIb−d)
(
(XIb−d)
(IIb−d)
1
R : (a)
* ; (b)
* ;
(c) H; (d) Me
O
N
1
2
Scheme 2. The synthesis of 2-R -5-R -6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-ones (I) and (II).
1
In general, the method is multipurpose for both mononitration product (IIа). The structure of 2-R -5-
reaction stages but there are some exceptions. For, exam- methyl-6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-ones (II)
1
ple, nitration of 2-furyltriazole pyrimidine (XIа) resulted was ascribed according to IR and H NMR spectroscopy
in the dinitration product, namely, 2-(5'-nitrofur-2'-yl)- and element analysis.
5
-methyl-6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-one.
The next stage of the synthetic part involved the devel-
The use of other nitrating agents, such as acetyl nitrate in opment of synthesis of ZM-241385 structural analogues
acetic acid or trifluoroacetyl nitrate in trifluoroacetic on the basis of 1,2,4-triazolo[1,5-a]pyrimidines. At the
acid, did not lead to the regioselective formation of first step, 6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7-one
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2017

6-NITROTRIAZOLO[1,5-a]PYRIMIDINES AS PROMISING STRUCTURES
425
(
Iс) was treated with phosphoryl chloride in pyridine to was unstable and quickly hydrolyzed to the starting triazo-
give 6-nitro-7-chloro-1,2,4-triazolo[1,5-a]pyrimidine lopyrimidone (Ic), it was used at the next stage immedi-
XII) (Scheme 3) [27]. Since the chloro derivative (XII) ately after isolation without purification [28].
(
R3
Cl
NH
N
O
NO2
NO2
NO2 POCl3
N
N
N
N
N
N
N
3
N
N
R -NH
t° Py
Py
N
N
H
(Ic)
(XII)
(IV), (V)
R3: (IV)−Et; (V)−Cl
*
Scheme 3. The synthesis of 7-alkylamino-6-nitro-1,2,4-triazolo[1,5-a]pyrimidines (IV), (V).
The reaction of chlorotriazolopyrimidine (XII) (pK = 5.99 and 6.53 respectively), did not demon-
i
with primary amines in the presence of pyridine strate in vivo a significant antiseptic activity. Particu-
resulted in 7-alkylamino-6-nitro-1,2,4-triazolo[1,5- larly, when compound (IIb) was injected in mice with
a]pyrimidines (IV), (V) (Scheme 3). The data of element septic shock, we did not observe an increase in the via-
1
13
analysis and H, C NMR and IR spectra of amines (IV) bility if compared with placebo. Possibly, these results
and (V) supported the proposed structures. A character- can be explained by a Zwitter ion structure of the het-
1
istic feature of the H NMR spectra of these compounds erocycles, which prevented the formation of stable
was a broad signal (δ 9.83–10.07 ppm) corresponding to hydrogen bonds with the receptors (Scheme 4).
the resonance of the NH proton and a two-proton
O
multiplet (δ 4.39–4.48 ppm) of the N-CH group.
2
N
N
NO2
Such a spectral pattern unambiguously evidenced the
N
presence of a (–NH–CH –) fragment.
2
+
N
N−
H
Results of Biological Tests
(IIb)
The results of the in vivo testing of the compounds
under study partly agreed with the calculated data.
Screening of their biological activity was performed on
a model of septic shock induced in mice by the injec-
tion of Salmonella typhosa lipopolysaccharide (LPS)
Scheme 4. Heterocycle (IIb) as a Zwitter ion.
On the basis of the analysis of viability of mice with
septic shock in screening experiments we sorted out
some promising compounds for the assessment of
their effect on pathological symptoms of acute respira-
tory distress syndrome upon Klebsiella pneumoniae
infection. This inflammatory injury of lungs is a severe
life threatening syndrome, which often accompanies
septic inflammation.
(
Sigma-Aldrich). For the experiment, 130 mice were
taken. The compounds under study (Ia), (Ic), (IIb–d),
and (III)–(VI) were dissolved in DMSO and injected
intraperitoneally twice a day at a daily dose of
5
0 mg/kg (a previously found effective dose of the
prototype, triazavirin) [29]. Hydrocortison hemisuc-
cinate was used as a reference drug. The dynamics of
mouse viability in the test and control groups was
monitored for 3 days. The results were presented as a
portion of survived mice (%) (Table 2).
All the selected compounds were studied in combi-
nation with meropenem, which displays an antibacte-
rial effect. Particularly, upon the injection of heterocy-
cles (III) and (VI) (to a larger degree, р = 0.03) and
Compound (Ia), which according to the results of
quantum chemical calculations was only inferior to
ZM-241385 and (V), demonstrated the highest activ-
ity (80% viability of mice with induced septic shock)
(
Ia) (р < 0.05) we observed a decrease in the extravas-
cular lung water, which correlated with a weaker sys-
temic inflammation associated with the migration of a
larger quantity of leukocytes into the inflammation
lesion.
(
Table 1). Compound (V) displayed the best affinity
among the heterocycles tested (with the exception of
triazolotriazine-based ZM-241385) was also signifi-
To summarize, ZM-241385 structural analogues,
cantly active in animals preventing the death from sep- triazolo[1,5-a]pyrimidine derivatives, were synthe-
tic shock in 60% animals. However, heterocycles (IIb) sized, a virtual prognosis model of their action was
and (IV), which had a satisfactory computed affinity developed in respect to the А2А receptor, and their sig-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 4 2017

4
26
SAVATEEV et al.
nificant biological activity against the septic condition 149.1 (C7), 152.5 (C6), 154.0 (C2). Found, %: С 43.65
was found. Н 2.01 N 28.30. C H N O . Calc., %: С 43.73 Н 2.04
9
5
5
4
N 28.33.
-Alkylamino-6-nitro-1,2,4-triazolo[1,5-a]pyrim-
idines (IV), (V). Phosphoryl chloride (4.6 mL,
.05 mol) was added to a suspension of 6-nitrotri-
7
EXPERIMENTAL
Chemical Part
0
1
13
azolo[1,5-a]pyrimidine-7-one (1.81 g, 0.01 mol) in
absolute MeCN (30 mL) and the mixture was heated
to 60°C. Dry pyridine (1.64 mL, 0.02 mol) was added
and the reaction mixture was refluxed for 3 h and
evaporated in vacuum at 35°C. The residue was
washed with hexane cooled to 5°C and dissolved in
CH Cl cooled to 5°C. To the resulting solution, tri-
H and C NMR spectra (δ, ppm, J, Hz) were reg-
istered on a Bruker Avance II spectrometer (400 and
1
00 MHz respectively) at 25°C; Me Si as an internal
4
standard; DMSO-d and CDCl as solvents. Element
6
3
analysis was performed on a Perkin Elmer 2400 CHN
analyzer. The reactions were monitored by TLC on
Silufol UV-254 plates, EtOAc as an eluent. Melting
points were measured on Staffordshire ST15 0SA
apparatus. Compounds (Ic), (IIc), (IId), (III), and
2
2
ethylamine (1.53 mL, 0.011 mol) was added at 5–7°C
and followed by the addition of the corresponding
alkylamine (0.01 mol) at a temperature below 10°C.
The mixture was stirred for 2 h at room temperature
and washed with water (3 × 50 mL). The organic
phase was dried with Na SO and evaporated to dry-
(
VI) were synthesized as described in [26, 28, 30–32].
Heterocycle ZM-241385 was used as a virtual struc-
ture for the calculation of the affinity to the А ade-
2а
2
4
nosine receptor in silico. However, it was not used as a
ness. The residue was dissolved in refluxing EtOAc
reference in the in vivo experiments.
(
3 × 30 mL) and the combined extracts were washed
2
-(3'-Pyridyl)-5-methyl-6-nitrotriazolo[1,5-a]pyrimi-
with water (3 × 50 mL). The organic phase was dried
with Na SO and the product was purified by flash
din-7-one (IIb). 2-(3’-Pyridyl)-5-methyltriazolo[1,5-
a]pyrimidin-7-one (2.27 g, 0.01 mol) was added to a
vitriol oil (10 mL) under cooling with ice and the sus-
pension was stirred for 15 min under cooling with ice.
2
4
chromatography eluting with EtOAc.
7-n-Butylamino-6-nitro-1,2,4-triazolo[1,5-a]pyrim-
Seventy-percent nitric acid (0.83 mL, 0.013 mol) was idine (IV). Light yellow powder. Yield 57%. mp 118–
1
added to the suspension at 5–10°C. Cooling was 120°C. H NMR (CDCl ): 0.96 (t, 3 H, J 7.2, CH ),
3
3
removed and the reaction mixture was stirred at room
temperature for 30 min. The reaction mixture was kept
overnight at room temperature and then poured onto
ice upon stirring. The resulting light yellow precipitate
1
.43–1.49 (mм, 2 H, СН ), 1.75–1.81 (m, 2 Н, СН ),
.39 (dt, 2 Н, J 6.8, J 6.8, N–CH ), 8.28 (s, 1 H,
2
2
4
1
2
2
13
H2), 9.24 (s, 1 H, H5), 9.83 (br m, 1 H, NH). C
NMR (CDCl ): 13.4 (CH ), 19.5 (CH ), 32.6 (CH ),
3
3
2
2
was filtered off and recrystallized from DMSO to give
4
6.6 (N-CH ), 118.6 (C6), 146.1 (C7), 153.0 (C5),
1
2
9
(
5% of the product; mp 293–295°C. H NMR
1
56.2 (C2), 157.5 (C3a). Found, %: С 45.70 Н 5.26 N
CF COOD): 2.76 (s, 3 Н, CH ), 8.26 (dd, 1 Н, J 6.0,
3 3 1
3
5.41. C H N O . Calc., %: С 45.76 Н 5.12 N 35.57.
9
12
6
2
J 6.0, H5'), 8.95 (d, 1 H, J 6.0, H4'), 9.23–9.30 (m,
2
13
6-Nitro-7-(2'-(4''-chlorophenyl)ethylamino)-1,2,4-
1
H, H6'), 9.52 (s, 1 H, H2'). С NMR (СF COOD):
3
triazolo[1,5-a]pyrimidine (V). Yellow crystals. Yield
2
0.4 (CH ), 130.2 (C5), 130.5 (C5'), 131.8 (C2), 142.4
3
1
4
0%. mp 189–191°C. H NMR (DMSO-d ): 3.06 (t,
(
C2'), 145.1 (C4'), 147.5 (C6'), 151.8 (C7), 153.0
6
2
H, J 7.6, Ph-CH ), 4.45–4.51 (br m, 2 H, N-CH ),
(
C3a), 155.8 (C6), 160.0 (C3'). Found, %: С 45.49 Н
2
2
3
3
.41 N 28.80. C H N O ⋅ H O. Calc., %: С 45.52 Н 7.32 (d, 2 Н, J 8.4, 2 × CH), 7.35 (d, 2 Н, J 8.4, 2 ×
1
1
8
6
3
2
CH), 8.64 (s, 1 H, H2), 9.19 (s, 1 H, H5), 10.07 (br m,
.47 N 28.96.
-(2'-Furyl)-6-nitrotriazolo[1,5-a]pyrimidin-7-one
Ia). 3-(2'-Furyl)-5-amino-1,2,4-triazole (1.5 g,
1
3
1
4
H, NH). C NMR (DMSO-d ): 35.9 (Ph-CH ),
6 2
2
6.9 (N-CH ), 119.6 (C6), 128.3 (C2', C6'), 130.6
(
0
2
(
C3', C5'), 131.2 (C4'), 137.2 (C1'), 146.1 (C7), 152.7
C5), 156.2 (C2), 157.4 (C3a). Found, %: С 48.89 Н
.01 mol) was mixed with ethyl 2-nitro-3-ethoxyacry-
(
3
late (1.7 mL, 0.0105 mol) and the mixture was heated
.63 N 26.45. C H ClN O . Calc., %: С 48.99 Н 3.48
at 70°C for 30 min. The reaction mixture was cooled to
13 11 6 2
room temperature and 2M sodium carbonate (5 mL, N 26.37.
0
4
.01 mol) was added. The suspension was stirred for
5 min at room temperature, and the precipitate was
Biological: In Vivo Experiments
filtered off, suspended in water (7 mL), and 12 M
hydrochloric acid (0.8 mL, 0.01 mol) was added. The
Mice were taken from a certified nursery “PLZh
Rappolovo.” During the quarantine the animal mate-
rial was studied in a state veterinary laboratory GGBU
LMVL to give a negative response to salmonellosis.
Manipulations with the animals were performed in
gray precipitate was filtered off and dried on air to give
1
8
7% of the product; mp 286–287°C. H NMR
(
DMSO-d ): 6.71 (dd, 1 Н, J 3.4, J 1.7, Н3'), 7.20 (d,
6 1 2
1
Н, J 3.4, Н2'), 7.94 (d, 1 Н, J 1.7, Н4'), 9.27 (s, 1 Н,
13
Н5). C NMR (DMSO-d ): 112.3 (С3'), 112.4 (C2'), accordance with the guidelines on humane treatment
6
1
25.1 (C3a), 144.5 (C1’), 145.4 (C4’), 148.0 (C5), of laboratory animals in biological experiments.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 4
2017

6-NITROTRIAZOLO[1,5-a]PYRIMIDINES AS PROMISING STRUCTURES
427
Statistical analysis
For the statistical processing, the data was entered
to a МSExcel 2010 program for accumulation and
preparation for the analysis. The analysis was per-
formed using statistical package programs Statistica
(
StatSoft, United States). The data was presented as a
mean (M) and error in mean (± m). Nonparametric
ANOVA test was used. The differences were regarded
as significant at р ≤ 0.05.
ACKNOWLEDGMENTS
The work was supported by the Russian Scientific
Foundation, project no. 14-13-01301.
100.0
62.3
74.0
55.9
63.5
84.9
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Translated by E. Shirokova
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Vol. 43
No. 4
2017