Synthesis and antiviral activity of nonannulated tetrazolylpyrimidines

Article abstract of DOI:10.1007/s10593-021-02922-6

[Figure not available: see fulltext.] Nonannulated tetrazolylpyrimidines in the structure of which the heterocyclic fragments are separated by hydrazinocarbonylmethyl, methylpyrazolyl groups or a sulfur atom were synthesized. Some of these compounds showed moderate in vitro activity against H1N1 subtype of influenza A virus. The selectivity index of the anti-influenza action of {5-[(4,6-dimethylpyrimidin-2-yl)sulfanyl]-1H-tetrazol-1-yl}acetic acid, which has very low cytotoxicity, was twice as high as the selectivity index of the reference drug rimantadine.

Full text of DOI:10.1007/s10593-021-02922-6

DOI 10.1007/s10593-021-02922-6
Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
Synthesis and antiviral activity
of nonannulated tetrazolylpyrimidines
Vladimir А. Ostrovskii1*, Gevorg G. Danagulyan2,3, Olga M. Nesterova1,
Yulia N. Pavlyukova1, Vladimir V. Tolstyakov1, Olga S. Zarubina1,
Pavel А. Slepukhin4, Yana L. Esaulkova5, Anna А. Muryleva5,
Vladimir V. Zarubaev5, Rostislav E. Trifonov1
1 Saint Petersburg State Institute of Technology (Technical University),
26 Moskovsky Ave., Saint Petersburg 190013, Russia; e-mail: va_ostrovskii@mail.ru
2 Russian–Armenian (Slavonic) University,
123 Hovsep Emin St., Yerevan 0051, Armenia; e-mail: gevorg.danagulyan@rau.am
3 Technological Center of Organic and Pharmaceutical Chemistry,
National Academy of Sciences of the Republic of Armenia,
26 Azatutyan Ave., Yerevan 0014, Armenia; e-mail: gdanag@email.com
4 Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences,
22/20 Sofyi Kovalevskoi St., Yekaterinburg 620108, Russia; e-mail: slepukhin@ios.uran.ru
5 Saint Petersburg Pasteur Research Institute of Epidemiology and Microbiology,
14 Mira St., Saint Petersburg 197101, Russia; e-mail: zarubaev@gmail.com
Submitted February 25, 2021
Accepted March 8, 2021
Translated from Khimiya Geterotsiklicheskikh Soedinenii,
2021, 57(4), 448–454
Nonannulated tetrazolylpyrimidines in the structure of which the heterocyclic fragments are separated by hydrazinocarbonylmethyl,
methylpyrazolyl groups or a sulfur atom were synthesized. Some of these compounds showed moderate in vitro activity against H1N1
subtype of influenza A virus. The selectivity index of the anti-influenza action of {5-[(4,6-dimethylpyrimidin-2-yl)sulfanyl]-1H-tetrazol-
1-yl}acetic acid, which has very low cytotoxicity, was twice as high as the selectivity index of the reference drug rimantadine.
Keywords: pyrimidines, tetrazoles, biological activity, linkers, properties, structure, synthesis.
The relevance of the development of new antiviral
drugs, as a rule, is determined by the presence or absence
of prophylactic vaccines against newly emerging infections,
as well as the ability of viruses to escape the host immune
response and develop drug-resistant strains.1 The current
situation has sharply exacerbated the problem of the
availability of drugs against highly pathogenic variants of
the influenza virus and the SARS2 coronavirus, the cause
of COVID-19. The solution of this problem is determined
by the effectiveness of the search for new drugs for the
treatment of influenza and associated infectious diseases.
Compounds of various structures are used in the treatment
of this viral disease (Fig. 1). In this context, drugs that
contain fragments of heterocyclic compounds, azoles and
azines, in their active pharmaceutical ingredients (API) are
of particular interest.
In 2014, riamilovir (Triazavirin) was registered as a drug
for the treatment of influenza in the Russian Federation.2
Triazavirin has recently been proven effective in treating
the new coronavirus infection and is well tolerated by
patients with COVID-19.3 Triazavirin API, 7-(methylsulfanyl)-
3-nitro[1,2,4]triazolo[5,1-c]triazin-4(1H)-one belongs to
annulated azoloazines, which indirectly indicates the
prospect of searching for new agents for influenza
chemotherapy in the ranks of biannulated (''hybrid'')
heterocyclic compounds based on azoles and azines.
Derivatives of tetrazole and pyrimidine are among the most
sought after scaffolds in modern medicinal chemistry.4,5 In
particular, it was shown that some tetrazole derivatives
exhibit pronounced activity against rhinoviruses6a and
various strains of influenza A virus.6b In this regard, the
synthesis and study of biological activity of binuclear
0009-3122/21/57(4)-0448©2021 Springer Science+Business Media, LLC
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Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
Figure 1. Active pharmaceutical ingredients of drugs used for influenza chemotherapy.
heterocyclic compounds containing both tetrazolyl and
pyrimidyl fragments in their structure are of interest. In the
case of annulated tetrazolylpyrimidines, the possibility of
azidoazomethine-tetrazole isomerism should be considered.7,8
For some represantives of this series, the equilibrium can
be shifted toward the open-chain azide form with
significant cytotoxicity.
In this study, a series of nonannulated tetrazolyl-
pyrimidines was synthesized, and their structure and anti-
influenza activity were investigated as well. Fragments of
different nature were chosen as linker groups connecting
the tetrazole and pyrimidine heterocycles – methylene, hydra-
zinylcarbonylmethyl, pyrazolyl groups and a sulfur atom.
This set of linkers would allow us to assess the prospects of
given structural types in regards to biological activity.
At the first stage of our study, we attempted to
synthesize 4,6-dimethyl-2-(5-phenyl-2H-tetrazol-2-yl)pyri-
midine (3) by direct hetarylation of the sodium salt of
5-phenyltetrazole (1) by 2-chloro-4,6-dimethylpyrimidine
(2). However, it was not possible to isolate the target
tetrazolylpyrimidine 3 from the reaction mixture. Instead of
nonannulated tetrazolylpyrimidine 3, a compound was
formed the properties of which were identical to those of
5,7-dimethyl-3-phenyl[1,2,4]triazolo[4,3-a]pyrimidine (4)
described earlier.9 A possible reason for the discovered
phenomenon is the thermolytic recyclization of interme-
diate 3 accompanied by the elimination of an N2 molecule
and the formation of annulated triazolylpyrimidine 4
(Scheme 1).
Considering this experience, we focused our efforts on
the synthesis of nonannulated tetrazolylpyrimidines in the
structure of which heterocyclic fragments are linked by
linker groups such as hydrazinocarbonylmethyl, pyrazolyl,
as well as a sulfur atom. To form a linking bridge in the
target compounds, we used classical methods such as
acylation of the terminal nitrogen atom of the NHNH2
group with carboxylic acid halides, hydrazinolysis of
esters, and the substitution of the chlorine atom in the
corresponding hetaryls for a sulfur atom by the action of
hetarylthiones.10 Hydrazides 7a,b were obtained by
acylation of 2-hydrazinylpyridines (5a) or 2-hydrazinyl-
4,6-dimethylpyrimidine (5b) with 5-phenyltetrazol-2-yl-
acetic acid chloride (6) (Scheme 2).
1-(4,6-Dimethylpyrimidin-2-yl)-3-[(5-phenyl-2H-tetrazol-
2-yl)methyl]-1H-pyrazol-5-ol (9) was accessed by hydra-
zinolysis of ethyl 3-oxo-4-(5-phenyl-2Н-tetrazol-2-yl)-
Scheme 2
R
Scheme 1
N
Et3N
+
NHNH2
O
N
MeCN, , 4 h
N
N
N
Cl
R
N
5a,b
6
R
N
N
H
N
N
N
N
R
H
N
N
O
7a R = H (48%)
b R = Me (78%)
449

Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
butanoate (8) by 2-hydrazinyl-4,6-dimethylpyrimidine (5b)
the intramolecular hydrogen bond O(1)–H(1)···N(6) with
the distance O(1)···N(6) 2.599 Å. At the same time, the
tetrazole and pyrazole fragments of the molecule at the
tetrahedral carbon atom are turned in relation to each other
at an angle of 88.4° which makes the conformation of this
compound propeller-like. A solid-state structural feature is
the packing of molecules in the form of stacks along the 0a
axis in such a way that the pyrimidine fragments of
neighboring molecules form interatomic π–π contacts at a
distance that is 0.05 Å less than the sum of the van der
Waals radii of the atoms of these fragments.
(Scheme 3). According to X-ray structural analysis data,
the pyrazole fragment of compound 9 exists in the aromatic
form – a hydroxy group is present at position 5. The
rendering of the structures of the molecules of compounds
7b and 9 according to X-ray structural analysis data is
shown in Figure 2.
Scheme 3
2-[(1-Phenyl-1H-tetrazol-5-yl)sulfanyl]pyrimidine (12a)
was obtained by hetarylation of 1-phenyl-5-sulfanyl-
tetrazole (10a) with 2-chloropyrimidine (11). Likewise,
{5-[(4,6-dimethylpyrimidin-2-yl)sulfanyl]-1H-tetrazol-1-yl}-
acetic acid (12b) was synthesized from 1-carboxymethyl-
5-sulfanyltetrazole (10b) and 2-chloro-4,6-dimethyl-
pyrimidine (2) (Scheme 4).
Scheme 4
According to the results of X-ray structural analysis,
compounds 7b and 9 are nonplanar systems. The torsion
angle НN(5)N(4)H of the hydrazine fragment is 110°. The
nitrogen atom in the pyrimidine ring has a trigonal pyramid
configuration. The phenyl substituent lies practically in the
same plane as the azole fragment, while the planes of the
heterocycles intersect at a significant (81°) angle to each
other. The peculiarities of molecular packing in the solid
state are determined by the presence of intermolecular
hydrogen bonds with the participation of protons of the
hydrazine fragment. A feature of the structure of compound
9 is its existence in the 5-hydroxypyrazole form, rather than
that of 2,3-dihydropyrazol-3-one. In this case, the
conformation of the compound turned out to be locked by
The prediction of the activity of nonannulated
tetrazolylpyrimidines 7a,b, 9, and 12a,b, performed using
the PASS computer system,11 indicates low (Pa 0.28–0.33)
probability of antiviral activity for the compounds of this
sample. However, low values of Pa factor are not always a
verdict since they may indirectly indicate the novelty of the
evaluated object.12 In this study, we investigated the in
vitro antiviral activity of nonannulated tetrazolyl-
pyrimidines 7a,b, 9, and 12a,b in the Laboratory of
Experimental Virology of Saint Petersburg Pasteur
Research Institute of Epidemiology and Microbiology. The
study was carried out using the MDCK cell culture (ATCC
CCL-34) against influenza virus strain A/Puerto Rico/8/34
(H1N1). Table 1 shows the values of 50% cytotoxic
concentration (CC50), 50% inhibitory concentration (IC50),
and selectivity index SI (the ratio of CC50 to IC50).
Rimantadine was used as the reference drug. Compounds
with a selectivity index of 10 and higher were considered
promising.
The data in Table 1 illustrate that compounds 7a,b, 9,
and 12a did not show activity against influenza virus strain
A/Puerto Rico/8/34 (H1N1). The best results were obtained
for compound 12b, the SI of which is twice that of the
reference drug (rimantadine), but, in contrast to it,
compound 12b has significantly lower cytotoxicity. This
result indirectly indicates the promise of further study of
compounds of this group and an expanded search for
substances with pronounced antiviral activity among non-
annulated tetrazolylpyrimidines the heterocyclic fragments
Figure 2. The molecular structures of compound 7b and 9 with
atoms represented as thermal vibration ellipsoids of 50%
probability.
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Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
Table 1. Antiviral properties of nonannulated tetrazolyl-
kept at this temperature for 8 h. The mixture was then
cooled, the formed precipitate was filtered off and dried in
a stream of air. Yield 0.81 g (61%), colorless crystals,
mp 259°С (decomp., PhMe–EtOH, 1:1) (decomp. temp.
258°C (PhMe–EtOH, 1:1)9). Rf 0.47 (CCl4–i-PrOH, 4:1,
25°С). IR spectrum, ν, cm–1: 3048 (С–H), 1630 (С6Н5),
1518 (C4N2H), 1448(CN4), 764 (C4N2H). 1H NMR
spectrum, δ, ppm: 2.17 (3Н, s, 5-СН3); 2.56 (3H, s, 7-CH3);
6.83 (1H, s, H pyrimidine); 7.51–7.65 (5Н, m, H Ph).
13C NMR spectrum, δ, ppm: 19.9 (C-5 CH3); 24.8 (C-7
CH3); 111.7 (C-6); 128.3 (C Ph); 129.7 (C Ph); 130.5
(C Ph); 131.5 (C Ph); 144.9 (C-3); 145.8 (C-9); 154.8
(C-7); 164.6 (C-5). Found, m/z: 225.1135 [М+H]+.
С13Н13N4. Calculated, m/z: 225.1135. Found, %: C 69.22;
H 5.28; N 24.80. С13Н12N4. Calculated, %: C 69.62;
H 5.39; N 24.98.
2-Hydrazinopyrimidine (5a). N2H4·H2O (1.1 ml,
14.0 mmol) was added with stirring at 10°С to a solution of
2-chloropyrimidine (11) (3.6 g, 25 mmol) in EtOH (15 ml).
The precipitate that formed after a few hours was filtered
off and dried in a stream of air. Yield 1.23 g (80%),
colorless crystals, mp 112–113°С (EtOH–H2O, 1:1)
(mp 112–113°С (EtOH–H2O, 1:1)17). Rf 0.11
(CCl4–i-PrOH, 4:1, 25°С). IR spectrum, ν, cm–1: 3255
(N–H val), 1602, 1577, 798 (C4N2H def). 1H NMR
spectrum, δ, ppm: 3.52 (1Н, br. s, NH); 6.58–6.60 (1Н, m,
H Ar); 6.97 (2Н, br. s, NH2); 8.27–8.29 (2H, m, H Ar).
13C NMR spectrum, δ, ppm: 110.9 (C-5); 158.3 (С-2);
164.8 (С-4,6). Found, m/z: 111.0665 [М+H]+. С4Н7N4.
Calculated, m/z: 111.0665. Found, %: C 44.28; H 5.12;
N 51.02. С4Н6N4. Calculated, %: C 44.63; H 5.49; N 50.88.
2-Hydrazino-4,6-dimethylpyrimidine (5b). N2H4·H2O
(6 ml, 124 mmol) was added with stirring at 10°С to a
solution of 2-chloro-4,6-dimethylpyrimidine (2) (3.6 g,
25 mmol) in EtOH (40 ml). The precipitate that formed
after a few hours was filtered off and dried in a stream of
air. Yield 2.4 g (70%), colorless crystals, mp 164–165°С
(EtOH–H2O, 1:1) (mp 165°С (EtOH–H2O, 1:1)18). Rf 0.54
(CCl4–i-PrOH, 4:1, 25°С). IR spectrum, ν, cm–1: 3307 (N–H,
val), 1371 (C–CH3), 1596, 1467, 794 (C4N2H def).
1H NMR spectrum, δ, ppm: 2.22 (6H, s, 2CH3); 3.36 (1H,
br. s, NH); 6.38 (1H, s, H Ar); 7.88 (2H, br. s, NH2).
13C NMR spectrum, δ, ppm: 23.9 (2CH3); 109.7 (C-5);
164.8 (С-2); 167.2 (C-4,6). Found, %: C 52.33; H 7.80;
N 40.54. С6Н10N4. Calculated, %: C 52.16; H 7.30;
N 40.55.
pyrimidines 7a,b, 9, and 12a,b against
influenza A H1N1 virus in MDCK cell cultures
Compound
СС50, μg/ml
IC50, μg/ml
SI
>300
>300
53.9
>300
>300
60
>300
>300
>30
170
31
1
1
7a
7b
2
9
12a
2
10
5
12b
Rimantadine
12
of which are separated by a sulfur atom. The synthesis and
study of the antiviral activity of analogs of compound 12b
containing various functional groups at the nitrogen atom
in position 1 of the tetrazole ring deserve further
development. Importantly, the influenza virus used in the
study is resistant to rimantadine (SI 5). The results of the
study of tetrazolylpyrimidine 12b indicate that this
compound may have a different mechanism of interaction
with the target as compared to adamantane derivatives.
This makes the evaluated group of compounds promising
as prototypes of anti-influenza drugs with an alternative
mechanism of action in comparison with existing drugs for
influenza therapy.
To conclude, nonannulated tetrazolylpyrimidines can be
recommended for in vivo evaluation of antiviral activity.
Experimental
IR spectra were registered on a Shimadzu 8400-FTIR
spectrometer in KBr pellets. 1H and 13C NMR spectra were
acquired on a Bruker Avance III spectrometer (400 and
100 MHz, respectively) in DMSO-d6 (compounds 5a,b, 6,
7a,b, 8, 9, 12a,b) and CDCl3 (compound 4) at 25°С with
TMS as internal standard. Elemental analysis was
performed on a Leco CHNS-932 elemental analyzer. Mass
spectra were recorded on a Bruker maXis impact ultra-high
resolution quadrupole time-of-flight mass spectrometer
(electrospray ionization, MeOH solvent). Melting points
were determined by the capillary method on a Büchi
M-560 apparatus with a heating rate of 1°C/min in the
melting range. Assessment of the purity of synthesized
compounds was done by TLC on Macherey-Nagel
Alugram Xtra SIL G plates, visualization in UV light
(254 nm).
The physiochemical properties of 1-phenyl-5-sulfanyl-
tetrazole (10a) and other commercial reagents, solvents,
and materials correspond to the data given in the catalogs
of Sigma-Aldrich, Merck, Fluka. 5-Phenyltetrazole sodium
salt (1),13 2-chloro-4,6-dimethylpyrimidine (2),14 1-carb-
oxymethyl-5-sulfanyltetrazole (10b),15 and 2-chloro-
pyrimidine (11)16 were obtained following known or
modified methods. The properties of compounds 1, 2, 10b,
and 11 correspond to those described previously.13–16
5,7-Dimethyl-3-phenyl[1,2,4]triazolo[4,3-a]pyrimidine
(4). 2-Chloro-4,6-dimethylpyrimidine (2) (0.86 g, 6.0 mmol)
was added with stirring to a suspension of 5-phenyl-
tetrazole sodium salt (1) (1.0 g, 6.0 mmol) in DMSO
(20 ml). The reaction mixture was heated to 150°С and
2-(5-Phenyltetrazol-2-yl)acetic acid chloride (6).
Crushed PCl5 (6.75 g, 32 mmol) was added to crushed
2-(5-phenyltetrazol-2-yl)acetic acid (6.0 g, 29 mmol). The
reaction mixture was heated in a boiling water bath
(reaction flask was closed by a distillation drip adapter with
a calcium chloride tube) until the mixture was homogenized
and gas evolution stopped. Upon completion of the
reaction, boiling hexane (75 ml) was added to the hot
reaction mixture, and the contents of the flask was cooled
to 10°C. Acid chloride 6 was filtered off, washed on a filter
with chilled hexane, dried for 1 h under reduced pressure
over P2O5 in a pistol dryer heated with CHCl3 vapor. Yield
6.43 g (96%), colorless crystals, mp 103–104°С (mp 103–
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Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
104°С (hexane)19). IR spectrum, ν, cm–1: 2985, 2945 (С–Н),
(2Н, s, СН2); 6.57** (1H, s, H pyrimidine); 6.71* (1H, s,
H pyrimidine); 7.57–7.60 (3H, m, H Ph); 8.06–8.09 (2H,
m, H Ph); 8.90** (1H, s, NHC4N2H4); 9.09* (1H, s,
NHC4N2H4); 9.79* (1H, s, NHC(O)); 10.40** (1H, s, NHC
(O)). 13C NMR spectrum, δ, ppm: 23.9** (2CH3); 24.0*
(2CH3); 53.9* (CH2); 54.1** (CH2); 111.9** (C-5
pyrimidine); 112.9* (C-5 pyrimidine); 126.8 (C Ph); 127.3
(C Ph); 129.8 (C Ph); 131.1 (C Ph); 162.9 (C-2
pyrimidine); 164.5** (C=O); 164.7 (C tetrazole); 167.6**
(C-4,6 pyrimidine); 168.1 (C-4,6 pyrimidine); 169.2*
(C=O). Found, m/z: 325.1530 [М+H]+. С15Н17N8O.
Calculated, m/z: 325.1520. Found, %: С 55.43; Н 4.82;
N 34.25. С15Н16N8O. Calculated, %: С 55.55; Н 4.97;
N 34.55.
1777 (C=O), 1450, 1278, 1148, 1073, 1039, 1022, 923
1
(СN4), 729, 688 (C6H5). H NMR spectrum, δ, ppm: 5.84
(2Н, s, CH2); 7.53–7.49 (3Н, m, H Ph); 8.17–8.14 (2Н, m,
H Ph). 13C NMR spectrum, δ, ppm: 60.7 (CH2); 126.6
(C Ph); 127.2 (C Ph); 129.2 (C Ph); 131.0 (C Ph); 166.2
(C tetrazole); 166.5 (C=O). Found, %: C 48.31; H 3.20;
N 25.60. C9H7СlN4O. Calculated, %: C 48.55; H 3.17;
N 25.17.
N'-(Pyrimidin-2-yl)-2-(5-phenyl-2H-tetrazol-2-yl)aceto-
hydrazide (7a), a mixture of isomers E/Z in a 3.6:1 ratio.
Et3N (0.97 g, 9.60 mmol) was added with stirring to a
solution of 2-hydrazinopyrimidine (5a) (1.0 g, 9.08 mmol)
in MeCN (20 ml). The reaction mixture was cooled to
0–5°С, and 2-(5-phenyltetrazol-2-yl)acetic acid chloride
(6) (2.0 g, 8.98 mmol) was added with stirring. The reaction
mixture was heated under reflux for 4 h. At the end
heating, the solvent was removed under reduced pressure.
The residue was washed with distilled H2O (2×20 ml) and
dried in a stream of air. Yield 1.3 g (48%), colorless
crystals, mp 235–236°С (DMF–H2O, 1:1). Rf 0.52 (CCl4–
i-PrOH, 4:1, 25°С). IR spectrum, ν, cm–1: 3278 (NH val),
3033 (C–H val), 1689 (C=O val), 1448, 1043 (CN4), 819,
788 (C4N2H def). 1H NMR spectrum, δ, ppm: 5.64* (2H, s,
CH2); 5.66** (2Н, s, СН2); 6.80–6.82** (1H, m,
H pyrimidine); 6.95–6.97* (1H, H pyrimidine); 7.55–
7.60** (5H, m, H Ar); 8.02–8.09* (5Н, m, H Ar); 8.40–
8.42** (2H, m, H Ar); 8.53–8.54* (2H, m, H Ph); 9.17**
(1H, s, NHC4N2H4); 9.32* (1H, s, NHC4N2H4); 9.84* (1H,
s, NHC(O)); 10.45** (1H, s, NHC(O)). 13C NMR
spectrum, δ, ppm: 53.9* (CH2); 54.1** (CH2); 113.3**
(C-5 pyrimidine); 114.4* (C-5 pyrimidine); 126.8 (C Ph);
127.2 (C Ph); 129.8 (C Ph); 131.1 (C Ph);158.6 (C-2
pyrimidine); 163.0** (C=O); 163.2* (C=O); 164.6**
(C-4,6 pyrimidine); 164.7* (C-4,6 pyrimidine); 169.4
(C tetrazole). Found, m/z: 297.1219 [М+H]+. С13Н13N8O.
Calculated, m/z: 297.1207. Found, %: С 52.77; Н 4.12;
N 37.49. С13Н12N8O. Calculated, %: С 52.70; Н 4.08;
N 37.82.
Ethyl 3-oxo-4-(5-phenyl-2Н-tetrazol-2-yl)butanoate
(8). Ethyl 4-chloro-3-oxobutanoate (10.0 g, 61 mmol) and
benzyltriethylammonium chloride (0.01 g, 0.044 mmol)
were added with stirring at 80°С to a suspension of
5-phenyltetrazole sodium salt (1) (10.2 g, 61 mmol) in
MeCN (150 ml). The reaction mixture was stirred at 80°С
for 3 h and cooled to room temperature. The formed
precipitate was filtered off and dried in a stream of air.
Yield 10.65 g (56%), beige crystals, mp 75–76°С (PhMe–
EtOH, 1:1). Rf 0.8 (CCl4–i-PrOH, 4:1, 25°С). IR spectrum,
ν, cm–1: 1715, 1749 (С=О), 1530 (С6Н5), 1452, 1044
1
(СN4). H NMR spectrum, δ, ppm (J, Hz): 1.22 (3Н, t,
J = 6.9, СН3); 3.92 (2Н, s, 2-СН2); 4.15 (2Н, q, J = 6.9,
СН2); 6.10 (2Н, s, 4-СН2); 7.56–7.59 (2Н, m, H Ph); 8.06–
8.10 (3Н, m, H Ph). 13C NMR spectrum, δ, ppm: 14.4
(CH3); 46.6 (C-2); 61.2 (CH2); 61.3 (C-4); 126.8 (C Ph);
127.1 (C Ph); 129.8(C Ph); 131.1 (C Ph); 164.7
(C tetrazole); 166.8 (C(1)=O); 195.9 (C(3)=O). Found, m/z:
297.0958 [М+Na]+. С13Н14N4NaO3. Calculated, m/z:
297.0958. Found, %: C 56.89; H 5.01; N 20.27.
С13Н14N4O3. Calculated, %: C 56.93; H 5.15; N 20.43.
1-(4,6-Dimethylpyrimidin-2-yl)-3-[(5-phenyl-2Н-
tetrazol-2-yl)methyl]-1Н-pyrazol-5-ol (9). Ethyl 3-oxo-
4-(5-phenyl-2H-tetrazol-2-yl)butanoate (8) (1.16 g,
4.23 mmol) was added to a solution of 2-hydrazino-
4,6-dimethylpyrimidine (5b) (0.58 g, 4.23 mmol) in
i-PrOH (50 ml). The reaction mixture was heated under
reflux for 8 h. The mixture was concentrated to half the
original volume by evaporation under reduced pressure, the
remaining mixture was cooled to room temperature, and
10% aqueous NaOH (20 ml) was added. The formed
precipitate was filtered off, the filtrate was acidified with
HCl to pH 2–3. The formed precipitate was filtered off and
dried in a stream of air. Yield 0.97 g (66%), beige crystals,
mp 175–176°С (PhMe). Rf 0.58 (CCl4–i-PrOH, 4:1, 25°С).
IR spectrum, ν, cm–1: 1448, 1040 (CN4), 784 (C4N2Н def).
1H NMR spectrum, δ, ppm: 2.50 (6H, s, 2CH3); 5.71 (1H,
s, H Ar); 5.93 (2H, s, CH2); 7.28 (1H, s, H Ar); 7.56–7.58
(3H, m, H Ph); 8.06–8.08 (2H, m, H Ph); 12.27 (1H, s,
OH). 13C NMR spectrum, δ, ppm: 23.8 (2CH3); 51.8 (CH2);
87.0 (C-4 pyrazole); 118.2 (C-5 pyrimidine); 126.8 (C Ph),
127.3 (C Ph); 129.8 (C Ph); 131.1 (C Ph); 147.4 (C-3
pyrazole); 156.6 (C-2 pyrimidine); 158.1 (C-5 pyrazole);
164.7 (C tetrazole); 168.8 (C-4,6 pyrimidine). Found, m/z:
349.1527 [М+H]+. С17Н17N8O. Calculated, m/z: 349.1520.
N'-(4,6-Dimethylpyrimidin-2-yl)-2-(5-phenyl-2H-tetra-
zol-2-yl)acetohydrazide (7b), a mixture of isomers E/Z in
a 2.9:1 ratio. Et3N (0.44 g, 4.31 mmol) and 2-hydrazino-
4,6-dimethylpyrimidine (5b) (0.6 g, 4.35 mmol) were
added with stirring to a solution of 2-(5-phenyltetrazol-
2-yl)acetic acid chloride (6) (0.96 g, 4.31 mmol) in MeCN
(20 ml). The reaction mixture was heated under reflux for
4 h. The mixture was concentrated to half the original
volume by evaporation under reduced pressure. The
remaining mixture was cooled, the formed precipitate was
filtered off, washed on a filter with distilled H2O (2×20 ml),
and dried in a stream of air. Yield 1.1 g (78%), colorless
crystals, mp 228°С (DMF–H2O, 1:1). Rf 0.54 (CCl4–
i-PrOH, 4:1, 25°С). IR spectrum, ν, cm–1: 3307 (N–H val),
3033 (C–H val), 1683 (C=O val), 1450, 1041 (CN4), 819,
1
788 (C4N2H def). H NMR spectrum, δ, ppm: 2.26** (6H,
s, 2CH3); 2.33* (6H, s, 2CH3); 5.61* (2H, s, CH2); 5.62**
* Hereinafter in the Experimental, one asterisk (*) denotes the signals of
the Z-isomer, two asterisks (**) denote the signals of the E-isomer.
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Chemistry of Heterocyclic Compounds 2021, 57(4), 448–454
Found, %: С 58.35; Н 4.71; N 32.36. С17Н16N8O.
Calculated, %: С 58.61; Н 4.63; N 32.17.
Multiskan FC photometer (Thermo Fisher Scientific, USA)
at a wavelength of 540 nm. Based on the obtained data, the
CC50 value was calculated, that is, the concentration of the
compound leading to a decrease in optical density by half
compared to the wells without addition of compounds.
The study of the antiviral activity of compounds 7a,b,
9, 12a,b was carried by determining the reduction of the
degree of cytopathic effect. The experiments used influenza
virus strains A/Puerto Rico/8/34 (H1N1) and B/Florida/4/2006.
The studied compounds in various concentrations were
added to the cells in the wells of the plate, incubated for 1 h
at 36°C in 5% CO2, then the cells were infected with the
virus at a dose of 0.01 TCID50 per cell. The cells were
incubated for 72 h at 36°C in 5% CO2, after which the cell
viability assay was performed using the methyl tetrazolium
assay as described above. Based on the obtained data, a
50% inhibitory concentration was calculated for each
compound, that is, the concentration that reduces the
degree of viral cell destruction by 50%.
X-ray structural analysis of compounds 7b and 9 was
carried out on an Xcalibur 3 automatic 4-circle diffracto-
meter equipped with a CCD detector. All calculations were
carried out in the Olex software shell using the SHELX
software package.21 The full set of X-ray structural data for
compounds 7b and 9 was deposited at the Cambridge
Crystallographic Data Center (deposits CCDC 1818619
and CCDC 2047135, respectively).
2-[(1-Phenyl-1H-tetrazol-5-yl)sulfanyl]pyrimidine (12a).
Et3N (0.85 g, 8.42 mmol) and 2-chloropyrimidine (11)
(1.1 g, 9.60 mmol) were added with stirring to a solution of
1-phenyl-1Н-tetrazole-5-thiol (10a) (1.5 g, 8.42 mmol) in
MeCN (10 ml). The reaction mixture was heated under
reflux for 4 h. The mixture was concentrated to half the
original volume by evaporation under reduced pressure, the
remaining reaction mixture was cooled, and H2O (50 ml)
was added. The formed precipitate was filtered off and
dried in a stream of air. Yield 1.0 g (46%), beige crystals,
mp 76°С (PhMe–EtOH, 1:1). Rf 0.17 (CCl4–i-PrOH, 4:1,
25°С). IR spectrum, ν, cm–1: 1554 (С6Н5), 1444, 1045
(СN4), 765 (C4N2Н def). 1H NMR spectrum, δ, ppm: 7.30–
7.32 (1Н, m, H pyrimidine); 7.56–7.57 (3Н, m, H Ph); 7.69–
7.71 (2Н, m, H pyrimidine); 8.58–8.59 (2Н, m, H Ph).
13C NMR spectrum, δ, ppm: 119.8 (C-5); 125.7 (С Ph);
130.0 (С Ph); 131.2 (С Ph); 133.9 (С Ph); 148.3 (С-4,6);
159.3 (C-2); 167.1 (C tetrazole). Found, m/z: 257.0616
[М+Н]+. С11Н9N6S. Calculated, m/z: 257.0604. Found, %:
C 51.59; H 3.28; N 32.44; S 12.62. С11Н8N6S. Calculated,
%: C 51.55; H 3.15; N 32.79; S 12.51.
{5-[(4,6-Dimethylpyrimidin-2-yl)sulfanyl]-1H-tetrazol-
1-yl}acetic acid (12b). Et3N (1.27 g, 12.57 mmol) and
chloro-4,6-dimethylpyrimidine (2) (0.89 g, 6.24 mmol)
were added with stirring to a solution of 2-(5-sulfanyl-1Н-
tetrazol-1-yl)acetic acid (10b) (1.0 g, 6.25 mmol) in MeCN
(25 ml). The reaction mixture was heated under reflux with
stirring for 4 h. The solvent was removed under reduced
pressure. The oily residue was dissolved in H2O (20 ml).
The resulting solution was acidified with HCl to pH 2–3
and extracted with EtOAc (3×20 ml). The combined
extracts were washed with H2O and dried over MgSO4.
EtOAc was removed under reduced pressure. Yield 1.1 g
(66%), beige crystals, mp 92°С (PhMe). Rf 0.2 (CCl4–
i-PrOH 4:1, 25°С). IR spectrum, ν, cm–1: 2995 (О–Н),
1743 (СН2–СООН), 1423, 1037 (СN4 val), 883 (C4N2Н
This work was supported by the Russian Foundation for
Basic Research (projects 20-53-00039-Bel_a and 20-53-
05010 Arm_а, 20RF-138).
X-ray structural analysis for compounds 7b and 9 was
carried out on the equipment of the Center for Collective
Use ''Spectroscopy and Analysis of Organic Compounds''
of Postovsky Institute of Organic Synthesis of the Ural
Branch of the Russian Academy of Sciences.
1
References
def). H NMR spectrum, δ, ppm: 2.31 (6Н, s, 2СН3); 5.38
(2Н, s, СН2); 7.15 (1H, s, H pyrimidine); 13.72 (1Н, br. s,
ОН). 13C NMR spectrum, δ, ppm: 23.7 (2CH3); 49.2 (CH2);
118.8 (C-5); 148.5 (C-2); 165.3 (C tetrazole); 167.5
(C-4,6); 169.1 (C=O). Found, m/z: 267.0681 [М+H]+.
С9Н11N6O2S. Calculated, m/z: 267.0659. Found, %:
C 40.97; H 4.03; N 31.35; S 12.05. С9Н10N6O2S.
Calculated, %: C 40.60; H 3.79; N 31.56; S 12.04.
The study of the biological activity of compounds
7a,b, 9, 12a,b. A series of threefold dilutions (300–
3.7 μg/ml) were prepared from the studied compounds. The
dilutions were introduced into the wells of plates with a
monolayer of MDCK cells. The plates were incubated for
72 h at 36°C in 5% CO2. The analysis of cell survival was
carried out using the methyl tetrazolium (MTT) assay:
MTT solution was added to the wells, wherein by the
action of mitochondrial enzymes it transforms into an
insoluble violet formazan derivative.20 The plates with cells
were kept at 36°C in an atmosphere of 5% CO2 for 2 h. The
precipitate was dissolved in DMSO (0.1 ml per well). The
optical density in the wells was measured on a Thermo
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