Synthesis and properties of 5-aryl-3-diazo-3H-pyrazoles and 3-aryl-1H-pyrazole-5-diazonium salts. Preparation and cytolytic activity studies of 2-arylpyrazolo-[5,1-c][1,2,4]benzotriazines

Article abstract of DOI:10.1007/s10593-019-02406-8

[Figure not available: see fulltext.] A comparative analysis of physicochemical properties and reactivity of 3-aryl-1H-pyrazole-5-diazonium tetrafluoroborates and 5-aryl-3-diazo-3H-pyrazoles in azo coupling reactions is presented. It is shown that diazonium salts have higher reactivity compared to the respective 3-diazopyrazoles, which is in agreement with their physicochemical properties. Heterocyclization of the synthesized azo compounds provided 2-arylpyrazolo[5,1-c][1,2,4]benzotriazines, which were screened for antitumor activity against human uterine endothelium cancer cells (HeLa cell line) and human skin fibroblasts using the MTT assay and flow cytometry. It was found that all of the tested compounds exhibited moderate to high cytotoxic activity. The best results were obtained with 6,8-dimethoxy-2-phenylpyrazolo-[5,1-c][1,2,4]benzotriazine.

Full text of DOI:10.1007/s10593-019-02406-8

DOI 10.1007/s10593-019-02406-8
Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
Synthesis and properties of 5-aryl-3-diazo-3H-pyrazoles
and 3-aryl-1H-pyrazole-5-diazonium salts.
Preparation and cytolytic activity studies of 2-arylpyrazolo-
[5,1-c][1,2,4]benzotriazines
Dar'ya L. Alekseeva1, Viktoriya Yu. Rakhimova1, Artem S. Minin1–3,
Anna V. Belousova1,4, Elena V. Sadchikova1,2*
1 Ural Federal University named after the first President of Russia B. N. Yeltsin,
19 Mira St., Yekaterinburg 620002, Russia; e-mail: e.v.sadchikova@urfu.ru
2 Ural State Medical University,
32 Dekabristov St., Yekaterinburg 620026, Russia; e-mail: e.v.sadchikova@urfu.ru
3 M. N. Mikheev Institute of Metal Physics, Ural Branch of the Russian Academy of Sciences,
18 Sofii Kovalevskoy St., Yekaterinburg 620108, Russia; e-mail: calamatica@gmail.com
4 Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences,
106 Pervomayskaya St., Yekaterinburg 620049, Russia; e-mail: an77777778@gmail.com
Submitted July 23, 2018
Accepted after revision November 18, 2018
Translated from Khimiya Geterotsiklicheskikh Soedinenii,
2018, 54(12), 1145–1152
A comparative analysis of physicochemical properties and reactivity of 3-aryl-1H-pyrazole-5-diazonium tetrafluoroborates and 5-aryl-
3-diazo-3H-pyrazoles in azo coupling reactions is presented. It is shown that diazonium salts have higher reactivity compared to the
respective 3-diazopyrazoles, which is in agreement with their physicochemical properties. Heterocyclization of the synthesized azo
compounds provided 2-arylpyrazolo[5,1-c][1,2,4]benzotriazines, which were screened for antitumor activity against human uterine
endothelium cancer cells (HeLa cell line) and human skin fibroblasts using the MTT assay and flow cytometry. It was found that all of
the tested compounds exhibited moderate to high cytotoxic activity. The best results were obtained with 6,8-dimethoxy-2-phenylpyrazolo-
[5,1-c][1,2,4]benzotriazine.
Keywords: 2-arylpyrazolo[5,1-c][1,2,4]benzotriazines, 3-diazopyrazoles, pyrazole-5-diazonium salts, antitumor activity, azo coupling,
diazotation, heterocyclization.
Large scale in vitro and in vivo studies of natural
biologically active compounds and their synthetic analogs
have resulted in the discovery of many new, clinically
important antitumor drugs. Despite significant advances in
therapy, cancer remains the second most common cause of
mortality in the world, after infectious diseases that cause
70% of all deaths.1 For this reason, the search for new
anticancer drugs remains an essential task for modern
science.
heterocyclic systems showing antimicrobial2 and
anxiolytic3 activity, as well as for the synthesis of drugs
suitable for treatment of central nervous system disorders.4
Some of the most significant products obtained on their
basis are pyrazolo[5,1-c][1,2,4]triazine derivatives, which
bear some structural similarity with purine bases, enabling
their use as antimetabolites in antiviral5 and antitumor
therapy.6
Despite the broad synthetic applications of azoles
containing diazo motif in their structure, many
experimental studies do not specify the exact form of these
Diazopyrazoles and pyrazolediazonium salts are of
interest as key reagents in the construction of new
0009-3122/18/54(12)-1145©2018 Springer Science+Business Media, LLC
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Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
109.4–110.7
107.8–109.7
7.80–7.96
compounds used in reactions, complicating systematic
8.00–8.13
analysis and prediction of the outcome of subsequent
synthetic steps.7 We have previously studied methods for
the preparation, properties,8 and reactivity of 5-substituted
4-diazoimidazoles and the respective 4-substituted
imidazole-5-diazonium salts in the typical reactions of
aromatic diazonium salts.9
Ar
Ar
145.8–147.1
149.8–151.0
+
+
N
N
N
N
N
H
2a–d
N
N–
N
12.20–13.00
3a d
–
122.7–124.1
112.6–113.4
N2 = 2271–2280 cm–1
max = 256262 nm
N2 = 2140–2152 cm
max = 274–279 nm
1
The goal of this work was to study the properties of
5-substituted 3-diazopyrazoles and related pyrazole-5-di-
azonium salts, to use them as reagents for preparing a series
of new pyrazolo[5,1-c][1,2,4]benzotriazines, as well to
evaluate the antitumor activity of the obtained compounds.
The diazotation of 1Н-pyrazol-5-amines 1a–d was
performed by treating suspensions of amines 1a–d in
48% aqueous HBF4 solution with an aqueous NaNO2
solution, while carefully maintaining the temperature
between –5 and 0°С. 3-Aryl-1Н-pyrazole-5-diazonium
tetrafluoroborates 2a–d were isolated as individual
compounds in high yields. The suspensions of compounds
2a–d in CHCl3 were subsequently neutralized with aqueous
NaHCO3 solution to рН 4–5 at 0°С, providing the
respective 5-aryl-3-diazo-3H-pyrazoles 3a–d as individual
compounds (Scheme 1).10
a Ar = Ph, b Ar = 4-MeC6H4, c Ar = 4-MeOC6H4, d Ar = 4-ClC6H4
Figure 1. The spectral characteristics (IR, UV, H and 13C NMR
1
spectra) of compounds 2a–d and 3a–d (NMR chemical shifts are
given in ppm).
case of 3-aryl-1Н-pyrazole-5-diazonium tetrafluoroborates
2a–d. The frequency of stretching vibrations of the diazo
group can serve as a specific indicator for the relative
electrophilicity: the higher the frequency, the higher is the
electrophilicity of the respective group and the more
reactive such compounds will be toward nucleophiles.
1H NMR spectra of 5-aryl-3-diazo-3H-pyrazoles 3a–d
lacked NH proton signals and contained a singlet due to the
proton bonded to the С-4 carbon atom of pyrazole ring at
7.80–7.96 ppm. At the same time, the spectra of the
respective 3-aryl-1Н-pyrazole-5-diazonium salts 2а–d
featured signals of acidic protons in the range of 12.20–
13.00 ppm and the Н-4 proton signal was shifted downfield
to 8.00–8.13 ppm. In the case of unsubstituted pyrazoles,
the signal of this proton appeared at 6.25 ppm.11 The
mutual conversion between the different forms of diazo
compounds was also investigated using 1H NMR
Scheme 1
R1
R1
3'
4'
6'
2'
1'
5'
NaNO2, HBF4
3
4
H2O
5 0°C, 45 min
77 84%
+
N
N
NH2
N
N
5
N
H
N
1
H
–
spectroscopy. When H NMR spectra were acquired with
BF4
1a–d
2a–d
the addition of 0.5 equiv, 1 equiv, and an excess of
deuterated trifluoroacetic acid (СF3CO2D) to a solution of
compound 3d in DMSO-d6, the signal of the proton bonded
to the С-4 carbon atom of pyrazole ring was shifted
downfield by ~0.2 ppm. At the same time, all of the
observed signals matched the spectrum of the individual
diazonium salt 2d. It should be noted that under the
conditions of gradual acidification, the intensity of Н-4
signal for diazo compound 3d did not decrease and the
respective signal for salt 2d did not increase. Thus, there
was no direct prototropic equilibrium between compounds
3d and 2d. When 0.5 equiv of СF3CO2D were added to
DMSO-d6 solution of compound 3d and the temperature
was gradually increased from –40 to 30°С, we observed
neither the formation of an intermediate product, nor
gradual generation of diazonium salt 2d, but only
instantaneous conversion of the diazo compound from one
form to the other. This means that diazo compound 3d did
not directly convert to salt 2d. Analogously to our earlier
results from the studies of acid-base transformations in the
series of 4-diazoimidazoles, it can be proposed that this
equilibrium involves at least one additional chemical
species.12
R1
3'
4'
2'
1'
5'
NaHCO3
5
6'
4
CHCl3 H2O
5 0°C, 5 10 min
74 85%
+
N
N
3
N
N
3a–d
a R1 = H, b R1 = Me, c R1 = OMe, d R1 = Cl
The structures of the synthesized 1H-pyrazole-5-di-
azonium salts 2a–d and 3-diazo-3H-pyrazoles 3a–d were
studied by using UV and IR spectra, one-dimensional
1H and 13С NMR spectroscopy, as well as two-dimensional
1Н–13С HMBC experiments (Fig. 1). UV spectra recorded
in MeCN showed absorption maxima in the range of 256–
262 nm for 3-aryl-1Н-pyrazole-5-diazonium tetrafluoro-
borates 2a–d and in the range of 274–279 nm for 5-aryl-
3-diazo-3H-pyrazoles 3a–d. Compared to our previously
published characterization of imidazole derivatives,8 a 36–
42 nm hypsochromic shift was observed for the diazonium
salts and 56–59 nm hypsochromic shift for the diazo com-
pounds. IR spectra of compounds 3a–d showed a strong
narrow absorption band in the range of 2140–2152 cm–1
due to stretching vibrations of the diazo group. The IR
absorption band due to streching vibrations in the
diazonium group was shifted to 2271–2280 cm–1 in the
The assignment of carbon signals in 13С NMR spectra of
compounds 2a–d and 3a–d was based on complex analysis
of two-dimensional NMR spectra, including 1Н–13С
HMBC experiment. The observed correlations allowed us to
1146

Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
96.3–97.9
7.66–8.05
R1
Scheme 2
2a–d
Method I
3'
R2
4'
R1
89.9–91.8
6.67–6.93
162.3–164.5
4'
3'
AcOH
rt, 1–7 h
84 98%
2'
1'
5'
147.1–147.4
2'
1'
5'
6'
2
3
MeO
OMe
p-TsOH
3
6'
4
R2
3a
4a,b
(10 mol %)
N
N
N
5
6''
N
N
N
1''
2''
AcOH
80 90°C, 3–8 h
77 94%
5''
4''
OMe
9a
N
N
144.6–145.4
H
9
5a
MeO
5a–h
R2
3''
6
Method II
8
MeO
7
3a–d
152.8–154.2
6a–h
CHCl3
rt, 7–9 days
65 83%
a R1 = R2 = H; b R1 = Me, R2 = H; c R1 = OMe, R2 = H; d R1 = Cl, R2 = H;
e R1 = H, R2 = OMe; f R1 = Me, R2 = OMe; g R1 = R2 = OMe; h R1 = Cl, R2 = OMe
unequivocally interpret the chemical shifts of quaternary
carbon atoms. 13C NMR signal of С-5 carbon atom bonded
to the diazonium group showed a downfield shift to 122.7–
124.1 ppm relative to the C-3 carbon atom at the diazo
group (112.6–113.4 ppm). It should be also noted that in all
of the studied compounds the carbon atom bonded to the
diazo moiety showed an upfield shift of 13C NMR signal by
~10 ppm in the diazonium salts and by ~20 ppm in
diazopyrazoles, compared to the typical range for
sp2-hybridized carbon atoms in unsubstituted rings.10 The
downfield shift of Н-4 proton signal and С-5 carbon signal
in the spectra of 3-aryl-1Н-pyrazole-5-diazonium salts 2a–d
compared to the spectra of diazopyrazoles 3a–d pointed to
a decreased electron density in the heteroaromatic ring,
which enhanced the electrophilic properties of the
diazonium group and, as a consequence, increased its
reactivity toward nucleophilic agents.
We explored the reactivity of 3-aryl-1Н-pyrazole-5-di-
azonium tetrafluoroborates 2a–d and the respective 5-aryl-
3-diazo-3H-pyrazoles 3a–d in azo coupling reactions with
1,3-dimethoxybenzene (4a) and 1,3,5-trimethoxybenzene
(4b) (Scheme 2). It was shown that 1Н-pyrazole-5-di-
azonium salts 2a–d reacted with polymethoxyarenes 4a,b
at room temperature in glacial AcOH over 4–7 and 1–3 h,
respectively. These reactions provided azo compounds 5a–h
in 84–98% yields. It should be noted that the reactivity of
5-aryl-3-diazo-3H-pyrazoles 3a–d in azo coupling
reactions was substantially lower than that of the respective
diazonium salts 2a–d. The reaction in CHCl3 required 7–
9 days and gave azo compounds 5a–h in yields that were
on average 15–20% lower. It is important to emphasize that
chromatographic analysis of the reaction mixture did not
reveal the presence of demethylation products that were
generated in an analogous transformation of imidazoles.8 In
general, the reactivity of compounds 2a–d and 3a–d in azo
coupling reactions was somewhat higher compared to
imidazole derivatives, but lower than observed for triazole
derivatives.13
shift of the С-3a carbon signal in 13C NMR spectra of
compounds 6a–h (to 147.1–147.4 ppm), compared to the
signal of С-5 carbon atom in azo compounds 5a–h (162.3–
164.5 ppm), which occurred due to the formation of a
1
conjugated π-bond system. The analysis of Н–13С HMBC
spectra allowed to clearly identify the quaternary carbon
atoms, as well as the carbon atoms bonded with hydrogen
atoms. For example, the chemical shift values of С-3 and С-2
carbon atoms for the respective pairs of compounds 5a–h and
6a–h were established from cross peaks with the aromatic
Н-2',6' protons, as well as the Н-4 (compounds 5a–h) and
Н-3 (compounds 6a–h) protons of the pyrazole ring that
showed spin-spin coupling. 13C NMR signals of carbon
atoms directly bonded with hydrogen atoms were identified
from the presence of direct spin-spin coupling constants in the
range of 179.0–184.2 Hz for the С-4 atoms (compounds 5a–h)
and С-3 atoms (compounds 6a–h) of the pyrazole ring.
Thus, the observed physicochemical characteristics of
compounds 2a–d and 3a–d showed good correlation with the
synthetic studies aimed at exploring their reactivity at the
terminal nitrogen atom, as well as the data of X-ray structural
analysis for related compounds.15 These findings enable the
use of physicochemical characteristics for the design of
synthetic strategies involving five-membered nitrogen
heterocycles containing diazo functionality in their
structures.
The current pharmaceutical screening methodology NCI-60
was adopted in 1990 by the National Cancer Institute, USA
and includes testing of compounds on an array of 60
human cell lines derived from various tumors.16 However,
regardless of the spectrum of physiological activity, the
investigation of biological properties of newly synthesized
compounds should be started from the evaluation of their
cytotoxicity.17
In the current work, we performed cytotoxicity evalua-
tion of the obtained compounds according to colorimetric
MTT assay.18 The cell lines selected for testing were the
well-known HeLa cells often used for such studies and
human skin fibroblasts.
The structure of compounds 5a–h was confirmed using
physicochemical methods of analysis and by conversion to
the respective pyrazolo[5,1-c][1,2,4]benzotriazines 6a–h,
which were formed by intramolecular cyclization of azo
compounds 5a–h (Scheme 2). The mechanism of this
transformation has been considered in the literature.14 The
formation of heterocyclization products was indicated by
the absence of NH proton signal and also by the upfield
It should be noted that biological activity studies of
compounds 6a–h were complicated by the fact that not all
of them were sufficiently soluble in water or in phosphate
buffer solution serving as the basis of DMEM cell culture
medium. In order to improve the solubility in water and to
sterilize the solutions, all compounds were treated by
ultrasonication. The cytotoxic activity determination and
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Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
comparison of pyrazolo[5,1-c][1,2,4]benzotriazines 6a–h
was based on a series of experiments in which several test
concentrations of the study compounds were used: 0.065
and 0.016 mmol/l in phosphate buffer and 0.016 mmol/l in
1% aqueous DMSO solution. The appropriate amount of
pure DMSO solution was used as a blank for the
compounds that were introduced into the growth medium
in the form of DMSO solutions.
The best results in the experiments on HeLa tumor cells
were obtained with compounds 6e,f,h that were dissolved
in phosphate buffer (0.065 mmol/l concentration), although
they did not exhibit strong antitumor activity when
dissolved in 1% aqueous DMSO solution at 0.016 mmol/l
concentration, except for compound 6e. When studying the
effect of the same compounds at analogous concentrations
on human skin fibroblasts, strong toxicity was not observed
except for compounds 6f,h at 0.065 mmol/l concentration.
Compound 6e showed strong activity against HeLa cells
both at 0.016 mmol/l concentration in 1% aqueous DMSO
solution and at 0.065 mmol/l concentration in phosphate
buffer.
The most cytotoxic compound 6e was further
characterized using flow cytometry method (Fig. 2). Cell
death according to the apoptotic mechanism was observed,
which may be of interest to medicine, as apoptotic cell
death is less harmful to the surrounding tissues than tumor
necrosis.19
Figure 2. The light-scattering dot diagram obtained for compound
6e with flow cytofluorimetry method.
Table 1. The cytotoxic activity of compound 6e
on HeLa cell culture (fraction of dead cells, %)
Concentration of compound
in phosphate buffer, mmol/l
Type of cell death
Control
0.030
53.5
1.4
0.015
24.6
1.2
0.003
19.3
1.1
Early apoptosis
Early necrosis
Late necrosis
13.2
0.2
0.3
0.3
0.2
0.1
1
PerkinElmer LAMBDA 35 spectrophotometer. Н, 13С, and
1H–13C HMBC NMR spectra were acquired on a Bruker
Avance II 400 instrument (400 and 100 MHz) in DMSO-d6,
using TMS as internal standard. Melting points were
determined on a Stuart SMP30 melting point apparatus.
Elemental analysis was performed on a PerkinElmer 2400
Series II elemental analyzer. The reaction progress and
individuality of the obtained compounds were controlled
by TLC method on Sorbfil UV-254 plates (sorbent type –
STKh-1А silica gel), eluent СHCl3–EtOH (3:1, 10:1) for
compounds 2a–d and 3a–d or hexane–EtOAc (1:3) for
compounds 5a–h and 6a–h.
The cytotoxic action of compound 6e was studied on
HeLa tumor cell culture (Table 1). The study compound
stimulated the apoptosis of cells, but changing the
concentration of the compound decreased the percentage of
cells undergoing apoptosis.
Thus, we have performed a study of the cytolytic pro-
perties of pyrazolo[5,1-c][1,2,4]benzotriazines synthesized
by an azo coupling reaction of 3-aryl-1H-pyrazole-
5-diazonium tetrafluoroborates and 5-aryl-3-diazo-3H-
pyrazoles with 1,3-dimethoxybenzene or 1,3,5-trimeth-
oxybenzene followed by cyclization of the intermediates.
The MTT assay was performed on HeLa tumor cells and
human skin fibroblasts under various conditions. It was
found that DMSO solutions of 2-aryl-6,8-dimethoxy-
pyrazolo[5,1-c][1,2,4]benzotriazines containing phenyl,
p-tolyl, and 4-methoxyphenyl groups showed strong
cytotoxic activity, which was not limited to tumor cells, but
also affected healthy cells. However, it should be noted that
6,8-dimethoxy-2-phenylpyrazolo[5,1-c][1,2,4]benzotriazine
showed relatively strong toxic effects against HeLa cells
(the surviving fraction of HeLa cells was 47%), while the
effect against human skin fibroblasts was weak (the
surviving fraction was 93%), which is clearly noteworthy.
It was experimentally established that tumor cell death
occurred according to the apoptotic route and the tested
6,8-dimethoxy-2-phenylpyrazolo[5,1-c][1,2,4]benzotriazine
did not cause tumor cell necrosis.
The starting 3-aryl-1H-pyrazole-5-amines 1–d were
synthesized according to published procedures.20
Synthesis of 3-aryl-1H-pyrazole-5-diazonium tetrafluoro-
borates 2a–d (General method). A cooled suspension of
3-aryl-1H-pyrazol-5-amine 1a–d (1.0 mmol) in aqueous
48% solution of HBF4 (3 ml) was treated by slow dropwise
addition of ice-cold NaNO2 (83 mg, 1.2 mmol) solution in
H2O (1 ml). The reaction mixture was stirred for 45 min on
an ice bath, while maintaining the temperature between
–5 and 0°С. The completion of the reaction was observed
by TLC as absence of the starting amine 1a–d. The
precipitate was filtered off and air-dried.
3-Phenyl-1H-pyrazole-5-diazonium tetrafluoroborate
(2a). Yield 207 mg (80%), beige powder, mp 129–130°С
+
(decomp.). IR spectrum, ν, cm–1: 3222 (N–H), 2275 (N2 ).
1
UV spectrum, λmax, nm (log ε): 260 (4.26). H NMR
spectrum, δ, ppm (J, Hz): 7.55–7.61 (3H, m, H-3',4',5');
7.90 (2H, d, J = 7.3, H-2',6'); 8.10 (1H, s, H-4); 12.80 (1Н,
br. s, NH). 13C NMR spectrum, δ, ppm: 110.2 (C-4); 122.7
(C-5); 126.4 (C-2',6'); 126.8 (C-4'); 129.5 (C-3',5'); 130.4
(C-1'); 147.1 (C-3).
Experimental
IR spectra were recorded on a Bruker Alpha FT-IR
spectrometer for thin layers of samples using a ZnSe ATR
accessory. UV spectra were recorded for MeCN solutions on a
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Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
3-(p-Tolyl)-1H-pyrazole-5-diazonium tetrafluoroborate
(2H, d, J = 7.9, H-2',6'). 13C NMR spectrum, δ, ppm: 20.8
(CH3); 108.7 (C-4); 112.6 (C-3); 125.6 (C-2',6'); 129.4
(C-3',5'); 129.9 (C-4'); 137.1 (C-1'); 151.0 (C-5).
(2b). Yield 210 mg (77%), cream-colored powder, mp 131–
132°С (decomp.). IR spectrum, ν, cm–1: 3217 (N–H), 2280
+
(N2 ). UV spectrum, λmax, nm (log ε): 257 (4.38). 1H NMR
3-Diazo-5-(4-methoxyphenyl)-3H-pyrazole (3с). Yield
148 mg (74%), yellowish-brown powder, mp 119–120°С.
IR spectrum, ν, cm–1: 2140 (=N+=N-). UV spectrum, λmax, nm
(log ε): 279 (4.42). 1H NMR spectrum, δ, ppm (J, Hz): 3.85
(3H, s, ОCH3); 7.07 (2H, d, J = 8.7, H-3',5'); 7.82 (2H, d,
J = 8.7, H-2',6'); 7.96 (1H, s, H-4). 13C NMR spectrum,
δ, ppm: 55.5 (OCH3); 107.8 (C-4); 112.5 (C-3); 114.2
(C-3',5'); 125.3 (C-1'); 127.0 (C-2',6'); 150.8 (C-5); 159.0
(C-4').
5-(4-Chlorophenyl)-3-diazo-3H-pyrazole (3d). Yield
160 mg (78%), yellow powder, mp 117–118°С. IR spectrum,
ν, cm–1: 2152 (=N+=N-). UV spectrum, λmax, nm (log ε):
274 (4.15). 1H NMR spectrum, δ, ppm (J, Hz): 7.50 (2H, d,
J = 8.6, H-3',5'); 7.90 (1H, s, H-4); 7.98 (2H, d, J = 8.6,
H-2',6'). 13C NMR spectrum, δ, ppm: 109.7 (C-4); 113.4
(C-3); 127.3 (C-2',6'); 128.9 (C-3',5'); 131.5 (C-4'); 132.2
(C-1'); 149.8 (C-5).
Synthesis of 3-aryl-5-[(2,4-dimethoxyphenyl)diazenyl]-
and 3-aryl-5-[(2,4,6-trimethoxyphenyl)diazenyl]-1H-pyra-
zoles 5a–h. Method I. A suspension of diazonium salt 2a–d
(1.0 mmol) in glacial AcOH (4 ml) was stirred at room
temperature and treated by the addition of 1,3-dimethoxy-
benzene (4a) (0.14 ml, 1.1 mmol) or 1,3,5-trimethoxy-
benzene (4b) (185 mg, 1.1 mmol), respectively. The
reaction mixture was further stirred under these conditions
until the disappearance of the starting diazonium salt 2a–d
(1–7 h, control by TLC). The precipitate was then filtered
off, washed with Et2O (10 ml), and air-dried.
Method II. A solution of diazo compound 3a–d
(1.0 mmol) in СHCl3 (5 ml) was stirred at room
temperature and treated by the addition of 1,3-di-
methoxybenzene (4a) (0.14 ml, 1.3 mmol) or
1,3,5-trimethoxybenzene (4b) (219 mg, 1.3 mmol),
respectively. The reaction mixture was further stirred under
these conditions until the disappearance of the starting
diazo compound 3a–d (7–9 days, control by TLC). The
precipitate was then filtered off, washed with Et2O (10 ml),
and air-dried.
spectrum, δ, ppm (J, Hz): 2.38 (3H, s, CH3); 7.40 (2H, d,
J = 8.0, H-3',5'); 7.80 (2H, d, J = 8.0, H-2',6'); 8.09 (1H, s,
H-4); 12.20 (1Н, br. s, NH). 13C NMR spectrum, δ, ppm:
21.0 (CH3); 109.4 (C-4); 123.2 (C-4'); 123.8 (C-5); 126.4
(C-2',6'); 130.1 (C-3',5'); 140.7 (C-1'); 146.7 (C-3).
3-(4-Methoxyphenyl)-1H-pyrazole-5-diazonium tetra-
fluoroborate (2c). Yield 225 mg (78%), yellow powder,
mp 125–126°С (decomp.). IR spectrum, ν, cm–1: 3247 (N–H),
+
2271 (N2 ). UV spectrum, λmax, nm (log ε): 262 (5.01).
1H NMR spectrum, δ, ppm (J, Hz): 3.84 (3H, s, OCH3);
7.12 (2H, d, J = 8.7, H-3',5'); 7.87 (2H, d, J = 8.7, H-2',6');
8.00 (1H, s, H-4); 12.60 (1Н, br. s, NH). 13C NMR
spectrum, δ, ppm: 55.6 (OCH3); 109.4 (C-4); 115.1
(C-3',5'); 118.2 (C-1'); 124.1 (C-5); 128.2 (C-2',6'); 146.5
(C-3); 161.2 (C-4').
3-(4-Chlorophenyl)-1H-pyrazole-5-diazonium tetrafluoro-
borate (2d). Yield 246 mg (84%), beige powder, mp 128–
129°С (decomp.). IR spectrum, ν, cm–1: 3199 (N–H), 2275
+
1
(N2 ). UV spectrum, λmax, nm (lg ε): 256 (4.13). H NMR
spectrum, δ, ppm (J, Hz): 7.65 (2H, d, J = 8.5, H-3',5');
7.91 (2H, d, J = 8.5, H-2',6'); 8.13 (1H, s, H-4); 13.00 (1Н,
br. s, NH). 13C NMR spectrum, δ, ppm: 110.7 (C-4); 123.6
(C-5); 125.3 (C-4'); 128.4 (C-2',6'); 129.7 (C-3',5'); 135.4
(C-1'); 145.8 (C-3).
Synthesis of 5-aryl-3-diazo-3H-pyrazoles 3a–d (General
method). A stirred suspension of 3-aryl-1H-pyrazole-5-di-
azonium salt 2a–d (1.0 mmol) in CHCl3 (10 ml) was
cooled to 0°С and treated over 10 min by slow dropwise
addition of ice-cold aqueous 10% NaHCO3 solution until
pH 4–5 was reached. After the addition of the NaHCO3
solution was complete, the two-phase mixture was further
stirred at a temperature between –5 and 0°С for 5–10 min.
If some suspended solids were present, the mixture was
filtered. The yellow organic layer was then separated and
the aqueous layer was additionally extracted with CHCl3
(2-3 × 3 ml) until complete removal of diazo compound
from the aqueous phase, which was determined by
qualitative test using m-phenylenediamine. The combined
CHCl3 extracts were dried over anhydrous Na2SO4 and the
solvent was removed by evaporation at reduced pressure.
Caution! Products 3a–d can undergo explosive
decomposition when initiated by friction.
3-Diazo-5-phenyl-3H-pyrazole (3a). Yield 145 mg
(85%), yellow powder, mp 107–108°С. IR spectrum, ν, cm–1:
2150 (=N+=N–). UV spectrum, λmax, nm (log ε): 276 (4.42).
1H NMR spectrum, δ, ppm (J, Hz): 7.32–7.34 (1H, m,
H-4'); 7.45 (2H, d, J= 7.5, H-3',5'); 7.87 (1H, s, H-4); 7.95
(2H, d, J = 7.5, H-2',6'). 13C NMR spectrum, δ, ppm: 109.3
(C-4); 113.0 (C-3); 125.7 (C-2',6'); 127.8 (C-1'); 128.9
(C-3',5'); 132.6 (C-4'); 151.0 (C-5).
5-[(2,4-Dimethoxyphenyl)diazenyl]-3-phenyl-1H-pyra-
zole (5a). Yield 280 mg (91%, method I), 210 mg (68%,
method II), orange powder, mp 105–106°С. 1H NMR
spectrum, δ, ppm (J, Hz): 3.88 (3H, s, OCH3); 3.99 (3H, s,
OCH3); 6.64 (1H, dd, J = 9.0, J = 2.4, H-5''); 6.77 (1H, d,
J = 2.4, H-3''); 6.85 (1H, s, H-4); 7.35–7.39 (1H, m, H-4');
7.44–7.48 (2H, m, H-3',5'); 7.63 (1H, d, J = 9.0, H-6'');
7.84 (2H, d, J = 7.8, H-2',6'); 9.27 (1Н, br. s, NH).
13C NMR spectrum, δ, ppm: 55.4 (OCH3); 56.3 (OCH3);
91.3 (С-4); 99.6 (С-3''); 106.4 (С-5''); 117.4 (С-6''); 125.1
(С-3',5'); 128.0 (С-1'); 128.5 (С-2',6'); 129.9 (С-4'); 136.4
(С-1''); 145.4 (С-3); 158.3 (С-2''); 162.3 (С-5); 163.4
(С-4''). Found, %: С 65.98; Н 5.29; N 18.24. C17H16N4O2.
Calculated, %: C 66.22; H 5.23; N 18.17.
3-Diazo-5-(p-tolyl)-3H-pyrazole (3b). Yield 150 mg
(81%), yellow-orange powder, mp 117–118°С. IR spect-
rum, ν, cm–1: 2141 (=N+=N–). UV spectrum, λmax, nm (log ε):
274 (4.46). 1H NMR spectrum, δ, ppm (J, Hz): 2.33 (3H, s,
CH3); 7.25 (2H, d, J = 7.9, H-3',5'); 7.80 (1H, s, H-4); 7.84
5-[(2,4-Dimethoxyphenyl)diazenyl]-3-(p-tolyl)-1H-pyra-
zole (5b). Yield 288 mg (89%, method I), 226 mg (70%,
method II), yellow-orange powder, mp 153–154°С.
1H NMR spectrum, δ, ppm (J, Hz): 2.37 (3H, s, CH3); 3.88
1149

Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
(3H, s, OCH3); 3.99 (3H, s, OCH3); 6.56 (1H, dd, J = 8.8,
90.6 (C-4); 91.7 (C-3'',5''); 125.3 (C-3',5'); 126.8 (С-1'');
127.1 (C-1'); 129.6 (C-2',6'); 138.0 (C-4'); 145.1 (C-3);
154.3 (C-2'',6''); 162.0 (С-4''); 163.3 (C-5). Found, %:
С 64.82; Н 5.78; N 15.76. C19H20N4O3. Calculated, %:
C 64.76; H 5.72; N 15.90.
J = 2.0, H-5''); 6.67 (1H, d, J = 2.0, H-3''); 6.74 (1H, s,
H-4); 7.22 (2H, d, J = 7.8, H-3',5'); 7.65 (1H, d, J = 8.8,
H-6''); 7.68 (2H, d, J = 7.8, H-2',6'); 8.64 (1Н, br. s, NH).
13C NMR spectrum, δ, ppm: 20.8 (CH3); 55.7 (OCH3); 56.1
(OCH3); 90.6 (С-4); 99.2 (С-3''); 106.3 (С-5''); 117.3
(С-6''); 125.2 (С-3',5'); 126.7 (С-1'); 129.5 (С-2',6'); 136.0
(С-4'); 137.9 (С-1''); 144.8 (С-3); 158.4 (С-2''); 163.4
(С-5); 163.6 (С-4''). Found, %: С 66.89; Н 5.55; N 17.27.
C18H18N4O2. Calculated, %: C 67.07; H 5.63; N 17.38.
5-[(2,4-Dimethoxyphenyl)diazenyl]-3-(4-methoxy-
phenyl)-1H-pyrazole (5с). Yield 285 mg (84%, method I),
220 mg (65%, method II), yellow powder, mp 131–132°С.
1H NMR spectrum, δ, ppm (J, Hz): 3.81 (3H, s, OCH3);
3.87 (3H, s, OCH3); 3.99 (3H, s, OCH3); 6.55 (1H, dd,
J = 8.8, J = 2.4, H-5''); 6.67 (1H, d, J = 2.4, H-3''); 6.73
(1H, s, H-4); 6.96 (2H, d, J = 8.3, H-3',5'); 7.66 (1H, d,
J = 8.8, H-6''); 7.72 (2H, d, J = 8.3, H-2',6'); 8.79 (1Н, br. s,
NH). 13C NMR spectrum, δ, ppm: 55.3 (OCH3); 55.7
(OCH3); 56.2 (OCH3); 90.1 (С-4); 99.2 (С-3''); 106.3
(С-5''); 114.4 (С-3',5'); 117.4 (С-6''); 122.1 (С-1'); 126.8
(С-2',6'); 136.1 (С-1''); 144.7 (С-3); 158.5 (С-4'); 159.5
(С-2''); 163.6 (С-4''); 164.5 (С-5). Found, %: С 63.74; Н 5.31;
N 16.61. C18H18N4O3. Calculated, %: C 63.89; H 5.36;
N 16.56.
3-(4-Methoxyphenyl)-5-[(2,4,6-trimethoxyphenyl)-
diazenyl]-1H-pyrazole (5g). Yield 327 mg (89%, method I),
260 mg (71%, method II), orange powder, mp 115–116°С.
1H NMR spectrum, δ, ppm (J, Hz): 3.82 (3H, s, OCH3);
3.85 (6H, s, OCH3); 3.89 (3H, s, OCH3); 6.31 (2H, s,
H-3'',5''); 6.67 (1H, s, H-4); 6.96 (2H, d, J = 8.5, H-3',5');
7.73 (2H, d, J = 8.5, H-2',6'); 9.78 (1Н, br. s, NH).
13C NMR spectrum, δ, ppm: 55.3 (OCH3); 55.6 (OCH3);
56.2 (2OCH3); 89.9 (C-4); 91.7 (C-3'',5''); 114.4 (C-3',5');
122.4 (С-1''); 126.7 (C-2',6'); 126.8 (C-1'); 144.8 (C-3);
154.1 (C-2'',6''); 159.4 (C-4'); 161.6 (С-4''); 163.6 (C-5).
Found, %: С 61.83; Н 5.55; N 15.09. C19H20N4O4.
Calculated, %: C 61.95; H 5.47; N 15.21.
3-(4-Chlorophenyl)-5-[(2,4,6-trimethoxyphenyl)diazen-
yl]-1H-pyrazole (5h). Yield 358 mg (96%, method I), 287 mg
1
(77%, method II), orange powder, mp 181–182°С. H NMR
spectrum, δ, ppm (J, Hz): 3.85 (6H, br. s, OCH3); 3.89 (3Н,
s, OCH3); 6.30 (2H, s, H-3'',5''); 6.80 (1H, s, H-4); 7.42 (2H,
d, J = 8.3, H-3',5'); 7.83 (2H, d, J = 8.3, H-2',6'); 9.64 (1Н,
br. s, NH). 13C NMR spectrum, δ, ppm: 55.7 (OCH3); 56.3
(2OCH3); 91.6 (C-4); 91.8 (C-3'',5''); 126.8 (С-1''); 127.1
(C-3',5'); 129.1 (C-2',6'); 129.3 (C-4'); 132.9 (C-1'); 144.9
(C-3); 154.5 (C-2'',6''); 162.2 (С-4''); 162.4 (C-5). Found,
%: С 57.96; Н 4.58; Cl 9.53; N 14.98. C18H17ClN4O3.
Calculated, %: C 57.99; H 4.60; Cl 9.51; N 15.03.
3-(4-Chlorophenyl)-5-[(2,4-dimethoxyphenyl)diazenyl]-
1H-pyrazole (5d). Yield 323 mg (94%, method I), 247 mg
(72%, method II), orange powder, mp 115–116°С.
1H NMR spectrum, δ, ppm (J, Hz): 3.88 (3H, s, OCH3);
3.97 (3H, s, OCH3); 6.65 (1H, dd, J = 8.8, J = 2.3, H-5'');
6.78 (1H, d, J = 2.3, H-3''); 6.93 (1H, s, H-4); 7.54 (2H, d,
J = 8.5, H-3',5'); 7.64 (1H, d, J = 8.8, H-6''); 7.91 (2H, d,
J = 8.5, H-2',6'); 10.35 (1Н, br. s, NH). 13C NMR spectrum,
δ, ppm: 55.8 (OCH3); 56.2 (OCH3); 91.8 (С-4); 99.2
(С-3''); 106.5 (С-5''); 117.5 (С-6''); 127.1 (С-3',5'); 129.0
(С-2',6'); 129.1 (С-4'); 133.0 (С-1'); 136.1 (С-1''); 144.6
(С-3); 158.7 (С-2''); 162.5 (С-5); 163.9 (С-4''). Found, %:
С 59.38; Н 4.21; Cl 10.42; N 16.53. C17H15ClN4O2.
Calculated, %: C 59.57; H 4.41; Cl 10.34; N 16.34.
3-Phenyl-5-[(2,4,6-trimethoxyphenyl)diazenyl]-1H-pyra-
zole (5e). Yield 322 mg (95%, method I), 281 mg (83%,
method II), orange powder, mp 164–165°С. 1H NMR
spectrum, δ, ppm (J, Hz): 3.84 (6H, s, OCH3); 3.88 (3H, s,
OCH3); 6.30 (2H, s, H-3'',5''); 6.75 (1H, s, H-4); 7.31–7.34
(1H, m, H-4'); 7.40–7.46 (2H, m, H-3',5'); 7.80 (2H, d,
J = 7.8, H-2',6'); 9.40 (1Н, br. s, NH). 13C NMR spectrum,
δ, ppm: 55.8 (OCH3); 56.3 (2OCH3); 91.1 (C-4); 91.8
(C-3'',5''); 125.4 (C-3',5'); 126.8 (С-1''); 128.5 (C-4'); 129.1
(C-2',6'); 130.0 (C-1'); 145.3 (C-3); 154.4 (C-2'',6''); 162.1
(С-4''); 163.2 (C-5). Found, %: С 63.74; Н 5.31; N 16.62.
C18H18N4O3. Calculated, %: C 63.89; H 5.36; N 16.56.
3-(p-Tolyl)-5-[(2,4,6-trimethoxyphenyl)diazenyl]-1H-
pyrazole (5f). Yield 345 mg (98%, method I), 256 mg
(73%, method II), orange-red powder, mp 199–200°С.
1H NMR spectrum, δ, ppm (J, Hz): 2.37 (3H, s, CH3); 3.83
(6Н, s, OCH3); 3.88 (3H, s, OCH3); 6.31 (2H, s, H-3'',5'');
6.70 (1H, s, H-4); 7.23 (2H, d, J = 7.5, H-3',5'); 7.68 (2H,
d, J = 7.5, H-2',6'); 9.32 (1Н, br. s, NH). 13C NMR
spectrum, δ, ppm: 20.9 (CH3); 55.7 (OCH3); 56.3 (2OCH3);
Synthesis of 2-arylpyrazolo[5,1-c][1,2,4]benzotriazines
6a–h (General method). Azo compound 5a–h (1 mmol) was
dissolved in glacial АсОН (5 ml), then p-TsOH (17 mg,
10 mol %) was added. The reaction mixture was stirred at
80–90°С until the disappearance of the starting azo
compound 5a–h (3–8 h, control by TLC). The solution was
concentrated to two thirds of the volume by evaporation at
reduced pressure, and the reaction mixture was cooled. The
precipitate that formed was filtered off, recrystallized from
EtOH, and dried.
8-Methoxy-2-phenylpyrazolo[5,1-c][1,2,4]benzotriazine
(6a). Yield 218 mg (79%), orange powder, mp 206–207°С.
1H NMR spectrum, δ, ppm (J, Hz): 4.03 (3H, s, ОCH3);
7.29 (1H, dd, J = 9.1, J = 2.5, Н-7); 7.42–7.46 (1Н, m,
H-4'); 7.49–7.52 (2H, m, H-3',5'); 7.63 (1H, d, J = 2.5,
H-9); 7.83 (1H, s, Н-3); 8.11 (2H, d, J = 7.3, H-2',6'); 8.39
(1Н, d, J = 9.1, H-6). 13C NMR spectrum, δ, ppm: 56.6
(OCH3); 94.2 (C-9); 97.8 (C-3); 117.6 (C-7); 126.4
(C-3',5'); 127.2 (C-5a); 128.9 (C-2',6'); 129.3 (C-1'); 131.6
(C-4'); 132.3 (C-6); 133.9 (C-9a); 147.2 (C-3a); 154.0
(C-2); 164.4 (C-8). Found, %: С 69.58; Н 4.42; N 20.25.
С16Н12N4O. Calculated, %: С 69.55; Н 4.38; N 20.28.
8-Methoxy-2-(p-tolyl)pyrazolo[5,1-c][1,2,4]benzotri-
azine (6b). Yield 273 mg (94%), red-brown powder, mp 189–
1
190°С. H NMR spectrum, δ, ppm (J, Hz): 2.42 (3H, s,
CH3); 4.12 (3H, s, OCH3); 7.31 (2H, d, J = 8.0, H-3',5');
7.34 (1H, dd, J = 9.1, J = 2.4, H-7); 7.74 (1H, s, Н-3); 7.78
(1H, d, J = 2.4, H-9); 8.03 (2H, d, J = 8.0, H-2',6'); 8.46
(1Н, d, J = 9.1, H-6). 13C NMR spectrum, δ, ppm: 21.0
1150

Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
(CH3); 56.8 (OCH3); 94.2 (C-9); 97.6 (C-3); 117.8 (C-7);
6.83 (1H, br. s, H-9); 7.10 (2H, d, J = 8.8, H-3',5'); 7.33
(1H, br. s, H-7); 7.76 (1H, s, Н-3); 8.09 (2H, d, J = 8.8,
H-2',6'). 13C NMR spectrum, δ, ppm: 55.1 (OCH3); 56.5
(OCH3); 56.6 (OCH3); 86.3 (C-7); 96.3 (C-3); 98.0 (C-9);
114.3 (C-3',5'); 124.1 (C-5a); 126.7 (C-9a); 127.7 (C-2',6');
127.9 (C-1'); 147.4 (C-3a); 154.1 (C-2); 158.8 (C-8); 160.2
(C-4'); 165.7 (C-6). Found, %: С 64.35; Н 4.84; N 16.46.
С18Н16N4O3. Calculated, %: С 64.28; Н 4.79; N 16.66.
2-(4-Chlorophenyl)-6,8-dimethoxypyrazolo[5,1-c]-
[1,2,4]benzotriazine (6h). Yield 273 mg (80%), orange
126.3 (C-3',5'); 127.4 (C-5a); 128.9 (C-1'); 129.6 (C-2',6');
132.5 (C-6); 134.0 (C-9a); 139.1 (C-4'); 147.4 (C-3a);
154.2 (C-2); 164.5 (C-8). Found, %: С 70.15; Н 4.82;
N 19.48. С17Н14N4O. Calculated, %: С 70.33; Н 4.86;
N 19.30.
8-Methoxy-2-(4-methoxyphenyl)pyrazolo[5,1-c]-
[1,2,4]benzotriazine (6c). Yield 254 mg (83%), bordeaux
1
powder, mp 219–220°С. H NMR spectrum, δ, ppm (J, Hz):
3.85 (3H, s, OCH3); 4.09 (3H, s, OCH3); 7.09 (2H, d,
J = 8.2, H-3',5'); 7.36 (1H, dd, J = 9.1, J= 2.4, H-7); 7.70
(1H, d, J = 2.4, H-9); 7.84 (1H, s, Н-3); 8.10 (2H, d,
J = 8.2, H-2',6'); 8.46 (1Н, d, J = 9.1, H-6). 13C NMR
spectrum, δ, ppm: 55.1 (OCH3); 56.5 (OCH3); 94.2 (C-9);
96.9 (C-3); 114.2 (C-3',5'); 117.3 (C-7); 124.1 (C-1'); 127.2
(C-5a); 127.7 (C-2',6'); 132.2 (C-6); 133.9 (C-9a); 147.2
(C-3a); 154.0 (C-2); 160.2 (C-4'); 164.3 (C-8). Found, %:
С 66.49; Н 4.54; N 18.42. С17Н14N4O2. Calculated, %:
С 66.66; Н 4.61; N 18.29.
1
flakes, mp 230–231°С. H NMR spectrum, δ, ppm (J, Hz):
4.09 (3H, s, OCH3); 4.11 (3H, s, OCH3); 6.76 (1H, s, H-9);
7.28 (1H, s, H-7); 7.50 (2H, d, J = 7.0, H-3',5'); 7.77 (1H, s,
Н-3); 8.13 (2H, d, J = 7.0, H-2',6'). 13C NMR spectrum,
δ, ppm: 56.6 (OCH3); 56.7 (OCH3); 86.4 (C-7); 97.4 (C-3);
98.4 (C-9); 126.9 (C-5a); 127.9 (C-9a); 128.0 (C-3',5');
128.9 (C-2',6'); 130.5 (C-1'); 133.9 (C-4'); 147.4 (C-3a);
152.8 (C-2); 158.9 (C-8); 165.8 (C-6). Found, %: С 59.84;
Н 3.77; Cl 10.63; N 16.26. С17Н13ClN4O2. Calculated, %:
С 59.92; Н 3.85; Cl 10.40; N 16.44.
Biological testing of compounds 6a–h. The cultures of
HeLa cells and human skin fibroblasts were obtained from
the cell culture collection of the Institute of Cytology,
Russian Academy of Sciences (Saint Petersburg, Russia).
The cell cultures were supported in cell culture flasks
(Eppendorf, Germany) in DMEM medium (Sigma-Aldrich,
USA) with the addition of 10% of fetal bovine serum
(Biolot, Russia) and 0.5% of gentamycin.
The MTT assay was performed according to the
following procedure: the cells were inoculated in a 96-well
plate (Eppendorf, Germany) and cultivated for 1 day until
reaching a concentration of approximately 105 cells/ml,
after which a solution of the study compound 6a–h in
distilled water, phosphate buffer, or DMSO was added
(concentrations of 0.065 or 0.016 mmol/l). The blank
samples were distilled water, phosphate buffer, or DMSO,
respectively. After incubation for 72 h, the growth medium
was replaced and 0.1 mg/ml concentration of methylthi-
azolyldiphenyltetrazolium bromide (Biolot, Russia) was
added to the medium. Incubation with the dye was
continued for 4 h, after which the medium was removed
and the dye was extracted from the cells using DMSO. The
color intensity was determined using a BioTek ELx808
absorbance microplate reader (Thermo Fisher Scientific,
USA) at 540 nm wavelength.
The cytotoxic activity of compounds (determined as
necrotic and apoptotic effects) was measured with an FC 500
Series flow cytometer (Beckman Coulter, USA) and an
Annexin V-FITC Apoptosis Detection Kit (Abcam, United
Kingdom) containing FITC-labeled antibodies against
annexin and the DNA stain propidium iodide. Cells for the
study were cultured in 24-well plates to a concentration of
104 cells/ml, after which the study compounds 6a–h were
added to the medium as solutions in phosphate buffer (pH
7.5). Incubation with the study compound was continued for
48 h, then cells were separated from the plate using a
Trypsin-Versene solution (Biolot, Russia). The cell
suspension was further studied using a flow cytometer
according to the manufacturer's manual.
2-(4-Chlorophenyl)-8-methoxypyrazolo[5,1-c][1,2,4]-
benzotriazine (6d). Yield 242 mg (78%), yellow powder,
1
mp 216–217°С. H NMR spectrum, δ, ppm (J, Hz): 4.11
(3H, s, OCH3); 7.45 (1H, dd, J = 9.0, J = 2.5, H-7); 7.64
(2H, d, J = 8.4, H-3',5'); 7.82 (1H, d, J = 2.5, H-9); 8.05
(1H, s, Н-3); 8.23 (2H, d, J = 8.4, H-2',6'); 8.54 (1H, d,
J = 9.0, H-6). 13C NMR spectrum, δ, ppm: 56.5 (OCH3);
94.4 (C-9); 97.9 (C-3); 117.4 (C-7); 127.1 (C-5a); 127.9
(С-3',5'); 128.7 (C-2',6'); 130.4 (С-1'); 132.3 (C-6); 133.9
(C-9a); 134.0 (С-4'); 147.1 (C-3a); 152.8 (C-2); 164.4 (C-8).
Found, %: С 61.69; Н 3.46; Cl 11.54; N 18.08. С16Н11ClN4O.
Calculated, %: С 61.84; Н 3.57; Cl 11.41; N 18.03.
6,8-Dimethoxy-2-phenylpyrazolo[5,1-c][1,2,4]benzotri-
azine (6e). Yield 236 mg (77%), light-yellow powder,
1
mp 229–230°С. H NMR spectrum, δ, ppm (J, Hz): 4.09
(3H, s, OCH3); 4.11 (3H, s, OCH3); 6.74 (1H, d, J = 2.5,
H-9); 7.30 (1H, d, J = 2.5, H-7); 7.41–7.44 (1H, m, H-4');
7.48–7.52 (2H, m, H-3',5'); 7.71 (1H, s, Н-3); 8.13 (2H, d,
J = 7.3, H-2',6'). 13C NMR spectrum, δ, ppm: 56.3 (OCH3);
56.4 (OCH3); 86.3 (C-7); 96.9 (C-3); 98.0 (C-9); 126.1
(C-3',5'); 126.6 (C-5a); 127.7 (C-9a); 128.5 (C-2',6'); 128.9
(С-4'); 131.5 (C-1'); 147.2 (C-3a); 153.9 (C-2); 158.8
(C-8); 165.6 (C-6). Found, %: С 66.47; Н 4.56; N 18.37.
С17Н14N4O2. Calculated, %: С 66.66; Н 4.61; N 18.29.
6,8-Dimethoxy-2-(p-tolyl)pyrazolo[5,1-c][1,2,4]benzo-
triazine (6f). Yield 282 mg (88%), yellow powder, mp 245–
1
246°С. H NMR spectrum, δ, ppm (J, Hz): 2.42 (3H, s,
CH3); 4.09 (3H, s, OCH3); 4.11 (3H, s, OCH3); 6.74 (1H, s,
H-9); 7.29 (2H, d, J = 7.5, H-3',5'); 7.30 (1H, s, H-7); 7.66
(1H, s, Н-3); 8.01 (2H, d, J = 7.5, H-2',6'). 13C NMR
spectrum, δ, ppm: 20.5 (CH3); 56.3 (OCH3); 56.4 (OCH3);
86.3 (C-7); 96.5 (C-3); 97.9 (C-9); 126.0 (C-3',5'); 126.6
(C-5a); 127.7 (C-9a); 128.7 (C-1'); 129.1 (C-2',6'); 138.6
(C-4'); 147.2 (C-3a); 154.0 (C-2); 158.8 (C-8); 165.5 (C-6).
Found, %: С 67.30; Н 4.94; N 17.37. С18Н16N4O2.
Calculated, %: С 67.49; Н 5.03; N 17.49.
6,8-Dimethoxy-2-(4-methoxyphenyl)pyrazolo[5,1-c]-
[1,2,4]benzotriazine (6g). Yield 276 mg (82%), yellow
1
powder, mp 220–221°С. H NMR spectrum, δ, ppm (J, Hz):
3.85 (3H, s, OCH3); 4.08 (3H, s, OCH3); 4.10 (3H, s, OCH3);
1151

Chemistry of Heterocyclic Compounds 2018, 54(12), 1145–1152
8. Sadchikova, E. V.; Mokrushin, V. S. Rus. Chem. Bull., Int.
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Chem. 1987, 52, 5538.
1
1
compounds 2a, 3b as well as Н, 13С, and Н–13С HMBC
NMR spectra of compounds 2a, 3b, 5a,e, and 6a,g is
available at the journal website at http://link.springer.com/
journal/10593.
11. Pretsch, E.; Bühlmann, P.; Affolter, C. Structure
Determination of Organic Compounds. Tables of Spectral
Data; Springer-Verlag: Berlin, 2000, p. 104, 186.
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