Synthesis of mycostatics based on 4-aryldiazenyl-3,5-dimethylpyrazoles

Article abstract of DOI:10.1007/s11172-021-3193-4

The condensation of 3-arylhydrazinylidenepentane-2,4-diones with hydrazine hydrate, 2-hydroxyethylhydrazine, benzylhydrazine hydrochloride, and 4-hydrazinylbenzenesulfonamide hydrochloride gave 4-aryldiazenyl-3,5-dimethylpyrazoles. An alternative route to

Full text of DOI:10.1007/s11172-021-3193-4

Russian Chemical Bulletin, International Edition, Vol. 70, No. 6, pp. 1124—1130, June, 2021
1124
Synthesis of mycostatics based on 4-aryldiazenyl-3,5-dimethylpyrazoles*
O. G. Khudina,a A. E. Ivanova,a Ya. V. Burgart,a N. A. Gerasimova,b N. P. Evstigneeva,b and V. I. Saloutina
a I. Ya. Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences,
22/20 ul. S. Kovalevskoi, 620137 Ekaterinburg, Russian Federation.
Fax: +7 (343) 374 5954. E-mail: saloutin@ios.uran.ru
bUral Research Institute of Dermatovenereology and Immunopathology,
8 ul. Shcherbakova, 620076 Ekaterinburg, Russian Federation
The condensation of 3-arylhydrazinylidenepentane-2,4-diones with hydrazine hydrate,
2-hydroxyethylhydrazine, benzylhydrazine hydrochloride, and 4-hydrazinylbenzenesulfonamide
hydrochloride gave 4-aryldiazenyl-3,5-dimethylpyrazoles. An alternative route to the synthesis
of N-substituted 4-aryldiazenylpyrazoles is based on the alkylation of NH-pyrazoles with halo-
alkanes. The synthesized compounds were tested for antimicrobial activity against eight patho-
genic dermatophytes, yeast-like fungi of the Candida genus and the bacteria Neisseria gonor-
rhoeae. The structure—activity relationship analysis showed that 4-tolyldiazenylpyrazoles
bearing H, AcO(CH2)4, or HO(CH2)4 substituents at the N(1) atom have the highest myco-
static activity against all the dermatophyte strains under study. However, 4-aryldiazenyl-3,5-
dimethylpyrazoles proved to be quite cytotoxic against the McCoy B cell line.
Key words: 3-arylhydrazinylidenepentane-2,4-dione, condensation, hydrazines, 4-aryldi-
azenyl-3,5-dimethylpyrazoles, alkylation, antimycotic and antigonorrhoeae activities, cytotoxicity.
The 4-aryldiazenyl-3,5-dimethylpyrazole scaffold has
phenyl)diazenyl]-3,5-dimethyl-1H-pyrazol-1-yl}-3-oxo-
N-arylpropanamides were shown to be able to effectively
inhibit the growth of the Gram-negative bacteria Escher-
ichia coli and the Gram-positive bacteria Staphylococcus
aureus, and they also exhibited high antifungal activity
against Aspergillus niger.2 4-Aryldiazenyl-3,5-dimethyl-
pyrazoles containing an 4-arylthiazole substituent at the
N(1) atom possess fungicidal activity against Candida
a great potential for the design of antimicrobial agents.1—4
For example, 4-[(3,5-dimethyl-1H-pyrazol-4-yl)diazenyl]-
benzenesulfonamide exhibited stronger antifungal activity
against the yeast-like fungi Candida albicans compared to
nystatin.1 This compound showed also high antibacterial
activity against the Gram-negative bacteria Escherichia
coli and Pseudomonas aeruginosa. 3-{4-[(3,4-Difluoro-
Antibacterial and
antimycotic activity
Antibacterial and
antimycotic activity
Antimycotic activity
Antibacterial activity
* Dedicated to Academician of the Russian Academy of Sciences V. N. Charushin on the occasion of his 70th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1124—1130, June, 2021.
1066-5285/21/7006-1124 © 2021 Springer Science+Business Media LLC

Synthesis of 4-aryldiazenyl-3,5-dimethylpyrazoles
Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
1125
Scheme 1
albicans.3 4-Hetaryldiazenyl-3,5-dimethylpyrazole con-
taining the antipyrine moiety and the N-benzenesulfon-
amide substituent exhibited, apart from antibacterial
activity, an anti-inflammatory effect comparable to that
of indomethacin, but did not have an ulcerogenic
side effect.4
A series of 4-aryldiazenyl-3,5-dimethylpyrazoles 3e—k
substituted at the N(1) atom were synthesized by the al-
kylation of N-unsubstituted pyrazoles 2 with haloalkanes
in the presence of K2CO3 (Scheme 2). Pyrazole 3e was
Scheme 2
The prospects of the use of the 4-aryldiazenyl-3,5-di-
methylpyrazole scaffold for the design of new antimycotics
have provided the impetus for us to synthesize new deriv-
atives of this series and evaluate their antifungal activity.
The condensation of 3-arylhydrazinylidenepentane-
2,4-diones 1а—c with hydrazine hydrate in methanol at
50 C afforded N(1)-unsubstituted 4-aryldiazenyl-3,5-
dimethylpyrazoles 2a—c (Scheme 1), which have been
previously5 tested for antibacterial activity against Escheri-
chia coli, Bacillus cereus, and Proteus vulgaris. We synthe-
sized these compounds in order to evaluate their anti-
mycotic and antigonorrhoeae effects.
The heating of arylhydrazones 1b,c with 2-hydroxy-
ethylhydrazine under reflux in ethanol gave derivatives of
pyrazoles 2a—c, heterocycles 3a,b containing the 2-hydr-
oxyethyl substituent at the N(1) atom. Pyrazole 3a was
prepared previously6 in acetic acid; however only the
melting point of this compound was reported, whereas the
spectroscopic data were absent. We fully characterized this
compound. Pyrazoles 3c,d were synthesized by the con-
densation of arylhydrazone 1c with benzylhydrazine and
4-hydrazinylbenzenesulfonamide hydrochlorides in acetic
acid in the presence of sodium acetate (see Scheme 1).
3
e
f
g
h
R
X
Me
3
i
R
X
H
OMe
OMe
(CH2)4OAc
(CH2)4OAc OMe
(CH2)3CF3
(CH2)3CF3
(CH2)2CF3
j
Et
Et
H
OMe
k

Khudina et al.
1126
Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
prepared previously7 by the alkylation of N-unsubstituted
pyrazole 2a with 4-bromobutyl acetate followed by the
removal of the acyl protecting group under acidic conditions
and the formation of 4-hydroxybutylpyrazole 3l described
previously.7 N(1)-Substituted 4-aryldiazenylpyrazoles
3f—h were synthesized by the alkylation of NH-pyrazoles
2b,c with 4-bromobutyl acetate and 1-iodoethane.
Organofluorine compounds have attracted increasing
interest because electron-withdrawing fluorine atoms in
substrates modulate various biological activities,8,9 in
particular antimycotic activity, as evidenced by the use of
fluorine-containing agents (fluconazole and flucytosine)
for the treatment of different fungal diseases.
Scheme 3
Therefore, we introduced fluorine-containing sub-
stituents into 4-aryldiazenyl-3,5-dimethylpyrazoles. Thus,
the alkylation of pyrazoles 2b,c with 1-iodo-4,4,4-tri-
fluorobutane and 1-iodo-3,3,3-trifluoropropane gave
N(1)-trifluoroalkyl-substituted 4-aryldiazenylpyrazoles
3i—k (see Scheme 2).
the trans-azo form. The largest upfield shifts are observed
for the o-protons of the aromatic ring (by 0.76 ppm) and
the Me group (by 0.83 and 0.58 ppm). This may be due to
the effect of the aromatic ring current on the Me groups
and the anisotropic field effect, induced by the pyrazole
ring, on the o-protons of Ph groups.
We evaluated the antimycotic and antibacterial activ-
ity of pyrazoles 2 and 3 against eight test strains of patho-
genic dermatophyte fungi, the yeast-like fungi Candida
albicans and the obligate bacterial pathogen Neisseria
gonorrhoeae (Table 1). Fluconazole and spectinomycin
were used as the positive control for the antimycotic and
antibacterial assessment.
Products 3 can exist in the trans-azo and cis-azo forms.
The 1H and 19F NMR spectroscopic studies showed that,
immediately after the dissolution in CDCl3, pyrazoles 3
exist in the sterically most favorable trans-azo form A
(Scheme 3). However, after the storage in CDCl3 for 3 h
or longer, the 1H NMR spectrum of compound 3i shows
the presence of 4.8% of the cis-azo form B (see the
Experimental).
In the 1H NMR spectra of the cis-azo form, the signals
of all protons are significantly shifted upfield compared to
Table 1. Antimycotic and antibacterial activity of pyrazoles 2 and 3 (MIC is the minimum inhibitory concentration)
Com-
pound
MIC/g mL–1
T.
T.
T.
T.
T.
T.
E.
M.
C.
N.
rubrum
gypseum tonsurans violaceum interdigitale schoenleinii floccosum canis albicans gonorrhoeae
a
2a
2b
2c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
6.25
25.00
7.80
50.00 >200.00
100.00 100.00
>200.00 >200.00
>200.00 >200.00
3.12
100.00
25.00
200.00
200
>200
>200.00 >200.00
6.25
25.00
31.20
6.25
50.00
12.50
25.00
25.00
12.50
25.00
6.25
25.00
7.80
100.00
50.00
25.00
50.00
7.80 1000.00
50.00 >200.00
100.00 >200.00
50.00
50.00
—
25.00
250.00
125.00
125.00
a
7.80
31.20
—
62.50
100.00
>200.00
>200.00
>200.00
12.50
100.00
50.00
12.50
100.00
>200.00
>200.00
>200.00
12.50
100.00
25.00
12.50
50.00
50.00
a
a
—
—
>200.00
>200.00
>2000
12.50
—
—
a
a
>200.00 >200.00 >200.00
>200.00 >200.00 >200.00
12.50
50.00
25.00
12.50
100.00 >200.00 >200.00
200.00 >200.00 >200.00
200.00 >200.00 >200.00
12.50
1.56
>200.00
12.50
>250.00
a
12.50
100.00
50.00
25.00
50.00
6.25 >200.00
—
—
a
a
—
>200.00
25.00
>200.00
25.00
100.00 >200.00
25.00
50.00
200.00
125.00
a
a
—
25.00 >200.00
—
>200.00
>200.00
>200.00
25.00
125.00
>200.00
>200.00
12.50
50.00
62.50
a
a
>200.00
—
—
>200.00
>200.00
6.25
—
a
a
—
a
3.12
3.12
12.50
6.25
25.00
0.78
12.50
3.12
50.00
1.56
—
b
Flucon-
azole
Spectino-
mycin
6.25
1.95
1.56
—
b
b
b
b
b
b
b
b
b
—
—
—
—
—
—
—
—
—
16
a Not tested.
b Data are absent.

Synthesis of 4-aryldiazenyl-3,5-dimethylpyrazoles
Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
1127
Table 2. Anti-Candida activity of pyrazoles 2a and 3l
Com-
pound
MIC/g mL–1
C. crusei
C. glabrata
C. parapsilosis
C. dubliniensis
C. guilliermondii
C. tropicalis
2a
3l
>200
100
100
200
100
200
>200
200
3.12
50
50
>200
100
100
25
200
100
>200
Fluconazole
The evaluation of the antifungal activity of pyrazoles
2 and 3 showed that pyrazoles 2a,c and 3e,l possess high
or moderate antimycotic activity against strains of patho-
genic dermatophyte fungi. Thus, pyrazoles 3e,l were found
to be able to suppress the growth of the strain of the fungi
T. rubrum comparable to that of fluconazole with the mini-
substituents in methoxyphenyldiazenylpyrazoles 3c,d,j,k
leads to the loss of fungistatic activity.
4-Hydroxybutyl-substituted pyrazole 3l and N-unsub-
stituted pyrazoles 2a,b also displayed weak activity
(MIC = 50 g mL–1) against the yeast-like fungi C. albi-
cans (see Table 1). For compounds 2a and 3l, the range
was extended by additional six strains of clinically sig-
nificant species of yeast-like fungi of the Candida genus
(Table 2). It was found that these compounds retained
weak activity only against the strain C. dubliniensis.
The evaluation of antibacterial activity of pyrazoles 2
and 3 against the bacteria Neisseria gonorrhoeae (see Table
1) revealed only weak activity of compound 3i.
The assessment of the cytotoxic effect of pyrazoles
2a—c and 3a,d,g,i,l using the McCoy B cell line (Table 3)
demonstrated that pyrazoles 2c and 3l exhibiting high
fungistatic activity against strains of pathogenic dermato-
phyte fungi are highly toxic and cause the death of
50% of McCoy B cells in the concentration range of
0.98—15.6 g mL–1. In the series of the tested compounds,
pyrazoles 2a and 3a,d exhibited the lowest cytotoxicity.
At a concentration of 125 g mL–1, these compounds
caused the death of 50% of McCoy B cells.
mum inhibitory concentration (MIC) of 3.12 g mL–1
;
compounds 2a,c, with MIC of 6.25—7.8 g mL–1. The
inhibitory activity of pyrazole 2a against T. gypseum is
comparable to that of the reference sample (MIC =
= 6.25 g mL–1), whereas analogues 3e,l were less effec-
tive (MIC = 12.5 g mL–1). The activity against T.
Tonsurans, comparable to that of fluconazole, was found for
pyrazoles 2a,c (MIC = 6.25—7.8 g mL–1), whereas com-
pounds 3e,h showed lower activity (MIC = 12.5 g mL–1).
Compounds 2 and 3 were less effective against T. viola-
ceum, T. interdigitale, T. schoenleinii, E. floccosum,
M. canis, and C. albicans than fluconazole. However, it
should be noted that pyrazoles 2a,c exhibited high activity
(MIC = 6.25—7.8 g mL–1) against E. floccosum; com-
pound 3l, against T. schoenleinii; heterocycles 2c and 3e,
against M. canis. In the series of the synthesized pyrazoles,
compounds 2a and 3e,l, were found to be the most active
(MIC = 12.5 g mL–1) as inhibitors of the growth of the
fungi T. violaceum; product 3e exhibited the highest in-
hibitory activity against T. interdigitale.
The structure—activity relationship analysis showed
that pyrazoles 2a and 3e,l containing the tolyl substituent
along with H, AcO(CH2)4, or HO(CH2)4 groups at the
N(1) atom are the most active compounds (MIC =
= 3.12—25 g mL–1) against all the dermatophyte strains
under study. The absence of a substituent at the N(1) atom
is favorable for the antimycotic activity, because all pyr-
azoles 2a—c were found to be able to inhibit the growth
of most of the dermatophyte strains with MIC from high
to moderate. Their activity decreases in the following
series of derivatives: tolyl (2a), methoxyphenyl (2b), and
phenyl (2c). Pyrazoles 3g,h containing the Et group at the
N(1) atom in the ring and H or OMe substituents in the
aromatic moiety displayed moderate antimycotic activity
against some fungi. Compounds 3a,b,f,i bearing phenyl
and methoxyphenyldiazenyl substituents along with
HO(CH2)2, AcO(CH2)4, or CF3(CH2)3 groups at the
pyrazole ring exhibited weak activity. The presence of
CF3(CH2)3, CF3(CH2)2, PhCH2, and 4-NH2SO2C6H4
In summary, we performed the synthesis of 4-aryldi-
azenyl-3,5-dimethylpyrazoles by the condensation of
3-arylhydrazinylidenepentane-2,4-diones with hydrazines
and by the alkylation of N-unsubstituted pyrazoles with
haloalkanes. The biological evaluation demonstrated that
Table 3. Cytotoxicity of pyrazoles 2 and 3
a
a
a
Compound
IC90
IC50
125
IC10
2a
2b
2c
3a
3d
3g
3i
3l
250
125
1.9
250
250
62.5
31.2
0.49
62.5
62.5
15.6
31.2
7.8
62.5
0.98
125
125
62.5
62.5
31.2
31.2
46.8
15.6
b
b
Spectinomycin
—
—
>500
a IC90, IC50, and IC10 are the concentrations that
induce 90, 50, and 10% cell death, respectively.
b Data are absent.

Khudina et al.
1128
Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
134.69, 138.61, 142.56, 147.80, 160.78. MS, found: m/z 275.1508
[M + H]+. Calculated for C14H19N4O2+: 275.1503.
3,5-dimethyl-4-[(4-methylphenyl)diazenyl]-1H-pyrazole
(2a) has the optimal activity—toxicity relationship.
Synthesis of pyrazoles 3c,d. A mixture of arylhydrazone 1c
(2 mmol), benzylhydrazine dihydrochloride (410 mg, 2.1 mmol)
or 4-hydrazinylbenzenesulfonamide hydrochloride (470 mg,
2.1 mmol), and AcONa•3H2O (300 mg, 2.2 mmol) in AcOH
(10 mL) was refluxed for 12—14 h. The reaction products were
precipitated with water, filtered off, washed with water, and dried.
Pyrazole 3c was purified by column chromatography using
ethyl acetate—hexane (5 : 1) as the eluent. Compound 3d was
washed with MeCN and recrystallized from EtOH.
1-Benzyl-4-[(4-methoxyphenyl)diazenyl]-3,5-dimethyl-1H-
pyrazole (3c). Yellow powder, yield was 64%, m.p. 111—113 C.
IR, /cm–1: 2923 (C—H); 1600, 1581, 1553, 1498 (C=C).
1H NMR (CDCl3), δ: 2.50 (s, 3 H, CH3); 2.53 (s, 3 H, CH3);
3.87 (s, 3 H, OCH3); 5.28 (s, 2 H, CH2); 6.97 (d, 2 H, C6H4,
J = 9.1 Hz); 7.14 (d, 2 H, Ph, J = 8.2 Hz); 7.27—7.35 (m, 3 H,
Ph); 7.77 (d, 2 H, C6H4, J = 9.1 Hz). 13C NMR (CDCl3), δ:
9.97 (CH3), 13.96 (CH3), 53.00 (NCH2), 55.48 (OCH3), 114.00,
123.29, 126.69, 127.72, 128.79, 135.28, 136.41, 138.21, 142.49,
147.88, 160.76. MS, found: m/z 321.1712 [M + H]+. Calculated
for C19H21N4O+: 321.1710.
4-{4-[(4-Methoxyphenyl)diazenyl]-3,5-dimethyl-1H-pyr-
azol-1-yl}benzenesulfonamide (3d). Yellow powder, yield was
53%, m.p. 267—268 C. IR, /cm–1: 3289, 3181 (NH2); 3023
(C—H); 1599, 1584, 1558, 1508 (C=C). 1H NMR (DMSO-d6),
δ: 2.49 (s, 3 H, CH3); 2.71 (s, 3 H, CH3); 3.85 (s, 3 H, OCH3);
7.10 (d, 2 H, C6H4, J = 9.0 Hz); 7.52 (br.s, 2 H, NH2); 7.80 (d, 2 H,
C6H4, J = 9.0 Hz); 7.86 (d, 2 H, C6H4, J = 8.6 Hz); 7.99 (d, 2 H,
C6H4, J = 8.6 Hz). 13C NMR (DMSO-d6), δ: 11.02 (CH3), 14.09
(CH3), 55.52 (OCH3), 114.41, 123.35, 124.23, 126.87, 135.69,
139.56, 141.16, 142.69, 142.89, 147.03, 160.92. MS, found: m/z
386.1280 [M + H]+. Calculated for C18H20N5O3S+: 386.1281.
Synthesis of pyrazoles 3f—k (general procedure). A mixture
of pyrazole 2b,c (1.5 mmol), haloalkane (3 mmol), and К2CO3
(0.31 g, 2.25 mmol) in MeCN (or acetone in the synthesis of
compound 3k) (10 mL) was refluxed for 6—12 h. The precipitate
was filtered off, and the mother liquor was concentrated. The
products were purified by column chromatography using a hex-
ane—ethyl acetate mixture (4 : 1) as the eluent.
Experimental
The NMR spectra were recorded on a Bruker AVANCE III
500 spectrometer in CDCl3 and DMSO-d6 (1H, 500 MHz rela-
tive to SiMe4; 19F, 470 MHz relative to C6F6; 13C, 126 MHz
relative to the signal of the solvent (C 77 for CDCl3, C 39.5 for
DMSO-d6)). The IR spectra were measured on a Perkin—Elmer
Spectrum Two Fourier-transform infrared spectrometer equipped
with an attenuated total reflectance diamond crystal (ATR) ac-
cessory in a range of 4000—400 cm–1. Electrospray-ionization
mass spectra were obtained on a MaXis Impact HD quadrupole
time-of-flight mass spectrometer (Bruker Daltonik GmbH) for
solutions in CHCl3—MeOH (0.2 : 1.6) or CH2Cl2—MeOH
(0.05 : 1.6) at a flow rate of 180 L–1 h–1 using a modified pre-set
method of small-molecule infusion. The mass calibration was
performed using external HPC (high precision calibration) and
a G1969-85000 calibration solution (Agilent Technologies). The
melting points were measured in open capillary tubes using
a Stuart SMP30 melting point apparatus. Column chromato-
graphy was performed on Macherey-nagel silica gel 60 (0.063—
0.2 mm). The progress of the reactions was monitored by TLC
on ALUGRAM® Xtra SIL G/UV254 plates.
3,5-Dimethyl-4-[(4-methylphenyl)diazenyl]-1H-pyrazole
(2a), 3,5-dimethyl-4-phenyldiazenyl-1H-pyrazole (2b), and
3,5-dimethyl-4-[(4-methoxyphenyl)diazenyl]-1H-pyrazole (2c)
were synthesized by known procedures.5 4-{3,5-Dimethyl-4-[(4-
methylphenyl)diazenyl]-1H-pyrazol-1-yl}butyl acetate (3e) and
4-{3,5-dimethyl-4-[(4-methylphenyl)diazenyl]-1H-pyrazol-1-
yl}butan-1-ol (3l) were synthesized by procedures described
previously.7
Synthesis of pyrazoles 3a,b. A mixture of 3-arylhydrazinyli-
denepentane-2,4-dione 1b,c (2 mmol) and 2-hydroxyethylhy-
drazine (0.167 g, 2.2 mmol) in EtOH (10 mL) was refluxed for
12—14 h. The reaction mixture was concentrated. The products
were purified by column chromatography using CHCl3—EtOH
(45 : 1) as the eluent.
2-{3,5-Dimethyl-4-phenyldiazenyl-1H-pyrazol-1-yl}ethan-
1-ol (3a). Yellow powder, yield was 97%, m.p. 105—107 C
(MeCN) (cf. lit. data6: m.p. 105—106 C). IR, /cm–1: 3249
4-{4-[(4-Methoxyphenyl)diazenyl]-3,5-dimethyl-1H-pyr-
azol-1-yl}butyl acetate (3f). Orange oil, yield was 78%. IR,
/cm–1: 2954 (C—H), 1734 (MeCO2); 1600, 1501 (C=C).
1H NMR (CDCl3), δ: 1.65—1.71 (m, 2 H, CH2); 1.89—1.95
(m, 2 H, CH2); 2.05 (s, 3 H, CH3CO2); 2.49 (s, 3 H, CH3); 2.57
(s, 3 H, CH3); 3.87 (s, 3 H, OCH3); 4.06 (t, 2 H, OCH2,
J = 7.2 Hz); 4.09 (t, 2 H, NCH2, J = 6.5 Hz); 6.97 (d, 2 H, C6H4,
J = 9.0 Hz); 7.77 (d, 2 H, C6H4, J = 9.0 Hz). MS, found: m/z
345.1923 [M + H]+. Calculated for C18H25N4O3+: 345.1921.
1-Ethyl-3,5-dimethyl-4-phenyldiazenyl-1H-pyrazole (3g).
Brown powder, yield was 63%, m.p. 38—39 C. IR, /cm–1: 2987
(C—H); 1548, 1503 (C=C). 1H NMR (CDCl3), δ: 1.43 (t, 3 H,
CH3, J = 7.3 Hz); 2.51 (s, 3 H, CH3); 2.58 (s, 3 H, CH3); 4.08
(q, 2 H, NCH2, J = 7.3 Hz); 7.36 (t, 1 H, Ph, J =7.2 Hz); 7.45
(t, 2 H, Ph, J = 7.5 Hz); 7.78 (dd, 2 H, Ph, J1 = 8.0 Hz, J2 = 0.9 Hz).
13C NMR (CDCl3), δ: 9.73 (CH3), 13.94 (CH3), 15.18 (CH3),
43.92 (NCH2), 121.71, 128.86, 129.20, 135.13, 137.92, 142.38,
153.65. MS, found: m/z 229.1449 [M + H]+. Calculated for
C13H17N4+: 229.1448.
1
(OH), 2866 (C—H); 1555, 1508 (C=C). H NMR (CDCl3), δ:
2.50 (s, 3 H, CH3); 2.60 (s, 3 H, CH3); 3.41 (br.s, 1 H, OH);
4.04 (t, 2 H, OCH2, J = 4.9 Hz); 4.13 (t, 2 H, NCH2, J = 4.9 Hz);
7.37 (tt, 1 H, Ph, J1 = 7.2 Hz, J2 = 1.1 Hz); 7.4 (t, 2 H, Ph,
J = 7.7 Hz); 7.78 (dd, 2 H, Ph, J1 = 8.3 Hz, J2 = 1.1 Hz).
13C NMR (CDCl3), δ: 9.80 (CH3), 13.98 (CH3), 50.13 (NCH2),
61.45 (OCH2), 121.77, 128.88, 129.41, 134.96, 139.40, 142.83,
153.52. MS, found: m/z 245.1397 [M + H]+. Calculated for
C13H17N4O+: 245.1397.
2-{3,5-Dimethyl-4-[(4-methoxyphenyl)diazenyl]-1H-pyr-
azol-1-yl}ethan-1-ol (3b). Yellow powder, yield was 67%, m.p.
109—111 C. IR, /cm–1: 3250 (OH), 2944—2868 (C—H); 1603,
1584, 1556, 1499 (C=C). 1H NMR (CDCl3), δ: 2.49 (s, 3 H,
CH3); 2.58 (s, 3 H, CH3); 3.43 (br.s, 1 H, OH); 3.87 (s, 3 H,
OCH3); 4.03 (t, 2 H, OCH2, J = 4.9 Hz); 4.13 (t, 2 H, NCH2,
J = 4.9 Hz); 6.97 (d, 2 H, C6H4, J = 9.0 Hz); 7.77 (d, 2 H, C6H4,
J = 9.0 Hz). 13C NMR (CDCl3), δ: 9.75 (CH3), 13.88 (CH3),
50.11 (NCH2), 55.47 (OCH3), 61.39 (OCH2), 113.99, 123.29,
1-Ethyl-4-[(4-methoxyphenyl)diazenyl]-3,5-dimethyl-1H-
pyrazole (3h). Orange powder, yield was 52%, m.p. 72—74 C.

Synthesis of 4-aryldiazenyl-3,5-dimethylpyrazoles
Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
1129
IR, /cm–1: 2968 (C—H); 1600, 1579, 1551, 1501 (C=C).
1H NMR (CDCl3), δ: 1.43 (t, 3 H, CH3, J = 7.3 Hz); 2.50 (s, 3 H,
CH3); 2.56 (s, 3 H, CH3); 3.86 (s, 3 H, OCH3); 4.08 (q, 2 H,
NCH2, J = 7.3 Hz); 6.97 (d, 2 H, C6H4, J = 9.0 Hz); 7.77 (d, 2 H,
C6H4, J = 9.0 Hz). 13C NMR (CDCl3), δ: 9.69 (CH3), 13.87
(CH3), 15.19 (CH3), 43.85 (NCH2), 55.47 (OCH3), 113.97,
123.20, 134.87, 137.12, 142.11, 147.93, 160.63. MS, found: m/z
259.1553 [M + H]+. Calculated for C14H19N4O+: 259.1553.
3,5-Dimethyl-4-phenyldiazenyl-1-(4,4,4-trifluorobutyl)-1H-
pyrazole (3i). Brown oil, yield was 68%. IR, /cm–1: 2953
(C—H); 1555, 1503 (C=C); 1148—1130 (C—F). Immediately
after the dissolution in CDCl3, the trans-azo form was observed.
1H NMR (CDCl3), δ: 2.11—2.20 (m, 4 H, (CH2)2); 2.50 (s, 3 H,
CH3); 2.60 (s, 3 H, CH3); 4.12 (t, 2 H, NCH2, J = 6.5 Hz); 7.38
(tt, 1 H, Ph, J1 = 7.4 Hz, J2 = 1.1 Hz); 7.47 (t, 2 H, Ph, J = 7.5 Hz);
7.78 (dd, 2 H, Ph, J1 = 8.3 Hz, J2 = 1.1 Hz) (trans). 19F NMR
(CDCl3), δ: 95.67 (t, CF3, J = 10.4 Hz) (trans). 13C NMR
(CDCl3), δ: 9.74 (CH3); 13.95 (CH3); 22.50 (q, CH2CH2CF3,
J = 3.0 Hz); 30.96 (q, CH2CH2CF3, J = 29.3 Hz); 47.27 (NCH2);
121.76; 126.8 (q, CF3, J = 276.0 Hz); 128.89; 129.42; 135.14;
138.48; 142.81; 153.53 (trans). After 3 h, a mixture of cis/trans-
azo forms (1 : 20) was observed. 1H NMR (CDCl3), δ: 1.67
(s, 3 H, CH3); 2.02 (s, 3 H, CH3); 2.01—2.04 (m, 4 H, (CH2)2);
3.97 (t, 2 H, NCH2, J = 6.5 Hz); 7.02 (dd, 2 H, Ph, J1 = 8.3 Hz,
J2 = 1.1 Hz); 7.19 (tt, 1 H, Ph, J1 = 7.5 Hz, J2 = 1.1 Hz); 7.34
(t, 2 H, Ph, J = 7.5 Hz) (cis). 19F NMR (CDCl3), δ: 95.62
(t, CF3, J = 10.4 Hz) (cis). MS, found: m/z 311.1476 [M + H]+.
Calculated for C15H18F3N4+: 311.1478.
4-[(4-Methoxyphenyl)diazenyl]-3,5-dimethyl-1-(4,4,4-tri-
fluorobutyl)-1H-pyrazole (3j). Orange powder, yield was 98%,
m.p. 77—79 C. IR, /cm–1: 2961 (C—H); 1603, 1583, 1557,
1504 (C=C), 1137 (C—F). 1H NMR (CDCl3), δ: 2.11—2.20
(m, 4 H, (CH2)2); 2.49 (s, 3 H, CH3); 2.57 (s, 3 H, CH3); 3.87
(s, 3 H, OCH3); 4.10 (t, 2 H, NCH2, J = 6.5 Hz); 6.97 (d, 2 H,
C6H4, J = 9.0 Hz); 7.78 (d, 2 H, C6H4, J = 9.0 Hz). 19F NMR
(CDCl3), δ: 95.69 (t, CF3, J = 10.4 Hz). 13C NMR (CDCl3), δ:
9.72 (CH3); 13.86 (CH3); 22.55 (q, CH2CH2CF3, J = 3.0 Hz);
30.96 (q, CH2CH2CF3, J = 29.3 Hz); 47.23 (NCH2); 55.49
(OCH3); 114.02; 123.31; 126.8 (q, CF3, J = 276.0 Hz); 134.92;
137.65; 142.60; 147.84; 160.83. MS, found: m/z 341.1590
[M + H]+. Calculated for C16H20F3N4O+: 341.1584.
4-[(4-Methoxyphenyl)diazenyl]-3,5-dimethyl-1-(3,3,3-tri-
fluoropropyl)-1H-pyrazole (3k). Yellow powder, yield was 36%,
m.p. 75—77 C. IR, /cm–1: 2966—2925 (C—H); 1602, 1581,
1557, 1503 (C=C), 1141 (C—F). 1H NMR (CDCl3), δ: 2.49
(s, 3 H, CH3); 2.59 (s, 3 H, CH3); 2.68—2.78 (m, 2 H, CH2);
3.87 (s, 3 H, OCH3); 4.27 (t, 2 H, NCH2, J = 7.2 Hz); 6.97
(d, 2 H, C6H4, J = 9.0 Hz); 7.78 (d, 2 H, C6H4, J = 9.0 Hz).
19F NMR (CDCl3), δ: 96.07 (t, CF3, J = 10.4 Hz). MS, found:
m/z 327.1433 [M + H]+. Calculated for C15H18F3N4O+: 327.1427.
Microbiological evaluation of compounds 2 and 3 was per-
formed at the Ural Research Institute of Dermatovenereology
and Immunopathology. The antimicrobial properties were
evaluated by the serial dilution method recommended by the
Clinical and Laboratory Standards Institute (CLSI, 2014). The
following test strains were used: strains of the dermatophyte
fungi Trichophyton rubrum (RCPF F 1408), Trichophyton men-
tagrophytes var. gypseum (RCPF F 1425), Trichophyton tonsurans
(RCPF F 1396/228), Trichophiton violaceum (RCPF F 1211),
Trichophyton mentagrophytes var. interdigitale (RCPF F 1459/
11044), Trichophyton schoenleinii (RCPF F 235/25), Epider-
mophyton floccosum (RCPF F 1659/17), Microsporum canis
(RCPF F 1643/1585); stains of the yeast-like fungi Candida
albicans (RCPF Y 401/NCTC 885/653), Candida krusei (RCPF
Y 1472/310), Candida glabrata (RCPF Y 1486/47), Candida
parapsilosis (RCPF Y 1579/296), Candida dubliniensis (RCPF Y
1307), Candida guilliermondii (RCPF Y 1373), Candida tropica-
lis (RCPF Y 1513/784) from the Russian Collection of Pathogenic
Fungi of the Kashkin Research Institute of Medical Mycology
of the North-Western State Medical University named after I.
I. Mechnikov (St. Petersburg, Russia); the reference strain
Neisseria gonorrhoeae (NCTC 12700 /ATCC 49226) from the
National Collection of Type Cultures (NCTC, UK).
The agar medium Complegon (Russia) supplemented with
a 1% growth additive (Russia, registration certificate number
FSR 2007/00163) was used as a culture medium for gonococci;
for fungi, the Sabouraud Dextrose Broth (SDB, Research Center
of Pharmacotherapy, St. Petersburg, registration certificate
number FSR 2012/14080) was used. Microorganisms were iden-
tified with an accuracy of 99.9% using a BioMerieux VITEK MS
MALDI-TOF Plus mass spectrometer by time-of-flight mass
spectrometry of matrix-anchored bacterial proteins. Inocula were
prepared using a BioMerieux DensiCHEK Plus densitometer;
the optical density (OD) was 0.5 McFarland, which corresponds
to 1.5•108 CFU mL–1. A suspension of microorganisms for N.
gonorrhoeae and C. albicans was prepared from an overnight
culture; for dermatophytes, from a two-week culture pre-homo-
genized in physiological sterile saline. The inoculum dose for
gonococci was 105 CFU mL–1; for fungi, 106 CFU mL–1. The
antimycotic activity of the chemicals was evaluated by a micro-
method.10 The antibacterial activity against N. gonorrhoeae was
assessed using serial twofold dilutions on agar (gold standard).11
Agar-based culture medium served as the growth medium; the
liquid state of the medium was maintained at 52 C in a BioSan
WB-4 MS water bath-thermostat. The tested chemicals were
dissolved in DMSO. The initial 1000 g mL–1 dilution was
prepared using sterile distilled water; serial twofold dilutions
(from 250—200 g mL–1) were prepared using growth culture
media. Dermatophytes were incubated for up to 14 days at 27 C;
yeast-like fungi of the Candida genus, for 24 h; gonococci, for
24 h at 37 C in a wet chamber of a Sanyo MCO-15AC CO2
incubator (CO2 5.0%). In each assay, the positive and negative
controls were employed. The minimum inhibitory concentration
was assessed visually as the lowest concentration, which inhibits
culture growth. The degree of antimicrobial activity of the
chemicals was determined according to the recommended
criteria.10 Chemically pure fluconazole and spectinomycin were
used as the reference analogues.
Evaluation of cytotoxicity of active compounds using the cell
culture McCoy. The cytotoxic effect of the newly synthesized
chemicals was evaluated using the transplantable fibroblast-like
McCoy B cells, which were obtained from the Russian Collection
of Cell Cultures of Vertebrates (RCCCV) of the Institute of
Cytology of the Russian Academy of Sciences (St. Petersburg,
Russia). The concentrations of compounds required to inhibit
90, 50, and 10% cell growth (IC90, IC50, and IC10, respectively)
were determined.
The cells were grown in DMEM culture medium using
HEPES salts and supplemented with L-glutamine (Biolot, Russia)
and 10% fetal bovine serum (standard quality (FBS), GE
Healthcare, Austria). Sterile 96-well cell culture plates were used.
A cell suspension (3•104 cells per well) was seeded in 96-well

Khudina et al.
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Russ. Chem. Bull., Int. Ed., Vol. 70, No. 6, June, 2021
plates, and the plates were incubated at 37 C (5.0% CO2) in an
incubator until a monolayer formed. Serial dilutions of solutions
of the chemicals were prepared in sterile 96-well plates at con-
centrations of 500—0.25 g mL–1. Dimethyl sulfoxide was used
as the solvent for chemicals; DMEM, as the dilution medium.
After removal of the incubation medium, solutions of the
chemicals were added to the wells, and the plates were incu-
bated for 72 h at 37 C in a CO2 incubator. All concentrations
were tested in triplicate. A negative control (culture medium)
and the DMSO solvent control were used in experiments. The
solutions of the tested compounds were removed, the plates were
washed with physiological sterile saline, and a solution (200 L)
containing 1 mg mL–1 NBT (USA) was added. The incubation
was performed for 30 min at 37 C (5.0% CO2). The results were
visually assessed using a LOMO BIOLAM P2-1 inverted micro-
scope. Purple-colored cells in the monolayer were counted as
live; yellow-colored cells, as dead. Apart from the color, the
morphology of the cells in the monolayer was evaluated.
Russ. J. Gen. Chem., 2019, 89, 2314; DOI: 10.1134/
S1070363219110240.
2. M. Shukla, D. S. Seth, H. Kulshreshtha, J. Appl. Chem., 2013,
2, 1484; www.joac.info.
3. K. Kaur, V. Kumar, V. Beniwal, V. Kumar, K. R. Aneja,
V. Sharma, S. Jaglan, Med. Chem. Res., 2015, 24, 3863; DOI:
10.1007/s00044-015-1429-2.
4. A. A. Bekhit, H. M. A. Ashour, A. A. Guemei, Arch. Pharm.
Chem. Life Sci., 2005, 338, 167; DOI: 10.1002/ardp.200400940.
5. S. A. Sankpal, M. B. Deshmukh, P. V. Anbhule, D. K.
Salunkhe, K. N. Alsundkar, P. P. Patil, D. R. Chandam, S. D.
Jagadale, A. G. Mulik, S. S. Rokade, J. Chem. Pharm. Res.,
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10.1016/j.jfluchem.2010.03.003.
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The work was performed within the framework of the
state assignment (No. AAAA-A19-119011790130-3) using
facilities of the Joint Use Center "Spectroscopy and
Analysis of Organic Compounds" (JUC SAOC) of the
I. Ya. Postovsky Institute of Organic Synthesis of the Ural
Branch of the Russian Academy of Sciences.
No human or animal subjects were involved in this
research.
10. A. A. Kubanova, Zh. V. Stepanova, T. A. Gus´kova, T. V.
Pushkina, L. Yu. Krylova, I. B. Shilova, A. S. Trenin,
Rukovodstvo po provedeniyu doklinicheskikh issledovanii lekar-
stvennykh sredstv [Guidelines for Preclinical Trials of Medicinal
Products], Ed. A. N. Mironov, Grif i K, Moscow, 2012, Part
1, 578 (in Russian).
11. Clinical and Laboratory Standards Institute (CLSI). Perfor-
mance Standards for Antimicrobial Susceptibility Testing. 24th
Ed. CLSI Document M100-S24, 2014, 34, 84.
The authors declare no conflict of interest, financial
or otherwise.
References
Received February 15, 2021;
in revised form April 5, 2021;
accepted April 8, 2021
1. A. H. Moustafa, H. A. El-Sayed, S. M. El Rayes, H. A.
Morsy, S. H. Abd-allah, H. F. Ismail, M. M. Abd El-Aal,