Multiple biological active 4-aminopyrazoles containing trifluoromethyl and their 4-nitroso-precursors: Synthesis and evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112768

4-Nitroso-3-trifluoromethyl-5-alkyl[(het)aryl]pyrazoles were synthesized via one-pot nitrosation of 1,3-diketones or their lithium salts followed by treatment of hydrazines. Reduction of nitroso-derivatives made it possible to obtain 4-amino-3-trifluorome

Full text of DOI:10.1016/j.ejmech.2020.112768

European Journal of Medicinal Chemistry 208 (2020) 112768
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Multiple biological active 4-aminopyrazoles containing
trifluoromethyl and their 4-nitroso-precursors: Synthesis and
evaluation
,
, b
Yanina V. Burgart a b, Natalia A. Agafonova a, Evgeny V. Shchegolkov a
,
Olga P. Krasnykh c, Svetlana O. Kushch a, Natalia P. Evstigneeva d, Natalia A. Gerasimova d,
Vera V. Maslova c, Galina A. Triandafilova c, Sergey Yu. Solodnikov c, Maria V. Ulitko b,
Galina F. Makhaeva e, Elena V. Rudakova e, Sophia S. Borisevich f, Natalia V. Zilberberg d,
Nikolai V. Kungurov d, Victor I. Saloutin a *, Oleg N. Chupakhin a
,
b,
, b
a Postovsky Institute of Organic Synthesis of the Ural Branch of the Russian Academy of Science, S. Kovalevskoi St., 22, Ekaterinburg, 620108, Russia
b Ural Federal University Named After the First President of Russia B.N. Yeltsin, Mira St. 19, Ekaterinburg, 620002, Russia
c Perm National Research Polytechnic University, Komsomolsky Av., 29, Perm, 614990, Russia
d Ural Research Institute for Dermatology, Venereology and Immunopathology, Shcherbakova St., 8, Ekaterinburg, 620076, Russia
e Institute of Physiologically Active Compounds of the Russian Academy of Sciences, Severny Proezd 1, Chernogolovka, 142432, Russia
f Ufa Institute of Chemistry of Russian Academy of Science, Octyabrya Av., 71, Ufa, 450078, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Nitroso-3-trifluoromethyl-5-alkyl[(het)aryl]pyrazoles were synthesized via one-pot nitrosation of 1,3-
diketones or their lithium salts followed by treatment of hydrazines. Reduction of nitroso-derivatives
made it possible to obtain 4-amino-3-trifluoromethylpyrazoles chlorides. According to computer-aided
calculations, all synthesized compounds are expected to have acceptable ADME profile for drug
design. Tuberculostatic, antibacterial, antimycotic, antioxidant and cytotoxic activities of the compounds
were evaluated in vitro, while their analgesic and anti-inflammatory action was tested in vivo along with
acute toxicity studies. N-Unsubstituted 4-nitrosopyrazoles were the most effective tuberculostatics (MIC
Received 8 July 2020
Received in revised form
10 August 2020
Accepted 15 August 2020
Available online 23 August 2020
Keywords:
to 0.36
m
g/ml) and antibacterial agents against Streptococcus pyogenes (MIC to 7.8
mg/ml), Staphylococcus
Trifluoromethyl-containing 4-amino- and 4-
nitrosopyrazoles
4-Hydroxyiminopyrazolines
ADME calculations
Tuberculostatic
Antibacterial
Antimycotic
Antioxidant
Cytotoxic
aureus, S. aureus MRSA and Neisseria gonorrhoeae (MIC to 15.6
m
g/ml). 4-Nitroso-1-methyl-5-
phenylpyrazole had the pronounced antimycotic action against a wide range of fungi (Trichophyton
rubrum, T. tonsurans, T. violaceum, T. interdigitale, Epidermophyton floccosum, Microsporum canis with MIC
0.38e12.5
test, ORAC/AAPH
m
g/ml). N-Unsubstituted 4-aminopyrazoles shown high radical-scavenging activity in ABTS
and oxidative erythrocyte hemolysis assays. 1-Methyl-5-phenyl-3-
trifluoromethylpyrazol-4-aminium chloride revealed potential anticancer activity against HeLa cells
(SI > 1351). The pronounced analgesic activity was found for 4-nitroso- and 4-aminopyrazoles having
phenyl fragment at the position 5 in “hot plate” test. The most of the obtained pyrazoles had a moderate
acute toxicity.
Analgesic activity
Acute toxicity
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
it appears to be a perspective scaffold for designing biologically
active molecules. The functionalization of the pyrazole backbone
Although a pyrazole moiety is rarely found in natural products,
allowed multifunctional compounds to be obtained that are able to
bind to biological targets in different ways, leading to various
pharmaceutical activities [1e5]. There is an increasing number of
drug discovery programs that are using aminopyrazoles as a ver-
satile framework [6,7]. Many of bioactive pyrazoles are 4-amino-
derivatives, e.g., pyrazolone analgesic-antipyretics (amino-
phenazone, metamizole, etc.), which are known to interact with
* Corresponding author. Postovsky Institute of Organic Synthesis of the Ural
Branch of the Russian Academy of Science, S. Kovalevskoi St., 22, Ekaterinburg,
620108, Russia.
E-mail address: saloutin@ios.uran.ru (V.I. Saloutin).
https://doi.org/10.1016/j.ejmech.2020.112768
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
several biological targets (Fig. 1). Recently, N-myristoyltransferase
inhibitor was found to be useful for treatment of the deadly African
trypanosomiasis (sleeping sickness) caused by Trypanosoma brucei
[8]. 4-Aminopyrazole chemotype is present in compounds that are
currently in clinical trial, for example, AT7519 as a potent inhibitor
of several CDK [9] and AT9283 as a multitargeted kinase inhibitor
with potent aurora kinase activity [10].
nitropyrazoles with hydrogen (at p ¼ 810 kPa) in the presence of
Pd/C [42,43], zinc in acetic acid [44], iron in hydrochloric acid or
potassium borohydride under the action of CuCl or SnCl2 [45]. This
approach is made complicated by the need for the preliminary
synthesis of 4-nitropyrazoles via nitration with HNO3/H2SO4 at
high temperature; and the process may occur non-selectively
[42,43].
Moreover, 1,2,4,5-functionalized 4-aminopyrazoles possess
antibacterial [11e13], antifungal [11,13], cytotoxic [14] actions and
show inhibitory activity against urease [15]. 4-Aminopyrazoles were
used as initial synthons for the synthesis of Sildenafil (Viagra®)
(Fig. 1) and its derivatives [16], antineoplastic and antibiotic drug
Formycin A [17], and bioactive 4-hetarylated derivatives [18], acyla-
mides [19], pyrazolo[4,3-d]pyrimidines [20,21], pyrazoloxadiazoles
[22], pyrazolo[4,3-c][1,2,6]benzothiadiazocine [23], pyrazolo[3,4-f]
pyrrolo[1,2-a][1,4][]diazepines [24] and 4-(het)arylazopyrazoles [25]
as disperse dyes. Therefore, 4-aminopyrazole core may serve as an
advantageous starting point for different drug discovery programs.
The literature data show a variety of synthetic routes to obtain
4-aminopyrazoles. The convenient precursors to their synthesis are
4-nitrosopyrazoles, because the reduction of the nitroso group can
be carried out under mild conditions, without affecting the other
functional groups in the molecule [18,19,21,26e28]. There is
another approach, which is based on the catalytic reduction of 4-
nitropyrazoles [16,18,19,23,27], but it requires preliminary nitra-
tion of pyrazoles to be carried out under rather severe conditions
[19,27,29]. An example of 4-arylazopyrazoles reduction into amino
derivatives is known [25]. By the ThorpeeZiegler reaction, it is
possible to obtain 4-aminopyrazoles having (het)aroyl
[11e14,22,30e34] or cyano [13,15,21,35] substituent at the position
5. However, this method requires using hardly accessible initial
reagents. The original process is described for 4-aminopyrazole
synthesis via the reaction of enaminones with diazonium tetra-
fluoroborate [36].
There are no data available on the application of 4-
nitrosopyrazoles for polyfluoroalkyl-containing 4-aminopyrazoles
synthesis. However, this approach may be regarded as very
promising so long as both introduction and reduction of the
nitroso-group are carried out under mild conditions ensuring effi-
ciency and selectivity of the processes. The feasibility of this
approach can be evidenced by the synthesis of 4-amino-5-phenyl-
4-trifluoromethylpyrazole via one-pot nitrosation of 4,4,4-
trifluoro-1-phenylbutane-1,3-dione [46] or its lithium salt [47]
followed by simultaneous cyclization and reduction with hydrazine
hydrate.
Herein, we have developed the effective approaches to the
synthesis of trifluoromethyl-containing 4-aminopyrazoles using 4-
nitrosopyrazoles as precursors (Fig. 2). In view of the multi-
targeted nature on many pyrazole-containing drugs, we have
assessed new trifluoromethylated pyrazoles as causing some of the
most expected types of biological activity, such as antibacterial
(including antituberculosis), antimycotic, antitumor, analgesic and
anti-inflammatory ones; and we have determined their radical-
scavenging activity as well.
Although 4-nitrosopyrazoles obtained in our study are viewed
mainly as intermediates for synthesis of amino-derivatives, we paid
special attention to the assessment of their biological activity. To
the best of our knowledge, the release of nitric oxide (NO) using
nitrosopyrazoles has thus far been unreported. However, it can be
expected that these compounds are capable of performing such a
function. Nitric oxide is known to be an important regulator and
mediator involved in many physiological processes, e.g., vasodila-
tion, neurotransmission, decreasing platelet aggregation, immune
system reactions, regulation of smooth muscle tone, memory state,
etc. [48,49]. Nitric oxide metabolism disorders play a crucial role in
the pathogenesis of a variety of serious conditions, such as septic
shock, arterial hypertension, coronary heart disease, stroke, heart
failure, diabetes mellitus, neurodegenerative diseases [50,51].
Multiple research have been done [52,53] on designing
Although fluorinated pyrazoles are, undoubtedly, promising for
being used in the pharmaceutical and agrochemical industries
[37,38], there is no or very few information on fluorine-containing
analogues in literature. The present studies are encouraged by the
discovery of selective COX-2 inhibitors among (trifluoromethyl)
pyrazole derivatives, e.g., drug celecoxib [39e41].
4-Amino-3-trifluoromethylpyrazoles having alkyl, aryl or acyl
substituents were obtained through the catalytic reduction of 4-
Fig. 1. Drugs based on 4-aminopyrazole derivatives.

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
3
In contrast to 1,1,1-trifluoropentane-2,4-dione 1e [64], its
lithium salt 2e reacted with hydrazine hydrate to yield 4-
nitrosopyrazole 5h immediately, while with methyl hydrazine
resulted in two isomeric 3-CF3- and 5-CF3-4-nitrosopyrazoles 5i
and 6 at a ratio of 2 to 1 (Scheme 2), which were separated by a
column chromatography. The same isomeric mixture has been
obtained by D.A. Fletcher et al. [66] based on 1,1,1-trifluoropentane-
2,4-dione, but this process required more time.
The synthesis of 4-nitrosopyrazoles bearing N-aryl substituent
proceeded ambiguously. Earlier, we have isolated 5-hydroxy-4-
hydroxyimino-5-trifluoromethyl-1-phenylpyrazoline by one-pot
reaction of 1,1,1-trifluoropentane-2,4-dione 1e with nitrosating
reagent (sodium nitrite in acetic acid) and phenyl hydrazine [64].
Nitrosation of lithium 1,2-diketonates 2a,d and cyclization with
aryl hydrazines carried out as a one-pot reaction also resulted in the
formation of 5-hydroxypyrazolines 7a,b (Scheme 3). However,
cyclocondensation of preliminary obtained 2-hydroxyimino-1,3-
diketones 3a,d with aryl hydrazines allowed us to synthesize 4-
nitrosopyrazoles 5j,k. 2-Hydroxyimino-1,3-diketones 3a,d reacted
with aryl hydrazine to form 5-CF3-pyrazoline 7a,c similarly to
three-component synthesis. Another cyclization direction can be
explained by changing of medium acidity due to the presence of
lithium cations obviously affecting as a buffer. We failed to dehy-
drate 5-hydroxypyrazolines 7a-c owing to they seemed to exist as
stable hydroxyimine tautomers.
Fig. 2. The structural modification for 4-amino- and 4-nitroso-3-trifluoromethyl pyr-
azoles and assessed biological effects.
combinational NO-NSAIDs having an anti-inflammatory moiety
and the source of the NO molecule.
We also recognize a rather useful aspect of studying the bio-
logical activity of nitroso- and aminopyrazoles, because nitroso-
and amino-compounds are the metabolites of many medicines
[54e57]. In this regard, the study of biological activity of this class
of compounds is of great importance.
2. Results and discussion
2.1. Chemistry
All pyrazoles 5a-k, 6 have a characteristic color from bright blue
to dark green for nitroso compounds. However, it may be typical for
N-unsubstituted pyrazoles 5a-d to have nitroso-oxime tautom-
erism. The structure of 4-nitrosopyrazole 5c was confirmed by X-
Ray diffraction analysis (XRD) (Fig. 3, CCDC 2006858). Albeit there
are no substituents at the nitrogen atom position in this compound,
the position of hydrogen atom allowed us to attribute the com-
pound to 3-CF3-isomer. Analysis of XRD data showed that the ox-
ygen atom O2 of nitroso group turns toward trifluoromethyl
substituent and binds with proton H7A0 of the furan ring of another
pyrazole molecule (the distance is 2.669 Å, not shown in Fig. 3). The
structure of compound 5c forms dimeric chains, which are bound
by the intermolecular hydrogen bond (IHB) between the nitrogen
atom N2 of one pyrazole molecule and the proton H1’ of another
molecule (the bond length is 2.191 Å).
4-Nitrosopyrazoles bearing alkyl, (het)aryl, carboxyl sub-
stituents at the positions 3 and 5 are known to be obtained by
cyclization of 2-hydroxyimino-1,3-diketones with hydrazines [58].
The reduction of 4-nitrosopyrazoles into 4-amino derivatives was
realized with hydrogen in the presence of Pd/C catalyst [59] or zinc
in acetic acid [60]. The photosensitized reduction of 4-
nitrosopyrazoles was described, with titanium dioxide being used
as a photocatalyst in the presence of trimethylamine [61]. Occa-
sionally, heating of 2-hydroxyimino-1,3-diketones with excess of
hydrazine hydrate immediately resulted in 4-aminopyrazole,
because cyclization and reduction occurred simultaneously [62].
To synthesize 4-nitroso-3-polyfluoroalkylpyrazoles 5, we
applied an earlier introduced one-pot method based on the nitro-
sation of 1,3-diketones 1 and the subsequent condensation of in-
The 3-CF3-regioisomeric structure of N-substituted 4-
nitrosopyrazoles 5g,k was proved by XRD data (Fig. 4, CCDC
2007108 and Fig. 5, CCDC 2006860, respectively). The crystalline
structure of compound 5g comprises two crystallographically in-
dependent molecules. Binding of these molecules was found to
termediates 2-hydroxyimino-1,3-diketones
3 with hydrazines
[63,64]. In this work, in addition to 1,3-diketones 1, we used their
lithium salts 2, which are intermediates in Claisen synthesis of
polyfluoroalkyl-1,3-diketones 1 from esters of polyfluorocarboxylic
acids and methyl ketones under the action of lithium hydride [65].
It was found that lithium phenyl- and thienyl-1,3-diketonates
2a,b and diketone 1b in the reactions of successive nitrosation
and cyclization with hydrazine hydrate firstly result in 3-hydroxy-
4-nitrosopyrazolines 4a,b, which then produce 4-nitrosopyrazoles
5a,b after dehydration (Scheme 1). Moreover, dehydration of
compound 4a was carried out under heating, while product 4b was
dehydrated on silica gel during flash-chromatography. Unlike the
previous transformations, furanyl- and tolyl-1,3-diketones 1c,d and
their lithium salts 2c,d yielded 4-nitrosopyrazoles 5c,d immedi-
ately. The using of methyl hydrazine in one-pot nitrosation and
cyclization of 1,3-diketones 1a-c and their lithium salts 2a-c
resulted in 4-nitrosopyrazoles 5e-g.
It should be noted that, in earlier studies [64], the products 4a
and 5a,d would be obtained from 4-aryl-1,1,1-trifluorobutane-2,4-
dione 1a following an analogous three-component method. In the
present research, however, these products were synthesized based
on lithium diketonate 2a. Comparing the products yields, a
conclusion can be made that using lithium salt 2a is more efficient
than using diketone 1a.
occur through CeHep-stacking between the proton H1Ab of one
molecule and the furan ring of another molecule, with the bond
lengths of 2.803 Å. Molecules of pyrazole 5k having a phenyl-
sulfamide fragment form dimers in the crystals due to the IHB
formation between amino- and sulfo-groups (the bond length is
2.071 Å).
According to 19F NMR spectroscopy, the trifluoromethyl group of
all 4-nitrosopyrazoles 5a,b, d-j resonated at
d 97 … 100 ppm (in
DMSO‑d6). It was consistent with the values for 3-CF3-4-
nitrosopyrazoles 5c,k, for which the XRD was made. In contrast
to 3-CF3-isomers 5, the signal from trifluoromethyl substituent of
5-CF3-pyrazole 6 was revealed in a lower field at
d 105 ppm.
In contrast to published data [46,47], no one-pot formation of 4-
aminopyrazoles was observed in our three- and two-component
syntheses. We carried out the reduction of nitroso group by SnCl2
in hydrochloric acid to yield trifluoromethyl-containing 4-
aminopyrazoles 8a,b,e,f,j,k as aminium chlorides (Scheme 4). To
synthesize 4-aminopyrazoles as a base, we tried out various con-
ditions. Heating of 4-nitrosopyrazole 5a in sodium dithionite water
solution resulted in 4-aminopyrazole 9a in 63% yield. We carried

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Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
Scheme 1. Synthesis of 3-hydroxy-4-nitrosopyrazolines and 4-nitrosopyrazoles.
Scheme 2. Synthesis of methyl-containing 3-CF3- and 5-CF3-4-nitrosopyrazoles.
Scheme 3. Synthesis of aryl-containing 4-nitrosopyrazoles and 5-CF3-5-hydroxypyrazolines.
out reduction of the same compound 5a with hydrogen in the
presence of Pd/C and the yield of amine 9a was 90%. However, the
disadvantages of this process were the necessity of using an auto-
clave and the high cost of catalyst. The most convenient method for
carrying out reduction of 4-nitrosopyrazole 5a turned out to be the
application of Zn/AcOH system; and amine 9a was synthesized in
96% yield, with practically no purification required. The remaining
4-aminopyrazoles 9b,d,f were obtained using this technique. The
only suitable method to yield 5-furanyl-substituted aminopyrazole
9g was reduction of pyrazole 5g with hydrogen in the presence of
Pd/C catalyst, so far as other conditions resulted in the formation of
a mixture of compounds that was difficult to characterize.
The salt form of compound 8e was unambiguously confirmed by
XRD (Fig. 6, CCDC 2006861).
2.2. In silico ADME studies
We have carried out the calculations of ADME parameters for

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
5
In column 2 (#star), the total number of descriptors is indicated,
which are going beyond the permitted values of the various
physico-chemical and structural parameters. The maximum
allowed disagreement cannot be more than five, and all the com-
pounds comply with this requirement. The most of the structures
have only one fault: the compounds do not contain definite groups
capable of metabolic reactions. The maximum number of faults
(three) are revealed in isomeric dimethyl-substituted 4-
nitrosopyrazoles 5i, 6. 1-[4-(Methanesulfonyl)phenyl]-4-nitroso-
5-phenylpyrazole 5j has two faults. All disagreements are con-
nected with the structural peculiarities of the compounds.
It is evident from Table 1 that all the synthesized compounds
follow the Lipinski’s rule and display favorable pharmacokinetic
profiles for all descriptors. The molecular masses and calculated
number of the allowed donor and acceptor hydrogen bonds of the
investigated compounds with water correspond to permitted
values. Estimation of octanol-water partition coefficient (lip-
ophilicity) examined by QPlogPo/w value showed good absorption.
Polar surface area (PSA), which should not be more than 200 Å, is
another key property linked to drug bioavailability and the values
for the target molecules are in the range of 41.0e131.6 Å. The
maximum PSA is observed for pyrazoline 7c having the sub-
stituents in both aryl fragments. Note, the presence of bulky moi-
eties in pyrazoles 5j,k, 7a-c, 8j,k results in the reduced Caco-2 cell
permeability parameter (QPPcaco) and aqua solubility parameter
(QPlogS). Blood/Brain partition coefficient (QPlogBB) value, which
is a measure of the ability of a drug to cross the blood-brain barrier,
is in the acceptable range for all molecules. Further, human serum
albumin binding co-efficient (QPlogKhsa) also is one of the key
factors, and the predicted values for the compounds are in the
range of ꢀ0.5 … 0.3. The number of possible metabolic reactions for
synthesized pyrazoles is no more than two; while for the de-
rivatives with phenyl substituent 5a,e,j, 8a,e,j and 9a,e it is zero.
The predicted values of human oral absorption (HOA) of the com-
pounds are in the range of 66e100%, which indicates their possi-
bility of good or high oral bioavailability. The reduction of HOA
parameter (<80%) is observed for pyrazoles 5k, 7b,c, 8k bearing
bulky moieties. Thus, all the compounds can be considered as
“drug-like” and are expected to have an acceptable ADME profile.
Fig. 3. ORTEP view of molecule 5c.
theoretical evaluation of physico-chemical and pharmaco-kinetic
characteristics, which would predict whether the synthesized
compounds can be applied as medicines [67]. For the investigated
compounds, pharmacological activity was forecast and the drug-
like level determined using the QSAR approach in subprogram
QikProp (software Schrodinger Small-Molecule Drug Discovery
Suite) [68].
In silico ADME properties and other pharmaco-kinetical prop-
erties viz. Partition coefficient of n-octanol and water, aqua solu-
bility, polar surface area, number of rotatable bonds, number of
hydrogen bond donors/acceptors, caco-2 cell permeability, human
serum albumin binding, blood/brain partition coefficient and hu-
man oral absorption of all synthesized compounds were computed
by using the QikProp program. The most valuable parameters are
presented in Table 1. Highlighted in red is the range of minimum
and maximum recommended values (in software limits) corre-
sponding to 95% of the known drugs.
2.3. Biology
Considering the multi-targeted nature of many pyrazolo-
containing drugs, we have evaluated new polyfluoroalkyl-4-
nitroso- and -4-amino-derivatives for a number of more expected
types of biological activity applying mainly a phenotypic screening
approach, which allows most of the possible biological targets and
metabolizing enzymes to be involved in a single screen.
2.3.1. Tuberculostatic, antibacterial and antimycotic activity
Powerful tuberculostatics are known among the substituted
pyrazoles [69], therefore a series of synthesized compounds was
tested for their antituberculosis action. The investigation was car-
ried out on Mycobacterium tuberculosis strain H37Rv via the mea-
surement of the minimum inhibitory concentration (MIC) which is
necessary to inhibit bacterial growth (reference drugs are isoniazid
and pyrazinamide). The results are presented in Table 2. The
studied pyrazoles were found to have tuberculostatic activity
ranging from moderate to high. 4-Nitrosopyrazoles 5 (MIC 0.36 …
12.5
m
g/ml) revealed higher activity compared to amino-
g/ml). N-unsubstituted
substituted analogues 8 (MIC 6.2 … 12.5
m
4-nitrosopyrazoles bearing phenyl, methyl and thienyl at the C5
position were the most active. It is worth noting that introduction
of the methyl substituent to N1 (leading to more lipophilic com-
pounds, Table 1, QPlogPo/w and PSA values) attenuates
Fig. 4. ORTEP view of molecule 5g.

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Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
bacteriostatic activity (MIC 125 mg/ml).
The analysis of structure - activity relationship showed that N-
unsubstituted nitrosopyrazoles 5a-c,h possessed a wide spectrum
of antibacterial actions. Besides, their inhibitory effect against
gram-positive bacteria was more evident (MIC 7.8 … 62.5
mg/ml)
than against gram-negative microorganisms (MIC 15.6 … 500
mg/
ml). Among N-substituted 4-nitrosopyrazoles, only compounds 5g,j
could be marked, which have a moderate activity against gram-
positive Staphylococcus aureus and Staphylococcus aureus MRSA
(MIC 15.6 … 31.2 mg/ml). 4-Hydroxyiminopyrazoline 7a exhibited a
similar antibacterial effect.
N-substituted 4-aminopyrazoles 8e,f,j,k did not show antibac-
terial effects, but N-unsubstituted analogues 8a,b proved to have
weak activity against N. gonorrhoeae (MIC 62.5 mg/ml).
Antifungal activity evaluations for compounds 5a-c,e-k, 6, 7a,
8a,b,e,f,k were carried out against 9 fungal strains (Table 3). Itra-
conazole and Fluconazole were used as positive control. 4-
Nitrosopyrazoles 5b,c,e-h, 6 were found to show antimycotic ac-
tivity ranging from moderate to pronounced. Compound 5e
showed the most effect in relation to seven strains, and it inhibited
Scheme 4. Reduction of 4-nitrosopyrazoles.
the growth of Epidermophyton floccosum at MIC 0.38
chophyton tonsurans, at MIC 3.12 g/ml, Trichophyton schoenleinii
and Trichophiton violaceum, at MIC 6.25 g/ml. The last mentioned
mg/ml, Tri-
m
tuberculostatic effect of both nitroso-derivatives (5b vs 5f; 5c vs 5g;
5h vs 5i) and amino-derivatives (8a vs 8e).
m
strain was sensitive to pyrazole 5g at the same concentration.
Compound 5g also proved to inhibit the growth of seven derma-
The high tuberculostatic activity for some of the obtained
compounds prompted us to investigate their antibacterial action in
relation to a broader range of microorganisms (Table 2) as well as
their antimycotic activity (Table 3). In addition, pyrazoles, including
trifluoromethyl-containing ones, are known to show promise as
antibacterial agents [37,70]. The testing of pyrazoles series against
11 strains of microorganisms revealed a high activity of N-unsub-
tophytes strains albeit at a lower concentration (MIC 6.25 … 50
ml). Nitrosopyrazoles 5b,c,e-g and 6 showed a moderate activity in
relation to Trichophyton rubrum (MIC 12.5 g/ml), while com-
pounds 5h, 7 were found to be half as active. The growth of
Microsporum canis inhibited by pyrazoles 5c,g at MIC 12.5 g/ml,
and compounds 5b,f,h, at MIC 25 g/ml. Pyrazole 5e demonstrated
moderate activity against Trichophyton mentagrophytes var.
mg/
m
m
m
stituted 4-nitrosopyrazoles 5a-c,h (MIC 7.8
Streptococcus pyogenes. Compounds 5c,h possessed a moderate
action against Neisseria gonorrhoeae (MIC 15.6 g/ml), pyrazoles
mg/ml) in relation to
a
interdigitale (MIC 12.5
mg/ml), and compounds 5f,g showed a
m
weaker effect (MIC 25
gypseum and Candida albicans were less sensible to pyrazoles.
Pyrazoles 5c,e had a moderate activity in relation to dermatophyte
mg/ml). Trichophyton mentagrophytes var.
5b,g had the same activity against Staphylococcus aureus, and het-
erocycle 5c, in relation to Staphylococcus aureus MRSA. These values
were higher or comparable to Spectinomycin activity. Compounds
5a,b,h showed a moderate action against Proteus vulgaris (MIC
31.2 mg/ml), while only pyrazole 5a had the identical activity in
relation to Shigella flexneri. Heterocycle 5a demonstrated a weak
inhibitory action against Klebsiella pneumoniae, Citrobacter braakii
T. gypseum (MIC 25
weak action (MIC 50
inhibited by heterocycles 5c,f at MIC 25
at MIC 50 g/ml. Nitrosopyrazoles 5a,i,j,k were not active against
m
g/ml), while compounds 5b,f,g,h exhibited
g/ml). Pathogenic yeast-like C. albicans
g/ml and compounds 5b,h
m
m
m
the tested fungi strains.
and Serratia marcescens growth (MIC 62.5 mg/ml). The same activity
4-Hydroxyiminopyrazoline 7a showed a weak antimycotic ac-
was observed for pyrazoles 5a,c in relation to Escherichia coli.
Almost all compounds were inactive against Pseudomonas aerugi-
tivity in relation to Trichophyton rubrum (MIC 25
mg/ml) and Tri-
chophyton tonsurans (MIC 50 g/ml) strains. 4-Aminopyrazoles
m
nosa, except for nitrosopyrazole 5a that showed
a weak
Fig. 5. ORTEP view of molecule 5k.

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
7
the data are heterogeneous. Compound 5a, the best in the anti-
bacterial test systems (Table 2), is not active against any tested fungi
strains, with the exception of some weak action against C. albicans.
However, its closest analogues 5b-5h inhibit the growth of all the
tested fungi. With the exception of compound 5f, all the NO-
derivatives carrying any substituent at N1 or N2 are not active or
have MIC values above 200 mg/ml against C. albicans.
The interesting data were obtained for compound 5h and its
derivatives, having a methyl group at different N-atoms in the
pyrazole ring, namely compounds 5i (N1eMe) and 6 (N2eMe).
Although isomers 5i and 6 have very narrow calculated values of
the lipophilicity-related parameters (QPlogPo/w and PSA, Table 1)
and their antibacterial activities against the tested strains appear
very similar (Table 2), the spectrum of their antifungal activity is
quite distinct. N2eMe-isomer 6 is, in toto, much more effective
than N1eMe one 5i (Table 3). A less lipophilic N-unsubstituted
compound 5h showed at least some activity against all the tested
strains, while more lipophilic analogue 5i can be regarded as
inactive.
Fig. 6. ORTEP view of molecule 8e.
The data obtained may imply the contribution of 4-NO sub-
stituent into the antifungal activity of fluorinated pyrazoles re-
ported here. Various compositions, which are based on nitrogen
monoxide-releasing material, are in use and/or under study as
means for the treatment of bacterial and fungal infections,
8a,b,e,f,k were found to have no fungicidal action.
Overall, 4-NH2-derivatives 8a-k are not active regardless of
other substituents in the pyrazole core. In 4-NO-pyrazoles subset,
Table 1
In-silico ADME predictions of compounds 5e9.
Compound #starsa #rotb mol MWc donor HBd accept HBe QPlogPo/wf PSAg
QPlogSh QPPcacoi
ꢀ6.5 <25 poor
QPlog BBj QPlog Khsak #metl HOA, %m
Min
Max
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
6
7a
7b
7c
8a
8b
8e
8f
0
5
1
1
0
0
1
0
0
1
3
2
1
3
0
0
0
1
0
1
0
1
0
1
0
0
1
0
0
0
15
1
1
1
1
1
1
1
1
1
2
3
1
3
4
5
1
1
1
1
2
3
1
1
1
1
1
1
130.0
725.0
241.2
247.2
231.1
255.2
255.2
261.2
245.2
179.1
193.1
395.4
410.4
193.1
335.3
413.4
428.4
228.2
234.2
242.2
248.2
382.4
397.4
227.2
233.2
241.2
241.2
247.2
231.2
0.0
6.0
1.0
1.0
1.0
1.0
0.0
0.0
0.0
1.0
0.0
0.0
2.0
0.0
2.0
2.0
4.0
2.5
2.5
1.5
1.5
1.5
3.5
2.5
2.5
2.5
1.5
1.5
1.5
2.0
20.0
2.5
2.5
3.0
2.5
2.5
2.5
3.0
2.5
2.5
6.5
7.0
2.5
3.7
7.7
8.2
2.0
2.0
2.0
2.0
6.0
6.5
2.0
2.0
2.0
2.0
2.0
2.5
ꢀ2.0
6.5
2.5
2.5
1.9
2.8
3.1
3.0
2.4
1.2
1.8
3.0
2.5
1.7
3.7
2.5
1.7
1.8
1.8
2.5
2.5
2.8
2.0
1.9
1.8
2.1
2.6
2.6
2.0
7.0
ꢀ3.0
ꢀ1.5
1.5
1
8
0
1
1
1
0
1
1
1
1
0
1
1
1
1
2
0
1
0
1
0
1
0
1
1
0
1
1
<25 is poor
>80 is high
96
200.0 0.5 >500 great 1.2
58.6
59.9
69.2
58.7
47.3
47.7
56.1
62.5
49.4
83.4
ꢀ3.5
1091
1021
908
1090
2099
2053
1920
782
1814
439
112
1666
1301
84
ꢀ0.1
0.0
ꢀ3.4
ꢀ2.8
ꢀ4.0
ꢀ3.8
ꢀ3.8
ꢀ3.0
ꢀ1.7
ꢀ2.3
ꢀ4.7
0.0
ꢀ0.1
ꢀ0.3
0.1
0.1
0.0
ꢀ0.2
ꢀ0.5
ꢀ0.4
ꢀ0.1
0.2
95
91
ꢀ0.1
ꢀ0.1
0.2
0.3
0.2
100
100
100
100
85
96
92
78
94
100
76
69
83
82
ꢀ0.1
0.2
ꢀ0.7
ꢀ1.4
0.1
ꢀ0.3
ꢀ1.8
ꢀ1.9
0.4
111.5 ꢀ5.4
48.9
65.9
ꢀ2.2
ꢀ4.8
ꢀ0.4
0.3
103.6 ꢀ5.6
0.1
131.6 ꢀ4.8
59
ꢀ0.1
ꢀ0.1
ꢀ0.2
0.1
52.8
54.0
41.5
42.0
77.0
ꢀ1.9
ꢀ1.9
ꢀ2.5
ꢀ2.5
ꢀ4.1
341
322
655
646
142
36
0.5
0.7
92
0.8
0.0
92
8j
ꢀ0.1
ꢀ0.9
0.4
0.5
0.4
0.3
0.1
ꢀ0.1
ꢀ0.2
0.0
80
66
83
82
8k
9a
9b
9d
9e
9f
104.9 ꢀ4.1
52.0
53.7
52.7
41.0
41.4
49.6
ꢀ2.0
ꢀ2.0
ꢀ2.6
ꢀ2.8
ꢀ2.7
ꢀ2.1
348
326
344.1
674
660
621
85
0.7
0.1
93
0.8
0.1
92
9g
0.7
ꢀ0.1
89
a
#stars e Number of property or descriptor values that fall outside the 95% range of similar values for known drugs. The following properties and descriptors are included
in the determination of #stars: MW, dipole, IP, EA, SASA, FOSA, FISA, PISA, WPSA, PSA, volume, #rotor, donorHB, accptHB, glob, QPpolrz, QPlogPC16, QPlogPoct, QPlogPw,
QPlogPo/w, logS, QPLogKhsa, QPlogBB, #metabol;
b
#rot - Number of rotatable bonds;
mol_MW e Molecular weight of the molecule;
donorHB e Number of hydrogen bond donors;
accptHB e Number of hydrogen bond acceptors;
QPlogPo/w eLogarithm of partition coefficient between n-octanol and water;
PSA e Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms (Polar Surface Area);
QPlogS e Predicted aqueous solubility;
QPPCaco e Predicted apparent Caco-2 cell permeability in nm/sec;
QPlogBB e Predicted brain/blood partition coefficient;
QPlogKhsa e Prediction of binding to human serum albumin;
#metab e Number of likely metabolic reactions;
HOA Percentage of human oral absorption.
c
d
e
f
g
h
i
j
k
l
m

8
Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
particularly, of those where Candida albicans species are involved
[71,72].
Therefore, we focused our attention primarily on the evalu-
ation of the ability of some synthesized compounds to produce
nitric oxide in vitro using the Griess reagent for detection both in
the presence and in the absence of rat blood plasma
(Supplementary data, Table S1 and Figure S2). The tested nitroso-
derivatives 5b, e and f, which have different activities against
Candida albicans, appeared as NO-producing compounds at
similar degree. Therefore, it is likely to be the presence of the
thienyl group at C5 (as in the active compounds 5b and 5f)
instead of phenyl (as in the inactive compound 5e) that may
serve as an important structural feature ensuring C. albicans in-
hibition rather than the nitric oxide (NO) releasing property.
In the presence of plasma, nitrite ion was not detected in the
cases of nitroso-derivatives 5b, e and f, while the samples con-
taining isosorbide dinitrate (Isacardin) and sodium nitroprus-
side, the known NO-releasing compounds, produced an evident
signal in the presence of plasma. Therefore, it was concluded that
the chemical transformations, which would lead to NO release
from compounds 5b,e and f, are apparently hindered or even
precluded by some of the plasma components. Absence of the
signal in samples 5b,e and f with plasma as a consequence of any
involvement of following events (e.g. NO removal from the media
or nitrite-ion oxidation reaction which is always a concern for
Griess reagent test [73]) may be cautiously excluded. The details
of NO-release and its quantitative estimation are to be studied
further.
To discuss the possible mechanism of antimicrobial action of
synthesized compounds, we used literature data. Searching for a
suitable biological target for determining the antitubercular ac-
tion of highly active 4-nitrosopyrazoles 5a-c, h,i, it was found
that the nitrobenzothiazinone compounds (BTZ) having prom-
ising antitubercular effect act as prodrugs, which, upon activa-
tion by reduction of the nitro substituent to an electrophilic
nitroso moiety, induce suicide inhibition of the enzyme decap-
renylphosphoryl-b
-D-ribose-20-oxidase (DprE1) of Mtb [55].
Nitroso-(het)aromatic compounds are known to can react with
the essential cysteine Cys 387 residue in DprE1 to form a covalent
semimercaptal adduct [74e78]. DprE1 mediates biosynthesis of
decaprenylphosphoryl-b-D-arabinose (DPA), which is needed for
assembly of the complex cell wall of mycobacteria [79,80]. If we
draw an analogy with the antitubercular action of BTZ as a DprE1
inhibitor, we should also add that the second significant struc-
tural fragment in BTZ molecule, i.e. the electron-acceptor CF3-
substituent, is also present in 4-nitrosopyrazoles [81].
Therefore, we suppose that the high antitubercular activity of
4-nitrosopyrazoles 5a-c, h,i may also be due to the ability to
inhibit DprE1. Reduction of the nitroso substituent to hydrox-
yimino or amino groups is known to reduce the anti-tuberculosis
effect [76], which we also observe for compounds 5a and 8a.
Toward antimicrobial (antibacterial and antifungal) activity
we think that the nitroso group also makes a significant contri-
bution to this activity, since only 4-nitrosopyrazoles 5 have
shown both the antibacterial and antifungal effect. It is assumed
that these compounds can act like antimicrobial agents of the
nitrofuran group, the exact mechanism of action of which is
unknown. For example, nitrofurazone inhibits several bacterial
enzymes, especially those involved in the aerobic and anaerobic
degradation of glucose and pyruvate [82]. The metabolism of
topically applied nitrofurazone is thought to be by 5-nitro
reduction to generate a reactive species which can covalently
bind to cellular macromolecules [83]. In addition, 4-
nitrosopyrazoles can act like the nitroimidazole class (for
example, metronidazole, ornidazole et al.) that inhibits nucleic

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
9
Table 3
Antifungal activity of pyrazoles 5e8.
a
No
Compounds
MIC (
m
g/ml) for fungi strains inhibition
%
0
T. rubrum
T. gypseum
T. tonsurans
T. violaceum
T. interdigitale
E. floccosum
M. canis
C. albicans
1
2
3
4
5
6
7
8
5a
5b
5c
5e
5f
5g
5h
5i
5j
5k
6
7a
8a
8b
8e
8f
>200
12.5
12.5
12.5
12.5
12.5
25
>200
200
200
12.5
25
>200
200
200
>200
200
>200
50
>200
50
>200
n/tb
n/t
6.25
n/t
6.25
n/t
>200
50
50
12.5
25
25
>200
50
50
0.38
50
12.5
50
200
200
200
100
100
>200
200
200
>200
200
0.19
3.12
>200
25
12.5
100
25
100
50
100
100
87.5
100
87.5
100
0
25
50
25
25
50
3.12
50
n/ac
25
50
50
12.5
50
12.5
25
n/a
50
50
>200
200
200
25
>200
>200
200
200
200
200
200
100
100
>200
>200
>200
200
200
0.095
>100
>200
200
200
100
100
>200
200
200
>200
200
>100
100
>200
>200
>200
>200
50
>200
>200
>200
>200
>200
>100
>100
9
>200
>200
100
0
0
10
11
12
13
14
15
16
17
18
19
25
37.5
37.5
0
100
>200
>200
>200
>200
>200
0.06
12.5
50
100
>200
>200
>200
>200
>200
0.19
>100
>200
>200
200
>200
>200
0.19
100
0
0
0
0
8k
Itraconazole
Fluconazole
0.06
6.25
a
Percentage of fungi for which the compound has MIC ꢁ62.5
mg/ml (from number of tested fungi).
b
c
n/t-not tested.
n/a-inactive.
acid synthesis by forming reduced nitroso forms, which disrupt the
DNA of microbial cells [84].
fluorescence intensity increases. In these tests, Quercetin was used
as a reference antioxidant. The results also are shown in Table 4.
In the ABTS test, all 4-nitrosopyrazoles 5 showed very weak
radical-scavenging activity or did not show it at all. This result is
quite unexpected because nitroso compounds are characterized
with the radical binding activity, and some of them are used as spin
traps [93] or as inhibitors of radical polymerization [94]. The ability
of 4-hydroxyimino-5-hydroxy-1-phenylpyrazoline 7a to bind the
ABTS radical was significantly higher (TEAC ¼ 0.44). The intro-
duction of a methanesulfonyl group into the 1-phenyl substituent
(Ar ¼ C6H4SO2Me-4) reduced the antiradical activity for pyrazoline
7b; whereas, the presence of a p-tolyl moiety instead of the phenyl
in N-phenylsulfamide-substituted pyrazoline 7c led to an increase
in TEAC value to 0.48.
The maximum antiradical activity was shown by N-unsub-
stituted 4-aminopyrazolones 9a,b,d and their hydrochlorides 8a,b
(Scheme 4, R1 ¼ H), which, in contrast to nitroso-containing pre-
cursors 5, showed high TEAC values exceeding that of Trolox (with
the exception of salt 8a, for which TEAC ¼ 0.58). Moreover, free
amines 9a,b,d proved to be rather fast antioxidants, the maximum
binding of the ABTS radical was observed after 10 min. Introducing
methyl (8e,f,j, 9e) or methanesulfophenyl (9g) group to the nitro-
gen atom at position 2 of the pyrazole ring led to an almost com-
plete absence of antiradical activity for the corresponding 4-
aminopyrazoles 8e,f,j and 9e,g.
However, it is obvious that for the 4-nitrosopyrazoles 5, not only
the nitroso group itself is responsible for the antimicrobial action,
but also its combination with other structural fragments, since high
antibacterial activity is shown by pyrazoles 5a-c,h, which have a
combination of NO and NH fragments in the cycle, and pyrazoles
5e-g, which combine NO and NMe fragments, have a more pro-
nounced antifungal effect compared to N-unsubstituted analogues
of 5a-c.
2.3.2. Radical-scavenging activity
Among pyrazole derivatives, high effective antioxidants have
been found [85,86]. The most known of them is Edaravone (3-
methyl-1-phenyl-2-pyrazolin-5-one; Radicut, Mitsubishi Tanabe
Pharma Corporation) used for the treatment of the acute stage of
cerebral infarction [87,88] and for amyotrophic lateral sclerosis
treatment [89]. In both indications, it is believed that the effect of
drug is developed due to its ability to scavenge free radicals and
thus prevent oxidative stress related damage to neurons.
Antioxidant activity of the synthesized pyrazoles 5e9 was
evaluated using two radical-scavenging assays: the ABTS and
ORAC-FL tests.
The ABTS assay is based on spectrophotometric determination
of a decrease in absorbance of a stable dark green ABTS cation-
radical (ABTS·þ) solution after its interaction with an antioxidant
compound [90]. The measurements were performed as previously
described in detail [91]. Trolox was used as a reference antioxidant,
and ascorbic acid as a positive control. The results were expressed
as TEAC values (Trolox equivalent antioxidant capacity) and IC50
values and are presented in Table 4.
Additionally, a set of compounds was evaluated in the oxygen
radical absorbance capacity (ORAC-FL) assay with 2,20-azobis-(2-
methylpropionamidine) dihydrochloride (AAPH) as a free peroxyl
radical generator [92]. In this method, fluorescein (FL) is used as a
fluorescent probe. The method is based on measuring the decrease
in the intensity of fluorescence with time, which characterizes the
degree of decay of the fluorescent probe under the influence of
peroxyl radicals. In the presence of antioxidants, the degree of
decay of the fluorescent probe decreases and, accordingly, the
According to ORAC assay, all the tested compounds did not
exceed the antioxidant capacity of Quercetin at both tested con-
centrations. Interestingly, the inverse concentration e activity
relationship was reproducibly observed for salt 8b and its base 9b:
the higher concentration (50
mM) provided less antiradical pro-
tection than the lower one (10 mМ), which is, possibly, a sign of
nonlinear relationship of concentration and radical-trapping ac-
tivity in this assay.
Overall, the results on antiradical activity for the most active
hydroxyimino- and aminoderivatives 7b,c, 8a,b and 9a,b obtained
in both tests are in a fairly good correspondence (TEAC or IC50
values in ABTS test and % of fluorescence restored at 10 mM in ORAC
test). However, compound 8e, one of the best in ORAC assay, does
not scavenge ABTS radicals.
A study of N-unsubstituted NO-derivatives 5a,b, and d in the

Table 4
Radical-scavenging activity of pyrazoles 5e9.
ꢂ
N
Compound
ABTS-scavenging activity*
ORAC/AAPH-assay
Oxidative erythrocyte hemolysis assay
Antiradical activity, TEAC** value (n ¼ 3, mean SEM, 1 h)
IC50
,
m
M (mean SEM, n ¼ 3)
Antiradical activity, fluorescence restored, %***
Relative protection values4*
10 mМ
50 mМ
50 mМ
400 mМ
1
2
3
4
5
6
7
5a
5b
5c
5d
5e
5g
5h
n.a.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
ꢀ80.40
ꢀ56.25
n/t
ꢀ60.05
3.00
n/t
ꢀ119.35
ꢀ98.65
n/t
ꢀ99.80
16.15
n/t
0.99
1.18
n/t
1.03
1.00
n/t
1.05
1.12
n/t
1.03
1.02
n/t
0.02 0.003
0.02 0.003
0.02 0.002
0.01 0.002
0.02 0.002
n.a.
n/t
n/t
n/t
n/t
8
9
7a
7b
7c
8a
8b
8e
8f
8j
8k
9a
9b
9d
9e
9g
0.44 0.02
0.16 0.02
0.48 0.03
0.58 0.04
1.22 0.05
n.a.
0.1 0.004
n.a.
n/t
1.35 0.06
1.10 0.05
1.43 0.11
n.a.
0.04 0.005
1.0
48.6 2.5
169
45.3 2.2
41.8 2.5
13.3 1.0
n.d.
n.d.
n.d.
n/t
14.1 0.9
17.6 1.0
13.4 1.2
n.d.
n/t
n/t
n/t
n/t
6
30.60
38.45
63.10
52.50
38.20
n/t
49.15
59.40
60.20
14.00
79.05
n/t
1.03
1.09
1.03
n/t
1.19
n/t
1.14
1.08
0.01
n/t
1.11
n/t
10
11
12
13
14
15
16
17
18
19
20
21
22
23
23
n/t
n/t
n/t
n/t
14.55
59.70
52.60
n/t
n/t
n/t
n/t
n/t
87.60
47.15
62.75
21.80
n/t
n/t
n/t
n/t
n/t
90.05
1.18
1.10
1.11
n/t
n/t
n/t
1.17
0.02
0.01
n/t
n/t
n/t
n.d.
Trolox
Ascorbic acid
Quercetin
20.1 1.4
21.6 1.9
n/t
n/t
n/t
n/t
n/t
0.98 0.02
n/t
0.023 0.005
0.045 0.01
*data are presented as means SEM, n ¼ 3; n.a. e not active; n.d. - not determined; n/t e not tested; **TEAC (Trolox equivalent antioxidant capacity) was determined from the ratio of the slopes of the concentrationꢀresponse
curves, test compound/Trolox; ***% value is an average for two independent experiments for each concentration; 4*data are presented as following: for the tested compound e means of two independent experiments; for
Quercetin - means standard deviations for four independent experiments.

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
11
Fig. 8. Relative viability of human dermal fibroblasts upon the action of compounds
5a,e,h, 7c, 8a,e,f,j compared to CPT.
Fig. 7. Relative viability of HeLa cells upon the action of compounds 5a,e,h, 7c, 8a,e,f,j
compared to Camptothecin (CPT).
during an oxidative stress, in the presence of tested compounds
better consistent with the results of ABTS scavenging assay rather
than ORAC test data.
ABTS test showed low radical-scavenging activity, while a decrease
in the fluorescence time was observed in the ORAC test in the
presence of these compounds, i.e. the degree of decay of the fluo-
rescent probe increased, which may indicate to increase in the
content of free radicals in the tested probe. Such results are likely to
reflect the difference in the basics of these two test systems and
emphasize the need for applying several test systems to make the
antiradical/antioxidant activity assessment.
The results for N1-unsubstituted 4-aminoderivatives 9a,b and
their hydrochlorides 8a,b obtained in both assays support the
importance of free pyrazole NH group for rendering the radical-
scavenging properties of compounds designed.
The antioxidant activity of the compounds was additionally
evaluated on a biological target by oxidative erythrocyte hemolysis
assay at 50 and 400 mМ in order to assess the ability to prevent
damage to the membranes of erythrocytes. AAPH was used as a free
radical initiator. Procedure was based on the described earlier with
some variations [95,96] (Table 4).
All the tested compounds, with three exceptions, are not active.
Aminoderivatives 9a,b and the salt 8a are not active at 50 mМ while
provide an excellent protection at 400 mМ, compared to Quercetin.
Compounds 7b,c, 8e,k, do not show any activity at both tested
concentrations. NO-derivatives 5a,b,d and e do not produce any
damage to erythrocyte membranes or any protection.
2.3.3. Cytotoxic activity
Nitroso-containing compounds are known for possessing both
antitumor [97] and carcinogen activity [98], therefore it was sig-
nificant to investigate the cytotoxicity that the synthesized pyr-
azoles cause in normal and tumor cells. We studied a cytotoxic
activity of compounds 5a,e,h, 7c, 8a,e,f,j on a culture of human
dermal fibroblasts of 5e7 passages and on a transplantable culture
of human cervical carcinoma HeLa cells, with Doxorubicin (DXR)
and Camptothecin (CPT) used as reference drugs. The IC50 values
and the selectivity index (SI) were determined for the compounds
having cytostatic activity.
All compounds, except for 8j, showed dose-depended cytotoxic
effect on HeLa cells in concentrations from 0.1
(Fig. 7).
mM to 100 mM
Pyrazole 8j was found to be a low-toxic compound for the
proliferative HeLa cells, while the substances 5e, 5h, 8a, 8f showed
cytotoxicity at a concentration of 10 … 100
showed pronounced cytotoxic action in relation to HeLa cells at 1 …
100 M. Among the investigated compounds, 4-aminopyrazole 8e
(Fig. 7) had the most cytotoxicity (at 0.1 … 100 M).
mM. Pyrazoles 5a, 7c
m
m
The concentration values causing 50% inhibition of population
growth of cells (IC50) were determined for compounds 5a,e,h, 7c,
8a,e,f. Data are given in Table 5. The most cytotoxicity in relation to
HeLa cells was found in 4-aminopyrazole 8e (IC50 ¼ 0.074 0.005
mМ), which was essentially greater than that of CPT
(IC50 ¼ 1.66 0.97 mМ). Compounds 5a (IC50 ¼ 1.86 0.07 mМ) and
8a (IC50 ¼ 1.86 0.12 mМ) were found to have an effect at the level
of CPT, while pyrazoline 7c (IC50 ¼ 7.23 0.04 mМ) showed cyto-
toxicity comparable to the known cytostatic drug DXR
Therefore, albeit the fact that a radical source used in erythro-
cyte hemolysis assay was the same as one applied in ORAC test (i.a.
AAPH), the overall data regarding erythrocyte membrane stability
Table 5
IC50 of compounds for HeLa cells and human dermal fibroblasts (HDF).
Entry Compound IC50
,
mМ in relation to cell line
Selectivity index (SI)
HeLa
HDF
(IC50
¼
4.25
0.55 mМ). 4-Nitrosopyrazoles 5e,h and 4-
1
2
3
4
5
6
7
8
9
10
5a
5e
5h
7c
8a
8e
8f
8j
CPT
DXR
1.86 0.07
63.86 5.74
61.59 7.01
7.23 0.04
1.86 0.12
0.074 0.005
48.68 3.27
n/a
1.42 0.13
49.46 3.31
n/ac
99.30 8.56
n/a
n/a
n/a
n/a
5.38 0.48
2.72 0.26
0.76a
aminopyrazole 8f had weaker cytotoxic activity against HeLa cells.
4-Nitrosopyrazole 5h and 4-aminopyrazoles 8a,e,j were found
to have no cytotoxic activity in relation to human dermal fibroblasts
(HDF). Pyrazoles 5e and 7c showed a moderate cytotoxicity on
0.77a
>1.62
13.73a
>53.76b
>1351b
>2.05b
e
viability of HDF at a concentration of 10 … 100 mM, while pyrazole
5a had significant toxicity at a concentration of 0.1 … 100 mM
(Fig. 8).
Excepting substances 5a,e, 7c, the investigated compounds had
a low toxicity against HDF. Sharp increasing in cytotoxicity at a
concentration of 10 mM was typical for compounds 5e, 7c. Pyrazole
5a with IC50 ¼ 1.42 0.13 mМ was found to have the most cytotoxic
activity exceeding that of CPT (IC50 ¼ 5.38 0.48 mМ) and of DXR
(IC50 ¼ 2.72 0.26 mМ) (Table 5).
1.66 0.97
4.25 0.55
3.24a
0.64a
a
SI determined as the ratio of IC50 median value in the normal human fibroblasts
divided by median for HeLa IC50
.
b
- SI range is determined as the ratio of maximal tested not cytotoxic concen-
tration against normal human fibroblasts divided by median for HeLa IC50
.
c
n/a e inactive.

12
Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
Table 6
associated with higher toxicity values when they are compared
with ones for the corresponding N-methyl-substituted analogues
(5a vs 5e; 5b vs 5f and 5c vs 5g). A similar trend has been described
for 3-trifluoromethyl-1H-pyrazol-3-oles containing H or a methyl
group at N1 [102]. Substitution of phenyl group at C5 for heteryl (2-
furanyl and 2-thienyl) leads to increasing the toxicity for N1eH
derivatives in both, nitroso- and amino-pyrazoles, sets (5a vs 5b
and 5c; 8a vs 8b; 9a vs 9b), while the data is not consistent and
sufficient for drawing an analogous suggestion in case of N1eMe
derivatives (5e vs 5f or 5g; 8a vs 8e). Expected LD50 values exceed
300 mg/kg for 4-hydroxyiminopyrazolines 7b and 7c, as well as for
compound 5k. Overall acute toxicity data for tested compounds
does not correlate with cytotoxicity data against HDF.
Acute toxicity of 4-nitroso-, 4-hydroxyimino- and 4-amino-derivatives.
ꢂ
N
Compound
Dose, mg/kg
Number of survived mice from
3 taken in the experiment
1
2
5a
5b
150
75
30
2
0
2
0
1
3
3
2
2
0
1
3
3
3
1
0
0
3
3
2
3
0
3
3
2
3
5c
300
150
150
150
300
300
150
75
300
300
300
150
150
75
10
60
150
150
75
10
70
100
4
5
6
7
8
5d
5e
5f
5g
5h
Due to the high acute toxicity, the compounds 5b, 5c, 5h, 8b and
9b were excluded from further in vivo evaluation.
9
5k
7b
7c
8a
8b
10
11
12
13
2.3.5. Analgesic and anti-inflammatory activity
Pyrazole fragment is present in
a numerous of anti-
inflammatory and analgesic compounds which act via modulation
of different biological targets [70]. An analgesic activity of synthe-
sized pyrazoles was evaluated in “hot plate” test [104,105] in rats
(Table 7). A latency period was measured at one or two time points:
1 h and, for some compounds, also 2 h after intraperitoneal injec-
tion of the sample.
For almost all of the tested pyrazole derivatives high analgesic
effects were revealed which are comparable to the effects known
for Diclofenac and Metamizole. When measured at both time
points, latent period prolongation values (%) remain approximately
the same during the experiment (7b, 9a) or are markedly increased
at 2nd hour when compared to the corresponding values at the 1st
hour (5f,g and 7c). Metabolic activation of compounds in vivo or
pharmacokinetics related issues could be implied based on such
data. Substitution of phenyl group at C5 for hetaryl (5e vs 5f and 5g)
practically abolishes analgesic activity at 1st hour after the
injection.
Despite the limited number of corresponding pairs tested, it
seems that 4-aminoderivatives are more potent than 3-
nitrosoderivatives (5a vs 8a and 5e vs 8e). Slight improvement of
the activity is achieved via NeMe-substitution: compare the data
for the corresponding pairs of NH and NeMe derivatives (5e vs 5a,
8e vs 8a). Therefore, this modification seems to be favorable from
both activity and toxicity (Table 6) standpoints.
4-Нydroxyiminopyrazolines 7b,c showed an encouraging com-
bination of activity (at both 1st and 2nd h) and toxicity data (ex-
pected LD50 > 300 mg/kg).
Several compounds which showed a considerable level of ac-
tivity in hot plate test (>50% latent period prolongation compared
to a vehicle treated control) were further evaluated in carrageenan
induced hind paw edema test in rats for anti-inflammatory prop-
erties. The paw volume was measured at 1st, 3rd and 5th h after
carrageenan injection. All the tested compounds were inactive at
5th hour. Only 4-aminoderivative 8e showed considerable level of
activity at both 1st and 3rd h. Its N-demethylated analogue 8a was
active at 1st hour. 4-Nitrosoderivative 5a showed mild activity at
3rd h. The remaining data for all tested compounds indicated the
absence of activity in the applied model. Unexpectedly, NO- or NH2-
groups in the molecule of celecoxib result in compounds (5k and
8k) which are inactive in both tests.
14
15
16
17
8e
8k
9a
9b
18
19
Diclofenac
Metamizole
LD50 250, i/p, mouse [103]
Thus, 4-amino-1-methanesulfophenyl-5-phenylpyrazole 8j
showed low toxicity in relation to proliferative HeLa cells and HDF.
4-Nitrosopyrazoles 5e,h and 4-aminopyrazole 8f were found to
have a weak anticancer action compared to the known cytostatic
drugs and they did not have significant negative influence on HDF.
4-Nitrosopyrazole 5a showed equal toxicity against HeLa and HDF.
4-Hydroxyiminopyrazoline 7c possessed cytotoxic action against
both HeLa comparable to CPT and DXR, while its toxicity was lower
in relation to HDF in comparison with the same drugs. 4-
Aminopyrazole 8a was found to have action near to that of CPT,
and compound 8e had cytotoxicity against HeLa two orders of
magnitude greater than CPT. These compounds were of low toxicity
for HDF, which allowed us to consider them as potential anticancer
drugs.
Analysis of literature data on the antitumor mode of 4-
aminopyrazole derivatives showed that they exhibit a high affin-
ity for the cyclin-dependent kinase family [99e101]. Therefore, it
can be assumed that for the 4-aminopyrazoles 8a,e synthesized in
this study, the most likely mode of action is to inhibit CDK1 and
CDK2.
2.3.4. Acute toxicity
Prior to the in vivo assessment, the compounds were evaluated
for acute toxicity in mice, and some structural features associated
with toxicity of the compounds were analyzed.
According to OECD Guideline for testing of chemicals, minimal
amount of animals, i.e. three mice, was involved in a single dose
evaluation. When administered intraperitoneally (i/p), the tested
nitroso- and amino-pyrazoles show different levels of toxicity
depending on the substituents around the main pyrazole core
(Table 6). The expected LD50 values for most of the tested com-
pounds are equal or greater than 150 mg/kg or, for some of them,
greater than 300 mg/kg. The representatives of 4-aminoderivatives
subset appear to be slightly more toxic than the ones containing a
4-nitroso-group: 5a vs 8a and 5k vs 8k. For a subset of 4-nitroso-
derivatives, a non-substituted nitrogen atom in the pyrazole core is
It is worth taking into consideration all the advantageous
properties of 4-amino-subset representatives. 8a and 8e showed
very promising activity in both tests in vivo. Additionally, 8e was
cytotoxic against HeLa cells (IC50 0.07 mM) while not toxic against
normal fibroblasts cell line.
An analgesic effect of different pyrazole core containing com-
pounds may be mediated through a range of biological targets

Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
13
Table 7
Anti-inflammatory and analgesic activity pyrazoles 5e9.
Compound
Dose, mg/kg
Anti-inflammatory activity, swelling inhibition, %
Analgesic activity, latent period prolongation,%
After 1 h
After 3 h
After 1 h
After 2 h
5a
5d
5e
5f
5g
5k
7b
7c
8a
8e
8k
9a
15
15
15
15
15
25
15
15
15
15
15
15
10
15
n/a
n/t
n/a
n/t
n/t
n/t
n/a
n/a
32.0**
n/t
n/a
n/t
n/t
n/t
n/a
n/a
n/a
51.7***
n/a
80.2**
29.8*
n/a
n/a
53.7***
58.9***
91.8***
100.74*
n/a
n/t
n/a
n/t
43.8**
484*
n/t
44.3*
96.55*
n/t
44.8*
54.3**
n/t
32.9*
n/t
n/t
n/t
n/t
n/t
56.7*
66.7*
Diclofenac
Metamizole
53.9 7.1a
n/a
61.8 10.8a
n/a
57.1 9.2b
59.8e93.8d
79.9 16,2c
n/t e not tested; n/a e inactive.
*p < 0.05; **p < 0.01; ***p < 0.001; 4*p < 0.0001;5*p < 0.00001.
a
Median for 3 different independent experiments standard deviation.
median for 5 different independent experiments standard deviation.
median for 6 different independent experiments standard deviation.
values range for 4 independent experiments.
b
c
d
which involve COX inhibition [39], radical scavenging activity [106],
modulation of TRPA1 channels [107], activating endogenous
opioidergic circuits [108] and other targets which list is particularly
long for an exhaustively studied drug Metamizole [109]. Therefore,
additional study is required for particular biological targets eluci-
dation. For aminoderivatives 8e and 8b which have shown both
analgesic and reasonable anti-inflammatory activities, targets
associated with arachidonic acid metabolism may be considered as
primary ones for further investigation. Representatives of NO-
derivatives 5a,e,f,g and compounds 7b,c were active in the hot
plate test, but were found to lack anti-inflammatory activity.
Additional tests which help to elucidate whether central or pe-
ripheral component dominates in their analgesia effect, are going to
be the next step of investigation to be carried out for the best
compounds.
2.3.6. “Structure-activity-toxicity” relationships of trifluoromethyl-
containing 4-nitroso-, 4-hydroxyimino- and 4-aminopyrazoles
N-Unsubstituted 4-nitrosopyrazoles bearing phenyl, methyl and
thienyl at the C5 position were the most effective tuberculostatics
Fig. 9. Structure-activity- toxicity chart for 4-amino- and 4-nitroso-3-trifluoromethyl pyrazoles.

14
Y.V. Burgart et al. / European Journal of Medicinal Chemistry 208 (2020) 112768
with MIC for 4-nitroso-5-phenylpyrazole 5a less than 0.5
m
g/ml.
fibroblasts depended on substituents combination. We found
among 4-nitrosopyrazoles the substances that showed cytotoxicity
to the both cell lines and compounds with more selective anti-
cancer action. 4-Aminopyrazoles were found to be cytotoxic
against cancer cells.
The acute toxicity evaluation of the pyrazoles showed that most
of them had a moderate toxicity. However, the introduction of
thienyl moiety at the position 5 significantly increased the acute
toxicity both for 4-nitroso- and 4-amino-derivatives.
The pronounced analgesic activity was revealed for 4-nitroso-
and 4-aminopyrazoles having phenyl fragment at the position 5;
and the amino-derivatives were found to be more effective than
nitroso-analogues. 4-Hydroxyimino-5-hydroxypyrazolines also
had a considerable analgesic action. It was revealed that only 4-
amino-5-phenylpyrazoles had the appreciable anti-inflammatory
activity among all the series of the tested compounds.
Although acute toxicity of compound 5a is not more than Isoniazid
one, it has a low potential for further development due to its high
cytotoxicity toward human dermal fibroblasts (HDF).
A promising tuberculostatic activity was shown by 4-nitroso-5-
methylpyrazole 5h. It was not toxic in relation to HDF, but its acute
toxicity was rather high.
4-Nitroso-1-methyl-5-phenylpyrazole 5e was found to have the
pronounced antimycotic action in combination with the moderate
cytotoxic effect on HDF and acute toxicity in vivo.
4-Hydroxyimino-5-hydroxy-1-arylpyrazolines 7b,c have good
analgesic and antitumor activities at the level of the corresponding
reference drugs Diclofenac and Camptothecin while their acute
toxicity was lower than that for the reference medicines. The data
obtained on the biological action of substances from the given
subset indicate that they certainly deserve further study.
4-Aminopyrazoles 8a,e have high analgesic, antiradical and
antitumor effects combined with lack of cytotoxicity in relation to
HDF. Their acute toxicity is comparable with the action of Diclofe-
nac and Camptothecin. Such combination of properties allows the
corresponding chemotype to serve as a starting point for multi-
targeted therapy design. Toxicity related concerns may be well
addressed during the structure optimization process. Small size of
the molecules and favorable calculated parameters (Table 1) leave
enough space for structure build-up strategies. A free amino-group
attached to a heterocyclic core is regarded as a soft metabolic spot
which may lead to more toxic derivatives in vivo [110]. Therefore,
one of the directions for structure optimization can be based on the
prodrug design approach leading to derivatives with significantly
safer toxicity profile.
The analysis of structure-activity-toxicity relationships showed
that there is a research potential for studying the 4-amino-3-
trifluoromethyl-5-phenylpyrazoles
and
4-hydroxyimino-5-
hydroxy-1-arylpyrazolines aimed at further chemical modifica-
tions and biological action optimization.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
Some data related to structure-activity relationship for two
subsets e 4-amino- and 4-nitroso-3-trifluoromethyl pyrazoles e
are summarized in Fig. 9. It is evident that all three points of
structural diversity explored in this work (Fig. 2) provide an op-
portunity to modulate biological spectra as well as to tune the level
of activity. Functional groups at the position C4 (NO and NH2) seem
to determine the type of action to a greater extent than substituents
at the positions N1 and C5. However, variation of the substituents R
and R1 allows toxicity, selectivity and analgesic activity tuning.
This work was supported by the Russian Science Foundation
(Grant No. 16-13-10255). Analytical studies (elemental analysis, IR
and NMR spectroscopy, XRD analysis) were carried out using
equipment of the Spectroscopy and Analysis of Organic Compounds
Center for Joint Use at the Postovsky Institute of Organic Synthesis
of the Russian Academy of Sciences (Ural Branch). The radical-
scavenging activity with ABTS test was performed with in the
framework of the State Assignment IPAC RAS 2019 (# 0090-2019-
0005).
We thank N. Yu. Pomortseva for assistance in manuscript
preparation.
3. Conclusions
In summary, we developed simple and efficient methods for
synthesis of 3-trifluoromethyl-containing 4-nitroso- and 4-
aminopyrazoles bearing the various substituents at the position 1
and 5 as well as for synthesis of 4-hydroxyiminopyrazolines.
According to calculated ADME parameters, all obtained pyrazole
derivatives could be considered as potentially possessing favorable
pharmacokinetic profile. The synthesized compounds revealed
multiple bioactivity depending on the nature of peripheral sub-
stituents at the pyrazole core.
The 4-nitrosopyrazoles with unsubstituted NH group had the
high tuberculostatic and antibacterial activity, while the pro-
nounced antimycotic action was typical for methyl-substituted
analogues. 4-Aminopyrazoles did not reveal the essential antibac-
terial and antimycotic activity.
The N-unsubstituted 4-aminopyrazoles and their hydrochlo-
rides were found to have a high ability to scavenge the free radicals,
while N-substituted analogues were inactive or significantly less
active. Regardless of N-substituent, 4-nitrosopyrazoles were prac-
tically inactive in the ABTS, ORAC tests and erythrocyte membrane
stability assay. Some of 4-hydroxyiminopyrazolines possessed a
moderate antiradical action.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112768.
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