Synthesis and biological evaluation of polyfluoroalkylated antipyrines and their isomeric O-methylpyrazoles

Article abstract of DOI:10.2174/1573406414666181106145435

Background: Formally belonging to the non-steroidal anti-inflammatory drug class pyrazolones have long been used in medical practices. Objective: Our goal is to synthesize N-methylated 1-aryl-3-polyfluoroalkylpyrazolones as fluorinated analogs of antipyrine, their isomeric O-methylated derivatives resembling celecoxib structure and evaluate biological activities of obtained compounds. Methods: In vitro (permeability) and in vivo (anti-inflammatory and analgesic activities, acute toxicity, hyperalgesia, antipyretic activity, “open field” test) experiments. To suggest the mechanism of biological activity, molecular docking of the synthesized compounds was carried out into the tyrosine site of COX-1/2. Results: We developed the convenient methods for regioselective methylation of 1-aryl-3- polyfluoroalkylpyrazol-5-ols leading to the synthesis N-methylpyrazolones and O-methylpyrazoles as antipyrine and celecoxib analogs respectively. For the first time, the biological properties of new derivatives were investigated in vitro and in vivo. Conclusion: The trifluoromethyl antipyrine represents a valuable starting point in design of the lead series for discovery new antipyretic analgesics with anti-inflammatory properties.

Full text of DOI:10.2174/1573406414666181106145435

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Medicinal Chemistry, 2019, 15, 521-536 521
RESEARCH ARTICLE
Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
and their Isomeric O-Methylpyrazoles
Natalya Agafonovaa, Evgeny Shchegolkov*a, Yanina Burgarta, Victor Saloutina, Alexandra
Trefilovab, Galina Triandafilovab, Sergey Solodnikovb, Vera Maslovab, Olga Krasnykhb, Sophia
Borisevichc and Sergey Khursanc
aPostovsky Institute of Organic Synthesis of the Ural Branch of the Russian Academy of Sciences, S. Kovalevskoy Str.,
22, Ekaterinburg 620990, Russia; bPerm National Research Polytechnic University, Komsomolsky Av., 29, Perm
614990, Russia; cUfa Institute of Chemistry of the Russian Academy of Sciences, Octyabrya Av., 71, Ufa 450078, Russia
Abstract: Background: Formally belonging to the non-steroidal anti-inflammatory drug class
pyrazolones have long been used in medical practices.
Objective: Our goal is to synthesize N-methylated 1-aryl-3-polyfluoroalkylpyrazolones as fluori-
nated analogs of antipyrine, their isomeric O-methylated derivatives resembling celecoxib struc-
ture and evaluate biological activities of obtained compounds.
A R T I C L E H I S T O R Y
Methods: In vitro (permeability) and in vivo (anti-inflammatory and analgesic activities, acute to-
xicity, hyperalgesia, antipyretic activity, “open field” test) experiments. To suggest the mechanism
Received: March 30, 2018
Revised: October 19, 2018
Accepted: October 30, 2018
of biological activity, molecular docking of the synthesized compounds was carried out into the
tyrosine site of COX-1/2.
DOI:
10.2174/1573406414666181106145435
Results: We developed the convenient methods for regioselective methylation of 1-aryl-3-
polyfluoroalkylpyrazol-5-ols leading to the synthesis N-methylpyrazolones and O-methylpyrazoles
as antipyrine and celecoxib analogs respectively. For the first time, the biological properties of
new derivatives were investigated in vitro and in vivo.
Conclusion: The trifluoromethyl antipyrine represents a valuable starting point in design of the
lead series for discovery new antipyretic analgesics with anti-inflammatory properties.
Keywords: Methylation; 1H-Pyrazol-5-ols; Polyfluoroalkyl-containing antipyrine; Analgesic and anti-inflammatory activities;
Toxicity; Permeability; Molecular docking; COX-1/2 inhibitor.
1. INTRODUCTION
pyrazolone drugs are comparatively safe in terms of general
toxicity and gastrointestinal tract (GIT) damage. Main side
effects associated with pyrazolones include individual
allergic reactions and hematological disorders [2, 3]. One of
the latter risks (agranulocytosis) was the reason for MET,
one of the most effective non-opioid analgesics ever,
withdrawal from the market in the number of countries.
Later it was shown that metamizole side effects were
possibly overestimated and its short-term use in the hospital
setting is safer if compared with other popular analgesics [4,
5], while the risk of drug induced agranulocytosis may be
managed by genetics based personalized approach to
patients’ treatment [6].
Formally belonging to the non-steroidal anti-inflam-
matory drug class, pyrazolones have long been used in medi-
cal practice. The parent compound in the series - antipyrine
(AP) – was synthesized by Knorr in 1883 [1]. After its
release to the market, the structure of AP was further
optimized in order to improve bioavailability and efficacy as
well as to diminish undesirable side effects. Therefore,
aminophenazone, propyphenazone, dipyrone (a.k.a. metami-
zole, MET) (Fig. 1a) and other pyrazolone scaffold based
drugs (PSBDs) were obtained (see DrugBank). Most of them
are still used by medical doctors and/or veterinarians
worldwide as anti-inflammatory analgesics and antipyretics.
In spite of serious limitations due to adverse drug reactions,
In the end of 1980-s, one of the AP metabolites, 3-
methyl-1-phenyl-5-pyrazolone (Fig. 1b) was approved under
INN edaravone for stroke recovery treatment as a free radical
acceptor [7, 8]. Over the last decade, edaravone was
additionally authorized by different agencies in several
countries (including FDA in 2017) for amyotrophic lateral
sclerosis treatment [9].
*Address correspondence to this author at the Postovsky Institute of Organic
Synthesis of the Ural Branch of the Russian Academy of Sciences,
S.
Kovalevskoy
Str.,
22,
Ekaterinburg
620990,
Russia;
E-mail: schegolkov@ios.uran.ru
1573-4064/19 $58.00+.00
© 2019 Bentham Science Publishers

522 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
R1
X
O
Me
CF3
O
Me
HN
N
N
N
N
N
Me
R2
Coxib derivatives (c)
Antipyrine derivatives (a)
Edaravone (b)
Celecoxib R1 = Me, R2 = SO2NH2
Mavacoxib R1 = F, R2 = SO2NH2
Antipyrine X = H
Propyphenazone X = CH(Me)2
Aminophenazone X = NMe2
Metamizole X = N(Me)CH2SO3H
Fig. (1). AP and its derivatives (a), edaravone (b), coxib derivatives (c).
Pyrazolones are characterized by mild to low level of
anti-inflammatory activity which contradicts their strong
analgesic and antipyretic effects. Unlike other NSAIDs, they
poorly inhibit COX1 / COX2 on their active sites alternati-
vely acting via sequestering radicals that initiate COX
catalytic activity or reducing oxidized COX protein [10].
Other mechanisms pyrazolones biological actions include,
for example, the ability of MET to manifest its analgesic
properties partially via cannabinoid system. The drug is
rapidly metabolized to the products which can be further N-
acylated by arachidonic acid. Formed arachidonoyl amides
effectively bind to cannabinoid receptors thus producing
analgesic effect [11]. Recently, it was additionally identified
that analgesic action of MET and other pyrazolones is
mediated by TRPA1 channel affecting the susceptibility to a
variety different stimulus [12].
RF
O
O
Me
N
N
N
N
Me
Me
R
compounds 2
AP
Me
RF
OMe
CF3
N
N
N
N
R
The multi-target nature of PSBDs seems to create a space
for enhancing their anti-inflammatory activity while
retaining the superior analgesic properties. The attempts to
further optimize the PSBDs structures are constantly made in
order to improve therapeutic outcomes in traditional
NSAIDs area [13, 14] as well as to modulate different new
targets [15, 16].
SO2NH2
CEL
compounds 3
Fig. (2). Structures derived from the replacements in the molecules
of AP and CEL.
2. MATERIALS AND METHOD
2.1. Chemical Part
We expect that to achieve progress in this area
fluorinated derivatives of pyrozolones should be studied,
since the effectiveness of introducing fluorine to various
compounds have shown to be high [17]. For example, the
incorporation of fluoroalkylated substituents into the
structures of organic compounds increases their solubility,
lipophilicity, bioavailability, and metabolic stability [18, 19].
Besides, due to high electronegativity of fluorine atom, the
fluorinated compounds are capable of forming strong
intermolecular fluorine-hydrogen like nucleotide-DNA
bonds [20-27]. Recently, we have shown that polyfluorinated
salicylic acids and their derivatives have stronger anti-
inflammatory activity than the known non-fluorinated drug
aspirin [28]. Moreover, the first registered selective COX-2
inhibitors which entered the clinical and veterinarian practice
are the fluorine containing pyrazoles – celecoxib (CEL) [29,
30] and mavacoxib [31] (Fig. 1c).
Melting points were measured in open capillaries on
“Stuart SMP30” melting point apparatus and uncorrected.
The IR spectra were recorded on “Perkin Elmer Spectrum
One FT-IR” by diffuse reflection accessory (DRA) and
“Thermo Nicolet 6700 FT-IR” spectrometers at 4000-400
cm-1 using the “Frustrated total internal reflection” method
(FTIR). The 1H (13C) NMR spectra were registered on
“Bruker Avance 500” spectrometer, 500 MHz (125 MHz)
relative to SiMe4. The 19F NMR spectra were obtained on
“Bruker Avance 500” spectrometer (470 MHz) using C6F6 as
an internal standard. The chemical shifts were converted
from C6F6 to CCl3F. The microanalyses (C, H, N) were
carried out on “Perkin Elmer PE 2400” series II elemental
analyzer. All crystal structures were solved by direct
methods followed by Fourier synthesis with SHELXS-97
[32] and refined with full-matrix least-squares methods for
all non-hydrogen atoms with SHELXL-97 [32] software
packages. The registration of absorption was carried out
analytically by the model of multi-facet crystal using a
program “CrysAlisPro 1.171.29.9”.
Here we report the synthesis and biological activity of N-
methylated 1-aryl-3-polyfluoroalkylpyrazolones as fluori-
nated analogs of AP and their isomeric O-methylated
derivatives resembling CEL structure (Fig. 2, compounds 2
and 3, respectively).

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 523
2.2.2. In vivo Experiments
A single crystal of compound 2b was obtained by
crystallization from the diethyl ether. The X-ray studies were
performed on “Xcalibur 3 CCD” diffractometer with
graphite monochromator, φ\ω scanning, λMo-Kα 0.71073 Å
radiation, T 295 K. Main crystallographic data for 2b:
C12H11F3N2O, M = 256.23, space group P21/c, monoclinic, a
= 12.3613(15), b = 8.3457(9), c = 12.3094(16) Å, β =
105.077(11)°, V = 1226.2(3) Å3, Z = 4, Dcalc = 1.388 g·cm-
3, µ(Mo-Kα) = 0.121 mm-1, 168 refinement parameters, 2990
reflections measured, 1163 unique reflections which were
used in all calculations. The final R is 0.044. CCDC
1584916 contains the supplementary crystallographic data
for this compound.
2.2.2.1. Carrageenan-induced Paw Edema Model
Anti-inflammatory activity was studied in Sprague-
Dawley rats (3 male and 3 female animals per group) in the
common carrageenan-induced paw edema model [33, 34].
The investigated compounds (solutions in 1% starch
mucilage) were intraperitoneally (ip) administered at the
dose of 15 mg/kg to treated group rodents 30 mins before the
injection of 0.1 ml of freshly prepared 1% carrageenan (λ-
carrageenan, type IV; Sigma Aldrich) solution in the right
hind paw plantar surface. The negative control group
consisted of rodents treated with 1% starch mucilage
solution only. Diclofenac (DIF) (Hemofarm, Serbia, 10
mg/kg in 1% starch mucilage solution) or MET sodium
(Moschempharm after N.A. Semashko, Russia, 15 mg/kg)
were used as a reference drugs. Paw volumes were measured
oncometrically with water plethysmometer (TSE Volume
Meter, Germany) before carrageenan administration (at “zero
time”) and at 1st, 3rd and 5th h after its injection.
A single crystal of compound 3c was obtained by
crystallization from the MeOH. The X-ray studies were
performed on “Xcalibur 3 CCD” diffractometer with
graphite monochromator, φ\ω scanning, λCu-Kα 1.54184 Å
radiation, T 295 K. Main crystallographic data for 3c:
C11H8F3N3O3, M = 287.20, space group P21/c, monoclinic, a
= 8.305(6), b = 11.807(5), c = 12.080(8) Å, β = 94.22(6)°, V
= 1181.4(13) Å3, Z = 4, Dcalc = 1.615 g·cm-3, µ(Cu-Kα) =
0.133 mm-1, 214 refinement parameters, 2044 reflections
measured, 1704 unique reflections which were used in all
calculations. The final R is 0.044. CCDC 1584917 contains
the supplementary crystallographic data for this compound.
To assess an anti-inflammatory activity of the sample at
certain time point, first, the percentage of an edema
increment (i.e. relative volume change for the inflamed paw
vs “zero time” value) both in the negative control group
(Pneg) and in the treated group (Ptr ) were calculated for this
time point. Then, swelling inhibition I was calculated
according to the formula: 100% (Pneg-Ptr)/Pneg and considered
as a measure of anti-inflammatory activity of the sample.
2.2. Biological Part
2.2.2.2. The Hot plate Test
2.2.1. Parallel Artificial Membrane Permeability Assay
(PAMPA) in vitro
The hot plate test was conducted according to established
guidelines [33, 34] on Sprague-Dawley rats (3 male and 3
female rodents per group). The compounds were
intraperitoneally administered in the form of suspensions in
1% starch mucilage (15 mg/kg). Negative control group
animals received vehicle only (1% starch mucilage). DIF
(Hemofarm, Serbia, 10 mg/kg in 1% starch mucilage
solution) or MET sodium (Moschempharm after N.A.
Semashko, Russia, 15 mg/kg) were used as a reference
drugs. Rats were placed on an electrically heated to 50 ºC
plate (Hot plate 60200 series, TSE-systems, Germany) in a
plexiglas cylindrical restrainer (19 cm diameter x 30 cm).
The nociceptive response time was measured by observing
the appearance of rats’ movements (e.g. jumping, hind paws
licking or shaking). Maximal cutoff time was set as 30s
regardless of the response. Data are expressed as a relative
change of the latency time in the treated group animals
compared to the corresponding value in the negative control
group rats.
PAMPA was performed per the protocol for GIT test
described in Instruction Manual for PAMPA Explorer (Pion,
USA). Stock solutions of all compounds and standard drugs
were prepared by dissolving them in 1 ml of DMSO to gain
50 mM concentration; 5 µl of each stock solution was further
diluted in PBS according to the protocol and pH-map
(pH=7.4, 6.2, 5.0). Firstly, the diluted solutions of all tested
compounds were transferred into the UV sensitive plate
(reference plate) to read the signals using Tecan Infinite
M1000 PRO (Tecan, Switzerland). Secondly, donor
compartment of Sandwich Stirwell Plate equipped with
magnetic stirrers was column by column filled with the
above mentioned diluted solutions. Lipid solution consisting
of lecithin dissolved in dodecane (10% v/v), was gently
dispensed on each membrane back side (5 µl per well). The
Acceptor Sink Buffer (ASB) was dispensed into all wells of
acceptor compartment. The acceptor plate was placed at the
top of the donor plate; the whole sandwich was covered by
lid and placed into Gut-Box with a wet sponge to maintain a
high relative humidity thus minimizing evaporation. The
assembly was allowed to incubate for 2 h and at 40 µm
unstirred water layer (UWL).
2.2.2.3. The Tail Flick Test
Analgesic activity was evaluated by measuring the
sensitivity of Sprague-Dawley rats to heat beam applied to
their tails with the Tail Flick instrument (TSE-system,
Germany). To prevent tissue damage in the study the beam
intensity was set to 25 % and cut-off time was limited to 60
seconds. The light beam exerting radiant heat was directed to
the proximal third of the restrained animal tail. The time
between tail exposure to radiant heat and escape reaction
appearance was measured [33-35]. The day before the
experiment was conducted, baseline testing was performed.
After 2 h, the sandwich was removed from the Gut-Box
and disassembled, both acceptor and donor compartments
were separately transferred to the UV sensitive plates, and
spectra were read. The results were processed with PAMPA
Explorer Software.

524 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
The rats with response time between 4 and 8 seconds were
chosen and included into three groups (3 male and 3 female
animals per group) for ip administration of one of the
following: 1% starch mucilage (vehicle), 2a (15 mg/kg in
1% starch mucilage solution) and MET sodium
(Moschempharm after N.A. Semashko, Russia) (163 mg/kg
in 1% starch mucilage). The latency time measurement was
performed at 1st and 2nd h after compound injection [33-35].
vehicle (1% starch mucilage solution). 1 h after injection,
mice were one by one placed in the center of a white circular
arena (diameter 63 cm, OOO Open Science, Russia) and
their movements were recorded for 5 min with digital
camera. The following behavioral patterns were analyzed:
line crossing (in two peripheral sectors), central square
entries, rearing (with and without support), grooming,
defecation, urination, number and duration of fading and
holes inspection. Numbers of patterns and duration of
actions in the treated and control groups were analyzed
statistically.
Mean percentages of the latency time vs baseline were
calculated for the control and treated group rodents and
compared to each other statistically.
2.2.2.7. Acute Toxicity Evaluation
2.2.2.4. The Mechanical Hyperalgesia Test
OECD recommendations [41] and guidelines for pre-
clinical study of medicinal products [34] were used to set
acute toxicity evaluation. Three CD-1 mice per one dose
were applied. The tested compounds in 1% starch mucilage
solution were injected ip. Animals were observed during 14
days, the number of deaths was counted.
The mechanical hyperalgesia was evaluated on Sprague-
Dawley rats in Randall-Selitto test [33-36]. Three groups of
animals (3 male and 3 female rodents per group) received
either 1% starch mucilage (vehicle), 2a (15 mg/kg in 1%
starch mucilage solution) or reference drug DIF (Hemofarm,
Serbia, 10 mg/kg) ip 30 mins before 0.1 ml of carrageenan
solution injection. The latter was administered to the hind
paw of each rat to induce inflammation. One day prior the
experiment, habituation of animals to the instrument was
conducted and paws of rats were manipulated with the
Randall-Selitto Advanced instrument (TSE-system, Ger-
many). The paws sensitivity was tested 1 h after carrageenan
injection. The mechanical pressure was applied to the dorsal
surface of the paw until the animal withdrew. At that
moment maximal pressure amount (P) was recorded. The
threshold was set to 530 P regardless of the response. Mean
P values in treated groups were compared with one in the
control group.
2.2.3. Statistical Analysis
The data were analyzed by a statistical software package
GraphPad Prism 6 using the «Multiplet tests» approach. The
values were considered significantly different at p <0.05.
3. EXPERIMENTAL
3.1. Synthesis of Pyrazolols
Pyrazololes 1a-g have been obtained from the
corresponding 4-polyfluoroalkyl-3-oxo esters with substi-
tuted hydrazines by the known method [42].
3.1.1. 1-Phenyl-3-trifluoromethyl-1H-pyrazol-3-ol (1a)
2.2.2.5. Evaluation of the Antipyretic Activity
Yield 81%, off-white powder, mp 193–194 °С (lit. [43],
mp 191–192 °С).
Antipyretic activity was evaluated in yeast pyrexia model
described in [37, 38], with slight modifications. The rectal
temperatures of Sprague-Dawley rats were measured by
digital thermometer (A&D, Japan). Fever was induced by
subcutaneous administration of 20% (w/v) baker’s yeast
suspended in 0.9% saline solution (20 ml/kg) at the
interscapular region. After 6 h from yeast injection, the rectal
temperatures (T6.0) were registered again and animals which
showed the temperature growth not less than 1.0 °C were
used for further experiment. Three group of rats (3 male and
3.1.2. 1-(4-Methylphenyl)-3-trifluoromethyl-1H-pyrazol-5-
ol (1b)
1
Yield 60%, colorless crystals, mp 218-219 °С. Н NMR
(DMSO-d6): δ 2.35 (s, 3H, Me), 5.91 (s, 1H, CH), 7.31, 7.57
(both d, 4H, C6H4, J 8.3 Hz), 12.33 (s, 1H, OH). 19F NMR
(DMSO-d6): δ -62.0 (s, CF3).
3.1.3. 1-(4-Nitrophenyl)-3-trifluoromethyl-1H-pyrazol-5-ol
(1c)
3
female animals per group) were intraperitoneally
administrated either 2a (30 mg/kg in 1% starch mucilage
solution) or Paracetamol (“Medisorb”, Russia, 100 mg/kg
1% in starch mucilage solution) or 1% starch mucilage
solution as vehicle control. Rectal temperatures for all rats
were measured at 7th, 7.5 and 8.5 h after yeast injection.
1
Yield 65%, orange crystals, mp 125 °С subl. Н NMR
(DMSO-d6): δ 5.99 (s, 1H, CH), 8.11, 8.38 (both d, 4H,
C6H4, J 9.2 Hz), OH is not observed. 19F NMR (DMSO-d6):
δ -62.6 (s, CF3).
Relative rectal temperature changes for each group at
3.1.4. 1-(3-Chlorophenyl)-3-trifluoromethyl-1H-pyrazol-5-
ol (1d)
each time point were calculated: ΔTrect
=
(Tex–
T6.0)/T6.0·100%, where Tex– rectal temperature at the
corresponding time point of the experiment, T6.0–temperature
at 6th h after yeast administration.
1
Yield 85%, colorless crystals, mp 210-211 °С. Н NMR
(DMSO-d6): δ 5.96 (s, 1H, CH), 7.46, 7.55, 7.75, 7.80 (all m,
4H, C6H4), 12.77 (s, 1H, OH). 19F NMR (DMSO-d6): δ -62.3
(s, CF3).
2.2.2.6. Open Field Test
The open field test was conducted for evaluating
exploratory, anxiety-related behavior and general activity of
CD-1 mice [33, 39, 40]. Two groups of animals (5 male and
5 female mice per group) were ip administrated with either
2a (suspension in 1% starch mucilage, 15 mg/kg) or the
3.1.5. 3-Trifluoromethyl-1-(3-trifluoromethylphenyl)-1H-
pyrazol-5-ol (1e)
1
Yield 73%, colorless crystals, mp 204-205 °С. Н NMR
(DMSO-d6): δ 5.99 (s, 1H, CH), 7.77, 8.12 (both m, 4H,

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 525
C6H4), 12.89 (s, 1H, OH). 19F NMR (DMSO-d6): δ -62.4 (s,
3F, CF3), -61.6 (s, 3F, CAr–CF3).
CF3). Anal. calcd. for C11H9F3ClN2O1.5. С, 46.25; Н, 3.18; N,
9.81. Found: С, 46.26; Н, 2.95, N, 9.60.
3.1.6. 1-Benzyl-3-trifluoromethyl-1H-pyrazol-5-ol (1f)
3.2.5. 1-Methyl-5-trifluoromethyl-2-(3-trifluoromethylphe-
nyl)-1,2-dihydro-3H-pyrazol-3-one (2e)
Yield 65%, white powder, mp 223-224 °C (lit. [44], mp
224.5-225.5 °C).
Yield 60%, light-yellow crystals, mp 56-58 °С. IR
(DRA): ν 1672 (C=O), 1610, 1587, 1492, 1446 (С=С), 1268-
1172 (С–F) cm-1. 1Н NMR (CDCl3): δ 3.23 (s, 3Н, Me), 6.10
3.1.7. 3-Pentafluoroethyl-1-phenyl-1H-pyrazol-3-ol (1g)
(s, 1H, CH), 5.15 (s, 2H, CH2), 7.64-7.67 (m, 4H, C6H4). 13
C
Yield 70%, off-white powder, mp 210-211°С (lit. [45] in
Supplementary, mp 209-210°С).
NMR (CDCl3): δ 37.33 (q, Me, J 1.2 Hz), 102.82 (q, CH, J
2.8 Hz), 119.00 (q, CF3, J 271.4 Hz), 123.45 (q, CF3, J 272.6
Hz), 121.26 (q, Co, J 3.8 Hz), 124.57 (q, Cp, J 3.5 Hz),
132.20 (q, Cm, J 33.2 Hz), 127.89, 130.25, 133.84 (Co’, Cp’,
Ci), 147.00 (q, C—CF3, J 39.1 Hz), 163.35 (C=O). 19F NMR
(CDCl3): δ -64.3 (s, 3F, CF3), -63.8 (s, 3F, CAr-CF3). Anal.
calcd. for C12H8F6N2O. С, 46.46; Н, 2.60; N, 9.03. Found: С,
46.51; Н, 2.69, N, 8.93.
3.2. Typical Procedure for N-methylation of pyrazoles
1a–g
A 3.15 g, 25 mmol of dimethyl sulfate was added to 5
mmol of pyrazoles 1a-g. The flask was immersed in the oil
bath and heated at 120-130 °С for 6-7 h. After cooling, 30 ml
of ether was added and the resulting precipitate was isolated
and washed by 10% sodium hydroxide. The residue was
purified by column chromatography (eluent – CHCl3).
3.2.6. 2-Benzyl-1-methyl-5-trifluoromethyl-1,2-dihydro-3H-
pyrazol-3-one (2f)
Yield 50%, crystallizing oil, IR (FTIR): ν 1676 (C=O),
3.2.1.
1-Methyl-2-phenyl-5-trifluoromethyl-1,2-dihydro-
1
3H-pyrazol-3-one (2a)
1606, 1576, 1498 (С=С), 1260-1142 (С–F) cm-1. Н NMR
(CDCl3): δ 3.36 (s, 3Н, Me), 5.15 (s, 2H, CH2), 6.02 (s, 1H,
CH), 7.20-7.21, 7.32-7.37 (both m, 5H, Ph). 13C NMR
(CDCl3): δ 35.00 (q, Me, J 1.5 Hz), 45.46 (CH2), 98.41 (q,
CH, J 2.6 Hz), 118.85 (q, CF3, J 270.9 Hz), 126.90, 128.26,
129.05, 134.90 (Ph), 141.19 (q, C—CF3, J 39.1 Hz), 163.04
(C=O). 19F NMR (CDCl3): δ -63.9 (s, CF3). Anal. calcd. for
C12H11F3N2O. С, 56.25; Н, 4.33; N, 10.93. Found: С, 56.34;
Н, 4.37, N, 10.85.
Yield 77%, colorless crystals, mp 138-139 °С (lit. [46],
mp 138-139 °С).
3.2.2. 1-Methyl-2-(4-methylphenyl)-5-trifluoromethyl-1,2-
dihydro-3H-pyrazol-3-one (2b)
Yield 58%, colorless crystals, mp 122-123оС. IR (DRA):
ν 1660 (C=O), 1613, 1584, 1515 (С=С), 1257-1171 (C–F)
1
cm-1. Н NMR (DMSO-d6): δ 2.37 (s, 3H, p-Me), 3.18 (s,
3.2.7. 1-Methyl-5-pentafluoroethyl-2-phenyl-1,2-dihydro-
3H-pyrazol-3-one (2g)
3H, Me), 6.25 (s, 1H, CH), 7.29, 7.36 (both d, 4H, C6H4, J
8.3 Hz). 13C NMR (DMSO-d6): δ 20.61 (s, p-Me), 36.33 (s,
Me), 100.42 (q, CH, J 2.6 Hz), 119.11 (q, CF3, J 271.0 Hz),
125.72, 129.81, 130.23, 137.86 (C6H4), 143.09 (q, C—CF3, J
38.5 Hz), 162.02 (C=O). 19F NMR (DMSO-d6): δ -62.2 (s,
CF3). Anal. calcd. for C12H11F3N2O. С, 56.25; Н, 4.33; N,
10.93. Found: С, 56.24; Н, 4.16, N, 10.86.
Yield 71%, orange crystals, mp 82-83 °С (lit. [46], mp
82-83 °С).
3.3. Typical Procedure for O-methylation of Pyrazoles
1a–g
3.2.3.
2-(4-Nitrophenyl)-1-methyl-5-trifluoromethyl-1,2-
A mixture of 5 mmol of pyrazoles 1a-g, 0.63 g, 5 mmol
of dimethyl sulfate and 1.04 g, 7.5 mmol of K2CO3 in 10 ml
of acetonitrile was refluxed for 6-7 h. After cooling, 30 ml of
water was added and the solution was extracted with 2x30ml
ether. The ether was removed and the residue was purified
by appropriate method.
dihydro-3H-pyrazol-3-one (2c)
Yield 68%, yellow crystals, mp 133-136°С. IR (DRA): ν
1695 (C=O), 1591, 1521, 1493 (С=С), 1171, 1143 (C–F) cm-
1
1. Н NMR (CDCl3): δ 3.25 (s, 3H, Me), 6.13 (s, 1H, CH),
7.65, 8.39 (both d, 4H, C6H4, J 9.0 Hz). 13C NMR (CDCl3): δ
38.03 (q, Me, J 1.2 Hz), 103.88 (q, CH, J 2.7 Hz), 118.90 (q,
CF3, J 271.6 Hz), 123.48, 125.02, 138.83, 145.87 (C6H4),
148.72 (q, C—CF3, J 39.1 Hz), 163.37 (C=O). 19F NMR
(CDCl3): δ -64.4 (s, CF3). Anal. calcd. for C11H8F3N3O3. С,
46.00; Н, 2.81; N, 14.63. Found: С, 46.08; Н, 2.85, N, 14.44.
3.3.1. 5-Methoxy-1-phenyl-3-trifluoromethyl-1H-pyrazole
(3a)
Yield 73%, yellow oil [46].
3.3.2. 5-Methoxy-1-(4-methylphenyl)-3-trifluoromethyl-1H-
pyrazole (3b)
3.2.4. 2-(3-Chlorophenyl)-1-methyl-5-trifluoromethyl-1,2-
dihydro-3H-pyrazol-3-one hemihydrate (2d)
Yield 80%, yellow oil. IR (FTIR): ν 1593, 1568, 1521,
1497 (C=C, C=N), 1250-1155 (C-F) cm-1. 1Н NMR (CDCl3):
δ 2.38 (s, 3H, p-Me), 3.96 (s, 3H, Me), 5.93 (s, 1H, CH),
7.24, 7.53 (both d, 4H, C6H4, J 8.4 Hz). 13C NMR (CDCl3): δ
21.02 (p-Me), 59.18 (Me), 83.97 (q, CH, J 2.0 Hz), 121.06
(q, CF3, J 268.9 Hz), 122.84, 129.50, 135.19, 137.55 (C6H4),
141.53 (q, C—CF3, J 38.5 Hz), 155.51 (C–OMe). 19F NMR
(CDCl3): δ -64.4 (s, CF3). Anal. calcd. for C12H11F3N2O. С,
56.25; Н, 4.33; N, 10.93. Found: С, 56.22; Н, 4.18, N, 11.15.
Yield 57%, light-yellow crystals, mp 85-87°С. IR
(DRA): ν 1681 (C=O), 1661, 1587, 1480 (С=С), 1259-1177
(С–F) cm-1. Н NMR (DMSO-d6): δ 3.21 (s, 3Н, Me), 6.35
1
(s, 1H, CH), 7.38-7.41, 7.50-7.61 (all m, 4H, C6H4). 13C
NMR (DMSO-d6): δ 36.91 (q, Me, J 1.3 Hz), 101.18 (q, CH,
J 2.8 Hz), 119.02 (q, CF3, J 271.2 Hz), 123.59, 124.82,
127.75, 130.98, 133.53, 134.19 (C6H4), 144.70 (q, C—CF3, J
38.5 Hz), 162.29 (C=O). 19F NMR (DMSO-d6): δ -62.3 (s,

526 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
85.4 (t, 3F, CF3, J 2.5 Hz). Anal. calcd. for C12H9F5N2O. С,
49.32; Н, 3.10; N, 9.59. Found: С, 49.10; Н, 2.98, N, 9.52.
3.3.3. 5-Methoxy-1-(4-nitrophenyl)-3-trifluoromethyl-1H-
pyrazole (3c)
Yield 75%, yellow crystals, mp 111-112 °С. IR (FTIR): ν
4. RESULTS AND DISCUSSIONS
4.1. Chemistry
1611, 1598, 1580, 1519, 1495 (C=C, C=N), 1224-1155 (C-F)
1
cm-1. Н NMR (CDCl3): δ 4.08 (s, 3H, Me), 6.00 (s, 1H,
CH), 8.02, 8.32 (both m, 4H, C6H4). 13C NMR (CDCl3): δ
59.74 (Me), 85.00 (q, CH, J 1.6 Hz), 120.60 (q, CF3, J 269.4
Hz), 121.80, 124.64, 142.75, 145.86 (C6H4), 143.30 (q, C—
CF3, J 38.9 Hz), 156.52 (C–OMe). 19F NMR (CDCl3): δ -
64.9 (s, CF3). Anal. calcd. for C11H8F3N3O3. С, 46.00; Н,
2.81; N, 14.63. Found: С, 46.10; Н, 2.84, N, 14.49.
The synthesis of trifluoromethyl-substituted AP 2a was
described in 1958 without proof of its structure [47]. Later,
its analog containing the second trifluoromethyl-group in
phenyl substituent was obtained [48], but it also had been not
characterized. However, the methylation of 1-aryl-3-
polyfluoroalkyl-1H-pyrazol-5-ols 1 may lead to formation of
isomeric O-methylated products.
3.3.4. 1-(3-Chlorophenyl)-5-methoxy-3-trifluoromethyl-1H-
pyrazole (3d)
Recently we developed [46] the convenient approaches to
selective O- and N-methylation of 1-aryl-3-polyfluoroalkyl-
1H-pyrazol-5-ols 1. The best conditions for chemoselective
N-methylpyrazolones 2 synthesis were found to be a heating
of pyrazolols 1a-g with 5 equiv of dimethyl sulphate while
O-methylpyrazoles 3 can be obtained as the only products by
refluxing of compounds 1a-g with 1 equiv of dimethyl
sulphate and 1.5 equiv of K2CO3. Herein, using these
procedures, we synthesized a series of NMe-substituted
pyrazolones 2a-g and OMe-containing pyrazoles 3a-g
(Scheme 1) with the various polyfluoroalkyl and aryl
substituents.
Yield 62%, orange crystals, mp 47-49 °С. IR (DRA): ν
1589, 1568, 1496, 1477 (C=C, C=N), 1246-1119 (C-F) cm-1.
1Н NMR (DMSO-d6): δ 4.03 (s, 3H, Me), 6.51 (s, 1H, CH),
7.51, 7.57, 7.68, 7.75 (all m, 4H, C6H4). 13C NMR (DMSO-
d6): δ 60.13 (Me), 85.48 (q, CH, J 1.7 Hz), 120.99 (q, CF3, J
268.8 Hz), 120.99, 122.06, 127.58, 130.98, 133.46, 138.26
(C6H4), 140.95 (q, C—CF3, J 37.8 Hz), 156.09 (C–OMe). 19
F
NMR (DMSO-d6): δ -62.3 (s, CF3). Anal. calcd. for
C11H8F3ClN2O. С, 47.76; Н, 2.91; N, 10.13. Found: С,
47.53; Н, 2.89, N, 9.98.
3.3.5.
5-Methoxy-3-trifluoromethyl-1-(3-trifluoromethyl-
phenyl)-1H-pyrazole (3e)
A chemical shift of methyl group at the heteroatom is a
convenient criterion for distinction of isomers 2 and 3. Thus,
the H NMR spectra of compounds 2a-g contain the signals
Yield 70%, colorless crystals, mp 43-45 °С. IR (FTIR): ν
1
1601, 1575, 1509, 1464 (C=C, C=N), 1256-1150 (C—F) cm-
1. Н NMR (CDCl3): δ 4.02 (s. 3H, Me), 5.97 (s, 1H, CH),
1
of NMe fragment at δ 3.2…3.3 ppm, while the signals of
OMe group are observed at δ 3.8…4.1 ppm in pyrazoles 3a-
g. Moreover, the IR spectra of N-isomers 2a-g have a high-
frequency absorption band of carbonyl group at 1695-1660
cm-1 in contrast to O-methylated products 3a-g.
7.59, 7.93, 8.02 (all m, 4H, C6H4). 13C NMR (CDCl3): δ
59.50 (Me), 84.50 (CH), 120.82 (q, CF3, J 269.2 Hz), 123.61
(q, CAr—CF3, J 272.5 Hz), 119.27 (q, Co, J 3.8 Hz), 123.29
(q, Cp, J 3.6 Hz), 125.44, 129.63, 138.23 (Co’, Cp’, Ci),
131.59 (q, Cm, J 33.1 Hz), 142.52 (q, C—CF3, J 38.9 Hz),
155.96 (C–OMe). 19F NMR (CDCl3): δ -64.6 (s, 3F, CF3), -
63.8 (s, 3F, CAr—CF3). Anal. calcd. for C12H8F6N2O. С,
46.16; Н, 2.60; N, 9.03. Found: С, 45.98; Н, 2.61, N, 8.80.
3.3.6. 1-Benzyl-5-methoxy-3-(trifluoromethyl)-1H-pyrazole
(3f)
Yield 55%, yellow oil. IR (FTIR): ν 1606, 1571, 1499,
1
1456, 1431 (C=C, C=N), 1134-1245 (C—F) cm-1. Н NMR
(CDCl3): δ 3.81 (s, 3H, Me), 5.10 (s, 2H, CH2), 5.73 (s, 1H,
CH), 7.15, 7.24 (both m, 5H, Ph). 13C NMR (CDCl3): δ
51.27 (Me), 59.02 (CH2), 83.22 (q, CH, J 1.7 Hz), 121.08 (q,
CF3, J 268.7 Hz), 121.52, 127.87, 128.65, 135.86 (Ph),
140.64 (q, C—CF3, J 38.3 Hz), 155.44 (C—OMe). 19F NMR
(CDCl3): δ -64.1 (s, CF3). Anal. calcd. for C12H11F3N2O. С,
56.25; Н, 4.33; N, 10.93. Found: С, 56.30; Н, 4.25, N, 10.70.
Fig. (3). The ORTEP view of molecules 2b and 3c.
We managed to grow up the monocrystals for both
isomers and carried out the X-Ray analysis of NMe
pyrazolone 2b (Fig. 3, CCDC 1584916) and OMe pyrazole
3c (Fig. 3, CCDC 1584917). This clearly confirmed the
regioisosteric structure of pyrazoles 2 and 3.
3.3.7. 5-Methoxy-3-pentafluoroethyl-1-phenyl-1H-pyrazole
(3g)
The development of convenient methods for pyrazoles 2
and 3 synthesis allowed us to study their biological
properties as potential NSAIDs.
Yield 75%, yellow oil. IR (FTIR): ν 1597, 1569, 1510,
1484 (С=С, C=N), 1214-1137 (С–F) cm-1. 1Н NMR
(CDCl3): δ 3.97 (s, 3Н, Me), 5.95 (s, 1H, CH), 7.31-7.35,
7.42-7.46, 7.67-7.69 (all m, 5H, Ph). 13C NMR (CDCl3): δ
59.24 (Me), 85.23 (CH), 110.59 (tq, CF2, J 251.0, 39.1 Hz),
118.85 (qt, CF3, J 286.0, 38.0 Hz), 122.80, 127.54, 128.96,
137.70 (Ph), 140.39 (t, C—C2F5, J 28.7 Hz), 155.82 (C—
OMe). 19F NMR (CDCl3): δ -115.1 (q, 2F, CF2, J 2.5 Hz), -
4.2. Biological Evaluation
The goal of our study was to obtain the fluorinated AP
derivatives which a) have enhance analgesic potency, b)
elicit anti-inflammatory effect and c) are less toxic or have

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 527
RF
O
5 equiv Me2SO4
N
N
RF
OH
Me
R
R
2a-g
N
N
heat or
reflux,
6-7 h
R
1a-g
RF
OMe
N
N
1 equiv Me2SO4,
1.5 equiv K2CO3,
MeCN
3a-g
RF = CF3, R = Ph (a), C6H4Me-4 (b), C6H4NO2-4 (c), C6H4Cl-3 (d),
C6H4CF3-3 (e); CH2Ph (f),
RF = C2F5, R = Ph (g).
Scheme 1. Synthesis of N- and O-methylated pyrazoles.
Fig. (4). Permeability Pe (10-6 cm/s) – lipophilicity (СlogP) relationship for AP and its derivatives.
favorable pharmacokinetics which would allow lowering the
therapeutic doses and, hence, diminishing the undesirable
side effects. Several factors, such as the “multi-targetness” of
pyrazolone scaffold, diverse profiles of biological action for
non-fluorinated AP derivatives and different patterns of
metabolic activation of the PSBDs [49, 50] may imply that
the biological evaluation in vivo could be more informative
for the lead series elucidation. One of the most beneficial
methods to identify a multi-target lead compound as well as
any relevant pro-drugs is phenotypic screening since all the
involved targets and the metabolic activation factors can be
engaged at ones in the in vivo experiment.
compounds 2a-g, 3a-g fall into the Lipinski rule of five
because their molecular weights are not higher 500 Da, the
predicted values of lipophilicity (AlopP) do not exceed 5, the
quantity of hydrogen bonds (donor or acceptor) is no more
than 2, rotating bonds are no more than 4, and at last, the
calculated polar surface areas of the structures are no more
than 70 Å. This means that the synthesized compounds
comply the “rules of thumb” applied in drug discovery,
including the Lipinski's rule of five [54] and for compounds
1a, 2a,f even more strict criteria - the “rule of three” [55].
4.2.2. Permeability Study in vitro
Further, in in vitro experiment we evaluated the impact
of fluorine atoms on the AP derivatives permeability. The
ability of AP and synthesized compounds 2a,c,g and 3a,c to
passively diffuse through the membranes was assessed in
Parallel Artificial Membrane Permeability Assay (PAMPA)
[56, 57] at pH values simulating the media in different parts
of the GIT (5.0, 6.2 and 7.4). Ketoprofen was used as a
reference drug with high permeability at acidic pH. Addi-
tionally, lipophilicity, represented as СlogPs, was calculated
in ChemBioDraw Ultra® 13.0.2.302. The lipophilicity–
permeability relationship of the tested compounds is pre-
sented in the diagram below (Fig. 4).
For the sake of this research, we have chosen the mixed
strategy which includes preliminary evaluation of the limited
set of derivatives in simple models of pain and inflammation
(hot plate test and carrageenan-induced edema test
respectively). The test involving carrageenan was supposed
to be performed for only those compounds which were
active in the hot plate test since analgesic activity evaluations
were considered as a primary goal.
4.2.1. ADME Analysis
To determine the possibility of synthesized compounds
theoretical hitting into «drug like space» ADME analysis
was performed [51, 52]. The compounds bioavailability was
assessed with Maestro Elements 2.4 (QikProp) program [53].
The calculation results are presented in Table 1. The
The figure shows that the compounds 2a,c,g and 3c
penetrate through an artificial membrane much better than
AP at all pH values. Thus, substitution of methyl group to

528 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
Table 1. ADME parameters.
Hydrogen Bond
Polar Sur-
face Area,
Ǻ2
ID
Molecular
Weight, Da
Lipophilicity
(AlogP)
Rotatable
Bond
Human Oral
Absorptione
c
QPlogPo/w
QPlogKpd
Ligand
Donor Acceptor
Constrains
500.0a
300.0b
5
3
10
3
5.00
3.00
140.00
60.00
10
3
> 80 is high
-2.0 – 6.5
-8.0 – -1.0
< 25% is
poor
DIF
CEL
SC560
1a
296.15
327.90
352.75
227.20
242.20
256.23
287.20
276.65
310.20
256.23
292.20
242.20
256.23
287.20
276.65
310.20
256.23
292.21
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
2
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3.67
4.06
5.55
2.55
2.86
3.35
2.75
3.52
3.80
2.87
3.35
3.48
3.97
3.38
4.15
4.42
3.49
3.97
56.16
86.41
27.05
40.88
26.93
26.93
70.07
26.93
26.93
26.93
26.90
27.05
27.05
70.19
27.05
27.05
27.05
27.05
4
5
4
2
2
2
3
2
3
3
3
3
3
4
3
4
4
4
4.50
3.36
5.98
3.44
2.17
2.56
1.65
2.92
3.37
2.78
3.09
4.33
4.65
3.36
4.83
5.34
4.70
5.00
-1.87
-3.20
-0.64
-1.57
-1.69
-1.89
-3.60
-1.86
-1.91
-1.51
-1.63
-0.81
-1.01
-2.72
-0.98
-1.04
-0.65
-0.75
100.0
92.4
100.0
100.0
100.0
100.0
82.9
2a
2b
2c
2d
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
2e
2f
2g
3a
3b
3c
3d
3e
3f
3g
[a] rules of five; [b] rules of three; [c] predict octanol/water partition coefficient; [d] predict skin permeability, log Kp; [e] predict human oral adsorption;
CF3 group in pyrazolone ring of AP (compound 2a) on
average allowed 15 times permeability increase. More
impressive difference is observed when the data for O-me-
thylated pyrazole 3c is compared with one for corresponding
N-derivative (compound 2c). Unexpectedly, for derivative
3a we observed low signals in both donor and acceptor
compartments far below the level in the reference solution.
Since 3a is fairly stable in the assay conditions, its
localization in the lipid membrane may serve as a putative
explanation for its disappearance from the compartments.
Moreover, the 3a lipophilicity is the highest among all
compounds tested in PAMPA. While high lipophilicity can
lead to GIT absorption complication, it may be favorable for
the tissue distribution of the compound [58]. Overall, the
PAPMA data demonstrates that fluorinated derivatives
possibly have an advantage in terms of bioavailability and
pharmacokinetic properties compared with AP.
vivo efficacy evaluation, the compounds were assessed first
in the acute toxicity test in CD-1 mice (3 rodents per dose).
The obtained data (Table 2) allows us to conclude that LD50
values for the most of the synthesized fluorinated AP
derivatives are expected to be significantly over 150 mg/kg
(ip), e.g. all compounds are substantially less toxic than DIF
[59]. Compounds 1a, 2a,b, g seem to be more toxic than AP
and MET (see Table 2).
When compared the data for 2a vs 3a, 2b vs 3b may
imply that O-methylated derivatives 3 are slightly less toxic
than the corresponding N-methylated derivatives 2. Both
isomeric pyrazoles 2e and 3e, having meta-CF3-group in aryl
substituent, are not toxic at the dose of 300 mg/kg. Two
routes of administration (ip and per os ) were tested for the
2a compound and application of the latter leads to
significantly lower level of acute toxicity (expected LD50
600 mg/kg).
>
Based on the toxicity data and the perspective to easily
elucidate the most promising compounds due to rather strict
threshold, the dose of 15 mg/kg was chosen for synthesized
compounds biological activity evaluation. In order to avoid
4.2.3. Acute Toxicity Study
In order to briefly estimate the toxicity of the synthesized
compounds and to choose an appropriate dose for their in

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 529
Table 2. Anti-inflammatory, analgesic activity and acute toxicity of fluorinated pyrazoles in vivo.
Anti-inflammatory Activity,
Analgesic Activity,
Dose,
Compound
Latent Period
Acute Toxicity a
Swelling Inhibition, %
mg/kg
Prolongation,%
After 1 h
After 3 h
After 5 h
1a
2a
15
15
n/t
n/t
n/t
54.5 **
300 (2)
56.8 *
n/a
51.1 ***
29.0 *
n/a
128.0 ***
150 (0)
225 (0)
300 (2)
300 b (0)
600 b (0)
10
25.69 *
59.3 ***
2b
2c
2d
2e
2g
3a
15
15
15
15
15
15
n/a
n/a
n/a
-32.52 *
n/a
n/a
n/a
n/a
n/a
n/a
n/a
82.6 ***
20.5 §
n/a
150 (0)
300 (1)
150 (0)
n/a
150 (0)
n/a
n/a
n/a §§
150 (0)
300 (0)
n/a §§§
n/a
n/a
51.1 ****
82.9 **
150 (0)
300 (1)
n/a
150(0)
300 (0)
600 (0)
0
n/t
n/t
n/t
n/t
n/t
n/t
n/t
n/t
n/t
53.3*, 82.4с,***
60.2 ***
3b
3c
15
15
300 (0)
150 (0)
n/a
n/aс
3e
15
15
10
n/t
n/ad
n/t
n/ad
n/t
n/ad
n/a
150 (0)
300 (0)
MET
DIF
59.8 - 93.8e
44.0 – 70.9g
LD50 2160-3340,
mice, ip [61]
53.9 ± 7.1f
61.8 ± 10.8f
48.5 ± 13.1f
LD50 74,
mice, ip [59]
n/a – not active; n/t – not tested; [a] represented as a dose, mg/kg (number of mice which did not survive of the three ones taken in the experiment); [b] tested at per os route; [c]
measured 120 min after sample administration; [d] data from two independent experiments; [e] interval of values obtained in five different independent experiments; [f] median for
five different independent experiments ± standard deviation; [g] interval of values obtained in four different independent experiments; § trend is observed: p=0.05; §§ trend is
observed: 36.3%, p=0.08;§§§ trend is observed: 30.4%, p=0.07;* p<0.05; ** p<0.01; ***p<0.001; ****p<0.0001
compounds degradation in the GIT, the samples were
administered intraperitoneally.
The CF3-AP 2a elicits the most prominent anti-
nociceptive effect exceeding such of MET at the same dose
(15 mg/kg). The lower dose of 2a (10 mg/kg) produces
approximately two-times weaker analgesic effect. Being
administered at the dose of 15 mg/kg the N-methylated
derivatives 2a, b are more potent than the corresponding O-
methylated isomers 3a,b, although compound 3a exhibits a
fairly strong analgesic effect (82.9 % latency time prolon-
gation). Interestingly, the 3a dose decrease to 10 mg/kg leads
to notably weaker effect (53.3%) at the 1st h after the
injection, however, at the 2nd h the activity restores to 82.4%
4.2.4. Analgesic Activity Study
Some of the synthesized compounds were first assessed
for analgesic activity in the hot plate test in rats [33] at the
dose of 15 mg/kg (Table 2). Since AP high doses are
required for achieving an anti-nociceptive response in the hot
plate test [34, 62], more effective pyrazolone analgesic MET
was chosen as a reference drug.

530 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
A
B
Fig. (5). Analgesic activity of 2a, represented as mean values±SD: A. in the “tail flick” test (2a at 15 mg/kg, n=6; MET at 163 mg/kg, n=5);
B. in the Randall-Selitto test of carrageenan-induced hyperalgesia in rats (2a at 15 mg/kg, n=6; DIF at 10 mg/kg, n=6). **p<0.01 compared
to the vehicle treated control.
latency time. Such data may imply the compounds pharma-
cokinetics profile further investigation importance or meta-
bolic activation occurrence. The parent pyrazole 1a, which
was tested for the structure-activity analysis, demon-strated
evident analgesic activity: a dose of 15 mg/kg caused the
latent period prolongation by 54.5%.
the oxygen in 3a (Fig. 2). The aromatic ring at the position 5
in CEL can be substituted while retaining significant
biological activity level for corresponding derivative [63].
The analgesic property of the CF3-AP 2a was further
assessed in the tail flick test and inflammation induced
hyperalgesia model (mechanical pressure on rat paw).
The structure-activity analysis showed that polyfluoro-
alkyl substituent elongation causes partial loss of analgesic
activity (C2F5-AP 2g vs CF3-AP 2a). Additionally, substi-
tuent in aryl fragment of pyrazoles 2 and 3 negatively
influences their anti-nociceptive properties. Thus, com-
pounds 2a, 3a, having unsubstituted phenyl moiety, are the
most potent. Electron-withdrawing substituents in both para-
and meta-positions of aryl fragment abolish the activity for
all tested compounds (2c, 2d and 2e vs 2a; 3c and 3d vs 3a).
The presence of an electron-donating methyl group
substantially reduces the activity (2b vs 2a; 3b vs 3a).
In the tail flick test the anti-nociceptive effect was
evaluated by measuring the latency time after application of
the radian hit to the proximal third of the rat tail one and two
hours after the samples injection [33]. The level of activity
was estimated as percentage to the baseline value. The
compound 2a was injected in the chosen screening dose of
15 mg/kg. MET was used as a reference drug at the higher
dose (163 mg/kg, which is approx. 0.05LD50) in order to
ensure its analgesic action in the experiment conditions.
Both CF3-AP 2a and MET exhibited anti-nociceptive effect
one hour after the injection (Fig. 5). After 2 h time point
MET partially retained the effect (p<0.05 compared to
control) while 2a did not.
Overall, the correlation between anti-nociceptive pro-
perties and permeability/ lipophilicity is absent for the
synthesized compounds: all of them (both active and not
active) are more lipophilic than AP. This may imply that the
membrane permeability is not the limiting factor for
exhibiting analgesic activity by synthesized pyrazolone
derivatives.
In the hyperalgesia test [33] an inflammatory pain was
induced by carrageenan injection in the rats hind paw. A
classical and effective NSAID DIF (10 mg/kg) produced
significant decrease in the severity of the inflammation
induced hyperalgesia: the latency time increased more than
twice (p<0.01) compared with not-treated control. The effect
of 2a (15 mg/kg) may be described as a tendency in
increasing the latency (p<0.1 compared to the vehicle treated
control) (Fig. 5).
PSBDs are quite rapid and extensively metabolized in
vivo and most of their biological effects are often ascribed to
their metabolites [11-13, 48-49]. The obtained patterns of the
structure-activity relationship for the studied compounds
may be attributed - at least partially - to the influence of
substituents on rate and directions of the metabolic
transformations which occur in vivo.
4.2.6. Anti-Inflammatory Activity
The carrageenan induced hind paw edema model in rats
was used for synthesized compounds anti-inflammatory
activity primary evaluation [33]. The test was completed
mainly with the compounds which were active in the hot
plate test. The measurements of paw volume were conducted
at the 1st, 3rd and 5th h after carrageenan injection.
The compound 3a, being not toxic at 600 mg/kg, elicits
quite significant anti-nociceptive effect at the dose of 15
mg/kg, above an average value for MET at the same dose.
This may open an avenue for design of non-toxic structural
CEL analogues via bulky alkyl or aromatic substitution at

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 531
Fig. (6). Changes in rectal temperature of rats. Each point represents the mean values ± SD for 6 rats. Vehicle (control), paracetamol (100
mg/kg) and compound 2a (30 mg/kg) were administered 6 h after the brewer's yeast injection. Temperatures were measured 60, 90 and 270
min after, ΔTrec,% calculated as it is described in the experimental section. *p < 0.05; **p < 0.01; ***p<0.001; ****p<0.0001;
*****p<0.00001 compared with control; §p <0.05 compared with paracetamol.
Pre-treatment of rats with a set of the synthesized
compounds and with MET at the 15 mg/kg dose did not lead
to any statistically valid results with few exceptions (Table
2). The p-nitrophenyl derivative 2c (15 mg/kg) produced
slight pro-inflammatory effect at the 3rd h. The CF3-AP 2a
(15 mg/kg) exhibited anti-inflammatory activity comparable
with such of DIF administered in lower dose (10 mg/kg). At
the same time when 2a was introduced in 10 mg/kg dose it
appeared to be significantly less active than DIF at the same
dose.
Antipyretic property of the leading compound 2a (at the
dose of 30 mg/kg) was assessed in rats with fever induced by
subcutaneous injection of brewer’s yeast. Paracetamol was
used as a reference drug at the dose of 100 mg/kg [33]. The
tested compound 2a significantly reduced the rats rectal
temperature at all time points on the level of paracetamol (p<
0.05 if the data for both samples with control is compared)
(Fig. 6).
The tests which were used for evaluating the compounds
analgesic properties, were based on the animals pain-
avoiding behavioral reactions. The behavioral component
interpretation may influence the outcomes of the nociceptive
threshold evaluation. In order to estimate if the obtained
analgesic activity data had any correlation with to the direct
influence of the leading compound on the animals behavioral
patterns, the open field test in mice was performed [33, 65].
Relatively high doses of AP and propyphenazone (50
mg/kg, subcutaneously) are required in order to achieve at
least mild paw edema inhibition development in the
carrageenan model in mice [14]. For AP given to rats orally
in the same test settings, median ED50 value was 432.9
mg/kg [64]. In this regard the obtained results for 2a (more
than 50% inhibition during nearly three hours after
inflammation induction) look very encouraging and
supporting the chosen direction of the AP structure
optimization.
The obtained data revealed that CF3-AP 2a at the dose of
15 mg/kg does not influence mice locomotion, anxiety and
exploratory behavior. Such result supports the conclusion
that compound 2a increases the pain thresholds in the
applied test systems via true analgesic properties.
Thus, only CF3-AP 2a, being the most active in the hot
plate test, elicit an anti-inflammatory effect. All the
compounds with lower analgesic activity appeared not to be
potent. This data may reflect the fact that even though the
anti-nociceptive and anti-inflammatory properties of
synthesized compounds most probably arise from
modulating different sets of targets, they may partially
overlap.
Thus, CF3-AP 2a shows all of the targeted anti-
inflammatory, analgesic and antipyretic effects are on the
higher level than those of DIF and MET. The antipyretic for
the tested compound seems to be even higher that of
paracetamol.
4.3. Possible Mechanism
4.2.7. Additional CF3-AP 2a Evaluation
Lack of correlation between the in vivo data for
synthesized compounds anti-inflammatory and analgesic
activities may result from the modulating the function of
biological targets distinct sets which are not completely
overlapping for the two types of activity. It seems that the
leading compound 2a may successfully modulate both sets
According to the reported ED50 data, PSBDs are
generally more effective fever reducers than acetylsalicylic
acid and cause ulcerogenic effect at significantly higher
doses than the latter [64]. Consequently, antipyretic activity
would be a valid property for new pyrazolone derivatives.

532 Medicinal Chemistry, 2019, Vol. 15, No. 5
Agafonova et al.
Table 3. Molecular docking results of the pyrazoles 2, 3 into the tyrosine active site of COX-1/2.
COX-1 (PDB code 3N8Y)
ΔGbind (kcal/mol)
COX-2 (PDB code 3LN1)
ID Liganda
|LE|b
ΔGbind (kcal/mol)
|LE|b
ACD
DIF
SC-560
CEL
2a
-11.56
-10.51
-10.45
−
0.52
0.55
0.44
−
-11.47
-10.86
−
0.52
0.57
−
-12.77
-8.78
-7.84
-7.59
-7.81
-8.49
-9.06
-6.31
-9.44
0.49
0.52
0.44
0.38
0.43
0.40
0.53
0.32
0.45
-9.44
-8.85
-7.52
-9.77
-9.25
-8.74
-7.27
-9.72
0.55
0.47
0.38
0.54
0.44
0.46
0.36
0.46
2b
2c
2d
2e
3a
3c
3e
[a] ID ligand correspond to Protein Data Bank; [b] Ligand efficiency (LE = ΔGbind/Nheavy atoms).
exhibiting anti-inflammatory, analgesic and, additionally,
antipyretic effects.
has been proven to provide mixed results for both central and
peripheral analgesics [33]. The anti-nociceptive effects
produced by 2a at the same dose of 15 mg/kg in the three
pain models reflect their mechanisms of action producing
different outcomes on the distinct pain induction and
development paths used in these models, which supports the
“multi-targetness” of this compound. The overall effective
carrageenan induced edema reduction and a clear trend to
produce anti-hyperalgesic activity in the Randal-Sellito test
may imply the the compound 2a or its metabolites
participation in the prostaglandine synthesis inhibition [69,
70].
This is consistent with the fact that the elucidation of
pathways and targets involved in the PSBDs biological
activity is still widely debated. The hypotheses vary from
considering the parent compounds the major biological
effects contributors [12] to almost completely attributing
biological activity to its metabolites [11, 49, 50]. The list of
putative targets which are involved in the pyrazolones
biological effect is expanding as well.
The key prostaglandin synthesis enzymes (COX-1 and
COX-2) inhibition is the main mechanism of action for
traditional NSAIDs. It was believed that inhibition of COX-2
activity may be an important contributor to the PSBDs
biological activity [66]. Further it was discovered that the
their site of action may not involve the the enzyme active
site , as the metabolic products possibly interfere with the
enzyme catalytic activity initiation via radicals scavenging
[10]. In this regard, an edaravone represents an interesting
case since despite that it is structurally one of the AP’s
metabolites (norantipyrine) it may be partially responsible
for the AP biological activity. Edaravone found to be
effective when administered intrathecally to treat inflam-
matory-induced pain in rats but not in case of the heat
induced pain in the tail flick test. Additionally, edaravone
was proven to prevent the development of neuropathic pain
[67, 68].
In order to investigate if the synthesized compounds may
inhibit the COX enzymes, we carried out a molecular
docking into the COX1/COX2 enzymes active sites. Two
cyclooxygenase enzyme isoforms, COX-1 and COX-2 (PDB
[71] codes 3N8Y [72] and 3LN1 [73]), in a complex with
arachidonic acid (ACD) as native ligand and well-known
СОХ inhibitors, DIF, SC-560 [74] and CEL, were used as
the basic biological targets for estimation of tested
compounds action and efficiency/selectivity. The compounds
2a-e, 3a,c,e docking into the COX-1 and COX-2 tyrosine
sites was performed and the estimated binding energy
(ΔGbind) as well as ligand efficiencies of these derivatives
were compared with those of native ligand and COX
inhibitors (Table 3).
The ligands 2a-e, 3a,c,e are located into the tyrosine site
at the top of hydrophobic channel COX-1 near Tyr385 (Fig.
7) and form H-bond bridges with some amino acids and π-π
stacking with aromatic amino acids.
On the list of the putative targets cannabinoid system
enzymes may also be found. Among the latest data there are
evidences that PSBDs block the signaling pathway activated
by oxidative stress by-products by stimulating the TRPA1
channel which is reviled in inflammatory and neuropathic
pain models [12].
The docking scores for all compounds were ranging
between -7.27 to -9.72 units in accordance to results
calculated for COX-1 (Table 3). The obtained values were
higher in comparison to those of ACD and DIF. However,
the 2a,d, 3a,e ligand efficiencies were consistent with the
ones of reference drug DIF (0.55). The docking scores
The hot plate and especially tail flick tests are considered
to be very effective for centrally acting analgesic drugs
efficacy and potency evaluation, while the hyperalgesia test

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines
Medicinal Chemistry, 2019, Vol. 15, No. 5 533
Fig. (7). The location of the compounds 2a,e, 3a,e into the tyrosine binding site of COX-1/2: (For additional figures, please see
Supplementary material).
obtained by 2a-e, 3a,c,e docking into the COX-2 tyrosine
site lie in the interval from -6.31 to -9.06 units. The COX-2
hydrophobic channel is wider than of the COX-1 one, and
ligands are located in the middle of the channel near trans-
membrane domain (Fig. 7).
ation leading to the N-methylpyrazolones and O-methyl-
pyrazoles synthesis as AP and CEL analogs respectively. For
the first time the biological properties of new derivatives
were confirmed in vitro (permeability through the biological
membranes) and in vivo (anti-inflammatory, analgesic
activities, acute toxicity). The trifluoromethyl-AP was found
to be active in a full set of applied testing systems: hot plate,
tail flick, mechanical hiperalgesia (Randall-Selitto),
carrageenan-induced inflammation and yeast-induced
pyrexia. At the same time this compound did not influence
mice locomotor activity, anxiety and exploratory behavior
assessed in the open field test. The trifluoromethyl-AP 2a
represents a valuable starting point in design of the lead
series for new antipyretic analgesics with anti-inflammatory
properties discovery.
Calculation results confirm that COX1/2 are likely not
the primary targets of the synthesized compounds. As a
further step, major metabolites elucidation for 2a would be
required and their synthesis as well as biological activity
evaluated.
CONCLUSION
We have developed the convenient methods for
regioselective 1-aryl-3-polyfluoroalkylpyrazol-5-ols methyl-

534 Medicinal Chemistry, 2019, Vol. 15, No. 5
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PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
Laboratory animals (Sprague Dawley rats and CD-mice)
were obtained from the Animal Unit ‘Pushino’ at the M.M.
Shemyakin and Yu.A. Ovchinnikov from Institute of
Bioorganic Chemistry RAS (Russia). The animals were
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environment, in propylene cages (Bioskape), on standard
bedding (Rehofix MK 2000, J. Rettenmaier&Söhne,
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CONFLICT OF INTEREST
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otherwise.
ACKNOWLEDGEMENTS
This work was supported by the Russian Science
Foundation (Grant No. 16-13-10255).
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