Synthesis, structure-activity relationships, and in vitro antibacterial and antifungal activity evaluations of novel pyrazole carboxylic and dicarboxylic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.03.033

A series of pyrazole-3-carboxylic acid and pyrazole-3,4-dicarboxylic acid derivatives were synthesized, the structures were confirmed by their NMR (¹H and¹³C) and FT-IR spectra, and elemental analyses. The antibacterial and antifungal activities of the compounds against five bacterial and five fungal pathogens were screened using modified agar well diffusion assay. Most of the molecules have inhibitory effects on both standard and clinical Candida albicans strains. However, only the molecules 8, 10, 21, and 22 demonstrate some inhibitory effects on Candida parapsilosis, Candida tropicalis, and Candida glabrata strains. The structure-antifungal activity relationships of the compounds on the C. albicans strains were investigated by electron-conformational method. The pharmacophores and antipharmacophores responsible for the inhibition and non-inhibition of the C. albicans strains were obtained by electronic and geometrical characteristics of the reactive fragments of the molecules. These fragments along with the associated parameters can be used in designing the future more potent antifungal agents. It has been shown that both the positions of electronegative atoms like F and O in the pyrazole substituents and the amount of the associated charges on such atoms are crucial in regulating the strength of antifungal activity for the C. albicans strain.

Full text of DOI:10.1016/j.ejmech.2014.03.033

European Journal of Medicinal Chemistry 78 (2014) 86e96
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structureeactivity relationships, and in vitro antibacterial
and antifungal activity evaluations of novel pyrazole carboxylic and
dicarboxylic acid derivatives
a
a
b
b
c
d,
_
*
ꢀ
ꢀ
Samet Mert , Rahmi Kasımogulları , Tuba Iça , Ferdag Çolak , Ahmet Altun , Salim Ok
a Department of Chemistry, Faculty of Arts and Sciences, Dumlupinar University, 43100 Kutahya, Turkey
b Department of Biology, Faculty of Arts and Sciences, Dumlupinar University, 43100 Kutahya, Turkey
c Department of Physics, Fatih University, 34500 B. Cekmece, Istanbul, Turkey
d School of Earth Sciences, The Ohio State University, 43210 Columbus, OH, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazole-3-carboxylic acid and pyrazole-3,4-dicarboxylic acid derivatives were synthesized,
the structures were confirmed by their NMR (1H and 13C) and FT-IR spectra, and elemental analyses. The
antibacterial and antifungal activities of the compounds against five bacterial and five fungal pathogens
were screened using modified agar well diffusion assay. Most of the molecules have inhibitory effects on
both standard and clinical Candida albicans strains. However, only the molecules 8, 10, 21, and 22
demonstrate some inhibitory effects on Candida parapsilosis, Candida tropicalis, and Candida glabrata
strains. The structureeantifungal activity relationships of the compounds on the C. albicans strains were
investigated by electron-conformational method. The pharmacophores and antipharmacophores
responsible for the inhibition and non-inhibition of the C. albicans strains were obtained by electronic
and geometrical characteristics of the reactive fragments of the molecules. These fragments along with
the associated parameters can be used in designing the future more potent antifungal agents. It has been
shown that both the positions of electronegative atoms like F and O in the pyrazole substituents and the
amount of the associated charges on such atoms are crucial in regulating the strength of antifungal
activity for the C. albicans strain.
Received 11 January 2014
Received in revised form
9 March 2014
Accepted 12 March 2014
Available online 13 March 2014
Keywords:
Antibacterial
Antifungal activity
Pyrazole-3-carboxylic acid
Pyrazole-3,4-dicarboxylic acid
Structureeactivity relationship (SAR)
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Modification of the structural profile by altering the 1-, 3-, or 4-
position substituent in pyrazole ring affects some bioactivities
The synthesis of pyrazole derivatives that contain a five-
membered heterocyclic organic compound with two adjacent ni-
trogen atoms has great interest in agrochemical, pharmaceutical,
and chemical industries [1]. For example, they possess a wide range
of bioactivities [1, 2], including antiviral [3], anti-inflammatory [4],
anticonvulsant [5], anticancer [6], insecticidal [7], and antifungal [8,
9] activities. In recent years, several drugs including patented ones
are developed from the pyrazole derivatives of five-membered ring.
For instance, celecoxib demonstrates anti-inflammation effect and
inhibits COX-2; rimonabant functions as cannabinoid receptor and
is utilized in obesity treatment; fomepizole inhibits alcohol de-
hydrogenase; and sildenafil inhibits phosphodiesterase (Fig. 1).
remarkably [2,10]. The incorporation of trifluoromethyl groups into
organic molecules, including pyrazole derivatives, has a potential to
modify the bioactivities [11e15].
In continuation of our research efforts of the discovery of novel
pyrazole derivatives [16e18], herein we describe synthesis, anti-
bacterial, and antifungal activities of a series of novel pyrazole
carboxylic acid and dicarboxylic acid derivatives. The structuree
activity relationships of the Candida albicans strains have also been
studied in terms of electronic and geometrical characteristics by
using electron-conformational method. This comprehensive
approach of combining the experimental and quantum chemical
studies give a chance to predict and to design further novel
derivatives.
Nitro group is known to lower solubility of compounds. There-
fore, nitro compounds are rarely considered in bioactivity mea-
surements. Since the polar groups present in the novel pyrazole
derivatives counteract the insolubility effect of the nitro group, the
* Corresponding author.
E-mail addresses: ok.12@osu.edu, oksalim@hotmail.com (S. Ok).
http://dx.doi.org/10.1016/j.ejmech.2014.03.033
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
87
2. Results and discussion
2.1. Chemistry
In the current report, the starting compound (1) was synthesized
via the reaction of furandiones with hydrazones by heating in sol-
ventless media [19], and its acid chloride (2) was obtained from the
reaction with SOCl2 [16]. Then a novel pyrazole-3,4-dicarboxylic
acid (3) was prepared from the basic hydrolysis of 1 at a high yield
(88%). IR spectrum of 3 showed a broad absorption band from 2500
to 3500 cmꢁ1 due to OH stretching of eCOOH. The absorption bands
associated with other functional groups appeared in the expected
regions and the absorptionvalues were consistent with our previous
reports and literature [16,19]. Pyrazole 3,4-dicarboxylic acid (3) was
easily converted to its acid chloride (4) reacted with excess SOCl2 in
solventless media again. The method is easy to perform and gives
the product in high yield (82%). Reaction of 1 with anhydrous hy-
drazine led to the formation of a pyrazolo-pyridazine derivative (5)
in about 63% yield (See Scheme 1). In this reaction, -NH2 groups of
hydrazine attacks to carbonyl carbons of 1 and in the second stage
cyclization occurs with the removal of 1 mol water and 1 mol
ethanol. Characteristic NH stretching bands are observed at
3361 cmꢁ1 and 3380 cmꢁ1 for compound 5. Considering the re-
actions and the final products in the current study, the important IR
peaks are CH (aromatic), C]O (amide), NH2, C]C, C]N. The
stretching of aromatic CH groups shows frequencies around
3060 cmꢁ1 [20]. Broad bands of the NH stretching indicate down-
ward wave-numbers [21]. The IR signals of C]C and C]N appear as
a region rather than single sharp peaks. This is explained by several
motions such as in plane vibration of C]N [22].
Fig. 1. Several drug molecules which include pyrazole scaffold.
present pyrazole derivatives with a nitro group become soluble in
DMSO at 25 ꢀC, allowing antifungal and antibacterial activity
measurements of the present molecules.
1H and 13C NMR spectroscopy were employed for clarifying
chemical structures of the derivatives while FT-IR was applied as a
complementary technique for determining their structure via
monitoring the frequencies of the characteristic functional groups
such as (C]O). The molecular weight of the novel molecules were
confirmed by elemental analysis. The bioactivity evaluations were
done by both standard strains and species obtained from patients.
1
The H NMR results depict the successful synthesis of the mol-
ecules. In the 1H NMR spectrum of the compound 5 the peaks
belonging to NH groups are observed as broad singlet at 12.57 ppm.
The compound 6 was obtained from the decarboxylation of 3 at
Scheme 1. The synthetic route of pyrazole carboxylic acid derivatives.

88
S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
elevated temperature. In this reaction, decarboxylation occurs at
the 3 position of the pyrazole ring as previously mentioned in our
studies [23]. Compound 6 also shows a singlet peak in the 1H NMR
spectrum at 8.23 ppm attributed to CH proton at the 3 position of
the pyrazole ring. Then novel pyrazole-3-carboxamides 7e12 and
pyrazole-3,4-dicarboxamides 13e22 were synthesized from the
reaction of acid chloride 2 with the corresponding aniline de-
rivatives. The signals of the aromatic protons appear between
8.25 ppm and 6.80 ppm in the 1H NMR spectra. The aromatic
protons show various multiplicities such as doublets due to several
couplings. The peaks belonging to NH groups are observed in the
region between 12.50 ppm and 10.00 ppm. The down-field shift of
NH signal is attributed to deshielding because of electron with-
drawing ability of the adjacent benzene ring. The 1H resonances of
aliphatic groups such as CH2 and CH3 are observed in the region
between 0.65 ppm and 4.15 ppm. The couplings between these
groups led to multiplicities of the resonances.
NMR spectra are also consistent with similar results in the case of
1H NMR spectra.
2.2. Antibacterial and antifungal activities
In the present contribution, 21 different pyrazole derivatives
were synthesized, the antibacterial and antifungal effects on
various bacteria (both gram positive and negative) and fungi were
analyzed (Table 1). None of the 21 derivatives depicted antimicro-
bial effect on gram negative bacteria (P. aeruginosa, Escherichia coli).
This is attributed to intrinsic resistance of the gram negative bac-
teria. The membrane of the gram negative bacteria functions as a
barrier and prevents penetration of the derivative molecules [25].
In order to figure out the antimicrobial effect of the 21 compounds,
Staphylococcus aureus, MRSA, and E. faecalis strains were utilized.
Some of the derivatives exhibited effect on S. aureus and MRSA. The
21 molecules did not show any effect on the other gram positive
bacteria, E. faecalis.
The species S. aureus cause several infections including endo-
carditis, osteomyelitis, and arthritis. S. aureus is one of the major
reasons of hospital originated nosocomial infections. Methicillin-
resistant S. aureus (MRSA) shows resistance towards both methi-
cillin and oxacilline. Hospital originated MRSAs having potential to
form epidemics are serious threats to public health. Methicillin
resistance found in Staphylococcus, an intrinsic resistance, cover not
In the 13C NMR spectrum of compound 6 only one C]O peak
was observed at 163.52 ppm. Finally, ureide derivatives 23e25
were prepared from the acid dichloride 4 with urea and thiourea
derivatives. The 13C NMR and 1H NMR spectra of ureides (23e25)
are also in full agreement with the proposed structures (See
Experimental section). In 13C NMR spectra characteristic C]O
peaks related to ureas were observed at w153 ppm and C]S peaks
were observed at w178 ppm. The 13C NMR spectra indicate the
signals of aromatic carbons are between 140.00 ppm and
120.00 ppm. The peaks of C]O groups appear closer to
160.00 ppm. All the carbons bonded to N give signals shifted down-
field with respect to aromatic carbons, consistent with literature
[24]. The 13C NMR signals of eCF3 groups appear around
w125.00 ppm while the fluorinated carbon of the benzene ring
gives a downfield signal higher than 150.00 ppm. The 13C NMR
signals of aliphatic groups appear between 45.00 ppm and
12.00 ppm. 13C NMR peaks of aliphatics next to benzene ring are
relatively shifted with respect to their analogues without benzene
in the neighborhood. Such shifts due to benzene rings in the 13C
only all b-lactam antibiotics, but also depict resistance against most
of the antimicrobial agents used in MRSAs [26]. For this reason,
there is need for alternative agents.
Compounds 6 and 23 have MIC value of 50
mg/ml on S. aureus.
The same compounds have MIC values of 25 and 50
mg/ml towards
MRSA, respectively. In order to focus on antifungal effects of the
molecules of interest, the strains of Candida glabrata, C. albicans,
Candida tropicalis, Candida parapsilosis were tested. Some of the
molecules have remarkable effect on C. albicans. C. albicans are
found as commensal species in various organs of human beings and
animals including oral cavity, intestinal tracts, and vaginal cavity.
Table 1
Minimum inhibitory concentration of strains used in agar well diffusion methods.
Compound
Minimum inhibitory concentration (MIC)
Gram negative bacteria
m
g/mLa
Gram positive bacteria
Fungi
Pa
Ec
Es
Sa
MRSA
Cg
Ca*
Ca**
Ct
Cp
5
6
7
8
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
50
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
50
e
e
e
25
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
50
e
e
e
e
e
e
e
50
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
50
25
50
25
25
25
25
50
25
50
50
25
50
25
e
e
e
e
50
e
e
e
e
e
e
e
e
e
e
e
e
e
50
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
50
25
e
e
e
e
50
50
25
50
50
50
50
e
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
e
e
e
e
e
e
25
25
25
25
25
25
25
25
25
25
25
a
Pa: P. aeruginosa, Ec: E. coli (Agricultural Research Center Culture Collection), Es: Enterococcus spp. (Vancomycin resistance enterococ), Sa: S. aureus, MRSA: Methicillin
resistance S. aureus, Cg: C. glabrata, Ca*: C. albicans ESOGÜ (Eskisehir Osmangazi University), Ca**: C. albicans (Agricultural Research Center Culture Collection), Ct: C. tropicalis,
Cp: C. parapsilosis.

S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
89
this purpose, we utilized the semi-empirical PM5 quantum
chemistry method [39]. All the energetically accessible con-
formers of each compound are included in the ECM study as
separate compounds since the molecules may not be found
in their lowest energy conformer while interacting with the
bio-receptor.
Moreover, C. albicans may cause diseases in human beings with
weak immune system, having cytotoxic therapy, using antibiotics
with broad spectrum [27].
The random usage of antifungals for medical treatment and
prolific purposes caused various resistance developments in some
fungi species. Some C. albicans species may also show resistance
towards antifungal drugs. Candida resistance arise from either
mutations in the genes responsible for the synthesis of ERGH en-
zymes affected by the drugs or the extreme expression of CDR1,
CDR2, MDR1 type efflux pumping genes. Due to those difficulties
and problems in C. albicans infection treatments, there is need for
alternative medical treatments [28].
(b) Each compound is expressed as a square matrix called
electron-conformational matrices of contiguity (ECMC).
ECMC is symmetric with respect to the diagonal elements.
Hence, we only demonstrate the upper part of ECMC in this
contribution. The diagonal elements of ECMC are chosen
from electronic atomic characteristics of each atom in a
molecule. In this current report, the diagonal elements are
the atomic charges (Qi). The non-diagonal elements of ECMC
are of two kinds, one of which is for chemical bonds and the
other one is for chemically non-bonded atoms. Bond orders
(Bij) and interatomic distances (Rij) are utilized here for the
chemically bonded and non-bonded atoms, respectively.
(c) ECMC of one of the active compounds is chosen as a tem-
plate. Each submatrix of the selected ECMC is compared with
the each submatrix of the rest of ECMCs within some flexi-
bility limits to reveal the submatrix present in the ECMCs of
all active compounds but absent in the ECMCs of all the
inactive ones. The found submatrix is called as electron-
conformational submatrix of contiguity (ECSC) and corre-
sponds to the activity feature, in other words, to the phar-
macophore of the investigated bioactivity. When the ECMC
of one of the inactive or weakly active compounds is chosen
as a template, inactivity or weak activity features can also be
extracted.
In the current study, all the molecules (excluding 19) showed
inhibition effect on the standard C. albicans strain (See Table 1). The
C. albicans’ values towards the molecules of interest are within the
range of 25e50 mg/ml. The molecules 5, 6, 14e20 did not exhibit
inhibition on the clinical isolate of C. albicans. The susceptibility
differences between the strains may be based on the origin of yeast.
The clinical isolates were obtained from a hospitalized patient at
the Eskis¸ ehir Osmangazi University hospital, while standard strain
was taken from culture collection. The molecules 7, 8, 10e13 have
MIC values of 50
MIC values of 25
m
g/ml, while the other molecules 9, 21e25 have
mg/ml. C. parapsilosis, following C. albicans, is the
second most isolated yeast from blood cultures in the hospitals
[29]. C. parapsilosis causes the invasive candida infection. The
infection caused by C. parapsilosis is related to prostatic tool and
catheter in addition to nosocomial dispersion [29]. The MIC values
of C. parapsilosis in the present study two compounds 21 and 22 are
50 and 25
molecules 8 and 22 showed antimicrobial effects with MIC value of
50 g/ml. In inhibiting C. glabrata, only molecule 10 had the effect
with MIC value of 50 g/ml. The current results are compared to the
results obtained by the antibacterial and antifungal agents. Cefo-
taxime (CTX; 30 g/disc) for bacteria and nystatin (100 U/disc) for
mg/ml, respectively. In the case of C. tropicalis strain, only
m
In searching ECSC, only three flexibility limits are used initially:
one for diagonal elements, one for bond parameters, and one for
interatomic distances since each matrix contains huge number of
elements [n$(n þ 1)/2, where n is the number of atoms in the
molecule], and entering different limits for each parameter is
impractical. After revealing the ECSC on a molecule, the flexibility
range of its each element is determined finely. The molecule-in
parameters in the ECSC are not in the middle of their flexibility
ranges in most cases. As we express the parameters and their limits
relative to the middle of the flexibility ranges, the features given in
this study are template independent. However, we demonstrate the
ECSCs on some selected compounds to relate them with the mo-
lecular fragments.
m
m
yeast were used as positive control agents. Cefotaxime was not
tested against P. aeroginosa and Enterococcus sp. Cefotaxime did not
show activity against MRSA while it exhibited antimicrobial activity
against S. aureus (35 mm) and E. coli (45 mm). Nystatin depicted
inhibition against C. albicans, C. tropicalis, C. parapsilosis on 19 mm
diameter while against C. glabrata on 24 mm.
The molecules of the current report have the highest inhibition
effect on the standard and clinical isolates of C. albicans. The com-
pounds reported herein are promising candidates for further in-
vestigations infinding inhibitors of various bacteria and fungi species.
The number of molecules demonstrating strong, weak and no
inhibition effect on Ca* (Table 1) is six (9, and 21e25), six (7, 8, and
10e13), and nine (5, 6, and 14e20), respectively. The feature that is
found in the strongly inhibiting molecules and absent in the mole-
cules demonstrating weak and no inhibition effects (SIF*) is
considered responsible for demonstrating strong inhibitory effect
on Ca* and shown in Fig. 2(a). This feature is formed by four atoms,
two of which are chemically bonded. The molecular fragment
constituting SIF* is different in 9 from the other strongly inhibiting
molecules (21e25) (Fig. 2(b) and (c)). In the fluorinated molecules
like 9, although it is essential to have a CF3 moiety at the para po-
sition for the strong inhibitory effect, this is not alone enough. The
other parameters entering SIF* must also associate to the molecules
within the given limits in SIF*. For example, CH3 moiety of the e
(CO)eOCH2CH3 substituent in 9 enters SIF*. When this substituent is
replaced by a fluorinated carboxylic acid as in 15, the molecule does
not include SIF* anymore and thus becomes none inhibitory. In 21e
25, when the R0 and X substituents (Scheme 1) include N, O or S
atom, the parameters associated to the atom G of SIF* are satisfied
and thus they inhibit Ca* strongly (Fig. 2(c)). 13e20 is weakly or
none inhibitory as they contain CH3 and F as the R0 substituent.
2.3. Structureeantifungal activity relations
The present molecules demonstrate antifungal activities at
different strengths (Table 1) on both the clinical isolate of C. albicans
(Ca*) and the standard C. albicans (Ca**) strain. In this study, the
structureeactivity relationships (SARs) are investigated by using
electron-conformational method (ECM) known also as electron-
topological method (ETM) [30e38]. ECM deals with the fine de-
tails of the molecules, i.e., geometrical as well as electronic pa-
rameters of each atom and bond in a molecule, rather than global
characteristics, and thus extracts more information compared with
the majority of the other SAR methods. Classical SAR methods can
investigate the compounds that are structurally alike. However,
ECM can handle diverse molecular structures simultaneously since
each atom and each bond in the molecules are represented by
numbers rather than atomic types. The main steps of ECM are as
follows.
(a) The molecular structure of each compound is determined by
geometry optimizations and conformational analyses. For

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S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
Fig. 2. Strongly inhibiting feature of Ca* (SIF*) and its realization on 9 and 23. Diagonal elements are atomic charges (Q) whereas non-diagonal elements are either bond orders (B,
italic numbers) or interatomic distances (R, plain numbers).
The feature that is found in the weakly inhibiting molecules and
absent in the molecules demonstrating strong and no inhibition
effects (WIF*) is considered responsible for demonstrating weak
inhibitory effect on Ca* and shown in Fig. 3(a). This property is
formed by three chemically non-bonded atoms. It is realized when
the eCF3 moiety is at the ortho (7 or 13, Fig. 3(b)) or meta (8)
position rather than the para (9) position unlike the strongly
inhibiting molecules. The feature also enters the molecules in the
presence of F atom (rather than eCF3) on the benzene ring irre-
spective of its position (10e12).
The feature that is found in the none-inhibitory molecules and
absent in the molecules demonstrating strong and weak inhibition
effects (NIF*) is considered responsible for breaking the inhibitory
effect on Ca* and shown in Fig. 4(a). This feature is formed by three
chemically non-bonded atoms. Especially, the parameters of NH or
OH moiety attached to the >C]O group (see 5 in Fig. 4(b)) are very
crucial for the inhibitory activity demonstration. Although all the
molecules of interest have such a NH or OH moiety, they become
none inhibitory only if the associating parameters are within the
values given in Fig. 4(a).
The number of molecules demonstrating strong, weak and no
inhibition effect on Ca** (Table 1) is fourteen (6, 8e11, 13, 16, 18, and
20e25), six (5, 7, 12, 14, 15, and 17), and one (19), respectively. As
there is only one none inhibitory compound on Ca**, the feature
responsible for breaking the antifungal activity cannot be deter-
mined with the present molecule set. The presence of several
related none inhibitory pyrazole derivatives for the Ca** strain is
necessary for obtaining more conclusive structureeactivity
relationships.
The feature that is found in the strongly inhibiting molecules
and absent in the molecules demonstrating weak and no inhibition
effects (SIF**) is considered responsible for demonstrating strong
inhibitory effect on Ca** and shown in Fig. 5(a). This feature is
formed by four chemically non-bonded atoms. In all compounds,
the atoms entering SIF** (shown in Fig. 5(b) on 9) are common and
their coordinates are very similar to each other. However, their
charges are very sensitive to the type of substituent attached to the
>C]O moieties. Therefore, the strong inhibition effect on Ca** arise
fully from electronic structure of the atoms in the feature and it is
difficult to assess if a molecule inhibits Ca** strongly without per-
forming any quantum chemical calculation by just substituent-
based analysis.
Fig. 3. Weakly inhibiting feature of Ca* (WIF*) and its realization on 7.

S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
91
Fig. 4. None inhibiting feature of Ca* (NIF*) that breaks antifungal activity and its
realization on 5.
Fig. 6. Weakly inhibiting feature of Ca** (WIF**) and its realization on 5.
The feature found in the weakly inhibiting molecules and absent
3. Experimental protocols
in the molecules demonstrating strong and no inhibition effects
(WIF**) is considered responsible for demonstrating weak inhibi-
tory effect on Ca** and shown in Fig. 6(a). This property is estab-
lished by three chemically non-bonded atoms. As in none
inhibitory feature on Ca*, the parameters of NH or OH moiety
attached to the >C]O group (see 5 in Fig. 6(b)) appear to be crucial
for demonstrating the weak inhibitory activity on Ca**.
3.1. Materials and methods
Some aniline derivatives was distilled (m-toluidine) or recrys-
tallized (p-toluidine, m-nitroaniline and p-nitroaniline) before use;
all other reagents are commercially available and were used
without further purification. The solvents used were of analytical
grade. Melting points were determined in open glass capillaries
using Barnstead Electrothermal 9200 melting point apparatus
(Electrothermal Co, Essex, UK) and are uncorrected. Infrared
spectra (IR) were recorded on Bruker Optics, Vertex 70 Fourier
Transform Infrared Spectrometer (FT-IR) equipped with an ATR
(Attenuated Total Reflection) device and the data are reported in
reciprocal centimeters (cmꢁ1) (Bruker Optik GmbH, Ettlingen,
Germany). 1H NMR and 13C NMR spectra were scanned on Jeol-
500 MHz spectrometer (Jeol, Tokyo, Japan) using tetramethylsilane
(TMS) as internal standard and using one or two of the following
solvents, DMSO-d6 and CDCl3. Chemical shifts are given in d, ppm.
Splitting patterns were designated as follows: s: singlet; d: doublet;
t: triplet; q: quartet; m: multiplet. Elemental analyses (C, H, and N)
were performed on a Leco CHNS-932 elemental analyser (LECO
Corporation, Saint Joseph, Michigan, USA). Follow up of the re-
actions and checking the purity of the compounds was made by
thin layer chromatography (TLC) on silicagel precoated aluminum
sheets (Kieselgel 60F 254 of E. Merck, Darmstadt, Germany).
Compounds were visualized by Camag TLC devices (Camag, Upland,
CA, USA) UV (254 and 366 nm). Compounds 1 and 2 were prepared
following previously published reaction conditions [16,19].
3.2. Synthesis
3.2.1. 1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylic
acid (3)
A solution of 1 (0.381 g, 1 mmol) and sodium hydroxide (0.1 g,
Fig. 5. Strongly inhibiting feature of Ca** (SIF**) and its realization on 9.
2.5 mmol) in 30 mL water was refluxed at 100 ꢀC for 3 h. After

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S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
solution was cooled down to room temperature it was stirred for a
while by adding HCl solution (1.5 mL d. HCl in 20 mL water). The
white precipitate was filtered and washed with water again. The
residual solid was crystallized in EtOH/H2O (1:3) mixture to afford
title compound 3 as white needles. Yield: 88%; m.p.: 207e208 ꢀC; IR
Mixture was refluxed at 140 ꢀC 3 h and the solvent was evaporated
under vacuum. The crude product so obtained was washed with
water and recrystallized from an appropriate solvent.
3.2.6. Ethyl 1-(3-nitrophenyl)-5-phenyl-3-((2-(trifluoromethyl)
phenyl)carbamoyl)-1H-pyrazole-4-carboxylate (7)
(n
, cmꢁ1): 3224e2474 (OH, COOH), 3069 (CH, aromatic), 1707 (C]
O, acid),1607e1427 (C]C and C]N); 1H NMR (500 MHz, DMSO-d6)
dH (ppm): 8.21-7.34 (m, 9H, ArH); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 164.07 and 163.30 (C]O, acid), 147.94 (CeNO2), 145.19,
(pyrazole C-3), 145.06 (pyrazole C-5), 120.70 (pyrazole C-4), 139.36,
131.98, 130.84, 130.46, 129.94, 128.69, 127.92, 123.54, 116.39. Anal.
calcd. for C17H11N3O6 (353.29 g/mol): C, 57.80; H, 3.14; N, 11.89.
Found: C, 57.68; H, 3.17; N, 11.85.
Synthesized from 2 (0.4 g, 1 mmol) and 2-(trifluoromethyl)
aniline (0.262 ml, 2 mmol) according to the general procedure. The
crude product was purified by crystallization from methanol. Yield:
89%; m.p.: 161e162 ꢀC; IR ( , cmꢁ1): 3415 and 3126 (NH), 3100 (CH,
n
aromatic), 2990 (CH, aliphatic), 1696 (C]O, amide), 1592e1451
(C]C and C]N), 1350 (NeO sym.), 1248 and 1067 (CeOeC asym.
and sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.43 (s, 1H,
NH), 8.27e7.42 (m, 13H, ArH), 4.13 (q, J ¼ 7.1 Hz, 2H, OCH2), 1.06 (t,
J ¼ 7.1 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) dC (ppm): 162.32
(C]O, ester), 160.32 (C]O, amide), 147.76 (CeNO2), 146.86 (pyr-
azole C-3), 145.35 (pyrazole C-5), 125.15 (CF3), 120.62 (pyrazole C-
4), 60.66 (OCH2), 13.61 (CH3), 139.01, 134.88, 133.22, 131.77, 130.57,
130.18, 129.77, 129.63, 128.46, 127.42, 127.12, 126.52, 124.93, 123.38,
114.37. Anal. calcd. for C26H19F3N4O5 (524.45 g/mol): C, 59.54; H,
3.65; N, 10.68. Found: C, 59.48; H, 3.69; N, 10.69.
3.2.2. 1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarbonyl
dichloride (4)
3 (0.353 g, 1 mmol) and excessive amount of thionyl chloride
(SOCl2) was heated in 80 ꢀC for 5 h. After the completion of the
chlorination, excess SOCl2 were evaporated. Finally, the crude
product was recrystallized from a mixture of ether/hexane (5:1).
Yield: 82%; m.p.: 58e60 ꢀC; IR ( , cmꢁ1): 3084 (CH, aromatic), 1754
n
(C]O, acyl), 1623e1451 (C]C and C]N). 1H NMR (500 MHz,
DMSO-d6) dH (ppm): 8.21e7.33 (m, 9H, ArH); 13C NMR (125 MHz,
DMSO-d6) dC (ppm): 164.04 and 163.26 (C]O, acyl), 147.91 (Ce
NO2), 144.94 (pyrazole C-3), 144.84 (pyrazole C-5), 120.66 (pyrazole
C-4), 139.27, 131.95, 130.84, 130.38, 129.96, 128.70, 127.73, 123.54,
116.33. Anal. calcd. for C17H9Cl2N3O4 (390.18 g/mol): C, 52.33; H,
2.32; N, 10.77. Found: C, 52.24; H, 2.38; N, 10.81.
3.2.7. Ethyl 1-(3-nitrophenyl)-5-phenyl-3-((3-(trifluoromethyl)
phenyl)carbamoyl)-1H-pyrazole-4-carboxylate (8)
Synthesized from 2 (0.4 g, 1 mmol) and 3-(trifluoromethyl)
aniline (0.276 ml, 2 mmol) according to the general procedure. The
crude product was purified by crystallization from methanol.
Yield: 92%; m.p.: 144e145 ꢀC; IR ( , cmꢁ1): 3281 and 3216 (NH),
n
3045 (CH, aromatic), 2992 (CH, aliphatic), 1693 and 1660 (C]O,
amide), 1605e1448 (C]C and C]N), 1341 (NeO sym.), 1247 and
1069 (CeOeC asym. and sym.); 1H NMR (500 MHz, DMSO-d6) dH
(ppm): 11.06 (s, 1H, NH), 8.27e7.39 (m, 13H, ArH), 4.10 (q,
J ¼ 7.1 Hz, 2H, OCH2), 1.00 (t, J ¼ 7.1 Hz, 3H, CH3); 13C NMR
(125 MHz, DMSO-d6) dC (ppm): 161.71 (C]O, ester), 160.21 (C]O,
amide), 147.96 (CeNO2), 147.75 (pyrazole C-3), 145.47 (pyrazole C-
5), 125.43 (CF3), 120.50 (pyrazole C-4), 60.46 (OCH2), 13.57 (CH3),
139.40, 138.92, 131.73, 130.62, 130.22, 130.10, 129.82, 129.65,
129.33, 128.46, 127.31, 123.39, 120.29, 115.84, 113.66. Anal. calcd.
for C26H19F3N4O5 (524.45 g/mol): C, 59.54; H, 3.65; N, 10.68.
Found: C, 59.45; H, 3.68; N, 10.66.
3.2.3. 2-(3-nitrophenyl)-3-phenyl-5,6-dihydro-2H-pyrazolo[3,4-d]
pyridazine-4,7-dione (5)
0.381 g (1 mmol) compound 1 was dissolved in 10 ml dry
toluene, and anhydrous hydrazine was added at 1/1 mol rate. The
mixture was refluxed at 110 ꢀC about 5 h. Precipitate yellow product
was filtered and purified from ethanol/H2O (2:1) mixture by crys-
tallization. Yield: 63%; m.p.: 307e308 ꢀC; IR ( , cmꢁ1): 3317 and
n
3189 (NH), 3056 (CH, aromatic), 1661 (C]O), 1607e1441 (C]C and
C]N), 1346 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm):
12.57 (br, s, 2H, 2NH), 8.30e7.19 (m, 9H, ArH); 13C NMR (125 MHz,
DMSO-d6) dC (ppm): 164.74 and 161.37 (C]O, pyridazine dione),
149.36 (CeNO2), 147.61 (pyrazole C-3),145.01 (pyrazole C-5), 121.28
(pyrazole C-4), 139.82, 132.44, 130.71, 130.06, 128.20, 127.92, 127.74,
122.34, 119.98. Anal. calcd. for C17H11N5O4 (349.30 g/mol): C, 58.45;
H, 3.17; N, 18.32. Found: C, 58.38; H, 3.18; N, 18.35.
3.2.8. Ethyl 1-(3-nitrophenyl)-5-phenyl-3-((4-(trifluoromethyl)
phenyl)carbamoyl)-1H-pyrazole-4-carboxylate (9)
Synthesized from 2 (0.4 g, 1 mmol) and 4-(trifluoromethyl)an-
iline (0.256 ml, 2 mmol) according to the general procedure. The
crude product was purified by crystallization from methanol. Yield:
3.2.4. 1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-4-carboxylic acid
(6)
84%; m.p.: 174e175 ꢀC; IR ( , cmꢁ1): 3267 and 3197 (NH), 3057 (CH,
n
0.353 g (1 mmol) pyrazole-3,4 dicarboxylic acid (3) was heated
at 250 ꢀC until gas exiting finished. Solid at the bottom was washed
with ether and water, respectively. The crude product was purified
aromatic), 2935 (CH, aliphatic), 1696 and 1667 (C]O, amide),
1606e1448 (C]C and C]N), 1320 (NeO sym.), 1213 and 1062 (Ce
OeC asym. and sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm):
11.09 (s, 1H, NH), 8.27e7.39 (m, 13H, ArH), 4.10 (q, J ¼ 7.1 Hz, 2H,
OCH2), 1.00 (t, J ¼ 7.1 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 161.68 (C]O, ester), 160.29 (C]O, amide), 148.11 (CeNO2),
147.75 (pyrazole C-3), 145.51 (pyrazole C-5), 123.72 (CF3), 120.49
(pyrazole C-4), 60.45 (OCH2), 13.60 (CH3), 142.22, 138.92, 131.73,
130.63, 130.23, 129.82, 128.46, 127.31, 126.15, 126.11, 123.39, 119.71,
113.58. Anal. calcd. for C26H19F3N4O5 (524.45 g/mol): C, 59.54; H,
3.65; N, 10.68. Found: C, 59.42; H, 3.63; N, 10.72.
from xylene by crystallization. Yield: 70%; m.p.: 203e204 ꢀC; IR (
n,
cmꢁ1): 3100e2550 (OH, COOH), 1675 (C]O, acid), 1608e1444 (C]
C and C]N), 1346 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH
(ppm): 8.23 (s, 1H, pyrazole CH]N), 8.18e6.94 (m, 9H, ArH); 13C
NMR (125 MHz, DMSO-d6) dC (ppm): 163.52 (C]O, acid),147.77 (Ce
NO2), 145.58 (pyrazole C-3), 143.05 (pyrazole C-5), 120.16 (pyrazole
C-4), 139.73, 131.52, 130.59, 129.49, 128.90, 128.24, 126.07, 122.81,
114.95. Anal. calcd. for C16H11N3O4 (309.28 g/mol): C, 52.14; H, 3.58;
N, 13.59. Found: C, 52.05; H, 3.63; N, 13.60.
3.2.9. Ethyl 3-((2-fluorophenyl)carbamoyl)-1-(3-nitrophenyl)-5-
phenyl-1H-pyrazole-4-carboxylate (10)
3.2.5. General procedure for the synthesis of pyrazole-3-
carboxamides and pyrazole-3,4-dicarboxamides (7e22)
Synthesized from 2 (0.4 g, 1 mmol) and 2-fluoroaniline
1 mmol 2 or 4 was dissolved in approximately 10 mL of dry
xylene and for pyrazole-3-carboxamides 2 mmol, and for pyrazole-
3,4-dicarboxamides 4 mmol aryl amine was added to this solution.
(0.214 ml, 2 mmol) according to the general procedure. The crude
product was purified by crystallization from toluene. Yield: 90%;
m.p.: 141e142 ꢀC; IR ( , cmꢁ1): 3295 and 3186 (NH), 3054 (CH,
n

S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
93
aromatic), 2979 (CH, aliphatic), 1669 (C]O, amide), 1616e1451
3.2.13. 1-(3-nitrophenyl)-5-phenyl-N3,N4-bis(3-(trifluoromethyl)
phenyl)-1H-pyrazole-3,4-dicarboxamide (14)
(C]C and C]N), 1347 (NeO sym.), 1214 and 1075 (CeOeC asym.
and sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.56 (s, 1H,
NH), 8.27e7.25 (m, 13H, ArH), 4.12 (q, J ¼ 7.0 Hz, 2H, OCH2), 1.03 (t,
J ¼ 7.1 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) dC (ppm): 162.10
(C]O, ester), 159.74 (C]O, amide), 155.78 (CeF), 153.32 (CeNO2),
147.75 (pyrazole C-3), 147.55 (pyrazole C-5), 120.50 (pyrazole C-4),
60.55 (OCH2), 13.57 (CH3), 145.42, 139.00, 131.69, 130.59, 130.21,
129.76, 128.45, 127.46, 124.40, 123.33, 115.84, 115.65, 113.79. Anal.
calcd. for C25H19FN4O5 (474.44 g/mol): C, 63.29; H, 4.04; N, 11.81.
Found: C, 63.21; H, 4.07; N, 11.80.
Synthesized from 4 (0.39 g, 1 mmol) and 3-(trifluoromethyl)
aniline (0.552 ml, 4 mmol) according to the general procedure. The
crude product was purified by crystallization from toluene. Yield:
73%; m.p.: 210e211 ꢀC; IR ( , cmꢁ1): 3371 (NH), 3073 (CH, aro-
n
matic), 1683 and 1660 (C]O, amide), 1601e1447 (C]C and C]N),
1356 (NeO sym.); 1H NMR (500 MHz, DMSO-d6/CDCl3) dH (ppm):
11.88 and 10.56 (s, 2H, 2NH), 8.23e7.23 (m, 17H, ArH); 13C NMR
(125 MHz, DMSO-d6/CDCl3) dC (ppm): 161.52 and 159.94 (C]O,
amide), 148.14 (CeNO2), 147.65 (pyrazole C-3), 143.46 (pyrazole C-
5), 125.10 and 125.06 (CF3), 120.37 (pyrazole C-4), 139.45, 139.30,
138.36, 131.15, 130.37, 130.17, 129.78, 129.61, 129.47, 128.64, 128.31,
124.59, 123.19, 123.13, 122.94, 122.90, 121.16, 121.13, 120.12, 120.08,
118.85. Anal. calcd. for C31H19F6N5O4 (639.50 g/mol): C, 58.22; H,
2.99; N, 10.95. Found: C, 58.11; H, 3.02; N, 10.97.
3.2.10. Ethyl 3-((3-fluorophenyl) carbamoyl)-1-(3-nitrophenyl)-5-
phenyl-1H-pyrazole-4-carboxylate (11)
Synthesized from
2 (0.4 g, 1 mmol) and 3-fluoroaniline
(0.149 ml, 2 mmol) according to the general procedure. The crude
product was purified by crystallization from toluene. Yield: 95%;
m.p.: 154e155 ꢀC; IR (
n
, cmꢁ1): 3275 and 3214 (NH), 3032 (CH,
3.2.14. 1-(3-nitrophenyl)-5-phenyl-N3,N4-bis(4-(trifluoromethyl)
phenyl)-1H-pyrazole-3,4-dicarboxamide (15)
aromatic), 2992 (CH, aliphatic), 1670 (C]O, amide), 1607e1446
(C]C and C]N), 1331 (NeO sym.), 1221 and 1086 (CeOeC asym.
and sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.93 (s, 1H,
NH), 8.27e6.95 (m, 13H, ArH), 4.11 (q, J ¼ 7.1 Hz, 2H, OCH2), 1.01 (t,
J ¼ 7.1 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) dC (ppm): 163.26
(CeF), 161.70 (C]O, ester), 160.03 (C]O, amide), 148.19 (CeNO2),
147.74 (pyrazole C-3), 145.45 (pyrazole C-5), 120.47 (pyrazole C-4),
60.43 (OCH2), 13.60 (CH3), 140.41, 140.30, 138.92, 131.72, 130.63,
130.52, 130.42, 130.21, 129.80, 128.46, 127.33, 123.37, 115.54, 113.57.
Anal. calcd. for C25H19FN4O5 (474.44 g/mol): C, 63.29; H, 4.04; N,
11.81. Found: C, 63.18; H, 4.08; N, 11.85.
Synthesized from 4 (0.39 g, 1 mmol) and 4-(trifluoromethyl)
aniline (0.512 ml, 4 mmol) according to the general procedure. The
crude product was purified by crystallization from toluene. Yield:
84%; m.p.: 218e219 ꢀC; IR ( , cmꢁ1): 3342 (NH), 3006 (CH, aro-
n
matic), 1671 (C]O, amide), 1607e1447 (C]C and C]N), 1346 (Ne
O sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 11.10 and 10.94 (s,
2H, 2NH), 8.31e7.17 (m,17H, ArH); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 161.38 and 160.22 (C]O, amide), 148.25 (CeNO2), 144.66
(pyrazole C-3), 144.56 (pyrazole C-5), 125.75 and 125.65 (CF3),
120.58 (pyrazole C-4), 142.86, 142.29, 139.36, 132.09, 131.08, 130.15,
129.24,129.12, 128.55, 127.70, 126.45, 126.27, 123.88, 123.59, 120.98,
120.85, 119.53. Anal. calcd. for C31H19F6N5O4 (639.50 g/mol): C,
58.22; H, 2.99; N, 10.95. Found: C, 58.16; H, 3.04; N, 10.98.
3.2.11. Ethyl 3-((4-fluorophenyl)carbamoyl)-1-(3-nitrophenyl)-5-
phenyl-1H-pyrazole-4-carboxylate (12)
Synthesized from
(0.198 ml, 2 mmol) according to the general procedure. The crude
2 (0.4 g, 1 mmol) and 4-fluoroaniline
3.2.15. N3,N4-bis(2-fluorophenyl)-1-(3-nitrophenyl)-5-phenyl-1H-
pyrazole-3,4-dicarboxa-mide (16)
product was purified by crystallization from toluene. Yield: 88%;
m.p.: 144e145 ꢀC; IR (
n
, cmꢁ1): 3276 and 3216 (NH), 3061 (CH,
Synthesized from 4 (0.39 g, 1 mmol) and 2-fluoroaniline
(0.428 ml, 4 mmol) according to the general procedure. The crude
product was purified by crystallization from toluene. Yield: 78%;
aromatic), 2970 (CH, aliphatic), 1674 (C]O, amide), 1625e1448
(C]C and C]N), 1341 (NeO sym.), 1250 and 1081 (CeOeC asym.
and sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.77 (s, 1H,
NH), 8.27e7.20 (m, 13H, ArH), 4.10 (q, J ¼ 7.1 Hz, 2H, OCH2), 1.01 (t,
J ¼ 7.1 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) dC (ppm): 161.79
(C]O, ester), 159.66 (C]O, amide), 157.14 (CeF), 148.40 (CeNO2),
147.75 (pyrazole C-3), 145.35 (pyrazole C-5), 120.43 (pyrazole C-4),
60.41 (OCH2), 13.62 (CH3), 138.96, 135.07, 131.68, 130.61, 130.20,
129.78, 128.46, 127.39, 123.31, 121.63, 121.55, 115.48, 115.26, 113.58.
Anal. calcd. for C25H19FN4O5 (474.44 g/mol): C, 63.29; H, 4.04; N,
11.81. Found: C, 63.19; H, 4.09; N, 11.87.
m.p.: 213e215 ꢀC; IR ( , cmꢁ1): 3371 (NH), 3061 (CH, aromatic),
n
1660 (C]O, amide), 1614e1452 (C]C and C]N), 1347 (NeO sym.);
1H NMR (500 MHz, DMSO-d6) dH (ppm): 11.31 and 10.51 (s, 2H,
2NH), 8.31e7.12 (m, 17H, ArH); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 164.99 and 164.66 (CeF), 160.85 and 160.12 (C]O, amide),
147.94 (CeNO2), 146.67 (pyrazole C-3), 143.21 (pyrazole C-5),
120.79 (pyrazole C-4), 139.18, 131.95, 130.70, 130.30, 129.62, 128.53,
128.38, 127.04, 126.35, 124.65, 124.63, 124.48, 123.61, 123.54, 118.79,
116.12, 115.96, 115.58, 115.42. Anal. calcd. for
C29H19F2N5O4
(539.49 g/mol): C, 58.22; H, 2.99; N, 10.95. Found: C, 58.15; H, 3.01;
N, 10.97.
3.2.12. 1-(3-nitrophenyl)-5-phenyl-N3,N4-bis(2-(trifluoromethyl)
phenyl)-1H-pyrazole-3,4-dicarboxamide (13)
Synthesized from 4 (0.39 g, 1 mmol) and 2-(trifluoromethyl)
aniline (0.524 ml, 4 mmol) according to the general procedure. The
crude product was purified by crystallization from methanol. Yield:
3.2.16. N3,N4-bis(3-fluorophenyl)-1-(3-nitrophenyl)-5-phenyl-1H-
pyrazole-3,4-dicarboxa-mide (17)
Synthesized from 4 (0.39 g, 1 mmol) and 3-fluoroaniline
(0.298 ml, 4 mmol) according to the general procedure. The crude
product was purified by crystallization from toluene. Yield: 86%;
65%; m.p.: 164e165 ꢀC; IR ( , cmꢁ1): 3380 (NH), 3036 (CH, aro-
n
matic), 1695 and 1662 (C]O, amide), 1591e1453 (C]C and C]N),
1348 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 11.09
and 10.47 (s, 2H, 2NH), 8.33e7.40 (m, 17H, ArH); 13C NMR
(125 MHz, DMSO-d6) dC (ppm): 161.31 and 160.94 (C]O, amide),
147.83 (CeNO2),146.43 (pyrazole C-3),143.10 (pyrazole C-5),126.09
and 126.05 (CF3), 120.68 (pyrazole C-4), 139.05, 135.13, 134.68,
133.39, 132.87, 131.79, 130.57, 130.13, 129.57, 129.48, 129.45, 128.31,
128.08, 127.39, 126.63, 126.59, 126.48, 123.49,122.57, 122.36, 118.58.
Anal. calcd. for C31H19F6N5O4 (639.50 g/mol): C, 58.22; H, 2.99; N,
10.95. Found: C, 58.15; H, 3.04; N, 10.95.
m.p.: 228e229 ꢀC; IR ( , cmꢁ1): 3355 and 3199 (NH), 3058 (CH,
n
aromatic), 1682 and 1656 (C]O, amide), 1611e1445 (C]C and C]
N),1349 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.98
and 10.77 (s, 2H, 2NH), 8.29e6.89 (m, 17H, ArH); 13C NMR
(125 MHz, DMSO-d6) dC (ppm): 161.17 and 161.05 (CeF), 160.78 and
159.74 (C]O, amide),147.89 (CeNO2), 144.29 (pyrazole C-3),144.24
(pyrazole C-5), 120.49 (pyrazole C-4), 139.04, 131.75, 130.72, 130.48,
130.41, 130.36, 130.29, 129.83, 129.74, 128.90, 128.75, 128.21, 127.46,
123.49, 120.21, 116.47, 116.46, 115.06, 115.04. Anal. calcd. for

94
S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
C29H19F2N5O4 (539.49 g/mol): C, 58.22; H, 2.99; N, 10.95. Found: C,
58.12; H, 3.03; N, 10.93.
(CeNO2), 144.07 (pyrazole C-3), 140.04 (pyrazole C-5), 120.49
(pyrazole C-4), 139.51, 138.97, 131.73, 130.74, 130.28, 130.11, 129.79,
128.81, 128.42, 127.21, 126.61, 125.19, 123.55, 120.10, 118.55, 118.15,
114.77, 113.20. Anal. calcd. for C29H19N7O8 (593.50 g/mol): C, 58.69;
H, 3.23; N, 16.52. Found: C, 58.57; H, 3.19; N, 16.52.
3.2.17. N3,N4-bis(4-fluorophenyl)-1-(3-nitrophenyl)-5-phenyl-1H-
pyrazole-3,4-dicarboxa-mide (18)
Synthesized from 4 (0.39 g, 1 mmol) and 4-fluoroaniline
(0.396 ml, 4 mmol) according to the general procedure. The crude
product was purified by crystallization from toluene. Yield: 74%;
3.2.21. 1-(3-nitrophenyl)-N3,N4-bis(4-nitrophenyl)-5-phenyl-1H-
pyrazole-3,4-dicarboxa-mide (22)
m.p.: 232e233 ꢀC; IR ( , cmꢁ1): 3362 and 3216 (NH), 3082 (CH,
n
Synthesized from 4 (0.39 g, 1 mmol) and 4-nitroaniline (0.564 g,
4 mmol) according to the general procedure. The crude product was
purified by crystallization from ethanol/DMF (3:1). Yield: 89%;
aromatic), 1688 and 1660 (C]O, amide), 1633e1451 (C]C and C]
N), 1344 (NeO sym.); 1H NMR (500 MHz, DMSO-d6/CDCl3) dH
(ppm): 11.71 and 10.37 (s, 2H, 2NH), 8.17e6.90 (m, 17H, ArH); 13C
NMR (125 MHz, DMSO-d6/CDCl3) dC (ppm): 161.21 and 159.59 (C]
O, amide), 158.40 and 157.72 (CeF), 148.05 (CeNO2), 147.41 (pyr-
azole C-3), 143.64 (pyrazole C-5), 120.32 (pyrazole C-4), 139.32,
135.03, 133.78, 131.27, 130.32, 130.23, 129.65, 128.94, 128.54, 128.19,
123.32, 123.26, 123.14, 121.63, 121.56, 118.83, 115.50, 115.32, 115.16.
Anal. calcd. for C29H19F2N5O4 (539.49 g/mol): C, 58.22; H, 2.99; N,
10.95. Found: C, 58.13; H, 3.02; N, 10.98.
m.p.: 320e321 ꢀC; IR ( , cmꢁ1): 3339 and 3214 (NH), 3049 (CH,
n
aromatic), 1671 (C]O, amide), 1634e1450 (C]C and C]N), 1324
(NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 11.29 and
11.17 (s, 2H, 2NH), 8.32e7.40 (m, 17H, ArH); 13C NMR (125 MHz,
DMSO-d6) dC (ppm): 161.27 and 159.89 (C]O, amide), 147.91,
145.02 and 144.09 (CeNO2), 144.58 (pyrazole C-3), 144.48 (pyrazole
C-5), 120.59 (pyrazole C-4), 142.81, 142.44, 138.92, 131.80, 130.77,
129.89, 129.80, 128.83, 127.12, 125.06, 124.75,123.65, 120.40, 120.12,
119.03. Anal. calcd. for C29H19N7O8 (593.50 g/mol): C, 58.69; H,
3.23; N, 16.52. Found: C, 58.55; H, 3.27; N, 16.56.
3.2.18. 1-(3-nitrophenyl)-5-phenyl-N3,N4-di-m-tolyl-1H-pyrazole-
3,4-dicarboxamide (19)
Synthesized from 4 (0.39 g, 1 mmol) and freshly distilled m-
toluidine (0.437 ml, 4 mmol) according to the general procedure.
The crude product was purified by crystallization from toluene.
3.2.22. General procedure for the synthesis of diureide derivatives
(23e25)
A mixture of the acid dichloride 4 (0.39 g, 1 mmol) and N-alkyl
ureas or thioureas (2 mmol) was refluxed at 140 ꢀC in xylene for 6 h.
After solvent had been evaporated, the formed crude product was
recrystallized from methanol or ethanol.
Yield: 95%; m.p.: 183e184 ꢀC; IR (
n
, cmꢁ1): 3351 and 3248 (NH),
3061 (CH, aromatic), 2971 (CH, aliphatic), 1659 (C]O, amide),
1618e1456 (C]C and C]N), 1345 (NeO sym.); 1H NMR (500 MHz,
DMSO-d6) dH (ppm): 10.84 and 10.48 (s, 2H, 2NH), 8.28e6.87 (m,
17H, ArH), 2.31 and 2.26 (s, 6H, 2CH3); 13C NMR (125 MHz, DMSO-
d6) dC (ppm): 160.29 and 159.82 (C]O, amide), 147.91 (CeNO2),
144.43 (pyrazole C-3), 144.39 (pyrazole C-5), 120.34 (pyrazole C-4),
21.21 (CH3), 139.18, 139.02, 138.16, 137.99, 137.95, 131.60, 130.67,
129.95, 129.62, 128.68, 128.60, 128.56, 127.89, 124.91, 124.21, 123.31,
121.24, 120.30, 119.74, 117.95, 116.49. Anal. calcd. for C31H25N5O4
(531.56 g/mol): C, 70.04; H, 4.74; N, 13.18. Found: C, 69.95; H, 4.77;
N, 13.22.
3.2.23. N3,N4-bis(methylcarbamoyl)-1-(3-nitrophenyl)-5-phenyl-
1H-pyrazole-3,4-dicarboxa-mide (23)
Synthesized from 4 (0.39 g, 1 mmol) and N-methylurea (0.153 g,
2 mmol) according to the general procedure. The crude product was
purified by crystallization from methanol. Yield: 72%; m.p.: 220e
221 ꢀC; IR ( , cmꢁ1): 3319 (NH), 3054 (CH, aromatic), 2970 (CH,
n
aliphatic), 1708 and 1671 (C]O, ureide), 1637e1451 (C]C and C]
N),1348 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 12.37
and 11.39 (s, 2H, 2NH), 10.88 and 9.02 (s, 2H, 2NH), 8.34e7.36 (m,
9H, ArH), 2.86 and 2.66 (d, J ¼ 6.8 Hz, 6H, 2CH3); 13C NMR (125 MHz,
DMSO-d6) dC (ppm): 162.53 and 162.15 (C]O, amide), 153.77 and
153.36 (C]O, ureide),147.96 (CeNO2), 147.68 (pyrazole C-3),143.26
(pyrazole C-5), 120.75 (pyrazole C-4), 26.21 (NCH3), 138.92, 131.93,
130.95, 130.54, 129.70, 128.87, 128.32, 123.54, 116.07. Anal. calcd. for
3.2.19. 1-(3-nitrophenyl)-5-phenyl-N3,N4-di-p-tolyl-1H-pyrazole-
3,4-dicarboxamide (20)
Synthesized from 4 (0.39 g, 1 mmol) and p-toluidine (0.451 g,
4 mmol) according to the general procedure. The crude product was
purified by crystallization from toluene. Yield: 88%; m.p.: 197e
198 ꢀC; IR ( , cmꢁ1): 3347 (NH), 3029 (CH, aromatic), 2973 (CH,
n
aliphatic), 1669 (C]O, amide), 1608e1451 (C]C and C]N), 1345
(NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm): 10.89 and
10.52 (s, 2H, 2NH), 8.27e7.09 (m, 17H, ArH), 2.28 and 2.24 (s, 6H,
2CH3); 13C NMR (125 MHz, DMSO-d6) dC (ppm): 160.02 and 159.83
(C]O, amide), 147.87 (CeNO2), 144.59 (pyrazole C-3), 144.41 (pyr-
azole C-5), 120.36 (pyrazole C-4), 20.53 and 20.47 (CH3), 139.17,
136.55, 135.65, 133.28, 132.46, 131.62, 130.63, 129.97, 129.55, 129.09,
128.59, 127.95, 123.29, 120.81, 120.10, 119.29. Anal. calcd. for
C21H19N7O6 (465.42 g/mol): C, 54.19; H, 4.11; N, 21.07. Found: C,
54.10; H, 4.15; N, 21.09.
3.2.24. N3,N4-bis(ethylcarbamoyl)-1-(3-nitrophenyl)-5-phenyl-1H-
pyrazole-3,4-dicarboxa-mide (24)
Synthesized from 4 (0.39 g, 1 mmol) and N-ethylurea (0.179 g,
2 mmol) according to the general procedure. The crude product was
purified by crystallization from ethanol. Yield: 65%; m.p.: 227e
C
31H25N5O4 (531.56 g/mol): C, 70.04; H, 4.74; N, 13.18. Found: C,
70.01; H, 4.78; N, 13.21.
228 ꢀC; IR ( , cmꢁ1): 3355 and 3226 (NH), 3062 (CH, aromatic), 2971
n
(CH, aliphatic), 1702 and 1659 (C]O, ureide), 1603e1450 (C]C and
C]N), 1347 (NeO sym.); 1H NMR (500 MHz, DMSO-d6) dH (ppm):
12.40 and 11.55 (s, 2H, 2NH), 10.42 (br, s, 2H, 2NH), 8.33e7.35 (m,
9H, ArH), 3.26 and 3.16 (pentet, J ¼ 6.8 Hz, 4H, 2NHCH2CH3), 1.12
and 1.05 (t, J ¼ 7.0 Hz, 6H, 2CH3); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 163.52 and 161.66 (C]O, amide), 152.80 and 152.44 (C]O,
ureide), 148.07 (CeNO2), 144.64 (pyrazole C-3), 142.86 (pyrazole C-
5), 120.18 (pyrazole C-4), 34.30 and 34.08 (NHCH2), 15.03 and 14.70
(CH3), 138.95, 131.07, 130.65, 130.56, 130.09, 128.97, 127.22, 123.41,
119.60. Anal. calcd. for C23H23N7O6 (493.47 g/mol): C, 55.98; H,
4.70; N, 19.87. Found: C, 55.88; H, 4.74; N, 19.91.
3.2.20. N3,N4,1-tris(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-
dicarboxamide (21)
Synthesized from 4 (0.39 g, 1 mmol) and 3-nitroaniline (0.558 g,
4 mmol) according to the general procedure. The crude product was
purified by crystallization from ethanol/DMF (3:1). Yield: 81%;
m.p.: 291e292 ꢀC; IR ( , cmꢁ1): 3314 (NH), 3006 (CH, aromatic),
n
1665 (C]O, amide), 1609e1451 (C]C and C]N), 1342 (NeO sym.);
1H NMR (500 MHz, DMSO-d6) dH (ppm): 11.20 and 11.09 (s, 2H,
2NH), 8.82e7.42 (m, 17H, ArH); 13C NMR (125 MHz, DMSO-d6) dC
(ppm): 161.19 and 159.80 (C]O, amide), 147.96, 147.87 and 144.26

S. Mert et al. / European Journal of Medicinal Chemistry 78 (2014) 86e96
95
3.2.25. N3,N4-bis(ethylcarbamothioyl)-1-(3-nitrophenyl)-5-phenyl-
1H-pyrazole-3,4-dicarbox amide (25)
inhibit only the standard and the clinical C. albicans strains, while
the other Candida species remained unaffected by the derivatives.
However, only the molecules 8, 10, 21, and 22 showed some
inhibitory effects on C. parapsilosis, C. tropicalis, and C. glabrata
strains. The structure-antifungal activity relationships of the com-
pounds on the C. albicans strains investigated by using electron-
conformational method (ECM). ECM study reveals that the esti-
mation of the antifungal activity is not fully possible only with
substituent-based considerations. In addition to geometric ar-
rangements of some groups revealed by the present ECM study,
their electronic parameters like the charges on atoms and the bond
order values are also crucial for the intensity of the antifungal
activity.
Among the presently studied molecules, the compounds 9, and
21e25 are strongly antifungal on both the clinical isolate of
C. albicans and the standard C. albicans strain, analogous to anti-
fungal drugs amphotericin B, ampicillin, ciprofloxacin, fluconazole,
streptomycin, nystatin, bifonazole, and miconazole [41e43]. In
terms of MIC values, the presently studied pyrazole derivatives
have comparable antifungal activities on C. albicans with the pre-
viously studied pyrazole derivatives of the class (1,4)-naph-
Synthesized from 4 (0.39 g, 1 mmol) and N-ethylthiourea
(0.213 g, 2 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 71%;
m.p.: 242e244 ꢀC; IR ( , cmꢁ1): 3376 and 3237 (NH), 3036 (CH,
n
aromatic), 2973 (CH, aliphatic), 1653 (C]O, ureide), 1602e1453
(C]C and C]N), 1343 (NeO sym.); 1H NMR (500 MHz, DMSO-d6)
dH (ppm): 11.69 and 10.49 (s, 2H, 2NH), 10.46 (br, s, 2H, 2NH), 8.31e
7.36 (m, 9H, ArH), 3.67 and 3.56 (pentet, J ¼ 7.1 Hz, 4H,
2NHCH2CH3), 1.21 and 1.14 (t, J ¼ 7.1 Hz, 6H, 2CH3); 13C NMR
(125 MHz, DMSO-d6) dC (ppm): 179.07 and 178.23 (C]S, thiourea),
162.58 and 159.81 (C]O, ureide), 147.91 (CeNO2), 145.07 (pyrazole
C-3), 142.18 (pyrazole C-5), 120.35 (pyrazole C-4), 39.57 (NHCH2),
13.25 (CH3), 138.62, 131.40, 130.71, 130.16, 129.89, 128.91, 126.69,
123.62, 118.31. Anal. calcd. for C23H23N7O4S2 (525.60 g/mol): C,
52.56; H, 4.41; N, 18.65; S, 12.20. Found: C, 52.43; H, 4.45; N, 18.66;
S, 12.17.
3.3. In vitro antibacterial and antifungal activities of the molecules
In the current study, 5 bacteria and 5 yeast species were used as
test organisms. Among these species, ATCC 10145, NRLL 3704, ATCC
25923, NRRL 12983 were utilized as standard strains. Vancomycine
resistant Enterococcus faecium, Methicillin-resistant S. aureus
(MRSA), C. albicans, C. glabrata and C. tropicalis (clinical isolates)
were obtained from patients, and were provided by the Microbi-
ology Laboratory of the Faculty of Medicine at the Eskisehir
Osmangazi University, Turkey. C. parapsilosis (clinical isolates) was
obtained from patients at the Microbiology Laboratory of Evliya
Çelebi Hospital of the Faculty of Medicine, Kütahya Dumlupinar
University, Turkey.
thoquinono-[3,2-c]-1H-pyrazoles,
(1,4)-naphthohydroquinone-
[3,2-c]-1H-pyrazoles, halogenated 4-[1H-imidazol-1-yl(phenyl)
methyl]-1,5-diphenyl-1H-pyrazoles, pyrazole-1-carbothioamides,
1,3-disubstituted indeno[1,2-c]pyrazoles, and 4-arylidene pyr-
azoles [41e45]. However, they are more antifungal on C. albicans
than the majority of pyrazoline and pyrazole derivatives with
indoline and quinoxaline substituents [46]. Therefore, the present
molecules constitute a new class of potent antifungal drugs.
Acknowledgment
Bacterial and fungal cultures of test organisms were maintained
on Nutrient Agar and Saboraud Dextrose Agar slants at 4 ꢀC,
respectively, and were sub-cultured in petri dishes prior to use.
Antimicrobial activity analysis of test compounds was carried out
according to modified agar well diffusion assay [40]. The com-
pounds were dissolved first in Dimethylsulfoxide (DMSO, Merck)
with the initial concentration of 2.5 mg/ml. Each solution was
diluted two-fold with DMSO. These solutions were used in MIC test.
Fifteen milliliters of the specified molten agar (45 ꢀC) were poured
into sterile Petri dishes. The cell suspensions containing 108 CFU/
mL cells for bacteria, 107 CFU/mL cells for yeasts were prepared and
evenly spread onto the surface of the agar plates of Nutrient Agar
(Merck 1.05450) for bacteria and Saboraud Dextrose Agar (CM0041,
Oxoid) medium for yeast using sterile swab sticks. The plates were
dried aseptically at 35 ꢀC for about 40 min in an incubator. The agar
well method for the estimation of MIC values (the lowest concen-
tration of compounds required to inhibit the growth of the tested
microorganisms) was applied to evaluate the antimicrobial activity.
The authors would like to express their gratitude to the Research
Funds of Dumlupınar and Fatih Universities (project numbers: BAP
2010e14 and P50011102_Y (1679), respectively). Also, the authors
would like to thank Department of Physics, Faculty of Arts and
Sciences, Dumlupınar University for FTIR measurements. S.O. is
grateful to King Fahd University of Petroleum and Minerals
(KFUPM) where he performed NMR measurements of some of the
reported compounds.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.033.
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