2-(4-phenyl-5-pyridin-2-yl-4H-1,2,4-triazol-3-yl)cyclohexanecarboxylic acid and its DMSO solvate: Synthesis, crystal structure and biological activity

Article abstract of DOI:10.1007/s10870-011-0017-7

A new 1,2,4-triazole derivative, 2-(4-phenyl-5-pyridin-2-yl-4H-1,2,4- triazol-3-yl)cyclohexanecarboxylic acid, C₂₀H₂₀N ₄O₂ (I), and its dimethyl sulfoxide solvate 1:1 (II) have been synthesized and their crystal

Full text of DOI:10.1007/s10870-011-0017-7

J Chem Crystallogr (2011) 41:880–885
DOI 10.1007/s10870-011-0017-7
ORIGINAL PAPER
2-(4-Phenyl-5-pyridin-2-yl-4H-1,2,4-triazol-3-
yl)cyclohexanecarboxylic Acid and Its DMSO Solvate:
Synthesis, Crystal Structure and Biological Activity
•
Liliana Mazur Anna E. Koziol Bozena Modzelewska-Banachiewicz
•
•
_
•
Marzena Ucherek Michał Zimecki Jolanta Artym
•
Received: 6 December 2010 / Accepted: 31 January 2011 / Published online: 15 February 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract A new 1,2,4-triazole derivative, 2-(4-phenyl-5-
pyridin-2-yl-4H-1,2,4-triazol-3-yl)cyclohexanecarboxylic
acid, C20H20N4O2 (I), and its dimethyl sulfoxide solvate
1:1 (II) have been synthesized and their crystal structure
was established. Compound (I) was screened for its anti-
proliferative and antiinflammatory activity. Structural
analysis indicated the substantial difference between two
symmetry independent molecules in (I) and this in (II), it
manifests in the relative orientation of pyridine/phenyl and
triazole rings, as well as in the orientation of carboxyl
group with respect to cyclohexane ring. The molecules A
and B in the crystal (I) form two hydrogen-bonded chains
through O–Hcarboxyl and Ntriazole atoms, giving separate
catemers of symmetry independent molecules. The cate-
mer of (IA) running along the 21 axis is homochiral, while
the catemer (IB) is racemic—formed about the c glide
plane. In the crystalline solvate (II) complexation of
(I) with DMSO induced enantiomeric self-resolution.
Obtained crystals are racemic twins, in which each part is
built of one enantiomer of (I) having the relative config-
uration 11S,12R or 11R,12S. A pair of host–guest mole-
cules is linked by the O–Hcarboxyl_ODMSO hydrogen bond.
Keywords Crystal structure Á 1,2,4-triazole Á
Racemate Á DMSO solvate
Introduction
The growing interest in synthesis and structures of 1,2,4-
triazole and its derivatives results from their applications in
the synthesis of other heterocyclic compounds or as ligands
in coordination chemistry [1–3]. Triazole derivatives have
a wide range of biological applications, which include
notably antitumorial, antifungal, antimicrobial, antiviral,
antidepressant or herbicidal activities [4–7].
In order to obtain more detailed information on the
molecular conformation and patterns of hydrogen bonds of
the title compound in the solid state, which may be of value
in a structure–activity analysis, an X-ray analysis of 2-(4-
phenyl-5-pyridin-2-yl-4H-1,2,4-triazol-3-yl)cyclohexane-
carboxylic acid (I) and its 1:1 DMSO solvate (II) has been
undertaken. So far, no crystal structure of 4H-1,2,4-triazole
derivative having cyclohexanecarboxylic acid as a sub-
stituent has been described in the literature (CSD, Ver.
5.32) [8].
Experimental
L. Mazur (&) Á A. E. Koziol
Department of Crystallography, Faculty of Chemistry, Maria
Curie-Sklodowska University, 20-031 Lublin, Poland
e-mail: lmazur2@op.pl
Instrumentation
All reagents and chemicals were purchased from com-
mercial sources and used without further purification. IR
spectrum was recorded on a FT-IR Perkin Elmer 1725X
spectrophotometer (KBr pellet, 4,000–400 cm-1). 1H
NMR spectrum was recorded at room temperature on
Bruker Avance (300 MHz) apparatus using DMSO-d6 as a
B. Modzelewska-Banachiewicz Á M. Ucherek
Department of Organic Chemistry, Faculty of Pharmacy,
Nicolaus Copernicus University, 85-067 Bydgoszcz, Poland
M. Zimecki Á J. Artym
Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, 53-114 Wroclaw, Poland
123

J Chem Crystallogr (2011) 41:880–885
881
solvent and TMS as an internal standard. The apparent
resonance multiplicity is described as: s = singlet and
m = multiplet. Elemental analysis was carried out with a
CHN Perkin-Elmer 2400 analyzer. Melting point was
measured on a Mel TEMP 1002D apparatus and is given
uncorrected.
In the absence of significant anomalous scattering effect it
was impossible to establish unambiguously the absolute
crystal structure (I). Crystals of (II) were twinned and
the DMSO molecules in their structure were disordered.
Thus, determination of the relative configuration only is
possible [11].
Single-crystal X-ray data were collected at room tem-
perature on a four-circle Kuma KM4 diffractometer using
Synthesis
˚
graphite monochromated Cu Ka radiation (k = 1.54178 A)
and x/2h scan mode [9]. Crystallographic details as well as
parameters of data collection and refinement are summa-
rized in Table 1. The structures were solved by direct
methods using the SHELXS97 program and refined by the
full-matrix least-squares on F2 using SHELXL97 [10]. In
the crystal net of (II) the DMSO molecule is disordered
between two positions with the site occupancy factors (sof’s)
of 0.815 (6):0.185 (6). All non-H atoms in (I) and (II) were
refined with anisotropic displacement parameters. All H
atoms were positioned geometrically and constrained with
N-Phenylpicolinehydrazonamide (10 mmol) and cis-1,2-
cyclohexanedicarboxylic anhydride (10 mmol) were dis-
solved in anhydrous diethyl ether. The reaction was carried
out in ambient temperature for 14 days. The resulting
precipitate was collected by filtration, washed with diethyl
ether to remove the unreacted anhydride, and heated in
chloroform under reflux for 5 min. The insoluble residue
was dissolved in 2% sodium hydroxide, and the resulting
solution was heated under reflux for 2 h. The solution was
cooled and acidified with hydrochloric acid solution. The
product (I) was crystallized from ethanol. Yield: 45%.
M.p.: 224–226 °C. Anal. Calcd. for C20H20N4O2: C, 68.55;
H, 6.33; N, 15.99%. Found: C, 68.76; H, 6.09; N, 15.59%.
IR (KBr, cm-1): 1700 (C=O), 2855 (Haliph), 2936 (Harom).
˚
O–H bond distances of 0.82 A and C–H distances being in
˚
the range of 0.93–0.98 A; a riding model was used during the
refinement process. The displacement parameters of the H
atoms were Uiso(H) = 1.2 Ueq(C/O).
Table 1 Crystal data and structure refinement details for (I) and (II)
(I)
(II)
Formula
C20H20N4O2
348.4
C20H20N4O2 C2H6OS
426.54
Formula weight (g mol-1
Crystal system
Space group
)
Orthorhombic
Pca21
Orthorhombic
P212121
˚
a (A)
28.330 (6)
10.548 (2)
12.130 (2)
3624.8 (12)
8
9.626 (2)
˚
b (A)
9.955 (2)
˚
c (A)
23.230 (5)
2226.1 (8)
4
3
V (A )
˚
Z
Calculated density (g cm-3
)
1.277
1.273
Absorption coefficient (mm-1
)
0.687
1.540
F(000)
1472
904
Crystal size (mm)
Crystal color and form
h Range (°)
0.36 9 0.19 9 0.09
Colorless plate
3.12–80.44
0.32 9 0.29 9 0.25
Colorless prism
3.81–80.26
Indexes ranges
0 B h B 36; -13 B k B 13; 0 B l B 15
8043/4165
-12 B h B 12; 0 B k B 12; 0 B l B 29
5159/4811
Reflections collected/unique
(Rint = 0.1116)
4165/1/469
(Rint = 0.0844)
4811/0/283
Data/restrains/parameters
Goodness-of-fit on F2
0.948
0.977
Final R indices (I [ 2r(I))
R1 = 0.0418;
R1 = 0.0579;
wR2 = 0.0708
R1 = 0.1667;
wR2 = 0.1528
R1 = 0.1616;
R indices (all data)
wR2 = 0.0945
0.18/-0.23
wR2 = 0.1987
0.26/-0.24
-3
)
˚
Largest diff. peak and hole (e A
123

882
J Chem Crystallogr (2011) 41:880–885
1H NMR (300 MHz, DMSO-d6, ppm), d: 12.1 (1H, s,
COOH); 7.3–8.3 (9H, m, arom); 3.2–1.0 (10H, m, aliph).
Crystals of compound (I), suitable for X-ray analysis, were
obtained by recrystallization from methanol solution at
room temperature. Single crystals of its DMSO solvate (II)
were obtained from DMSO solution by slow evaporation of
solvent.
(Nunc). Production of cytokines was induced by an addi-
tion of lipopolysaccharide (LPS) from E. coli, serotype
0111:34 (3 9 106 E.U. mg-1) (Sigma) at a dose of 1 lg/
mL. The studied compound (I) was added to the cultures
at concentrations of 10 and 100 lg/mL. As a control,
appropriate dilutions of DMSO in the culture medium were
used. After 24 h incubation in a cell culture incubator the
supernatants were harvested and kept at -20 °C until
cytokine determination. The activities of tumor necrosis
factor alpha (TNF-a) and interleukin 6 (IL-6) were deter-
mined using bioassays [13, 14].
Biological Screening
Determination of Cell Toxicity
Human peripheral blood mononuclear cells (PBMC)
(2 9 105/0.2 mL/well in 96-well plates) were suspended in
a culture medium, referred later to as the culture medium,
consisting of: RMPI 1640, 10% fetal calf serum, L-gluta-
mine, sodium pyruvate, 2-mercaptoethanol and antibiotics
(streptomycin and penicillin). The studied compound
(I) was initially dissolved in DMSO and subsequently in
the culture medium and added to the cultures at concen-
trations of 1–100 lg/mL. As a control, appropriate dilu-
tions of DMSO in the culture medium were used. Cell
survival was determined by MTT colorimetric method [12]
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
razolium bromide) (Sigma). The mean optical density (OD)
value at 550/630 nm from quadruplicate wells standard
error (SE) was analysed.
Results and Discussion
Crystal Structure
The title compound and its solvate crystallize in the ortho-
rhombic noncentrosymmetric space groups Pca21 (I) and
P212121 (II), respectively. There are two independent mol-
ecules (A and B) in the asymmetric unit of (I) (Fig. 1),
whereas the asymmetric part of (II) consists of one molecule
of 4H-1,2,4-triazole derivative and one DMSO solvent
Proliferation of Human Peripheral Blood Mononuclear
Cells to Phytohemagglutinin A (PHA)
Venous blood was taken into heparinized syringes and
diluted two-fold with phosphate buffered saline (PBS).
Blood was applied onto LymphoprepÒ (Polfa, Kutno,
Poland) and centrifuged for 25 min at 8009g. Cells from
the interphase were washed 3 times with PBS and re-sus-
pended at a density of 2 9 106/mL in the culture medium.
The cells were placed then in 96-well, flat-bottom culture
plates (Nunc) (2 9 105/100 lL/well). Phytohemagglutinin
(PHA) (Sigma) was used at concentration of 5 lg/mL. The
studied compound was used at concentrations of 1, 10 and
100 lg/mL. As a control, appropriate dilution of DMSO in
the culture medium was used. The cultures were incubated
for 72 h in a cell culture incubator. The degree of cell
proliferation was measured by a colorimetric method [12].
The results were presented as a mean OD value from
quadruplicate wells SE.
Determination of Cytokine Production in Whole Blood
Cell Cultures
Venous blood from a single donor was diluted fivefold with
RPMI 1640 medium and placed in 24-well culture plates
Fig. 1 The perspective view of molecules (I) with 30% probability
displacement ellipsoids
123

J Chem Crystallogr (2011) 41:880–885
883
moiety (Fig. 2). Dimethyl sulfoxide in (II), as in many other
crystalline solvates [15, 16], is partly disordered, with two
positions occupied by the S1 atom and the H-atoms of
methyl groups. The positions of O1s, C1s and C2s atoms are
not affected by the disorder. Moreover, complexation of
(I) with DMSO induces enantiomeric self-resolution,
forming homochiral domains (with enantiomer 11S,12R or
11R,12S) in twinned crystals [17]. It is an interesting
example of spontaneous resolution of racemic compound by
an achiral guest. A very similar behaviour was observed
for 2,7-bis(trifluoromethyl)tricyclo[4.3.1.13,8]-undecane-
syn-2,syn-7-diol [18] and cyclo-bis(2-amino-7-cyano-4-
methoxyindan-2-carboxylic) acid [19]. It is very likely that
the polarity of solvent influences the racemic resolution
process which was observed in other cases [20, 21].
Table 2 Selected geometric parameters
(I)
(II)
A
B
˚
Bond lengths (A)
N2–N1
1.394 (4)
1.311 (4)
1.363 (4)
1.373 (4)
1.307 (4)
1.300 (5)
1.203 (5)
1.385 (4)
1.372 (5)
1.313 (5)
1.385 (5)
1.377 (5)
1.304 (5)
1.257 (7)
1.189 (8)
N2–C3
1.308 (4)
1.371 (4)
1.372 (4)
1.313 (4)
1.322 (4)
1.197 (4)
N4–C3
C5–N4
N1–C5
C1–O1
C1–O2
Torsion angles (°)
N1–C5–C22–N21
C12–C11–C1–O1
137.0 (4)
171.9 (4)
147.0 (4)
-153.3 (4)
-35.3 (5)
145.9 (5)
The central 1,2,4-triazole ring of (I) is substituted at the
C3, N4 and C5 atoms by 2-cyclohexanecarboxylic acid,
phenyl and 2-pyridyl groups, respectively. Bond lengths and
angles within the triazole ring are not equal (Table 2) but
comparable (within 3r) in all three conformers and similar
to those observed in related structures [22, 23]. A small
difference in the geometry of triazole rings is observed only
for N1–N2 bonds, which may result from different interac-
tion patterns. The pyridyl and phenyl rings are twisted with
respect to the 1,2,4-triazole plane; interplanar angles are
43.9 (3), 80.6 (3)° (molecule IA), 31.8 (3), 69.7 (3)° (mol-
ecule IB) and 24.0 (5) and 88.7 (5)° (II), respectively. The
second conformational difference between the molecules
(IA), (IB) and (II) is in the orientation of carboxyl group
(Fig. 3). The relative orientation of the carboxyl O1(H) and
chiral C12 atoms of the cyclohexane ring is described by the
O1–C1–C11–C12 torsion angle (Table 2) indicating both
trans or cis conformations. These differences depend on a
pattern of intermolecular hydrogen bonds in which O1
atoms act as donors.
The most characteristic feature of crystal (I) is the
presence of two alternating ribbons (Fig. 4), build sepa-
rately by the symmetry independent molecules. Each rib-
bon is linked by the O1–H1_N1 hydrogen-bonds
(Table 3) in the C(8) chain motif [24]. As shown in Fig. 4,
molecules (IA) form a catemer around the two-fold screw
axis, while the catemer of molecules (IB) runs about the
c-glide plane. Thus, the first one is enantiopure and the
second is racemic. Further, weak intermolecular C–H_N/
O hydrogen bonds link adjacent chains of the molecules
(IA) and (IB).
As a result of the racemic resolution by solvent, in the
crystal structure of (II) the host molecule, i.e. 4H-1,2,4-
triazole derivative, is one of enantiomers (11S,12R or
11R,12S), depending on the part of twinned crystal. Two
chemical components, host and guest, are linked by a very
short and nearly linear O1–H1_O1s hydrogen bond
(Table 3; Fig. 5). Moreover, the guest DMSO molecules in
(II) play a vital role in the supramolecular aggregation
participating, both as an acceptor and donor, in an extended
net of weak C–H_O and C–H_N hydrogen bonds to host
molecules (Fig. 5; Table 3).
Biological Activity
Compound (I) was tested for its potential activity to affect
proliferative response of human peripheral blood mono-
nuclear cells (PBMC) to phytohemagglutinin (PHA) and
lipopolysaccharide (LPS)-induced tumor necrosis factor
alpha (TNF-a) and interleukin 6 (IL-6) production by
human whole blood cell cultures. In addition, the effect of
(I) on PBMC survival in 24 h culture at the concentration
range of 5–100 lg/mL was tested.
Fig. 2 The asymmetric unit of (II). Displacement ellipsoids are
drawn at the 30% probability level. Dashed lines indicate hydrogen
bonds
The results showed that (I) caused about 40.5% inhibi-
tion of the proliferative response of PBMC to PHA at
123

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J Chem Crystallogr (2011) 41:880–885
Fig. 3 The molecular fit through the triazole atoms of molecules (IA)
(open lines), (IB) (full lines) and (II) (dashed lines)
Fig. 5 The chain formed by the O–H_O and C–H_O/N hydrogen
bonds between host and guest molecules in (II). Dashed lines indicate
hydrogen bonds
100 lg/mL and a similar inhibition (43.3%) of TNF-a
production at 10 lg/mL. IL-6 production was not affected.
Importantly (I) was completely devoided of cell toxicity up
to the concentration of 100 lg/mL. Therefore, both the
inhibition of cell proliferation and TNF-a production could
not be due to its toxicity. In conclusion, the preliminary
results indicate a weak antiproliferative and a moderate
antiinflammatory property of the compound.
Supplementary Material
Fig. 4 The crystal structure of (I) viewed along the b axis. The
catemers of molecules (IA) and (IB) are marked. Dashed lines
indicate hydrogen bonds
CCDC-802411 and CCDC-802412 contains the supplemen-
tary crystallographic data for this paper. Copies of available
materials can be obtained free of charge on application to
CCDC, 12UnionRoad,CambridgeCB21EZ,UK(fax:(?48)
1223-336-033; e-mail: data_request@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk/data_request/cif).
Table 3 Hydrogen-bonding geometry
D–H H_A D_A
\D–H_A
(°)
˚
(A)
˚
(A)
˚
(A)
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
(I)
O1A–H1A_N1A(i)
O1B–H1B_N1B(ii)
C25B–H25B_O2B(iii) 0.93
(II)
O1–H1_O1s
C24–H24_O1s(iv)
C26–H26_O1s(v)
C13–H13a_O1s(vi)
C1s–H1S2_N1(iv)
C2s–H2S3_N2(iv)
C33–H33_O1(vii)
0.82
0.82
1.92
1.90
2.43
2.688 (5) 157
2.706 (5) 167
3.285 (6) 153
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0.82
0.93
0.93
0.97
0.96
0.96
0.93
1.85
2.65
2.71
2.67
2.49
2.63
2.54
2.660 (7) 172
3.554 (6) 162
3.536 (6) 150
3.540 (7) 150
3.357 (7) 151
3.415 (7) 139
3.368 (7) 148
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