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)cyclohexanecarboxylic 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 substituent has been described in the literature (CSD, Ver. 5.32) [8].

Experimental

Instrumentation

All reagents and chemicals were purchased from commercial 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 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.

Single-crystal X-ray data were collected at room temperature on a four-circle Kuma KM4 diffractometer using graphite monochromated Cu Kα radiation (λ = 1.54178 Å) and ω/2θ scan mode [9]. Crystallographic details as well as parameters of data collection and refinement are summarized in Table 1. The structures were solved by direct methods using the SHELXS97 program and refined by the full-matrix least-squares on F 2 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 O–H bond distances of 0.82 Å and C–H distances being in the range of 0.93–0.98 Å; a riding model was used during the refinement process. The displacement parameters of the H atoms were U iso(H) = 1.2 U eq(C/O).

Table 1 Crystal data and structure refinement details for (I) and (II)
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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].

Synthesis

N-Phenylpicolinehydrazonamide (10 mmol) and cis-1,2-cyclohexanedicarboxylic anhydride (10 mmol) were dissolved 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). 1H NMR (300 MHz, DMSO-d6, ppm), δ: 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.

Biological Screening

Determination of Cell Toxicity

Human peripheral blood mononuclear cells (PBMC) (2 × 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-glutamine, 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 concentrations of 1–100 μg/mL. As a control, appropriate dilutions 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 tetrazolium bromide) (Sigma). The mean optical density (OD) value at 550/630 nm from quadruplicate wells ± standard error (SE) was analysed.

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 Lymphoprep® (Polfa, Kutno, Poland) and centrifuged for 25 min at 800×g. Cells from the interphase were washed 3 times with PBS and re-suspended at a density of 2 × 106/mL in the culture medium. The cells were placed then in 96-well, flat-bottom culture plates (Nunc) (2 × 105/100 μL/well). Phytohemagglutinin (PHA) (Sigma) was used at concentration of 5 μg/mL. The studied compound was used at concentrations of 1, 10 and 100 μg/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 (Nunc). Production of cytokines was induced by an addition of lipopolysaccharide (LPS) from E. coli, serotype 0111:34 (3 × 106 E.U. mg−1) (Sigma) at a dose of 1 μg/mL. The studied compound (I) was added to the cultures at concentrations of 10 and 100 μg/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-α) and interleukin 6 (IL-6) were determined using bioassays [13, 14].

Results and Discussion

Crystal Structure

The title compound and its solvate crystallize in the orthorhombic noncentrosymmetric space groups Pca21 (I) and P212121 (II), respectively. There are two independent molecules (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 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].

Fig. 1
figure 1

The perspective view of molecules (I) with 30% probability displacement ellipsoids

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Fig. 2
figure 2

The asymmetric unit of (II). Displacement ellipsoids are drawn at the 30% probability level. Dashed lines indicate hydrogen bonds

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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 3σ) 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 interaction 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)° (molecule 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.

Table 2 Selected geometric parameters
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Fig. 3
figure 3

The molecular fit through the triazole atoms of molecules (IA) (open lines), (IB) (full lines) and (II) (dashed lines)

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The most characteristic feature of crystal (I) is the presence of two alternating ribbons (Fig. 4), build separately by the symmetry independent molecules. Each ribbon 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).

Fig. 4
figure 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

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Table 3 Hydrogen-bonding geometry
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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).

Fig. 5
figure 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

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Biological Activity

Compound (I) was tested for its potential activity to affect proliferative response of human peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA) and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) 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 μg/mL was tested.

The results showed that (I) caused about 40.5% inhibition of the proliferative response of PBMC to PHA at 100 μg/mL and a similar inhibition (43.3%) of TNF-α production at 10 μg/mL. IL-6 production was not affected. Importantly (I) was completely devoided of cell toxicity up to the concentration of 100 μg/mL. Therefore, both the inhibition of cell proliferation and TNF-α 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

CCDC-802411 and CCDC-802412 contains the supplementary crystallographic data for this paper. Copies of available materials can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (+48) 1223-336-033; e-mail: data_request@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk/data_request/cif).