Synthesis, Crystal Structure and Antimicrobial Activity of Ethyl 2-(1-cyclohexyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate①

MUHAMMAD Neem Ahme SHAHID Hmee KHAWAJA Ansr Ysin IFZAN Arsh IHSAN-ul -Hq SAFEENA Zfr MUHAMMAD Nwz Thir

a (Department of Chemistry, The University of Azad Jammu and Kashmir, Muzaffarabad 13100, Pakistan)

b (Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan)

c (Department of Pharmacy,Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad 45320, Pakistan)

d (University of Sargodha, Department of Physics, Sargodha)

1 INTRODUCTION

Now a days research is focused on the synthesis of new and safe therapeutic agents of clinical importance. 1,2,3-triazoles are enjoying their importance day by day in organic synthesis, biology and material sciences. Significance of 1,4,5-trisubstituted 1,2,3-triazole and their derivatives is of key importance due to their lots of biological activities[1]like antibacterial, antifungal, antimalarial, antiviral[2],anticonvulsant[3], antidepressant and anticancer[4-6].Recently, many 1,4,5-trisubstituted 1,2,3-triazoles have shown novel properties[7]because of their extensive structural diversity. Currently, more structurally diverse 1,2,3-triazoles can be easily synthesized by CuAAC method using one-pot strategy. In view of the emerging biological and synthetic importance of 1,4,5-trisubstituted 1,2,3-triazoles, we have designed and synthesized some novel 1,2,3-triazoles and their derivatives using one-pot, three component reaction with high yield.

2 EXPERIMENTAL

2.1 Materials and instruments

Melting point was determined on a Stuart SMP3 melting point apparatus. IR spectrum was recorded on a Nicolet FTIR SDX spectrometer. The1H and13C NMR spectra were recorded on a JEOL JNM-ECA 300 instrument in CDCl3. TMS was used as internal standard and J values are given in Hz.HRMS was obtained on a Bruker micrOTOF-Q II spectrometer. 1-Copper(I) phenylethyne was prepared according to the procedure reported in references[8,9]. Reaction was monitored by thin layer chromatography (TLC) using F254silica gel. All chemicals were purchased from commercial suppliers and were dried and purified where necessary.

2.2 Theoretcial calculations

All the calculations were performed by DFT(B3LYP)[10,11]method by taking the crystal structure coordinates as an initial geometry with 6-311G basis set[12]using PC GAMEES/Firefly Version 8.0.0[13]together with the Gabedit graphical user interface[14].All geometric parameters were visualized with Chemcraft[15].

2.3 Synthesis

Ethoxalyl chloride (0.968 g, 0.5 mmol) was added to a suspension of 1-azidocyclohexane (0.075 g, 0.6 mmol) and 1-copper(I) phenylethyne (0.082 g, 0.5 mmol) in PhCl (2 mL). The resulting mixture was stirred at room temperature for 4 h and then passed through a column chromatography (silica gel,EtOAc in petroleum ether (60～90 °C ) to gave the product (I) as a white solid. Crystals suitable for crystallographic study were grown by slow evaporation of an ethanol:ethyl acetate solution at room temperature. m.p.: 97～99 °C, yield = 85%, IR (KBr)ν 3057, 2996, 2960, 2938, 2859, 1746, 1671, 1477,1449, 1290, 1277, 1221, 1202, 992, 975, 789, 735,705 cm-1;1H-NMR δ 7.55～7.44 (m, 5H), 4.93～4.81 (m, 1H), 3.77 (q, 2H, J = 7.20 Hz), 2.24～1.95(m, 6H), 1.82～1.76 (m, 1H), 1.57～1.26 (m, 3H),0.98 (t, 3H, J = 7.20 Hz);13C-NMR δ 177.8, 161.1,152.6, 129.9, 129.5, 129.1, 128.6, 127.2, 62.8, 60.9,33.0, 25.3, 25.0, 13.2 ppm HRMS (ESI-TOF) (m/z):calculated for C18H21N3O3, [M+H]+328.1656; found 328.1654.

2.4 X-ray crystallography

C18H21N3O3, Mr= 327.38, crystal size 0.36mm ×0.28mm × 0.26mm, monoclinic, space group P21/n,a = 12.8167(9), b = 8.0966(6), c = 16.7079(9) Å, β =98.716(2), Z = 4, V = 1713.8(2) Å3. ρcalc= 1.269 g/cm³, μ= 0.088 mm-1and F(000) = 696. Data were collected at 296(2) K on a Bruker Kappa Apex II CCD diffractometer using MoKα radiation. 14207 intensities 1.87≤θ≤26.9°. The structure was solved by direct methods, full-matrix least-squares refinement[16]on F² and 218 parameters for 3748 unique intensities (Rint= 0.025). The structure was not only solved through direct methods but also refined by full-matrix least-squares using SHELX-97[16]. All non-hydrogen atoms were refined anisotropically.

Scheme 1. Synthesis of the title compound (I)

3 RESULTS AND DISCUSSION

3.1 Crystal structure

The molecular structure of the title compound (I)shown in Fig. 1 is closely related to that of ethyl 2-[1-(3-methylbutyl)-4-phenyl-1H-1,2,3-triazol-5-yl]-2-oxoacetate[17]with iso-pentyl group replaced by cyclohexyl group. In the molecule, the cyclohexyl group adopts the chair conformation. The dihedral angle between the mean planes of the triazole and phenyl rings is 51.03°. In the crystal, molecules related by inversion are paired into dimers via C(14)–H(14)···O(1) (2.631 Å) and C(15)···O(2)–C(16) interactions[18-20], and the latter is indicated by the short distance of 3.188 Å (Fig. 2).

Fig. 1. Molecular structure of (I). Anisotropic displacement ellipsoids are drawn at 20% probability level. H atoms are shown as spheres of arbitrary radii

Fig. 2. Dimer formed via C–H…O and C–O…C interactions (dashed lines)in the crystal structure of the compound (I).

3.2 Optimized geometry

The optimized geometry parameters, namely the selected bond lengths and bond angles, experimental and calculated by the B3LYP/6-311G level, are listed in Table 1. From the theoretical results, the expected variations of the calculated values against the experimental ones were observed. Comparing the experimental and calculated bond lengths and bond angles, it is noticed that calculated values are always higher than the experimental ones.Theoretical structure also showed identical bond lengths (both experimental and calculated) for the C–C bonds of the aryl and cyclohexyl rings.

By comparing the theoretical and experimental data, several observations can be made. The experimental N(1)–N(2) and N(2)–N(3) bond lengths are 1.330 and 1.323 Å which are calculated as 1.360 and 1.368 Å respectively. The N(3)–C(8) bond length is 1.355 Å and calculated as 1.375 Å which is close to the experimental value and smaller than the normal C–N single bond length of about 1.47 Å[21,22]. From the calculated results it was concluded that theoretical and experimental geometric parameters were in good agreement. The X-ray structure of the title compound was compared with its optimized counterpart by superimposing experimental structure over the theoretical (Fig. 3); and minor conformational discrepancies were observed.

Table 1. Bond Lengths (Å) and Bond Angles (º) for the Title Compound (I)Determined by X-ray Diffraction and B3LYP Calculations using the 6-311G Basis Set

Fig. 3. Atom-by-atom superimposition of the structure calculated (red)by B3LYP/6-311G on the X-ray structure (blue) of the title compound (I)

4 ANTIMICROBIAL ACTIVITY

In this study the antibacterial activity of the compound was observed using disc diffusion method[23,24]against various pathogenic bacteria as S. typhimurium, M. luteus and B. bronchiseptica.Different fungal strains like Aspergilus niger, Mucor species and Aspergilus flavus were also used to check the antifungal potential of the synthesized compound. The values of antibacterial and antifungal activities of compound I are listed in Tables 2 and 3, respectively. Graphical results of the antibacterial and antifungal activities are shown in Fig. 4a and 4b.

4.1 Antibacterial assay

Bacterial inoculum was prepared in nutrient broth.It was prepared by dissolving 2 grams of nutrient broth in 100 mL of water and the pH was maintained to 7. Nutrient agar medium was used for the growth of bacterial strains that were produced by dissolving 2 grams in 100 mL of water. A filter paper disc of 6 mm in size was prepared from whatman no. 1 filter paper. Media, filter paper discs along with other apparatus required in this assay were autoclaved for sterilization. After autoclaving, the whole experiment was carried out in microbiology safety cabinet.Solidified plate of nutrient agar was labeled and respective bacterial strain was streaked. Then solution of the compound in DMSO was absorbed on disc which was placed on respective place in petri plate. This petri plate was incubated for 24 hours at 28 °C. Zones of inhibition were measured in mm after 24 hours. Cefixime-USP was used as the standard drug and DMSO as the negative control.

Fig. 4a. Graphical representation of antibacterial activities of compound I

Fig. 4b. Graphical representation of antifungal activities of compound I

Table 2. Antibacterial Activity Data of the Title Compound (I)

Table 3. Antifungal Activity Data of the Title Compound (I)

4.2 Antifungal assay

Nutrient agar medium was used for the growth of bacterial strains produced by dissolving 2 grams in 100 mL of water. Fungal cultures were grown on SDA medium at pH = 6.5. Sabouraud dextrose agar(SDA) was prepared by dissolving 6.5 grams in 100 mL of distilled water and the pH value was adjusted to 6.5. Whatman no. 1 filter paper was used for the preparation of circular discs of 6 mm size. All apparatus required in the assay were autoclaved for sterilization. An experiment was carried out in microbiological safety cabinet. SDA media were poured on petri plate and left for solidification. The filter paper disc was used for the absorption of sample on disc. Contamination was prevented by wrapping the petri plate with parafilm. After that,the plate was incubated for 24 hours at 28 . On next day, the inhibition zones were measured. Terbinafine was used as the standard drug and DMSO as the negative control.

5 CONCLUSION

In this study, a novel 1,4,5-trisubstituted triazole was synthesized and mainly characterized by X-ray diffraction. The experimental and theoretical structure parameters of the title compound I were compared and showed that the experimental X-ray structure is close to its optimized counterpart. Actimicrobial activity results showed that the synthesized compound displayed good antibacterial and anti-fungal activities against three different bacterial and fungal strains.

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