Synthesis , Spectroscopic Characterization and Antimicrobial Activity of Some New 2-Substituted Imidazole Derivatives

The reaction of imidazole-2-thione derivative 1 with 2-chloro-N-p-tolylacetamide afforded the corresponding 2-(1H-imidazol-2-ylthio)-N-p-tolylacetamide 2. Reaction compound 2 with different reagents such as p-chlorobenzaldehyde and p-chlorophenyl diazonium chloride afforded the corresponding arylidene derivative 3 and hydrazone derivative 6. Reactions of 2 with carbon disulfide in dimethylformamide (DMF) in one equivalent potassium hydroxide afforded intermediate potassium sulphide salt 8, which treatment with dilute hydrochloric acid and phenacyl bromide afforded the corresponding 2-[p-tolylcarbamoyl]ethanedithioic acid 9 and 3-[benzo-ylmethylthio]-N-p-tolyl-3-thioxo-propaneamide 10. While the reaction 2 with carbon disulphide in the presence of two equivalent potassium hydroxide in DMF gave non-isolated potassium salt 11, which was allowed to react with halogenated compounds namely ethyl chloroacetate and methyl iodide afforded the corresponding 3, 3-bis[(ethoxycarbonyl)methylthio]-N-p-tolylacrylamide 12 and 3,3bis-(methylthio)-N-p-tolylacrylamide 13 respectively. Reaction 2 with phenyl isothiocyanate in basic DMF yielded the intermediate potassium sulphide salt 18. Acidification 18 with dilute hydrochloric acid afforded the corresponding thiocarbamoyl derivative 19. Treatment of intermediate 18 with methyl iodide, phenacyl bromide and ethylchloroacetate afforded the 3-anilino-3-(methylthio)-N-p-tolylacrylamide 20, 2-(1,3-thiazol-2(3H)-ylidene)-N-p-tolylacetamide 21 and 2-(4-oxo-3phenyl-1,3-thiazolidin-2-ylidene)-N-p-tolylacetamide 22 respectively. The structure of the newly synthesized compounds has been confirmed by elemental analysis and spectra data. Synthesized compounds 2, 3, 6, 13, 15a, 15b, 17, 20, 21, 22 and 23 were screened for their antibacterial activities in vitro against Gram-positive (Staphylococcus aureus and Bacillus subtilis), Gram-negative (Pseudomonas aeuroginosa and Escherichia coli ) and antifungal activities against (Aspergillus fumigates, Syncephalastrum racemosum, Geotrichum candidum and Candida albicans).


Introduction
Aromatic heterocycles are valuable synthetic templates for the preparation of new compounds with specific biological, pharmaceutical and material properties.The pursuit of these properties requires efficient synthetic routes that allow rapid construction of diverse aromatic heterocycles with defined substitution patterns.Therefore, αoxoketen dithioacetals and thiocarbamyl as the organic synthetic intermediates have been widely used in the formation of alicyclic, aromatic and heterocyclic compounds [1]- [4].In view of the above and in continuation of our studies on the synthesis of heterocyclic compounds exhibiting biological activity, we report here the synthesis of some novel heterocycles compounds incorporating imidazole moiety from α-oxoketen dithioacetals and thiocarbamyl.

Experiment
All melting points were determined in open glass capillaries on a Gallen kamp apparatus and are uncorrected.IR spectra (cm −1 ) were recorded on a Pye-Unicam spectrophotometer type 1200 using KBr discs.1H-NMR spectra were recorded on a Varian EM-390 (90 MHz) spectrometer using TMS as an internal standard and dimethyl sulphoxide (DMSO-d6) as a solvent.Chemical shifts were expressed in δ (ppm) values and mass spectra were determined on Finnigan Incos 500 (70 ev).Elemental analyses were determined using a Parkin-Elmer 240C Microanalyzer.The microanalyses were performed at the Microanalytical Unit, Faculty of Science, Cairo University.

3-Anilino-2-[1-(4-chlorophenyl)-4,5-diphenyl-1H-imidazol-2-ylthio]-3-(methylthio)-N-ptolylacrylamide 20
To a stirred solution of potassium hydroxide (0.01 mol) in DMF (20 ml) was added compound 2 (0.01mol).After the mixture was stirred for 30 min, phenyl isothiocyanate (0.01 mol) was added to the resulting mixture.Stirring was continued for 6 h, and then methyl iodide (0.01 mol) was added and stirring was continued for 6 h.The reaction mixture was poured onto ice-cold water.The solid product that formed was collected by filtration, dried and recrystallized from ethanol to give compound 20 as yellow crystals.Yield: 62%.M.p.: 234˚C -236˚C; IR (KBr, cm  (0.01 mol) and phenyl isothiocyanate (0.01 mol).The reaction mixture was stirred at room temperature for 6 h, and then treated with phenacyl bromide and/or ethyl chloroacetate (0.01 mol) and the stirring was continued at room temperature for further 10 h.The reaction mixture was poured into 50 ml of cold water.The result solid products were collected by filtration and recrystallized from a mixture of ethanol/DMF (

Antimicrobial Assays
Synthesized compounds 2, 3, 6, 13, 15a, 15b, 17, 20, 21, 22 and 23 were screened for their antimicrobial activities in vitro against two species of Gram-positive bacteria, namely Staphylococcus aureus (RCMB 0100010) and Bacillus subtilis (RCMB 010067), two Gram-negative bacteria, namely Pseudomonas aeuroginosa (RCMB 010043) and Escherichia coli (RCMB 010052) and against four species of fungi, namely Aspergillus fumigatus (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotrichum candidum (RCMB 05097) and Candida albicans (RCMB 05036).The antibacterial and antifungal activities were determined by means of inhibition % ± standard deviation at a concentration of 100 μg/ ml of tested samples [5]- [7].Optical densities of antimicrobial were measured after 24 hours at 37˚C to bacteria and measured after 48 hours at 28˚C to fungal using a multidetection microplate reader at the Regional Center for Mycology and Biotechnology (Sun Rise-Tecan, USA at 600 nm) Al-Azhar University.Ampicillin, gentamicin and amphotericin B were used as references to evaluate the potency of the tested compounds under the same conditions.

Chemistry
The synthetic procedures adopted to obtain the target compounds are depicted in Schemes 1-4.S-Alkylation of 1-(4-chlorophenyl)-4,5-diphenyl-1H-imiazole-2-thione 1 with 2-chloro-N-p-tolylacetamide afforded the corresponding 2-(1H-imidazol-2-ylthio)-N-p-tolylacetamide derivative 2. The assignment of structure 2 was based on both elemental analysis and spectral data.1H-NMR spectrum of 2 in (DMSO-d6) revealed signals at 2.24 ppm corresponding to CH 3 group and a single at 4.11 ppm for CH 2 group.Moreover, mass spectrum showed a molecular ion peak at m/z 510 corresponding to a molecular formula C 30 H 24 ClN 3 OS.Further evidence for the structure of compound 2 was obtained through studying their chemical reactivity via some chemical reactions.Thus, interaction of compound 2 with p-chlorobenzaldehyde yielded the arylidene derivative 3 (Scheme 1).1H-NMR spectrum in (DMSO-d6) of 3 show the disappearance of CH 2 protons observed with the respective starting precursors 2 at δ 4.11 ppm, and the appearance multiple signals in the region at δ 6.41 -7.27 ppm corresponding to  the aromatic protons together with 1H benzylidene C=CH proton.Mass spectrum of 3 showed a molecular ion peak at m/z 632 corresponding to the molecular formula C 37 H 27 Cl 2 N 3 OS (see experimental section).
The structure of the pyrazole derivatives 4 and 5 were established on the basis of analytical and spectral data.
The IR spectrum of 4 showed the disappearance of absorption band of C=O group and appearance of new absorption band of NH at 3238 cm −1 .Mass spectrum of 4 showed a molecular ion peak at m/z 644 corresponding to the molecular formula C 37 H 27 Cl 2 N 5 S. Diazotization of p-chloroaniline followed by coupling with active methylene group in compound 2 in pyridine yielded the hydrazone form 6 rather than the azo form 7 based on spectral data [10].The 1H-NMR spectrum of compound 6 recorded in (DMSO-d6) revealed a signal at δ 8.58 ppm, which could be attributed to hydrazone NH group (Scheme 1).
While the reaction 2 with carbon disulphide in the presence of two equivalent potassium hydroxide in DMF to give non-isolated potassium salt 11, which was allowed to react with halogenated compounds namely ethylchloroacetate [13] and methyl iodide [14] afforded the corresponding 3,3-bis[(ethoxycarbonyl)methylthio]-N-p-tolylacrylamide 12 and 3,3-bis-(methylthio)-N-p-tolylacrylamide 13 respectively.We suggest a mechanism for the formation of 12 in which the intermediate I is obtained first, then elimination of potassium chloride (Figure 1).
The structure of synthesis compound 12 and 13 ware elucidated on the basis of the elemental analysis and spectral data.For example, 1H-NMR spectra in (DMSO-d6) of 12 displayed two multiple at 1.13 -1.23 and 4.04 -4.13 for ethoxy protons of two carboethoxy group and two single at 4.33 and 4.37 ppm for two methylene protons.On the other hand, 1H-NMR spectrum (DMSO-d6) of 13 showed single signal at δ 2.28 ppm for 6 protons of two similar methyl protons.The mass spectrum of compound 13 showed a molecular ion peak at m/z 614 corresponding to a molecular formula C 33 H 28 ClN 3 OS 3 (Scheme 2).
Moreover, condensation of 13 with hydrazine hydrate afforded the corresponding 3, 3-dihydrazino-N-p-tolylacrylamide derivative 14.The structure of 14 was identified as the reaction product on the basis of its elemental analysis and spectral data.The 1H-NMR spectrum of 14 showed a multiple signals in the region at δ 7.09 -7.56 ppm corresponding to the aromatic protons together with the NH proton, two single signals at δ 4.84 ppm and 4.89 corresponding to the two NH 2 protons, and another two single signals at δ 10.33 and δ 10.51 ppm assignable to two NH protons.Mass spectrum of 14 showed a molecular ion peak at m/z 582 corresponding to a molecular formula C 31 H 28 ClN 7 OS.In addition, the condensation of 13 with suitable amine namely o-phenylenediamine, and o-aminophenol [15] in refluxing absolute ethanol to afford the corresponding 2-(1H-benzimidazol-2-yl)-and 2-(1,3-benzoxazol-2-yl)-N-p-tolylacetamide 15a, b respectively (Scheme 3).The structures of compounds 15a, b were established and confirmed by their elemental analysis and spectral data (see experimental section).The formation of 15 a, b were assumed to proceed through nucleophilic attack of the two -NH2 group in o-phenylenediamine,or NH,OH groups in o-aminophenol to the ethylenic double bond in the compound 13 followed by elimination of two moles of methyl mercaptan (Figure 2).S,S-acetals 13 was converted to corresponding S,N-acetals by reacting with appropriate primary.Thus, reaction of 13 with p-chloroaniline afforded ketene N,S-acetals 16 (Scheme 3).The assignment of the structure of 16 was based on spectral data.The IR spectrum of 16 showed absorption bands at 3246, 3191 cm −1 for two NH.Its 1H-NMR spectrum (DMSO-d6) of 16 showed single signal at δ 2.39 ppm for SCH 3 protons, δ 7.09 -7.62 ppm corresponding to the aromatic protons together with the NH proton and single signals at δ 10.29 ppm assignable to NH.The mass spectrum of 16 showed a molecular ion peak at m/z 693 corresponding to the molecular formula C 38 H 30 Cl 2 N 4 OS 2 .
Furthermore, the reaction of 13 with Glycine in ethanol containing triethylamine [16] afforded the corresponding 1H-pyrrole-2-carboxylic acid derivatives 17 (Scheme 3).The assignment of the structure of 17 was based on spectral data.The IR spectrum of 17 showed absorption bands at 3436 cm −1 (OH) and 3247, 3184 cm −1 (NH).Its 1H-NMR spectrum (DMSO-d6) single signal at δ 2.29 ppm for SCH 3 protons, δ 7.09 -7.47 ppm corresponding to the aromatic protons together with the 2NH protons.The mass spectrum of 17 showed a molecular ion peak at m/z 623 corresponding to the molecular formula C 34 H 27 Cl N 4 O 2 S 2 .
2-(1H-imidazol-2-ylthio)-N-p-tolylacetamide 2 was utilized as a key intermediate for the synthesis of thiocarbamoyl derivative 19 via its reaction with phenyl isothiocyanate.Thus, reaction 2 with phenyl isothiocyanate in DMF in the presence of an equimolar amount of potassium hydroxide yielded the non-isolable intermediate potassium sulphide salt 18. Acidification of the potassium salt 18 with dilute hydrochloric acid afforded the corresponding thiocarbamoyl derivative 19, which can exist in two tautomeric thione-thiol forms A and B (Scheme 4).Assignment of the product 19 was based on elemental analysis and spectral data.1H-NMR spectrum displayed multiple signals at δ 7.05 -7.59 ppm for aromatic protons and two single signals at δ 10.40 and 10.20 ppm assignable to two NH protons.Mass spectrum showed a molecular ion peak at m/z 645 corresponding to a molecular formula C 37 H 29 ClN 4 OS 2 .
Treatment of the non-isolable potassium sulfide salt 18 with methyl iodide [17] afforded the ketene N, Sacetal 20.The structure of 20 was established on the basis of its elemental analysis and spectral data.Its IR spectrum showed absorption bands at 3246, 3191 cm −1 due to two NH groups.On addition 1H NMR spectrum (DMSO-d6) displayed single signal at δ 2.54 ppm for SCH 3 .The mass spectrum showed a molecular ion peak at m/z 659 corresponding to a molecular formula C 38 H 31 ClN 4 OS 2 (Scheme 4).
On the other hand, reaction of 18 with phenacyl bromide and ethyl chloroacetate [18] afforded 2-(3, 4-diphenyl-1,3-thiazol-2(3H)-ylidene)-and 2-(4-oxo-3-phenyl-1,3-thiazolidin-2-ylidene)-N-p-tolylacetamide 21 and 22 respectively.The structures of compounds 21 and 22 were established and confirmed by their elemental analysis and spectral data.The 1H-NMR spectrum of 21 showed a multiple signals in the region at δ 7.05 -7.59 ppm corresponding to the aromatic protons together with the H-5 protons of the thiazole ring and a single signal at δ 10.40 ppm for NH proton.Mass spectrum of 21 revealed a molecular ion peak at m/z 745 corresponding to a molecular formula C 45 H 33 ClN 4 OS 2 .The IR spectrum of 22 showed absorption bands at 1726 cm −1 due to CO of thiazolidinone ring.The 1H-NMR spectrum of 22 showed a single signal equivalent to two protons at  4.13 ppm which represent the CH 2 protons of the thiazolidinone ring.The Claisene Schmidt condensation of thiazolidin-5-one 22 with benzaldehyde [19] in DMF and in the presence of a catalytic amount of piperidine afforded arylidene derivatives 23 (Scheme 4).The structures of latter products were confirmed based on elemental analysis and spectral data (see experimental section).
In general, most of the tested compounds revealed better activity against the Gram-positive bacteria rather than the Gram-negative bacteria.Compounds 3, 13 and 15a exhibited excellent antibacterial activity against the tested organisms while compounds 15b, 17, 20, 21 and 23 showed moderate antibacterial activity against the tested organisms and compound 2, 6 and 22 showed weak antibacterial activity against the tested organisms.In addition, all test compounds were found to be inactive against Pseudomonas aeuroginosa (RCMB 010043).

Conclusion
In this paper, we report the synthesis of 2-(1H-imidazol-2-ylthio)-N-p-tolylacetamide 2. The active methylene moiety of compound 2 was allowed to react with CS 2 and/or phenyl isothiocyanate in dimethylformamide in the presence of potassium hydroxide and yielded the non-isolable intermediate potassium sulphide salt 8, 11 and 18, which is used as intermediate to synthesis series of novel substituted imidazole derivatives in good yield.Synthesized compounds 2, 3, 6, 13, 15a, 15b, 17, 20, 21, 22 and 23 were evaluated for antibacterial and antifungal activities.Most of the tested compounds revealed better activity against the Gram-positive rather than the Gramnegative bacteria.Compound 13 exhibited excellent antibacterial activity against Staphylococcus aureus (RCMB 0100010), Bacillus subtilis (RCMB 010067) and Escherichia coli (RCMB 010052).Compounds 13, 15a exhibited excellent antifunger activity, which is better than the amphotericin B against Syncephalastrum racemosum (RCMB 05922).

Figure 3 .
Figure 3. Graphical representation of the antibacterial activity of tested compounds compared to ampicillin and gentamicin.

Figure 4 .
Figure 4. Graphical representation of the antifungal activity of tested compounds compared to amphotericin B.

Table 1 .
Antibacterial evaluation of the some synthesized compounds.

Table 2 .
Antifungal evaluation of the some synthesized compounds.