Synthesis and antimicrobial activity of some new diphenylamine derivatives.

In search of new leads toward potent antimicrobial agent, an array of novel derivatives of 2-hydrazinyl-N-N, diphenyl acetamide has been synthesized from the chloroacetylation reaction of diphenylamine (DPA). For this, a series of DPA derivatives were prepared by replacing chlorine with hydrazine hydrate in alcoholic medium and 2-hydrazino-N, N-diphenylacetamide was synthesized. The 2-hydrazino-N, N-diphenylacetamide was further subjected to reaction with various aromatic aldehydes in presence of glacial acetic acid in methanol. The synthesized compounds were characterized by their IR, 1HNMR spectral data and elemental analysis. The compounds were screened for antibacterial and antifungal activity by cup plate method. 2-(2-Benzylidenehydrazinyl)-N, N-diphenylacetamide (A1); 2-(2-(3-methylbenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A5) and 2-(2-(2-nitrobenzylidine) hydrazinyl)-N, N-diphenyl-acetamide compounds (A7) showed significant antimicrobial as well as antifungal activity. Diphenylamine compounds may be explored as potent antimicrobial and antifungal compounds.

Diphenylamine (DPA) has been used as an antimicrobial agent since a long time due to the presence of nitrogen between two rings. DPA (C6H5)2NH exists as a colorless solid at ordinary room temperature, but often exist in yellow color compound due to oxidized impurities.[1] DPA is the most common stabilizer used in smokeless single base powders.[2] Ring alkylated derivatives of DPA are also useful in the manufacture of rubber product, which exhibit the antioxidant nature of aniline derivatives.[3] In combination with sulfur, it gives phenothiazine, a precursor to neuroleptic and antihistaminic drugs which include chlorpromazine, promethazine, trifluoperazine, thioridazine, mesoridazine, and levomepromazine.[4]

Diphenylamine derivatives are reported to show a variety of biological activities such as antihelmentic, e.g. amoscanate,[5] analgesic and antiinflammatory, e.g. diclofenac, tolfenamic acid,[6] anticonvulsant, e.g. retigabine.[7]

Several researchers revealed that DPA and its derivatives may be used as a potent stabilizer in nitrocellulose containing explosives and propellants. DPA and its derivatives are also used in the production of dyes, photographs chemicals and pharmaceuticals. Epidermal growth inhibitor activity of 2,4 dicarboxamide and its conjugates with DPA has been proved recently.[8] N-β-dimethylaminoethyl derivatives of DPA are known to have potent antihistamine activity.[9] Literature review suggests that still focus is to be laid on synthesis of DPA having fewer side-effects and potent activity.[1011121314] The presence of diphenylamino function is assumed to be responsible for antimicrobial activity with fewer side-effects. In the present communication, we report the synthesis of some new DPA compounds having 2-hydrazino-N, N-diphenylacetamide moiety.[1234567815] The structures of the various synthesized compounds were assigned on the basis of IR, 1HNMR spectral data and elemental analysis. The compounds were also screened for antimicrobial effect [Figures 1 and 2].

Figure 1

Structure of some diphenyl amine derivatives

Figure 2

Synthetic scheme of reaction. A1: R = 2,4,6-tri Cl, A2: R = 4-Cl, A3: R = 2-OH, A4: R = 4-NO2, A5: R = 3-OCH3A6: R = 3-Cl, A7: R = 2-NO2, A8: R = 2,3-CH3, A9: R = 3-NO2

Experimental

Melting points were determined by open capillary tubes and were uncorrected. The IR spectra were recorded in KBr discs on Fourier transform infrared spectrophotometer (8300 Shimadzu). The 1HNMR spectra were recorded in CDCl3 Broker jeol at 300 MHz spectrophotometer using trap mass spectrometer (TMS) as an internal standard. The 1H chemical shifts are reported as parts/million downfield from TMS. The elemental analysis was performed on Carlo Erba-1108 element analyzer. The purity of the compounds was routinely checked by TLC using silica gel-G (Merck). Column chromatography was performed on silica gel (Merck 60-120 msh).

General procedure for the synthesis of substituted aromatic aldehydes derivatives of 2-hydrazino-N, N-diphenylacetamide

Procedure for the synthesis of 2-chloro-N, N-diphenylacetamide

Diphenylamine (0.04 M) was dissolved in toluene (200 ml) and chloroacetylchloride (0.04 M) was added to it. The reaction mixture was refluxed for 4 h, then poured into crushed ice and finally added to the reaction mixture and kept overnight for precipitation of the product. The precipitate was filtered, washed with cold water, dried, and recrystallized from ethanol.

Procedure for the synthesis of 2-Hydrazino-N, N-Diphenylacetamide

2-chloro N-N diphenylacetamide (0.002 M) was stirred in methanol (100 ml) and 0.004 M of hydrazine hydrate was added to this. The reaction mixture was refluxed for 48 h under careful observation, followed by its keeping for overnight in the refrigerator. The product was filtered and recrystallized with ethanol.

Yield-88%; M.P-60-65°C; IR (KBr, cm−1): 3242 (NH Str.); 3139 (NH2); 3031 (CH Str. Aro.); 2978 (CH Str. Alip.); 1630 (C = O Str.); 1411 (C-H Bend); NMR (ppm): 8.68 (1H, NH); 9.06 (1H, NH); 7.58-6.14 (10H, Aro.); 3.43 (3H, CH2).

2-(2-Benzylidenehydrazinyl)-N, N-diphenylacetamide (A1)

Yield-89%; M.P-84-88°C; IR (KBr, cm−1):3431 (NH Str.); 3048 (C-H Str. Aro.); 2949 (CH Str. Alip.); 1623 (C = O Str.); 1574 (C = N Str.). NMR Spectra (ppm): 8.68 (1H, NH); 8.28-6.84 (15H, Aro.); 3.23 (3H, =CH); 2.54 (2H, CH2).

Synthesis of 2-(2-(4-Chlorobenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A2)

Yield-82%; M.P-182-184°C; IR (KBr, cm−1): 3432 (NH Str.); 3047 (CH Str. Aro.); 2941 (CH Str. Alip.); 1623 (C = O Str.); 1399 (C-N Str.); NMR Spectra (ppm): 8.62 (1H, NH); 7.26-7.92 (14H, Aro.); 3.81 (1H, =CH); 2.55 (2H, CH2).

Synthesis of 2-(2-(2-hydroxybenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A3)

Yield-87%; M.P-210-215°C; IR (KBr, cm−1): 3043 (CH Str. Aro.); 2915 (CH Str. Alip.); 1623 (C = O str.); 1572 (C = N Str.); 386 (C-N Str.); 1114 (C-O Str.); NMR Spectra (ppm): 8.88 (1H, NH); 11.23 (1H, OH); 7.91-6.94 (14H, Aro.); 3.28 (1H, =CH); 2.56 (2H, CH2)

Synthesis of 2-(2-(4-nitrobenzylidine) hydrazinyl)-N, N-diphenyl-acetamide (A4)

Yield-79%; M.P-275-280°C; IR (KBr, cm−1): 3431 (NH Str.); 3045, 3112 (CH Str. Aro.); 2932 (CH Str. Alip.); 1627 (C = O str.); 1522 (N = O Str.); 1597 (C = N Str.); 1345 (C-N Str.). NMR Spectra (ppm): 8.81 (1H, NH); 8.35-7.01 (14H, Aro.); 3.23 (1H, =CH); 2.54 (2H, CH2).

2-(2-(3-methylbenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A5)

Yield-88%; M.P-58-60°C; IR (KBr, cm−1): 3404 (NH Str.); 3031 (CH Str. aro.); 2918 (CH Str. Alip.); 1671 (C = O str.); 1597 (C = N Str.); 1370 (C-N Str.); NMR (ppm): 8.61 (1H, NH); 8.13-7.28 (14H, Aro.); 3.37 (1H, =CH); 2.53 (2H, CH2); 2.59 (3H, CH3).

Synthesis of 2-(2-(4-Chlorobenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A6)

Yield-81%; M.P-130-134°C; IR (KBr, cm−1): 3432 (NH Str.), 3061 (CH Str. aro.); 2954 (CH Str. Alip.); 1626 (C = O str.); 1490 (C = N Str.); 1318 (C-N Str.). NMR (ppm): 8.64 (1H, NH); 8.05-7.44 (14H, Aro.); 3.35 (1H, =CH); 2.55 (2H, CH2).

Synthesis of 2-(2-(2-nitrobenzylidine) hydrazinyl)-N, N-diphenyl-acetamide (A7)

Yield-84%; M.P-178-180°C; IR (KBr, cm−1): 3433 (NH Str.); 3042 (CH Str. aro.); 2923 (CH Str. Alip.); 1624 (C = O str.); 1528 (N = O Str.); 1570 (C = N Str.); 1345 (C-N Str.). NMR (ppm): 9.01 (1H, NH); 8.25-7.15 (14H, Aro.); 3.63 (1H, =CH); 2.55 (2H, CH2).

2-(2-(2,3-dimethoxybenzylidene) hydrazinyl)-N, N-diphenyl-acetamide (A8)

Yield-81%; M.P-170-172°C; IR (KBr, cm−1): 3411 (NH Str.); 3003 (CH Str. aro.); 2962 (CH Str. Alip.); 1660 (C = O str.); 1598 (C = N Str.); 1344 (C-N Str.); NMR (ppm): 8.6 (1H, NH); 8.12-7.22 (14H, Aro.); 3.40 (1H, =CH); 2.53 (2H, OCH3); 2.55 (2H, CH2).

Synthesis of 2-(2-(3-nitrobenzylidine) hydrazinyl)-N, N-diphenyl-acetamide (A9)

Yield-89%; M.P-175-178°C; IR (KBr, cm−1):3401 (NH Str.); 3084 (CH Str. aro.); 2926 (CH Str. Alip.); 1671 (C = O str.); 1522 (N = O Str.); 1491 (C = N Str.); 1354 (C-N Str.). NMR Spectra (ppm): 8.86 (1H, NH); 8.74-6.95 (14H, Aro.); 3.34 (1H, =CH); 2.55 (2H, CH2). The element analyses of all the derivatives were performed and are presented in Table 1.

Table 1

Elemental analysis of synthesized compounds

Results and Discussion

Antimicrobial activity

The newly synthesized compounds (A1 − 9) were screened by cup plate method for their antibacterial activity against 2 g positive bacteria viz., Bacillus pumilis (ATCC7061), Bacillus subtilis (ATCC6051) and 2 g negative bacteria viz., Escherichia coli (ATCC35218), Proteus vulgaris (ATCC6380).[910] The stains were procured from Microbiology R and D Laboratory, Department of Pharmacy, IFTM University, Moradabad, India. As standard drug, chloramphenicol was used because of its use in the treatment of a number of bacterial infections. In general, chloramphenicol is considered a prototypical broad spectrum antibiotic, and it is both cheap and easy to manufacture so easy to obtain. It is frequently an antibiotic of choice in the developing world. The agar medium was procured from Hi Media Laboratories Ltd., Mumbai, India. Using a standard procedure the subculture, base layer medium, agar medium and peptone water were performed. Discs having a measurement of 6.25 mm in diameter were punched from Whatman No. 1 filter paper. The test compounds were prepared in different concentrations using dimetylsulfoxide (DMSO). Solutions of the test compounds were prepared by dissolving 5 mg each in 5 ml of DMSO at a concentration of 1000 μg/ml. Volumes of 0.05 ml and 0.1 ml of each compound were used for testing. The cups each of 9 mm diameter were made by scooping out medium with a sterilized cork borer in a petridish, that was streaked with the organisms.

The solutions of each test compound (0.05 ml and 0.1 ml) were added separately in the cups and petridishes were subjected to incubate for 24 h at 37°C. As a standard drug for both the type of strain, chloramphenicol was selected, and aliquots of concentration (200 and 1000 μg/ml) were prepared in water, separately. Parallely, in order to maintain the control group, 0.1 ml of DMSO was added, and this group did not exhibit any sign of inhibition. Zones of inhibition produced by each compound were measured (in mm) and the results of antibacterial studies are presented in Table 1.

The cup plate method using potato-dextrose agar medium was used for screening of antifungal activity against fungal strains, including Aspergellus niger, Rhizopus oryaze and Aspergellus flavus. The growth medium was purchased from Hi Media Laboratories Ltd., Mumbai. Chloramphenicol was obtained as gift sample from crystal Pharma, West Mulund, Mumbai. Fluconazole as standard drug was procured as gift sample from Triveni Interchem Pvt. Ltd., Vapi, India. The nutrient broth medium and other subculture were prepared by method stated in Indian pharmacopoeia. DMSO (100 μg/ml) was used to make solubilize synthesized compounds. As stated above, 0.05 ml and 0.1 ml aliquots of each synthesized compounds were used for screening antifungal activity. Fluconazole (200 μg/ml and 1000 μg/ml) was used in the antifungal activity evaluation. DMSO was used as control and no sign of zone of inhibition were observed. Zone of inhibition produced by each compound was measured in mm. The findings of antibacterial and antifungal evaluations are presented in Tables 2 and 3, respectively.

Table 2

Antibacterial activity of diphenylamine derivatives

Table 3

Antifungal activity of diphenylamine derivatives

The screening results revealed that the compounds A1-9 exhibited significant antimicrobial activity. Specially, the compounds A2 and A4 only exhibited mild antimicrobial activity on the stains of P. vulgaris. The compounds A3 and A9 showed significant antimicrobial activity on the stains of B. pumilis, B. subtilis and E. coli. The compound A1 showed the highest potential against fungal strains of Rhizopus oryzae and A. niger. The synthesized compounds A4 and A9 did not show any sighn of inhibition against A. flavus. All the strains used at concentration of 1 mg/ml (0.01 ml dose) exhibited remarkable antimicrobial and antifungal activities when comparison was made with standard antimicrobial and antifungal drugs as chloramphenicol and fluconazole respectively.

Conclusion

The study revealed the fact that compounds with electron releasing groups such as methoxy, methyl and hydroxyl showed significant antibacterial activity than the others compounds synthesized. The synthesized compounds are having functional moiety such as, chloro groups have exhibited more antifungal activity in comparison to the others. The findings suggested that the DPA derivatives have excellent scope for further development as commercial antimicrobial agent. Further experiments were needed to elucidate their mechanism of action.