Synthesis, Spectral Characterization, Antimicrobial and Theoretical Calculation of Some 4-(tosylamino)benzohydrazide Derivatives

The novel synthesis of benzohydrazide derivatives were synthesized by condensation reaction of 4(tosylamino)benzohydrazide and substituted benzaldehyde. The synthesized compounds were characterized by IR, NMR and Mass spectral analysis. The benzohydrazide derivatives 1-4 were tested for antibacterial and antifungal activities. HOMO-LUMO analysis were carried out theoretically using Gaussian-03 package at DFT/B3LYP/6_31G (d, p) method. Dipole moment (D), polarisability (α), and hyperpolarisability (β) values show that the synthesized molecules possess electronic properties.


Introduction
The work of an organic and medicinal chemist is centered on the discovery of new lead compounds with specific medicinal properties. It includes the development of more effective and safer analogues of these new and existing lead compounds. Thus, the main interest of an organic chemist normally lies in conceiving an ideal structure for the needed drug with very minimal or negligible adverse effect usually based on theoretical consideration and in constructing a plausible way for the strategical synthesis towards that target drug.
N-((Pyridin-4-yl)methylnaphthalene-2-amine was synthesized by means of a reaction in which stoichiometric amounts were used of the reagents, viz., 2-naphthylamine,4-pyridylcarboxyaldehyde, anhydrous ethanol to obtain the (Z)-N-((Pyridin-4-yl)methylene)naphthalen-1amine and their later reduction with NaBH 4 produced the required compound [1]. 2-(N-naphthylamido)benzoic acid was synthesized by a solution of phthalic anhydride in acetic acid was added to a solution of naphthylamine in acetic acid and the mixture was stirred at room temperature overnight. The light purple precipitate was filtered, washed with a cold distilled water and air dried [2]. 2-[(4-N,Ndimethylamino)benz-2-ylideneamino]-5(6)-methylbenzimidazole was synthesized by using 2-amino-5(6)-methylbenzimidazole, 4-N, N-(dimethylamino) benzaldehyde in absolute ethanol and the mixture was stand for 20 hrs at room temperature. The yellow precipitate was filtered, washed with distilled water and air dried [3][4][5]. Obafemi and Akinpelu have synthesized 2-oxo-1,2dihydroquinoxaline-6-sulfonyl azide derivatives and 2,3-dioxo-1, 2, 3, 4-tetrahydroquinoxaline-6-sulfonyl azide derivatives and studied their antimicrobial activity against gram-positive and gram-negative bacterial strains [6]. Both the compounds showed broad spectral activity against the bacterial strains. A series of oxazolidinone derivatives carrying sulfonyl group was synthesized and their antibacterial activity was evaluated by Cui et al. and Topala et al. have prepared four new cholesteryl carbamates exhibiting liquid crystal properties and evaluated their thermodynamic properties by differential scanning calorimetry [7,8]. Reflux of tosyl chloride with ptolylurea for about 30 minutes using pyridine as a base and poured the reaction mixture into 10 mL of cold water and stirred until the product crystallizes and filtered off the solid and wash several times with water to get the corresponding derivatives [9].

Synthesis of ethyl-4-(tosylamino)benzoate
A mixture of ethyl-4-aminobenzoate (5 mmol), tosyl chloride (5 mmol) and triethylamine (5 mmol) in 50 mL of methanol was refluxed for about 2 hrs. The reaction is monitored by TLC. After completion of reaction, it was poured into water and extracted with three 50 mL portion of ether. The combined ether extract was then washed well with 3% sodium bicarbonate solution and dried over anhydrous sodium sulphate. The afforded product was purified by column chromatography using benzene: chloroform (6:4) as an eluent. This upon evaporation and subsequent recrystallization furnished the product ethyl-4-(tosylamino)benzoate in pure form (m.p. 242-246; yield 95%).

Synthesis of 4-(tosylamino)benzohydrazide
A mixture of ethyl-4-(tosylamino)benzoate (5 mmol) and hydrazine hydrate (5 mmol) in methanol was refluxed for about 10 hrs (Scheme 1). The reaction was monitored by TLC. After completion of reaction, it was poured into ice cold water to remove the excess of hydrazine hydrate. The solid was filtered and dried. The synthesized compound was further purified by column chromatography using benzene: chloroform mixture (6:4) as an eluent. This upon evaporation and recrystallization furnished the product 4-(tosylamino) benzohydrazide in pure form.

Synthesis of 4-(tosylamino)benzohydrazide derivatives
A mixture of 4-(tosylamino)benzohydrazide (5 mmol), substituted benzaldehyde (5 mmol) and 3 drops of acetic acid in methanol was refluxed for about 2-3 hrs (Scheme 2). The reaction is monitored by TLC. After completion of reaction, excess of solvent was removed. The final mass was poured into ice cold water to remove the excess of the aldehyde. The residue obtained was purified by column chromatography using benzene: chloroform mixture (8:2) as an eluent. This upon evaporation and subsequent recrystallization furnished the product 4-(tosylamino) benzohydrazide derivatives in pure form.

Spectral measurements
IR spectra were recorded in AVATAR-330 FT-IR spectrophotometer (Thermo Nicolet) and only noteworthy absorption levels (reciprocal centimeters) are listed. 1 H NMR spectra were recorded on BRUKER AMX operating at 500 MHz and the 13 C NMR spectra were recorded in the same instrument and the operating frequency is 106 MHz. All NMR measurements were made on 5 mm NMR tubes. For recording 1 H NMR spectra, solutions were prepared by dissolving about 10 mg of the compound was dissolved in 0.5 mL of CDCl3 (or) DMSO. While for recording 13 C NMR spectra, about 50 mg of the compound was dissolved in the same volume of the solvent. Here, tetramethylsilane (TMS) was used as an internal standard. Mass spectra were recorded on VARIAN CP-3800 GC Mass Spectrometer (EI mode).

Antimicrobial studies
Antibacterial studies: The following Gram-positive and Gramnegative strains have been used for the study [19,20].
Determination of antibacterial activity by disc-diffusion method: Nutrient agar plates were prepared under steriled conditions and incubated overnight to detect contamination. About 0.2 mL of working stock culture was transferred into separate nutrient agar plates and spreaded thoroughly using a glass spreader. Whatmann No. 1 discs (6 mm in diameter) were impregnated in the test compounds dissolved in DMSO (200 μg/mL) for about half an hour. Commercially available drug disc (Ciprofloxacin 10 μg/disc) was used as positive reference standard. Negative controls were also prepared by impregnating the disc of same size in DMSO solvent. The discs were placed on the inoculated agar plates and incubated at 37 ± 1°C for about 18-24 hours. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism.

Computational details
The quantum chemical calculations were performed using the Gaussian-03 package [21]. Computations of the vertical excitations, difference density plots and optimization of the ground and excited states were performed using density functional theory (DFT) and time-dependent DFT (TD-DFT) using B3LYP/6-31G (d,p) basis set, respectively. The ground and excited states HOMO and LUMO frontier orbital's of 4-(tosylamino) benzohydrazide derivatives were calculated by methods at the B3LYP/6-31G(d,p) level.

IR spectral analysis of compounds 1, 2, 3 and 4
The FT-IR spectra were recorded in the region of 4000-400 cm -1 .

NMR spectral analysis of 4-(tosylamino)benzohydrazide (1)
1 H NMR spectrum of compound 1, the signals observed in the region of 7.10-7.86 ppm with an expected integral value. Therefore, these signals are unambiguously assigned to aromatic protons. The methyl proton signal is observed in the region of 2.29 ppm as a singlet. A hydrazide N-H proton is observed at 10.61 ppm and sulfonyl attached N-H proton is observed at 9.75 ppm. The NH 2 protons signal are merged with DMSO solvent peak. 13 C NMR spectrum of compound 1 the ipso carbons are absorbed in the higher frequency region compared to the aromatic carbons. In compound 1 the signal appeared in the region 118.33-129.80 ppm is due to the aromatic carbons. The phenyl ipso carbons C-1, C-4, C-8 and C-11 are observed at 127.61, 144.08, 135.76 and 140.37 ppm respectively. A signal appeared at 165.86 ppm is due to carbonyl carbon. Methyl carbon signal is appeared at 20.77 ppm.

NMR spectral analysis of N'-(4-chlorobenzylidene)-4-(tosylamino) benzohydrazide (3)
1 H and 13 C NMR spectra of compound 3 the signals appeared at 7.11-8.30 ppm is assigned to aromatic protons. There are two singlets appeared at 10.75 and 11.86 ppm with one proton integral values. These signals are due to sulfonyl group attached N-H proton and hydrazide N-H proton respectively. The methyl protons signal is appeared at 2.29 ppm. The 13C NMR spectrum the signals appeared in the region 118.14-146.76 ppm is due to the aromatic carbons. The phenyl ipso carbons C-1, C-4, C-8, C-11, C-16 and C- 19

NMR spectral analysis of N′-(4-methoxybenzylidene)-4-(tosylamino) benzohydrazide (4)
1 H and 13 C NMR spectra of compound 4 the signals observed in the region of 6.95-8.26 ppm are assigned to aromatic protons. Further a singlet appeared at 11.70 ppm with one proton integral value. This signal is due to hydrazide N-H proton. There are two singlets appeared in the aliphatic region at 2.27 and 3.74 ppm. These signals are assigned to methyl and methoxy protons respectively. There are two signals resonated at 20.77, 55.33 ppm. These signals are assigned to methyl carbon and methoxy carbon of the aromatic moiety. The signals appeared at 148.22 and 163.02 ppm, these signals are assigned to aldehyde (N=CH) carbon and carbonyl carbon respectively.

Mass spectral analysis of compounds 1, 2, 3 and 4
EI Mass spectra were recorded on VARIAN CP-3800 Gas Chromatography for compounds 1, 2, 3 and 4 and it is displayed in Figure 3. The obtained molecular ion peak (m/z) of the compounds 1,

Antimicrobial studies
The preliminary antimicrobial activity (Figures 4 and 5) of the compounds 1-4 are examined using disc diffusion method. The bacterial strains viz., Escherichia coli, Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa and fungal strains viz., Candida albicans, Aspergillus niger, Mucor and Rhizopus sp . are used in this study. Dimethyl sulphoxide is used as a control while Ciprofloxacin and Amphotericin B are used as a reference for bactrial and fungal studies respectively.
The antibacterial studies revealed that the reported compounds 2, 3 and 4 against E. coli, S. typhi, P. aeruginosa and S. aureus shows considerable inhibition activity whereas compound 1 did not show any inhibition activity against the bacterial strains. The antifungal studies of compounds 1, 2, 3 and 4 indicates that all the compounds exhibit moderate to maximum activity against the reported fungal strains.
The antimicrobial studies concluded that the prepared compound 1, 2, 3 and 4 shows more antifungal inhibition activity than the antibacterial inhibition activity. Further the compound 4 shows excellent antifungal and antibacterial inhibition activity. This may be due to electronic effect (+ I effect) exerted by methoxy group.

HOMO-LUMO molecular orbital studies
The electron density of HOMO-LUMO molecular orbital of all molecules 2, 3 and 4 contributed the entire molecule except the methyl substituted aryl ring and the corresponding energies are tabulated in Table 1. HOMO molecular orbital picture of all molecules show that the bonding character is more in the aryl ring whereas anti-bonding character is more in the LUMO orbital of 2, 3 and 4.   Optimized structures of 2-4: Optimized structures of various compounds are shown in Figure 6.
Energy gap values show that intramolecular charge transfer takes place within the molecule. Dipole moment (D), polarisability (α), and hyperpolarisability (β) values show that the synthesized molecules possess electronic properties.