Hydroxytriazene Derived from Sulphanilamide: Spectrophotometric and Biological Applications

In this investigation, we report synthesis, spectrophotometric application and antimicrobial activities of 3-hydroxy-3-(4-chlorophenyl)-1-(4sulphonamido)phenyltriazene(HCNT) and its Fe(III) complex [Fe(HCNT)2(H2O)2]. The complex has been synthesized by traditional as well as mechanochemical routes. These compounds have been characterized and screened for antimicrobial activity against bacterial strains i.e. E. coli, S. aureus, S. pyogenes, P. aeruginosa and fungal strains i.e. A.clavatus, A. niger, C. albicans using brothmicrodilution method. The results indicate that the compounds may serve as better bactericides compared to fungicides and the molar composition of iron(III) complex was found 1:2 (Fe:HCNT) by spectrophotometric study.


INTRODUCTION
Hydroxytriazenes have been synthesized and used as analytical reagents for spectrophotometric determination during last many years [1][2] . This is an organic compound which have linear -N(OH)-N=Nmoiety and reported as a good chelating agent having nitrogen and oxygen donor atoms. This class of compounds and their metal complexes has been studied for many biological activities such as wound healing 3 , antimicrobial 4-7 , insecticidal 8 , anti-inflammatory 7,[9][10][11] , antioxidant 12 , analgesic [13][14] , antidiabetic [15][16] activities etc. Hydroxytriazene's derivatives have been also used for photo-induced green synthesis of azo dyes [17][18] . Literature survey revealed that the molecules bearing sulphonamide moiety play promising role in modern drug discovery and medicinal chemistry. The sulfonamide group is considered as a pharmacophore because this group showed a broad spectrum of biologically activities. Hydroxytriazenes have good chelating ability to form complex with Fe(III) ions. Only few reports were found on spectrophotometric determination of Fe(III) metal using sulpha drug based hydroxytriazene 19 . In view of this, sulphanilamide based hydroxytriazene and its [Fe(HCNT) 2 (H 2 O) 2 ] (Fe-HCNT) complex have been synthesized using traditional as well as mechanochemical route and screened for antimicrobial activity.
Step-I: Reduction of 4-chloronitrobenzene 4-chloronitrobenzene (3.15 g) was dissolved in a solution of alcohol (14 mL) and distilled water (14 mL) and stirred. In this mixture, saturated solution of 1.06 g of ammonium chloride was added and warm up about 50°C. After then, zinc dust (4 g) was added in small lots with continuous stirring and temperature was maintained between 55°C to 60°C. When the addition of zinc dust was completed, the stirring was continued for some more time and then filtered. The obtained filtrate (4-chlorophenylhydroxylamine) was chilled in freeze and used in Step-III.
Step-II: Diazotization of Sulphanilamide Sulphanilamide (1.72 g) was dissolved in a solution of con. HCl and distilled water. This solution was placed on ice bath and saturated solution of sodium nitrite was added with continuous stirring and maintained temperature around 0-5°C. The diazotized product 4-sulfamoylbenzenediazonium chloride was obtained which was directly used for coupling with 4-chlorophenylhydroxylamine.
Step-III: Coupling of 4-sulfamoylbenzenediazonium chloride with 4-chlorophenylhydroxylamine 4-Chlorophenylhydroxylamine solution was kept on ice bath to maintain temperature around 0°C. After then, 4-sulfamoylbenzenediazonium chloride solution was added drop wise in hydroxylamine with continuous stirring. Saturated sodium acetate solution was used to maintain pH (5-6) of reaction mixture. After the completion of reaction, the crude product was precipitated out. The obtained crude product was recrystallized using ethyl alcohol. All the steps of synthesis are described in scheme1.

Conventional method
In a 100 mL beaker, HCNT (0.65 g) was dissolved in methanol and in another beaker Fe(NO 3 ) 3 .9H 2 O salt (0.40 g) was dissolved in double distilled water. A small amount of tris buffer solution was added to methanolic solution of HCNT to adjust pH 6 and then Fe(III) ions solution was added to HCNT solution dropwise with continuous stirring. Resultant instant black colour was appeared in the solution. When addition of Fe(III) ions was completed, the mixture was stirred for another 30 minutes. The obtained product (Fe-HCNT) was washed and filtered and then recrystallized using ethyl alcohol.

Mechanochemical method
Mechanochemistry deals with either liquid assisted grinding (LAG) or ball milling. The Fe(III) complex has been prepared by ball milling method using SPEX 8000M High Energy Ball Mill. The respective quantities of Fe(NO 3 ) 3 .9H 2 O (0.40 g) and HCNT (0.65 g) were taken to prepare solvent free complex. Mixture was grinded in the mortar pestle initially and transferred to stainless steel vial and grind for 30 min in ball mill. After grinding black coloured product (Fe-HCNT-M) was collected and washed with chilled distilled water and dried.

Spectrophotometric determination of Fe(III) with HCNT
All spectrophotometric measurements have been done at room temperature. Following solutions were prepared for spectrophotometric determination of Fe(III).

Fe(III) solution
A stock solution of Fe(NO 3 ) 3 .9H 2 O (1×10 -2 M) was prepared in double distilled water containing 1-2 drops of conc. nitric acid. Standardization was done by complexometric titration using EDTA and sulphosalicylic acid as an indicator at pH 2.5. This stock solution was used to prepare solution of different concentration as required.

Buffer solution (a)
Tris buffer solution: 100 mL of 1% solution of tris buffer was prepared by dissolving 1 g of tris buffer in double distilled water.
Perchloric acid solution: 100 mL of 1% HClO 4 solution was prepared by diluting 1 mL of HClO 4 in double distilled water.
procedures Determination of working wavelength: To determine the suitable working wavelength, the solution of Fe(III) and HCNT were prepared in 1:10 molar ratio respectively, having 4×10 -5 M Fe(III) and 4×10 -4 M HCNT. Absorption spectrum of the [Fe(HCNT) 2 (H 2 O) 2 ] complex as well as HCNT were recorded in the wavelength region 300-500 nm against reagent blank and working wavelength was found 402 nm. The maximum absorbance of HCNT reagent was obtained at 354 nm Figure1.

Effect of pH on absorbance
A series of a solutions were prepared in the molar ratio 1[Fe(III)]:10 [HCNT] having same concentration used in working wavelength deter mination with different pH range and absorbance was measured at 402 nm. Effect of pH on absorbance was studied in pH range 1 to 9. constant and maximum absorbance was found in the pH range 5 to 6.5 and the colour of developed complex was black Figure 2.

Effect of time on absorbance
The effect of time on absorbance of Fe-HCNT complex formation was studied under optimum conditions. The absorbance of Fe-HCNT complex was measured for 35 minute. This study suggests that formed Fe-HCNT complex was stable with time and did not decomposed Figure 3.
is sensitive and satisfactory for the determination of Fe(III) from solution.

Determination of molar composition of Fe-HCNT complex
In the present research work two methods were used for established molar composition of Fe-HCNT complex namely Job's and Mole ratio method.

Job's method
Job's method of continuous variation is based on change in absorbance with variation in concentration of both reagent and metal by keeping total volume constant.

Validation of Lambert -Beer law
The validation of Lambert-Beer law has been studied by measuring the absorbance of solution by varying Fe(III) and HCNT concentration under optimum conditions. For this study, a set of solution containing Fe(III) and HCNT in molar ratio 1:10 have been prepared. This law was studied in 2×10 -5 M to 4×10 -4 M concentration range Figure 4.

Sandell's sensitivity
The molar absorptivity of the Fe-HCNT complex was calculated from Lambert-Beer law's plot and it is found to be e=1915.82 dm 3 mol -1 cm -1 . Using this value Sandell's sensitivity was calculated which is 29.14 µgcm -2 . This value shows that the method

Mole ratio method
Mole ratio method of Yoe and Jones is based on the constant metal concentration and varying reagent concentration. A series of a solution has been prepared having Fe(III):HCNT in molar ratio range 1:0.5 to 1:10 in which concentration of Fe(III) kept constant i.e. 4×10 -5 M and HCNT concentration has been varied from 2×10 -5 M to 4×10 -4 M. The absorbance was measured at 402 nm under optimum conditions. By this method, the complex composition was again established as 1:2 [Fe(III):HCNT] that was agreed with Job's method. The stability constant of Fe-HCNT complex has also been calculated by this method. The calculated stability constant (β) and free energy were found 4.0257×10 10 and -60481.99 J Mole -1 respectively, for Fe-HCNT complex Figure 6.

Biological activity Antimicrobial Activity
Antimicrobial activities have been assessed by broth micro dilution method 23 and MIC values were determined against bacterial strains viz. E. coli, S. aureus, S. pyogenes and P. aeruginosa and fungal strains viz. A. clavatus, A. niger and C. albicans. MIC is a lowest concentration of antimicrobial drug that inhibits the visible growth of microorganisms after incubation. In this assay, Mueller-Hinton agar has been used as nutrient to grow the drug suspension 24 . The stock solution has been prepared of 2000 µg/mL and dilutionsin DMSO were made. The concentration of samples has been taken 1000, 500 and 250 µg/mL in primary screening and active samples were diluted to 200, 100 and 50 µg/mL in secondary screening. The inoculum size of test microbes has been adjusted to 10 8 Cfu/ mL. The tubes were incubated at 37°C for overnight and visible growth of tested microorganisms was observed. The reported antimicrobial activity was compared with standard drugs, viz, Ampicillin, Chloramphenicol and Greseofulvin. The obtained results are shown in Table 2.

Characterization of solid metal complexes
Solid complex Fe-HCNT (prepared by conventional method) and Fe-HCNT-M (prepared by mechanochemical method) have been characterized by physical properties (Table 1)  This analysis measured the temperature difference between the sample and a thermally inert reference material. This analysis deals with the enthalpy change. DTA curve is a plot of the differential temperature (T sample -T reference ) against temperature. In DTA curve downward peak was observed which shows reaction is exothermic in nature Figure 8.

Antimicrobial activity
The obtained result indicates that compounds are good antibacterial agent than antifungal. Compounds show good antibacterial result against S. aureus bacterial strain compared to standard drug. The metal complex, Fe-HCNT-M which was synthesized by mechanochemical route was showing good activity against E. colias compared to Fe-HCNT because ball milling increases surface area of the metal complexes which responsible for increasing in biological activity.

CONCLUSION
In summary, 3-hydroxy-3-(4-chlorophenyl) -1-(4-sulphonamido)phenyltriazene and its Fe(III) complex have been synthesized via conventional as well as mechanochemical route and characterized by various spectral techniques. The obtained result indicates that complex composition is [Fe(HCNT) 2 (H 2 O) 2 ] and octahedral geometr y of the complex is proposed. The synthesized compounds were found moderate to good antibacterial agent than antifungal agent. The mechanochemical synthesis has been carried at low cost and increased product yield compared to traditional method.

ACKNOwLEDgEMENT
The authors acknowledge SAIF, Chandigarh for NMR spectra, MNIT, Jaipur for mass spectra, Department of Physics, MLS University, Udaipur for TGA-DTA analysis and Department of Chemistry, MLS University, Udaipur for IR spectra. Authors are also obliged to Microcare Laboratory, Surat (Gujrat) for antimicrobial assay.

Conflicts of Interest
The authors declare no conflict of interest.