Synthesis, Characterization and Study of Anti-Bacterial Activity of Some New Bis-Heterocyclic Derivatives

through the reaction of an aromatic ketone with different aldehydes. The second step involves the reaction of chalcones derivatives ( D1-D3 ) with compound (B) in the presence of copper (I) to obtain a 1,2,3-triazoline compound ( H5 – H7 ). Different spectroscopy methods, such as FT-IR, 1 H-NMR, and 13 C-NMR, were used to identify synthesized compounds. The potential antibacterial activity of the synthesized heterocyclic derivatives has been finally tested as a third stage, using two types of bacteria ( Staphylococcus aureus and Escherichia coli ).


INTRODUCTION
Sulpha medicines, commonly referred to as sulphonamides, are chemical substances with a sulphonamide moiety (-SO2NH-)  in their structure. Sulpha medications were designed to be used as antimicrobial agents in the past (García Ruano et al., 2008), and they remain commonly employed today as preventive and restorative mixtures as a function against various bacterial contaminations in different application areas like eye diseases, flu, meningitis, actinomyces contaminations, and urinary lot infections (Ebrahimi et al., 2013). The sulphonamide is also functioning as an anti-microbial to treat incurable diseases, such as a specialist inhibitor against cancer cells , antagonist against thyroid (Zafar et al., 2021), antagonist against hypoglycemia, antagonist against inflammation , and several other uses in various sectors (Ibrahim et al., 2014). Sulphonamide compounds are designed similarly to para-amino benzoic corrosives, which call for the conjunction of folate in bacterial cells. Para benzoic acid is shielded from exposure by sulphonamides . The Chemistry of Heterocycles describes the 1,2,3-triazole as an unsaturated, aromatic, five-membered, excessive nitrogen heterocycle with a six-electron ring structure made up of two double-bonded carbon atoms and three regular nitrogen atoms. Three categories are used to categorize them: monocyclic 1,2,3-triazoles, benzotriazoles, and 1,2,3-triazolium salts 9 . Monocyclic 1,2,3-triazoles are further split into three subclasses based on the location of the NH proton. While the nonaromatic 4H-1,2,3triazole is not in equilibrium, the aromatic 1H-and 2H-1,2,3-triazoles are, both in the gas phase and solution. Additionally, in this research, some new 1,2,3-triazole derivatives of dapsone, have been prepared .

MATERIALS AND METHODS Experimental General Considerations:
All solvents and chemicals were of the highest analytical grade and used as supplied from commercial sources. The infrared spectra were characterized with FT-IR spectrophotometer (FTIR-8400s, Shimadzu). Nuclear magnetic resonance (NMR) spectra were characterized using Bruker 400 MHz spectrometers at ambient temperature.

Preparation of Azo Compounds (A) (Khalil A et al., 2019):
Take (0.02mol,2.72g) from the aromatic amine compounds (p-amino acetophenone) and dissolved in the beaker containing 4 mL of concentrated HCl and 10 mL of distilled H2O and then the solution was cooled in an ice water bath. The NaNO2 solution was prepared in another beaker by dissolving (0.02 mol., 1.38 g) in 5ml of distilled H2O and also cooled at (0 0 c ) and then added slowly to solution one at the same temperature with constant stirring by the magnetic stirrer. The formed Diazonium salt solution was maintained at (0 0 c) and added drop-wise to (0.02mol., 5 g) Dapsone solution prepared in 10% sodium hydroxide solution. The pH was kept between (8-9) at temperature (0 0 c) and then the mixture was stirred for 30 min. The final product was precipitated, filtered out and washed with distilled water several times and then recrystallized with ethanol.  155.88,151.21,137.26,131.64,129.31,128.80,128.43,122.74,121.82,116.05,26.35,40.76,40.58,40.10,39.98,39.8 8,39.76.

The first prepared azo compound (A) by reaction
Aromatic amine 4-amino acetophenone was converted to diazonium chlorides with concentrated hydrochloric acid and sodium nitrite got in a solid state in good yield, The azide derivative of the azo dye (B) is formed by first forming the azo dapson diazonium ion, then proceeding with the reaction via an azide attack on the diazonium ion, as suggested by Huisgen and Ugi(Huisgen & Ugi, 1956). the amine group (-NH2) of the dapson is linked to the benzene ring, this provides a strong driving force for the reaction to occur with high yield. The chalcones derivatives (D1, D2 and D3) have been synthesized by the Claisen-Schmidt condensation strategy which is the most popular one, with simplicity and higher yields as compared to other traditional methods. It is done between a ketone containing alpha-hydrogen (aceto-phenone) and aromatic aldehyde. Later by adopting 1,3-Dipolar cycloadditions reaction of the azide derivative with the prepared chalcones (D1, D2 and D3), using the copper chloride (CuCl) as a catalyst, we prepared a kind of heterocyclic compounds (H5, H6 and H7) linking two important classes in medicinal chemistry. These reactions have simple conditions and straightforward work-up steps.
All prepared compounds were characterized by 1 H NMR and 13 C NMR spectroscopy. The 1 H NMR spectrum of D1 in DMSO-d6 (Fig. 1) shows the multiple signals in the range of 7.95 -7.35 ppm for CH in the benzene ring. The spectrum also shows a doublet at (6.75-6.67) for CH=CH. For Compound D2, the 1 H NMR spectrum in DMSO-d6 (Fig. 2) shows multiple signals in the range of 7.91 -7.36 ppm for CH in the benzene ring. The spectrum also shows a doublet at (6.70-6.67) ppm for CH=CH group. The single signal at 3.02 ppm for CH3 group. The 1 H NMR spectrum of D3 in DMSO-d6 (Fig. 3) shows multiple signals in the range of 7.98 -7.35 ppm for CH in the benzene ring. The spectrum also shows a doublet at (6.91-6.88) ppm for CH=CH group. The single signal at 9.58 ppm for OH group.  The 1 H NMR spectrum of H5 in DMSO-d6 (Fig. 4) shows multiple signals in the range of 8.28 -7.28 ppm for CH in the benzene ring. The spectrum also shows doublet at (5.66 and 4.31 ppm) for CH in the triazoline ring. The methyl group appeared at 2.57 ppm. For Compound H6, the 1 H NMR spectrum in  shows the multiple signals in the range 8.35 -7.25 ppm for CH in benzene ring. The 1 H NMR spectrum of H7 in DMSO-d6 (Fig. 6) shows the multiple signals in the range 8.33 -7.28 ppm for CH in benzene ring. The spectrum also shows doublet at (6.86 and 4.36 ppm) for CH in triazoline ring. The acetyl group appeared at 2.57 ppm. The single signal at 8.35 ppm for OH group.
The mass spectrum of [C85H42C2N10O6] shows a molecular ion peak [M+] at / 1078.00 which is in accordance with the proposed formula of the compound (H5) (Fig.7).

Antibacterial Study of The Prepared Heterocyclic:
The synthesized heterocyclic compounds were tested using Escherichia coli as Gram-negative and Staphylococcus aureus as Gram-positive bacteria for their anti-bacterial efficacy by a diffusion method in a Mueller-Hinton agar medium. After 24hrs of incubation, the inhibition zones were measured. The anti-bacterial activity of the synthesized compounds (H5, H6 and H7) was studied as a function against one gram-positive bacteria Staphylococcus aureus and one gramnegative bacteria Escherichia coli. The anti-bacterial activity has been initially checked as the recognized growth inhibition zones by disk-diffusion approach employing Mueller Hinton Broth (MBH) environment. Then, Minimum inhibitory concentrations (MIC) were recognized for the produced compounds. DMSO was employed as a negative control for anti-bacterial activity. The observed minimum inhibitory concentration (MIC) anti-bacterial data of the synthesized compounds (H5, H6 and H7). The antibacterial activity findings showed that the tested compounds exhibited various degrees of inhibition against Grampositive and Gram-negative bacteria both tested microorganisms were found to be the sensitive bacteria. All prepared compounds increase their effectiveness with increasing concentration. Compounds (H5, H6 and H7) and the dapsone-1,2,3-triazoles skeleton observed the best anti-bacterial activity against Staphylococcus aureus with MIC values Beside this, compounds (H6 and H7) showed the most anti-bacterial activity against Staphylococcus aureus with MIC value 32 μg/mL. in Figure 9. On the other hand, also, compounds (H5, H6 and H7) give moderate activity against the selected bacteria Escherichia coli. are given in Figure 8.