Synthesis, Characterization, and In-Silico Studies of Some Novel Phenylhydrazone Derivatives as Potential Agents for Antimicrobial Activities ^†

Abstract

Antimicrobial chemotherapeutic failure as a result of pathogenic resistance stain is great concern across the globe, there is need to search for an effective antimicrobial agent from synthetic sources to overcome emergent of microbial resistant in clinical practice. The phenylhydrazone derivatives were scientifically found to have wide application in the field of drug discovery due to their anticancer, anti-tubercular, antibacterial, and antifungal activities. The (E)-Substituted-N-(phenylhydrazones) derivatives were obtained by a condensation reaction between substituted acetophenone and substituted phenyl hydrazine through a one-step reaction, resulting of five (5) novel compounds such as HS1 (E)-1-(1-(4-bromophenyl)ethylidene)-2-(2,4-dinitrophenyl)hydrazine), HS2(E)-1-(1-(4-bromophenyl)ethylidene)-2-(4-nitrophenyl)hydrazine), HS3(E)-1-(4-nitrophenyl)-2-(1-(3-nitrophenyl)ethylidene)hydrazine), HS4(E)-1-(2,4-dinitrophenyl)-2-(1-(3-nitrophenyl)ethylidene)hydrazine), and HS5 (E)-1-(1-(3-nitrophenyl)ethylidene)-2-phenylhydrazine) and the in-silico prediction of physicochemical properties were found within Lipinski’s rule of five and the synthesized compounds were established by structurally elucidations on the basis of FTIR, 1D and 2D NMR spectral analysis and the newly synthesized compounds were then subjected to antimicrobial assessment for an in vitro test evaluation using the inhibition zone technique, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC).

1. Introduction

Infectious diseases caused by pathogenic agents have been a major problem for humans that have accounted for the death of millions of people annually worldwide [1]. Report shown that, antimicrobial drug resistance also claims numbers of lives a results of misuses and overuse of antimicrobial drugs accessible over the counter and the mortality rate may likely increases in 2050 if majors is not taken [2]. Recently, pathogenic infectious disease caused by the coronavirus (COVID-19) became one of the top causes of death across the globe and more than 70% who were asymptomatic by this infectious diseases caused unnecessary anxiety [3]. Antimicrobial resistance (AMR) poses drawn an urgent needs for public health problem associated with human life threatening and economic challenge around the globe [4]. AMR occurs when pathogens change over time with no longer responding to antimicrobial drugs agent leading to infection hard to treat [5].

Hydrazones compounds are class of organic compounds synthetically formed by the replacement of oxygen of the carbonyl group to carbon–nitrogen double bonds (R-CH=N-) [6]. Hydrazone can also be a Schiff base compound and their complexes attracted a numbers of attention over the last few years due to their potential active ingredient in pharmaceutical industries such as antimicrobial, antibacterial, antifungal, anti-inflammatory, etc. [7].

2. Materials and Methods

Most of the glassware used for these experiments was made of Pyrex and the glassware was cleaned and rinsed with an organic solvent and dried in an oven before use to avoid the contamination of the products. Some of this equipment was sourced from the Department of Pharmaceutical and Medicinal Chemistry and Pharmaceutical Microbiology, Ahmadu Bello University (ABU), Zaria.

2.1. Reagents, Solvents, and Standard Drugs

All the solvents and reagents used for this research are of analytical grade and were used without further purification. They included Phenyl-hydrazine, 4-bromoacetophenone, 4-nitrophenylhydrazine, Sodium hydroxide (20%), substituted Acetophenone, Hydrazine hydrate, 2,4-diphenylhydrazine, glacial acetic acid, distilled water, and some organic solvents such as methanol, ethanol absolute, n-hexane, ethyl acetate (EtOAc), chloroform (CHCl₃), and dichloromethane (DCM).

2.1.1. Characterization

The synthesized of Phenylhydrazone derivatives were characterized to ascertained the quality of the work using the series shown below

2.1.2. Physical Appearance of Phenylhydrazone Derivatives

The color and appearance of the synthesized phenylhydrazone compound derivatives were observed and recorded.

2.1.3. Solubility Test of Phenylhydrazone Derivatives

The solubility of synthesized hydrazone was tested and observed in dichloromethane only.

2.1.4. Melting Point Determination of Phenylhydrazone Derivatives

All melting points of the synthesized hydrazone derivative compounds were determined using a Gallenkamp melting point apparatus and were uncorrected at the Department of Pharmaceutical and Medicinal Chemistry of Ahmadu Bello University (A.B.U) Zaria.

2.1.5. UV Lamp Determination of Phenylhydrazone Derivatives

The spot was developed and viewed under a UV lamp.

2.1.6. In Silico Mechanistic Studies

The in-silico physicochemical properties were evaluated using Swiss ADME where the molecular weight, hydrogen bond donor, hydrogen bond acceptor, number of rotatable bond and MLogP value of the synthesized hybrids phenylhydrazone derivatives were predicted based on theoretical oral bioavailability and Lipinski’s rule of five criteria as guided for the choice of molecule. All the five compounds were found to be within the acceptable range of the five set criteria thereby can be consider as potential drugs candidate for antimicrobial agent, Table 1 [8].

2.1.7. Spectroscopic Analysis

The proton magnetic resonance (¹H NMR) and 13C spectral analysis were recorded on a Brucker (Billerica, MA, USA) 300 and 400 MHz instrument in DMSOd6 using tetramethylsilane (TMS) as an internal standard (University of Pretoria, Republic of South Africa). The infrared spectra of the compounds were recorded on an Agilent FTIR Spectrophotometer (Multi-user A.B.U, Zaria, Nigeria).

Detailed structural analyses of the synthesized compounds were performed using Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR), which include proton (¹H) NMR, carbon-13 (¹³C) NMR, correlated spectroscopy (COSY), HSQC, and HMBC.

The FT-IR data are reported in terms of the frequency of absorption in cm⁻¹ while the data for ¹H NMR and ¹³C NMR are reported as chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublet, m = multiplet), and integration (J).

2.2. Chemistry

In this current study, (E)-1-phenyl-2-(1-phenylethylidene) hydrazone derivatives (1–5) were obtained by following the synthetic protocols as mentioned in Scheme 1 below, where an equimolar of both the substituted acetophenone and phenylhydrazone was dissolved in a 100 mL round flask containing 30 mL of absolute ethanol with a few drops of glacial acetic acid as a catalyst, and under refluxed for 3 hours at 65–75 °C (Scheme 1) [9]. The progress of the reactions was monitored by thin-layer chromatography (TLC) using hexane and ethyl acetate (1:1) and the reaction mixture was filtered off using vacuum machine filtration, and dried to obtained the desire product [10,11].

The purity of the compounds were checked by TLC using the solvent ratio of hexane—ethyl acetate (1:1) and their structures were confirm using spectral data (IR, ¹H NMR) and elemental analysis.

3. Results and Discussion

The final products obtained are given below in Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5.

3.1. In-Silico Parameter of Substituted Phenyl Hydrazones

The in-silico parameters of the synthesized compounds are shown in Table 1 below

Table 1. In-silico prediction of physicochemical parameters for phenylhydrazone derivatives.

	Lipinski’s Rule Of Five
Code	Mol. Wt. a (g/mol)	HbA	HbD	nRB	LogP	Lipinski b	GIA	BA Score
HS1	379.17	5	1	5	5.22	Yes	High	0.55
HS2	334.17	3	1	4	4.79	Yes	High	0.55
HS3	300.27	5	1	5	4.46	Yes	High	0.55
HS4	345.27	7	1	6	4.89	Yes	Low	0.55
HS5	255.27	3	1	4	4.03	Yes	High	0.55
Ciprofloxacin	331.34	5	2	3	1.98	Yes	High	0.55
Terbinafine	291.43	1	0	4	4.88	Yes	High	0.55

(^a) Molecular weight in g/mol, (^b) Lipinski rule of 5 [12] (Mwt ≤ 500, MLogP ≤ 4.15, N or O ≤ 10, NH or OH ≤ 5 and number of rotatable bonds ≤ 10; GIA, gastrointestinal absorption; BA, bioavailability.

Table 1. In-silico prediction of physicochemical parameters for phenylhydrazone derivatives.

	Lipinski’s Rule Of Five
Code	Mol. Wt. a (g/mol)	HbA	HbD	nRB	LogP	Lipinski b	GIA	BA Score
HS1	379.17	5	1	5	5.22	Yes	High	0.55
HS2	334.17	3	1	4	4.79	Yes	High	0.55
HS3	300.27	5	1	5	4.46	Yes	High	0.55
HS4	345.27	7	1	6	4.89	Yes	Low	0.55
HS5	255.27	3	1	4	4.03	Yes	High	0.55
Ciprofloxacin	331.34	5	2	3	1.98	Yes	High	0.55
Terbinafine	291.43	1	0	4	4.88	Yes	High	0.55

(^a) Molecular weight in g/mol, (^b) Lipinski rule of 5 [12] (Mwt ≤ 500, MLogP ≤ 4.15, N or O ≤ 10, NH or OH ≤ 5 and number of rotatable bonds ≤ 10; GIA, gastrointestinal absorption; BA, bioavailability.

3.2. Yield and Some Physical Properties of Substituted Phenylhydrazones

The yield and some physical properties, such as the range of melting points and the crystal color of the synthesized compounds, are presented below in

Table 2. Phenylhydrazone synthetic compounds and their properties.

Code	Mol. Formula	Mol. Wt. g/mol	Color	M.P. (°C)	Rf Value	Yield (%)
HS1	C14H11BrN4O4	379.17	Pink	213–215	0.91	40.00
HS2	C14H12BrN3O2	334.17	Brown	220–222	0.85	67.00
HS3	C14H12N4O4	300.27	Sandy brown	213–215	0.83	67.00
HS4	C14H11N5O6	345.27	Yellow	210–212	0.87	89.00
HS5	C14H13N3O2	255.27	Reddish Brown	110–112	0.89	81.00

HS = hydrazone, Mol. = molecular, Wt. = weight, M.P. = melting point, g/mol = gram per mole, Rf = retardation factor.

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4. Conclusions

Five (5) new compounds of (E)-Substituted-N-(phenylhydrazones) derivatives were synthesized and obtained by one step condensation reaction with an excellent yield of 40–89% where all compounds were structurally elucidated and confirmed by spectral analysis.