Statistical Analysis of Antimicrobial Data of 2-[2-(Aroyl)aroyloxy]methyl-1,3,4 Oxadiazolesanalogues Using ANOVA

 

Fares HezamAl-Ostoot^1,3, Vidya, R. ², Zabiulla¹, Yasser Hussein Eissa Mohammed¹,

Mahima Jyothi¹, Pallavi. H.M¹, Shaukath Ara Khanum¹*

¹Department of Chemistry, Yuvaraja’s College, University of  Mysore, Mysuru, Karnataka, India.

²Department of Statistics, Yuvaraja’s College, University of Mysore, Mysuru, Karnataka, India.

³Department of  Biochemistry, Faculty of Education and Science, University of Albaidha, Albaidha, Yemen.

*Corresponding Author E-mail: shaukathara@yahoo.co.in

Heterocyclic chemistry has become one of the most important fields of research in pharmaceutical industry due to their many fold applications. Amongst all, heterocyclic molecules containing nitrogen and oxygen (like oxadiazole ring system) have shown most potent biological activities. Microorganisms are strictly attendant with the fitness and well-being of human beings. The present treatments of bacterial and fungal infections are a bit unsatisfactory, owing to rapidly developing drug resistance and side effects. The improved of antibacterial and antifungal agents results in resistant to drugs.A series of substituted Oxadiazoles analogues 4a-jwere synthesized and screened for their antibacterial and antifungal activities to evaluate zone of inhibition. The significant effect ofin-vitroantimicrobial and antifungal activities of compounds 4a-jwere studied using one way ANOVA. It was shown that the variables 4a-j series weresignificant. Further pairwise comparison of  significant differences between the variables were analyzed using Tukey HSD. The data revels that compound 4a with 2-methyl and 3-chloro group in phenyl and benzoyl ring respectively, have shown excellent activity and more potent among the 4a-j series.

 

 

 

 

 

 

INTRODUCTION:

The evolution of “Antimicrobial resistance” (AMR) is perplexing and serious health problem. The resistance gained by bacteria against antibiotics makes increasingly difficult the treatment of infection diseases [1, 2]. Thus, it is necessary to develop alternatives for the treatment of infections caused by microorganisms [3]. The treatment of infectious diseases still remain a challenging task because of combination of factors such as an alarming increase in number of multi-drug-resistant microbial pathogens. Nitrogen containing compounds are considered promising drug candidates due to their broad range of activity, low toxicity and decreased resistance development by the target cell [4-6]. Heterocyclic compounds play an important role in an untiring effort aimed at developing new antimicrobial and antitumor agents with new mechanism of action [7]. The place of oxadiazoles derivatives in biomedical research, highlighting their versatile biological properties, the mode of action and Structure Activity Relationship (SAR) studies for a variety of antimicrobial, anti-tubercular, anticancer activity along with some enzyme inhibitory activities [8]. 1,3,4-Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen atoms in a five-membered ring. It is derived from furan by substitution of two methylene groups (=CH) with two pyridine typesnitrogen (-N=) [9, 10]. However,1,3,4-oxadiazole are better known and more widely studied by researchers because of their many important chemical and biological properties [11-14]. Through five-membered aromatic heterocyclic 1,3,4-oxadiazoles have attracted considerable interest in recent decades due to their broad spectrum of pharmaceutical and biological activities [15]. Among heterocyclic compounds, nitrogen containing compounds like 1,3,4 oxadiazoles have exhibited broad spectrum antitumor activity with an IC50 concentration range from 7.21 mMto 25.87 mMagainst the HeLa cancer cell lines [16] and with an IC50 of 9.3 mM against the DU145 cancer cell lines [17]. Oxadiazole analogs also inhibit bacterial growth by inhibiting DNA replication or DNA transcription [18].Many compounds containing this scaffold display antimicrobial [19], antiviral [20], anti-inflammatory[21], antihypertensive [22],  analgesic [23], anticonvulsant[24],  antidiabetic[25] and antitubercular activities [26]. They have also attracted attention in medicinal chemistry as potential therapeutic agents for the treatment of cancer [27] and HIV infections [28]. Recently our group has demonstrated that compounds containing oxadiazole have induced cytotoxicity and antiproliferative effect on various cancer cell lines [29,30].In this paper, we have applied ANOVA techniques that reveals the significance of in-vitro antifungal activity of compounds 4a-j.Further we applied Tukey HSD for the pairwise comparison of  significant differences between these compounds when the result shown significance differences among different compounds.

 

RESULTS AND DISCUSSION

The reaction sequence for the title compounds is outlined in scheme. Compounds (1a–j) to (3a–j) have been prepared as previously reported by our group [16–18]. Compounds (3a–j) react with triethylorthoformate under go intramolecular cyclization, to afford substituted 2-[2-aroylaroyloxymethyl]-1,3,4-oxadiazoles (4a–j). The antimicrobial activities of synthesized compounds were screened against eight bacteria and four fungi using in vitro disc diffusion method. The results revealed that most of the synthesized compounds exhibited antimicrobial activities against Staphylococcus aureus, S. aureus (MRSA), Enterobacter aero genes, Micrococcus luteus, Klebsiella pneumonia, Salmonella typhimurium, S. paratyphi-B, Proteus vulgaris, Candida albicans, Botyritiscinerea, Malassesiapachydermatis, and C. krusei organisms. The results are summarized in Table 1. Compounds (4a), (4d), (4g), (4h), and (4j) showed good activity more than standard drug against S. aureus. Compound 4a with methyl and chloro groups at the para position in phenyl ring and the meta position in benzoyl ring, respectively showed excellent activity against both Gram-positive and Gram-negative bacteria among all synthesized compounds as compared with the standard. Among the compounds (4g–j) in which chloro group is substituted in the phenyl ring compounds (4g), (4h) and (4j) show good activity against S.aureus. Compound (4a) showed significant antifungal activity against B.cinerea and C.krusei. In contrast, com- pounds (4b) and (4i) with bromo and (4c) with methoxy groups exhibited lowest activity and this can be attributed to the bulkiness of bromo and methoxy groups which might render the molecule to penetrate through the cell wall. Thus compounds (4a), (4e), (4g), (4h) and (4j) showed good activity against S. aureus. In comparison to compound (4d), the presence of chloro group in the benzoyl ring of compounds (4a) and (4e) increased the potency against S.aureus by one fold. Interestingly the presence of chloro group in the phenyl ring in compounds (4g), (4h) and (4j) increased the potency against S.aureus by two fold. In comparison to compound 4d, in 4a the potency is increased by two fold against bacteria S.paratyphi-B, three fold by S. aureus (MRSA) and S. typhimurium, four fold by M.luteus and K pneumonia and fivefold by E. aerogenes. Besides, the potency of compound (4a) is increased by two fold against fungi B. cinerea, three fold by C.krusei and four fold by C.albicans compared to compound (4d). In general, compound (4a) showed excellent activity than standard drugs for most of the tested bacteria and fungi.

 

EXPERIMENTAL:

Chemicals were purchased from Aldrich Chemical Co.TLC was performed on aluminum-backed silica plated with visualization by UV-light. Melting points were determined on a Thomas Hoover capillary melting point apparatus with a digital thermometer. IR spectra were recorded in Nujol on FT-IR Shimadzu 8300 spectrometer and 1H NMR spectra were recorded on a Bruker 400 MHz spectrometer in CDCl₃.Chemical shifts were recorded in parts per million downfield from tetramethylsilane. Mass spectra were obtained with a VG70-70H mass spectrometer and the elemental analysis (C, H, and N) was performed on Elementar Vario EL III elemental analyzer. The synthesis of the hitherto unreported title com- pounds is as outlined in scheme in 65–73.5% yield. Hydroxy benzophenones (1a–j) on reaction with ethyl chloroacetate affords ethyl (2-aroylaryloxy)acetates (2a–j) in excellent yield,which on treatment with hydrazine hydrate yields corresponding 2-(2-aroylaryloxy)acetohydrazides (3a–j) [16–18]. Intramolecular cyclization of (3a–j) with triethylorthoformate resulted substituted 2-[2-(aroyl)aroyloxy]methyl-1,3,4-oxadiazoles(4a–j).

 

Synthesis of 2-[2-(3-chlorobenzoyl)-4-methylphenoxy] methyl-1,3,4-oxadiazole (4a)

A suspension of compound (3a) (0.52 g, 1.6 mmol) in triethylorthoformate (10 mL) was refluxed until (3a) disappeared. A solid was separated on cooling, which was filtered off, dried and recrystallized from ethanol to afford compound (4a). Compounds (4b–h) were synthesized analogously starting with derivatives (3b–h), respectively.

 

4a: Yield 72%. Mp 116–118°C; IR(Nujol):1648 (C=N), 1665 cm–1 (C=O); 1H NMR (CDCl3): δ 2.3 (3 H, s, CH3), 4.55 (2 H, s, CH2), 6.85–7.7 (7 H,m,Ar-H), 9.4 (1 H, s, oxadiazole-H). MS: m/z 328.5(M+,78), 301.5(49.5), 299.5(52.5), 285.5(36.5), 139.5 (100),   111.5   (13.5).   Anal.   calcd.   for  C₁₇H₁₃ClN₂O₃(328.5): C, 62.11; H, 3.99; Cl, 10.78; N, 8.52.  Found: C, 62.09; H, 3.95; Cl, 10.75; N, 8.50%.

 

MATERIALS  AND METHODS  FOR THE ANTIMICROBIAL ACTIVITY

Streptomycin and ciprofloxacin (Sigma) were used as positive controls against bacteria. Fluconazole and ketoconazole (Himedia, Mumbai) were used as positive controls against fungi. The following Gram-positive bacteria were used for the experiments; S. aureus (MTCC 7443), S. aureus (MRSA) (MTCC 84), E. aerogenes (MTCC 111), M. lu-teus (MTCC 1538). The Gram-negative bacteria included, K. pneumonia (MTCC 109), S.typhimurium (MTCC 2488), S. paratyphi-B (MTCC 733), P. vulgaris (MTCC 321). In addition, fungi C.albicans (MTCC 227), B.cinerea (MTCC 2880), M.pachydermatis,C. krusei (MTCC 231) were also used for the experiments. All cultures were obtained from the Department of Microbiology, Manasagangotri, Mysore. Bacterial inoculums were prepared by growing cells in Mueller Hinton Broth (MHA) (Himedia) for 24 h at 37°C. These cell suspensions were diluted with sterile MHB to provide initial cell counts of about 104 CFU/ml. The filamentous fungi were grown on sabouraud dextrose agar (SDA) slants at 28°C for 10 days and the spores were collected using sterile doubled distilled water and homogenized. Antibacterial activity was carried out using a disc diffusion method [20]. Petri plates were prepared with 20 mL of sterile Mueller Hinton Agar (MHA) (Himedia, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. The tests were conducted at 1000 µg/disc. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvent. Streptomycin (10µg/disc) was used as positive control. The plates were incubated for 24h at 37°C for bacteria and 48h at 27°C for fungi. Zone of inhibition was recorded in millimeters and the experiment was repeated twice.

 

STASTISTICAL ANALYSIS

All results were analysed using descriptive statistical techniques such as mean and standard deviation. One-way ANOVA was employed to test the significance and was considered statistically significant. Tukey’s multiple-range test was performed for multiple-parameter analysis. All statistical analysis was performed by SPSS statistical software version 11 (SPSS Inc. Chicago, USA).

 

Antibacterial Activity

Eleven compounds studied with different concentration levels on both gram positive and gram negative bacteria, formed four subsets in accordance with the zone of inhibition values. A one-way ANOVA was conducted to compare the effect of In-vitro antibacterial activity of compounds (4a-j). From Table 1,we have found a statistically significant result. Since the result was significant, we computed a Turkey HSD test to compare the compound pair wise. From Table 1 it is observed that the in-vitro antibacterial activity of compounds 11 (Streptomycin) and 4a significantly different from all other compounds. But Streptomycin is used as standard to correlates the lead compound from the series (4a-j). It is evident from the ANOVA that the compound (4a) with 2-methyl  and 3-chloro group at phenyl and benzoyl ring respectively, exhibit significantly high antibacterial activity compare to the all other synthesized compound in the series (4a-j) and also with standard.

 

 

Fig 1. Box -plots  represent Zone of inhibition in mm of 11compounds 4a-j.

 

Table 1:In-vitro antibacterial activity of compounds 4a-j

Compounds	Zone of inhibition in mm								Mean	SD
	Gram positive bacteria				Gram negative bacteria
	S. aureus	S. aureus (MRSA)	E. aerogenes	M. luteus	K. pneumonia	S. typhimurium	S. paratyphi B	P. vulgaris
(4a)	19	17	22	23	25	26	29	27	23.50d	4.07
(4b)	10	9	9	10	11	10	9	9	9.63a	0.74
(4c)	11	9	9	10	9	10	7	11	9.50a	1.31
(4d)	18	9	11	14	14	16	10	11	12.88abc	3.14
(4e)	16	13	14	14	14	13	12	10	13.25abc	1.75
(4f)	15	11	15	13	11	17	9	12	12.88abc	2.64
(4g)	23	14	13	19	13	20	11	16	16.13c	4.16
(4h)	21	13	17	18	11	20	9	13	15.25bc	4.37
(4i)	12	10	10	11	10	12	7	9	10.13ab	1.64
(4j)	23	15	18	16	14	23	9	17	16.88c	4.64
Streptomycin	17	22	24	26	22	25	19	24	22.38d	3.07

Each value represents mean ± SE. Zone of inhibition mean ± SD followed by different letters with in a column indicate significance differences according to ANOVA and (Turkey HSD) test (p<0.05)

 

Antifungal Activity

Ten compounds (4a-j), along with standard ketanazole were studied with different concentration levels on four different fungi. From Table 2it is observed that there is significant difference between on in-vitro antifungal activity of the series 4a-j. A one-way ANOVAtool was applied to compare and compute the effect of in-vitro antifungal activity of compounds (4a-j). From Table 2,we have found that the result showing statistically significant. Since the result was significant, we computed a Turkey HSD test to compare the compound pair wise. Table 2 as well as shows that the4ais more significantly different from all other compounds on in-vitro antifungal activity of the series 4a-j along with standard (ketanozole). Furthermore, from the ANOVAtechnique it evident that the compound (4a) with 2-methyl  and 3-chloro group at phenyl and benzoyl ring respectively, exhibit significantly excellent antifungal activity compare to the all other synthesized compound in the series (4a-j) and also with standard.

 

 

Fig 2. Box-plots  represent Zone of inhibition in mm of 11compounds 4a-j

 

 

 

 

 

Table 2:In-vitro antifungal activity of compounds 4a-j

Each value represents mean ± SE. Zone of inhibition mean ± SD followed by different letters with in a column indicate significance differences according to ANOVA and (Turkey HSD) test (p<0.05)

 

 

 

 

 

 

 

CONCLUSION:

A series of novel biologically active substituted Oxadiazole analogs 4a-j were synthesized and screened for antibacterial and antifungal activities against eight bacteria and four fungi,  then  screening data is subjected to statistical analysis using one way ANOVA technique, the data revealed that title compounds 4a-jare good  antibacterial agent than antifungal. Further the data also revealed that compound 4a with 2-methyl  and 3-chloro group of phenyl and benzoyl ring respectively, have shown excellent activity and more potent among the series 4a-j. These results showed that synthesized compound 4a might be a potential antibacterial agent in near future.

 

ACKNOWLEDGEMENTS:

Fares Hezam Al-Ostoot gratefully acknowledges the financial support provided by the Government of Yemen  and University of Albaidha. Zabiulla gratefully acknowledges the financial support provided by the Department of Science and Technology, New Delhi, Under INSPIRE-Fellowship scheme [IF140407].Yasser Hussein Eissa Mohammed gratefully acknowledges the financial support provided by the Government of Yemen  and University of Hajjah. Shaukath Ara Khanum thankfully acknowledges the financial support provided by VGST, Bangalore, under CISEE Programme [Project sanction order: No. VGST/ CISEE /282].

 

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Received on 12.12.2017         Modified on 20.01.2018

Accepted on 21.02.2018         © AJRC All right reserved