Benzofurano-isatins: Search for antimicrobial agents

1 Introduction

Human struggle against the affliction of disease, decay and death is eternal. The deterioration of human population due to an enhanced prevalence of infectious diseases is becoming a global problem. The contemporary treatment of infectious diseases involves administration of a multidrug regimen over a long period of time, which has led to the rapid emergence of multidrug-resistant strains plus a high level of patient noncompliance (Chambhare et al., 2003). The rising prevalence of multidrug resistant superbugs like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) continues to provide impetus for the search and discovery of novel antimicrobial agents. A potential approach to overcome this resistance problem is to design new and innovative agents with a completely different mode of action so that no cross-resistance with the present therapeutics can occur (Khan et al., 2005).

Among an extensive diversity of heterocycles that have been explored for developing pharmaceutically important molecules, benzofuran derivatives have played an important role in medicinal chemistry. Benzofuran derivatives have drawn considerable attention due to their profound physiological and chemotherapeutic properties. Benzofuran has a variety of activities like antimicrobial (Khan et al., 2005; Alper-Hayta et al., 2008), anti-inflammatory (Jadhav et al., 2008), anticancer (Mahboobi et al., 2007; Asoh et al., 2009), antihistaminergic (Gfesser et al., 2005; Peschke et al., 2006; Cowart et al., 2005) and anticholinesterase (Luo et al., 2005; Belluti et al., 2005). Several attempts were made to study the effects of different functional groups on the homocycle and/or the heterocycle for bioactivity. On the other hand isatin, an indole derivative possesses good antimicrobial activity (Pandeya et al., 1999; Pandeya and Sriram, 1998; Mirjana et al., 2006). In the view of biological importance of these two moieties, the present work was undertaken to synthesize a new series of benzofurano-substituted isatins and to evaluate their possible anti-microbial activity.

2 Rationale and hypothesis

The chemistry of benzofurans available in a large number of natural products has attracted widespread interest due to their biological activities and their potential applications as pharmacological agents. Several benzofuran ring systems bearing various substituents at the C-2 position are widely distributed in nature. There are well known natural products having related benzofuran ring structures, the most recognized benzofurans are ailanthoidol, amiodarone and bufuralol compounds. Ailanthoidol, a neolignan with a 2-arylbenzofuran skeleton, was isolated from the Chinese herbal medicine Zanthoxylum ailanthoides (Fuganti and Serra, 1998). It has been reported that neolignans and lignans possess a variety of biological activities such as anticancer, antiviral, immunosuppressive, antioxidant, antifungal and antifeedant activities (Kao and Chern, 2001). Cicerfuran, another antifungal benzofuran derivative, was first obtained from the roots of wild species of chickpea, Cicer bijugum, reported to be a major factor in the defence system against Fusarium wilt (Aslam et al., 2009) (Fig. 1).

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Figure 1

Reported and proposed structure 3(a–p).

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Benzofuran derivatives are of special interest to natural product researchers for their biological activities and potential applications as pharmacological agents, e.g. corsifuran C (Xiao et al., 2008; Toshio et al., 2004; Kuete et al., 2009). Specifically, several benzofuran ring systems bearing various substituents at C-2 and C-3 positions are widely distributed in nature, the stilbenoids structural compounds, e.g. an oligostilbene derivative viniferin, are known to possess antimicrobial, antiviral, antioxidant, antifungal, and antitumor activities (Lin and Yao, 2006; Kim et al., 2008).

Isatin an endogenous compound identified in many organisms shows a wide range of biological activities (Pandeya et al., 2005). The isatin ring is a prominent structural moiety found in several pharmaceutically active compounds. This is mainly due to the easy synthesis and the importance of pharmacological activity. Therefore, the synthesis and selective functionalization of isatins have been the focus of active research area over the years (Lian-Shun et al., 2011). Isatin derivatives are reported to show antibacterial (Praveen et al., 2011) and antifungal (Amalraj et al., 2003) activities. Methisazone, for example plays an important role as prophylactic agent against several viral diseases (Foye and Sethi, 2002).

Prompted by the above-mentioned results, it was planned to combine two biologically active pharmacophore benzofuran and substituted isatins for the construction of some benzofuran–isatins. These combinations were suggested in an attempt to investigate the possible synergistic influence of such structure hybridizations on the anticipated activity, hoping to discover a new lead structure that would have a significant antimicrobial activity at very small concentration.

3 Chemistry

In the present work, 16 novel derivatives of benzofurano-substituted isatins were synthesized 3(a–p). The synthetic strategies adopted to obtain the target compounds are depicted in Scheme 1. The 5 and 7 substituted-isatins 2(a–p) and benzofuran-2-carbohydrazide 1 were prepared as per the reported methods given by Henry and Blatt (1964) and Marvel and Heirs (1941), respectively. Finally 5 and 7 substituted-isatins 2(a–p) and benzofuran-2-carbohydrazide 1 were refluxed in absolute ethanol with 2–3 drops of glacial acetic acid as a catalyst to afford corresponding N′-(5 and 7 substituted-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide 3(a–p). All the synthesized compounds were characterized by their physical (Table 1), analytical, and spectral data.

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Scheme 1

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Table 1 Physical data of benzofurano-isatins 3(a–p).

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Compounds	Molecular formula	Molecular weight	Yield (%)	Melting point (°C)
3a	C17H11N3O3	305.29	76.64	320–322
3b	C17H10BrN3O3	384.18	73.12	312–314
3c	C17H10ClN3O3	339.73	75.47	318–320
3d	C17H10FN3O3	323.28	64.45	339–341
3e	C18H13N3O3	319.31	74.16	315–317
3f	C18H13N3O4	335.31	70.15	320–322
3g	C17H10N4O5	350.29	72.60	306–308
3h	C17H11N3O4	321.29	71.14	311–314
3i	C17H10BrN3O3	384.18	71.40	316–318
3j	C17H10ClN3O3	339.73	75.58	321–323
3k	C17H11N3O4	321.29	70.57	315–317
3l	C18H13N3O3	319.31	71.23	313–315
3m	C18H13N3O4	335.31	69.76	319–321
3n	C19H15N3O4	349.34	63.12	329–331
3o	C17H10N4O5	350.29	71.34	309–311
3p	C17H10FN3O3	323.28	69.15	335–337

^aElemental analysis for C, H, N were within 0.4% of the theoretical values.

The mechanism of the reaction involves in the synthesis of N′-(5 and 7 substituted-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazides 3(a–p) is nucleophilic addition of benzofuran-2-carbohydrazide to 5 and 7 substituted-isatins to give N′-(5 and 7 substituted-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazides as shown in Scheme 2.

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Scheme 2

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4 Result and discussion

All the synthesized compounds were characterized by their physical and spectral data. The IR spectra of all the compounds 3(a–p) showed appearance of band at 3290–3180 cm⁻¹ due to amidal –NH– stretch. The title compounds were also confirmed by the appearance of bands at 1724–1697 cm⁻¹ due to >C⚌O stretch of isatins. The ¹H NMR (DMSO-d₆) spectrum of compounds 3(a–p) exhibited proton absorption singlet at 11.64–12.85 ppm and 10.97–10.01 ppm due to CONH- and indole –NH–, respectively. All other aromatic protons were observed as multiplet in the range 8.21–5.75 ppm. The ¹³C NMR and mass spectra of the compounds 3(a–p) were in agreement with the proposed structures.

In the present research we planned to combine two biologically active moieties such as benzofuran and isatin to observe influence over the biological activity since several benzofuran ring systems are known to possess significant antimicrobial activity as reported by Xiao et al. (2008), Toshio et al. (2004) and Kuete et al. (2009). Similarly isatin moiety also possesses good antimicrobial activity as described by Pandeya et al. (1999), Pandeya and Sriram (1998), Mirjana et al. (2006). Newly prepared compounds 3(a–p) were screened for their antibacterial and antifungal activity. MICs were recorded as the minimum concentration of a compound that inhibits the growth of tested microorganisms. The MIC values are generally within the range of 31.25–500 μg/mL against all evaluated strains. The results of the in vitro antibacterial activity screening of the novel series of N′-(5-bromo-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazides 3(a–p) are summarized in Table 2, against the Gram-positive bacteria S. aureus (ATCC-25923) and Bacillus subtilis (ATCC 6633), the Gram-negative bacteria Escherichia coli (ATCC-25922) and Pseudomonas vulgaris (ATCC-27853). Anti-microorganism tests with 16 compounds have established some interesting structure–activity relationships. Compound 3o with a nitro group at C-7 of isatins, displayed excellent antibacterial activities against E. coli and P. vulgaris with MIC values of 31.25 μg/mL as compared to the positive control drugs. The presence of fluoro group at the C-7 position, compound 3p, also displayed good activities against B. subtilis, E. coli and P. vulgaris with an MIC value of 31.25 μg/mL as compared to standard drugs. Surprisingly both 3o and 3p are less active against S. aureus.

Table 2 Minimum inhibitory concentration (MIC) of benzofuran–isatins 3(a–p).

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Compounds	MIC corresponding effects on micro-organism (μg/mL)
	Antibacterial activity				Antifungal activity
	Gram positive		Gram negative
	S. aureus	B. subtilis	E. coli	P. vulgaris	C. albicans	A. niger
3a	500	250	500	500	500	250
3b	250	250	250	250	250	250
3c	125	62.50	125	125	250	125
3d	125	62.50	62.50	62.50	250	62.50
3e	500	250	250	250	500	250
3f	250	250	250	500	500	250
3g	62.50	62.50	125	125	250	62.50
3h	125	62.50	125	125	250	125
3i	125	62.50	125	125	250	62.50
3j	62.50	125	62.50	125	250	62.50
3k	62.50	125	125	125	250	125
3l	500	250	250	250	500	250
3m	125	62.50	125	62.50	500	250
3n	125	62.50	125	62.50	500	250
3o	250	62.50	31.25	31.25	62.50	31.25
3p	250	31.25	31.25	31.25	62.50	31.25
Ampicillin	0.48	3.90	3.90	3.90	–	–
Norfloxacin	0.48	3.90	0.12	62.50	–	–
Fluconazole	–	–	–	–	0.98	1.95

Among the tested series 3c, 3d, 3i, 3j, 3k, 3m, 3n exhibited moderate antibacterial activity against Gram positive bacteria (S. aureus and B. subtilis) as well as Gram negative bacteria (E. coli and P. vulgaris) with MIC values of 62.50–125 μg/mL. However, rest of the compounds in the series were found to have less or poor activity against tested micro-organisms as compared to the standard drugs. It is interesting to note that the introduction of an electron withdrawing substituent to the aromatic ring at C5 and C7 positions resulted in compounds with an excellent antibacterial activity as it is confirmed by 3o and 3p. However, an electron releasing substituent gives compounds with poor activity, as it is observed in compounds 3e and 3i.

All the synthesized N′-(5-bromo-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazides 3(a–p) were also screened for antifungal activity against two fungal species Candida albicans and Aspergillus niger. Compounds 3o and 3p displayed good activity (31.25 μg/mL) against A. niger. The results showed that compounds 3c, 3d, 3g, 3i, 3j, 3m, 3n showed moderate activity (62.50–125 μg/mL) when compared to fluconazole against A. niger. In case of C. albicans, except 3o and 3p, all the tested compounds 3(a–p) displayed weak anti-fungal activities as compared to standard (fluconazole).

5 Conclusion

In the present research, we report the synthesis and in vitro antimicrobial activity of a new series of novel benzofurano–isatins 3(a–p). In general, the results of the in vitro antibacterial activity are also encouraging, as out of 16 compounds tested, compound 3o with a nitro group at C-7 of isatins exhibited significant antibacterial activity against E. coli and P. vulgaris with MIC values of 31.25 μg/mL. Similarly the presence of fluoro group at the C-7 position, compound 3p, also displayed good activities against B. subtilis, E. coli and P. vulgaris with an MIC value of 31.25 μg/mL as compared to standard drugs. In case of antifungal activity compounds 3o and 3p displayed moderate activity (31.25 μg/mL) against A. niger when compared to fluconazole against A. niger. It is worth mentioning that the combination of two biologically active moieties benzofurano and isatin profoundly influences the biological activity. Possible improvements in the antimicrobial activity can be further achieved by slight modifications in the substituents and/or additional structural activity investigations to have good antifungal activity. Further developments on this subject to understand their mechanistic interactions are currently in progress.

6 Experimental

All chemicals and solvents were supplied by Merck, S.D. Fine Chemical Limited, Mumbai. All the solvents were distilled and dried before use. The reactions were monitored with the help of thin-layer chromatography using pre-coated aluminium sheets with GF₂₅₄ silica gel, 0.2 mm layer thickness (E. Merck). Melting points of the synthesized compounds were recorded on the Veego (VMP-MP) melting point apparatus. IR spectrum was acquired on a Shimadzu Infra Red Spectrometer, (model FTIR-8400S). Both ¹H NMR (DMSO) and ¹³C NMR (DMSO) spectra of the synthesized compounds were performed with Bruker Avance-II 400 NMR Spectrometer operating at 400 MHz in SAIF, Punjab University (Chandigarh). Chemical shifts were measured relative to internal standard TMS (δ: 0). Chemical shifts are reported in δ scale (ppm). Mass (FAB) spectra of the synthesized compounds were recorded at MAT 120 in SAIF, Punjab University.

7 Antimicrobial activity

All the test compounds were evaluated for the antibacterial activity against (Koneman et al., 1997; NCCLS, 2002) the Gram positive S. aureus (ATCC-25923) and B. subtilis (ATCC 6633); the Gram-negative bacteria E. coli (ATCC-25922) and Pseudomonas aeruginosa (ATCC-27853); and for antifungal activity against two pathogenic fungi viz. A. niger and C. albicans. All the synthesized compounds were evaluated for antimicrobial activity against four bacterial and two fungal strains against norfloxacin and fluconazole as standard. Antimicrobial susceptibility testing was performed by the standardized disc diffusion and the agar dilution methods of the National Committee for Clinical Laboratory Standards. Inhibitory zone diameters were measured on Nutrient Agar (NA) for bacteria and Potato Dextrose Agar (PDA) for fungi, with conventional metrical filter paper discs (6 mm in diameter) containing specified doses of compounds.