Nematicidal, Larvicidal and Antimicrobial Activities of Some New Mannich Base Imidazole Derivatives

Purpose: To synthesize Mannich base imidazole derivatives, and evaluate their antimicrobial, nematicidal and larvicidal properties . Methods: Compounds 1a-g and 2a-g were prepared using a Mannich condensation method. The chemical structures of compounds 2a-g were confirmed by Fourier transform infrared spectroscopy (IR), proton nuclear magnetic resonance ( 1 H-NMR), carbon nuclear magnetic resonance ( 13 C-NMR), and mass spectrometry (MS) and elemental analyses. Compounds 1a-f and 2a-f were screened for antimicrobial properties using an agar diffusion method. The nematicidal activity of the compounds was evaluated against juvenile Meloidogyne javanica as test organism while larvicidal activity was assessed against the urban mosquito, Culex. Quinquefasciatus, using a standard bioassay protocol. Results: Compounds 1b, 1g, 2e and 2g were highly active against a few bacterial organisms compared with the reference antibacterial, ciprofloxacin while the antifungal activity of compound 2d was high compared with the reference, clotrimazole. Compounds 1c, 1e, 1g, and 2e showed high toxicity levels of larvicidal activity based their half maximal lethal dose (LD 50 ) values. Compounds 1d, 1e, 1f, 1g, 2d and 2e were highly toxic to nematodes. Conclusion: Compounds 1b, 1g, 2e and 2g may be useful as lead molecules for the development of new classes of larvicidal, nematicidal and antimicrobial agents.


INTRODUCTION
Culex is an important genus of mosquito that acts as a vector for several serious diseases, including filariasis, west Nile virus, dangue fever, yellow fever, chikungunya and other diseases caused by encephalitides. Nematodes are tiny worms, some of which are plant parasites. It is thought that nematode infections play an important role in the predisposition of a host plant to invasion by secondary pathogens. Edible plants attacked by nematodes show retarded growth and development, resulting in loss of quality and quantity of the harvest.
Currently employed nematicides are slated for tighter regulations and less use due to environmental problems and human and animal health concerns. The optimum methods of controlling mosquito larvae and nematodes involve the use of insecticides such as various organophosphates, and natural and synthetic heterocyclic products. New environmentally safe and biodegradable insecticides that specifically target mosquitoes and nematodes are urgently needed.
Naturally occurring and synthetic imidazole derivatives are an important class of heterocycles that are known to exhibit various biological activities [1]. The imidazole nucleus is a major component in a variety of drugs, including angiotensin II receptor antagonists, anti-inflammatory agents, protein kinase inhibitors, and fungicides [2]. Imidazole also plays important roles in biochemical processes [3]. Many substituted imidazoles are used as fungicides and herbicides, plant growth regulators and therapeutic agents [4]. Imidazole is common compounds of a large number of biologically and medicinally significant substances [5,6], including anticonvulsant [7] and monoamine oxidase (MAO) inhibitors [8].
The Mannich reaction is commonly employed to develop agricultural chemicals such as plant growth regulators [9] and is an important tool in the synthesis and modification of biologically active compounds [10]. It provides a convenient access to many useful synthetic building blocks because amino groups can be easily converted into a variety of other functionalities [11]. Mannich bases often exhibit significant biological properties including antimicrobial [12], cytotoxic [13] and anticancer [14].
The present investigation focuses on a series of imidazole compound in a single molecular framework and examines their larvicidal, nematicidal, antibacterial and antifungal activities.

EXPERIMENTAL Materials
All the melting points were recorded in open capillary tubes and are uncorrected. IR spectra were recorded in KBr on a FT-IR Shimadzu 8201pc and ¹H NMR spectra were recorded on a Brucker DRX-300 MHZ. Elemental analysis (C, H and N) were undertaken using an Elementer analyzer model vario EL III. The purity of the compounds was checked by thin layer chromatography (TLC) with silica gel plates.

Imidazole
(0.1 mol), thiosemicarbazide hydrochloride (0.1 mol), and benzaldehyde (0.1 mol) were added in ethanol solvent (20 mL). The reaction mixture was refluxed 5h with temperature 60 o C. Then the mixture was poured over crushed ice. The precipitate was obtained in few min. then the precipitate was collected by filtration. The precipitate was dried and recrystallized from suitable alcohols. The above procedure was followed by all the remaining compounds 1a-1g.
Scheme 1: Synthetic route of compounds 1a-1g and 2a-2g The precipitate was dried and recrystallized from suitable alcohols. The above procedure was followed by all the remaining compounds 2a-2g.
Each compound was tested at a concentration of 100 μg/mL in DMSO. Ciprofloxacin was used as the standard. The zone of inhibition was measured after 24 h incubation at 37 ºC ( Table  2). The minimum inhibitory concentration (MIC) was considered to be the lowest concentration that completely inhibited growth on agar plates.

In vitro antifungal screening
Compounds 1a-1g and 2a-2g were evaluated for their in vitro antifungal activity Aspergillus niger, Candia albicans, Aspergillus fumigatus, Cryptococcus neoformans and Microsporum audouinii (recultured) using an agar diffusion method [17,18] with Sabouraud's dextrose agar (Hi-Media). Each compound was tested at a concentration of 100 μg/mL in DMSO. Clotrimazole was used as the standard. The zone of inhibition was measured incubated at 37 °C for 24 h and MICs were determined.

Evaluation of larvicidal activity
The assessment of larvicidal activity of synthesized test compounds 1a-1g and 2a-2g was tested against the urban mosquitoes Culex. Quinquefasciatus using standard bioassay protocol [19]. Egg rafts of mosquito were obtained from a drainage system. The eggs were reared under standard insectary conditions at ambient temperature (29 ± 3 °C) and relative humidity (80 ± 5 %), 12:12 light: dark photoperiod and fed with ground shrimp feed daily.
Larval development was monitored for 7 days. The second and third stage larvae were collected at the tip of a pasture pipette and placed in cotton bud to remove excess water and transferred gently to the test vial (10/vial) by tapping. The larval mortality was observed using various concentrations of synthesized compounds (10,20,30, and 40 µg/mL).

Assessment of nematicidal activity
For the determination of nematicidal activity, juveniles of Meloidogyne javanica were used as test organism [19]. Assay system was prepared with 2 ml Milli Q water containing different concentrations (10, 20, 30 and 40 µg/mL) of synthesized test compounds 1-6 in glass tubes.
Ten juveniles of M. javanica were transferred in test, positive (with 2 % methanol) and negative (without vehicle) control tubes. Mortality was observed under a zoom stereomicroscope after 24 h of exposure.

Statistical analysis
The mean of the results was calculated based on at least 3 independent evaluations and the standard deviations (SD) were also calculated using Microsoft Excel. All LD 50 values were calculated from the corresponding sigmoidal dose-response curve according to best fit shapes based on at least five reaction points using the Microsoft Office Excel 2007 software (Microsoft, Redmond, WA, USA).

Antibacterial activity
Compounds 1a-g and 2a-2g were evaluated for in vitro antibacterial activity against Staphylococcus aureus, Escherichia coli, Enterococcus.
faecalis. Pseudomonas aeruginosa and Klebsiella pneumoniae using conventional agar dilution procedures. Ciprofloxacin was used as a positive control. Inhibition zones were measured and compared against those of controls. The bacterial zones of inhibition are given in Table 2.
Compared with ciprofloxacin, compound 1b was highly active against S. aureus, and 1g exhibited an equivalent activity (0.5 μg/mL) against E. faecalis and greater activity (0.25 μg/mL) against P. aeruginosa. Compound 2e was highly active against K. pneumonia and 2f showed equivalent activities (0.5 μg/mL) against E. coli and E. feacalis. Compound 2g was highly active (0.5 μg/mL) against P. aeruginosa. The MIC values are summarised in Table 3.

Antifungal activity
Compounds 1a-g and 2a-g were evaluated in terms of their in vitro antifungal activity against Aspergillus niger, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans (recultured), and Microsporum audouinii using an agar diffusion method. The fungal activity of each compound was compared with that of clotrimazole as positive control. The fungal zones of inhibition are given in Table 2. Compounds 2d and 2g (0.25 μg/mL) were highly active against C. albicans. Compound 2d was equipotent active (1 μg/mL) against M. audouinii compared with clotrimazole (MIC, 2 μg/ml).

Larvicidal activity
The larvicidal activity of the test compounds is listed in Table 4.

DISCUSSION
We synthesised and characterised 14 new Mannich base imidazole derivatives (1a-g) and (2a-g) as outlined in Scheme 1. The physical data of these compounds are given in Table 1.
The Mannich base condensation reaction proceeds via an attack by benzaldehyde at a secondary amine. During this reaction, one mole of water was eliminated and the resulting thiazolidine-4-one products were purified by column chromatography using an eluent of hexane: chloroform (1:4) The chemical structure of each new compound was confirmed by IR, ¹H NMR, 13  C NMR spectra of 1a contained important peaks at 182.7 and 75.3 ppm, corresponding to C=S and CH carbon atoms, respectively. The mass spectrum of 1a contained a molecular ion peak m/z 247.28, thereby confirming its molecular mass. Similar spectral data and corresponding molecular masses were obtained for compounds (1b-1g).
Similarly, the IR spectrum of compound 2a showed absorption bands at 3287.20, 2932.97, 1623.21 and 456.98 cm -1 corresponding to NH 2 , NH, C=O and CH groups respectively. The ¹H NMR spectrum of 2a showed broad signals at 9.50, 7.86 and 2.21 ppm, corresponding to NH 2 , CH and NH protons respectively. The 13 C NMR spectrum of compound 2a showed important peaks at 157.9 and 75.9 ppm, corresponded to C=O and CH carbon atoms respectively. The mass spectrum of 2a contained a molecular ion peak m/z 231.28, which is consistent with its molecular mass. Similar spectral data and corresponding molecular masses were obtained for compounds (2b-2g).
The synthesized imidazole derivatives were evaluated in terms of their antimicrobial, larvicidal and, nematicidal activities. Consistent with their expected structure -activity relationship, compounds 1a-1g and 2a-2g were biologically active. The presence of imidazole nucleus and the para substitution of the phenyl ring contribute to the observed activities.
The chemical structure in Figure 1 show that the 4-substituted phenyl ring acts as a lipophilic domain. The C=S group in thiosemicarbazone and the C=O group in semicarbazone form hydrogen bonds with the NH groups in thiosemicarbazone and semicarbazone act as hydrogen bonding domain. Therefore, the imidazole ring is an essential pharmacophore that determine biological activity.

CONCLUSION
A new series of imidazole derivatives has been synthesized. Some of them possess strong larvicidal, nematicidal, antibacterial and antifungal activities, and thus are capable of serving as potential lead molecules for the development of clinically useful antimicrobial, larvicidal and nematicidal agents.