Synthesis, studies and in-vitro antibacterial activity of N-substituted 5-(furan-2-yl)-phenyl pyrazolines

1 Introduction

Bacterial infections such as food poisoning, salmonellosis and diarrhea are caused by multidrug-resistant Gram-positive and Gram-negative Aeromonas hydrophila, Yersinia enterocolitica, Listeria monocytogenes and Staphylococcus aureus pathogens (Vivekanandhan et al., 2002). One million people in the subtropical regions of the world are infected and 20,000 deaths every year due to these parasitic bacterial infections. The spread of drug resistance Aeromonas is of concern because recent surveys indicate the emergence of these organisms as primary human pathogens and pose a public health risk and an etiological agent of gastroenteritis in epidemics. It is speculated that resistance to multiple antibiotics in Aeromonas isolate may be mediated by several conducible enzymes under the selection pressure of certain widely prescribed antibiotics. Resistance to β-lactam antibiotics is due to the production of multiple inducible, chromosomally encoded β-lactames (Goňi-Urriza et al., 2000a,b) Multiple antibiotic resistances among Aeromonas sp. have been reported from many parts of the world (WHO, 2006; Goňi-Urriza et al., 2000b). Amoxicillin, norfloxacin, ciprofloxacin are the principal drugs of choice in the treatment of bacterial infection since they are effective against extraintestinal and intestinal wall infections (Datta et al., 1974), but these are associated with several side effects such as nausea, metallic taste, dizziness, hypertension, etc. as well as resistance that have been reported (Alauddin and Smith, 1962). The development of nitrogen-containing five-membered heterocycles, pyrazolines is well known, representing a class of compounds of great importance of pyrazoline derivatives that possess biological activities like, antidepressant (Prasad et al., 2005), anticonvulsant (Amnerkar and Bhusari, 2010) antimicrobial (Ozdemir et al., 2007), analgesic (Aysel Gursoy et al., 2000), and antitumor (Brzozwskim et al., 2000), activities and also serve as human acyl-CoA: cholesterol acyltransferase inhibitors (Jeong et al., 2004). After the pioneering works of Fischer and Knövenagel in the late 19th century, 8 the reaction of α,β-unsaturated aldehydes and ketones with hydrazines became one of the most popular methods for the preparation of 2-pyrazolines (Sammour, 1964; Bhatnagar and George, 1968; Aubagnac et al., 1969; Andotra et al., 1993; Bilgin et al., 1994 and Mishriky et al., 1996). As a result, numerous substituted 2-pyrazolines have been synthesized, which has made possible structure–activity relationship investigations of these substances. In view of the therapeutic importance of these pyrazole heterocycles, the goal is to investigate the synthesis of pyrazoline. The present work is an extension of our ongoing efforts toward the development and identification of new pyrazole molecules to act as antibacterial agents. Here in we describe the synthesis of new pyrazoline derivative from various chalcones and screened for antibacterial Activity.

2 Experimental

3 Results and discussion

The present study describes, the synthesis and antibacterial evaluation of some pyrazoline derivatives 2a–2d that were tested for in vitro antibacterial activity using the disc-diffusion method by measuring and the diameter of the zone of inhibition measured in mm. It was found that all the compounds were screened in vitro for their antibacterial activity against a variety of Gram-positive, Gram-negative bacterial strains, such as A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus emerging pathogens responsible for gastrointestinal. Looking at the structure–activity relationship, compounds 2a, 2b, 2c and 2d exhibit significant activities while 2b and 2d exhibit good activity as compared to standard known antibiotics, such as Gentamicin (10 mcg) and Tetracycline (30 mcg) that were used for comparison purposes and the antibacterial screening data are recorded in Tables 1 and 2. On the basis of the above observations, modification will be done to improve antibacterial activity.

Assignment of selected characteristic IR bands provides significant indications for the formation of the cyclized pyrazoline analogues of the phenyl hydrazine 2a–2d. In starting material alkoxy chalcones, 1a–1d (C⚌O) and (CH⚌CH), functional absorbed in the expected region; (C⚌O) at 1603–1754 cm⁻¹ and (CH⚌CH) at 1552–1598 cm⁻¹, respectively. The IR spectra of the compounds showed ν(C⚌N) stretching at 1585–1596 cm⁻¹ and ν(N–N) stretching vibration at 1445–1493 cm⁻¹, respectively, which also confirm the formation of desired pyrazoline compounds.

The ¹H NMR spectra (400 MHz, CDCl₃), the signals corresponding to the double hydrogens (H-2 and 3) at δ 7.43–7.52, and 7.38–7.91 alkoxy chalcones 1a–1d were found missing altogether which indicates the involvement of the enone moiety during the cyclization reactions and the coupling value of J = 15.2–15.7 Hz between theses hydrogens describes the trans geometry around the C-2 and C-3 double bonds. The aromatic protons were located at δ 6.7–7.9, respectively. The downfield resonance of the H-3 as compared to H-2 could be ascribed to the electron deficient nature of the β-carbon in the enone moiety. The major feature of this spectrum was the signals of the pyrazoline 2a–2d ring proton (H-x) and (H-a and b) which were found in the region at δ 5.23–5.25 (1H, dd, J_xa = 6.5–6.7 Hz, J_xb = 11.2–11.8 Hz) and 3.23–3.82 ppm (1H, dd, J_ax = 6.5–6.7 Hz, J_ab = 16.1–17.1 Hz) describes the trans relationship between H_X and H_b & H_a are geminally placed at C-4,which clearly describes the inter-relationship between the H-x, b and a. The proposed expression 2a–2d the strong deshielding of the C₅ (H-a and b) protons compared with the C₄ (H-x) protons of the pyrazoline ring can be assumed due to its structure Fig. 2. The protons belonging to the aromatic ring and the other cyclic groups were observed with the expected chemical shift and integral values.

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Figure 2

Schematic diagram indicating the synthesis of compounds 2a–2d.

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Finally, ¹³C NMR (400 MHz, CDCl₃) spectra of all compounds were recorded in DMSO and spectral signals are in good agreement with the probable structures. The carbon of C⚌O and C⚌C displayed signals at 191.2–191 and 141.5–144.6 ppm in 1a–1d. The methylene groups for 1c and 2c were resonating at δ 55.2–55.4 ppm (CH₃) and δ 72.2–72.4 ppm (OCH₂); the downfield resonance of the former suggests their placement near an electronegative oxygen atom. The signals are due to the aromatic carbons and the carbon at 1-N substituted aliphatic group. The C₄ and C₅ carbon of pyrazoline resonated at 42.3–68.3 and 62.4–77.4 ppm, respectively. The downfield resonance of former as compared to C-4 could be attributed to its benzylic nature and proximity to nitrogen atom. The other resonates were showed at their usual position in Section 2.

Characteristic peaks were observed in the mass spectra of all compounds, which followed the similar fragmentation pattern. The spectrum of the compound 2a showed a molecular ion peak (M⁺) at m/z 345. The characteristic peaks observed within the mass spectra of pyrazoline compounds are given in experimental section.

It may be concluded that this study describes the general method for the synthesis of some pyrazolines linked through the 5-aryl ring under normal conditions. These results show that compounds 2b and 2d are better antibacterial agents against A. hydrophila, Y. enterocolitica, L. monocytogenes, and S. aureus as compared to Gentamicin and Tetracycline. Thus the accumulation of the pyrazoline derivatives will better antibacterial agents for therapeutic use.

4 Conclusions

In the present studies, this research examined the antibacterial activities of the pyrazoline derivatives from alkoxy chalcones that were found to be more active among all pyrazoline compounds while 2b and 2d displayed as good antibacterial agents as compared to Gentamicin and Tetracycline. The molecular structure of these active compounds showed property of pyrazoline; these compounds might be more efficacious drugs against these bacteria and their biological effects which could be helpful in designing more potent antibacterial agents.

5 Pharmacology

All the synthesized compounds of bis-pyrazoline derivatives were evaluated for in vitro antibacterial activity by using disk diffusion method and the Minimum Inhibitory Concentration (MIC) as compared to antibiotics positive controls. The results of antibacterial activity and Minimum Inhibitory Concentration (MIC) are summarized in Tables 1 and 2 respectively.

5.2

5.2 In vitro antibacterial activities

In vitro antibacterial activities of pyrazoline 2a–2d derivatives were carried out using the culture of A. hydrophila, Y. enterocolitica, L. monocytogenes, and S. aureus by the disc-diffusion method using nutrient broth medium[contained (mcg/ml): beef extract 1 g; peptone 5 g; pH 7.0]. In the disc-diffusion method, sterile paper discs (0.5 mm) impregnated with compound dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 mcg/mL were used. Gentamicin and Tetracycline were used as the standard drugs, where as DMSO poured disk was used as negative control. Then, the paper discs impregnated with the solution of the compounds were tested placed on the surface of the media inoculated with the microorganism. The plates were incubated at 18 h, at 37 °C. After incubation, the diameters of the inhibition zones were measured. The pyrazoline derivatives were further checked by MIC method and results are presented in Tables 1 and 2. The in vitro studies result showed that compounds 2b and 2e are of highest activity against A. hydrophila, Y. enterocolitica, L. monocytogenes, S. aureus among all the pyrazolines when compared to antibiotics. The susceptibility of the bacteria to the test compounds were determined by the formation of an inhibitory zone after 48 h of incubation at 37 °C. The molecular structure of these active compounds showed enhanced activity. The distinct differences in the antibacterial property of these compounds further justify the purpose of this study. The importance of such work lies in the possibility that the new compounds might be more efficacious drugs against bacteria for which a thorough investigation regarding the structure–activity relationship, toxicity and their biological effects which could be helpful in designing more potent antibacterial agents for therapeutic use (Fig. 1).

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Figure 1

Schematic diagram indicating the ring conformation of compounds 2a–2d.

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