Synthesis of new pyrazolyl-2, 4-thiazolidinediones as antibacterial and antifungal agents

Background Thiazolidine-2, 4-diones (TZDs) have become a pharmacologically important class of heterocyclic compounds since their introduction in the form of glitazones into the clinical use for the treatment of type 2 diabetes. TZDs lower the plasma glucose levels by acting as ligands for gamma peroxisome proliferators-activated receptors. In addition, this class of heterocyclic compounds possesses various other biological activities such as antihyperglycemic, antimicrobial, anti-inflammatory, anticonvulsant, insecticidal, etc. TZDs are also known for lowering the blood pressure thereby reducing the chances of heart failure and micro-albuminuria in the patients with type 2 diabetes. Results We have described herein the synthesis of three series of compounds, namely, ethyl 2-((Z)-5-((3-aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-2, 4-dioxothiazolidin-3-yl)acetates (4), methyl 2-((Z)-5-((3-aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-2, 4-dioxothiazolidin-3-yl)acetates (5), and 2-((Z)-5-((3-aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-2, 4-dioxothiazolidin-3-yl)acetic acids (6). The compounds 4 and 5 were synthesized by Knoevenagel condensation between 3-aryl-1-phenyl-1H-pyrazole-4-carbaldehydes (1) and ethyl/methyl 2-(2, 4-dioxothiazolidin-3-yl)acetates (3, 2) in alcohol using piperidine as a catalyst. The resultant compounds 4 and 5 having ester functionality were subjected to acidic hydrolysis to obtain 6. All the new compounds were tested for their in vitro antibacterial and antifungal activity. Conclusions Knoevenagel condensation approach has offered an easy access to new compounds 4-6. Antimicrobial evaluation of the compounds has shown that some of the compounds are associated with remarkable antifungal activity. In case of antibacterial activity, these were found to be effective against Gram-positive bacteria. However, none of the compounds were found to be effective against Gram-negative bacteria.


Background
Natural antibiotic compounds have become essential to current health care system, assisting and complementing the natural immune system against microbial pathogens. As conventional antibiotics are often abused to treat microbial infections, some microorganisms have developed tolerance to these antibiotics. Because of the appearance of antibiotic-resistant strains, the continuous development of novel efficient antibiotic agents is more crucial than ever [1][2][3]. So, the medical community faces a serious problem against infections caused by the pathogen bacteria and needs an effective therapy and search for novel antimicrobial agents. Synthetic organic chemistry has always been a vital part of highly integrated and multidisciplinary process of various drug developments. In this context, this study was designed to evaluate antimicrobial properties of new pyrazole derivatives containing thiazolidindiones.
On the other hand, thiazolidines are also known for their potential biological activities. The varied biological activities of rhodanines (2-thioxo-thiazolidin-4-one) and their analogs have been known from the beginning of twentieth century. Rhodanines and 2, 4-thiazolidinediones (TZDs) have become a pharmacologically important class of heterocyclic compounds since the introduction of various glitazone and epalrestat into clinical use for the treatment of type II diabetes and diabetic complications [25]. Several studies have been reported that TZDs have acquired much importance because of their diverse pharmaceutical applications such as antihyperglycemic [26], bactericidal [27], pesticidal [28], fungicidal [29], insecticidal [30], anticonvulsant [31], tuberculostatic [32], anti-inflammatory [33] etc.
Different possibilities of heterocyclic modifications with a wide spectrum of pharmacological propertiesare the most important grounds for investigation of this class of compounds. There have been many reports in literature depicting that the presence of heterocyclic moieties such as thiazole, pyrazole, flavone, chromone, sultam, and furan at fifth position proves to be more potent and efficacious than a simple aryl group [34][35][36][37][38][39]. Although there are not many TZDs fused to pyrazoles, a number of them are incorporated into a wide variety of therapeutically important compounds possessing a broad spectrum of biological activities. In a recent article, pyrazolyl-2, 4-TZDs have been reported as antiinflammatory and neuroprotective agents.
All other compounds 4b-h, 5b-h, and 6b-h were prepared adopting the similar methodology. The physical data of all compounds 4-6 have been summarized in Table 1.
The structures of all compounds 4a-h, 5a-h, and 6a-h were established by the spectral (IR, NMR {see additional files 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 and 24}, Mass) and elemental analysis. For example, IR spectrum of the compound 4a exhibited characteristic absorption bands at 1736 and 1690 cm -1 because of carbonyl groups of ester and TZD. The 1 H NMR spectrum of the product 4a (see additional files 1) showed three characteristic singlets at δ 8.213, δ 7.963, and δ 4.473 because of C(5)-H of pyrazole ring, =CH and -NCH 2 , respectively, apart from other aromatic signals. Besides these the aliphatic region also showed the characteristic quartet and triplet due to -OCH 2 CH 3 at δ 4.248 and δ 1.301, respectively. The product 6a was characterized by careful comparison of the IR and 1 H NMR spectra (see additional file 17) with those of the 4a. An important characteristic feature in 1 H NMR spectrum of 6a was disappearance of the triplet and quartet in the aliphatic region which was present in the spectrum of 4a.

In vitro antifungal activity
All the 24 compounds were tested for their in vitro antifungal activity against two fungi, namely, Aspergillus niger and Aspergillus flavus. Standard antibiotic, namely, Fluconazole, was used for comparison with antifungal activity shown by compounds 4a-h, 5a-h, and 6a-h. A careful analysis of percentage mycelial growth inhibition revealed that almost all the newly synthesized compounds showed comparable antifungal activity with commercial antibiotics Fluconazole as shown in Table 2.
In case of Gram-positive bacteria, compounds 4h, 5b, 5h, 6a, 6b, and 6h were found to be most effective against S. aureus with zone of inhibition ranging between 18.6 mm and 20.0 mm and the compounds 5h, 6a, and 6b were most effective against B. subtillis with zone of inhibition ranging between 19.3 mm and 21.0 mm (Table 3).

Conclusions
We have described herein an efficient and convenient synthesis of three series of pyrazolyl-2, 4-TZDs (4-6) by Knoevenagel condensation. All the 24 compounds synthesized were characterized by spectral and elemental analytical data and evaluated for their in vitro antifungal and antibacterial activities. Results of the antifungal activity were found to be comparable with the reference compound. On the other hand, antibacterial activity was best observed for Gram-positive bacteria only, none of the compounds showed activity against Gram-negative bacteria.
All other derivatives 6b-h were synthesized by adopting the similar procedure.

In vitro antibacterial activity
The antibacterial activity of synthesized compounds was evaluated by the agar well-diffusion method. All the cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 × 10 8 cfu/mL. 20-mL of Mueller Hinton agar medium was poured into each Petri plate and the agar plates were swabbed with 100 μL inocula of each test bacterium and kept for 15 min for adsorption. Using sterile cork borer of 8-mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100-μL volume with concentration of 4.0 mg/mL of each compound reconstituted in the dimethylsulphoxide (DMSO). All the plates were incubated at 37°C for 24 h. Antibacterial activity of each synthetic compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (Hi Antibiotic zone scale). DMSO was used as a negative control whereas ciprofloxacin was used as a positive control. This procedure was performed in three replicate plates for each organism [53].

Determination of MIC
MIC is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. MIC of the various compounds against bacterial strains was tested through a macro dilution tube method as recommended by NCCLS [54]. In this method, various test concentrations of synthesized compounds were made from 128 to 0.25 μg/mL in sterile tubes no. 1 to 10. 100-μL sterile Mueller Hinton Broth (MHB) was poured in each sterile tube followed by addition of 200 μL test compound in tube 1. Twofold serial dilutions were carried out from the tube no. 1 to the tube no. 10 and excess broth (100 μL) was discarded from the last tube no. 10. To each tube, 100 μL of standard inoculums (1.5 × 10 8 cfu/mL) was added. Ciprofloxacin was used as control. Turbidity was observed after incubating the inoculated tubes at 37°C for 24 h.

In vitro antifungal activity
The antifungal activity of the synthesized compounds was evaluated by poisoned food technique. The molds were grown on Sabouraud dextrose agar (SDA) at 25°C for 7 days and used as inocula. 15 mL of molten SDA (45°C) was poisoned by the addition of 100 μL volume of each compound having concentration of 4.0 mg/mL, reconstituted in the DMSO, poured into a sterile Petri plate and allowed it to solidify at room temperature. The solidified poisoned agar plates were inoculated at the centre with fungal plugs (8-mm diameter), obtained from the actively growing colony and incubated at 25°C for 7 days. DMSO was used as the negative control whereas fluconazole was used as the positive control. The experiments were performed in triplicates. Diameter of the fungal colonies was measured and expressed as percent mycelial inhibition determined by applying the formula [55].
Inhibition of mycelial growth % = (dc − dt)/dc × 100 where dc average diameter of fungal colony in negative control plates, dt average diameter of fungal colony in experimental plates.