Synthesis and antimicrobial evaluation of new 1,4-dihydro-4-pyrazolylpyridines and 4-pyrazolylpyridines

Background Dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates (1,4-DHP) have now been recognized as vital drugs. Some of these derivatives such as amlodipine, felodipine, isradipine, etc. have been commercialized. In view of wide range of biological properties associated with 1,4-DHP and owing to the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicrobial evaluation of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g). Results Synthesis of a series of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) has been accomplished by multicomponent cyclocondensation reaction of ethyl acetoacetate, 3-aryl-1-phenyl pyrazole-4-carboxaldehyde (1a-g) and ammonium acetate. The dihydropyridines 2a-g were smoothly converted to new diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g) using HTIB ([Hydroxy (tosyloxy)iodo]benzene, Koser's reagent) as the oxidizing agent. The antimicrobial studies of the title compounds, 2a-g &3a-g, are also described. Graphical abstract Synthesis of a series of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g), their aromatization using HTIB ([Hydroxy(tosyloxy)iodo]benzene, Koser's reagent) to afford new diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g), and antimicrobial studies of 2a-g and 3a-g are reported.

In addition to above, aromatization of 1,4-DHP has also attracted considerable attention in recent years as Böcker has demonstrated that metabolism of the above drugs involves a cytochrome P-450 catalysed oxidation in the liver [11].

Chemistry
The synthetic scheme used for the synthesis of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g) is outlined in Scheme 1. Synthesis of the title compounds 2a-g was accomplished by multicomponent cyclocondensation reaction of ethyl acetoacetate, 3-aryl-1-phenyl-pyrazole-carboxaldehyde (1a-g) and ammonium acetate in ethanol. The purity of the compounds was checked by TLC and elemental analysis. Spectral data (IR, 1 H NMR (see additional files 1, 2, 3, 4 and 5, mass) of the newly synthesized compounds 2a-g were in full agreement with their proposed structures. The IR spectra of compounds 2a-g exhibited characteristic peak at approximately 1697 cm -1 because of the presence of ester group (-COOEt), and peak due to -N-H stretch appeared in the region 3300-3317 cm -1 . In 1 H NMR of compounds 2a-g, the protons of C 4 -H and -NH of the dihydropyridine ring resonate between δ 5 and 6 ppm.
A plausible mechanism for the oxidation of dihydropyridines 2 to 3 is outlined in Scheme 2. The probable mechanism might involve the attack by N-H on PhI (OH)OTs, leading to the formation of intermediate 4.  Scheme 1 Synthesis of 1,4-DHP (2) and aromatization of 2 to 3 using HTIB.

Pharmacology
All the synthesized compounds, 2a-g and 3a-g, were evaluated in vitro for their antibacterial activity against two gram-positive bacterial strains, Staphylococcus aureus &Bacillus subtilis and two gram-negative bacteria, namely, Escherichia coli and Pseudomonas aeruginosa and their activities were compared with a well-known commercial antibiotic, ciprofloxacin. In addition, the synthesized compounds were also evaluated for their antifungal activity against Aspergillus niger &Aspergillus flavus and their antifungal potential was compared to reference drug, fluconazole. Compounds possessed variable antibacterial activities against Gram-positive bacteria, S. aureus, B. subtilis. However, the compounds in this series were not effective against any Gram-negative bacteria, neither against E. coli nor against P. aeruginosa. Results of antibacterial evaluation are summarized in Table 1.
Compounds 2a-g and 3a-g showed zones of inhibition ranging between 14 and 20 mm. On the basis of the zones of inhibition produced against the test bacteria, compounds 2b and 3a were found to be most effective against S. aureus, showing the maximum zones of inhibition at 18 and 20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B. subtilis. The remaining compounds showed fair activity against gram-positive bacterial strains (Table 1). In the whole series, the MIC (minimum inhibitoty concentration) values of various tested chemical compounds ranged between 64 and 256 μg/mL against gram-positive bacteria. Compounds 2b and 3a displayed good antibacterial activity with the lowest MIC value, 64 μg/ml against S. aureus. Three compounds, 3a, 3e and 3g possessed antibacterial activity with MIC value of 64 μg/mL against B. subtilis ( Table 2).
From the overall result it is evident that compound 3a could be identified as the most biologically active member within this study with good antifungal and antibacterial profile.

Conclusions
and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (3a-g) has been synthesized with the hope of discovering new structure leads. Compounds 2b and 3a were found to be most effective against S. aureus showing the maximum zones of inhibition of 18 and 20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B. subtilis. Moreover, six compounds 2a, 2d, 2g, 3a, 3c and 3d showed more than 50% mycelial growth inhibition against A. niger whereas compounds, 2a, 2e, 2f, 3a, 3d and 3f were found to be active against A. flavus; however, no compound was found superior over the reference drug. Scheme 2 Proposed mechanism for the oxidation of 2 to 3.  Finally, compound 3a could be identified as the most biologically active member within this study with an interesting antibacterial and antifungal profile.

Chemical synthesis
Melting points were taken in open capillaries and are uncorrected. IR spectra were recorded on Perkin-Elmer IR spectrophotometer. The 1 H NMR spectra were recorded on Brucker 300 MHz instrument. The chemical shifts are expressed in ppm units downfield from an internal TMS standard. 3-Aryl-1-phenylpyrazole-4-carboxaldehydes (1a-h), needed for the present study, were synthesized by Vilsmeier-Haack reaction according to the literature procedure [19].

Test microorganisms
Total six microbial strains were selected on the basis of their clinical importance in causing diseases in humans. Two Gram-positive bacteria (S. aureus MTCC 96 and B. subtilis MTCC 121); two Gram-negative bacteria (E. coli MTCC 1652 and P. aeruginosa MTCC 741) and two fungi (A. niger and A. flavus) the ear pathogens isolated from the patients of Kurukshetra [21], were used in the present study for the evaluation of antimicrobial activities of the chemical compounds. All the cultures were procured from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh. The bacteria and fungi were subcultured on Nutrient agar and Sabouraud's dextrose agar (SDA), respectively, and incubated aerobically at 37°C.

In vitro antibacterial activity
The antibacterial activities of compounds, 2a-g and 3ag, were 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 then loaded with a 100 μL volume with concentration of 2.0 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 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 [22,23].

Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. MIC of the compounds against bacterial strains was tested through a macrodilution tube method as recommended by NCCLS [24]. In this method, various test concentrations of chemically synthesized compounds were made from 256 to 1 μg/ mL in sterile tubes, 1-10. 100 μL sterile Mueller Hinton Broth was poured in each sterile tube, and followed by addition of 200 μL test compound in tube 1. Twofold serial dilutions were carried out from tubes 1 to 10, and excess broth (100 μL) was discarded from the tube 10. To each tube, 100 μL of standard inoculum (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.