Synthesis and in vitro antimicrobial evaluation of 4 H-pyrazolopyran, -benzopyran and naphthopyran derivatives of 1 H -pyrazole

A new series of eight derivatives each of 4-pyrazolyl-4 H -pyrazolopyran, -benzopyran and sixteen derivatives of naphthopyran has been synthesized by one-pot base-catalyzed cyclocondensation reactions of 1-phenyl-3-(het)aryl-pyrazole-4-carbaldehyde, malononitrile and substituted pyrazolin-5-ones or dimedone or naphthols respectively. All the synthesized compounds were subjected to in vitro antimicrobial screening against a panel of pathogenic strains of bacteria and fungi. Some of the compounds were found to be equipotent or more potent than commercial antibiotics against most of employed strains.


Introduction
The 4H-Pyran nucleus is a fertile source of biologically important molecules possessing a wide spectrum of biological and pharmacological activities, such as antimicrobial, 1 antiviral, 2 mutagenicity, 3 antiproliferative, 4 sex pheromone, 5 antitumor, 6 cancer therapy 7 and central nervous system activity. 8Some of these compounds are widely employed as cosmetics and pigments and as potential biodegradable agrochemicals. 9Therefore, the synthesis of such compounds has attracted strong interest.
In recent years, 4-functionally substituted 1,3-diarylpyrazole derivatives have received considerable attention due to their wide range of useful biological properties, which include antimicrobial, [10][11][12] anti-inflammatory (COX-2 inhibitor and ulcerogenic activity), 11 antitubercular, 12 antitumor, 13,14 antiangiogenesis, 14 anti-parasitic 15 and antiviral activity. 16A literature survey 17 revealed that a number of 4H-pyran derivatives have been synthesized using various aldehydes but not a single reference has been found where 1,3-diaryl pyrazole-4carbaldehydes are used.Thus, in a view to obtain more biologically potent heterocyclic systems, containing therapeutically active moieties pyran and pyrazole, and in continuation of our work 18 on biologically active heterocyclic compounds, we report herein the synthesis of some new substituted 4H-pyrazolopyran, -benzopyran and naphthopyran derivatives of pyrazole via a Multi-Component Reaction (MCR) approach.The constitutions of all the products were confirmed using elemental analysis, FT-IR, 1 H NMR and 13 C NMR spectroscopy.All synthesized compounds were screened for in vitro antimicrobial activity against eight human pathogens, of which three gram positive bacterial pathogens Streptococcus pneumoniae, Clostridium tetani, Bacillus subtilis, three gram negative bacterial pathogens Salmonella typhi, Vibrio cholerae, Escherichia coli and two fungal pathogens Aspergillus fumigatus and Candida albicans, using broth microdilution MIC (Minimum Inhibitory Concentration) method. 19

Results and Discussion
A series of 4-pyrazolyl-4H-pyrazolopyran 6a-h, -benzopyran 7a-h and naphthopyran 8a-h, 9a-h derivatives has been synthesized by one-pot three-component cyclocondensation reaction of 1phenyl-3-(het)aryl-pyrazole-4-carbaldehyde 1a-h, malononitrile 2 and substituted pyrazolin-5ones 3 or dimedone 4 or naphthols 5a-b respectively, in the presence of piperidine as catalyst.The mixture refluxing under ethanol or acetonitrile gives moderate to good yield (50-76%) (Scheme 1).A mechanism for the formation of the pyran derivatives is outlined in Scheme 2. The reaction occurs via an in situ initial formation of the heterylidenenitrile, containing the electron-poor C=C double bond, from the Knoevenagel condensation between pyrazole-4carbaldehyde and malononitrile by loss of water molecules.Finally, Michael addition of 3 or 4 or 5 to the initially formed unsaturated nitrile, i.e. nucleophilic attack of hydroxyl moiety to the cyano moiety affords cyclized pyran derivatives 6a-h, 7a-h, 8a-h and 9a-h.
The structures of all the new synthesized compounds were established by 1 H NMR, 13 C NMR and FT-IR spectral data and molecular weight of some selected compounds confirmed by mass spectrometry.NMR spectroscopy is especially useful to elucidate the structures of products i.e. 1 H NMR (DMSO-d 6 ) spectrum of 6a exhibited a singlet peak at δ 4.59 for H4 and δ 6.91 ppm for NH 2 of the pyran ring.Aromatic protons of 6a resonate as multiplets at around δ 6.98-7.96ppm and a deshielded aromatic singlet at δ 8.31 ppm stands for H5 of pyrazole ring.A singlet at δ 1.79 ppm and δ 12.18 ppm stands for methyl and secondary amine of the fused pyrazole ring respectively. 13C NMR of 6a exhibited a distinctive signal at δ 10.4 ppm for methyl of fused pyrazole ring and δ 27.2 ppm for C4 of the pyran ring.All the aromatic carbons of 6a showed signals around δ 114.5-151.2ppm in the 13 C NMR spectra.Moreover, distinctive signals at δ 161.6 ppm for C2, δ 57.8 ppm for C3, δ 98.4 ppm for C5, δ 154.7 ppm for C6 and δ 120.9 ppm for CN of pyran ring in the 13 C NMR spectra confirms the structure 6a.
The IR spectrum of compound 6a exhibited characteristic absorption bands at 3410 and 3385 cm -1 (asym.& sym.str.) for -NH 2 and 2210 cm -1 for -CN functional group respectively.The presence of band around 1220 cm -1 of cyclic ether linkage supports the formation of pyran derivative 6a.Further, the structure of selected compounds, 6e, 7e, 8f and 9h were confirmed by its mass spectral studies.The mass spectra detected the expected molecular ion signals corresponding to respective molecular formula of synthesized compounds.Mass spectra of compound 6e gave molecular ion peak at 425.1 (M + +1) corresponding to molecular formula C 24 H 20 N 6 O 2 .
Similarly, all these compounds were characterized on the basis of spectral studies.All spectroscopic data have been given in experimental section.All the compounds were screened for their antibacterial and antifungal activity.

Antimicrobial screening
The in vitro antimicrobial activity of all the synthesized compounds was carried out by broth microdilution method. 19Mueller Hinton broth was used as nutrient medium to grow and dilute the compound suspension for the test bacteria and Sabouraud Dextrose broth used for fungal nutrition.Inoculum size for test strain was adjusted to 10 8 CFUmL -1 (Colony Forming Unit per milliliter) by comparing the turbidity.The strains employed for the activity were procured from (MTCC -Micro Type Culture Collection) Institute of Microbial Technology, Chandigarh.
The compounds 6a-h, 7a-h, 8a-h and 9a-h were screened for their antibacterial activity against Bacillus subtilis (MTCC 441), Clostridium tetani (MTCC 449), Streptococcus pneumoniae (MTCC 1936), Escherichia coli (MTCC 443), Salmonella typhi (MTCC 98) and Vibrio cholerae (MTCC 3906) as well as antifungal activity against Aspergillus fumigatus (MTCC 3008) and Candida albicans (MTCC 227).DMSO was used as vehicle to get desired concentration of compounds to test upon microbial strains.The lowest concentration, which showed no visible growth after spot subculture was considered as MIC for each compound.Ampicillin, chloramphenicol, ciprofloxacin, gentamicin and norfloxacin were used as standard antibacterial drugs, whereas griseofulvin and nystatin was used as standard antifungal drugs.The protocols were summarized in Table 1.
The examination of the data (Table 1) reveals that most of the compounds showed excellent antibacterial and antifungal activity when compared with ampicillin and griseofulvin.Against Gram positive pathogen S. pneumoniae, compounds 8b and 9a were found to be more efficient than chloramphenicol and ciprofloxacin where as, 8c and 9h were more potent, and 7f, 8g and 9f were found to exhibit comparable activity, to ampicillin.The compounds 9f and 8g possess comparable activity to ciprofloxacin, and 6a, 6d-g, 7b, 7f, 8a-b, 8d-e, 8h, 9c-d and 9g-h were found equally potent to ampicillin, towards C. tetani.The compounds 8b and 9a shows better activity where as, 9h is equally active compared to ciprofloxacin and chloramphenicol and compounds 8c, 8g, 9f shows better activity, and 6a-b, 6d, 7a, 7e-f, 8a, 8d-e, 8h, 9c-d and 9g found equally potent, to ampicillin, against B. subtilis.

ISSN 1551-7012
Page 367 © ARKAT USA, Inc. Towards Gram negative strain S. typhi, compounds 8b, 9h and 9a were equally active to ciprofloxacin and chloramphenicol where as, compounds 8g, 9c and 9f were found better active and 8c is equally active than ampicillin.The Compounds 8b, 9a and 9h found equipotent to ciprofloxacin and chloramphenicol where as, compounds 6b, 8c, 8g and 9f are comparably active to ampicillin against V. cholerae.The compounds 8b shows better, and 7f, 8g, 8h, 9c and 9d were found to exhibit comparable activity to ampicillin towards E. coli.The compounds 8b and 8g were highly active against all the the tested Gram positive and negative pathogens.The remaining compounds showed moderate to good activity to inhibit the growth of bacterial pathogens and are all less effective than ampicillin.Against fungal pathogen C. albicans, compound 6d found better activity where as, 6a, 6c, 6g, 7b-c, 7e, 7g, 8b, 8d, 8h, 9b, 9d, 9f and 9g were found to be equipotent compared to griseofulvin.None of the tested compounds found to be potent against A. fumigatus compared to standard drugs.

Conclusion
A new series of substituted 4-pyrazolyl-4H-pyrazolopyran 6a-h, -benzopyran 7a-h and naphthopyran 8a-h, 9a-h derivatives has been synthesized via an MCR approach and was characterized by elemental and spectral analysis.This synthetic strategy allows the construction of relatively complicated oxygen containing fused heterocyclic system as well as the introduction of various (hetero)aromatic substitutions into 4-position of pyran system.It can be concluded from antimicrobial screening (Table 1), against panel of human pathogens, that most of the synthesized naphthopyran derivatives 8a-h, 9a-h was found to be highly active, compared to pyrazolopyran 6a-h and benzopyran 7a-h, against bacterial pathogens.Among them, compounds 8b, 8g, 9a, 9f and 9h were found to be the most active against the microorganisms employed for antibacterial activity.Antifungal activity of the compounds shows that most of the compounds found to be potent against C. albicans compared to A. fumigatus.It is worth mentioning that minor change in molecular configuration of these compounds profoundly influences the activity.

Experimental Section
General.All the reagents were obtained commercially and used with further purification.Solvents used were of analytical grade.All melting points were taken in open capillaries and are uncorrected.Thin-layer chromatography (TLC, on aluminium plates coated with silica gel 60F 254 , 0.25 mm thickness, Merck) was used for monitoring the progress of all reactions, purity and homogeneity of the synthesized compounds; eluent-toluene:ethyl acetate::7:3.UV radiation and iodine were used as the visualizing agents.Elemental analysis (% C, H, N) was carried out by Perkin-Elmer 2400 series-II elemental analyzer at Sophisticated Instrumentation Centre for Applied Research & Training (SICART), Vallabh Vidyanagar and all compounds are within ±0.4% of theory specified.The IR spectra were recorded in KBr on a Perkin-Elmer Spectrum GX FT-IR Spectrophotometer and only the characteristic peaks are reported in cm -1 . 1 H NMR and 13 C NMR spectra were recorded in DMSO-d 6 on a Bruker Avance 400F (MHz) spectrometer using solvent peak as internal standard at 400 MHz and 100 MHz respectively.Chemical shifts are reported in parts per million (ppm).Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer.1-Phenyl-3-(het)aryl-pyrazole-4-carbaldehydes 1a-h were prepared by Vilsmeier-Haack reaction of acetophenone (het)arylhydrazones (Scheme 1). 20

Scheme 2 .
Scheme 2. Plausible mechanistic pathway of the synthesis of pyran derivatives.

Table 1 .
Antimicrobial activity of the compounds