Synthesis and Evaluation of Substituted 4,4a-Dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-oneasAntimicrobialAgent

A series of pyrano[4,3-b][1]benzopyranones ( 7a–t ) were synthesized through hetero-Diels-Alder reaction of substituted 3-formyl-chromones( 5 )withenolethers( 6 ),characterizedbyIR, 1 HNMR, 13 CNMR,andmassspectraltechniques.Allthecompoundswere evaluated for antimicrobial activity against various bacterial and fungal strains, found to possess significant inhibitory potential, particularly, compounds bearing electron withdrawing group - fluoro such as 7i and 7h . Compounds were also tested and displayed a significant inhibitory potential against methicillin-resistant Staphylococcus aureus analysis: calculated for 65.74 H 5.86% and 65.70


Introduction
Despite decades of extensive progress in treatment and prevention, infectious diseases remain a major cause of death and are responsible for worsening the living conditions of many millions of people around the world [1].Additionally, resistance to known antibiotics is also a serious problem and presents a challenge for the medicinal chemists to develop new effective molecular entities against pathogenic microorganism resistant to available current treatments [2].Chromones are an important class of heterocyclic molecules naturally occurring, and synthetic analogs are found to display a wide range of pharmacological activities such as antimicrobial, anticancer, neuroprotective, HIV-inhibitory, antifungal activities, and antioxidant [3][4][5][6][7][8][9].Natural products such as aposhaerin A (1), isolated from Aposhaeria sp.possess remarkable antibacterial activity [10].Recently, we have reported that 3-(5-phenyl-3H- [1,2,4]dithiazol-3-yl-) chromen-4-ones (2) possess significant antibacterial activity against Shigella flexneri (Figure 1) [11].
Taking cognizance of high antimicrobial activity of both chromone and pyran derivatives, it was decided to synthesize chromone fused pyrans and evaluate against various pathogenic bacterial and fungal strains. (2) Figure 1: Chromone based compounds as antimicrobial agents.
Figure 2: Pyran based compounds as antimicrobial agents.

Results and Discussion
2.1.Chemistry.Substituted pyrano [4,3-b][1]benzopyranones were synthesized by the hetero-Diels-Alder reaction of substituted 3-formylchromones (5a-j) with excess of enol ethers (6) in dichloromethane at room temperature [19][20][21][22].All the purified products were characterized by rigorous spectroscopic techniques (IR, 1 H NMR, 13 C NMR, and mass) and elemental analysis (Scheme 1, Table 1).Finally, the structure of compound 7k was confirmed by X-ray crystallography (Figure 3) [23]. 1 H NMR spectrum of 7k displayed doublet of C9-H at  7.91 with  = 6 Hz, and C1-H showed up as a doublet at  7.51 with  = 1.2 Hz.Resonances of C3-H and C4a-H appeared as a multiplet at  5.16-5.10.C4-Ha resonance appeared at  2.53 as dd with  gem = 12.9 Hz and  = 6.9 Hz; C4-Hb showed up as a dt with  gem = 12.9 Hz and  = 9.9 Hz at  2.30. 13C NMR revealed a quaternary carbon resonance at 181.8 ppm attributed to carbonyl carbon (C10), which was further corroborated by a strong characteristic band at 1668 cm −1 in the IR spectrum.Further, the mass spectrum of 7k (ESI) showed the highest ion peak at / 247 (M + +1).The stereochemistry of product 7k (endo) was assigned on the basis of NMR spectral evidence.C4-Hb showed vicinal coupling constants of 6.9 and 2.4 Hz which can be attributed to axial-equitorial relationships with C4a-H and C3-H, whereas C4-Ha showed vicinal coupling of ∼10.0 Hz with both C4a-H and C3-H indicating its diaxial relationship with both neighbouring protons, and this alludes to cis-relationship between C4a-H and C3-H; this trans-diaxial relationship was further confirmed by X-ray crystallographic structure determination of 7k (Figure 3) [20][21][22].The corresponding exo-isomers (8a-t, traces) were detected by 1 H NMR of some column fractions, and the ratio of endo/exo was determined from NMR of crude reaction mixture (4 : 1 approximately).The endo and exo approaches leading to compounds 7 and 8 are shown in Figure 4.

Antibacterial Activity.
All the synthesized compounds (7a-t) were screened for their antibacterial potential in triplicate against two Gram-positive bacteria, Staphylococcus aureus (MTCC96), Bacillus subtilis (MTCC2451), and three gram-negative bacteria, Escherichia coli (MTCC 82), Pseudomonas aeruginosa (MTCC 2642), and Salmonella typhimurium (MTCC 1251), by using disc diffusion method [24].The activity of compounds was determined in comparison to standard antibiotic discs of amoxicillin (5 g) and ciprofloxacin (10 g).Minimum inhibitory concentration (MIC) in g/mL of compounds exhibiting activity (Table 2) was determined by using serial tube dilution method [25].All tested compounds were found to exert prominent antibacterial activity against both gram-positive and gram-negative bacterial strains.Compound 7i showed comparable potent inhibitory activity with positive controls against various bacterial strains such as MIC 0.48 against both S. aureus and E. coli, whereas MIC 1.12 against both B. subtilis and P. aeruginosa.Compound 7d showed high inhibitory potential against gram-negative bacterial strain E. coli with MIC 1.56, followed by MIC 1.82 against both B. subtilis and S. aureus, and MIC 12.5 against P. aeruginosa and S. typhi.Compounds 7e and 7n showed good activity against B. subtilis with MIC 6.25, whereas compound 7j showed activity against E. coli and S. aureus with MIC 6.25 and 12.5, respectively.Compounds 7m and 7q showed significant activity against S. aureus and B. subtilis with MIC 6.25 and 12.5, respectively.Compounds 7n and 7o showed promising inhibitory activity against both S. aureus and E. coli, whereas compounds 7j and 7q showed inhibitory potential against P. aeruginosa with MIC 12.5.The literature reports reveal that these types of tricyclic compounds have been isolated from a strain of Chaetomium funicola, which act as potent broad-spectrum metallo-lactamase inhibitors [26].Active compounds were further evaluated against bacterial resistant strains such as methicillin resistant staphylococcus aureus (MRSA), a clinically isolate obtained from PGIMER, Chandigarh, and Klebsiella pneumoniae (MTCC 530) by using disk-diffusion assay.Compounds were found to be active against MRSA and completely inactive against Klebsiella pneumoniae.Minimum inhibitory concentration (MIC) in mg/mL of compounds exhibiting activity (Table 3) was determined by using serial tube dilution method [25].All the compounds were found to be active against resistant bacterial strain MRSA, whereas compound 7j showed a maximum activity in comparison to other compounds.

Antifungal Activity.
All synthesized compounds 7a-t were tested against five reference fungal strains: Aspergillus niger (MTCC 1344), Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), Cryptococcus gastricus (MTCC 1715), and Microsporum gypseum (MTCC 4490) by using disc diffusion method [24].Moreover, the compounds were found to exert prominent antifungal activity against various fungal strains, specially, against A. niger, S. cerevisiae, and C. albicans (Table 4).Compound 7h showed significant inhibitory activity with MIC 2.4, whereas, compounds 7d, 7g, 7j, and 7m exhibit good inhibitory potential against A. niger with MIC < 15.Compound 7j posseses maximum inhibitory Compounds bearing electron withdrawing groups such as -fluoro and -chloro at chromone ring were found to display high activity against both bacterial and fungal strains, whereas substitution with electron donating group led to a decrease in activity.According to Craig's plot, these -fluoro, -chloro, and -bromo groups are lipophilic in nature, having high -values; from the literature, it was found that lipophilicity is essential for the compound permeability across the microbes cell membrane [27].Therefore, compounds having these lipophilic groups exhibit valuable inhibitory potential; similarly, compounds having bulkier or lipophilic group such as Oi-Bu-at position 3 of the fused pyran ring were found to be more active than -OEt against various pathogenic bacterial strains.Disubstitution with electron withdrawing groups such as -fluoro and -chloro on chromone ring showed moderate inhibitory activity against both bacterial and fungal strains.

Conclusion
Variously substituted pyrano [4,3-b][1]benzopyrans (7a-t) were synthesized through the hetero-Diels-Alder reaction [4 + 2] of substituted 3-formylchromones (5) with excess of enol ethers (6) in dichloromethane at room temperature.Compounds bearing electron withdrawing groups such as -fluoro and -chloro at chromone ring were found to display high activity against both bacterial and fungal strains such as compound 7i which showed excellent antibacterial activity and compound 7h which displayed promising antifungal activity.All active compounds were also evaluated against bacterial resistant strain MRSA and found to posseses good inhibitory potential, particularly, compound 7j.These "lead" compounds can be taken under consideration for further antimicrobial development and their mode of action.

Experimental
4.1.General.Starting materials and reagents were purchased from commercial suppliers and used after further purification (crystallization/distillation).Bruker (400 MHz), JEOL AL-300 FT (300 MHz), and NMR spectrometer were used to record 1 H NMR (300 MHz and 400 MHz) and 13 C NMR (75 MHz and 100 MHz) spectra, and chemical shifts () are reported as downfield displacements from tetramethylsilane (TMS) used as an internal standard, and coupling constants () are reported in Hz.IR spectrum was recorded with Shimadzu FT-IR-8400S and Bruker spectrophotometers on KBr pellets.Mass spectrum, EI, and ESI methods were recorded on Shimadzu GCMS-QP-2000A and Bruker Daltonics Esquire 300 mass spectrometer, respectively.Elemental analyses were carried out on a Thermoelectron EA-112 elemental analyzer and are reported in percent abundance.

Antifungal Activity.
All synthesized compounds 7a-t were tested against five reference fungal strains, Aspergillus niger (MTCC 1344), Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), Cryptococcus gastricus (MTCC 1715), and Microsporum gypseum (MTCC 4490) by using disc diffusion method [20][21][22].The antifungal activity of synthesized compounds was determined by observing the zone of inhibition in comparison to the standard antifungal discs (fluconazole and griseofulvin).Test compounds were dissolved in DMSO to make a stock solution of 1 mg/mL.The fresh subculture of strains in normal saline was added to the sterile assay medium (Sabouraud Dextrose agar with chloramphenicol) at 40-45 ∘ C and mixed well.The medium was poured into each of the petri dishes.Sterile discs of diameter 6 mm were placed on the medium.20 L of each test solution was added to the previously marked discs and the media were allowed to stand for 5 min.The petri dishes were kept aside for 1 h, and then incubated at 28 ∘ C for 48 h.Zone of inhibition was measured, and the average of the three readings was calculated; DMSO was also used as negative control.The MIC of active compounds (zone of inhibition) was determined by serial tube dilution method [25].Different dilutions of test compounds (1.9 g/mL-500 g/mL) were made from stock solution, 1 mL nutrient broth was taken in each test tube, and 20 L of standard strains was added to previously marked test tubes.

Table 1 :
Reaction time (h) and yield (%) of various purified products.
potential against S. cerevisiae with MIC 2.9, whereas compounds 7b, 7h, 7i, and 7t displayed good inhibitory potential with MIC < 15.Compounds 7f, 7n, and 7r showed promising activity against C. albicans with MIC < 15.Compound 7q is found to display high antifungal activity as compared to standard drug with MIC 20.5 against C. gastricus.