Synthesis of Chromen-2one , Pyrano [ 3 , 4c ] chromene and Pyridino [ 3 , 4c ] chromene Derivatives as Potent Antimicrobial Agents

In an attempt for development of new antimicrobial agents; new series of chromen-2-one, pyrano[3,4-c]chromene and pyridino[3,4c]chromene derivatives bearing a diazo moiety were synthesized. Chromen-2-one derivatives were synthesized via treatment of 5-(aryldiazo) salicylaldehyde with different type of active methylene derivatives. Pyrano[3,4-c]chromene and pyridino[3,4-c]chromene derivatives were synthesized via treatment of chromen-2-one derivatives with another active methylene derivatives. The synthesized compounds were evaluated for their expected antimicrobial activity; where, the majority of these compounds showed potent antibacterial and antifungal activities against the tested strains of bacteria and fungi.


INTRODUCTION
3][4][5] Chromen-2-one moiety is being the parent scaffold in many of biological derivatives.Some of chromene skeleton are well established as antimicrobial agents such as Novobiocin and Chlorobiocin. [6]Recently, chromene derivatives have great interesting due to their potential application such as anti-HIV, [7] antitubercular, [8] antioxidant, [9] anticancer, [10] cytotoxic agents, [11] antidyslipidemic agent, [12] antileishmanial, [13] anti-inflammatory agents. [14]17][18] Bacterial and fungal resistance is one of the serious medical problems.Also, the levels rate of resistance is increasing to classical antibiotics.21][22] In light of these facts, the present study was designed to synthesize new chromene derivatives and evaluate their antimicrobial activity.As a trial to obtain novel class of antibacterial and antifungal agents, various groups were introduced into the target compounds

Chemistry
Treatment salicylaldehyde derivative 1 [23] with ethyl acetoacetate in boiling EtOH/DMF led to the formation of chromen-2-one derivative 2 as yellow solid in high yield (Scheme 1).Its IR spectrum showed absorption bands at: 1740, 1650 cm -1 corresponding to 2C=O functional groups. 1 H NMR revealed signals at: δ = 2.86 and 8.87 ppm corresponding to CH3 and chromene-H4, respectively.In N 1 H NMR spectrum displayed in addition to the aromatic proton signals, two doubles signals at: δ = 3.93 and 4.16 ppm integrated for two protons due to the two CH protons.Moreover, its 13 C NMR confirmed the structure.
On the same manner, interaction of 1 with 2-cyano-N'-(4-methoxybenzylidene)acetohydrazide afforded the corresponding iminochromene derivative 10.IR spectrum of 10 showed bands for amino and carbonyl groups at: 3301 and 1660 cm -1 , respectively. 1  (RCMB02593), Candida albicans (RCMB 05035), and Penicillium marneffei (RCMB 01267).Agar-diffusion method [26] was used for the determination of the preliminary screening of antibacterial activity.The newly synthesized target compounds were evaluated against clinical isolates of standard strains of fungi by the broth dilution method according to NCCLs [27] Ampicillin, Gentamycin and Amphotericin B were used as reference drugs for Gram-positive bacteria, Gram-negative bacteria and fungi, respectively.The results were recorded for each tested compounds as the average diameter of inhibition zones of bacterial growth around the discs in mm.The inhibition zone diameters, attributed to the tested original concentration (5 mg/mL) as a preliminary test, are shown in Table 1.
Concerning the antimicrobial activity of chromene derivatives 2, 5, 8, 9a,b, 10, 11, the results displayed those derivatives showed moderate to good activities against most of all the screened organisms.Using the general structure provided in Figure 1, certain aspects of the structure activity relationships for these compounds can be more clearly highlighted.
The results revealed that compound 2 showed antimicrobial activity nearly equipotent to the reference drug against all screened organisms.While compound 8 showed antimicrobial activity nearly equipotent to the reference drug only against gram -ve bacteria.Chromen-2-one 5, 9a,b, 10 and 11; X was NH group and R were cyano or carboxamide derivatives.Regarding the effect of R group, compound 5 showed high antimicrobial activity against all screened organisms.Compound 9b and 10 showed good antimicrobial activities only against gram +ve bacteria.Compound 11 showed high antimicrobial activity only against gram -ve bacteria.Compound 9a showed high antimicrobial activity only against fungi.Using the general structure provided in Figure 1, compound 6 showed high antimicrobial activity against all screened bacteria while compound 7 showed good activity only against fungi.
Concerning the antimicrobial activity of pyridino[3,4-c]chromene derivatives 12-15: The presence of pyridino [3,4c]chromene moiety resulted in the highest antimicrobial activity among all the compounds investigated in this study.The presence of pyridino [3,4-c]-chromene moiety exhibited the highest antibacterial activity against most of the organisms; pyridino[3,4-c]-chromene moiety showed results greater than the reference drug against most of the Table 1.Antimicrobial activities of the synthesized compounds against the pathological Gram +ve bacteria, Gram -ve bacteria and fungi expressed as inhibition diameter zones in millimeters Compd. No.

MINIMUM INHIBITORY CONCENTRATIONS AGAINST GRAM POSITIVE BACTERIA, GRAM NEGATIVE BACTERIA AND FUNGI
The minimal inhibitory concentrations (MICs) for the promising compounds were determined using twofold serial dilution method [27] MICs results (µg/mL) of the most promising derivatives 2, 3, 5, 6, 9a, and 11-15 are presented in Table 2.The majority of synthesized compounds showed varying degrees of inhibition against the test panel of species.

CONCLUSION
Chromen-2-one, pyrano [3,4-c]chromene and pyridino[3,4-c]chromene derivatives bearing a diazo moiety were synthesized in order to evaluate their antibacterial and antifungal activities.Regarding the effect of each derivative against bacterial and fungal strains, results of antimicrobial activity in this study revealed that: the majority of these    compounds showed potent antibacterial and antifungal activities against the tested strains of bacteria and fungi.Pyridino [3,4-c]chromene moiety resulted in the highest antimicrobial activity among all the compounds investigated in this study.Pyridino [3,4-c]chromene moiety showed results greater than the reference drug against most of the organisms.

Experimental Section
All melting points are recorded on digital Gallen Kamp MFB-595 instrument and may be uncorrected.The IR spectra (KBr) (cm -1 ) were measured on a JASCO spectrophotometer. 1 H NMR spectra were recorded on Bruker spectrometers (at 500 MHz) and are reported relative to solvent signals in deuterated dimethylsulfoxide (DMSO-d6). 13C NMR spectra were recorded on Bruker Spectrometers (at 125 MHz) in deuterated dimethylsulfoxide (DMSO-d6).The purity of the synthesized compounds was monitored by TLC.Elemental analyses were carried out by the Microanalytical Research Center, Faculty of Science, Cairo University.Analytical results for C, H and N were within -/+ 0.4 of the calculated values.The antimicrobial screening and minimal inhibitory concentrations of the tested compounds were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
.7±0.1 21.9±0.1 26.4±0.222.6±0.3organisms.Using the general structure provided in Figure 2, a moderate difference in antimicrobial activity is noted between the tested pyridino[3,4-c]chromene derivatives, this indicate that the main effect related to the presence of the pyridino[3,4-c]chromene moiety.The comparison between the antimicrobial activity of pyridino[3,4-c]chromene and standard reference drugs against the used Gram positive, Gram negative bacteria and fungi is represented graphically in Figure 3.

Figure 3 .
Figure 3.The comparison between the antimicrobial activities of pyridino[3,4-c]chromene and standard drug against the used Gram +ve, Gram -ve bacteria and fungi.