Synthesis and anti-microbial / anti-malarial activity of a new class of chromone-dihydroquinazolinone hybrid heterocycles

A new series of chromone-2,3-dihydroquinazolin-4-one hybrid heterocycles are synthesized from chromone-2-carbaldehydes by coupling with 2-aminoarylamides and hydrazides without oxidizing agents. The newly synthesized products exhibited moderate to good antimicrobial activity.


Introduction
Chromones and their derivatives are well-known naturally occurring oxygen heterocyclic compounds which exhibit important biological functions in nature.2][3] Simple, nonhydroxylated chromones were discovered to be selective inhibitors of p56lck tyrosine kinase. 4Thus, a simple, efficient synthesis of chromone molecular hybrids remains an important research topic.
Following the above observations, the study was extended to 2-aminobenzohydrazides 14a-c which were synthesized using commercially available isatoic anhydride 12 and different hydrazine's 13a-c in DMF (Scheme 3).The reaction of 4-oxo-4H-chromene-2-carbaldehyde 9a-f with 2-aminobenzohydrazide 14a gave imine intermediates 15a-f and the desired cyclized products 16a-f were not observed even under forcing experimental conditions (Scheme 4).Literature survey supported the imine formation from aldehydes with 2aminobenzohydrazide. 51 The IR (solid, KBr) spectrum of 2-amino-N'-[(4-oxo-4H-chromen-2-yl)methylene]benzohydrazide 15a exhibited absorption bands at 3466 cm -1 (NH, primary amine), 3351 cm -1 (NH, secondary amine), 1738 cm -1 (CO, chromone) and 1663 cm -1 (CO, amide).The 1 H NMR (400 MHz, DMSO-d6) spectrum of 15a showed twelve signals corresponding to thirteen protons.The aldehyde proton of 9a had disappeared.D2O exchange analysis showed exchangeable protons at δH 12.2 (s, 1H, amide NH) and δH 6.52 ppm (s, 2H, NH2).Presence of two amine protons suggested the involvement of only the hydrazide NH2 in the reaction.The absence of a proton in the aliphatic region suggested that the compound formed was an imine, but not the expected cyclic product.The 13 C NMR (100 MHz, DMSO-d6) of 15a showed seventeen signals between δC 111.71 ppm and 176.84 ppm.The absence of aliphatic carbons correlates with the absence of aliphatic protons in 1 H NMR. The positive ESI-MS and HRMS data of 15a showed the molecular ion at m/z 308.1 [M+H] + and 308.1046 [M+H] + , respectively.From the HSQC data of 15a, the absence of correlations for the protons at δH 12.2 and 6.52 ppm confirmed that these are not attached to any carbon.This data supported the D2O exchange information.The HSQC data showed that there are ten methine groups and seven quaternary carbons, two of them corresponding to carbonyl carbons.HOMO COSY of 15a showed two singlets at δH 8.25 and 6.72 ppm which did not show any correlations.From the 1 H NMR study on 4-oxo-4H-chromene-2-carbaldehydes, the proton at δH 6.72 ppm was assigned to the H-3 olefin proton.The other proton at δH 8.25 ppm which correlated with the carbon at δC 138.7 ppm (HSQC) was predicted to be from an imine.This was supported by the double bond equivalence (DBE) from HRMS data.
The results can be explained based on the difference in nucleophilicity of the NH2 of hydrazide and aniline.The lone pair of electrons on the amino group corresponding to aniline 14a are conjugated with the aromatic nuclei and also the carbonyl group at the ortho position.Hence, these electrons are not freely available for the reaction when compared to the lone pair of electrons on the NH2 of hydrazide 14a.
Further reaction was carried out using the N-substituted 2-aminobenzohydrazides 14b,c.Interestingly, the compounds obtained were the desired cyclic products 19a-f (Scheme 5).In the 1 H NMR (400 MHz, DMSO-d6) spectra of the obtained compounds 19a-f, a characteristic signal of H-2 was observed at δH 5.93 -6.41 ppm and the corresponding carbon signal was observed at δC 71.4 -72.2 ppm in 13 C NMR.In the case that the free NH2 of hydrazide 14a was substituted with a phenyl 14b (where the lone pair is conjugated with the phenyl ring), the NH of the amide participated in the reaction, resulting in cyclized compounds having two heterocyclic moieties.

Biological Activity In vitro antibacterial activity
The hybrid molecules synthesized were tested against Gram-positive bacteria Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC 442), and Gram-negative bacteria Escherichia coli (MTCC 443), and Pseudomonas aeruginosa (MTCC 741) (Table 1).Compounds 11b, 19a-c, 20b showed very good activity against the gram-positive organism Staphylococcus aureus compared to the standard drug Ampicillin.The compound 19b exhibited higher activity against gram-negative organism Escherichia coli when compared with the drug Ampicillin.

Antimalarial activity
The antimalarial activity of the synthesized compounds was evaluated and compared with standard drugs Chloroquine and quinine.Activity of all the synthesized compounds was found to be lower than the standard drugs.

Conclusion
In summary, we have developed a simple, efficient and convenient method for the synthesis of novel chromone / 2,3-dihydroquinazolin-4-one hybrid heterocycles by coupling of 2-aminobenzamide / 2-aminobenzohydrazides with chromone-2-carbaldehydes.Some of the synthesized compounds were found to exhibit moderate to very good antimicrobial activity.Hence, derivatives of chromone / 2,3-dihydroquinazolin-4-ones can be utilized in the future for the development of potent antimicrobial drugs.

Experimental Section
General.Electrospray ionization and tandem mass spectrometry experiments were performed using a triple quadrupole mass spectrometer (PE Sciex model API 3000).The positive and negative electrospray data were obtained by switching the capillary voltage between +5000 and −4500 V, respec vely.For HRMS, UPLC-TOF-MS system consisted of an Acquity™ Ultra Performance Liquid Chromatography system and Micromass LCT Premier XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA) equipped with an ESI lock spray source for accurate mass values.Leucine-enkephalin was used as reference compound, was introduced via the lock spray channel.
The NMR experiments were performed on Varian spectrometers operating at 400 and 500 MHz in DMSO-d6 at 30 o C. The 1 H chemical shift values were reported on the δ scale in ppm, relative to TMS (δ = 0.00) and the 13 C chemical shift values were reported relative to DMSO (δ = 40 ppm) as internal standard.Standard pulse sequences provided by Varian were used for distortionless enhancement by polarization transfer (DEPT), gradient double quantum filtered correlation spectroscopy (gDQCOSY), and gradient heteronuclear single quantum coherence spectroscopy (gHSQC).Biological activity measurements were performed at M/s Microcare Laboratories, Surat, India.Antibacterial activity: Minimum inhibitory concentration (MIC) assay of the hybrid molecules synthesized was done by broth dilution method in tubes for macro dilution and in plates for micro dilution.Muller Hinton broth was used as nutrient medium to grow and dilute the drug suspension for the test bacteria.DMSO was used as diluent to get the desired concentration of synthesized compounds.Standard drugs ampicillin and ciprofloxacin were used for comparison.
Antifungal activity was performed against the fungal strains Candida albicans (MTCC 227), Aspergillus niger (MTCC 282) and Aspergillus clavatus (MTCC 1323).Fungal growth was done with Sabourauds dextrose broth at 28.8 o C in aerobic condition for 48 hrs.2% DMSO and sterilized distilled water were used as negative control and Griseofulvin (1 U strength) was used as positive control.Results were recorded in the form of primary and secondary screening.Antimalarial Activity.The in vitro antimalarial assay was carried out in 96 well microtitre plates according to the micro assay protocol reference.Chloroquine and Quinine were taken as the reference drug for comparison.
General procedure for synthesis of 4-oxo-4H-chromen-2-carbaldehydes (9a-c) 1,4-dioxane (10 volumes), 2-methyl-4H-chromene-4-ones (7a-c) (160 mg, 1.0 mmol) and a catalytic amount of hydrogen peroxide were placed in a three-necked flask and selenium dioxide (177.5 mg, 1.6 mmol) was added under stirring and heated to 100-105 o C.After completion of the reaction (16 h.; TLC monitoring), the reaction mass was cooled to 25-35 o C and the selenium salts were removed by filtration.The filtrate was concentrated and the crude compound was purified by silica gel column chromatography.Elution of the column with ethyl acetate/petroleum ether 15:85 gave compounds 9a-c in 60% yield.

Table 1 .
Antimicrobial activity results of the synthesized compounds Minimal inhibition concentration (µg/ml)