Microwave assisted synthesis, spectral studies and antimicrobial activities of some 2′,4′-difluorophenyl chalcones

Some 2′,4′-difluorophenyl chalcones have been synthesized under microwave irradiation using aldol condensation between 2,4-difluoroacetophenone and substituted benzaldehydes using catalytic amount of hydroxyapatite. The yields of the chalcones are more than 85 %. The purities of these synthesized chalcones were examined by their physical constants and spectroscopic data. The UV absorption maxima (λmax, nm), infrared stretches (ν, cm -1 ) of CO, fingerprint region of CHip/op, CH=CHop, C=Cop modes, NMR chemical shifts (δ, ppm) of vinyl proton, carbon and carbonyl carbons have been assigned and correlated with Hammett substituent constants, F and R parameters using single and multi-regression analysis. From the statistical analysis the effect of substituent on the above spectral frequencies can be discussed. The antimicrobial activities of these synthesized chalcones have been screened using Bauer-Kirby method.

Presence of the reactive keto group and the vinylenic group in the chalcone and their analogues possesses the antioxidant activity [20]. Compounds that associated with the antioxidant properties are hydroxyl and phenyl group. Oxidative stress which is caused by the free radical damage is help to deal with the antioxidants [21]. Spectroscopic data is useful for study of effects of substituents on the specified spectral group frequencies using Hammett substituent constants, F and R parameters by single and multi-regression analysis.
From the results the ground state molecular equilibration can be predicted. Recently Subramanian et. al. [22,23] have studied the synthesis and spectral correlations of some heterocyclic chalcones and they observed satisfactory correlations. Sathiyamoorthy et al., [24][25][26] also studied the same with the some iodo and hydroxy substituted phenyl chalcones. With a view to above there is no report available for microwave assisted synthesis, the study of substituent effects and screening of antimicrobial activities of 2′,4′-diflurophenyl chalcones in the past in literature. Therefore the authors have taken efforts for microwave assisted synthesis of 2′,4′-difluorophenyl chalcones, study of substituent effects and screening of antimicrobial activities using Bauer-Kirby [27] method.

1. Materials and Methods
All the chemicals involved in the present investigation, have been procured from Sigma-Aldrich and E-Merck chemical companies. Melting points of all chalcones have been determined in open glass capillaries on SUNTEX melting point apparatus and are uncorrected. The UV spectra of all the chalcone, synthesized, have been recorded with ELICO-BL222 spectrophotometer ( max nm) in spectral grade methanol solvent. Infrared spectra (KBr, 4000-400 cm -1 ) have been recorded on AVATAR-300 Fourier transform spectrophotometer. The NMR spectra were recorded in Bruker AV400 NMR spectrometer operating at 400 MHz has been utilized for recording 1 H NMR spectra and 100 MHz for 13 C spectra in CDCl 3 solvent using TMS as internal standard.

Synthesis of 2′,4′-fluorophenyl chalcones
An appropriate equimolar quantities of 2,4-difluoracetophenone (2 mmol), substituted benzaldehydes (2 mmol), methanol (10 mL) and 1 g hydroxyapatite was added and the mixture was stirred at room temperature for 5 min. Methanol was evaporated to give a homogeneous solid. About 5 mL of water was added to this solid and the mixture was irradiated by microwave for 5-6 minutes (Scheme 1) [22]. After completion of reaction, dichloromethane (20 mL) was added, followed by simple filtration. The solution was concentrated and to purified by re-crystallization. The synthesized chalcones are characterized by their physical constants, IR, 1 H and 13 C NMR and Mass spectral data. Analytical and Mass spectral data are presented in Table 1.

1. Spectral linearity
In the present investigation the Hammett spectral linearity of these synthesized chalcones has been studied by evaluating the substituent effects on the group frequencies.
The Hammett σ I , σ R constants, F and R parameters produced satisfactory correlations excluding H and 4-CH 3 substituents. When included these substituents in correlations, they reduced the correlation coefficients considerably and leads to fail.
The failure in correlation is due to the incapable of inductive and resonance effects of substituents on the absorption and is associated with the resonance-conjugative structure shown in Figure 1. The multi regression analysis of these frequencies of all ketones with inductive, resonance and Swain -Lupton's [36] constants produce satisfactory correlations as evident in equations (2 and 3).

1. 2. IR spectral study
The assigned carbonyl stretching frequencies (cm -1 ) of s-cis and s-trans isomers of present study are presented in Table 2 and the corresponding conformers are shown in Fig. 2. The stretching frequencies for carbonyl absorption are assigned based on the assignments made by Hays and Timmons [37] for s-cis and s-trans conformers at 1690 and 1670 cm -1 , respectively. These data have been correlated with Hammett substituent constants and Swain-

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Lupton's constants [36] and are presented in Table 3. In this correlation the structure parameter Hammett equation employed is as shown in the following equation: where ν is the carbonyl frequencies of substituted system and ν 0 is the corresponding quantity of unsubstitued system; σ is a Hammett substituent constant, which in principle is characteristics of the substituent and ρ is a reaction constant which is depend upon the nature of the reaction. The results of single parameter statistical analysis of carbonyl frequencies of s-cis conformers with all Hammett substituent constants, F and R parameters were poor correlated The failure in correlation is due the conjugation between the substituent and the carbonyl group in chalcones and is associated with the resonance conjugative structure shown in Figure 1. The s-trans conformer gave satisfactory correlation for constants, F and R parameters excluding H, 3-Br, 2-OCH 3 , 4-CH 3 substituents. If these substituents were included in the correlation, they reduced the correlation significantly.
The correlation of CH in-plane with Hammett substituent constants, F and R parameters were poor correlated and out of plane modes with Hammett σ and σ + constants shown satisfactory correlations. The σ I , σ R constants and F and R parameters has poorly correlated. The CH=CH and C=C out of plane modes with all Hammett substituent constants, F and R parameters were fail in correlation, This is due to the conjugation between the substituent and the vinyl group in chalcones as shown in Figure 1.
In view of the inability of some of the  constants to produce individually satisfactory correlations, it was thought that worthwhile to seek multiple correlations involving either  I and  R constants or Swain-Lupton's [36] F and R parameters. The correlation equations for scis, s-trans and deformation modes are given in equations 5-16.

1. 1 H NMR spectral study
The 1 H NMR spectra of synthesized chalcones have been recorded using deuteriochloroform employing tetramethylsilane (TMS) as internal standard. The ethylenic protons signals of the chalcones were assigned from their spectra. They were calculated as AB or AA' or BB' systems respectively. The lower chemical shifts (ppm) obtained for Hα and higher chemical shifts (ppm) obtained for Hβ in this series of ketones. The vinyl protons give an AB pattern and the β-proton doublets were well separated from the signals of the aromatic protons. The assigned vinyl proton chemical shifts δ(ppm) of all ketones were presented in Table 2. In nuclear magnetic resonance spectra, the proton or the 13 C chemical shifts (δ) depends on the electronic environment of the nuclei concerned. The assigned vinyl proton chemical shifts (ppm) have been correlated with reactivity parameters using Hammett equation in the form of Log δ = Log δ 0 + ρσ … (17) where δ 0 is the chemical shift of unsubstitued ketones.

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The remaining Hammett sigma constants and R parameters were fail correlations. The failure in correlation is due to the reason stated earlier.
Some of the single parameter correlation were fail in correlation for vinyl protons. While seeking the multi-correlation by the application of Swain-Lupton's [36] treatment to the relative chemical shifts of H α and H β with F and R values is successful with resonance, inductive effect generates the multi regression equations 18-21.

1. 4. 13 C NMR spectral study
Scientists and physical organic chemists [1,4,[22][23][24][25][26][31][32][33][34][35], have made extensive study of 13 C NMR spectra for a large number of different ketones and styrenes. The assigned vinyl C α , C β and carbonyl carbon chemical shifts are presented in Table 2. The results of statistical analysis are given in Table 3. The C α chemical shifts (ppm) gave satisfactory correlation with Hammett σ + constants. The correlation of these chemical shifts with remaining Hammett constant, F and R parameters gave poor correlation. The chemical shifts (ppm) of C β and CO carbons with Hammett substituent constants, F and R parameters gave poor correlation. This is due to the reason stated earlier and associated with resonance conjugative structure shown in Figure.

2. 2. Antifungal activity
The antifungal activity of the substituted styryl 2,4-difluorophenyl ketones have been measured using Baue-Kirby zone of inhibition method [27]. The measured antifungal activity of the synthesized chalcones were shown in Fig. 5, for Plates (1)(2)(3)(4). Analysis of the zone of inhibition as given in Table 5 and the Clustered column Chart given in Fig. 6. The 2-OCH 3, substituted compound has shown more antifungal activity than other substituents against the fungal specie Mucor species, under investigation. The 3-NO 2 substituent compound has shown equal antifungal activity against the above two fungal species.

CONCLUSION
Some 2′,4′-difluorophenyl chalcones have been synthesized by condensation of 2,4difluoroacetophenone and substituted benzaldehydes using microwave irradiation in the presence of hydroxyapatite catalyst. This reaction protocol offers a simple, easier work-up procedure and good yields. The chalcones have been characterized by their physical constants, spectral data. The UV, IR, NMR spectral data of these chalcones has been correlated with Hammett substituent constants, F and R parameters. From the results of statistical analyses the effects of substituent on the spectral data have been studied. The antimicrobial activities of all synthesized chalcone have been studied using Bauer-Kirby method.