1-Indanone chalcones and their 2 , 4-Dinitrophenylhydrazone derivatives : Synthesis , physicochemical properties and in vitro antibacterial activity

Chalcones are natural biocides. Several publications appear every year covering the synthesis of chalcones because they exhibit an array of pharmacological activities. In this study, some condensation reactions of 1-Indanone with substituted benzaldehydes were carried out under different reaction conditions. The chalcone products were converted to their corresponding 2,4Dinitrophenylhydrazone derivatives and evaluated against five gram-positive and eight gram-negative bacteria for their in vitro antibacterial property. Antimicrobial activity was observed against many of the tested strains, with zones of inhibition ranging from 10 to 28 mm. In many cases, the hydrazone derivatives were more active than their chalcone precursors. The best results were obtained against gram-negative bacteria for most of the compounds. Compound 1b was the most active with its minimum inhibitory concentrations (MICs) against six strains of bacteria ranging from 15.6 to 31.3 μg/ml, hence, could be developed as an antibacterial agent against infections caused by some gramnegative bacteria such as Pseudomonas aeruginosa and Salmonella typhimurium.

From the chemical point of view, they are α, β-Unsaturated ketones which are readily obtained via synthetic routes in the laboratory, by base-catalyzed condensation of aromatic (or heteroaromatic) aldehydes with an acetophenone (or its analogs) in aqueous alcohol (this is the classical Claisen Schmidt condensation reaction), (Furnis et al., 2004) (Scheme 1).Bases that have been used include NaOH, LiOH, Ba(OH) 2 and Na 2 CO 3 in water (Zhang et al, 2003).They have been prepared also under ultrasound irradiation of alcohol solutions of mixtures of benzaldehydes and acetophenones, catalyzed by KOH and KF-Al 2 O 3 (Li, 2002).
Hydrazones (aryl-, heteroaryl, acyl and sulfonyl) have been reported to exhibit diverse biological properties such as anti-tubercular, anti-malarial, antimicrobial, antitumor, anti-inflammatory, anticonvulsant, analgesic, etc, activities and possess varied analytical applications (Rollas and Küçükgüzel, 2007;Ajani et al., 2010;de Oliveira et al., 2011;Suvarapu et al., 2012).It is pertinent to point out here that the development of drug resistance in human pathogens against commonly used antibacterial drugs, resulting in relapse of disease, has necessitated the search for new antimicrobial agents from both natural and synthetic sources.Screening of synthetic organic compounds for antimicrobial activities is important for finding potential new compounds for therapeutic use.The in vitro sensitivity testing of antibacterial agents against pathogenic bacteria is very important because the results are useful in carrying out studies of animal models of infection (in vivo studies) (Greenwood, 1981).It has been pointed out that in vitro tests could not usually "be developed that would make it possible to (quantitatively) predict the efficacy of any antibiotic against any specific infection in vivo".The results of in vivo studies are utilized on the long run by practicing physicians to determine treatment recommendations for patients with infections (Greenwood, 1981;Zak et al., 1985).
In this paper, we wish to report the syntheses of 1-Indanone chalcone analogs using different reaction conditions, their corresponding 2,4-Dinitrophenylhydrazone derivatives and the assessment of their effect on some pathogenic bacteria.

Chemistry
The purity of all described compounds was checked by melting point (m.p.) and thin layer chromatography (TLC) (E.Merck Kieselgel 60 F254).
Melting points (uncorrected) were determined using a gallenkamp melting point machine.R f values were determined using silica gel F 254 TLC plates (Merck), developed with n-Hexane:ethyl acetate (2:1) and observed under UV light (λ = 254 and 366 nm).The infrared (IR) spectra were recorded as KBr pellets using a Bruker IFS 13v Fourier transform infrared spectroscopy (FT-IR) spectrometer. 1H NMR and 13 C NMR were recorded on a Bruker 400 MHz AVANCE spectrometer.The data were obtained from CDCl 3 solutions.Chemical shifts are given in the δ scale (ppm) using tetramethylsilane as an internal standard.The elemental analysis (C, H, N) of the compound was performed using a Carlo Erba-1108 elemental analyzer.

Microbiology: Antibacterial sensitivity testing
The antibacterial activities of the compounds were determined using the agar-well diffusion method as described by Russell and Furr (1977) and Akinpelu and Kolawole (2004).

Minimum inhibitory concentration (MIC)
The MICs of the compounds and the reference antibiotic were Scheme 2. Synthesis of 1-Indanone chalcones and hydrazone derivatives.
4b: R 1 = R 2 = H; R 3 = OH 5a: R 1 = H; R 2 = OH; R 3 = H 5b: R 1 = H; R 2 = OH; R 3 = H 6a: R 1 = H; R 2 = OCH 3 ; R 3 = OH 6b: R 1 = H; R 2 = OCH 3 ; R 3 = OH 7a: R 1 = NO 2 ; R 2 = R 3 = H 7b: R 1 = NO 2 ; R 2 = R 3 = H Method a: KOH/MeOH, rt (used to synthesize 1a -3a, 6a); Method c: SOCl 2 /ethanol (used to synthesize 4a, 5a, 7a).Method b: Microwave-assisted synthesis (used to synthesize 4a -6a, 1b -5b, 7b); Method d: conventional heating (used to synthesize 6b) determined using the method of Akinpelu and Kolawole (2004).Dimethyl sulfoxide was used as negative control.Two milliliter of different concentrations of the test solution was added to 18 ml of pre-sterilized molten nutrient agar at 40°C to give final concentration regimes of 0.0313 and 2.0 mg/ml for the test compound and 0.0157 and 1.0 mg/ml for the standard antibiotics.The medium was then poured into sterile Petri dishes and allowed to set.The surfaces of the media were allowed to dry under a laminar flow before streaking with 18 h old bacterial cultures.The plates were later incubated at 37°C for up to 72 h after which they were examined for the presence or absence of growth.The MIC was taken as the lowest concentration of the test compound and standard antibiotics that will prevent the growth of the susceptible test bacteria.

Chemistry
The 1-Indanone chalcones (2a -7a) were synthesized by a Claisen-Schmidt type of reaction in which various substituted benzaldehydes were reacted with 1-Indanone, using acid or base catalysts (Scheme 2).Benzaldehydes with hydroxyl substituents gave very low yields of the corresponding indanone chalcones when the conventional base catalyzed (methanolic KOH) condensation method was used, hence acid catalyzed procedure was employed for the synthesis of the chalcones.Compound 6a was synthesized in high yield using microwave assisted method in the presence of a mixture of acetic acid and conc.H 2 SO 4 .This method, however, did not work well for the synthesis of Compounds 4a and 5a (where charring was observed).A different method was then employed for the synthesis of the two compounds, whereby HCl (generated in situ by adding a small quantity of SOCl 2 (thionyl chloride) to the alcoholic reaction medium) was used as a catalyst.
The base-catalyzed reaction of benzaldehyde with 1-Indanone yielded the dimer of the expected chalcone(1a), as reported from the reaction under basic condition (Berthelette et al., 1997).The corresponding chalcone hydrazones were synthesized from the condensation reaction of the chalcones with 2,4-DNP (2,4-) in methanol using conventional heating or microwave irradiation.The reaction of 2,4-DNP with the chalcones was rapid (30 s to 5 min).Only one of the carbonyl functional groups of Compound 1a reacted with the 2,4-DNP.The second carbonyl functional group was not assesible for the nucleophile (2,4-DNP) due to steric hindrance.The physicochemical properties of the compounds are listed in Table 1.
All compounds have been characterized on the basis of spectral analysis ( 1 H-NMR, 13 C-NMR and IR) and elemental analysis.Assignments of 13 C-NMR resonances of the compounds were deduced from the analysis of the Attached Proton Test (APT) and Distortionless Enhancement by Polarization Transfer (DEPT) experiments.The spectroscopic data for all the synthesized compounds are in agreement with their structures.In the 1 H-NMR spectra, the signals for the aromatic protons appeared in the region δ 6.65-8.05ppm, while those of the methylene protons (CH 2 ) of the indanone ring showed at δ 3.80 to 4.07 ppm.The 1 H-NMR spectrum of Compound 1a was similar to the data reported by Berthelette et al. (1997).
The 13 C-NMR spectra of the chalcones similarly gave expected signals and the expected number of carbon atoms (CH, CH 2 , CH 3 and quatenary carbons).The signal for the -CH 2 -carbon of the indanone moiety appeared at around δ 32.00 ppm.The carbonyl carbon appeared around δ 194.00 ppm for all the compounds except Compound 1a that showed two signals for the C=O at δ 208.18 and δ 206.19 ppm, while the spiro-carbon signal showed at δ 70.67 ppm.
In the IR spectra of the chalcones, the OH group showed around 3455 to 3491 cm -1 as a broad band, while absorption bands showed between 1665 and 1700 cm -1 for the C=O functional group and between 1573 and 1625 cm -1 for the C=C functional group.

Antimicrobial evaluation
The antimicrobial susceptibility tests of the 1-Indanone chalcones and their corresponding 2,4dinitrophenylhydrazone derivatives were performed using the agar-well diffusion method against thirteen strains of gram-positive and gram-negative bacteria.The activity of the compounds against the microorganisms was assessed through the zone of inhibition and the MICs, whose values are shown in Table 2.A known antibiotic (tetracycline) was used for comparison.
The results showed that the compounds at a concentration of 200 µg/ml showed zones of inhibition ranging from 10 to 28 mm.The results further indicated that the fourteen synthesized compounds showed antimicrobial activity against P. aeruginosa, Salmonella typhimurium and Shigella flexineri (all gram-negative) strains.The MICs of the compounds against the P. aeruginosa strain ranged from 16.5 to 250 µg/ml and from 16.5 to 1000 µg/ml for S. typhimurium and S. flexineri strains.
In many cases, the hydrazone derivatives appear to be more active than their chalcone precussors, except for Compound 3 and in some other cases (Table 2).Compound 1b exhibited the best activity with the lowest MIC values for four gram-negative bacterial strains (15.6 µg/ml) and two gram-positive strains (31.3 µg/ml).Comparing the activities of the compounds against the strains of bacteria, we can notice that they were globally more active against the gramnegative bacteria.P. aeruginosa is the most sensitive organism to the synthesized compounds and the activity order is 4a > 6a = (1a) >3a = 7a > 2a = 5a for the chalcones and (1b) = 5b > 6b =7b > 2b > 3b = 4b for their corresponding hydrazones.
In the past several years, there has been an increasing use of quantitative structure-activity relationship (QSAR) to predict the biological activities of various organic molecules.One method that has been extensively employed involves the QSAR approach together with multivariate data analysis, combined with statistical design (Vasanthanathan et al., 2006).This approach is an attempt to show that there is a relationship between biological activities of compounds and structural or molecular descriptors such as physicochemical, thermodynamic, electronic, topological or geometrical parameters (Podunavac-Kuzmanović et al., 2008).The importance of the lipophilic or hydrophobic nature of bioactive compounds has been brought into fore by the tremendous progress in the use of QSAR methods.The penetration of bioactive compounds through the apolar cell membranes is modified by lipophilicity, characterized by the partition coefficient (log P).A measure of hydrophobicity/lipophilicity is the octanol/water partition coefficient Clog P.
In this work, Clog P was calculated using ChemDraw Ultra 8.0 software (CambridgeSoft Corporation).The results obtained are given in Table 3.The calculated values of log P for the hydrazones were higher than for the corresponding chalcones.However, the dimeric Compounds 1a and 1b have the highest values.Taking the calculated lipophilicity (Clog P) values for the hydrazones and comparing with their corresponding activity (MIC) values for the most sensitive organism, P. aeruginosa, it could be seen that they are not of the same increasing order: Clog P: 1b > 2b > 3b > 7b > 4b = 5b >6b MIC (P.aeruginosa)-activity: 1b = 5b > 6b = 7b >
In a similar manner, the molar refractivity (MR -which represents size and polarizability) describing steric effects was calculated using ChemDraw Ultra 8.0 software and the results are included in Table 3.The largest molecule having the largest CMR and log P values (1b) has the best antimicrobial activity.This compound may be viewed as having a cup-like structure in which the bulky phenyl groups form the rim of the cup (Figure 1).

Conclusion
Different methods have been successfully employed in the synthesis of some 2-Arylidene-1-indanone derivatives in good yields.The compounds have been characterized using IR, 1 H and 13 C NMR and elemental analysis and their effects on some pathogenic bacteria evaluated.They exhibited broad spectrum antibacterial activity.

Figure 1 .
Figure 1.Three dimensional structure of Compounds 1a and b, respectively.

Table 1 .
Physicochemical properties and catalytic method of synthesis of the 1-Indanone chalcones.

Table 3 .
Calculated Clog P and MR values.