Green Synthesis of Chalcones and Microbiological Evaluation

A green method was developed for the synthesis of chalcones using glycerin as solvent. Subsequently, the potential microbiology activity of these molecules was evaluated by testing them against the Gram-positive bacteria Staphylococcus aureus (S. aureus) ATCC 19095 and Enterococcus faecalis (E. faecalis) ATCC 4083, the Gram-negative bacteria Escherichia coli (E. coli) ATCC 29214 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027, and the fungus Candida albicans (C. albicans), which includes ATCC 62342 and three clinical strains of C. albicans from human oral cavities. The results showed that some chalcones exhibited moderate inhibitory activity, the most prominent being those acting against the fluconazole-resistant strains of C. albicans.


Introduction
Major problems in public health are associated with fungal and bacterial infections, mainly when these microorganisms are resistant.This situation is becoming increasingly common, even with the wide variety of antimicrobial drugs and their medicinal-chemical evolution, owing to the indiscriminate use of these drugs by people.Consequently, medicines used cannot combat pathogenic organisms, thereby placing limitations on options for treatment. 1Studies show that more than 70% of pathogenic bacteria develop resistance to at least one antibiotic available for clinical use. 2 To circumvent this, it is necessary to obtain substances that are able to be used as prototypes of drugs, with pharmacological activity similar to or greater than the original ones and which have an effect on resistant organisms. 3,4Advances in the development of analytical techniques purification and organic synthesis can contribute to this process, making it possible to obtain a variety of molecules with highest purity. 5ne of the most studied classes of molecules are the chalcones, which are α,β-unsaturated ketones with two aromatic rings.Many studies were performed with these molecules and a range of activities were reported, such as anti-inflammatory, 6 antifungal, antibacterial, 7 antileishmanial, antiparasitic, 8 antitubercular 9 and antioxidant activity. 10Due to their structure, these molecules are very versatile, as they may contain different aromatic rings, fused or not, with heteroatoms in their structure and different substituents. 11These molecules can be obtained from natural products or by synthesis. 6The Claisen-Schmidt reaction, which is the condensation of an aromatic ketone with an aromatic aldehyde in presence of a catalyst, such as NaOH, Ba(OH) 2 , KOH, AlCl 3 or HCl, 7 is the most widely used method for the synthesis of chalcones.Furthermore, chalcones can be used as intermediates in different organic reactions to obtain pyrazoles, 12,13 isoxazoles, 14 and thiazoles. 15mong several lines of study, the search for methodologies that include a green process has been attracting attention in the scientific community in order to be environment friendly, 16 as the investment in techniques that reduce the demand for chemical waste using alternative technologies, such as sonochemistry 13,15,17 and microwave technology, 18,19 reusable catalysts, 20 renewable solvents or even solvent-free reactions. 21An example of a renewable solvent is glycerin, a byproduct of biodiesel production.With a significant increase in the demand for this fuel, the amount of glycerin produced also increased, becoming an inconvenience.Aimed at alleviating this situation, it became necessary to find applications for the same. 22An alternative is to increase its industrial value by employing glycerin as a raw material for the production of glycerol carbonate, which is used as a polar solvent, emulsifier for cosmetics, and a source of new polymeric materials and fuel additives. 23,24][27][28] In this context, our group developed a methodology for the synthesis of chalcones using glycerin as a solvent, and subsequently, these molecules were tested for in vitro antimicrobial activity against two representatives of Gram-positive bacteria, Staphylococcus aureus (S. aureus) ATCC 19095 and Enterococcus faecalis (E.faecalis) ATCC 4083; two Gram-negative bacteria, Escherichia coli (E.coli) ATCC 29214 and Pseudomonas aeruginosa (P.aeruginosa) ATCC 9027; and, four strains of yeasts: Candida albicans (C.albicans) ATCC 62342 and three clinical strains from the human oral cavity by the broth microdilution method according to the guidelines of the National Committee for Clinical and Laboratory Standards (NCCLS) for yeasts (M27-A3) and bacteria (M7-A7).

Experimental
All solvents and reagents used in the synthesis were obtained from Sigma-Aldrich Co., St. Louis, MO, USA, and used without further purification.Reagents for the microbial assays included Mueller-Hinton broth (BD, Sparks, MD, USA) and RPMI-1640 (Sigma, St Louis, MO, USA).Progress of the reactions was monitored on a Shimadzu 2010 gas chromatograph by flame ionization (GC-FID) using a split/splitless injector and a HP-1 30 m × 0.32 mm × 0.25 μm column.Analyses by Fourier transform infrared spectroscopy (FTIR) were obtained using attenuated total reflection (ATR) on an Agilent Technologies Cary 600 series equipment, and mass spectra were obtained on a Shimadzu GC-MS-QP 2010SE mass spectrometer-coupled gas chromatograph (GC-MS) with an AOC-20i automatic injector and Rtx-5MS 30 m × 0.25 mm × 0.25 μm column. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX 400 spectrometer (400.13MHz for 1 H and 100.48MHz for 13 C) at 300 K and the melting points (mp) were determined on apparatus Fisatom 430 with mercury thermometer, using capillary tubes to the samples.
The biological assays were performed at the School of Dentistry, and chemical analyzes at the Center of Chemical, Pharmaceutical and Food Sciences, both at the Federal University of Pelotas, except the NMR experiments, conducted at the Chemical Institute at the Federal University of Santa Maria (Brazil).

General procedure for chalcones 3a-n
A solution of 5 mmol of acetophenone or 2-acetylthiophene 1, 5 mmol of aldehyde 2 and 6 mmol of sodium hydroxide (NaOH) in 5 mL of glycerin was stirred overnight, neutralized with 0.5% HCl and filtered.The solid residue was recrystallized from ethanol to obtain the purity product.

Antimicrobial screening
The in vitro antimicrobial activity of each compound and the standard drugs were determined against two representatives of Gram-positive bacteria, S. aureus ATCC 19095 and E. faecalis ATCC 4083; two Gram-negative bacteria E. coli ATCC 29214 and P. aeruginosa ATCC 9027; and, four strains of yeasts: C. albicans ATCC 62342 and three clinical strains from the human oral cavity by the broth microdilution method according to the guidelines of NCCLS for yeasts (M27-A3) and bacteria (M7-A7).
Microbial strains were primarily inoculated for overnight growth.An aliquot of the colonies was directly suspended in saline solution until the turbidity matched of the 0.5 McFarland standard and adjusted to give 5 × 10 5 CFU mL −1 of final microorganisms.Mueller-Hinton broth (BD, Sparks, MD, USA) was used as a nutrient medium to grow and dilute the compound suspension for the test bacteria and Roswell Park Memorial Institute medium (RPMI) 1640 (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with 3-(N-morpholino) propanesulfonic acid (MOPS) was used for fungal nutrition.Dilutions of tetracycline and fluconazole were used as the reference compounds for comparing the data between independent experiments and as indicators for assessing the relative level of inhibition for the samples tested.Dimethyl sulfoxide (DMSO) was used as the diluent/vehicle to obtain the desired concentrations of the compounds and standard drugs.
The diluted samples were serially transferred in duplicate to 96-well microplates, and 100 μL microbial inoculums were added to achieve a final volume of 200 μL and concentrations ranging from 1 to 500 μg mL −1 .The final concentration of DMSO in the assay did not exceed 0.5%.Vol. 26, No. 6, 2015   Controls for microbial viability, sterility of the medium and sterility of the extract were also carried out.The plates were incubated at 37 °C for 24 h for bacteria and 48 h for fungi.Plates were read at 630 nm for bacteria or 590 nm for fungi, prior to and after incubation.The antimicrobial effect was characterized by half maximal inhibitory concentration (IC 50 ) values, the concentration that affords 50% inhibition of bacterial/fungal growth relative to the growth control, and minimal inhibitory concentration (MIC) values, the minimal concentration of a substance that completely inhibits the bacterial or fungal growth.IC 50 values were determined from logarithmic graphs of growth inhibition versus concentration.After determining the MIC, the minimal microbicidal concentration (MMC) was determined.Aliquots of 20 μL from the wells were plated on Mueller-Hinton agar for bacteria or Sabouraud dextrose agar for fungi and incubated at 37 °C for 24 h.MMC was defined as the lowest concentration of each compound that resulted in no cell growth on the surface of the plates.

Results and Discussion
The chalcones 3a-n were synthesized from an equimolar mixture of acetophenone or 2-acetylthiophene and one of a series of eight aldehydes in glycerin using NaOH as a catalyst with stirring at room temperature, as shown in Scheme 1.
Our group has been working with chalcones 10,29,30 because they are very versatile molecules and present activities against different microorganisms as described previously.The molecules chosen started with two aryl ketones, acetophenone and 2-acetylthiophene, and seven substituted benzaldehydes, where most are substituted in the 4-position to compare the influence of the groups on the activity of each compound.Furthermore, we wanted to evaluate the influence of the thiophenyl group, compared with the phenyl group, on the antimicrobial activities.Although the structure of these chalcones are published, we think it is important that their activity be evaluated against the bacteria S. aureus, E. faecalis, E. coli, and P. aeruginosa and the fungi C. albicans and three clinical strains of this fungi from human oral cavities.
Generally, chalcones are obtained using an alcohol (methanol [36][37][38] or ethanol 10,39,40 ) as the solvent.Glycerin was employed as a solvent in this work because it had similar properties to the cited solvents, such as polarity and organic functionality.
The yields of the synthesized chalcones 3a-n are listed in Table 1, as well as their experimentally determined melting point (mp) and those found in the literature.Chalcones were obtained in good yields and with high purity, which is important for subsequent tests.All compounds exhibited purity up to 95% by GC-FID analysis and had their respective molecular ions identified by GC-MS.
We present advantages observed over other earlier published methods, such as that the reaction occurs at room temperature 36,37 and the solvent used is less toxic. 10,39,40he improved mild and convenient protocol described here is an economical and efficient method for carrying out the Claisen-Schmidt reaction, resulting in high purity molecules in good yields (73-94%).The main advantage of this method is the employment of glycerin, a byproduct of biodiesel, often discarded as a consequence of its high production.The biofuel industries are unable to absorb the amount of glycerin for the preparation of chalcones, molecules of pharmaceutical interest.
To confirm the structures of the chalcones, they were analyzed by GC-MS, FTIR and 1 H and 13 C NMR spectroscopy.All compounds showed a molecular ion corresponding to the molecular mass, and both fragments were consistent with the chalcones structures.Analyses by FTIR identified the carbonyl group (band at 1640-1480 cm −1 ), the double bond and aromatic bonds (bands at 1720-1640 cm −1 and 840-700 cm −1 , respectively).Generally, the carbonyl group is identified by a band at 1820-1600 cm −1 ; however, conjugation with the aromatic rings caused a decrease in the absorption wavelength.The 1 H NMR spectrum showed the isomer with the E configuration in these molecules owing to the presence of two doublets with J ca. 15 Hz at approximately 7.35-7.99ppm, indicating that the Cα−Cβ hydrogens are in the trans configuration.
Despite all the molecules having been identified and having their structures confirmed, some melting points did not agree with those previously determined.We think that this may have occurred due to different form crystallization of chalcones during the purification process.
Data of chalcones 3a-n  The synthesized chalcones were tested against the Gram-positive bacteria S. aureus ATCC 19095 and E. faecalis ATCC 4083, Gram-negative bacteria E. coli ATCC 29214 and P. aeruginosa ATCC 9027, and four strains of the yeast C. albicans ATCC 62342 and three clinical strains from the human oral cavity and were compared with the reference drugs fluconazole, chloramphenicol and tetracycline.The results for the microbial assays are listed in Table 2.
Analyzing the results obtained, the IC 50 , the concentration of product which inhibits 50% of the growth of microorganisms compared with positive control was determined, which was calculated by nonlinear regression using GraphPad Prism®.The MIC was also determined, corresponding to the minimal concentration able to inhibit 100% of microorganism growth, and the MMC.Chalcones which showed no inhibition at a concentration higher than 500 μg mL −1 have a negative sign (−) in the Table 2, and they were not evaluated for MMC.
Generally, the fungi were more susceptible to the chalcones tested.Antifungal assays determined that (E)-1,3-diphenylprop-2-en-1-one 3a in contact with C. albicans and the strains C. albicans 1, 2 and 3 showed IC 50 of 76.6 μg mL −1 , 29.5 μg mL −1 , 141.4 μg mL −1 and 134.3 μg mL −1 , respectively.Despite the fact that all values were lower than that for the reference drug fluconazole (IC 50 21.2μg mL −1 ), chalcone 3a showed a MIC of 250 μg mL −1 against C. albicans 1, whereas fluconazole had a value greater than 500 μg mL −1 .This is a positive result taking into account that this strain was resistant to fluconazole.
Analyzing the results, it can be observed that the p-methoxy group (chalcone 3b) was more effective against C. albicans than the analog with a m-methoxy group (chalcone 3g).Furthermore, molecules with smaller atoms in the aryl ring, i.e., the hydrogen in chalcone 3a and the p-fluoro in chalcone 3c, showed better results when compared with other chalcones without activity, which had a p-bromo, p-chloro or p-methyl group in the structure.
The tested chalcones exhibited IC 50 higher than fluconazole against strains of C. albicans, but some Apparently, more electronically stable molecules showed better results against fungi strains.In chalcones 3a-g, with an aryl moiety corresponding to the ketone used in the synthesis, compounds 3a-c proved to be the most promising.Chalcone 3a has two aryl groups lacking substituents in its structure, which allows resonance between the double bonds without interference.An activating group, such as methoxy, in the para position in chalcone 3b increased the electronic density and stability of the ring, which may have been responsible for the decrease in IC 50 values.The methoxy group in the meta position increased the IC 50 values, most likely because there is no uniform electronic distribution in the ring, and the molecule was less stable.
In particular, in chalcones 3h-n, with a thiophenyl moiety corresponding to the ketone used in the synthesis, the sulfur atom in a five-membered ring changes the properties of the molecules.Chalcone 3i, for example, with a methoxy group in the para position, did not exhibit activity against fungi, unlike its analog with an aryl moiety, chalcone 3b.A similar situation appeared in chalcones with halogens and a methoxy group in the para position, which exhibited better activity compared with analogs with an aryl moiety.The presence of such leads to a less stable molecule, and atoms with high electronic density can counterbalance this loss of stability.
Recently, Shakil et al., 41 showed some analogs similar to those described in our work and evaluated their fungicidal activity against two phytopathogenic fungi: Rhizoctonia solani and Sclerotium rolfsii.The results suggested that chalcones with a p-fluoro in the aryl ring corresponding to the precursor ketone were highly effective as fungicides against R. solani.Chalcones 3c and 3j have a p-fluoro substituent in their structure, but in the ring corresponding to the benzaldehyde precursor.
Another study, reported by Sivakumar et al., 36 obtained a series of chalcones that was tested for bacterial adhesion and slime production on coated polymeric biomaterials against S. aureus, E. coli and P. aeruginosa.The results showed that bacterial adhesion decreased considerably on all the coated surfaces with 3-hydroxy-4-methoxychalcone.As with the chalcone 3b mentioned in our study, the p-methoxy substituent showed better results against fungi strains.
With respect to antibacterial assays, the best result for IC 50 was exhibited with compound 3k, with a value of 13.9 μg mL −1 against E. faecalis, however, a value higher than chloramphenicol (IC 50 3.9 μg mL −1 ) and tetracycline (IC 50 1 μg mL −1 ).Chalcone 3n was the compound that exhibited activity against the major bacteria, with an IC 50 of 219.1 μg mL −1 against S. aureus, 441.9 μg mL −1 against P. aeruginosa and 338.5 μg mL −1 against E. faecalis.All values are higher than those for the reference drugs.
The E. faecalis strain of bacteria was the most susceptible to the chalcones tested, where compound 3g showed an IC 50 of 425.9 μg mL −1 , 3h showed an IC 50 of 471 μg mL −1 , 3j showed an IC 50 of 30.2 μg mL −1 and 3n showed an IC 50 of 338.5 μg mL −1 , as well as chalcone 3k, which showed an aforementioned IC 50 of 13.9 μg mL −1 .
Chalcones 3h-n exhibited better results than chalcones 3a-g against bacteria, most likely because of the presence of a thiophenyl group in their structure.Among them, the chalcones with p-fluoro and p-bromo substituents showed better IC 50 values, and these atoms can be responsible for the improved results.Two previous studies comment about the better results with chalcones containing thiophene.Molecules containing a 5-chlorothiophene moiety and a halogen-substituted phenyl ring were synthesized by Kumar et al. 42 Results indicated that the tested compounds exhibited a range of inhibition values against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the Gram-negative bacteria Xanthomonas campestris and Escherichia coli.The authors attribute the elevated antimicrobial activity to the p-fluoro substituent on the phenyl ring.
Rizvi et al. 43 reported the synthesis of chalcones from 2-chloro-6-methyl-3-formylquinoline, 2-chloro-6methoxy-3-formylquinoline and a series of aryl aldehydes.All compounds were screened for antimicrobial activity and the authors observed that compounds with substituted heteroaryl groups, such as 2,5-dichloro-3-methylthiophene, showed antibacterial activities almost equivalent to the standard chloramphenicol against three bacterial strains, Escherichia coli, Micrococcus luteus and Staphylococcus aureus.
To continue the work, the chalcones that exhibited promising results will be tested for their cytotoxicity to determine the concentrations at which the exhibited activity will not be toxic.Fluconazole is toxic at a concentration of > 69 μg mL −1 .A cell viability study will illustrate whether chalcones are toxic at concentrations that inhibit microbial growth.

Conclusions
According to the results presented, the method to synthesize chalcones using glycerin as a renewable solvent was efficient, because the products were obtained in good yields and with excellent purity.In microbiological assays, chalcone 3b showed the lowest IC 50 value of 14.8 μg mL −1 against C. albicans 1, whereas fluconazole presents an IC 50 of 21.21 μg mL −1 .Chalcone 3n showed activity against the largest number of bacteria, with an IC 50 of 219.1 μg mL −1 against S. aureus, 441.9 μg mL −1 against P. Aeruginosa and 338.5 μg mL −1 against E. faecalis; however, all these values are higher than that for the reference drug.Ultimately, chalcone 3a had MIC of 250 μg mL −1 against C. albicans 1, whereas fluconazole showed MIC of > 500 μg mL −1 ; therefore, this compound may be regarded as a promising agent against this strain, which is resistant to fluconazole.Subsequently, cytotoxicity tests will be conducted to determine cell viability with these molecules, because fluconazole is cytotoxic at concentrations above 69 μg mL −1 .

Table 2 .
In vitro antimicrobial activities of compounds and reference substances against human pathogens