Synthesis of 4-hydroxy-3-methylchalcone from Reimer-Tiemann reaction product and its antibacterial activity test

A 4-hydroxy-3-methylchalcone has been synthesized from 4-hydroxy-3-methylbenzaldehyde as the Reimer-Tiemann reaction product. This research consists of three steps involve synthesize of 4-hydroxy-3-methylbenzaldehyde from ortho-cresol, synthesize of chalcone derivatives from 4-hydroxy-3-methylbenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde or vanillin for the comparison, the last is antibacterial activity test of both chalcone derivatives against Escherichia coli (negative gram) and Staphylococcus aureus (positive gram) bacteria using disc diffusion method. Results of Reimer-Tiemann reaction is 4-hydroxy-3-methylbenzaldehyde compound in an orange colour solid form which has 43% yields and melting point 110-114°C. A 4-hydroxy-3-methylbenzaldehyde then reacted with acetophenone in a base condition and form 4-hydroxy-3-methylchalcone compound in a yellow colour solid form which has 40% yields and melting point 83-86°C. The antibacterial activity of the 4-hydroxy-3-methylchalcone against gram-positive bacteria Staphylococcus aureus is better than the 4-hydroxy-3-methoxychalcone.


INTRODUCTION
Chalcone is an open chain flavonoid group which has two aromatic ring containing unsaturated α,βcarbonyl three carbons system as a connector [12] . During this time, chalcone is synthesized from acetophenone with benzaldehyde and its derivatives by Claisen-Schmidt condensation reaction [6] . According to researches [2][8] a 4-hydroxy-3-methoxychalcone have been synthesized from the reaction of acetophenone and 4-hydroxy-3-methoxybenzaldehyde or vanillin. Chalcone derived from vanillin has a better antibacterial activity due to the influence of its two substituents. In this study, synthesis of 4-hydroxy-3-methylchalcone will be done by reacting acetophenone and 4-hydroxy-3methylbenzaldehyde. The 4-hydroxy-3-methylbenzaldehyde compound used is obtained from Reimer-Tiemann formylation reaction of ortho-cresol and chloroform in a base condition. Then the antibacterial activity of this compound will be compared with the 4-hydroxy-3-methoxychalcone obtained from the reaction of acetophenone and vanillin. The 4-hydroxy-3-methylchalcone has a lipophilic methyl group, so it is easier to bind and form complexes with cell membranes that can cause damage to the membrane. Therefore, the antibacterial activity of 4-hydroxy-3-methylchalcone would be greater than 4-hydroxy-3-methoxychalcone.

Synthesis of 4-hydroxy-3-methylbenzaldehyde
A total of 15 g of sodium hydroxide (NaOH) dissolved in 75 mL of distilled water was added to 5.15 mL ortho-cresol (50 mmol) in 100 mL of absolute ethanol. The reaction mixture was heated at 60°C. After the reaction lasts 5 minutes, the addition of 20 mmol of chloroform was carefully started. Chloroform was added excess because it is easily evaporated. Stirring was continued for 1 hour after the addition of chloroform was completed. Ethanol was removed, and 1 M of HCl was added to neutralize the excess NaOH to pH = 2-3. Then the solution was separated by extraction method using ethyl acetate for 3 times. After that, ethyl acetate fraction was added by anhydrous Na2SO4 and filtered before evaporated by reducing the pressure. The reaction mixture is further purified by column chromatography with silica gel. The product obtained were analyzed using spectrophotometer FT-IR, GC-MS, and H-NMR [5][10] [11] .

2.2
Synthesis of Chalcone Derivative A total of 1.7 mL of Acetophenone (14.68 mmol) and 9 mL of NaOH 60% were added to 2 g of 4hydroxy-3-methylbenzaldehyde (14.68 mmol) in 15 mL absolute ethanol. The mixture was heated at 70°C for 3 hours and every hour monitored by TLC. Ethanol was removed and 1 M HCl was added to pH = 1. The reaction mixture was separated by extraction method using ethyl acetate. Then the organic phase obtained was added by anhydrous Na2SO4 to bind water that still exists in the organic phase. Solvents were distilled off and then the reaction mixture was further purified by column chromatography on silica gel. The formed solids were then analyzed using spectrophotometer UV-VIS, FT-IR, and LC-MS. Under the same procedure, acetophenone was reacted with vanillin to obtain a 4hydroxy-3-methoxychalcone [2][8] .

2.3
Antibacterial Activity Test Nutrient agar media as much as 15 mL was poured into a petri dish and left to solid. All bacterial planting process was done in Laminar Air Flow. A total of 0.1 mL of diluted bacterial suspension was inoculated on the nutrient agar medium which had been solidified. Furthermore, the disc paper (6 mm diameter) was inserted into the dilute synthesized product solution as a test solution into various concentrations for 1 minute until it was fully diffused on the disc paper. The disc paper containing the test solution was placed in the agar medium. The test medium which has been ready, was incubated for 24 hours in an incubator at 37°C. The clear zone around the disc were a zone of inhibition which measured using a vernier caliper in millimeters.

Synthesis of 4-hydroxy-3-methylbenzaldehyde
The 4-hydroxy-3-methylbenzaldehyde has been synthesized from ortho-cresol through Reimer-Tiemann formylation reaction. The reaction is shown in Figure 1 below. Chromatogram of the product mixture developed by GC-MS method showed the presence of 3methylsalicylaldehyde (m/z 136) at the retention time (RT) of 12.658 minutes with an abundance of 2% and 4-hydroxy-3-methylbenzaldehyde (m/z 136) at RT of 25,068 minutes as much as 53%.

Synthesis of Chalcone Derivatives
The 4-hydroxy-3-methylchalcone and 4-hydroxy-3-methoxychalcone as its comparative compound have been synthesized from the reaction of acetophenone and 4-hydroxy-3-methylbenzaldehyde while its comparative compound from 4-hydroxy-3-methoxybenzaldehyde or vanillin through Claisen-Schmidt condensation reaction. The reaction is shown in Figure 3  The reaction product obtained has been purified by column chromatography. It was then analyzed using spectrophotometer UV-VIS, FT-IR, and LC-MS. The presence of 4-hydroxy-3-methylchalcone was proven by UV-VIS spectrum which showed two peaks gained at a wavelength 353 nm (1 st Band) and 253 nm (2 nd Band). These results prove that the product obtained is a group of flavonoid chalcone. The UV-VIS spectrum of chalcone has a characteristic that consists of two bands in the 340-390 nm range (1 st band) and 230-270 nm (weak, as the 2 nd band) (Markham, 1988). The UV-VIS spectrum of 4-hydroxy-3-methylchalcone is shown in Figure 4 below.
A dominant peak at the chromatogram ( Figure 6) with a retention time (RT) of 2,6 minutes developed by LC-MS method was indicating that there was only one constituent in the product. From the mass spectrum of the product, that constituent was identified and characterized as 4-hydroxy-3metylchalcone (m/z 238). The 4-hydroxy-3-methylchalcone obtained in a yellow color solid form at room temperature, has a yield of 40%, abundance of >95%, and has a melting point of 83-86 o C.

Figure 6. Chromatogram and Mass Spectrum of 4-hydroxy-3-methylchalcone
Meanwhile, as a comparison, the product obtained from the reaction of acetophenone and vanillin also gave similar results to the 4-hydroxy-3-methylchalcone. The UV-VIS spectrum of the reaction products of acetophenone and vanillin compounds showed two peaks at the wavelength of 363 nm (1 st Band) and 261 nm (2 nd Band). Moreover, a dominant peak at the retention time of 3.8 minutes on the chromatogram and mass spectrum developed by LC-MS method was indicating that the product was 4-hydroxy-3-methoxychalcone (m/z 254). The 4-hydroxy-3-methoxychalcone obtained in a yellow color solid form at room temperature, has a yield of 76%, abundance of >95%, and has a melting point of 78 o C.

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The 12th Joint Conference on Chemistry IOP Publishing IOP Conf. Series: Materials Science and Engineering 349 (2018) 012036 doi:10.1088/1757-899X/349/1/012036 their relation to each substituent character (σ). The value of σ on each substituent shows the resultant between induction and mesomery influence on a compound [4] . In the two chalcone derivatives, position of the -CH3 and -OCH3 substituents is meta to the aldehyde group. At the meta position, the -CH3 group has a value of σm = -0.07 and the -OCH3 group has a value of σm = +0.12. It should be noted that if the value of σ is positive, it means the substituent is electron withdrawing group. Conversely, if the value of σ is negative then the substituent is electron releasing group [4] .
Electron withdrawing substituents also can be called as a ring activator group. This is because it can increase the reactivity of the aromatic ring. Since the aromatic ring of vanillin is more active than the aromatic ring in the 4-hydroxy-3-methylbenzaldehyde, the vanillin is more reactive with the enolate ions derived from the acetophenone. Because it is easy to react, the yield is much more than a compound with an electron releasing substituent like 4-hydroxy-3-methylbenzaldehyde.

Antibacterial Activity Test
The antibacterial activity test of 4-hydroxy-3-methylchalcone against Escherichia coli (negative gram) and Staphylococcus aureus (Positive gram) bacteria have been done by disc diffusion method. In this test, peptone and yeast extract were used as bacterial growth medium. Before the antibacterial test, the bacterial stock was incubated in a nutrient broth medium for about 3 hours to obtain an equivalent of 0.5 Mc Farland turbidity. Results of antibacterial activity test of chalcone derivative are shown in table 1 below. The antibacterial mechanism of flavonoid can occur through the formation of complexes with proteins by forming hydrogen bonds, covalent bonds, and hydrophobic effects. This is related to the ability of flavonoid antibacterial compounds in disabling microbial solvents, enzymes, and envelope cell proteins [7] .
When the antibacterial activity of the 4-hydroxy-3-methylchalcone is compared with the 4-hydroxy-3methoxychalcone and the unsubstituted chalcone, antibacterial activity of the 4-hydroxy-3methylchalcone against gram-positive bacteria Staphylococcus aureus is better than the comparable compounds. This is because besides the simpler cell wall structure, the thick layer of peptidoglycan contains lipoteichoic acid that is hydrophobic. This lipoteichoic acid connects to the outer layers of the bacterial cell wall and the cell membrane layer that lies beneath a thick layer of peptidoglycan [3] . The presence of lipoteichoic acid causes the lipophilic 4-hydroxy-3-methylcchalcone will more easily bond and form complexes with cell membranes that can cause damage to the membrane. Nevertheless, the increase in antibacterial activity of flavonoid compounds especially chalcone is not only influenced by the type of substituent or functional group attached to the aromatic ring. However, the functional group position in the aromatic ring also influences the increase in antibacterial activity of chalcone [1] .

CONCLUSION
Results of Reimer-Tiemann reaction is 4-hydroxy-3-methylbenzaldehyde compound in an orange color solid form which has 43% yields and melting point 110-114 o C. A 4-hydroxy-3methylbenzaldehyde then reacted with acetophenone in a base condition and form 4-hydroxy-3methylchalcone compound in a yellow colour solid form which has 40% yields and melting point 83-86 o C. The 4-hydroxy-3-methoxychalcone compound obtained in 76% yields. The antibacterial activity of the 4-hydroxy-3-methylchalcone against gram-positive bacteria Staphylococcus aureus is better than the 4-hydroxy-3-methoxychalcone.