Antibacterial Activity for Synthesized Coumarin Derivatives and a Coumarin Component of Lime Peel (Citrus aurantifolia)

In this study, we investigated the antibacterial activity of the coumarin component isolated from lime peel and coumarin derivatives synthesized using various techniques against eight types of food-poisoning bacteria. The minimum inhibitory concentration (MIC) for the 3b [5,7-dihydroxy-4-trifluoromethylcoumarin] derivative was measured as 1.5 mM in Bacillus cereus, Micrococcus luteus, Listeria monocytogenes, and Staphylococcus aureus subsp. aureus; that for the 3c [7-hydroxy-4-trifluoromethylcoumarin] derivative was 1.7 mM in Enterococcus facium; and that for the 3n [dicoumarol] derivative was 1.2 mM in L. monocytogenes. These results confirmed that coumarin derivatives with CF3 and OH substituents had enhanced antibacterial activity.


Introduction
Coumarin is a component involved in plant defense reactions, is a substance that is present in plants, and exhibits various physiological activities.Since ancient times, plant resources such as this have been used to prevent or treat diseases, and in modern times, they are being used in various ways in terms of natural resource use and development.
Among fragrant fruits, lime peel, which is inexpensive and convenient to purchase, was used as the material for this study.The activity of coumarin derivatives damages cell membranes and shows high activity in Gram-positive and -negative bacteria, but it has been reported to have particularly strong antibacterial activity against Gram-negative bacteria [40][41][42].In measuring the anti-inflammatory inhibitory activity, it was reported that 6-geranyloxycoumarin had inhibitory effects of 68.9 and 72.6% on the production of interleukin (IL)-6 at 1 and 10 µM, respectively [3].In addition, as a result of examining the anti-inflammatory effect of geranyloxycoumarin derivatives, natural products, such as 7-geranyloxycoumarin (1 µM/cm 2 ) and 8-acetoxt geranyloxycoumarin (1 µM/cm 2 ), reduced the edema rate by about 50% [4].
However, detailed studies on the structure-activity relationship (SAR) and new derivatives for these results are lacking.Accordingly, natural coumarin and geranyloxycoumarin were extracted from lime peel (Citrus aurantifolia) containing coumarin, separated, and purified, and their structures were analyzed using GC-MS, IR, 1 H-NMR, 13 C-NMR, and 19 F-NMR.We intended to separate the substances and produce coumarin derivatives and geranyloxycoumarin derivatives through synthesis and semi-synthesis.
In this study, the antibacterial activities of coumarin derivatives, geranyloxycoumarin derivatives, and lime peel were confirmed, and it is expected that these results can be used as basic data for antibacterial research on food-poisoning bacteria.
First, 2 kg of dried lime peel was used in the extraction with 10 L of EtOH at room temperature.The extraction was performed twice, and the extract was vacuum concentrated using a rotary evaporator at 40 • C.Then, 235.8 g of the resulting EtOH extract was mixed with 4 L of n-hexane and then vortexed at 500 rpm and 50-60 • C for 6 h.The n-hexane layer was isolated, and 34.63 g of concentrate was obtained.A total of 4 L of ethyl acetate was added to the concentrate from which ethanol was completely removed, and after stirring at 50 • C for 6 h, the dissolved layer was separated and concentrated under a 40 • C water bath with the vacuum rotary evaporator, obtaining 15.36 g of concentrate.The remaining residue undissolved in ethyl acetate was then stored in cold storage at 2-4 • C for the purpose of the next study.

Isolation of Lime Peel Components from n-Hexane Layer
Silica gels were placed in the column tube, to which 10 g of lime concentrate from the nhexane layer was added after dilution with 30 mL of n-hexane.Then, 50 mL of fresh hexane was added (three times), and the column tube was filled with sea sand up to approximately 3 cm.The eluate was produced in the following order: This was performed to isolate the following components.

Synthesis of Coumarin Derivatives
General coumarin synthesis can be achieved by the Perkin [43,44], Pechmann [45], and Knoevenagel [46,47] reactions.In this study, the following coumarin derivatives with the CF 3 functional group were synthesized using the Pechmann reaction method (    Our first goal was to obtain O-alkylated geranyloxycoumarin derivatives through the reaction between hydroxycoumarin and geranyl bromide, and between K 2 CO 3 and acetone, reacted at room temperature for 5 h to obtain a yield of 35% (Table 2, entry 2).As a result of the optimized conditions, 7-hydroxycoumarin (3d) and geranyl bromide (4) under Cs 2 CO 3 and CH 3 CN were reacted for 3 h at room temperature to obtain 5d in a 93% yield (Table 2, entry 6).Next, we obtained compounds 5oa, 5ob, and 5oc as a result of reacting 4-hydroxycoumarin with geranyl bromide as an optimization condition to obtain O-alkylated 4-geranyloxycoumarin.During the formation of these compounds, O-alkylated 4-geranyloxycoumarin 5oc was obtained by reacting A-form coumarin with geranyl bromide by tautomeric ketoenol forms by base.The yield of O-alkylated 4-geranyloxycoumarin that was obtained was 18%.On the other hand, C-alkylated coumarin 5oa was reacted with 2 equivalents of geranyl bromide (4) and keto form B to obtain an 11% yield.In addition, compound 5ob was obtained in a 35% yield by hydrolysis and decarboxylation from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).from 5oa.We also confirmed that 5ob was obtained by adding 5oa to acetonitrile, to which water was added with stirring at rt for 9 h.Using these results, 4-hydroxycoumarin and cinnamyl alcohol were reacted with water and a palladium catalyst to obtain C-alkylated coumarin and diallylated products, which were then hydrolyzed and decarboxylated [48] (Figure 1).In this result, the reaction of the different types of hydroxy coumarin and geranyl bromide under the given conditions produced various novel coumarin derivatives (Table 3).The substances isolated from lime peel are as follows.Confirmation of the structure of this material is provided in the Supplementary Materials (Supplementary Table S1).

Synthesis of Coumarin Derivatives and Geranyloxycoumarin Derivatives Common Synthesis Method of Coumarin Derivatives
Ethyl trifluoromethyl acetoacetate (20.6 mmol) and phenol derivative (18.2 mmol) were added to a 50 mL round-bottom flask, and the mixture was maintained at below 10 • C. The mixture was stirred while adding 10 mL of sulfuric acid slowly for 30 min.Next, the mixture was stirred at room temperature for 18~26 h and then slowly poured into beaker containing 80.0 g of iced water, whose temperature was maintained at ≤10 • C. The resulting precipitate was filtered, washed with 10 mL of cold water (four times), and dried in air.The crude solid was purified using column chromatography with CH 2 Cl 2 /n-hexane (5:1, v/v) as the eluent.Confirmation of the structure of this material is provided in the Supplementary Materials (Supplementary Table S2).
(E)- Distilled water (2.6 mmol) was added to a stirred mixture of 5oa (2.3 mmol) and powdered cesium carbonate (2.5 mmol) in acetonitrile (30 mL), and the stirring was continued at rt for 9 h.The reaction progress was monitored using TLC.The resulting mixture was filtered, and the filtrate was concentrated.The crude product was fractionated on a silica gel column using n-hexane/CH 2 Cl 2 (3:1, v/v) to give the product 5ob as a colorless liquid.

Test Strain
The following microorganisms were used to evaluate the antibacterial activity: five strains of Gram-positive bacteria (B.cereus, M. luteus, E. faecium, L. monocytogenes, S. aureus subsp.aureus) and three strains of Gram-negative bacteria (Salmonella enteritidis, Shigella boydii, E. coli) as the common pathogens.The strains used in this study were obtained from the Korean Collection for Type Cultures (KTCT) and Korean Culture Center of Microorganisms (KCCM).

Microbial Culture
The obtained pathogenic microorganisms were cultured under standard microbial culture conditions, and after passaging in each medium, a preculture was performed at 37 • C and 150 rpm for 12 h.Each microbial strain was then cultured up to 6.4 × 10 6 CFU/mL for subsequent analyses.The composition of each medium for evaluating the antimicrobial activity is listed in Table 4.The final concentration of coumarin was set to 10 mg/mL, and the inhibitory activities for B. cereus, M. luteus, L. monocytogenes, and E. faecium were evaluated to test the activity of the indicator strains according to the concentration of coumarin derivatives.For each medium and optimal temperature, the indicator strains were cultured in a shaking incubator for 12 h.Thereafter, a 1% culture solution of each indicator strain was added to 0.8% soft agar, and 20 mL of agar was solidified in a Petri dish.The test plate for antibacterial activity had 8 mm wells, and 50 µL of a sample was loaded into each well and cultured at the optimal temperature for 12 h.The indicator strains showing transparent circular colonies were selected and their diameters were measured.The results are expressed as the measured values of the screening test (-: no activity; +: inhibition zone >5-10 mm; ++: inhibition zone >11-15 mm; +++: inhibition zone >16-20 mm; and ++++: inhibition zone >20 mm).
DMSO was used as the negative control to investigate the effect of inhibiting proliferation on the solvent.

Minimum Inhibitory Concentration (MIC)
The final concentration of coumarin was set to 0.09, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 mg/mL, and the inhibitory activities for M. luteus, L. monocytogenes, E. faecium, and B. cereus were evaluated to determine the MIC of the indicator strains according to the concentration of coumarin derivatives.
For each medium and optimal temperature, the indicator strains were cultured in a shaking incubator for 12 h.To 0.8% soft agar, 1% culture solution of each indicator strain was added, and 20 mL of agar was solidified in a Petri dish.The test plate for antimicrobial activity had 8 mm wells, and 50 µL of a sample was loaded into each well and cultured at the optimal temperature for 12 h.The lowest concentration at which the transparent circular colonies appeared was considered as the MIC.The results are expressed by converting the concentration in mg/mL into a molar concentration.
DMSO was used as the negative control to investigate the effect of inhibiting proliferation on the solvent.DMSO was used as the negative control to investigate the effect of inhibiting proliferation on the solvent.

Minimum Inhibitory Concentration (MIC) of Coumarin Derivatives
To measure the MIC of the coumarin derivative with antibacterial activity, the final concentration of coumarin was adjusted to 0.09, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 mg/mL, and then the inhibitory activity was measured.The results are expressed by converting the concentration in mg/mL into a molar concentration.
According to Table 7, the MIC of 3b was 1.5 mM in B. cereus, M. luteus, L. monocytogenes, and S. aureus subsp.aureus.The MIC of 3c was 1.7 mM in E. faecium, and that of 3n was 1.2 mM in L. monocytogenes.As a result of the MIC measurement, the antibacterial activity was confirmed in the 3b and 3c derivatives.These results confirmed that coumarin derivatives with CF 3 and OH substituents had antibacterial activity against food-poisoning bacteria.
The antibacterial screening of coumarin derivatives, geranyloxycoumarin derivatives, and the coumarin component of lime peel was initiated on eight species of food-poisoning bacteria, and the MIC of the coumarin derivative with good activity was measured.As a result of the MIC measurement, antibacterial activity was confirmed in the 3b and 3c derivatives.These results confirm that coumarin derivatives with CF 3 and OH substituents had antibacterial activity against food-poisoning bacteria.
Antibacterial activity against Enterococcus faecalis has been reported only for 1,2,3triazole-coumarin derivatives [16].However, the results of the antibacterial screening conducted in this study confirm that coumarin derivatives exhibited antibacterial effects on B. cereus, S. aureus subsp.aureus, M. luteus, S. enteritidis, and S. boydii.
In addition, the water-soluble coumarin quaternary ammonium chloride was synthesized against Gram-negative E. coli and Gram-positive B. subtilis bacteria, and it was reported that no antibacterial activity was observed [52].However, the antibacterial screening results of compound 3a showed that it exhibited activity toward E. coli.As a result of this experiment, the MIC of the 3b derivative was measured at a concentration of 2.9 mM in E. coli.
The results obtained herein imply that 3b and 3c derivatives, which contain CF 3 substituents, can be utilized as natural antibacterial substances, as their antibacterial activity was confirmed.We believe that the large-scale synthesis of coumarin derivatives with antibacterial activity will be possible if the various derivative synthetic compounds we have proposed are utilized in further research.
Accordingly, in this study, we attempted to identify a method with a high recovery rate using a coumarin synthesis method and then confirmed the antibacterial activity using a coumarin derivative.These results are expected to provide useful information on antibacterial activity using coumarin derivatives that are water soluble and contain fluorine (F).In the future, it is expected that useful conditions to achieve a better antibacterial activity can be found by synthesizing additional derivatives utilizing structure-activity relationships.

Table 4 .
Growth conditions of pathogenic bacteria.

Table 7 .
Minimum inhibitory concentration (MIC) of coumarin derivatives (mM).Statistical analysis was performed using the ANOVA 26 VERSION SPSS program.The mean and standard deviation were calculated for each item, and significance was verified using two-way ANOVA (p < 0.05).Values presented are means ± standard errors from three independent experiments.