Sonochemistry in organic synthesis: A convenient method for the preparation of halo-bicyclic molecules with keten addition reaction and biological properties of Dimethyl 7,7-dichloro-8-oxabicyclo[4.2.0]oct-3-ene-3,4- dicarboxylate

doi:10.21203/rs.3.rs-2095109/v1

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Sonochemistry in organic synthesis: A convenient method for the preparation of halo-bicyclic molecules with keten addition reaction and biological properties of Dimethyl 7,7-dichloro-8-oxabicyclo[4.2.0]oct-3-ene-3,4- dicarboxylate

https://doi.org/10.21203/rs.3.rs-2095109/v1

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A convenient method for the preparation of halo-bicyclic molecules likely to show biological activity, from keten addition reaction with dimethyl cyclohexa-1,4-diene-1,2-dicarboxylate (3) and dichloroketene, respectively, under ultrasound irradiation, is reported. First, 1,4-diene 3 had been synthesized with Diels-Alder addition, and then the ketene addition reaction was carried out. Ultrasonic and non-ultrasonic conditions in the ketene addition reaction were investigated. Then, lactones (8 and 9) were obtained from ketene adducts. The structures of the molecules were illuminated by IR, and NMR spectroscopy techniques, especially Cosy spectroscopy. The test compounds improved antioxidant ability from 9.48% to 69.29% at concentration rates of 12.5-200 mg/L. The newly synthesized dichloro lactone 8 showed good antibacterial activity against gram-negative and –positive bacteria and also, antifungal activity. The antibiofilm activity of dichloro lactone 8 investigated and it showed 99.92 and 99.14% biofilm inhibiton activity againts Staphylococcus aureus and Pseudomonas aeruginosa, respectively at concentration of 250 mg/L. Moreover, it was displayed 100% Escherichia coli viability inhibition at concentrations of 125 and 250 mg/L.

Ketene addition

halo-bicyclic molecules

sonochemistry

antioxidant

antimicrobial

DNA cleavage

biofilm inhibition

Ketene addition reactions are one of the used methods in the synthesis of cyclobutanon (Snider 1988; Sengül et al. 2000; Kishali et al. 2011; Yılmaz et al. 2017; Krepski and Hassner 1978). Cyclobutanones are valuable synthetic intermediates that participate in a variety of new and useful synthetic transformations. Alkenes perform cycloaddition reactions with highly electrophilic ketenes such as dichloroketene in high yield. These reactions can be occurred in different methods under different media at room temperature, under reflux temperature of solvent in an N₂ atmosphere. Sometimes metallic Zn and in some cases Zn-Cu couple are used in the addition reaction. We used Zn-Cu in ketene addition reactions (Sengül et al. 2000; Brady 1981).

In addition, sonochemistry is very effective in some reactions. In cases where the reaction does not occur or the yield is low, the reaction can be performed in an ultrasonic bath (Mason 1997). In recent years, the use of ultrasound in chemical reactions has been quite common. Ultrasound is defined as sound beyond the frequency that the human ear can respond to. The normal range of hearing is between 16 Hz and about 18 kHz, and ultrasound is generally considered to be between 20 kHz and 100 MHz. By applying ultrasound, expansion occurs with the compression of the liquid, a sudden drop in pressure, and the formation of bubbles of oscillating gaseous matter. The changes that occur within these bubbles are still being studied. But what is known is that the use of an ultrasonic bath reduces the activation energy, increases the reaction rate, and allows non-existent reactions to occur. The reaction, which does not occur under normal conditions, reacts in the ultrasonic bath, and an increase in yield can be observed. There are many examples in the literature on this subject (Phakhodee et al.2020; Dadhania et al. 2011; Singh and Mangaonkar 2018; da Silveira Pinto and de Souza 2017; Kuik et al. 2017; Vidal et al. 2016; Qin and Schneider 2016; Mitchell 1995). Mitchell (1995) saw the yields and time of ketene addition reaction of indene, cyclopentene, and norbornene when the reaction was made under sonication.

On the other hand, halo-ketone and halo-lactones are also important molecules that show biological activity. There are also carbonyl groups and halogens as functional groups in the active ingredients of drugs available in the market (Drège et al. 2005; Trost et al. 2005; Austin et al. 2006). In particular, chlorine atom is widely used in the synthesis of drugs such as meningitis, cholera, plague, typhoid fever, bacterial skin infections, respiratory and nervous system problems (Fang et al. 2019). In addition, lactones are molecules with very different biological activities. For example, a-methylene-g-lactones are anti-leukemic agents (Gach and Janecka 2014). Terpenoids, which have a lactone functional group, have cytotoxic, anti-inflammatory, antimicrobial, anticancer and antimalarial activities (Surowiak et al. 2021). While designing the molecular structure, it was especially desired to be small and bicyclic, and to have chlorine and carbonyl groups.

Based on these studies, we aimed to increase the yield of the keten addition reaction to diene 3, which has a low yield, and to synthesize halo-bicyclic molecules. The next step is to investigate the biological activities of the synthesized molecules.

General

Infrared spectra were recorded on the Perkin Elmer Win First® Satellite. The ¹H and ¹³C NMR spectra were recorded on the Bruker Ultrashield Plus Biospin GmbH 400 MHz spectrometer. Column chromatography was performed on silica gel (60-mesh, Merck), TLC was carried out on Merck 0.2 mm silica gel 60F₂₅₄ analytical aluminum plates. All the chemicals and reagents were of analytical grade and obtained from either Sigma or Merck.

Diels-Alder Addition Reaction

34 cm³ %13 1,3-bütadiene 1 (in THF) and 7.24 g (51 mmol) Dimethyl acetylene dicarboxylate (DMDA) (2) were stirred at room temperature for 2 days. After the reaction is finished, the solvent was removed. Then, 1,4-diene 3 and DMDA were separated with chromatography on 50 g of silica gel which eluted with ethyl acetate/hexane (1:3). 8.48 g dimethyl cyclohexa-1,4-diene-1,2-dicarboxylate (3) (%85) was obtained at the end of the purification.

Dimethyl cyclohexa-1,4-diene-1,2-dicarboxylate (3, C₁₀H₁₂O₄) ¹H NMR (400 MHz, CDCl₃): d = 5.70 (s, 2H), 3.77 (s, 6H), 2.99 (s, 4H) ppm; ¹³C NMR (100 MHz, CDCl₃): d = 167.9, 132.2, 122.2, 51.7, 27.2 ppm.

Keten Addition Reaction

A 500 cm³three-necked flask equipped with a condenser, addition funnel, and N₂ inlet was charged with 1,4-cyclohexadiene (3) (5 g, 30 mmol), Zn–Cu (2.94 g, 45 mmol) and anhydrous diethyl ether (250). The suspension was stirred under N₂ and a solution of trichloroacetyl chloride (8.19 g, 45 mmol) and phosphoryl trichloride (6.91 g, 45 mmol,) in diethyl ether (50 cm³) was added dropwise over 2 h. When the addition of the solution was completed, the mixture was refluxed with stirring for 24 h. The reaction mixture was then filtered on celite and the unreacted zinc was washed with 100 cm³ of diethyl ether. The ethereal solution was washed with saturated NaHCO₃, and water and dried with MgSO₄. The solvent was then removed in vacuo. The residue was chromatographed through a silica gel column (50 g) eluting with ethyl acetate/hexane (1:9). As a result of the reaction, dichloro ketone 4 is formed in 20% yield, monochloro ketone 5 is formed in 10% yield, while 60% of the starting compound 3 remains.

The second reaction was carried out at the ether's reflux temperature by increasing the reaction medium's temperature under the reaction conditions described above. When the reaction temperature is increased, aromatic product 6 is formed with a yield of 75 percent as the main product, while dichloro ketone 4 is formed with a yield of 10 percent.

In the third reaction, the reaction was carried out using an ultrasonic bath. In the reaction made in the ultrasonic bath, dichloro ketone 4 was formed with an 80 percent yield.

rac-Dimethyl 7,7-dichloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (4, C₁₂H₁₂Cl₂O₅)

Colorless liquid, ¹H NMR (400 MHz, CDCl₃): d = 4.11-4.05 (m, 1H), 3.64 (s, 3H), 3.63 (s, 3H), 3.38-3.33 (m, 1H), 2.88-2.83 (A part of AB system, dd, 1H), 2.81-2.76 (A’ part of A’B’ system, dd, 1H), 2.57-2.50 (B part of AB system, ddd, 1H, J= 17.31, 7.77, 2.64 Hz), 2.36-2.29 (B’ part of A’B’ system, ddd, 1H, J= 17.33, 17.23, 2.62 Hz); ¹³C NMR (100 MHz, CDCl₃): d = 195.6, 167.1, 134.9, 133.9, 87.4, 52.8, 52.3, 44.4, 25.7, 23.1; IR: = 2956.13 (-C-H), 1798.77-1710.33 cm^-1 (-C=O); MS: m/z = 306, (M⁺,-OCH₃), 275 (M⁺, -OCH₃) 244, (M⁺, -CO), 216 (M⁺, -CO), 188.

rac-Dimethyl 7-chloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (5, C₁₂H₁₃ClO₅)

Colorless liquid, ¹H NMR (400 MHz, CDCl₃): d = 5.13-5.10 (dd, 1H, J= 9.41, 2.71 Hz), 3.81-3.80 (m, 7H), 3.23-3.15 (m, 1H), 2.84-2.78 (A part of AB system, dd, 1H, J= 17.59, 2.9 Hz), 2.65-2.62 (m, 2H), 2.44-2.37 (B part of AB system, ddt, 1H, J= 17.61, 7.73, 1.78 Hz); ¹³C NMR (100 MHz, CDCl₃): d = 202.1, 167.4, 134.7, 133.9, 63.1, 52.7, 52.4, 29.3, 23.6, 23.1; IR: = 2955.23, 1792.88, 1718.35, 1435.92, 1372.33, 1247.77, 1045.03; MS: m/z = 272, 241 (M⁺,-OCH₃), 214 (M⁺,-OCH₃), 177 (M⁺,-Cl), 149 (M⁺, -CO), 121 (M⁺, -CO).

Dimethyl phthalate (6, C₁₀H₁₀O₄) ¹H NMR (400 MHz, CDCl₃): d = 7.67-7.46 (AA’BB’ system, 4H), 3.84 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d = 167.9, 131.6, 130.8, 128.7, 52.6.

Dechlorination Reaction

To a vigorously stirring suspension of Zn (563 mg, 8.7 mmol) in 50 cm³ of glacial acetic acid at room temperature, was added dropwise a solution of dichloroketone 3 (1.33 g, 4.3 mmol) in 50 cm³ of acetic acid. After the addition was completed, the temperature was raised to 100 °C and maintained for 20 h. The reaction mixture was cooled and treated with diethyl ether, and the zinc residue was filtered. The ethereal layer was washed with water and a saturated solution of sodium bicarbonate to remove acetic acid and then it was dried with MgSO₄. The solvent was removed in an evaporator. Chromatography of the residue on 50 g of silica gel eluting with diethyl ether/hexane (1:1) afforded mono-chloro bicyclic ketone 5 and bicyclic ketone 7 as a colorless liquid.

rac-Dimethyl 7-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (7, C₁₂H₁₄O₅)

Colorless liquid, ¹H NMR (400 MHz, CDCl₃): d = 3.81-3.77 (m, 1H), 3.79 3.76 (s, 3H), 3.76 (s, 3H), 3.63-3.56 (m, 1H), 3.34-3.2 (ddd, 1H, J= 18.3, 9.15, 3.73), 2.96-2.88 (m, 1H), 2.85-2.80 (dd, 1H, J= 16.86, 2.64, 0.51 Hz), 2.68-2.65 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): d = 209.1, 168.9, 167.4, 137.0, 133.6, 56.1, 51.9, 51.3, 29.1, 23.6, 21.8; IR: = 2955.62, 2920.51, 2854.52, 1714.28, 1435.19, 1258.34; MS: 238 (M⁺,-OCH₃), 207 (M⁺,-OCH₃), 178 (M⁺, -CO), 150 (-CO), 119.

The reaction of rac-Dimethyl 7,7-dichloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (4) with MCPBA

rac-Dimethyl 7,7-dichloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (4) (500 mg, 1.6 mmol) was dissolved in 150 cm³ of chloroform, 957 mg, (5.57 mmol) 70% meta-chloro perbenzoic acid (MCPBA) was added, and then the reaction was stirred at room temperature for 24 h. The reaction mixture was added to 15 cm³ NaHSO₃ solution (624 mg) and the mixture was stirred for 15 min. The organic layer was separated and then washed with saturated aqueous NaHCO₃ (100 cm³), dried with MgSO₄, and concentrated to give dichlorolactones 8.

rac-Dimethyl 3,3-dichloro-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-5,6-dicarboxylate (8, C₁₂H₁₂Cl₂O₆)

Colorless liquid, ¹H NMR (400 MHz, CDCl₃): d = 5.12-5.03 (m, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.17-3.11 (m, 1H), 3.05-2.98 (dd, 1H, J= 19.56, 6.63, 1.73 Hz) , 2.92-2.83 (m, 2H), 2.43-2.35 (ddt, 1H, J= 18.53, 8.4, 2.52 Hz); ¹³C NMR (100 MHz, CDCl₃): d = 166.9, 132.4, 130.5, 82.5, 73.7, 52.5, 47.7, 28.5, 24.4; IR: = 3006.34, 2955.43, 2900.81, 2900.81, 2650.49, 2291.00, 2165.39, 2101.50, 1795.92, 1720.87, 1678.20, 157285, 1412.42, 1378.95; MS : m/z = 288 (M⁺,-OCH₃), 257 (M⁺,-CO), 221 (M⁺,-OCH₃), 193 (M⁺,-CO), 177 (M⁺,-O), 149.

The reaction of rac-Dimethyl 7-chloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (5) with MCPBA

rac-Dimethyl 7-chloro-8-oxobicyclo[4.2.0]oct-3-ene-3,4-dicarboxylate (5) (500 mg, 1.83 mmol) was dissolved in 150 cm³ of chloroform, 1.1 g, 6.38 mmol) 70% MCPBA was added, and then the reaction was stirred at room temperature for 24 h. The reaction mixture was added to 15 cm³ NaHSO₃ solution (624 mg) and the mixture was stirred for 15 min. The organic layer was separated and then washed with saturated aqueous NaHCO₃ (100 cm³), dried with MgSO₄, and concentrated to give lactones 9.

rac-Dimethyl 3-chloro-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-5,6-dicarboxylate (9, C₁₂H₁₃ClO₆)

Colorless liquid, ¹H NMR (400 MHz, CDCl₃): d = 4.77-4.75 (m, 2H), 3.73 (s, 3H), 3.72 (s, 3H), 2.99-2.78 (m, 3H), 2.75-2.58 (dd, 1H, J= 18.46, 7.36 Hz), 2.28-2.20 (ddt, 1H, J= 18.43, 8.91, 2.72 Hz) ¹³C NMR (100 MHz, CDCl₃): d = 170.8, 167.6, 166.9, 133.6, 130.9, 73.7, 57.1, 52.4, 37.4, 28.7, 23.3, IR: = 2954.46, 2920.81, 2650.37, 1785.03, 1715.19, 1663.11, 1434.2, 1259.24, 1190.95, 1149.83,1057.54, 1012.07; MS: m/z = 288, (M⁺,-OCH₃), 257 (M⁺,-OCH₃), 222 (M⁺,-CO), 193 , (M⁺,-O), 177.

DPPH Free Radical Scavenging Activity

Different concentrations of 500 µL of dichlorolactone 8 was added into 2 mL of DPPH solution. The mixture was then left at room temperature for 30 minutes. After 30 minutes, absorbance was measured in a spectrophotometer at 517 nm. Trolox and Ascorbic acid were used as standard and the same protocol was applied for them (Agırtaş et al. 2015). The free radical scavenging effect of dichlorolactone 8 was calculated according to the formula (1):

Control: DPPH solution without compound, Sample: Compound and DPPH solution

DNA Cleavage Activity

The DNA fragmentation activity of dichlorolactone 8 was evaluated by agarose gel electrophoresis after the interaction of the compound with the pBR322 plasmid DNA. Agarose gel electrophoresis experiments were performed in 0.5x TBE (Tris Borate EDTA) buffer at room temperature. Plasmid DNA was incubated with compound of different concentrations at 37 °C for 60 min. After that, loading dye was added to the mixtures and the mixtures were electrophoresed using 1% agarose gel at 80 V for 45 minutes and the results were visualized with UV light after the electrophoresis run.

Antimicrobial Activity

The in-vitro antimicrobial activities of dichlorolactone 8 were investigated by micro-dilution method. For the antimicrobial activity of the synthesized compound, Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Enterococcus hirae (ATCC 10541), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Legionella pneumophila subsp. pneumophila (ATCC 33152) used as bacterial strains and also Candida parapisilosis (ATCC 22019) and Candida tropicalis (ATCC 750) used as fungal strains. Before testing, the strains were seeded in Nutrient Broth and incubated overnight. Two-fold serial dilutions of compound at certain concentrations were done in U-based 96-well ELISA well plates. An equal amount of test microorganisms was then added to the wells containing the medium. The plates were then incubated at 37 °C overnight. After the incubation period, MIC (minimum inhibition concentration) was determined as the lowest values of the compound inhibiting strains growth (Agırtaş et al. 2015).

Bacterial Viability Inhibitory Activity

E. coli was inoculated into Nutrient Broth and incubated for 24 hours at 37 °C on a shaker at 120 rpm. At the end of the period, it was centrifuged at 5000 rpm for 5 minutes and then the pellet was washed with sterile 0.9% NaCl solution (isotonic). The washing process was repeated twice. The bacterial cell was incubated with different concentrations of dichlorolactone 8 8 in sterile Eppendorf tubes at 37 °C for 90 minutes on a shaker. After incubation, it was diluted with sterile 0.9% NaCl and inoculated into solid medium and incubated at 37 °C for 24 hours. The same procedures were applied to the control group as well. Colonies formed after incubation were counted and the percent inhibition of cell viability was calculated with the following equation (2) (Gonca et al 2021).

Biofilm Inhibition

S. aureus and P. aeruginosa strains were used for biofilm inhibition study. Equal volumes of dichlorolactone 8 but at different concentrations was again inoculated with equal amounts of S. aureus and P. aeruginosa, followed by incubation at 37 °C for 72 hours. After the time, the contents of the plates were emptied and then gently washed twice and then incubated at 80 °C for half an hour to dry. Then, crystal violet dye was added into all wells and incubated for 60 min. At the end of the period, the wells were washed twice with distilled water to remove unbound dye. Then, ethanol was added to the wells and their absorbance was measured at 595 nm (Gonca et al 2021). The following formula 3 was used to calculate the percent biofilm inhibition.

First, 1,4-diene (3) obtained from the Diels-Alder reaction of 1,3-butadiene (1) and dimethyl acetylene dicarboxylate (2) was synthesized with 85% in our study (Scheme 1) Simsek et al. 2001). Next, dichloroketene was added to the 1,4-diene by [2 + 2] cycloaddition reaction (Scheme 2) (Snider 1988; Sengül et al. 2000; Kishali et al.2011; Yılmaz 2017; Krepski and Hassner 1978; Brady 1981). The structure of the formed products was determined by spectroscopic methods. It was observed that the yield and the products formed changed as the reaction conditions changed.

When the reaction is carried out at room temperature, dichloro ketone 4 results in about 20% yield and monochloro ketone 5 in 10% yield, while 1,4-diene 3 remains unreacted in 60% yield (Scheme 2). The reaction time was gradually extended up to two weeks. It was observed that the yield of dichloro ketone 4 did not increase, on the contrary, 1,4-diene 3 was converted to an aromatic compound. Next, the reaction temperature was increased to the reflux temperature of the ether (Scheme 2). It was observed that dichloro ketone 4 with 10% yield and with 75% yield aromatic product 6 were formed. In the reaction, ideal conditions were sought by changing the mole amounts of the reactants, but it was seen that the result did not change. Finally, the ultrasonic bath was used in the reaction and it was observed that the dichloro ketone 4 was formed as a single product in 4 hours (Scheme 2). It was observed that both the reaction time decreased (4 hours) and the yield of dichloro ketone 4 increased (80%) significantly in the reaction performed in the ultrasonic bath.

In the ¹H NMR spectrum of dichloro ketone 4, the AB system of two methylene protons, two methine protons, and two methyl protons confirm the structure. In the ¹³C NMR spectrum of dichloro ketone 4, the presence of the carbonyl group and the dichlor-bonded quaternary carbon atom confirm the structure. The methine proton attached to the chlorine atom seen at 5.13–5.10 ppm in the ¹H NMR spectrum of the monochloro ketone molecule and also the methine carbon at 73.5 ppm seen in the ¹³C NMR confirms the structure. The AA'BB' system was observed at 7.67–7.46 ppm in the ¹H NMR spectrum of aromatic compound 6. Methine proton gave a signal at 5.13–5.10 ppm in the ¹H NMR spectrum of monochloro ketone 5, methine carbon gave a signal at 63.1 ppm in the ¹³C NMR spectrum. These results are also consistent with the structure.

In the next step, we aimed to reduce the chlorine of dichloro ketone 4 (Scheme 3). Two products (Monochloroketone 5 with 40% conversion and ketone 7 with 60% conversion) were formed from the reaction of Zn-Cu with dichloro ketone 4 in acetic acid. Since the reduction product 7 obtained was decomposed very quickly, problems were encountered during the characterization processes and spectroscopic analyzes. The methylene protons signaling at 3.81–3.77 in the ¹H NMR spectrum of ketone 7 and the methylene carbon at 51.9 ppm in ¹³C NMR confirms the structure.

In the last step, the transition from dichloro ketone 4 and monochloro ketone 5 to their respective lactones was made and chloro lactones were obtained in high yields (Scheme 4).

In the NMR spectrum of dichlorolactone 8, the methine proton attached to the oxygen signaled at 5 ppm, the methine carbon attached to the oxygen signaled at 73.7 ppm, and the quaternary carbon attached to the chlorines signaled at 82.5 ppm. These results are in agreement with the structure. In the NMR spectrum of monochlorolactone 9, the methine proton attached to oxygen and the proton of the chloro-bonded methine carbon overlapped at 5 ppm, while the lactone carbon signaled at 170.8 ppm.

Antioxidant Activity

Free radicals are substances that are defined as the transport of one or more unpaired high-energy electrons in the outer orbital of atoms or molecules and therefore can easily react with other substances. As a result of these reactions, cell membrane, protein, enzyme, RNA and DNA damage may occur. Depending on these damages, cell death or cell cancer may occur. Substances that reduce the harmful effects of free radicals formed as a result of oxygen metabolism in the cell and even prevent their formation are called antioxidants (MacDonald-Wicks et al. 2006). Due to these properties, interest in natural or synthetic antioxidants is increasing day by day. In this study, the antioxidant activity of the newly synthesized dichlorolactone 8 was investigated. The results are presented in Fig. 1. At 12.5 mg/L, DPPD scavenging activity was found to be 9.48%. DPPH scavenging activity increased from 28.41–46.03% when the concentration of dichlorolactone 8 increased from 50 mg/L to 100 mg/L. The highest DPPH scavenging inhibition was 69.29% complete at 200 mg/L. According to these results, the newly synthesized dichlorolactone 8 can be used as an antioxidant agent.

DNA cleavage

The interaction of compounds with DNA molecule is very significant in biological systems because understanding the interaction of these compounds with DNA simplifies more effective and selective antibiotic and anticancer agent design. In gel electrophoresis, matrix is performed as a support material and allows the separation of DNA molecules according to their sizes. Mixtures of agarose polyacrylamide or agarose can be utilized as matrix material. It is the most widely studied process in routine as the agarose gel preparation is fast and simple. The agarose gel procedure is based upon specifying DNA molecule and its form. It is also observed the rearrangements in DNA form because of alterations in the DNA structure. DNA cleavage test was carried out using pBR322 DNA to investigate cleavage ability of the compound at different concentrations. The result of DNA cleavage property of dichlorolactone 8 is depicted in Fig. 2. As can be clearly seen in Fig. 2, dichlorolactone 8 showed excellent DNA cleave property and plasmid DNA was completely fragmented at all tested concentrations. Mashrai et al. (2017) studied the DNA binding ability of synthesized steroidal lactones. It was seen that cytotoxicity experiments (MTT assay) of the steroidal lactones have been identified showing potential anticancer behavior of IC₅₀ = 19.27 against the MCF-7 cell line. Elaine et al. reported antileukemic properties of sesquiterpene lactones (2018). Gach et al. studied the role of oxidative stress in anticancer activity of sesquiterpene lactones (2015). Our results showed that dichlorolactone 8 can be used as a DNA targeting molecule in anticancer and antibiotic studies after toxicological test systems.

Antimicrobial Activity

Bacteria use some molecules as signal molecules. The most studied class of signaling molecules for gram-positive and gram-negative bacteria is γ-butyro-lactones (Pomini and Marsaioli 2008). In this study, the antimicrobial activity of dichlorolactone 8 was tested and the antimicrobial activity was evaluated by MICs. The results are presented in Table 3. According to these results, it was determined that dichlorolactone 8 was more effective against gram positives than gram negatives. The antibacterial MICs values were found to be 32 mg/L for E. faecalis, 64 mg/L for E. hirae and S. aureus, 128 mg/L for P. aeruginosa and E. coli, and 256 mg/L for L. pneumophila, while the antifungal MICs value of C. parapisilosis and C. tropicalis were determined as 128 mg/L. According to these results, E. faecalis and L. pneumophila were found to be the most sensitive and the most resistant microorganisms to dichlorolactone 8, respectively. Our results indicated that dichlorolactone 8 can be used as antimicrobial agent.

Table 3

Antimicrobial activity
Microorganisms		MIC Values (mg/L)
Microorganisms		2
E. coli		128
L. pneumophila subsp. pneumophila		256
P. aeruginosa		128
S. aureus		64
E. hirae		64
E. faecalis		32
C. tropicalis		128
C. parapisilosis		128

Biofilm Inhibition

Bacterial biofilms are complex structures that show dynamic changes formed by a glycoproteic three-dimensional matrix produced by microorganisms for their proliferation, survival and adaptation to changing environmental conditions and the bacterial population embedded in it. These high-level microbial structures are almost ubiquitous. Biofilms can be formed on non-living and living surfaces and can be composed of homogeneous or heterogeneous microorganism communities. Other compositions in the biofilm layer are; 1–2% globular glycoproteins and other proteins, 1–2% EPS, 1–2% phospholipids, lipids, nucleic acids (Flemming and Wingender 2006). However, these proportion may change according to the microbial strain, their physiological properties, the structure of the growth environment, the type of fluid, and general physical features (Padera 2006). In this study, biofilm formation inhibition of dichlorolactone 8 was tested against S. aureus and P. aeruginosa. Biofilm inhibition results are shown in Figs. 4 and 5. For P. aeruginosa, the rate of biofilm inhibition at 62.5 mg/L was 79.35%. When the concentration of dichlorolactone 8 increased from 125 mg/L to 250 mg/L, biofilm inhibition of P. aeruginosa was determined to increase from 90.58 to 99.14%. Biofilm inhibition for S. aureus at 62.5, 125 and 250 mg/L was 91.17, 96.91 and 99.92%, respectively. Mousaa et al. (2022) investigated the antibiofilm inhibition of green materials based epoxy acrylate againts. It was claimed that the highest antibiofilm inhibition % of A1.6 and D1.6 sample was found as 84.43%, and 77.08% for P. aeruginosa, and also followed as 78.58% and 58.74% for Proteus mirabilis, as 77.81%, and 75.52% for Salmonella typhi and as 75.30% and 72.59% for S. aureus, respectively. The results showed that dichlorolactone 8 can be used as a biofilm inhibition agent in various industries such as food, environment and health.

Cell Viability Inhibition

The microbial cell viability inhibition activity of the newly synthesized and characterized dichlorolactone 8 was tested. For this test, E. coli was exposed to dichlorolactone 8 at different concentrations for 90 minutes and then seeded onto solid media. The sample to which test compound was not added was accepted as the control. E. coli viability inhibition activity was determined by counting the colonies formed afterwards. The results are shown in Fig. 6. When compared to the control, E. coli was inhibited by 99.99% at a concentration of 62.5 mg/L. The inhibition of E. coli viability was determined as 100% at concentrations of 125 mg/L to 250 mg/L. Ji et al. (2022) indicated that they studied antimicrobial activity of the water-borne bisphenol A type epoxy thermoset (WE51/BC919) using S. aureus and E. coli. The antimicrobial ability of WE51/BC919 was found as 89.4 and 97.8% for S. aureus and E. coli, respectively. Islam et al. (2020) investigated in their study to determine antimicrobial ability of epoxy-based nanocomposite coatings doped with nanosilica, zinc oxide (ZnO) and titanium oxide (TiO₂) against E. coli and S. aureus. They found that all of the tested compounds significantly inhibited growth of E. coli and S. aureus after 2 h and 4 h exposure. These findings showed good similarity with our results. The results obtained from our study showed that the newly synthesized compound can be used for microbial inhibition in pharmaceutical industries.

In this paper, a good alternative procedure for ketene addition reactions using the sonochemistry techniques was shown. The main advantages of using ultrasound in the ketene addition reaction compared to conventional methodologies are the reduction in reaction time and improvement in yield. When we do the ketene addition reaction without using an ultrasonic bath, the product yield is very low (20%). A significant increase (80%) in the efficiency of the reaction was observed when ultrasonic bath was used. In addition, new halo-ketone and halo-lactone molecules were synthesized and presented to the literature in this study. In this study, several biologic activities of dichlorolactone 8 were also investigated. The sample displayed good antioxidant activity and demonstrated good antimicrobial property against test microbial strains. It was also had perfect DNA cleavage and biofilm inhibition properties. Microbial cell viability inhibition assay confirmed that compound had 100% cell viability inhibition property against E. coli. Therefore, dichlorolactone 8 could be employed as a promising applicant for antimicrobial applications in drug industry after following studies.

Acknowledgements The authors are indebted to Mersin University [2019-3-AP4-3774] for their financial support to this work.

Author informations

Mersin University, Department of Chemistry, Faculty of Arts and Sciences, TR-33343 Yenisehir, Mersin, Turkey

Hazal Sezen and Nermin Şimşek Kuş

Mersin University, Food Processing Programme, Technical Science Vocational School, TR-33343 Yenisehir, Mersin, Turkey

Sadin Özdemir

3Technical Science Vocational School, Department of Laboratory and Veterinary Health, Mersin University, TR-33343 Yenisehir, Mersin, Turkey

Gülşah Tollu

Contributions

Nermin Şimşek Kuş and Sadin Özdemir were involved in the methodology, software, investigation, structural analysis, and writing the original draft. Hazal Sezen and Gülşah Tollu took part in conducting the experiments and evaluating the results.

Corresponding author

Correspondence to Nermin Şimşek Kuş.

Ethics declarations

Conflict of Interest

The authors declare no competing interests.

Ethical Approval

Funding

This study was financially supported by the grant from Mersin University [2019-3-AP4-3774].

Supplementary Information

Below is link to the electronic supplementary material.

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Scheme 1 to 4 are available in the Supplementary Files section.

No competing interests reported.

supplementary.docx
SCHEME1.png
Scheme 1 The synthesis of 1,4-diene (3)
SCHEME2.png
Scheme 2 Ketene addition reactions
SCHEME3.png
Scheme 3 Reduction reaction
SCHEME4.png
Scheme 4 The synthesis of lactones

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Sonochemistry in organic synthesis: A convenient method for the preparation of halo-bicyclic molecules with keten addition reaction and biological properties of Dimethyl 7,7-dichloro-8-oxabicyclo[4.2.0]oct-3-ene-3,4- dicarboxylate

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Abstract

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Introduction

Experimental

Results And Discussion

Antioxidant Activity

DNA cleavage

Antimicrobial Activity

Biofilm Inhibition

Cell Viability Inhibition

Conclusions

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References

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Additional Declarations

Supplementary Files

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