New bicyclic brominated furanones as potent autoinducer-2 quorum-sensing inhibitors against bacterial biofilm formation

Article abstract of DOI:10.1016/j.ejmech.2017.05.037

Bacterial behaviors such as virulence factor secretion and biofilm formation are critical for survival, and are effectively regulated through quorum sensing, a mechanism of intra- and interspecies communication in response to changes in cell density and species complexity. Many bacterial species colonize host tissues and form a defensive structure called a biofilm, which can be the basis of inflammatory diseases. Periodontitis, a chronic inflammatory disease affecting the periodontium, is caused by subgingival biofilms related to periodontopathogens. In particular, Fusobacterium nucleatum is a major co-aggregation bridge organism in the formation and growth of subgingival biofilms, linking the early and late colonizers in periodontal biofilms. According to our previous study, the intergeneric quorum-sensing signal molecule autoinducer-2 (AI-2) of F. nucleatum plays a key role in intra- and interspecies interactions of periodontopathogens, and may be a good target for periodontal biofilm inhibition. Recently, brominated furanones produced by the macroalga Delisea pulchra were shown to inhibit biofilm formation via AI-2, and have been investigated toward the goal of increasing the inhibition effect. In this study, we describe the synthesis of new bromofuranone analogs, i.e., 3-(dibromomethylene)isobenzofuran-1(3H)-one derivatives, and demonstrate their inhibitory activities against biofilm formation by periodontopathogens, including F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia.

Full text of DOI:10.1016/j.ejmech.2017.05.037

Accepted Manuscript
New bicyclic brominated furanones as potent autoinducer-2 quorum-sensing
inhibitors against bacterial biofilm formation
Ji Su Park, Eun-Ju Ryu, Linzi Li, Bong-Kyu Choi, B. Moon Kim
PII:
S0223-5234(17)30398-7
10.1016/j.ejmech.2017.05.037
EJMECH 9467
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 12 May 2017
Accepted Date: 14 May 2017
Please cite this article as: J.S. Park, E.-J. Ryu, L. Li, B.-K. Choi, B.M. Kim, New bicyclic brominated
furanones as potent autoinducer-2 quorum-sensing inhibitors against bacterial biofilm formation,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.037.
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ACCEPTED MANUSCRIPT
Periodontopathogens
New bicyclic
brominated furanones
Biofilm
formation

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New bicyclic brominated furanones as potent autoinducer-2
quorum-sensing inhibitors against bacterial biofilm formation
Ji Su Parka,†, Eun-Ju Ryub,†, Linzi Lia, Bong-Kyu Choi*,b, B. Moon Kim*,a
aDepartment of Chemistry, College of Natural Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
Seoul 08826, Republic of Korea
bDepartment of Oral Microbiology and Immunology and Dental Research Institute, School of Dentistry, Seoul
National University, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea
†These two authors contributed equally to this work.
*Corresponding authors
B. Moon Kim:
Department of Chemistry, College of Natural Sciences
Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826
Republic of Korea
E-mail: kimbm@snu.ac.kr
Tel: +82 2-880-6644
Fax: +82 2-872-7505
B-K. Choi:
Department of Oral Microbiology and Immunology, School of Dentistry
Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul 03080
Republic of Korea
E-mail: bongchoi@snu.ac.kr
Tel: +82 2 740 8640
Fax: +82 2 743 0311

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Abstract
Bacterial behaviors such as virulence factor secretion and biofilm formation are critical for survival, and are
effectively regulated through quorum sensing, a mechanism of intra- and interspecies communication in
response to changes in cell density and species complexity. Many bacterial species colonize host tissues and
form a defensive structure called a biofilm, which can be the basis of inflammatory diseases. Periodontitis, a
chronic inflammatory disease affecting the periodontium, is caused by subgingival biofilms related to
periodontopathogens. In particular, Fusobacterium nucleatum is a major co-aggregation bridge organism in
the formation and growth of subgingival biofilms, linking the early and late colonizers in periodontal biofilms.
According to our previous study, the intergeneric quorum-sensing signal molecule autoinducer-2 (AI-2) of F.
nucleatum plays a key role in intra- and interspecies interactions of periodontopathogens, and may be a good
target for periodontal biofilm inhibition. Recently, brominated furanones produced by the macroalga Delisea
pulchra were shown to inhibit biofilm formation via AI-2, and have been investigated toward the goal of
increasing the inhibition effect. In this study, we describe the synthesis of new bromofuranone analogs, i.e., 3-
(dibromomethylene)isobenzofuran-1(3H)-one derivatives, and demonstrate their inhibitory activities against
biofilm formation by periodontopathogens, including F. nucleatum, Porphyromonas gingivalis, and Tannerella
forsythia.
Keywords: Quorum sensing, bacterial biofilm, AI-2 inhibitors, brominated furanones

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I. Introduction
Quorum sensing (QS) is the process of cell-to-cell communication in microorganisms mediated by small
signaling molecules referred to as autoinducers, which are secreted by bacteria and fungi [1-3]. Bacteria can
effectively regulate numerous phenotypes, including virulence factor expression, bioluminescence, and biofilm
formation, in response to their changes in population density through recognition of the threshold reached by
autoinducers, the so-called quorum, via intracellular or intercellular communication [4]. In particular, formation
of a biofilm can not only enhance the viability of bacteria in response to antibiotics but can also be the basis of
the development of chronic inflammatory diseases, including endocarditis, cystic fibrosis, osteomyelitis, chronic
urinary tract infections, chronic prostatitis, and periodontal diseases [5].
QS molecules can be classified into three major types: oligopeptides, acyl-homoserine lactones (AHLs,
autoinducer-1), and autoinducer-2 (AI-2). Oligopeptides and AHLs are used by gram-negative and gram-
positive bacteria, respectively, in intraspecies communication [6]. AI-2 is a universal QS molecule secreted by
both gram-negative and gram-positive bacteria [7-9], and plays a critical role in the virulence of pathogenic
bacteria and in biofilm formation [10]. AI-2 mediates both intra- and interspecies communication, and can thus
be a good target for the regulation of bacterial infection. Accordingly, AI-2 inhibitors are ideal compounds for
bacterial biofilm inhibition in multispecies bacterial communities.
More than 700 species of bacteria have been found in the human oral cavity, which establish mixed-species
communities [11]. Among these oral bacteria, periodontopathogens, including Aggregatibacter
actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Tannerella forsythia, and
spirochetes, form subgingival biofilms and cause periodontitis, which is a chronic inflammatory disease. Since
AI-2 is an interspecies QS molecule that controls intergeneric signaling, it may induce oral biofilm formation
and increase the bacterial virulence of periodontopathogens [12-14]. Furthermore, Kolenbrander et al. [15]
suggested that, compared to commensal bacteria, periodontopathogens produce much higher levels of AI-2; such
high concentrations would enhance pathogen biofilm maturation, leading to periodontitis. In particular, F.
nucleatum is known as a major co-aggregation bridge organism that connects late pathogenic colonizers and
early commensal colonizers in a periodontal biofilm [15-19]. We previously reported that the level of F.
nucleatum AI-2 was reduced by QS inhibitors (QSIs), including (5Z)-4-bromo-5-(bromomethylene)-2(5H)-
furanone and D-ribose [20]. Because of the diversity of oral bacteria, it is hard to selectively eliminate
periodontopathogens without disturbing oral commensals. Therefore, oral biofilm formation regulation using

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QSIs is one of the most hopeful protective means for preserving oral health [21].
Brominated furanones produced from the macroalga Delisea pulchra are known to prevent microbial
colonization [22, 23]. In addition, these compounds have been shown to inactivate LuxS, which is required for
AI-2 synthesis, and to inhibit the QS activity of various bacterial species [4, 24-27]. Recently, Yang et al. [28]
reported a bicyclic version of brominated furanones that could potentially reduce their toxicity while retaining
their biofilm inhibitory activities. However, compared with reported monocyclic brominated furanones, the
inhibitory activities of the bicyclic furanones were relatively low [28]. These results suggest that modification of
the exocyclic vinyl position, which is an essential structural element for the inhibition of LuxS [29], is not an
appropriate strategy for the development of effective QSI’s. Therefore, discovery of new potent and safer
brominated furanone candidates is still desired. In this study, to improve the QS inhibitory activity and biofilm
inhibition efficacy of periodontal bacteria, we report the synthesis and evaluation of 3-
(dibromomethylene)isobenzofuran-1(3H)-one derivatives, which have different ring sites from the existing
bicyclic compound. In particular, we investigated the structure-activity relationships of the biofilm inhibition
effects of various new furanone derivatives prepared with new ring structures and possible side chains on the
new ring.
2. Results and discussion
2.1. Chemistry
We focused on the design and synthesis of new bicyclic brominated furanone derivatives with reduced
toxicity while retaining or enhancing their biofilm inhibitory activities. As shown in Table 1, we synthesized two
kinds of bicyclic brominated furanone derivatives: one through the modification of the ring structure and the
other through introduction of a side chain onto the benzene ring. The ring structural component of furanones has
been reported to be important for the inhibition of biofilm formation by bacteria [28]. Thus, compounds with
different ring structures (1–5) were designed for evaluation of the ring structure effect on the biofilm inhibitory
activity. Another set of compounds possessing side chains on the benzene ring (6–9d) were designed with the
aim of investigating the influence of the side chain length on the benzene ring, since previous reports have
shown that monocyclic furanones with chain lengths of two to six atoms show good inhibitory activities [30, 31].

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According to the Ramirez olefination procedure [32], brominated compounds with various ring structures
(10–14) shown in Table 1 were synthesized from the corresponding commercially available compounds in one
step. The yields of the products ranged from low to moderate (14–65% yields). Although the reactions
proceeded for cyclopropane- and cyclobutane-1,2-dicarboxylic anhydrides, the olefination products decomposed
rapidly at room temperature, presumably due to the unstable nature of the anhydrides based on the angle strains
of the hydrocarbon ring [33].
For the preparation of brominated furanone derivatives containing alkoxy groups on the benzene ring, such
as 16a–16e and 18a–18d, starting materials containing the alkoxy derivatives 7a–7e and 9a–9d were prepared
from the corresponding phenol derivatives through the use of a reported synthetic method [34]. These precursors
(7a–7e and 9a–9d) were obtained at moderate to good yields over four steps (45–77%). Through the Ramirez
olefination with anhydrides possessing side chains on the benzene ring (6–9d), brominated compounds 15–18d
were produced with 70–83% regioselectivity. Similar to a study that examined the regioselectivity of Wittig
reactions for mono-substituted phthalic anhydrides [35], this result showed that the electronic effects of the
substituents appear to have a large influence on the regioselectivity of the olefination. Brominated compounds
15–18d were obtained in overall yields ranging from 16% to 22%. Although small amounts of minor
regioisomers were produced in olefination with the anhydrides 6–9d, we focused on the major product because
of its better inhibitory effect on biofilm formation. All target compounds were separated and purified through
the use of column chromatography. The structures of the desired products were confirmed by spectroscopic
analyses, including 1H, 13C, heteronuclear multiple bond correlation (HMBC), and mass spectrometry (MS).
2.2. Biological evaluation
In our preliminary experiments, in order for us to evaluate the inhibitory activities based on the ring structural
component of the inhibitors, brominated furanone compounds with different ring structures (10–14) were
screened for their inhibitory effects on the biofilm formation of F. nucleatum at 0.2 and 2 µM concentrations
(Fig. S1). Since it was confirmed in previous studies that the reference furanone compound R1, (Z)-4-bromo-5-
(bromomethylene)furan-2(5H)-one, has neither bactericidal effect nor cytotoxicity against host cells at 2 µM
[20], the following experiments were performed starting at 2 µM. As shown in Fig. S1, bromofuranones
containing a benzene ring (10) and a 5-membered ring (13) showed higher inhibitory activities at 0.2 and 2 µM
compared to other compounds examined.

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According to previous reports [30, 31], the biofilm inhibitory activities of furanones were dependent on the
side chain structure. Therefore, we synthesized brominated furanone derivatives (15-18d) based on compound
10, which showed good inhibitory activity, and investigated the biofilm inhibitory effects according to the
variation of the side chains. Compounds 15-18d were evaluated along with compounds 10-14 at 0.02 and 0.2
µM to investigate the activities at lower concentrations. Compounds with 5-membered ring structure (13) and a
short side chain on the benzene ring (15 and 17) showed high inhibitory effects on the biofilm formation of F.
nucleatum. However, as shown in Fig. S2, the inhibitory activities of compounds except for 13, 15 and 17 were
worse than that of compound R1 as the increase of the chain length rather decreased the inhibitory activity.
2.2.1. Inhibitory effect of new furanone compounds on AI-2 activity
To investigate whether the inhibitory effects of highly active compounds (13, 15 and 17) on biofilm formation
arise from the inhibition of AI-2 activity, these furanone compounds were evaluated for their inhibitory activities
on the AI-2 activity at 2 µM. AI-2 activities were assessed using Vibrio harveyi BB170, an AI-2 reporter strain,
by measuring the AI-2-mediated bioluminescence of the bacteria. As shown in Fig. 1, compounds 13, 15, and 17
at 2 µM significantly reduced the bioluminescence of V. harveyi BB170 induced by F. nucleatum AI-2, but their
inhibitory activities appeared to be lower than that of the compound R1 (Fig. 1A). Through an additional
bacterial growth test, we confirmed that the compound R1 significantly inhibited the growth of V. harveyi
BB170 (Fig. 1B), resulting in the reduction of bioluminescence. However, the newly synthesized furanone
compounds did not affect the planktonic growth of V. harveyi BB170. This revealed that the inhibitory effect of
highly active compounds on biofilm formation was caused by inhibition of the AI-2 activity without affecting
the planktonic growth of V. harveyi BB170.
Through F. nucleatum biofilm formation assay and AI-2 activity test, it was found that 5-membered ring
structure (13) and benzene derivatives having a short side chain (15 and 17) exhibit good inhibitory effects on
biofilm formation through the inhibition of AI-2 activity. Therefore, three most effective compounds (13, 15 and
17) were selected.
2.2.2 Inhibitory effect of the new furanone compounds on the biofilm formation of major periodontopathogens
To evaluate the biofilm formation inhibitory effect of the three new furanone compounds (13, 15, and 17)
with high inhibitory activity on the major periodontopathogens, F. nucleatum, P. gingivalis, and T. forsythia

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were grown on a cover slip in the absence or presence of the compounds, followed by observation using a
confocal laser-scanning microscope. The biofilm formed by each group was also analyzed by measuring the
biomass and average thickness. As shown in Fig. 2 and Table 2, the furanone compounds 13, 15, and 17 at a
concentration of 2 µM remarkably reduced the biofilm formation of periodontopathogens induced by F.
nucleatum AI-2. The biomass of the biofilm formed by F. nucleatum, P. gingivalis, and T. forsythia was
quantified by dividing the value of the total fluorescence intensity by the area after culture alone or with 10%
semi-purified F. nucleatum AI-2 in the absence or presence of the compound R1 or new furanone compounds
(13, 15, and 17) at 2 µM. The average depth of the biofilm formed on glass cover slips was analyzed using the
Carl Zeiss LSM 700 program. The inhibitory effects of all three compounds on the biofilm formation of F.
nucleatum, P. gingivalis, and T. forsythia were comparable to those of the compound R1.
To assess the effects of the compounds at lower concentrations, the biofilm formation was evaluated by
crystal violet staining. As shown in Fig. 3, compounds 13, 15, and 17 significantly reduced the biofilm
formation of F. nucleatum, P. gingivalis, and T. forsythia induced by F. nucleatum AI-2. Compounds 13, 15,
and 17 significantly inhibited the biofilm formation of F. nucleatum, and their inhibitory activities were higher
at all concentrations tested (0.002–2 µM) than that of the compound R1. In the case of P. gingivalis, compound
17 showed a higher inhibitory effect on biofilm formation than the compound R1 at all concentrations tested,
whereas compounds 13 and 15 showed higher inhibitory activity at the concentration of 0.02 µM and 0.2 µM,
respectively. Compared to the compound R1, compounds 17, 13, and 15 showed higher inhibitory activity of T.
forsythia biofilm formation at 0.002–0.2 µM, 0.2 µM, and 0.2 and 2 µM, respectively. The inhibitory activities
of compounds 13 and 17 did not follow a dose-dependent tendency, which varied according to the bacterial
species. Although AI-2 is a universal signaling molecule, the structure of the AI-2 receptor may be slightly
different among bacterial species [4, 36]. The furanone compounds may act as partial agonists or antagonists
depending on the concentration in accordance with the different structures of AI-2 receptors [37-39]. AI-2
receptors or transporters have been reported in several species of bacteria, including LsrB and LsrR in
Escherichia coli and Salmonella Typhimurium, AgrC in Staphylococcus aureus, TlpB in Helicobacter pylori,
and LuxP/Q in Vibrio sp. [40]. However, the AI-2 receptors of periodontopathogens have only been reported in
A. actinomycetemcomitans [41, 42]. To clarify the interaction between the receptors and their ligands, the
structure of an AI-2 sensor in periodontopathogens needs to be identified. Furthermore, the inhibitors may
inhibit other adhesin-related factors of periodontopathogens induced by AI-2, resulting in further inhibition of
biofilm formation; this hypothesis also remains to be tested. It is notable that the inhibitory activities of 13, 15,

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and 17 against F. nucleatum biofilm formation are superior to those of the reference furanone compound R1 at a
concentration as low as 2 nM. As F. nucleatum mediates the colonization of periodontopathogens, including P.
gingivalis, T. forsythia, and Treponema denticola, the inhibition of biofilm formation can hamper the
development of a pathogenic biofilm, and thus prevent periodontitis [19, 20, 43, 44].
2.2.3. Effect of the new furanone compounds on planktonic bacterial growth
Traditional antibacterial agents aim to kill or inhibit bacterial growth. However, QSIs generally target
virulence expression without bacterial growth inhibition. To confirm whether the inhibitory effect of the new
furanone compounds on the biofilm formation of periodontopathogens is not attributed to bacterial growth
inhibition, we assessed the effects of the compounds on planktonic bacterial growth. As shown in Fig. 4, the
compounds did not inhibit the growth of F. nucleatum, P. gingivalis, and T. forsythia at the highest concentration
tested (2 µM) in the biofilm formation assay.
2.2.4. Cytotoxicity evaluation of the new furanone compounds
To evaluate whether the new furanone compounds are toxic to host cells, a cytotoxicity test was performed in
three types of human cells: the monocytic cell line THP-1, human gingival fibroblasts (HGFs), and the human
oral keratinocyte cell line HOK-16B. As shown in Fig. 5, the compounds did not affect host cell viability at 2
µM.
2.2.5. Effect of furanone compounds on the host cell immune response
To confirm whether the new compounds induce an inflammatory response in host cells, the expression levels
of proinflammatory cytokines, including interleukin (IL)-6 and IL-8, were analyzed by real-time reverse
transcription-polymerase chain reaction (RT-PCR) after treating THP-1 cells and HGFs with the furanone
compounds. Fig. 6 shows that the new furanone compounds (13, 15, and 17) did not induce an inflammatory
response, whereas the reference compound R1 or lipopolysaccharide (LPS; 1 µg/mL) significantly induced the
gene expression of IL-6 and IL-8 in THP-1 cells (Fig. 6A and 6B). In HGFs, the new furanone compounds (13,
15, and 17) did not induce the gene expression of IL-6 and IL-8 (Fig. 6C and 6D), whereas the reference
compound R1 induced the gene expression of IL-8, and LPS (1 µg/mL) induced the gene expression of both
cytokines.

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3. Conclusion
We synthesized 3-(dibromomethylene)isobenzofuran-1(3H)-one derivatives and tested their inhibitory effects
against the AI-2-mediated QS and biofilm formation of periodontopathogens. Our results demonstrate that
bicyclic furanone derivatives containing a five-membered ring (13) or methylbenzene moieties (15 and 17)
significantly inhibited the F. nucleatum AI-2 activity and biofilm formation of F. nucleatum, P. gingivalis, and
T. forsythia without a bactericidal effect. The inhibitory activities of 13, 15, and 17 were similar to or better than
that of the reference compound R1. Our new furanone compounds showed neither cytotoxicity nor induction of
proinflammatory cytokine expression, whereas the reference compound R1 showed cytotoxicity and induced the
expression of proinflammatory cytokines such as IL-6 and IL-8 in human cells.
In addition, our results highlight the importance of the structural element of a bicyclic compound for its
effective biofilm inhibition activity. First, compound 13, which has a similar ring structure form to AI-2,
showed good inhibition activity. Second, we found that the inhibitory activity was good when the compound
had a short side chain. In particular, compound 17, in which the methyl group was introduced at a similar
position to that of the existing monocyclic brominated furanone, exhibited the highest inhibitory activity. As the
widespread use of traditional antibacterial agents such as disinfectants and antibiotics can lead to the
development of drug-resistant strains of bacteria and even kill beneficial bacteria, the new compounds 13, 15,
and 17 can be good candidates for prevention of the biofilm formation of periodontopathogens. The effect of
these compounds on other bacteria involved in infectious diseases remains to be assessed.
4. Experimental part
4.1. Chemistry
1
The H, 13C NMR-spectra and heteronuclear multiple bond correlation (HMBC) were measured with an
Agilent 400-MR DD2 Magnetic Resonance System (400 MHz) and a Varian/Oxford As-500 (500 MHz)
spectrophotometer. Chemical shifts were measured as parts per million (δ values) from tetramethylsilane as an
internal standard at probe temperature in CDCl3 or DMSO-D6 for neutral compounds. Coupling constants are
provided in Hz, with the following spectral pattern designations: s, singlet; d, doublet; t, triplet; q, quartet; quint,
quintet; m, multiplet; br, broad; app, apparent. Melting points were determined on an Electro-thermal IA 9100
apparatus without correction. Reactions that needed anhydrous conditions were carried out in flame-dried

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glassware under positive pressure of dry N2 using standard Schlenk line techniques. Evaporation of solvents was
performed at reduced pressure using a rotary evaporator. TLC was performed using silica gel 60F254 coated on
aluminum sheet (E. Merck, Art.5554). Column chromatography was performed on silica gel (Merck. 7734 or
9385 Kiesel gel 60), and eluent was mentioned in each procedure. Elemental analyses were performed on a
Thermo Scientific model Flash 2000 instrument (Waltham, MA, USA) and the results are within ±0.4% of the
theoretical values. High resolution mass spectra (HRMS) were recorded on a ThermoFinnigan LCQ™ Classic,
Quadrupole Ion-Trap Mass Spectrometer. HPLC analyses were carried out on an Agilent HP1100 system (Santa
Clara, CA, USA), composed of an auto sampler, quaternary pump, photodiode array detector (DAD), and HP
Chemstation software. The separation was carried out on a poroshell 120 EC-C18 column 4.6 × 50 mm i.d. (2.7
µm particle size) with 0.1% TFA in water (A) and acetonitrile (B) as a mobile phase at a flow rate of 1 mL/min
at 20 °C. Method: 100% A and 0% B (0 min), 50% A and 50% B (5 min), 5% A and 95% B (15 min), 5% A and
95% B (22 min), 100% A and 0% B (23 min), 100% A and 0% B (25 min). Details are given below for one
example of each reaction type; the syntheses and characterization of analogues are reported in the
Supplementary Information. All materials were obtained from commercial supplier and used without further
purification unless otherwise noted.
4.1.1. General procedure for the preparation of 3 or 4-alkoxyphthalic anhydride (7a–7d, 9a–9d)
Following a reported procedure [34], we added H2SO4 (0.054 mL, 1.02 mmol) to a stirred solution of 3- or 4-
hydroxyphthalic acid (1.09 g, 6.00 mmol) in 12 mL of MeOH and the reaction was stirred at reflux overnight.
Solvent was removed under reduced pressure and the solid residue obtained was dissolved in dichloromethane
(40 mL) and washed with water (20 × 3 mL). The combined organic layer was dried over anhydr MgSO4,
filtered and concentrated under reduced pressure to provide crude dimethyl 4-hydroxyphthalate. The crude
dimethyl 3- or 4-hydroxyphthalate was dissolved in 3 mL of acetone and K2CO3 (1.3 g, 9.5 mmol) was added to
the solution. The mixture was stirred at room temperature for 1 h. Alkyl iodide (5.7 mmol) was added, and the
mixture was stirred at reflux overnight. K2CO3 was removed by filtration and the solvent was removed under
reduced pressure to provide dimethyl 3- or 4-alkoxyphthalate.
A solution of dimethyl 3- or 4-alkoxyphthalate in 3 mL of acetone was treated with an aq solution of NaOH
(0.34 g, 8.4 mmol) in 2 mL water and then the resulting solution was stirred at room temperature overnight.
After evaporation of the solvent, the mixture was acidified with 6 M HCl to pH 2 and concentrated under
reduced pressure. Then, the 3- or 4-alkoxyphthalic acid was dissolved in 4 mL of Ac2O and the solution was
heated at reflux for 18 h. Upon cooling to room temperature, the resulting solution was concentrated by rotary
evaporation and then the residue was purified with flash column chromatography. The desired products 7a-7d

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and 9a–9d were obtained.
4.1.1.1. 4-Ethoxyphthalic anhydride (7a). Yield 0.83 g (72%). 1H NMR (DMSO-d6, 500 MHz) δ 7.96 (d, J = 8.4
Hz, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.45 (dd, J = 8.4, 2.3 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 1.37 (t, J = 7.0 Hz,
3H). 13C NMR (DMSO-d6, 125 MHz) δ 164.92, 163.1, 162.6, 134.0, 127.2, 123.1, 122.5, 109.6, 64.9, 14.3.
1
4.1.1.2. 4-Propoxyphthalic anhydride (7b). Yield 0.86 g (70%). H NMR (DMSO-d6, 500 MHz) δ 7.97 (d, J =
8.4 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.4, 2.3 Hz, 1H), 4.16 (t, J = 6.5 Hz, 2H), 1.81 – 1.72 (m,
2H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 165.1, 163.1, 162.7, 134.0, 127.2, 123.1,
122.5, 109.6, 70.5, 21.7, 10.2.
4.1.1.3. 4-Butoxyphthalic anhydride (7c). Yield 0.97 g (74%). 1H NMR (DMSO-d6, 500 MHz) δ 7.97 (d, J = 8.4
Hz, 1H), 7.56 (d, J = 2.3 Hz, 1H), 7.47 (dd, J = 8.5, 2.2 Hz, 1H), 4.20 (t, J = 6.5 Hz, 2H), 1.79 – 1.68 (m, 2H),
1.51 – 1.38 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 165.1, 163.1, 162.7, 134.0,
127.2, 123.2, 122.5, 109.6, 68.8, 30.3, 18.6, 13.6.
4.1.1.4. 4-Pentyloxyphthalic anhydride (7d). Yield 0.98 g (70%). 1H NMR (DMSO-d6, 500 MHz) δ 7.96 (d, J =
8.4 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 1.82 – 1.69 (m,
2H), 1.47 – 1.28 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H).
1
4.1.1.5. 3-Ethoxyphthalic anhydride (9a). Yield 0.61 g (53%). H NMR (DMSO-d6, 500 MHz) δ 7.92 (dd, J =
8.5, 7.4 Hz, 1H), 7.58 (dd, J = 16.4, 7.9 Hz, 2H), 4.31 (q, J = 7.0 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H). 13C NMR
(DMSO-d6, 125 MHz) δ 163.2, 160.6, 156.9, 138.7, 133.0, 120.2, 116.8, 116.4, 65.0, 14.3.
4.1.1.6. 3-Propoxyphthalic anhydride (9b). Yield 0.58 g (47%). 1H NMR (DMSO-d6, 500 MHz) δ 7.92 (dd, J =
8.4, 7.4 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 1.85 – 1.75 (m, 2H),
1.02 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 163.2, 160.6, 157.1, 138.7, 133.0, 120.3, 116.8,
116.4, 70.5, 21.7, 10.1.
1
4.1.1.7. 3-Butoxyphthalic anhydride (9c). Yield 0.59 g (45%). H NMR (DMSO-d6, 500 MHz) δ 7.91 (dd, J =
8.4, 7.4 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 7.3 Hz, 1H), 4.24 (t, J = 6.4 Hz, 2H), 1.82 – 1.71 (m, 2H),

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1.54 – 1.42 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 163.2, 160.5, 157.1, 138.7,
133.0, 120.2, 116.8, 116.4, 68.8, 30.3, 18.5, 13.6.
1
4.1.1.8. 3-Pentyloxyphthalic anhydride (9d). Yield 0.64 g (46%). H NMR (DMSO-d6, 500 MHz) δ 7.91 (dd, J
= 8.4, 7.4 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 4.23 (t, J = 6.5 Hz, 2H), 1.79 (dd, J = 14.1,
7.3 Hz, 2H), 1.52 – 1.30 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 163.2, 160.5,
157.1, 138.7, 133.0, 120.2, 116.8, 116.4, 69.1, 28.0, 27.4, 21.8, 13.9.
4.1.2. General procedure for the preparation of bicyclic brominated furanone derivatives (10– 18d)
Triphenylphosphine (1.60 g, 6.00 mmol) was dissolved in 6 mL of THF and the solution was cooled to 0 °C
under N2. A solution of CBr4 (1.00 g, 3.00 mmol) in THF (1.00 mL per mmol PPh3) was added to the THF (6
mL) solution of PPh3 and the reaction was stirred until the color changed to yellow. Triethylamine (TEA) (1.1
mL, 6.0 mmol) was added dropwise to the mixture over 5 min, after which a solution of an anhydride compound
(1–9d, 1.00 mmol) in THF (1.0 mL per mmol phthalic anhydride) was incrementally added. The reaction was
stirred for 1 h at 0 °C, after which it was allowed to warm to room temperature and stirred overnight. The
reaction was quenched by addition of sat aq NH4Cl solution (30 mL). The phases were then separated, and the
crude product was extracted with hexane from the aqueous layer (30 × 3 mL). The combined organic layers
were concentrated under reduced pressure. The residue was dissolved in Et2O (25 mL) and filtered over a Celite
pad (7.0 × 3.0 cm). The resulting filtrate was concentrated by rotary evaporation and the crude products were
purified through the use of silica gel column chromatography (5–15% EtOAc in hexane). The desired products
were obtained.
4.1.2.1. 3-(Dibromomethylene)isobenzofuran-1(3H)-one (10). White solid, yield 0.091 g (30%). m.p. 136 –
138 °C; 1H NMR (DMSO-d6, 500 MHz) δ 8.32 (d, J = 8.0 Hz, 1H, H-4), 7.95 (d, J = 7.6 Hz, 1H, H-7), 7.90 (t, J
= 7.7 Hz, 1H, H-5), 7.75 (t, J = 7.5 Hz, 1H, H-6); 13C NMR (DMSO-d6,125 MHz) δ 164.0 (C-1), 145.5 (C-3),
135.8 (C-3a), 135.6 (C-5), 131.4 (C-6), 126.0 (C-7), 125.3 (C-7a), 123.7 (C-4), 77.3 (=CBr2). Anal. Calcd. for
C9H4Br2O2: C, 35.57; H, 1.33. Found: C, 35.62; H, 1.34. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C9H4Br2NaO2+: 324.8470; found 324.8471. HPLC purity: 99.9%.

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1
4.1.2.2. 3-(Dibromomethylene)hexahydroisobenzofuran-1(3H)-one (11). Colorless oil, yield 0.12 g (40%). H
NMR (CDCl3, 500 MHz) δ 3.31 – 3.19 (m, 1H), 2.98 (t, J = 6.8 Hz, 1H), 2.22 (t, J = 14.3 Hz, 2H), 1.72 (d, J =
15.0 Hz, 2H), 1.35 – 1.05 (m, 4H). 13C NMR (CDCl3, 125 MHz) δ 174.1, 158.2, 68.9, 40.7, 40.0, 26.5, 23.0,
22.3. Anal. Calcd. for C9H10Br2O2: C, 34.87; H, 3.25. Found: C, 34.89; H, 3.24. HRMS (ESI) m/z: Anal. calcd.
for [M+Na]+ C9H10Br2NaO2+: 330.8940; found 330.8942. HPLC purity: 97.8%.
4.1.2.3. 3-(Dibromomethylene)-4,5,6,7-tetrahydroisobenzofuran-1(3H)-one (12). White solid, yield 0.20 g
(65%). m.p. 128 – 130 °C;1H NMR (DMSO-d6, 500 MHz) δ 2.74 – 2.69 (m, 2H), 2.16 – 2.11 (m, 2H), 1.72 –
1.68 (m, 2H), 1.63 – 1.59 (m, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 166.5, 149.7, 148.6, 131.2, 78.4, 25.0,
21.3, 20.5, 20.1. Anal. Calcd. for C9H8Br2O2: C, 35.10; H, 2.62. Found: C, 35.19; H, 2.60. HRMS (ESI) m/z:
Anal. calcd. for [M+Na]+ C9H8Br2NaO2+: 328.8783; found 328.8786. HPLC purity: 99.0%.
4.1.2.4. 3-(Dibromomethylene)hexahydro-1H-cyclopenta[c]furan-1-one (13). Yellowish oil, yield 0.071 g (24%).
1H NMR (DMSO-d6, 500 MHz) δ 3.50 – 3.48 (m, 2H), 2.04 – 1.82 (m, 4H), 1.73 – 1.66 (m, 1H), 1.39 – 1.30 (m,
1H). 13C NMR (DMSO-d6, 125 MHz) δ 176.5, 155.4, 68.0, 45.8, 43.8, 32.2, 30.8, 25.6. Anal. Calcd. for
C8H8Br2O2: C, 32.47; H, 2.72. Found: C, 32.50; H, 2.80. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C8H8Br2NaO2+: 316.8783; found 316.8780. HPLC purity: 99.5%.
4.1.2.5. 3-(Dibromomethylene)-3,4,5,6-tetrahydro-1H-cyclopenta[c]furan-1-one (14). Brown solid, yield 0.080
1
g (27%). m.p. 120 – 122 °C; H NMR (DMSO-d6, 500 MHz) δ 2.96 – 2.91 (m, 2H), 2.48 – 2.39 (m, 4H). 13C
NMR (DMSO-d6, 125 MHz) δ 162.5, 162.2, 146.5, 140.9, 79.4, 29.5, 28.1, 25.1. Anal. Calcd. for C8H6Br2O2: C,
32.69; H, 2.06. Found: C, 32.71; H, 2.06. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C8H6Br2NaO2+:
314.8627; found 314.8625. HPLC purity: 97.3%.
4.1.2.6. 3-(Dibromomethylene)-5-methylisobenzofuran-1(3H)-one (15). White solid, yield 0.076 g (24%). m.p.
1
128 – 130 °C; H NMR (DMSO-d6, 500 MHz) δ 8.18 (s, 1H, H-4), 7.87 (d, J = 7.9 Hz, 1H, H-7), 7.60 (d, J =
7.8 Hz, 1H, H-6), 2.52 (s, 3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 164.0 (C-1), 146.9 (C-5), 145.5 (C-3),
136.3 (C-3a), 132.6 (C-6), 126.0 (C-7), 124.0 (C-4), 122.9 (C-7a), 77.0 (=CBr2), 22.0 (-CH3). Anal. Calcd. for
C10H6Br2O2: C, 37.77; H, 1.90. Found: C, 37.78; H, 2.03. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C10H6Br2NaO2+: 338.8627; found 338.8629. HPLC purity: 98.9%.

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4.1.2.7. 3-(Dibromomethylene)-5-ethoxyisobenzofuran-1(3H)-one (16a). White solid, yield 0.70 g (20%). m.p.
1
156 – 158 °C; H NMR (DMSO-d6, 500 MHz) δ 7.90 (d, J = 8.5 Hz, 1H, H-7), 7.76 (s, 1H, H-4), 7.31 (d, J =
7.0 Hz, 1H, H-6), 4.20 (q, J = 6.8 Hz, 2H, -OCH2), 1.39 (t, J = 6.9 Hz, 3H, -CH3). 13C NMR (DMSO-d6, 125
MHz) δ 164.3 (C-5), 163.6 (C-1), 145.2 (C-3), 138.2 (C-3a), 128.0 (C-7), 118.4 (C-6), 117.3 (C-7a), 108.7 (C-4),
77.4 (=CBr2), 64.6 (-OCH2), 14.3 (-CH3). Anal. Calcd. for C11H8Br2O3: C, 37.97; H, 2.32. Found: C, 37.97; H,
2.39. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C11H8Br2NaO3+: 368.8732; found 368.8731. HPLC purity: >
99.9%.
4.1.2.8. 3-(Dibromomethylene)-5-propoxyisobenzofuran-1(3H)-one (16b). White solid, yield 0.69 g (19%). m.p.
1
121 – 123 °C; H NMR (DMSO-d6, 500 MHz) δ 7.89 (d, J = 8.5 Hz, 1H, H-7), 7.75 (s, 1H, H-4), 7.31 (d, J =
8.5 Hz, 1H, H-6), 4.09 (t, J = 6.4 Hz, 2H, -OCH2), 1.84 – 1.75 (m, 2H, -CH2), 1.00 (t, J = 7.4 Hz, 3H, -CH3). 13C
NMR (DMSO-d6, 125 MHz) δ 164.4 (C-5), 163.5 (C-1), 145.1 (C-3), 138.1 (C-3a), 127.9 (C-7), 118.5 (C-6),
117.3 (C-7a), 108.7 (C-4), 77.4 (=CBr2), 70.2 (-OCH2), 21.8(-CH2), 10.3(-CH3). Anal. Calcd. for C12H10Br2O3:
C, 39.81; H, 2.78. Found: C, 39.85; H, 2.88. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C12H10Br2NaO3+:
382.8889; found 382.8887. HPLC purity: 97.6%.
4.1.2.9. 3-(Dibromomethylene)-5-butoxyisobenzofuran-1(3H)-one (16c). White solid, yield 0.79 g (21%). m.p.
1
127 – 129 °C; H NMR (DMSO-d6, 500 MHz) δ 7.90 (d, J = 8.5 Hz, 1H, H-7), 7.76 (s, 1H, H-4), 7.31 (d, J =
8.5 Hz, 1H, H-6), 4.14 (t, J = 6.4 Hz, 2H, -OCH2), 1.80 – 1.68 (m, 2H, -CH2), 1.50 – 1.39 (m, 2H, -CH2), 0.94 (t,
J = 7.4 Hz, 3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 164.4 (C-5), 163.5 (C-1), 145.2 (C-3), 138.2 (C-3a),
128.0 (C-7), 118.4 (C-6), 117.3 (C-7a), 108.7 (C-4), 77.4 (=CBr2), 68.4 (-OCH2), 39.5 (-CH2), 30.4 (-CH2), 18.6
(-CH2), 13.6 (-CH3). Anal. Calcd. for C13H12Br2O3: C, 41.52; H, 3.22. Found: C, 41.62; H, 3.32. HRMS (ESI)
m/z: Anal. calcd. for [M+Na]+ C13H12Br2NaO3+: 396.9045; found 396.9047. HPLC purity: 98.6%.
4.1.2.10. 3-(Dibromomethylene)-5-(pentyloxy)isobenzofuran-1(3H)-one (16d). White solid, yield 0.74 g (19%).
m.p. 100 – 102 °C; 1H NMR (DMSO-d6, 500 MHz) δ 7.88 (d, J = 8.5 Hz, 1H, H-7), 7.74 (s, 1H, H-4), 7.30 (d, J
= 8.6 Hz, 1H, H-6), 4.12 (t, J = 6.4 Hz, 2H, -OCH2), 1.81 – 1.72 (m, 2H, -CH2), 1.46 – 1.29 (m, 4H, -CH2), 0.90
(t, J = 7.1 Hz, 3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 164.4 (C-5), 163.5 (C-1), 145.1 (C-3), 138.1 (C-3a),
127.9 (C-7), 118.4 (C-6), 117.3 (C-7a), 108.7 (C-4), 77.4 (=CBr2), 68.7 (-CH2), 28.1 (-CH2), 27.5 (-CH2), 21.8 (-

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CH2), 13.9(-CH3). Anal. Calcd. for C14H14Br2O3: C, 43.11; H, 3.62. Found: C, 43.18; H, 3.70. HRMS (ESI) m/z:
Anal. calcd. for [M+Na]+ C14H14Br2NaO3+: 410.9202; found 410.9203. HPLC purity: 99.2%.
4.1.2.11. 3-(Dibromomethylene)-7-methylisobenzofuran-1(3H)-one (17). White solid, yield 0.83 g (26%). m.p.
1
138 – 140 °C; H NMR (DMSO-d6, 500 MHz) δ 8.18 (d, J = 7.9 Hz, 1H, H-4), 7.77 (t, J = 7.7 Hz, 1H, H-5),
7.55 (d, J = 7.5 Hz, 1H, H-6), 2.61 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 164.0 (C-1), 145.2 (C-3), 139.8
(C-7), 136.1 (C-3a), 135.2 (C-5), 132.9 (C-6), 122.8 (C-7a), 121.3 (C-4), 76.5 (=CBr2), 16.9 (-CH3). Anal. Calcd.
for C10H6Br2O2: C, 37.77; H, 1.90. Found: C, 37.78; H, 1.86. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C10H6Br2NaO2+: 338.8627; found 338.8628. HPLC purity: 99.8%.
4.1.2.12. 3-(Dibromomethylene)-7-ethoxyisobenzofuran-1(3H)-one (18a). White solid, yield 0.66 g (19%). m.p.
1
141 – 143 °C; H NMR (DMSO-d6, 500 MHz) δ 7.87 (d, J = 7.8 Hz, 1H, H-4), 7.82 (t, J = 8.0 Hz, 1H, H-5),
7.32 (d, J = 8.2 Hz, 1H, H-6), 4.24 (q, J = 6.9 Hz, 2H, -OCH2), 1.38 (t, J = 7.0 Hz, 3H, -CH3). 13C NMR
(DMSO-d6, 125 MHz) δ 161.4 (C-1), 157.8 (C-7), 145.1 (C-3), 137.8 (C-5), 137.7 (C-3a), 115.3 (C-4), 114.7
(C-6), 111.8 (C-7a), 76.7 (=CBr2), 64.6 (-OCH2), 14.3 (-CH3). Anal. Calcd. for C11H8Br2O3: C, 37.97; H, 2.32.
Found: C, 37.91; H, 2.42. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C11H8Br2NaO3+: 368.8732; found
368.8733. HPLC purity: 99.8%.
4.1.2.13. 3-(Dibromomethylene)-7-propoxyisobenzofuran-1(3H)-one (18b). White solid, yield 0.65 g (18%). m.p.
1
101 – 103 °C; H NMR (DMSO-d6, 500 MHz) δ 7.87 (d, J = 7.8 Hz, 1H, H-4), 7.82 (t, J = 8.0 Hz, 1H, H-5),
7.32 (d, J = 8.3 Hz, 1H, H-6), 4.13 (t, J = 6.4 Hz, 2H, -OCH2), 1.85 – 1.73 (m, 2H, -CH2), 1.01 (t, J = 7.4 Hz,
3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 161.4 (C-1), 157.9 (C-7), 145.1 (C-3), 137.8 (C-5), 137.7 (C-3a),
115.4 (C-4), 114.8 (C-6), 111.9 (C-7a), 76.7 (=CBr2), 70.1 (-OCH2), 21.8 (-CH2), 10.2 (-CH3). Anal. Calcd. for
C12H10Br2O3: C, 39.81; H, 2.78. Found: C, 39.79; H, 2.78. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C12H10Br2NaO3+: 382.8889; found 382.8891. HPLC purity: 96.2%.
4.1.2.14. 3-(Dibromomethylene)- 7-butoxyisobenzofuran-1(3H)-one (18c). White solid, yield 0.68 g (18%). m.p.
86 – 88 °C; 1H NMR (DMSO-d6, 500 MHz) δ 7.88 (d, J = 7.8 Hz, 1H, H-4), 7.82 (t, J = 8.0 Hz, 1H, H-5), 7.33
(d, J = 8.3 Hz, 1H, H-6), 4.18 (t, J = 6.4 Hz, 2H, -OCH2), 1.80 – 1.66 (m, 2H, -CH2), 1.53 – 1.37 (m, 2H, -CH2),
0.94 (t, J = 7.4 Hz, 3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 161.4 (C-1), 157.9 (C-7), 145.1 (C-3), 137.9

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(C-5), 137.7 (C-3a), 115.4 (C-4), 114.8 (C-6), 111.9 (C-7a), 76.7 (=CBr2), 68.5 (-CH2), 30.4 (-CH2), 18.6 (-CH2),
13.7 (-CH3). Anal. Calcd. for C13H12Br2O3: C, 41.52; H, 3.22. Found: C, 41.57; H, 3.23. HRMS (ESI) m/z: Anal.
calcd. for [M+Na]+ C13H12Br2NaO3+: 396.9045; found 396.9047. HPLC purity: 97.7%.
4.1.2.15. 3-(Dibromomethylene)-7-(pentyloxy)isobenzofuran-1(3H)-one (18d). White solid, yield 0.62 g (16%).
m.p. 72 – 74 °C; 1H NMR (DMSO-d6, 500 MHz) δ 7.89 (d, J = 7.8 Hz, 1H, H-4), 7.83 (t, J = 8.0 Hz, 1H, H-5),
7.34 (d, J = 8.3 Hz, 1H, H-6), 4.18 (t, J = 6.5 Hz, 2H, -OCH2), 1.84 – 1.70 (m, 2H, -CH2), 1.50 – 1.28 (m, 4H, -
CH2CH2), 0.89 (t, J = 7.2 Hz, 3H, -CH3). 13C NMR (DMSO-d6, 125 MHz) δ 161.4 (C-1), 157.9 (C-7), 145.1 (C-
3), 137.9 (C-5), 137.7 (C-3a), 115.4 (C-4), 114.8 (C-6), 111.9 (C-7a), 76.6 (=CBr2), 68.8 (-CH2), 28.0 (-CH2),
27.5 (-CH2), 21.8 (-CH2), 13.9 (-CH3). Anal. Calcd. for C14H14Br2O3: C, 43.11; H, 3.62. Found: C, 43.14; H,
3.62. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C14H14Br2NaO3+: 410.9202; found 410.9201. HPLC purity:
99.2%.
4.2. Biological Assay
4.2.1 Bacteria culture
AI-2 reporter strain, Vibrio harveyi BB170 (ATCC BAA-1117) was cultured aerobically at 30 °C in
autoinducer bioassay (AB) medium consisting of 0.30 M sodium chloride, 0.050 M magnesium sulfate, 0.2%
casamino acids, 10 mM potassium phosphate (pH 7.0), 1.0 mM L-arginine and 2% glycerol with shaking. F.
nucleatum (ATCC 25586) and P. gingivalis (ATCC 33277) were cultured anaerobically (10% H2, 10% CO2, 80%
N2) in brain heart infusion broth supplemented with vitamin K (0.2 mg/mL) and hemin (10 mg/mL) at 37 °C. T.
forsythia (ATCC 43037) was cultured anaerobically in new oral spirochete (NOS) broth (ATCC medium 1494)
supplemented with N-acetylmuramic acid (0.01 µg/mL) and vitamin K (0.2 mg/mL) at 37 °C.
4.2.2 Purification of AI-2
AI-2 of F. nucleatum was partially purified as described previously [20]. In brief, F. nucleatum which was
cultured overnight was diluted 1:20 with fresh culture medium. After incubating the culture mixture until the
late logarithmic phase (OD660 nm = 0.7), the culture supernatants were collected by centrifugation at 10,000× g
at 4 °C. The culture supernatants were passed through 0.2 µm pore-size membrane filters (Sartorius Stedium

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Biotech, Goettingen, Germany) and the filtrates were subsequently passed through a Centricon YM-3 3-kDa
exclusion filter (Millipore, Bedford, MA). Then the filtrates were chromatographed on a C18 Sep-Pak reverse-
phase column (Waters Co., Milford, MA) according to the manufacturer’s instructions.
4.2.3. AI-2 activity test
As QS molecules stimulates the luciferase operon (lux gene) to express luciferase of V. harveyi BB170,
bioluminescence of V. harveyi BB170 was determined as AI-2 activity. V. harveyi BB170 was diluted to a
concentration of 1 × 106 bacteria/ml in fresh AB medium. Then the bacterial suspension was mixed with 10%
(vol/vol) of partially purified AI-2 of F. nucleatum in the presence or absence of the furanone compounds (the
reference compound R1 and new synthesized furanone analogs) of various concentrations, and incubated for 1-6
h at 30 °C under aerobic condition with shaking. The reference compound R1 was (5Z)-4-bromo-5-
(bromomethylene)-2(5H)-furanone. The bioluminescence was measured using a luminometer (GloMax-Multi
detection system, Promega, Madison, WI, USA) and the value was converted into a percentage out of untreated
control value.
4.2.4. Biofilm formation assay
Biofilm formation assay was performed by crystal violet staining and confocal laser scanning microscopy.
Bacterial suspensions were added to 24-well plates with round glass slips (12 mm radius) in the presence of F.
nucleatum AI-2 (10% vol/vol) and the furanone compounds (the reference furanone compound and new
synthesized furanone analogs)at various concentrations. The initial number of F. nucleatum, P. gingivalis, and T.
forsythia was 2 × 107 bacteria/mL, 2 × 108 bacteria/mL, and 2 × 108 bacteria/mL, respectively and incubated
for 48 h under anaerobic condition (10% H2, 10% CO2, 80% N2). After 48 h, biofilms formed on the glass slips
were washed with phosphate buffered saline three times and stained with 1% crystal violet for 10 min and
destained with 1 mL of acetone–alcohol (20:80, vol/vol). The absorbance at 595 nm of the destaining solution
containing crystal violet was measured using a microplate reader (Wallac Victor3 microtiter, PerkinElmer Life
Sciences, Waltham, MA).
Biofilm which was formed on the glass slips was stained by the Live/Dead-BacLight bacterial viability kit
(Invitrogen, Grand Island, NY) and then observed using a confocal scanning laser microscope (Carl Zeiss LSM
700, Germany) at a magnification of 1000× and quantified by measuring fluorescence intensity and average
thickness of the biofilm using Carl Zeiss LSM 700 program. Biomass was determined by dividing total intensity

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by the area where the fluorescence was obtained.
4.2.5. Planktonic bacterial growth test
F. nucleatum, P. gingivalis, and T. forsythia were grown anaerobically at 37 °C in the presence or absence of
the furanone compounds (the reference furanone compound and new synthesized furanone analogs) without
addition of F. nucleatum AI-2. V. harveyi BB170 was incubated aerobically at 30 °C in the presence or absence
of the QSIs without addition of AI-2. Bacterial growth was monitored by measuring absorbance at 600 nm using
a spectrophotometer
4.2.6. Cytotoxicity test
Human monocytic cell line (THP-1), human gingival fibroblasts (HGFs) and Human oral keratinocyte cell
line (HOK-16B) were used to evaluate the cytotoxicity of the compounds on host cells. THP-1 cells were
culured in RPMI 1640 media (Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum
(Hyclone, Waltham, MA, USA), 2.05 mM L-glutamate and antibiotics (100 units/mL penicillin, 100 ㎍/mL
streptomycin). HGFs were cultured in DMEM media (Hyclone, Waltham, MA, USA) supplemented with 4.0
mM L-glutamine, 4500 mg/L Glucose, sodium pyruvate, antibiotics (100 units/mL penicillin, 100 ㎍/mL
streptomycin) and 10% fetal bovine serum. HOK-16B cells were cultured in Keratinocyte Basal Medium (Lonza,
Walkersville, MD, USA) supplemented with insulin, epidermal growth factor, bovine pituitary extract,
hydrocortisone and gentamicin sulfate amphotericin. Cultured THP-1 cells (1 × 105 cells/well) were seeded in 96
well microtiter plate and incubated in the presence of the compounds for 24 h. HGFs (2 × 104 cells /well) and
HOK-16B (5 × 104 cells/well) were seeded in 96 well plates and grown until the confluence of 85%. The cells
were then treated with the compounds and incubated for 24 h. The cell viability was evaluated using the Cell
Counting Kit-8 (CCK-8, DOJINDO, Kumamoto, Japan) according to the manufacturer's protocol.
4.2.7. Evaluation the effect on host immune response
Effects of the compounds on host immune response was evaluated using real-time PCR. THP-1 cells (1 × 106
cells/well) and HGFs (2 × 105 cells/well) in 6 well plates were incubated at 37 °C for 24 h in the presence or
absence of the compounds or lipopolysaccharides (LPS, 1 µg/ml) known as endotoxin of gram-negative bacteria
that elicits strong immune responses in host cells. After 24 h incubation, RNA from THP-1 cell and HGFs cells

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was extracted using Easy-Blue total RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea) according
to the manufacturer's protocol. cDNA samples (2 µL) synthesized from the extracted RNA (1 µg) using a M-
MLV Reverse Transcription kit (Promega, Madison, WI) were mixed with each primer pairs (10 pM) and Power
SYBR Green Master Mix (Applied Biosystems, Warrington, UK) in a 20 µl reaction volume. The mixtures were
subjected to ABI PRISM 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using
the following thermocycling: amplification for 40 cycles composed of a denaturation step at 95 °C for 15 sec, an
annealing and extension step at 60 °C for 1 min. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene,
the housekeeping gene, was used as a reference gene for normalization of gene expression level of inflammatory
cytokines including interleukin 6 (IL-6) and interleukin 8 (IL-8). The sequences of the primers used in this study
were as follows: 5´-GTG GCC AGC CGA GCC-3´ and 5´-TGA AGG GGT TGA TGG CA-3´ for GAPDH; 5´-
GAT TCA ATG AGG AGA CTT GCC TGG-3´, 5´-GCA GAA CTG GAT CAG GAC TTT-3´ for IL-6 ; 5´-CTG
TGT GAA GGT GCA GTT TTG C-3´ and 5´- AAC TTC TCC ACAACC CTC TGC-3´ for IL-8.
4.2.8. Statistical analysis
Statistical analysis was performed using Student’s t test. Statistically significant differences between the
control and AI-2 or between AI-2 and furanone compound-treated groups were analyzed. A p value of <0.05
was considered statistically significant.
5. Appendix A. Supplementary data.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.
6. Acknowledgments
This study was supported by a grant (HI12C0675) from the Korea Healthcare Technology R&D, Ministry of
Health and Welfare, Republic of Korea.
7. References
[1] P. Albuquerque, A. Casadevall, Quorum sensing in fungi–a review, Med. Mycol., 50 (2012) 337-345.

ACCEPTED MANUSCRIPT
[2] W.C. Fuqua, S.C. Winans, E.P. Greenberg, Quorum sensing in bacteria: the LuxR-LuxI family of cell
density-responsive transcriptional regulators, J. Bacteriol., 176 (1994) 269.
[3] M.E. Taga, B.L. Bassler, Chemical communication among bacteria, Proc. Natl. Acad. Sci., 100 (2003)
14549-14554.
[4] W.R. Galloway, J.T. Hodgkinson, S.D. Bowden, M. Welch, D.R. Spring, Quorum sensing in Gram-negative
bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways, Chem. Rev., 111 (2010) 28-67.
[5] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections,
Science, 284 (1999) 1318-1322.
[6] A.K. Bhardwaj, K. Vinothkumar, N. Rajpara, Bacterial quorum sensing inhibitors: attractive alternatives for
control of infectious pathogens showing multiple drug resistance, Recent Pat Antiinfect Drug Discov, 8 (2013)
68-83.
[7] M.J. Federle, Autoinducer-2-based chemical communication in bacteria: complexities of interspecies
signaling in: Bacterial Sensing and Signaling, Karger Publishers, 16 (2009) 18-32.
[8] A.H. Rickard, R.J. Palmer, D.S. Blehert, S.R. Campagna, M.F. Semmelhack, P.G. Egland, B.L. Bassler, P.E.
Kolenbrander, Autoinducer 2: a concentration dependent signal for mutualistic bacterial biofilm growth, Mol.
Microbiol., 60 (2006) 1446-1456.
[9] S. Schauder, K. Shokat, M.G. Surette, B.L. Bassler, The LuxS family of bacterial autoinducers: biosynthesis
of a novel quorum sensing signal molecule, Mol. Microbiol., 41 (2001) 463-476.
[10] A. Jayaraman, T.K. Wood, Bacterial quorum sensing: signals, circuits, and implications for biofilms and
disease, Annu Rev Biomed Eng., 10 (2008) 145-167.
[11] B.J. Paster, I. Olsen, J.A. Aas, F.E. Dewhirst, The breadth of bacterial diversity in the human periodontal
pocket and other oral sites, Periodontol. 2000, 42 (2006) 80-87.
[12] P.E. Kolenbrander, R.N. Andersen, D.S. Blehert, P.G. Egland, J.S. Foster, R.J. Palmer, Communication
among oral bacteria, Microbiol. Mol. Biol. Rev., 66 (2002) 486-505.
[13] P.D. Marsh, M.V. Martin, M.A. Lewis, D. Williams, Oral Microbiology, Elsevier Health Sciences, 2009.
[14] J. Frias, E. Olle, M. Alsina, Periodontal pathogens produce quorum sensing signal molecules, Infect.
Immun., 69 (2001) 3431-3434.
[15] P.E. Kolenbrander, R.J. Palmer, S. Periasamy, N.S. Jakubovics, Oral multispecies biofilm development and
the key role of cell–cell distance, Nat. Rev. Microbiol., 8 (2010) 471-480.
[16] D.J. Bradshaw, P.D. Marsh, G.K. Watson, C. Allison, Role of Fusobacterium nucleatum and coaggregation

ACCEPTED MANUSCRIPT
in anaerobe survival in planktonic and biofilm oral microbial communities during aeration, Infect. Immun., 66
(1998) 4729-4732.
[17] P.E. Kolenbrander, R.J. Palmer, A.H. Rickard, N.S. Jakubovics, N.I. Chalmers, P.I. Diaz, Bacterial
interactions and successions during plaque development, Periodontol. 2000, 42 (2006) 47-79.
[18] K. Hojo, S. Nagaoka, T. Ohshima, N. Maeda, Bacterial interactions in dental biofilm development, J. Dent.
Res., 88 (2009) 982-990.
[19] T. Okuda, E. Kokubu, T. Kawana, A. Saito, K. Okuda, K. Ishihara, Synergy in biofilm formation between
Fusobacterium nucleatum and Prevotella species, Anaerobe, 18 (2012) 110-116.
[20] Y.-J. Jang, Y.-J. Choi, S.-H. Lee, H.-K. Jun, B.-K. Choi, Autoinducer 2 of Fusobacterium nucleatum as a
target molecule to inhibit biofilm formation of periodontopathogens, Arch. Oral Biol., 58 (2013) 17-27.
[21] D.G. Cvitkovitch, Y.-H. Li, R.P. Ellen, Quorum sensing and biofilm formation in Streptococcal infections, J.
Clin. Invest., 112 (2003) 1626-1632.
[22] T.B. Rasmussen, M. Manefield, J.B. Andersen, L. Eberl, U. Anthoni, C. Christophersen, P. Steinberg, S.
Kjelleberg, M. Givskov, How Delisea pulchra furanones affect quorum sensing and swarming motility in
Serratia liquefaciens MG1, Microbiology, 146 (2000) 3237-3244.
[23] M. Hentzer, M. Givskov, Pharmacological inhibition of quorum sensing for the treatment of chronic
bacterial infections, J. Clin. Invest., 112 (2003) 1300-1307.
[24] J. Pan, D. Ren, Quorum sensing inhibitors: a patent overview, Expert Opin Ther Pat, 19 (2009) 1581-1601.
[25] M. Hentzer, H. Wu, J.B. Andersen, K. Riedel, T.B. Rasmussen, N. Bagge, N. Kumar, M.A. Schembri, Z.
Song, P. Kristoffersen, Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors, EMBO
J., 22 (2003) 3803-3815.
[26] J. Lönn Stensrud, F. Petersen, T. Benneche, A.A. Scheie, Synthetic bromated furanone inhibits
autoinducer 2 mediated communication and biofilm formation in oral streptococci, Oral Microbiol. Immunol.,
22 (2007) 340-346.
[27] H. Wu, Z. Song, M. Hentzer, J.B. Andersen, S. Molin, M. Givskov, N. Høiby, Synthetic furanones inhibit
quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice, J.
Antimicrob. Chemother., 53 (2004) 1054-1061.
[28] S. Yang, O.A. Abdel-Razek, F. Cheng, D. Bandyopadhyay, G.S. Shetye, G. Wang, Y.-Y. Luk, Bicyclic
brominated furanones: a new class of quorum sensing modulators that inhibit bacterial biofilm formation,
Bioorg. Med. Chem., 22 (2014) 1313-1317.

ACCEPTED MANUSCRIPT
[29] T. Zang, B.W. Lee, L.M. Cannon, K.A. Ritter, S. Dai, D. Ren, T.K. Wood, Z.S. Zhou, A naturally occurring
brominated furanone covalently modifies and inactivates LuxS, Bioorg. Med. Chem. Lett., 19 (2009) 6200-6204.
[30] J.C. Janssens, H. Steenackers, S. Robijns, E. Gellens, J. Levin, H. Zhao, K. Hermans, D. De Coster, T.L.
Verhoeven, K. Marchal, Brominated furanones inhibit biofilm formation by Salmonella enterica serovar
Typhimurium, Appl. Environ. Microbiol., 74 (2008) 6639-6648.
[31] H.P. Steenackers, J. Levin, J.C. Janssens, A. De Weerdt, J. Balzarini, J. Vanderleyden, D.E. De Vos, S.C. De
Keersmaecker, Structure–activity relationship of brominated 3-alkyl-5-methylene-2 (5H)-furanones and
alkylmaleic anhydrides as inhibitors of Salmonella biofilm formation and quorum sensing regulated
bioluminescence in Vibrio harveyi, Bioorg. Med. Chem., 18 (2010) 5224-5233.
[32] S.G. Newman, V. Aureggi, C.S. Bryan, M. Lautens, Intramolecular cross-coupling of gem-dibromoolefins:
a mild approach to 2-bromo benzofused heterocycles, Chem. Commun., (2009) 5236-5238.
[33] L. Eberson, L. Landström, Studies on cyclic anhydrides IV. Rate constants for the hydrolysis of some
cyclic anhydrides exhibiting ring strain, Acta Chem. Scand., 26 (1972) 239.
[34] F. De Nanteuil, E. Serrano, D. Perrotta, J. Waser, Dynamic Kinetic Asymmetric [3+ 2] Annulation
Reactions of Aminocyclopropanes, J. Am. Chem. Soc., 136 (2014) 6239-6242.
[35] A. Allahdad, D.W. Knight, An Investigation of the Wittig Reaction between a Series of Mono-substituted
Phthalic Anhydrides and Ethoxycarbonylmethyl idenetri- phenylphosphorane, J. Chem. Soc. Perkin trans. I,
(1982) 1855.
[36] W.-L. Ng, B.L. Bassler, Bacterial quorum-sensing network architectures, Annu. Rev. Genet., 43 (2009) 197.
[37] D. Martinelli, G. Grossmann, U. Séquin, H. Brandl, R. Bachofen, Effects of natural and chemically
synthesized furanones on quorum sensing in Chromobacterium violaceum, BMC Microbiol., 4 (2004) 1.
[38] K.M. Smith, Y. Bu, H. Suga, Library screening for synthetic agonists and antagonists of a Pseudomonas
aeruginosa autoinducer, Chem. Biol., 10 (2003) 563-571.
[39] L. Yujie, X. Geng, Y.-c. Huang, Y. Li, K. Yang, L. Ye, X. Chen, G. Zhao, C. Yin, The effect of brominated
furanones on the formation of Staphylococcus aureus biofilm on PVC, Cell Biochem. Biophys., 67 (2013) 1501-
1505.
[40] M. Guo, S. Gamby, Y. Zheng, H.O. Sintim, Small molecule inhibitors of AI-2 signaling in bacteria: state-
of-the-art and future perspectives for anti-quorum sensing agents, Int J Mol Sci, 14 (2013) 17694-17728.
[41] H. Shao, D. James, R.J. Lamont, D.R. Demuth, Differential interaction of Aggregatibacter (Actinobacillus)

ACCEPTED MANUSCRIPT
actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2, J. Bacteriol ., 189 (2007) 5559-5565.
[42] D. James, H. Shao, R.J. Lamont, D.R. Demuth, The Actinobacillus actinomycetemcomitans ribose binding
protein RbsB interacts with cognate and heterologous autoinducer 2 signals, Infect. Immun., 74 (2006) 4021-
4029.
[43] P.-F. Liu, W. Shi, W. Zhu, J.W. Smith, S.-L. Hsieh, R.L. Gallo, C.-M. Huang, Vaccination targeting surface
FomA of Fusobacterium nucleatum against bacterial co-aggregation: Implication for treatment of periodontal
infection and halitosis, Vaccine, 28 (2010) 3496-3505.
[44] R. Huang, M. Li, R.L. Gregory, Bacterial interactions in dental biofilm, Virulence, 2 (2011) 435-444.
Table 1. Synthesis of bicyclic brominated furanone derivatives.

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Table 2. Biomass and depth of periodontopathogen biofilms
*p < 0.05 compared with the untreated group (Control), and #p < 0.05 compared with the F. nucleatum AI-2-
treated value in the absence of furanone compounds.
Fig. 1. Effect of furanone compounds on AI-2 activity and V. harveyi growth. (A) AI-2 activity was
assessed by measuring bioluminescence using the AI-2 reporter strain V. harveyi BB170. V. harveyi BB170 (1 ×
106 bacteria/mL) was incubated for 6 h with 10% semi-purified F. nucleatum AI-2 in the absence or presence of
furanone compounds (13, 15, and 17) and the reference furanone compound (R1) at a concentration of 2 µM.
The bioluminescence of V. harveyi BB170 was measured using a luminometer and the value was converted to a
percentage of the control value. *p < 0.05 compared to the value of the F. nucleatum AI-2-treated group. (B) V.
harveyi BB170 was grown aerobically for 9 h in the absence of F. nucleatum AI-2 at 30°C in the presence of the
reference furanone compound (R1) or new furanone compounds (13, 15, and 17) at a concentration of 2 µM.
Planktonic bacterial growth was monitored by measuring absorbance at 600 nm using a spectrophotometer. *p <
0.05 compared to the untreated group (cont). The experiments were performed three times in triplicate and
representative data are shown.

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Fig. 2. Biofilm images showing the inhibitory effects of furanone compounds on the biofilm formation of
F. nucleatum, P. gingivalis, and T. forsythia. (A) F. nucleatum (2 × 107 bacteria/mL), (B) P. gingivalis (2 ×
108 bacteria/mL), and (C) T. forsythia (2 × 108 bacteria/mL) were cultured with semi-purified F. nucleatum AI-2
in the absence or presence of the reference furanone compound (R1) or new furanone compounds (13, 15, and
17) at 2 µM under an anaerobic condition at 37°C. After 48 h incubation, each biofilm formed on glass cover
slips was stained with the live/dead-BacLight bacterial viability kit followed by observation using a confocal

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laser-scanning microscope at 1000× magnification. The control group was the biofilm of each bacterium
cultured without F. nucleatum AI-2 and inhibitors.
Fig. 3. Inhibitory effects of furanone compounds on the biofilm formation of periodontopathogens at
various concentrations. (A) F. nucleatum (2 × 107 bacteria/mL), (B) P. gingivalis (2 × 108 bacteria/mL), and
(C) T. forsythia (2 × 108 bacteria/mL) were cultured with 10% semi-purified F. nucleatum AI-2 in the presence
of the reference furanone compound (R1) or new furanone compounds (13, 15, and 17) at various

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concentrations for 48 h. Biofilm formation was assessed by crystal violet staining. *p < 0.05 compared with
the non-treated value, and #p < 0.05 compared to the F. nucleatum AI-2-treated value in the absence of
furanone compounds. The experiments were performed three times in triplicate and representative data are
shown.
Fig. 4. Effects of furanone compounds on bacterial growth. (A) F. nucleatum, (B) P. gingivalis, and (C) T.
forsythia were grown for 48 h under anaerobic conditions at 37°C in the presence of the reference furanone
compound (R1) or new furanone compounds (13, 15, and 17) at a concentration of 2 µM. Bacterial growth was
monitored by measuring absorbance at 600 nm using a spectrophotometer.
Fig. 5. Effects of furanone compounds on host cell viability. (A) THP-1 cells (1 × 105 cells/well), (B) HGFs
(2 × 104 cells /well), and (C) HOK-16B cells (5 × 104 cells/well) cultured in 96-well plates were treated with the
reference furanone compound (R1) and new furanone compounds (13, 15, and 17) for 24 h, and the cell viability
was detected using Cell Counting Kit-8. The control was the non-treated group. The experiments were
performed three times in triplicate and representative data are shown.