New Pyrimidinone-Fused 1,4-Naphthoquinone Derivatives Inhibit the Growth of Drug Resistant Oral Bacteria

Background: Dental caries is considered to be a preventable disease, and various antimicrobial agents have been developed for the prevention of dental disease. However, many bacteria show resistance to existing agents. Methods/Principal Findings: In this study, four known 1,4-naphthoquinones and newly synthesized 10 pyrimidinone-fused 1,4-naphthoquinones, i.e. KHQ 701, 702, 711, 712, 713, 714, 715, 716, 717 and 718, were evaluated for antimicrobial activity against Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Actinomyces viscosus and Fusobacterium nucleatum. Pyrimidinone-fused 1,4-naphthoquinones were synthesized in good yields through a series of chemical reactions from a commercially available 1,4-dihydroxynaphthoic acid. MIC values of KHQ 711, 712, 713, 714, 715, 716, 717 and 718 were 6.25–50 μg/mL against E. faecalis (CCARM 5511), 6.25–25 μg/mL against E. faecium (KACC11954) and S. aureus (CCARM 3506), 1.56–25 μg/mL against S. epidermidis (KACC 13234), 3.125–100 μg/mL against S. mutans (KACC16833), 1.56–100 μg/mL against S. sobrinus (KCTC5809) and P. gingivalis (KCTC 5352), 3.125–50 μg/mL against A. viscosus (KCTC 9146) and 3.125–12.5 μg/mL against F. nucleatum (KCTC 2640) with a broth microdilution assay. A disk diffusion assay with KHQ derivatives also exhibited strong susceptibility with inhibition zones of 0.96 to 1.2 cm in size against P. gingivalis. Among the 10 compounds evaluated, KHQ 711, 712, 713, 715, 716 and 717 demonstrated strong antimicrobial activities against the 9 types of pathogenic oral bacteria. A pyrimidin-4-one moiety comprising a phenyl group at the C2 position and a benzyl group at the N3 position appears to be essential for physiological activity. Conclusion/Significance: Pyrimidinone-fused 1,4-naphthoquinones synthesized from simple starting compounds and four known 1,4-naphthoquinones were synthesized and showed strong antibacterial activity to the 9 common oral bacteria. These results suggest that these derivatives should be prospective for the treatment of dental diseases caused by oral bacteria, including drug-resistant strains.


Introduction
Two major dental diseases in the world are dental caries and periodontal disease, both of which are caused by various bacteria in the oral cavity [1]. Dental caries is a common oral disease that usually develops the formation of plaque biofilms on tooth surfaces, and the causative agents are Gram-positive bacteria such as Streptococcus mutans and Streptococcus sobrinus, as well as some

General Experimental Details for Synthesis of Compounds
All chemical reagents were purchased from Sigma-Aldrich, Tokyo Chemical Industry, Alfa Aesar and Acros Organics, and were used without further purification. All glassware was thoroughly dried in a convection oven. Reactions were monitored using thin-layer chromatography (TLC). Commercial TLC plates (silica gel 60 F 254 , Merck Co.) were developed and the spots were visualized under UV light at 254 or 365 nm. Silica gel column chromatography was performed with silica gel 60 (particle size 0.040−0.063 mm, Merck Co.). Extra pure-grade solvents for column chromatography were purchased through Samchun Chemicals and Duksan Chemicals. 1 H and 13 C NMR spectra were collected with a JEOL ECX-400 spectrometer (at 300 MHz for 1 H NMR and 75 MHz for 13 C NMR), and the chemical shifts were recorded with respect to tetramethylsilane, Si(CH 3 ) 4 , as an internal reference or were referenced to the residual proton peaks of the deuterated solvent. Mass spectrometry (MS) spectra were recorded using a Bruker compact ESI quadrupole-TOF ultra-high-resolution liquid chromatograph/mass spectrometer.
The minimum bactericidal concentration was obtained using a microdilution assay according to the standard with little modification. 5 After bacteria were cultured in the same manner as in the MIC test, bacterial cultures were inoculated onto the agar plates and incubated at 37 • C for 24-72 h, and the bacterial colonies were counted. The lowest concentration of compound that inhibited the growth of bacteria was considered to be the minimum bactericidal concentration. The experimental and control groups were assigned to the wells in triplicate.

Disk Diffusion Method
Broth inocula with 0.5 McFarland standard were spread onto the sterile plate by cotton swabs. P. gingivalis was cultured in tryptic soy broth supplemented with 10% defibrinated horse blood and brain heart infusion (BHI) at 37 • C for 24 h in anaerobic conditions (10% H 2 , 10% CO 2 , and balanced N 2 ). Then, 8-nm sterile paper discs were impregnated with KHQ 711-718 100 µg/mL was added, and the sizes of the inhibition zones were measured after 24 h [26,27].

Statistical Analysis
All experiments were performed at least three times, and data are represented as mean ± S.D.

Results
With the pharmacophore hybridization strategy already described, a series of novel pyrimidinone or pyrimidindione-fused 1,4-naphthoquinones were synthesized (Figures 1 and 2). the bacterial colonies were counted. The lowest concentration of compound that inhibited the growth of bacteria was considered to be the minimum bactericidal concentration. The experimental and control groups were assigned to the wells in triplicate.

Disk Diffusion Method
Broth inocula with 0.5 McFarland standard were spread onto the sterile plate by cotton swabs. P. gingivalis was cultured in tryptic soy broth supplemented with 10% defibrinated horse blood and brain heart infusion (BHI) at 37 °C for 24 h in anaerobic conditions (10% H2, 10% CO2, and balanced N2). Then, 8-nm sterile paper discs were impregnated with KHQ 711-718 100 μg/mL was added, and the sizes of the inhibition zones were measured after 24 h [26,27].

Statistical Analysis
All experiments were performed at least three times, and data are represented as mean ± S.D.

Broth Dilution Method
The
Interestingly, the positive control, oxytetracycline (0.8 cm), showed less antibacterial activity than the fused compounds, while KHQ 714 showed the strongest activity.

Time Kill Assay
To confirm the antibacterial effects of the fused compounds, we constructed growth curves, and the fused compounds exhibited concentration-dependent inhibitory activities against P. gingivalis. Interestingly, even concentrations between 1.56-12.5 µg/mL of fused compounds completely blocked bacterial growth ( Figure 3A-H)

Time Kill Assay
To confirm the antibacterial effects of the fused compounds, we constructed growth curves, and the fused compounds exhibited concentration-dependent inhibitory activities against P. gingivalis. Interestingly, even concentrations between 1.56-12.5 μg/mL of fused compounds completely blocked bacterial growth ( Figure 3A-H)

Discussion
Dental disease is one of the most prevalent public health concerns. The problems caused by dental caries affect all age groups, and treatment is both expensive and labor-intensive. [1] Dental caries and periodontal diseases are infectious caused by common oral bacteria including Lactobacillus spp., Streptococcus spp. and Actinomyces spp., which usually form plaque biofilms on the tooth surfaces.
E. faecalis is an opportunistic pathogen that is frequently isolated from asymptomatic and persistent endodontic infections, especially from failed root canals undergoing retreatment [29]. E. faecalis is a better survivor than other root canal microbes being able to resist to many antibacterial agents [30].
Among the bacteria included in the present study, viridians streptococci; S. sobrinus and S. mutans were the most representative human cariogenic bacteria and are also moderately resistant to antibiotics [26]. Therefore, controlling or even reducing the levels of these pathogens is a key step in the prevention and treatment of these diseases [1,4].
Analyzing the structure-activity relationship from the results of the evaluation of the antimicrobial activities of the synthetic compounds, the pharmacophore would be a 1,4-naphthoquinone fused with a pyrimidin-4-one moiety having a phenyl group at the C2 position and a benzyl group at the N3 position.
Based on the antibacterial activity of synthetic derivatives, disk diffusion method and time kill assay were performed against P. gingivalis because P. gingivalis is one of the strains that exhibited strong susceptibility and is important for causing the dental caries.
The disk diffusion method has been shown that KHQ 711, 712, 713, 714, 715, 716, 717, and 718 were able to inhibit P. gingivalis strain growth compare to oxytetracycline (Table 3). Moreover, as shown in Figure 3A-H, all these derivatives at 1 2 MIC concentrations, significantly inhibited P. gingivalis until 18 h of incubation.
In conclusion, we synthesized 10 new pyrimidinone-fused 1,4-naphthoquinones and evaluated the antimicrobial activities of synthetic compounds against oral bacteria. Of the tested compounds, KHQ 711, 712, 713, 715, 716 and 717 showed the strongest antimicrobial activity against P. gingivalis. Therefore, these results suggest that these synthetic compounds with proven antimicrobial effects, KHQ 711, 712, 713, 715, 716 and 717 could be useful for the treatment of dental disease.