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In vitro osteogenic differentiation and antibacterial potentials of chalcone derivatives Daheui Choi, Jin Chan Park, Ha Na Lee, Ji-Hoi Moon, Hyo-won Ahn, Kwangyong Park, and Jinkee Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00288 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Molecular Pharmaceutics

In vitro osteogenic differentiation and antibacterial potentials of chalcone derivatives Daheui Choi1, Jin Chan Park2, Ha Na Lee2, Ji-Hoi Moon4,5, Hyo-won Ahn3, Kwangyong Park2,*, and Jinkee Hong1,* 1 Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea 2 School of Chemical Engineering and Material Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea 3 Department of Orthodontics, School of Dentistry, Kyung Hee University, 26 Kyungheedaero, Dongdaemun-gu, Seoul 02447, Republic of Korea 4 Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea 5 Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea.

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Abstract Chalcone derivatives have been investigated for therapeutic agent such as anticancer, antioxidant and anti-inflammatory fields. In this study, we also have synthesized 4 different type of chalcone derivatives and demonstrated in vitro bioactivities. We divided into 2 groups of chalcones on the basis of similar substituents at aromatic rings and tested cell viability and proliferation potentials, indicating that methoxy substituent on A ring could enhance cytotoxicity and anti-proliferation potential depending on chalcone concentration. We also investigated osteogenic differentiation of C2C12 cells by ALP staining, the early marker for osteogenesis, which is demonstrated that the chalcones could not only induce activity of BMP-2, but also inhibit Noggin activity that is BMP antagonist. In addition, chalcone bearing hydroxyl groups at the 2-, 4-, and 6-position on the A ring inhibited treptococcus mutans growth, a major causative agent of dental caries. Therefore, we concluded that the chalcone derivatives synthesized in this research can be good candidates for therapeutic agent promoting bone differentiation, with an expectation of inhibiting S. mutans, in dentistry.

Key words: Chalcone derivatives, cytotoxicity, osteogenic differentiation and antibacterial

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Molecular Pharmaceutics

1. Introduction Chalcones (1,3-diaryl-2-propen-1-ones) are one of the series of flavonoids, which are produced by natural plants or chemical syntheses. The bioactivities of chalcone derivatives have been discovered in diverse application fields, mainly for therapeutic agents, such as antifungal1, antibacterial2, anti-inflammatory3, antioxidant4, anticancer5 or antiobesity properties6. Chalcones also serve as intermediate for the biosynthesis of natural compounds, especially open-chain precursors for flavonoids in nature7. Chalcones have a skeleton of α, βunsaturated ketone with two phenyl rings. In previous studies, the bioactivies of chalcones were found to be highly dependent on the modification of the functional groups of the phenyl rings8, 9. Therefore, syntheses of chalcone derivatives are necessary to discover appropriate biomedical applications and figure out the structure–activity relationship. Bone has the intrinsic potency to regenerate and remodel as part of fracture in the response of injury site. The bone homeostasis is maintained continually remodeling and restoring to replace to the new bone through the adult life10. However, in case of large bone defects caused by tumor, trauma or infection could be retarded to restore to original state, which is still major healthcare challenge11, 12. In addition, for enhancement of bone regeneration, allogeneic bone grafting which is obtained from living donors are implanted into patients, which concerns to induce immunological reaction13. Therefore, intrinsic retention or stimulation of osteogenic differentiation potential on injury site is required to be effective to regenerate bone mass. Bone Morphogenetic Protein 2 (BMP-2) is the member of subfamily of the Transforming Growth Factor-β (TGF-β) which is well-known growth factor to induce osteogenesis of preostoblast or stem cells14, most effective material in stimulation of bone regeneration. However, there are some difficulties involved in using BMP-2 for osteogenesis, 3

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such as high costs and low stability in reconstitution state15. In order to promote BMP-2 activity and functions, some small molecules which are contained aromatic rings have been developed, inhibiting degradation of genes that is involved in BMP-2 signaling pathway16, 17. For the osteogenic differentiation of chalcones to cells, in our knowledge, few publications have been reported for synthetic chalcone derivatives. For example, X. R. Ortolan et al., have been demonstrated the osteogenic potential of chalcone for bone defect in rat calvaria model, showing bone repair on the injured site18. In this study, we synthesized four different trihydroxychalcone derivatives shown in Figure 1b. Size and hydrophilicity of the chalcones were varied by introducing hydroxyl (OH) or methoxy (OMe) groups on A and B ring. Osteogenic differentiation and antibacterial properties of the chalcone derivatives, which were classified into two groups, group A and group B, depending on the substituents on A ring, were investigated. We used C2C12 (mouse myoblast cell line), mesenchymal precursor cells, to determine osteogenic differentiation property of the trihydroxychalcone derivatives. We also examined their effect on the growth of Streptococcus mutans, which is a major pathogenic agent of dental caries.

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Molecular Pharmaceutics

2. Experimental section 2.1. Synthesis of chalcone derivatives 2’,4’,6’-Trihydroxyacetophenone and 4’-hydroxybenzaldehyde were purchased from AlfaAesar. Benzaldehyde (1), dimethyl sulfate, and chloromethyl methyl ether were purchased from

Sigma-Aldrich

Co.

Ltd.

(St.

Louis,

MO,

USA).

2’-Hydroxy-4’,6’-

di(methoxymethoxy)acetophenone (A) and 2’-hydroxy-4’,6’-dimethoxyacetophenone (B) were prepared by the reaction of 2’,4’,6’-trihydroxyacetophenone with chloromethyl methyl ether or dimethyl sulfate in the presence of K2CO3 in acetone at 50 °C, as previously described with a modification (Figure 2)19. 4’-(Methoxymethoxy)benzaldehyde (2) was prepared, as previously described20. Trihydroxychalcone derivatives A1, A2, B1 and B2 were synthesized by Claisen–Schmidt condensation of the corresponding acetophenones with benzaldehydes as following21. Ethanol (200 mL) was added to acetophenone A or B (3.9 mmol), benzaldehyde 1 or 2 (4.3 mmol), and KOH (4.0 mmol) in a round-bottomed flask under nitrogen atmosphere. After stirring for 72 h at room temperature, the reaction mixture was quenched with 1 % aqueous HCl. The organic solution was extracted with Ethyl acetate (EtOAc), washed with water and saturated aqueous NaCl, dried over MgSO4, and concentrated under vacuum. The crude product was purified by recrystallization from ethanol to yield the corresponding chalcones A1’, A2’, B1 and B2’ as a cubic-shaped yellow crystalline solid in 68.7, 65.2, 78.3 and 60.3% isolated yields respectively. HCl (3 %, 100 mL) and ethanol (100 mL) were added to the chalcones A1’, A2’ or B2’ (1.0 g). The solution was heated for 15 min at 70 °C and then cooled to room temperature. The crude product was extracted with EtOAc, washed with water and saturated aqueous NaCl, dried over MgSO4, and concentrated under vacuum. The product was purified 5

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by column chromatography to yield the corresponding 2’,4’,6’-trihydroxychalcone derivatives A1, A2 and B2 as a yellow powder in 78.5, 60.2 and 62.8 % isolated yields respectively. Structure confirmation after synthesizing was conducted via 1H-MNR,

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C-

MNR, FT-IR and UV-vis absorption in our previous report21. 2.2. Cell culture C2C12 (Mouse myoblast cell line) was obtained from National Cancer Center (Republic of Korea). The cells were used within 15-17 passages. For media preparation, 10% of Fetal Bovine Serum (FBS, WELGENE, Gyeongsan-si, South Korea) and 1% of Antibiotics (Gibco, Grand Island, NY, USA.) were added into the high glucose Dulbecco Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA.) as followed our previous work22. Cells were seeded on 100 mm dishes and maintained at 37°C and 5% CO2. The medium was changed every 3 days. Upon reaching cell confluency up to 95%, C2C12 was detached from the dishes with 0.05% Trypsin (Gibco, Grand Island, NY, USA.) dissolved in 1× Phosphate Buffered Saline (PBS, Gibco, Grand Island, NY, USA.). 2.3. Proliferation and viability test For measuring cell proliferation depending on concentration of chalcone, the C2C12 was seeded on well plate at the density of 1.3×102 cells/cm2. After 1 day of incubation, cell culture media was replaced with chalcone (group A and B) treated medium. Dimethyl sulfoxide (DMSO) dissolved chalcone (3 mg/mL) was diluted with cell culture media for adjusting to 5 µM-500 µM. The chalcone treated medium was used 5% FBS contained cell culture media and changed every 3 days. The increased cell number was measured by 3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay with a standard cell number curve. 6

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Molecular Pharmaceutics

In order to measure viability of C2C12 at high chalcone concentration, the cells were seeded on well plate at the density of 1.3×104 cells/cm2. After 1 day of culture, chalcone solutions were treated and cells were cultured for 24 hours. For checking DMSO effect on cells, we also treated cytotoxicity of DMSO. The average amount of DMSO corresponded with each chalcone concentration was employed to C2C12 for viability. The amounts of DMSO we added are 8.34 µL for 200 µM, 20.87 µL for 500 µM, 41.74 µL for 1 mM and 83.48 µL for 2 mL in 5 mL of cell culture media. The viability was measured by MTT assay. 2.4. Osteogenic differentiation of C2C12 and ALP staining For inducing osteogenic differentiation, C2C12 was seeded on well plates at cell number of 1.3×102 cells/cm2. After 1 day of incubation, osteogenic differentiation medium which is comprised of 100 ng/mL or 300 ng/mL of Recombinant Human Bone Morphogenetic Protein-2 (BMP-2, Genoss, Suwon-si, South Korea) mixed with cell culture media containing 10% of FBS was replaced and maintained for 8 days to differentiate myoblast to osteoblast linage. For chalcone (group A and group B) treated groups, 500 µM of A1 and A2 and 250 µM of B1 and B2 were treated to as described above. 100 mg/mL of BMP-2 was added to chalcone treated groups. The medium was replaced every 4 days. After 8 days of incubation, cells were measured by Alkaline Phosphatase (ALP) staining for checking osteogenic differentiation. In case of Recombinant Human Noggin (NOG, Peprotech, Rocky Hill, NJ, USA) treatment to C2C12 for osteogenic differentiation, 5% of FBS containing cell culture media was used. 50 µM of group A and 25 µM of group B were added to each well with/without BMP-2 (100 ng/mL) or NOG (100 ng/mL). The medium was changed every 4 days. After 8 days of incubation, Alkaline Phosphatase (ALP) staining was carried out. For ALP staining, C2C12 was washed with PBS 3 times and fixed with 10% Formalin neutral 7

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buffer solution (Sigma, St. Louis, MO, USA) for 20 min at room temperature. The ALP staining was carried out following Burstone’s staining protocol. Briefly, for preparation of substrate working solution, 5 mg of Naphthol AS-MS phosphate (Sigma, St. Louis, MO, USA) was dissolved in 250 µL of Dimethylformamide (DMF, Daejung, Siheung-si, South Korea). The Tris-hydrochloric acid buffer (Santacruz Biotech., CA, USA) was adjusted at pH 8.74. Sequentially, 250 µL of Naphthol AS-MS phosphate-DMF solution, 25 mL of Trishydrochloric acid buffer and 25 mL of DI water were mixed together. Finally, 30 mg of fast red violet (Sigma, St. Louis, MO, USA) was added into the mixture and vortexed mildly (substrate working solution). The substrate working solution was treated each well plate and wait for 30 min at incubator for enough staining of cells to red color. After staining, the cells were washed with PBS 3 times. In order to quantitively analyze ALP staining, the ALP stained area deducted from microscopic images are were calculated using Image J software (n=3). 2.5. Antibacterial test S. mutans GS-5 was grown at 37 °C anaerobically (85% N2, 10% H2, and 5% CO2) in brain heart infusion broth (BHI, Difco Laboratories, Detroit, MI, USA). To examine the effect of the chalcone derivatives on the bacterial growth, a 24 h culture of S. mutans was adjusted to OD600 of approximately 0.1 in fresh medium. The bacterial suspension was then dispensed (100 µL per well) into wells of polystyrene 96-well plates containing various concentrations of the test agents diluted in BHI (100 µL). The final concentrations of chalcone derivatives ranged from 0.001 mM to 1.0 mM. The blank control comprised BHI medium without the bacterial inoculum. An inoculated BHI medium without test agents (0 mM) was also included. The plates were incubated under anaerobic condition at 37°C. At various time points between 8

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Molecular Pharmaceutics

0 and 24 h, the OD600 was recorded for every sample (n=1). 2.6. Statistical analysis Numerical data obtained by ALP staining, MTT assay, and cell proliferation analysis was represented in a graph with mean value and standard deviation. Differences between two groups were compared by two-tailed, paired t-test. Differences between several groups were analyzed by 2-way ANOVA. P values smaller than 0.05 was marked (*p