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Biol. Pharm. Bull. 28(6) 1120—1122 (2005)
Vol. 28, No. 6
Antibacterial Activity of Dipropofol and Related Compounds Masahiro OGATA,*,a,c,1) Kanae TUTUMIMOTO SATO,b Takao KUNIKANE,a Kentaro OKA,a Masako SEKI,a Shiro URANO,c Keiichi HIRAMATSU,b and Toyoshige ENDOa a
Kyoritsu University of Pharmacy; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan: b Juntendo University; 2–1–1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan: and c Shibaura Institute of Technology; 3–9–14 Shibaura, Minato-ku, Tokyo 108–8548, Japan. Received January 20, 2005; accepted March 5, 2005 Phenolic compounds, in general, exhibit antioxidant and antibacterial activities. We studied antimicrobial activity of the phenolic antioxidants, propofol (2,6-diisopropylphenol), tocopherol, eugenol, butylated hydroxyanisole (BHA), and several of their dimer compounds. Dipropofol (dimer of 2,6-diisopropylphenol) showed strong antibacterial activity against gram-positive strains including methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE), while propofol and other monomeric and dimeric phenols having methyl or tert-butyl groups showed no remarkable activity. The results indicated that the dimeric structure of 2,6-diisopropylphenol moiety may play an important role in the antibacterial activity. Key words dipropofol; antimicrobial activity; vancomycin resistant Enterococci (VRE); antioxidant; methicillin resistant Staphylococcus aureus (MRSA)
During the past decade, nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in hospitals have become a serious clinical problem.2) A glycopeptide antibiotic, vancomycin, has been used for the treatment of infections due to MRSA. Enterococci, which have some a part of the normal faecal flora of humans and animals have generally emerged as important nosocomial pathogens. Acquired resistance to vancomycin and teicoplanin in enterococci is due to the replacement of the cell wall component DAla–D-Ala replaced by the depsipeptide D-Ala–D-lactate. This substitution leads to the formation of modified peptidoglycan precursors, for which glycopeptides exhibit 1000-fold lower binding affinities.3) In most clinical isolates of enterococci, resistance is due to acquisition for plasmids that are often transferable to other gram-positive bacteria by conjugation.4) Recently, MRSA strain which has vanA gene with high resistance to vancomycin (minimum inhibitory concentration (MIC) 128 m g/ml), has been reported in clinical isolates.5) Therefore, a new anti-MRSA antibiotic, which differs from vancomycin in its mode of action, is clinically of interest. In the course of our screening system for new anti-MRSA and anti-vancomycin resistant Enterococci (VRE) antibiotics from some phenolic compounds, we investigated the antibacterial activity of propofol (2,6-diisopropylphenol), tocopherol, eugenol, butylated hydroxyanisole (BHA), and several of their dimer compounds. Dipropofol, which is an oxidative compound of propofol, showed inhibitory activity against gram-positive bacteria. The present study deals with antibacterial activities of propofol, dipropofol and related phenols. MATERIALS AND METHODS Chemicals 2,6-Di-tert-butylphenol, 2,6-dimethylphenol, and 2,6-diisopropylphenol (propofol) were purchased from Sigma-Aldrich Japan K.K. (Tokyo). BHA and eugenol were purchased from Tokyo Kasei Kogyo Co. (Tokyo). a -Tocopherol was purchased from E. Merck (Darmstadt, Germany). Muller-Hinton broth and agar were purchased from Nissui Pharmaceutical Co. (Tokyo). Synthesis of Dipropofol Propofol (1 g) was dissolved in ∗ To whom correspondence should be addressed.
CH2Cl2 (10 ml) and stirred with CuCl(OH) · tetramethylethylenediamin (TMEDA) (16 mg) for 5 h at room temperature.6) The reaction product was extracted with AcOEt and the solvent was evaporated to afford a resultant residue which was dissolved in ethanol, and heated with Na2S2O4 (1 g) for 20 min. The separated precipitate was collected on a filter and was crystallized from hexane to give the dimeric propofol, dipropofol (950 mg, 95%) as a white solid. FAB-MS m/z: 355 (MH). 1H-NMR (500 MHz, CDCl3, d , ppm): 1.32 (24H, d, J6.7 Hz, 8CH3), 3.20 (4H, m, J6.7 Hz, 4 CH), 7.20 (4H, s, 4H-Ar). HR-FAB-MS: 355.2643 (Calcd for C24H35O2: 355.2637). mp 108 °C. Synthesis of DiBHA BHA (10 g) was dissolved in pyridine (10 g) and heated with FeSO4 (100 mg) and 31% H2O2 (20 g3) at 60 °C for 30 h. The reaction product was extracted with AcOEt and distilled by steam distillation. The residue was recrystallized from ethanol to give diBHA (3.52 g, 35%) as a white solid. FAB-MS m/z: 359 (MH). 1 H-NMR (500 MHz, CDCl3, d , ppm): 1.43 (18H, s, 2tertbutyl), 3.77 (6H, s, 2OCH3), 6.60 (2H, s, H-4, 4), 6.96 (2H, s, H-6, 6). mp 223 °C.7) Synthesis of Di(2,6-di-t-butylphenol) 2,6-Di-t-butylphenol (500 mg) was dissolved in CH2Cl2 (10 ml) and stirred with CuCl(OH) · TMEDA (16 mg) for 2 h at room temperature. The reaction product was extracted with AcOEt. After removal of solvent, the reactant was dissolved in ethanol, and heated with Na2S2O4 (2 g) for 2 h. The precipitate was collected on a filter and crystallized from hexane to give di(2,6di-t-butylphenol) (481 mg, 96%) as a white solid. FAB-MS m/z: 411 (MH). 1H-NMR (500 MHz, CDCl3, d , ppm): 1.47 (36H, s, 4tert-butyl), 5.23 (2H, s, 2–OH), 7.36 (4H, s, 4H-Ar). mp 112 °C.8) Synthesis of Di(2,6-dimethylphenol) 2,6-Dimethylphenol (500 mg) was dissolved in CH2Cl2 (10 ml) and mixed with CuCl(OH) · TMEDA (16 mg) at room temperature at 5 h. The reaction product was extracted with AcOEt and evaporated. The reactant was dissolved in ethanol, Na2S2O4 (2 g) was added and the mixture was heated for 2 h. The precipitate was collected in a filter and crystallized from hexane to give di(2,6-dimethylphenol) (462 mg, 93%) as a white solid. FAB-MS m/z: 243 (MH). 1H-NMR (500 MHz,
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© 2005 Pharmaceutical Society of Japan
June 2005
Fig. 1.
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Structures of Phenolic Compounds Assessed
CDCl3, d , ppm): 2.10 (12H, s, 4CH3), 6.41 (2H, s, 2 –OH), 7.22 (4H, s, 4H-Ar). mp 118 °C.8) Synthesis of Dieugenol Eugenol (10 g) was dissolved in pyridine (10 g) and mixed with FeSO4 (100 mg) and 31% H2O2 (20 g3) at 60 °C for 24 h. The reaction product was extracted with AcOEt and distilled by steam distillation. The residue was recrystallized from ethanol to give dieugnol (3.56 g, 35.6%) as a white solid. FAB-MS m/z: 327 (MH). 1 H-NMR (500 MHz, CDCl3, d , ppm): 3.33 (4H, d, J7.3 Hz, 2H-7, 7), 3.82 (6H, s, 2OCH3), 5.06 (4H, m, J9.8, 17.1, 1.9 Hz, 2H-9, 9), 5.95 (2H, m, J9.8, 17.1, 6.7 Hz, H-8, 8), 6.69 (2H, s, H-2, 2), 6.74 (2H, s, H-6, 6). mp 93 °C.9) Antibacterial Susceptibility The minimum inhibitory concentrations (MICs) of test compounds were determined by an agar double dilution method. An overnight bacterial culture was diluted 100-fold (to approximately 106 cfu/ml) and the suspension was inoculated on Muller-Hinton agar plates prepared with each agar. The MICs were determined after overnight culture at 37 °C.10) RESULTS AND DISCUSSION It is well known that phenolic compounds exhibit antioxidant and antimicrobial activities. Propofol (2,6-diisopropylphenol), a rapid, short-acting general anesthetic that is widely employed in the induction and maintenance of anesthesia, has antioxidant activity,11) but its antimicrobial activity is unknown. We therefore studied the antibacterial activities of propofol and other phenolic compounds having antioxidant activity (Fig. 1), in the phenolic compounds we tested, only dipropofol showed antibacterial activity against Staphylococcus aureus and Bacillus subtilis (Table 1). Antimicrobial activities (MICs) of dipropofol were compared with those of vancomycin against standard bacteria. The results are shown in Table 2. The antibacterial spectrum of dipropofol was recognized against gram-positive bacteria but not against gram-negative as vancomycin.
Table 1. Antibacterial Activities of Phenolic Compounds against S. aureus, B. subtilis and E. coli Compound
S. aureus 209P
B. subtilis PC219
E. coli K12
128 128 128 128 128 128 128 128 2 128 128
128 128 128 128 128 128 128 128 4 128 128
128 128 128 128 128 128 128 128 128 128 128
a -Tocopherol BHA Eugenol Propofol 2,6-Dimethylphenol 2,6-Di-tert-butylphenol DiBHA Dieugenol Dipropofol Di(2,6-dimethylphenol) Di(2,6-di-tert-butylphenol)
Table 2. Antibacterial Activities of Dipropofol and Vancomycin against Standard Strains Organism Staphylococcus aureus FDA 209-P S. aureus SMITH S. epidermidis IFO-12648 Micrococcus luteus ATCC-9341 Enterococcus faecalis ATCC-21212 Escherichia coli NIHJ JC-2 E. coli 445 Citrobacter freundii ATCC-8090 Proteus mirabilis IFO-3849 P. vulgaris OX-19 Morganella morganii IID KONO Serratia marcescens IFO-12648 Enterobacter cloacae IFO-133535 E. aerogenes NCTC-10006 Pseudomonas aeruginosa 46001 P. aeruginosa E-2 Acinetobacter calcoaceticus IFO-12552
Dipropofol 2 2 2 2 2 128 128 128 128 128 128 128 128 128 128 128 128
Vancomycin 0.5 1 1 0.5 2 128 128 128 128 128 128 128 128 128 128 128 128
Next, we studied the antibacterial activity of dipropofol against the antibiotic resistant strains MRSA and VRE (Table 3). Dipropofol has strong activity against VRE (MIC
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Table 3. Antibacterial Activities of Dipropofol and Vancomycin against MRSA and VRE Organism MRSA N315 IR94 MRSA N315 IR94-HR-1 MRSA 70 E. faecalis (vanA) E. faecium (vanA) E. faecalis (vanB) E. faecium (vanB) E. gallinarum (vanC) E. casseliflavas (vanC)
Dipropofol
Vancomycin
2 2 2 4 2 4 4 4 4
0.5 1 0.5 128 128 32 128 16 32
dence, it was suggested that dipropofol seems to be a useful compound for a hospital infection such as Linezolid, because of its strong antibacterial activity against VRE and MRSA. Mechanistic studies are in progress. Acknowledgments We thank Kazue Kunikane, Naomi Huse and Aki Ogata for helpful assistance. This work was supported in part by a research grant from the KANPO Science Foundation, Tokyo, Japan, the Ministry of Education, Culture, Sports, Science and Technology, Japan and the New Energy and Industrial Technology Development Organization of Japan. REFERENCES AND NOTES
2—4 m g/ml), and MRSA at the same level as standard strains. In general, phenol (at high concentration) acts as a protoplasm toxin to destroy the cell wall system and to precipitate protein in cells, whereas at low concentration it inhibits the multiplication of enzymes in vitro. However, phenol and some phenolic compounds such as cresol, thymol, and epigallocatehin gallate, have no antimicrobial activity against MRSA and VRE at low concentration (100 m g/ml) (data not shown).12,13) In our investigations of the structure–activity relationships of some phenolic antioxidants, dipropofol showed strong antibacterial activity against gram-positive strains including MRSA and VRE, while propofol and other monomeric and dimeric phenols having methyl or tert-butyl groups showed no remarkable activity. Also, dipropofol dimethylester (3,3,5,5-tetraisopropyl-4,4-dimethoxybipenyl) did not inhibit bacteria growth (data not shown). The results indicated that the dimeric structure of 2,6-diisopropylphenol moiety may play an important role of the antibacterial activity. We concluded that the mode of action of dipropofol was mediated by inhibition of protein synthesis or an amino acid incorporation system.14) However, interaction of the hydroxyl group, isopropyl group, and dimeric skeleton in the expression of antibacterial activity is not clear. Based on the evi-
1) 2) 3) 4) 5) 6)
7) 8) 9) 10) 11) 12) 13) 14)
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