Synthesis, Characterization, and Antibacterial Activities of Novel

Biomacromolecules 2005, 6, 514-520

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Synthesis, Characterization, and Antibacterial Activities of Novel Methacrylate Polymers Containing Norfloxacin Bekir Dizman,† Mohamed O. Elasri,‡ and Lon J. Mathias*,† Department of Polymer Science and Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, Mississippi 39406-0076

Biomacromolecules 2005.6:514-520. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/23/19. For personal use only.

Received September 30, 2004

A novel methacrylate monomer containing a quinolone moiety was synthesized and homopolymerized in N,N-dimethylformamide (DMF) by using azobisisobutyronitrile (AIBN) as an initiator. The new monomer was copolymerized with poly(ethylene glycol) methyl ether methacrylate (MPEGMA) in DMF using the same initiator. The monomer, homopolymer, and copolymer were characterized by elemental analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), FTIR, 13C NMR, and 1H NMR. The antibacterial activities of the monomer as well as polymers were investigated against Staphylococcus aureus and Escherichia coli, which are representative of Grampositive and Gram-negative bacteria, respectively. All compounds showed excellent antibacterial activities against these two types of bacteria. The antibacterial activities were determined using the shaking flask method, where 25 mg/mL concentrations of each compound were tested against 105 CFU/mL bacteria solutions. The number of viable bacteria was calculated by using the spread plate method, where 100 µL of the incubated antibacterial agent in bacteria solutions were spread on agar plates and the number of viable bacteria was counted after 24 h of incubation period at 37 °C. Introduction Fluoroquinolones are a group of synthetic antimicrobial agents that exhibit excellent potencies and a broad spectrum of activity against a variety of Gram-positive and Gramnegative bacteria as well as mycoplasmas.1-3 They are widely used in human and veterinary medicine for the treatment of infectious diseases.4-5 The antibacterial activity of fluoroquinolones depends on the bicyclic heteroaromatic pharmacophore as well as the nature of the peripheral substituents and their spatial relationship.1 There has been extensive research on the structureactivity relationships of antimicrobial fluoroquinolones.1,6-10 In general, the C-3 carboxy and C-4 keto moieties are required for interactions with DNA bases.1 N-1 and C-8 moieties should be relatively small and lipophilic to enhance self-association.11-13 The amino and fluoro substituents have been shown to be best at the C-5 and C-6 positions.1 Norfloxacin is a member of the fluoroquinolone antibiotics, which are derived from nalidixic acid. It is one of the secondgeneration quinolone agents, which shows very good antimicrobial activity against various types of bacteria.1 It is a specific inhibitor of DNA gyrase, a bacterial type II topoisomerase, which unwinds the supercoiled DNA prior to replication and transcription.4 Norfloxacin also controls DNA superhelicity and plays important roles in various cellular processes such as the efficiency of replication.14 * To whom correspondence should be addressed. Tel: +1-601-266-4871. Fax: +1-601-266-5504. E-mail: [email protected]. † Department of Polymer Science. ‡ Department of Biological Sciences.

Increasing effort has been made during the last two decades to synthesize polymeric systems in order to prevent microbial infections on polymeric materials used in biomedical devices. One approach to controlling infection is to incorporate antimicrobial agents within the polymers. Traditionally, incorporation of the antimicrobial agents into polymers is achieved by either grafting or coating drug agents on the surface of polymers by chemical or physical means15-18 or by physically entrapping the active agent within a polymer matrix or micelle.19,20 An alternative approach is to have polymeric carriers incorporating drug moieties either in the backbone of the polymer or as terminal and pendant groups on the polymer chain. Recently, norfloxacin was conjugated to mannosylated dextran in order to increase the drug’s uptake by cells, enabling faster access to microorganisms.21,22 Studies showed that norfloxacin could be enzymatically cleaved from the polymer-drug conjugate.21 In another study, the synthesis of polyurethanes having antimicrobial norfloxacin drug in the backbone was carried out; their antimicrobial activities are currently being tested.23 Yoon et al. synthesized poly(acrylated quinolone) using norfloxacin as a pendant group on the polymer chain and observed very good antibacterial activities against several Gram-positive and Gram-negative bacteria. They also compounded these polymers with other ordinary synthetic polymers such as low-density polyethylene and poly(methyl methacrylate) and were able to reduce the viable cell number significantly on contact.24 In this work, we report the facile synthesis of a new methacrylate monomer and its polymers containing norfloxacin as a pendant group on the polymer chain. 3-(Acryloy-

10.1021/bm049383+ CCC: $30.25 © 2005 American Chemical Society Published on Web 11/19/2004

Methacrylate Polymers Containing Norfloxacin

loxy)-2-hydroxypropyl methacrylate (AHM) was reacted with norfloxacin (NOR) via Michael addition reaction. This is a very easy method for chemical incorporation of an antimicrobial drug onto a monomer. The homopolymer of the new monomer (AHM-NOR) as well as its copolymer with poly(ethylene glycol) methyl ether methacrylate (MPEGMA) were synthesized using conventional free-radical solution polymerization. MPEGMA was chosen as the comonomer since it is both hydrophilic (it improves water solubility) and biocompatible. Antimicrobial activities of the monomer, homopolymer, and copolymer were tested against S. aureus and E. coli. Experimental Section Materials and Bacterial Strains. Norfloxacin was purchased from Sigma-Aldrich Corporation. 3-(Acryloyloxy)2-hydroxypropyl methacrylate (AHM) and poly(ethylene glycol) methyl ether methacrylate (MPEGMA) were purchased from Aldrich Chemical Co. All solvents used in synthesis were purchased from Acros Chemical Company, Fisher, or Aldrich Chemical Co. Azobisisobutyronitrile (AIBN) was obtained from Aldrich and recrystallized from methanol three times before use. All other chemicals were used as received. Tryptic soy agar (TSA) was purchased from Difco Laboratories. It contained 15.0 g pancreatic digest of casein, 5.0 g enzymatic digest of soybean meal, 5.0 g sodium chloride, and 15.0 g agar. Tryptic soy broth (TSB) was also purchased from DifcoLaboratories. It contained 17.0 g pancreatic digest of casein, 3.0 g enzymatic digest of soybean meal, 2.5 g dextrose, 5.0 g sodium chloride, and 2.5 g dipotassium phosphate. Bacterial strains used for antimicrobial activity tests included S. aureus RN4220 and E. coli TOP10 strain. The strains were kept at - 80 °C in a freezer. Measurements. 13C and 1H NMR spectra were collected on a Varian 300 MHz NMR using CDCl3 and DMSO-d6 solvents. FTIR spectra were recorded on a Nicholet 5DX using pressed KBr pellets or NaCl pellets. Thermal analyses were performed on a TA Instruments 9900 analyzer equipped with 910 differential scanning calorimeter and 952 thermal gravimetric analyzer cells using heating rates of 10 °C/min under nitrogen purge. Elemental analysis results were obtained from Quantitative Technologies, Inc. Molecular weights and molecular weight distributions were measured using size exclusion chromatography (SEC) on a system equipped with four styrene gel mixed-bed columns using tetrahydrofuran (THF) or 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as eluent. Polystyrene and poly(methyl methacrylate) standards were used for calibration in case of using THF and HFIP, respectively. Synthesis of AHM-NOR Monomer. 3-(Acryloyloxy)-2hydroxypropyl methacrylate (AHM, 6.428 g, 0.03 mole) and norfloxacin (NOR, 9.580 g, 0.03 mole) were mixed in 60 mL N,N-dimethylformamide (DMF) in a 100 mL roundbottom flask. The flask was closed with a rubber septum, and the mixture was stirred at 40 °C for 24 h. The reaction mixture was cooled, precipitated into deionized water, and extracted with dichloromethane (CH2Cl2). Excess CH2Cl2

Biomacromolecules, Vol. 6, No. 1, 2005 515

was evaporated by a rotary evaporator, and the final mixture was precipitated into diethyl ether. A solid yellow product was obtained, which was dried in a vacuum oven to remove trapped solvents to give AHM-NOR monomer (12.020 g, 0.0225 mol) in 75% yield. The melting temperature of the monomer was 118-119 °C. The monomer was soluble in DMF and dimethyl sulfoxide (DMSO) at 40 °C. Synthesis of AHM-NOR Homopolymer. A mixture of AHM-NOR monomer (1.073 g, 2 mmol) and AIBN (0.0143 g, 0.09 mmol) was stirred in DMF (5 mL) in a 25 mL roundbottom-flask at 40 °C for 24 h. The reaction mixture was cooled and precipitated into a 50/50 (v/v) water/acetone mixture. The solid yellow product was filtered, and then reprecipitated from DMF into ethanol. The solid product was filtered and washed with water and ethanol, respectively. Finally, the product was dried in a vacuum oven to remove trapped solvents and gave AHM-NOR homopolymer (0.873 g) in 82% yield. Synthesis of AHM-NOR/MPEGMA Copolymer. A mixture of AHM-NOR monomer (0.534 g, 1 mmol), poly(ethylene glycol) methyl ether methacrylate (MPEGMA, 1.425 g, 3 mmol), and AIBN (0.0066 g, 0.04 mmol) was stirred in DMF (10 mL) in a 25 mL round-bottom-flask at 40 °C for 24 h. The reaction mixture was cooled and precipitated into diethyl ether. The sticky yellow product was redissolved in chloroform (CHCl3), filtered, and reprecipitated into diethyl ether. Finally, the diethyl ether was poured and the yellow sticky product was dried in a vacuum oven to remove trapped solvents and gave AHM-NOR/MPEGMA copolymer (1.627 g) in 83% yield. Antibacterial Assessment. The antibacterial activity tests were performed using the shaking flask method 24 and the number of viable cells was counted utilizing the spread plate method.25 S. aureus and E. coli were streaked out on TSA plates and incubated at 37 °C for 24 h. A representative colony was lifted off with a wire loop and placed in 5 mL of TSB, which was then incubated with shaking at 37 °C for 24 h. At this stage, the cultures of S. aureus and E. coli each contained approximately 109 colony-forming units (CFU) per mL. Cultures of S. aureus and E. coli containing 107 CFU/mL were prepared by dilution with TSB, and these were used for antimicrobial tests. The antibacterial activities of the new monomer, homopolymer, and copolymer were determined by testing 25 mg/ mL concentration of the compounds against these two types of bacteria using the aforementioned methods. Only one concentration of the polymers was tested since these polymers were not soluble in TSB. The polymers were mixed with TSB. The homopolymer was in a powder form, whereas the copolymer was a sticky material. Since both polymers were not soluble in water, they formed suspensions upon mixing with TSB. Each suspension containing antimicrobial agent was mixed with 105 CFU of the test organism in a 10 mL culture tube (Falcon). The tubes were incubated at 37 °C for 24 h. The test was repeated at least three times for each antimicrobial agent. Samples were taken from each tube and diluted with TSB. The diluted solutions were spread on agar plates and the plates were incubated at 37 °C for 24 h.

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Scheme 1. Synthesis of the AHM-NOR Monomer

The number of bacterial cells was calculated by multiplying the number of colonies with the dilution factors. Results and Discussion Characterization of the Monomer and Polymers. The AHM-NOR monomer was prepared according to the synthetic route shown in Scheme 1. Michael addition of the secondary amine group of norfloxacin to the acrylate group of 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM) occurred in excellent yield. It is worth mentioning that AHM contains a small amount of a 2-substituted isomer (lee than 5%), which is present throughout the reactions and which also undergoes Michael addition to give an isomeric adduct. The monomer synthesis was followed by both 13C NMR and 1H NMR. Figure 1 shows 13C NMR spectra of AHM, norfloxacin, and the AHM-NOR monomer. The chemical shifts for the vinyl -CH and -CH2 groups of the acrylate on AHM at 128.05 and 131.91 ppm, respectively, disappear as a result of the Michael addition of the secondary amine group of norfloxacin to the acrylate double bond. The carbonyl peak of the acrylate on AHM also shifts from

Figure 1.

13C

Dizman et al.

166.17 to 172.09 ppm upon addition. The new methylene peaks formed after the reaction, corresponding to carbons located between the carbonyl group of the starting acrylate part of AHM and the secondary amine group of norfloxacin, show up at 32.76 and 49.40 ppm. The appearance of these peaks (as well as the disappearance of the acrylate double bond peaks) indicates that the Michael addition reaction was accomplished successfully. The peaks for piperazine methylene groups, which are R and β to the secondary amine group of norfloxacin, also shift from 46.07 and 51.47 ppm to 52.78 and 54.11 ppm, respectively. All of the other peaks of norfloxacin and AHM are seen at the same chemical shifts in the AHM-NOR monomer spectrum. 1 H NMR was also utilized to follow the monomer synthesis. Figure 2 shows the 1H NMR of norfloxacin, AHM, and the AHM-NOR monomer. The acrylate double bond peaks at 5.85, 6.35, and 6.47 ppm disappear as the reaction proceeds to completion. The methylene group R to the secondary amine of norfloxacin shifts upfield from 2.89 to 2.67 ppm. All of the other peaks from norfloxacin and AHM are observed at the same chemical shifts in the AHM-NOR spectrum. The elemental analysis results of the monomer are also in a very good agreement with the calculated values. ANAL Calculated for C26H32FN3O8: C, 58.53%; H, 6.05%; F, 3.56%; N, 7.88%; O, 23.99%. Found: C, 58.04%; H, 5.92%; N, 7.71%. Scheme 2 shows the synthetic route for the AHM-NOR homopolymer and AHM-NOR/MPEGMA copolymer. The homopolymer and copolymer were synthesized via free radical polymerization in DMF using AIBN as an initiator. 13 C NMR and 1H NMR were also used to follow the homopolymerization and copolymerization reactions. The disappearance of the double bond peaks as well as the other monomer peaks were observed upon polymerization in 13C

NMR spectra of NOR (in DMSO-d6), AHM and the AHM-NOR monomer (in CDCl3).


Figure 2.

1H


NMR spectra of norfloxacin (in DMSO-d6), AHM, and the AHM-NOR monomer (in CDCl3).

Scheme 2. Synthesis of the AHM-NOR Homopolymer (a) and AHM- NOR/MPEGMA Copolymer (b)

NMR (data not shown). Figure 3 illustrates 1H NMR spectra of the AHM-NOR monomer, AHM-NOR homopolymer, and AHM-NOR/MPEGMA copolymer. The methacrylate double bond peaks of the AHM-NOR monomer at 5.53 and 6.07 ppm in 1H NMR disappeared upon polymerization. All of the other monomer peaks were observed in both homopolymer and copolymer. The additional peaks from the MPEGMA unit of the copolymer showed up at 0.90, 1.73, 3.38, 3.55, 3.65, and 4.08 ppm (peaks b, a, f, c, e, and d, respectively, in Figure 4). 1H NMR was also used for the determination of the copolymer composition. The peaks c, e, and f in the MPEGMA unit and peak g in the AHM-NOR unit (all peaks are shown in Figure 4) were integrated and the ratio (c +e)/ (f + g) was used to determine the copolymer composition. The ratio was found to be 5/1 and the copolymer composition was calculated to be 57% of MPEGMA unit and 43% AHMNOR unit. The p and r values (number of repeat units) for the copolymer were found to be 38 and 29, respectively.

The molecular weights and molecular weight distributions for the homopolymer and copolymer are shown in Table 1 below. The molecular weight of the homopolymer was measured using HFIP as eluent, whereas the copolymer molecular weight was measured using THF. Intermediate molecular weights were obtained with the homopolymer and copolymer. FTIR spectroscopy was used for the analysis of the monomer and its polymers. Figure 5 illustrates the FTIR spectra of norfloxacin, the monomer, homopolymer, and copolymer. In the monomer spectrum, the new broad peak at around 1200 cm-1 is due to both the C-N stretch of the bond formed during the Michael addition and C-O stretch of the secondary alcohol group on AHM. The peak showing up at around 1750 cm-1 in the monomer spectrum is broader and more intense compared to the peak in the norfloxacin spectrum due to the additional CdO stretching of AHM. The same behavior is seen in the polymer spectrum as well. This peak becomes even more intense in the copolymer due to

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Figure 3. 1H NMR of the AHM-NOR homopolymer (in DMSO-d6), AHM-NOR/MPEGMA copolymer, and AHM-NOR monomer (in CDCl3) (from top to bottom).

Figure 4. The assignments of the peaks of the AHM-NOR-MPEGMA copolymer in 1H NMR. Table 1. Molecular Weights and Molecular Weight Distributions of the Polymers polymer

Mn

Mw

PDI

homopolymer copolymer

15 508 33 804

25 698 61 898

1.66 1.83

the CdO peak of the MPEGMA ester. In the copolymer spectrum, the appearance of a new peak around 1100 cm-1 is due to C-O-C stretching of ester and ether parts of MPEGMA. It is also observed that the peak intensity in the 2800-3000 cm-1 region increases in the copolymer as a result of an increase in C-H antisymmetric and symmetric stretching due to MPEGMA methylene units. Thermogravimetric analysis and differential scanning calorimetry were used for thermal analysis of all compounds. Figure 6 shows the TGA thermograms of norfloxacin, the AHM-NOR monomer, and the polymers. The decomposition

onset temperatures for the homopolymer and the copolymer are 270-280 °C. The thermal behavior of the polymers is mainly due to cleavage and/or degradation of norfloxacin. The monomer has a lower decomposition onset temperature (200 °C), but both the monomer and polymers can be used for applications where thermal stability is important. One example is the use of these polymers as coatings on biomedical devices, which are sterilized at high temperatures (120 °C) and pressures for a certain period of time (20-30 min) prior to their use in vivo. TGA-DTA and DSC were also useful for the determination of the melting point of the monomer, which was found to be 118-119 °C from the first derivatives of both DTA and DSC thermograms. DSC analysis of the polymers did not show any melting or glass transitions. Antimicrobial Assessment. The screening of the compounds for antimicrobial activity was done using S. aureus and E. coli as test organisms since they represent Grampositive and Gram-negative bacteria, respectively. S. aureus and E. coli are also two of the most common nosocomial (originating in a hospital) pathogens.26,27 The shaking flask method was utilized here in order to determine the antimicrobial activities of the monomer and polymers. In accordance with this method, each antimicrobial agent (25 mg/ mL) was mixed with a certain number of bacteria (5 × 105 CFU/mL) in a flask (culture tube) and the flask was incubated at 37 °C for 24 h. Then, a 100 µL sample was taken from each tube and spread onto agar plates. The agar plates were incubated at 37 °C for 24 h, and the number of viable bacteria on the plates was counted at the end of incubation period. The number of viable bacteria and percent reduction of the number of bacteria are shown in Table 2. The monomer and polymers tested in this work showed excellent antimicrobial activities. The percent reduction was



Figure 5. FTIR spectra of norfloxacin, the AHM-NOR monomer, AHM-NOR homopolymer (KBr pellets prepared), and AHM-NOR/MPEGMA copolymer (a film prepared on a NaCl pellet).

Figure 6. TGA thermograms of norfloxacin, the AHM-NOR monomer, AHM-NOR homopolymer, and AHM-NOR-MPEGMA copolymer. Table 2. Antibacterial Activity Results Measured by Shaking Flask Method bacteria

S. aureus (Gram positive)

E. coli (Gram negative)

sample

no. of viable bacteria

% reduction

no. of viable bacteria

% reduction

blank monomer homopolymer copolymer

2.98 × 109 660 620 20

100 100 100

2.88 × 109 10 0 20

100 100 100

expressed as 100% since 7 orders of magnitude reduction was observed for the compounds. Although the monomer and homopolymer showed better antimicrobial activities against E. coli than S. aureus, the difference was not significant. The copolymer showed better antimicrobial activity against S. aureus compared to the monomer and homopolymer. Even though there is not a considerable difference, the better activity of the copolymer is probably due to the partial solubility of the copolymer in water. In the future, the copolymers with higher MPEGMA comonomer units can be synthesized to obtain water-soluble polymers to improve the antibacterial activity. The norfloxacin is used as an antibiotic and has a very good antibacterial

activity with a very small MIC value (around 1 µg/mL against S. aureus and E. coli). Since the new antibacterial polymers synthesized in this work were not soluble in water, it was impossible to compare their antibacterial activities to that of norfloxacin. The lethal action of fluoroquinolone biocides is an outcome of their ability to inhibit DNA synthesis by promoting cleavage of bacterial DNA in the DNA-enzyme complexes of DNA-gyrase and type IV topoisomerase. The specific Gram-positive bacterial activity is associated with inhibition of DNA type IV topoisomerase while that of Gram-negative activity is related to inhibition of DNA gyrase.28 DNA-gyrase is responsible for introducing negative supercoils into DNA and for relieving tortional stress expected to accumulate ahead of transcription or replication processes. Topoisomerase IV, on the other hand, provides a potent unlinking activity. The fluoroquinolones do not simply eliminate enzyme function; they actively poison cells by trapping two topoisomerases on DNA as drug/enzyme/DNA complexes in which double-strand DNA breaks are held together by protein.29,30 One of the important steps in the antimicrobial activity of the fluoroquinolones is their absorbance into the nuclear region of the bacteria, where DNA is located. The fluoroquinolones should be hydrophilic enough to pass through the hydrophilic porin channels in Gram-negative bacteria, whereas in Gram-positive bacteria they should be lipophilic enough in order to pass through bacterial cell wall. Most of the fluoroquinolones have a good balance between the two because they show a wide spectrum of activity. In this work, we modified the norfloxacin structure by reacting it with AHM. The new monomer, as well as the homo- and copolymers, will certainly have different diffusivity characters and different antibacterial activities. To show any activity against S. aureus and E. coli, either the new compounds should be absorbed into the bacterial nuclear region or norfloxacin should be cleaved from the compounds and diffuse inside the bacteria. In the case of the monomer, both of these events can occur, whereas with polymers, it seems that the cleavage and diffusion of norfloxacin are more likely to happen than the diffusion of the polymers into the nuclear region since the polymers are much larger and less mobile.

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However, the actual mechanism of action of these new compounds has not been determined. There are a few possibilities for the cleavage of norfloxacin or active groups such as norfloxacin from the polymers. The ester bonds in the polymers can hydrolyze to give small antibacterial molecules. There are two ester bonds in the polymers for possible hydrolysis. Another possibility is that the Michael addition can be reversed to release norfloxacin. Norfloxacin and other fluoroquinolones are not dangerous to eukaryotic cells at low concentrations. The new polymers containing norfloxacin are not expected to have toxicities to humans; however, a toxicity study for these polymers should be carried out before their use in vivo. Although allergies may be developed for the methacrylate monomers, in general, this should not be a concern for the polymers. The methacrylate polymers cause fewer allergic responses compared to the methacrylate monomers; however, this issue should also be clarified before any internal use of these polymers. Conclusions The new methacrylate monomer containing norfloxacin as a pendant group has been synthesized and homopolymerized by free radical solution polymerization. The monomer also copolymerized with MPEGMA to give a more hydrophilic polymer. The monomer and polymers showed excellent antimicrobial activities against S. aureus and E. coli. These results indicate that the new monomer and polymers have potential as potent antimicrobial agents although mode of activity is not clear. Since these agents are relatively stable to high temperatures, they can be used for medical and biomaterial applications requiring thermal sterilization. Acknowledgment. This material is based upon work supported by the National Science Foundation under MRSEC (Grant No. DMR 0213883). We also thank the NSF-MRI program (Grant No. DMR-0079450) for funding to upgrade and expand the NMR capability at USM. References and Notes (1) Fang, K.; Chen, Y.; Sheu, J.; Wang, T.; Tzeng, C. J. Med. Chem. 2000, 43, 3809. (2) Aeschlimann, J. R.; Dresser, L. D.; Kaatz, G. W.; Rybak, M. J. Antimicrob. Agents Chemother. 1999, 43 (2), 335. (3) Hooper, D. C.; Wolfson, J. S. Quinolone Antimicrobial Agents, 2nd ed.; American Society of Microbiology: Washington, DC, 1995.

Dizman et al. (4) Parshikov, I. A.; Freeman, J. P.; Lay, J. J. O.; Beger, R. D.; Williams, A. J.; Sutherland, J. B. Appl. EnViron. Microbiol. 2000, 66 (6), 2664. (5) Golet, E. M.; Alder, A. C.; Hartmann, A.; Ternes, T. A.; Giger, W. Anal. Chem. 2001, 73, 3632. (6) Koga, H.; Itoh A.; Murayama, S.; Suzue, S.; Irikura, T. J. Med. Chem. 1980, 23 (12), 1358. (7) Kondo, H.; Sakamoto, F.; Inoue, Y.; Tsukamato, G. J. Med. Chem. 1989, 32 (3), 679. (8) Ledoussal, B.; Bouzard, D.; Coroneos, E. J. Med. Chem. 1992, 35 (1), 198. (9) Chen, Y.; Fang, K.; Sheu, J.; Hsu, S.; Tzeng, C. J. Med. Chem. 2001, 44, 2374. (10) Oliphant, C. M.; Green, G. M. Am. Fam. Physician 2002, 65 (3), 455. (11) Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Pihuleac, E.; Nordeen, C. W.; Maleczka, R. E.; Pernet A. G. J. Med. Chem. 1985, 28, 1558. (12) Chu, D. T. W.; Fernandes, P. B.; Pernet A. G. J. Med. Chem. 1986, 29, 1531. (13) Domagala, J. M.; Heifetz, C. L.; Hutt, M. P.; Mich, T. F.; Nichols, J, B.; Solomon, M.; Worth, D. F. J. Med. Chem. 1988, 31, 991. (14) Hwangbo, H. J.; Lee, Y.; Park, J. H.; Lee, Y. R.; Kim, J. M.; Yi, S.; Kim, S. K. Bull. Korean Chem. Soc. 2003, 24 (5), 579. (15) Trooskin, S. Z.; Dontetz, A. P.; Harvey, R. A.; Greco, R. S. Surgery 1985, 97, 547. (16) Modak, S. M.; Sampath, L.; Fox, C. L.; Benvenisty, A.; Nowygrod, R.; Reemstmau, K. Surg. Gynecol. Obstert. 1987, 164, 143. (17) Bach, A,; Schmidt, H.; Bottiger, B.; Schreiber, B.; Bohrer, H.; Motsch, J.; Martin, E.; Sonntag, H. G. J. Antimicrob. Chemother. 1996, 37, 315. (18) Phaneuf, M. D.; Ozaki, C. K.; Bide, M. J.; Quist, W. C.; Alessi, J. M.; Tannenbaum, G. A.; Logerfo, F. W. J. Biomed. Mater. Res. 1993, 27, 233. (19) Schierholz, J. M.; Rump, A.; Pulverer, G. Arzneim.-Forsch. 1997, 47, 70. (20) Lowman, A. M.; Peppas, N. A.; Macromolecules 1997, 30, 4959. (21) Roseeuw, E.; Coessens, V.; Schacht, E.; Vrooman, B.; Domurado, D.; Marchal, G. J. Mater. Sci.: Mater. Med. 1999, 10, 743. (22) Coessens, V.; Schacht, E.; Domurado, D. J. Controlled Release 1997, 47, 283. (23) Yang, M.; Santerre, J. P. Biomacromolecules 2001, 2, 134. (24) Moon, W.; Kim, J. C.; Chung, K.; Park, E.; Kim, M.; Yoom, J. J. Appl. Polym. Sci. 2003, 90, 1797. (25) Merianos, J. J. Disinfection, Sterilization, and PreserVation, 4th ed.; Lea and Febiger: Pennsylvania, 1991; p 225. (26) Kenawy, E.; Abdel-Hay, F. I.; El-Raheem, A.; El-Shanshoury, R.; El-Newehy, M. H. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2384. (27) Hazziza-Laskar, J.; Helary, G.; Sauvet, G. J. Appl. Polym. Sci. 1995, 58, 77. (28) Hooper, D. C. In Mandell, Dougles, and Bennett’s Principles and Practice of Infectious Diseases, 5th ed.; Mandel, G. L.; Bennett, J. E.; Dolin, R., Eds.; Churchill Livingstone: Philadelphia, 2000; p 404. (29) Drlica, K. Curr. Opin. Microbiol. 1999, 2 (5), 504. (30) Fournier, B.; Zhao, X.; Lu, T.; Drlica, K.; Hooper, D. C. Antimicrob. Agents Chemother. 2000, 44 (8), 2160.

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