Ind. Eng. Chem. Res. 1998, 37, 2873-2877
2873
Monomeric and Polymeric N-Halamine Disinfectants M. W. Eknoian, J. H. Putman, and S. D. Worley* Department of Chemistry, Auburn University, Auburn, Alabama 36849
A new series of N-halamine compounds have been synthesized and tested in aqueous solution and on various substrates for efficacy at inactivating bacteria. The compounds, which are 3-halo4-[[(alkylacryl)oxy]methyl]-4-ethyl-2-oxazolidinones, are inexpensive to synthesize and can be polymerized to form polymeric N-halamine compounds. They are efficient biocides in their monomeric form in aqueous solution, and when polymerized, they can be coated on glass, plastic, and fibrous materials for use as surface-active disinfection agents. Introduction Disinfection is the most important process used for preventing the spread of diseases or contamination of sterile environments. In order for a disinfectant to be commercially viable, it must be economical, kill organisms efficiently, and not harm the environment in which it is present. Current disinfection agents include heavymetal cations, pyridinium and quaternary ammonium salts, gaseous agents, and oxidizing agents. The most common biocides in use are sources of free halogen, ozone, or chlorine dioxide; most of these materials tend to be corrosive, have short lifetimes in water, have been linked to cancer in laboratory animals (Brodtman and Russo, 1979; Vogt and Regli, 1981; Cotruvo, 1980), and may react with organic material to produce toxic compounds such as trihalomethanes having known or unknown health risks (Worley and Williams, 1988). Recently, new N-halamines have been synthesized in these laboratories, which stabilize free halogen in water and do not decompose in water to form toxic byproducts (Tsao et al., 1991). There is a need for new biocidal compounds which do not possess the limitations inherent in the disinfectants currently employed. Recently synthesized such compounds are polymeric N-halamines which have limited or no solubility in water, leach small amounts of free halogen into the water supply, produce little or no toxic byproducts, and stabilize the free halogen in the water (Sun et al., 1994). The most effective polymeric Nhalamine prepared to date is a modified polystyrene, wherein a halogenated hydantoin moiety has been incorporated onto the polymer backbone (1; see Figure 1). This polymer is insoluble in water and needs short contact times to kill organisms, and its biocidal activity can be regenerated once exhausted by simply flowing an aqueous solution of free halogen through it. Currently, work in these laboratories has focused upon the development of a new class of monomers, which are biocidal once halogenated. These monomers can be used as disinfectants alone or can be polymerized to form a partially water-soluble polymeric N-halamine. These monomers are substituted oxazolidinones (2) which are reacted with several acryloyl chloride derivatives (Scheme 1) to give the desired monomeric compounds (3a-d). The monomers can then homopolymer* To whom correspondence should be addressed. Telephone: 334-844-6980. Fax: 334-844-6959. E-mail: worlesd@ mail.auburn.edu.
Figure 1. Structure of a cyclic N-halamine polymer.
Scheme 1. Synthesis of Oxazolidinone N-Halamine Derivatives
ize (Scheme 2) to give polymeric oxazolidinones (4) which are then activated by halogenating (4-Cl). It has been shown (Worley and Williams, 1988) that substituted oxazolidinones, when halogenated, are stable in water for prolonged periods of time and are efficient
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2874 Ind. Eng. Chem. Res., Vol. 37, No. 7, 1998 Scheme 2. Synthesis of Polymeric N-Halamines
biocides toward numerous organisms. These compounds are inexpensive to synthesize and have considerable potential for use in various disinfection applications. Also, since these compounds can be polymerized, they can be used as concentrated disinfectants since a high molecular weight polymer can contain numerous N-halamine moieties. This paper will discuss the synthesis and biocidal data for these monomeric and polymeric N-halamines. Experimental Methods 4-(Hydroxymethyl)-4-ethyl-2-oxazolidinone (2). To a one-neck, round-bottom flask were added 13.7 g (0.115 mol) of 2-amino-2-ethyl-1,3-propanediol (Aldrich Chemical Co., Milwaukee, WI), 17.5 mL (0.144 mol) of diethyl carbonate, and 0.10 g (0.0019 mol) of sodium methoxide. The reagents were heated to 110 °C and refluxed with stirring for 48 h. The reflux condenser was then removed and replaced with a distillation head. The ethanol was then removed by simple distillation, and the thick residue was cooled to room temperature. A total of 100 mL of ethyl acetate was then added to the residue, and the solution was vigorously stirred. Slowly a white solid formed. The solid was vacuum filtered to give 13.34 g (80% yield) of 4-(hydroxymethyl)4-ethyl-2-oxazolidinone as a white powder. The recrystallized product (from chloroform) exhibited a melting point of 75-77 °C and the following spectral characteristics: 250-MHz 1H NMR (CDCl3) δ 0.91 (t, 3H, J ) 7.5 Hz), 1.53-1.65 (q, 2H), 3.31 (s, 2H), 3.92 (d, 1H, J ) 2.4 Hz), 4.13 (d, 1H, J ) 2.4 Hz), 5.83 (br, 1H), 7.46 (br, 1H); 13C NMR (CDCl3) δ 7.7, 28.2, 62.6, 66.2, 71.2, 160.8; IR (KBr) 3316, 3245, 2967, 1727 cm-1; MS m/z 145. 4-[(Acryloxy)methyl]-4-ethyl-2-oxazolidinone (3a). A total of 3.10 g (0.021 mol) of the 4-(hydroxymethyl)4-ethyl-2-oxazolidinone prepared as described above, 2.0 g (0.022 mol) of acryloyl chloride (Aldrich Chemical Co., Milwaukee WI), and 25 mL of chloroform were mixed in a one-neck, round-bottom flask. The heterogeneous solution was heated to reflux, with stirring, for 6 h, at which time all of the solid had dissolved. The solution was cooled to room temperature, and the solvent was
removed under reduced pressure. A total of 4.10 g (98% yield) of 4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone was obtained as a yellow oil. The product exhibited the following spectral data: 1H NMR (CDCl3) δ 0.88 (t, 3H, J ) 1.8 Hz), 1.53-1.66 (q, 2H), 3.99-4.18 (m, 4H), 5.83 (d, 1H, J ) 2.4 Hz), 5.98-6.10 (m, 1H), 6.34 (d, 1H, J ) 4.1 Hz), 7.24 (br, 1H); 13C NMR (CDCl3) δ 7.4, 28.4, 60.2, 67.2, 71.0, 127.6, 132.1, 159.7, 165.7; IR (NaCl) 3229, 3015, 2969, 1753 cm-1; MS m/z 199. 3-Chloro-4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone (3a-Cl). A total of 1.0 g (0.005 mol) of 4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone, 1.1 g (0.01 mol) of tert-butyl hypochlorite, and 5.0 mL of methylene chloride were added to a one-neck, round-bottom flask. The solution was stirred at room temperature for 30 min and the solvent removed under reduced pressure. A total of 1.17 g (100% yield) of 3-chloro-4-[(acryloxy)methyl]4-ethyl-2-oxazolidinone was obtained as a clear oil. The product had the following spectral data: 1H NMR (CDCl3) δ 0.93 (t, 3H, J ) 1.8 Hz), 1.54-1.78 (q, 2H), 4.10-4.23 (m, 4H), 5.84 (d, 1H, J ) 2.4 Hz), 5.98-6.10 (m, 1H), 6.32 (d, 1H, J ) 4.2 Hz); 13C NMR (CDCl3) δ 6.6, 24.7, 65.9, 67.7, 71.0, 127.2, 132.6, 159.6, 165.3; IR (NaCl) 2969, 1783 cm-1; MS m/z 233, 235. 3-Bromo-4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone (3a-Br). A total of 1.0 g (0.005 mol) of 4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone and 50 mL of a 0.1 N sodium hypobromite solution were stirred together at room temperature for 30 min in a sealed flask. The solution was then extracted with three 50-mL portions of methylene chloride, and the organic layer was washed with a saturated sodium chloride solution and dried over sodium sulfate. The solvent was removed under reduced pressure to give 1.40 g (100% yield) of 3-bromo4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone as a clear oil. The product exhibited the following spectral data: 1H NMR (CDCl ) δ 0.90 (t, 3H, J ) 2.0 Hz), 1.58-1.81 3 (q, 2H), 4.15-4.27 (m, 4H), 5.80 (d, 1H, J ) 2.3 Hz), 6.00-6.15 (m, 1H), 6.37 (d, 1H, J ) 4.5 Hz); 13C NMR (CDCl3) δ 7.6, 25.6, 64.9, 68.3, 71.7, 123.2, 136.6, 159.1, 165.5; IR (NaCl) 2970, 1775 cm-1; MS m/z 277, 279. 4-(Crotonoxymethyl)-4-ethyl-2-oxazolidinone (3b). A total of 3.1 g (0.021 mol) of 4-ethyl-4-(hydroxymethyl)2-oxazolidinone, 2.3 g (0.022 mol) of trans-crotonyl chloride, and 25 mL of chloroform were mixed in a oneneck, round-bottom flask. The procedure employed was identical with that previously discussed. A total of 4.20 g (98% yield) of pure product was obtained as a clear oil. The product exhibited the following spectral data: 1H NMR (CDCl ) δ 0.90 (t, 3H, J ) 1.8 Hz), 1.48-1.67 3 (q, 2H), 1.83 (d, 3H, J ) 3.6 Hz), 3.97-4.19 (m, 4H), 5.79 (d, 1H, J ) 3.6 Hz), 6.90-6.99 (m, 2H); 13C NMR (CDCl3) δ 7.3, 18.1, 28.3, 60.2, 66.7, 70.9, 121.7, 146.3, 159.6, 166.0; IR (NaCl) 3229, 3020, 2960, 1760 cm-1; MS m/z 213. 3-Chloro-4-(crotonoxymethyl)-4-ethyl-2-oxazolidinone (3b-Cl). A total of 1.1 g (0.005 mol) of 4-(crotonoxymethyl)-4-ethyl-2-oxazolidinone, 1.1 g (0.01 mol) of tert-butyl hypochlorite, and 5.0 mL of methylene chloride were added to a one-neck, round-bottom flask. The solution was stirred at room temperature for 30 min and the solvent removed under reduced pressure. A total of 1.20 g (97% yield) of 3-chloro-4-(crotonoxymethyl)-4-ethyl-2-oxazolidinone was obtained as a clear oil. The product had the following spectral data: 1H NMR (CDCl3) δ 0.80 (t, 3H, J ) 1.8 Hz), 1.58-1.76 (q,
Ind. Eng. Chem. Res., Vol. 37, No. 7, 1998 2875
2H), 2.01 (d, 3H, J ) 3.6 Hz), 4.10-4.25 (m, 4H), 5.81 (d, 1H, J ) 3.6 Hz), 6.95 (d, 1H, J ) 3.6 Hz); 13C NMR (CDCl3) δ 7.3, 18.1, 28.3, 60.2, 66.7, 70.9, 121.7, 146.3, 159.6, 166.0; IR (NaCl) 3020, 2960, 1780 cm-1; MS m/z 246, 248. 4-[[(2′-Methylacryl)oxy]methyl]-4-ethyl-2-oxazolidinone (3c). A total of 3.1 g (0.021 mol) of 4-ethyl-4(hydroxymethyl)-2-oxazolidinone, 2.3 g (0.022 mol) of 2-methylacryloyl chloride, and 25 mL of chloroform were mixed in a one-neck, round-bottom flask. The procedure employed was identical with that previously discussed. A total of 2.56 g (60% yield) of pure product was obtained as a brown oil. The product gave the following spectral data: 1H NMR (CDCl3) δ 0.92 (t, 3H, J ) 1.8 Hz), 1.46-1.65 (q, 2H), 1.93 (s, 3H), 4.12-4.25 (m, 4H), 5.55 (s, 1H), 6.07 (s, 1H), 7.06 (s, 1H); 13C NMR (CDCl3) δ 7.2, 18.0, 27.7, 60.2, 65.6, 70.8, 126.6, 135.4, 160.4, 166.8; IR (NaCl) 3230, 3010, 2960, 1740 cm-1; MS m/z 213. 3-Chloro-4-[[(2′-methylacryl)oxy]methyl]-4-ethyl2-oxazolidinone (3c-Cl). A total of 1.1 g (0.005 mol) of 4-[[(2′-methylacryl)oxy]methyl]-4-ethyl-2-oxazolidinone, 1.1 g (0.01 mol) of tert-butyl hypochlorite, and 5.0 mL of methylene chloride were added to a one-neck, round-bottom flask. The solution was stirred at room temperature for 30 min and the solvent removed under reduced pressure. A total of 1.20 g (97% yield) of 3-chloro-4-[[(2′-methylacryl)oxy]methyl]-4-ethyl-2-oxazolidinone was obtained as a yellow oil. The product had the following spectral data: 1H NMR (CDCl3) δ 1.02 (t, 3H, J ) 1.8 Hz), 1.42-1.60 (q, 2H), 2.10 (s, 3H), 4.324.52 (m, 4H), 5.80 (s, 1H), 6.10 (s, 1H); 13C NMR (CDCl3) δ 7.2, 18.0, 27.7, 60.2, 65.6, 70.8, 126.6, 135.4, 160.4, 166.8; IR (NaCl) 3010, 2960, 1757 cm-1; MS m/z 246, 248. 4-[[(3′,3′-Dimethylacryl)oxy]methyl]-4-ethyl-2oxazolidinone (3d). A total of 3.1 g (0.021 mol) of 4-ethyl-4-hydroxymethyl-2-oxazolidinone, 2.6 g (0.022 mol) of 3,3-dimethylacryloyl chloride, and 25 mL of chloroform were mixed in a one-neck, round-bottom flask. The procedure employed was identical with that previously discussed. A total of 4.10 g (90% yield) of pure product was obtained as a yellow oil. The product exhibited the following spectral data: 1H NMR (CDCl3) δ 0.98 (t, 3H, J ) 1.2 Hz), 1.65-1.71 (q, 2H), 1.92 (s, 3H), 2.17 (s, 3H), 4.04-4.23 (m, 4H), 5.71 (s, 1H), 6.43 (s, 1H); 13C NMR (CDCl3) δ 7.6, 20.6, 27.7, 28.7, 60.3, 66.1, 71.3, 115.2, 159.0, 159.5, 166.2; IR (NaCl) 3215, 3020, 2970, 1755 cm-1; MS m/z 227. 3-Chloro-4-[[(3′,3′-dimethylacryl)oxy]methyl]-4ethyl-2-oxazolidinone (3d-Cl). A total of 1.24 g (0.005 mol) of 4-[[(3′,3′-dimethylacryl)oxy]methyl]-4-ethyl-2oxazolidinone, 1.1 g (0.01 mol) of tert-butyl hypochlorite, and 5.0 mL of methylene chloride were added to a oneneck, round-bottom flask. The solution was stirred at room temperature for 30 min and the solvent removed under reduced pressure. A total of 1.30 g (93% yield) of 3-chloro-4-[[(3′,3′-dimethylacryl)oxy]methyl]-4-ethyl2-oxazolidinone was obtained as a yellow oil. The product had the following spectral data: 1H NMR (CDCl3) δ 1.02 (t, 3H, J ) 1.2 Hz), 1.65-1.71 (q, 2H), 2.02 (s, 3H), 2.30 (s, 3H), 4.14-4.33 (m, 4H), 5.81 (s, 1H); 13C NMR (CDCl3) δ 7.6, 20.6, 27.7, 28.7, 60.3, 66.1, 71.3, 115.2, 159.0, 159.5, 166.2; IR (NaCl) 3020, 2970, 1785 cm-1; MS m/z 226, 228. Poly[4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone] (4). A total of 1.0 g (0.005 mol) of 4-[(acryloxy)-
methyl]-4-ethyl-2-oxazolidinone, 0.01 g (6.1 × 10-5 mol) of 2,2′-azobis(isobutyronitrile), and 10 mL of anhydrous N,N-dimethylformamide were added to a two-neck, round-bottom flask. The reagents were purged with nitrogen for 15 min, and the flask was sealed and heated to 70 °C for 10 h with stirring. The viscous solution was cooled to room temperature and was drop-added into 200 mL of water. The polymer immediately precipitated out and was filtered and dried in a vacuum oven to give 0.9 g (90% yield) of pure product as an amorphous solid. The product, poly[4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone], exhibited the following spectral data: 13C NMR (CDCl3) δ 7.1, 27.6, 30.8, 35.7, 59.2, 60.9, 67.1, 69.8, 158.1, 162.3; IR (neat) 3400, 2937, 1757 cm-1. Poly[3-chloro-4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone] (4-Cl). A total of 0.5 g (0.0025 mol) of poly[4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone], prepared identically with that discussed above, 0.3 g (0.0028 mol) of tert-butyl hypochlorite, and 10 mL of methylene chloride were added to a one-neck, round-bottom flask. The reagents were stirred vigorously for 30 min and the solvent removed under reduced pressure. The residue was dried in a vacuum oven to give 0.58 g (100% yield) of poly[3-chloro-4-[(acryloxy)methyl]-4-ethyl-2-oxazolidinone] as an amorphous solid. The product exhibited the following spectral data: 13C NMR (CDCl3) δ 7.5, 26.6, 31.8, 35.7, 59.2, 60.3, 69.1, 70.0, 159.1, 160.3; IR (neat) 3400, 2937, 1780 cm-1. Bacterial Efficacies. The bacterial efficacies of the compounds against S. aureus (ATCC 6538) in chlorinedemand-free water were determined by using techniques employed previously in these laboratories and described in extensive detail (Williams et al., 1987). Solutions containing 106 CFU/mL final cell densities of bacteria were prepared and treated with the various disinfectant compounds at 5 mg/L total chlorine concentration, which was determined by titration using a stock solution with sodium thiosulfate. Aliquots were removed at several predetermined times, and the active halogen was quenched by sterile 0.02 N sodium thiosulfate. Serial dilutions were made into sterile saline, and three 25-µL aliquots of each dilution were applied to the dried surface of a Petri dish containing nutrient agar. After incubation at 37 °C for 48 h, the three replicates for each dilution were counted and averaged. Control samples containing no disinfectant, or in some cases unchlorinated precursor compounds, were handled in the same manner. In all cases the two types of control experiments yielded plates which contained confluent growth too numerous to count, indicating that the bacterial samples were viable. Inactivation of the organism was considered to be at least 99.9999% when no colonies were detected in the thiosulfate-quenched aliquots. Coating Protocol. The general method for coating polymeric materials onto various substrates is as follows: A total of 1.0 g of the unchlorinated polymer was dissolved in 50 mL of cyclohexanone and the solution filtered to remove any undissolved polymer particles. The substrate to be coated was washed, autoclaved, and dried at 100 °C before the polymer solution was added. Enough of the polymeric solution was added to coat the surface of the substrate without running over the sides; then the material with the polymer solution coating it was heated in an oven set at 80 °C until the solvent was removed. In all cases, the coating was clear and
2876 Ind. Eng. Chem. Res., Vol. 37, No. 7, 1998 Table 1. Efficacies of N-Halamine Compounds against S. aureus in Aqueous Solution
compound
pH
temp (°C)
contact time for 6-log inactivation of S. aureus (min)
3a-Cl 3a-Br 3b 3c 3d 4-Cl
7 7 7 7 7 7
22 22 22 22 22 22
