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APPLIED CHEMISTRY A Facile Synthetic Approach to Imidazolidinone Biocides D. B. Elrod and S. D. Worley* Department of Chemistry, Auburn University, Auburn, Alabama 36849
A synthetic route has been developed for the production of novel N-halamine disinfectants. These compounds are derivatives of 1,3-dichloro-2,2,5,5-tetramethylimidazolidin-4-one (DC). The synthetic route chosen consists of producing a 2,2,5,5-tetrasubstituted-imidazolidin-4-thione with identical substituents at the 2- and 5-positions, followed by hydrolysis of the cyclic thione to the corresponding acyclic aminothioamide, condensation with a ketone resulting in the formation of a 2,2,5,5-tetrasubstituted-imidazolidin-4-thione having different substituents at the 2- and 5-positions, oxidation to the corresponding 2,2,5,5-tetrasubstituted-imidazolidin-4-one, and finally, chlorination to the desired N-halamine derivative. These derivatives are more nonpolar than is DC, and thereby may possess more antimicrobial activity against microorganisms containing cell membranes of considerable lipophilicity than does DC. Preliminary results presented in this work show that these new derivatives are bactericidal, with some more so than is DC against Staphylococcus aureus. Potential applications may exist for these compounds in the disinfection of nonpolar media such as oils, paints, and lubricants as well as for aqueous solutions of organisms containing lipophilic cell membranes. Introduction The goal of research in these laboratories over the past 2 decades has been to synthesize and test organic N-halamine monomers, polymers, and copolymers which are more stable and which possess antimicrobial activity against a wider spectrum of microorganisms than those available commercially, or those previously produced by other laboratories. Review articles on both N-halamine monomers1 and polymers2 have appeared. The most recent monomeric disinfectant compounds developed in these laboratories are the halogenated 2,2,5,5-tetramethylimidazolidin-4-one (D series) compounds consisting of MC (1-chloro-2,2,5,5-tetramethylimidazolidin-4-one), DC (1,3-dichloro-2,2,5,5-tetramethylimidazolidin-4-one), DB (1,3-dibromo-2,2,5,5-tetramethylimidazolidin-4-one), and DBC (1-bromo-3-chloro2,2,5,5-tetramethylimidazolidin-4-one) (Figure 1).3 The D series compounds have been tested by Tsao and coworkers for stability in dry storage and in aqueous solution as well as for biocidal efficacy against both Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) under varying conditions of pH, temperature, and amount of halogen demand. In other experiments DC and MC were shown to prevent the establishment and growth of algae in water, while DC and DBC exhibited great potential as disinfectants for use on hard surfaces such as stainless steel.3 Recently, an interest has developed in these laboratories in the production of N-halamine compounds which could inactivate microorganisms possessing cell membranes that are rather lipophilic in nature, such as mycobac* To whom correspondence should be addressed. Tel.: 334-844-6980. FAX: 334-844-6959. E-mail: worlesd@ mail.auburn.edu.
Figure 1. Structures of the D series compounds.
teria, and against which the previously described compounds, particularly DC and MC, have only modest biocidal activity. The syntheses of 2,2,5,5-tetrasubstituted-imidazolidin-4-thiones have been reported.4-7 All of these cyclic thiones contain substituents on the 2-position which are the same as those at the 5-position, as is the case for the cyclic thione precursor to the D series compounds produced in these laboratories.3 One of the goals of this study was to discover a reaction or series of reactions to produce 2,2,5,5-tetrasubstituted-imidazolidin-4-thiones possessing substituents at the 2-position that were different from those at the 5-position. Modifying one of the methods of Christian,7 Tsao and co-workers outlined a procedure utilizing 2 mol of acetone, ammonium sulfide, ammonium chloride, and potassium cyanide in aqueous solution to produce 2,2,5,5-tetramethylimidazolidin-4-thione (TMIT) (Scheme 1).3 This method was then used in the attempted synthesis of cyclic thiones with different substituents at the 2- and 5-positions by
10.1021/ie9902479 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/11/1999
Ind. Eng. Chem. Res., Vol. 38, No. 11, 1999 4145 Scheme 1. Synthetic Pathway for the Production of DC Derivatives
replacing 2 mol of the same ketone with 1 mol each of two different ketones. This method was not successful, resulting in the formation of a mixture of cyclic thione products.8 Asinger and co-workers have described the production of a stable, white solid, 2-amino-2-methylthiopropamide
(ATA 1) (Scheme 1), which was synthesized as an intermediate in the eventual preparation of amino acid derivatives. This compound was produced through the acid hydrolysis of 2,2,5,5-tetramethylimidazolidin-4thione (TMIT).9 Asinger et al. also described the synthesis of 2,2,5-trisubstituted-imidazolidin-4-thiones by
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reacting different R-aminothioamides (not produced as just described) with various aldehydes.10 Therefore, it was thought that ATA 1 could be condensed with various ketones to produce a variety of desired 2,2,5,5tetrasubstituted-imidazolidin-4-thiones containing different substituents at the 2- and 5-positions. This method proved successful, resulting in the synthesis of a series of tetrasubstituted-imidazolidin-4-thiones (Scheme 1), which could be oxidized to their corresponding 2,2,5,5-tetrasubstituted-imidazolidin-4-one analogues. Another possible method would be the reversal of the ring cyclization and oxidation steps; ATA 1 would first be oxidized to 2-amino-2-methylpropanamide, ATA 1(O), and then be condensed with the appropriate ketones to produce the same ketones as those in the ATA 1 series (Scheme 1). In this work, the ketones of the ATA 1 series were chlorinated to produce the desired N-halamine derivatives of DC. Several N-halamine derivatives were synthesized, but only four could be isolated as solids, the others being isolated as viscous oils. Only the solid compounds will be presented for discussion. The four solid N-halamines were 1,3-dichloro-2,5,5-trimethyl-2propylimidazolidin-4-one (1-Cl), 1,3-dichloro-2,5,5-trimethyl-2-phenylimidazolidin-4-one (2-Cl), 1,3-dichloro5,5-dimethyl-2-(spirocyclopentyl)imidazolidin-4-one (3Cl), and 1,3-dichloro-5,5-dimethyl-2-(spirocyclohexyl)imidazolidin-4-one (4-Cl) (Scheme 1). These new derivatives are less polar than DC, so it was postulated that they might be more biocidal against microorganisms with more lipophilic cell membranes, specifically mycobacteria such as Mycobacterium tuberculosis. Furthermore, these compounds could be useful in the disinfection of nonpolar media such as paints, oils, and lubricants. Testing of the compounds in nonpolar media is underway elsewhere, but in this work preliminary data concerning the efficacies of the compounds against the bacterium S. aureus in aqueous solution will be presented. Testing is also underway elsewhere comparing the bactericidal activity of the new DC derivatives to the parent molecule, DC, against more lipophilic microorganisms such as Mycobacterium terrae, a surrogate to M. tuberculosis. Experimental Methods All reagents and solvents were purchased from Fisher Scientific Co. or Aldrich Chemical Co. and used without further purification unless otherwise noted. Melting points (mp) were determined on a Mel-Temp melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded using a Perkin-Elmer 297 spectrophotometer, the compounds being mounted in KBr pellets. 1H and 13C NMR spectra were recorded on a Bruker 250 MHZ spectrophotometer operating at 20 ( 1 °C. Chemical shifts are reported in parts per million (ppm) relative to the internal standard tetramethylsilane (TMS) on the δ scale. Mass spectra (MS) were recorded using a Finnigan 3300 spectrometer (EI or DCI mode). Elemental analyses were performed by Atlantic Microlab Inc., Norcross, GA. 2,5,5-Trimethyl-2-propylimidazolidin-4-thione (1a). 2,2,5,5-Tetramethylimidazolidin-4-thione, TMIT, was prepared by the method outlined by Tsao and coworkers.3 ATA 1 was synthesized by a procedure similar to that of Asinger et al.9 First, 1.18 g (0.01 mol) of ATA 1 were transferred to a 50-mL round-bottom flask and dissolved in an excess of 2-pentanone. Molecular sieves
were added, and the flask was fitted with a condenser and drying tube. The reaction mixture was heated to 90-100 °C with stirring for 2 days. The flask was allowed to cool, and the solution was separated from the molecular sieves by vacuum filtration. The filtrate was extracted between chloroform and 1 N HCl solution. The aqueous layer was retained, and its pH was adjusted to 9-10 with concentrated NaOH solution. The aqueous layer was extracted with chloroform, and the organic layer was subsequently evaporated under reduced pressure, yielding a brown-orange solid. The crude solid was purified by recrystallization from hexane, followed by further recrystallization from ethanol/water. Then, 0.70 g (38%) of white solid was isolated. mp 87-88 °C. 1H NMR (CDCl3) δ: 0.92-0.98 (t, 3H, J ) 7.5 Hz), 1.351.48 (m, 11H), 1.63-1.69 (t, 2H, J ) 5.0 Hz), 2.02 (br s, 1H), 10.10 (br s, 1 H). 13C NMR (CDCl3) δ: 14.18, 17.54, 28.35, 29.90, 30.56, 44.56, 70.67, 81.09, 207.75. IR (KBr): 3322, 3137, 2967, 1537, 1462, 1429, 1377, 1365 cm-1. MS (EI) m/z: 186. 2,5,5-Trimethyl-2-phenylimidazolidin-4-thione (2a). When acetophenone was substituted as the ketone and an analogous procedure to that discussed for 1a was used, 0.91 g (41%) of a white solid was isolated. mp 149150 °C. 1H NMR (CDCl3) δ: 1.17 (s, 3H), 1.56 (s, 3H), 1.76 (s, 3H), 2.26 (br s, 1H), 7.28-7.55 (m, 5H), 10.77 (br s, 1H). 13C NMR (CDCl3) δ: 28.20, 29.37, 31.59, 71.40, 82.37, 125.16, 128.06, 128.70, 144.63, 209.42. IR (KBr): 3287, 3123, 2977, 1518, 1447, 1380, 1373, 1360 cm-1. MS (EI) m/z: 220. 5,5-Dimethyl-2-(spirocyclopentyl)imidazolidin4-thione (3a). When cyclopentanone was used as the ketone and an analogous procedure to that discussed for 1a was employed, 0.71 g (39%) of a white solid was isolated. mp 183-184 °C. 1H NMR (CDCl3) δ: 1.34 (s, 6H), 1.75-2.03 (m, 8H), 2.12 (br s, 1H), 10.20 (br s, 1H). 13C NMR (CDCl ) δ: 23.98, 28.94, 40.53, 70.83, 88.42, 3 207.94. IR (KBr) 3281, 3123, 2967, 2872, 1524, 1464, 1456, 1433, 1379, 1360, 1329 cm-1. MS (EI) m/z: 184. 5,5-Dimethyl-2-(spirocyclohexyl)imidazolidin-4thione (4a). With cyclohexanone used as the ketone and an analogous procedure to that discussed for 1a employed, 1.40 g (71%) of white solid were isolated. mp 183-184 °C. 1H NMR (CDCl3) δ: 1.47 (s, 6H), 1.591.79 (m, 10H), 1.87 (br s, 1H), 9.67 (br s, 1H). 13C NMR (CDCl3) δ: 23.28, 24.81, 30.69, 39.60, 70.49, 80.76, 208.5. IR (KBr): 3322, 3141, 2977, 2928, 2863, 1520, 1458, 1441, 1379, 1356 cm-1. MS (EI) m/z: 198. 2,5,5-Trimethyl-2-propylimidazolidin-4-one (1b). 1a (1.86 g (0.01 mole)) was dissolved in 12.48 mL of 2 N NaOH solution. The reaction mixture was placed in an ice bath, and 5.7 mL of 30% hydrogen peroxide were added dropwise via an addition funnel. The temperature of the reaction mixture was maintained between 0 and 15 °C. After the addition, the solution was evaporated, and the resulting solid was boiled in chloroform. Any insoluble material was separated by vacuum filtration, and the filtrate was evaporated under reduced pressure. The product (0.92 g (54%)) was isolated without recrystallization. mp 72-74.5 °C. 1H NMR (DMSO-d6) δ: 0.84-0.90 (t, 3H, J ) 7.5 Hz), 1.12 (s, 3H), 1.17 (s, 3H), 1.24 (s, 3H), 1.27-1.42 (m, 4H), 2.42 (br s, 1H), 8.14 (br s, 1H). 13C NMR (DMSO-d6) δ: 14.22, 16.83, 27.63, 28.06, 29.59, 45.64, 58.24, 71.84, 178.06. IR (KBr): 3349, 3191, 2965, 2872, 1707, 1663, 1464, 1426, 1383, 1360 cm-1. MS (DCI) m/z: 170. Anal. Calcd for C9H18N2O: C, 63.49; H, 10.66; N, 16.45.
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Found: C, 63.49; H, 10.62; N, 16.56. 2,5,5-Trimethyl-2-phenylimidazolidin-4-one (2b). 2a 0.98 g ((0.0045 mol)) was dissolved in about 50 mL of 2 N NaOH solution (the increased amount of NaOH solution in this case is due to the poor solubility of this derivative in basic solution). The reaction mixture was placed in an ice bath, and 2.54 mL of 30% hydrogen peroxide were added dropwise via an addition funnel. The reaction was performed and product isolated exactly as that just described for 1b. The crude solid was purified by recrystallization from water, and 0.59 g (65%) of a white solid was isolated. mp 145-146 °C. 1H NMR (CDCl3) δ: 1.09 (s, 3H), 1.43(s, 3H), 1.71 (s, 3H), 2.22 (br s, 1H), 7.27-7.53 (m, 5H), 8.94 (br s, 1H). 13C NMR (CDCl3) δ: 25.85, 26.84, 32.61, 60.05, 74.68, 74.68, 124.77, 127.48, 146.35, 181.34. IR (KBr): 3343, 3200, 3067, 2975, 2959, 1705, 1661, 1495, 1449, 1387, 1373, 1304 cm-1. MS (DCI) m/z: 204. Anal. Calcd for C12H16N2O: C, 70.56; H, 7.89; N, 13.71. Found: C, 70.66; H, 7.93; N, 13.71. 5,5-Dimethyl-2-(spirocyclopentyl)imidazolidin4-one (3b). 3a (0.92 g (0.005 mol)) was dissolved in 6.24 mL of 2 N NaOH solution and reacted with 2.85 mL of 30% hydrogen peroxide. The addition of hydrogen peroxide and isolation of product was performed using a procedure analogous to that previously described for 1b. The resulting crude solid was purified by recrystallization from acetone, and 0.39 g (46%) of a white solid was isolated. mp 164-165 °C. 1H NMR (CDCl3) δ: 1.34 (s, 6H), 1.76-1.88 (m, 8H), 2.10 (br s, 1H), 8.23 (br s, 1H). 13C NMR (CDCl3) δ: 23.32, 26.72, 41.30, 59.72, 81.02, 180.78. IR (KBr): 3291, 3193, 2973, 1682, 1472, 1435, 1385, 1363, 1352, 1327 cm-1. MS (EI) m/z: 168. Anal. Calcd for C9H16N2O: C, 64.25; H, 9.59; N, 16.65; Found: C, 64.00; H, 9.64; N, 16.54. 5,5-Dimethyl-2-(spirocyclohexyl)imidazolidin-4one (4b). 4a (0.99 g (0.005 mol)) was employed using an analogous procedure to that described for 1b. The crude solid produced was purified by recrystallization from ethanol, and 0.39 g (43%) of a white solid was isolated. mp 196-198 °C. 1H NMR (CDCl3) δ: 1.35 (s, 6H), 1.39-1.70 (m, 10H), 1.85 (br s, 1H), 7.90 (br s, 1H). 13C NMR (CDCl ) δ: 23.08, 24.78, 28.02, 40.68, 59.03, 3 72.54, 180.24. IR (KBr): 3181, 3075, 2936, 2851, 1696, 1456, 1437, 1379, 1360, 1340 cm-1. MS (EI) m/z: 182. Anal. Calcd for C10H18N2O: C, 65.90; H, 9.95; N, 15.37. Found: C, 65.78; H, 9.96; N, 15.32. 1,3-Dichloro-2,5,5-trimethyl-2-propylimidazolidin4-one (1-Cl). 1b (0.34 g (0.002 mol)) was transferred to a 50 mL round-bottom flask and dissolved in methylene chloride. Then, 0.48 g (0.0044 mole) of tert-butyl hypochlorite was added, and the flask was stoppered, wrapped in aluminum foil, and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, the crude solid was dissolved in hexane, and any insoluble material (unchlorinated ketone) was separated via vacuum filtration. The filtrate was evaporated under reduced pressure, leaving 0.36 g (75%) of a white solid. mp 35-36 °C. 1H NMR (CDCl3) δ: 0.90-0.95 (t, 3H, J ) 7.5 Hz), 1.37 (s, 3H), 1.45 (s, 3H), 1.53 (s, 3H), 1.66-1.74 (m, 4H). 13C NMR (CDCl3) δ: 13.66, 15.80, 21.96, 22.80, 24.93, 38.69, 65.93, 86.06, 169.96. IR (KBr): 2961, 2932, 2872, 1740, 1464, 1456, 1381, 1368, 1323, 1304 cm-1. An iodometric titration determined that the N-halamine contained 93% of its theoretical total chlorine content.
1,3-Dichloro-2,5,5-trimethyl-2-phenylimidazolidin-4-one (2-Cl). 2b (0.22 g (0.001 mole)) was transferred to a 50-mL round-bottom flask and dissolved in methylene chloride. Then, 0.24 g (0.0022 mol) of tertbutyl hypochlorite was added, and an analogous procedure to that described for 1-Cl was employed, resulting in the isolation of 0.24 g (83%) of a white solid. mp 114115 °C. 1H NMR (CDCl3) δ: 1.42 (s, 3H), 1.53 (s, 3H), 2.01 (s, 3H), 7.40-7.50 (m, 5H). 13C NMR (CDCl3) δ: 21.16, 23.62, 24.65, 66.29, 86.34, 127.63, 128.31, 129.47, 135.54, 170.12. IR (KBr): 2984, 1736, 1495, 1449, 1372, 1304 cm-1. An iodometric titration determined that the N-halamine contained 91% of its theoretical total chlorine content. 1,3-Dichloro-5,5-dimethyl-2-(spirocyclopentyl)imidazolidin-4-one (3-Cl). 3b (0.34 g (0.002 mol)) was transferred to a 50-mL round-bottom flask and dissolved in methylene chloride. Then, 0.48 g (0.0044 mol) of tertbutyl hypochlorite was added, and an analogous procedure to that described for 1-Cl was employed, resulting in the isolation of 0.42 g (89%) of a white solid. mp 2830 °C. 1H NMR (CDCl3) δ: 1.37 (s, 6H), 1.71-1.84 (m, 4H), 1.94-2.05 (t, 2H, J ) 14.5 Hz) 2.22-2.30 (t, 2H, J ) 11 Hz). 13C NMR (CDCl3) δ: 22.70, 26.04, 34.72, 66.49, 94.21, 169.26. IR (KBr): 2978, 2936, 2872, 1742, 1458, 1433, 1381, 1362, 1337, 1318 cm-1. An iodometric titration determined that the N-halamine contained 93% of its theoretical total chlorine content. 1,3-Dichloro-5,5-dimethyl-2-(spirocyclohexyl)imidazolidin-4-one (4-Cl). 4b (0.20 g (0.001 mol)) was transferred to a 50-mL round-bottom flask and dissolved in methylene chloride. Then, 0.24 g (0.0022 mol) of tertbutyl hypochlorite was added. When employing the procedure similar to that for 1-Cl, 0.21 g (84%) of a white solid was isolated. mp 78-79 °C. 1H NMR (CDCl3) δ: 1.31 (s, 6H), 1.43-1.47 (t, 2H, J ) 5.0 Hz), 1.792.05 (m, 8H). 13C NMR (CDCl3) δ: 21.64, 22.96, 23.65, 33.27, 65.25, 83.82, 169.51. IR (KBr): 2967, 2946, 2909, 2851, 1730, 1458, 1381, 1364, 1318 cm-1. An iodometric titration determined that the N-halamine contained 91% of its theoretical total chlorine content. Bactericidal Activity Against S. aureus for DC Derivatives in pH 7.0 Buffer. A solution of the N-halamine to be tested was prepared. The amount of solid sample to be tested was weighed and transferred to a volumetric flask. The sample was then dissolved in sufficient pH 7.0 buffer to obtain the desired concentration of N-halamine in solution. An aliquot of the sample solution was then titrated using the iodometric method to determine the exact concentration of Nhalamine present. Bacterial suspensions (0.5 mL) containing about 107108 CFU (colony forming units) per mL of S. aureus ATCC 6538 were transferred to test tubes containing 4.5 mL of the N-halamine solution. After carefully measured contact times, 1 mL of a mixture was transferred to its respective test tube containing 1 mL of sterile 0.02 N thiosulfate solution to “quench” the bactericidal activity of the N-halamine solution by neutralizing all of the combined and free chlorine in solution. This quenched sample was then serially diluted with sterile pH 7.0 buffer. Finally, three 0.025mL aliquots of each dilution (100-105) were transferred to a sectioned agar plate. A control sample containing only the bacterial suspension was tested using the same procedure. All plates were incubated at 37 °C for 2448 h, and then plate counts were determined. The
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bactericidal activity of the N-halamine solution was determined in terms of a log reduction. Longevity of Bactericidal Activity against S. aureus for DC Derivatives at Maximum Concentration. Each N-halamine compound to be tested was dissolved in pH 7.0 buffer at its maximum concentration. The flasks containing the N-halamine solutions were sealed and placed on the lab bench at room temperature. The solutions were tested periodically (once every 1-2 weeks) against a bacterial suspension containing S. aureus ATCC 6538 as previously described. This was continued for each N-halamine solution until it no longer exhibited adequate bactericidal activity. Minimum Concentration of DC Derivatives Required for a 7-8 Log Reduction of S. aureus in pH 7.0 Buffer at 5- and 10-min Contact Times. Several samples of each N-halamine to be tested were prepared in pH 7.0 buffer. Each stock solution was titrated using the iodometric method to determine its respective total chlorine concentration. These stock solutions were then diluted to obtain a wide range of concentrations (from very dilute to near maximum solubility) for each Nhalamine solution. Each diluted solution of a given N-halamine compound was tested against a bacterial suspension containing ∼107-108 CFU/mL of S. aureus ATCC 6538 at room temperature, as previously described, at both 5- and 10-min contact times. This experiment was repeated until the minimum effective concentration range for each compound was determined. Results and Discussion Although some of the yields of the thione derivatives were low, only one cyclized product, the expected product, was obtained in each case. The interesting result from the chosen reaction scheme is that it is essentially the conversion from one cyclic thione derivative to another. A variety of new cyclic thione derivatives were synthesized by condensing the acyclic aminothioamide, ATA 1, with various ketones, but it was found that the largest straight-chain alkyl group which could be placed at the 2-position was pentyl from the reaction of ATA 1 with 2-heptanone (not discussed in this work). A phenyl group could be placed at the 2-position by the reaction of ATA 1 with acetophenone. It was found that conversion of the new cyclic thiones to their corresponding ketone analogues, thereby producing the desired 2,2,5,5-tetrasubstituted-imidazolidin4-one derivatives, was preferable to oxidation of ATA 1 to the acyclic aminoamide, ATA 1(O), followed by reaction with the same ketones used to produce the ATA 1 series cyclic ketones. The former method produced higher yields than the latter, as might be expected since the nucleophilicity of the acyclic aminothioamide, ATA 1, should be higher than that of the acyclic aminoamide, ATA 1(O). The cyclic ketones were converted to their corresponding N-halamines by chlorination with tert-butyl hypochlorite in methylene chloride. Although chlorination could have been achieved in aqueous solution,3 an organic solvent and chlorinating reagent were chosen to ensure that the desired N-halamine remained in solution during the course of chlorination. The valid question arises: Why is a nonpolar Nhalamine derivative possessing different substituents at the 2- and 5-positions necessary since the method for producing an N-halamine derivative with identical and
Table 1. Longevity of Biocidal Activity against S. aureus at Maximum Concentration (2-Cl)
0-16 days 16-26 days 26-40 days
(4-Cl)
0-10 days 10-17 days 17-25 days
(1-Cl)
0-27 days 27-34 days 34-41 days
(3-Cl)
0-40 days 40-46 days 46-52 days
9 log to 1 log/5 mina 9 log to 3 log/10 min 0 log/5 min 3 log/10 min 0 log/5 min 0 log/10 min 8 log/5 min 8 log to 4 log/5 min 8 log to 7 log/10 min 4 log to 0 log/5 min 7 log to 0 log/10 min 8 log to 7 log/5 min 7-8 log to 1-2 log/5 min 7-8 log to 4-5 log/10 min 0 log/5 min 0 log/10 min 7-8 log/5 min 7-8 log to 1 log/5 min 7-8 log to 3 log/10 min 0 log/5 min 1 log/10 min
a The bactericidal activity steadily declined between the days indicated.
nonpolar substituents at these positions is facile and well-established?3,7 This synthesis has been attempted in these laboratories. The largest N-halamine derivatives produced by the literature method with identical cyclic substituents at the 2- and 5- positions were 1,3dichloro-2,5-bis(spirocyclopentyl)imidazolidin-4-one and 1,3-dichloro-2,5-bis(spirocyclohexyl)imidazolidin-4one.8 The former compound was isolated as a viscous oil which was difficult to handle and therefore not viable for practical purposes. The latter was isolated as a white solid, but the compound was so nonpolar, and hence so water-insoluble, that an accurate measurement of its solubility in aqueous solution using the iodometric method was impossible. Therefore, the goal of the research discussed herein was to produce an Nhalamine derivative in which two or three of the four substituents at the 2- and 5-positions were made as small as possible (i.e., methyl) to enhance water solubility, while the other one or two substituents were made as large as possible for enhanced penetration into the cell membranes of lipophilic microorganisms. It should be noted that alkyl substituents are necessary adjacent to the N-Cl moieties to stabilize the N-Cl bond; H is not a viable substituent.3 The four solid N-chloramine derivatives were tested for maximum solubility in pH 7.0 buffer at room temperature. It was observed that these derivatives were far less soluble in aqueous solution than their parent compound, DC (1920 ppm). The order of water solubility for the four derivatives were 3-Cl, 321.2 ppm > 1-Cl, 177.6 ppm > 4-Cl, 78.7 ppm > 2-Cl, 10.1 ppm. The above solutions containing the four derivatives were tested for bactericidal activity against S. aureus ATCC 6538 at 5- and 10-min contact times after being freshly prepared, and then periodically tested until they were no longer effective (Table 1). In general, the more soluble the compound was in pH 7.0 buffer, the longer that it was effective as a bactericide. 3-Cl retained its bacterial effectiveness for 40 days, followed by 1-Cl, 4-Cl, and finally, 2-Cl for less than 16 days. However, 4-Cl is about 8 times more water-soluble than 2-Cl, yet the two compounds retained effective bactericidal activity for about the same amount of time. Therefore, the molec-
Ind. Eng. Chem. Res., Vol. 38, No. 11, 1999 4149 Table 2. Minimum Concentration of DC Derivatives for a 7-8 log Reduction of S. aureus at 5- and 10-min Contact Times compound
conc. of cmpd./ conc. of Cl+
log reduction/ contact time
(4-Cl)
6-8 ppm/1.7-2.3 ppm 9-12 ppm/2.5-3.4 ppm
7-8 log/10 min 7-8 log/5 min
(3-Cl)
18-22 ppm/5.4-6.6 ppm 34-36 ppm/10.2-10.8 ppm
7-8 log/10 min 7-8 log/5 min
DC
22-24 ppm/7.4-8.1 ppm 43-45 ppm/14.5-15.1 ppm
7-8 log/10 min 7-8 log/5 min
(1-Cl)
50-51 ppm/14.8-15.1 ppm 102-104 ppm/30.3-30.9 ppm
7-8 log/10 min 7-8 log/5 min
ular structure of the compounds (the only difference between these derivatives are the substituents at the 2-position) could also be a factor in regard to longevity of bactericidal activity. All of the compounds seemed to decline sharply in their bactericidal activity as they approached one-half of their original titratable concentration of Cl+. This was expected because at this concentration the compounds have converted primarily to their mono-chlorinated form, behaving now as derivatives of MC with chlorine only attached to the amine nitrogen. Since the N-Cl bond of an amine functionality is much more stable than the corresponding amide, little or no free chlorine is produced, and the contact time required for effective inactivation of bacteria is much longer.3 Compounds 4-Cl, 3-Cl, 1-Cl, and DC were tested to determine the minimum concentration of each required for a 7-8 log reduction of S. aureus ATCC 6538 at both 5- and 10-min contact times. It was decided that 2-Cl would be excluded from any further bactericidal testing in aqueous solution due to its relatively low solubility. From this experiment, the order of bactericidal activity against S. aureus could be determined. The resulting order was the following: 4-Cl > 3-Cl > DC > 1-Cl (Table 2). It should be remembered that bactericidal activity is organism-dependent. The order against other microorganisms could be different than the one shown above. The test results in progress for mycobacteria will be reported in due course. Conclusion A new synthetic route has been developed for the production of new 1,3-dichloro-2,2,5,5-tetrasubstituted-
imidazolidin-4-one derivatives. The methods were straightforward, and although some of the product yields were low, the schemes selected avoided the possibility of side product formation and resulted in only the desired product being obtained. The four solid N-halamine compounds synthesized are more nonpolar than their parent compound, DC. Hence, these new monomeric disinfectants may possess enhanced antimicrobial activity against microorganisms possessing cell walls of considerable lipophilicity. This work shows that these new derivatives are bactericidal. Two of the three derivatives tested displayed greater bactericidal activity against S. aureus than did DC. Testing of the new N-halamine derivatives for bactericidal efficacy against more lipophilic microorganisms is ongoing. Possible applications for these compounds may also lie in the disinfection of nonpolar media such as paints, oils, and lubricants. Literature Cited (1) Worley, S. D.; Williams, D. E. Halamine Water Disinfectants. CRC Crit. Rev. Environ. Control 1988, 18, 133. (2) Worley, S. D.; Sun, G. Biocidal Polymers. Trends Polym. Sci. 1996, 4, 364. (3) Tsao, T.-C.; Williams, D. E.; Worley, C. G.; Worley, S. D. Novel N-Halamine Disinfectant Compounds. Biotechnol. Prog. 1992, 7, 60. (4) Gatewood, E.; Johnson, J. B. The Interaction of Hydrogen Sulfide with Certain Amino and Amino Acid Nitriles. J. Am. Chem. Soc. 1928, 50, 1422. (5) Abe, K. Organic Sulfur Compounds. I. The Action of Hydrogen Cyanide, Ammonia, and Hydrogen Sulfide on Saturated Ketones. Sci. Rep. Tokyo Bunrika Daigaka, Sect. A 1934, 2, 1. (6) Bucherer, H.; Brandt, W. U ¨ ber Cyanhydrine. J. Prakt. Chem. 1934, 140, 129. (7) Christian, J. D. 4-Imidazolidinethiones. J. Org. Chem. 1957, 22, 396. (8) Naquib, I. Synthesis and Kinetic Study of Novel Water Disinfectants. Ph.D. Dissertation, Auburn University, Auburn, AL, 1991. (9) Asinger, F.; Scha¨fer, W.; Kersten, H.; Saus, A. Zur Kenntnis der Hydrolyse Substituierter Imidazolidin-thione-(4). Monatsh. Chem. 1967, 98, 1843. (10) Asinger, F.; Scha¨fer, W.; Meisel, H.; Kersten, H.; Saus, A. Zur Darstellung von Imidazolidin-thionen-(4) und Imidazolin-(3)thionen-(5). Monatsh. Chem. 1967, 98, 338.
Received for review April 5, 1999 Revised manuscript received August 4, 1999 Accepted August 4, 1999 IE9902479
