which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent.
Biocontrol Science, 2013, Vol. 18, No. 4, 183−191
Original
Synthesis of Thiazole Derivatives and Evaluation of Their Antiamoebic Activity and Cytotoxicity AKIHIRO SHIRAI＊, TOSHIYUKI ENDO, HIDEAKI MASEDA, AND TAKESHI OMASA Department of Biological Science and Technology, Biosystems Engineering, Institute of Technology and Science, The University of Tokushima, 2-1, Minamijosanjima-cho, Tokushima 770-8506, Japan Received 9 March, 2013/Accepted 23 May, 2013
Five ethyl（5-alkyl-2-amino-1,3-thiazol-4-yl）acetates（designated compounds 4a-e）incorporating octyl, decyl, dodecyl, tetradecyl, and hexadecyl alkyl chains, respectively, were prepared by reacting 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters（3a-e）with thiourea in dried acetonitrile. Compounds 3a-e were synthesized by reacting alkylated ethyl acetoacetates with bromine. The newly synthesized compounds were characterized by mass spectrometry, NMR, and elemental analysis. Compounds 4a-c demonstrated good in vitro antiamoebic activity against Acanthamoeba polyphaga exposed to 10 mg L−1 for 6 h at 28℃. Compound 4b showed the highest antiamoebic activity among the tested compounds, comparable to that of chlorhexidine dihydrochloride（CHX）, decreasing the number of viable cells to below the detection limit of 1 cell mL−1. The activity of compounds 4a and 4c was similar to that of the commercial antifungal agent fluconazole（Flu）. The cytotoxic and hemolytic activity of the compounds was assayed against human neonate dermal fibroblasts and sheep erythrocytes, respectively. Compounds 4a-c were less cytotoxic than Flu and CHX. Our results suggest that compound 4b, which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent. Key words：Thiazole derivatives / Antiamoebic / Cytotoxicity / Acanthamoeba.
INTRODUCTION Species belonging to the genus of free-living protozoa known as Acanthamoeba can be found in most soil, dust, and aquatic environments（Stevens and Tyndall, 1977）. Although almost all Acanthamoeba are nonpathogenic, they have been known to cause a potentially blinding keratitis upon invading the cornea （Anger and Lally, 2008）. Analysis of clinical and patient questionnaire data used to investigate the frequency, outcome, and risk factors associated with Acanthamoeba keratitis in England from 1992 to 1996 indicated that the infection was most common in contact lens wearers（93％ of patients）, and of these patients, 84％ used soft contact lenses（Radford et al., Corresponding author. Tel：+81-88-656-7519（No FAX） E-mail : shirai（a） bio.tokushima-u.ac.jp ＊
1998）. The recent increase in the incidence of Acanthamoeba keratitis relates not only to an increase in the number of soft contact lens users（Verani et al., 2009）, but also to widespread noncompliance with recommended rubbing and rinsing procedures（Gray et al., 1995; Radford et al., 1995）. In addition, there are disconcerting differences in commercially available contact lens cleaning and soaking solutions with respect to amoebicidal activity against Acanthamoeba （Silvany et al., 1990）. For example, while a solution containing chlorhexidine（5 mg L−1, CHX）was found to be effective with a 30 min treatment, neither polyaminopropyl biguanide nor polyquaternium-1 were effective, even after an 8 h treatment. The antiamoebic activity of eight commercially available Japanese contact lens solutions was also investigated（Kobayashi et al., 2011）. Only four of the tested solutions were effective against trophozoites after treatment for 4 h,
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but all 8 solutions were ineffective against cysts. Seven of the 8 solutions tested contained polyhexamethylene biguanide（PHMB）at concentrations of 1.0-1.1 mg L−1. Therefore, for solutions that do not use neutralizers, biguanide biocides such as CHX and PHMB must be used, which are relatively more active against trophozoites. However, as biguanides are less effective against mature cysts, potentially toxic concentrations must be used to completely kill Acanthamoeba. This problem has spurred investigations into the amoebicidal properties of plant extracts（Degerli et al., 2012; Tepe et al., 2012） . These studies resulted in the isolation of a plant extract that was effective against trophozoites at a concentration of more than 16 mg L−1. A relatively long treatment time of 48 h was required to kill trophozoites, and the plant extract was not effective against cysts, even after a 72-h exposure at the maximum tested concentration of 32 mg L−1（Tepe et al., 2012）. The present study focused on the development of new low-molecular-weight antiamoebic agents with an amoebicidal activity comparable to that of the biguanide disinfectants commonly used in contact lens care solutions. The objective of the study was to facilitate development of more effective eye drops for use in treating Acanthamoeba keratitis. Thiazoles and their derivatives have recently attracted increased research interest due to their antibacterial （Rudolph et al., 2001）, antitubercular（Aldo and Granaiola, 2001）, and antimicrobial（Liaras, 2011; Nitin, 2012）activities. The thiazole derivative 2（4-thiazolyl）-benzimidazole is commonly used as an antifungal agent and as a food preservative. The biocides described in this paper are based on simple thiazole derivatives that incorporate a hydrophobic alkyl group and an ethyl ester group into a thiazole skeleton. An amino group at the 2-position of the thiazole ring also allows for modification with various substituents, such as amino acids, peptides, or saccharides. The added hydrophobic alkyl group（octyl, decyl, dodecyl, tetradecyl, or hexadecyl）is important because it imparts higher antimicrobial activity. Using ethyl acetoacetate（1）as the starting reagent, the newly described TEE-n series thiazol e derivatives were synthesized through a cyclization reaction with thiourea involving 3 synthetic steps. Although the thiazole derivative intermediates carry unreacted amino groups before the modification reaction, the antiamoebic activity of the simple structural derivatives was investigated using stocks of Acanthamoeba polyphaga stored in our laboratory. A. polyphaga is suitable for the analysis of antiamoebic activity because it is reportedly more resistant to disinfectants than the corneal infection-causing species A. castellanii（Silvany et al., 1990）. Of the derivatives examined in the present study,
thiazole 4b, which contains a decyl alkyl group, showed amoebicidal activity comparable to that of CHX and better than that of fluconazole（Flu）, which is a chemotherapeutic agent used in the clinical treatment of Acanthamoeba keratitis. In addition, the antiamoebic thiazole derivatives described here demonstrated low cytotoxicity against human dermal fibroblasts and sheep erythrocytes.
MATERIALS AND METHODS Chemistry All reagents used for compound synthesis were commercially available reagent grade and were used without further purification. Tetrahydrofuran（THF）as a reaction solvent was freshly distilled with benzophenone and particles of metallic sodium to yield absolute THF. Similarly, chloroform was purified through distillation after pre-drying with calcium chloride. Acetonitrile was dried for 3 days using type 4A molecular sieves. Fluconazole, which is an effective ingredient included in antiamoebic eye drops for clinical use, was purchased from LKT Laboratories, Inc.（USA）for comparison of commercially available agents. Chlorhexidine dihydrochloride（CHX, Sigma Chemical Co., USA）was used as an alternative to polyhexamethylene biguanide hydrochloride, which is an active constituent in commercial contact lens disinfection solutions（Fig.1）. Instruments All reactions were monitored by thin layer chromatography（TLC）using precoated Merck silica gel 60 F254 plates（thickness＝0.25 mm）, and the Rf values for the synthesized compounds were determined from the TLC plates. NMR spectra were recorded with a JEM-EX 400 NMR spectrometer（JOEL, Japan）using tetramethylsilane as an internal standard. The purity of compounds was checked by TLC and by the melting point, which was determined using a micro-melting apparatus
FIG. 1. Comparison of the chemical structures of fluconazole （Flu）and chlorhexidine dihydrochloride（CHX）.
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FIG. 2. Synthesis of ethyl（5-alkyl-2-amino-1,3-thiazol-4-yl）acetates 4a-e.
（Fisher Scientific, USA）. Mass spectra were recorded with a QP1000 EI-MS system（Shimadzu, Japan）. Elemental analyses were performed using a Micro Corder JM10 instrument（J-Science Lab Co., Ltd., Japan） , and the results were consistent with theoretical values to within±0.3％. Optical rotation values for compounds 3a-e were determined using a DIP-360 digital polarimeter （Jasco, Japan）. The crude compounds were purified through silica gel（mesh 70-230）using flash column chromatography. Synthesis of thiazole derivatives As shown in Figure 2, a total of five ethyl（5-alkyl-2amino-1,3-thiazol-4-yl）acetates（4a-e）possessing various alkyl groups at the 5-position were synthesized by reacting the brominated compounds 3a-e with thiourea using a modification of a previously reported synthesis method（Shivarama et al., 2003）. The 4-substituted-3-oxobutyric acid ethyl esters 2a-e and their brominated forms 3a-e were prepared according to previously methods reported by Hamzě et al.（2005） and Svendsen and Boll（1973）, respectively. All reactions were conducted under a stream of nitrogen. Preparation of 3-oxobutyric acid ethyl esters alkylated at the 4-position（2a-e） All ethyl acetoacetate alkylation reactions were performed at 4℃ with mechanical stirring. Sodium hydride（NaH, 1.08 g, 45 mmol, 1.5 equiv.）was added to a solution of ethyl acetoacetate（3.90 g, 30 mmol, 1.0 equiv.）diluted with anhydrous THF（50 mL）. After stirring for 15 min, 1.6 mol L –1 n-butyl lithium in n-hexane（n-BuLi, 27.9 mL, 44.8 mmol, 1.5 equiv.） was added to the solution in a drop-wise manner. After stirring for 15 min, 1.0 equiv. of n-octyl bromide, n-decyl bromide, n-dodecyl bromide, n-tetradecyl bromide, or n-hexadecyl bromide dissolved in 5 mL of anhydrous THF was added in a drop-wise fashion and the suspension was stirred for 1 h. After quenching with saturated ammonium chloride solution, most of the organic solvent in the reaction mixtures was evaporated. The ethyl acetate layer was extracted from the residue and
washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification by flash chromatography on silica gel using a gradient elution of n-hexane/ethyl acetate（100：1, v/v）yielded the intended compounds（2a-e）. Preparation of 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters（3a-e） Bromine（10 mmol, 1.0 equiv.）was added in a dropwise manner to a solution of each of the 2-series compounds（10 mmol, 1.0 equiv.）in 20 mL of CHCl3, and the reaction mixture was stirred for 1 h at 4℃. The solvent was evaporated and the residue was dissolved in ethyl acetate/H2O（1：1）, after which the organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification by flash chromatography on silica gel using a gradient elution of n-hexane/ethyl acetate（200：1, v/v）yielded the intended compounds（3a-e）. Preparation of ethyl（5-alkyl-2-amino-1,3-thiazol4-yl）acetates（4a-e） Compounds 3a-e（8.0 mmol, 1.0 equiv.）and thiourea（12 mmol, 1.5 equiv.）were dissolved in 40 mL of dried acetonitrile. The mixture was refluxed for 6 h, after which the reaction was quenched with 10 mL of saturated sodium bicarbonate solution. The organic solvent was removed under reduced pressure, after which additional water was added and the extraction process was performed with ethyl acetate. The resulting organic phase was dried with sodium sulfate and removed under reduced pressure. The purified thiazole compounds（4a-e）were obtained by silica gel column chromatography using a gradient elution of hexane/ ethyl acetate（6：1-1：1, v/v）. Amoeba cultures and growth conditions Stocks of A. polyphaga ATCC 30871 that had been stored in our laboratory were used in this study. Trophozoites were grown at 28℃ for 3-4 d under static culture conditions in a tissue culture flask（65 mL, cultivation dimensions 25 cm2, Orange Scientific）containing
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5 mL of PYGC liquid broth（1％（w/v）proteose peptone, 1％（w/v）yeast extract, 1％（w/v）glucose, 0.95％（w/v）L-cysteine hydrochloride, 0.5％（w/v） NaCl, 10 mM Na2HPO4, 5 mM KH2PO4, 2 mM MgCl2, （Shirai et al., 2000）. The culture and 0.4 mM CaCl2） supernatant was then discarded and the amoeba cells adhering to the bottom of the flask were resuspended in 5 mL of fresh 1/2 PYGC medium. After incubation at 28℃ for approximately 14 d under static culture conditions with periodic changing of the culture medium, the cell suspension was centrifuged（1000×g, 5 min）, the supernatant was discarded, and the amoeba cells were resuspended in freshly prepared physiological saline. The number of amoeba cells was determined using a hemocytometer（Depth＝0.1 mm, Erma Inc., Japan）, and prepared to 5×103 cells mL−1 with fresh physiological saline. In vitro antiamoebic activity assay Antiamoebic tests were performed using A. polyphaga ATCC 30871 trophozoites suspended in physiological saline as described above. A 0.9 mL volume of a 20 mg L −1 solution of synthesized compound（4a-e）or comparative control agent（Flu or CHX）dissolved in physiological saline including 0.2％ DMSO was added to the cell suspension（0.9 mL）in a 24-well tissue culture plate（Techno Plastic Products, Switzerland）. The final biocide and DMSO concentrations were 10 mg L −1 and 0.1％, respectively. The mixture was incubated at 28℃ for 6 h under static culture conditions. Subsequent to the addition of 0.2 mL of 5％ Tween 80, the adherent cells were dislodged by pipetting the mixture vigorously in the well. The number of surviving cells was determined by incubating 1-mL aliquots of physiological saline stepwise dilutions for 7 days at 28℃ on Bacto agar TM plates（Difco Laboratories, USA）onto which 0.1 mL of heated Escherichia coli NBRC 12713 was spread. Surviving cells formed plaques on the plates, and the plaques were counted daily during the course of the 7-d incubation（Khunkitti et al., 1996）. Antiamoebic activity was determined as the inactivation percentage using the following equation： Inactivation percentage（％） ＝100−（Nt / N0×100） where N t represents the biocide-treated sample plaque count and N0 represents the non-biocide control sample plaque count taken before the incubation. To prepare the heated E. coli suspension, 5 mL of a bacterial suspension precultivated in nutrient broth （Difco Laboratories, USA）was inoculated into 1 L of nutrient broth prepared at a 2-fold concentration. After incubation for 24 h at 37℃ with shaking, the bacteria
were collected by centrifugation（5000×g, 10 min） and washed twice with 25 mL of Page’ s amoeba saline （PAS, in 1 L of distilled water: NaCl, 120 mg; MgSO4·7H2O, 4 mg; CaCl2, 4 mg; Na2HPO4, 142 mg; （Page, 1967）. General KH2PO4,136 mg; pH 6.8-7.0） physiological saline may be used instead of PAS. The washed E. coli cells were then resuspended in 25 mL of PAS in a 500 mL flask and heated at 70℃ for 90 min in a water bath shaker（90 strokes min−1）. The heated cells were stored at −80℃. In vitro cytotoxicity assay The IC50 was defined as the concentration of TEE-n that induces a 50％ reduction in the number of viable human neonate dermal fibroblasts, and was determined by plotting cell viability（％）against the TEE-n concentration. Likewise, the concentration necessary to produce 50％ hemolysis（HC50）was determined from plots of hemoglobin released by erythrocytes treated with various concentrations of tested compounds. For all cytotoxicity experiments, solutions of TEE-n and comparative agents were initially dissolved in 100％ DMSO at a high concentration. The human neonate dermal fibroblast line NB1RGB （Riken Cell Bank, Japan）was used for cytotoxicity assays. Cells were cultured in minimal essential medium alpha（α-MEM）and Dulbecco’ s modified Eagle’ s medium（D-MEM）enriched with 10％ fetal bovine serum and 60 mg L–1 of kanamycin sulfate. Cell viability was assessed using an MTT assay（Cell Counting Kit-8, Dojindo Molecular Technologies, Inc., Japan）. Cells were preincubated with α-MEM, collected by centrifugation（150×g, 5 min）, resuspended and diluted to 5 ×104 cells mL −1 with D-MEM, and distributed in a 96-well culture plate（100μL in each well）. The plate was incubated at 37℃ in a humid atmosphere with 5％ CO2 for 2-3 days until the cells reached confluence. A 50μL volume of the supernatant was removed from each well, after which 50μL of a 1.5-fold serial dilution of one of the synthesized compounds（4a-c）or comparative agents（Flu or CHX）was added and the plate was incubated at 37℃ for 1 h. Next, 10μL of MTT solution was added to each well and the plate was incubated at 37℃ for 40 min. The solution in each well was removed and placed into a microcentrifuge tube, centrifuged at 11,800×g for 10 min at 4℃, and the absorbance was measured at 450 nm. The percent viability was determined by comparing the absorbance of test wells with the absorbance of wells in which cells were incubated in the presence of 10,000 mg L –1 sodium dodecyl sulfate（0％ viability）. The 100％ viability control consisted of cells incubated in medium containing 0.1％ DMSO. Data for the assay of compounds 4d and 4e were excluded due to insolu-
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TABLE 1. 1H- and 13C-NMR data and appearance, mass, m.p., Rf, and total yield for the compound 4a. Appearance
m.p.
Yellowish crystal
55-56℃
＋ EI-MS（calcd / found） ［M］
Rfa
Total yield（％）
298.44 / 298
0.31
36
Elemental analysis（％） Anal. calcd. for C15H26N2O2S：C, 60.37; H, 8.78; N, 9.39. Found：C, 60.54; H, 9.05; N, 9.24. H-NMR（400 MHz, CDCl3）δ
1
, 1.54（2H, m, -CH2CH2CH2-）, 2.53 0.88（3H, t, J ＝ 6.8 Hz, -CH2CH2CH3）, 1.26-1.38（13H, m, -CH（CH 2 2） 5CH3, -OCH2CH3） （2H, t, J ＝ 7.6 Hz, CCH2CH2-）, 3.59（2H, s, CCH2CO-）, 4.23（2H, q, J ＝ 7.1 Hz, -OCH2CH3）, 8.82（2H, bs, CNH2） C-NMR（100 MHz, CDCl3）δ
13
14.1, 14.2, 22.6, 25.8, 28.9, 29.1, 29.2, 30.5, 31.6, 31.8, 62.3, 122.7, 125.4, 168.0, 168.3 The value of Rf was determined by TLC plates precoated with silica gel using the mixture solvent（n-hexane： ethyl acetate ＝ 1：1, v/v）as the mobile phase.
a
bility of these compounds in D-MEM at a concentration of 10 mg L−1. Hemolytic toxicity was determined on sheep red blood cells（Nippon Biotest Laboratories, Japan）using the serial 1.5-fold dilution method（Shirai et al., 2005）.
RESULTS AND DISCUSSION Synthesis of ethyl（5-alkyl-2-amino-1,3-thiazol4-yl）acetates As shown in Figure 2, the α-position of ethyl acetoacetate 1 was alkylated at 4℃ by adding sodium hydride and n-butyl lithium, followed by addition of 1-bromoalkane（n-octyl bromide, n-decyl bromide, n-dodecyl bromide, n-tetradecyl bromide, or n-hexadecyl bromide）, giving yields for 2a-e of 31％-48％. Using this replacement reaction it is possible to synthesize thiazole series compounds 4a-e, which contain alkyl groups of various carbon lengths at the 5-position. The α-position in the alkyl compounds 2a-e was subsequently bromo-substituted, giving yields for 3a-3e of 84％-98％. The five alkylated 1,3-thiazole acetates （4a-e）were derived by heating the α-bromo-substituted compounds with thiourea in anhydrous ethyl alcohol, giving yields of 43％-76％. Confirmation of the structures of the 2-4 series of synthesized thiazole derivatives was obtained using NMR, mass spectrometry, and elemental analysis. Data regarding the appearance, melting point（m.p.）, EI-MS, R f data, total yield, elemental analysis, and 1H- and 13 C-NMR analyses are shown in Table 1. Compound 4a produced 1 H-NMR peaks at δ 0.88（CH 3 corresponding to the terminus of the octyl group）, 1.26-1.38 （polymethylene chain and CH3 corresponding to the ethyl ester group）, 1.54（CH2 corresponding to the methylene of the polymethylene chain）, 2.53（CH2 at the 5-position of the 1,3-thiazole ring） , 3.59（CH2 at the α-position of the ethyl acetate group）, 4.23（CH 2
FIG. 3. Antiamoebic activity of thiazole compounds 4a-e, Flu, and CHX against A. polyphaga ATCC 30871. Cells were exposed to each compound at a concentration of 10 mg L−1 for 6 h at 28℃. Values are the mean and standard deviation obtained from three independent experiments. ＊P＜0.05 （t-test）and ＊＊P＜0.01（t-test）as compared to the control in the absence of biocides. ＃P＜0.05（t-test）as compared to Flu.
corresponding to the ethyl ester group）, and a broad peak at 8.82（NH 2）. The mass spectrum of 4a indi＋ ＝298. Our results showed cated its exact mass,［M］ that the synthesized TEE-n series were the intended compounds. Antiamoebic activity Compounds 4a-e, Flu, and CHX were evaluated for antiamoebic activity using A. polyphaga ATCC 30871. Cells were treated at a concentration of 10 mg L−1 for 6 h at 28℃. The results are shown in Figure 3. The TEE-n series compounds 4a-c, as well as CHX, showed significant amoebicidal activity as compared to that of the untreated control: percent inhibition＝53±8.9, 100
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±0, 45±7.7, and 100±0 for 4a, 4b, 4c, and CHX, respectively. No significant inhibition of A. polyphaga was observed with compounds 4d, 4e, and Flu: percent inhibition＝42±22, 39±12, and 34±11, respectively. Interestingly, the amoebicidal activity of compound 4b was significantly higher than that of Flu, a fungicidal agent included in clinical antiamoebic eye drops. The activity of 4b was similar to that of CHX, reducing the number of viable A. polyphaga to less than 1 cell mL−1. These results indicate that compound 4b is the most potent of the series 4 antiamoebic compounds and is more potent than the clinically used agent Flu, even though the structure of 4b consists only of a simple thiazole ring, decyl group, and ethyl ester moiety. Generally, the relatively low-molecular-weight alkyl chains such as those incorporated into the synthesized compounds described in this report are associated with molecular hydrophobicity. The molecular hydrophobicity of simple surfactants containing alkyl chains, such as quaternary ammonium salts, increases with alkyl chain length（Maeda et al., 1998）. The antibacterial potency of surfactants has been linked to physicochemical parameters such as critical micelle concentration and molecular hydrophobicity（Devínsky et al., 1985; Maeda et al., 1998; Shirai et al., 2005）. As Figure 3 indicates, as the length of the TEE-n alkyl chain increased（e.g., from octyl to decyl）, the Rf value determined by silica gel TLC also increased, from 0.31 to 0.32（Table 1 and Appendices）, coincident with enhanced amoebicidal activity. Therefore, the amoebicidal mechanism of the TEE-n series compounds can be attributed to an interaction between the hydrophobic alkyl chain and the amoeba cell membrane. The amoebicidal activity of the compounds containing longer alkyl chains（e.g., dodecyl, tetradecyl, and hexadecyl groups）was lower than that of the compounds containing shorter alkyl chains（Fig.3）. The lower activity of these compounds may be related to a decrease in the effective concentration resulting from their low solubility in physiological saline. Remarkably, compound 4b, which contains a decyl group, decreased the number of viable A. polyphaga to less than 1 cell mL−1. Azole type antimicrobial compounds such as fluconazole, ketoconazole, and itraconazole are used clinically as antifungal agents（Sheehan et al., 1999）. The antifungal activity of these agents results from competitive inhibition of CYP51, an enzyme involved in sterol biosynthesis（Aoyama et al., 1984）, and the antifungal mechanism through which azole agents affect membrane-associated proteins is believed to be related to their solubility in the eukaryotic lipid layer. The targets of azole agents are different from the targets of CHX, which targets the cytoplasmic membrane and is classi-
fied as a cationic surfactant like the quaternary ammonium salts. A previous report demonstrated that 5 mg L −1 of chlorhexidine diacetate induces leakage of pentose, indicative of damage to the A. castellanii cytoplasmic membrane（Turner et al., 2004）. The series of thiazole derivatives we synthesized contain no cationic moieties. The amoebicidal activity of 4b, which is comparable to that of CHX, could thus be attributed to a synergistic effect resulting from insult to the cell membrane due to hydrophobic interaction between the membrane and the long alkyl chain of the thiazole compound combined with inhibition of enzymes following passage of the compound through the membrane. Future studies should evaluate the amoebicidal activity of the synthesized thiazole derivatives against cysts. Because Acanthamoeba trophozoites can form highly resistant cysts in response to adverse conditions, the cysts tend to be more resistant to disinfectants than the trophozoites（Beattie et al., 2003）. Regrettably, compounds 4a-c exhibited inconsistent biological activity. Compound 4b demonstrated no antibacterial activity against E. coli at a concentration of 500 mg L −1 as assessed using a general MIC assay, but did inhibit Staphylococcus aureus with an MIC of 3.9 mg L−1（data not shown）. Furuhata et al.（2010） reported that of 34 bacterial strains isolated from stock solutions in contact lens storage cases, 21 were nonfermenting gram-negative bacilli. For thiazole compounds to be used in contact lens stock solutions, their antibacterial potency must therefore be improved, especially with respect to their activity against gram-negative bacteria. Fortunately, the compounds we synthesized have an amino group at the 2-position of the thiazole ring, enabling further chemical modification（e.g., modification with amino acids or peptides, or glycosylation） that may improve their antimicrobial activity. Cytotoxicity The potential toxicity of TEE-n（4a-e）against human fibroblasts and sheep erythrocytes was evaluated and compared to that of Flu and CHX（Table 2）. The IC50 values for compounds 4b and 4c against human fibroblasts were 53±5.0 and 130±0.9 mg L–1, respectively. These values were higher than those of Flu and CHX, which exhibited IC50 values of 32±10 and 24±5.4 mg L–1, respectively. No cytotoxic activity was observed with compound 4a at concentrations as high as 150 mg L−1. The cytotoxicity of compounds 4a-c, in which the alkyl chain length increases from 8 to 12, respectively, increased with increasing alkyl chain length. A similar relationship between alkyl chain length and hemolytic activity has been reported for cationic biocides incorporating long alkyl chains（Shirai et al., 2005; Kourai et al., 2006）.
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TABLE 2. Cytotoxicity of the TEE-n series（4a-e）, and of Flu and CHX as agents used for comparison against human neonate dermal fibroblasts and sheep red blood cells. Cytotoxic activity（mg L−1）a TEE-n series 4a
4b
4c
Comparative agents 4d
IC50
＞150
53±5.0
130±0.9
n.t.
HC50
＞150
120±5.7
＞150
＞150
b
4e
Flu
CHX
n.t.
32±10
24±5.4
＞150
＞300
＞300
Cytotoxic activity of compounds were determined by the 50％ inhibitory concentration（IC50）against human neonate dermal fibroblasts, and by the 50％ hemolytic concentration（HC50）in sheep erythrocytes within concentrations at which agents can be possibly dissolved. Values are the mean ± S.D. obtained from three independent experiments. bnot tested. a
We were unable to determine the IC 50 values for compounds 4d and 4e due to their low solubility in the growth medium we used. The concentration of compound 4b necessary to induce hemolysis in 50％ of erythrocytes（HC50）was 120±5.7 mg L–1. Within the range of concentrations that could be dissolved in the test buffer, compounds 4a, 4c-e, Flu, and CHX showed no hemolytic activity. Although the hemolytic toxicity of compound 4b was the highest among the compounds we tested, its HC50 was much higher than that of benzalkonium chloride（11 mg L−1）used widely in food industry and hospitals as reported previously by our group（Kourai et al., 2006）. Our data thus suggest that compounds 4a-c are safer than Flu and CHX.
CONCLUSION Here, we reported on the synthesis and characterization of the in vitro antiamoebic activity and cytotoxicity of five thiazole derivatives. The results of both antiamoebic activity（Fig.3）and cytotoxicity（Table 2） assays suggest that thiazole derivative 4b, which is a low-molecular-weight compound consisting of 2-aminothiazole, a decyl group, and ethyl acetate, is a stronger and safer amoebicidal agent than the other TEE-n series compounds we synthesized or the commonly used commercial agents Flu and CHX.
Appendices Chemistry The results of mass spectral, NMR, and elemental analyses of 2a-e and 3a-e are found in the appendices of Shirai et al.（2013）. This section includes data only for the appearance, yield, m.p., and Rf for 2a-e and 3a-e. 4-Octyl-3-oxobutyric acid ethyl ester（2a） Yellowish liquid; yield: 48％; R f（n-hexane：ethyl acetate＝4：1, v/v）: 0.64.
4-Decyl-3-oxobutyric acid ethyl ester（2b） Yellowish liquid; yield: 46％; R f（n-hexane：ethyl acetate＝4：1, v/v）: 0.64. 4-Dodecyl-3-oxobutyric acid ethyl ester（2c） White crystal; yield: 43％; m.p.: 23-25℃; R f （n-hexane：ethyl acetate＝4：1, v/v）: 0.64. 4-Tetradecyl-3-oxobutyric acid ethyl ester（2d） White crystal; yield: 33％; m.p.: 33-34℃; R f （n-hexane：ethyl acetate＝4：1, v/v）: 0.63. 4-Hexadecyl-3-oxobutyric acid ethyl ester（2e） White crystal; yield: 31％; m.p.: 43-44℃; R f （n-hexane：ethyl acetate＝4：1, v/v）: 0.63 （±）-4-Octyl-4-bromo-3-oxobutyric acid ethyl ester（3a） 25 ; Brownish oily; yield: 98％;［α］ D 0.00（c 0.67 in CHCl3） ethyl acetate＝4：1, v/v） : 0.63. R（n-hexane: f （±）-4-Decyl-4-bromo-3-oxobutyric acid ethyl ester（3b） 25 ; Brownish oily; yield: 92％;［α］ D 0.00（c 0.80 in CHCl3） ethyl acetate＝4：1, v/v） : 0.65. R（n-hexane: f （＋）-4-Dodecyl-4-bromo-3-oxobutyric acid ethyl ester（3c） 25 Brownish oily; yield: 95％;［α ］D ＋0.30（c 0.67 in ethyl acetate＝4：1, v/v）: 0.67. CHCl3）; R（n-hexane: f （＋） -4-Tetradecyl-4-bromo-3-oxobutyric acid ethyl ester（3d） 25 Brownish oily; yield: 84％;［α ］D ＋0.75（c 1.06 in ethyl acetate＝4：1, v/v）: 0.70. CHCl3）; R（n-hexane: f （±） -4-Hexadecyl-4-bromo-3-oxobutyric acid ethyl ester（3e） 25 Yellowish oily; yield: 85％;［α］ ; D 0.00（c 1.00 in CHCl3） ethyl acetate＝4：1, v/v） : 0.72. R（n-hexane: f
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Ethyl（5-octyl-2-amino-1,3-thiazol-4-yl）acetate （4a） Data summarized in Table 1. Ethyl（5-decyl-2-amino-1,3-thiazol-4-yl）acetate （4b） White crystal; yield: 75％; m.p.: 56-58℃; R f （n-hexane: ethyl acetate＝1：1, v/v）: 0.32; 1H-NMR （400 MHz, CDCl3, Me4Si, δin ppm）: 0.84（3H, t, J＝ 7.0 Hz, decyl group CH3）, 1.17-1.37（17H, m, decyl , 1.51-1.59（2H, m, group（CH2） 7 and ethyl group CH3） , 2.58（3H, s, CCH3） , 2.65（2H, t, J decyl group CH2） ＝7.6 Hz, CCH2）, 3.63（2H, s, CH2CO）, 4.12（2H, q, J ＝7.2 Hz, COOCH 2）; 13C-NMR（100 MHz, CDCl 3, Me4Si, δin ppm）: 14.1, 14.2, 18.8, 22.7, 26.4, 29.1, 29.3, 29.3, 29.5, 29.5, 31.7, 31.9, 34.7, 61.0, 135.6, ＋ : 142.6, 162.8, 170.3; MS （calcd/found）［M］ 325.51/325; Anal. calcd. for C17H30N2O2S： C, 62.54; H, 9.26; N, 8.58. Found: C, 62.39; H, 9.06; N, 8.62. Ethyl（5-dodecyl-2-amino-1,3-thiazol-4-yl）acetate （4c） White crystal; yield: 43％; m.p.: 57-59°C; R f （n-hexane: ethyl acetate＝1：1, v/v）: 0.33; 1H-NMR （400 MHz, CDCl3, Me4Si, δin ppm）: 0.81（3H, t, J＝ 6.3 Hz, dodecyl group CH 3）, 1.16-1.25（21H, m, , 1.48-1.54 dodecyl group（CH2） 9 and ethyl group CH3） , 2.57（3H, s, CCH3）, 2.62 （2H, m, dodecyl group CH2） , 3.63（2H, s, CH2CO）, 4.09 （2H, t, J＝7.6 Hz, CCH2） （2H, q, J＝7.2 Hz, COOCH2）; 13C-NMR（100 MHz, CDCl3, Me4Si, δin ppm）: 14.1, 14.2, 18.9, 22.7, 26.4, 29.1, 29.3, 29.3, 29.5, 29.6, 29.6, 29.6, 31.7, 31.9, 34.7, 61.0, 135.6, 142.6, 162.7, 170.3; MS（calcd/ ＋ : 353.56/353; Anal. calcd. for f o u n d ）［ M ］ C19H34N2O2S: C, 64.36; H, 9.67; N, 7.90. Found: C, 64.42; H, 9.55; N, 7.97. Ethyl（5-tetradecyl-2-amino-1,3-thiazol-4-yl） acetate（4d） White crystal; yield: 55％; m.p.: 63-65℃; R f （n-hexane: ethyl acetate＝1：1, v/v）: 0.34; 1H-NMR （400 MHz, CDCl3, Me4Si, δin ppm）: 0.85（3H, t, J＝ 6.8 Hz, tetradecyl group CH3）, 1.21-1.29（25H, m, , 1.52tetradecyl group（CH2） 11 and ethyl group CH3） 1.60（2H, m, tetradecyl group CH 2）, 2.60（3H, s, CCH3）, 2.66（2H, t, J＝7.6 Hz, CCH2）, 3.65（2H, s, , 4.14（2H, q, J＝7.2 Hz, COOCH2） ; 13C-NMR CH2CO） （100 MHz, CDCl3, Me4Si, δin ppm）: 14.1, 14.2, 18.3, 22.7, 26.1, 29.1, 29.3, 29.4, 29.5, 29.5, 29.6, 29.6, 29.7, 29.7, 31.5, 31.9, 33.9, 61.1, 135.4, 142.9, 164.1, ＋ : 381.62/381; Anal. 170.0; MS（calcd/found） ［M］ calcd. for C21H38N2O2S: C, 65.92; H, 10.01; N, 7.32. Found: C, 65.69; H, 9.82; N, 7.28.
Ethyl（5-hexadecyl-2-amino-1,3-thiazol-4-yl） acetate（4e） White crystal; yield: 45％; m.p.: 72-74℃; R f （n-hexane: ethyl acetate＝1：1, v/v）: 0.35; 1H-NMR : 0.86（3H, t, J＝ （400 MHz, CDCl3, Me4Si, δin ppm） 6.7 Hz, hexadecyl group CH3）, 1.16-1.26（29H, m, , 1.53 hexadecyl group（CH2） 13 and ethyl group CH3） （2H, m, hexadecyl group CH2）, 2.67（3H, s, CCH3）, 2.67（2H, t, J＝7.4 Hz, CCH2）, 3.72（2H, s, CH2CO）, 4.15（2H, q, J＝7.2 Hz, COOCH 2）; 13C-NMR（100 MHz, CDCl3, Me4Si, δin ppm）: 14.1, 14.2, 18.4, 22.6, 26.1, 29.1, 29.2, 29.3, 29.3, 29.4, 29.4, 29.5, 29.7, 29.7, 29.8, 29.8, 31.6, 31.8, 34.1, 61.4, 135.5, 142.2, ＋ : 409.67/409; 163.1, 170.5; MS（calcd/found） ［M］ Anal. calcd. for C23H42N2O2S: C, 67.27; H, 10.31; N, 6.82. Found: C, 67.03; H, 10.16; N, 6.54. REFERENCES Andreani, A., Granaiola, M., Leoni, A., Locatelli, A., Morigi, R., and Rambaldi, M.（2001）Synthesis and antitubercular activity of imidazo［2,1-b］thiazoles. Eur. J. Med. Chem., 36, 743-746. Anger, C., and Lally, J.M.（2008）Acanthamoeba: a review of its potential to cause keratitis, current lens care solution disinfection standards and methodologies, and strategies to reduce patient risk. Eye Contact Lens, 34, 247-253. Aoyama, Y., Yoshida, Y., and Sato, R.（1984）Yeast cytochrome P-450 catalyzing lanosterol 14α-demethylation. II. Lanosterol metabolism by purified P-450（14DM）and by intact microsomes. J. Biol. Chem., 259, 1661-1666. Degerli, S., Tepe, B., Celiksoz, A., Berk, S., and Malatyali, E. （2012）In vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites. Exp. Parasitol., 131, 20-24. Devínsky, F., Lacko, I., Mlynarcˇ ík, D., Racˇ anský, V., and Krasnec, Ĺ .（1985）Relationship between critical micelle concentration and minimum inhibitory concentrations for some none-aromatic quaternary ammonium salts and amine oxides. Tenside Detergents, 22, 10-15. Furuhata, K., Ishizaki, N., Kawakami, Y., and Fukuyama, M.（2010）Bacterial contamination of stock solutions in storage cases for contact lens, and the disinfectant-resistance of isolates. Biocontrol Sci., 15, 81-85. Gray, T.B., Cursons, R.T., Sherwan, J.F., and Rose, P.R. （1995）Acanthamoeba, bacterial, and fungal contamination of contact lens storage cases. Br. J. Ophthalmol., 79, 601-605. Hamzě, A., Rubi, E., Arnal, P., Boisbrun, M., Carcel, C., Salom-Roig, X., Maynadier, M., Wein, S., Vial, H., and Calas, M.（2005）Mono- and bis-thiazolium salts have potent antimalarial activity. J. Med. Chem., 48, 3639-3643. Holla, B.S., Malini, K.V., Rao, B.S., Sarojini, B.K., and Kumari, N.S.（2003）Synthesis of some new 2,4-disubstituted thiazoles as possible antibacterial and anti-inflammatory agents. Eur. J. Med. Chem., 38, 313-318. Kobayashi, T., Gibbon, L., Mito, T., Shiraishi, A., Uno, T., and Ohashi, Y.（2011）Efficacy of commercial soft contact lens disinfectant solutions against Acanthamoeba. Jpn. J. Ophthalmol., 55, 547-557. Kourai, H., Yabuhara, T., Shirai, A., Maeda, T., and
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