Turk J Biochem 2018; 43(3): 220–227

Research Article

Gülhan Turan-Zitouni, Betül Kaya Çavuşoğlu*, Begüm Nurpelin Sağlık and Ulviye Acar Çevik Synthesis and antimicrobial activities of some novel compounds Bazı Yeni Tiyazol Bileşiklerinin Sentezi ve Antimikrobiyal Aktiviteleri https://doi.org/10.1515/tjb-2017-0093 almost four-fold better than against C. albi- Received March 29, 2017; accepted June 12, 2017; previously cans with MIC90 value of 1.95. ­published online November 28, 2017 Conclusion: The current study contributed to the knowl- Abstract edge of the antimicrobial activity of thiazole bearing compounds. Objective: The advent of resistant pathogenic microorgan- Keywords: Antibacterial activity; activity; Thi- isms against current antimicrobial drugs prompted scien- ; Broth microdilution method. tists to investigate novel molecules with new mechanisms. In this paper, some new 2-[2-[4-(ethyl/phenyl)cyclohex- ylidene]hydrazinyl]-4-(4-substitutedphenyl)thiazole Özet (2a–2o) derivatives were synthesized and studied for their antimicrobial activities. Amaç: Mevcut antimikrobiyal ilaçlara karşı dirençli Materials and methods: The title compounds (2a–2o) were patojen mikroorganizmaların ortaya çıkışı bilim insanla- obtained via the reaction of 4-(ethyl/phenyl)cyclohexane- rını farklı mekanizmalara sahip yeni molekülleri keşfet- 1-one with appropriate phenacyl bromide in ethanol at meye sevk etmiştir. Bu çalışmada, bazı yeni 2-[2-[4-(etil/ room temperature. The chemical structures of the com- fenil)sikloheksiliden]hidrazinil]-4-(4-sübstitüefenil) pounds were elucidated by FT-IR, 1H-NMR, 13C-NMR, tiyazol (2a–2o) türevleri sentezlenmiş ve antimikrobiyal HRMS and elemental analysis. Antimicrobial activity of etkileri araştırılmıştır. the compounds was measured by using broth microdi- Metot: Final bileşikleri (2a–2o) 4-(etil/fenil)siklohek- lution method. Chloramphenicol and ketoconazole were san-l-on ile uygun fenaçil bromürlerin oda ısısında etanol used as reference drugs. içinde reaksiyona sokulmasıyla elde edilmiştir. Bileşik- Results: Among the synthesized compounds, 2-[2-(4-phe- lerin kimyasal yapıları FT-IR, 1H-NMR, 13C-NMR, HRMS nylcyclohexylidene)hydrazinyl]-4-phenylthiazole (2h) spektrum verileri ve elementel analiz kullanılarak aydın- and 2-[2-(4-phenylcyclohexylidene)hydrazinyl]-4-(4-chlo- latılmıştır. Antimikrobiyal aktivite çalışmaları broth mik- rophenyl)thiazole (2l) have been found to exhibit potency rodilüsyon yöntemi ile tespit edilmiştir. Kloramfenikol ve ketokonazol referans ilaç olarak kullanılmıştır. Bulgular: Sentezlenen bileşikler arasında, 2-[2-(4-fenilsik- loheksiliden)hidrazinil]-4-feniltiyazol (2h) ve 2-[2-(4-fenil- *Corresponding author: Betül Kaya Çavuşoğlu, Anadolu University, siklohekziliden)hidrazinil]-4-(4-klorofenil)tiyazol (2l) Faculty of Pharmacy, Department of Pharmaceutical Chemistry, türevlerinin 1.95 MIC değeri ile C. albicans’a karşı keto- Eskişehir, Turkey, e-mail: [email protected] 90 konazolden yaklaşık dört kat daha etkili olduğu tespit Gülhan Turan-Zitouni: Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Eskişehir, Turkey edilmiştir. Begüm Nurpelin Sağlık and Ulviye Acar Çevik: Anadolu University, Sonuç: Bu çalışma tiyazol taşıyan bileşiklerin antimikro- Faculty of Pharmacy, Department of Pharmaceutical Chemistry, biyal etki gösterdiğini desteklemiştir. Eskişehir, Turkey; and Anadolu University, Faculty of Pharmacy, Doping and Narcotic Compounds Analysis Laboratory, 26470 Anahtar Kelimeler: Antibakteriyel aktivite; Antifungal Eskişehir, Turkey aktivite; Tiyazol; Broth mikrodilüsyon yöntemi. Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds 221

Introduction Chemicals (Merck KGaA, Darmstadt, Germany). All melting points (m.p.) were determined by MP90 digital One of the most significant current discussions in the melting point apparatus (Mettler Toledo, OH, USA) and world is the therapy of infectious diseases due to the were uncorrected. All reactions were monitored by thin- increasing appearance of resistant pathogenic micro- layer chromatography (TLC) using Silica Gel 60 F254 TLC organisms against present antibacterial and antifungal plates (Merck KGaA, Darmstadt, Germany). Spectroscopic drugs [1, 2]. Recent research, thus, has tended to focus data were recorded with the following instruments: IR, on developing new effective antimicrobial agents that Shimadzu Affinity 1S spectrophotometer (Shimadzu, act through different mechanisms than the conventional Tokyo, Japan); NMR, Bruker DPX 500 NMR spectrometer drugs, particularly for the treatment of the of (Bruker Bioscience, Billerica, MA), in DMSO-d6, using hospitalized and immunosuppressed patients [3]. There- TMS as internal standard; M + 1 peaks were determined fore, the disclosure of novel and powerful antibacterial by Shimadzu LC/MS ITTOF system (Shimadzu, Tokyo, and antifungal drugs is very necessary. Japan). Elemental analyses were performed on a Per- Considering antimicrobial agents with innovative kin-Elmer EAL 240 elemental analyser (Perkin-Elmer, mode of actions, various heterocyclic rings have attracted Norwalk, USA). a great interest over the years owing to their different biological activities. Among diverse heterocyclic com- pounds, and their derivatives are crucial scaf- General procedure for synthesis folds in medicinal chemistry. In many pharmaceutically of the compounds active compounds and natural products such as including thiamin and penicillin G, thiazole ring composes the scaf- General procedure for the synthesis of 2-[4-(ethyl/ fold of core molecular structure. Thiazole compounds are phenyl)cyclohexylidene]hydrazine-1-carbothioamide accompanied with improved lipophilicity and are metabo- derivatives (1a, 1b) lized via known biochemical reactions [4]. The enthusiasm for thiazoles is because of their potential natural action 4-(Ethyl/phenyl)cyclohexane-1-one (29 mmol) was dis- and magical physicochemical characteristics thus, some solved in ethanol (100 mL). Thiosemicarbazide (29 mmol) many potent drugs such as sulfathiazole (antimicrobial and a catalytic amount of acetic acid were added and the drug) and abafungin (antifungal drug) contain a thiazole reaction mixture was refluxed for 2 h. After completion ring. Thiazole and its derivatives are important pharma- of reaction, the mixture was cooled, precipitated product cophore and they have a broad range of biological activi- was filtered and recrystallized from ethanol [24, 25]. ties including antimicrobial [5–10], antitumor [11, 12], anti-inflammatory [13], anti-cancer [14], anti-tubercular [15, 16], antiviral [17], antioxidant [18], anti HIV [19], anti- General procedure for the synthesis of hypertensive [20], antischizophrenia [21], antiallergic [22] 4-(4-substitutedphenyl)-2-[2-[4-(ethyl/phenyl) and analgesic activity [23]. Besides that, it was clear that cyclohexylidene]hydrazinyl]thiazole (2a–2o) thiazoles have gotten significant consideration because of their effective properties as antimicrobial agents. Compounds 1a or 1b (2 mmol) and appropriate phenacyl Based on the above-mentioned findings to recognize bromide (2 mmol) were dissolved in ethanol (25 mL). The new candidates that may be with a great value in design- reaction mixture stirred at room temperature for 1–8 h. ing new, potent and selective antimicrobial agents, we After TLC screening, precipitated product was filtered and report in this paper the synthesis and antimicrobial activ- recrystallized from ethanol. ity of some new thiazole derivatives. 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(p-meth- ylphenyl)thiazole (2a) Yield 65%. m.p. 187°C. IR (KBr, −1 cm ): ʋmax 3309 (N–H stretching), 3105–3017 (aromatic Materials and methods C–H), 2953–2842 (aliphatic C–H), 1602–1445 (C=N and C=C 1 stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ 0.91 (t,

Chemicals 3H, CH3), 1.03–1.43 (m, 5H, CH2, cyclohexyl-H), 1.88–1.95 (m, 3H, cyclohexyl-H), 2.16–2.35 (m, 3H, cyclohexyl-H),

All chemicals were purchased from Sigma-Aldrich Chemi- 2.31 (s, 3H, CH3), 7.22 (d, J = 8.3 Hz, 2H, Ar-H), 7.31 (s, 1H, cals (Sigma-Aldrich Corp., St. Louis, MO, USA) and Merck thiazole-H), 7.71 (d, J = 8.3 Hz, 2H, Ar-H), 8.20 (1H, s, NH). 222 Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds

13 C-NMR (125 MHz, DMSO-d6, ppm) δ 11.97 (CH3), 21.26 175.58 (C). For C17H20ClN3S calculated: 61.15% C, 6.04% H,

(CH3), 26.82, 31.64, 32.21, 32.82, 34.37, 37.28 (CH2), 105.2 10.62% Cl, 12.59% N, 9.60% S; found: 61.28% C, 6.05% H, (CH-thiazole), 125.98, 126.23, 129.08, 129.61 (CH), 129.98, 10.64% Cl, 12.56% N, 9.61% S. HRMS (m/z): [M + H]+ calcd

130.2 (C ), 149.1 (C ), 153.20(C=N). For C18H23N3S calculated: for C17H20ClN3S: 334.1139; found 334.1130. 68.97% C, 7.40% H, 13.41% N, 10.23% S; found: 69.12% C, 7.38% H, 13.43% N, 10.21% S. HRMS (m/z): [M + H]+ calcd 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4-fluo- for C18H23N3S: 314.1685; found 314.1685. rophenyl)thiazole (2e) Yield 68%. m.p. 195°C. IR (KBr, −1 cm ): ʋmax 3318 (N–H stretching), 3108–3028 (aromatic 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4-meth- C–H), 2958–2842 (aliphatic C–H), 1624–1487 (C=N and C=C 1 oxyphenyl)thiazole (2b) Yield 64%. m.p. 187°C. IR stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ 0.90 (t, −1 (KBr, cm ): ʋmax 3324 (N–H stretching), 3105–3017 (aro- 3H, CH3), 1.01-1.39 (m, 5H, CH2, cyclohexyl-H), 1.85–1.93 (m, matic C–H), 2953–2842 (aliphatic C–H), 1608–1455 (C=N 3H, cyclohexyl-H), 2.18–2.34 (m, 3H, cyclohexyl-H), 7.21– 1 and C=C stretching). H-NMR (500 MHz, DMSO-d6, ppm) 7.25 (m, 3H, Ar-H), 7.86–7.89 (m, 2H, Ar-H), 10.84 (s, 1H, NH). 13 δ 0.89 (t, 3H, CH3), 1.05–1.43 (m, 5H, CH2, cyclohexyl-H), C-NMR (100 MHz, DMSO-d6, ppm) δ 11.97 (CH3), 26.74 (CH),

1.88–2.37 (m, 6H, cyclohexyl-H), 3.78 (s, 3H, OCH3), 6.97 28.69 (CH), 31.63, 32.84, 34.40, 37.19 (CH2), 103.35, 115.74 (d, J = 9.1 Hz, 2H, Ar-H), 7.07 (s, 1H, thiazole-H), 7.76 (d, (CH-thiazole), 115.92, 127.86, 127.92, 132.02 (CH) 155.86, 13 J = 8.5 Hz, 2H, Ar-H), 10.75 (s, 1H, NH). C-NMR (100 MHz, 160.99, 162.93, 170.66 (C). For C17H20FN3S calculated: 64.32%

DMSO-d6, ppm) δ 11.97 (CH3), 26.82, 28.67, 31.64, 32.83, C, 6.35% H, 5.99% F, 13.24% N, 10.10% S; found: 64.45% C, + 34.37, 38.13, 39.66 (CH2), 55.60 (OCH3), 114.42, 114.90, 6.36% H, 5.97% F, 13.26% N, 10.12% S. HRMS (m/z): [M + H]

127.38 (CH). For C18H23N3OS calculated: 65.62% C, 7.04% calcd for C17H20FN3S: 318.1435; found 318.1457. H, 12.75% N, 4.86% O, 9.73% S; found: 65.72% C, 7.02% H, 12.77% N, 4.85% O, 9.74% S. HRMS (m/z): [M + H]+ calcd for 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4-nitro-

C18H23N3OS: 330.1635; found 330.1641. phenyl)thiazole (2f) Yield 70%. m.p. 181°C. IR (KBr, −1 cm ): ʋmax 3311 (N–H stretching), 3078–3013 (aromatic 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4- C–H), 2997–2858 (aliphatic C–H), 1612–1479 (C=N and 1 bromophenyl)thiazole (2c) Yield 68%. m.p. 204°C. IR C=C stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ −1 (KBr, cm ): ʋmax 3307 (N–H stretching), 3087-3004 (aro- 0.87–0.92 (m, 3H, CH3), 1.05–2.33 (m, 8H, CH2, cyclohexyl- matic C–H), 2951–2856 (aliphatic C–H), 1604–1481 (C=N H), 2.03–3.01 (m, 3H, cyclohexyl-H), 8.09–8.34 (m, 4H, 1 13 and C=C stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ Ar-H), 8.62 (s, 1H, thiazole-H), 10.97 (s, 1H, NH). C-NMR

0.90 (t, 3H, CH3), 1.24–1.34 (m, 5H, CH2, cyclohexyl-H), 1.59– (125 MHz, DMSO-d6, ppm) δ 11.85 (CH3), 12.13 (CH2), 26.81,

2.35 (m, 6H, cyclohexyl-H), 7.64–7.70 (m, 4H, Ar-H), 8.35 28.20, 31.63, 34.39, 38.08 (CH2), 108.62 (CH-thiazole), (s, 1H, thiazole-H), 10.89 (s, 1H, NH). 13C-NMR (100 MHz, 121.16, 124.14, 126.74, 129.18, 129.55 (CH), 146.55, 147.50,

DMSO-d6, ppm) δ 11.85 (CH3), 12.13 (CH2), 28.11, 29.57, 32.22, 170.96 (C). For C17H20N4O2S calculated: 59.28% C, 5.85% H,

34.33, 37.28 (CH2), 38.23 (CH), 117.85 (CH-thiazole), 128.33, 16.27% N, 9.29% O, 9.31% S; found: 59.36% C, 5.84% H, 128.50, 128.64, 132.18 (CH), 132.37, 132.28, 153.44, 175.58 16.30% N, 9.31% O, 9.32% S. HRMS (m/z): [M + H]+ calcd for

(C). For C17H20BrN3S calculated: 53.97% C, 5.33% H, 21.12% C17H20N4O2S: 345.1380; found 345.1373. Br, 11.11% N, 8.48% S; found: 53.81% C, 5.31% H, 21.17% Br, 11.09% N, 8.47% S. HRMS (m/z): [M + H]+ calcd for 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4-

C17H20BrN3S: 378.0634; found 378.0630. cyanophenyl)thiazole (2g) Yield 69%. m.p. 187°C. −1 IR (KBr, cm ): ʋmax 3317 (N–H stretching), 3095–3025 2-[2-(4-Ethylcyclohexylidene)hydrazinyl]-4-(4-chlo- (aromatic C–H), 2984–2858 (aliphatic C–H), 2358 (C≡N rophenyl)thiazole (2d) Yield 66%. m.p. 189°C IR (KBr, stretching), 1600–1435 (C=N and C=C stretching). 1H-NMR −1 cm ): ʋmax 3304 (N–H stretching), 3098–3006 (aromatic (500 MHz, DMSO-d6, ppm) δ 0.90 (t, 3H, CH3), 1.26–1.32

C–H), 2995–2872 (aliphatic C–H), 1604–1487 (C=N and C=C (m, 5H, CH2, cyclohexyl-H), 1.60–2.35 (m, 6H, cyclohexyl- 1 stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ 0.91 (t, H), 7.99 (d, J = 8.3 Hz, 2H, Ar-H), 8.21 (d, J = 8.4 Hz, 2H, 13 3H, CH3), 1.24–1.29 (m, 3H, CH2, cyclohexyl-H), 1.58–2.32 Ar-H), 8.55 (s, 1H, thiazole-H), 10.71 (s, 1H, NH). C-NMR

(m, 8H, cyclohexyl-H), 7.59 (d, J = 8.6 Hz, 2H, Ar-H), 8.03 (100 MHz, DMSO-d6, ppm) δ 11.85 (CH3), 12.13 (CH2), 28.09,

(d, J = 8.5 Hz, 2H, Ar-H), 8.34 (s, 1H, thiazole-H), 10.97 (s, 32.21, 34.31, 37.28, 38.22 (CH2), 96.62 (CH), 112.22 (C), 119.23 13 1H, NH). C-NMR (100 MHz, DMSO-d6, ppm) δ 11.85 (CH3), (CN), 120.41, 126.98, 127.32, 133.13 (CH), 133.49, 179.5 (C),

12.13 (CH2), 28.11, 29.57, 32.22, 34.33, 37.28 (CH2), 38.23 (CH), 138.16. For C18H20N4S calculated: 66.64% C, 6.21% H, 17.27% 117.78 (CH-thiazole), 128.05, 129.46, 132.94 (CH), 133.55, N, 9.88% S; found: 66.45% C, 6.22% H, 17.29% N, 9.85% S. Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds 223

+ HRMS (m/z): [M + H] calcd for C18H20N4S: 325.1481; found 33.82, 34.44 (CH), 34.67 (C), 104.65 (CH-thiazole), 120.85 325.1488. (C), 126.57, 128.02, 130.27, 132.39 (CH), 146.35, 170.67 (C).

For C21H20BrN3S calculated: 59.16% C, 4.73% H, 18.74% 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-phe- Br, 9.86% N, 7.52% S; found: 59.25% C, 4.73% H, 18.70% nylthiazole (2h) Yield 75%. m.p. 199°C. IR (KBr, cm−1): Br, 9.88% N, 7.50% S. HRMS (m/z): [M + H]+ calcd for

ʋmax 3381 (N–H stretching), 3061–3007 (aromatic C–H), C21H20BrN3S: 426.0634; found 426.0631. 2920–2856 (aliphatic C–H), 1608–1444 (C=N and C=C 1 stretching). H-NMR (500 MHz, DMSO-d6, ppm) δ 1.57–1.73 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4-­ (m, 2H, cyclohexyl-H), 1.94–2.01 (m, 2H, cyclohexyl- chlorophenyl)thiazole (2l) Yield 70%. m.p. 175°C. IR (KBr, −1 H), 2.05–2.08 (m, 1H, cyclohexyl-H), 2.39–2.45 (m, 2H, cm ): ʋmax 3336 (N–H stretching), 3116–3012 (aromatic C–H), cyclohexyl-H), 2.85 (t, 1H, cyclohexyl-H), 3.18 (d, 1H, 2927–2841 (aliphatic C–H), 1635–1485 (C=N and C=C stretch- 1 cyclohexyl-H), 7.20 (t, J = 6.8 Hz, 1H, Ar-H), 7.87 (s, 1H, ing). H-NMR (500 MHz, DMSO-d6, ppm) δ 1.57–1.72 (m, 3H, Ar-H), 7.26–7.31 (m, 5H, Ar-H), 7.41 (t, 2H, Ar-H), 7.85 cyclohexyl-H), 1.91–2.01 (m, 1H, cyclohexyl-H), 2.04–2.09 (d, J = 7.5 Hz, 2H, Ar-H), 10.97 (br s, 1H, NH). 13C-NMR (m, 2H, cyclohexyl-H), 2.37–2.44 (m, 1H, cyclohexyl-H),

(125 MHz, DMSO-d6, ppm) δ 27.38, 33.19, 34.45, 35.02 2.85–2.88 (m, 1H, cyclohexyl-H), 3.17–3.19 (m, 1H, cyclohexyl-

(CH2), 43.03 (CH), 126.00, 126.57, 127.20, 127.93, 128.83, H), 7.20–7.23 (m, 1H, Ar-H), 7.27–7.32 (m, 5H, Ar-H), 7.45 (d,

129.04 (CH), 146.37, 170.54 (C). For C21H21N3S calculated: J = 8.30 Hz, 2H, Ar-H), 7.88 (d, J = 8.45 Hz, 2H, Ar-H), 10.98 (br 13 72.59% C, 6.09% H, 12.09% N, 9.23% S; found: 72.80% C, s, 1H, NH). C-NMR (125 MHz, DMSO-d6, ppm) δ 27.36, 33.17, + 6.07% H, 12.10% N, 9.24% S. HRMS (m/z): [M + H] calcd 34.44, 37.21 (CH2), 43.02 (CH), 104.54 (CH-thiazole), 126.57, for C21H21N3S: 348.1529; found 348.1546. 127.20, 127.69, 128.83, 129.05, 129.35, 132.24 (CH), 141.0, 146.36,

170.68 (C). For C21H20ClN3S calculated: 66.04% C, 5.28% H, 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(p- 9.28% Cl, 11.00% N, 8.40% S; found: 65.88% C, 5.27% H, methylphenyl)thiazole (2i) Yield 69%. m.p. 202°C. 9.26% Cl, 11.03% N, 8.41% S. HRMS (m/z): [M + H]+ calcd for −1 IR (KBr, cm ): ʋmax 3334 (N–H stretching), 3116–3021 C21H20ClN3S: 382.1139; found 382.1132. (aromatic C–H), 2972–2853 (aliphatic C–H), 1606–1457 1 (C=N and C=C stretching). H-NMR (500 MHz, DMSO-d6, 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4- ppm) δ 1.58–1.74 (m, 2H, cyclohexyl-H), 1.96–2.03 (m, fluorophenyl)-thiazole (2m) Yield 68%. m.p. 182°C. −1 2H, cyclohexyl-H), 2.40–2.45 (m, 4H, cyclohexyl-H), 2.35 IR (KBr, cm ): ʋmax 3329 (N–H stretching), 3096–3008

(s, 3H, CH3), 2.86 (t, 1H, cyclohexyl-H), 7.18–7.33 (m, 9H, (aromatic C–H), 2976–2858 (aliphatic C–H), 1610–1489 13 1 Ar-H), 7.73 (d, J = 8.1 Hz, 1H, Ar-H), 11.09 (s, 1H, NH). C- (C=N and C=C stretching). H-NMR (500 MHz, DMSO-d6,

NMR (125 MHz, DMSO-d6, ppm) δ 21.26 (CH3), 27.43, 33.18, ppm) δ 1.60–1.68 (m, 2H, cyclohexyl-H), 1.97–2.09 (m, 3H,

33.82, 34.43, 34.99, 37.62 (CH2), 42.99 (CH), 126.00, 126.25, cyclohexyl-H), 2.39–2.47 (m, 2H, cyclohexyl-H), 2.85–2.88 126.58, 126.69, 127.19, 128.69, 128.84, 129.62, 129.99 (CH), (m, 1H, cyclohexyl-H), 3.17–3.20 (m 1H, cyclohexyl-H),

137.34, 146.34 (C), 170.42 (C–N). For C22H23N3S calculated: 7.18–7.32 (m, 8H, Ar-H), 7.87–7.90 (m, 2H, Ar-H), 11.07 (s, 13 73.09% C, 6.41% H, 11.62% N, 8.87% S; found: 73.26% C, 1H, NH). C-NMR (125 MHz, DMSO-d6, ppm) δ 27.42, 33.17, + 6.42% H, 11.65% N, 8.89% S. HRMS (m/z): [M + H] calcd 34.44, 36.77 (CH2), 43.01 (CH), 103.53 (CH-thiazole), 115.81, for C22H23N3S: 362.1685; found 362.1711. 115.98 (2CH), 126.01 (CH), 126.58, 127.20 (2CH), 127.95, 128.01 (2CH), 128.84, 131.58 (2CH), 146.34, 155.43, 161.09, 163.03,

2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4- 170.61 (C). For C21H20FN3S calculated: 69.01% C, 5.52% H, methoxyphenyl)thiazole (2j) [26] 5.20% F, 11.50% N, 8.77% S; found: 68.92% C, 5.50% H, 5.21% F, 11.52% N, 8.76% S. HRMS (m/z): [M + H]+ calcd for

2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4- C21H20FN3S: 366.1435; found 366.1443. bromophenyl)thiazole (2k) Yield 65%. m.p. 207°C. −1 IR (KBr, cm ): ʋmax 3309 (N–H stretching), 3105–3024 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4- (aromatic C–H), 2972–2869 (aliphatic C–H), 1610–1479 nitrophenyl)thiazole (2n) Yield 72%. m.p. 225°C. IR 1 −1 (C=N and C=C stretching). H-NMR (500 MHz, DMSO-d6, (KBr, cm ): ʋmax 3311 (N–H stretching), 3113–3015 (aro- ppm) δ 1.59–1.72 (m, 2H, cyclohexyl-H), 1.91–2.11 (m, 3H, matic C–H), 2947–2849 (aliphatic C–H), 1595–1469 cyclohexyl-H), 2.38–2.48 (m, 2H, cyclohexyl-H), 2.85–2.87 (C=N and C=C stretching), 1504–1338 (NO2 stretching). 1 (m, 1H, cyclohexyl-H), 3.17–3.19 (m, 1H, cyclohexyl-H), H-NMR (500 MHz, DMSO-d6, ppm) δ 1.57–1.73 (m, 2H, 7.18–7.32 (m, 6H, Ar-H), 7.52–7.80 (m, 4H, Ar-H), 10.99 (s, cyclohexyl-H), 1.97–2.01 (m, 2H, cyclohexyl-H), 2.05–2.09 13 1H, NH). C-NMR (125 MHz, DMSO-d6, ppm) δ 2 7. 3 7, 3 3 .1 7, (m, 1H, cyclohexyl-H), 2.40–2.45 (m, 2H, cyclohexyl-H), 224 Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds

2.85–2.89 (m, 1H, cyclohexyl-H), 3.18–3.21 (m, 1H, Antimicrobial activity assay cyclohexyl-H), 7.21–7.24 (m, 1H, Ar-H), 7.27–7.32 (m, 4H, Ar-H), 7.66 (s, 1H, thiazole-H), 8.12 (d, J = 8.7 Hz, 2H, Antimicrobial activity studies were performed according Ar-H), 8.28 (d, J = 8.6 Hz, 2H, Ar-H), 11.08 (s, 1H, NH). to the following guides CLSI reference M07-A9 broth micro­ 13 C-NMR (125 MHz, DMSO-d6, ppm) δ 27.39, 33.16, 34.45, dilution method [27] for bacterial strains and EUCAST

35.03 (CH2), 43.02 (CH), 108,69 (CH-thiazole), 124.57 definitive (EDef 7.1) method [28] for fungal strains. Tested (2CH), 126.58 (CH), 127.20 (2CH), 128.83 (2CH), 141.44, microorganism strains were: Escherichia coli (ATCC 35218)

146.34, 146.57, 148.94, 155.17, 170.98 (C). For C21H20N4O2S (E. coli 1), Escherichia coli (ATCC 25922) (E. coli 2), Klebsiella calculated: 64.27% C, 5.14% H, 14.28% N, 8.15% O, 8.17% pneumoniae (NCTC 9633), Pseudomonas aeuroginosa (ATCC S; found: 64.38% C, 5.12% H, 14.30% N, 8.17% O, 8.14% 27853), Salmonella typhimurium (ATCC 13311) Staphylococ- + S. HRMS (m/z): [M + H] calcd for C21H20N4O2S: 393.1380; cus aureus (ATCC 25923), Candida albicans (ATCC 24433), found 393.1380. Candida glabrata (ATCC 90030), Candida krusei (ATCC

6258) and Candida parapsilosis (ATCC 22019). MIC90 read- 2-[2-(4-Phenylcyclohexylidene)hydrazinyl]-4-(4- ings were accomplished twice for all compounds. As refer- cyanophenyl)thiazole (2o) Yield 74%. m.p. 202°C. IR ence drugs, chloramphenicol and ketoconazole were used. −1 (KBr, cm ): ʋmax 3307 (N–H stretching), 3111–3032 (aro- matic C–H), 2978–2883 (aliphatic C–H), 1600–1435 (C=N 1 and C=C stretching). H-NMR (500 MHz, DMSO-d6, ppm) Broth microdilution assay δ 1.62–1.70 (m, 2H, cyclohexyl-H), 1.98–2.07 (m, 3H, cyclohexyl-H), 2.32-2.37 (m, 2H, cyclohexyl-H), 2.82–2.87 Mueller-Hinton broth (Difco) was used to produce the bacte- (m, 1H, cyclohexyl-H), 3.16–3.19 (m, 1H, cyclohexyl-H), rial strains. The strains were incubated at 37 °C for 24h. The 7.17–7.22 (m, 4H, Ar-H), 7.27–7.30 (m, 5H, Ar-H), 7.73 (d, yeasts were produced in RPMI after night long incubation J = 8.1 Hz, 1H, Ar-H), 11.09 (s, 1H, NH). 13C-NMR (125 MHz, at 37 °C. The inoculation of test microorganisms adjusted to

DMSO-d6, ppm) δ 21.26, 27.37, 33.18, 34.45 (CH2) 43.02 (CH), match the turbidity of a Mac Farland 0.5 standard tube as 102.83(CH-thiazole), 126.02 (CH), 125.97 (2CH), 126.57 determined with a spectrophotometer. For antibacterial and (2CH), 127.20 (2CH), 128.83(2CH), 129.60, 137.22, 146.36, antifungal assays, the final inoculum size was 0.5–2.5 × 105

170.43 (C). For C22H20N4S calculated: 70.94% C, 5.41% H, cfu/mL. For test, the two-fold serial dilutions technique 15.04% N, 8.61% S; found: 71.12% C, 5.42% H, 15.00% utilized and test was carried out in Mueller–Hinton broth + N, 8.63% S. HRMS (m/z): [M + H] calcd for C22H20N4S: and RPMI at pH = 7. As controls, the last well on the micro- 373.1481; found 373.1477. plates including only inoculated broth was held. In order to

Table 1: Antibacterial activity of the compounds (2a–o) as MIC values (mg/mL).

Compound A B C D E F

2a >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2b >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2c >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2d >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2e >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2f >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2g >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2h >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2i >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2j >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2k >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2l >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2m >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2n >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2o >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL Chloramphenicol ≤1.95 μg/mL ≤1.95 μg/mL 3.9 μg/mL 250 μg/mL ≤1.95 μg/mL 15.62 μg/mL

A: Escherichia coli (ATCC 35218), B: Escherichia coli (ATCC 25922), C: Klebsiella pneumoniae (NCTC 9633), D: Pseudomonas aeuroginosa (ATCC 27853), E: Salmonella typhimurium (ATCC 13311), F: Staphylococcus aureus (ATCC 25923). Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds 225

Table 2: Antifungal activity of the compounds (2a–o) as MIC values (mg/mL).

Compound A B C D

2a >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2b >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2c >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2d >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2e >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2f >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2g >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2h ≤1.95 μg/mL 3.9 μg/mL ≤1.95 μg/mL 1.95 μg/mL 2i >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2j >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2k >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2l ≤1.95 μg/mL 15.6 μg/mL ≤1.95 μg/mL ≤1.95 μg/mL 2m 125 μg/mL 125 μg/mL ≤1.95 μg/mL 1.95 μg/mL 2n >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL 2o >1 mg/mL >1 mg/mL >1 mg/mL >1 mg/mL Ketoconazole 7.8 μg/mL ≤1.95 μg/mL ≤1.95 μg/mL ≤1.95 μg/mL

A: Candida albicans (ATCC 24433), B: Candida glabrata (ATCC 90030), C: Candida krusei (ATCC 6258), D: Candida parapsilosis (ATCC 22019).

present the MIC expressed in μg/mL, the last well with no equimolar quantities of compound 1a and 1b with appropri- growth of microorganism was registered. DMSO was used to ate phenacyl bromide in ethanol as solvent eventuated in the dissolve compounds for both the antibacterial and antifun- formation of the final compounds (2a–2o). gal assays. Further dilutions of the compounds and control Some characteristics of the synthesized compounds drugs in test medium were equipped in the range of 1000, are shown in Table 3. Structures of the obtained com- 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9 and 1.95 μg/mL con- pounds were confirmed by FT-IR, 1H-NMR, 13C-NMR, centrations with Mueller–Hinton broth, RPMI and Middle HRMS and elemental analysis. Brook medium. The finished plates were incubated for 24 h. In the IR spectra of the compounds (2a–2o), some sig- At the end of this period, resazurin (20 μg/mL) was added nificant specific bands were observed at 3381–3304 cm−1 into each well to control whether the growth in wells. After and 1635–1435 cm−1 belong to N–H, C=N and C=C bonds. 2 h incubation of completed plates including each micro- In the 1H NMR spectra of all final compounds, the organism, MIC90 values were confirmed with a microplate protons of cyclohexyl ring were observed at 1.01–3.18 ppm. reader at 590 nm excitation, 560 nm emission. Each experi- A singlet peak due to thiazole ring was resonated at ment in the antimicrobial assays was performed twice. The 7.07–8.62 ppm. The other protons in aromatic region were

MIC90 values are listed in Tables 1 and 2. observed at the range of 6.91–8.34 ppm. The signals cor- respond to N–H residue were observed at about 10.71– 11.09 ppm. The other aromatic and aliphatic protons were observed at the expected regions. Results and discussion In the 13C NMR spectrum of the compounds, the signals

belonging to –CH2–CH3 group in compounds 2a–2g, were Chemistry determined at 11.85–12.14 ppm. The carbon atoms belong- ing to cyclohexyl ring were determined at 21.26–43.03 ppm.

In order to develop new effective anti-microbial agents, we The signals belonging to C2 carbon of thiazole ring were synthesized a novel series of 4-(4-substitutedphenyl)-2-[2-[4- resonated at 159.29–175.58 ppm all the other aromatic and (ethyl/phenyl)cyclohexylidene] hydrazinyl]thiazole (2a–2o) aliphatic carbons were observed at expected regions. and studied their antibacterial and antifungal activities. The final compounds were synthesized according to the steps as shown in Scheme 1. Firstly, 1-[4-(ethyl/phenyl)cyclohex- Antimicrobial activity ylidene] thiosemicarbazide derivatives (compound 1a and 1b) were synthesized by the reaction of 4-(ethyl/phenyl) MICs were recorded as the minimum concentra- cyclohexanone with thiosemicarbazide. The reaction of tion of a compound that inhibits the growth of tested 226 Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds

R1 R1 H i + N NH2 H2N S S N O NH NH2 1a, 1b

R1 O R1 Br + R2 ii S N S NH NH2 N NH N R2

2a–2o

R1=C2H5– or C6H5–

Scheme 1: Synthesis of the compounds. Reactans and reagents: (i) EtOH, acetic acid, reflux, 2 h, (ii) EtOH, 1–8 h, room temperature.

Table 3: Some properties of the compounds. approximately four-fold better than a reference drug against C. albicans with MIC values of 1.95. In addition, Compound R R m.p. (°C) Yield (%) Molecular formula 90 1 2 ­compound 2h, 2l and 2m exhibited equal level of activity Ethyl CH 187 65 C H N S 2a 3 18 23 3 with ketoconazole against C. krusei and C. ­parapsilosis. 2b Ethyl OCH 184 64 C H N OS 3 18 23 3 Also compound 2h, 2l and 2m showed comparable activ- 2c Ethyl Br 204 68 C H BrN S 17 20 3 ities against C. glabrata and the other compounds were 2d Ethyl Cl 189 66 C17H20ClN3S found less active than the reference agent used. 2e Ethyl F 189 68 C17H20FN3S

2f Ethyl NO2 181 70 C17H20N4O2S Since, the addition of halogen substituents (in par-

2g Ethyl CN 189 69 C18H20N4S ticular F, Cl, Br, I) will increase the lipophilicity of mole- 2h Phenyl H 199 75 C H N S 21 21 3 cules and as consequence increase penetration to the 2i Phenyl CH 202 69 C H N S 3 22 23 3 bacterial cell [29], it appears that nonsubstitution or sub- 2j Phenyl OCH 192 72 C H N OS 3 22 23 3 stitution on para position with chloro or fluoro on phenyl 2k Phenyl Br 207 65 C21H20BrN3S

2l Phenyl Cl 175 70 C21H20ClN3S ring attached to the thiazole moiety contribute to the out-

2m Phenyl F 182 68 C21H20FN3S standing antifungal activity.

2n Phenyl NO2 225 72 C21H20N4O2S

2o Phenyl CN 202 74 C22H20N4S microorganisms. The results are summarized in Tables 1 Conclusion and 2. The antibacterial assessment showed that the In the present study, we synthesized a new series of compounds possess no useful inhibitory action. On the 4-(4-substitutedphenyl)-2-[2-[4-(ethyl/phenyl)cyclohex- other hand, some of the compounds tested illustrated ylidene] hydrazinyl]thiazole (2a–2o) derivatives and screen remarkable antifungal activity when compared with for their antibacterial and antifungal activity. According to reference drug, ketoconazole. In the antifungal activ- the results, it was observed that some of the compounds ity, compound 2h with nonsubstituted phenyl ring, exhibited remarkable effects. Among the them, compound compound 2l with para-chloro substituent on phenyl 2h with nonsubstituted phenyl ring and compound 2l with ring and compound 2m with para-fluoro substituent chloro substituent at para position on phenyl ring were on phenyl ring exhibited­ significant antifungal activity found to be the most promising antifungal agents with MIC against tested fungi species. According to results, it is values of 1.95 that is approximately four-fold better than the clear that besides nonsubstituted derivative, halogen reference drug chloramphenicol against C. albicans. substituted derivatives possessed enhanced antimicro- bial activity. In comparing their MIC values with that of Conflict of interest statement: The authors declare no ­chloramphenicol, compound 2h and 2l, showed potency conflicts of interest. Gülhan Turan-Zitouni et al.: Antimicrobial evaluation of thiazole compounds 227

15. Jeankumar VU, Kotagiri S, Janupally R, Suryadevara P, Sridevi References JP, Medishetti R, et al. Exploring the gyrase ATPase domain for tailoring newer anti-tubercular drugs: hit to lead optimization 1. Leeb M. Antibiotics: a shot in the arm. Nature 2004;431:892–3. of a novel class of thiazole inhibitors. Bioorg Med Chem Lett 2. O’Connell KM, Hodgkinson JT, Sore HF, Welch M, Salmond GP, 2015;23:588–601. Spring DR. Combating multidrug-resistant bacteria: current 16. Mathew V, Keshavayya J, Vaidya VP, Giles D. Studies on synthe- strategies for the discovery of novel antibacterials. Angew Chem sis and pharmacological activities of 1, 2, 4-triazolo [3, 4-b] 1, 3, 2013;52:10706–33. 4-thiadiazoles and their dihydro analogues. Arch Pharm Chem 3. Silver LL. Challenges of antibacterial discovery. Clin Microbiol Life Sci 2009;342:210–22. Rev 2011;24:71–109. 17. Li Z, Khaliq M, Zhou Z, Post CB, Kuhn RJ, Cushman M. Design, 4. Taori K, Paul VJ, Luesch H. Structure and activity of largazole, a synthesis, and biological evaluation of antiviral agents targeting potent antiproliferative agent from the floridian marine cyano- flavivirus envelope proteins. J Med Chem 2008;51:4660–71. bacterium Symploca sp. J Am Chem Soc 2008;130:1806–7. 18. Jaishree V, Ramdas N, Sachin J, Ramesh B. In vitro antioxi- 5. Koti RS, Kolavi GD, Hegde VS, Khaji IM. Vilsmeier Haack reaction dant properties of new thiazole derivatives. J Saudi Chem Soc of substituted 2-acetamidothiazole derivatives and their antimi- 2012;16:371–6. crobial activity. Ind J Chem 2006;45B:1900–4. 19. Bell FW, Cantrell AS, Hogberg M, Jaskunas SR, Johansson NG, 6. Guzeldemirci NU, Kucukbasmac O. Synthesis and antimicrobial Jordan CL, et al. Phenethylthiazolethiourea (PETT) compounds, a activity evaluation of new 1,2,4- and 1,3,4-thiadia- new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis zoles bearing imidazo[2,1-b] thiazole moiety. Eur J Med Chem and basic structure–activity relationship studies of PETT ana- 2010;45:63–8. logs. J Med Chem 1995;38:4929–36. 7. Bondock S, Khalifa W, Fadda AA. Synthesis and antimicrobial 20. Patt WC, Hamilton HW, Taylor MD, Ryan MJ, Taylor DG Jr, Con- evaluation of some new thiazole, thiazolidinone and thiazoline nolly CJ, et al. Structure–activity relationships of a series of derivatives starting from 1-chloro-3,4-dihydronaphthalene- 2-amino-4-thiazole containing renin inhibitors. J Med Chem 2-carboxaldehyde. Eur J Med Chem 2007;42:948–54. 1992;35:2562–72. 8. Holla BS, Karegoudar P, Karthikeyan MS, Prasad DJ, Mahal- 21. Jaen JC, Wise LD, Caprathe BW, Tecle H, Bergmeier S, Humblet inga M, Kumari NS. Synthesis of some novel 2,4-disubstituted CC, et al. 4-(1,2,5,6- Tetrahydro-1-alkyl-3-pyridinyl)-2-thiazo- thiazoles as possible antimicrobial agents. Eur J Med Chem lamines: a novel class of compounds with central dopamine 2008;43:261–7. agonist properties. J Med Chem 1990;33:311–17. 9. Pandeya SN, Sriram D, Nath G, DeClerq E. Synthesis, antibacte- 22. Hargrave KD, Hess FK, Oliver JT. N-(4-Substitutedthiazolyl) rial, antifungal and anti-HIV activities of Schiff and Mannich oxamic acid derivatives, new series of potent, orally active bases derived from isatin derivatives and N-[4-(40 - chlorophe- antiallergy agents. J Med Chem 1983;26:1158–63. nyl) thiazol-2-yl] thiosemicarbazide. Eur J Pharm Sci 1999;9: 23. Carter JS, Kramer S, Talley JJ, Penning T, Collins P, Graneto MJ, 25–31. et al. Synthesis and activity of sulfonamide-substituted 4,5-dia- 10. Zitouni GT, Kaplancıklı ZA, Yıldız MT, Chevallet P, Kaya D. ryl thiazoles as selective cyclooxygenase-2 inhibitors. Bioorg Synthesis and antimicrobial activity of 4-phenyl/cyclohexyl-5- Med Chem Lett 1999;9:1171–4. (1-phenoxyethyl)-3-[N-(2-thiazolyl)acetamido]thio-4H–1,2,4- 24. Richard JH, Robertson A, Ward J. Experiments on the synthesis of derivatives. Eur J Med Chem 2005;40:607–13. rotenone and its derivatives. Part XV. J Chem Soc 1948:1610–2. 11. Youssef AM, Malki A, Badr MH, Elbayaa RY, Sultan AS. Synthesis 25. Turan-Zitouni G, Chevallet P, Erol K, Boydağ BS. Synthesis of and anticancer activity of novel and benzothia- some chroman derivatives and preliminary investigation on zole derivatives against HepG2 liver cancer cells. Med Chem their vasodilatory activity. Farmaco 1997;52:569–71. 2012;8:151–62. 26. Pirbasti FG, Mahmoodi NO. Facile synthesis and biological 12. Łączkowski KZ, Misiura K, Świtalska M, Wietrzyk J, Łączkowska assays of novel 2,4-disubstituted hydrazinyl-thiazoles analogs. AB, Fernández B, et al. Synthesis and in vitro antiproliferative Mol Divers 2016;20:497–506. activity of thiazole-based nitrogen mustards. The hydrogen 27. Clinical and Laboratory Standards Institute. Methods for Dilution bonding interaction between model systems and nucleobases. Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobi- Anti-Cancer Agents Med Chem 2014;14:1271–81. cally; Approved Standard—Ninth Edition. CLSI document M07-A9. 13. Sharma RN, Xavier FP, Vasu KK, Chaturvedi SC, Pancholi SS. 28. EUCAST Definitive Document EDef 7.1: method for the determina- Synthesis of 4-benzyl-1,3-thiazole derivatives as potential anti- tion of broth dilution MICs of antifungal agents for fermentative inflammatory agents: an analogue-based drug design approach. yeasts. J Enzyme Inhib Med Chem 2009;24:890–7. 29. Pliška V, Testa B, van de Waterbeemd H. Lipophilicity in drug 14. Nicolaou KC, Roschanger F, Vourloumis D. Chemical biology of action and toxicology. Methods and principles in medicinal epothilones. Angew Chem Int Ed 1998;37:2014–45. chemistry. Hoboken: John Wiley and Sons, 2008.