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Research Article

Synthesis and anticandidal evaluation of new benzothiazole derivatives with hydrazone moiety

Leyla YurttaşDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey, Correspondence[email protected]
,
Zafer Asım KaplancıklıDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey,
,
Gamze GögerDepartment of Pharmacognosy, Graduate School of Health Sciences, Anadolu University, Eskişehir, Turkey, and
&
Fatih DemirciFaculty of Health Sciences, Anadolu University, Eskişehir, Turkey
Pages 714-720 | Received 26 Feb 2015, Accepted 12 May 2015, Published online: 06 Aug 2015

Abstract

In this study, we have performed the synthesis of new N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–s) bearing azole moiety and hydrazone group in a lipophilic structural framework. The target compounds were prepared by a three step synthetic procedure starting from 2-mercaptobenzothiazole. The structures of the target compounds were elucidated by IR, 1H NMR, 13C NMR spectra and elemental analysis. The antifungal activity of the obtained compounds has been determined against a number of clinic and fluconazole-resistant Candida strains by using microdilution method. Compounds (3a–3s) exhibited anticandidal activity in different ratios varying between the range of MIC: 50 and 200 µg/mL.

Introduction

Infectious diseases constitute an important part of human pathologies. In recent years, all over the world fungal infections, especially caused by Candida have increased tremendously. The main reasons for this situation are weakening of the immune system, due to a variety of diseases (AIDS), drugs (anticancer agents) and organ transplantations as well as enhancement of microorganisms resistance to some of the existing drugs. There are different approaches in treatment but, chemotherapeutic agents are still unavoidableCitation1–3.

Fungi, differing from bacteria, are in eukaryotic nature, which resembles to human cell. This situation makes antifungal therapy more difficult and challenging. The polyene amphotericin B, lipopeptide caspofungin and terbinafine as well as some azole compounds are most commonly used drugs in the treatment of fungal infections by acting with different mechanisms of actionCitation4–6. In fungal therapy, tolciclate and tolnaftate are the other effective antifungal entities, which also include sulfur atom in their structureCitation7. However, the fungicidal activity of sulfurated compounds has been known since long beforeCitation8.

Benzothiazoles exist in the structure of several naturally bioactive compounds and commercially available pharmaceutical productsCitation9–16. The activity mechanism of the benzothiazole ring is considering it as analogues of adenine and guanine bases of nucleic acids accordingly they can easily interact biopolymers in the organisms. The inhibition of nucleic acids is one of the mechanism of action for antimicrobial activity that DNA–drug complexes inhibits DNA-dependent RNA polymerase, thus the formation of mRNA blocks and antimicrobial activity occurCitation17,Citation18. Until today, activity studies have revealed mostly 2-substituted benzothiazoles. It has recognized that substitutions from second position of the ring determine activity, while substitutions at the other position of the ring play effectiveness gradeCitation19–24. In the literature, 2-mercaptobenzothiazoles (MBT) have been reported with a number of biological activityCitation25–28 and in particular with antifungal activityCitation29–32. Scientist Sidoova and her research group have studied a large number of 2-alkylthio-6-aminobenzothiazol derivatives since 1979Citation33–38. They have determined some of these derivatives to show anticandidous, antifungal and anti-yeast activity by inhibiting yeast – mycelial conversion of Candida albicans and also by inhibiting ergosterol biosynthesis in C. albicans. Besides, in same study lipophilicity spreading has been stated as one of the most important conditions of antifungal efficiencyCitation39–41.

In the light of these experiences and informations, we have designed new 2-mercaptobenzothiazole compounds containing a hydrazone structure, which are another important pharmacophoric group for antifungal activity. We combined these two scaffolds with an alkyl chain, of which the lipophilicity is known to increase the antifungal activity. Newly synthesized 19 N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–s) were tested against 10 clinical and resistant Candida strains using in vitro broth microdilution techniques.

Materials and methods

Chemistry

All compounds and chemicals were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO) and Merck (Darmstadt, Germany) if not othervise indicated. Melting points were determined using an Electrothermal 9300 digital melting point apparatus (Electrothermal, Essex, UK) and were uncorrected. All the reactions were monitored by thin-layer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany). Spectroscopic data were recorded with the following instruments: FTIR, Perkin Elmer Spectrum 100 (Perkin Elmer Inc., Waltham, MA); 1H NMR, Bruker DPX 400 MHz spectrometer (Bruker Bioscience, Billerica, MA); 13C NMR, Bruker DPX 100 MHz spectrometer (Bruker Bioscience, Billerica, MA); M + 1 peaks were determined by Agilent MS Trap SL mass spectrometer (Agilent technologies, Palo Alto, CA) and Bruker Daltonics Microtof II (Bruker Bioscience, Billerica, MA). Elemental analyses were performed on a Thermo Finnigan EA1112 series Flash elemental analyser (Thermo Finnigan, Milano, Italy). Some of the results were given as Supplementary data.

General procedure for the synthesis N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanohydrazide derivatives (1–26)

The obtained hydrazide compound 4-[(benzothiazol-2-yl)thio]butanoylhydrazide (2 mmol) (2) and appropriate aldehyde derivative (2.4 mmol) were refluxed in ethanol for 2 h. After standing overnight in a cool place, final products precipitated as crystal in reaction medium. The raw product was filtered and washed with excess ethanol (Scheme 1).

Scheme 1. The synthesis of the compounds.

Scheme 1. The synthesis of the compounds.

N′-benzylidene-4-[(benzothiazol-2-yl)thio]butanoylhydrazide (3a)

72–74% yield; mp 111–112 °C. IR νmax (cm−1): 3292 (amide N–H), 1675 (amide C=O), 1347–1032 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.07–2.14 (m, 2H, CH2), 2.41 and 2.83 (2t, 2H, J: 7.2 Hz, CH2), 3.42–3.49 (m, 2H, CH2), 7.33–7.48 (m, 5H, Ar–H), 7.59–7.61 (m, 1H, Ar–H), 7.66–7.69 (m, 1H, Ar–H), 7.84–7.86 (m, 1H, Ar–H), 7.95 and 8.15 (2s, 1H, CH=N), 7.98–8.02 (m, 1H, Ar–H), 11.30 and 11.42 (2s, 1H, NH). For C18H17N3OS2 calculated: (%) C 60.82, H 4.82, N 11.82; found: (%) C 60.71, H 4.72, N 11.93. MS [M + 1]+: m/z 356.

N′-(2-methylbenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3b)

69–71% yield; mp 105–108 °C. IR νmax (cm−1): 3173 (amide N–H), 1659 (amide C = O), 1333–1141 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.08–2.15 (m, 2H, CH2), 2.38–2.45 (m, 4H, CH and CH3), 2.83 (t, 1H, J: 6.8 Hz, CH), 3.36–3.48 (m, 2H, CH2), 7.16–7.39 (m, 3H, Ar–H), 7.43–7.48 (m, 1H, Ar–H), 7.66 (d, 1H, J: 7.6 Hz, Ar–H), 7.78 (d, 1H, J: 8.5 Hz, Ar–H), 7.84–7.87 (m, 1H, Ar–H), 7.97–8.02 (m, 1H, Ar–H), 8.24 and 8.43 (2s, 1H, CH = N), 11.24 and 11.42 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 19.67, 20.03, 24.86, 25.42, 31.61, 33.15, 33.28, 33.50, 121.77, 121.80, 122.37, 122.42, 125.05, 125.10, 126.49, 126.73, 126.76, 126.80, 126.97, 126.99, 129.94, 130.24, 131.49, 131.53, 132.85, 132.94, 135.22, 137.05, 137.31. For C19H19N3OS2 calculated: (%) C 61.76, H 5.18, N 11.37; found: (%) C 61.68, H 5.26, N 11.46. MS [M + 1]+: m/z 370.

N′-(3-methylbenzylidene-4-[(benzothiazol-2-yl) thio]butanoylhydrazide (3c)

72–75% yield; mp 113–115 °C. IR νmax (cm−1): 3173 (amide N–H), 1657 (amide C = O), 1333–1124 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.07–2.14 (m, 2H, CH2), 2.40–2.47 (m, 4H, CH and CH3), 2.85 (t, 1H, J: 7.2 Hz, CH), 3.36–3.47 (m, 2H, CH2), 7.22–7.35 (m, 3H, Ar–H), 7.48 (s, 1H, Ar–H), 7.66 (d, 2H, J: 7.8 Hz, Ar–H), 7.84–7.87 (m, 2H, Ar–H), 7.97–8.02 (m, 1H, Ar–H), 8.24 and 8.43 (2s, 1H, CH = N), 11.27 and 11.42 (2s, 1H, NH). C19H19N3OS2 calculated: (%) C 61.76, H 5.18, N 11.37; found: (%) C 61.65, H 5.24, N 11.39. MS [M + 1]+: m/z 370.

N′-(4-methylbenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3d)

75–78% yield; mp 129–131 °C. IR νmax (cm−1): 3179 (amide N–H), 1661 (amide C = O), 1310–1130 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.09–2.16 (m, 2H, CH2), 2.38–2.47 (m, 4H, CH and CH3), 2.87 (t, 1H, J: 6.9 Hz, CH), 3.39–3.45 (m, 2H, CH2), 7.43–7.48 (m, 2H, Ar–H), 7.66 (d, 2H, J: 7.6 Hz, Ar–H), 7.78 (d, 1H, J: 8.5 Hz, Ar–H), 7.95–8.0 (m, 3H, Ar–H), 8.25 and 8.42 (2s, 1H, CH = N), 11.28 and11.43 (2s, 1H, NH). C19H19N3OS2 calculated: (%) C 61.76, H 5.18, N 11.37; found: (%) C 61.69, H 5.32, N 11.43. MS [M + 1]+: m/z 370.

N′-(2-methoxybenzylidene-4-[(benzothiazol-2-yl)thio]butanoylhydrazide (3e)

75–78% yield; mp 133–134 °C. IR νmax (cm−1): 3182 (amide N–H), 1667 (amide C = O), 1340–1022 (C–N and C–O). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.09–2.15 (m, 2H, CH2), 2.38 and 2.45 (2t, 2H, J: 7.2 Hz, CH2), 3.39–3.44 (m, 2H, CH2), 3.74 and 3.76 (2s, 3H, OCH3), 7.18–7.43 (m, 3H, Ar–H), 7.87 (d, 1H, J: 7.8 Hz, Ar–H), 7.95 (d, 2H, J: 8.6 Hz, Ar–H), 7.99-8.02 (m, 2H, Ar–H), 8.25 and 8.43 (2s, 1H, CH = N), 11.26 and 11.44 (2s, 1H, NH). C19H19N3O2S2 calculated: (%) C 59.20, H 4.97, N 10.90; found: (%) C 59.25, H 5.09, N 11.08. MS [M + 1]+: m/z 386.

N′-(3-methoxybenzylidene-4-[(benzothiazol-2-yl) thio]butanoylhydrazide (3f)

66–69% yield; mp 115–118 °C. IR νmax (cm−1): 3181 (amide N–H), 1662 (amide C = O), 1329–1045 (C–N and C–O). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.04–2.11 (m, 2H, CH2), 2.40 and 2.80 (2t, 2H, J: 7.2 Hz, CH2), 3.38–3.45 (m, 2H, CH2), 3.74 and 3.75 (2s, 3H, OCH3), 6.91–6.97 (m, 1H, Ar–H), 7.15–7.44 (m, 5H, Ar–H), 7.80–7.83 (m, 1H, Ar–H), 7.90 and 8.10 (2s, 1H, CH = N), 7.93–7.98 (m, 1H, Ar–H), 11.30 and 11.41 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.89, 25.40, 31.54, 33.14, 33.29, 33.49, 55.80, 55.82, 111.82, 112.23, 116.14, 116.73, 119.83, 120.56, 121.77, 121.81, 122.37, 122.42, 125.05, 125.10, 126.97, 130.51, 135.22, 135.24, 136.35, 136.46, 143.22, 146.57, 153.45, 160.17, 160.19, 167.20, 167.27, 168.45, 174.22. For C19H19N3O2S2 calculated: (%) C 59.20, H 4.97, N 10.90; found: (%) C 59.27, H 5.06, N 11.02. MS [M + 1]+: m/z 386.

N′-(4-methoxybenzylidene-4-[(benzothiazol-2-yl)thio]butanoylhydrazide (3g)

66–69% yield; mp 138–139 °C. IR νmax (cm−1): 3177 (amide N–H), 1659 (amide C = O), 1336–1021 (C–N and C–O). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.08–2.14 (m, 2H, CH2), 2.41 and 2.82 (2t, 2H, J: 7.6 Hz, CH2), 3.41–3.45 (m, 2H, CH2), 3.73 and 3.75 (2s, 3H, OCH3), 7.23–7.56 (m, 4H, Ar–H), 7.80–7.83 (m, 2H, Ar–H), 7.93 and 8.12 (2s, 1H, CH = N), 7.93–7.98 (m, 2H, Ar–H), 11.30 and 11.43 (2s, 1H, NH). C19H19N3O2S2 calculated: (%) C 59.20, H 4.97, N 10.90; found: (%) C 59.30, H 5.09, N 11.07. MS [M + 1]+: m/z 386.

N′-(2-bromobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3h)

70–72% yield; mp 108–109 °C. IR νmax (cm−1): 3181 (amide N–H), 1674 (amide C = O), 1338–1197 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.03–2.07 (m, 2H, CH2), 2.39 and 2.78 (2t, 2H, J: 7.4 Hz, CH2), 3.41–3.47 (m, 2H, CH2), 7.13–7.41 (m, 3H, Ar–H), 7.69–7.78 (m, 2H, Ar–H), 7.92 and 8.11 (2s, 1H, CH = N), 7.93–7.99 (m, 3H, Ar–H), 11.31 and 11.40 (2s, 1H, NH). C18H16BrN3OS2 calculated: (%) C 49.77, H 3.71, N 9.67; found: (%) C 49.75, H 3.70, N 9.63. MS [M + 1]+: m/z 434.

N′-(3-bromobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3i)

72–75% yield; mp 131–134 °C. IR νmax (cm−1): 3177 (amide N–H), 1666 (amide C = O), 1335–1074 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.04–2.10 (m, 2H, CH2), 2.40 and 2.82 (2t, 2H, J: 7.6 Hz, CH2), 3.39–3.43 (m, 2H, CH2), 7.22–7.48 (m, 2H, Ar–H), 7.58–7.79 (m, 3H, Ar–H), 7.91 and 8.12 (2s, 1H, CH = N), 7.94–7.98 (m, 3H, Ar–H), 11.30 and 11.41 (2s, 1H, NH). C18H16BrN3OS2 calculated: (%) C 49.77, H 3.71, N 9.67; found: (%) C 49.73, H 3.75, N 9.61. MS [M + 1]+: m/z 434.

N′-(4-bromobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3j)

68–70% yield; mp 158–161 °C. IR νmax (cm−1): 3283 (amide N–H), 1668 (amide C = O), 1336–1003 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.05–2.09 (m, 2H, CH2), 2.39 and 2.79 (2t, 2H, J: 7.6 Hz, CH2), 3.38–3.45 (m, 2H, CH2), 7.29–7.35 (m, 1H, Ar–H), 7.40–7.50 (m, 4H, Ar–H), 7.78–7.83 (m, 2H, Ar–H), 7.88 and 8.09 (2s, 1H, CH = N), 7.94–7.98 (m, 1H, Ar–H), 11.33 and 11.45 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.82, 25.36, 31.57, 33.13, 33.28, 33.46, 121.78, 121.81, 122.40, 122.44, 123.45, 123.77, 125.07, 125.11, 126.98, 129.11, 129.50, 130.89, 132.36, 132.45, 132.69, 134.18, 134.32, 135.22, 142.13, 145.39, 153.45, 167.18, 167.29, 168.51, 174.26. For C18H16BrN3OS2 calculated: (%) C 49.77, H 3.71, N 9.67; found: (%) C 49.57, H 3.91, N 9.74. MS [M + 1]+: m/z 434.

N′-(2-chlorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3k)

73–76% yield; mp 116–118 °C. IR νmax (cm−1): 3283 (amide N–H), 1666 (amide C = O), 1332–1045 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.07–2.11 (m, 2H, CH2), 2.42 and 2.82 (2t, 2H, J: 6.8 Hz, CH2), 3.40–3.46 (m, 2H, CH2), 7.26–7.58 (m, 5H, Ar–H), 7.80–7.93 (m, 3H, Ar–H), 8.31 and 8.52 (2s, 1H, CH = N), 11.47 and 11.64 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.85, 25.33, 31.52, 33.11, 33.24, 33.50, 121.77, 121.82, 122.38, 130.88, 131.69, 132.02, 132.15, 132.21, 133.49, 133.71, 133.87, 135.22, 135.38, 139.38, 142.52, 153.44, 158.94, 167.20, 167.20, 168.61, 174.36. For C18H16ClN3OS2 calculated: (%) C 55.45, H 4.14, N 10.78; found: (%) C 55.56, H 4.07, N 10.71. MS [M + 1]+: m/z 390.

N′-(3-chlorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3l)

68–70% yield; mp 120–122 °C. IR νmax (cm−1): 3178 (amide N–H), 1668 (amide C = O), 1336–1076 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.09–2.12 (m, 2H, CH2), 2.43 and 2.84 (2t, 2H, J: 6.9 Hz, CH2), 3.41–3.45 (m, 2H, CH2), 7.29–7.51 (m, 3H, Ar–H), 7.85–7.90 (m, 5H, Ar–H), 8.31 and 8.52 (2s, 1H, CH = N), 11.48 and 11.65 (2s, 1H, NH). C18H16ClN3OS2 calculated: (%) C 55.45, H 4.14, N 10.78; found: (%) C 55.52, H 4.09, N 10.73. MS [M + 1]+: m/z 390.

N′-(4-chlorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3m)

76–78% yield; mp 153–155 °C. IR νmax (cm−1): 3180 (amide N–H), 1661 (amide C = O), 1335–1086 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.06–2.10 (m, 2H, CH2), 2.41 and 2.80 (2t, 2H, J: 7.6 Hz, CH2), 3.39–3.46 (m, 2H, CH2), 7.31–7.59 (m, 4H, Ar–H), 7.59 and 7.67 (d, 2H, J: 8.6 Hz, Ar–H), 7.81–7.84 (m, 1H, Ar–H), 7.91 and 8.12 (2s, 1H, CH = N), 7.95–7.99 (m, 1H, Ar–H), 11.34 and 11.46 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.84, 25.37, 31.57, 33.14, 33.27, 33.47, 121.79, 122.41, 125.08, 127.0, 128.89, 129.28, 129.48, 129.55, 129.78, 130.71, 133.87, 133.99, 134.71, 135.0, 135.23, 142.06, 145.34, 153.46, 167.30, 168.52, 174.27. For C18H16ClN3OS2 calculated: (%) C 55.45, H 4.14, N 10.78; found: (%) C 55.53, H 4.10, N 10.70. MS [M + 1]+: m/z 390.

N′-(2-fluorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3n)

69–71% yield; mp 123–125 °C. IR νmax (cm−1): 3282 (amide N–H), 1666 (amide C = O), 1334–1091 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.05–2.10 (m, 2H, CH2), 2.44 and 2.82 (2t, 2H, J: 7.4 Hz, CH2), 3.39–3.44 (m, 2H, CH2), 7.56–7.64 (m, 4H, Ar–H), 7.89–7.94 (m, 4H, Ar–H), 7.92 and 8.15 (2s, 1H, CH = N), 11.37 and 11.49 (2s, 1H, NH). C18H16FN3OS2 calculated: (%) C 57.89, H 4.32, N 11.25; found: (%) C 57.68, H 4.15, N 11.40. MS [M + 1]+: m/z 373.

N′-(3-fluorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3o)

69–71% yield; mp 117–120 °C. IR νmax (cm−1): 3285 (amide N–H), 1668 (amide C = O), 1328–1078 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.03–2.11 (m, 2H, CH2), 2.40 and 2.81 (2t, 2H, J: 7.2 Hz, CH2), 3.38–3.45 (m, 2H, CH2), 7.15–7.49 (m, 6H, Ar–H), 7.79–7.83 (m, 1H, Ar–H), 7.91 and 8.13 (2s, 1H, CH = N), 7.92–7.98 (m, 1H, Ar–H), 11.34 and 11.46 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.85, 25.34, 31.47, 33.12, 33.24, 33.46, 113.16, 113.38, 113.51, 113.73, 116.92, 117.13, 117.38, 121.76, 121.80, 122.36, 122.43, 123.70, 123.93, 123.96, 125.05, 125.10, 126.97, 127.0, 131.40, 131.48, 131.54, 135.21, 137.47, 137.55, 137.59, 137.68, 141.93, 141.96, 145.28, 153.44, 161.86, 164.27, 167.18, 167.26, 168.61, 174.37. For C18H16FN3OS2 calculated: (%) C 57.89, H 4.32, N 11.25; found: (%) C 57.68, H 4.15, N 11.40. MS [M + 1]+: m/z 373.

N′-(4-fluorobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3p)

69–71% yield; mp 141–142 °C. IR νmax (cm−1): 3282 (amide N–H), 1668 (amide C = O), 1396–1128 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.04–2.09 (m, 2H, CH2), 2.43 and 2.83 (2t, 2H, J: 7.2 Hz, CH2), 3.39–3.47 (m, 2H, CH2), 7.37–7.45 (m, 3H, Ar–H), 7.59–7.65 (m, 2H, Ar–H), 7.94 and 8.16 (2s, 1H, CH = N), 7.95–7.98 (m, 3H, Ar–H), 11.38 and 11.49 (2s, 1H, NH). C18H16FN3OS2 calculated: (%) C 57.89, H 4.32, N 11.25; found: (%) C 57.72, H 4.15, N 11.20. MS [M + 1]+: m/z 373.

N′-(2-nitrobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3q)

72–75% yield; mp 118–120 °C. IR νmax (cm−1): 3203 (amide N–H), 1668 (amide C = O), 1518, 1333 (NO2), 1309–1103 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.03–2.14 (m, 2H, CH2), 2.45 and 2.86 (2t, 2H, J: 7.6 Hz, CH2), 3.40–3.46 (m, 2H, CH2), 7.34–7.45 (m, 2H, Ar–H), 7.79–7.99 (m, 4H, Ar–H), 7.98–8.29 (m, 3H, CH = N and Ar–H), 11.58 and 11.68 (2s, 1H, NH). C18H16N4O3S2 calculated: (%) C 53.99, H 4.03, N 13.99; found: (%) C 53.85, H 4.10, N 13.87. MS [M + 1]+: m/z 401.

N′-(3-nitrobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3r)

70–72% yield; mp 140–143 °C. IR νmax (cm−1): 3223 (amide N–H), 1681 (amide C = O), 1527, 1352 (NO2), 1352–1077 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.07–2.16 (m, 2H, CH2), 2.47 and 2.88 (2t, 2H, J: 7.6 Hz, CH2), 3.43–3.49 (m, 2H, CH2), 7.29–7.45 (m, 2H, Ar–H), 7.79–7.99 (m, 4H, Ar–H), 7.97–8.25 (m, 3H, CH = N and Ar–H), 11.55 and 11.71 (2s, 1H, NH. C18H16N4O3S2 calculated: (%) C 53.99, H 4.03, N 13.99; found: (%) C 53.88, H 4.11, N 13.80. MS [M + 1]+: m/z 401.

N′-(4-nitrobenzylidene-4-[(benzothiazol-2-yl)thio] butanoylhydrazide (3s)

74–78% yield; mp 158–160 °C. IR νmax (cm−1): 3223 (amide N–H), 1674 (amide C = O), 1510, 1338 (NO2), 1338–1004 (C–N). 1H NMR (400 MHz, DMSO-d6, ppm) δ 2.06–2.12 (m, 2H, CH2), 2.43 and 2.83 (2t, 2H, J: 7.6 Hz, CH2), 3.39–3.46 (m, 2H, CH2), 7.28–7.44 (m, 2H, Ar–H), 7.77–7.96 (m, 4H, Ar–H), 7.99–8.26 (m, 3H, CH = N and Ar–H), 11.56 and 11.69 (2s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, ppm) δ 24.79, 25.26, 31.47, 33.09, 33.20, 33.47, 121.76, 121.81, 122.39, 122.44, 124.61, 124.69, 125.06, 125.13, 126.98, 128.11, 128.55, 135.21, 140.97, 141.21, 141.41, 144.17, 148.17, 148.43, 153.41, 167.27, 168.88, 174.62. For C18H16N4O3S2 calculated: (%) C 53.99, H 4.03, N 13.99; found: (%) C 53.88, H 4.11, N 13.80. MS [M + 1]+: m/z 401.

Anticandidal assay

The anticandidal properties of compounds (3a–3s) were evaluated by the microdilution method according to the reported standard procedure using Beckman Coulter Biomek 4000 apparatusCitation42,Citation43. Tested Candida strains are C. albicans (ATCC 24433), C. albicans (Fluconazole resistant), Candida glabrata (ATCC 66032), C. glabrata (Fluconazole resistant), Candida tropicalis (ATCC 750), C. tropicalis (isolate obtained from Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey), Candida krusei (ATCC 6258), C. krusei (Fluconazole resistant), Candida parapsilosis (ATCC 22109), C. parapsilosis (Fluconazole resistant). Amphotericin B, fluconazole and ketoconazole were used as positive controls.

All Candida strains were inoculated on Patoto Dextrose Agar (PDA) prior the experiments at 37 °C. After incubation, grown microorganisms were inoculated with sterile saline % 0.85. And then standardized turbitometrically (McFarland No: 0.5) to 5 × 103 CFU per well in RPMI medium under sterile conditions. Serial dilution series were prepared in 100 µL RPMI medium with an equal amount of the test samples. After serial dilution 100 µL each microorganism suspension was pipetted into each well and incubated at 37 °C for 24 h. Positive growth controls (to assess the presence of turbidity) were performed in wells not containing antifungal. In addition negative growth control (medium) was applied in 96-well plate. MIC was defined as the lowest concentration which did not result in any visible growth of the yeast compared with the growth in the control plate (MIC, in µg/mL).

Results and discussion

Chemistry

The target N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–3s) were prepared by a three step synthetic procedure. Initially, 2-mercaptobenzothiazole was reacted with ethyl 4-chlorobutanoate using potassium carbonate as base in ethanol at reflux conditions for six hours to give ethyl 4-[(benzothiazol-2-yl)thio]butanoate (1) in 74% yield. The obtained ester compound was further treated with hydrazine hydrate to form 4-[(benzothiazol-2-yl)thio]butanoylhydrazide (2). Condensation of the hydrazide with variously substituted benzaldehydes gave the corresponding arylidene hydrazides (3a–3s). All the final compounds were well characterized by IR, 1H NMR, 13C NMR, MASS spectroscopic data and elemental analyses results.

In the IR spectra of the compounds, peaks which were seen at 3173–3285 cm−1 proved N–H bond of the hydrazide groups. Additionally, bands belongs to carbonyl (C = O) function of this group were seen at 1657–1674 cm−1. Otherwise, bands belonging to C–N, C–O bonds were detected at 1004–1396 cm−1. Besides, two sharp bands were seen at about 1350 and 1550 cm−1 in the spectra of the compounds 3q, 3r and 3s which have nitro function on phenyl ring. In the NMR spectra of the compounds, peaks belong to alkyl protons were detected as triplet and multiplets at about 2.03–3.49 ppm. Azomethine protons and amine protons were determined at about 7.88–7.52 and 11.24–11.71 ppm, respectively and as two singlets confirming E/Z isomers mixture as reported in literatureCitation44. In the 13C NMR spectra, all alkyl carbons were seen as two different peaks, carbonyl carbon was detected at about 174 ppm. In aromatic region, the peaks were seen at estimated areas but the assignments could not be determined, clearly. Additionally, mass spectral data of the compounds demonstrated that the data were compatible with of the molecular weight of the compounds and the calculated values of elemental analysis were determined in the range of ±4%.

Anticandidal activity

Candida species are the most common reason of opportunistic fungal infections in the world. Candidiasis is the term that refers to the presence of Candida species and it has a particular importance due to life-threating feature in some cases. There are five Candida species responsible for the 90% of human candidiasis: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. kruseiCitation45.

Although, the treatment options are expanded with the increase of fungal infections, the unconscious and common use of antifungal agents revealed one or multi drug-resistant against fungal pathogens. Especially, it is crucial to synthesize new and effective drugs against resistant pathogens. Accordingly, new N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–3s) were synthesized and tested against fluconazole resistant C. glabrata, C. parapsilosis and C. krusei. The antifungal activities of the obtained compounds were evaluated using Beckman Coulter Biomek 4000 apparatus and according to microdilution method.

All of the compounds were tested against all the indicated Candida spp. and they exhibited a spectrum of activity having MIC values of 50–200 µg/mL, whereas standard drugs MIC’s were found between 0.25 and 64 µg/mL (). The compounds did not show significant antifungal activity. Among the microorganisms, C. tropicalis (ATCC 750) was determined as the most resistant Candida spp. against to the compounds. Compound 3r including 2-nitrophenyl moiety can be evaluated as the most active compound. Compound 3r inhibited fluconazole resistant C. parapsilosis at 50 µg/mL concentration, all the other compounds effected same microorganism at 100 µg/mL concentration. Compounds 3a, 3i, 3m, 3n, 3p and 3r exhibit half potency of the standard drug fluconazole against C. parapsilosis (ATCC 22109). Most of the compounds were effected C. albicans (ATCC 24433) and C. tropicalis (ATCC 750) strains at least. Besides, all of the compounds showed lower MIC values against fluconazole resistant C. glabrata, C. parapsilosis and C. krusei than clinic or commercial isolates of these strains. In general evaluation, compounds 3q, 3r and 3s bearing 2,3,4-nitro substituents have come into prominence among the others.

Table 1. Anticandidal activities of the compounds (3a–3s) against Candida species (µg/mL) of the compounds.

The compounds did not show high antifungal activity, contrary to expectations. Alternatively, anticandidal efficiency could be increased with the synthesis of similar compounds bearing new substituents and combination studies. Additionally, it would be beneficial to test the compounds against different fungus species in further studies.

Conclusion

In this study, we have reported the synthesis, structural elucidation and anticandidal activities of N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–3s). The target compounds have obtained with a three step reaction starting from 2-mercaptobenzothiazole and the structures of the final compounds have been elucidated with IR, NMR, mass spectroscopy data and elemental analysis results. The antifungal activities of the target compounds have been determined against different Candida spp. by using microdilution method. As a result of the antifungal activity evaluation the pathogen C. tropicalis (ATCC 750) was identified as the most resistant microorganism against the compounds among the tested panel of pathogens and compounds 3q, 3r and 3s bearing 2-nitro, 3-nitro and 4-nitro substituents on phenyl moiety have attracted attention with higher anticandidal activity.

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Declaration of interest

The authors declare that this article content has no conflict of interest.

This study was supported within the Anadolu University Scientific Research Project, Eskişehir, Turkey (Project no: 1406S066).

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