Home Medicine Novel tetrazole derivatives: synthesis, anticholinesterase activity and cytotoxicity evaluation
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Novel tetrazole derivatives: synthesis, anticholinesterase activity and cytotoxicity evaluation

  • Leyla Yurttaş EMAIL logo , Zafer Asım Kaplancıklı , Halide Edip Temel and Gülşen Akalın Çiftçi
Published/Copyright: November 16, 2016
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 42 Issue 2

Abstract

Objective(s)

The synthesis of new N′-arylidene-4-[(1-phenyl-1H-tetrazole-5-yl)thio]butanoylhydrazide derivatives (1–26) and investigation of their potential anticholinesterase (AChE), butyrylcholinesterase (BuChE) enzyme inhibition activities and also cytotoxic properties on mouse embryonic fibroblast cells (NIH/3T3) were aimed in this work.

Materials and methods

The target compounds were prepared by a three step synthetic procedure using 1-phenyl-1H-tetrazole-5-thiol and ethyl 4-chlorobutanoate as starting materials. The structures of the obtained compounds were elucidated by IR, 1H-NMR, 13C-NMR spectra and elemental analysis data. The enzyme inhibition and cytotoxic activities were determined according to Ellman and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] methods, respectively.

Results

Compounds 14, 15 and compound 18 exhibited the highest inhibitory activity on AChE and BuChE enzymes. Additionally, compounds 4, 5, 8 and 16 exhibited the lowest cytotoxicity against NIH/3T3 cells.

Conclusion

Compounds 14, 15 and 18 bearing 2-nitro, 3-nitro and 3-hydroxy substituents have showed selective enzyme inhibitory activities.

Özet

Amaç

Bu çalışmada, yeni N-ariliden-4-[(1-fenil-1H-tetrazol-5-il)tiyo]bütanoilhidrazit (1–26) türevlerinin sentezi ve bu bileşiklerin asetilkolinesteraz (AChE) ve bütirilkolinesteraz (BuChE) enzimleri üzerindeki potansiyel antikolinesteraz etkileri ile fare embrionik fibroblast hücreleri (NIH/3T3) üzerindeki sitotoksik etkilerinin incelenmesi amaçlanmıştır.

Metod

Hedeflenen bileşikler, başlangıç maddeleri olarak 1-fenil-1H-tetrazol-5-tiyol ve etil 4-klorobütanoatın kullanıldığı üç basamaklı sentez prosedürü ile hazırlanmışlardır. Elde edilen bileşiklerin yapıları IR, 1H-NMR, 13C-NMR spektrumları ve elemental analiz verileri kullanılarak aydınlatılmıştır. Enzim inhibisyonu aktivitesi ve sitotoksik aktivite Ellman ve MTT metodlarına göre belirlenmiştir.

Bulgular

14, 15 ve 18 nolu bileşikler AChE ve BuChE enzimleri üzerinde en yüksek inhibitör aktiviteyi göstermişlerdir. Ayrıca 4, 5, 8 ve 16 nolu bileşikler NIH/3T3 hücrelerine karşı en düşük sitotoksisiteyi göstermişlerdir.

Sonuç

2-Nitro, 3-nitro ve 3-hidroksi sübstitüentlerini içeren 14, 15 ve 18 nolu bileşikler selektif enzim inhibitor aktivite göstermişlerdir.

Keywords: Tetrazole; Hydrazone; Anticholinesterase; AChE; BuChE; Cytotoxicity
Anahtar Kelimeler: Tetrazol; Hidrazon; Antikolinesteraz; AChE; BuChE; Sitotoksisite

Introduction

Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder of the central nervous system and primarily older people suffer from AD [1]. The disease is characterized by cognitive dysfunction which is associated with a reduction in cholinergic transmission in cortical and hippocampal neurons [2]. Cholinergic neurotransmission is mediated by the neurotransmitter acetylcholine (ACh) which is rapidly hydrolyzed to choline and acetate after its presynaptic release by the enzyme acetylcholinesterase [3], [4]. In this way, AD therapy is mainly managed with acetylcholinesterase inhibitors (AChEIs) like donepezil, rivastigmine, galantamine and also the N-methyl-D-aspartate-receptor (NMDA) antagonist memantine, currently [5], [6]. Tetrazole ring is an important pharmacophoric structure which can serve as a bioisosteric moiety of carboxylic group in biologically active molecules, because both groups possess comparable acidities and sizes [7], [8], [9], [10], [11]. In particular, 5-substituted thiotetrazoles and 1,5-disubstituted tetrazoles have been used in the synthesis of pharmacologically active drugs [12]. A lot of studies dealing with the synthesis of new tetrazole derivatives exhibiting diverse biological activities as hypotensive, antimicrobial, antiviral, antiallergic, cytostatic, nootropic are published [13], [14], [15]. Tetrazole bearing compounds were also reported as anticholinesterase inhibitors in the literature [16], [17], [18]. The approach of replacing the ester group with five-membered rings such as tetrazoles, triazoles to discover more potent and metabolically stable cholinomimetic ligands could be confirmative [19].

Hydrazones also constitute an important class of biologically active drug molecules for new drug development [20]. They has attracted the attention of medicinal chemists due to their wide range of pharmacological properties such as antimicrobial, antimycobacteria, anticonvulsant, analgesic, antiinflammatory, antiplatelet, antitubercular, and antitumoral [21], [22]. Moreover, hydrazone bearing molecules with cholinesterase inhibition activity have also been reported in recent literature [23], [24], [25].

In the view of the above literature findings and as an extension of our previous study [26] we have synthesized new N′-arylidene-2-[(tetrazol-5-yl)thio]butanohydrazide derivatives (1–26) and evaluated their inhibitor activity on AChE and BuChE enzymes and their cytotoxic profiles against NIH/3T3 cell line.

Materials and methods

Chemistry

All compounds and chemicals were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO, USA) and Merck (Darmstadt, Germany) if not otherwise 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, USA); 1H-NMR, Bruker DPX 500 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA); 13C-NMR, Bruker DPX 125 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA); M + 1 peaks were determined by Agilent MS Trap SL mass spectrometer (Agilent technologies, Palo Alto, CA, USA) and Bruker Daltonics Microtof II (Bruker Bioscience, Billerica, MA, USA). Elemental analyses were performed on a Thermo Finnigan EA1112 series Flash elemental analyser (Thermo Finnigan, Milano, Italy).

General procedure for the synthesis N′-arylidene-2-[(tetrazol-5-yl)thio]butanohydrazide derivatives (1–26)

The synthesized hydrazide compound, 2-[(tetrazol-5-yl)thio]butanoylhydrazide (B, 2 mmol), appropriate aldehyde/ketone derivative (2 mmol) and catalytic amount of acetic acid were refluxed in ethanol for 2 h. After standing reaction mixture overnight in a cool place, final products were precipitated as crystals in reaction medium. The raw material was filtered and washed with excess ethanol to acquire final products (1–26).

N′-benzylidene-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (1)

Yield 73%. m.p. 135°C. IR (KBr) ν (cm−1): 3341 (amide N-H), 1673 (amide C=O), 1564–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.09 (m, 2H, -CH2), 2.38 and 2.80 (2t, 2H, J=7.35, 7.25 Hz, -CH2), 3.42 (t, 2H, J=7.15, -S-CH2), 7.37 (t, 1H, J=7.60, 7.10 Hz, Ar-H), 7.43 (t, 1H, J=7.04, 7.19 Hz, Ar-H), 7.51 (d, 2H, J: 7.01 Hz, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.91–7.95 (m, 1H, Ar-H), 8.37 and 8.54 (2s, 1H, -CH=N), 11.51 and 11.65 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.1, 31.4, 33.0, 33.2, 33.4, 125.8, 125.9, 127.9, 128.2, 130.0, 130.9, 131.2, 131.3, 132.1, 133.4, 134.4, 140.2, 143.5, 154.8, 155.3, 168.4. For C18H18N6OS calculated: 59.00% C, 4.95% H, 22.93% N; found: 59.96% C, 4.88% H, 22.97% N. MS [M + 1]+: m/z 367.

N′-(2-methylbenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (2)

Yield 70%. m.p. 106°C. IR (KBr) ν (cm−1): 3345 (amide N-H), 1669 (amide C=O), 1568–1411 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.08 (m, 2H, -CH2), 2.35–2.51 (m, 4H, CH3 and -CH), 2.77 (t, 1H, J=7.30 Hz, -CH), 3.40–3.45 (m, 2H, -S-CH2), 7.20–7.30 (m, 3H, Ar-H), 7.66–7.78 (m, 6H, Ar-H), 8.25 and 8.41 (2s, 1H, -CH=N), 11.24 and 11.40 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.6, 19.9, 24.6, 25.2, 31.4, 33.0, 33.2, 33.4, 125.8, 125.9, 127.0, 127.4, 130.6, 130.9, 131.3, 131.9, 132.1, 132.2, 133.4, 133.5, 134.4, 137.7, 138.0, 143.2, 145.9, 155.8, 169.1, 174.9. For C19H20N6OS calculated: 59.98% C, 5.30% H, 22.09% N; found: 59.95% C, 5.24% H, 22.13% N. MS [M + 1]+: m/z 381.

N′-(3-methylbenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (3)

Yield 71%. m.p. 102°C. IR (KBr) ν (cm−1): 3348 (amide N-H), 1670 (amide C=O), 1574–1405 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.32–2.38 (m, 4H, -CH and -CH3), 2.78 (t, 1H, J=7.28 Hz, CH), 3.43–3.44 (m, 2H, -S-CH2), 7.21–7.34 (m, 2H, Ar-H), 7.41–7.51 (m, 2H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.84 and 8.10 (2s, 1H, -CH=N), 11.30 and 11.41 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 21.5, 21.6, 24.6, 25.2, 31.3, 33.0, 33.2, 33.4, 125.2, 125.6, 125.8, 125.9, 128,3, 128.5, 129.9, 131.3, 131.7, 131.9, 132.0, 134.4, 135.5, 135.6, 139.3, 139.4, 144.3, 147.4, 155.7, 155.8, 169.2, 174.9. For C19H20N6OS calculated: 59.98% C, 5.30% H, 22.09% N; found: 59.93% C, 5.22% H, 22.15% N. MS [M + 1]+: m/z 381.

N′-(4-methylbenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (4)

Yield 70%. m.p. 107°C. IR (KBr) ν (cm−1): 3343 (amide N-H), 1674 (amide C=O), 1565–1414 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.33–2.38 (m, 4H, -CH and -CH3), 2.77 (t, 1H, J=7.24 Hz, CH), 3.33–3.45 (m, 2H, -S-CH2), 7.21–7.26 (m, 2H, Ar-H), 7.51–7.58 (m, 2H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.94 and 8.10 (2s, 1H, -CH=N), 11.25 and 11.35 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 21.6, 24.6, 25.2, 31.3, 33.0, 33.2, 33.4, 125.8, 125.9, 127.9, 128.2, 130.6, 131.3, 131.9, 132.8, 132.9, 134.4, 140.8, 141.0, 144.1, 147.4, 155.7, 155.8, 169.1, 174.8. For C19H20N6OS calculated: 59.98% C, 5.30% H, 22.09% N; found: 59.92% C, 5.26 % H, 22.16% N. MS [M + 1]+: m/z 381.

N′-(2-methoxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (5)

Yield 68%. m.p. 142°C. IR (KBr) ν (cm−1): 3336 (amide N-H), 1674 (amide C=O), 1558–1426 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.08 (m, 2H, -CH2), 2.34 and 2.77 (2t, 2H, J=7.08 Hz, -CH2), 3.40–3.44 (m, 2H, -S-CH2), 3.84 and 3.85 (2s, 3H, OCH3), 6.96 and 7.0 (2t, 1H, J=7.30 Hz, Ar-H), 7.09 (t, 1H, J=6.85 Hz, Ar-H), 7.39 (t, 1H, J=7.84 Hz, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.74–7.80 (m, 1H, Ar-H), 8.31 and 8.49 (2s, 1H, -CH=N), 11.28 and 11.41 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.2, 31.4, 33.0, 33.2, 33.4, 56.5, 113.0, 121.9, 122.0, 123.5, 125.8, 125.9, 126.4, 126.6, 131.3, 131.9, 132.4, 134.4, 139.7, 142.7, 155.8, 158.9, 169.0, 174.8. For C19H20N6O2S calculated: 57.56% C, 5.08% H, 21.20% N; found: 57.51% C, 5.02% H, 21.31% N. MS [M + 1]+: m/z 397.

N′-(3-methoxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (6)

Yield 69%. m.p. 129°C. IR (KBr) ν (cm−1): 3346 (amide N-H), 1678 (amide C=O), 1563–1407 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.37 and 2.79 (2t, 2H, J=7.32 Hz, -CH2), 3.41–3.45 (m, 2H, -S-CH2), 3.77 and 3.79 (2s, 3H, OCH3), 6.99 (t, 1H, J=7.25 Hz, Ar-H), 7.19–7.25 (m, 2H, Ar-H), 7.31–7.37 (m, 1H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.94 and 8.11 (2s, 1H, -CH=N), 11.26 and 11.43 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.1, 24.6, 25.2, 31.3, 33.0, 33.2, 33.4, 55.9, 56.0, 112.3, 112.7, 116.7, 117.3, 120.4, 121.1, 125.8, 125.9, 131.1, 1313, 131.9, 132.0, 1134.4, 137.0, 137.1, 143.9, 147.2, 155.7, 155.8, 160.9, 169.2, 175.0. For C19H20N6O2S calculated: 57.56% C, 5.08% H, 21.20% N; found: 57.61% C, 5.01% H, 21.30% N. MS [M + 1]+: m/z 397.

N′-(4-methoxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (7)

Yield 74%. m.p. 118°C. IR (KBr) ν (cm−1): 3345 (amide N-H), 1672 (amide C=O), 1570–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.08 (m, 2H, -CH2), 2.35 and 2.76 (2t, 2H, J=7.32 Hz, -CH2), 3.39–3.45 (m, 2H, -S-CH2), 3.79 and 3.80 (2s, 3H, OCH3), 6.95–7.01 (m, 1H, Ar-H), 7.57–7.63 (m, 3H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.92 and 8.08 (2s, 1H, -CH=N), 11.19 and 11.28 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.1, 24.6, 25.2, 31.4, 33.0, 33.2, 33.4, 56.1, 115.4, 115.5, 125.8, 125.9, 128.1, 129.5, 129.8, 131.3, 131.9, 134.4, 143.9, 147.2, 155.7, 155.7, 155.8, 161.9, 162.1, 168.9, 174.7. For C19H20N6O2S calculated: 57.56% C, 5.08% H, 21.20% N; found: 57.59% C, 5.04% H, 21.25% N. MS [M + 1]+: m/z 397.

N′-(2-bromobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (8)

Yield 79%. m.p. 134°C. IR (KBr) ν (cm−1): 3342 (amide N-H), 1670 (amide C=O), 1565–1411 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.38 and 2.80 (2t, 2H, J=7.26 Hz, -CH2), 3.38–3.45 (m, 2H, -S-CH2), 7.33–7.47 (m, 2H, Ar-H), 7.66–7.67 (m, 6H, Ar-H), 7.89–7.93 (m, 1H, Ar-H), 8.34 and 8.50 (2s, 1H, -CH=N), 11.54 and 11.68 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.0, 31.4, 33.0, 33.1, 33.4, 124.4, 125.9, 126.0, 128.2, 124.4, 129.3, 129.5, 131.3, 131.9, 132.6, 132.9, 134.3, 134.4, 134.5, 142.4, 145.5, 155.8, 169.4, 175.1. For C18H17BrN6OS calculated: 48.55% C, 3.85% H, 18.87% N; found: 48.57% C, 3.89% H, 18.82% N. MS [M + 1]+: m/z 445.5.

N′-(3-bromobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (9)

Yield 75%. m.p. 138°C. IR (KBr) ν (cm−1): 3338 (amide N-H), 1668 (amide C=O), 1558–1403 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.38 and 2.79 (2t, 2H, J=7.30 Hz, -CH2), 3.39–3.44 (m, 2H, -S-CH2), 7.36–7.42 (m, 1H, Ar-H), 7.59–7.60 (m, 1H, Ar-H), 7.66–7.67 (m, 6H, Ar-H), 7.84 and 7.88 (2brs, 1H, Ar-H), 7.94 and 8.11 (2s, 1H, -CH=N), 11.44 and 11.55 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.5, 25.1, 31.2, 33.0, 33.1, 33.3, 123.4, 125.9, 126.0, 127.0, 130.1, 131.3, 131.9, 132.2, 133.5, 134.4, 138.0, 142.4, 142.4, 155.8, 175.1. For C18H17BrN6OS calculated: 48.55% C, 3.85% H, 18.87% N; found: 48.51% C, 3.92% H, 18.81% N. MS [M + 1]+: m/z 445.5.

N′-(4-bromobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (10)

Yield 76%. m.p. 160°C. IR (KBr) ν (cm−1): 3338 (amide N-H), 1675 (amide C=O), 1569–1406 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.37 and 2.78 (2t, 2H, J=7.32 Hz, -CH2), 3.38–3.45 (m, 2H, -S-CH2), 7.59–7.66 (m, 9H, Ar-H), 7.94 and 8.11 (2s, 1H, -CH=N), 11.38 and 11.49 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.1, 31.4, 33.0, 33.1, 33.3, 124.1, 124.3, 125.8, 125.9, 129.8, 130.1, 131.3, 131.9, 133.0, 133.1, 134.4, 134.8, 142.9, 146.1, 155.8, 169.3, 175.1. For C18H17BrN6OS calculated: 48.55% C, 3.85% H, 18.87% N; found: 48.59% C, 3.82% H, 18.81% N. MS [M + 1]+: m/z 445.5.

N′-(2-fluorobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (11)

Yield 75%. m.p. 112°C. IR (KBr) ν (cm−1): 3339 (amide N-H), 1669 (amide C=O), 1571–1408 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.38 and 2.79 (2t, 2H, J=7.32 Hz, -CH2), 3.40–3.45 (m, 2H, -S-CH2), 7.22–7.31 (m, 2H, Ar-H), 7.44–7.49 (m, 1H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.83–7.89 (m, 1H, Ar-H), 8.19 and 8.38 (2s, 1H, -CH=N), 11.44 and 11.55 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.1, 31.3, 32.0, 33.2, 33.4, 117.1, 117.2, 122.0, 123.1, 125.8, 125.9, 126.0, 126.1, 126.2, 127.3, 127.5, 131.3, 131.9, 132.8, 132.9, 133.1, 133.2, 134.4, 136.8, 136.9, 140.0, 159.8, 161.0, 161.1, 163.0, 163.1, 169.3, 175.1. For C18H17FN6OS calculated: 56.24% C, 4.46% H, 21.86% N; found: 56.27% C, 4.50% H, 21.81% N. MS [M + 1]+: m/z 385.

N′-(3-fluorobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (12)

Yield 74%. m.p. 107°C. IR (KBr) ν (cm−1): 3343 (amide N-H), 1672 (amide C=O), 1565–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.38 and 2.79 (2t, 2H, J=7.36 Hz, -CH2), 3.41–3.45 (m, 2H, -S-CH2), 7.22–7.28 (m, 1H, Ar-H), 7.45–7.54 (m, 2H, Ar-H), 7.66–7.67 (m, 6H, Ar-H), 7.95 and 8.15 (2s, 1H, -CH=N), 11.42 and 11.52 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.1, 31.3, 33.0, 33.2, 33.4, 113.6, 113.8, 114.0, 114.2, 117.5, 117.7, 124.3, 124.5, 125.8, 125.9, 131.3, 131.9, 132.1, 132.2, 134.4, 138.1, 138.2, 142.7, 145.0, 155.8, 162.9, 164.8, 169.4, 175.2. For C18H17FN6OS calculated: 56.24% C, 4.46% H, 21.86% N; found: 56.20% C, 4.52% H, 21.89% N. MS [M + 1]+: m/z 385.

N′-(4-fluorobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (13)

Yield 71%. m.p. 124°C. IR (KBr) ν (cm−1): 3343 (amide N-H), 1668 (amide C=O), 1556–1402 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.98–1.99 (m, 2H, -CH2), 2.16 and 2.81 (2t, 2H, J=7.20 Hz, -CH2), 3.41–3.45 (m, 2H, -S-CH2), 7.22–7.28 (m, 1H, Ar-H), 7.46–7.57 (m, 2H, Ar-H), 7.66–7.67 (m, 6H, Ar-H), 7.97 and 8.14 (2s, 1H, -CH=N), 11.33 and 11.43 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.1, 31.6, 33.0, 33.5, 33.4, 113.6, 113.8, 114.0, 116.2, 117.5, 117.7, 124.3, 124.5, 125.8, 125.9, 130.3, 131.9, 132.1, 132.2, 134.4, 137.1, 138.2, 140.7, 145.0, 155.8, 162.9, 165.8, 169.4, 175.2. For C18H17FN6OS calculated: 56.24% C, 4.46% H, 21.86% N; found: 56.19% C, 4.55% H, 21.91% N. MS [M + 1]+: m/z 385.

N′-(2-nitrobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (14)

Yield 69%. m.p. 133°C. IR (KBr) ν (cm−1): 3337 (amide N-H), 1670 (amide C=O), 1567–1405 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.40 and 2.78 (2t, 2H, J=7.34 Hz, -CH2), 3.40–3.45 (m, 2H, -S-CH2), 7.66–7.67 (m, 6H, Ar-H), 7.74–7.82 (m, 1H, Ar-H), 8.01–8.09 (m, 2H, Ar-H), 8.35 and 8.55 (2s, 1H, -CH=N), 11.62 and 11.76 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.1, 24.5, 25.0, 31.4, 32.0, 33.1, 33.3, 56.8, 125.7, 125.8, 125.9, 129.2, 129.3, 129.6, 130.1, 131.3,131.6, 132.8, 132.9, 134.4, 134.7, 135.0, 139.4, 142.7, 149.4, 149.5, 155.7, 155.8, 169.6, 175.3. For C18H17N7O3S calculated: 52.55% C, 4.16% H, 23.83% N; found: 52.60% C, 4.22% H, 23.90% N. MS [M + 1]+: m/z 412.

N′-(3-nitrobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (15)

Yield 67%. m.p. 156°C. IR (KBr) ν (cm−1): 3341 (amide N-H), 1675 (amide C=O), 1560–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.11 (m, 2H, -CH2), 2.40 and 2.82 (2t, 2H, J=7.34 Hz, -CH2), 3.40–3.45 (m, 2H, -S-CH2), 7.66–7.67 (m, 5H, Ar-H), 7.69–7.76 (m, 1H, Ar-H), 8.10–8.26 (m, 3H, Ar-H), 8.44 and 8.51 (2s, 1H, -CH=N), 11.55 and 11.69 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.5, 25.0, 31.2, 33.1, 33.3, 122.1, 122.2, 125.8, 125.9, 131.3, 131.7, 131.9, 133.9, 134.3, 141.9, 149.6, 155.8, 175.3. For C18H17N7O3S calculated: 52.55% C, 4.16% H, 23.83% N; found: 52.63% C, 4.21% H, 23.85% N. MS [M + 1]+: m/z 412.

N′-(4-nitrobenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (16)

Yield 72%. m.p. 208°C. IR (KBr) ν (cm−1): 3342 (amide N-H), 1669 (amide C=O), 1563–1411 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.09–2.11 (m, 2H, -CH2), 2.41 and 2.83 (2t, 2H, J=7.27 Hz, -CH2), 3.40–3.45 (m, 2H, -S-CH2), 7.66–7.67 (m, 6H, Ar-H), 7.90–7.96 (m, 2H, Ar-H), 8.08–8.30 (m, 2H, Ar-H and -CH=N), 11.63 and 11.73 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.0, 31.4, 33.1, 33.3, 33.9, 34.5, 125.2, 125.8, 125.9, 128.8, 129.1, 131.3, 131.9, 134.4, 141.7, 141.9, 144.8, 148.9, 175.4. For C18H17N7O3S calculated: 52.55% C, 4.16% H, 23.83% N; found: 52.61% C, 4.23% H, 23.89% N. MS [M + 1]+: m/z 412.

N′-(2-hydroxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (17)

Yield 72%. m.p. 143°C. IR (KBr) ν (cm−1): 3339 (amide N-H), 1670 (amide C=O), 1564–1402 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.39 and 2.75 (2t, 2H, J=7.30 Hz, -CH2), 3.35–3.46 (m, 2H, -S-CH2), 6.81–6.92 (m, 2H, Ar-H), 7.21–7.30 (m, 2H, Ar-H), 7.50–7.62 (m, 2H, Ar-H), 7.66–7.67 (m, 3H, Ar-H), 8.26 and 8.34 (2s, 1H, -CH=N), 10.12 and 11.16 (2s, 1H, -OH), 11.29 and 11.66 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.5, 25.0, 31.3, 32.9, 33.1, 33.2, 56.85, 117.3, 117.5, 119.8, 120.5, 121.2, 125.8, 125.9, 127.9, 130.1, 131.2, 131.8, 132.0, 132.2, 132.5, 134.3, 134.4, 142.3, 147.9, 155.7, 155.8, 157.7, 158.7, 169.0, 174.5. For C18H18N6O2S calculated: 56.53% C, 4.74% H, 21.97% N; found: 56.59% C, 4.78% H, 21.84% N. MS [M + 1]+: m/z 383.

N′-(3-hydroxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (18)

Yield 71%. m.p. 152°C. IR (KBr) ν (cm−1): 3339 (amide N-H), 1671 (amide C=O), 1567–1412 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.10 (m, 2H, -CH2), 2.36 and 2.77 (2t, 2H, J=7.30 Hz, -CH2), 3.34–3.44 (m, 2H, -S-CH2), 6.79–6.81 (m, 2H, Ar-H), 7.02–7.14 (m, 1H, Ar-H), 7.19–7.25 (m, 1H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.89 and 8.05 (2s, 1H, -CH=N), 9.61 and 9.63 (2s, 1H, -OH), 11.26 and 11.36 (2s, 1H, N-H). 13C NMR (125 MHz, DMSOd6) δ (ppm): 24.5, 25.1, 31.3, 33.0, 33.2, 33.4, 113.7, 113.8, 118.2, 118.4, 119.3, 119.9, 125.9, 126.0, 131.1, 131.3, 131.9, 132.0, 134.4, 136.8, 138.9, 144.2, 147.4, 155.7, 155.7, 159.0, 169.1, 174.8. For C18H18N6O2S calculated: 56.53% C, 4.74% H, 21.97% N; found: 56.52% C, 4.79% H, 21.88% N. MS [M + 1]+: m/z 383.

N′-(4-hydroxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (19)

Yield 69%. m.p. 175°C. IR (KBr) ν (cm−1): 3338 (amide N-H), 1673 (amide C=O), 1569–1413 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.33 and 2.74 (2t, 2H, J=7.30 Hz, -CH2), 3.34–3.44 (m, 2H, -S-CH2), 6.78–6.82 (m, 2H, Ar-H), 7.45–7.51 (m, 2H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.86 and 8.03 (2s, 1H, -CH=N), 9.89 and 9.91 (2s, 1H, -OH), 11.10 and 11.12 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.1, 24.6, 25.2, 31.4, 33.0, 33.2, 33.4, 56.9, 116.8, 125.8, 125.9, 126.5, 129.6, 130.0, 131.3, 131.9, 134.4, 144.4, 147.7, 155.7, 155.8, 160.5, 160.7, 158.7, 168.8, 174.6. For C18H18N6O2S calculated: 56.53% C, 4.74% H, 21.97% N; found: 56.60% C, 4.79% H, 21.87% N. MS [M + 1]+: m/z 383.

N′-(3,4-dimethoxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (20)

Yield 69%. m.p. 78°C. IR (KBr) ν (cm−1): 3342 (amide N-H), 1671 (amide C=O), 1569–1412 (C=C and C=N), 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.35 and 2.78 (2t, 2H, J=7.18 Hz, -CH2), 3.40–3.46 (m, 2H, -S-CH2), 3.76–3.80 (m, 6H, OCH3), 6.97–7.02 (m, 1H, Ar-H), 7.13–7.17 (m, 1H, Ar-H), 7.24–7.29 (m, 1H, Ar-H), 7.66–7.67 (m, 4H, Ar-H), 7.68–7.70 (m, 1H, Ar-H), 7.90 and 8.07 (2s, 1H, -CH=N), 11.21 and 11.29 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.13, 24.7, 25.3, 31.3, 32.9, 33.2, 33.4, 56.2, 56.3, 56.4, 56.8, 109.4, 109.7, 112.6, 112.7, 122.0, 122.8, 125.8, 125.9, 128.3, 131.2, 131.9, 133.9, 132.2, 144.2, 147.6, 150.4, 150.7, 151.8, 152.0, 155.7, 155.8, 168.9, 174.7. For C20H22N6O3S calculated: 56.32% C, 5.20% H, 19.70% N; found: 56.38% C, 5.24% H, 19.82% N. MS [M + 1]+: m/z 427.

N′-(3,5-dimethoxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (21)

Yield 69%. m.p. 164°C. IR (KBr) ν (cm−1): 3345 (amide N-H), 1673 (amide C=O), 1566–1418 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.07–2.10 (m, 2H, -CH2), 2.37 and 2.79 (2t, 2H, J=7.27 Hz, -CH2), 3.34–3.44 (m, 2H, -S-CH2), 3.76 and 3.77 (2s, 6H, OCH3), 6.53–6.56 (m, 1H, Ar-H), 7.80 and 7.83 (dd, 2H, J=2.24 and 7.86 Hz, Ar-H), 7.66–7.67 (m, 4H, Ar-H), 7.67–7.69 (m, 1H, Ar-H), 7.89 and 8.07 (2s, 1H, -CH=N), 11.36 and 11.44 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.2, 31.2, 32.9, 33.1, 33.2, 33.6, 56.1, 56.2, 102.8, 103.3, 105.6, 105.8, 125.8, 125.9, 126.0, 131.2, 131.3, 131.9, 132.0, 132.2, 137.5, 134.3, 137.6, 142.3, 143.8, 155.7, 155.8, 162.1, 169.2, 175.0. For C20H22N6O3S calculated: 56.32% C, 5.20% H, 19.70% N; found: 56.36% C, 5.28% H, 19.80% N. MS [M + 1]+: m/z 427.

N′-(3-methyl-4-methoxybenzylidene)-4- [(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (22)

Yield 72%. m.p. 125°C. IR (KBr) ν (cm−1): 3336 (amide N-H), 1674 (amide C=O), 1574–1417 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.05–2.09 (m, 2H, -CH2), 2.15 and 2.17 (2s, 3H, CH3), 2.34 and 2.76 (2t, 2H, J=7.31 Hz, -CH2), 3.34–3.45 (m, 2H, -S-CH2), 4.39 and 4.40 (2s, 3H, OCH3), 6.95–7.0 (m, 1H, Ar-H), 7.40–7.50 (m, 2H, Ar-H), 7.65–7.66 (m, 5H, Ar-H), 7.88 and 8.04 (2s, 1H, -CH=N), 11.16 and 11.26 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 16.6, 24.6, 25.2, 31.3, 33.0, 33.2, 33.4, 56.2, 111.5, 111.6, 125.8, 125.9, 127.4, 127.5, 127.8, 128.0, 129.4, 129.8, 131.3, 131.9, 134.4, 144.3, 147.4, 155.7, 155.8, 160.1, 160.3, 168.9, 174.7. For C20H22N6O2S calculated: 58.52% C, 5.40% H, 20.47% N; found: 58.56% C, 5.47% H, 20.51% N. MS [M + 1]+: m/z 411.

N′-(3-nitro-4-hydroxybenzylidene)-4- [(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (23)

Yield 72%. m.p. 125°C. IR (KBr) ν (cm−1): 3339 (amide N-H), 1669 (amide C=O), 1568–1404 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.37 and 2.77 (2t, 2H, J=7.31 Hz, -CH2), 3.35–3.46 (m, 2H, -S-CH2), 7.16–7.20 (m, 5H, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.84–7.89 (m, 2H, Ar-H), 7.94 and 8.10 (2s, 1H, -CH=N), 8.10 and 8.16 (2s, 1H, Ar-H), 9.89 and 9.91 (2s, 1H, -OH), 11.34 and 11.47 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.6, 25.2, 31.3, 33.0, 33.3, 56.9, 120.8, 120.9, 124.8, 125.1, 125.8, 125.9, 127.1, 127.2, 131.2, 131.8, 131.9, 133.6, 134.0, 134.4, 147.4, 135.6, 138.3, 138.4, 142.3, 145.3, 154.3, 154.6, 155.7, 155.8, 169.2, 174.9. For C20H22N6O2S calculated: 58.52% C, 5.40% H, 20.47% N; found: 58.56% C, 5.47% H, 20.51% N. MS [M + 1]+: m/z 411.

N′-(3,4,5-trimethoxybenzylidene)-4- [(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (24)

Yield 70%. m.p. 123°C. IR (KBr) ν (cm−1): 3337 (amide N-H), 1674 (amide C=O), 1576–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.10 (m, 2H, -CH2), 2.37 and 2.79 (2t, 2H, J=7.30 Hz, -CH2), 3.39–3.46 (m, 2H, -S-CH2), 3.69 and 3.70 (2s, 3H, OCH3), 3.82 and 3.87 (2s, 6H, OCH3), 6.95 and 6.98 (2s, 2H, Ar-H), 7.66–7.69 (m, 5H, Ar-H), 7.89 and 8.07 (2s, 1H, -CH=N), 11.35 and 11.39 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.7, 25.2, 31.2, 32.9, 33.2, 56.7, 56.7, 60.9, 105.0, 105.3, 125.8, 125.9, 131.3, 131.9, 134.4, 135.9, 147.3, 154.5, 169.1, 174.9. For C21H24N6O4S calculated: 55.25% C, 5.30% H, 18.41% N; found: 55.28% C, 5.39% H, 18.50% N. MS [M + 1]+: m/z 457.

N′-(4-benzyloxybenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (25)

Yield 65%. m.p. 159°C. IR (KBr) ν (cm−1): 3337 (amide N-H), 1670 (amide C=O), 1572–1422 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.06–2.09 (m, 2H, -CH2), 2.34 and 2.76 (2t, 2H, J=7.25 Hz, -CH2), 2.54 (s, 2H, CH2), 3.38–3.45 (m, 2H, -S-CH2), 7.05 and 7.08 (2d, 2H, J=7.50 Hz, Ar-H), 7.35 (t, 2H, J=7.10 Hz, Ar-H), 7.41 (t, 2H, J=7.32 Hz, Ar-H), 7.47 (d, 2H, J=7.62 Hz, Ar-H), 7.58 (d, 1H, J=7.64 Hz, Ar-H), 7.63 (d, 1H, J=8.10 Hz, Ar-H), 7.66–7.69 (m, 4H, Ar-H), 7.91 and 8.08 (2s, 1H, -CH=N), 11.19 and 11.29 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 24.8, 25.2, 31.3, 32.9, 33.3, 70.2, 106.0, 106.3, 11.2, 112.7, 118.6, 119.2, 120.1, 121.6, 121.9, 124.6, 125.8, 125.9, 131.3, 131.9, 134.4, 135.9, 147.3, 154.5, 169.2, 174.8. For C25H24N6O2S calculated: 63.54% C, 5.12% H, 17.78% N; found: 63.59% C, 5.20% H, 17.84% N. MS [M + 1]+: m/z 473.

N′-(4-izopropylbenzylidene)-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanoylhydrazide (26)

Yield 68%. m.p. 123°C. IR (KBr) ν (cm−1): 3338 (amide N-H), 1672 (amide C=O), 1571–1410 (C=C and C=N). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.19–1.21 (m, 6H, CH3), 2.07–2.10 (m, 2H, -CH2), 2.37 and 2.77 (2t, 2H, J=7.32 Hz, -CH2), 2.88–2.91 (m, 1H, CH), 3.41–3.45 (m, 2H, -S-CH2), 7.25 and 7.31 (2d, 2H, J=7.45 Hz, Ar-H), 7.55 (d, 1H, J=7.20 Hz, Ar-H), 7.60 (d, 1H, J=7.12 Hz, Ar-H), 7.66–7.67 (m, 5H, Ar-H), 7.95 and 8.12 (2s, 1H, -CH=N), 11.27 and 11.35 (2s, 1H, N-H). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 19.14, 24.3, 24.6, 25.2, 31.4, 33.0, 33.2, 33.4, 34.0, 34.1, 125.8, 125.9, 127.9, 128.0, 128.3, 131.3, 131.8, 131.9, 130.2, 133.3, 134.4, 144.1, 147.4, 151.6, 151.9, 155.7, 169.0, 174.8. For C21H24N6OS calculated: 61.74% C, 5.92% H, 20.57% N; found: 61.70% C, 5.81% H, 20.51% N. MS [M + 1]+: m/z 409.

Biochemistry

AChE and BuChE inhibitory activity

AChE and BuChE inhibitory activity was determined by Ellman’s [27] method with minor modifications. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and tested at maximum final concentration 80 μg/mL. Twenty microliter of enzyme (electric eel AChE or equine serum BuChE 1 U/mL), 10 μL sample were added to 2.4 mL buffer and the mixture was incubated at 37°C for 15 min. After 15 min incubation, 50 μL of 0.01 M 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and 20 μL of 75 mM acetylthiocholine iodide (ATCI) or 25 mM butyrylthiocholine iodide (BTCI) were added and the final mixture was incubated at room temperature for 30 min. Blank was prepared using 10 μL of DMSO instead of the test sample with all other procedures similar to those used in the case of the sample mixture. Absorbances were measured at 412 nm and 37°C using polystyrene cuvettes with spectrophotometer (Shimadzu UV-1700). Experiment was done in triplicate. Data are expressed as mean ± standart deviation (SD). Donepezil and galanthamine were used as standard drugs. The inhibition (percent) of AChE or BuChE was calculated using the following equation.

I (%)=100−(ODsample/ODcontrol)×100

Cytotoxicity

In vitro cytotoxicity of the synthesized compounds was assessed by using standard MTT bioassay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] against NIH/3T3 mouse embryonic fibroblast cells at 24 h of drug administration [28]. This time interval was determined by our preliminary studies [29]. NIH/3T3 cells were cultured in 96-well flat-bottom plates at 37°C for 24 h (2×104 cells per well). All of the compounds were DMSO individually and added to culture wells at varying concentrations (50–200 µg/mL), the highest final DMSO concentration was under 0.1% which had no effect on the cell viability [26]. After 24 h drug incubation at 37°C, 20 µL MTT solution (5 mg/mL MTT powder in PBS) was added to each well. Than 3 h incubation period was maintained in the same conditions. Purple formazan was occurred at the end of the process which is the reduction product of MTT agent by the mitochondrial dehydrogenase enzyme of intact cells. Formazan crystals were dissolved in 100 µL DMSO and the absorbance was read by ELISA reader (OD570 nm). The percentage of viable cells was calculated based on the medium control. Every concentration was repeated in three wells and IC50 values were defined as the drug concentrations that reduced absorbance to 50% of control values by using logarithmic graphics.

Results and discussion

Chemistry

In the present study, some N′-arylidene-4-[(1-phenyl-1H-tetrazol-5-yl)thio]butanohydrazide derivatives (1–26) were synthesized and some characteristics of the compounds were presented in Table 1. Target compounds were obtained at three steps as can be seen from Scheme 1. Initially, 1-phenyl-1H-tetrazole-5-thiol and ethyl 4-chlorobutanoate were refluxed with potassium carbonate in acetone to give ethyl 4-[(1-phenyl-1H-tetrazole-5-yl)thio]butanoate compound (A). Then, this ester compound was reacted with hydrazine hydrate to reach its corresponding hydrazide (B). To obtain final hydrazone compounds, compound B was reacted with substituted benzaldehydes. Structure elucidations of the final compounds were performed with IR, 1H NMR, 13C-NMR and MS spectroscopic methods and elemental analyses. In the IR spectra of all compounds characteristic nitrogen-hydrogen, carbon-oxygen bonds were observed at about 1668–1674 cm−1 region due to amide function. Also bands belong to amino group were seen at 3336–3341 cm−1 region compounds as expected. In the 1H-NMR spectra of the compounds, due to E/Z isomerization, most of the protons were resonated two different regions of the spectrum. The proton of the azomethine group was observed as two singlet peaks at about 7.84–8.54 ppm. Similarly, the signal belongs to the amine proton of this group was seen as two singlets at 11.10–11.76 ppm in the lowest field of the spectrum. The alkyl protons were also affected by the isomerization and mostly they were seen as multiplet peaks. –SCH2-protons were resonated at 3.33–3.46 ppm as two triplets or a multiplet peak. -COCH2-protons were observed as two triplets and at about 2.16–2.83 ppm. –C-CH2C- protons were seen as multiplet peak due to vicinal protons and also isomeric feature of the molecules. All other aromatic protons owing to phenyl ring and alkyl protons owing to methyl, methoxy substituents of the ring were determined at expected areas of the spectrum. In 13C-NMR spectra of the compounds, because of isomerism signals belong to alkyl carbons were resonated as two different peaks. The signals which were detected at highest value range 168.4–175.4 ppm were assigned for carbonyl carbon. All compounds demonstrated satisfactory elemental analyses results within ± 0.4%. In the MS spectra, the electron spraying technique with positive polarity mode was applied and M + 1 peaks were detected as base peak in agreement with the molecular weights.

Table 1:

Some characteristics of the compounds (1–26).

CompoundsRMolecular formulaMolecular weightM.P. (°C)
1HC18H18N6OS366135
22-CH3C19H20N6OS380106
33-CH3C19H20N6OS380102
44-CH3C19H20N6OS380107
52-OCH3C19H20N6O2S396142
63-OCH3C19H20N6O2S396129
74- OCH3C19H20N6O2S396118
82-BrC18H17BrN6OS444134
93-BrC18H17BrN6OS444138
104-BrC18H17BrN6OS444160
112-FC18H17FN6OS384112
123-FC18H17FN6OS384107
134-FC18H17FN6OS384124
142-NO2C18H17N3-7O3S411133
153-NO2C18H17N3-7O3S411156
164-NO2C18H17N3-7O3S411208
172-OHC18H18N6O2S382143
183-OHC18H18N6O2S382152
194-OHC18H18N6O2S382175
203,4-DiOCH3C20H22N6O3S42678
213,5-DiOCH3C20H22N6O3S426164
223-CH3,4-OCH3C20H22N6O2S410125
233-NO2,4-OHC18H17N7O4S427157
243,4,5-TriOCH3C21H24N6O4S456123
254-BenzyloxyC25H24N6O2S472158
264-IsopropylC21H24N6OS408123
Scheme 1: Synthesis of the compounds.Reagents and conditions: (i) K2CO3. reflux; (ii) NH2NH2.H2O. EtOH. r.t.; (iii) Ar-CHO. EtOH. reflux.
Scheme 1:

Synthesis of the compounds.

Reagents and conditions: (i) K2CO3. reflux; (ii) NH2NH2.H2O. EtOH. r.t.; (iii) Ar-CHO. EtOH. reflux.

Biochemistry

All of the compounds were tested to determine their anticholinesterase activity and cytotoxicity. The anticholinesterase activity of the compounds for AChE and BuChE enzymes were determined using, donepezil and galanthamine as standard drugs (Table 2). Compounds exhibited weak anticholinesterase activity as compared to standard drugs. Although the half maximal inhibitory concentrations of donepezil (3×10−3 µg/mL) and galanthamine (0.56 µg/mL) to inhibit 50% of the indicated enzymes have been defined at lower concentrations, the compounds have been studied at higher concentrations (max conc.: 80 µg/mL) to determine IC50 of them and also to identify their comparatively enzyme inhibition percentages at 80 µg/mL. However IC50 of the compounds 1–5, 7, 9, 10, 13, 20, 22–25 could not find on AChE at the highest test concentration. Additionally, compound 16 and 21 could not be analyzed for enzyme inhibitory activity due to solving problem. The lowest IC50 values have been determined for compounds 14 and 15 on AChE enzyme and for compound 18 on BuChE enzyme. The % enzyme inhibition results and IC50 results have been in consistency. Compounds 14 and 15 possessing the lowest IC50 values on AChE enzyme, have caused 91.97 and 98.51% inhibition on same enzyme. Moreover, compounds 11, 12, 17, 18 and 26 inhibited AChE enzyme over than 60%. Similarly, compound 18 possessing the highest IC50 value on BuChE have exhibited the maximum inhibition percentage (51.26%) on the enzyme. Most of the other compounds failed to show any inhibitory activity on BuChE at the highest tested dose along with solvation problem.

Table 2:

IC50 values of the title compounds (µg/mL) on AChE and BuChE enzymes and against NIH/3T3 cell line.

CompoundsAChE % inhibition (80 µg/mL)IC50a(µg/mL)BuChE % inhibition (80 µg/mL)IC50a(µg/mL)IC50b(µg/mL)
148.26 ± 0.99> 80––93.33±2.87
247.72± 0.69>80––108.33±2.89
337.99± 2.87>8013.08±2.50>8098.33±7.63
423.81±3.58>80––>200
532.70±2.48>80––>200
651.81±1.5078.50± 1.686.56±0.80>8093.33±15.27
746.2±0.83>808.37±1.75>80106.67±15.30
857.56±2.8075.69±2.37––>200
948.16±2.23>80––113.33±41.63
1038.95±1.62>80––128.33±10.40
1160±2.6077.25±1.066.02±0.50>8078.33±7.64
1266.02±2.0576±120.02±0.72>8098.33±7.63
1315.10±3.34> 80––136.67±15.30
1491.97±3.7254.67± 0.58––55.0±10.0
1598.51±0.3542.5±3.54––51.67±2.89
16––4.63±0.7>80>200
1765.37±3.4177±120.02±0.72>8088.33±16.07
1864.59±2.1276.5±2.8255.2±3.0551.26±1.37105.0±5.0
1957.82±0.6776.48±2.21––106.67±2.89
2028.78±2.27>80––66.67±7.64
21––––173.33±11.54
2223.63±4.94>80––98.33±12.58
235.58±1.44>80––133.33±30.55
2414.39±1.85>8014.94±0.24>80123.33±5.77
256.24±0.80>8015.44±1.11>8095.0±13.23
2664.32±0.0673.45±2.1613.94±1.00>80103.33±5.77
Donepezilnt3×10−3± 0.28×10−3nt1.26±0.14nt
Galanthaminent0.56±0.04nt7.28±2.48nt

  1. –, Not active; nt, non tested. aThe half maximal inhibitory concentration of the compounds to inhibit 50% of the indicated enzymes. bThe half maximal inhibitory concentration of the compounds to inhibit 50% of the mouse fibroblast cells (NIH/3T3).

The cytotoxicity of the compounds were determined against NIH/3T3 normal cells and IC50 values were detected as the half maximal inhibitory concentration of the compounds to inhibit 50% of the mouse fibroblast cell proliferations and results were given in Table 2. Accordingly, compounds 4, 5, 8 and 16 exhibited the highest potential showing with the lowest cytotoxicity against NIH/3T3 cells. These compounds did not inhibited 50% of the cells on the concentrations used and the IC50 values could not be calculated. The IC50 values for the compounds 2, 7, 9, 10, 13, 18, 19, 21, 23, 24 and 26 have been determined higher than 100 µg/mL which can be interpreted with moderate cytotoxicity. Furthermore when we evaluate the most active compounds, the IC50 values of the compounds 14 and 15 on AChE enzyme and compound 18 on BuChE were found lower than cytotoxic dose.

Discussion

The title tetrazole-hydrazone compounds (1–26) were obtained from various aldehydes and the main structure of the molecules differs by benzaldehyde moiety possessing methyl, methoxy, bromo, nitro, fluoro, hydroxy, benzyloxy and isopropyl substitutions. Regarding with substituent effect and position, 2-nitro and 3- nitro groups have positively contributed anticholinesterase activity of the compounds (Compound 14 and 15). A similar finding was observed in our previous study [30] that 3-nitro substituent caused an obvious increase of AChE enzyme inhibitory activity. However, there was not seen same situation for 3-methyl and 3-chloro substituents reported in the mentioned paper. In another reported literature, fluoro substituted derivatives showed higher AChE enzyme inhibitory activity than methyl substituted derivatives as in our study [31]. Mostly, methyl and methoxy substituents caused activity increase [32], [33], for our compounds this information has not been valid.

Considering BuChE inhibitory activity, compound 18 with 3-hydroxy substituent showed the highest enzyme inhibition activity differently from the literatures [17], [34]. In these studies, the positive effect of fluoro substituent was focused supporting the percentage inhibitions finding at 80 µg/mL of BuChE enzyme. Also, benzyl moiety which presents in the structure of the anticholinesterase drug donepezil contributed enzyme inhibitory activity on both enzymes [35].

Compounds 4, 5, 8 and 16 were determined as the least cytotoxic derivatives which possessed 4-methyl, 2-methoxy, 2-bromo and 4-nitro functions, respectively. Similar finding was seen in the previous study that nitro function caused a decrease in cytotoxic activity against healthy cell line NIH/3T3 [36]. The comparative interpretation of the cytotoxic activity and enzyme inhibitory activity of the compounds could not be done due to unlike and incompatible activity results.

Conclusion

New N′-arylidene-4-[(1-phenyl-1H-tetrazol-5-yl)thio]buta noylhydrazide derivatives (1–26) were synthesized and their anticholinesterase activities were screened on AChE and BuChE enzymes and cytotoxic activities were evaluated against NIH/3T3 cell line. According to the activity results, none of the compounds showed anticholinesterase activity as much as standard drugs. Compound 14 with 2-nitro phenyl and compound 15 with 3-nitro phenyl moieties on AChE and compound 18 bearing 3-hydroxy phenyl moiety on BuChE have been determined as the most active compounds with the highest inhibitory activity. Additionally, compounds 4, 5, 8 and 16 exhibited the lowest cytotoxicity against NIH/3T3 cells.

Corresponding author: Dr. Leyla Yurttaş, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey, Tel.: +90 222 335 05 80 – ext. 3783, Fax: +90 222 335 07 50

  1. Conflict of interest: The authors confirm that this article content has no conflict of interest.

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Received: 2016-02-08
Accepted: 2016-06-14
Published Online: 2016-11-16
Published in Print: 2017-04-01

©2017 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/tjb-2016-0207
Keywords for this article
Tetrazole; Hydrazone; Anticholinesterase; AChE; BuChE; Cytotoxicity

Articles in the same Issue

  1. Frontmatter
  2. Research Articles
  3. Robust background normalization method for one-channel microarrays
  4. Is there a relation between Murine double minute 2 T309G polymorphism and lung cancer risk in the Turkish population?
  5. Synthesis and biological evaluation of some new pyrimidine bearing 2,5-disubstituted 1,3,4-oxadiazole derivatives as cytotoxic agents
  6. Characterization and expression of dax1 during embryonic and gonad development in the carp (Cyprinus carpio)
  7. Implications of Stisa2 catalytic residue restoration through site directed mutagenesis
  8. Vitamin D receptor polymorphisms and related biochemical parameters in various cancer species
  9. Novel tetrazole derivatives: synthesis, anticholinesterase activity and cytotoxicity evaluation
  10. Superoxide Dismutase 1 (SOD 1) A251G Polymorphism
  11. Elevated serum ubiquitin-proteasome pathway related molecule levels in attention deficit hyperactivity disorder
  12. Alteration of protein localization and intracellular calcium content due to connexin26 D50A and A88V mutations
  13. Comparison of two inference approaches in Gaussian graphical models
  14. Proteomic analysis of erythropoietin-induced changes in neuron-like SH-SY5Y cells
  15. UVB-irradiated indole-3-acetic acid induces apoptosis via caspase activation
  16. A novel immobilization matrix for the biosensing of phenol: self assembled monolayers of calixarenes
  17. Opinion Papers
  18. Free radical area needs a radical change
  19. Possible mechanisms of transmissible cancers in Tasmanian devils
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