Synthesis and Study of α-Glucosidase Inhibitory, Antimicrobial and Antioxidant Activities of Some Benzimidazole Derivatives Containing Triazole, Thiadiazole, Oxadiazole, and Morpholine Rings

Article abstract of DOI:10.1007/s10593-015-1637-1

A new series of 2-(4-bromobenzyl)- and 2-(4-fluorobenzyl)-1H-benzimidazole derivatives containing 1,2,4-triazole, 1,3,4-thiadiazole, 1,3,4-oxadiazole, and morpholine rings has been synthesized. The structures of the newly synthesized compounds were confirmed by ¹H and ¹³C NMR and mass spectra, and all substances have been screened for their α-glucosidase inhibitory, antimicrobial and antioxidant activities. Hydrazide, oxadiazole, thiosemicarbazide, and 1,2,4-triazole-3-thiol derivatives showed very good ABTS scavenging activities (IC₅₀ 1.94-4.79 μM). Oxadiazole and thiosemicarbazide derivatives also revealed notable DPPH scavenging activity. 5-[(2-(4-Bromobenzyl)-1H-benzimidazol-1-yl)methyl]-1,3,4-oxadiazole-2-thiol was found to be more potent than acarbose. 2-(4-Fluorobenzyl)-1H-benz- imidazole was effective against both Gram-positive and Gram-negative bacteria, especially, M. smegmatis.

Full text of DOI:10.1007/s10593-015-1637-1

DOI 10.1007/s10593-015-1637-1
Chemistry of Heterocyclic Compounds, Vol. 50, No. 12, March, 2015 (Russian Original Vol. 50, No. 12, December, 2014)
SYNTHESIS AND STUDY OF α-GLUCOSIDASE INHIBITORY,
ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SOME
BENZIMIDAZOLE DERIVATIVES CONTAINING TRIAZOLE,
THIADIAZOLE, OXADIAZOLE, AND MORPHOLINE RINGS
1
1
2
*
E. Menteşe , S. Ülker , and B. Kahveci
A new series of 2-(4-bromobenzyl)- and 2-(4-fluorobenzyl)-1H-benzimidazole derivatives containing
1,2,4-triazole, 1,3,4-thiadiazole, 1,3,4-oxadiazole, and morpholine rings has been synthesized. The
1
13
structures of the newly synthesized compounds were confirmed by H and C NMR and mass spectra,
and all substances have been screened for their α-glucosidase inhibitory, antimicrobial and antioxidant
activities. Hydrazide, oxadiazole, thiosemicarbazide, and 1,2,4-triazole-3-thiol derivatives showed very
good ABTS scavenging activities (IC50 1.94-4.79 µM). Oxadiazole and thiosemicarbazide derivatives
also revealed notable DPPH scavenging activity. 5-[(2-(4-Bromobenzyl)-1H-benzimidazol-1-yl)methyl]-
1,3,4-oxadiazole-2-thiol was found to be more potent than acarbose. 2-(4-Fluorobenzyl)-1H-benz-
imidazole was effective against both Gram-positive and Gram-negative bacteria, especially,
M. smegmatis.
Keywords: benzimidazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole, 1,2,4-triazole, antimicrobial activity,
antioxidant activity, α-glucosidase inhibition.
The emergence of resistant bacteria to antibiotics is a major health problem [1, 2]. Infectious diseases
remain a major cause of mortality. In particular, tuberculosis causes almost three million deaths every year, and
approximately one-third of the world's population has been infected with the pathogen Mycobacterium
tuberculosis [2, 3]. Therefore, there is an immediate need for the development of potent new antimycobacterial
agents [4].
The synthesis of α-glucosidase inhibitors is an important area of medicinal chemistry. α-Glucosidase
plays a key role in carbohydrate metabolism and glycoprotein biosynthesis. Inhibition of this enzyme, therefore,
can play an important role in the treatment of degenerative diseases, such as type-II diabetes mellitus.
Interference with the enzymatic action of intestinal α-glucosidase would delay the glucose absorption and
decrease postprandial hyperglycemia. In addition, α-glucosidase inhibition is a promising strategy for
development of novel antiHIV agents because glycosylation of viral envelope proteins is essential for virus
infectivity [5, 6].
_______
*To whom correspondence should be addressed, e-mail: emre.mentese@erdogan.edu.tr.
1Recep Tayyip Erdoğan University, Fener St., Rize 53100, Turkey.
2Karadeniz Technical University, Farabi St., Trabzon 61080, Turkey; e-mail:bahittin@ktu.edu.tr.
_________________________________________________________________________________________
Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 12, pp. 1820-1831, December, 2014.
Original article submitted July 4, 2014; revision submitted August 25, 2014.
0009-3122/15/5012-1671©2015 Springer Science+Business Media New York
1671

During the last decades, great attention has been focused on the synthetic antioxidants due to their
biological importance and because they generally are less expensive than many natural antioxidants [7]. Free
radicals play a negative role in human life: overproduction of free radicals results in many diseases, such as
diabetes, cardiovascular diseases, septic shock, cancer, Parkinson's disease, Alzheimer's disease, and some other
neural disorders [8-10].
Five-membered nitrogen-containing heterocyclic compounds continue to play an important role in
medicinal chemistry [11-13]. Each year, a large number of drug candidates that contain heterocyclic ring are
synthesized by researchers [14]. Among those compounds, benzimidazole derivatives are represented by a wide
variety of medicines, such as albendazole, thiabendazole, omeprazole, carbendazim, mebendazole, and
timoprazole [15-17], while in nature the benzimidazole moiety is found in the structure of vitamin B12 [18].
Different heterocyclic rings (like 1,2,4-triazole, 1,3,4-thiadiazole, and oxadiazole) linked to the benzimidazole
moiety have been recognized as important pharmacophores, and such ring assemblies have shown diverse
biological activities, such as antibacterial, anti-inflammatory, antiulcer, antioxidant, antitumor, lipase inhibition,
glutaminyl cyclase inhibition, antienterovirus, and anticancer activities [19-26]. Also, it has been found that
insertion of halogen atoms, such as fluorine, chlorine, and bromine, in the structures of heterocyclic compounds
leads to an increase of biological activity [13, 27, 28]. Literature survey reveals that many benzimidazole
derivatives possess appreciable antimycobacterial activity [29-33]. Moreover, benzimidazoles bearing
thiosemicarbazide and triazole ring have demonstrated considerable antioxidant actitity [34, 35]. In addition, α-
glucosidase inhibitory studies of benzimidazole derivatives are limited [36].
In our previous studies, we have synthesized some bioactive benzimidazole derivatives. Some of the
compounds were shown to demonstrate important antitumor, antioxidant, and lipase inhibition activities [34, 37,
38]. In continuation of our work towards the synthesis of bioactive heterocyclic compounds, we report here the
preparation of some new benzimidazole derivatives bearing the triazole, thiadiazole, oxadiazole, and
morpholine ring and the biological evaluation of their α-glucosidase inhibitory, antimicrobial, and antioxidant
properties.
The synthetic strategies for obtaining the target compounds are outlined in Schemes 1 and 2. The
starting iminoester hydrochlorides 1a,b were synthesized by the Pinner method [39]. 2-Substituted benz-
imidazoles 2a,b [40, 41] were prepared by the reaction of o-phenylenediamine with iminoesters 1a,b according
to the procedures published earlier [38]. The reaction of compounds 2a,b with methyl bromoacetate in the
presence of K2CO3 yielded esters 3a,b. Treatment of the latter with morpholine produced 2-(2-(4-halo-benzyl)-
1H-benzimidazol-1-yl)-1-(morpholin-4-yl)ethanones 4a,b. Compounds 5a,b were prepared by refluxing esters
3a,b with hydrazine hydrate in ethanolic solution. The ring closure reaction of the synthesized hydrazides 5a,b
with carbon disulfide in the presence of KOH resulted in the formation of 2-mercapto-1,3,4-oxadiazoles 6a,b.
Thiosemicarbazides are known to be useful compounds for the synthesis of different heterocyclic ring
systems containing nitrogen and sulfur [42]. In this work, thiosemicarbazides 7a-d were prepared by the
nucleophilic addition of acid hydrazides 5a,b to methyl isothiocyanate (for compounds 7a,b) or ethyl iso-
thiocyanate (for compounds 7c,d). The synthesis of 5-{[2-(4-halobenzyl)-1H-benzimidazol-1-yl]methyl}-
4-alkyl-4H-1,2,4-triazole-3-thiols 8a-d was realized through intramolecular cyclization of thiosemicarbazides
7a-d in the presence of 2 N aqueous NaOH solution under reflux. On the other hand, the cyclization of
compounds 7a-d with cold concentrated sulfuric acid resulted in the formation of 5-{[2-(4-halobenzyl)-
1H-benzimidazol-1-yl]methyl}-N-alkyl-1,3,4-thiadiazol-2-amines 9a-d. The structures of all the new
compounds were confirmed by 1H and 13C NMR spectroscopy and mass spectrometry.
The scavenging activity of the synthesized compounds was evaluated by the 2,2'-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS) assay. ABTS is easily converted to a stable radical cation that reacts
with various antioxidants [43-46]. Firstly, each compound was tested at the initial concentration of 70 µM in the
assay mixture. Several dilutions from this concentration were made and tested on the decolorization
of the ion radical ABTS•+, and the concentration extinguishing the absorbance at 734 nm was evaluated.
1672

NH2
N
.
HCl
NH
OEt
BrCH2CO2Et
NH2
N
H
MeOH, room temp., 12 h
K2CO3, Me2CO
10 h
R1
R1
1a,b
2a,b
H
N
N
N
N
N
O
O
N
EtO
R1
O
R1
O
3a,b
4a,b
.
NH2NH2 H2O
EtOH, , 8 h
N
N
N
N
CS2
N
H
N
KOH, EtOH
R1
N
, 4 h
O
H2N
O
R1
SH
5a,b
6a,b
R2N=C=S
EtOH, , 2 h
N
N
N
N
H
H
HS
S
N
N
N
N
H
R1
R1
O
O
HN
R2
HN
R2
7a–d
1. NaOH,
H2O, , 2 h
2. HCl, H2O
1. conc. H2SO4,
40 min
2. NH3, H2O
N
N
N
N
N
N
N
R1
N
R1
S
N
R2
R2
N
H
HS
8a–d
9a–d
1–6 a R1 = Br, b R1 = F; 7–9 a,b R1 = Br, c,d R1 = F, a,c R2 = Me, b,d R2 = Et
1673

Results were expressed in Trolox equivalents of antioxidant activity (μM TEAC). Compounds 5a,b, 6a,b, 7a-d,
and 8a-d showed very good radical scavenging properties (Table 1). These compounds exhibited higher
scavenging activity than the reference antioxidant Trolox. Other tested compounds showed no activity.
The free radical scavenging activity of the synthesized compounds was evaluated by the colorimetric
decrease in absorbance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) resulting from chemical trapping of unpaired
electron. Initially, each compound was assessed at the initial concentration of 70 µM in the reaction mixture.
Results are expressed as percentage decrease with respect to the control values. Several dilutions of the initial
solution were made and assayed accordingly to obtain the concentration of the sample required to scavenge
50% of DPPH free radical (IC50) applying a suitable regression analysis of the mean values (Table 1).
Compounds 6a,b and 7a-d showed good scavenging activity. On the other hand, compounds 8a,b showed weak
activity, while compounds 5a,b showed no activity.
Data on the antimicrobial activity of the newly synthesized compounds are presented in Table 2. The
tested compounds not included in Table 2 were found to be ineffective. Only compound 2b was effective
against both Gram-negative and Gram-positive bacteria. Compound 2b also showed very good activity against
M. smegmatis (30 mm inhibition zone). Compounds 5a and 6a were very effective against E. faecalis (15 and
17 mm inhibition zone, respectively). Only compound 6b exhibited moderate antifungal activity (22 mm
inhibition zone with S. cerevisiæ).
TABLE 1. DPPH and ABTS Radical Scavenging Activities of the
Synthesized Compounds
Compound
DPPH method: IC50, µM
ABTS method: TEAC, µM
—*
—
1.55 ± 0.07
1.58 ± 0.24
1.51 ± 0.22
1.86 ± 0.02
1.68 ± 0.09
1.53 ± 0.26
1.72 ± 0.13
1.41 ± 0.07
1.78 ± 0.06
1.56 ± 0.19
1.36 ± 0.07
1.55 ± 0.09
—
5a
5b
6a
16.14 ± 1.143
10.95 ± 0.43
9.69 ± 4.22
11.93 ± 1.59
8.98 ± 1.26
10.84 ± 2.23
29.16 ± 10.85
59.26 ± 36.61
—
6b
7a
7b
7c
7d
8a
8b
8c
75.05 ± 1.86
7.57 ± 0.60
8d
Trolox
_______
*Not determined.
All compounds were evaluated with regard to α-glucosidase activity. Compounds 2a,b, 4a and 6a
showed anti-α-glucosidase activity at various concentrations. No significant inhibitory effect was detected for
the other benzimidazoles. Among the tested compounds, oxadiazolethiol 6a showed the best α-glucosidase
inhibition. The compound inhibited α-glucosidase activity by 99.5% at a concentration of 100 µM. Acarbose, a
known α-glucosidase inhibitor used as an antidiabetic drug, showed an inhibitory effect of 93.0% at the same
concentration (Table 3).
In conclusion, a series of new 2-(4-bromobenzyl)- and 2-(4-fluorobenzyl)-1H-benzimidazole
derivatives with 1,2,4-triazole, 1,3,4-thiadiazole, 1,3,4-oxadiazole, and morpholine ring-containing substituents
has been obtained. Among these compounds were found effective free radical scavengers and antibacterial
(effective against Gram-positive, Gram-negative, and mycobacteria) and antifungal agents, as well as
α-glucosidase inhibitors. The results provide an incentive for further search of potential biologically active
heterocyclic compounds containing the benzimidazole ring.
1674

TABLE 2. Antimicrobial Activity of the Synthesized Compounds
(Inhibition Zone, mm)
Compound
E. c.* Y. p.
P. a.
K. p.
S. a.
B. c.
E. f.
M. s. C. a.
S. c.
10
—
—
—
—
—
—
—
—
10
—
—
—
6
—*2
—
—
—
—
—
—
—
—
18
—
—
—
14
—
—
—
—
—
—
—
—
15
—
—
—
6
6
—
—
—
15
17
—
—
—
—
10
—
—
—
30
10
—
—
—
—
—
12
15
—
35
—
—
—
—
—
—
—
—
—
—
—
—
—
25
—
—
8
2b
—
—
—
—
—
—
—
—
18
—
—
—
—
—
12
8
—
—
—
4a
8
4b
—
5a
6a
—
—
—
—
35
—
—
—
—
—
—
22
10
6b
7a
—
9c
—
9d
Ampicillin
Streptomycin
Fluconazole
DMSO
15
—
—
—
—
—
>25
—
_______
*Bacteria tested: E. c. – Escherichia coli ATCC 25922, Y. p. – Yersinia
pseudotuberculosis ATCC 911, P. a. – Pseudomonas æruginosa ATCC
43288, K. p. – Klebsiella pneumoniæ ATCC 13833, S. a. –
Staphylococcus aureus ATCC 25923, B. c. – Bacillus cereus 702 Roma,
E. f. – Enterococcus fæcalis ATCC 29212, M. s. – Mycobacterium
smegmatis ATCC 607. Fungi tested: C. a. – Candida albicans ATCC
60193, S. c. – Saccharomyces cerevisiæ RSKK 251.
*2No inhibition.
TABLE 3. Inhibition of α-Glucosidase by the Newly Synthesized Compounds
Compound
Inhibition, % (at 100 µM)
IC50, µM
69.4 ± 4.9
16.4 ± 5.8
11.8 ± 2.7
99.5 ± 0.5
93.0 ± 0.5
82.85 ± 3.76
nd*
2a
2b
nd
4a
4.67 ± 0.58
11.16 ± 0.23
6a
Acarbose
_______
*Not determined.
EXPERIMENTAL
1H and 13C NMR spectra were recorded on a Varian Mercury spectrometer (400 and 100 MHz,
respectively) in CDCl3 (compounds 4a,b) or DMSO-d6 (other compounds) using TMS as internal standard. ESI
mass spectra were registered on a Thermo Scientific Quantum Access max LC-MS spectrometer with HESI-II
probe. The elemental compositions were determined on a Carlo Erba 1106 CHN-analyzer. Melting points were
determined in capillary tubes on a Büchi oil-heated melting point apparatus and are uncorrected. All reactions
were monitored by TLC using precoated aluminum sheets (silica gel 60 F 254, 0.2 mm thickness) and EtOAc–
hexane, 3:1, as eluent. All the chemicals were supplied by Merck, Aldrich, or Fluka.
Synthesis of Methyl 2-[2-(4-Halobenzyl)-1H-benzimidazol-1-yl]acetates 3a,b (General Method). A
mixture of compound 2a,b (0.01 mol), dry K2CO3 (4.14 g, 0.03 mol), and methyl bromoacetate (1.07 ml,
1675

0.011 mol) in acetone (30 ml) was stirred at room temperature for 10 h. After the completion of the reaction,
the product was precipitated by the addition of water. The precipitate was filtered off, dried, and recrystallized
from Me2CO–H2O, 1:1.
Methyl 2-[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]acetate (3a). Yield 94%, white powder,
1
mp 168-169°C (Me2CO–H2O, 1:1). H NMR spectrum, , ppm: 3.53 (3H, s, OCH3); 4.38 (2H, s, CH2); 5.32
(2H, s, NCH2), 7.14-7.23 (4H, m, H Ar); 7.42-7.48 (3H, m, H Ar); 7.54-7.57 (1H, m, H Ar). 13C NMR
spectrum,  (ppm): 32.5 (CH2); 44.8 (NCH2); 52.6 (OCH3); 110.6; 119.07; 120.2; 122.1; 122.5; 131.6 (2C);
136.1; 136.4; 142.6 (C Ar); 153.8 (C=N); 168.7 (C=O). Mass spectrum, m/z (Irel, %): 361 [M(81Br)+H]+ (19),
359 [M(79Br)+H]+ (20). Found, %: C 57.85; H 4.63; N 7.48. C18H17BrN2O2. Calculated, %: C 57.92; H 4.59;
N 7.51.
Methyl 2-[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]acetate (3b). Yield 88%, white powder,
mp 140-141°C (Me2CO–H2O, 1:1). 1H NMR spectrum, , ppm (J, Hz): 3.60 (3H, s, OCH3); 4.30 (2H, s, CH2);
5.28 (2H, s, NCH2); 7.16-7.26 (4H, m, H Ar); 7.35-7.39 (2H, m, H Ar); 7.52 (1H, d, J = 4.8, H Ar); 7.66 (1H, d,
J = 4.8, H Ar). 13C NMR spectrum, , ppm (J, Hz): 32.47 (CH2), 44.95 (NCH2), 52.80 (OCH3), 110.7; 115.6 (d,
JC–F = 18.0, C Ar); 119.2; 122.3; 131.4; 131.5; 133.2; 136.2; 142.7 (C Ar); 154.3 (C=N); 161.63 (d,
J
C–F = 240.0, F–C); 168.93 (C=O). Mass spectrum, m/z (Irel, %): 299 [M+H]+ (100). Found, %: C 69.17; H 5.40;
N 8.93. C18H17FN2O2. Calculated, %: C 69.22; H 5.49; N 8.97.
Synthesis of 2-[2-(4-Halobenzyl)-1H-benzimidazol-1-yl]-1-(morpholin-4-yl)ethanones 4a,b (General
Method). A mixture of compound 3a,b (0.01 mol) and morpholine (5 ml) was allowed to reflux for 19 h. After
the reaction was completed, the solvent was evaporated under reduced pressure. The crude residue was
recrystallized from EtOH–H2O, 1:1.
2-[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]-1-(morpholin-4-yl)ethanone (4a). Yield 65%, white
1
powder, mp 172-173°C. H NMR spectrum, , ppm (J, Hz): 3.32-3.36 (2H, m, NCH2); 3.51-3.54 (2H, m,
NCH2); 3.62-3.64 (4H, m, OCH2); 4.24 (2H, s, CH2); 4.63 (2H, s, NCH2); 7.08-7.14 (4H, m, H Ar); 7.23-7.26
13
(2H, m, H Ar); 7.41 (1H, d, J = 8.0, H Ar); 7.76 (1H, d, J = 8.0, H Ar). C NMR spectrum, , ppm: 34.1
(CH2); 42.4 (NCH2); 44.5; 45.2 (NCH2); 66.2; 66.7 (OCH2); 108.7; 119.8; 121.1; 122.3; 122.9; 130.3; 131.9;
134.9; 135.9; 142.4 (C Ar); 152.6 (C=N); 164.0 (C=O). Mass spectrum, m/z (Irel, %): 416 [M(81Br)+H]+ (100),
414 [M(79Br)+H]+ (90). Found, %: C 57.92; H 4.80; N 10.08. C20H20BrN3O2. Calculated, %: C 57.98; H 4.87;
N 10.14.
2-[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]-1-(morpholin-4-yl)ethanone (4b). Yield 60%, white
1
powder, mp 159-160°C. H NMR spectrum, , ppm (J, Hz): 3.32-3.36 (2H, m, NCH2); 3.52-3.55 (2H, m,
NCH2); 3.65-3.66 (4H, m, OCH2); 4.14 (2H, s, CH2); 5.19 (2H, s, NCH2); 7.08-7.14 (4H, m, H Ar); 7.28-7.31
(2H, m, H Ar); 7.38 (1H, d, J= 8.0, H Ar); 7.53 (1H, d, J= 8.0, H Ar). 13C NMR spectrum, , ppm (J, Hz): 32.5
(CH2); 42.3 (NCH2); 44.6; 45.1 (NCH2); 66.4 (2OCH2); 110.6; 115.5 (d, JC-F = 25.0, C Ar); 118.8; 121.7; 122.1;
131.2; 131.4; 133.4; 133.9; 136.5; 142.5 (C Ar); 152.6 (C=N); 161.6 (d, JC-F = 242.0, C-F); 165.3 (C=O). Mass
spectrum, m/z (Irel, %): 354 [M+H]+ (100). Found, %: C 67.91; H 5.62; N 11.80. C20H20FN3O2. Calculated, %:
C 67.97; H 5.70; N 11.89.
Synthesis of 2-[2-(4-Halobenzyl)-1H-benzimidazol-1-yl]acetohydrazides 5a,b (General Method). A
mixture of compound 3a,b (0.01 mol) and hydrazine monohydrate (1.27 ml, 0.025 mol) in ethanol (15 ml) was
refluxed for 8 h. After the reaction was completed, the mixture was kept overnight in the refrigerator, and a
white solid precipitated. The crude product was filtered off, dried, and recrystallized from EtOH.
2-[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]acetohydrazide (5a). Yield 88%, white crystals,
1
mp 214-215°C. H NMR spectrum, , ppm: 4.32 (2H, s, CH2); 4.45 (2H, s, NH2, exchanged with D2O); 4.90
(2H, s, NCH2); 7.20-7.27 (4H, m, H Ar); 7.29-7.33 (1H, m, H Ar); 7.43-7.64 (3H, m, H Ar); 9.60 (1H, s, NH,
exchange, D2O). 13C NMR spectrum, , ppm: 32.8 (CH2); 45.1 (NCH2); 110.6; 119.2; 120.3; 122.1; 122.4;
131.8; 131.9; 136.2; 137.00; 142.8 (C Ar); 154.4 (C=N); 166.5 (C=O). Mass spectrum, m/z (Irel, %): 361
[M(81Br)+H]+ (90), 359 [M(79Br)+H]+ (100). Found, %: C 53.41; H 4.15; N 15.53. C16H15BrN4O. Calculated,
%: C 53.50; H 4.21; N 15.60.
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2-[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]acetohydrazide (5b). Yield 82%, white crystals,
1
mp 195-196°C (EtOH). H NMR spectrum, , ppm (J, Hz): 4.38 (2H, s, CH2); 4.43 (2H, s, NH2, exchanged
with D2O); 4.96 (2H, s, NCH2); 7.19-7.28 (4H, m, H Ar); 7.41-7.45 (2H, m, H Ar); 7.49 (1H, d, J = 8.0, H Ar);
13
7.64 (1H, d, J = 8.0, H Ar); 9.63 (1H, s, NH, exchanged with D2O). C NMR spectrum, , ppm (J, Hz): 32.6
(CH2); 45.0 (NCH2); 110.6; 115.7 (d, JC-F = 20.0, C Ar); 119.1; 122.1; 122.4; 131.5; 133.6; 136.2; 142.8 (C Ar);
154.7 (C=N); 161.6 (d, JC–F = 240.0, CF); 166.5 (C=O). Mass spectrum, m/z (Irel, %): 299 [M+H]+ (100). Found,
%: C 64.35; H 5.01; N 18.72. C16H15FN4O. Calculated, %: C 64.42; H 5.07; N 18.78.
Synthesis of 5-[(2-(4-Halobenzyl)-1H-benzimidazol-1-yl)methyl]-1,3,4-oxadiazole-2-thiols 6a,b
(General Method). A solution of KOH (0.56 g, 0.01 mol) in water (5 ml) and CS2 (0.61 ml, 0.01 mol) were
added to a solution of compound 5a,b (0.01 mol) in ethanol (5 ml) and then the mixture was refluxed for 4 h.
The mixture was first yellow, then turned to green, and then light-yellow as the reaction progressed. After the
reaction was completed, the mixture was cooled to room temperature and neutralized with diluted HCl (4 N) to
pH 5-6. The mixture was left to cool down, and the precipitated product was filtrated, washed with H2O, and
recrystallized from EtOH.
5-[(2-(4-Bromobenzyl)-1H-benzimidazol-1-yl)methyl]-1,3,4-oxadiazole-2-thiol (6a). Yield 75%,
1
white crystals, mp 247-248°C. H NMR spectrum, , ppm: 4.35 (2H, s, CH2); 5.74 (2H, s, NCH2); 7.23-7.33
(4H, m, H Ar); 7.41-7.54 (2H, m, H Ar); 7.59-7.68 (2H, m, H Ar); 14.40 (1H, br. s, SH). 13C NMR spectrum, ,
ppm: 32.5 (CH2); 38.7 (NCH2); 110.9; 119.3; 120.5; 122.8; 123.1; 131.7; 131.8; 135.7; 136.2; 142.4 (C Ar);
153.9; 159.6 (C=N); 178.4 (C=S). Mass spectrum, m/z (Irel, %): 403 [M(81Br)+H]+ (100), 401 [M(79Br)+H]+
(90). Found, %: C 50.84; H 3.22; N 13.93. C17H13BrN4OS. Calculated, %: C 50.88; H 3.27; N 13.96.
5-[(2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl)methyl]-1,3,4-oxadiazole-2-thiol (6b). Yield 72%,
1
white crystals, mp 232-233°C. H NMR spectrum, , ppm: 4.42 (2H, s, CH2); 5.80 (2H, s, NCH2); 7.17-7.22
(2H, m, H Ar); 7.31-7.37 (4H, m, H Ar); 7.67-7.69 (2H, m, H Ar); 13.00 (1H, br. s, SH). 13C NMR spectrum, ,
ppm (J, Hz): 32.3 (CH2); 38.7 (NCH2); 110.8; 115.68 (d, JC–-F = 20.0, C Ar); 122.7; 123.1; 131.2; 131.3; 132.9;
135.8; 142.5 (C Ar); 154.1; 159.6 (C=N); 161.6 (d, JC–F = 240.0, CF); 178.4 (C=S). Mass spectrum, m/z (Irel, %):
341 [M+H]+ (100). Found, %: C 59.93; H 3.81; N 16.42. C17H13FN4OS. Calculated, %: C 59.99; H 3.85; N 16.46.
Synthesis of 4-Alkyl-1-{2-[2-(4-halobenzyl)-1H-benzimidazol-1-yl]acetyl}thiosemicarbazides 7a-d
(General Method). A mixture of an appropriate acid hydrazide 5a,b (0.01 mol) in ethanol (15 ml) and the
corresponding isothiocyanate (0.013 mol) was refluxed for 2 h. The solution was cooled, and a white solid
appeared. The precipitated product was filtered off and recrystallized from EtOH–H2O, 2:1.
1-{2-[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]acetyl}-4-methylthiosemicarbazide (7a). Yield 75%,
1
white crystals, mp 226-227°C. H NMR spectrum, , ppm: 2.97 (3H, s, CH3); 4.27 (2H, s, CH2); 4.99 (2H, s,
NCH2); 7.22-7.31 (4H, m, H Ar); 7.33-7.59 (4H, m, H Ar); 8.14 (1H, s, NH, exchanged with D2O); 9.42 (1H, s,
NH, exchanged with D2O); 10.34 (1H, s, NH, exchanged with D2O). 13C NMR spectrum, , ppm: 31.5 (NCH3);
32.8 (CH2); 44.9 (NCH2); 110.7; 119.2; 120.3; 122.2; 122.5; 131.8 (2C); 131.9; 136.2; 136.9; 142.8 (C Ar);
154.3 (C=N); 167.1 (C=O). Mass spectrum, m/z (Irel, %): 434 [M(81Br)+H]+ (100), 432 [M(79Br)+H]+ (90).
Found, %: C 49.96; H 4.16; N 16.15. C18H18BrN5OS. Calculated, %: C 50.01; H 4.20; N 16.20.
1-{2-[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]acetyl}-4-ethylthiosemicarbazide (7b). Yield 72%,
white crystals, mp 218-219°C. 1H NMR spectrum, , ppm (J, Hz): 0.90 (3H, t, J = 6.8, CH3); 3.08-3.11 (2H, m,
CH2); 3.94 (2H, s, CH2); 4.67 (2H, s, NCH2); 6.89-6.99 (4H, m, H Ar); 7.02-7.29 (4H, m, H Ar); 7.82 (1H, s,
NH, exchanged with D2O); 9.01 (1H, s, NH, exchanged with D2O); 10.00 (1H, s, NH, exchanged with D2O).
13C NMR spectrum, , ppm: 14.9 (CH3); 32.6 (CH2); 44.8 (NCH2); 110.7; 115.8; 119.0; 120.4; 122.4; 122.7;
131.6; 131.7; 131.9; 135.0; 136.6; 142.3 (C Ar); 154.3 (C=N); 167.1 (C=O). Mass spectrum, m/z (Irel, %): 448
[M(81Br)+H]+ (100), 446 [M(79Br)+H]+ (90). Found, %: C 51.05; H 4.48; N 15.61. C19H20BrN5OS. Calculated,
%: C 51.12; H 4.52; N 15.69.
1-{2-[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]acetyl}-4-methylthiosemicarbazide (7c). Yield 72%,
1
white crystals, mp 220-222°C. H NMR spectrum, , ppm: 3.07 (3H, s, CH3); 4.39 (2H, s, CH2); 5.15 (2H, s,
NCH2); 7.27-7.49 (4H, m, H Ar); 7.51-7.74 (4H, m, H Ar); 8.26 (1H, s, NH, exchanged with D2O); 9.53 (1H, s,
1677

13
NH, exchanged with D2O); 10.45 (1H, s, NH, exchanged with D2O). C NMR spectrum, , ppm (J, Hz): 31.5
(CH3); 32.6 (CH2); 44.9 (NCH2); 110.7; 115.7 (d, JC–F = 20.0, C Ar); 119.1; 122.1; 122.4; 131.3; 131.4; 131.5;
136.2; 142.7 (C Ar); 154.6 (C=N); 160.9 (d, JC–F = 240.0, CF); 167.14 (C=O). Mass spectrum, m/z (Irel, %): 372
[M+H]+ (100). Found, %: C 58.16; H 4.83; N 18.82. C18H18FN5OS. Calculated, %: C 58.21; H 4.88; N 18.86.
1-{2-[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]acetyl}-4-ethylthiosemicarbazide (7d). Yield 82%, white
crystals, mp 140-41°C. 1H NMR spectrum, , ppm (J, Hz): 1.22 (3H, t, J = 7.2, CH3); 3.54-3.57 (2H, m, CH2);
4.32 (2H, s, CH2); 5.04 (2H, s, NCH2); 7.19-7.29 (4H, m, H Ar); 7.31-7.47 (2H, m, H Ar); 7.54 (1H, d, J = 8.0,
H Ar); 7.65 (1H, d, J = 8.0, H Ar); 8.19 (1H, s, NH, exchanged with D2O); 9.39 (1H, s, NH, exchanged with
D2O); 10.37 (1H, s, NH, exchanged with D2O). 13C NMR spectrum, , ppm (J, Hz): 15.0 (CH3); 32.5; 39.4
(CH2); 44.9 (NCH2); 110.7; 115.7 (d, JC–F = 20.0, C Ar); 119.1; 122.0; 122.1; 122.4; 131.3; 131.5; 133.5; 136.1;
142.7 (C Ar); 154.7 (C=N); 161.6 (d, JC–F = 241.0, CF); 167.0 (C=O). Mass spectrum, m/z (Irel, %): 386 [M+1]+
(100). Found, %: C 59.14; H 5.18; N 18.12. C19H20FN5OS. Calculated, % C: 59.20; H 5.23; N 18.17.
Synthesis of 4-Alkyl-5-{[2-(4-halobenzyl)-1H-benzimidazol-1-yl]methyl}-4H-1,2,4-triazole-3-thiols 8a-d
(General Method). A mixture of thiosemicarbazide 7a-d (0.01 mol) in 2 N aqueous NaOH (10 ml) was
refluxed for 2 h. The solution was cooled and acidified to pH 5-6 with 37% HCl. The precipitated product was
filtered off, washed with water, and recrystallized from EtOH.
5-{[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]methyl}-4-methyl-4H-1,2,4-triazole-3-thiol (8a). Yield
75%, white powder, mp 286-287°C. 1H NMR spectrum, , ppm: 3.53 (3H, s, CH3); 4.33 (2H, s, CH2); 4.72 (2H,
13
s, NCH2); 7.26-7.31 (4H, m, H Ar); 7.52-7.66 (4H, m, H Ar); 13.59 (1H, s, NH). C NMR spectrum, , ppm:
30.6 (CH3); 32.8 (CH2); 41.0 (NCH2); 111.0; 111.7; 119.4; 119.6; 122.5; 122.7; 122.9; 131.9; 132.0; 137.0
(C Ar); 149.1 (triazole C-5); 154.5 (benzimidazole C-2); 168.2 (triazole C-3). Mass spectrum, m/z (Irel, %): 416
[M(81Br)+H]+ (100), 414 [M(79Br)+H]+ (90). Found, %: C 52.05; H 3.83; N 16.82. C18H16BrN5S. Calculated, %:
C 52.18; H 3.89; N 16.90.
5-{[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]methyl}-4-ethyl-4H-1,2,4-triazole-3-thiol (8b). Yield
80%, white powder, mp 288-290°C. 1H NMR spectrum, , ppm (J, Hz): 1.14 (3H, t, J = 6.8, CH3); 4.05 (2H, q,
J = 6.8, CH2); 4.34 (2H, s, CH2); 5.77 (2H, s, NCH2); 7.26-7.51 (4H, m, H Ar); 7.53-7.67 (4H, m, H Ar); 13.65
(1H, s, NH). 13C NMR spectrum, , ppm: 13.6 (CH3); 32.6 (CH2); 39.0; 40.7 (NCH2); 110.8; 119.3; 120.4;
122.4; 122.8; 131.6; 131.7; 135.9; 136.4; 142.7 (C Ar); 148.2 (C-5 triazole); 154.0 (C-2 benzimidazole); 167.5
(C-3 triazole). Mass spectrum, m/z (Irel, %): 430 [M(81Br)+H]+ (100), 429 [M(79Br)+H]+ (90). Found, %:
C 53.21; H 4.28; N 16.30. C19H18BrN5S. Calculated, %: C 53.28; H 4.24; N 16.35.
5-{[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]methyl}-4-methyl-4H-1,2,4-triazole-3-thiol (8c). Yield
1
77 %, white powder, mp 259-261°C. H NMR spectrum, , ppm: 2.76 (3H, s, CH3); 4.32 (2H, s, CH2); 5.67
(2H, s, NCH2); 7.08-7.20 (4H, m, H Ar); 7.31-7.34 (2H, m, H Ar); 7.51-7.53 (2H, m, H Ar); 14.05 (3H, s, NH,
13
exchanged with D2O). C NMR spectrum, , ppm (J, Hz): 30.6 (CH3); 32.4 (CH2); 39.3 (NCH2); 111.0; 115.6 (d,
JC–F = 20.0, C Ar); 119.3; 122.4; 122.7; 131.3; 131.4; 133.4; 136.1; 142.9 (C Ar); 148.9 (C-5 triazole); 154.5
(C-2 benzimidazole); 161.7 (d, JC–F = 246.0, CF); 168.1 (C-3 triazole). Mass spectrum, m/z (Irel, %): 354
[M+H]+ (100). Found, %: C 61.10; H 4.49; N 19.75. C18H16FN5S. Calculated, %: C 61.17; H 4.56; N 19.82.
5-{[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]methyl}-4-ethyl-4H-1,2,4-triazole-3-thiol (8d). Yield
83%, white powder, mp 265-266°C. 1H NMR spectrum, , ppm (J, Hz): 1.12 (3H, t, J = 7.2, CH3); 4.02 (2H, q,
J = 7.2, CH2); 4.34 (2H, s, CH2); 5.75 (2H, s, NCH2); 7.10-7.14 (2H, m, H Ar); 7.24-7.34 (4H, m, H Ar); 7.55
(1H, d, J = 8.0, H Ar); 7.65 (1H, d, J = 8.0, H Ar); 13.61 (1H, s, NH). 13C NMR spectrum, , ppm (J, Hz): 13.6
(CH3); 32.4 (CH2); 39.0; 39.1 (NCH2); 110.8; 115.5 (d, JC–F = 21.0, C Ar); 119.3; 122.4; 122.8; 131.2; 131.3;
136.0; 142.7 (C Ar); 148.2 (C-5 triazole); 154.3 (C-2 benzimidazole); 161.6 (d, JC–F = 241.0, CF); 167.5 (C-3
triazole). Mass spectrum, m/z (Irel, %): 368 [M+H]+ (100). Found, %: C 62.05; H 4.87; N 19.02. C19H18FN5S.
Calculated, %: C 62.11; H 4.94; N 19.06.
Synthesis of N-Alkyl-5-{[2-(4-halobenzyl)-1H-benzimidazol-1-yl]methyl}-1,3,4-thiadiazol-2-amines
9a-d (General Method). A mixture of thiosemicarbazide 7a-d (0.01 mol) in concentrated sulfuric acid (10 ml)
was stirred for 10 min and then left for another 30 min at room temperature. The resulting solution was poured
1678

slowly into ice-cold water, neutralized to pH 8 with ammonia, and the precipitated product was filtered off,
washed with water, and recrystallized from EtOH.
5-{[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]methyl}-N-methyl-1,3,4-thiadiazol-2-amine
(9a).
Yield 95%, white powder, mp 169-170°C. 1H NMR spectrum, , ppm (J, Hz): 2.77 (3H, d, J = 4.0, CH3); 4.31
(2H, s, CH2); 5.68 (2H, s, NCH2); 7.15-7.22 (4H, m, H Ar); 7.46-7.48 (2H, m, H Ar); 7.52-7.58 (2H, m, H Ar);
7.65 (1H, q, J = 4.0, NH, exchanged with D2O). 13C NMR spectrum, , ppm: 31.7 (CH3); 32.6 (CH2); 42.4
(NCH2); 110.7; 119.2; 120.3; 122.4; 122.7; 131.7; 131.8; 135.3; 136.5; 142.7 (C Ar); 153.1 (C-2 thiadiazole);
153.4 (C-2 benzimidazole); 170.8 (C-5 thiadiazole). Mass spectrum, m/z (Irel, %): 416 [M(81Br)+H]+ (100), 414
[M(79Br)+H]+ (95). Found, %: C 52.11; H 3.83; N 16.85. C18H16BrN5S. Calculated, %: C 52.18; H 3.89;
N 16.90.
5-{[2-(4-Bromobenzyl)-1H-benzimidazol-1-yl]methyl}-N-ethyl-1,3,4-thiadiazol-2-amine (9b). Yield
1
92%, white powder, mp 163-164°C. H NMR spectrum, , ppm (J, Hz): 1.08 (3H, t, J = 7.2, CH3); 3.12-3.19
(2H, m, CH2); 4.13 (2H, s, CH2); 5.67 (2H, s, NCH2); 7.15-7.24 (4H, m, H Ar); 7.45-7.47 (2H, m, H Ar);
13
7.52-7.57 (2H, m, H Ar); 7.67 (1H, t, J = 5.2, NH, exchanged with D2O). C NMR spectrum, , ppm: 14.6
(CH3); 32.6 (CH2); 40.1; 42.4 (NCH2); 110.7; 119.2; 120.3; 122.4; 122.7; 131.6; 131.7; 135.3; 136.5; 142.7
(C Ar); 153.0 (C-2 thiadiazole); 153.4 (C-2 benzimidazole); 169.9 (C-5 thiadiazole). Mass spectrum, m/z (Irel,
%): 430 [M(81Br)+H]+ (100), 428 [M(79Br)+H]+ (90). Found, %: C 53.21; H 4.18; N 16.20. C19H18BrN5S.
Calculated, %: C 53.28; H 4.24; N 16.35.
5-{[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]methyl}-N-methyl-1,3,4-thiadiazol-2-amine (9c). Yield
93 %, white powder, mp 167-168°C. 1H NMR spectrum, , ppm (J, Hz): 2.76 (3H, d, J = 8.0, NCH3); 4.33 (2H,
s, CH2); 5.68 (2H, s, NCH2); 7.08-7.19 (4H, m, H Ar); 7.32-7.34 (2H, m, H Ar); 7.52-7.53 (2H, m, H Ar); 7.60
13
(1H, t, J = 5.0, NH exchanged with D2O). C NMR spectrum, , ppm (J, Hz): 31.7 (CH3); 32.4 (CH2); 42.5
(NCH2); 110.7; 115.7 (d, JC–F = 21.0, C Ar); 119.2; 122.3; 122.7; 131.2; 131.3; 135.3; 142.8 (C Ar); 153.2 (C-2
thiadiazole); 153.7 (C-2 benzimidazole); 161.6 (d, JC–F = 240.0, CF); 170.8 (C-5 thiadiazole). Mass spectrum,
m/z (Irel, %): 354 [M+H]+ (100). Found, %: C 61.10; H 4.51; N 19.75. C18H16FN5S. Calculated, %: C 61.17;
H 4.56; N 19.82.
5-{[2-(4-Fluorobenzyl)-1H-benzimidazol-1-yl]methyl}-N-ethyl-1,3,4-thiadiazol-2-amine
(9d).
1
Yield 95 %, white powder, mp 178-179°C. H NMR spectrum, , ppm (J, Hz): 1.08 (3H, t, J = 6.8, CH3);
3.14-3.18 (2H, m, CH2); 4.32 (2H, s, CH2); 5.67 (2H, s, NCH2); 7.08-7.12 (2H, m, H Ar); 7.16-7.20 (2H, m,
H Ar); 7.30-7.34 (2H, m, H Ar); 7.51-7.58 (2H, m, H Ar); 7.66 (1H, t, J = 4.8, NH, exchanged with D2O).
13C NMR spectrum, , ppm (J, Hz): 14.6 (CH3); 32.4 (CH2); 37.7; 42.4 (NCH2); 110.6; 110.7; 115.6 (d,
JC–F = 21.0, C Ar); 119.2; 122.3; 122.7; 131.1; 131.2; 131.3; 135.3; 142.7 (C Ar); 153.00 (C-2 thiadiazole);
153.7 (C-2 benzimidazole); 161.6 (d, JC–F = 241.0, CF); 169.8 (C-5 thiadiazole). Mass spectrum, m/z (Irel, %):
368 [M+H]+ (100). Found, %: C 62.05; H 4.86; N 19.02. C19H18FN5S. Calculated, %: C 62.11; H 4.94; N 19.06.
ABTS Radical Scavenging Activity Assay. The antioxidant capacity of the synthesized compounds
was estimated by the ABTS assay [45-47], modified by Kim et al. [47]. ABTS (Fluka Chemika) was dissolved
in water to obtain 7.0 mM stock solution. The ABTS cation radical was generated in the reaction of the ABTS
stock solution with 2.45 mM K2S2O8 (Sigma) solution in water. The mixture was stored in the dark at room
temperature for 18 h before use. For the assay, the ABTS solution was diluted in phosphate buffered saline
(PBS) solution (100 mM potassium phosphate buffer (pH 7.4) containing 150 mM NaCl). The concentration of
the resulting blue-green ABTS solution was adjusted with PBS to an absorbance of 0.400 ± 0.005 at 734 nm.
The sample solution of 10 µl was added to 190 µl of the resulting blue-green ABTS radical solution. The
mixture was incubated in the dark at room temperature for 10 min. After incubation, the decrease of absorbance
at 734 nm was measured using a Molecular Devices SpectraMax-M5 multiplate reader. The control consisted of
10 µl of 50% aqueous MeOH and 190 µl of ABTS radical solution.
DPPH radical scavenging activity was determined using a modified method of Brand-Williams et al.
[48]. DPPH (2,2-diphenyl-1-picrylhydrazyl, Aldrich-Germany) (100 µM) was dissolved in 80% aqueous
methanol. Ten µl (1.5 nmol) of the synthesized compounds was added to 190 µl of the methanolic DPPH
1679

radical solution in a microtiter plate. The plate was shaken vigorously and allowed to stand at room temperature
in the dark for 30 min. The decrease in absorbance of the resulting solution was recorded at 517 nm. The
control consisted of 10 µl of 50% aqueous MeOH and 190 µl of DPPH radical solution. The DPPH radical
scavenging activity of the synthesized compounds was expressed as percent compared to the standard
compound Trolox. The DPPH radical stock solution was prepared fresh daily.
Antimicrobial Activity Assay. The test microorganisms were obtained from the Hifzissihha Institute of
Refik Saydam (Ankara, Turkey). All the newly synthesized compounds were weighed and dissolved in DMSO to
prepare extract stock solutions of 20 mM. The antibacterial and antifungal assays were performed in Mueller–Hinton
broth (Difco) at pH 7.3 and buffered yeast nitrogen base (Difco) at pH 7.0. Brain heart infusion broth (Difco) was
used for M. smegmatis, which was incubated for 48-72 h at 35°C [49]. Ampicillin (10 µg), streptomycin (10 µg),
and fluconazole (5 µg) were used as standard antibacterial and antifungal drugs, respectively. DMSO was used as
solvent control. Five-millimeter diameter wells were cut from the agar using a sterile corkborer, and 50 µl of each
test compound solution was delivered into the wells. The Petri dishes were incubated for 18 h at 35°C. Antimicrobial
activity was evaluated by measuring the zone of inhibition against the test organisms.
α-Glucosidase inhibition assay was performed spectrophotometrically. α-Glucosidase from Saccharomyces
cerevisiæ (Sigma-Aldrich) was dissolved in phosphate buffer (pH 6.8, 50 mM). The test compounds were dissolved
in 80% MeOH. In 96-well microtiter plates, 20 μl of test compound solution, 20 μl of enzyme (20 mU/ml), and 135
μl of buffer were added and the whole incubated for 15 min at 37°C. After incubation, 25 μl of p-nitrophenyl-
α-D-glucopyranoside (2 mM, Sigma-Aldrich) were added, and the change in absorbance was monitored for 30 min
at 400 nm. The test compound was replaced by 80% aqueous MeOH (7.5% final concentration) as control. Acarbose
(Sigma-Aldrich) was used as a standard inhibitor [50].
The authors gratefully acknowledge financial support from the Recep Tayyip Erdoğan University, BAP,
Turkey, through Project 2013.102.02.10.
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