Synthesis and biological activity of novel thiourea derivatives as carbonic anhydrase inhibitors

Article abstract of DOI:10.3109/14756366.2013.879656

A new series of chiral thiourea derivatives (5a-5c) and thiourea containing benzimidazole moieties (9b-9e) were synthesized from different amino acids (L-valine, L-isoleucine, L-methionine, L-phenylalanine, and D-phenylglycine). The compounds were charact

Full text of DOI:10.3109/14756366.2013.879656

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J Enzyme Inhib Med Chem, Early Online: 1–6
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.879656
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RESEARCH ARTICLE
Synthesis and biological activity of novel thiourea derivatives as
carbonic anhydrase inhibitors
Neslihan Korkmaz1, Oday A. Obaidi1, Murat Senturk2, Demet Astley1, Deniz Ekinci3, and Claudiu T. Supuran4
2
1Department of Chemistry, Science Faculty, Ege University, Izmir, Turkey, Department of Chemistry, Art and Science Faculty, Department of
3
Agricultural Biotechnology, Agrı Ibrahim Cecen University, Agri, Turkey, Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz
˘
4
Mayıs University, Samsun, Turkey, and Neurofarba Department, University of Florence, Florence, Italy
Abstract
Keywords
A new series of chiral thiourea derivatives (5a–5c) and thiourea containing benzimidazole
moieties (9b–9e) were synthesized from different amino acids (L-valine, L-isoleucine, L-
methionine, L-phenylalanine, and D-phenylglycine). The compounds were characterized and
tested against the two most studied members of the pH regulatory enzyme family, carbonic
anhydrase (CA, EC 4.2.1.1). KI values of the novel compounds were measured in the range of
3.4–73.6 mM for hCA I isozyme and 8.7–1.44.2 mM for hCA II isozyme, respectively. Phenol was
also tested as standard in order to understand the structure activity relationship and the
clinically used sulfonamide acetazolamide was tested for comparison reasons. All of the
compounds exhibited competitive inhibition with 4-nitrophenylacetate as substrate.
Amino acid, benzimidazole, biological activity,
carbonic anhydrase, thiourea
History
Received 14 November 2013
Accepted 27 December 2013
Published online 25 March 2014
Introduction
CA inhibitors (CAIs) act as promiscuous inhibitors of most
isozymes with physiological/pathological relevance resulting in
undesired side effects10–14. So far inhibitory effects of different
sulfonamide derivatives, phenols, anions, and drugs have been
Benzimidazole derivatives incorporating thiourea moieties play
important roles in medical field with various pharmacological
activities such as antimicrobial, antiviral, antidiabetic, and
anticancer activity1. The potency of these clinically useful drugs
in the treatment of microbial infections and other activities
encouraged the development of some more potent and significant
compounds2,3. Benzimidazoles are remarkably effective com-
pounds, and extensive biochemical and pharmacological studies
have confirmed that these molecules are effective against various
strains of microorganisms. The development of new antimicrobial
and anticancer therapeutic agents is one of the fundamental goals
investigated against many CAs15–19
.
However, it is still important to discover further classes of
potential CAIs in order to develop novel compounds with distinct
inhibition profiles as compared to the known molecules.
Therefore, in this study, we focused on the synthesis, character-
ization, and CA inhibitory properties of benzimidazole deriva-
tives of thiourea compounds.
In this study, a new series of chiral thiourea derivatives (5a–c)
(Figure 1) and chiral thiourea containing benzimidazol derivatives
(9b–e) (Figure 2) were synthesized in good yields, and the
structures of the compounds were confirmed by IR, 1H-NMR,
13C-NMR, and elemental analysis. In addition, CA inhibitory
activity of the compounds was evaluated.
in medicinal chemistry4,5
.
Carbonic anhydrase (CA; carbonate hydrolase, EC 4.2.1.1)
enzymes play important roles in several physiological and
pathological processes6. Sixteen CA isoforms have been identi-
fied in mammals that differ in subcellular localization and
catalytic activity7. CA isoforms take part in several vital
biological processes such as acid–base balance, electrolyte
secretion, carbon dioxide and ion transport, bone resorption,
General procedure for the synthesis of N-Boc-amide
derivatives (3a–c)
respiration, ureagenesis, gluconeogenesis, and lipogenesis6–10
.
To a solution of N-Boc-amino acid (2a–c) (1 mmol) in 15 ml THF,
Inhibitors or activators of these enzymes have several medical 2-aminophenol (1 mmol) and a slight excess of N,N0-dicyclohex-
applications, such as diuretics, in the treatment of glaucoma, in ylcarbodiimide (1.2 mmol) were added. The mixture was stirred
the management of several neurological disorders, including under inert atmosphere at room temperature overnight. The
epilepsy, possibly in the treatment of Alzheimer’s disease. insoluble dicyclohexylurea was removed by filtration and the
However, it is relatively difficult to design agents (inhibitors or solvent was evaporated. Evaporation of the solvent provided a
activators) with specificity or selectivity for any of these isoforms, residue which was purified by column chromatography using
and many pharmacological agents belonging to the class of the dichloromethane and methanol (25:1) to give 3a–c.
CA inhibitory activity
Address for correspondence: Dr. Demet Astley, Department of
Chemistry, Science Faculty, Ege University, Izmir, Turkey. E-mail:
chemiker80@hotmail.com
Compounds 5a–c and 9b–d, as well as the standard, clinically
used CAI acetazolamide (AZA), have been tested for the

2
N. Korkmaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
R
Figure 1. Synthesis of compounds 5a–c.
H
N
R
NH
2
DCC, THF
RT
HN
HO
+
NH
O
Boc
O
Boc
OH
OH
2-aminophenol
R= CH(CH3)2, 2a
CH2CH(CH3)2,2b
CH2Ph, 2c
R= CH(CH3)2, 3a
CH2CH(CH3)2,3b
CH2Ph, 3c
H3PO4(85%)
THF, RT
O
S
R
H
N
R
H
N
N
NH
PhCONCS
Acetone, reflux
H
NH
2
O
O
OH
OH
R= CH(CH3)2, 5a
R= CH(CH3)2, 4a
CH2CH(CH3)2,5b
CH2Ph, 5c
CH2CH(CH3)2,4b
CH2Ph, 4c
R
Figure 2. Synthesis of compounds 9b–e.
R
NH2
HO
+
NH
NH
DCC,THF
RT, N2 (atm.)
NH
O
Boc
NH2
O
Boc
NH2
6(b-e)
R= -CH2CH(CH3)2,(2b)
-CH2Ph,(2c)
Acetone,
72°C,
8hr.
-CH2CH2-S-CH3,(2d)
-Ph,(3e)
N
R
N
R
H3PO4(85%)
THF,RT,24hr.
NH2
N
H
NH
N
H
Boc
8(b-e)
7(b-e)
NCS
Acetone,
reflux
CF3
F3C
N
R
H
N
NH
CF3
N
H
S
9(b-e)
CF3
inhibition of two cytosolic ubiquitous isozymes of human origin,
that is, hCA I and hCA II (Table 1).
The following should be noted regarding the CA inhibitory
data of Table 1:
(1) The thiourea derivatives investigated here showed moderate
were less effective than compound 5c. This trend also shows
the attenuator potency of the benzimidazole moiety. As these
compounds do not possess any of the zincanchoring groups
present in known CAIs, presumably such compounds may
bind in the coumarin/phenol binding site.
to strong inhibitory properties against the slow cytosolic (2) Similar to hCA I inhibition data, compound 9b acted as the
isoform hCA I. Compound 9b exhibited the lowest inhibition
of this isoform, with KI value of 73.6 mM. Compounds 5a–c
were much more effective inhibitors against hCA I, with KI-s
in the range of 3.4–7.6 mM. These results demonstrate the
contribution of the hydroxyl groups to the inhibition efficacy.
Interestingly, benzimidazole containing compounds 9b–d
weakest inhibitor against the ubiquitous and dominant rapid
cytosolic isozyme hCA II. However, 5a–c derivatives acted
as more effective hCA II inhibitors with a comparable
potency as the reference compounds phenol (KI: 5.5 mM).
The most potent inhibitor was compound 5a (KI: 8.7 mM).
This trend again demonstrates the attenuator potency of the

DOI: 10.3109/14756366.2013.879656
Novel thiourea derivatives as carbonic anhydrase inhibitors
3
Table 1. hCAs I and II inhibition data with studied compounds, phenol
and acetazolamide, by an esterase assay with 4-nitrophenylacetate as
substrate.
solution was extracted with ethyl acetate. The organic phase was
dried using Na2SO4 and evaporated under vacuum and pure
products (4a–c) were obtained.
2-Amino-N-(2-hydroxyphenyl)-3-methylbutanamide
(4a):
KI (mM)*
Yellow solid, 77% yield, mp ¼ 140–142 ꢀC, IR (KBr): 3411
(–OH), 3329(NH2), 3274(NH2), 2966(C–H), 2918(C–H),
1676(C¼O), 1596(C¼O), 1525(ArC¼C), 1456(ArC¼C),
Inhibitor
hCA I
hCA II
Phenol
5a
5b
5c
9b
9c
9d
AZA
10.2y
3.4
4.3
5.50y
8.7
1284(C–O), 1004(C–N), 751(mono subs. Ar) cmꢁ1 1H-NMR
.
(CDCl3, ꢀ ppm) 9.81 (s, 1H), 7.11 (td, J ¼ 8.4 Hz and 1.2 Hz, 1H),
7.02 (dd, J ¼ 8 Hz and 1.2 Hz, 1H) 6.95 (dd, J ¼ 8 Hz and 1.6 Hz,
1H), 6.84 (td, J ¼ 8.8 Hz and 1.6 Hz, 1H), 3.47 (d, J ¼ 3.6 Hz, 1H),
2.46 (m, 1H), 1.07 (d, J ¼ 7.2 Hz, 3H) and 0.9 (d, J ¼ 6.8 Hz, 3H).
14.3
22.1
44.2
26.5
12.9
0.37z
7.6
73.6
62.3
46.1
36.2z
2-Amino-N-(2-hydroxyphenyl)-4-methylpentanamide
(4b):
Yellow oily product, 85% yield, IR (KBr): 3287(–OH),
1596(C¼O),
3064(ArC–H),
2963(C–H),
1429(ArC¼C),
1695(C¼O),
*Mean from at least three determinations. Errors in the range of ꢂ3% of
the reported value (data not shown).
yFrom Ref. 7.
1499(ArC¼C),
1282(C–O),
1105(C–N),
750(mono subs Ar). 1H-NMR (CDCl3, ꢀ ppm), 9.81 (bs, 1H),
7.12 (td, J ¼ 8 Hz and 1.6 Hz, 1H), 7.02 (d, J ¼ 8 Hz, 1H), 6.95
(d, J ¼ 6.8 Hz, 1H), 6.84 (td, J ¼ 7.2 Hz and 0.8 Hz,1H), 3.52
zFrom Ref. 23.
benzimidazole moiety. Also, hydroxyl group containing (d, J ¼ 3.6 Hz, 1H), 2.16 (m, 2H), 2.09 (s, 1H), 1.37 (m, 1H), 1.15
compounds 5a–c were much more effective compared to (m, 1H), 1.05 (d, J ¼ 6.8 Hz, 3H) and 0.93 (t, 7.6 Hz, 3H).
hydrophobic group containing compounds 9b–d.
2-Amino-N-(2-hydroxyphenyl)-3-phenylpropanamide
(4c):
White solid, 86% yield, mp ¼ 170–173 ꢀC, IR (KBr): 3399
(ꢁOH), 3296(NH), 3083(ArC–H), 2978(C–H), 1647(C¼O),
1614(C¼O), 1588(ArC¼C), 1542(ArC¼C), 1454(ArC¼C),
1282(C–O), 1102(C–N), 748.6(mono subs. Ar), 700(mono subs.
Ar). 1H-NMR (CDCl3, ꢀ ppm), 9.69 (bs, 1H), 7.36–7.23 (m, 5H),
7.11 (t, J ¼ 7.6 Hz, 1H), 7.18 (d, J ¼ 8 Hz, 1H), 6.93 (dd, J ¼ 8,
1.6 Hz, 1H), 6.83 (td, J ¼ 8, 1.6 Hz, 1H) 3.81 (s, 1H), 3.35 (dd,
J ¼ 14, 3.6 Hz, 1H), 2.85 (J ¼ 13.6, 8.8 Hz, 1H).
Experimental
All chemicals were obtained commercially from Sigma-
Aldrich (Bornem, Belgium). IR spectra were recorded on
a Perkin-Elmer 100 FTIR spectrometer and all 1H-NMR and
13C-NMR spectra were recorded using an Oxford NMR 400 MHz
spectrometer using TMS as the internal standard d-values in ppm
at ambient temperature. Melting points were measured on variable
heater.
Tert-butyl 1-(2-hydroxyphenylamino)-3-methyl-1-oxobutan-2- Synthesis of thiourea derivatives (5a–c)
ylcarbamate (3a): Yellow oily product, 73% yield, IR (KBr):
About 1 mmol of potassiumthiocyanide (KSCN) was stirred in
3291(–OH), 3070(ArC–H), 2970(C–H), 2934(C–H), 1680(C¼O),
1
1286(C–O), 1169(N–C), 749(mono subs. Ar). H-NMR (CDCl3,
15 ml dried acetone and 1 mmol benzoyl chloride was added
dropwise under an argon atmosphere. Precipitation was observed
immediately. The reaction mixture was refluxed until the color of
the solution turned yellow (approximately 2 h). Then, the reaction
mixture was cooled to room temperature and an amide (4a–b)
solution in 5 ml dried acetone was added dropwise to the reaction
flask which was then refluxed for 2 h. Reaction was monitored by
TLC. The solution was filtered and evaporation of the solvent
from the filtrate provided a residue which was crystallized from
CH2Cl2: hexane to give bright yellow crystals.
1612(C¼O), 1532(ArC¼C), 1505(ArC¼C), 1499(ArC¼C),
ꢀ ppm) 8.67 (s, 1H), 8.59 (bs, 1H), 7.08 (d, J ¼ 8 Hz, 2H), 6.98
(dd, J ¼ 8.4 Hz and 1.2 Hz, 1H), 6.82 (td, J ¼ 8 Hz and 1.2 Hz,
1H), 5.19 (d, J ¼ 8 Hz, 1H), 4.42 (t, J ¼ 7.2 Hz, 1H), 2.24 (m, 1H),
1.46 (s, 9H), 1.05 (d, J ¼ 6.4 Hz, 3H) and 1.01 (d, J ¼ 6.8 Hz).
Tert-butyl 1-(2-hydroxyphenylamino)-4-methyl-1-oxopentan-
2-ylcarbamate (3b): Yellow oily product, 53% yield, IR (KBr):
3291(–OH), 3049(ArC–H), 2965(C–H), 2933(C–H), 1694(C¼O),
1660(C¼O),
1524(ArC¼C),
1450(ArC¼C),
1256(C–O),
N-(1-(2-hydroxyphenylamino)-3-methyl-1-oxobutan-2-ylcar-
bamothioyl)benzamide (5a): Bright yellow crystal, 79% yield,
mp ¼ 164.5–165.8 ꢀC, IR (KBr): 3308(–OH), 3248(N–H),
3071(ArC–H), 2968(C–H), 2981(C–H), 1670(C¼O amide),
1645(C¼O), 1531(ArC¼C), 1520(ArC¼C), 1453(ArC¼C),
1263(C–O), 1162(N–C), 1093(C¼S), 748(mono subs. Ar),
1
1168(C–N) and 738(mono subst. Ar). H-NMR (CDCl3, ꢀ ppm)
8.74 (bs, 1H), 7.08 (m, 2H), 6.98 (d, J ¼ 7.6 Hz, 1H), 6.79
(t, J ¼ 7.2 Hz, 1H), 5.73 (d, J ¼ 8 Hz, 1H), 4.18 (m, 1H), 1.96
(m, 1H), 1.6 (m, 1H), 1.44 (s, 9H), 1.01 (d, J ¼ 6.8 Hz, 3H), 0.93
(d, J ¼ 7.6 Hz, 3H).
Tert-butyl 1-(2-hydroxyphenylamino)-1-oxo-3-phenylpropan-
2-ylcarbamate (3c): White solid, 51% yield, mp ¼ 126–128 ꢀC,
IR (KBr): 3360(N–H), 3311(N–H), 3064(ArC–H), 2978(C–H),
714(mono subs. Ar). 1H-NMR (CDCl3,
d ppm) 11.23
(d, J ¼ 7.2 Hz, 1H), 9.11 (s, 1H), 8.44 (s, 1H), 7.84 (d,
J ¼ 7.6 Hz, 2H), 7.64 (t, J ¼ 1.2 Hz, 1H), 7.52 (t, J ¼ 8 Hz, 2H),
7.17 (d, J ¼ 1.6 Hz, 1H), 7.14 (t, J ¼ 6.8 Hz, 1H), 7.09
(d, J ¼ 1.6 Hz, 1H), 6.87 (t, J ¼ 6.8 Hz, 1H), 4.93 (t, J ¼ 7.2 Hz,
1H), 2.52 (m, 1H) and 1.15 (d, J ¼ 6.8 Hz, 6H). 13C-NMR
(CDCl3, d ppm) 169.7, 167.3, 148.7, 134.1, 131.6, 129.4, 127.8,
127.4, 125.4, 122.9, 120.8, 119.4, 65.7, 30.6, 19.6, and 18.6. Anal.
Calc. for C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31; S, 8.63.
Found: C, 61.57; H, 5.88; N, 10.60; S, 7.73%.
1668(C¼O),
1616(C¼O),
1599(C¼O),
1543(ArC¼C),
1455(ArC¼C), 1279(C–O), 1165(C–N), 750(mono subs Ar).
1H-NMR (CDCl3, ꢀ ppm), 8.47 (bs, 1H), 8.22 (bs, 1H), 7.33–7.22
(m, 5H), 7.11–7.07 (m, 1H), 6.97 (dd, J ¼ 7.6, 1.2 Hz, 1H), 6.84–
6.77 (m, 2H).
Deprotection reaction of 2-N-Boc-amide derivatives
(4a–c)
N-(1-(2-hydroxyphenylamino)-4-methyl-1-oxopentan-2-ylcar-
About 1 mmol N-Boc-amide (3a–b) was dissolved in 2 ml THF bamothioyl)benzamide (5b): Yellow solid, 79% yield,
and 2 ml 85% H3PO4 was added dropwise to the solution. The mp ¼ 161–163 ꢀC, IR (KBr): 3269(N–H), 3063(ArC–H),
reaction mixture was stirred overnight at room temperature. 2961(C–H),
2927(C–H),
1666(C¼O),
1599(C¼O)
To terminate the reaction, 5 ml distilled water was added and the 1520(ArC¼C), 1498(–ArC¼C), 1458(–ArC¼C), 1166(C–O)
reaction mixture was neutralized with saturated NaOH. The 1157(N–C), 1084(C¼S), 745(mono subs. Ar), 723(mono subs.

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J Enzyme Inhib Med Chem, Early Online: 1–6
1
Ar). H-NMR (CDCl3, d ppm) 11.22 (d, J ¼ 7.6 Hz, 1H), 9.09 (t.d, 1H, J ¼ 14, 6.8 and 7.2 Hz), 2.00 (t.d, 1H, J ¼ 13.6, 6.8 and
(s, 1H), 8.4 (s, 1H), 8.29 (bs, 1H), 7.86 (d, J ¼ 8 Hz, 2H), 7.64 3.6 Hz), and 1.44 (s, 9H). 13C NMR (CDCl3, 100 MHz, ꢀ ppm)
(t, J ¼ 7.2 Hz, 1H), 7.53 (t, J ¼ 8 Hz, 2H), 7.12 (t, J ¼ 7.6 Hz, 2H), 170.79, 156.29, 141.10, 127.50, 125.82, 123.52, 119.18, 117.58,
7.08 (d, J ¼ 7.2 Hz, 1H), 6.86 (t, J ¼ 7.6 Hz, 1H), 4.99 80.76 54.41, 31.59, 30.54, 28.57, and 15.59.
(t, J ¼ 7.6 Hz, 1H), 2.32 (m, 1H), 1.72 (m, 1H), 1.67 (s, 1H), Tert-butyl 2-(2-aminophenylamino)-2-oxo-1-phenylethylcar-
1.40 (m, 1H), 1.11 (d, J ¼ 6.8 Hz, 3H), and 1.01 (t, J ¼ 7.2 Hz, bamate (6e): Yellow oily product, 69% yield, IR (KBr): 3324.7,
3H). 13C-NMR (CDCl3, d ppm), 181.3, 169.7, 167.3, 148.8, 3274.3, 2976.9, 2964.3, 2930.3, 1685.1, 1498.1, 1458.6, 1366.2,
134.1, 131.6, 129.4, 127.8, 127.5, 125.4, 122.9, 120.8, 119.6, 1246.9, 1167.5, 1052.6, 886.2, and 698.7. 1H-NMR (CDCl3,
64.7, 36.5, 25.4, 15.9, and 11.4. Anal. Calc. for C20H23N3O3S: 400 MHz, ꢀ ppm) 8.04 (s, 1H), 7.42 (d, 2H, J ¼ 7.6 Hz), 7.32
C, 63.32; H, 6.01; N, 10.90; S, 8.32. Found: C, 61.93; H, 6.03; N, (d, 3H, J ¼ 5.6 Hz), 7.09 (d, 1H, J ¼ 7.6 Hz), 6.98 (t, 1H, J ¼ 15.6
10.54; S, 7.72%.
and 7.6 Hz), 6.68 (dd, 2H, J ¼ 14 and 7.6 Hz), 5.94 (d, 1H,
J ¼ 6.4 Hz), 5.37 (bs, 1H), 3.66 (s, 2H), and 1.41 (s, 2H).
N-(1-(2-hydroxyphenylamino)-1-oxo-3-phenylpropan-2-ylcar-
bamothioyl)benzamide (5c): White solid, 65% yield, mp ¼ 164– 13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 169.61, 155.70, 141.24,
167 ꢀC IR (KBr): 3262(N–H), 3063(ArC¼C), 2955(C–H), 138.14, 137.35, 128.71, 129.30, 128.71, 127.65, 127.48, 126.09,
2922(C–H),
1532(ArC¼C), 1515(ArC¼C), 1498(ArC¼C), 1220(C–O),
1667(C¼O),
1600(C¼O),
1580(ArC¼C), 123.40, 117.65, 80.64, 59.28, and 28.56.
1
1162(C–N), 743(mono subs. Ar), 714(mono subs. Ar). H-NMR
Synthesis of 2-N-Boc-amide derivative of benzimidazoles
(7b–e)
(CDCl3, ꢀ ppm) 11.31 (d, J ¼ 6.8 Hz, 1H), 9.04 (s, 1H), 8.1 (s,
1H), 7.93–6.79 (m, 14H), 5.36 (dd, J ¼ 14.8, 8 Hz, 1H), 3.37 (dd,
J ¼ 13.6, 6.4 Hz, 1H), and 3.29 (dd, J ¼ 14, 8.4 Hz, 1H). 13C-
NMR (CDCl3, ꢀ ppm), 180.8, 169.11, 168.85, 149.07, 137.13,
133.72, 132.76, 130.09, 129.27, 129.09, 128.91, 127.38, 126.23,
125.78, 123.56, 119.59, 116.28, 60.39, and 38.12. Anal. Calc. for
C23H21N3O3S: C, 65.85; H, 5.05; N, 10.02; S, 7.64. Found: C,
64.66; H, 5.40; N, 9.14; S, 6.89%.
N-Boc-amide derivative (6b–e) was dissolved in 20 ml of acetic
acid and the solution was stirred at 72 ꢀC for 8 h. The acetic acid
was removed under reduced pressure and the crude compound
was purified by column chromatography (hexane/EtOAc, 3:2 by
volume) to afford a white solid.
Tert-butyl 1-(1H-benzo[d]imidazol-2-yl)-2-methylbutylcarba-
mate (7b): White solid product, 75% yield, m.p ¼ 226–228 ꢀC, IR
(KBr): 3202.9, 3060.8, 2966.8, 2985.8, 2877.5, 1674.8, 1623.3,
1584.7, 1487.5, 1456.1, 1318.2, 1173.4, 1011.6, 930.9, 877.8,
Synthesis of N-Boc-amide derivatives (6b–e)
The general procedure described for compounds (3a–c) was and 740.5. 1H-NMR (DMSO-d6, 400 MHz, ꢀ ppm) 12.10
carried out by using phenylenediamine (1 mmol). The resulting (bs, 1H), 7.55 (d, 1H, J ¼ 6.8), 7.45 (d, 1H, J ¼ 7.2 Hz), 7.13
residue was purified by column chromatography using (hexane/ (dd, 2H, J ¼ 12 and 5.6 Hz), 4.63 (t, 1H, J ¼ 16.4 and 8 Hz),
ethyl acetate (EtOAc), 3:2) to give product (6b–e).
1.95 (s, 1H), 1.48 (m, 1H, J ¼ 13.6, 6.8 and 4 Hz), 1.37 (s, 9H),
Tert-butyl 1-(2-aminophenylamino)-3-methyl-1-oxopentan-2- 0.85 (t, 3H, J ¼ 14.8 and 7.6 Hz), and 0.74 (d, 3H, J ¼ 6.8 Hz).
ylcarbamate (6b): Yellow oily product, 72% yield, IR (KBr): 13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 156.70, 155.32, 122.51,
3488.1, 3419.6, 3354.2, 3304.8, 3033.2, 2966.8, 2632.7, 2310.9, 137.52, 117.25, 80.22, 54.88, 38.83, 28.54, 25.82, 15.86,
1673.6, 1660.5, 1625.9, 1592.9, 1525.5, 1460.3, 1392.5, 1314.4, and 11.19.
1165.8, 1020.7, 987.8, 811.9, and 746.2. 1H-NMR (CDCl3, Tert-butyl 1-(1H-benzo[d]imidazol-2-yl)-2-phenylethylcarba-
400 MHz, ꢀ ppm) 7.85 (bs, 1H), 7.20 (d, 1H, J ¼ 7.2 Hz), 7.03 (d, mate (7c): White solid product, 68% yield, IR (KBr): 3315.7,
1H, J ¼ 16 and 7.6 Hz), 6.75 (dd, 2H, J ¼ 12 and 7.6 Hz), 5.21 (d, 2930.2 2916.8, 1677.9, 1634.9, 1530.1, 1522.4, 1455.3, 1367.2,
1H, J ¼ 7.6 Hz), 4.79 (t, 1H, J ¼ 9.6 and 7.6), 4.06 (t, 1H, J ¼ 15.2 1363.8, 1273.5, 1169.4, 1017.2, 730.1, and 697.9. 1H-NMR
and 7.2), 3.85 (s, 2H), 1.99 (m, 1H, J ¼ 6.4 and 3.6 Hz), 1.44 (s, (DMSO-d6, 400 MHz, ꢀ ppm) 8.19 (bs, 1H), 7.49 (dd, 2H, J ¼ 6
9H), 1.36 (t, 1H, J ¼ 7.6 and 4 Hz), and 1.03 (d, 3H, J ¼ 6.8 Hz). and 3.6 Hz), 7.32 (d, 1H, J ¼ 4 Hz), 7.23 (d, 4H, J ¼ 4 Hz), 7.12
13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 171.57, 156.63, 141.45, (dd, 2H, J ¼ 3.6 and 1.2 Hz), 4.99 (d, 1H, J ¼ 5.6 Hz), 3.34 (dd,
127.32, 126.19, 118.76, 117.30, 80.16, 60.08, 28.57, 25.20, 37.27, 1H, J ¼ 14 and 5.2 Hz), 3.08 (dd, 1H, J ¼ 9.6 Hz), 1.71 (s, 1H),
15.79, and 11.38.
and 1.28 (s, 9H). 13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 155.93,
Tert-butyl 1-(2-aminophenylamino)-1-oxo-3-phenylpropan-2- 155.83, 138.81, 129.89, 128.72, 126.86, 122.13, 78.76, 51.53,
ylcarbamate (6c): Yellow oily product, 66% yield, IR (KBr): 34.04, and 28.84.
3283.2, 2963.2, 2935.3, 2852.4, 1663.9, 1661.5, 1527.3, 1366.1, Tert-butyl 1-(1H-benzo[d]imidazol-2-yl)-3-(methylthio)pro-
1342.3, 1332.5, 1256.9, 1167.3, 1019.3, 872.4, and 748.8. 1H- pylcarbamate (7d): White solid product, 82% yield, IR (KBr):
NMR (DMSO-d6, 400 MHz, ꢀ ppm) 9.19 (bs, 1H), 7.29 (dd, 3H, 3298.3, 3072.7, 2916.8, 1634.9, 1527.4, 1457.6, 1439.5, 1367.1,
J ¼ 15.2 and 7.6 Hz), 7.20 (t, 1H, J ¼ 13.2 and 6 Hz), 7.03 (dd, 1362.4, 1307.8, 1270.5, 1250.7, 1165.2, 1043.4, 1171.4, 1024.7,
1
2H, J ¼ 22 and 8 Hz), 6.89 (dd, 2H, J ¼ 15.2 and 7.6 Hz), 6.52 (dd, 867.5, and 760.1. H-NMR (DMSO-d6, 400 MHz, ꢀ ppm) 12.15
2H, J ¼ 14.8 and 7.2 Hz), 4.77 (s, 2H), 4.34 (d, 1H, J ¼ 5.6 Hz), (bs, 1H), 7.33 (d, 2H, J ¼ 8 Hz), 7.12 (d, 2H, J ¼ 3.6 Hz), 4.90 (d,
3.31 (s, 1H), 3.03 (dd, 1H, J ¼ 13.6 and 5.2 Hz), 2.88 (t, 1H, 1H, J ¼ 6.4 Hz), 3.32 (s, 1H), 2.50 (t, 2H, J ¼ 14.4 and 6.8 Hz),
J ¼ 22.4 and 9.2 Hz), 1.34 (s, 9H). 13C-NMR (DMSO-d6, 2.19 (td, 1H, J ¼ 14, 6.4 and 6.8 Hz), 2.07 (t, 1H, J ¼ 15.2 and
100 MHz, ꢀ ppm) 171.35, 156.21, 143.34, 138.65, 130.01, 7.6), 1.37 (s, 3H) and 1.21 (s, 9H). 13C-NMR (CDCl3, 100 MHz, ꢀ
128.75, 126.97, 126.63, 123.35, 116.65, 116.22, 78.87, 56.94, ppm) 156.05, 143.69, 134.87, 122.43, 121.67, 112.00, 78.89,
38.14, and 28.86.
Tert-butyl1-(2-aminophenylamino)-4-(methylthio)-1-oxobu-
49.17, 34.13, 30.50, 28.88, and 15.38.
Tert-butyl 1-(1H-benzo[d]imidazol-2-yl)-3-(methylthio)pro-
tan-2-ylcarbamate(6d): Yellow oily product, 71% yield, IR pylcarbamate (7e): White solid product, 76% yield, mp ¼ 195–
(KBr): 3285.3, 3072.7, 2976.8, 2934.3, 2928.8, 1664.3, 1634.9, 198 ꢀC, IR (KBr): 3408.7, 2975.8, 2964.8, 2874.5, 1671.2, 1622.4,
1533.2, 1500.1, 1457.6, 1366.4, 1250.7, 1166.3, 1049.9, 1042.1, 1585.6, 1439.6, 1247.8, 1163.9, 1052.6, 750.1 and 740.5.
1
1018.8, 860.5, and 748.1. H-NMR (CDCl3, 400 MHz, ꢀ ppm) 1H-NMR (DMSO-d6, 400 MHz, ꢀ ppm) 12.22 (bs, 1H), 7.76 (s,
8.19 (bs, 1H), 7.17 (d, J ¼ 8 Hz, 1H), 7.01 (t, 1H, J ¼ 8 and 1H), 7.54 (d, 1H, J ¼ 6.8 Hz), 7.41 (d, 1H, J ¼ 6.4 Hz), 7.25 (td,
3.6 Hz), 6.72 (t, 2H, J ¼ 8 and 6.8 Hz), 5.53 (bs, 1H), 4.41 (d, 1H, 1H, J ¼ 8.4, 7.2 and 6.4 Hz), 7.23 (dd, 2H, J ¼ 12.8 and 6 Hz),
J ¼ 6.8 Hz), 3.86 (s, 2H), 2.59 (t, 2H, J ¼ 14.4 and 7.2 Hz), 2.16 7.12 (d, 2H, J ¼ 0.8 Hz), 5.99 (d, 1H, J ¼ 3.6 Hz) and 1.37 (s, 9H).

DOI: 10.3109/14756366.2013.879656
Novel thiourea derivatives as carbonic anhydrase inhibitors
5
13C-NMR (DMSO-d6, 100 MHz, ꢀ ppm) 157.34, 154.97, 140.86, 130.78, 129.84, 128.88, 125.26, 122.71, 122.55, 112.12, 54.52,
129.04, 128.16, 127.96, 122.36, 79.27, 28.84, and 53.95.
and 39.86.
1-(1-(1H-benzo[d]imidazol-2-yl)-2-phenylethyl)-3-(3,5-bis(tri-
fluoromethyl)phenyl) thiourea (9c): Bright yellow oily product,
65% yield, mp ¼ 175–177 ꢀC, IR (KBr): 3267.4, 2968.9, 1663.7,
1542.1, 1458, 1382.7, 1277.1, 1179.6, 1133.1, 1000.6, 955.4,
886.7, 848.1, 745.1, and 681.3. 1H-NMR (CDCl3, 400 MHz, ꢀ
ppm) 8.92 (bs, 1H), 7.43 (s, 3H), 7.32 (d, 2H, J ¼ 9.6 Hz), 7.16 (s,
2H), 5.80 (s, 1H), 2.31 (t, 1H, J ¼ 6.8 Hz), 2.08 (s, 1H), 1.84 (s,
1H), 1.54 (t, 1H, J ¼ 13.6 and 5.6 Hz), 1.26 (m, 1H, J ¼ 10.8, 6.8
and 3.2 Hz) and 1.12 (d, 3H, J ¼ 6 Hz). 13C-NMR (CDCl3,
100 MHz, ꢀ ppm) 182.22, 156.33, 139.49, 131.52, 131.19, 124.32,
124.10, 123.83, 121.60, 118.52, 59.32, 40.13, 26.66, 14.38, and
11.26.
1-(1-(1H-Benzo[d]imidazol-2-yl)-3-(methylthio)propyl)-3-(3,5-
bis(trifluoromethyl) phenyl)thiourea (9d): Bright yellow oily
product, 69% yield, mp ¼ 105–108 ꢀC, IR (KBr): 3247.2, 2968.9,
2981.5, 2892.1, 1738.5, 1642.4, 1542.1, 1457.9, 1381.1, 1280.7,
1180.5, 1372.5, 1265.2, 1130.6, 886.9, and 681.1. 1H-NMR
(CDCl3, 400 MHz, ꢀ ppm) 9.10 (bs, 1H), 7.99 (s, 1H), 7.63 (s,
1H), 7.47 (s, 1H), 7.34 (s, 1H), 7.20 (dd, 2H, J ¼ 5.6 and 3.6 Hz),
6.14 (s, 1H), 2.76 (td, 1H, J ¼ 13.2 and 5.6 Hz), 2.65 (td, 1H,
J ¼ 12.8 and 6.4 Hz), 2.47 (d, 2H, J ¼ 6.8 Hz) and 1.95 (s, 1H).
13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 182.23, 156.46, 139.46,
131.64, 131.30, 123.99, 123.60, 121.61, 118.54, 52.79, 34.43,
30.93, and 15.50.
Deprotection of 2-N-Boc-amide derivative benzimidazoles
(8b–e)
The general procedure described for compound (4a–c) was
carried out with benzimidazole derivative (8b–e).
1-(1H-Benzo[d]imidazol-2-yl)-2-methylbutan-1-amine (8b):
White solid product, 70% yield, mp ¼ 162–164 ꢀC, IR (KBr):
2965.4, 2928.7, 1625.9, 1592.8, 1519.9, 1447.9, 1392.3, 1342.4,
1311.3, 1272.9, 1149.7, 1016.5, 839.6, 768.1, 742.1 and 609.8.
1H-NMR (CDCl3, 400 MHz, ꢀ ppm) 7.54 (d, 2H, J ¼ 5.2 and
2.8 Hz), 7.16 (dd, 2H, J ¼ 7.2 and 4 Hz), 5.75 (bs, 2H), 4.21
(d, 1H, J ¼ 6 Hz), 2.01 (s, 1H), 1.54 (m, 1H) and 1.24 (s, 1H).
13C-NMR (CDCl3, 100 MHz, ꢀ ppm) 157.35, 138.49, 122.42,
115.19, 55.67, 40.82, 24.82, 15.76, and 11.69.
1-(1H-Benzo[d]imidazol-2-yl)-2-phenylethanamine (8c): White
solid product, 88% yield, mp ¼ 169–172 ꢀC, IR (KBr): 3167.2,
2930.4, 2776.3, 1452.8, 1274.8, 1121.5, 764.3, 751.2, 749.8 and
1
700.09. H-NMR (CDCl3, 400 MHz, ꢀ ppm) 8.20 (bs, 1H), 7.48
(s, 2H), 7.22 (d, 2H, J ¼ 7.2 Hz), 7.12 (dd, 5H, J ¼ 18.4 and
6.8 Hz), 7.10 (dd, 2H, J ¼ 3.2 and 1.2 Hz), 4.27 (t, 1H, J ¼ 7.6 and
5,6 Hz), 3.25 (dd, 1H, J ¼ 13.2 and 6 Hz), 2.96 (dd, 1H, J ¼ 13.2
and 7.6 Hz). 13C-NMR (DMSO-d6, 100 MHz, ꢀ ppm) 159.32,
139.45, 129.94, 128.80, 126.74, 121.83, 43.81, and 52.67.
1-(1H-Benzo[d]imidazol-2-yl)-3-(methylthio)propan-1-amine
(8d): Yellow oily, 66% yield, IR (KBr): 3321.3, 2922.1, 2966.4,
2918.8, 1675.3, 1575.5, 1437.2, 1271.8, 1252.4, 1022.1, 752.4,
and 743.4. 1H-NMR (CDCl3, 400 MHz, ꢀ ppm) 7.55 (dd, 2H,
J ¼ 6 and 3.2 Hz), 7.21 (dd, 2H, J ¼ 7.2 and 2.8 Hz), 4.99 (s, 2H),
4.42 (t, 1H, J ¼ 13.6 and 6.8), 2.80 (td, 1H, J ¼ 14.8 and 2.8 Hz),
2.56 (t, 2H, J ¼ 7.2 and 2.8 Hz), 2.30 (td, 1H, J ¼ 13.2 and 6.8),
2.16 (s, 1H), and 2.03 (s, 3H). 13C-NMR (CDCl3, 100 MHz, ꢀ
ppm) 157.27, 138.47, 122.70, 115.27, and 49.92.
1-((1H-Benzo[d]imidazol-2-yl)(phenyl)methyl)-3-(3,5-bis(tri
fluoromethyl)phenyl)thiourea (9e): Bright yellow oily product,
66% yield, mp ¼ 104–106 ꢀC, IR (KBr): 3277.3, 3031.4, 1704.9,
1662.6, 1541.6, 1457.7, 1382.5, 1277.9, 1179.5, 1132.1, 957.8,
1
885.8 and 739.9. H-NMR (CDCl3, 400 MHz, ꢀ ppm) 10.18 (bs,
1H), 9.69 (s, 2H), 7.75 (s, 2H), 7.40 (s, 1H), 7.32 (dd, 3H, J ¼ 8.8
and 5.6 Hz), 7.22 (dd, 2H, J ¼ 6.8 and 4.2 Hz), 7.19 (dd, 4H,
J ¼ 8.4 and 2.8 Hz), and 4.10 (d, 1H, J ¼ 6.4 Hz). 13C-NMR
(CDCl3, 100 MHz, ꢀ ppm) 181.89, 155.07, 140.03, 136.94,
131.98, 131.65, 131.31, 130.97, 129.47, 129.19, 124.48, 123.94,
123.65, 121.78, 118.32, and 57.05.
(1H-Benzo[d]imidazol-2-yl)(phenyl)methanamine (8e): White
solid product, 65% yield, mp ¼ 201–203 ꢀC, IR (KBr): 3380.7,
2935.3, 2925.1, 1564.2, 1413.6, 1275.3, 1312.4, 1019.7, 1012.2,
1
CA inhibition
750.1, 742.1 and 653.1. H-NMR (DMSO-d6, 400 MHz, ꢀ ppm)
7.84 (dd, 4H, J ¼ 6.8, 3.2 and 3.6 Hz), 7.30 (dd, 2H, J ¼ 7.6, 2 and
1.2 Hz), 7.20 (m, 1H, J ¼ 6.8, 2 and 1.2 Hz), 7.11 (dd, 2H, J ¼ 6.8,
4 and 3.2 Hz), 5.74 (s, 1H), 5.29 (s, 2H), 2.48 (t, 1H, J ¼ 4.4 and
2 Hz). 13C-NMR (DMSO-d6, 100 MHz, ꢀ ppm) 158.99, 144.59,
128.86, 127.60, 121.91, and 55.36.
Enzyme activity was determined spectrophotometrically by
following the change in absorbance at 348 nm of 4-nitropheny-
lacetate to 4-nitrophenylate over a period of 3 min at 25 ꢀC20–23
.
The enzymatic reaction contained 1.4 ml 0.05 M Tris-SO4 buffer
(pH 7.4), 1 ml 3 mM 4-nitrophenylacetate, 0.5 ml H2O and 0.1 ml
enzyme solution, in a total volume of 3.0 ml24. Inhibitory effects
of compounds 5a–c and 9b–d were compared with phenol and
AZA. Different inhibitor concentrations were used and all
compounds were tested in triplicate at each concentration used.
Control cuvette activity was acknowledged as 100% in the
absence of inhibitor. An Activity% – [Inhibitor] graph was drawn
for each inhibitor21–23. The curve-fitting algorithm allowed to
obtain the IC50 values, working at the lowest concentration of
Synthesis of thiourea derivatives (9b–e)
2-N-Boc-amide derivative benzimidazole (1 mmol) (8b–e) was
dissolved in 5 ml of dry THF. Then 1-isothiocyanato-3,5-
bis(trifluoromethyl)benzene (1 mmol) was added at 0 ꢀC to the
solution. The mixture was stirred for 10 min at 0 ꢀC then allowed
to reach room temperature, and stirred for a further 24 h. The
solvent was removed in vacuum and the resulting material was
purified by column chromatography (hexane/EtOAc, 3:2 by
volume) to get a bright viscous product.
substrate of 0.15 mM, from which KI values were calculated22–23
.
The catalytic activity of these enzymes was calculated from
Lineweaver-Burk plots, as reported earlier25, and represents the
mean from at least three different determinations. The CAI and II
isoenzymes used here were purified from human blood as
1-(1-(1H-Benzo[d]imidazol-2-yl)-2-methylbutyl)-3-(3,5-bis(tri-
fluoromethyl)phenyl) thiourea (9b): Bright yellow oily product,
65% yield, mp ¼ 195–197 ꢀC, IR (KBr): 3027.8, 2874.2, 1738.1,
1541.5, 1457.3, 1381.5, 1357.8, 1276.2, 1180.1, 1131.3, 886.3,
described earlier8–13,26–28
.
1
764.3 and 680.9. H-NMR (DMSO-d6, 400 MHz, ꢀ ppm) 12.42
Conclusion
(bs, 1H), 10.32 (s, 1H), 8.76 (d, 1H, J ¼ 7.6 Hz), 8.29 (s, 2H), 7.75
(s, 1H), 7.59 (d, 1H, J ¼ 6.8 Hz), 7.47 (d, 1H, J ¼ 6.8 Hz), 7.21 In summary, synthesis of chiral thiourea containing benzimidazol
(dd, 5H, J ¼ 8 and 7.6 Hz), 7.19 (dd, 2H, J ¼ 8 and 6.8 Hz), 4.04 derivatives 5a–c and the first synthesis of 9b–d have been
(t, 1H, J ¼ 7.6 and 6.8 Hz), 3.45 (dd, 1H, J ¼ 14 and 6.8 Hz) and achieved. In addition, several benzimidazol compounds including
3.34 (dd, 1H, J ¼ 13.6 and 6.8 Hz). 13C-NMR (DMSO-d6, novel derivatives have been assayed for the inhibition of the
100 MHz, ꢀ ppm) 180.88, 154.38, 142.34, 137.80, 131.10, physiologically relevant human CA isozymes hCA I and II.

6
N. Korkmaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
12. Demirdag R, Comakli V, Senturk M, et al. Purification and
characterization of carbonic anhydrase from sheep kidney and
effects of sulfonamides on enzyme activity. Bioorg Med Chem 2013;
21:1522–5.
13. Ekinci D, Ceyhun SB, Sentu¨rk M, et al. Characterization
and anions inhibition studies of an a-carbonic anhydrase from the
teleost fish Dicentrarchus labrax. Bioorg Med Chem 2011;19:
744–8.
14. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for
medicinal chemistry. Bioorg Med Chem 2007;15:4336–50.
15. Reis O, Eymur S, Reis B, Demir AS. Direct enantioselective aldol
reactions catalyzed by a proline-thiourea host-guest complex. Chem
Commun 2009;9:1088–90.
16. Demirdag R, Yerlikaya E, Senturk M, et al. Heavy metal ion
inhibition studies of human, sheep and fish a-carbonic anhydrases.
J Enzym Inhib Med Chem 2013;28:278–82.
These compounds showed inhibition constants in the range of
3.4–73.6 mM for hCA I and 8.7–44.2 mM for hCA II. In general,
the compounds had comparable inhibitory activity with the
clinically used sulfonamide AZA. Interaction of most CA
isozymes with several types of phenols, such as simple phenol
and its substituted derivatives, clioquinol, salicyclates, and some
of their derivatives has been recently investigated. Here, we
extend these earlier investigations to a novel series of chiral
thiourea containing benzimidazol derivatives. The novel benzi-
midazols represent a promising class of CAIs and the results
discussed in this study may help medicinal chemists to design new
analogs with enhanced activity and other tailored properties.
Declaration of interest
17. Balaydin HT, Soyut H, Ekinci D, et al. Synthesis and carbonic
anhydrase inhibitory properties of novel bromophenols including
natural products. J Enzym Inhib Med Chem 2012;27:43–50.
18. Ekinci D, Ceyhun SB, Senturk M, et al. Characterization and anions
inhibition studies of an a-carbonic anhydrase from the teleost fish
Dicentrarchus labrax. Bioorg Med Chem 2011;19:744–8.
19. Ekinci D, Cavdar H, Talaz O, et al. NO-releasing esters show
carbonic anhydrase inhibitory action against human isoforms I and
II. Bioorg Med Chem 2010;18:3559–63.
20. Ekinci D, Cavdar H, Durdagi S, et al. Structure-activity relationships
for the interaction of 5,10-dihydroindeno[1,2-b]indole derivatives
with human and bovine carbonic anhydrase isoforms I, II, III, IV and
VI. Eur J Med Chem 2012;49:68–73.
21. Alp C, Ekinci D, Gultekin MS, et al. A novel and one-pot synthesis
of new 1-tosyl pyrrol-2-one derivatives and analysis for carbonic
anhydrase inhibitory potencies. Bioorg Med Chem 2010;18:
4468–74.
22. Ekinci D, Senturk M, Kufrevioglu OI. Salicylic acid derivatives:
synthesis, features and usage as therapeutic tools. Expert Opin Ther
Pat 2011;21:1831–41.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
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