Synthesis and evaluation of new thiadiazole derivatives as potential inhibitors of human carbonic anhydrase isozymes (hCA-I and hCA-II)

Article abstract of DOI:10.3109/14756366.2013.873038

2-[[5-(2,4-Difluoro/dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio] acetophenone derivatives (3a-s) were designed as human carbonic anhydrase isozymes (hCA-I and hCA-II) inhibitors and synthesized. hCA-I and hCA-II were purified from erythrocyte cells by

Full text of DOI:10.3109/14756366.2013.873038

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2015; 30(1): 32–37
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.873038
!
RESEARCH ARTICLE
Synthesis and evaluation of new thiadiazole derivatives as potential
inhibitors of human carbonic anhydrase isozymes (hCA-I and hCA-II)
1
1
2
1
3
Mehlika Dilek Altintop , Ahmet Ozdemir , Kaan Kucukoglu , Gulhan Turan-Zitouni , Hayrunnisa Nadaroglu , and
Zafer Asim Kaplancikli
1
,4
1
2
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskisehir, Turkey, Faculty of Pharmacy, Department of
3
Pharmaceutical Chemistry, Ataturk University, Erzurum, Turkey, Erzurum Vocational Training School, Department of Food Technology,
4
Ataturk University, Erzurum, Turkey, and Graduate School of Health Sciences, Department of Pharmaceutical Chemistry, Anadolu University,
Eskisehir, Turkey
Abstract
-[[5-(2,4-Difluoro/dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio] acetophenone derivatives Carbonic anhydrase, hydratase activity,
3a–s) were designed as human carbonic anhydrase isozymes (hCA-I and hCA-II) inhibitors
Keywords
2
(
thiadiazole
and synthesized. hCA-I and hCA-II were purified from erythrocyte cells by the affinity
chromatography. The inhibitory effects of 18 newly synthesized acetophenones on hydratase
activity of these isoenzymes were studied in vitro. The average IC50 values of the new
compounds for hydratase activity ranged from 0.033 to 0.14 mM for hCA-I and from 0.030 to
History
Received 30 October 2013
Revised 20 November 2013
Accepted 27 November 2013
Published online 25 March 2014
0
.11 mM for hCA-II. Among the newly synthesized compounds, 2-[[5-(2,4-dichlorophenylamino)-
,3,4-thiadiazol-2-yl]thio]-4 -bromoacetophenone (3n) can be considered as a promising hCA-II
0
1
inhibitor owing to its selective and potent inhibitory effect on hCA-II.
Introduction
Inhibition of CA isoenzymes is a promising approach for
pharmacologic intervention in a variety of disorders such as
glaucoma, epilepsy, obesity, and cancer. In the last decades,
CA inhibition has also emerged as a major focus of pharmaceut-
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of zinc-
containing metalloenzymes, present in prokaryotes and eukary-
otes, being encoded by five distinct, evolutionarily unrelated gene
families: the a-, b-, g-, ꢀ-, and "-CAs. 16 different a-CA isoforms
were isolated and characterized in mammals, where they play
crucial physiological roles. Some of them are cytosolic (CA I, CA
II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV,
CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are
mitochondrial, and CA VI is secreted in saliva and milk. Three
acatalytic forms are also known, the CA related proteins (CARP),
ical research for the design of anti-infective agents with a novel
1–9
mechanism of action
.
Thiadiazole has received considerable attention as a privileged
scaffold due to its significant therapeutic potential. The sulfur
atom of thiadiazole ring imparts improved liposolubility and the
mesoionic nature of 1,3,4-thiadiazoles also allows these com-
pounds to cross cellular membranes and interact with biological
10,11
1
–4
targets with distinct affinities
. Several derivatives of 5-amino-
CARP VIII, CARP X and CARP XI
.
1
,3,4-thiadiazole-2-sulfonamide have been synthesized and used
as carbonic anhydrase inhibitors. One of them is acetazolamide
AAZ) (N-(5-sulfamoyl-1,3,4-thiadiazole-2-yl)acetamide), which
CAs, which catalyze the interconversion of carbon dioxide and
water to bicarbonate and protons, have attracted a great deal
of interest as important targets for drug discovery due to their
essential roles in crucial physiological processes connected with
respiration and transport of CO /bicarbonate between metaboliz-
2
ing tissues and lungs, pH and CO2 homeostasis, electrolyte
secretion in a variety of tissues/organs, biosynthetic reactions
(such as gluconeogenesis, lipogenesis and ureagenesis), bone
resorption, calcification, tumorigenicity and many other physio-
4
logic or pathologic processes .
(
12–15
is used in the treatment of glaucoma (Figure 1)
. Some
researchers have reported the potency of the SH moiety as a zinc-
binding function in the design of carbonic anhydrase inhibitors
and carried out considerable research for the synthesis and
evaluation of 5-amino-1,3,4-thiadiazole-2-thiol derivatives as
16–18
carbonic anhydrase inhibitors
.
On the basis of afore-mentioned findings, herein we
report the synthesis and biological evaluation of a series of 2-
[[5-(2,4-difluoro/dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]
acetophenone derivatives as hCA-I and hCA-II inhibitors.
Methods
Address for correspondence: Dr. Zafer Asim Kaplancikli, Faculty of Chemistry
Pharmacy, Department of Pharmaceutical Chemistry, Anadolu
All reagents were purchased from commercial suppliers and
used without further purification. Melting points (m.p.) were
University, 26470 Eskisehir, Turkey. Tel: +90 222 3350580/3776. Fax:
90 222 3350750. E-mail: zakaplan@anadolu.edu.tr
+

DOI: 10.3109/14756366.2013.873038
Inhibitors of human carbonic anhydrase isozymes 33
N
N
Anal. Calcd. for C H F N O S : C, 47.05; H, 2.47; N, 13.72.
1
6 10 2 4 3 2
O
Found: C, 47.07; H, 2.45; N, 13.70.
0
H C
C
N
H
SO NH
2
2-[[5-(2,4-Difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
fluoroacetophenone (3c)
3
S
2
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.94 (s, 2H), 7.07–7.11
6
AAZ
(
(
m, 1H), 7.32–7.42 (m, 3H), 8.12–8.15 (m, 2H), 8.24–8.29
m, 1H), 10.16 (s, 1H).
Figure 1. Chemical structure of the clinically used sulfonamide
acetazolamide (AAZ).
þ
MS (ESI) (m/z): [Mþ1] 382
Anal. Calcd. for C H F N OS : C, 50.39; H, 2.64; N, 11.02.
1
6
10
3
3
2
Found: C, 50.41; H, 2.65; N, 11.00.
determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected.
Proton nuclear magnetic resonance ( H-NMR) spectra were
recorded on a Bruker 500 MHz spectrometer (Bruker, Billerica,
0
2
-[[5-(2,4-difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
chloroacetophenone (3d)
1
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.94 (s, 2H), 7.08–7.09
6
MA). Chemical shifts were expressed in parts per million (ppm) (m, 1H), 7.33–7.35 (m, 1H), 7.65 (d, J ¼ 8.5 Hz, 2H), 8.06
and tetramethylsilane was used as an internal standard. Mass (d, J ¼ 8.5 Hz, 2H), 8.25–8.27 (m, 1H), 10.17 (s, 1H).
þ
spectra were recorded on a Agilent LC-MSD-Trap-SL Mass
spectrometer (Agilent, Minnesota, MN). Elemental analyses were
MS (ESI) (m/z): [Mþ1] 398
Anal. Calcd. for C H ClF N OS : C, 48.30; H, 2.53;
1
6
10
2
3
2
performed on a Perkin Elmer EAL 240 elemental analyser N, 10.56. Found: C, 48.28; H, 2.54; N, 10.55.
Perkin-Elmer, Norwalk, CT).
(
0
2
-[[5-(2,4-difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
bromoacetophenone (3e)
General procedure for the synthesis of the compounds
-(2,4-Difluoro/dichlorophenyl)thiosemicarbazide (1a–b)
1
4
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.93 (s, 2H), 7.07–7.10
6
(
(
m, 1H), 7.31–7.36 (m, 1H), 7.79 (d, J ¼ 8.0 Hz, 2H), 7.97
d, J ¼ 8.5 Hz, 2H), 8.24-8.29 (m, 1H), 10.16 (s, 1H).
A mixture of 2,4-difluoro/dichlorophenyl isothiocyanate (0.1 mol)
and hydrazine hydrate (0.2 mol) in ethanol (30 ml) was stirred
at room temperature for 5 h and then filtered. The residue was
þ
MS (ESI) (m/z): [Mþ1] 443
19,20
Anal. Calcd. for C16
H
10BrF
N, 9.50. Found: C, 43.43; H, 2.30; N, 9.49.
N
2
3
OS
2
: C, 43.45; H, 2.28;
crystallized from ethanol
.
5-[(2,4-Difluoro/dichlorophenyl)amino]-1,3,4-thiadiazole-2(3H)-
thione (2a–b)
0
2-[[5-(2,4-difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
cyanoacetophenone (3f)
4
-(2,4-Difluoro/dichlorophenyl)thiosemicarbazide (1a–b) was
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.97 (s, 2H), 7.08–7.09
6
dissolved in a solution of sodium hydroxide in ethanol. Carbon
disulfide was then added while stirring and the reaction mixture
was heated under reflux for 10 h. The solution was cooled
and acidified to pH 4–5 with hydrochloric acid solution and
(
(
m, 1H), 7.33–7.34 (m, 1H), 8.06 (d, J ¼ 8.5 Hz, 2H), 8.19
d, J ¼ 8.5 Hz, 2H), 8.24–8.25 (m, 1H), 10.17 (s, 1H).
þ
MS (ESI) (m/z): [Mþ1] 389
2
1
Anal. Calcd. for C17
H
F
10
N
2
4
OS : C, 52.57; H, 2.60; N, 14.42.
2
crystallized from ethanol .
Found: C, 52.55; H, 2.59; N, 14.41.
2-[[5-(2,4-Difluoro/dichlorophenylamino)-1,3,4-thiadiazol-2-
yl]thio]acetophenone derivatives (3a–s)
0
2-[[5-(2,4-Difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
methylacetophenone (3g)
A mixture of 5-(2,4-difluoro/dichlorophenylamino)-1,3,4-thiadia-
zole-2(3H)-thione (2a–b) (0.05 mol) and appropriate phenacyl
bromide (0.05 mol) in the presence of potassium carbonate
(0.05 mol) in acetone was stirred at room temperature for 8 h.
The reaction mixture was filtered and crystallized from ethanol.
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 2.40 (s, 3H), 4.92 (s,
6
2
2
H), 7.07–7.11 (m, 1H), 7.33–7.39 (m, 3H), 7.94 (d, J ¼ 8.0 Hz,
H), 8.24–8.29 (m, 1H), 10.06 (s, 1H).
MS (ESI) (m/z): [Mþ1] 378
þ
Anal. Calcd. for C H F N OS : C, 54.10; H, 3.47; N, 11.13.
3
1
7
13
2
2
Found: C, 54.11; H, 3.45; N, 11.11.
2-[[5-(2,4-difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]
acetophenone (3a)
0
2-[[5-(2,4-Difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
methoxyacetophenone (3h)
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.96 (s, 2H), 7.07–7.11
6
(
(
1
m, 1H), 7.32–7.37 (m, 1H), 7.56–7.59 (m, 2H), 7.69
t, J ¼ 7.5 Hz, 1H), 8.04–8.06 (m, 2H), 8.24–8.29 (m, 1H),
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 3.87 (s, 3H), 4.89 (s,
6
2H), 7.06–7.12 (m, 3H), 7.33–7.37 (m, 1H), 8.01–8.04 (m, 2H),
8.24–8.29 (m, 1H), 10.15 (s, 1H).
0.17 (s, 1H).
MS (ESI) (m/z): [Mþ1] 364
2
þ
þ
MS (ESI) (m/z): [Mþ1] 394
Anal. Calcd. for C H F N OS : C, 52.88; H, 3.05; N, 11.56.
1
6
11
2
3
Found: C, 52.90; H, 3.04; N, 11.55.
Anal. Calcd. for C17
H
13
F
2
N
3
O
Found: C, 51.89; H, 3.31; N, 10.69.
2
S
2
: C, 51.90; H, 3.33; N, 10.68.
0
2-[[5-(2,4-Difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
nitroacetophenone (3b)
0
2-[[5-(2,4-Difluorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
methylsulfonylacetophenone (3i)
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 5.00 (s, 2H), 7.06–7.34
6
1
(
(
m, 2H), 8.22–8.27 (m, 3H), 8.37 (d, J ¼ 8.5 Hz, 2H), 10.17
MS (ESI) (m/z): [Mþ1]þ 409
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 3.32 (s, 3H), 4.99
6
s, 1H).
(s, 2H), 7.08–7.11 (m, 1H), 7.33–7.37 (m, 1H), 8.12 (d,
J ¼ 8.5 Hz, 2H), 8.23–8.28 (m, 3H), 10.18 (s, 1H).

3
4
M. D. Altintop et al.
J Enzyme Inhib Med Chem, 2015; 30(1): 32–37
þ
0
MS (ESI) (m/z): [Mþ1] 442
7 13 2 3 3 3
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
Anal. Calcd. for C H F N O S : C, 46.25; H, 2.97; N, 9.52. methylacetophenone (3p)
1
Found: C, 46.26; H, 2.95; N, 9.51.
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 2.41 (s, 3H), 4.93
6
(
7
(
s, 2H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.42 (dd, J ¼ 9.0, 2.5 Hz, 1H),
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]
acetophenone (3j)
.64 (d, J ¼ 2.0 Hz, 1H), 7.95 (d, J ¼ 8.5 Hz, 2H), 8.33
d, J ¼ 9.0 Hz, 1H), 9.94 (s, 1H).
þ
1
MS (ESI) (m/z): [Mþ1] 411
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.97 (s, 2H), 7.42
6
Anal. Calcd. for C H Cl N OS : C, 49.76; H, 3.19; N, 10.24.
1
7
13
2
3
2
(
dd, J ¼ 9.0, 2.5 Hz, 1H), 7.58 (t, J ¼ 7.5 Hz, 2H), 7.64 (d,
Found: C, 49.75; H, 3.20; N, 10.25.
J ¼ 2.5 Hz, 1H), 7.70 (t, J ¼ 7.5 Hz, 1H), 8.05 (d, J ¼ 7.0 Hz, 2H),
8
.33 (d, J ¼ 9.0 Hz, 1H), 9.95 (s, 1H).
þ
0
MS (ESI) (m/z): [Mþ1] 397
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
Anal. Calcd. for C H Cl N OS : C, 48.49; H, 2.80; N, 10.60. methoxyacetophenone (3r)
1
6
11
2
3
2
Found: C, 48.51; H, 2.79; N, 10.59.
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 3.86 (s, 3H), 4.90 (s,
6
2
8
H), 7.07–7.12 (m, 2H), 7.40–7.45 (m, 1H), 7.62-7.65 (m, 1H),
.03 (d, J ¼ 8.5 Hz, 2H), 8.33 (d, J ¼ 8.5 Hz, 1H), 9.94 (s, 1H).
0
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
nitroacetophenone (3k)
þ
MS (ESI) (m/z): [Mþ1] 427
1
Anal. Calcd. for C H Cl N O S : C, 47.89; H, 3.07; N, 9.86.
2
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 5.02 (s, 2H), 7.42
6
17 13
2
3
2
Found: C, 47.90; H, 3.05; N, 9.85.
(
(
dd, J ¼ 9.0, 2.5 Hz, 1H), 7.64 (d, J ¼ 2.5 Hz, 1H), 8.27
d, J ¼ 8.5 Hz, 2H), 8.32 (d, J ¼ 8.5 Hz, 1H), 8.39 (d, J ¼ 8.5 Hz,
0
2
H), 9.96 (s, 1H).
MS (ESI) (m/z): [Mþ1] 442
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
methylsulfonylacetophenone (3s)
þ
Anal. Calcd. for C H Cl N O S : C, 43.55; H, 2.28;
1
6
10
2
4
3
2
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 3.32 (s, 3H), 5.01
6
N, 12.70. Found: C, 43.53; H, 2.31; N, 12.69.
(
8
s, 2H), 7.42 (dd, J ¼ 9.0, 2.5 Hz, 1H), 7.65 (d, J ¼ 2.5 Hz, 1H),
.12 (d, J ¼ 8.0 Hz, 2H), 8.27 (d, J ¼ 8.5 Hz, 2H), 8.32
0
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
fluoroacetophenone (3l)
(d, J ¼ 9.0 Hz, 1H), 9.96 (s, 1H).
þ
MS (ESI) (m/z): [Mþ1] 475
1
Anal. Calcd. for C H Cl N O S : C, 43.04; H, 2.76; N, 8.86.
3
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.96 (s, 2H), 7.39–7.43
6
17 13
2
3
3
Found: C, 43.03; H, 2.74; N, 8.85.
(
(
m, 3H), 7.63 (d, J ¼ 2.5 Hz, 1H), 8.12-8.15 (m, 2H), 8.33
d, J ¼ 8.5 Hz, 1H), 9.95 (s, 1H).
þ
MS (ESI) (m/z): [M þ1] 415
Biochemistry
Anal. Calcd. for C H Cl FN OS : C, 46.38; H, 2.43; N,
2
1
6
10
2
3
Sepharose-4B, sulfanilamide, L-tyrosine, Tris, Na SO , protein
2
4
1
0.14. Found: C, 46.40; H, 2.41; N, 10.15.
assay reagents, and chemicals for electrophoresis were purchased
from Sigma-Aldrich Co. (St. Louis, MO). All other chemicals
were of analytical grade and obtained from Merck (Darmstadt,
Germany).
0
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
chloroacetophenone (3m)
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.95 (s, 2H), 7.42
6
(
2
dd, J ¼ 9.0, 2.5 Hz, 1H), 7.64–7.66 (m, 3H), 8.06 (d, J ¼ 8.5 Hz,
Purification of carbonic anhydrase isozymes (hCA-I and hCA-II)
from human erythrocytes by affinity chromatography
H), 8.33 (d, J ¼ 9.0 Hz, 1H), 9.95 (s, 1H).
þ
MS (ESI) (m/z): [Mþ1] 431
Fresh human blood was obtained from the blood center, Ataturk
ꢀ
Anal. Calcd. for C H Cl N OS : C, 44.61; H, 2.34; N, 9.75.
2
1
6
10
3
3
University, it was stored at 4 C used within 2–3 d at most. The
blood samples were centrifuged to separate erythrocytes at
Found: C, 44.60; H, 2.35; N, 9.72.
2
500 rpm for 15 min plasma and white blood cells which is the
0
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
bromoacetophenone (3n)
upper part the layers carefully removed and discarded. Then,
underlying erythrocytes were washed with 0.9% NaCl solution
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.94 (s, 2H), 7.42 twice and upper portions were also discarded. The erythrocytes
6
ꢀ
(
(
dd, J ¼ 9.0, 2.5 Hz, 1H), 7.64 (d, J ¼ 2.5 Hz, 1H), 7.79–7.81 were hemolyzed with distilled water at 0 C, it was stirred for half
ꢀ
an hour at 4 C. The hemolysate was centrifuged at 20 000rpm
for 30 min and cell membranes were separated. pH was adjusted to
m, 2H), 7.97-7.99 (m, 2H), 8.32 (d, J ¼ 9.0 Hz, 1H), 9.95 (s, 1H).
þ
MS (ESI) (m/z): [Mþ1] 476
Anal. Calcd. for C H BrCl N OS : C, 40.44; H, 2.12; 8.7 with solid Tris. So, the hemolysate was recovered to be applied
1
6
10
2
3
2
2
2,23
to the column
.
N, 8.84. Found: C, 40.42; H, 2.13; N, 8.85.
The affinity gel was prepared on Sepharose-4B matrix.
After Sepharose-4B was activated with CNBr, L-tyrosine was
covalently fitted. Then sulfanilamide was coupled to tyrosine
with diazotization reaction as a ligand. The hemolysate was applied
0
2-[[5-(2,4-Dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
cyanoacetophenone (3o)
1
H-NMR (500 MHz, DMSO-d ) ꢀ (ppm): 4.99 (s, 2H), 7.42 (dd, to the prepared Sepharose-4B-L-tyrosine-sulfanilamide affinity
6
J ¼ 9.0, 2.5 Hz, 1H), 7.63 (d, J ¼ 2.5 Hz, 1H), 8.06 (d, J ¼ 8.5 Hz, column equilibrated with 25 mM Tris-HCl/0.1 M Na SO (pH 8.7).
2
4
2
H), 8.19 (d, J ¼ 8.5 Hz, 2H), 8.32 (d, J ¼ 9.0 Hz, 1H), 9.94 The affinity gel was washed with 25 mM Tris-HCl/22 mM Na SO4
2
(s, 1H).
(pH 8.7). The human carbonic anhydrase (hCA I and hCA II)
isozymes were eluted with 1 M NaCl/25 mM Na HPO (pH 6.3)
Anal. Calcd. for C H Cl N OS : C, 48.46; H, 2.39; N, 13.30. and 0.1 M CH COONa/0.5 M NaClO (pH 5.6), respectively. All
þ
MS (ESI) (m/z): [Mþ1] 422
2
4
1
7
10
2
4
2
3
4
ꢀ
22,24
Found: C, 48.45; H, 2.41; N, 13.29.
procedures were performed at 4 C
.

DOI: 10.3109/14756366.2013.873038
Inhibitors of human carbonic anhydrase isozymes 35
2,4-difluoro/dichlorophenylisothiocyanates with hydrazine
Hydratase activity assay
hydrate. 5-[(2,4-Difluoro/dichlorophenyl)amino]-1,3,4-thiadia-
zole-2(3H)-thiones (2a–b) were synthesized via the ring closure
reaction of 4-(2,4-difluoro/dichlorophenyl)thiosemicarbazides
Carbonic anhydrase activity was determined using the Wilbur–
2
5,26
Anderson Method which was modified by Rickli and Sly
þ
.
This method, as a result hydration of CO is released H ions and
2
(
1a–b) with carbon disulfide in the presence of sodium hydroxide.
Finally, the nucleophilic substitution reaction of 5-[(2,4-difluoro/
dichlorophenyl)amino]-1,3,4-thiadiazole-2(3H)-thiones (2a–b)
the pH changes were determined by means of bromine thymol
blue indicator, based on measuring the elapsed time. Enzyme Unit
(EU) were calculated using the equation (to-tc/tc) to and tc are the
with appropriate phenacyl bromides afforded 2-[[5-(2,4-
difluoro/dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]aceto-
phenone derivatives (3a–s). Some physicochemical properties of
the compounds were given in Table 1.
times for pH change of the nonenzymatic and the enzymatic
reactions, respectively.
Inhibition assays
1
The structures of compounds 3a–s were confirmed by H-
1
It was studied with compounds 3a–s to calculate values of IC50 NMR, mass spectral data, and elemental analysis. In the H-NMR
of hCA-I and hCA-II enzymes on the hydratase activity at spectra of compounds 3a–s, the signal due to the S–CH protons
2
different concentrations while maintaining constant the substrate was observed in the region 4.89–5.02 ppm as a singlet peak. The
concentration. Activities of enzymes in the medium without signal due to the N–H proton attached to the thiadiazole ring
inhibitors were used as 100% activity. The activity % values occurred in the region 9.94–10.18 ppm as a singlet. The other
of enzymes were calculated by measuring hydratase activity in aromatic and aliphatic protons were observed at expected regions.
the presence of different concentrations of inhibitors. The IC50
value was calculated by utilizing graphs of % activity-[I] for each Table 1. Physicochemical properties of the compounds (3a–s).
.
27,28
inhibitor
0
ꢀ
Compound
R
R
Yield (%) M.p. ( C) Log P*
MR*
Protein determination
3
3
3
3
3
3
3
a
b
c
d
e
f
F
F
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
NO
F
Cl
Br
78
90
79
81
83
85
73
71
74
80
92
81
83
85
87
75
73
76
125
167
161
158
155
169
159
140
179
168
183
203
195
184
217
162
148
186
5.63
–
94.26
–
Protein assay is performed according to the Bradford method for
all purification steps. This method bases on the principle which
Coomassie brilliant blue G-250 was binding with proteins in
medium of phosphoric acid. Formed complex shows a maximum
absorbance at 595 nm. The sensitivity of this method is between 1
2
5.79
6.19
6.46
5.66
6.12
5.5
4.34
6.43
–
6.59
6.99
7.26
6.46
6.92
6.3
94.67
98.87
101.95
100.36
100.16
101.51
109.04
102.66
–
103.07
107.27
110.35
108.76
108.56
109.91
117.44
CN
CH
3
g
2
9
and 100 mg .
3h
OCH
3
SO CH
3
3
3
i
j
k
2
3
H
SDS polyacrylamide gel electrophoresis
NO
F
2
Then purifying hCA-I and-II isoenzymes from erythrocytes, 3l
3
m
3n
3o
Cl
Br
CN
CH
3
OCH
discontinuous 3-10% sodium dodecyl sulfate (SDS) polyacryl-
amide gel electrophoresis was performed in accordance Laemmli
3
and the purity of enzymes were checked .
0
3
3
3
p
r
s
3
Results and discussion
Cl SO CH
2
3
5.14
The synthesis of compounds 3a–s followed the general pathway
outlined in Scheme 1. Initially, 4-(2,4-difluoro/dichlorophe-
nyl)thiosemicarbazides (1a–b) were obtained by the reaction of
*
Log P and MR values were calculated using ChemDraw Ultra 7.0
Cambridge Soft Corporation, Cambridge, MA). Log P and MR values
of compounds 3b and 3k were not calculated.
(
R
R
H
S
H
N
i
R
N
C
S
R
N
C
NH2
1
a-b
ii
H
R
R'
R
N
N
N
N
iii
N
H
S
S
N
H
S
S
R
O
R
3a-s
2
a-b
Scheme 1. The synthetic route for the preparation of the thiadiazole derivatives (3a–s). Reagents and conditions: (i) NH
ii) (1) CS /NaOH, ethanol, reflux, 10 h; (2) HCl, pH 4–5; (iii) PhCOCH Br, K CO , acetone, rt, 8 h.
2
NH
2
ꢁ H
2
O, ethanol, rt, 5 h;
(
2
2
2
3

3
6
M. D. Altintop et al.
J Enzyme Inhib Med Chem, 2015; 30(1): 32–37
Table 2. Summary of purification for hCA-I and hCA-II from human erythrocyte.
Total activity
Activity
(EU/mL)
Protein
(mg/mL)
Specific activity
(EU/mg)
Purification steps
Volume mL
EU
%
Purification fold
Hemolysate
hCA-I
hCA-II
100
40
30
42.75
56.5
62.6
4275
2260
1875
100
52.9
43.9
155
0.082
0.045
0.28
689.0
1391.1
–
2460.7
4968.2
Table 3. IC50 values for the in vitro inhibition of hCA-I and hCA-II with Conclusion
compounds 3a–s.
In this article, we synthesized a series of 2-[[5-(2,4-difluoro/
dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]acetophenone
derivatives (3a–s) and evaluated their ability to inhibit carbonic
anhydrase isozymes (hCA-I and hCA-II). Although compounds
3a–s do not carry a sulfonamide group, an important pharmaco-
phore for hCA inhibitory activity, it is a remarkable finding
that compounds 3a–r exhibited significant inhibitory effects on
IC50 (mM)
hCA-II
Compound
hCA-I
hCA-I/hCA-II
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
r
s
0.1
0.11
0.055
0.074
0.055
0.056
0.080
0.078
0.084
0.10
0.098
0.096
0.11
0.044
0.062
0.072
0.054
0.056
0.030
*
1.82
1.49
1.05
1.21
1.11
1.0
0.058
0.068
0.089
0.078
0.11
hCA-I and hCA-II. Among the newly synthesized compounds,
0
2
-[[5-(2,4-dichlorophenylamino)-1,3,4-thiadiazol-2-yl]thio]-4 -
bromoacetophenone (3n) can be evaluated as a potential hCA-II
inhibitor due to its selective and potent inhibitory effect on
hCA-II.
1.31
1.4
0.14
0.111
0.128
0.05
0.071
0.049
0.068
0.033
0.036
0.043
*
1.13
1.33
1.45
1.61
1.79
1.94
1.61
1.56
1.43
*
Declaration of interest
The authors report no conflicts of interest.
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