Synthesis and carbonic anhydrase inhibitory properties of novel 1,4-dihydropyrimidinone substituted diarylureas

Article abstract of DOI:10.3109/14756366.2012.746972

A new series of 1,4-dihydropyrimidinone (DHPM) substituted diaryl urea and thiourea derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and II were evaluated. 4-Nitrophenyl-1,4-DHPM was prepared with dimedone, nitrobenzaldehyde and urea or thiourea and nitro group was reduced to amine derivative. The compound was reacted with isocyanates and isothiocyanates to get the final products. The results showed that all the synthesized compounds inhibited the carbonic anhydrase isoenzyme activity; 4c (IC50 = 66.23 μM for hCA I) and 4f (IC50 = 63.09 μM for hCA II) have the most inhibitory effect. The synthesized compounds are very bulky to be able to bind near the zinc ion and they much more probably bind as the coumarins and activators.

Full text of DOI:10.3109/14756366.2012.746972

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(1): 18–22
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.746972
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RESEARCH ARTICLE
Synthesis and carbonic anhydrase inhibitory properties of novel
1,4-dihydropyrimidinone substituted diarylureas
Fatma Celik1, Mustafa Arslan1, Emre Yavuz2, Dudu Demir2, and Nahit Genc¸er2
2
1Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, Sakarya, Turkey, and Department of Chemistry, Science and Art
Faculty, Balikesir University, Balikesir, Turkey
Abstract
Keywords
A new series of 1,4-dihydropyrimidinone (DHPM) substituted diaryl urea and thiourea
derivatives were synthesized and their inhibitory effects on the activity of purified human
carbonic anhydrase (hCA) I and II were evaluated. 4-Nitrophenyl-1,4-DHPM was prepared with
dimedone, nitrobenzaldehyde and urea or thiourea and nitro group was reduced to amine
derivative. The compound was reacted with isocyanates and isothiocyanates to get the final
products. The results showed that all the synthesized compounds inhibited the carbonic
anhydrase isoenzyme activity; 4c (IC50 ¼ 66.23 mM for hCA I) and 4f (IC50 ¼ 63.09 mM for hCA II)
have the most inhibitory effect. The synthesized compounds are very bulky to be able to bind
near the zinc ion and they much more probably bind as the coumarins and activators.
Carbonic anhydrase, dihydropyrimidinone,
enzyme inhibitor, urea
History
Received 27 July 2012
Revised 31 October 2012
Accepted 1 November 2012
Published online 16 January 2013
Introduction
method for the synthesis of DHPMs first reported by Biginelli14 in
1893 was using an aldehyde, a b-ketoester and urea (or thiourea)
under strongly acidic conditions, but the reaction suffered from
drawbacks such as long reaction time, low yields, etc. For this
transformation, several methods were improved such as using
zirconium hydrogen phosphate15, alumina sulphuric acid (ASA)16
and heteropoly acids17.
Ureido-substituted benzenesulphanilamides show very inter-
esting profile for the inhibition of several human CAs (hCAs)
such as hCAs I and II (cytosolic isoforms) and hCAs IX and XII
(transmembrane, tumour-associated enzymes). They mentioned
that the compounds have excellent inhibitory effects for all these
isoforms due to the urea moiety18. CA IX is highly expressed in
breast malignancies, and CA IX and CA XII are variably
expressed in breast cancer cell lines. Human breast cancers
provided definitive evidence of CA IX as an independent poor
prognostic biomarker for distant metatheses and survival. Lou
et al.19 have studied CA inhibitory activities in vitro on 4T1
mouse metastatic breast cancer cells.
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread zinc
metalloenzymes that catalyse the reversible hydration ofþcarbon
dioxide (CO2) to bicarbonate (HCOꢀ3 ) and a proton (H ) with
water1. CAs are ubiquitous enzymes present in prokaryotes and
eukaryotes which are encoded by four evolutionarily unrelated
gene families (the a-, b-, g- and ꢀ-CAs)2,3. There are 16 isozymes
which are characterized and many of them are involved in critical
physiological processes4. In humans, CAs are found in a variety of
tissues such as kidneys, lungs, eyes, skins, the nervous systems
and the gastrointestinal tract5. The different isozymes are found in
different parts of the cell and CA I and II are localized in the
cytosol6. Biological activities of this metalloenzyme family have
several medicinal applications such as treatment of glaucoma,
diuretics, in the management of several neurological disorders,
whereas several agents are in clinical evaluations as antiobesity or
antidrugs7.
In recent years, much attention has been focused on the
synthesis of 1,4-dihydropyrimidinone (DHPM) due to their
significant biological activities. The compounds have various
therapeutic and pharmacological properties such as calcium
channel modulators, antihypertensive agents, a1a-adrenergic
receptor antagonists8,9, antiviral, antitumour, antibacterial and
anti-inflammatory activities10,11. The DHPM core is also found in
many natural products and marine alkaloids and have been found
to be potent HIV gp-120CD4 inhibitors12.
In this study, a new series of 1,4-dihydropyrimidine substituted
diaryl urea and thiourea derivatives were synthesized and their
inhibitory effects on the activity of purified hCA I and II were
evaluated.
Materials and methods
1,4-Dihydropyrimidine substituted urea and thiourea derivatives
Multicomponent reactions are important for various reactions shown in Scheme 1 were synthesized and their effect was
in medicinal and organic chemistry13. The simple and direct examined on CA I and II. 4-Nitrophenyl-1,4-DHPM was prepared
with dimedone, nitrobenzaldehyde and urea or thiourea by ASA
acid catalyst. The compound was reduced to amine derivative
with tin (II) chloride in ethanol. The amine containing
Science and Art Faculty, Balikesir University, Cagis kampus, Balikesir
Address for correspondence: Nahit Genc¸er, Department of Chemistry,
dihyropyrimidinone compound was reacted with isocyanates
and isothiocyanates to get the products (4a–i) at high yields.
10145, Turkey. Tel: þ90 266 6121278. Fax: þ90 266 6121215. E-mail:
ngencer@balikesir.edu.tr

DOI: 10.3109/14756366.2012.746972
1,4-Dihydropyrimidinone substituted diarylureas 19
Scheme 1. Synthesis of 1,4-dihydropyrimi-
dine substituted urea and thiourea derivatives.
The prepared compounds were characterized by 1H-, referenced to the internal deuterated solvent. The elemental
13C-NMR, infrared (IR) and elemental analysis. The hydrogens analysis was carried out with a Leco CHNS-932 (St. Joseph,
attached to the nitrogen resonances between 8.00 and 10.40 ppm Michigan) instrument. Flash column chromatography was per-
1
and were indicated from the H-NMR spectra. The signals for formed using Merck silica gel 60 (230–400 mesh ASTM)
aromatic hydrogens are between 6.50 and 8.50 ppm. The (Darmstadt, Germany).
hydrogen next to the phenyl ring was observed around
5.00 ppm. From the 13C-NMR spectra, ketone and urea carbonyl General procedure 1: synthesis of 1,4-DHPM (2)
are seen between 200 and 150 ppm, respectively. In the IR
A mixture of 4-nitrobenzaldehyde (3 mmol), dimedone (3 mmol),
spectra of compounds, it was possible to observe the absorptions
urea or thiourea (4.5 mmol) and ASA catalyst (7% mmol) in
between 3250 and 3450 cmꢀ1 relating to NH stretching and
ethanol were finely mixed together in a test tube at 90 ꢁC for one
absorptions in 1650–1750 cmꢀ1 from urea carbonyl moiety
hour. After cooling, the reaction mixture was poured onto crushed
stretching.
ice (50 g) and stirred for 10 min. The precipitate was filtered under
suction and washed with cold water (20 mL) to remove excess
urea. Then, the solid was dissolved in ethanol and filtered to
General
All starting materials and reagents were purchased from remove the catalyst and purified further by recrystallization (hot
commercial suppliers. Reactions were monitored by TLC and ethanol).
TLC plates (Fluka, Taufkirchen, Germany) visualized with short-
1,3,7,7-Tetramethyl-4-(4-nitrophenyl)-3,4,7,8-tetrahydroquina-
1
wave UV fluorescence (k ¼ 254 nm). Melting points were taken zoline-2,5(1H,6H)-dione (2): yield 88%, m.p. 145.5 ꢁC; H-NMR
on a Yanagimoto Barnstead Electrothermal (Surrey, UK) micro- (DMSO-d6; 300 MHz): 8.16(2H,d), 7.45(2H,d), 5.56(H,s),
melting point apparatus and were uncorrected. IR spectra were 3.25(3H,s), 2.97(3H,s), 2.40(2H,d,d), 2.20(2H,d,d), 1.10(3H,s)
measured on a SHIMADZU Prestige-21 (200 VCE) (Kyoto, and 1.00(3H,s); 13C-NMR(DMSO-d6; 75 MHz):194.04, 153.43,
1
Japan) spectrometer. H- and 13C-NMR spectra were measured 153.05, 146.28, 132.84, 127.35, 123.40, 118.87, 110.01, 58.42,
on spectrometer at Varian Infinity Plus 300 and at 75 Hz 49.59, 40.31, 35.14, 33.03, 31.00 and 28.93; and IR (KBr,
(California), respectively. 1H- and 13C-chemical shifts are ꢁ, cmꢀ1): 3042, 2964, 2875, 1690 and 1580.

20 F. Celik et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 18–22
General procedure 2: reduction of nitro group (3)
7.45(2H,d), 7.40(H,d), 7.38(H,t), 7.35(H,t), 7.30(H,d), 7.25(H,t),
7.10(H,t), 5.20(H,s), 3.30(3H,s), 2.85(3H,s), 2.40(2H,d,d),
2.20(2H,d,d), 1.10(3H,s) and 0.98(3H,s); 13C-NMR(DMSO-d6;
75 MHz): 193.54, 180.03, 153.54, 153.15, 140.04, 139.50, 137.49,
128.99, 127.26, 124.99, 124.23, 124.05, 110.06, 65.69, 58.09,
49.71, 34.69, 33.02, 30.89, 28.85 and 28.58 and IR (KBr, ꢁ,
cmꢀ1): 3332, 3188, 1672 and 1593. Anal. calcd for C25H30N4OS:
C, 69.09; H, 6.96; N, 12.89 and S, 7.38. Found: C, 69.61; H, 7.37;
N, 13.51 and S, 7.63.
1-(4-Nitrophenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,5,6,
7,8-octahydroquinazolin-4-yl)phenyl)thiourea (4e): yield: 88%,
m.p.: 209.8 ꢁC; 1H-NMR (DMSO-d6; 300 MHz): 10.40(H,s),
10.20(H,s), 8.20(2H,d), 7.80(2H,d), 7.40(2H,s), 7.20(2H,d),
5.22(H,s), 3.20(3H,s), 2.80(3H,s), 2.60(2H,d,d), 2.10(2H,d,d),
1.10(3H,s) and 0.98(3H,s); 13C-NMR(DMSO-d6; 75 MHz):
193.66, 179.80, 153.81, 153.17, 146.91, 142.91, 139.04, 138.23,
127.36, 125.05, 124.17, 122.17, 109.98, 79.64, 58.04, 49.68,
34.70, 33.00, 30.91, 28.85 and 28.46 and IR(KBr, ꢁ, cmꢀ1): 3319,
3186, 1620 and 1506. Anal. calcd for C25H29N5O3S: C, 62.61; H,
6.09; N, 14.60 and S, 6.69. Found: C, 62.02; H, 6.27; N, 13.92 and
S, 6.18.
Compound 2 (3.24 mmol) and SnCl2 (16.22 mmol) in ethanol
(30 mL) were stirred under reflux for 4 h. When the reaction was
completed, the solvent was evaporated under reduced pressure
and the reaction mixture was diluted with water (15 mL), adjusted
to pH ¼ 10 with 1 M sodium hydroxide solution and extracted
with ethyl acetate (3 ꢂ 25 mL2). The organic phase was dried over
MgSO4 and filtered and purified.
4-(4-Aminophenyl)-1,3,7,7-tetramethyl-3,4,7,8-tetrahydroqui-
nazoline-2,5(1H,6H)-dione (3): recrystallized from ether to give
1
yellow crystals. Yield 75%, m.p. 180.6 ꢁC; H-NMR (300 MHz,
CDCl3, ꢂ, ppm): 7.37(2H,d), 6.98(2H,d), 5.38(H,s), 4.47 (2H,s),
3.25(3H,s), 2.97(3H,s), 2.40(2H,d,d), 2.20(2H,d,d), 1.10(3H,s)
and 1.00(3H,s); 13C-NMR(75 MHz, CDCl3, ꢂ, ppm): 194.04,
153.43, 153.05, 128.28, 127.84, 127.35, 123.40, 118.87, 110.01,
58.42, 49.59, 40.31, 35.14, 33.03, 31.00 and 28.93 and IR (KBr, ꢁ,
cmꢀ1): 3022, 2962, 2870, 1628 and 1582.
General procedure 3: synthesis of 1,4-DHPM substituted urea
and thiourea derivatives (4a–i)
A solution of 4-(4-aminophenyl)-1,3,7,7-tetramethyl-3,4,7,8-tet-
rahydroquinazoline-2,5(1H,6H)-dione (3) (0.644 mmol) and iso-
cyanate or isothiocyanate derivatives (0.704 mmol) in toluene
(20 mL) were stirred at 60 ꢁC for 20 h. When the reaction was
completed, the solvent was evaporated under reduced pressure
and recrystallized from ether.
1-(4-(1,3,7,7-Tetramethyl-5-oxo-1,2,3,4,5,6,7,8-octahydroqui-
nazolin-4-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)thiourea (4f):
yield: 80%, m.p.: 210.1 ꢁC; 1H-NMR (DMSO-d6; 300 MHz):
10.01(H,s), 10.08(H,s), 7.85(H,s), 7.70(H,d), 7.52(H,t), 7.45(H,d),
7.38(2H,d), 7.20(2H,d), 5.20(H,s),3.20(3H,s), 2.80(3H,s),
2.60(2H,d,d), 2.10(2H,d,d) and 1.10(3H,s);
13C-NMR
1-Phenyl-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,5,6,7,8-octa-
hydroquinazolin-4-yl)phenyl)urea (4a): yield: 82%, m.p:
150.6 ꢁC; 1H-NMR(CDCl3-d1; 300 MHz): 7.90(H,s), 7.60(H,s),
7.45(2H,d), 7.40(H,d), 7.38(H,t), 7.35(2H,d), 7.30(H,d), 7.25
(H,t), 7.10(H,t), 5.25(H,s), 3.30(3H,s), 2.80(3H,s), 2.40(2H,d,d),
2.20(2H,d,d), 1.10(3H,s) and 0.98(3H,s); 13C-NMR(CDCl3-d1;
75MHz): 195.53, 153.75, 153.49, 153.40, 139.14, 138.922,
134.99, 129.25, 127.32, 123.18, 120.33, 119.57, 110.91, 58.59,
49.78, 40.38, 34.90, 33.18, 31.13, 28.87 and 28.61 and IR(KBr, ꢁ,
cmꢀ1): 3340, 2956, 1662, 1595. Anal. calcd for C25H30N4O2:
C, 71.74; H, 7.22 and N, 13.39. Found: C, 71.62; H, 7.47 and
N, 13.72.
1-(4-Methoxyphenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,
5,6,7,8-octahydroquina zolin-4-yl)phenyl)urea (4b): yield: 85%,
m.p.: 134.2 ꢁC; 1H-NMR(CDCl3-d1) (300 MHz): 8.01(H,s),
7.62(H,s), 7.20(2H,d), 7.15(2H,d), 6.85(2H,d), 6.70(2H,d),
5.22(H,s), 3.80(3H,s), 3.30(3H,s), 2.80(3H,s), 2.40(2H,d,d),
2.20(2H,d,d), 1.10(3H,s) and 0.98(3H,s); 13C-NMR(CDCl3-d1;
75 MHz): 195.49, 160.47, 153.73, 153.44, 153.41, 140.44, 139.99,
138.82, 135.10, 129.87, 127.32, 120.09, 110.92, 58.58, 55.45,
49.76, 40.37, 34.87, 33.14, 31.08, 28.84 and 28.58 and IR(KBr, ꢁ,
cmꢀ1): 3307, 2954, 1595, and 1539. Anal. calcd for C26H32N4O3:
C, 66.62; H, 7.19 and N, 12.49. Found: C, 67.12; H, 7.57 and
N, 12.82.
1-(4-Fluorophenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,5,6,
7,8-octahydroquinazolin-4-yl)phenyl)urea (4c): yield: 90%, m.p.:
164.7 ꢁC; 1H-NMR(CDCl3-d1; 300 MHz): 7.80(H,s), 7.42(H,s),
7.35(2H,d), 7.30(2H,d), 7.10(2H,d), 6.92(2H,d), 5.20(H,s),
3.20(3H,s), 2.80(3H,s), 2.42(2H,d,d), 2.20(2H,d,d), 1.10(3H,s)
and 0.98(3H,s); 13C-NMR(CDCl3-d1; 75 MHz): 195.49, 165.47,
154.73, 153.44, 153.41, 145.44, 140.99, 138.82, 137.10,
129.87, 127.32, 122.09, 110.92, 58.58, 55.45, 49.76, 40.37,
34.87, 33.14, 31.08, 28.84 and 28.58 and IR(KBr, ꢁ, cmꢀ1):
3340, 2960, 1602 and 1504. Anal. calcd for C25H29FN4O2:
C, 68.79; H, 6.70 and N, 12.83. Found: C, 68.62; H, 6.37 and
N, 12.52.
(DMSO-d6; 75 MHz): 194.54, 183.03, 154.54, 153.15, 142.04,
140.50, 138.49, 129.99, 128.26, 124.99, 124.83, 124.75,
110.06, 65.69, 58.09, 49.71, 34.69, 33.02, 30.89, 28.85
and 28.58 and IR(KBr, ꢁ, cmꢀ1): 3320, 3170, 1615 and
1555. Anal. calcd for C26H29F3N4OS: C, 62.13: H, 5.82;
N, 11.15 and S, 6.38. Found: C, 62.52; H, 6.23; N, 12.02
and S, 6.78.
1-(3-Methoxyphenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,
5,6,7,8-octahydroquina zolin-4-yl)phenyl)thiourea (4g): yield:
82%, m.p.: 210.2 ꢁC; 1H-NMR (DMSO-d6; 300 MHz): 9.78
(H,s), 9.81(H,s), 7.40(2H,d), 7.30(H,s), 7.28(H,t), 7.20(2H,d),
6.98(H,d), 6.82(H,d), 5.20(H,s), 3.20(3H,s), 2.80(3H,s), 2.42
(2H,d,d), 2.20(2H,d,d), 1.10(3H,s) and 0.98(3H,s); 13C-NMR
(DMSO-d6; 75 MHz): 193.53, 179.83, 159.91, 153.52,
153.17, 141.14, 139.48, 137.56, 129.75, 127.24, 124.11, 116.12,
110.08, 58.11, 55.65, 49.73, 34.69, 33.01, 30.88, 28.87 and
28.55 and IR(KBr, ꢁ, cmꢀ1): 3161, 2960, 1660 and
1635. Anal. calcd for C26H32N4O2S: C, 67.21; H, 6.94;
N, 12.06 and S, 6.90. Found: C, 67.62; H, 7.27; N, 12.52
and S, 7.45.
1-(2,4-Dichlorophenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,
5,6,7,8-octahydroquinazolin-4-yl)phenyl)thiourea (4h): yield:
78%, m.p.: 210.9 ꢁC; 1H-NMR (DMSO-d6; 300 MHz): 10.01
(H,s), 10.10(H,s), 7.62(H,s), 7.42(H,s), 7.40(2H,d), 7.33(H,s),
7.20(2H,d), 5.20(H,s), 3.20(3H,s), 2.80(3H,s), 2.42(2H,d,d),
2.20(2H,d,d), 1.10(3H,s) and 0.98(3H,s); 13C-NMR(DMSO-d6;
75 MHz): 193.65, 180.11, 153.89, 153.21, 142.81, 139.03, 138.17,
134.11, 129.59, 128.89, 127.53, 124.44, 123.94, 122.12, 109.94,
58.01, 49.71, 34.72, 33.02, 30.91, 28.84 and 28.49 and IR(KBr,
ꢁ, cmꢀ1): 3188, 2960, 1660 and 1635. Anal. calcd for
C25H28Cl2N4OS: C, 59.64; H, 5.61; N, 11.13 and S, 6.37.
Found: C, 59.34; H, 5.67; N, 11.52 and S, 5.98.
1-(3-Fluorophenyl)-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,5,6,
7,8-octahydroquinazolin-4-yl)phenyl)thiourea (4i): yield: 80%,
m.p.: 184.2 ꢁC; 1H-NMR (DMSO-d6; 300 MHz): 10.01(H,s),
9.98(H,s), 7.52(H,d), 7.40(2H,d), 7.32(H,t), 7.22(2H,d), 7.20
(H,s), 6.98(H,d), 5.20(H,s), 3.20(3H,s), 2.80(3H,s), 2.42(2H,d,d),
2.20(2H,d,d) and 1.10(3H,s); 13C-NMR(DMSO-d6; 75 MHz):
193.66, 180.00, 164.04, 160.84, 153.86, 153.21, 141.91, 139.30,
1-Phenyl-3-(4-(1,3,7,7-tetramethyl-5-oxo-1,2,3,4,5,6,7,8-octa-
hydroquinazolin-4-yl)-phenyl)thiourea (4d): yield: 78%, m.p.:
1
211.7 ꢁC, H-NMR (DMSO-d6; 300 MHz): 9.76(H,s), 9.73(H,s),

DOI: 10.3109/14756366.2012.746972
1,4-Dihydropyrimidinone substituted diarylureas 21
Table 1. The IC50 (mM) values of compounds (4a–i).
137.86, 130.57, 127.41, 124.32, 119.57, 110.41, 58.01, 52.95,
49.69, 34.71, 33.01, 30.91, 28.84 and 28.48 and IR(KBr, ꢁ,
cmꢀ1): 3329, 2960, 1670 and 1591. Anal. calcd for
C25H29FN4OS: C, 66.34; H, 6.46; N, 12.38 and S, 7.08. Found:
C, 66.65; H, 6.67; N, 12.82 and S, 7.30.
Compounds
hCA I
hCA II
4a
4b
4c
4d
4e
4f
4g
4h
4i
135.26
153.15
66.23
136.11
197.70
105.86
164.13
132.19
130.30
153.01
156.96
142.11
88.94
155.28
63.09
169.71
151.09
89.21
Preparation of haemolysate and purification from red
blood cells
Blood samples (25 mL) were taken from healthy human
volunteers. They were anticoagulated with acid-citrate-dextrose,
centrifuged at 2000 g for 20 min at 4 ꢁC and the supernatant was
removed. The packed erythrocytes were washed three times with
0.9% NaCl and then haemolysed in cold water. The ghosts and any
intact cells were removed by centrifugation at 2000 g for 25 min at
4 ꢁC and the pH of the haemolysate was adjusted to pH 8.5 with
solid Tris-base. The 25 mL haemolysate was applied to an affinity
column containing L-tyrosine-sulphonamide-sepharose-4B20
equilibrated with 25 mM Tris–HCl/0.1 M Na2SO4 (pH 8.5). The
affinity gel was washed with 50 mL of 25 mM Tris–HCl/22 mM
Na2SO4 (pH 8.5). The hCA isozymes were then eluted with 0.1 M
NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M
NaClO4 (pH 5.6), which recovered hCA I and II, respectively.
Fractions of 3 mL were collected and their absorbance measured
at 280 nm.
The results showed that all the compounds (4a–i) inhibited
enzyme activity. The inhibition constants of the synthesized
compounds against CAs are given in Table 1. The following
structure–activity relationship observations can be drawn from
the data.
(i) The slow cytosolic isoform hCA I was weakly inhibited by
the 1,4-DHPM substituted diaryl urea and thiourea deriva-
tives with inhibition constants in the range
66.23–197.70 mM. The best hCA I inhibitor among the
newly synthesized and investigated compounds was 4-flouro
substituted derivative (4c). Fluorinated molecule can bind to
a hydrolytic enzyme as a covalent adduct with an active site
nucleophile, effectively binding as
a transition state
CA enzyme assay
analogue26. It is obviously clear that bulky groups (such as
nitro and methoxy) on the phenyl ring affect inhibition due
to steric effect and IC50 values are higher than 150 mM.
(ii) The second off target isoform, hCA II, which is in fact the
physiologically dominant cytosolic isozyme, was also
weakly inhibited by all the compounds, with inhibition
constants in the region of 63.09–169.71 mM. The best hCA
II inhibitor among the newly synthesized and investigated
compounds was 4-(triflouro substituted) phenyl derivative
(4f). From the values given in Table 1, flouro substituted
urea derivatives showed more inhibitory effect than nitro,
chloro, methoxy and unsubstituted ureas. Flourophenyl
sulphamate adducts were reported that the sulphomates
possess a rather variable binding pattern within the hCA II
CA activity was measured by the Maren method which is based
on determination of the time required for the pH to decrease from
10.0 to 7.4 due to CO2 hydration21. The assay solution was 0.5 M
Na2CO3/0.1 M NaHCO3 (pH 10.0) and phenol red was added as
the pH indicator. CO2-hydratase activity (enzyme units) was
calculated using the equation t0ꢀtc/tc where t0 and tc are the times
for pH change of the nonenzymatic and the enzymatic reactions,
respectively.
In vitro inhibition studies
For the inhibition studies of sulphanilamide, different concentra-
tions of these compounds were added to the enzyme. Activity
percentage values of CA for different concentrations of each
sulphanilamide were determined by regression analysis using
Microsoft Office 2000 Excel (New York, NY). CA enzyme
activity without a synthesized compounds solution was accepted
as 100% activity.
active site26,27
.
The clinically used sulphonamides are low micromolar to low
nanomolar inhibitors, whereas the compounds in this study are
millimolar CAIs. For hCA II, the clinical used sulphonamides are
stronger inhibitory effect than the synthesized compounds7.
Enzyme inhibition studies are important issue for drug design
and biochemical applications28–39. The results showed that 1,4-
dihydropyrimidine substituted urea and thiourea derivatives
inhibited the hCA I and II enzyme activities. The compounds
have weak inhibitory effects and may be taken for further
evaluation in vivo studies.
Results and discussion
For evaluation, the physiologically relevant human CA isozyme
hCA I and II inhibitory activity, several new diaryl urea and
thiourea compounds were subjected to CA inhibition assay with
CO2 as substrate.
Sulphonamides are coordinated to the zinc (II) ion within the
hCA active site, whereas its organic scaffold fills the entire
enzyme cavity, making an extensive series of van der Waals and
polar interactions with amino acid residues both at the bottom,
middle and entrance of the active site cavity22. Coumarins/
thiocoumarins may possess various tautomeric forms, such as the
zwitterionic benzo(thio)pyrylium phenoxides, which may bind
within the CA active site similarly to phenols, i.e. by enchoring to
the zinc-bound water molecule/hydroxide ion23. Coumarins
cannot bind enzyme effectively in the restricted space near
Zn2þ ion due to its bulky pendant group and exhibit unusual
binding mode not interacting with the metal ion of the
enzyme24,25. The synthesized compounds are very bulky to be
able to bind near the zinc ion and they much more probably bind
as the coumarins and activators.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
This study was supported by the Research Fund of the Sakarya
University (project no.: 2012-02-04-033).
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