Carbonic anhydrase inhibitory properties of some uracil derivatives

Article abstract of DOI:10.1080/14756366.2016.1235043

Inhibitors of carbonic anhydrase (CA) have been carried out in many therapeutic applications, especially antiglaucoma activity. In this study, we investigated some uracil derivatives (4–12) to inhibit human CA I (hCA I) and II (hCA II) isoenzymes. The K_I values of the compounds 4–12 are in the range of 0.085–428 μM for hCA I and of 0.1715–645 μM against hCA II, respectively. It is concluded from the kinetic investigations, all compounds used in the study act as competitive inhibitors with substrate, 4-NPA. Uracil derivatives are emerging agents for the inhibiton of carbonic anhydrase which could be used in biomedicine.

Full text of DOI:10.1080/14756366.2016.1235043

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Carbonic anhydrase inhibitory properties of some
uracil derivatives
Emir Alper Türkoğlu, Murat Şentürk, Claudiu T. Supuran & Deniz Ekinci
To cite this article: Emir Alper Türkoğlu, Murat Şentürk, Claudiu T. Supuran & Deniz Ekinci
(2017) Carbonic anhydrase inhibitory properties of some uracil derivatives, Journal of Enzyme
Inhibition and Medicinal Chemistry, 32:1, 74-77, DOI: 10.1080/14756366.2016.1235043
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1235043
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Date: 19 January 2017, At: 17:15

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 74–77
http://dx.doi.org/10.1080/14756366.2016.1235043
RESEARCH ARTICLE
Carbonic anhydrase inhibitory properties of some uracil derivatives
a
b
c
d
€
ꢀ
€
Emir Alper Turkoglu , Murat S¸enturk , Claudiu T. Supuran and Deniz Ekinci
b
aDepartment of Pharmaceutical Technology, Agri Ibrahim Cecen University, Agri, Turkey; Department of Chemistry, Agri Ibrahim Cecen
c
ꢁ
University, Agri, Turkey; Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Florence, Italy;
dDepartment of Agricultural Biotechnology, Ondokuz Mayis University, Samsun, Turkey
ABSTRACT
ARTICLE HISTORY
Received 15 August 2016
Revised 7 September 2016
Accepted 7 September 2016
Inhibitors of carbonic anhydrase (CA) have been carried out in many therapeutic applications, especially
antiglaucoma activity. In this study, we investigated some uracil derivatives (4–12) to inhibit human CA I
(hCA I) and II (hCA II) isoenzymes. The KI values of the compounds 4–12 are in the range of 0.085–428 mM
for hCA I and of 0.1715–645 mM against hCA II, respectively. It is concluded from the kinetic investigations,
all compounds used in the study act as competitive inhibitors with substrate, 4-NPA. Uracil derivatives are
emerging agents for the inhibiton of carbonic anhydrase which could be used in biomedicine.
KEYWORDS
Carbonic anhydrase;
hydroxyl; inhibitor; uracil
derivatives
Introduction
benzenesulfonamides15 and phenoles16. And uracil derivatives
have also been carried out to inhibit the catalytic activity of CAs17.
Uracil, one of the pyrimidine bases, is commonly present in ribo-
nucleic acid (RNA)1. It is chemically weak acid and exposed to two
tautomeric forms at pH 7.0. This amide-imidic acid tautomeric
shifts from lactam which is the amide tautomer to the imidic acid
tautomer is referred to as the lactim structure (Figure 1)2. Uracils
are used in the area of drug discovery due to their bioactivities,
drug similarity in the perspectives of synthetic accessibility and
ability. And it is reported that uracils show anti-viral and anti-
tumour characteristics in addition to bactericidal, herbicidal and
insecticidal features3.
Experimental
Orotic acid (9), isoorotic acid (10), 6-Amino 1,3-Dimethyluracil
(11), 5,6-Diamino 1,3-Dimethyluracil (12), and other chemicals
were obtained commercially from Sigma-Aldrich.
Purification of human carbonic anhydrase isozymes by affinity
chromatography
5-Fluorouracil (5-FU) (1) is an anti-tumour agent used for the
treatment of cancerous tumours in colon or/and breast4. Though
5-FU as a single agent has gained medical achievement, the mol-
ecule has been chemically modified with different processes to
create new derivatives. Therefore, modified syntheses can improve
their therapeutic indexes due to their well-known side effects2.
6-Amino-5-chlorouracil (2) and 6-amino-5-bromouracil (3) were the
first thymidine phosphorylase inhibitors to be generated. However,
their relatively less favourable IC50 values did not allow them to
be developed into drug candidates2.
Erythrocytes were purified from fresh human blood obtained from
€
the Blood Centre of the Research Hospital at Ataturk University.
The blood samples were centrifuged at 1500 rpm for 15 min and
the plasma and buffy coat were removed. The red cells were iso-
lated and washed twice with 0.9% NaCl, and hemolysed with 1.5
volumes of ice-cold water. The ghost and intact cells were
removed by centrifugation at 20 000 rpm for 30 min at 4 ꢀC. The
pH of the hemolysate was adjusted to 8.7 with solid Tris18. Firstly,
benzoyl chloride and stirred for four hours at room temperature in
CH2Cl2 cellulose. After the spacer arm cellulose added as a benzyl
group and finally diazotized sulfanilamide clamped to the para
position of benzyl group as ligand. The hemolysate was applied to
the prepared cellulose-benzyl-sulfanylamide affinity column equili-
brated with 25 mM Tris-HCl/0.1 M Na2SO4 (pH 8.7). The affinity gel
was washed with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7). The
human carbonic anhydrase (hCA I and hCA II) isozymes were
eluted with 1 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M
CH3COONa/0.5 M NaClO4 (pH 5.6), respectively. All procedures
were performed at 4 ꢀC16.
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes
involving in numerous vital biochemical/physiological processes5,6
7
based on reversible hydration/dehydration process of CO2/HCO3À
.
The enzyme is found in all living beings of the three domains of
life8 with six evolutionary gene families9. 15 CA isoenzymes
encoded by a-CA gene family described in humans10. Some of
these human CA isoenzymes such as CA I, II, III, VII and XIII are
cytosolic, some forms, CA IV, IX, XII and XIV, are membrane bound,
two ones as CA VA and VB are mitochondrial and CA VI is found
in saliva. The last three forms of human CAs (CA VIII, X and XI) are
determined as noncatalytic5.
CA inhibition
The testing of CA inhibitors is carried out for the treatment of
clinically important cases11. Many chemical ligands have been
Enzyme activity was determined spectrophotometrically by follow-
used to inhibit CAs such as anions12, bischalcones13, coumarins14, ing the change in absorbance at 348 nm of 4-nitrophenylacetate
€
CONTACT Murat S¸enturk
senturkm36@gmail.com; Claudiu T. Supuran
claudiu.supuran@unifi.it
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
75
to 4-nitrophenylate over a period of 3 min at 25 ꢀC19–21. The enzymes (Table 1), using the esterase activity of hCA I and II, with
4-NPA as substrate. Uracil derivatives 4 and 5 were synthesized
from commercially available uracil. For this purpose uracil was
converted to the brom uracil by using 1.1 Eq bromine in DMF at
120 ꢀC17. Condensation of brom uracil 2 with methanesulfonyl
chloride, p-toluenesulfonyl chloride and acetic anhydryde to afford
uracil derivatives 3 which has antitomour activity, 4, and 5 in com-
parable yield35,36, respectively. The other tested compounds 6–15
were purchased from Sigma-Aldrich.
(i) Against the slow cytosolic isozyme hCA I, compounds 4–8,
and 15 behave as weak inhibitors, with Ki values in the range of
316.2–464 mM, similarly to the structurally related compounds 13
and 14 (KIs of 795 and 4003 mM). It is interesting that compounds
9–12 were much better hCA I inhibitors as compared to the corre-
sponding compounds 7 and 9 from which it was derivated
(Figure 2). Kinetic investigations (Lineweaver–Burk plots, data not
shown) indicate that similarly to sulfonamides and inorganic
anions12–16, all the investigated natural compounds act as com-
petitive inhibitors with 4-NPA as substrate.
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 mL22. Inhibitory effects
of compounds 4–12 were compared with phenolic compounds
13–15. Different inhibitor concentrations were used and all com-
pounds 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 inhibitor22–24. The curve-fitting algorithm allowed to
obtain the IC50 values, working at the lowest concentration of sub-
strate of 0.15 mM, from which KI values were calculated25,26. The
catalytic activity of these enzymes was calculated from
Lineweaver–Burk plots, as reported earlier, and represent the
mean from at least three different determinations. The CA I and II
isoenzymes used here were purified from human blood as
described earlier19–24
.
(ii) A better inhibitory activity has been observed with com-
pounds 9–12 investigated here for the inhibition of the rapid cyto-
solic isozyme hCA II (Table 1). Structure-activity relationship (SAR)
is thus quite sharp for this small series of hydroxylic compounds:
compounds 9–12 are effective leads, with two mono or di hidroxy
moieties is already a submicromolar hCA II inhibitor. This effect is
maintained when different groups are present in the meta position
to the phenol OH moiety, such as in resorcinol. The best hCA II
Results and discussion
Some research groups have studied with phenol and several phen-
olic compound types on 12 mammalian CA isoenzymes to show
inhibitory properties of phenols27. We have extended previous
investigations with some phenolic compounds which are globally
used in food and medical industries. These kinds of compounds in
different phenolic structures with antioxidant characteristics are
essential in quenching reactive oxygen species28–29. These type of
compounds show several medical activities such as anticancer,
anticarcinogenic, antimutagenic, antibacterial, antiviral or antiin-
flammatory5. Phenol, phenolic compounds and hydroxybenzoic
acid derivatives are widely put into effect as prodrugs and/or
drugs. Salicylic acid possesses the ability to reduce fevers and ease
aches and pains. To date these medicinal features have been
Table 1. hCA I and II inhibition data some compounds, by an esterase assay
with 4-nitrophenylacetate as substrate.12b
Ã
KI (lM)
Compound
hCA I
428
10.83
57.76
49.51
316.2
0.7325
0.4585
0.085
hCA II
645
28.88
NE
4a
known and used as anti-inflammatory drug27–34
In the present study we have purified CA I and II (hCA I and
hCA II) from human erythrocytes and examined the in vitro inhib-
ition effects of above mentioned uracil derivatives 4–12 on these
.
5a
6a
7a
NE
8a
166.4
1.682
1.432
0.1715
0.2360
7.7
Orotic acid (9)
Isoorotic acid (10)
6-Amino 1,3-Dimethyluracil (11)
O
OH
5,6-Diamino 1,3-Dimethyluracil (12)
0.1035
13b
14b
15b
795
4003
10.2
NH
N
9.9
5.5
N
O
H
N
OH
aFrom Ref. 17. bFrom Ref. 27. NE: No-effect.
a
b
Ã
Mean from at least three determinations. Errors in the range of 1–3% of the
reported value (data not shown).
Figure 1. Tautomeric forms of Uracil: lactam-lactim.
O
O
O
O
O
O
Br
Br
NH
NH
NH
N
NH
HO
NH
N
O
N
O
HOOC
N
H
O
N
O
N
H
O
N
H
O
SO2CH3
SO2PhCH3
4
5
6
7
8
9
O
N
O
O
OH
H2N
H2N
HOOC
N
NH
OH
OH
H2N
N
O
N
O
N
O
H
OH
OH
10
11
12
13
14
15
Figure 2. Structure of tested compounds.

€
ꢀ
E. ALPER TURKOGLU ET AL.
76
inhibitor in this series of derivatives were compound 11 with a KI 10. Le Darz A, Mingot A, Bouazza F, et al. Fluorinated pyrroli-
dines and piperidines incorporating tertiary benzenesulfona-
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J Enzyme Inhib Med Chem 2015;30:737–45.
of 0.1715 mM.
In a recent study it was reported that catechol and resorcinol27
act as a CAI inhibitor, and could represent the starting point for a
new class of inhibitors that may have advantages for patients with
sulfonamide allergies. The sulfonamide zinc-binding group is thus
superior to the thiol one (from the thioxolone hydrolysis product)
for generating CA inhibitors with a varied and sometimes isozyme-
selective inhibition profile against the mammalian enzymes.
However, it is still important to explore further classes of potent
CAIs in order to detect compounds with different inhibition
profiles.
11. Masini E, Carta F, Scozzafava A, Supuran CT. Antiglaucoma
carbonic anhydrase inhibitors: a patent review. Expert Opin
Ther Patents 2013;23:705–16.
12. Orhan F, Senturk M, Supuran CT. Interaction of anions with
a
newly characterized alpha carbonic anhydrase from
Halomonas sp. J Enzyme Inhib Med Chem 2015. [Epub
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1100177.
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Compounds 3–12 used in this study affect the activity of CA
isozymes due to the presence of the different functional groups
(CH3, OH, Br, COOH, NH2, mesityl, and tosyl) present in their scaf-
fold. Therefore, our findings indicate another class of possible CAIs
of interest, in addition to the well-known inhibitors, the phenols/
biphenyl diphenols bearing bulky ortho moieties in their mole-
cules. Some hyroxylic compounds investigated here exhibited
effective hCA I and II inhibitory activity, in the low-micromolar
range, by the esterase method which usually gives KI-s an order of
magnitude higher as compared to the CO2 hydrase assay. These
findings point out that substituted hdroxylic compounds may be
used as leads for generating potent CAIs eventually targeting
other isoforms.
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carbonic anhydrase isoforms I, II and VI with thiamine and
thiamine-like molecules. J Enzyme Inhib Med Chem 2013;28:
316–19.
Disclosure statement
The authors report no declarations of interest
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