Synthesis of N-alkyl (aril)-tetra pyrimidine thiones and investigation of their human carbonic anhydrase I and II inhibitory effects

Article abstract of DOI:10.3109/14756366.2015.1113172

Tetrahydropyrimidine thiones, which are cyclic thiocarbamides derivatives, were synthesised from thiourea, β-diketones and substituted benzaldehydes. A tautomeric form of these derivatives incorporates the thiol functionality, which is known to interact w

Full text of DOI:10.3109/14756366.2015.1113172

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of N-alkyl (aril)-tetra pyrimidine thiones
and investigation of their human carbonic
anhydrase I and II inhibitory effects
Afsun Sujayev, Leyla Polat Kose, Emin Garibov, İlhami Gülçin, Vagif Farzaliev,
Saleh H. Alwasel & Claudiu T. Supuran
To cite this article: Afsun Sujayev, Leyla Polat Kose, Emin Garibov, İlhami Gülçin, Vagif Farzaliev,
Saleh H. Alwasel & Claudiu T. Supuran (2015): Synthesis of N-alkyl (aril)-tetra pyrimidine thiones
and investigation of their human carbonic anhydrase I and II inhibitory effects, Journal of
Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1113172
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1113172
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RESEARCH ARTICLE
Synthesis of N-alkyl (aril)-tetra pyrimidine thiones and investigation of
their human carbonic anhydrase I and II inhibitory effects
1
2
1
Afsun Sujayev , Leyla Polat Kose , Emin Garibov , Ilhami Gu¨lc¸in2,3, Vagif Farzaliev1, Saleh H. Alwasel3, and
_
Claudiu T. Supuran4,5
1Laboratory of Theoretical Bases of Synthesis and Action Mechanism of Additives, Institute of Chemistry of Additives, Azerbaijan National Academy
of Sciences, Baku, Azerbaijan, 2Department of Chemistry, Faculty of Sciences, Ataturk University, Erzurum, Turkey, 3Department of Zoology, College
4
of Science, King Saud University, Riyadh, Saudi Arabia, Dipartimento Di Chimica Ugo Schiff, Universita Degli Studi Di Firenze, Sesto Fiorentino
`
5
(Firenze), Italy, and Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Universita Degli Studi Di Firenze, Sesto
`
Fiorentino (Florence), Italy
Abstract
Keywords
Tetrahydropyrimidine thiones, which are cyclic thiocarbamides derivatives, were synthesised
from thiourea, b-diketones and substituted benzaldehydes. A tautomeric form of these
derivatives incorporates the thiol functionality, which is known to interact with metal ions from
metalloenzymes active sites, such as the carbonic anhydrases (CAs, EC 4.2.1.1) among others.
This is a superfamily of widespread enzymes, which catalyses a crucial biochemical reaction, the
reversible hydration of carbon dioxide to bicarbonate and protons (H+). The newly synthesised
N-alkyl (aril)-tetrahydropyrimidine thiones were tested for inhibition of the cytosolic human
isoforms I and II (hCA I and II). Both isoforms were effectively inhibited by the newly synthesised
thiones. Ki values were in the range of 218.5 23.9–261.0 41.5 pM for hCA I, and of
181.8 41.9–273.6 41.4 pM for hCA II, respectively. This under-investigated class of derivatives
may bring interesting insights in the field of non-sulphonamide CA inhibitors.
Carbonic anhydrase, enzyme inhibition,
pyrimidine, synthesis
History
Received 12 October 2015
Revised 22 October 2015
Accepted 23 October 2015
Published online 19 November 2015
Introduction
Indeed, the carbonic anhydrase (CA, EC. 4.2.1.1) is a
metalloenzyme that catalyses the hydration of carbon dioxide
(CO2) and the corresponding dehydration of bicarbonate
(HCOꢀ3 )6–10. This is a rather simple but crucial process, as the
products of this reaction are involved in fundamental physiological
processes, including pH and CO2 homeostasis, respiration, trans-
port of CO2/HCOꢀ3 between metabolising tissues and the lungs,
electrolyte secretion, gluconeogenesis, ureagenesis, lipogenesis,
bone calcification, tumorigenicity and some other pathologic and
physiologic processes11–16. There are six distinct and unrelated CA
classes, i.e., the a-, b-, g-, d-, z- and Z-CAs, which are involved in
crucial physiologic and pathologic processes in organisms all over
the phylogenetic tree17–21. All of them are metalloenzymes,
whereas a-, b-, d- and Z-CAs use Zn2+ ions at the active site,
the g-CAs are probably Fe2+ enzymes, but they are active also with
bound Zn2+ or Co2+ ions. On the other hand, the z-class uses Cd2+
The search of physiologically active compounds with various
applications expanded exponentially in the last period in the
search of drugs with better efficacy and less side effects1–4. The
pyrimidine-thiones possess relevant pharmacological activity, and
the development of optimal synthetic methods for some of their
classes is of interest5. The tetrahydropyrimidine thiones belong to
the cyclic thiocarbamide class, and their carboxylic acid deriva-
tives were poorly investigated to date. This is the reason why we
underwent the exploration of some synthetic strategies for a small
panel of such compounds. We report here the syntheses of allyl
6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carb-
oxylate (1), 2-(methacryloyloxy)ethyl 6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (2), allyl 4-(4-
(dimethylamino)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (3), 1-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)ethanone (4) and ethyl 6-ethoxy-2-
thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5).
Our interest in this type of heterocyclic compounds is motivated
by the fact that a tautomeric form of these thiones incorporates a
thiol functionality and heterocyclic thiols are known to act as
metalloenzyme inhibitors, binding to the metal centre of such
proteins.
or Zn2+ to perform the physiologic reaction catalysis22–26
.
To date, 16 different a-CA isoforms were discovered in
vertebrates. a-CAs are among the most effective catalysts known
in nature. The a-CAs are present in vertebrates, algae, protozoa,
and cytoplasm compartment of green plants, as well as in some
bacteria27–32. In humans, a-CAs are diffused in different tissues
including the blood, kidneys, gastrointestinal tract, reproductive
tract, the nervous central system, eyes, skin, lungs, etc.33–37. Two
cytosolic human CA isoforms (hCA I, and II) are widespread
throughout the human body and are drug targets for clinically
used diuretics, anticonvulsants and antiglaucoma drugs38–42. Five
of the vertebrate CA isoforms are cytosolic (CA I, II, III, VII and
_
Address for correspondence: Prof. Dr. Ilhami Gu¨lc¸in, Department of
Chemistry, Faculty of Science, Ataturk University, 25240- Erzurum,
Turkey. E-mail: igulcin@atauni.edu.tr; gulcin@ksu.edu.sa

2
A. Sujayev et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
XIII), some others (CA IV, IX, XII, XIV and XV) are membrane acetoacetate (1.37 mL, 0.01 mol) is added to it drop by drop.
bound, two isoenzymes (CA VA and VB) are mitochondrial, and After being dissolved in magnetic stirrer for 5 minutes, 4 -
one isoform (CA VI) is secreted in saliva43–48. It was recently (dimethylamino) benzaldehyde (1.49 mL, 0.01 mol) is added.
reported that CA XV isoform is not expressed in humans or in Upon being dissolved in the stirring rod within 30 minutes, TFAA
living primates. However, it is found in rodents and other higher catalyst is inserted. After determining that the reaction has been
vertebrates. Three acatalytic forms are known and called CA- fully completed, the solvent is evaporated. It is distilled in ꢀa1n
related proteins (CARP)49–51
.
ethyl alcohol solution. The yield is 1.8 g, m.p. 208 ꢁC. IR ꢀ, sm
:
The dimeric transmembrane glycoproteins hCA IX and XII are 1618, 1560, 1526 (Ar), 3112 (CH2 ¼ C), 785 (Ar-CH), 1722,
also isoforms having an extracellular active site and are markers 1701 (C(O)R), 1377, 1370 (CH3), 1651 (C¼C), 3242 (NH), 1314
for a broad spectrum of hypoxic tumors52–54. The overexpression (C–N).
of these isoforms contributes to the increased acidification of
extracellular hypoxic environment in solid tumours (pH: 6.8) and Synthesis of 1–(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydro-
normal tissues (pH: 7.4), thus promoting tumour cell survival in pyrimidin-5-yl)ethanone (4)
an acidic condition by decreasing uptake of weakly basic
Thiourea (1.52 g, 0.02 mol) is dissolved in 3:1 ratio of
anticancer drugs55. They also help tumour growth by supplying
acetylacetone (3 mL) and ethyl alcohol (1 mL), and acetylacetone
HCOꢀ3 to be used as substrate for cell growth as this anion is
(2.05 mL, 0.02 mol) is added to it drop by drop. After being
required in the synthesis of pyrimidine nucleotides such as
dissolved in magnetic stirrer for 5 minutes, benzaldehyde
cytosine, thymine, and uracil56. Thus, specifically targeting the
(2.03 mL, 0.02 mol) is added. Upon being dissolved in the stirring
tumour-related isozymes (hCA IX and XII) over the main off
rod within 30 minutes, TFAA catalyst is inserted. After
target isoenzymes hCA I, and II may be a new antitumor
determining that the reaction has been fully completed, the
approach, with a sulphonamide CA inhibitor in Phase I clinical
solvent is evaporated. It is distilled in an ethyl alcohol solution.
trials for the treatment of hypoxic tumors57–59. However, CA II is
The yield is 2.5 g, m.p. 203 ꢁC. IR ꢀ, smꢀ1: 1810–1951, 3000–
also a drug target for antiglaucoma agents, diuretics and high-
3100 (Ar), 1493 (Ar C–H), 1614 (Ar C–C), 703 (CH), 1675, 1703
altitude sickness among others60–62. The precise physiological
(C¼O), 3257 (NH), 1236 (C¼S).
role of CA I, a highly abundant protein in the blood and
gastrointestinal tract, is not well understood62.
Synthesis of ethyl 6-ethoxy-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylate (5)
Experimental
Thiourea (2.58 g, 0.0339 mol) is dissolved in 3:1 ratio of
acetylacetone (3 mL) and ethyl alcohol (1 mL), and diethyl
Chemistry
Synthesis of allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylate (1)
malonate (5.15 mL, 0.0339 mol) is added to it drop by drop.
After being dissolved in magnetic stirrer for 5 minutes, p-
tolualdehyde (4 mL, 0.0339 mol) is added to it. Upon being
dissolved in the stirring rod within 30 minutes, TFAA catalyst is
inserted. After determining that the reaction has been fully
completed, the solvent is evaporated. It is distilled in an ethyl
alcohol solution. The yield is 3.2 g. White crystalline with melting
temperature of 196 ꢁC is formed. IR ꢀ, smꢀ1: 1605 (Ar), 1743,
1710 (C(O)R), 3315–3463 (NH), 1236 (C¼S).
Thiourea (1.52 g, 0.02mol) is dissolved in 3:1 ratio of acetylace-
tone (3mL) and ethyl alcohol (1mL), and allyl acetoacetate
(2.74mL, 0.02 mol) is added to it drop by drop. After being
dissolved in magnetic stirrer for 5 minutes, tolualdehyde (2.36 mL,
0.02 mol) is added to it. Upon being dissolved in the stirring rod
within 30 minutes, trifluoroacetic acid (TFAA) catalyst is inserted.
The process of reaction is controlled through Sulifol UV-254 plate.
After determining the reaction has been fully completed, the
solvent is evaporated. It is distilled in ethyl alcohol solution. The
CA inhibition
yield is 2.15 g. White crystalline with melting temperature of Both cytosolic hCA isoenzymes were purified by Sepharose-4B-L
205 ꢁC is formed. IR ꢀ, smꢀ1: 3237 (NH), 1223 (C¼S), 786, 1545, tyrosine-sulphanilamide affinity separation technique with a
3107 (Ar-CH), 1651 (C¼C), 1727 1707 (C(O)R).
single purification step7,8. Sepharose-4B-L tyrosine-sulphanila-
mide affinity gel was prepared and used according to the previous
studies47,63. At the outset, the pH of the homogenate was adjusted
to 8.7 using solid Tris(hydroxymethyl)aminomethane. Then, an
aliquot of the supernatant was transferred to a previously prepared
affinity column. Then, the purification process was realised. The
protein flow in the column effluents was determined spectro-
photometrically at 280 nm as previously reported64–66. Both
isoenzymes purities were controlled by sodium dodecyl sul-
phate-polyacrylamide gel electrophoresis (SDS-PAGE) according
to Laemmli’s method67. SDS-PAGE was widely used in bio-
chemistry for separation of biological macromolecule including
proteins and enzymes according to their electrophoretic mobil-
ity29. SDS-PAGE was applied after purification of hCA I, and II
isoenzymes. Also, this method was previously described in the
literature68–72. Briefly, it was performed in acrylamide for the
running (10%) and the stacking gel (3%) containing SDS
(0.1%)73–76. A single band was observed for each hCA isoenzyme.
Both CA isoenzymes activities were determined in accordance
Synthesis of 2-(methacryloyloxy)ethyl 6-methyl-4-phenyl-2-
thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (2)
Thiourea (1.52 g, 0.02 mol) is dissolved in 3:1 ratio of
acetylacetone (3 mL) and ethyl alcohol (1 mL), and (methacry-
loyloxy) ethyl acetoacetate (3.82 mL, 0.02 mol) is added to it drop
by drop. After being dissolved in magnetic stirrer for 5 minutes,
benzaldehyde (2.03 mL, 0.02 mol) is added. After determining
that the reaction has been fully completed, the solvent is
evaporated. Processing of the reaction mixture was carried out
by washing the reaction mixture with ice water. Then the
precipitate was filtered, washed with 500 mL of water, dried and
recrystallized from ethanol (75 mL). The yield is 2.4 g, m.p.
211 ꢁC. IR ꢀ, smꢀ1: 3175 (NH), 1709 (C¼O), 1092 (C¼S), 756,
852, 974, 1230 (CH), 1608 (C¼C).
Synthesis of allyl 4-(4-(dimethylamino)phenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (3)
with the method of Verpoorte et al.77 described previously47,49
.
Thiourea (0.76 g, 0.01 mol) is dissolved in 3:1 ratio of The increasing absorbance at 348 nm of p-nitrophenylacetate
acetylacetone (3 mL) and ethyl alcohol (1 mL), and allyl (NPA) to p-nitrophenolate (NP) was recorded during a short

DOI: 10.3109/14756366.2015.1113172
N-alkyl (aril)-tetra pyrimidine thiones
3
period (3 minutes) at 25 ꢁC using a spectrophotometer (Shimadzu,
Cytosolic hCA I is expressed in the body and can be found in
UV-VIS Spectrophotometer, UVmini-1240). The quantity of high concentrations in the blood and gastrointestinal tract89. The
protein was spectrophotometrically determined at 595 nm during slow cytosolic isoform hCA I was effectively inhibited by newly
the purification steps according to the Bradford method78. Bovine synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) with Ki
serum albumin as a standard protein was used and has been values ranging of 218.5 23.9–312.6 61.1 p.m. (Table 1). On
described previously79–81. To determine the inhibition effect of the other hand, acetazolamide (AZA) is a medium potency hCA I
each N-alkyl (aril)-tetra pyrimidine thiones, an activity (%)–[N- inhibitor demonstrated a Ki of 369.4 68.5 p.m. However, the
alkyl (aril)-tetra pyrimidine thiones] graph was drawn. For most powerful hCA I inhibition was observed by allyl 6-methyl-2-
calculation of Ki values, three different N-alkyl (aril)-tetra thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1)
pyrimidine thiones concentrations were used. In these experi- with Ki value of 218.5 23.9 p.m. Also, AZA is an effective
ments, NPA was used as a substrate at five different concentra- antiglaucoma agent, but has a limited use due to numerous side
tions. Finally, the Lineweaver–Burk curves were drawn82. These effects that arise by inhibition of CAs other than those present in
drawings and calculations are described in detailed in other the eye ciliary processes leading to kidney stones, fatigue, and
works83,84
.
paresthesias86.
hCA II, the dominant physiologic isoform90,91, was potently
inhibited by all newly synthesised N-alkyl (aril)-tetra pyrimidine
thiones (1–5) with Kis in the range of 181.8 41.9–
273.6 41.4 p.m. Also, similar to hCA I, the most powerful
hCA II inhibition property was shown by allyl 6-methyl-2-thioxo-
4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1) with Ki
value of 155.4 25.9 p.m. Also, AZA, which was used as clinical
CA inhibitor, showed a Ki value of 271.8 54.5 p.m. This result
clearly showed that all newly synthesised N-alkyl (aril)-tetra
pyrimidine thiones (1–5) are rather effective inhibitors for the
cytosolic isoform hCA II. It was demonstrated that the CA II
inhibition is due to the ability of an inhibitor to mimic the
Results and discussion
The synthesis of the new compounds is reported in Scheme 1. The
reaction of substituted benzaldehydes, with methylene active
compounds such as b-diketones and thiourea in the presence of
NiCl2.6H2O led to the desired cyclic thioureas 1–5. The three-
component condensation reactions come to an end within 2–4 h at
65–70 ꢁC. The synthesised compounds were crystalline and their
structure was confirmed by spectral and physico-chemical
methods, among which were H and 13C NMR spectroscopies.
1
Inhibitors of carbonic anhydrase enzymes (CAIs) have a large
number of applications in therapy, such as anticancer, antiglau-
coma and anti-osteoporosis agents85. The classical CAIs are the
primary sulphonamides, which are in clinical use for more than 50
years as diuretics and systemically acting antiglaucoma drugs. In
addition to the established role of these CAIs as diuretics and
antiglaucoma agents, it has recently emerged that they have
potential as anticonvulsant, antiobesity, anticancer, antipain, and
anti-infective drugs86–88. Many types of CAIs have been reported
recently, together with their potential applications27,28. Some
newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5)
were screened for the inhibition of both human CA isoforms
involved in important physiologic or pathologic processes, i.e. the
cytosolic, hCA I, and II. Table 1 shows inhibition data of newly
synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) reported
here and acetazolamide AZA, which is a standard CA inhibitor
against hCA I and II. The following structure–activity relationship
may be noted regarding the inhibition data of Table 1.
Table 1. Inhibition data of human isoforms hCA I, and II with N-alkyl
(aril)-tetrathiopyrimidines 1–5.
KI (pM)
Compounds
hCA I
hCA II
1
2
3
4
218.5 23.9
261.0 41.5
312.6 61.1
219.8 30.8
231.0 44.3
369.4 68.5
155.4 25.9
190.2 32.8
273.6 41.4
181.8 41.9
197.3 30.6
271.8 54.5
5
AZA*
*Acetazolamide (AZA) was used as a standard inhibitor for all both CA
isoenzymes and demonstrated as nanomolar (nM) inhibition.
R
3
O
H
S
O
O
O
+
+
H N
NH
R
R
1
2
2
1
2H 0
2
NH
R
2
R
3
N
S
R
1
H
Compounds
1
R
R
R
3
1
2
CH
2
CH
CH
3
3
3
O
O
O
O
CH
2
3
CH
H
O
CH
2
CH
2
3
4
5
CH
CH
N(CH )
3 2
3
3
CH
H
H
3
OC H
OC H
2 5
2
5
Scheme 1. The synthesis route of new N-alkyl (aril)-tetra pyrimidine thiones (1–5).

4
A. Sujayev et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
tetrahedral transition state when binding to the catalytic Zn2+
located in the active site29.
14. Hisar O, Beydemir S, Gulcin I, et al. Effect of low molecular weight
plasma inhibitors of rainbow trout (Oncorhyncytes mykiss) on
human erythrocytes carbonic anhydrase-II isozyme activity in vitro
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35–9.
Conclusion
_
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erythrocyte carbonic anhydrase in vitro and in vivo. Biol Pharm Bull
This study clearly showed that the newly synthesised N-alkyl
(aril)-tetrapyrimidinethiones (1–5) possess effective inhibition
profiles against both CA I and II isoenzymes. Picomolar Ki values
were observed for all compounds against these two isoforms
(218.5 23.9–312.6 61.1 pM against hCA I, and 181.8 41.9–
273.6 41.4 pM against hCA II, respectively). These findings
signify that these derivatives may be used as leads for generating
interesting CAIs. The allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1) demonstrated the most
effective inhibition profile against hCA I and II isoenzymes.
2007;30:2257–61.
16. C¸ oban TA, Beydemir S, Gu¨lc¸in I, Ekinci D. The effect of ethanol on
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Bioorg Med Chem 2015;23:2598–605.
18. Vullo D, Isik S, Bozdag M, et al. 7-Amino-3,4-dihydro-1H-quinolin-
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19. Goc¸er H, Akıncıoglu A, Oztas¸kın N, et al. Synthesis, antioxidant,
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¨
˘
¨
Acknowledgements
IG and SHA would like to extend his sincere appreciation to the Research
Chairs Program at King Saud University for funding this research.
_
20. Gu¨l HI, Kucukoglu K, Yamali C, et al. Synthesis of 4-(2-
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Declaration of interest
The authors have declared no conflict of interest.
_
˘
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