Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines and investigation of their acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase I/II inhibitory and antioxidant activities

Article abstract of DOI:10.1080/14756366.2016.1198901

A series of tetrahydropyrimidinethiones were synthesized from thiourea, β-diketones and aromatic aldehydes, such as p-tolualdehyde, p-anisaldehyde, o-tolualdehyde, salicylaldehyde and benzaldehyde. These cyclic thioureas showed good inhibitory action agai

Full text of DOI:10.1080/14756366.2016.1198901

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-
tetrahydropyrimidines and investigation of their
acetylcholinesterase, butyrylcholinesterase,
carbonic anhydrase I/II inhibitory and antioxidant
activities
Emin Garibov, Parham Taslimi, Afsun Sujayev, Zeynebe Bingol, Songul
Çetinkaya, İlhami Gulçin, Sukru Beydemir, Vagif Farzaliyev, Saleh H. Alwasel
& Claudiu T. Supuran
To cite this article: Emin Garibov, Parham Taslimi, Afsun Sujayev, Zeynebe Bingol, Songul
Çetinkaya, İlhami Gulçin, Sukru Beydemir, Vagif Farzaliyev, Saleh H. Alwasel & Claudiu T.
Supuran (2016): Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines and
investigation of their acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase I/II
inhibitory and antioxidant activities, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1198901
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1198901
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1198901
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RESEARCH ARTICLE
Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines
and investigation of their acetylcholinesterase, butyrylcholinesterase,
carbonic anhydrase I/II inhibitory and antioxidant activities
1
2
1
2
2
Emin Garibov , Parham Taslimi , Afsun Sujayev , Zeynebe Bingol , Songul C¸etinkaya , Ilhami Gulc¸in
2,3
_
,
Sukru Beydemir2, Vagif Farzaliyev1, Saleh H. Alwasel2, 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, Firenze, Sesto
`
5
Fiorentino, Italy, and Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Universita Degli Studi Di Firenze, Florence,
`
Sesto Fiorentino, Italy
Abstract
Keywords
A series of tetrahydropyrimidinethiones were synthesized from thiourea, b-diketones and
aromatic aldehydes, such as p-tolualdehyde, p-anisaldehyde, o-tolualdehyde, salicylaldehyde
and benzaldehyde. These cyclic thioureas showed good inhibitory action against acetylcholine
esterase (AChE), butyrylcholine esterase (BChE), and human (h) carbonic anhydrase (CA)
isoforms I and II. AChE and BChE inhibitions were in the range of 6.11–16.13 and 6.76–15.68 nM,
respectively. hCA I and II were effectively inhibited by these compounds, with Ki values in the
range of 47.40–76.06 nM for hCA I, and of 30.63–76.06 nM for hCA II, respectively. The
antioxidant activity of the cyclic thioureas was investigated by using different in vitro
antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPHꢀ) radical scavenging, Cu2+ and
Fe3+ reducing, and Fe2+ chelating activities.
Acetylcholinesterase, antioxidant activity,
butyrylcholinesterase, carbonic anhydrase,
enzyme inhibition
History
Received 19 April 2016
Revised 1 June 2016
Accepted 4 June 2016
Published online 21 June 2016
Introduction
(due to the physiological pH values). Indeed, bicarbonate is a
substrate for several carboxylation enzymes involved in biosyn-
thetic pathways such as biosynthesis of urea, glucose, fatty acids
and possibly nucleotides7–10. The reaction not only provides a
means to control the physiological pH values, but also supplies
HCO3ꢁ ions for various physiological processes or metabolic,
biosynthetic pathways, as those mentioned above11–14. So far, six
unrelated genetic CA families have been discovered, namely, the
a-, b-, g-, d-, z- and Z-CAs. In many organisms, a large number of
isoforms of the CA family are present, which possess specialized
functions in various tissues and organs15–19. These CA families
are found in most living organisms. For example a-CAs are found
in humans and other mammals and divided into four broad
subgroups, which, in turn consist of several isoforms: the
cytosolic CAs (CA I, II, III, VII, and XIII), mitochondrial CAs
(CA VA and VB), secreted CA (CA VI), as well as membrane-
associated CAs (CA IV, IX, XII, XIV and XV)20–24. There are
three additional, acatalytic CA isoforms (CA VIII, X, and XI)
whose functions remain unclear. On the other hand, it was
reported that b-CAs exist in most prokaryotes and plant chloro-
plasts, g-CAs are present in methanogens and most bacteria.
d-CAs and z-CAs were found in diatoms25. Recently, the Z-CA
family was identified in organisms of the genus Plasmodium.
These enzymes previously thought to belong to the a-CA family,
were shown to possess unique features such as their metal ion
coordination pattern26–29, which motivates their designation as a
new genetic family, i.e., the Z-CAs. As such, CA inhibitors (CAIs)
Recently, the synthesis of some pyrimidine thiones derivatives
attracted great attention due to their biological and pharmaceut-
ical properties including analgesic, anti-staphylococcal, antiviral,
anti-tuberculosis, anti-fungal, and efficacy against some cardio-
vascular diseases1. A facile synthesis of these compounds
includes Biginelli reaction, when aliphatic/aromatic aldehydes,
urea/thiourea, acetylacetone, and various catalysts lead to the ring
closure with the formation of the desired heterocycle. One major
factor influencing the outcome and yields in the desired products
is the selection of the catalyst for Biginelli’s reaction.2 Synthesis
of 3,4-dihydropyrimidine-2(1H) thiones in a simple and useful
way is based on this one-pot, three-component condensation of
aldehydes, methylene active compounds and thiourea in acidic
media.1,2
The reversible hydration of carbon dioxide (CO2) to form
bicarbonate (HCO3ꢁ) and protons (H+) is a simple but very
important chemical reaction that can be performed by various
classes of carbonic anhydrases (CAs, EC 4.2.1.1).3–6 The
ꢁ
interconversion of CO2 and HCO3 is spontaneously balanced
to maintain the equilibrium between dissolved inorganic CO2, the
highly unstable carbonic acid (H2CO3) and carbonate (CO32ꢁ) of
ꢁ
which HCO3 is physiologically the most important species
_
Address for correspondence: Prof. Dr. Ilhami Gu¨lc¸in, Department of
Chemistry, Faculty of Science, Atatu¨rk University, 25240- Erzurum,
Turkey. E-mail: igulcin@atauni.edu.tr

2
E. Garibov et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
have a role as pharmaceutical agents in the treatment of various anhydrase inhibitor and antioxidant agents. Another main goal
pathophysiological conditions30–33. CAIs possessing different of this study, is compared their activities to related standard
inhibition mechanisms compared to the classical inhibitors were compounds including tacrine (for acetylcholinesterase and
reported in the last years34,35. The interest in CAIs is mainly butyrylcholinesterase), acetazolamide (for human carbonic anhy-
motivated by their pharmacological properties and clinical use as drase isoenzymes I and II) and BHA, BHT, a-tocopherol and
diuretics, antiglaucoma, antiobesity or antiepileptic agents, as trolox (for antioxidant activities).
well as anticancer agents and diagnostic tools36–38
.
The neurotransmitter acetylcholine (ACh) mediates its physio- Experimental
logical effects via a plethora of receptors expressed throughout the
Chemistry
central nervous system and the peripheral nervous system39,40
.
Elder persons suffering from Alzheimer’s disease (AD) have a Synthesis of 2-(methacryloyloxy)ethyl 6-methyl-2-thioxo-4-
low ACh level in the hippocampus and cortex, which is the main (p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1)
cause of AD17,41. Acetylcholinesterase (AChE, EC 3.1.1.7) and
Thiourea (0.76 g, 0.01 mol) was added to the solution of
butyrylcholinesterase (BChE, EC 3.1.1.8) are serine hydrolases
acetylacetone (5 mL) in ethyl alcohol (1 mL) and then 2-
that catalyze the hydrolysis of acetylcholine, thus regulating
(methacryloyloxy) ethyl acetoacetate (1.91 mL, 0.01 mol) was
cholinergic neurotransmission42–44. It is well known that both
added to the solution dropwise. After the mixture was stirred for
enzymes are two major forms of cholinesterases in mammalian
5 minutes, p-tolualdehyde (1.18 mL, 0.01 mol) was added and it
tissues. AChE is highly active for the hydrolysis of acetylcholine
was stirred for 30 min. Then TFAA (0.02 mL) was added to the
(ACh) and can regulate the concentration of ACh, which plays a
reaction mixture and it was stirred by heating at 70–75 ꢂC. The
key role in memory and learning as a central neurotransmit-
progress of the reaction was controlled by Sulifol UV 254 plate.
ter45,46. On the other hand, BChE is an endogenous enzyme
After determining the full completion of reaction, solution was
synthesized by the liver, which serves as a catalyzer for the
evaporated and is cleansed in ethyl alcohol solution. The white
hydrolysis of esters in choline47. In general, AChE displays a
crystalline having the melting temperature of 212 ꢂC is obtained.
greater affinity for ACh and thus has greater activity for its
The yield is 1.8 g.
hydrolysis as compared to BChE. Studies indicate that in the case
Eluent-ethanol: hexane (5:2). 1H NMR (300 MHz, (DMSO-d6,
of AD, the level of AChE in certain brain regions is significantly
d): 2.27 (s, 3H, CH3); 4.49 (d, 2H, CH2O); 5.06–5.17 (dd, 2H,
reduced and the BChE level is progressively increased, which is
CH2); 5.78 (m, 1H, ¼CH); 7.25 (d, 2H, 2CH-Ar); 7.30 (t, 2H,
responsible for the level of ACh. In fact, a portion of evidence
2CH-Ar); 7.77 (s, 1H, NH); 9.26 (1H, NH). 13C NMR (75 MHz,
suggests that the inhibition of BChE can raise ACh levels and
DMSO-d6) 18.34, 54.32, 64.18, 99.25, 117.43, 126.70, 127.79,
improves cognition in AD. Currently, the most efficacious
128.91, 133.43, 145.10, 149.56, 152.53, 165.38. IR ꢀ, smꢁ1: 3175
treatment approaches for AD are four cholinesterase inhibitors
(NH), 1709 (C¼O), 1092 (C¼S), 756, 852, 974, 1230 (CH), 1608
(C¼C).
including tacrine, rivastigmine, donepezil and galantamine48,49
.
All aerobic organisms have antioxidant defenses against free
radicals and reactive oxygen species (ROS), including antioxidant
enzymes and antioxidant food constituents to remove or repair the
damaged molecules50–52. Antioxidant compounds can scavenge
free radicals or ROS and increase shelf life by retarding the
process of lipid peroxidation, which is one of the major reasons
for deterioration of food and pharmaceutical products during
processing and storage53–55. Also, they protect the human body
from hazardous effects of free radicals and ROS. They retard the
progress of many chronic diseases as well as lipid peroxidation.
Hence, a need has appeared to identify alternative safe sources of
antioxidants56,57. Antioxidants are often added to foods and
pharmaceuticals for preservation from the radical chain reactions.
They act by inhibiting the initiation and propagation steps of
radical chain reactions. Thus they lead the termination of the
reaction and delay the oxidation process58,59. At the present time,
the most commonly used antioxidants are butylated hydroxyani-
sole (BHA), butylated hydroxytoluene (BHT), propylgallate, and
tert-butyl hydroquinone60–62. However, BHA and BHT have been
restricted by legislative rules due to doubts over their toxic and
carcinogenic effects63,64. Therefore, there is a growing interest in
natural and safer antioxidants for food applications, and a growing
trend in consumer preferences towards natural antioxidants, all of
which have given impetus to the attempts to explore new and safer
Synthesis of 1-(4-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)ethanone (2)
Synthesis of 1-(4-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)ethanone (2) was realized according to
the general procedure at 2.1 according to the literature69. 1H NMR
data are in agreement with data given in the literature70. 13C NMR
(75 MHz, DMSO-d6) 10.01, 19.33, 21.11, 30.19, 48.26, 54.01,
126.82, 129.50, 137.64, 141.76, 152.58, 194.78. IR ꢀ, smꢁ1
:
1810–1951, 3000–3100 (Ar), 1493 (Ar C-H), 1614 (Ar C-C), 703
(CH), 1675, 1703 (C¼O), 3257 (NH), 1236 (C¼S).
Synthesis of ethyl 6-methyl-2-thioxo-4-(o-tolyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3)
Compound 3 was also synthesized fallowing the procedure above
and its 1H- and 13C-NMR data are in agreement with data given in
the literature71.
Synthesis of ethyl 4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (4)
Synthesized according to the general procedure at given
1
above72. H NMR data are in agreement with data given in the
sources of antioxidants65–67
.
literature72. 13C NMR (75 MHz, DMSO-d6) 14, 15, 48, 61, 104,
115, 121, 122, 128, 154, 160, 167, 180. IR ꢀ, smꢁ1: 3370–3040
(NH), 1709 (C¼O), 1092 (C¼S), 756, 852, 974, 1230 (CH), 1608
(C¼C).
The interest on the synthesis of new such heterocyclic
compounds, containing multifunctional nitrogen and sulfur
atoms within the ring, and their application as antimicrobial and
antioxidant agents68, prompted us to design the new cyclic
thioureas (1–8), which were obtained for the first time by us based
on using trifluoroacetic acid (TFAA) as catalyst (Figure 1).
In the present study we describe the design, synthesis and
evaluation of some of cyclic thioureas (1–8) as effective
acetylcholinesterase, butyrylcholinesterase, human carbonic
Synthesis of allyl 4-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (5)
Compound 5 was synthesized according to the procedure
described above (‘‘Synthesis of 2-(methacryloyloxy)ethyl

DOI: 10.1080/14756366.2016.1198901
4,5-Disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines
3
CH3
CH3
O
O
CH3
S
O
OH
S
CH2
O
O
O
CH3
NH
NH
H3C
O
H3C
O
NH
H3C
O
H3C
H3C
NH
H3C
N
H
H3C
N
H
O
H3C
N
H
S
N
H
S
1
2
3
4
CH3
O
O
OH
O
O
O
OH
S
H2C
H2C
H2C
H3C
H3C
NH
O
NH
O
NH
O
NH
N
S
H3C
N
H
S
H3C
N
H
S
H3C
N
H
H
5
6
7
5
Figure 1. Chemical formula cyclic thioureas 1–8.
6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-
solution was adjusted to 8.7, using solid Tris. Then, supernatant
carboxylate (1)’’ section). White crystalline. M.p: 198 ꢂC. was transferred to the previously prepared Sepharose-4B-L-tyro-
1H NMR (300 MHz, (DMSO-d6, d): 1.79 (s, 3H, CH3); 2.22 sine-sulphanilamide affinity column77. Subsequently, the proteins
(s, 3H, CH3); 4.19 (d, 2H, CH2O); 5.12 (d, 1H, CH-Ar); 5.63–5.92 from the column were spectrophotometrically determined at
(s-s, 2H, ¼CH2); 7.20 (m, 5H, 5CH-Ar); 9.75 (s, 1H, NH); 9.19 280 nm78. For determination of the purity of the hCA isoenzymes,
(s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) 15.5, 54.6, 55.8, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
67.8, 106, 114, 116, 127, 133, 135, 158, 167, 180. IR ꢀ, smꢁ1
:
PAGE)79, having 10 and 3% acrylamide as an eluent and packing
3370–3040 (NH), 1635–1630 (C¼O), 1092 (C¼S), 756, 852, 974, gel, respectively, with 0.1% SDS80 was performed, through which a
1230 (CH), 1607–1608 (C¼C).
single band was observed for each isoenzyme.
CA isoenzymes activities were determined according to the
Synthesis of allyl 4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4- methods described by Verpoorte et al.81 and the methods reported
tetrahydropyrimidine-5-carboxylate (6)
previously82. Absorbance change at 348 nm from p-nitrophenyla-
cetate (NPA) to p-nitrophenolate (NP) was recorded by 3 min
intervals at the room temperature (25 ꢂC) using a spectrophotom-
eter (Shimadzu, UV-VIS Spectrophotometer, UVmini-1240,
Kyoto, Japan). Quantity of the protein was measured spectro-
photometrically at 595 nm during the purification steps according
to the Bradford method83 as reported previously. Bovine serum
albumin was used as a standard protein. An activity (%)-[Cyclic
thioureas] graph was depicted to determine the inhibition effect of
each cyclic thioureas. For Ki values, three different cyclic
thioureas were tested. NPA was used as a substrate at five
different concentrations, and Lineweaver–Burk curves84 were
drawn as described previously85.
Compound 6 was synthesized as described above the
section ‘‘Synthesis of 1-(4-(4-methoxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)ethanone (2)’’. M.p:
1
210 ꢂC. H NMR (300 MHz, (DMSO-d6, d): 1.75 (s, 3H, CH3);
4.67 (d, 2H, CH2O); 5.21–5.41 (dd, 2H, ¼CH2); 5.89 (m,
1H, ¼CH); 6.78 (d, 1H, CH-Ar); 6.90 (d, 1H, CH-Ar); 7.19 (t,
2H, CH-Ar); 7.65 (s, 1H, NH); 9.64 (s, 1H, NH); 9.83 (H, OH).
13C NMR (75 MHz, DMSO-d6) 24.43, 29.79, 48.16, 65.28, 83.51,
117.01, 118.26, 120.96, 125.84, 129.12, 132.71, 151.06, 155.00,
168.57. IR ꢀ, smꢁ1: 1618, 1560, 1526 (Ar), 3112 (CH2 ¼C), 785
(Ar-CH), 1722, 1701 (C(O)R), 1377, 1370 (CH3), 1651 (C¼C),
3242 (NH), 1314 (C-N).
In the third part of this study, the inhibitory effects of cyclic
thioureas (1–8) on AChE and BChE activities were determined
according to the Ellman test.86 Acetylthiocholine iodide (AChI) or
butyrylthiocholine iodide (BChI) were used as substrates for both
reactions. 5,50-Dithio-bis(2-nitro-benzoic)acid (DTNB) was used
Synthesis of 1-(4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)ethanone (7) and Allyl 6-methyl-4-
phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (8)
The compounds 7 and 8 were also synthesized according to above for the measurement of the AChE/BChE activities. Briefly,
procedure and their 1H- and 13C-NMR data are in agreement with 100 mL of Tris/HCl buffer (1.0 M and pH 8.0), 10 mL of cyclic
data given in the literature73,74
.
thioureas (1–8) solution dissolved in deionized water at different
concentrations and 50 mL AChE/BChE (5.32 10–3 EU) solution
were mixed and incubated for 10 min at 25 ꢂC. Then a portion of
DTNB (50 mL, 0.5 mM) was added. Subsequently, the reaction
was initiated by the addition of 50 mL of AChI/BChI (10 mM).
The hydrolysis of these AChI/BChI was monitored spectrophoto-
metrically by the formation of yellow 5-thio-2-nitrobenzoate
anion as the result of the reaction of DTNB with thiocholine,
Enzymes studies
CA isoenzymes (hCA I, and II) were purified by sepharose-4B-L-
a single
tyrosine-sulfanilamide affinity chromatography in
purification step75. Sepharose-4B-L-tyrosine-sulfanilamide was
prepared according to a reported method76. Thus, pH of the

4
E. Garibov et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
released by the enzymatic hydrolysis of AChI/BChI, at a techniques and elemental analysis. Valence vibrations of NH
wavelength of 412 nm48. For determination of the effects of bond in IR spectrum of synthesized compounds (1–8) were
cyclic thioureas (1–8) on AChE/BChE, cyclic thioureas (1–8) observed in 3370–3040 cmꢁ1 regions. Valence vibrations of the
concentrations were added into the reaction solution. AChE/ C¼O bond in acetyl group are compatible with 1635–1630 cmꢁ1
BChE activities were measured, and an experiment in the absence band.
of drug was used as control. The IC50 values were obtained from
Only some functional groups differ in their appearance.
activity (%) versus cyclic thioureas (1–8) concentration plots. For Carbon atoms in molecule in the 13C NMR spectrum of the
determination of Ki values in the media with cyclic thioureas same compounds have the following signals according to their
(1–8) as inhibitor, different ACh/BCh concentrations were used as electronic densities: 24, 29, 37, 51, 86, 117, 122, 125, 129, 132,
substrates.
141, 151, 179 (C¼S), 205 (C¼O) ppm. It is known from the
reaction mechanism that, enol form of aceto-acetic ether takes
part in the reaction.
Determination of antioxidant activities
Fe3+ reducing ability of cyclic thioureas (1–8), Fe3+(CNꢁ) to
6
Biological activities
Fe2+(CNꢁ)6 reduction method was used87. Briefly, various
concentrations of cyclic thioureas (1–8) (10–30 mg/mL) in Here, we report the inhibition effect of cyclic thioureas (1–8) on
0.75 mL of deionized H2O were added with 1.25 mL of phosphate two catalytically active hCA I, and II as well as against AChE, and
buffer (0.2 M, pH 6.6) and 1.25 mL of potassium ferricyanide BChE. Also, antioxidant activity of cyclic thioureas (1–8) was
[K3Fe(CN)6] (1%)88. Then, the solution was incubated at 50 ꢂC studied using four different antioxidant methods, including 1,1-
during 20 min. After incubation period, trichloroacetic acid was diphenyl-2-picrylhydrazyl (DPPHꢀ) radical scavenging, Cu2+ and
added (1.25 mL, 10%). Lastly, a portion of FeCl3 (0.5 mL, 0.1%) Fe3+ reducing, and Fe2+ chelating activities. We discovered
was transferred to this mixture and the absorbance value was nanomolar inhibition against these metabolic enzymes. Cyclic
enrolled at 700 nm in a spectrophotometer89,90. According to the thioureas 1, 4 and 8, which possesses a phenolic moiety in its
obtained results, when reduction capability increases, absorbance scaffolds demonstrated effective antioxidant activities. The
indicates greater value91.
enzymes inhibition and antioxidant activities data of cyclic
Cu2+ reducing capability was performed according to the thioureas (1–8) reported here are shown in Tables 1–3, and the
previous studies92–94. For this purpose, aliquots of CuCl2 solution following comments can be drawn from these data:
(0.25 mL, 0.01 M), ethanolic neocuproine solution (0.25 mL, (1) The cytosolic isoenzymes CA I, and II are examined in this
7.5 ꢃ 10ꢁ3 M) and NH4Ac buffer solution (0.25 mL, 1.0 M) were
transferred to a test tube, which contains cyclic thioureas (1–8) at
different concentrations (10–30 mg/mL). Total volume was
completed with distilled H2O to 2 mL and shaken vigorously.
Absorbance of samples was recorded at 450 nm after 30 min95.
Metal chelating ability of cyclic thioureas (1–8) was predicted
according to previous studies96–98. Fe2+-binding capacity of
cyclic thioureas (1–8) was spectrophotometrically recorded at
562 nm. In brief, to a mixture of FeCl2 (0.1 mL, 0.6 mM) cyclic
thioureas (1–8) were added at three different concentrations (10–
20 mg/mL) in methanol (0.4 mL). The reactions were started by
Ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p0-disulfo-
nic acid sodium salt) solution addition (0.1 mL, 5 mM). After
that, the solution was mixed and incubated at room temperature
for 10 min. Finally, absorbance value of the mixture was
quantified spectrophotometrically at 562 nm versus blank
study. Cytosolic hCA I isoenzyme is ubiquitously expressed
in body, and available in high concentrations in blood and
gastrointestinal tract106. It was demonstrated that CA I is
involved in retinal and cerebral edema. Also, inhibition of
CA I could be a valuable tool for fighting the condition107. It
is generally accepted that if Ki value of a tested compound is
less than 50 mM (Ki450 mM), that compound is considered to
be inactive against hCA I108. The results presented in Table 1
indicate that cyclic thioureas (1–8) had effective inhibition
profile against slow cytosolic hCA I isoform, and cytosolic
dominant rapid hCA II isoenzyme. The cytosolic hCA I
isoenzyme was inhibited by cyclic thioureas (1–8) in low
nanomolar levels, the Ki of which differed between
47.40 4.43 and 77.68 3.69 nM. On the other hand,
acetazolamide (AZA), considered being a broad-specificity
CA inhibitor owing to its widespread inhibition of CAs,
showed Ki value of 289.22 2.60 nM against hCA I. Among
sample99,100
.
DPPHꢀ radical scavenging activity was performed according to
our previous studies101–103. The solution of DPPHꢀ was prepared
daily, stored in a flask coated with aluminum foil and kept in the
dark at 4 ꢂC. In brief, fresh solution of DPPHꢀ (0.1 mM) was
prepared in ethanol104. Then, 1.5 mL of each cyclic thiourea (1–8)
in ethanol was added an aliquot of this solution (0.5 mL) (10–
30 mg/mL). These mixtures were mixed vigorously and incubated
in the dark for 30 min. Finally the absorbance value was recorded
at 517 nm in a spectrophotometer105
.
Results and discussion
The synthesis of compounds 1–8 was reported in Scheme 1. The
synthesis of cyclic thioureas related to the title compounds has
been reported in the literature69–73. By a similar approach, the
reaction of substituted p-tolualdehyde, p-anisaldehyde, o-tolual-
dehyde, salicylaldehyde and benzaldehyde with methylene active
compounds such as b-diketones and thiourea in the presence of
TFAA led to the desired cyclic thioureas (1–8). The three-
component condensation reactions occurred within 2.5–3.0 h, at
60–75 ꢂC. The synthesized compounds were crystalline and their
structures were confirmed by IR, 1H-, and 13C-NMR spectroscopy
Scheme 1. The synthesis route of the new cyclic thioureas (1–8).

DOI: 10.1080/14756366.2016.1198901
4,5-Disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines
5
the inhibitors, 1-(4-(2-hydroxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidin-5-yl)ethanone (7) was found to
be the best hCA I inhibitor with Ki of 47.40 4.43 nM. The
hCA I inhibition effects of all cyclic thioureas (1–8) were
found to be the greater than that of acetazolamide, which was
a clinically standard CA inhibitor.
neurotransmitter ACh into choline and acetate.
Cholinesterase inhibitors, including carbamates, have
gained much attention since they have been successfully
used in the treatment of a number of diseases involving
cholinergic dysfunction. A number of different types of
cholinesterase inhibitors are known and have been found to
affect these enzymes in a variety of ways112. In our study,
cyclic thioureas (1–8) were investigated for their ability to
inhibit AChE, which was the primary cholinesterase in the
(2) CA II, which generally exists in red blood cells in lower
concentrations in mammals, has approximately ten times
higher activity compare with CA I.109 The hCA II is not only
a very effective catalyst for interconversion between CO2
and HCO3110, it also shows some catalytic versatility,
participating in several other hydrolytic processes, which
presumably involve nonphysiological substrates111. Against
the physiologically dominant isoform hCA II, cyclic thio-
ureas (1–8) demonstrated Kis varying from 30.63 7.62 to
76.06 3.15 nM (Table 1). Among which the cyclic thiourea
2 was the best hCA II inhibitor (Ki: 30.63 7.62 nM). Thus,
these cyclic thioureas (1–8) demonstrated high hCA II
inhibition. They are probably interact with the distinct
hydrophilic and hydrophobic halves of the CA II active
site. The compound 2 [1-(4-(4-Methoxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)ethanone] shown the
most inhibition effect with Ki value of 30.63 7.62 nM
against cytosolic CA II. CA isoenzymes are physiological
very important enzymes. Recently, very intense studies were
performed on this subject.
Table 3. 1,1-Diphenyl-2-picrylhydrazyl (DPPHꢄ) radical scavenging, and
ferrous ion (Fe2+) chelating activities of cyclic thioureas (1–8).
DPPHꢄ scavenging
IC50
Metal chelating
Antioxidants
r2
IC50
r2
BHA
BHT
a-Tocopherol
Trolox
30.13
40.76
28.87
21.65
86.62
173.35
164.48
69.30
76.15
230.87
138.60
144.37
–
0.9622
0.9881
0.9924
0.9686
0.9743
0.9889
0.9628
0.9760
0.9938
0.9718
0.9833
0.9911
–
21.01
31.50
15.40
11.01
43.31
79.02
100.01
53.31
36.47
34.65
69.30
33.02
11.36
0.9622
0.9830
0.9932
0.9797
0.9638
0.9924
0.9921
0.9896
0.9968
0.9826
0.9722
0.9791
0.9888
1
2
3
4
5
6
7
8
EDTA
(3) AChE is localized at cholinergic synapses in vertebrates and
regulates neurotransmission through rapid hydrolysis of the
Table 1. Human carbonic anhydrase isoenzymes I and II, AChE and BChE enzymes inhibition values of compounds 1–8.
IC50 (nM)
hCA II
r2
Ki (nM)
hCA II
Compounds hCA I
r2
AChE
r2
BChE
r2
hCA I
AChE
BChE
1
2
3
4
5
6
7
8
61.38 0.9754
66.18 0.9613
69.23 0.9820
55.44 0.9788
82.01 0.9760
79.11 0.9800
74.67 0.9766
63.28 0.9660
99.71 0.9825 13.81 0.9882 49.74 0.9932
33.83 0.9819 14.31 0.9683 18.41 0.9521
65.51 0.9636 16.76 0.9385 23.29 0.9770
68.28 0.9757 14.36 0.9620 29.35 0.9965
78.75 0.9468 22.58 0.9525 43.88 0.9908
96.25 0.9476 16.44 0.9904 30.13 0.9736
80.39 0.9735 13.13 0.9795 43.91 0.9965
52.35 7.31
77.68 3.69
47.53 6.49
50.67 6.45
74.06 8.18
50.49 8.56
47.40 4.43
66.95 2.26
55.22 1.05
30.63 7.62
54.18 2.01
53.87 2.38
56.52 1.59 16.13 56.1 15.53 1.71
71.27 2.18 11.57 13.4 9.55 3.69
7.41 12.2 22.44 5.71
6.35 23.3
7.18 14.1
8.83 6.54
8.02 1.68
8.37 1.98
6.76 0.59
76.06 3.15
49.78 1.51
6.11 9.32 15.68 3.41
6.17 16.6 14.85 2.79
71.59 0.9670
9.36 0.9564 23.36 0.9901
AZA*
TAC**
262.50 0.9567 107.61 0.9496
–
–
–
–
–
23.84 0.9738 46.13 0.9643
289.22 2.60 135.48 4.59
–
–
–
16.27 42.9 22.49 7.59
–
–
–
–
*Acetazolamide (AZA) was used as a standard inhibitor for both hCA I and II isoenzymes.
**Tacrine (TAC) was used as a standard inhibitor for AChE and BChE enzymes.
Table 2. Fe3 + and Cu2 + reducing abilities of cyclic thioureas 1–8.
Fe3+-Fe2+reducing
Cu2+-Cu+ reducing
a
l
r2
l450
r2
700
BHA*
BHT*
2.046 0.039
1.398 0.007
1.715 0.014
1.266 0.011
0.816 0.006
0.138 0.006
0.126 0.004
0.671 0.009
0.806 0.014
0.166 0.006
0.192 0.005
0.153 0.005
0.9722
0.9718
0.9981
0.9828
0.9683
0.9924
0.9889
0.9877
0.9938
0.9880
0.9944
0.9911
2.126 0.004
1.709 0.003
1.202 0.009
1.177 0.014
0.507 0.011
0.123 0.003
0.109 0.003
0.768 0.017
0.479 0.003
0.137 0.004
0.125 0.003
0.105 0.004
0.9870
0.9822
0.9940
0.9917
0.9918
0.9644
0.9588
0.9921
0.9787
0.9910
0.9838
0.9958
a-Tocopherol*
Trolox*
1
2
3
4
5
6
7
8
*They were used as reference antioxidant molecules.

6
E. Garibov et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
body. According to our data, inhibitory effects of these cyclic
thioureas (1–8) revealed a significant elevation in the case of
AChE. AChE inhibitors inhibit the AChE from breaking
down ACh, thereby increasing both the level and duration of
action of the neurotransmitter ACh. Generally, these com-
pounds showed higher inhibition and higher lipophilicity.
Considering the results, all cyclic thioureas (1–8) expressed
significantly higher inhibition activity. All of cyclic thioureas
(1–8) derivatives had significantly higher AChE inhibitory
of these compounds was addicted to different concentration
(10–30 mg/mL). Cu2+ reducing capability of cyclic thioureas
(1–8) and positive controls at the same concentration (30 mg/
mL) showed the following order: BHA (2.126 0.004; r2:
0.9870)4BHT (1.709 0.003; r2: 0.9822)4a-Tocopherol
(1.202 0.009; r2: 0.9940) & Trolox (1.177 0.014; r2:
0.9917)44 (0.768 0.017; r2: 0.9921)41 (0.507 0.011;
r2: 0.9918) & 5 (0.479 0.003; r2: 0.9787). There was a
positive control between Fe3+ and Cu2+ reducing abilities.
activity than that of standard AChE inhibitors such as (7) DPPH test is generally used as the substrate to gauge free
Tacrine. Furthermore, the Ki values of cyclic thioureas (1–8)
and standard compound (tacrine) are summarized in Table 1.
As can be seen in the results obtained from Table 1, AChE
was effectively inhibited by cyclic thioureas (1–8), with Ki
values in the range of 6.11 9.32 to 16.13 56.1 nM.
However, all of cyclic thioureas (1–8) had almost similar
inhibition profiles. The most active one is 1-(4-(2-hydro-
xyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)ethanone (7) and showed a Ki value of 6.11 9.32 nM.
These results clearly indicate that newly synthesized com-
pounds as well as future similar derivatives may function as
drugs for the treatment of AD.
radical scavenging effectiveness of antioxidant molecules119
.
It is based on the reduction of a DPPH solution in alcohol in
the source of a hydrogen-donating antioxidant, owing to the
formation of non-radical form DPPH-H by the reaction120
.
Cyclic thioureas (1–8) have the ability to reduce steady
radical DPPH to yellow-colored DPPH-H. Table 3 defines a
crucial decrement (p50.01) in the concentration of DPPH
radical owing to the scavenging capability of cyclic thioureas
(1–8) and reference radical scavenging agents like Trolox,
a-Tocopherol, BHT and BHA. IC50 values were found as
21.65 mg/mL (0.9686) for trolox, 28.87 mg/mL (0.9924) for
a-Tocopherol, 30.13 mg/mL (0.9622) for BHA, 40.76 mg/mL
(0.9881) for BHT, 69.30 mg/mL (0.9760) for 4, 76.15 mg/mL
(0.9938) for 5, and 86.62 mg/mL (0.9743) for 1. A lower
EC50value showed a higher DPPH radical scavenging
activity121. DPPH exposed an absorbance at 517 nm, which
vanished after acceptation of an electron or hydrogen radical
from an antioxidant compound to become a steadier
(4) BChE is the major ACh hydrolyzing enzyme in peripheral
mammalian systems. It can either reside in the circulation or
adhere to cells and tissues and protect them from antic-
holinesterases, including insecticides and poisonous nerve
gases. Some cholinesterase inhibitors such as rivastigmine
and phenserine readily cross the blood-brain barrier to inhibit
cholinesterases in the central nervous system, leading to
diamagnetic molecule122
.
improved cognition in dementias113. It is well known (8) On the other hand, cyclic thioureas (1–8) had also effective
phenothiazines are known to inhibit cholinesterases, espe-
cially BChE114. In the present study, cyclic thioureas (1–8)
inhibited BChE in ranging of 6.76 0.59–22.44 5.71 nM.
Cyclic thiourea (6) was the best BChE inhibitor (Ki:
6.76 0.59 nM). However, all cyclic thioureas (1–8) demon-
strated higher BChE inhibition activity than that of Tacrine
(Ki: 22.49 7.59 nM).
Fe2+ ions chelating effect. The distinction between different
concentrations of cyclic thioureas (1–8) (10–30 mg/mL) and
the control value was fixed to be statistically important
(p50.01). Furthermore, it is found that IC50 values for
compounds (1–8) varied between 33.02–100.01 mg/mL
(Table 3). Whereas, IC50 values belonging to Fe2+ ions
chelating capacity of positive controls like BHT, BHA,
a-Tocopherol, Trolox, and was found in ranging from
11.01 mg/mL to 31.50 mg/mL. A lower EC50 value reflects a
higher Fe2+ ions binding activity. These results clearly
introduce that Fe2+ ions chelating effect of cyclic thioureas
(1–8) was close to trolox, a-tocopherol, BHA and BHT.
Fe2+ ions are the most efficient pro-oxidants in pharmacol-
ogy systems and food. Ferrozine can create complexes with
(5) Reducing capability of cyclic thioureas (1–8) was evaluated
by reduction of Fe4[(CN)6]3 to Fe4[(CN)6]2115. In this
technique, the presence of reductants would result in the
reduction of ferric ions (Fe3+) to ferrous ions (Fe2+ 116
.
)
Addition of free Fe3+ to the reduced molecule brings about
the formation of intensive Perl’s Prussian blue complex,
Fe4[Fe(CNꢁ)6]3, which has a strong absorbance at 700 nm117
.
Fe3+ reducing assay gets advantage of an electron chain
reaction where a ferric salt is utilized as an oxidant118. In
addition, the yellow color of the tested mixture changes into
diverse tons of green and blue by the ability of the
compounds. However, the effective reducing ability was
only demonstrated by cyclic thioureas, which possesses
Fe2+
Ferrozine-Fe2+ complex formation is a broken down, result-
ing in decrease in the red color of Ferrozine-
Fe2+ complex123
.
In the presence of Fe2+ chelating compounds,
a
.
phenolic hydroxyl group in their scaffold (1, 4 and 5). Conclusion
Cu2+ reducing capability of cyclic thioureas (1–8) and
The cyclic thioureas (1–8) used in the present study demonstrated
effective inhibition profiles against hCA isoforms, AChE and
BChE enzymes. Additionally, these compounds demonstrated
effective antioxidant activities using by different bioassays. These
compounds identified their potential CA isoenzymes, and AChE
and BChE enzyme inhibitors. In this study, nanomolar level of Ki
values was observed for all cyclic thioureas (1–8) and these
compounds can be selective inhibitor of both cytosolic CA I and
II isoenzymes and AChE and BChE enzymes. Also, they can be
used as novel antioxidants in applications including pharmaceut-
ical industry.
positive controls at the same concentration (30 mg/mL)
showed the following order: BHA (2.046 0.039; r2:
0.9722)4a -Tocopherol (1.715 0.014; r2: 0.9981)4BHT
(1.398 0.007; r2: 0.9718)4Trolox (1.266 0.011; r2:
0.9828)41 (0.816 0.006; r2: 0.9683) & 5 (0.806 0.014;
r2: 0.9938)44 (0.671 0.009; r2: 0.9877) (Table 2).
(6) The CUPRAC method is a rapid, simple, cost-effective,
selective, steady and versatile antioxidant assay useful for a
wide variety of phenolic compounds. CUPRAC reactions are
essentially complete within 30 min. Cu2+ reducing power of
cyclic thioureas (1–8) and positive controls are shown in
Table 2.
A positive relationship was found between
Declaration of interest
Cu2+ reducing power and different concentration of cyclic
thioureas (1–8). It was detected that Cu2+ reducing capacity The authors have declared no conflict of interest.

DOI: 10.1080/14756366.2016.1198901
4,5-Disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines
7
dimethoxy-bromophenol derivatives incorporating cyclopropane
moieties. J Med Chem 2015;58:640–50.
22. De Simone G, Alterio V, Supuran CT. Exploiting the hydrophobic
and hydrophilic binding sites for designing carbonic anhydrase
inhibitors. Expert Opin Drug Disc 2013;8:793–810.
IG and SA would like to extend his sincere appreciation to the
Research Chairs Program at King Saud University for funding this
research.
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8
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J Enzyme Inhib Med Chem, Early Online: 1–9
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