Synthesis and evaluation of 2,4,5-trisubstitutedthiazoles as carbonic anhydrase-III inhibitors

Article abstract of DOI:10.1080/14756366.2020.1786820

A series of 17 compounds (12–16 b) with 2,4,5-trisubstitutedthiazole scaffold having 5-aryl group, 4-carboxylic acid/ester moiety, and 2-amino/amido/ureido functional groups were synthesised, characterised, and evaluated for their carbonic anhydrase (CA)-III inhibitory activities using the size exclusion Hummel–Dreyer method (HDM) of chromatography. Compound 12a with a free amino group at the 2-position, carboxylic acid moiety at the 4-position, and a phenyl ring at the 5-position of the scaffold was found to be the most potent CA-III inhibitor (K_i = 0.5 μM). The presence of a carboxylic acid group at the 4-position of the scaffold was found to be crucial for the CA-III inhibitory activity. Furthermore, replacement of the free amino group with an amide and urea group resulted in a significant reduction of activity (compounds 13c and 14c, K_i = 174.1 and 186.2 μM, respectively). Thus, compound 12a (2-amino-5-phenylthiazole-4-carboxylic acid) can be considered as the lead molecule for further modification and development of more potent CA-III inhibitors.

Full text of DOI:10.1080/14756366.2020.1786820

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of 2,4,5-
trisubstitutedthiazoles as carbonic anhydrase-III
inhibitors
Bilal A. Al-Jaidi , Pran Kishore Deb , Soha Taher Telfah , Abdel Naser Dakkah ,
Yazan A. Bataineh , Qutaiba Ahmed Al Khames Aga , Mohammad A. Al-
dhoun , Ala’ Ali Ahmad Al-Subeihi , Haifa’a Marouf Odetallah , Sanaa K.
Bardaweel , Raghuprasad Mailavaram , Katharigatta N. Venugopala &
Anroop B. Nair
To cite this article: Bilal A. Al-Jaidi , Pran Kishore Deb , Soha Taher Telfah , Abdel Naser Dakkah ,
Yazan A. Bataineh , Qutaiba Ahmed Al Khames Aga , Mohammad A. Al-dhoun , Ala’ Ali Ahmad Al-
Subeihi , Haifa’a Marouf Odetallah , Sanaa K. Bardaweel , Raghuprasad Mailavaram , Katharigatta
N. Venugopala & Anroop B. Nair (2020) Synthesis and evaluation of 2,4,5-trisubstitutedthiazoles
as carbonic anhydrase-III inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1483-1490, DOI: 10.1080/14756366.2020.1786820
To link to this article: https://doi.org/10.1080/14756366.2020.1786820
© 2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1483–1490
https://doi.org/10.1080/14756366.2020.1786820
ORIGINAL ARTICLE
Synthesis and evaluation of 2,4,5-trisubstitutedthiazoles as carbonic
anhydrase-III inhibitors
Bilal A. Al-Jaidia,b , Pran Kishore Deba , Soha Taher Telfaha, Abdel Naser Dakkaha, Yazan A. Bataineha,
Qutaiba Ahmed Al Khames Agaa , Mohammad A. Al-dhouna, Ala’ Ali Ahmad Al-Subeihic,
Haifa’a Marouf Odetallaha, Sanaa K. Bardaweeld , Raghuprasad Mailavarame , Katharigatta N. Venugopalaf,g
and Anroop B. Nairf
b
aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman, Jordan; Faculty of Pharmacy, Yarmouk
c
d
University, Irbid, Jordan; Faculty of Pharmacy, Amman Arab University, Amman, Jordan; Department of Pharmaceutical Sciences, School of
e
Pharmacy, University of Jordan, Amman, Jordan; Pharmaceutical Chemistry Division, Sri Vishnu College of Pharmacy, Bhimavaram, India;
fDepartment of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia; Department of
g
Biotechnology and Food Technology, Durban University of Technology, Durban, South Africa
ABSTRACT
ARTICLE HISTORY
Received 24 March 2020
Revised 15 June 2020
Accepted 15 June 2020
A series of 17 compounds (12–16 b) with 2,4,5-trisubstitutedthiazole scaffold having 5-aryl group, 4-car-
boxylic acid/ester moiety, and 2-amino/amido/ureido functional groups were synthesised, characterised,
and evaluated for their carbonic anhydrase (CA)-III inhibitory activities using the size exclusion
Hummel–Dreyer method (HDM) of chromatography. Compound 12a with a free amino group at the 2-
position, carboxylic acid moiety at the 4-position, and a phenyl ring at the 5-position of the scaffold was
found to be the most potent CA-III inhibitor (Ki ¼ 0.5 lM). The presence of a carboxylic acid group at the
4-position of the scaffold was found to be crucial for the CA-III inhibitory activity. Furthermore, replace-
ment of the free amino group with an amide and urea group resulted in a significant reduction of activity
(compounds 13c and 14c, Ki ¼ 174.1 and 186.2 lM, respectively). Thus, compound 12a (2-amino-5-phenyl-
thiazole-4-carboxylic acid) can be considered as the lead molecule for further modification and develop-
ment of more potent CA-III inhibitors.
KEYWORDS
Carbonic anhydrase III
inhibitors; 2-amino-5-aryl-
thiazole; Hummel–Dreyer
method of chromatography
1. Introduction
turn lead to the disturbance of normal physiological functions16.
However, hCA inhibitors (hCAIs) are found to be associated with
various side effects mainly due to their lack of isoform specificity
and off-target activity. Thus, current research efforts are focussed
on the design and development of CA-specific inhibitors by using
both traditional non-spectral and spectroscopic experimental
approaches including recently reported innovative strategy of
using narrow-bore nano-electronspray ionisation emitters in tan-
dem with native mass spectrometry to enhance the accuracy of
Carbonic anhydrases (CAs, EC 4.2.1.1) are a well-known superfam-
ily of metalloenzymes, ubiquitously present in both prokaryotes as
well as eukaryotes1,2. CAs catalyse the reversible hydration of car-
bon dioxide (CO2) into bicarbonate (HCO3ꢀ) and proton (Hþ),
thereby involved in various important physiological and patho-
physiological processes including acid–base regulation, ion trans-
port, electrolyte secretions, biosynthetic reactions, and
calcifications3,4. There are eight genetically distinguished families
of CAs, viz. a, ꞵ, c, d, f, g, h, and i-CAs. In human, 15 isoforms of
a-CAs containing Zn(II) have been reported to date, where hCAs I-
III, VII, and XIII are cytosolic isoforms, hCAs IV, IX, XII, and XIV are
membrane-bound isoforms and hCAs VA and VB are mitochon-
drial isoforms, hCA VI is mainly secreted in saliva5,6. It should be
noted that hCAs VIII, X, and XI are characterised as a catalytic iso-
enzymes7. A large number of potential small-molecule inhibitors
have been developed and some are under current clinical trial
investigations targeting hCAs as potential therapeutic agents for
the treatment of various diseases including diuretics, glaucoma,
edema, obesity, osteoporosis, epilepsy, pain, malaria, and can-
cer8–15. It is worth mentioning that carboxylic acid group-contain-
ing environmental organic pollutants like perfluorinated alkyl
ligand-protein binding measurement17–19
.
The cytosolic enzyme hCA III is specifically found to be present
in liver, adipocytes, and skeletal muscle20. The hCA III is compara-
tively inefficient in catalysing CO2 hydration (ꢁ300-fold less than
hCA II)21. However, hCA III has been found to be involved in the
contraction of skeletal muscle and protection of cells from reactive
oxygen species (ROS) and oxidative stress22–25. The hCA III also
facilitates the regulation of adipogenesis in relation to peroxisome
proliferating-activated
receptor-gamma
2
(PPAR-c2)26.
Furthermore, hCA III is found to be a valuable biomarker in vari-
ous diseases like neuromuscular disease, sarcopenia, and hepatitis
B and C infections and liver carcinoma27–29. It should be noted
that most important classes of hCAIs contain sulphonamide (R-
substances (PFASs) do exhibit toxicity by inhibiting hCAs, which in SO2NH2), sulphamide (R-NH2-SO2NH2), sulphamate (R-OSO2NH2)
CONTACT Bilal A. Al-Jaidi
bilaljeaidi77@gmail.com,
bilal.aljaidi@yu.edu.jo;
pdeb@philadelphjia.edu.jo
Faculty of Pharmacy, Yarmouk University, P. O. Box – 566, Irbid 21163, Jordan;
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia
Pran Kishore Deb
prankishore1@gmail.com,
University, Amman, Jordan
ß 2020 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,
distribution, and reproduction in any medium, provided the original work is properly cited.

1484
B. A. AL-JAIDI ET AL.
functional groups which are found to be insensitive towards hCA and evaporated to get the solid residue. The solid residue thus
III18,30–32. Recently, an ureido-substituted benzene-sulphonamide
obtained was recrystallized with ethyl acetate, and hexane to get
(SLC 0111) has been reported to have potent hCA inhibitory activ-
ity (selective towards hCA IX/XII against hCA I and II), which is cur-
rently under Phase Ib/II clinical trials investigation for the
treatment of metastatic breast cancer33, but, this study did not
explore the selectivity profile against hCA III isoform. Thus, several
heterocyclic hCA III inhibitors from natural and synthetic origin
with different mechanism as compared to sulphonamides have
been discovered and are currently being investigated as potential
therapeutic agents for the treatment of diabetes and hyperlipid-
aemia20,31,32,34. In particular, Supuran et al. reported hCA III inhib-
ition profile of diverse sulphonamide containing molecules
including 2-aminothiadiazoles32. Same group of researchers fur-
ther reported that most potent inhibitors of hCA-III are mainly
anions like carbonate, cyanate, cyanide, thiocyanate, etc.31. In con-
tinuation of these observations, Alzweiri et al. reported promising
hCA-III inhibitory activities of carboxylic acid containing analogues
of benzoic acid and nicotinic acid35,36. Interestingly, vanillic acid
showed more potent hCA-III inhibitory activity as compared to the
the crystals of compounds 12–16 in quantitative yield (40–70%).
Compounds 12 and 16 were prepared according to the reported
procedure and their physical and spectral properties were found
to be on par with the literature report38,39
.
2.1.1.1. Methyl-2-amino-5-(p-tolyl)thiazole-4-carboxylate (13). Pale
yellow crystals, yield: 54%, M.P.: 201 ꢂC. FT-IR (V cmꢀ1): 3413, 3127,
1
1698, 1605, 1537, 1213, 996. H NMR (DMSO-d6, 400 MHz) d ¼ 2.31
(s, 3H, CH2), 3.62 (s, 3H, COCH3), 7.17 (d, 2H, NH2), 7.26 (m, 4H, Ar-
H). 13 C NMR (DMSO-d6, 400 MHz) d ¼ 20.79, 51.34, 128.14, 128.74,
129.19, 132.72, 135.22, 137.52, 162.63, 165.50. MS (ESI): m/z (%) ¼
249.1 (90) [M þ H]þ, 217.0 (100) [M-31]þ.
2.1.1.2.
Methyl-2-amino-5-(4-(methylsulfonyl)phenyl)thiazole-4-
carboxylate (14). White fluffy powder, yield: 42%, M.P.: 260 ꢂC. FT-
IR: 3470, 3256, 1725, 1536, 1257, 1203, 1137. 1H NMR (DMSO-d6,
400 MHz) d ¼ 3.25 (s, 3H, SO2CH3), 3.66 (s, 3H, COCH3), 7.47 (s, 2H,
acetazolamide37. Therefore, it was our thought of interest to syn- NH2), 7.65 (d, 2H, Ar-H), 7.89 (d, 2H, Ar-H). 13 C NMR (DMSO-d6,
thesise and evaluate a novel series of 2,4,5-trisubstitutedthiazole
derivatives with 5-aryl group, 4-carboxylic acid/ester moiety, and
2-amino/amido/ureido functional groups as possible hCA-
400 MHz) d ¼ 42.48, 51.66, 126.83, 129.83, 130.11, 136.34, 136.92,
139.81, 162.41, 166.68. MS (ESI): m/z (%) ¼ 312.7 (100) [M þ H]þ,
281.0 (90) [M-31]þ.
III Inhibitors.
2.1.2. General methods for the synthesis of 2-amino-5-substituted-
4-carboxylic acid derivatives (12a-16a)
2. Materials and methods
Compounds 12–16 (2 mM) were suspended in distilled water, and
1 M NaOH solution (2 mM) was added to the reaction. The reac-
tion mixture was warmed to 50 ꢂC until the solution became clear
and then the reaction mixture was kept in stirring at room tem-
perature for 5 h. The reaction mixture was acidified by 1M HCl
solution to pH 4 and the precipitate thus formed was collected by
filtration, dried, and recrystallized with methanol and dichlorome-
thane to get the hydrolysed compounds 12a–16a in quantitative
yield (45–80%). Compounds 12a and 16a were prepared accord-
ing to the reported procedure and their physical and spectral
properties were found to be on par with the literature report38.
2.1. Chemistry
The progress of reactions was routinely monitored by thin-layer
R
chromatography (TLC) on silica gel plates (pre-coated MerckV),
and spots were examined under the UV light (254 nm). Melting
points were measured by open capillaries using a Stuart Scientific
electro-thermal melting point apparatus (UK). FT-IR spectra were
recorded on Thermo-Nicolet Avatar FT-IR (Thermo Fisher Scientific,
Rockville, MD). 1H and 13 C NMR spectra were recorded on
400 MHz Avance Ultrashield spectrometer (Bruker, Ettingen,
Germany) in DMSO-d6 in part per million (d) using trimethylsilane
as an internal standard. Mass spectra were measured in a positive
ion mode using the electrospray ion trap (ESI) technique on a
Bruker Apex-4 instrument (Germany).
2.1.2.1. 2-Amino-5-(p-tolyl)thiazole-4-carboxylic acid (13a). White
fine crystals, yield: 80%, M.P.: 236 ꢂC. FT-IR: 3302, 2945, 1694, 1585,
1297, 1284, 994. 1H NMR (DMSO-d6, 400 MHz) d ¼ 2.30 (s, 3H,
ArCH3), 7.16 (bs, 2H, NH2), 7.26 (m, 4H, Ar-H).13C NMR (DMSO-d6,
400 MHz) d ¼ 20.49, 128.14, 128.74, 129.19, 132.72, 135.22, 137.52,
2.1.1. General methods for the synthesis of methyl 2-amino-5-sub-
stituted-4-carboxylates (12–16)
A mixture of appropriate aldehyde 1–5 (9 mM) solution in dry 162.63, 165.50. MS (ESI): m/z (%) ¼ 235.2 (100%) [M þ H]þ, 206.9
(85%) [M-17]þ.
diethyl ether (25 mL), and methyl dichloroacetate 6 (10 mM) was
kept in stirring at 0 ꢂC. A pre-cooled solution of sodium methox-
ide (10 mM) in dry methanol was added drop wise to the alde-
hyde solution. After the completion of the sodium methoxide
addition, the reaction mixture was kept in stirring at room tem-
2.1.2.2. 2-Amino-5-(4-(methylsulfonyl)phenyl)thiazole-4-carboxylic
acid (14a). Off-white crystals, yield: 74%, M.P.: 238 ꢂC. FT-IR: 3201,
1
2845, 1684, 1310, 1284, 974. H NMR (DMSO-d6, 400 MHz) d ¼ 3.23
perature for 4 h. Then the reaction mixture was evaporated to dry-
(s, 3H, SO2CH3), 7.39 (s, 2H, NH2), 7.65 (bs, 2H, Ar-H), 7.87 (bs, 2H,
ness, and the resulted creamy residue was suspended in distilled
Ar-H), 12.70 (bs, 1H, COOH). 13 C NMR (DMSO-d6, 400 MHz)
water followed by extraction with ethyl acetate. The ethyl acetate
d ¼ 43.45, 126.77, 128.44, 129.99, 136.71, 138.55, 139.56, 163.48,
extract was dried on anhydrous sodium sulphate and evaporated
166.49. MS (ESI): m/z (%) ¼ 299.0 (100%) [M þ H]þ, 281 (40%)
to yield the intermediate compounds 7–11 as creamy oily residue
[M-17]þ.
that was used for the next step without any farther purification.
Thiourea (9 mM) and the creamy residue (7–11) were dissolved
2.1.2.3.
2-Amino-5-(4-chloro-3-nitrophenyl)thiazole-4-carboxylic
in dry methanol and heated under reflux for 5 h and left under
stirring at room temperature for 12 h. The reaction mixture was
evaporated to dryness to get the yellow precipitate and sus-
pended in water, followed by extraction with ethyl acetate. The
acid (15a). Orange crystals, yield: 45%, M.P.: 163 ꢂC. FT-IR: 3107,
2841, 1645, 1585, 1147, 1032, 941. 1H NMR (DMSO-d6, 400 MHz)
d ¼ 7.24 (s, 2H, NH2), 7.44 (m, 2H, Ar-H), 8.73 (s, 1H, Ar-H). 13 C
ethyl acetate extract was dried on anhydrous sodium sulphate, NMR (DMSO-d6, 400 MHz) d ¼ 127.76, 128.10, 131.05, 131.42,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1485
135.21, 137.01, 154.45, 157.91, 163.61, 165.55. MS (ESI): m/z (%) ¼ yields. Compound 13b on further hydrolysis with sodium hydrox-
301 (15%) [M þ H]þ, 249 (80%) [M-52]þ.
ide resulted in the formation of compound 13c.
2.1.4.1. Methyl-2-(3-phenylureido)-5-(p-tolyl)thiazole-4-carboxylate
2.1.3. General methods for the synthesis of methyl-2-(4-substi-
(13b). Orange crystals, yield: 39%, M.P.: 147 ꢂC. FT-IR: 3278, 2949,
tuted-benzamido)-5-substituted-thiazole-4-carboxylate derivatives
(12 b, 14 b) and 2-(4-methoxybenzamido)-5-(4-(methylsulfonyl)phe-
nyl)thiazole-4-carboxylic acid (14c)
Compounds 12 and 16 (2 mM) were dissolved in dry tetrahydro-
furan, and triethylamine (2 mM) was added. Then 4-nitrobenzoyl
chloride (2.5 mM)/4-methoxybenzoyl chloride (2.5 mM) was grad-
1
1695, 1538, 1244, 1207, 996. H NMR (DMSO-d6, 400 MHz) d ¼ 2.38
(s, 3H, ArCH3), 3.67 (s. 3H, COCH3), 7.22 (d, 2H, Ar-H), 7.31 (m, 4H,
Ar-H), 7.46 (d, 3H, Ar-H), 9.07 (s, 1H, CONH), 10.89 (s, 1H, CONH).
13 C NMR (DMSO-d6, 400 MHz) d ¼ 16.02, 46.81, 113.37, 114.09,
118.22, 122.66, 123.97, 124.11, 129.28, 133.40, 133.51, 134.90,
146.91, 151.66, 157.62. MS (ESI): m/z (%) ¼ 368 (100%) [M þ H]þ,
ually added dropwise to the reaction mixture. The reaction mix- 336 (35%) [M-31]þ.
ture was kept in stirring at room temperature for 24 h. The solvent
was evaporated to dryness and then suspended in water, followed
by extraction with ethyl acetate. The organic layer extract was
dried on anhydrous sodium sulphate and evaporated to dryness.
The solid product thus obtained was recrystallized with ethyl acet-
ate, and hexane to get the target compounds 12b and 14b in
quantitative yields (70–77%). Compound 14b on further hydrolysis
with sodium hydroxide resulted in the formation of com-
pound 14c.
2.1.4.2. Methyl 5-(4-(methylsulfonyl)phenyl)-2-(3-phenylureido)thia-
zole-4-carboxylate (14d). White crystals, yield: 59%, M.P.: 143 ꢂC.
FT-IR: 3267, 3035, 1716, 1622, 1502, 1277, 1145, 961. 1H NMR
(DMSO-d6, 400 MHz) d ¼ 3.25 (3H, s, SOsCH3), 3.66 (s, 3H, COCH3),
7.09 (d, 2H, Ar-H), 7.82 (d, 2H, Ar-H), 7.99 (d, 2H, Ar-H), 8.16 (d, 2H,
Ar-H), 9.07 (s, 1H, CONH), 10.89 (s, 1H, CONH). 13 C NMR (DMSO-d6,
400 MHz) d ¼ 43.31, 51.72, 55.46, 113.92, 123.16, 126.76, 130.31,
130.62, 135.59, 136.42, 140.51, 157.12, 161.94, 162.93, 164.88. MS
(ESI): m/z (%) ¼ 432 (55%) [M þ H]þ.
2.1.3.1. Methyl-2-(4-nitrobenzamido)-5-substituted-thiazole-4-carb-
oxylate (12b). White fluffy crystals, yield: 41%, M.P.: 232 ꢂC. FT-IR:
2.1.4.3. Methyl-5-benzyl-2-(3-phenylureido)thiazole-4-carboxylate
1
3399, 3115, 1707, 1674, 1556, 1296, 1256, 863. H NMR (DMSO-d6, (16b). Off-white powder, yield: 33%, M.P.: 226 ꢂC. FT-IR: 3267,
1
3025, 1690, 1536, 1238, 1213, 900.16. H NMR (DMSO-d6, 400 MHz)
400 MHz) d ¼ 3.70 (s, 3H, COCH3), 7.44 (m, 3H, Ar-H), 7.52 (m, 2H,
Ar-H), 8.34 (m, 4H, Ar-H), 13.42 (bs, 1H, CONH). 13 C NMR (DMSO-
d ¼ 3.79 (s. 3H, COCH3), 4.43 (Ar-CH2), 7.28 (m, 7H, Ar-H), 7.41 (m,
3H, Ar-H), 8.91 (s, 1H, CONH), 10.67 (s, 1H, CONH). 13 C NMR
d6, 400 MHz) d ¼ 51.73, 123.70, 128.36, 128.89, 129.78, 129.85,
(DMSO-d6, 400 MHz) d ¼ 27.20, 46.84, 114.05, 118.15, 121.87,
130.09, 134.62, 137.05, 139.25, 149.83, 155.80, 162.15, 164.11. MS
(ESI): m/z (%) ¼ 284 (100%) [M þ H]þ, 351.9 (98%) [M-31]þ.
123.71, 123.83, 124.07, 129.83, 133.50, 134.90, 136.66, 146.84,
150.99, 157.71. MS (ESI): m/z (%) ¼ 368 (100%) [M þ H]þ.
2.1.3.2. Methyl-2-(4-methoxybenzamido)-5-(4-(methylsulfonyl)phe-
nyl)thiazole-4-carboxylate (14b). Pale yellow crystals, yield: 45%,
2.1.4.4. 2-(3-Phenylureido)-5-(p-tolyl)thiazole-4-carboxylic acid
(13c). White puffy powder, yield: 54%, M.P.: 226 ꢂC. FT-IR: 3395,
2947, 1685, 1538, 1207, 1187, 996. 1H NMR (DMSO-d6, 400 MHz)
d ¼2.32 (s, 3H, ArCH3), 7.00 (t, 1H, Ar-H), 7.20 (d, 2H, Ar-H), 7.28 (t,
2H, Ar-H), 7.35 (d, 1H, Ar-H), 7.47 (d, 2H, Ar-H), 10.34 (bs, 1H,
CONH), 11.17 (bs, 1H, COOH). 13 C NMR (DMSO-d6, 400 MHz)
d ¼ 28.01, 113.95, 118.04, 122.21, 123.71, 123.63, 124.42, 129.83,
133.50, 135.19, 136.66, 145.84, 150.84, 160.11. MS (ESI): m/z (%) ¼
353.9 (30%) [M þ H]þ, 307 (100%) [M-46]þ.
M.P.: 239 ꢂC. FT-IR: 3251, 2918, 1722, 1652, 1513, 1202, 1089. 1H
NMR (DMSO-d6, 400 MHz) d ¼ 3.29 (s, 3H, SO2CH3), 3.72 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 7.09 (d, 2H, Ar-H), 7.82 (d, 2H, Ar-H),
7.99 ((d, 2H, Ar-H), 8.16 (d, 2H, Ar-H),13.01 (s, 1H, CONH). 13 C NMR
(DMSO-d6, 400 MHz) d ¼ 43.31, 51.72, 55.46, 113.92, 123.16,
126.76, 130.31, 130.62, 135.59, 136.42, 140.51, 157.12, 161.94,
162.93, 164.88. MS (ESI): m/z (%) ¼ 447 (100%) [M þ H]þ.
2.1.3.3. 2-(4-Methoxybenzamido)-5-(4-(methylsulfonyl)phenyl)thia-
zole-4-carboxylic acid (14c). White puffy powder, yield: 44%, M.P.:
174 ꢂC. FT-IR: 3402, 3241, 2831, 1699, 1153, 1310, 1234, 1187. 1H
NMR (DMSO-d6, 400 MHz) d ¼ 3.29 (s, 3H, SO2CH3), 3.72 (s, 3H,
ArOCH3), 7.09 (d, 2H, Ar-H), 7.82 (d, 2H, Ar-H), 7.99 (d, 2H, Ar-H),
8.16 (d, 2H, Ar-H), 13.04 (s, 1H, COOH). 13 C NMR (DMSO-d6,
400 MHz) d ¼ 43.3, 55.46, 113.92, 123.16, 126.76, 130.31, 130.62,
135.59, 136.42, 140.51, 157.12, 161.94, 162.93, 164.88. MS (ESI): m/
z (%) ¼ 433 (30%) [M þ H]þ.
2.2. Carbonic anhydrase – III inhibition assay
2.2.1. Instrumentation
HPLC screening was conducted on the LC-2010A HT HPLC system,
coupled with column temperature controller, degasser, auto sam-
pler, and isocratic elution system, LC-solution software (to calculate
peak area), Shimadzu Corporation, Kyoto, Japan. Back pressure was
maintained around 6 MPA.
A Phenomenex BioSep-SEC s2000,
300 ꢃ 7.8 mm column was used for size exclusion chromatography.
2.1.4. Synthesis of methyl 5-(substituted)-2-(3-phenylureido)thia-
zole-4-carboxylate (13b, 14d and 16 b) and 2-(3-phenylureido)-5-
(p-tolyl)thiazole-4-carboxylic acid (13c)
Compound 13/14/16 (1.5 mM) was dissolved in dry dimethyl for-
mamide, and phenyl isocyanate (2.0 mM) was added to the reac-
tion. The reaction mixture was stirred at room temperature for
24 h. Solvent was evaporated to dryness, and the solid residue
thus obtained was recrystallized with ethyl acetate and hexane to
2.2.2. Materials
Bovine carbonic anhydrase, isozyme III (Biovar Ltd, Armenia) was
stored at 0 ꢂC in refrigerator until use. Enzyme was left for few
minutes at room temperature before analysis. HPLC-grade metha-
nol and acetonitrile (Fisher Scientific, Loughborough, UK) were
used without further purification. NaH2PO4.H2O (Gainland,
Deeside, UK), HCl (Carlo Erba, Italy), NaOH (Lonver, UK), and deion-
ised water were used for the preparation of mobile phase. All the
get the compounds 13b, 4d, and 16b, respectively, in quantitative required chemicals for the synthesis of target compounds

1486
B. A. AL-JAIDI ET AL.
(12–16b) were procured either from Fisher Scientific, 2.2.4.1. Optimisation of buffer concentration. The effect of buffer
concentration was investigated by systematically increasing the
concentration from 0.2 to 35 mM. The column was thermostatted
at 37 ꢂC throughout the chromatographic run. The mobile phase
composition was 10% acetonitrile in phosphate buffer with pH ¼
6. Optimum buffer concentration was considered to be 10 mM.
Loughborough, UK or Sigma-Aldrich, St. Louis, MO.
2.2.3. Mobile phase and sample preparation
Each target compounds (12–16b) was dissolved in a mixture of
acetonitrile: phosphate buffer, pH 6.5 (1:9 v/v) to obtain a final
concentration of 0.24 mM. Subsequently, filtration and degassing
by sonication were conducted for the mobile phase. Three differ-
ent enzyme (CA-III) concentrations of 1.065, 1.7, and 1.92 mM
were prepared from the stock solution (containing 0.865 mg in
10 mL of distilled water) by subsequent dilution method and
injected separately in each run in the chromatographic system. All
samples were used in duplicate. Vanillic acid was used as a posi-
tive control, and toluene was used as a negative control.
The mobile phase used for analysis was chosen according to
the optimised conditions obtained, where 10% of organic modi-
fier, 30 mM phosphate buffer (pH ¼ 6.5) and 37 ꢂC temperature
was used. Column was pre-equilibrated with the mobile phase for
50 min. The flow rate of mobile phase was maintained at 1 mL/
min during the run time. A wavelength of 230 nm (kmax) was
selected for the analysis of all the tested compounds.
2.2.4.2. Optimisation of mobile phase pH. The effect of pH was
investigated by increasing pH in 0.5 increment rate starting from
5.5 to 7.5. Column pH toleration was taken into consideration and
the maximum area of negative peak was chosen accordingly. The
column was thermostatted at 37 ꢂC. Optimum interaction was
found at pH ¼ 6.5.
2.2.4.3. Optimisation of organic solvent percent. The effect of
organic modifier proportion was investigated by increasing the
volume fraction of acetonitrile in the mobile phase by an incre-
ment of 5. Starting from 5 to 40%, the maximum area of negative
peak was chosen accordingly, indicating higher interaction
between enzyme and ligand. The column was thermostatted at
37 ꢂC. Optimum interaction was observed by using 10% aceto-
nitrile at a buffer concentration of 10 mM, and pH 6.5.
2.2.4.4. Optimisation of temperature. The column temperature
was gradually increased from 25 to 50 ꢂC (which is the maximum
tolerated temperature by the column). Selected temperatures
were: 25, 30, 35, 36, 37, 38, 40, 45, and 50 ꢂC. There was a sharp
increase in the interaction by increasing temperature, however,
increasing temperature could affect the column longevity, thereby
37 ꢂC was chosen as the optimum temperature.
2.2.4. Optimisation of HPLC ligand–enzyme interaction
The interaction between the enzyme and ligand in the
Hummel–Dreyer method (HDM) was optimised using vanillic acid
as a reference standard, while changing four experimental condi-
tions: buffer concentration, pH of the mobile phase, temperature,
and proportion of organic modifier, respectively. In order to study
the effect of a particular chromatographic condition, one of the
four conditions was varied, and the other three conditions were
kept constant. Each reading represents the average of duplicate
measurements and the one achieving the optimum interaction
3. Result and discussion
3.1. Chemistry
A series of 17 compounds with 2,4,5-trisubstitutedthiazole scaffold
between the enzyme and ligand was used for further consideration. were synthesised by following Schemes 1 and 2. Compounds
Scheme 1. Synthesis of 2-amino-5-substituted-4-carboxylic acid derivatives (12a–16a) and 2-amido-5-substituted-4-carboxylate/carboxylic acid derivatives (12 b, 14 b
and 14c). Reagents and conditions: (i) NaOCH3, ether, 0 ꢂC, stirring, 5 h; (ii) thiourea, methanol, reflux, 3 h; (iii) NaOH, H2O, stirring, 5 h; (iv) R2COCl, tretrahydrofuran
(THF), stirring, room temperature, 5 h; (v) NaOH, H2O, stirring, overnight.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1487
Scheme 2. Synthesis of 2-uriedothiazole derivatives (13b, 14d, 16b, 13c). Reagents and conditions: (i) DMF, room temperature, stirring, 24 h; (ii) NaOH, H2O, room
temperature, stirring, overnight.
7–11 were synthesised following well known Drazen’s reaction
Table 1. Carbonic anhydrase (CA-III) inhibitory activities of compounds 12–16b.
procedure40 by treating various aldehydes (1–5) with methyldi-
R1
R1
S
S
chloroacetate (6) in dry ether under basic condition using sodium
methoxide. Compounds 7–11 on further reaction with thiourea
under reflux condition in methanol resulted in the formation of
methyl 2-amino-5-substituted-4-carboxylate derivatives (12–16).
Hydrolysis of compounds 12–16 with aqueous sodium hydroxide
solution resulted in the formation of 2-amino-5-substituted-4-car-
boxylic acid derivatives (12a–16a). Compounds 12 and 14 on
reaction with 4-nitrobenzoyl chloride and 4-methoxybenzoyl chlor-
ide, respectively, at room temperature under basic medium (trie-
thylamine) resulted in the formation of compounds 12b, and 14b.
Compound 14b on hydrolysis with sodium hydroxide resulted in
the formation of compound 14c. Compounds 12, 16, 12a, and
16a were prepared according to the reported procedures and
their physical and spectral properties were found to be on par
with the literature report38.
R2
NH
R2
NH
HO
O
N
N
O
O
(12-16, 12b, 13b, 14b, 14d, 16b) (12a-16a, 13c, 14c)
Ki (mM)
Compound no.
R1
R2
CA-III
12
13
14
15
-C6H5
H
H
H
H
H
H
H
H
H
H
>500
>500
>500
>500
>500
0.5
18.9
86.6
81.2
-p-CH3-C6H4
-p-SO2CH3-C6H4
-p-Cl-m-NO2-C6H4
-CH2-C6H5
16
12a
13a
14a
15a
16a
12b
13b
14b
16b
13c
14c
14d
Vanillic acid
-C6H5
-p-CH3-C6H4
-p-SO2CH3-C6H4
-p-Cl-m-NO2-C6H4
-CH2-C6H5
Compounds 13, 14, and 16 on further reaction with phenyliso-
cyanate at room temperature under stirring resulted in the forma-
tion of compounds 13b, 14d, and 16b, respectively. Hydrolysis of
compounds 13b with sodium hydroxide resulted in the formation
of compound 13c.
>500
>500
>500
>500
>500
174.1
186.2
>500
6.8
-C6H5
-p-OCH3-C6H4CO
-C6H5
-p-NO2-C6H4CO
-C6H5
-p-CH3-C6H4
-p-SO2CH3-C6H4
-CH2-C6H5
-p-CH3-C6H4
-p-SO2CH3-C6H4
-p-SO2CH3-C6H4
–
-C6H5
-p-NO2-C6H4CO
3.2. Carbonic anhydrase – III inhibition assay
-C6H5
–
CA-III inhibitory activities of all the 17 target compounds (12–16b)
as shown in Table 1 were carried out by using size exclusion HDM
chromatography35–37. The HDM chromatography was preferred
over colorimetric assay of CA inhibitors, due to the susceptibility
of the colorimetric methods to the intrinsic acidic or alkaline prop-
erties of analytes (inhibitors), including the lower catalytic activity
of CA-III in CO2 hydration. Moreover, in HDM chromatographic
method, the dilution effect of the mobile phase not only provides
comparatively larger space for interaction between the macromol-
ecule (CA-III) and analytes, but also lowers the probability of self-
aggregation of macromolecules35–37,41. The HDM was further
optimised prior to use, where mobile phase was buffered to main-
tain the pH ¼ 6.5 to mimic the physiological condition and to
minimise the intrinsic properties of analytes. Moreover, acetonitrile
(10%) was used to facilitate the elution of analytes (inhibitors)
from the hydrophobic column as well as to prevent the adhesion
of macromolecule to the stationary phase of the column. A con-
the chromatographic run, as the increase in temperature could
not only influence the protein-ligand interaction but also affect
the longevity of the column.
The chromatographic analysis by HDM is basically based on
the measurement of the intensity of the formation of vacancy
(negative) peak due to the subtraction of CA-III bound portion of
analytes from the mobile phase. This method is also independent
of the enzyme catalytic activity, thereby considered as more sensi-
tive method for the estimation of inhibitory activities of analytes
as compared to the colorimetric assay35–37,41. Thus, weak inhibi-
tors created weak vacancy peaks independent of the enzyme con-
centrations. In case of potent inhibitors, intense vacancy peaks
directly proportional to the low concentrations of CA-III was
observed, followed by a steady state at higher concentrations of
stant optimised temperature of 37 ꢂC was maintained throughout CA-III solutions (Figure 1).

1488
B. A. AL-JAIDI ET AL.
uV
750000
500000
250000
0
-250000
-500000
0.0
2.5
5.0
7.5
10.0
12.5
min
Figure 1. Chromatogram showing vacancy peak (V.P), resulted after the injection of 1.7 mM CA-III solution, with mobile phase containing 0.24 mM of the com-
pound 12a.
Different concentrations of CA-III were injected in the chroma- has been observed that in case of carboxylic acid derivatives, con-
tographic system containing a constant concentration (0.24 mM) version of free amino group into amide and urea group in com-
of analytes (12a–16a) in the mobile phase. HPLC quantification pounds 13c and 14c resulted in a significant reduction of activity
was generated with Shimadzu’s LC-solution software coupled with (Ki ¼ 174.1 and 186.2 lM, respectively) as compared to the com-
Shimadzu instrument. Absorbance (A) as a response in terms of pounds 13a and 14a (Ki¼18.9 and 86.6 lM, respectively).
detector signal is obtained from the negative-peak area determin-
ation. This response was utilised to measure the concentration or
amount of the ligand bound with the CA-III enzyme [IB], by divid-
4. Conclusions
ing the area obtained for absorbance of negative peak by absorp-
tivity (a) of the standard ligand with known concentration
according to Beer–Lambert law:
In this work, a series of 17 compounds (12–16b) with 2,4,5-trisubsti-
tutedthiazole scaffold were synthesised and evaluated for their CA-
III inhibitory activities using size exclusion HDM chromatography.
Compound 12a with a free amino group at 2-position, carboxylic
acid moiety at 4-position, and a phenyl ring at 5-position of the
scaffold was found to be the most potent CA-III inhibitor (Ki
¼0.5 lM). Substitution at the para position of the phenyl ring of
the scaffold reduced the activity in case of the compounds
13a–15a; whereas, the replacement of phenyl ring with a bulky
benzyl ring completely abolished the activity (compound 16a,
Ki>500 lM). Interestingly, the presence of a carboxylic acid group
at the 4-position of the scaffold was found to be of prime import-
ance for CA-III inhibitory activity, probably by inducing electrostatic
interaction at the binding site of the enzyme; as evident from com-
pounds 12–16, 12b, 13b, 14b, 14d, and 16b having carboxylic
ester group at 4-position with no CA-III inhibitory activities
(Ki>500 lM). Furthermore, replacement of the free amino group
with amide and urea group resulted in a significant reduction of
activity (compounds 13c and 14c, Ki¼174.1 and 186.2 lM). Thus
compound 12a can be considered as a lead molecule for further
modification and development of more potent CA-III inhibitors.
A ¼ aBC
(1)
where A ¼ absorbance, a ¼ absorptivity, B ¼ cell bath length,
and C ¼ concentration.
The absorptivity (a) of the standard was then deduced from
the known concentration and absorbance of the standard using
Beer–Lambert law. The concentration of ligand bound to CA-III
(which represents the amount of ligand removed from the station-
ary phase as a result of binding to the enzyme) was measured
again using the same equation.
The inhibition constant (Ki) values were calculated according to
the previously reported37 following equation (2):
½IꢄðEꢀIBÞ
Ki ¼
(2)
IBÞ
where [I] is the concentration of the analyte in the mobile phase,
which is constant (0.24 mM) for all ligands; [IB] is the bound
amount of the inhibitor (mM); [E] is the amount of enzyme (mM)
injected in HPLC.
Compounds (12a–16a) with a free amino group at 2-position
and a carboxylic acid moiety at 4-position, having an aromatic Acknowledgements
ring at 5-position of the thiazole scaffold showed good CA-III
The authors are thankful to the Faculty of Pharmacy, Philadelphia
inhibitory activity. In particular, compound 12a with a phenyl ring
at 5-position was found to be the most potent CA-III inhibitor (Ki
¼ 0.5 lM) (Figure 1). Substitution at the para position of the phe-
nyl ring of the scaffold reduced the activity for compounds 13a,
14a, and 15a (Ki ¼ 18.9, 86.6, and 81.2 lM, respectively). However,
replacing the phenyl ring with a benzyl ring completely abolished
the activity for the compound 16a (Ki > 500 lM). Interestingly,
compounds 12–16, 12b, 13b, 14b, 14d, and 16b with carboxylic
ester group at 4-position did not show any activity (Ki > 500 lM),
indicating the importance of the carboxylic group in CA-III inhibi-
University, Jordan for providing the necessary laboratory facilities
to carry out this research project.
Disclosure statement
Authors declare that they have no competing interest.
Funding
tory activity for compounds 12a–16a probably by inducing elec- The work was supported by the Philadelphia University Research
trostatic interaction at the binding site of the enzyme. Further, it Grant (Project number: 107/16/425PU), Jordan.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1489
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ORCID
Bilal A. Al-Jaidi
Pran Kishore Deb
Qutaiba Ahmed Al Khames Aga
1165-008X
Sanaa K. Bardaweel
Raghuprasad Mailavaram
6126-5860
Katharigatta N. Venugopala
0680-1549
http://orcid.org/0000-0003-3308-8697
https://orcid.org/0000-0002-8650-2874
https://orcid.org/0000-0002-
https://orcid.org/0000-0002-4823-0708
https://orcid.org/0000-0001-
https://orcid.org/0000-0003-
Anroop B. Nair
https://orcid.org/0000-0003-2850-8669
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