1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone based α,β-unsaturated derivatives an alternate to non-sulfonamide carbonic anhydrase II inhibitors, synthesis via Sonogashira coupling, binding analysis, Lipinsk's rule validation

Article abstract of DOI:10.1016/j.bioorg.2018.11.031

A novel series of silyl-yne containing chalcone derivatives 5a-5j was synthesized by exploiting Sonogashira coupling reaction and Claisen-Schimdt condensation reaction. The synthesized derivative were characterized by spectroscopic and elemental analysis. The selective inhibition of carbonic anhydrases is considered critical in the field of medicinal chemistry because carbonic anhydrases exits in several isoforms. Synthesized compounds were subjected to carbonic anhydrase –II assay. Except 5j, the other derivatives exhibited better potential than standard acetazolamide. Compound 5e was found to be potent derivative in the series with IC₅₀ value 0.054 ± 0.001 μM. Binding analysis revealed that most potent derivative 5e binds in the active site of CA-II and single π-π stacking interaction was observed between ring structure of ligand and Phe129 having bond length 4.90 ?. Pharmacokinetics elicited that compounds obey Lipinski's rule and show significant drug score.

Full text of DOI:10.1016/j.bioorg.2018.11.031

Accepted Manuscript
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone based α,β-unsatura-
ted derivatives an alternate to non-sulfonamide carbonic anhydrase II inhibitors,
synthesis via Sonogashira coupling, binding analysis, Lipinsk’s rule validation
Jamaluddin Mahar, Aamer Saeed, Kevin D. Belfield, Fayaz Ali Larik, Pervaiz
Ali Channar, Mehar Ali Kazi, Qamar Abbas, Mubashir Hassan, Hussain Raza,
Sung-Yum Seo
PII:
S0045-2068(18)30470-X
https://doi.org/10.1016/j.bioorg.2018.11.031
YBIOO 2639
DOI:
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 May 2018
12 November 2018
20 November 2018
Please cite this article as: J. Mahar, A. Saeed, K.D. Belfield, F. Ali Larik, P. Ali Channar, M. Ali Kazi, Q. Abbas,
M. Hassan, H. Raza, S-Y. Seo, 1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone based α,β-unsaturated
derivatives an alternate to non-sulfonamide carbonic anhydrase II inhibitors, synthesis via Sonogashira coupling,
binding analysis, Lipinsk’s rule validation, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.031
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2]
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone
based
α,β-unsaturated
derivatives an alternate to non-sulfonamide carbonic anhydrase II inhibitors, synthesis via
Sonogashira coupling, binding analysis, Lipinsk’s rule validation
Jamaluddin Mahara,b, Aamer Saeeda, Kevin D. Belfieldb , Fayaz Ali Larik*a, Pervaiz Ali
Channara, Mehar Ali Kazic , Qamar Abbasd, Mubashir Hassane, Hussain Razae, Sung-Yum Seoe
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
b
Department of Chemistry, School of Optics/Center for Research and Education in Optics and
Lasers University of Central Florida, P.O. Box 162366 Orlando,FL 32816, United States
c Institute of Biochemistry, University of Sindh, Jamshoro-76080, Pakistan
dDepartment of Physiology, University of Sindh, Jamshoro, 76080, Pakistan
eDepartment of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea
Email: aamersaeed@yahoo.com and fayazali@chem.qau.edu.pk (F.A.L) Tel +92-51-9064-2128;
Fax: +92-51-9064-2241

Graphical Abstract

Abstract
A novel series of silyl-yne containing chalcone derivatives 5a-5j was synthesized by exploiting
Sonogashira coupling reaction and Claisen-Schimdt condensation reaction. The synthesized
derivative were characterized by spectroscopic and elemental analysis. The selective inhibition
of carbonic anhydrases is considered critical in the field of medicinal chemistry because carbonic
anhydrases exits in several isoforms. Synthesized compounds were subjected to carbonic
anhydrase –II assay. Except 5j, the other derivatives exhibited better potential than standard
acetazolamide. Compound 5e was found to be potent derivative in the series with IC50 value
0.054±0.001µM. Binding analysis revealed that most potent derivative 5e binds in the active site
of CA-II and single π-π stacking interaction was observed between ring structure of ligand and
Phe129 having bond length 4.90Å. Pharmacokinetics elicited that compounds obey Lipinski’s
rule and show significant drug score.
Keywords: Sonogashira coupling; Claisen-Schmidt reaction; Silyl yne chalcone derivtaives;
Carbonic anhydrase-II; Binding analysis; Lipinsk’s rule

1. Introduction
The carbonic anhydrases (CAs, EC 4.2.1.1) are widely distributed among prokaryotes and
eukaryotes. These are zinc containing metalloenzymes and are divided into distinct three gene
families. -CAs (present in vertebrates, eubacteria, algae and cytoplasm of
-
CAs (predominantly in eubacteria, algae and chloroplasts of both mono- as well as dicotyledons)
-CAs (mainly in Archaea and some eubacteria), respectively. In higher vertebrates,
carbonic anhydrases exists in various differen
-class CAs are very
relevant clinical family with 14 known isoforms found in mammalian tissues [1-5]. Carbonic II
anhydrase is the first member of zinc depended CAs which was thoroughly characterized in
1933. CA-II is a subcellular cystol bound enzyme and elicits high CO2 catalytic activity. CA-II is
involved in the catalysis of reaction between carbon dioxide and bicarbonate ion and thus play
critical role in physiological processes such as respiration and movement of carbon
dioxide/bicarbonate, homeostasis balance of CO2 and pH, biosynthetic reactions (such as
gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification and tumorigenicity
[6-8]. Up until now, the carbonic anhydrase II inhibitors are based on sulfonamides, sulfamate
and hydroxysulfamates [9-12]. However, sulfonamides have dominated the drug industry for the
designing of therapeutic agents for the inhibition of carbonic anhydrase II. Treatment of
glaucoma requires high doses of sulfonamide drugs and this sometimes results in some side
effects such as altered taste, malaise, fatigue, depression, and anorexia. Allergic adverse effect
has been observed in some human being who intake sulfonamide based drugs. Thus we
envisioned to seek potential small organic compounds based on non-sulfonamide moiety and
evaluate their role as inhibitors of carbonic anhydrase II enzyme.

α,β-unsaturated derivatives of carbonyl compounds are known as Chalcones. Chalcone
derivatives can be synthesized by using Claisen-Schmidt condensation reaction. Chalcone
derivatives serve as valuable synthon for the synthesis of wide variety of heterocycles. The
enone moiety in chalones play the crucial role in the synthesis of chalone derivatives. Chalcones
and their derivatives, whether synthetic or naturally occurring are an interesting and significant
group of molecules as they possess a wide range of pharmacological activities such as anti-
inflammatory, antimicrobial, antifungal, antibacterial, antioxidant, cytotoxic, anti-tumor,
anticancer, antimitotic, antileishmanial, anti-malarial, antitubercular, and antiviral [13-18].
A number of chalcone derivatives are reported to inhibit enzymes such as xanthine oxidase,
aldose reductase, epoxide hydrolase, protein tyrosine kinase, quinone reductase, monoamine
oxidase and lipoxygenase [19, 20]. Herein, we have designed the chalcone derivatives (Figure 1)
to explore their potential as inhibitors of carbonic anhydrase- II enzyme.
Figure 1. Structural features linked with synthesized molecule

2. Experimental
2.1 Materials and Methods
Fluorene, 4-iodophenol, acetyl chloride, trimethylamine, ethynyltrimethylsilane, copper(I)iodide,
bis(triphenylphosphine)palladium(II)dichloride, 4-(dimethylamino)benzaldehyde, 1-hydroxy-2-
naphthaldehyde. Reactions were carried out under a N2 atmosphere. THF was freshly distilled
from Na and benzophenone ketyl. AlCl3 was purified by sublimation before use. All other
1
reagents and solvents were used as received from commercial suppliers. H and 13C NMR
spectra were recorded on an NMR spectrometer at 300 and 75 MHz, respectively.
2.2 General procedure for the synthesis of 1-(2-hydroxy-5 ((trimethylsilyl)ethynyl)phenyl)
ethanone based α,β-unsaturated derivatives
Synthesis of 1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone based α,β-unsaturated
derivatives were achieved by using 1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone and
aromatic aldehydes in equivalent ratio, using piperidine (1.5 eq) as catalyst, in ethanol. Reaction
mixture was refluxed for 15 h until the reaction was completed, then reaction was cooled to room
temperature. Mixture was diluted with distilled water and neutralized with dilute HCl,
precipitated product was filtered and recrystallized from ethanol.
2.3 Experimental data
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-phenylprop-2-en-1-one (5a)
o
Yellow white solid; Yield: 81%; Rf: 0.69; m.p. 169-171 C;
FTIR (Neat, cm-1) max: 3380 (O-H), 3063 (C=C-H), 2928 (C-
Hstr), 2852 (C-Hstr), 1694 (Ar-C=O), 1603 (C=C αβ-
1
unsaturated), 1597(Aromatic, C=Cstr); 837 (Si-H); H-NMR (300 MHz, CDCl3) δ 8.29 (s, 1H),
8.22 (d, J = 15.0 Hz, 1H), 7.87 (d, J = 15 Hz, 1H), 7.76-7.43 (m, 6H), 6.91 (d, J = 7.5 Hz, 1H),

4.32 (s, 1H), 0.35 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 143.9, 137.0, 135.9,
134.2, 129.5, 129.0, 128.1, 125.2, 122.2, 120.6, 115.6, 106.7, 104.2, 0.2; MS (m/z, APCI): calcd
320.46 (M+), found 322.41 [M+2H]
3-(4-(dimethylamino)phenyl)-1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)prop-2-en-1-one
(5b)
Light yellow solid; Yield: 81%; Rf: 0.71 ; m.p. 175-176
oC; FTIR (Neat, cm-1) max: 3382 (O-H), 3061 (C=C-H),
2930 (C-Hstr), 2848 (C-Hstr), 1700 (Ar-C=O), 1601 (C=C
1
αβ-unsaturated), 1596(Aromatic, C=Cstr); 1370 (-CH3), 1276 (C-N), 839 (Si-C); H-NMR (300
MHz, CDCl3) δ 8.25 (s, 1H), 8.19 (d, J = 15.0 Hz, 1H), 7.86 (d, J = 15 Hz, 1H), 7.57 (d, J = 7.5,
Hz, 1H), 7.37 (d, J = 7.5, Hz, 1H), 6.90 (d, J = 7.5 Hz, 2H), 6.78 (d, J = 7.5 Hz, 2H), 3.99 (s,
1H), 2.93 (s, 6H), 0.23 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 151.1, 143.9, 137.0,
134.2, 129.9, 125.2, 123.8, 122.2, 120.6, 115.3, 112.7, 106.7, 104.2, 41.9, 0.2; MS (m/z, APCI):
calcd 363.25 (M+), found 365.25 [M+2H]
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (5c)
o
Yellow solid; Yield: 76%; Rf: 0.65 ; m.p. 173-174 C; FTIR
(Neat, cm-1) max: 3352 (O-H), 3090 (C=C-H), 2926 (C-Hstr),
2836 (C-Hstr), 1701 (Ar-C=O), 1629 (C=N), 1600 (C=C αβ-unsaturated), 1590(Aromatic,
C=Cstr); 1273 (C-N), 840 (Si-C);1H-NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 8.61 (d, J = 7.5 Hz,
1H), 8.15 (d, J = 15.0 Hz, 1H), 7.92 (d, J = 15.0 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H),
7.51 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 7.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 4.19 (s, 1H), 0.30 (s,
9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 149.7, 149.1, 139.9, 137.0, 135.8, 134.2, 131.2,

125.2, 123.4, 122.3, 120.6, 115.6, 106.7, 104.2, 0.15; MS (m/z, APCI): calcd 321.45 (M+), found
323.39 [M+2H]
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5d)
o
Yellow solid; Yield: 85%; Rf: 0.68 ; m.p. 172-173 C;
FTIR (Neat, cm-1) max: 3348 (O-H), 3092 (C=C-H),
2927 (C-Hstr), 2838 (C-Hstr), 1698 (Ar-C=O), 1627 (C=N), 1599 (C=C αβ-unsaturated), 1588
(Aromatic, C=Cstr); 1212 (C-O), 837 (Si-C); 1H-NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 7.95 (d,
J = 15 Hz, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.5 Hz, 1H), 7.32
13
(d, J = 15 Hz, 1H), 7.04 (d, J = 7.5 Hz, 2H), 3.95 (s, 1H), 3.81 (s, 3H), 0.35 (s, 9H); C-NMR
(75 MHz, CDCl3) δ 192.5, 161.1, 160.8, 143.9, 137.0, 134.2, 129.4, 128.7, 125.2, 122.2, 120.6,
115.6, 114.6, 106.7, 104.2, 56.0, 0.15; MS (m/z, APCI): calcd 340.48 (M+), found 343.48
[M+3H]
1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(4-(phenoxymethyl)phenyl)prop-2-en-1-one
(5e)
Yellow solid; Yield: 78 %; Rf: 0.74 ; m.p. 178-
o
179 C; FTIR (Neat, cm-1) max: 3369 (O-H),
3080 (C=C-H), 2928 (C-Hstr), 1703 (Ar-C=O),
1602 (C=C αβ-unsaturated), 1598(Aromatic, C=Cstr); 1465 (-CH2), 1215 (C-O), 840 (Si-C);1H-
NMR (300 MHz, CDCl3) δ 8.55 (s, 1H), 8.34 (d, J = 15 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.61
(d, J = 7.5 Hz, 2H), 7.49 (d, J = 15 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 7.5 Hz, 2H),
7.30 (d, J = 8.3 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 7.5 Hz, 2H), 5.16 (s, 2H), 0.38 (s,
9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 159.2, 143.9, 137.0, 135.0, 134.2, 129.5, 128.1,

127.1, 125.2, 122.2, 121.5, 120.6, 115.7, 115.6, 106.7, 104.2, 70.8, 0.2; MS (m/z, APCI): calcd
426.58 (M+), found 428.56 [M+2H]
1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(4-((4-methoxyphenoxy)methyl)phenyl)prop-
2-en-1-one (5f)
Yellow solid; Yield: 81 %; Rf: 0.73 ; m.p.
181-182 oC; FTIR (Neat, cm-1)
:
max
3363(O-H), 3078 (C=C-H), 2930 (C-Hstr),
1702 (Ar-C=O), 1600 (C=C αβ-unsaturated), 1590(Aromatic, C=Cstr); 1465 (-CH2), 1378 (-
CH3), 1213 (C-O), 835 (Si-C);1H-NMR (300 MHz, CDCl3) δ 8.80 (s, 1H), 8.12 (d, J = 15.0 Hz,
1H), 8.67 (d, J = 7.3 Hz, 1H), 8.64 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 15.0 Hz, 1H), 7.38 (d, J = 7.5
Hz, 2H), 7.13 (d, J = 7.3 Hz, 1H), 6.95 (s, 4H), 5.18 (s, 2H), 3.82 (s, 3H), 0.08 (s, 9H); 13C-NMR
(75 MHz, CDCl3) δ 192.5, 161.1, 154.7, 154.2, 143.9, 137.0, 137.0, 135.0, 134.2, 128.1, 127.1,
125.2, 122.2, 120.6, 117.1, 115.6, 115.1, 106.7, 104.2, 70.8, 56.0, 0.2; MS (m/z, APCI): calcd
456.61 (M+), found 459.47 [M+3H]
1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(pyren-1-yl)prop-2-en-1-one (5g)
Light red solid; Yield: 86 %; Rf: 0.73 ; m.p. 196-197
oC; FTIR (Neat, cm-1) max: 3352 (O-H), 3065 (C=C-
H), 2930 (C-Hstr), 1702 (Ar-C=O), 1607 (C=C αβ-
unsaturated), 1598(Aromatic, C=Cstr); 834 (Si-C);1H-NMR (300 MHz, CDCl3) δ 8.73 (s, 1H),
8.67 (d, J = 7.3 Hz, 1H), 8.61 (d, J = 7.5 Hz, 2H), 8.10 (d, J = 15.0 Hz, 1H), 8.06-7.79 (m,
5H),7.71 (s, 1H), 7.54 (d, J = 15.0 Hz, 1H), 7.35 (d, J = 7.5 Hz, 2H), 7.09 (d, J = 7.3 Hz, 1H)
3.80 (s, 1H), 0.33 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 138.0, 137.0, 134.2,
134.2, 131.6, 131.4, 129.5, 128.7, 128.3, 128.2, 128.2, 128.0, 127.8, 127.4, 125.2, 125.2, 124.9,

124.0, 123.6, 120.6, 115.6, 106.7, 104.2, 0.2; MS (m/z, APCI): calcd 444.60 (M+), found 445.59
[M+H]
1-(4-(4-(3-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-oxoprop-1-en-1-yl)-2,6-
dimethoxyphenoxy)butyl)pyrrolidine-2,5-dione (5h)
Red solid; Yield: 79 %; Rf: 0.52 ; m.p.
211-212 oC; FTIR (Neat, cm-1) max: 3328
(O-H), 3073 (C=C-H), 2928 (C-Hstr),
1702 (Ar-C=O), 1685 (C=O amide), 1605 (C=C αβ-unsaturated), 1596 (Aromatic, C=Cstr); 1373
(C-N), 841 (Si-C);1H-NMR (300 MHz, CDCl3) δ 7.98 (d, J = 15.0 Hz, 1H), 7.84 (d, J = 1.4 Hz,
1H), 7.57 (dd, J = 7.5, 1.4 Hz, 1H), 7.39 (d, J = 15.0 Hz, 1H), 6.93-6.87 (m, 3H), 4.09 (t, J = 7.6
Hz, 2H), 3.96 (s, 1H), 3.83 (s, 6H), 3.56 (t, J = 7.6 Hz, 2H), 2.87 (s, 4H), 1.74 (dq, J = 7.6, 5.6
Hz, 2H), 1.58 (tt, J = 7.5, 5.6 Hz, 2H), 0.35 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 178.2,
161.1, 153.1, 143.5, 140.9, 137.0, 134.2, 130.9, 125.2, 122.9, 120.6, 115.6, 106.7, 105.8, 104.2,
72.7, 56.8, 36.3, 28.4, 27.5, 26.8, 0.2; MS (m/z, APCI): calcd 549.69 (M+), found 555.69
[M+6H]
3-(4-(diethylamino)phenyl)-1-(2-hydroxy-5-((trimethylsilyl)ethynyl)phenyl)prop-2-en-1-one (5i)
Yellow solid; Yield: 83 %; Rf: 0.70 ; m.p. 178-179 oC;
FTIR (Neat, cm-1) max: 3333 (O-H), 3078 (C=C-H),
2928 (C-Hstr), 1702 (Ar-C=O), 1685 (C=O amide),
1608 (C=C αβ-unsaturated), 1592 (Aromatic, C=Cstr); 1460 (-CH2), 1368 (-CH3), 1278 (C-N),
836 (Si-C);1H-NMR (300 MHz, CDCl3) δ 8.18 (d, J = 15.0 Hz, 1H), 7.85 (d, J = 1.4 Hz, 1H),
7.76 (d, J = 7.5 Hz, 2H), 7.72 (dd, J = 7.5, 1.4 Hz, 1H), 7.42 (d, J =15 Hz , 1H), 6.90 (d, J = 7.5
Hz, 1H), 6.80 (d, J = 7.5 Hz, 2H), 3.96 (s, 1H), 3.61 (q, J = 6.3 Hz, 4H), 1.21 (t, J = 6.3 Hz, 6H),

0.23 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 148.8, 143.9, 137.0, 134.2, 131.9,
125.2, 123.0, 122.2, 120.6, 115.6, 110.5, 106.71104.2, 46.3, 13.0, 0.2; MS (m/z, APCI): calcd
391.58 (M+), found 393.57 [M+2H]
1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)-3-(pyridin-4-yl)prop-2-en-1-one (5j)
o
Yellow solid; Yield: 78%; Rf: 0.63 ; m.p. 175-176 C; FTIR
(Neat, cm-1) max: 3350 (O-H), 3092 (C=C-H), 2928 (C-Hstr),
1705 (Ar-C=O), 1630 (C=N), 1600 (C=C αβ-unsaturated), 1590(Aromatic, C=Cstr); 1267 (C-N),
835 (Si-C);1H-NMR (300 MHz, CDCl3) δ 8.59 (d, J = 7.4 Hz, 2H), 8.19 (d, J = 15.0 Hz, 1H),
8.04 (s, 1H), 7.90 (d, J = 15.0 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.37 (d, J = 7.4 Hz, 2H), 6.91
(d, J = 7.5 Hz, 1H), 4.34 (s, 1H), 0.34 (s, 9H); 13C-NMR (75 MHz, CDCl3) δ 192.5, 161.1, 151.5,
143.9, 143.2, 137.0, 134.2, 125.2, 122.2, 121.7, 120.6, 115.6, 106.7, 104.2, 0.2; MS (m/z, APCI):
calcd 321.45(M+), found 323.09 [M+2H]
2.4 Carbonic anhydrase assay
Carbonic anhydrase inhibition was measured as described previously with some modifications
[21]. The method is based on the principle that p-nitrophenyl acetate is hydrolyzed by Carbonic
anhydrase to form yellow colored p-nitrophenol which was measured spectrophotometrically.
Briefly, Reaction mixture contained 120 µL of 50 mM Tris-sulfate buffer (pH 7.6 containing 0.1
mM ZnCl2), 20 µL of inhibitor and 20 µL (50 U) bovine enzyme per well. Contents were well
mixed and pre-incubated at 25 °C for 10 min. substrate p-nitrophenyl acetate was prepared (6
mM stock using <5% acetonitrile in buffer and used fresh every time) and 40 µL was added per
well to achieve 0.6 mM concentration per well. Total reaction volume was made to 200 µL.
After30 min incubation at 25 °C contents were mixed and absorbance was measured at 348 nm
using a microplate reader. Acetazolamide was used as a reference inhibitor and tris-sulfate buffer

was used as negative control. Each concentration was analyzed in three independent
experiments. IC50 values were calculated by nonlinear regression using GraphPad Prism 5.0.
Inhibition (%)
Here, the B and S are the absorbances for the blank and samples.
2.5 Free radical scavenging assay
Radical scavenging activity was determined by modifying method by 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) assay [22]. The assay solution consisted of 100 mL of (150 mM) 2,2-
diphenyl-1 picrylhydrazyl (DPPH), 20 µL of increasing concentration of test compounds and the
volume was adjusted to 200 µL in each well. This reaction mixture was then incubated for 30
min at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The
measurements were carried out by using a micro plate reader (OPTIMax, tunable) at 517 nm.
The reaction rates were compared and the percent inhibition due to the presence of tested
inhibitors was calculated. Each concentration was analyzed in three independent experiments.
2.6 Selection of Carbonic anhydrase II from PDB
The crystal structure of carbonic anhydrase II was retrieved form the Protein Data Bank (PDB)
having PDBID 1V9E (www.rcsb.org). Energy minimization of target structure was carried out
by using conjugate gradient algorithm and Amber force field in UCSF Chimera 1.10.1. The
stereo-chemical properties, Ramachandran graph and values of Carbonic anhydrase II structure
were assessed by Molprobity server, while the hydrophobicity graph was generated by Discovery
Studio 4.1 Client. The protein architecture and statistical percentage values of helices, beta-
sheets, coils and turns were accessed by using online tool VADAR 1.8.
In-silico designing of synthesized ligands and Lipinski Rule validation

The synthesized ligand molecules (5a-5j) were sketched in drawing ACD/ChemSketch tool and
further minimized by visualizing software UCSF Chimera 1.10.1. The different online drug
assessment tools like Molinspiration (http://www.molinspiration.com/) and Molsoft
(http://www.molsoft.com/) were employed to predict the drug-likeness and biological properties
of these designed candidate molecules. The number of rotatable bonds, hydrogen bond acceptors
(HBA) and hydrogen bond donors (HBD) were also confirmed by PubChem
(https://pubchem.ncbi.nlm.nih.gov/). Moreover, Lipinski’s rule of five was analyzed using
Molsoft and Molinspiraion tools.
2.7 Molecular docking
The docking experiment against target protein and ligands was performed using PyRx docking
tool [23]. The grid box center values of (center_X= 11.6361, center_Y= 47.8016 and center_ Z=
22.1317) and size values were adjusted as (X= 46.4690, Y= 50.6562, and Z= 50.9719) for better
conformational position in the active region of target protein. The synthesized ligands (5a-5j)
were docked separately against carbonic anhydrase II with default exhaustiveness value = 8. The
predicted docked complexes were evaluated on the basis of lowest binding energy (Kcal/mol)
values and structure activity relationship (SAR) analyses. The three dimensional (3D) graphical
depictions of docked complexes were accomplished by Discovery Studio (2.1.0) and UCSF
Chimera 1.10.1 tool. The two dimensional graphical depictions of other complexes was
generated by LIGPLOT [24].
3. Results and Discussions
3.1 Chemistry
The synthesis of silyl group containing chalcone derivatives has been outlined in Scheme 1. 4-
Iodophenol 1 was used starting precursor. 1 was reacted under Friedel Crafts reaction conditions

to obtained acylated product 2. Ethenyl silyl group was reacted using Sonogashira coupling
protocol to afforded silylated product 3. In the last step various substituted aromatic aldehydes
were reacted with 3 to obtain the desired product (5a-5j) in good yield and high purity.

3.2 Carbonic anhydrase II inhibitory activity
Table 1 displays the results of carbonic anhydrase II inhibition assay in micromolar range. The
IC50 values of the compounds 5a-5j reveal that except 5j, other derivatives showed better
potential than the reference acetazolamide (IC50 0.998 ± 0.024 µM). Various substituents were
attached with chalcone linkage. Compounds 5c and 5j bear pyridine rings. In case of compound
5c the results of CA-II inhibition are better than 5j. Compounds 5b and 5i possess N,N-dimethyl
and N,N-diethyl groups and compound 5b expressed significant potential than 5i. Ether linkages
were also imparted in the chalcone derivatives such as 5d, 5e and 5f. Compound 5d contains
methoxy group at para position of phenyl ring and exhibited higher inhibition potency compared
to 5f. Derivative 6e also possessed ether moiety and showed better activity compared to 5d and
5e. Compound 5g contains perylene entity and it showed better activity compared to standard
acetazolamide. Molecule 5h contained multifunctional groups and showed good CA-II
inhibition.
Table. 1. Carbonic anhydrase II activity and radical scavenging activity of chalcone derivatives
(5a-5j)
Compound Carbonic anhydrase Radical Scavenging
Energy Values
(Kcal/mol)
(IC50 µM)
%
5a
5b
5c
0.356±0.008
0.093±0.002
0.033±0.008
39.708±1.538
-7.1
-7.3
-7.7
75.786±1.96
55.966±2.16

5d
5e
5f
0.205±0.004
0.054±0.001
0.577±0.013
0.093±0.002
0.992±0.024
0.131±0.003
0.910±0.022
43.035±1.66
94.312±3.65
97.251±3.766
10.731±0.14
59.422±2.3
-6.9
-7.7
-7.5
-8.7
-8.4
-7.3
-6.9
------
-----
5g
5h
5i
35.998±1.29
52.157±2.02
---------------
96.422 ±3.35
5j
Acetazolamide 0.998 ± 0.024
Vitamin C -------------
For calculation of IC50 six to eight concentrations were used. IC50 values were calculated by
nonlinear regression using GraphPad Prism 5.0.
3.3 Free radical scavenging
The synthesized chalcone series compounds were evaluated for DPPH free radical scavenging
ability. The compounds 5b, 5e, and 5f showed excellent % scavenging potency, other
compounds did not show significant radical scavenging potential even at high concentration
(100µg/mL) Table 1.
3.4 Structural assessment of carbonic anhydrase II
Carbonic anhydrase II (EC#: 4.2.1.1) is metal (Zn) containing protein which comprises 259
residues. The residual architecture of carbonic anhydrase II consists of 9% helices, 45% β sheets
and 45% coils. The X-Ray diffraction study confirmed its resolution 1.95Å, R-value 0.238 and
unit cell dimensions like length and angles of coordinates. The computational structure
assessment showed that the carbonic anhydrase II has unit cell length values (a=103.84,
b=104.82 and c=119.36) with angles (90°, 110.45° and 90°) for all α, β and γ dimensions

respectively. Furthermore, the Ramachandran graph and values also confirms the reliability and
efficacy of carbonic anhydrase II structure. The Ramachandran plots indicated that 93.8% of all
residues were present in favored regions and only six poor rotamers lies in unfavored regions
(Figure.2). This selected Ramachandran graph values showed the good accuracy of phi (φ) and
psi (ψ) angles among the coordinates of receptor molecules and most of residues plummeted in
acceptable region.
Figure.2 A) Crystal structure of bovine anhydrase II. B) Ramachandran graph accessed from
PDB.
3.5 Chemo-informatic properties and Lipinski Rule (RO5) evaluation of ligands
The designed ligands were analyzed computationally to predict the best ligand on the basis of
chemical and bio-molecular properties and RO5. The predicted chemo-informatic properties like
LogP, HBD, HBA, molar volume, polar surface area (PSA) and drug likeness values of ligand
molecules are mentioned in Table 1. It has been confirmed from previous research data that the

standard values for molecular weight (MW) and polar surface area (PSA) are (160 to 480 g/mol)
and (<89 Å2) respectively [24,]. The predicted results of compounds (5a-5j) showed good, MW
and PSA values which are comparable with standard values. RO5 also confirmed the therapeutic
potential of the ligands. Hydrogen-bonding capacity has been identified as an important
parameter for describing drug permeability. Research data revealed poor permeation is more
likely to be observed when the HBA and HBD are exceeded then 10 and 5 respectively [25]. The
chemo-informatics analysis justified that the designed compounds possess <10 HBA and <5
HBD. Moreover, their logP value were also comparable with standard value. However there are
plenty of examples available for RO5 violation amongst the existing drugs [26]. The predicted
chemo-informatic values of the designed ligand are mentioned in Table 1.
Table. 2 Chemo-informatics analysis of designed chemical compounds
Mol.
No.
No.
Mol.
PSA Mol.Vol Drug
Ligands
Wt(g/mol) HBA HBD Logp(mg/L) (A2) (cm3)
Score
0.37
0.10
1.02
0.38
0.34
0.30
0.74
0.76
0.15
0.85
320.12
363.17
321.12
350.13
426.17
456.18
446.17
549.22
391.20
321.12
2
2
3
3
3
4
2
7
2
3
1
1
1
1
1
1
1
1
1
1
4.75
4.87
3.70
4.84
6.46
6.55
8.48
4.64
5.81
3.70
29.76 346.65
32.57 396.20
39.28 342.12
37.31 378.50
36.99 451.62
44.53 483.47
29.33 511.01
82.67 593.53
32.50 434.51
39.19 341.95
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j

Abbrevation: HBA= No of hydrogen bond acceptor, HBD= No of hydrogen bond donor, LogP=
lippophilicity of partition coefficient, PSA= polar surface area,
3.5 Molecular docking and binding energy analysis
The docked complexes of all the compounds 5a-5j against carbonic anhydrase II were analyzed
separately and evaluated on the basis of minimum energy values and ligand interactions pattern.
Results showed that all compounds (5g and 5h) showed good binding energy value -8.7, and -8.4
kcal/mol, respectively and exhibited in the active region of target protein (Table 2). Furthermore,
5e showed -7.7 kcal/mol having good conformational position within the binding pocket of target
protein. Prior research showed that the standard error for Autodock is testified as 2.5 kcal/mol.
However, in all docking complexes the predicted energy values difference was less than standard
energy value. Although, the basic nucleus of all the synthesized compounds was similar,
therefore most of ligands possess good efficient energy values and have no big energy
fluctuations difference. The comparative docking analysis and inhibition constant (IC50) value
justified that 5e has good therapeutic potential as compared to all other compounds.
3.6 Binding analyses of synthesized compounds against carbonic anhydrase II
The ligands-protein binding analyses showed that 5e confined in the active binding pocket of
target protein as mentioned in Figure.3. The ligand structure showed the good conformational
position closely binds near the Zn+2 metal.

Figure. 3. Binding pocket of 5e within the active region of carbonic anhydrase II
The CA II has an active site cleft (15 Å in diameter and 15 Å deep), and contains a Zn2+ ion that
is coordinated in a tetrahedral geometry with three histidine residues (His94, His96 and His119)
and a water molecule/hydroxide ion. The 5e-receptor docked complex reveals the good
conformational state with hydrogen bond interactions within the receptor binding pocket. The
docking result of 5e receptor docked complex showed that three hydrogen bonds at Asp71 and
Gln91 respectively. The carbonyl oxygen of 5e interacts with Gln91 with bond distance 2.83Å
while benzyl methyl group form two hydrogen bonds against Asp71 with bond length 2.18 and
2.02Å, respectively. Single π-π stacking interaction was observed between ring structure of
ligand and Phe129 having bond length 4.90Å. Our docking results shows good correlation with
published research which strengthen our work and efficacy [26]. The 2D conformations and
binding pose and interactions with binding residues of all the candidate molecules are mentioned
in (Figures. S1-9).

Fig. 4 Docking interaction 5e with receptor molecule. A) The protein structure is represented in
khaki and yellow colors while the interacted residues are justified in light blue color. B) The
closer view of binding interaction. The ligand molecule is depicted in purple color while their
functional groups such as oxygen and sulphur are shown in red and yellow colors, respectively.
Amino acids are highlighted in black color and while light green color represent the hydrogen
bonds with distance mentioned in angstrom (Å). Two hydrogen bonds were observed at Asp71
and Gln91 positions in the target protein. Zinc metal is represented in cyan color.
4. Conclusions
Sonogashira coupling and Claisen-Schmidt reactions were employed to obtain silyl yne
1
containing chalcone derivatives 5a-5j. Synthesized compounds were characterized through H-
NMR and 13C-NMR spectroscopy. Compounds 5a-5j were screened against carbonic anhydrase-
II enzyme and antioxidant activity. The compounds displayed CA-II inhibition in micromolar
range and compound 6e having IC50 0.054±0.001µM and this showed several fold time better

potential than reference acetazolamide. Molecular docking studies were performed to explore the
binding affinity of potent ligand 5e in the active site of target protein and 5e showed good
binding affinity and hydrogen bonding and pi-pi stacking interactions were observed with amino
acid residues. Drug score and Lipinski’s rule ascertained that compound 5e could serve as
structural template and probably can be an alternate to sulfonamide drugs.
Conflict of interest
Authors declare no any conflict of interest
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1-(2-Hydroxy-5-((trimethylsilyl)ethynyl)phenyl)ethanone based α,β-unsaturated derivatives
an alternate to non-sulfonamide carbonic anhydrase II inhibitors, synthesis via
Sonogashira coupling, binding analysis, Lipinsk’s rule validation
a,b
a
b
a
*a

Jamaluddin Mahar , Aamer Saeed , Kevin D. Belfield , Pervaiz Ali Channar , Fayaz Ali Larik ,
Mehar Ali Kazic , Qamar Abbase, Mubashir Hassanb, Hussain Razab, Sung-Yum Seob
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
b
Department of Chemistry, School of Optics/Center for Research and Education in Optics and
Lasers University of Central Florida, P.O. Box 162366 Orlando,FL 32816, United States
c Nano Science and Catalysis Division, National Center for Physics, Quaid-i-Azam University
Campus, Islamabad, Pakistan
dDepartment of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea
eDepartment of Physiology, University of Sindh, Jamshoro, 76080, Pakistan
c Institute of Biochemistry, University of Sindh, Jamshoro-76080, Pakistan
Highlight
 Sonogashira coupling reaction was used to synthesized novel silicon containing
alkyne compounds
 Synthesized compounds were evaluated against carbonic anhydrase II enzyme
 Binding analysis and pharmacokinetics was explored