Novel N-(benzo[d]oxazol-2-yl)alkanamides; synthesis and carbonic anhydrase II inhibition studies

Article abstract of DOI:10.1002/jhet.3992

Carbonic anhydrase (CA II) inhibitors are very important therapeutic targets in drug design for treatment of neuropathic pain and in eradication of glaucoma, cancer, epilepsy, ulcer and obesity. In this study, some two2-substituted benzoxazoles (3a-j) wer

Full text of DOI:10.1002/jhet.3992

Received: 20 January 2020
DOI: 10.1002/jhet.3992
Revised: 2 March 2020
Accepted: 23 March 2020
A R T I C L E
Novel N-(benzo[d]oxazol-2-yl)alkanamides; synthesis and
carbonic anhydrase II inhibition studies
Aamer Saeed1
| Pervaiz A. Channar1 | Muhammad Arshad2
|
Hesham R. El-Seedi3 | Qamar Abbas4 | Mubashir Hassan5 | Hussain Raza6
Sung-Yum Seo6
|
1Department of Chemistry, Quaid-i-Azam
Abstract
University, Islamabad, Pakistan
2Chemistry Division Directorate of
Science, PINSTECH, Nilore, Pakistan
3Pharmacognosy Group, Department of
Medicinal Chemistry, Biomedical Center
(BMC), Uppsala University, Uppsala,
Sweden
4Department of Physiology, University of
Sindh, Jamshoro, Pakistan
5Institute of Molecular Biology and
Biotechnology (IMBB), The University of
Lahore, Lahore, Pakistan
Carbonic anhydrase (CA II) inhibitors are very important therapeutic tar-
gets in drug design for treatment of neuropathic pain and in eradication of
glaucoma, cancer, epilepsy, ulcer and obesity. In this study, some
two2-substituted benzoxazoles (3a-j) were developed as a new family of car-
bonic anhydrase II inhibitors by employing acyl thiourea chemistry via a
simple and expedient protocol and evaluated for CA II inhibitor activity
and radical scavenging ability. Compounds 3f and 3j were found to be the
most potent inhibitors, with IC50 values of 0.00564 and 0.00596 μM, respec-
tively which are several times better than that of the standard, acetazol-
amide (IC50 value 0.997 0.0586 μM). Docking experiments were carried
out against the carbonic anhydrase II crystal structure to better rationalize
the inhibitory activities of these new structures. Moreover, the results of a
DPPH radical scavenging assay showed that the antioxidant profile of com-
pound 3i is superior to those of other derivatives. The results have revealed
that derivatives 3f and 3j behave as CA-II inhibitors significantly better
than standard and 3i has good anti-oxidation potential.
6College of Natural Sciences, Department
of Biological Sciences, Kongju National
University, Gongju, Republic of Korea
Correspondence
Aamer Saeed, Department of Chemistry,
Quaid-i-Azam University, 45320,
Islamabad, Pakistan.
Email: aamersaeed@yahoo.com, asaeed@
qau.edu.pk
1 | INTRODUCTION
While examples of some bioactive molecules containing
benzoxazole from recent literature are mentioned in
Benzoxazole motif is amongst the most important five
membered nitrogen oxygen heterocycles fused to a benzene
ring exhibiting remarkable pharmacological activities.[1,2] It
is found in several naturally occurring bioactive molecules,
like UK-1,[3] salvianen,[4] AJI9561,[5] A-33853,[6]
caboxamycin,[7] and antimycobacterial pseudopteroxazole.[8]
Figure 1 shows examples of some marketed drugs con-
taining benzoxazole motif.
Figure 2 including the selective peroxisome proliferator-
activated receptor γ antagonist JTP-426467,[9c] and the
oestrogen receptor-β agonist ERB-041.[9d] Melatonin recep-
tor agonists,[9a] 5HT3 receptor agonists,[9b] are some addi-
tional examples. Furthermore, benzoxazoles have been
widely employed as versatile synthetic building blocks[3,10]
in synthesis of insecticides,[11] pharmaceuticals,[12] fluores-
cent probes,[13] and other functional materials.[14]
Carbonic
anhydrases
are
ubiquitous
zinc-
metalloenzymes principally responsible for the rapid revers-
ible conversion of CO2 to bicarbonate.[15–19] In higher verte-
brates, 16 isoforms of CA have been identified on the basis
of different cellular localizations and tissue distributions.
Present address: Hesham R. El-Seedi, International Research Center
for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013,
China; Al-Rayan Research and Innovation Center, Al-Rayan Colleges,
42541, Medina, Saudi.
J Heterocyclic Chem. 2020;1–13.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC
1

2
SAEED ET AL.
O
NH2
H
N
CH3
CH3
Cl
H
HO
N
N
O
N
O
HOOC
N
X
O
O
O
CH3
Chloroxazole
Boxazomycin B
X=Cl Benzoxaprofen
X=F Flunoxaprofen
HN
HO
O
HO
H3C
O
Cl
N
O
O
N
O O
O
CH3
H3CHN
COOH
H3C
N
CO2H
Cl
H3C
Tafamidis
Caboxamycin
Calcimycin
FIGURE 1 Some marketed drugs containing this nucleus
Cl
F
O
Cl
O
CH3
N
O
O
N
OH
NH
N
N
HO
N
NO2
H3C
CH3
antidiarrhetically active
ERB-041
JTP426467
O
N
O
S
N
S
O
N
N
R
O
O
N
NH
N
R=alkyl, aryl
R=alkyl, aryl,aralkyl
R
Ar
FIGURE 2 Bioactive compounds containing the benzoxazole core
These CA isozymes are involved in various physiological
processes, including CO2 homeostasis, pH regulation, elec-
trolyte secretion, bone resorption and calcification,
tumourigenicity, and many other metabolic processes.[15–19]
Overexpression of certain CA isozymes may lead to physio-
logical disorders such as hypoxic tumours, gastric ulcers,
glaucoma, and obesity.[20] Consequently, CA inhibitors
have attracted attention as safe and effective therapeutic
agents, and new molecules have been screened by many
research groups to identify potent inhibitors of CA.[21]
FIGURE 3 Design and architecture of carbonic anhydrase II
inhibitors synthesized
The presence of amide linkage further increases the
biological potential of benzoxazoles. Keeping in view
aforementioned importance of benxoxazole and amide
linkage,
a
new series of N-(benzo[d]oxazol-2-yl)
lengths confers diversity to these molecules (Figure 3).
The ability of these compounds to inhibit carbonic
anhydrase II activity was assessed as continuation of
our focus to design new classes of CA-II
inhibitors.[22,23]
alkanamides possessing both benzoxazole and amide
linkages in a single molecules were prepared in an
effort to prepare compounds with better biological
potential. The presence of alkyl chains (R) of various

SAEED ET AL.
3
2 | MATERIALS AND METHODS
2.1 | Chemistry
Calc. for C9H10N2O2S: C, 51.41; H, 4.79; N, 13.32; S,
15.25; found: C, 51.43; H, 4.82; N, 13.29; S, 15.27.
1-(2-Chloroacetyl)-3-(2-hydroxyphenyl)thiourea (2b)
Brown solid; Yield: 78%; Rf: 0.45 (n-hexane: ethyl acetate,
1:1); m.p.: 145ꢀC; FTIR (pure, ῡ cm−1): 3618 (OH), 3270,
All chemicals, reagents, and solvents were purchased from
Sigma-Aldrich Chemical Co. or Merck, Germany. The Rf
values were determined using aluminium pre-coated silica
gel plates Kiesel 60F254 from Merck (Darmstadt, Germany).
The melting points of the compounds were measured in
open capillaries using a Stuart melting point apparatus
(SMP3). The FTFTIR spectra recorded on FTS 3000 MX,
Bio-Rad Merlin (Excalibur Model) spectrophotometer as
pure compounds. The 1H and 13C NMR spectra were
acquired on a Bruker NMR spectrometer at 300 and
75.5 MHz using TMS as an internal standard. Mass spectra
were recorded on an Agilent Technologies 6890 N gas chro-
matograph equipped with an inert mass selective detector
(5973 mass spectrometer), and elemental analyses were
conducted using a LECO-183 CHNS analyzer.
3
3162 (NH), 2924, 2850 (Csp H), 1682 (C O), 1568, 1438
1
(Ar C C), 1283 (C S); H-NMR (300 MHz, DMSO-d6):
δ (ppm) 12.60 (s, 1H, CS NH CO), 11.22 (s, 1H,
Ar NH CS), 10.31 (s, broad, 1H, OH), 8.50 (t, 1H,
J = 6.9 Hz, Ar H), 7.07 to 7.03 (m, 1H, Ar H), 6.95 to
6.90 (m, 1H, Ar H), 6.83 to 6.78 (m, 1H, Ar H), 4.23 (s,
2H, CH2Cl); 13C-NMR (75.5 MHz, DMSO-d6): δ (ppm)
179.5 (C S), 177.0 (C O), 147.8, 131.2, 125.3, 121.1,
121.8, 116.0 (ArCs), 42.9 (CH2Cl); GC-MS: m/z
(%) = 244.01 (100) [M+ꢁ]; Anal. Calc. for C9H9ClN2O2S: C,
44.18; H, 3.71; N, 11.45; S, 13.10; found: C, 44.22; H,
3.74; N, 11.48; S, 13.09.
1-(2-Hydroxyphenyl)-3-pivaloylthiourea (2c)
Brown crystalline solid; Yield: 75%; Rf: 0.49 (n-hexane:
ethyl acetate, 1:1); m.p.: 125ꢀC to 126ꢀC; FTIR (pure, ῡ
2.1.1 | Synthesis of 1-acyl-
3-(2-hydroxyphenyl)thioureas (2a-j);
general procedure
cm−1): 3625 (OH), 3267, 3180 (NH), 2958, 2869 (Csp H),
3
1688 (C O), 1575, 1442 (Ar C C), 1293 (C S); 1H-
NMR (300 MHz, DMSO-d6): δ (ppm) 12.43 (s, 1H,
CS NH CO), 11.05 (s, 1H, Ar NH CS), 10.31 (s,
broad, 1H, OH), 8.52 (t, 1H, J = 6.9 Hz, Ar H), 7.06 to
7.01 (m, 1H, Ar H), 6.93 to 6.88 (m, 1H, Ar H), 6.81 to
6.77 (m, 1H, Ar H), 1.24 (s, 9H, C(CH3)3); 13C-NMR
(75.5 MHz, DMSO-d6): δ (ppm) 185.2 (C S), 173.9
(C O), 149.9, 130.7, 126.7, 123.4, 118.7, 115.4 (ArCs), 39.5
(C(CH3)3), 28.3 (C(CH3)3); GC-MS: m/z (%) = 252.09
(100) [M+ꢁ]; Anal. Calc. for C12H16N2O2S: C, 57.12; H,
6.39; N, 11.10; S, 12.71; found: C, 57.16; H, 6.41; N,
11.13; S, 12.75.
A solution of suitably substituted acid chloride (3 mmol) in dry
acetone (20 mL) was added dropwise to a stirred suspension of
potassium thiocyanate (3 mmol) in anhydrous acetone
(20 mL). The reaction mixture was stirred at room tem-
perature for 1 hour to afford acyl isothiocyanates (1a-j)
intermediates. To this a solution of 2-aminophenol
(3 mmol) in dry DMF (20 mL) was added, and the
resulting mixture was refluxed for 4 to 5 hours. After
cooling to room temperature, the reaction mixture was
poured onto crushed ice, and the precipitated solid
products were filtered off and thoroughly washed.
Recrystallization from ethanol afforded (2a-j) as
coloured crystals.
1-Butyryl-3-(2-hydroxyphenyl)thiourea (2d)
Grey solid; Yield: 79%; Rf: 0.46 (n-hexane:ethyl acetate,
1:1); m.p.: 133ꢀC; FTIR (pure, ῡ cm−1): 3612 (OH), 3269,
3
1-Acetyl-3-(2-hydroxyphenyl)thiourea (2a)
3170 (NH), 2937, 2848 (Csp H), 1660 (C O), 1577, 1446
1
Brown solid; Yield: 82%; Rf: 0.47 (n-hexane: ethyl acetate,
(Ar C C), 1295 (C S); H-NMR (300 MHz, DMSO-d6):
1:1); m.p.: 119ꢀC to 120ꢀC; FTIR (pure, ῡ cm−1): 3629
δ (ppm) 12.52 (s, 1H, CS NH CO), 11.47 (s, 1H,
Ar NH CS), 10.24 (s, broad, 1H, OH), 7.68 (d, 1H,
J = 7.0 Hz, Ar H), 7.54 (dd, 1H, J = 1.8 Hz, Ar H), 7.30
to 6.77 (m, 2H, Ar H), 2.41 (t, 2H, J = 6.5 Hz, COCH2),
1.66 to 1.60 (m, 2H, CH2), 0.84 (t, 3H, J = 6.0 Hz, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 187.8 (C S),
176.5 (C O), 150.1, 129.9, 124.3, 122.4, 120.7, 114.5
(ArCs), 38.3 (COCH2), 19.2, 13.1; GC-MS: m/z
(%) = 238.08 (100) [M+ꢁ]; Anal. Calc. for C11H14N2O2S: C,
55.44; H, 5.92; N, 11.76; S, 13.46; found: C, 55.40; H,
5.89; N, 11.79; S, 13.42.
3
(OH), 3273, 3167 (NH), 2960, 2869 (Csp H), 1685
(C O), 1560, 1435 (Ar C C), 1286 (C S); 1H-NMR
(300 MHz, DMSO-d6):
δ
(ppm) 12.73 (s, 1H,
CS NH CO), 11.31 (s, 1H, Ar NH CS), 10.10 (s,
broad, 1H, OH), 8.53 (t, 1H, J = 6.9 Hz, Ar H), 7.11 to
7.07 (m, 1H, Ar H), 6.93 to 6.88 (m, 1H, Ar H), 6.84 to
6.80 (m, 1H, Ar H), 2.42 (s, 3H, COCH3); 13C-NMR
(75.5 MHz, DMSO-d6): δ (ppm) 182.3 (C S), 175.6
(C O), 149.1, 129.7, 125.3, 122.2, 121.8, 115.8 (ArCs),
23.4 (CH3); GC-MS: m/z (%) = 210.05 (100) [M+ꢁ]; Anal.

4
SAEED ET AL.
1-(2-Hydroxyphenyl)-3-pentanoylthiourea (2e)
1-(2-Hydroxyphenyl)-3-pentadecanoylthiourea (2h)
Light grey solid; Yield: 85%; Rf: 0.50 (n-hexane:ethyl ace-
Light brownish solid; Yield: 74%; Rf: 0.35 (n-hexane:ethyl
tate, 1:1); m.p.: 160ꢀC; FTIR (pure, ῡ cm−1): 3635 (OH),
acetate, 1:1); m.p.: 207ꢀC; FTIR (pure, ῡ cm−1): 3640
3
3
3272, 3174 (NH), 2924, 2852 (Csp H), 1669 (C O),
(OH), 3267, 3173 (NH), 2930, 2865 (Csp H), 1678
1
1580, 1449 (Ar C C), 1297 (C S); H-NMR (300 MHz,
(C O), 1572, 1453 (Ar C C), 1305 (C S); 1H-NMR
DMSO-d6): δ (ppm) 12.76 (s, 1H, CS NH CO), 11.31 (s,
1H, Ar NH CS), 10.16 (s, 1H, OH), 8.51 (t, 1H,
J = 6.0 Hz, Ar H), 7.07 to 7.01 (m, 1H, Ar H), 6.93 to
6.90 (dd, J = 6.0 Hz, 1H, Ar H), 6.83 to 6.78 (m, 1H,
Ar H), 2.45 (t, 2H, J = 9.0 Hz, COCH2), 1.59 to 1.49 (m,
2H, CH2), 1.36 to 1.24 (m, 2H, CH2), 0.91 (s, 3H, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 177.7 (C S),
175.6 (C O), 149.1, 126.7, 126.3, 123.4, 118.7, 115.4
(ArCs), 35.8 (COCH2), 26.9, 22.1 (CH2), 14.1 (CH3); GC-
MS: m/z (%) = 252.09 (100) [M+ꢁ]; Anal. Calc. for
C12H16N2O2S: C, 57.12; H, 6.39; N, 11.10; S, 12.71;
found: C, 57.15; H, 6.41; N, 11.13; S, 12.75.
(300 MHz, DMSO-d6):
δ
(ppm) 12.70 (s, 1H,
CS NH CO), 11.55 (s, 1H, Ar NH CS), 10.15 (s,
broad, 1H, OH), 7.69 (d, 1H, J = 6.8 Hz, Ar H), 7.22 to
7.17 (m, 1H, Ar H), 6.84 to 6.78 (m, 1H, Ar H), 6.76 to
6.70 (m, 1H, Ar H), 2.30 (t, 2H, J = 6.4 Hz, COCH2),
1.61 to 1.55 (m, 2H, CH2), 1.28 (s, 22H, CH2), 0.88 (t, 3H,
J = 4.6 Hz, CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ (ppm)
175.2 (C S), 170.5 (C O), 148.2, 126.7, 125.4, 125.0, 122.5,
118.7 (ArCs), 35.2 (COCH2), 32.2, 29.0, 25.5, 23.9, 22.5
(CH2), 14.5 (CH3); GC-MS: m/z (%) = 392.25 (100) [M+ꢁ];
Anal. Calc. for C22H36N2O2S: C, 67.30; H, 9.24; N, 7.14; S,
8.17; found: C, 67.33; H, 9.26; N, 7.16; S, 8.19.
1-Heptanoyl-3-(2-hydroxyphenyl)thiourea (2f)
1-(2-Hydroxyphenyl)-3-hexadecanoylthiourea (2i)
Light brownish solid; Yield: 65%; Rf: 0.33 (n-hexane:ethyl
acetate, 1:1); m.p.: 215ꢀC; FTIR (pure, ῡ cm−1): 3669
(OH), 3279, 3185 (NH), 2937, 2858 (Csp H), 1665
(C O), 1583, 1454 (Ar C C), 1217 (C S); 1H-NMR
Brown solid; Yield: 80%; Rf: 0.39 (n-hexane:ethyl acetate,
1:1); m.p.: 177ꢀC; FTIR (pure, ῡ cm−1): 3645 (OH), 3275,
3
3
3181 (NH), 2928, 2856 (Csp H), 1654 (C O), 1574, 1445
1
(Ar C C), 1258 (C S); H-NMR (300 MHz, DMSO-d6):
δ (ppm) 12.68 (s, 1H, CS NH CO), 11.18 (s, 1H,
Ar NH CS), 10.60 (s, 1H, OH), 7.69 (d, 1H, J = 6.9 Hz,
Ar H), 7.56 to 7.50 (m, 1H, Ar H), 7.30 to 7.25 (m, 1H,
Ar H), 6.93 to 6.98 (m, 1H, Ar H), 2.51 (t, 2H,
J = 6.6 Hz, COCH2), 1.58 to 1.45 (m, 2H, CH2), 1.36 to
1.30 (m, 2H, CH2), 1.25 (s, 4H, CH2), 0.90 (s, 3H, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 177.8 (C S),
175.4 (C O), 151.9, 130.3, 129.8, 126.5, 123.4, 119.6
(ArCs), 37.3 (COCH2), 31.3, 28.5, 26.0, 22.8 (CH2), 14.5
(CH3); GC-MS: m/z (%) = 280.12 (100) [M+ꢁ]; Anal. Calc.
for C14H20N2O2S: C, 59.97; H, 7.19; N, 9.99; S, 11.44;
found: C, 59.99; H, 7.22; N, 9.94; S, 11.46.
(300 MHz, DMSO-d6):
δ
(ppm) 12.77 (s, 1H,
CS NH CO), 11.60 (s, 1H, Ar NH CS), 10.13 (s,
broad, 1H, OH), 7.66 (d, 1H, J = 6.7 Hz, Ar H), 7.25 to
7.20 (m, 1H, Ar H), 6.84 to 6.78 (m, 1H, Ar H), 6.74 to
6.69 (m, 1H, Ar H), 2.33 (t, 2H, J = 6.4 Hz, COCH2),
1.59 to 1.53 (m, 2H, CH2), 1.29 (s, 24H, CH2), 0.87 (t, 3H,
J = 4.6 Hz, CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ
(ppm) 177.4 (C S), 172.7 (C O), 148.2, 126.7, 125.4,
125.0, 122.5, 118.4 (ArCs), 37.0 (COCH2), 32.2, 29.0, 25.5,
23.9, 22.5 (CH2), 13.9 (CH3); GC-MS: m/z (%) = 406.27 (100)
[M+ꢁ]; Anal. Calc. for C23H38N2O2S: C, 67.94; H, 9.42; N,
6.89; S, 7.89; found: C, 67.96; H, 9.45; N, 6.93; S, 7.87.
1-(2-Hydroxyphenyl)-3-octanoylthiourea (2g)
1-(2-Hydroxyphenyl)-3-stearoylthiourea (2j)
Dark brown solid; Yield: 78%; Rf: 0.37 (n-hexane:ethyl ace-
Light brownish solid; Yield: 69%; Rf: 0.32 (n-hexane:ethyl
tate, 1:1); m.p.: 198ꢀC to 200ꢀC; FTIR (pure, ῡ cm−1): 3623
acetate, 1:1); m.p.: 248ꢀC; FTIR (pure, ῡ cm−1): 3649
3
3
(OH), 3280, 3179 (NH), 2927, 2854 (Csp H), 1693 (C O),
(OH), 3263, 3178 (NH), 2929, 2850 (Csp H), 1672
1
1580, 1449 (Ar C C), 1274 (C S); H-NMR (300 MHz,
(C O), 1602, 1437 (Ar C C), 1235 (C S); 1H-NMR
DMSO-d6): δ (ppm) 12.75 (s, 1H, CS NH CO), 11.59 (s,
1H, Ar NH CS), 10.17 (s, 1H, OH), 7.65 (d, 1H,
J = 6.8 Hz, Ar H), 7.27 to 7.21 (m, 1H, Ar H), 6.86 to
6.80 (m, 1H, Ar H), 6.77 to 6.71 (m, 1H, Ar H), 2.50 (t,
2H, J = 6.6 Hz, COCH2), 1.61 to 1.57 (m, 2H, CH2), 1.30 to
1.36 (m, 2H, CH2), 1.25 (s, 6H, CH2), 0.85 (t, 3H,
J = 4.4 Hz, CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ (ppm)
177.6 (C S), 171.3 (C O), 149.1, 126.8, 125.0, 125.0, 122.7,
118.4 (ArCs), 36.1 (COCH2), 31.6, 29.0, 25.7, 24.8, 22.5
(CH2), 14.3 (CH3); GC-MS: m/z (%) = 294.14 (100) [M+ꢁ];
Anal. Calc. for C15H22N2O2S: C, 61.19; H, 7.53; N, 9.52; S,
10.89; found: C, 61.17; H, 7.55; N, 9.54; S, 10.91.
(300 MHz, DMSO-d6):
δ
(ppm) 12.55 (s, 1H,
CS NH CO), 10.88 (s, 1H, Ar NH CS), 10.10 (s,
broad, 1H, OH), 7.67 (d, 1H, J = 6.9 Hz, Ar H), 7.27 to
7.22 (m, 1H, Ar H), 6.84 to 6.78 (m, 1H, Ar H), 6.77 to
6.72 (m, 1H, Ar H), 2.31 (t, 2H, J = 6.4 Hz, COCH2),
1.60 to 1.54 (m, 2H, CH2), 1.27 (s, 26H, CH2), 0.89 (t, 3H,
J = 4.7 Hz, CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ
(ppm) 180.0 (C S), 173.4 (C O), 147.7, 127.2, 126.0,
125.1, 122.5, 119.2 (ArCs), 36.9 (COCH2), 32.2, 29.0, 25.5,
23.9, 22.5 (CH2), 14.1 (CH3); GC-MS: m/z (%) = 434.30 (100)
[M+ꢁ]; Anal. Calc. for C25H42N2O2S: C, 69.08; H, 9.74; N,
6.44; S, 7.38; found: C, 69.11; H, 9.76; N, 6.47; S, 7.40.

SAEED ET AL.
5
2.1.2 | Synthesis of N-(benzo[d]oxazol-
2-yl)alkanamides (3a-j), general procedure
Ar H), 7.40 (dd, 1H, J = 6.1 Hz, Ar H), 6.80 to 6.76 (m,
2H, Ar H), 2.32 (t, 2H, J = 6.0 Hz, COCH2), 1.66 to 1.60
(m, 2H, CH2), 0.90 (t, 3H, J = 6.1 Hz, CH3); 13C-NMR
(75.5 MHz, DMSO-d6): δ (ppm) 169.3 (C O), 154.7
(C N), 149.5, 144.3, 123.5, 120.9, 114.5, 108.7 (ArCs),
37.6, 19.7 (CH2), 13.5 (CH3); GC-MS: m/z (%) = 204.09
(100) [M+ꢁ]; Anal. Calc. for C11H12N2O2: C, 64.69; H,
5.92; N, 13.72; found: C, 64.65; H, 5.88; N, 13.77.
The synthesized thioureas (2a-j) were heated above their
melting points on a sand bath and evolution of H2S gas
was noticed during fusion. On completion, marked by
TLC and cessation of H2S (in approx.30 minutes). The
solid products obtained were cooled to room temperature
and purified by re-crystallization from toluene.
N-(benzo[d]oxazol-2-yl)pentanamide (3e)
N-(benzo[d]oxazol-2-yl)acetamide (3a)
Dark brown solid; Yield: 79%; m.p.: 200ꢀC to 201ꢀC; FTIR
3
Dark brown solid; Yield: 69%; m.p.: 160ꢀC to 161ꢀC; FTIR
(pure, ῡ cm−1): 3320 (NH), 2937, 2858 (Csp H), 1670
(pure, ῡ cm−1): 3312 (NH), 2939, 2855 (Csp H), 1658
(C O), 1568 (C N); H-NMR (300 MHz, DMSO-d6): δ
3
1
(C O), 1568 (C N); 1H-NMR (300 MHz, DMSO-d6): δ
(ppm) 9.69 (s, broad, 1H, NH), 7.66 to 7.61 (m, 1H, Ar H),
7.34 to 7.30 (m, 1H, Ar H), 6.93 to 6.88 (m, 2H, Ar H),
2.31 (s, 3H, CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ
(ppm) 168.6 (C O), 158.2 (C N), 145.5, 143.3, 123.5,
121.7, 116.0, 108.7 (ArCs), 23.9 (CH3); GC-MS: m/z
(%) = 176.06 (100) [M+ꢁ]; Anal. Calc. for C9H8N2O2: C,
61.36; H, 4.58; N, 15.90; found: C, 61.32; H, 4.60; N, 15.87.
(ppm) 9.61 (s, broad, 1H, NH), 7.68 to 7.65 (m, 1H,
Ar H), 7.36 to 7.31 (m, 1H, Ar H), 6.95 to 6.89 (m, 2H,
Ar H), 4.00 (t, 2H, J = 6.0 Hz, CH2), 1.74 to 1.65 (m, 2H,
CH2), 1.49 to 1.36 (m, 2H, CH2), 0.92 (t, J = 6.0 Hz, 3H,
CH3); 13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 174.0
(C O), 158.3 (C N), 149.1, 144.5, 124.9, 123.4, 115.4,
109.9 (ArCs), 38.1, 26.9, 20.4 (CH2), 13.0 (CH3); GC-MS:
m/z (%)
=
218.11 (100) [M+ꢁ]; Anal. Calc. for
C12H14N2O2: C, 66.04; H, 6.47; N, 12.84; found: C,
66.07; H, 6.53; N, 12.80.
N-(benzo[d]oxazol-2-yl)-2-chloroacetamide (3b)
Brown crystalline; Yield: 72%; m.p.: 200ꢀC to 202ꢀC;
FTIR (pure, ῡ cm−1): 3309 (NH), 2937, 2858 (Csp H),
N-(benzo[d]oxazol-2-yl)heptanamide (3f)
3
1
1665 (C O), 1559 (C N); H-NMR (300 MHz, DMSO-
Grey Solid; Yield: 80%; m.p.: 205ꢀC to 206ꢀC; FTIR (pure,
3
d6): δ (ppm) 10.82 (s, broad, 1H, NH), 7.68 to 7.63 (m, 1H,
Ar H), 7.33 to 7.29 (m, 1H, Ar H), 6.95 to 6.91 (m, 2H,
Ar H), 4.20 (s, 2H, CH2Cl); 13C-NMR (75.5 MHz,
DMSO-d6): δ (ppm) 168.4 (C O), 155.9 (C N), 148.4,
142.6, 124.1, 122.8, 115.4, 110.1 (ArCs), 45.2 (CH2Cl); GC-
MS: m/z (%) = 210.02 (100) [M+ꢁ]; Anal. Calc. for
C9H7ClN2O2: C, 51.32; H, 3.35; N, 13.30; found: C,
51.35; H, 3.40; N, 13.22.
ῡ cm−1): 3314 (NH), 2937, 2858 (Csp H), 1690 (C O),
1653 (C N); 1H-NMR (300 MHz, DMSO-d6): δ (ppm)
11.05 (s, broad, 1H, NH), 7.60 to 7.54 (m, 1H, Ar H),
7.33 to 7.29 (m, 1H, Ar H), 6.74 to 6.69 (m, 2H, Ar H),
2.03 (t, 2H, J = 6.2 Hz, CH2), 1.79 to 1.71 (m, 2H, CH2),
1.45 to 1.33 (m, 6H, CH2), 0.85 (t, 3H, J = 6.5 Hz, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 167.3 (C O),
155.9 (C N), 149.5, 145.3, 125.0, 124.6, 118.5, 110.3
(ArCs), 36.0, 31.6, 28.6, 26.9, 22.3 (CH2), 13.9 (CH3); GC-
MS: m/z (%) = 246.14 (100) [M+ꢁ]; Anal. Calc. for
C14H18N2O2: C, 68.27; H, 7.37; N, 11.37; found: C,
68.25; H, 7.40; N, 11.33.
N-(benzo[d]oxazol-2-yl)pivalamide (3c)
Dark brown solid; Yield: 79%; m.p.: 194ꢀC to 196ꢀC; FTIR
(pure, ῡ cm−1): 3317 (NH), 2937, 2858 (Csp H), 1670
3
1
(C O), 1543 (C N); H-NMR (300 MHz, DMSO-d6): δ
(ppm) 10.09 (s, broad, 1H, NH), 7.63 (d, 1H, J = 7.2 Hz,
Ar H), 7.37 (dd, 1H, J = 6.0 Hz, Ar H), 7.31 to 7.26 (m,
2H, Ar H), 1.25 (s, 9H, CH3); 13C-NMR (75.5 MHz,
DMSO-d6): δ (ppm) 170.2 (C O), 157.2 (C N), 147.5,
143.3, 122.9, 121.7, 115.8, 109.2 (ArCs), 38.3 (C(CH3)3),
27.9 (C(CH3)3); GC-MS: m/z (%) = 218.11 (100) [M+ꢁ];
Anal. Calc. for C12H14N2O2: C, 66.04; H, 6.47; N, 12.84;
found: C, 66.09; H, 6.52; N, 12.80.
N-(benzo[d]oxazol-2-yl)octanamide (3g)
Dark brown powder; Yield: 73%; m.p.: 210ꢀC to 212ꢀC;
FTIR (pure, ῡ cm−1): 3308 (NH), 2937, 2858 (Csp H),
3
1
1678 (C O), 1659 (C N); H-NMR (300 MHz, DMSO-
d6): δ (ppm) 9.59 (s, broad, 1H, NH), 7.58 to 7.54 (m, 1H,
Ar H), 7.41 to 7.35 (m, 1H, Ar H), 6.88 to 6.73 (m, 2H,
Ar H), 2.94 (t, 2H, J = 7.5 Hz, CH2), 1.60 to 1.56 (m, 2H,
CH2), 1.25 (s, 8H, CH2), 0.88 (t, 3H, J = 6.5 Hz, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 172.4 (C O),
157.2 (C N), 148.3, 141.1, 126.4, 124.5, 119.0, 110.7
(ArCs), 36.3, 31.6, 29.0, 28.9, 24.8, 22.3 (CH2), 14.4 (CH3);
GC-MS: m/z (%) = 260.15 (100) [M+ꢁ]; Anal. Calc. for
C15H20N2O2: C, 69.20; H, 7.74; N, 10.76; found: C,
69.22; H, 7.78; N, 10.77.
N-(benzo[d]oxazol-2-yl)butyramide (3d)
Dark brown solid; Yield: 81%; m.p.: 184ꢀC to 186ꢀC; FTIR
(pure, ῡ cm−1): 3321 (NH), 2930, 2853 (Csp H), 1650
3
1
(C O), 1566 (C N); H-NMR (300 MHz, DMSO-d6): δ
(ppm) 9.92 (s, broad, 1H, NH), 7.65 (d, 1H, J = 7.2 Hz,

6
SAEED ET AL.
N-(benzo[d]oxazol-2-yl)pentadecanamide (3h)
(pH 7.6 containing 0.1 mM ZnCl2), 20 μL of inhibitor and
20 μL (50 U) bovine enzyme per well. Contents were
mixed well and pre-incubated at 25ꢀC for 10 minutes.
The solution of the substrate, p-nitrophenyl acetate, was
prepared fresh every time (6 mM stock using <5% aceto-
nitrile in buffer), and 40 μL of fresh solution was added
to each well to achieve a final concentration of 0.6 mM.
The total reaction volume was 200 μL. After 30 minutes
of incubation at 25ꢀC, the contents were mixed, and the
absorbance was measured at 348 nm using a microplate
reader. Acetazolamide was used as a reference inhibitor,
and Tri-sulfate buffer was used as a negative control.
Each concentration was analyzed in three independent
experiments. The IC50 values were calculated by
nonlinear regressions using GraphPad Prism 5.0.
Brown crystalline solid; Yield: 77%; m.p.: 220ꢀC to 222ꢀC;
FTIR (pure, ῡ cm−1): 3326 (NH), 2937, 2858 (Csp H),
3
1
1660 (C O), 1569 (C N); H-NMR (300 MHz, DMSO-
d6): δ (ppm) 11.31 (s, broad, 1H, NH), 7.68 to 7.54 (m, 1H,
Ar H), 7.30 to 7.25 (m, 1H, Ar H), 6.83 to 6.78 (m, 2H,
Ar H), 2.90 (t, 2H, J = 7.3 Hz, CH2), 1.61 to 1.55 (m, 2H,
CH2), 1.29 (s, 22H, CH2), 0.83 (t, 3H, J = 6.0 Hz, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 171.3 (C O),
157.7 (C N), 149.1, 141.5, 126.6, 123.9, 119.2, 109.4
(ArCs), 36.7, 32.3, 31.6, 28.6, 27.4, 24.2, 22.3, 20.1 (CH2),
14.1 (CH3); GC-MS: m/z (%) = 358.26 (100) [M+ꢁ]; Anal.
Calc. for C22H34N2O2: C, 73.70; H, 9.56; N, 7.81; found: C,
73.66; H, 9.59; N, 7.84.
N-(benzo[d]oxazol-2-yl)palmitamide (3i)
Dark brown solid; Yield: 74%; m.p.: 227ꢀC to 229ꢀC; FTIR
Inhibition ð%Þ = ½ðB−SÞ=Bꢂ × 100
(pure, ῡ cm−1): 3305 (NH), 2937, 2858 (Csp H), 1650
3
(C O), 1568 (C N); 1H-NMR (300 MHz, DMSO-d6): δ
(ppm) 9.25 (s, broad, 1H, NH), 7.65 to 7.60 (m, 1H, Ar H),
7.36 to 7.31 (m, 1H, Ar H), 6.92 (dd, 2H, J = 8.0 Hz,
Ar H), 2.88 (t, 2H, J = 7.3 Hz, CH2), 1.58 to 1.46 (m, 2H,
CH2), 1.28 (s, 24H, CH2), 0.87 (s, 3H, CH3); 13C-NMR
(75.5 MHz, DMSO-d6): δ (ppm) 166.3 (C O), 159.5 (C N),
150.6, 141.8, 126.6, 123.9, 114.6, 109.8 (ArCs), 35.2, 29.3,
22.3, 20.1 (CH2), 13.8 (CH3); GC-MS: m/z (%) = 372.28
(100) [M+ꢁ]; Anal. Calc. for C23H36N2O2: C, 74.15; H,
9.74; N, 7.52; found: C, 74.17; H, 9.77; N, 7.48.
Here, B and S are the absorbance of the blank sam-
ples and those with the test compounds, respectively.
2.3 | Free radical scavenging assay
The radical scavenging activities of the test compounds
were determined by modifying the method of a reported
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.[25] The assay
solution consisted of 100 mL of 150 mM DPPH, 20 μL of
solutions of increasing concentrations of test compounds,
and the volume of each was adjusted to 200 μL. The reac-
tion mixtures were then incubated for 30 minutes at
room temperature. Ascorbic acid (vitamin C) was used as
a reference inhibitor. The measurements were carried out
using a micro plate reader (OPTIMax, tunable) at
517 nm. The reaction rates were compared, and the per-
cent inhibition due to the presence of the test compound
was calculated. Each concentration was analyzed in three
independent experiments.
N-(benzo[d]oxazol-2-yl)stearamide (3j)
Dark brown solid; Yield: 79%; m.p.: 234ꢀC to 236ꢀC; FTIR
(pure, ῡ cm−1): 3315 (NH), 2927, 2849 (Csp H), 1680
3
1
(C O), 1567 (C N); H-NMR (300 MHz, DMSO-d6): δ
(ppm) 11.59 (s, broad, 1H, NH), 7.58 to 7.54 (m, 1H, Ar-
H), 7.30 to 7.24 (m, 1H, Ar H), 6.90 (dd, 2H, J = 8.0 Hz,
Ar H), 2.46 (t, 2H, J = 6.8 Hz, CH2), 1.60 to 1.54 (m, 2H,
CH2), 1.23 (s, 28H, CH2), 0.85 (t, 3H, J = 6.0 Hz, CH3);
13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 166.3 (C O),
158.3 (C N), 149.1, 140.9, 126.9, 122.7, 116.3, 110.3 (ArCs),
36.3, 29.4, 25.7, 22.1 (CH2), 13.9 (CH3); GC-MS: m/z
(%) = 400.31 (100) [M+ꢁ]; Anal. Calc. for C25H40N2O2: C,
74.95; H, 10.06; N, 6.99; found: C, 74.99; H, 10.11; N, 6.95.
2.4 | Computational methodology
2.4.1 | Retrieval of carbonic anhydrase II
from PDB
2.2 | Carbonic anhydrase assay
The three-dimensional (3D) crystal structure of carbonic
anhydrase II was retrieved from the Protein Data Bank
(PDB) with PDBID 1V9E (www.rcsb.org). Energy mini-
mization of the target structure was carried out using a
conjugate gradient algorithm and an Amber force field in
UCSF Chimera 1.10.1.[26] The stereo-chemical properties,
Ramachandran graph and values[27] of the carbonic
anhydrase II structure were assessed by the MolProbity
Carbonic anhydrase inhibition was measured as
described previously with some modifications.[24] The
method is based on the principle that p-nitrophenyl ace-
tate is hydrolysed by carbonic anhydrase to form p-
nitrophenol, which is yellow colored and can therefore
be measured spectrophotometrically. Briefly, the reaction
mixture contained 120 μL of 50 mM Tri-sulfate buffer

SAEED ET AL.
7
server,[28] while the hydrophobicity graph was generated
by Discovery Studio 4.1 Client.[29] The protein architec-
ture and statistical proportions of helices, beta sheets,
coils and turns were accessed by using the online tool
VADAR 1.8.[30]
(HBD) were also confirmed by PubChem (https://
pubchem.ncbi.nlm.nih.gov/). Moreover, adherence to
Lipinski's rule of five was analyzed using Molsoft and
MolinspIRaion tools.
2.4.3 | Molecular docking simulation
using PyRx
2.4.2 | In silico design of synthesized
compounds and Lipinski's rule validation
The PyRx docking tool was used to perform molecular
docking experiments for all the synthesized ligands (3a-j)
against carbonic anhydrase II.[31] To perform the docking
experiments, grid box dimension values were adjusted to
X = 11.6361, Y = 47.8016 and Z = 22.1317. A default
exhaustiveness value of 8 was used to obtain the finest
binding conformational pose of the protein-ligand docked
complexes. All compounds were docked separately
against the crystal structure of carbonic anhydrase II.
The predicted docked complexes were further evaluated
The synthesized ligands (3a-j) were sketched using the
ACD/ChemSketch tool and further minimized by visual-
izing software (UCSF Chimera 1.10.1). Different online
drug assessment tools, such as Molinspiration (http://
www.molinspFTIRation.com/) and Molsoft (http://www.
molsoft.com/), were employed to predict the drug-
likeness and biological properties of these designed can-
didate molecules. The numbers of rotatable bonds, hydro-
gen bond acceptors (HBA) and hydrogen bond donors
SCHEME 1 Synthetic route and structures of benzoxazoles (3a-j)

8
SAEED ET AL.
TABLE 1
synthesized compounds 3a-j
Carbonic anhydrase inhibitory activities of
on the basis of lowest binding energy (kcal/mol) values,
and their hydrogen bond and hydrophobic interaction
patterns were analyzed using Discovery Studio (4.1) and
UCSF Chimera 1.10.1.
3 | RESULTS AND DISCUSSION
3.1 | Chemistry
Compounds
R
IC50 (μM)
3a
CH3
0.06221 0.00382
0.29755 0.0013
0.1466 0.0056
0.12907 0.0085
0.0269 0.0032
0.00564 0.00016
0.0927 0.0024
0.0146 0.00094
0.06499 0.00298
0.00596 0.00021
0.997 0.0451
3b
C(CH3)3
CH2Cl
The strategy adopted for the synthesis of the title com-
pounds is depicted in Scheme 1. Acyl isothiocyanates (1a-
j) prepared by reaction of corresponding acid chlorides
with potassium thiocyanate, in dry acetone, were reacted
with o-aminophenol in N,N-dimethylformamide under
reflux to afford 1-acyl-3-(2-hydroxyphenyl)thioureas (2a-
j). The heterocyclization with concurrent expulsion of the
H2S was achieved by heating thiourea derivatives (2a-j)
at elevated temperature to furnish the desired N-(benzo
[d]oxazol-2-yl)alkanamides (3a-j) in good yields.
3c
3d
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)6CH3
(CH2)13CH3
(CH2)14CH3
(CH2)16CH3
3e
3f
3g
3h
3i
3j
Acetazolamide
3.2 | Characterization
strongest affinities, with IC50 values of 0.00564 and
0.00596 μM, respectively. The compounds with
chloromethyl (3c; IC50 = 0.1466 μM) and propyl (3d;
IC50 = 0.12907 μM) R groups showed weaker affinities.
Among the compounds in this series, 3b, with its bulky
t-Bu group (IC50 = 0.29755 μM), was least active. It
seems that the length of the alkyl chain has a significant
effect on the enzyme inhibition profile of the tested
compounds.
The elemental analyses and spectral data were in close
agreement with the proposed benzoxazole structures. In
the FTFTIR spectra of the prepared compounds, the dis-
appearance of the NH and OH peaks and the appearance
of absorption peaks in the range 1543 to 1659 cm−1 for
C N indicated the cyclization of the thiourea moieties to
benzoxazoles. The 1H NMR spectra of all compounds
exhibited a downfield proton for N H at 9.25 to
11.59 ppm. In the 13C NMR the carbonyl carbon
appeared at δ 166.3 to 174.0, while the C N carbon reso-
nated in the range of 154.7 to 159.5 ppm. The signal at
35.0 to 38.3 ppm was assigned to the CH2 adjacent to the
carbonyl, while the remainder of the carbons appeared in
their expected regions.
3.4 | Free radical scavenging
All the synthesized N-(benzo[d]oxazol-2-yl)alkanamides
(3a-j) were evaluated for DPPH free radical scavenging
ability. The compound 3i showed good % scavenging
potency, other compounds did not show significant radi-
cal scavenging potential even at high concentration
(100 μg/mL) (Table 2).
3.3 | Carbonic anhydrase inhibitory
activity
The title compounds (3a-j) were screened for in vitro
carbonic anhydrase II inhibition activities, and the
obtained results are summarized in Table 1. Examina-
tion of these data revealed that all newly synthesized
benzoxazoles were more potent, with IC50 values
ranging from 0.00564 to 0.29755 μM, than the stan-
dard inhibitor (acetazolamide, IC50 = 0.997 μM). The
R groups generally appear to influence the CA II
inhibitory activities. For instance, the analogues with
hexyl and heptadecyl groups (3f and 3j) showed the
3.5 | Structural assessment of carbonic
anhydrase II
Carbonic anhydrase II (EC#: 4.2.1.1) is a metal (Zn)-
containing protein that comprises 259 residues. The
residual architecture of carbonic anhydrase II consists
of 9% helices, 45% β sheets and 45% coils. An X-ray dif-
fraction study confirmed its structure to a resolution of
1.95 Å with an R value of 0.238, and unit cell

SAEED ET AL.
9
TABLE 2
synthesized compounds 3a-j
Antioxidant activity of
DPPH % activity concentrations (μg/mL)
Compounds
5
10
15
20
40
60
80
100
3a
35.37
28.52
7.21
38.95
30.85
8.15
40.81
39.11
9.31
43.67
48.51
11.81
32.97
32.64
30.65
30.34
16.41
27.25
12.11
81.31
58.52
61.52
13.17
48.75
45.41
44.62
44.37
22.96
39.41
21.21
89.22
67.57
74.43
16.89
59.27
51.63
52.23
51.28
30.26
54.39
26.29
90.52
73.69
81.59
21.04
67.01
58.83
58.71
57.41
37.28
61.58
31.24
94.36
88.27
93.74
86.42
90.91
83.36
76.18
80.72
43.81
98.11
35.36
95.97
3b
3c
3d
21.51
24.51
24.41
23.38
5.19
24.61
26.25
23.48
26.94
8.43
25.58
28.34
25.65
28.09
12.56
23.46
11.98
70.98
3e
3f
3g
3h
3i
11.03
7.38
18.99
8.16
3j
Vitamin C
45.83
63.51
Note: Results are presented as a mean of n = 3, SEM <3%.
FIGURE 4 A, Crystal structure of bovine anhydrase II. B, Ramachandran graph accessed from PDB
dimensions, such as lengths and angles of coordinates,
could be obtained. The computational structure assess-
ment showed that carbonic anhydrase II has unit cell
length values of a = 103.84, b = 104.82 and c = 119.36
with angles of 90ꢀ, 110.45ꢀ and 90ꢀ for all α, β and γ
dimensions, respectively. Furthermore, the Ram-
achandran graph and values also confirm the reliability
and efficacy of the carbonic anhydrase II structure. The
Ramachandran plots indicated that 93.8% of all resi-
dues were present in favored regions and that only six
rotamers lie in unfavourable regions (Figure 4). The
selected Ramachandran graph values showed the good
accuracy of the phi (φ) and psi (ψ) angles among the
coordinates of the receptor molecules, and most of the
residues fell in acceptable regions.
3.6 | Chemo-informatics properties and
adherence of the ligands to Lipinski's
rule (RO5)
The designed ligands were analyzed computationally to
predict the best ligand based on chemical and bio-
molecular properties and Lipinski's RO5. The predicted
chemo-informatics properties, such as LogP, HBD, HBA,
molar volume, polar surface area (PSA) and drug-likeness
values, of the ligand molecules are presented in Table 1.
Previous research has confirmed that the standard values
for molecular weight (MW) and PSA are 160 to 480 g/
mol and (<89 Å2), respectively.[32,33] The predicted
results of compounds 3a-j showed good MW and PSA
values that are comparable to the standard values. The

10
SAEED ET AL.
TABLE 3
Chemo-informatics analysis of designed chemical compounds (3a-j)
Ligands
3a
Mol. Wt (g/mol)
176.06
No. HBA
No. HBD
Mol. LogP (mg/L)
PSA (A2)
41.89
42.00
41.78
41.78
41.78
41.78
41.78
41.78
41.78
41.78
Mol.Vol (A3)
167.37
Drug Score
−0.58
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1.33
2.54
1.54
2.31
2.79
3.75
4.24
7.61
8.09
9.06
3b
218.11
232.97
−0.40
3c
210.02
180.22
−0.41
3d
204.09
204.01
−0.21
3e
218.11
221.92
−0.30
3f
246.14
257.73
−0.44
3g
260.15
275.64
−0.44
3h
3i
358.26
400.98
−0.44
372.28
418.89
−0.44
3j
400.31
454.70
−0.44
Abbreviations: HBA, No of hydrogen bond acceptor; HBD, No of hydrogen bond donor; LogP, lipophilicity of partition coefficient; LogS,
lipophilicity of water; MR, Molar refractivity; PSA, polar surface area; PZ, polarizability.
adherence of these compounds to the RO5 also confirmed
their therapeutic potential. Hydrogen-bonding capacity
has been identified as an important parameter for
describing drug permeability. Poor permeation is more
likely to be observed when the number of HBAs and
HBDs exceed 10 and 5, respectively.[34,35] The chemo-
informatics analysis indicated that all the designed com-
pounds possess <10 HBA and < 5 HBD. Moreover, their
LogP values were also comparable to the standard values.
Compounds 3h, 3i and 3j showed higher values of LogP.
However, there are many examples of RO5 violations
among existing drugs.[36,37] The predicted chemo-
informatics values of all the designed ligands are pres-
ented in Table 3.
3.7 | Molecular docking and binding
energy analysis
FIGURE 5 Docking energy values of the synthesized
compounds (3a-j) against the target protein
Molecular docking experiment is the significant compu-
tational approach to study the binding conformation
behaviour of ligands against target proteins.[38,39] The
docked complexes of all the benzoxazoles (3a-j) against
CA II were analyzed separately and evaluated on the
basis of their minimum energy values and ligand interac-
tion patterns. The results showed that compounds 3b, 3f,
3h, 3i and 3j showed good binding energy values of
−7.10, −6.70, −7.30, −7.50 and − 7.30 kcal/mol, respec-
tively (Figure 5). Prior research has shown that the SE for
Autodock is 2.5 kcal/mol (http://autodock.scripps.edu/).[40]
However, in all docking complexes, the predicted energy
value difference was less than that of the standard energy.
Although the basic core of each synthesized compound
was similar, most ligands possess good energy values, and
no large energy fluctuations were observed.
3.8 | Binding profiles of synthesized
compounds against CA II
Based on in vitro and binding energy valued 3f-CA II
docking complex was selected to interpret the binding
behavior of synthetic compounds. The ligand-protein
binding analyses displayed that 3f was confined in the
active binding pocket of the target protein, as mentioned
in Figure 6. CA II has an active site cleft (15 Å in diame-
ter and 15 Å deep) and contains a Zn2+ ion that is coordi-
nated in a tetrahedral geometry to three histidine
residues (His94, His96 and His119) and a water

SAEED ET AL.
11
FIGURE 6 Docking interactions 6B with the receptor molecule. A, The general overview of the docking depiction. The surface of the
protein structure is represented in brown, while the ligand is highlighted in purple. B, A closer view of the binding pocket interactions with
the ligand in the best conformational position. The ligand molecule is depicted in purple, while its functional groups, such as oxygen and
amino groups, are shown in red and blue, respectively. C, The docking complex is represented with the ligand conformation. Amino acids
are highlighted in green. D, The closer view of the docked complex. The residues involved in hydrogen bonds are shown in red, while
residues involved in hydrophobic interactions are labelled in purple with dotted lines showed the interactions, and the distances are given in
angstroms (Å). Hydrogen bonds were observed to both His63 and Pro199, while hydrophobic interactions were observed to Val141 and
Leu196 in the target protein. Zinc metal is shown in grey
molecule/hydroxide ion.[15] The compound 3f displayed a
good binding conformational position within the CA II
binding pocket having hydrogen and hydrophobic interac-
tions. The results of the 3f-receptor docked complex
showed that couple of hydrogen bonds were observed at
His63 and Pro199 residual positions. The hydrogen bonds
are more significant compared to hydrophobic interaction
to interpret the docking complexes. The oxygen moiety in
3f interacts with His63 with a bond distance of 2.15 Å,
while the amide group forms a hydrogen bond to Pro199
having bond length of 2.60 Å.[41] Two hydrophobic inter-
actions were also observed at Val141 and Leu196 having
bond lengths of 3.63 and 5.14 Å, respectively.[42] It has
been also observed that the bond distances of hydrogen
and hydrophobic interactions were also comparable with
standard values of bond lengths (<3 and < 5 Å), respec-
tively. The 2D conformation, binding pose and interac-
tions with the binding site residues for all the candidate
molecules are presented in Figure S1 to S10.
4 | CONCLUSIONS
In summary, 10 new benzoxazole derivatives have been
prepared in very good yields employing a facile and prac-
tical synthetic methodology. The compounds (3a-j) have

12
SAEED ET AL.
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been evaluated for their inhibition of carbonic anhydrase
II as well as for their antioxidant capacity. All N-(benzo
[d]oxazol-2-yl)alkanamides displayed significant micro-
molar enzyme inhibition activities; compounds 3f
(IC50 = 0.00564 μM) and 3j (IC50 = 0.00596 μM) were
found to be the most active inhibitors in the series. in sil-
ico docking and molecular simulations were performed
to understand the binding modes of these benzoxazoles
with CA II. The IR drug-likeness scores were estimated,
and all compounds obeyed Lipinski's rules. Our prelimi-
nary results revealed that compound 3f showed good
drug likeness and might be useful for further biomedical
applications.
ACKNOWLEDGEMENTS
We are thankful to the Department of Chemistry, Quaid-
i-Azam University Islamabad, Pakistan for providing
encouraging environment and facilities for research
work. M.F.A. and H.R.E. extend their appreciation to the
International Scientific Partnership Program (ISPP) at
King Saud University for funding this research work
through (ISPP-126).
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The authors declare no potential conflict of interest.
ORCID
Aamer Saeed https://orcid.org/0000-0002-7112-9296
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How to cite this article: Saeed A, Channar PA,
Arshad M, et al. Novel N-(benzo[d]oxazol-2-yl)
alkanamides; synthesis and carbonic anhydrase II
inhibition studies. J Heterocyclic Chem. 2020;1–13.
https://doi.org/10.1002/jhet.3992