Ethyl benzoate bearing pyrrolizine/indolizine moieties: Design, synthesis and biological evaluation of anti-inflammatory and cytotoxic activities

Article abstract of DOI:10.1016/j.bioorg.2019.103371

Two new series of ethyl benzoate bearing pyrrolizine and indolizine moieties 8–11 were synthesized and evaluated for their anti-inflammatory and anticancer activities. Among these derivatives, compounds 9a, 10b and 11b displayed in vivo anti-inflammatory

Full text of DOI:10.1016/j.bioorg.2019.103371

Journal Pre-proofs
Ethyl benzoate bearing pyrrolizine/indolizine moieties: Design, synthesis and
biological evaluation of anti-inflammatory and cytotoxic activities
Khalid M. Attalah, Ashraf N. Abdalla, Akhmed Aslam, Muhammad Ahmed,
Mohammed A.S. Abourehab, Naser A. ElSawy, Ahmed M. Gouda
PII:
S0045-2068(19)31501-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103371
YBIOO 103371
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
10 September 2019
7 October 2019
16 October 2019
Please cite this article as: K.M. Attalah, A.N. Abdalla, A. Aslam, M. Ahmed, M.A.S. Abourehab, N.A. ElSawy,
A.M. Gouda, Ethyl benzoate bearing pyrrolizine/indolizine moieties: Design, synthesis and biological evaluation
of anti-inflammatory and cytotoxic activities, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2
019.103371
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Ethyl benzoate bearing pyrrolizine/indolizine moieties: Design, synthesis and
biological evaluation of anti-inflammatory and cytotoxic activities
1
2,3
4
2
Khalid M. Attalah , Ashraf N. Abdalla , Akhmed Aslam , Muhammad Ahmed , Mohammed
1,5
6
7*
A.S. Abourehab , Naser A. ElSawy , Ahmed M. Gouda
1Department of Pharmaceutics, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi
Arabia;
2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-Qura University, Makkah
2
1955, Saudi Arabia;
3Department of Pharmacology, Medicinal and Aromatic plants research institute, National center for
research, Khartoum 2404, Sudan;
4Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University,
Makkah 21955, Saudi Arabia;
5Department of Pharmaceutics, Faculty of Pharmacy, Minia University, Minia 61519, Egypt;
6Department of Anatomy and Embryology, Faculty of Medicine, Zagazig University, Egypt;
7Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,
Egypt;
*
Correspondence
Ahmed M. Gouda: Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef 62514, Egypt.
Scopus ID: 26321547200
ORCID: 0000-0003-4527-8885
Tel.: (002)-011-26897483
Fax: (002)-082-2317958
E-mail address: ahmed.gouda@pharm.bsu.edu.eg or amsaid@uqu.edu.sa
Page 1 of 57

Graphical abstract
Highlights
1
2
. Two new series of ethyl benzoate bearing pyrrolizine/indolizine moieties were synthesized.
. The new compounds displayed anti-inflammatory and analgesic activities comparable to
ibuprofen, mediated by COX-1/2 inhibition, with improved GIT safety profile.
. The new compounds exhibited potent to moderate cytotoxicity against three (MCF-7, A2780
and HT29) cancer cell lines (IC = 0.02-23.35 µM).
3
5
0
4
5
. Compound 9a induced G1 cell cycle arrest and apoptosis in MCF-7 cells.
. The new compounds exhibited nice fitting into COX-1/2 and drug-likeness scores comparable
to licofelone and ketorolac.
Page 2 of 57

Abstract
Two new series of ethyl benzoate bearing pyrrolizine and indolizine moieties 8-11 were
synthesized and evaluated for their anti-inflammatory and anticancer activities. Among these
derivatives, compounds 9a, 10b and 11b displayed in vivo anti-inflammatory and analgesic
activity comparable to ibuprofen. The acute ulcerogenicity and histopathological studies revealed
better GIT safety profile than ibuprofen. Mechanistic study of these compounds revealed
inhibitory activity against COX-1/2 with preferential inhibition of COX-2. Evaluation of
cytotoxic activity of the new compounds using MTT assay revealed potent to moderate activity
against three human (MCF-7, A2780 and HT29) cancer cell lines (IC50 = 0.02-23.35 µM).
Compounds 9a, 10a,b and 11a,b exhibited high cytotoxic selectivity against MCF-7 cells (SI =
4
-84). Although the indolizine bearing derivatives 8-11b exhibited higher selectivity to COX-2
than their corresponding pyrrolizine analogs 8-11a, but they were less active and selective
against MCF-7 cells. Cell cycle analysis and annexin V-FITC/PI assay revealed G1 cell cycle
arrest and induction of apoptosis in MCF-7 cells by compound 9a. The docking study revealed
nice fitting of the new compounds into the active site of COX-1/2 with higher affinity to COX-2.
Compounds 8-11 displayed drug-likeness score in the range of 0.67-1.56 compared to 1.06 for
licofelone. These results suggested that compounds 9a, 10b and 11b could be promising agents
in future research as anti-inflammatory and anticancer agents.
Key words: pyrrolizine; indolizine; anti-inflammatory; cytotoxicity; cell cycle analysis;
apoptosis.
Page 3 of 57

1
. Introduction
Cancer-related inflammation and pain are of the most common symptoms in solid tumors. They
can be managed effectively with strong analgesic and anti-inflammatory agents [1,2]. Moreover,
a huge volume of research describing the relationship between inflammation and cancer has been
reported [3,4]. The upregulation of COX-2 gene was confirmed in several types of solid tumors
such as breast [5], ovarian [6], and colon [7] cancers. The overexpression of COX-2 in cancer
cells was associated with an increase in prostaglandin biosynthesis and inhibition of apoptosis
[
8]. Accordingly, it was not surprising that COX-2 inhibitors have antiproliferative activity
against different types of cancer [9]. Most of the research in this area focused on selective COX-
inhibitors, although selective COX-1 and nonselective COX inhibitors have also displayed
2
potential anticancer activity [10,11]. However, the exact mechanism of action of these inhibitors
is still unclear [9]. Moreover, the use of NSAIDs as chemopreventive or anticancer agents was
restricted by many limitations such as GIT, kidney and cardiovascular side effects [12]. These
problems encouraged the research to find new scaffolds with potential anti-inflammatory and
anticancer activities.
Pyrrolizine is one of the promising scaffolds in this field. Several pyrrolizines 1-3 (Fig. 1) have
displayed remarkable anti-inflammatory/anticancer activities [13-21]. Unlike other NSAIDs, the anti-
inflammatory activity of licofelone 1 was associated with gastric sparing properties [14] and
favorable cardiovascular profile [15]. The study of structure activity relationship (SAR) of licofelone
1
revealed that carboxylic acid and the two methyl groups are not essential for COX inhibition [13].
Cl
Ph
NC
O
NH2
O
HO
N
N
N
O
HN
O
OH
OH
1
(Licofelone)
2 (Ketorolac)
IC50 = 13 M (A549) [
IC50 = 140 nM (COX-2) [A5]
3
A5]
O
IC50 = 5.5 M (MCF-7)
IC50 = 4.7 M (COX-2) [A1]
Fig.1. Pyrrolizine-based anti-inflammatory agents.
Licofelone 1 (Fig. 1) displayed also potent cytotoxic activity against breast, colon, and prostate
cancer cell lines, mediated by induction of apoptosis [16-18]. This activity was mediated by the
Page 4 of 57

induction of apoptosis in cancer cells. Licofelone 1 inhibited the growth of the MCF-7 breast
cancer cells and COX-2 at nearly equal IC values [16,19]. These findings indicating that the
5
0
two activities could be achieved simultaneously at the same dose level.
Ketorolac 2 (Fig. 1) is currently marketed as an analgesic and anti-inflammatory agent. Due to its
potent analgesic activity, it was used as morphine sparing agent to alleviate cancer pain [20].
Moreover, ketorolac displayed potent anticancer activity against A549 cells with an IC value
50
around 13 µM [21]. Moreover, compound 3 was previously reported with ~50% in vivo anti-
inflammatory activity compared to ketorolac 2 [22]. Encouraged by these findings, compounds 3
was used as a lead compound in this study. Structural modifications of compound 3 were
performed to investigate its anti-inflammatory and anticancer potential.
Rational Design
Esterification of the carboxylic acid group in NSAIDs was previously used to overcome their
GIT toxicity [23]. Moreover, esterification of several NSAIDs was associated with improvement
of their pharmacokinetic profile, cellular permeability and anticancer activity [21,23]. The ester
derivatives of several NSAIDs have also displayed in vitro COX-1/2 inhibitory activity with high
selectivity to COX-2 [24,25]. Based on these findings, we have designed two new series of
nonacidic pyrrolizines and indolizine derivatives by masking the free carboxylic acid group and
expansion of the pyrrolidine ring in in compound 3, Fig. 2.
O
Ibupofen
Structure
O
moiety
extension
(
n = 1,2)
N
Hybridization
with ibuprofen
NC
NH2
Pyrrolizine/indolizine
Ring
expansion
(n = 1,2)
(n = 1,2)
N
O
tert-butyl
(n = 2,3)
HN
n
Me
Et
Cyclohexyl
Hexyl
Isobutyl
Masking by
O
Estrification
H
3
n = 1)
N
Propyl Pentyl Cyclopentyl
Isopropyl Isopentyl Isohexyl
O
(
preliminary
docking study
(n = 1-3)
Butyl
Neopentyl
Benzyl
Fig. 2. Rational design of the new compounds.
Page 5 of 57

A preliminary docking study was performed to evaluate binding affinity of seventeen ester
derivatives of compound 3 toward COX-1/2 (Table S1, supplementary data). The results
revealed high binding affinity toward COX-2 for the alkyl ester compared to compound 3.
Among these derivatives, the ethyl ester displayed the highest affinity to COX-2. The results also
revealed an increase in COX-2 selectivity on expansion of the pyrrolidine ring in compound 3 to
piperidine ring. On the other hand, expansion of pyrrolizine to azepine ring was associated with a
noticeable increase in binding affinity to COX-1, suggesting that the resulting pyrroloazepine
would be a nonselective COX inhibitor.
To improve dinging affinity of the new compounds toward COXs, extension of the chemical
structure of ethyl ester of compound 3 and its indolizine analog was considered. Both benzoyl
and benzylidene groups which can form additional hydrophilic/hydrophobic interactions with
COXs were selected for this study. Based on the structural difference between the two COXs,
increasing the molecular volume by benzoyl or benzylidene moieties could be used to increase
selectivity to COX-2 [24]. The preliminary docking study revealed higher affinity for benzoyl or
benzylidene derivatives toward COX-1/2 compared to compound 3 (Table S2, supplementary
data).. Moreover, hydrides of aryl propionic acid derived NSAIDs with pyrrolizines have proven
high safety in GIT. In this study we have designed two hybrids of ibuprofen with
pyrrolizine/indolizine to compare their anti-inflammatory and anticancer activities, Fig. 2.
2
2
. Results and discussion
.1. Chemistry:
Synthesis of the starting materials was outlined in Scheme 1. Compounds 5a-c and 7 were
synthesized according to the previous reports [26-28]. Moreover, compound 8a was prepared
from the reaction of 2-(pyrrolidin-2-ylidene)malononitrile 5a with ethyl 4-(2-
chloroacetamido)benzoate 7 in acetone according to our previous report [29].
Similarly, compounds 8b,c were obtained from the reaction of compound 7 with 2-(piperidin-2-
ylidene)malononitrile 5b and 2-(azepan-2-ylidene)malononitrile 5c, respectively. The two
compounds were prepared using the same reaction conditions applied for preparation of
compound 8a, Scheme 1. The expected mechanisms of cyclization of pyrrolizine and indolizine
nuclei were previously discussed [27,30]. The expected mechanism of cyclization of the
Page 6 of 57

pyrroloazepine 8c is essentially similar to those of pyrrolizine and indolizine derivatives 8a,b.
The reaction can take place in two steps. The first step involves dehydrohalogenation catalyzed
by potassium carbonate. The second step involves intramolecular cyclization to give
pyrroloazepine 8c. The spectral data are provided in supplementary data (Fig. S1-91).
-1
The IR spectrum of compound 8b revealed absorption band at 2211 cm assigned for cyano
-1
1
group and two stretching bands at 1699 and 1672 cm attributed to carbonyl groups. The H
NMR spectrum of compound 8b revealed four signals at δ 1.76-4.11 ppm attributed to protons of
the four methylene (CH ) groups of indolizine nucleus, a singlet signal at δ 5.35 ppm attributed
2
to the two protons of amino (NH ) group and two doublet signals were observed at δ 7.70-7.91
2
ppm, indicating protons of the para-substituted phenyl ring of compound 8b. Moreover, a singlet
signal was observed at δ 9.82 ppm indicating the amide proton (CONH).
NC
CN
NH
O
NH
a
NC
NC
n
a-c
n
a-c
N
NH2
O
5
4
c
O
N
N
Cl
HN
HN
n
n
NH2
HN
O
O
O
b
8
a,b
8a,b
O
O
CH3
CH3
4
a, 5a, 8a: n = 1
b, 5b, 8b: n = 2
O
O
O
O
4
CH3
CH3
4c, 5c, 8c: n = 3
6
7
Scheme 1. Reagents and conditions: (a) (CH ) SO , benzene, CH (CN) ; b) ClCH COCl, g. acetic acid,
3
2
4
2
2
2
○
CH COONa, rt, 2 h; (c) acetone, K CO , reflux (50-60 C), 24 h.
2
2
3
1
3
The C NMR spectrum of compound 8b revealed a signal at δ 14.71 ppm due to the methyl
CH ) group carbon. Four signals were observed at δ 19.00-45.84 ppm attributed to carbons of
(
3
the four methylene (CH ) groups of indolizine nucleus. Another signal at δ 60.84 ppm indicated
2
the methylene (CH ) group carbon of ethyl side chain. Moreover, two signals at δ 159.85 and
2
1
65.90 ppm attribute to amide and ester group carbons, respectively. DEPT C135 spectrum of
compound 8b revealed two signals (up) at δ 119.41 and 130.49 indicating the aromatic CH
Page 7 of 57

groups. Mass spectra of compound 8b revealed the molecular ion peak at m/z = 351 ([M-H]+,
00%)
1
-1
The IR spectrum of compound 8c revealed absorption band at 2212 cm assigned for cyano
-1
1
group and two stretching bands at 1706 and 1667 cm attributed to carbonyl groups. The H
NMR spectrum revealed a triplet signal at δ 1.28 ppm and singlet signal at δ 4.62 ppm attributed
to methyl (CH ) and amino (NH ) groups. Two doublet signals at δ 7.73 and 8.01 ppm indicated
3
2
aromatic protons of the para-substituted phenyl ring. One singlet signal at δ 10.35 ppm assigned
1
3
for the amide proton (CONH). The C NMR spectrum of compound 8c revealed five signals at δ
6.21-47.27 ppm attributed to the five methylene (CH ) groups carbons of the azepine ring.
2
2
Moreover, two signals at δ 159.23 and 165.64 ppm attribute to amide and ester group carbons,
respectively. DEPT C135 spectrum of compound 8c revealed two signals (up) at δ 118.98 and
1
29.29 indicating the aromatic CH group carbons. Mass spectra of compound 8c revealed the
+
molecular ion peak at m/z = 365 (M -H, 100%).
Compound 8c was obtained in poor yield (36%) compared to 62 and 54% for compounds 8a and
8
b, respectively. Moreover, the results of the preliminary docking study suggested that
compound 8c will have approximately equal affinity to COX-1/2. Accordingly, this work
focused mainly on derivatization of compounds 8a,b.
The Schiff bases 9a,b were obtained by refluxing compounds 8a,b with benzaldehyde, Scheme
-1
2
. The IR spectra revealed an absorption band at 2218 and 2211 cm assigned to the cyano
groups in compounds 9a and 9b, respectively. The absorption bands of the two carbonyl groups
-1
1
appeared in the ranges of 1672-1714 cm . The H NMR spectra of compounds 9a,b revealed the
appearance of new singlet signals at δ 9.13 and 9.15 ppm attributed to the benzylidene protons
(PhCH=N). The aromatic protons of the benzylidene moiety appeared at the range of δ 7.51-8.04
1
3
ppm. Moreover, C NMR spectra revealed three signals at the range of δ 158.61-166.23 ppm
attributed to the benzylidene (PhCH=N), amide (CONH) and ester (COOEt) group carbons.
DEPT C135 spectra of compounds 9a,b revealed five signals (up) at δ 118.68-132.76 ppm
attributed to aromatic (CH) carbons. Mass spectra of compounds 9a,b revealed the molecular
+
+
ion peaks at m/z = 426 (M ) and 439 ([M-H] ), respectively.
Page 8 of 57

O
NC
NC
NH
N
a
b
O
O
N
8a,b
N
HN
HN
n
n
O
CH3
O
CH3
9
a,b
10a,b
9
9
a, 10a: n = 1
b, 10b: n = 2
O
O
Scheme 2. Reagents and conditions: (a) benzaldehyde, absolute ethanol, g. acetic acid, reflux 6 h (70-
○
8
0 C); (b) benzoyl chloride, K CO , DCM, stir, rt, 24 h.
2
3
The amide derivatives 10a,b were obtained by benzoylation of compounds 8a,b, Scheme 2. The
-1
IR spectra of compounds 10a,b revealed a stretching band at 2220 and 2221 cm assigned to
cyano groups in compounds 10a and 10b, respectively. Two stretching band at the range of
-1
1
1
651-1717 cm attributed to the carbonyl groups of the two compounds. The H NMR spectra of
compounds 10a,b revealed two signals at the range δ 7.99-10.24 ppm attributed to the two
1
3
amide protons (Ph-NHCO and Ph-CONH). The C NMR spectra showed three signals at δ
57.69-169.59 ppm attributed to carbonyl carbons of the two amide (CONH) and ester (COOEt)
1
groups. DEPT C135 spectra of five signals (up) at the range of δ 118.62-133.36 ppm attributed to
the aromatic (CH) carbons. Mass spectra of compounds 10a,b revealed the molecular ion peaks
+
+
at m/z = 456 [(M+CH )-1] and 455 ([M-H] ), respectively.
3
The hybrids 11a,b were prepared from the reaction of the acid chloride of ibuprofen with
compounds 8a,b, Scheme 3. The IR spectrum of hybrids revealed absorption band at 2222 cm-1
-1
assigned to cyano groups and two stretching bands at the range of 1667-1724 cm attributed to
the carbonyl groups.
Page 9 of 57

O
a
Ibuprofen
Cl
O
NC
NH
b
O
N
8a,b
n
HN
O
CH3
1
1a,b
O
8a, c
O
NC
H
N
NC
H
O
C
N
O
N
O
N
N
HN
O
O
CH3
O
CH3
8a, 11a: n = 1
1
2
2
13
O
8b, 11b: n = 2
O
○
Scheme 3. Reagents and conditions: (a) SOCl , heat (70-80 C), 2 hr; (b) DCM, K CO stir, rt, 24 h; (c)
2
2
3,
oxalyl chloride, DCM, stir, rt, 4 hr.
1
The H NMR spectra of compounds 11a,b revealed the appearance of doublet signal at δ 0.78
and 0.88 ppm attributed to the two methyl (CH(CH ) ) groups of isobutyl moiety. Two new
3
2
doublets at δ 6.97-7.27 ppm were observed indicated the aromatic protons of the p-substituted
phenyl ring of ibuprofen moiety. Moreover, two singlet signals were observed at the range of δ
1
3
7
.39-10.26 ppm attributed to the amide protons. The C NMR spectra showed three signals at
the range of 157.74-177.51 ppm attributed to the three carbonyl carbons. DEPT C135 spectra of
compounds 11a,b revealed five new signals at δ 18.09-49.76 ppm indicated the aliphatic carbons
of ibuprofen moiety. Moreover, two new signals at the range of δ 127.40-129.98 ppm attributed
to the CH groups of the para-substituted phenyl of ibuprofen moiety. Mass spectra of
+
+
compounds 11a,b revealed the molecular ion peaks at m/z = 525 [M -H] and 554 [(M+CH )-
3
1
]+, respectively.
Moreover, the tricyclic diazepino[5,6-b]pyrrolizine 13 was also evaluated in the preliminary
docking study for its binding affinity to COX-1/2. The results of this study revealed high
potential selectivity toward COX-2 (Table S2, supplementary data). In attempt to prepare the
tricyclic diazepino[5,6-b]pyrrolizine 13, oxalyl chloride was reacted with compound 8a in DCM,
Scheme 3. Unfortunately, the reaction failed to give the expected product 13. Instead, a dimeric
product 12 was obtained which exhibited bad solubility in most of organic solvents. Due to its
Page 10 of 57

poor physical properties and the high molecular weight, compound 12 was excluded form
biological evaluation.
-1
The IR, spectrum of compound 12 revealed a stretching band at 2210 cm assigned to cyano group
-1
1
and two absorption band at 1667, 1707 cm attributed to carbonyl groups. The H NMR spectrum
13
revealed two singlet signals at δ 9.87 and 10.78 ppm attributed to the two amide protons. The C
NMR spectrum of compound 12 revealed three signals at δ 158.14, 161.69 and 165.80 ppm
attributed to the carbonyl carbons of amide, ester and oxalyl groups, respectively. DEPT C135
spectrum revealed δ 119.81 and 130.59 ppm indicated the aromatic CH groups of the para-
substituted phenyl rings. Mass spectrum revealed the molecular ion at m/z = 731 ([M+H]+).
2
2
.2. Biological evaluation
.2.1. Anti-inflammatory activity
Compounds 8-11 were evaluated for their in vivo anti-inflammatory activity using carrageenan
induced rat paw edema model [31]. According to this model, inhibition in carrageenan-induced
inflammation after treatment with the new compounds was used as a measure of their anti-
inflammatory activity. The test compounds and the reference drug (ibuprofen) were given orally
to Sprague Dawley rats at a dose of 0.48 mmol/kg. Results were outlined in Fig. 3.
Fig. 3. Change in rat paw edema thickness at 2h and 4h post-carrageenan injection; data expressed as mean
±
SEM, (n = 6); data were analyzed by One way ANOVA followed by Dunnett Multiple Comparisons
*
**
Test; statistically significant from control (P <0.05), statistically significant from control (P <0.01).
Page 11 of 57

The anti-inflammatory activity of the new compounds was calculated according to our previous
report [30]. The results were summarized in Table 1. The new compounds displayed 17.33-
2
8.62% inhibition of inflammation compared to 37.49% for ibuprofen at 2h post-carrageenan
injection. The activity was increased to 27.19-46.32% anti-inflammatory activity (4 h post-
carrageenan injection), compared to 53.78% anti-inflammatory activity for ibuprofen, Table 1.
Compounds 8a-c displayed 27.19-42.30% inhibition of inflammation (4h post-carrageenan
injection), where compound 8b was the most active. On the other hand, compounds 9a,b and
1
8
0a,b exhibited better anti-inflammatory activity (36.26-46.32%) than the parent compounds
a,b. Compounds 11a,b showed delayed anti-inflammatory activity, which reached 38.03% for
the pyrrolizine derivative 11a. Among the new compounds, the benzoyl derivative 10b was the
most active while compound 8c exhibited the lowest anti-inflammatory activity.
Table 1. Anti-inflammatory activity of compounds 8-11 and ibuprofen.
Comp.
No
Anti-inflammatory activity
a
Ulcer
index
(%)
b
2
h
4h
Control
-
-
-
8
a
21.77
25.80
17.33
24.19
27.82
26.61
28.62
16.12
19.35
37.49
38.07
42.30
27.19
44.72
40.49
36.26
46.32
38.03
35.89
53.78
2.76
4.66
nt
8
b
8
c
9
a
5.83
2.99
5.43
4.99
0
9
b
1
0a
0b
1a
1b
Ibu.
1
1
1
0
10.96
a
Anti-inflammatory activity = 100 x (1-L/L ); L is the mean change in paw thickness in rats treated with
t
c
t
tested compounds/ibuprofen (Ibu.); L is the mean increase in paw thickness in rats treated with vehicle;
c
b
sum of %incidence of ulcers/10, average number of ulcers and average severity of ulcers; (n = 6), nt, not
tested.
Generally, the new compounds 8-11 exhibited moderate to strong anti-inflammatory activity. The
degree of inhibition in edema thickness were statistically significant from control for most of the
Page 12 of 57

new compounds at 4h. The delayed anti-inflammatory activity of these compounds could be due to
pharmacokinetic issues.
2
2
.2.2. Analgesic activity
.2.2.1. Hot plate test
The new compounds were evaluated for their analgesic activity against thermal pain using hot
plate test according to the previous reports [26,30]. The latency time was determined at 2h and
4
h after administration of test compounds and ibuprofen (0.48 mmol/kg p.o.). The extension in
withdrawal latency was taken as an index for analgesic effect. The results were summarized in
Table 2.
The results revealed the ability of compound 8b to increase pain reaction time to 14.25 sec after
4
h in comparison with its control. This result was significant and comparable to ibuprofen
14.18 sec), Table 2. Moreover, compounds 9a and 11a increased pain reaction time after 4h to
3.40 and 13.42 sec, respectively. However, ibuprofen showed better effects than both
(
1
compounds 9a and 11a.Treatment with compound 10b increased pain reaction time to 15.30 sec
after 2 h, which persisted after 4 h (15.10 sec). In addition, compound 11b increased pain
reaction time to 15.10 sec.
Table 2. Analgesic effects of compounds 8-11 and ibuprofen using hot plate test.
Latency time ± SEM
Comp. No
0
h
2h
4h
Control
8.75±0.35 8.07±0.37
8.45±0.61 10.32±0.33
8.71±0.57 11.23±0.64
8.15±0.53 9.35±0.55
8.73±0.71 11.33±1.18
8.08±0.72 11.25±0.99
8.83±0.73 10.85±1.13
8.30±0.75
12.62±1.32
14.25±1.43**
9.68±0.75
13.40±1.68*
11.90±1.20
9.88±1.03
8
a
8
b
8
c
9
a
9
b
1
0a
0b
1a
1b
Ibu.
1
8.72±0.93 15.25±1.44** 15.30±1.23**
1
8.74±0.54 9.92±1.07
13.42±1.23*
15.10±1.31**
1
8.72±0.74 10.55±1.31
8.72±0.39 11.85±0.39* 14.18±1.28*
Page 13 of 57

Data were represented as means ± SEM, n = 6; data were analyzed by One way ANOVA followed by
Dunnett Multiple Comparisons Test; * statistically significant from control (P <0.05), ** statistically
significant from control (P <0.01).
Compounds 8a, 9b and 10a increased the reaction time, although these changes were not
significant compared to control. These results revealed that the new compounds 8b, 9a, 10b and
1
1a,b have analgesic activity comparable to or slightly higher than ibuprofen.
2
.2.2.2. Acetic acid-induced writhing test
The analgesic activity of the new compounds 8-11 were also evaluated by acetic acid induced
writhing test [22]. After treatment with test compounds, the decrease in number of acetic acid-
induced writhes compared to control was used as a measure of analgesic activity. The ability of
the test compounds to inhibit induced writhes was calculated in Table 3.
The results revealed 29.74-51.54% inhibition of acetic acid-induced writhes by test compounds
8
-11, compared to 62.33% for ibuprofen. Compounds 9a and 10b exhibited 50.44% and 51.54%
inhibition of abdominal writhes, respectively. Moreover, compound 11b displayed 47.58%
inhibition of the induced writhes which was higher than the parent compound 8b.
Table 3. Analgesic activity of compounds 8-11 and ibuprofen using acetic acid-induced writhing
test.
Comp.
No
Writhing
reflex ± SEM
Inhibition
%
Control 75.67±6.30
-
8
a
47.50±4.06**
43.50±4.62**
53.17±5.62*
37.50±5.72**
46.00±4.52**
51.17±5.91**
36.67±5.84**
42.50±4.37**
39.67±2.59**
28.50±2.73**
37.22
42.51
29.74
50.44
39.21
32.38
51.54
43.83
47.58
62.33
8
b
8
c
9
a
9
b
1
0a
0b
1a
1b
Ibu.
1
1
1
Page 14 of 57

Data expressed as mean number of writhes ± SEM; %Inhibition of abdominal writhes = 100*[(n – nʹ)/n],
where n is mean number of writhes in control group, nʹ is the mean number of writhes in test group; data
*
were analyzed by One way ANOVA followed by Dunnett Multiple Comparisons Test; statistically
*
*
significant from control (P <0.05), statistically significant from control (P <0.01).
2
.2.3. Acute ulcerogenicity studies
GIT toxicity is one of the major side effects of NSAIDs [12]. In this work, compounds 8-11 were
evaluated for their GIT side effects. The ulcerogenic liability of these compounds was evaluated
according to the previous report [22]. The test compounds 8-11 and ibuprofen were given to the
rats at a dose of 0.48 mmol/kg/day p.o. for 3 days. In the fourth day, the rats were sacrificed, and
their stomachs were removed and examined for any mucosal damage using magnifying lens (10x).
Number of mucosal damage (red spots) were counted and their severity were graded from 0-4,
where 0 for normal (no lesion) and 4 for severe lesions (Table S4, supplementary data). Ulcer
index was calculated for each of the tested compounds according to the previous report [22]. The
results were summarized in Table 1.
Examination of stomachs of control group revealed normal texture with no ulcerative spots. In
addition, only small to wide red spots were observed in the stomachs of rats treated with test
compounds 8-10 with no ulcerative spots. Stomachs of the rats treated with ibuprofen revealed
several spots ranging from hyperemia to slight injury. Compound 9b, which showed the highest
selectivity towards COX-2, displayed low ulcer index (2.99), compared to 10.96 for ibuprofen.
On the other hand, stomachs of the rats treated with compounds 11a,b showed normal stomach
texture, Table 1.
2
.2.4. Histopathological studies
To investigate the effects of the test compounds on histological structure of gastric wall, cross
sections in stomachs used in acute ulcerogenicity study were examined microscopically. These
sections were examined after staining with hematoxylin and eosin (H and E) according to the
previous report [26]. The results were presented in Figs. 4 and 5.
Stomach cross sections of the stomachs of control rats (received vehicle) showed normal
histological structure of gastric layers, while those treated with ibuprofen showed abnormal
histological structure of gastric layers with sloughing of gastric mucosa, Figs. 4 and 5.
Page 15 of 57

Fig. 4. Cross section of stomach of a rat (H & E stain X 100) treated with A) vehicle (control); B)
ibuprofen; C) compound 8a; D) compound 8b; E) compound 9a; F) compound 9b.
The cross sections of stomachs of rats treated with compound 8a showed normal histological
structure of gastric mucosa with focal lymphocytic infiltration, while those treated with
compound 8b showed also normal histological structure of gastric mucosa with some change in
submucosa.
Histopathological examination of cross sections of stomachs of rats treated with compound 9a
showed normal histological structure of gastric mucosa with some change in submucosa
specially at gastroesophageal junction. In addition, the cross sections of stomachs of the rats
treated with compound 9b showed normal histological structure of gastric mucosa with some
change in submucosa. Moreover, the cross sections of stomach of rats treated with compounds
1
0a,b showed normal histological structure of gastric mucosa with some change in submucosa,
while those treated with compounds 11a,b showed normal histological structure of gastric layers.
Page 16 of 57

Fig. 5. Cross section of stomach of a rat (H & E stain X 100) treated with A) vehicle (control); B)
ibuprofen; G) compound 10a; H) compound 10b; I) compound 11a; J) compound 11b.
2
.2.5. COX-1/2 inhibitory activity
The mechanism of the anti-inflammatory activity of the pyrrolizine derivatives 1 and 2 was
mediated by COX inhibition [14,21]. In this work, compounds 8-10 were evaluated for their
COX-1/2 inhibitory activity. The assay was performed according to the previous report [32]. The
results expressed in IC values and selectivity indices (SIs) were summarized in Table 4.
5
0
The tested compounds displayed inhibitory activity against COX-1 (IC = 5.52-63.85 µM), and
50
COX-2 (IC = 1.46-11.57 µM). Compounds 8a-c exhibited inhibitory activity against the two
50
COXs with preferential inhibition of COX-2 (SI = 3.69-8.65), where the indolizine analog 8b was
the most selective toward COX-2. Although compounds 8a and 8c showed nearly equal selectivity
for the two COXs, but compound 8a was nearly eight times more potent as COX-2 inhibitor.
Compounds 9a,b were less active in inhibiting COX-1 than their parent compounds 8a,b. As a
result their selectivity to COX-2 was markedly improved compared to the parent compounds.
Among the tested derivatives, compound 9b was the most selective to COX-2 (SI = 27.52).
Moreover, the benzoyl derivatives 10a,b showed better COX-2 inhibitory activity than their
corresponding Schiff bases 9a,b. Compound 10a showed twofold increase in SI compared to 9a,
Table 4.
Page 17 of 57

Table 4. COX-1/2 inhibitory activity of compounds 8-10, celecoxib and indomethacin.
a
IC (µM)
5
0
Comp. No
SI b
COX-1
COX-2
8
a
5.52±0.24 1.46±0.51
37.52±1.92 4.34±0.317
3.78
8.65
8
b
8
c
22.75±3.64 11.57±0.526 3.69
9
a
37.20±2.09 6.89±0.33
63.85±6.52 2.32±0.25
35.44±0.26 3.754±0.42
28.31±0.84 2.146±0.79
43.03±3.54 0.579±0.08
5.40
27.52
9.44
9
b
1
0a
0b
Celecoxib
1
13.19
74.32
0.02
Indomethacin 1.25±0.07 64.76±3.72
a
Values represent the mean of four determinations ± SEM, (n = 3).
b
selectivity index (SI) for COX-2 = IC against COX-1/IC against COX-2.
5
0
50
2
2
.2.6. Cytotoxic activity
.2.6.1. Cytotoxicity assay
Beside their anti-inflammatory activities, licofelone 1 and ketorolac 2 exhibited remarkable cytotoxic
activity against different types of solid tumors [16-19,21]. Encouraged by these findings, compounds
8-11 were investigated for their cytotoxicity against different human organ cancer cell lines,
including breast (MCF-7), ovarian (A2780) and colon (HT29) cancer cell lines. The three cancer
cell lines were selected based on the reported anticancer activity of licofelone [16-18]. The 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used in the
evaluation of cytotoxic activity of these compounds according to our previous reports [34,35].
The results were represented in Table 5. The resulted revealed potent to moderate cytotoxic
activities for the new compounds against the three cancer cell lines with IC values in the range of
50
0.02-23.35 µM, compared to lapatinib (IC50 = 6.80-12.67 µM). Among these derivatives,
compound 9a displayed the highest cytotoxic activity against MCF-7 (IC = 0.02 µM) cell line,
5
0
while compound 11b was the most active against A2780 and HT29 cells.
Page 18 of 57

Table 5. IC50 values of compounds 8-11 and lapatinib against MCF-7, A2780, HT29 and
MRC5 cell lines
NC
R2
N
O
n
HN
COOEt
IC (µM)
5
0
Comp. No n
MCF-7
A2780
HT29
MRC5
8
a
1
2
3
1
2
1
2
1
2
0.07±0.01 0.45±0.10 0.70±0.12 2.00±1.01
3.24±0.99 15.24±0.01 7.71±0.04 1.50±0.16
2.87±0.13 4.25±1.21 12.87±0.57 3.21±0.43
0.02±0.01 4.36±0.37 1.86±0.03 0.85±0.18
18.52±1.42 23.35±2.67 3.57±0.48 22.45±3.65
0.15±0.07 1.63±0.34 0.22±0.01 12.60±0.97
0.37±0.05 14.65±1.36 1.83±0.02 13.95±0.35
0.87±0.06 0.82±0.15 0.20±0.14 7.08±1.89
0.82±0.11 0.06±0.01 0.17±0.01 3.38±0.29
6.80±1.20 10.40±0.80 12.67±1.33 12.89±3.24
8
b
8
c
9
a
9
b
1
0a
0b
1a
1b
1
1
1
Lapatinib -
Results were presented as mean ± S.D. (n = 3), after 72 h treatment with test compounds or vehicle (control).
Compounds 8a-c displayed potent cytotoxic activity against the three cancer cell lines with IC50
values in the range of 0.07-15.24 µM. Among these three derivatives, compound 8a was the most
active as cytotoxic agent. It exhibited IC values in submicromolar range against the three cancer
50
cell lines. Among the three derivatives, compound 8b which showed the highest selectivity toward
COX-2 (Table 4), exhibited the lowest cytotoxic activity against both MCF-7 and A2780 cells.
The Schiff base 9a displayed more than nine hundred times higher cytotoxic activity than compound
9b against MCF-7 cells. Moreover, compound 9b was the least active as a cytotoxic agent against
both MCF-7 and A2780 cells. Moreover,
On the other hand, compounds 10a,b showed higher cytotoxicity against both A2780 and HT29 cells
compared to the corresponding Schiff bases 9a,b. Compound 10a exhibited higher cytotoxic activity
against the three cancer cells compared to the indolizine analog 10b. The two ibuprofen hybrids
11a,b displayed IC50 values in submicromolar ranges against the three cancer cell lines. The
Page 19 of 57

indolizine bearing derivative 11b showed more than 10 times higher cytotoxicity than compound 11a
against A2780 cells. These results indicated that pyrrolizine bearing derivatives 8-10a are more
active as cytotoxic agents than their corresponding indolizine analogs 8-10b against the three cancer
cell lines.
2
.2.6.2. Structure activity relationship (SAR)
Compound 8a displayed high cytotoxic activity against the three cancer cell lines (IC = 0.07-0.70
50
µM). Expansion of the pyrrolidine ring in compound 8a into piperidine was associated with
decrease in both COX-2 inhibitory activity and cytotoxicity against the three cancer cell lines.
However, expansion of piperidine ring in compound 8b to azepine resulted in a decrease in COX-2
inhibitory activity with an improvement in cytotoxicity against MCF-7 and A2780 cells, Tables 4
and 5.
Ring
expansion
Condensation with benzylidene moiety
enhanced activity against MCF-7 cells.
decreased cytotoxicity against
the three cancer cell lines
sharp decrease in cytotoxic activity
against the three cancer cell lines
replace benzylidene
by benzoyl
increased cytotoxicity
against A2780 and HT29
NC
Hybridization with
ibuprofen moiety
NH2
O
Ring
expansion
Ring
expansion
N
decreased cytotoxicity against
the three cancer cell lines
HN
O
8a
Et
O
Ring
enhanced activity
improved cytotoxicity
against HT29 cells
expansion
against A2780 cells
Fig. 6. SAR of cytotoxic activity of compounds 8-11.
Cytotoxic activity against MCF-7 cells was improved, while COX-2 inhibitory activity was
decreased on condensation of compound 8a with benzaldehyde. On the other hand, expansion of
the pyrrolizine ring in compound 9a to piperidine was associated with increase in an COX-2
inhibitory activity and decrease in growth inhibitory activity against the three cancer cell lines, Fig.
6.
Page 20 of 57

Moreover, benzoylation of compound 8a improved cytotoxicity against HT29 cells, while MCF-7
and A2780 cells became less sensitive. This activity was decreased on expansion of pyrrolizine
ring in compound 10a to piperidine, Fig. 6.
Hybridization of compounds 8a,b with ibuprofen moiety resulted in compounds that displayed
IC values at the submicromolar ranges against the tested cell lines. Ring expansion to piperidine
50
enhanced cytotoxic activity against A2780 cells, Fig. 6.
2
.2.6.3. Cytotoxic selectivity
The ideal anticancer agent must have high cytotoxic selectivity toward tumor cells. To evaluate
the cytotoxic selectivity of our new compounds 8-11, their growth inhibitory activity was
evaluated against a normal non-transformed human fibroblast cell line (MRC5). The study was
performed using the MTT assay [35]. The results expressed in IC values were summarized in
5
0
Table 5. Cytotoxic selectivity index (SI) was calculated for each of the new compounds by
dividing the IC value of the test compound against normal non-cancer MRC5 cells by its IC
50
5
0
value against cancer cells, Fig. 7.
Fig. 7. Selectivity indices (IC value against MRC5 cell line/ IC value against cancer cell line) of
5
0
50
compounds 8-11 toward MCF-7, A2780 and HT29 cancer cell lines.
Compound 8a displayed SI in the range of 3-29 toward the three cancer cell lines, while the
indolizine 8b and pyrroloazepine 8c were not selective. Compound 9a was 43 times more
selective for MCF-7 than the normal MCR5 cells, while compound 9b was only 6 times more
selective toward HT29 than MRC5 cells.
Page 21 of 57

A remarkable increase in cytotoxic selectivity was observed for compounds 10a,b compared to
the parent compounds 8a,b. They displayed SIs in the range of 1-84 toward the three cancer cell
lines. The highest selectivity of the two compounds was observed toward MCF-7 cells.
Moreover, compounds 11a,b were also more selective toward the three cancer cell lines than
MCR5 cells. Compound 11a displayed SI in the range of 8-35, while compound 11b was 4-56
times more selective toward the three cancer cell lines. Compounds 10a,b and 11a,b exhibited
higher selectivity toward the three cancer cell lines compared to lapatinib (SI = 1-2), Fig. 7.
2
.2.7. Cell cycle analysis
MCF-7 cells were chosen for further explanations of the mechanistic actions of the new
compounds as the highest selectivity observed was towards this cell line. For this, cell cycle
analysis of MCF-7 cells treated with compound 9a (24h) was performed using a Novocyte flow
cytometer (Acea Biosciences, USA). Compound 9a was selected for this study due to its high
cytotoxic activity (IC = 0.02 μM) and cytotoxic selectively (SI = 43). Cell cycle analysis was
5
0
performed according to our previous report [35]. The results were presented in Table 6 and Fig.
8
.
Table 6. Cell cycle analysis of MCF-7 cells treated with compound 9a (24h).
%
Cell Cycle
Stage/%
Control 0.1 µM 0.5 µM
1 µM
Sub G1
G1
3.0±0.9 1.6±0.4 1.9±0.2 1.7±0.4
49.6±0.6 57.6±1.2 60.1±1.5 56.6±1.4
28.5±1.6 16.8±0.3 15.8±1.6 18.0±1.7
17.2±1.5 18.5±1.0 17.6±2.2 17.8±1.6
S
G2/M
The distribution of different phases was analysed using the cell cycle algorithm of the
NovoExpress software and expressed as percentage of total gated single events. Compared to the
control, compound 9a increased the percentage of cells in the G1 phase at 0.1 µM. This increase
was also maintained at 0.5 and 1 µM and was mainly at the expense of cell accumulation in the S
stage.
Page 22 of 57

Fig. 8. Flow cytometry histograms showing the effect of compound 9a on cell cycle distribution of MCF-
7
0
cells after 24h treatment. X-axis: DNA content of 20,000 events, y axis: cell number. a): 0μM; b):
.1μM; c): 0.5μM; d): 1 μM. (n = 3). Experiment was repeated 3x.
2
.2.8. Annexin V-FITC/PI apoptosis assay
The anticancer activity of several NSAIDs was mediated by induction of apoptosis in different
types of cancer cell lines [9-11]. In addition, the anticancer activity of licofelone 1 and ketorolac 2
were mediated by induction of apoptosis in colon, prostate and osteosarcoma cancer cell lines
[17,18,36]. Accordingly, the ability of compound 9a to induce apoptosis in MCF-7 cells was
investigated in this work. Annexin V fluorescein isothiocyanate (FITC)/ propidium iodide (PI)
apoptosis assay was used in this study according to our previous reports [30,34]. The results were
presented in Fig. 9. Compound 9a induced a dose-dependent increase in the apoptotic events of
MCF-7 cells compared to control.
Page 23 of 57

Fig. 9. Evaluation of apoptosis-inducing activity of compound 9a in MCF-7 cells using annexin V
FITC/PI staining assay, cells were treated with compound 9a for 24h at: a) 0 μM, b) 0.1 μM, c) 0.5 μM,
d) 1 μM (n = 3). x-axis: annexin V/FITC, y-axis: PI. C1: necrosis, PI+/annexin V-); C2: (late apoptosis,
PI+/ annexin V+); C3: (living cells, PI-/annexin V-); C4: (early apoptosis, PI-/annexin V+).
The effect of compound 9a on apoptotic events in MCF-7cells was determined based on the results
of annexin V-FITC/PI assay. Compound 9a induced 5.8% increase in apoptotic events (late plus
early apoptosis) in MCF-7 cells at 0.1 μM, compared to the control (0%). A slight increase in
apoptotic evets to 6.4% and 6.9% was observed at 0.5 and 1 μM, respectively. These results indicated
that the compound 9a include apoptosis at a dose of ≤ 0.1 μM. Only, slight necrosis (0.5-1.1%) was
observed in the MCF-7 cells treated with compound 9a, compared to 4.1% for control, Fig. 10.
Fig. 10. Bar graph showing the effect of treatment with compound 9a (24h) on MCF-7 cells stained with
annexin V-FITC (x-axis), and PI. Data shown are % mean ± SD (n=3). Experiment was repeated 3x.
Page 24 of 57

2
2
.3. Computational studies
.3.1. Molecular docking studies
COXs inhibition is the main mechanism of action of NSAIDs [37]. Compounds 8-10 exhibited COX-
/2 inhibitory activity, Table 4. Based on these results, a comparative molecular docking study was
1
performed to evaluate binding affinities/modes of the new compounds 8-11 into the active site the two
enzymes. The study was performed using AutoDock 4.2 [38]. Crystal structure of both COX-1 [39]
and COX-2 [40]were obtained from protein data bank (http://www.rcsb.org/pdb/home/home.do).
Preparation of ligand, protein file and grid/docking parameters were done according to our
previous reports [41,42]. Visualization of binding interactions of the new compounds with different
+
amino acids in the active sites of COXs was generated by LigPlot [43] and Discovery Studio
Visualizer [44]. The binding free energies and inhibition constants of the new compounds were
also evaluated.
2
.3.1.1. Docking study into COX-1 enzyme
The native ligand (ibuprofen) of COX-1 (pdb: 1EQG) was initially re-docked into the active site of
the enzyme to validate the docking protocol [41,42]. The results revealed superimposition of
redocked ibuprofen above the co-crystallized ligand with RMSD of 0.87 Å. The new compounds
were docked into COX-1 and docking results were compared with that of compound 3, Table 7.
The results of the docking study revealed the ability of compounds 8-11 to form 1-4
conventional hydrogen bonds with ARG83, ARG120, VAL349, TYR355 and SER530 in the
active site of COX-1. The new compounds exhibited binding free energy (G ) in the range of
b
-
8.53 to -10.15 kcal/mol, compared to -8.43 kcal/mol for ibuprofen and -9.50 kcal/mol for
compound 3. The new compounds also displayed inhibition constants (K ) in the range of 36.43-
i
5
61.1 nM compared to 664 nM for ibuprofen. The results suggested that all of the new
compounds have higher inhibitory activity against COX-1 than ibuprofen, Table 7.
Compound 8c displayed the highest binding free energy to COX-1 with (G = -10.15
b
kcal/mol, K = 36.43 nM), while compound 8b exhibited the lowest affinity. Moreover,
i
compounds 9a,b and 10a,b displayed higher affinity to COX-1 than their parent compounds 8a,b.
On the other hand, compound 11a showed higher affinity to COX-1 than parent 8a, while the
indolizine analog 11b exhibited comparable affinity to the parent 8b.
Page 25 of 57

Table 7. Results of the docking of compounds 8-11 into ovine COX-1 (pdb: 1EQG) [39] in
comparison to compound 3 and the co-crystallized ligand (ibuprofen).
Lengthd
Atoms in H-bonding
Comp. No Gb a
Ki b
HBsc
In ligand
In COX-1
(Å)
8
a
-9.32
-8.53
-10.15
148.2 nM
3
COOEt
COOEt
NH2
COOEt
COOEt
NH2
PhNHCO
COOEt
COOEt
NH2
Ph-NHCO
Ph-NHCO
Ph-NHCO
Ph-NHCO
CN
NH of ARG120
2.00
2.04
2.14
1.96
2.14
3.08
1.70
1.74
1.85
2.16
2.23
2.36
2.00
2.22
1.94
1.97
1.80
1.91
2.56
2.12
2.84
2.22
1.99
1.80
1.89
1.71
1.79
1.83
2
NH of ARG120
2
NH of ARG120
8b
561.1 nM
36.43 nM
3
4
NH of ARG120
2
OH of TYR355
C=O of VAL349
OH of SER530
OH of TYR355
8
c
NH of ARG120
2
C=O of VAL349
OH of TYR355
9a
-9.03
-8.74
241.87 nM
393.91 nM
2
2
NH of ARG120
2
9b
NH of ARG120
2
OH of TYR355
1
1
1
0a
0b
1a
-9.52
-9.99
-9.95
104.59 nM
47.20 nM
50.89 nM
1
1
3
NH of ARG83
2
CN
NH of ARG83
2
Ph-NHCO
Ph-NHCO
CN
CN
CN
NH2
OH
OH
COOEt
C=O
NH of ARG120
2
OH of TYR355
OH of SER530
NHof ARG83
11b
-8.56
-9.50
535.29 nM
109.54 nM
2
4
NH of ARG83
2
3
C=O of MET522
OH of TYR355
NH of ARG120
2
NH of ARG120
2
Ibu.
-8.43
664 nM
3
NH of ARG120
COOH
COOH
NH of ARG120
2
OH of TYR355
a
b
c
d
Binding free energy; inhibition constant; number of hydrogen bonds; length in angstrom (Å).
2
.3.1.2. Docking study into COX-2 enzyme
To validate the docking procedures into COX-2 (pdb: 1CX2), the native ligand (SC-558) was
re-docked into the active site of the enzyme. The result revealed superimposition of SC-558
above the native SC-558 with RMSD of 1.56 Å. SC-588 displayed inhibition constant (Ki) of
1
2.52 nM. This value was slightly higher than its IC value (9.3 nM) against COX-2 [40].
50
Page 26 of 57

The new compounds 8-11 were docked into the active site of COX-2 and the results were
summarized in Table 8. The new compounds exhibited binding free energy in the range of -9.00-
1
2.39 kcal/mol, compared to -10.78 kcal/mol for SC-558. They displayed also inhibition constant
in the range of 0.83-253.45 nM. The results revealed the formation of 1-3 conventional hydrogen
bonds with amino acids in the active site of COX-2. Compound 9b displayed the highest affinity
for COX-2 with binding free energy of G of -12.39 and inhibition constant of 0.830 nM.
b
Moreover, compound 10b also displayed high binding free energy to COX-2 with G of -12.26
b
kcal/mol and inhibition constant of 1.03 nM. Due to their high affinities to COX-2, compounds
9
a,b and 10a,b exhibited higher selectivity for COX-2 than their parent compounds 8a,b.
Table 8. Results of the docking of compounds 8-11 into COX-2 (pdb: 1CX2) [40] in comparison
to compound 3 and the native co-crystallized SC-558.
Atoms in H-bonding
In ligand In COX-2
COOEt
Lengthc
Å)
Comp.
No

Gb a
K
i
HBsc
(
8a
-10.08
-10.05
-10.29
40.98 nM
43.3 nM
28.87 nM
2
NH of ARG513
2.86
2.49
1.95
2.01
2.08
2.09
2.14
2.79
2.97
2.99
2.56
2.97
2.29
2.57
2.93
2.35
2.78
2.86
2.45
2.68
1.96
1.98
2.21
2.25
2.88
2.08
1.97
2
NH2
NH2
C=O of VAL523
C=O of VAL523
8b
2
3
COOEt
NH2
NH of ARG513
C=O of VAL523
2
8c
COOEt
NH2
NH of ARG513
C=O of VAL523
NH of PHE518
2
9a
-11.79
2.26 nM
3
COOEt
COOEt
COOEt
COOEt
COOEt
PhNHCO
PhNHCO
CN
COOEt
PhNHCO
COOEt
PhNHCO
CN
NH of GLN192
2
NH of ILE517
NH of ILE517
9b
-12.39
-11.20
0.830 nM
6.15 nM
2
3
1
NH of HIS90
10a
OH of TYR355
NH of ARG120
2
OH of SER530
NH of PHE518
OH of TYR355
NH of ILE517
10b
-12.26
1.03 nM
3
11a
-10.43
-9.00
-9.01
22.54 nM
253.45 nM
247.26 nM
1
1
4
NH of ARG120
2
11b
NH of ARG120
2
3
COOEt
COOH
NH2
NH2
SO2
NH of ARG513
2
C=O of SER353
C=O of VAL523
C=O of VAL523
NH of HIS90
Sc-588
-10.78
12.52 nM
5
NH2
NH2
CO of LEU352
CO of GLN192
Page 27 of 57

CF3
CF3
NH of ARG120
NH of ARG120
3.22
3.58
a
b
c
d
Binding free energy; inhibition constant; number of hydrogen bonds; length in angstrom (Å).
Compounds 8a-c displayed higher affinity toward COX-2, where compound 8a exhibited the
highest affinity toward COX-2. These results was in agreement the in vitro COX-1/2 inhibitory
activities. Moreover, compounds 9a,b and 10a,b displayed very high affinity to COX-2 in
docking results. This was also matched with the results of in vitro COXs inhibitory activity.
Compound 9b 10b showed higher COX-2 inhibitory activity than their corresponding pyrrolizine
derivative 9a and 10a. These results were also in concordance with the results of COXs
inhibition assay. The correlation coefficient was calculated between selective indices (SIs) of the
new compounds to COX-2 (Table 4) and the calculated selectivity obtained by dividing the Ki
values of the new compounds against COX-1 by K values against COX-2 enzyme (Table S3,
i
supplementary data). The results revealed a correlation coefficient of 0.902 indicating strong
correlation between docking results and the in vitro results of COX-1/2 inhibition.
Although compound 3 has a free carboxylic acid group which can form multiple hydrogen bond
with COXs, but the ethyl ester 8a displayed higher affinity to COX-2 (G = -10.08 kcal/mol)
b
than compound 3 (G = -9.01). To understand the reasons behind this difference 2D LigPlot
b
view was generated for compounds 3 and 8a in the active site of COX-2, Fig. 11. The plot
showed different types of hydrogen bonding and hydrophobic interactions between the two
compounds and COX-2. The red circles and ellipses indicate protein residues which form similar
types of interactions with the two ligands. Compound 8a formed only two conventional
hydrogen bonds with COX-2 compared to four hydrogen bonds for compound 3. However
compound 8a displayed larger number of hydrophobic interactions with COX-2 than compound
3
, Fig. 11. These hydrophobic interactions could justify the higher affinity of compound 8a
toward COX-2 compared to compound 3.
Page 28 of 57

Fig. 11. LigPlot view of showing different types of binding interactions between compounds 3/8a and amino
acids in the active site of COX-2 (pdb: 1CX2): A) compound 3; B) compound 8a. Ligand bonds shown in
purple. The red circles and ellipses in each plot indicate protein residues that are in equivalent 3D positions and
form similar types of interactions with the two ligands, hydrophobic interactions shown in brick red dotted
lines and hydrogen bonding interactions shown in olive green dotted lines. Hydrogen atoms were omitted for
clarity.
The three indolizine bearing derivatives 8-10b displayed higher inhibitory activity and selectivity
(Table 4) against COX-2 than their pyrrolizine analogs 8-10a. Compound 8b displayed three
conventional hydrogen bonds with ARG120, VAL349 and TYR355 in COX-1, with bond length
in the range of 1.96-3.08 Å and one carbon hydrogen bond with MET522, Table 7. Different
types of hydrophobic interactions were also observed between the indolizine nucleus, phenyl
ring, and ethyl group in compound 8b with hydrophobic residues in COX-1, Fig. 12. On the
other hand, compound 8b displayed two conventional hydrogen bonds with COX-2. These bonds
were formed with VAL523 and ARG513 with bond length of 1.95 and 2.01 Å, respectively,
Table 8. Moreover, two carbon hydrogen bonds were observed with GLA192 and ARG513
amino acids. It showed several hydrophobic interactions with COX-2, Fig. 12. The high affinity
of compound 8b toward COX-2 could be attributed to the extra carbon hydrogen bonds and
stronger hydrophobic interactions observed with COX-2 compared to COX-1.
Page 29 of 57