Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety: design, synthesis, molecular docking, and biological evaluation as potential multi-target cytotoxic agents

Article abstract of DOI:10.1080/14756366.2021.1937618

In the present study, two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety were designed, synthesised, and evaluated for their cytotoxic activity. The benzamide derivatives 16a–e showed higher cytotoxicity than their corresponding Schiff b

Full text of DOI:10.1080/14756366.2021.1937618

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl
moiety: design, synthesis, molecular docking, and
biological evaluation as potential multi-target
cytotoxic agents
Ahmed M. Shawky, Nashwa A. Ibrahim, Ashraf N. Abdalla, Mohammed A. S.
Abourehab & Ahmed M. Gouda
To cite this article: Ahmed M. Shawky, Nashwa A. Ibrahim, Ashraf N. Abdalla, Mohammed A.
S. Abourehab & Ahmed M. Gouda (2021) Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl
moiety: design, synthesis, molecular docking, and biological evaluation as potential multi-target
cytotoxic agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1313-1333, DOI:
10.1080/14756366.2021.1937618
To link to this article: https://doi.org/10.1080/14756366.2021.1937618
© 2021 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Submit your article to this journal
View related articles
Published online: 21 Jun 2021.
Article views: 380
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1313–1333
https://doi.org/10.1080/14756366.2021.1937618
RESEARCH PAPER
Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety: design, synthesis,
molecular docking, and biological evaluation as potential multi-target
cytotoxic agents
Ahmed M. Shawkya,b, Nashwa A. Ibrahimc,d, Ashraf N. Abdallae,f, Mohammed A. S. Abourehabg,h and
Ahmed M. Goudac,d
b
aScience and Technology Unit (STU), Umm Al-Qura University, Makkah, Saudi Arabia; Central Laboratory for Micro-analysis, Minia University,
c
d
Minia, Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; Medicinal
e
Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt; Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; Department of Pharmacology and Toxicology, Medicinal and Aromatic Plants
Research Institute, National Center for Research, Khartoum, Sudan; Department of Pharmaceutics, Faculty of Pharmacy, Umm Al-Qura
University, Makkah, Saudi Arabia; Department of Pharmaceutics, Faculty of Pharmacy, Minia University, Minia, Egypt
f
g
h
ABSTRACT
ARTICLE HISTORY
Received 16 December 2020
Revised 23 May 2021
Accepted 26 May 2021
In the present study, two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety were designed,
synthesised, and evaluated for their cytotoxic activity. The benzamide derivatives 16a–e showed higher
cytotoxicity than their corresponding Schiff bases 15a–e. Compounds 16a,b,d also inhibited the growth
of MCF-7/ADR cells with IC50 in the range of 0.52–6.26 lM. Interestingly, the new compounds were less
cytotoxic against normal MRC-5 cells (IC50¼0.155–17.08 lM). Mechanistic studies revealed the ability of
compounds 16a,b,d to inhibit tubulin polymerisation and multiple oncogenic kinases. Moreover, com-
pounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells. The molecu-
lar docking analyses of compounds 16a,b,d into the active site in tubulin, CDK-2, and EGFR proteins
revealed higher binding affinities compared to the co-crystallised ligands. These preliminary results sug-
gested that compounds 16a,b,d could serve as promising lead compounds for the future development of
new potent anticancer agents.
KEYWORDS
Pyrrolizine; cytotoxicity;
apoptosis; kinase inhibitor;
cell cycle
HIGHLIGHTS
1. Two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moieties were synthesized.
2. Compounds 16a,b,d displayed the highest cytotoxicity against the three cancer cell lines.
3. Kinase profiling test revealed inhibition of multiple oncogenic kinases by compounds 16a,b,d.
4. Compounds 16a,b,d exhibited weak to moderate inhibition of tubulin-polymerization.
5. Compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells.
6. Docking studies revealed high binding affinities for compounds 16a,b towards tubulin and CDK-2.
CONTACT Ahmed M. Gouda
ahmed.gouda@pharm.bsu.edu.eg, amsaid@uqu.edu.sa
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef 62514, Egypt
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1314
A. M. SHAWKY ET AL.
could be an olefinic (ethenyl) group which restricts the free rota-
tion and keep the two aromatic rings in cis-conformation.
However, this type of restriction is absent in compounds 4–6,
where the two phenyl rings can adopt different orientations rela-
tive to each other (Figure 1).
Incorporation of tubulin-binding moieties such as the TMP moi-
ety into anticancer agents was succeeded as a new strategy to
produce multi-target anticancer agents (Figure 2). Compound 7, a
multi-target anticancer agent designed by combining the pharma-
cophoric groups which can target tubulin polymerisation and tyro-
sine kinase, simultaneously11. Biological evaluation of compound 7
revealed potent inhibitory activity against tubulin polymerisation,
vascular endothelial growth factor receptor-2 (VEGFR-2), and plate-
let-derived growth factor receptor b (PDGFR-b). Compound 7 also
displayed superior in vivo activity in reducing tumour size and vas-
cularity compared to sunitinib, and docetaxel11.
1. Introduction
Despite the presence of clinically effective anticancer agents, can-
cer is still one of the most leading causes of death in the world1.
In addition, the development of multidrug resistance to many of
the currently used anticancer drugs represents another challenge
in this field2. To overcome these problems, several approaches
have been developed and proved better efficacy in cancer treat-
ment. Of these approaches, combination therapy has gained
momentum in the treatment of different types of cancers3.
However, the high cost, toxicity, and high potential for drug–drug
interactions have limited the widespread use of combination ther-
apy in the treatment of cancers3,4
.
Recently, multi-target anticancer agents also emerged as a new
approach to cancer chemotherapy5–7. This approach attracted
much attention as it could provide a better alternative for com-
bination therapy with lower toxicity and fewer drug–drug inter-
action problems. Although most of the multi-target agents were
discovered by serendipity, several rationally designed multi-target
anticancer agents have also been reported7. The rational design of
these agents was achieved by combining pharmacophoric groups
Compound 8, another multi-target anticancer agent designed
by combining the pharmacophoric groups of the TBI (colchicine)
with those needed to inhibit HDAC12. Compound 8 displayed
potent cytotoxic activity (IC50¼2–105 nM) against a panel of can-
cer cell lines mediated by moderate inhibition of HDAC and tubu-
lin polymerisation and activity (Figure 2).
of two or more different anticancer drugs in a single scaffold5–7
Accordingly, the resulting multi-pharmacophore scaffold could hit
multiple targets, simultaneously.
.
Tubulin polymerisation is one of the promising targets in the
development of new anticancer agents8–10. Combretastatin A-4
(CA-4) 1, and its analogues 2, and 3 (Figure 1) are examples of
1.1. Rational design
Recently, we reported compounds 9a,b (Figure 3) among a series
tubulin polymerisation inhibitors (TBIs) that exhibited potent anti- of pyrrolizine derivatives with cytotoxic activity against MCF-7,
cancer activities. The mechanism of action of these compounds is
mediated by their binding to the colchicine binding site in tubulin
resulting in inhibition of the polymerisation8. In addition, the tri-
methoxybenzoyl derivatives 4–6 (Figure 1) were also reported as
TBIs with potent anticancer activities8–10. The mechanism of action
of compounds 4–6 depends also on the inhibition of tubulin
assembly resulting in suppression of microtubule formation8.
Considering the chemical structure of compounds 1–6, it was
observed that they all have similar pharmacophoric features which
include 3,4,5-trimethoxyphenyl (TMP) moiety attached through a
linker of 1–3 atom length to another substituted phenyl ring. A
2D pharmacophore of these TPIs 1–6 is generated in Figure 1.
The TMP moiety is considered as a tubulin-binding moiety and
plays an essential role in the antitubulin activity of compounds
1–6 [9]. In addition, the linker between the two aromatic rings
Figure 2. Rationally designed multi-target anticancer agents incorporating tubu-
lin binding moieties.
Figure 1. TMP bearing tubulin polymerisation inhibitors 1–6 and their pharmacophore features.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1315
Figure 3. Rational design and structural modifications of scaffold A.
A2780, and HT29 cell lines (IC50¼0.10–4.16 lM)13. Mechanistic Based on these results, 10 of the designed derivatives which
showed good scores were selected for the synthesis.
studies of compounds 9a,b revealed inhibitory activities against
COX-2 (IC50¼13.49 and 1.49 lM, respectively) and/or multiple
oncogenic kinases. However, the results of the MTT assay revealed
the ability of the two compounds to inhibit the growth of MCF-7
cells at a concentration lower than those required for COX-2 inhib-
ition. In addition, compound 9b exhibited poor selectivity towards
2. Experimental
2.1. Chemistry
All the chemical reagents and solvents used were purchased from
Sigma-Aldrich (St. Louis, MO). Solvents were dried according to
the literature when necessary. The purity of the new compounds
was checked with TLC using the benzene–ethanol mixture (9:1).
Melting points (m.p.) are uncorrected and were determined by IA
9100MK-Digital melting point apparatus. BRUKER TENSOR 37 spec-
trophotometer (Billerica, MA) was used to perform the infra-red
(IR) spectra of the new compounds, the spectra were recorded
using KBr disc and were expressed in wavenumber (cmꢀ1). The
proton magnetic spectra were recorded on BRUKER AVANCE III at
500 MHz (Faculty of Pharmacy, Umm Al-Qura University, Mecca,
Saudi Arabia) in CDCl3/DMSO-d6. The J constant is given in Hz.
The 13C NMR spectra of the new compounds in CDCl3/DMSO-d6
were done at 125 MHz. Mass spectra were recorded on Shimadzu
GCMS QP5050A spectrometer (Kyoto, Japan), at 70 eV (EI) at the
regional centre for mycology and biotechnology, Al-Azhar
University (Cairo, Egypt). Elemental analyses were done in the
microanalytical centre, Cairo University (Giza, Egypt). Compounds
1114, 13a–e15,16, 14a–e17, 1815, and 1915 were prepared according
to the previous report13. Copies of spectral data including IR, 1H
NMR, 13C NMR, 13C NMR, DEPT C135, and mass spectra for each of
the new compounds are provided in Supplementary
(Figs. S1–S141).
A2780 and HT29 cells (SI
¼
2.16 and 3.03, respectively).
Accordingly, we perform the current study to optimise the cyto-
toxic potential of compounds 9b and investigate other potential
targets which could contribute to their cytotoxic activities.
However, to keep the ability of the new derivatives targeting
oncogenic kinases, only small structural modifications in the scaf-
fold of compound 9a were allowed.
To optimise the cytotoxic potential of the new compounds,
scaffold A was designed by replacement of the 3-methylbutyli-
dene/benzylidene moieties in compounds 9a,b by trimethoxy-ben-
zylidene/benzoyl moieties (Figure 3). The new compounds
designed bearing these pharmacophoric features will be evaluated
for their ability to interfere with the activity of oncogenic kinases
and tubulin polymerisation.
The study of structure–activity relationship (SAR) of scaffold A
(Figure 3) was achieved through a series of structural modifica-
tions which include: (1) variation of the linker between the TMP
moiety and pyrrolizine nucleus to restrict/allow the free rotation
of the two moieties, (2) expansion of pyrrolidine ring to the six-
membered piperidine ring, and (3) variation of the type of sub-
stituents (R) on the phenyl ring (A) to include electron-donating/
electron-withdrawing groups.
designed based on these modifications (Supplementary data,
Tables S1 and S2).
A set of 52 derivatives were
For easy identification of different protons/carbons and their
chemical shifts, the atoms were numbered as illustrated in scaffold
A (Figure 3). The readers should also note that the indolizine
nucleus is numbered in a reverse direction.
In addition, a preliminary docking study was performed to
evaluate the binding affinities of the designed derivatives into the
binding site of colchicine in tubulin protein. The derivatives which
displayed high binding scores towards tubulin were also evaluated
for their binding affinities towards two of the oncogenic kinases
(CDK-2, and EGFR). The selection of these kinases was based on
the results of the mechanistic study of compound 9a13. Moreover,
the synthetic accessibility and drug-likeness scores of the
designed analogues were also evaluated. The results of these
2.1.1. General procedure (A) for the preparation of compound
15a–e
A mixture of pyrrolizine-5-carboxamides 14a–e (3 mmol) and 3,4,5-
trimethoxybenzaldehyde (0.8 g, 4 mmol), 0.5 mL glacial acetic acid
in absolute ethanol (30 mL) was stirred under reflux for 5 h
studies are provided in Supplementary data (Tables S1 and S2). (Scheme 1). The solvent was then evaporated under reduced

1316
A. M. SHAWKY ET AL.
Scheme 1. Synthesis of compounds 15a-e and 16a-e.
pressure. The solid obtained was collected and recrystallised from 278 (12), 277 (45), 249 (13), 222 (7), 196 (13), 195 (100), 194 (7),
acetone–chloroform (1:1).
186 (10), 174 (13), 168 (15), 154 (5), 139 (11), 119 (5), 122 (2), 107
(10), 92 (6), 77 (10). Anal. Calcd. for C25H24N4O4 (444.48): C, 67.55;
H, 5.44; N, 12.60. Found: C, 67.38; H, 4.98; N, 12.69.
2.1.1.1. 7-Cyano-N-phenyl-6-((3,4,5-trimethoxybenzylidene)amino)-
2,3-dihydro-1H-pyrrolizine-5-carboxamide (15a). The title com-
pound was prepared from the reaction of compound 14a (0.8 g,
2.1.1.2. 7-Cyano-N-(p-tolyl)-6-((3,4,5-trimethoxybenzylidene)amino)
3 mmol) with trimethoxy benzoyl chloride (0.8 g, 4 mmol) accord- -2,3-dihydro-1H-pyrrolizine-5-carboxamide (15b). The title com-
ing to the general procedure A. Compound 15a was obtained as pound was prepared from the reaction of compound 14b (0.84 g,
a yellow amorphous solid product, m.p. 242–4 ꢁC, yield 71%. 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) accord-
IRVmax/cmꢀ1 3271, 3236, 3183 (NH), 3076, 3024 (aromatic C–H), ing to the general procedure A. Compound 15b was obtained as
2967, 2941 (aliphatic C–H) 2211 (CN), 1672 (COs), 1609, 1578, 1556 a yellow amorphous solid product, m.p. 251–3 ꢁC, yield 78%.
(C═C, C═N), 1432, 1318, 1232 (C–N, C–O, C–C). 1H NMR (CDCl3, IRVmax/cmꢀ1 3275, 3232, 3179 (NH), 3070 (aromatic C–H), 2962,
500 MHz, d ppm): 2.57–2.63 (m, 2H, pyrrolizine CH2-2), 3.09 (t, 2H, 2926 (aliphatic C–H), 2211 (CN), 1668 (COs), 1611, 1576, 1552 (C═
J¼ 7.5 Hz, pyrrolizine CH2-1), 3.97 (s, 6H, 300-OCH3þ500-OCH3), 3.98 C, C═ N), 1459, 1337, 1233 (C–N, C–O, C–C). 1H NMR (CDCl3,
(s, 3H, 400-OCH3), 4.58 (t, 2H, J¼ 7.1 Hz, pyrrolizine CH2-3), 7.13 (t, 500 MHz, d ppm): 2.33 (s, 3H, 40-CH3), 2.54–2.60 (m, 2H, pyrrolizine
1H, J¼ 7.3 Hz, CH-40), 7.19 (s, 2H, Ph CH-200þCH-600), 7.35 (t, 2H, J¼ CH2-2), 3.06 (t, 2H, J¼ 7.4 Hz, pyrrolizine CH2-1), 3.94 (s, 6H, 300-
7.6 Hz, Ph CH-30þCH-50), 7.68 (d, 2H, J¼ 7.8 Hz, Ph CH-20þCH-60), OCH3þ500-OCH3), 3.95 (s, 3H, 400-OCH3), 4.54 (t, 2H, J¼ 7.1 Hz, pyr-
9.11 (s, H, N═CH), 10.68 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, d rolizine CH2-3), 7.12 (d, 2H, J¼ 7.7 Hz, Ph CH-30þCH-50), 7.16 (s, 2H,
ppm): 24.59 (pyrrolizine CH2-2), 25.46 (pyrrolizine CH2-1), 50.14 Ph CH-200þCH-600), 7.54 (d, 2H, J¼ 7.6 Hz, Ph CH-20þCH-60), 9.08 (s,
(pyrrolizine CH2-3), 56.35 (2C, 300-OCH3þ500-OCH3), 61.09 (400-OCH3), H, N═CH), 10.58 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, d ppm): d
84.71 (pyrrolizine C-7), 105.90 (2C, Ph CH-200þCH-600), 116.31 (pyr- 20.88 (CH3), 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1),
rolizine C-6), 117.71 (CN), 119.79 (2C, Ph CH-20þCH-60), 124.12 (Ph 50.13 (pyrrolizine CH2-3), 56.35 (2C, 300-OCH3þ500-OCH3), 61.09 (400-
CH-40), 129.04 (2C, Ph CH-30þCH-50), 130.75 (Ph C-100), 138.23 (pyr- OCH3), 105.87 (2C, Ph CH-200þCH-600), 116.36 (pyrrolizine C-7),
rolizine C-5), 139.40 (pyrrolizine C-7a), 142.18 (Ph CH-10), 148.19 117.82 (CN), 119.75 (2C, Ph CH-20þCH-60), 129.52 (2C, Ph CH-
(Ph C-400), 153.82 (2C, Ph C-300þC-500), 158.48 (PhNHCO) 159.52 (N═ 30þCH-50), 130.79 (pyrrolizine C-6), 133.74 (Ph C-100), 135.67 (pyrro-
CH). DEPT135 (CDCl3, 125 MHz, d ppm): d 24.59 (pyrrolizine CH2-2), lizine C-5), 139.23 (pyrrolizine C-7a), 142.13 (Ph C-10), 142.69 (Ph C-
25.47 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.35 (2C, 300- 40), 148.08 (Ph C-400), 153.80 (2C, Ph C-300þC-500), 158.39 (PhNHCO),
OCH3þ500-OCH3), 61.09 (400-OCH3), 105.90 (2C, Ph CH-200þCH-600), 159.39 (N═CH). DEPT135 (CDCl3, 125 MHz, d ppm): d 20.88 (CH3),
119.79 (2C, Ph CH-20þCH-60), 124.12 (Ph CH-40), 129.04 (2C, Ph CH- 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.13 (pyrroli-
30þCH-50), 159.52 (N═CH). MS (EI): m/z (%) 445 ([M þ 1]þ, 4), 444 zine CH2-3), 56.36 (2C, 300-OCH3þ500-OCH3), 61.09 (400-OCH3), 105.82
(Mþ, 12) 443 ([M–1]þ, 2), 367 (4), 352 (36), 322 (5), 308 (6), 294 (4), (2C, Ph CH-200þCH-600), 119.75 (2C, Ph CH-20þCH-60), 129.52 (2C, Ph

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1317
CH-30þCH-50). MS (EI): m/z (%) 460 ([M þ 2]þ, 3), 459 ([M þ 1]þ, 19), OCH3), 61.12 (400-OCH3), 105.95 (2C, Ph CH-200þCH-600), 120.90 (2C,
458 (Mþ, 72), 457 (7), 429 (4), 353 (21), 352 (100), 336 (4), 322 (7), Ph CH-20þCH-60), 129.05 (2C, Ph CH-30þCH-50), 159.75 (N═CH). MS
308 (7), 292 (11), 291 (54), 280 (2), 278 (3), 263 (8), 229 (4), 196 (3), (EI): m/z (%) 480 ([M þ 2]þ, 1), 479 ([M þ 1]þ, 1), 478 (Mþ, 4), 458
195 (21), 194 (2), 186 (9), 168 (9), 154 (3), 106 (2), 91 (2), 77 (4). (7), 353 (13), 352 (73), 323 (4), 322 (8), 311 (11), 308 (10), 292 (12),
Anal. Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. 291 (44), 278 (8), 263 (13), 252 (9), 229 (8), 222 (11), 196 (15), 195
Found: C, 68.53; H, 5.32; N, 12.67.
(100), 186 (20), 168 (28), 154 (12), 147 (6), 111 (4), 106 (5), 91 (7),
77 (12). Anal. Calcd. for C25H23ClN4O4 (478.93): C, 62.70; H, 4.84; N,
11.70. Found: C, 63.14; H, 5.07; N, 11.22.
2.1.1.3. 7-Cyano-N-(4-methoxyphenyl)-6-((3,4,5-trimethoxybenzyli-
dene)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15c). The
title compound was prepared from the reaction of compound 14c
2.1.1.5. N-(4-Bromophenyl)-7-cyano-6-((3,4,5-trimethoxybenzylide-
(0.9 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) ne)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15e). The
according to the general procedure A. Compound 15c was title compound was prepared from the reaction of compound 14e
obtained as a yellow amorphous solid product, m.p. 263–5 ꢁC, (1.04 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol)
yield 74%. IRVmax/cmꢀ1 3281, 3240, 3185 (NH), 3077, 3000 (aro- according to the general procedure A. Compound 15e was
matic C–H), 2942, 2841 (aliphatic C–H) 2208 (CN), 1674 (COs), obtained as a yellow amorphous solid product, m.p. 277–80 ꢁC,
1605, 1578, 1511 (C═ C, C═ N), 1334, 1234 (C–N, C–O). 1H NMR yield 68%. IRVmax/cmꢀ1 3172 (NH), 3063 (aromatic C–H), 2961,
(CDCl3, 500 MHz, d ppm): d 2.55–2.61 (m, 2H, pyrrolizine CH2-2),
2939, 2838 (aliphatic C–H) 2209 (CN), 1679 (COs), 1610, 1578, 1545
3.06 (t, 2H, J¼ 7.4 Hz, pyrrolizine CH2-1), 3.82 (s, 3H, 40-OCH3), 3.95 (C═C, C═N), 1418, 1334, 1232 (C–N, C–O, C–C). 1H NMR (CDCl3,
(s, 6H, 300-OCH3þ500-OCH3), 3.99 (s, 3H, 400-OCH3), 4.52 (t, 2H, J¼
500 MHz, d ppm): d 2.57–2.62 (m, 2H, pyrrolizine CH2-2), 3.08 (t,
7.1 Hz, pyrrolizine CH2-3), 6.88 (d, 2H, J¼ 8.6 Hz, Ph CH-30þCH-50),
7.28 (s, 2H, Ph CH-200þCH-600), 7.61 (d, 2H, J¼ 8.3 Hz, Ph CH-
20þCH-60), 8.96 (s, 1H, CONH), 10.70 (s, 1H, CONH). 13C NMR
(CDCl3, 125 MHz, d ppm): d 24.62 (pyrrolizine CH2-2), 25.48 (pyrroli-
zine CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (40-OCH3), 56.42 (2C,
300-OCH3þ500-OCH3), 61.20 (400-OCH3), 106.45 (2C, Ph CH-200þCH-600),
106.72 (pyrrolizine C-7), 114.16 (2C, Ph CH-30þCH-50), 114.77 (CN),
116.08 (pyrrolizine C-6), 118.05 (Ph C-10), 121.49 (2C, Ph CH-
20þCH-60), 129.60 (Ph C-100), 131.29 (pyrrolizine C-7a), 148.00 (pyr-
rolizine C-5), 153.66 (Ph C-400), 153.71 (2C, Ph C-300þC-500), 156.32
(Ph C-40), 158.06 (CONH), 160.30 (N═ CH). DEPT C135 (CDCl3,
125 MHz, d ppm): d 24.62 (pyrrolizine CH2-2), 25.48 (pyrrolizine
CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (40-OCH3), 56.41 (2C, 300-
OCH3þ500-OCH3), 61.20 (400-OCH3), 106.42 (2C, Ph CH-200þCH-600),
114.16 (2C, Ph CH-30þCH-50), 121.48 (2C, Ph CH-20þCH-60). MS (EI):
m/z (%) 474 (Mþ, 23), 449 (8), 422 (5), 396 (34), 383 (43), 361 (58),
332 (100), 320 (37), 261 (28), 190 (11), 120 (22), 93 (8), 69 (9). Anal.
Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found:
C, 65.44; H, 5.21; N, 12.15.
2H, J¼ 7.5 Hz, pyrrolizine CH2-1), 3.96 (s, 6H, 300-OCH3þ500-OCH3),
3.99 (s, 3H, 400-OCH3), 4.52 (t, 2H, J¼ 6.9 Hz, pyrrolizine CH2-3), 7.21
(s, 2H, Ph CH-200þCH-600), 7.44 (d, 2H, J¼ 7.7 Hz, Ph CH-30þCH-50),
7.59 (d, 2H, J¼ 8.2 Hz, Ph CH-20þCH-60), 9.01 (s, 1H, CONH), 10.77
(s, 1H, CONH). 13C NMR (CDCl3, 125 MHz, d ppm): d 24.64 (pyrroli-
zine CH2-2), 25.45 (pyrrolizine CH2-1), 50.15 (pyrrolizine CH2-3),
56.42 (2C, 300-OCH3þ500-OCH3), 61.20 (400-OCH3), 106.22 (2C, Ph CH-
200þCH-600), 106.72 (pyrrolizine C-7), 116.55 (CN), 117.54 (pyrrolizine
C-6), 121.28 (2C, Ph CH-20þCH-60), 130.46 (Ph C-40), 131.73 (Ph C-
100), 131.97 (2C, Ph CH-30þCH-50), 132.62 (pyrrolizine C-5), 137.34
(pyrrolizine C-7a), 148.41 (Ph C-10), 153.67 (Ph C-400), 153.78 (2C, Ph
C-300þC-500), 158.31 (PhNHCO), 160.29 (N═CH). DEPT C135 (CDCl3,
125 MHz, d ppm): d 24.64 (pyrrolizine CH2-2), 25.44 (pyrrolizine
CH2-1), 50.15 (pyrrolizine CH2-3), 56.40 (2C, 300-OCH3þ500-OCH3),
61.20 (400-OCH3), 106.18 (2C, Ph CH-200þCH-600), 121.25 (2C, Ph CH-
20þCH-60), 131.97 (2C, Ph CH-30þCH-50). MS (EI): m/z (%) 525
([M þ 3]þ, 6), 524 ([M þ 2]þ, 21), 523 ([M þ 1]þ, 8), 522 (Mþ, 14),
368 (28), 352 (100), 313 (22), 236 (13), 195 (17), 109 (16), 97 (26),
69 (35). Anal. Calcd. for C25H23BrN4O4 (523.38): C, 57.37; H, 4.43; N,
10.70. Found: C, 56.93; H, 4.57; N, 11.16.
2.1.1.4. N-(4-Chlorophenyl)-7-cyano-6-((3,4,5-trimethoxybenzylide-
ne)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15d). The
title compound was prepared from the reaction of compound
14d (0.9 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g,
4 mmol) according to the general procedure A. Compound 15d
was obtained as a yellow amorphous solid product, m.p. 271–3 ꢁC,
yield 64%. IRVmax/cmꢀ1 3277, 3234, 3177 (NH), 3065 (aromatic
C–H), 2996, 2939 (aliphatic C–H) 2208 (CN), 1676 (COs), 1611,
1577, 1547 (C═ C, C═ N), 1417, 1398, 1291 (C–N, C–O). 1H NMR
(CDCl3, 500 MHz, d ppm): 2.55–2.61 (m, 2H, pyrrolizine CH2-2), 3.07
(t, 2H, J¼ 7.4 Hz, pyrrolizine CH2-1), 3.94 (s, 6H, 300-OCH3þ500-
2.1.2. General procedure (B) for the preparation of compounds
16a–e
A mixture of pyrrolizine-5-carboxamides 14a–e (3 mmol) and 3,4,5-
trimethoxybenzoyl chloride (1 g, 4.4 mmol) in dry benzene (30 mL)
was stirred for 48 h at room temperature (Scheme 1). The reaction
mixture was filtered, set a side. The formed precipitate was recrys-
tallised from ethanol–acetone (1:1).
2.1.2.1. 7-Cyano-N-phenyl-6-(3,4,5-trimethoxybenzamido)-2,3-dihy-
OCH3), 3.96 (s, 3H, 400-OCH3), 4.53 (t, 2H, J¼ 7.1 Hz, pyrrolizine CH2- dro-1H-pyrrolizine-5-carboxamide (16a). The title compound was
3), 7.15 (s, 2H, Ph CH-200þCH-600), 7.28 (d, 2H, J¼ 7.8 Hz, Ph CH- prepared from the reaction of compound 14a (0.8 g, 3 mmol) with
30þCH-50), 7.60 (d, 2H, J¼ 7.8 Hz, Ph CH-20þCH-60), 9.08 (s, H, N═ 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the
CH), 10.67 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, d ppm): d 24.60 general procedure B. Compound 16a was obtained as a white
(pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.14 (pyrrolizine solid product, m.p. 253–5 ꢁC, yield 63%. IRVmax/cmꢀ1 3190, 3131
CH2-3), 56.37 (2C, 300-OCH3þ500-OCH3), 61.11 (400-OCH3), 105.95 (2C, (NHs), 3064, 3014 (aromatic C–H), 2968, 2909 (aliphatic C–H) 2224
Ph CH-200þCH-600), 116.18 (pyrrolizine C-7), 117.42 (CN), 120.90 (2C, (CN), 1656, 1639 (COs), 1584, 1567 (C═C, C═N), 1465, 1338, 1297
Ph CH-20þCH-60), 126.45 (pyrrolizine C-6), 128.98 (Ph C-100), 129.05 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, d ppm): 2.47–2.51 (m,
(2C, Ph CH-30þCH-50), 130.65 (Ph C-40), 138.87 (pyrrolizine C-5), 2H, pyrrolizine CH2-2), 3.04 (t, 2H, J¼ 7.4 Hz, pyrrolizine CH2-1),
139.60 (pyrrolizine C-7a), 142.34 (Ph C-10), 148.37 (Ph C-400), 153.85 3.73 (s, 3H, 400-OCH3), 3.84 (s, 6H, 300-OCH3þ500-OCH3), 4.32 (t, 2H,
(2C, Ph C-300þC-500), 158.46 (PhNHCO), 159.75 (N═ CH). DEPT135 J¼ 7.2 Hz, pyrrolizine CH2-3), 7.06 (t, H, J¼ 7.4 Hz, Ph CH-40), 7.31
(CDCl3, 125 MHz, d ppm): d 24.61 (pyrrolizine CH2-2), 25.45 (pyrroli- (t, 2H, J¼ 7.9 Hz, Ph CH-30þCH-50), 7.35 (s, 2H, Ph CH-200þCH-600),
zine CH2-1), 50.14 (pyrrolizine CH2-3), 56.37 (2C, 300-OCH3þ500- 7.58 (d, 2H, J¼ 7.7 Hz, Ph CH-20þCH-60), 9.78 (s, H, CONH), 10.34 (s,

1318
A. M. SHAWKY ET AL.
H, CONH). 13C NMR (DMSO-d6, 125 MHz, d ppm): d 24.88 (pyrroli- pyrrolizine CH2-3), 6.85 (d, 2H, J¼ 8.8 Hz, Ph CH-30þCH-50), 7.45 (s,
zine CH2-2), 25.71 (pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 2H, Ph CH-200þCH-600), 7.52 (d, 2H, J¼ 8.4 Hz, Ph CH-20þCH-60),
56.57 (2C, Ph 300-OCH3þ500-OCH3), 60.63 (400-OCH3), 84.98 (pyrroli- 10.69 (broad s, 2H, two CONHs). 13C NMR (DMSO, 125 MHz, d
zine C-7), 105.80 (2C, Ph CH-200þCH-600), 115.26 (CN), 119.06 (Ph C- ppm): d 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47
100), 119.75 (2C, Ph CH-20þCH-60), 124.16 (Ph CH-40), 128.80 (pyrroli- (pyrrolizine CH2-3), 55.66 (40-OCH3), 56.39 (2C, 300-OCH3þ500-OCH3),
zine C-5), 128.84 (pyrrolizine C-7a), 129.34 (2C, Ph CH-30þCH-50), 60.58 (400-OCH3), 84.33 (pyrrolizine C-7), 105.76 (2C, Ph CH-200þCH-
139.00 (pyrrolizine C-6), 141.23 (Ph C-10), 146.44 (Ph C-400), 153.21 600), 106.44 (CN), 114.35 (2C, Ph CH-30þCH-50), 116.20 (Ph C-10),
(2C, Ph C-300þC-500), 157.92 (PhNHCO), 166.38 (PhCONH). DEPT C135 120.89 (2C, Ph CH-20þCH-60), 121.46 (Ph C-100), 132.77 (pyrrolizine
(DMSO-d6, 125 MHz, d ppm): d 24.88 (pyrrolizine CH2-2), 25.71 C-5), 140.40 (pyrrolizine C-7a), 145.92 (pyrrolizine C-6), 152.32 (Ph
(pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 56.56 (2C, Ph 300- C-400), 152.96 (2C, Ph C-300þC-500), 155.57 (Ph C-40), 158.33
OCH3þ500-OCH3), 60.63 (400-OCH3), 105.78 (2C, Ph CH-200þCH-600), (PhNHCO), 166.33 (PhCONH). DEPT C135 (DMSO, 125 MHz, d ppm):
119.75 (2C, Ph CH-20þCH-60), 124.16 (Ph CH-40), 129.34 (2C, Ph CH- d 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47 (pyrroli-
30þCH-50). MS (EI): m/z (%) 460 (Mþ, 1), 373 (2), 368 (2), 367 (6), zine CH2-3), 55.66 (40-OCH3), 56.39 (2C, 300-OCH3þ500-OCH3), 60.58
344 (2), 266 (1), 169 (10), 195 (100), 173 (11), 167 (3), 147 (9), 145 (400-OCH3), 105.75 (2C, Ph CH-200þCH-600), 114.35 (2C, Ph CH-
(22), 122 (5), 117 (20), 106 (5), 105 (17), 93 (6), 91 (40), 78 (2). 30þCH-50), 120.89 (2C, Ph CH-20þCH-60). MS (EI): m/z (%) 492
Anal. Calcd. for C25H24N4O5 (460.48): C, 65.21; H, 5.25; N, 12.17. ([M þ 2]þ, 3), 491 ([M þ 1]þ, 14), 490 (Mþ, 52), 382 (4), 368 (100),
Found: C, 65.54; H, 5.17; N, 12.64.
341 (16), 267 (6), 195 (45), 123 (41), 77 (7). Anal. Calcd. for
C26H26N4O6 (490.51): C, 63.66; H, 5.34; N, 11.42. Found: C, 63.78; H,
5.61; N, 11.69.
2.1.2.2.
7-Cyano-N-(p-tolyl)-6-(3,4,5-trimethoxybenzamido)-2,3-
dihydro-1H-pyrrolizine-5-carboxamide (16b). The title compound
was prepared from the reaction of compound 14b (0.84 g,
2.1.2.4. N-(4-Chlorophenyl)-7-cyano-6-(3,4,5-trimethoxybenzamido)
3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) -2,3-dihydro-1H-pyrrolizine-5-carboxamide (16d). The title com-
according to the general procedure B. Compound 16b was pound was prepared from the reaction of compound 14d (0.9 g,
obtained as a white solid product, m.p. 265–7 ꢁC, yield 66%. 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol)
IRVmax/cmꢀ1 3187, 3112 (NHs), 3038 (aromatic C–H), 2968, 2870 according to the general procedure B. Compound 16d was
(aliphatic C–H) 2221 (CN), 1656, 1638 (COs), 1586, 1549 (C═C, C═ obtained as a white solid product, m.p. 281–3 ꢁC, yield 61%.
N), 1466, 1389, 1263 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, d IRVmax/cmꢀ1 3193, 3108 (NHs), 3062, 3006 (aromatic C–H), 2969,
ppm): 2.23 (s, 3H, Ph-CH3), 2.46–2.54 (m, 2H, pyrrolizine CH2-2), 2937 (aliphatic C–H) 2220 (CN), 1644 (COs), 1586, 1524 (C═C, C═
3.03 (t, 2H, J¼ 7.3 Hz, pyrrolizine CH2-1), 3.74 (s, 3H, 400-OCH3), 3.85 N), 1440, 1296, 1235 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, d
(s, 6H, 300-OCH3þ500-OCH3), 4.31 (t, 2H, J¼ 7.0 Hz, pyrrolizine CH2- ppm): 2.47–2.51 (m, 2H, pyrrolizine CH2-2), 3.04 (t, 2H, J¼ 7.4 Hz,
3), 7.10 (d, 2H, J¼ 7.9 Hz, Ph CH-30þCH-50), 7.35 (s, 2H, Ph CH- pyrrolizine CH2-1), 3.73 (s, 3H, 400-OCH3), 3.84 (s, 6H, 300-OCH3þ500-
200þCH-600), 7.47 (d, 2H, J¼ 7.8 Hz, Ph CH-20þCH-60), 9.65 (s, H, OCH3), 4.31 (t, 2H, J¼ 7.1 Hz, pyrrolizine CH2-3), 7.33 (s, 2H, Ph CH-
CONH), 10.32 (s, H, CONH). 13C NMR (DMSO-d6, 125 MHz, d ppm): 200þCH-600), 7.37 (d, 2H, J¼ 8.8 Hz, Ph CH-30þCH-50), 7.63 (d, 2H, J¼
d 20.87 (CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 8.8 Hz, Ph CH-20þCH-60), 9.94 (s, H, CONH), 10.31 (s, H, CONH). 13C
49.63 (pyrrolizine CH2-3), 56.63 (2C, 300-OCH3þ500-OCH3), 60.64 (400- NMR (DMSO-d6, 125 MHz, d ppm): d 24.90 (pyrrolizine CH2-2),
OCH3), 85.07 (pyrrolizine C-7), 105.89 (2C, Ph CH-200þCH-600), 25.69 (pyrrolizine CH2-1), 49.62 (pyrrolizine CH2-3), 56.58 (2C, 300-
115.21 (CN), 119.32 (Ph C-100), 119.77 (2C, Ph CH-20þCH-60), 127.09 OCH3þ500-OCH3), 60.63 (400-OCH3), 84.98 (pyrrolizine C-7), 105.82
(pyrrolizine C-5), 128.70 (pyrrolizine C-7a), 129.69 (2C, Ph CH- (2C, Ph CH-200þCH-600), 115.23 (CN), 118.67 (Ph C-40), 121.29 (Ph C-
30þCH-50), 133.23 (Ph C-10), 136.47 (pyrrolizine C-6), 141.36 (Ph C- 100), 121.41 (2C, Ph CH-20þCH-60), 127.70 (pyrrolizine C-5), 128.87
40), 146.32 (Ph C-400), 153.24 (2C, Ph C-300þC-500), 157.69 (PhNHCO), (pyrrolizine C-7a), 129.21 (2C, Ph CH-30þCH-50), 138.03 (pyrrolizine
166.40 (PhCONH). DEPT C135 (DMSO-d6, 125 MHz, d ppm): d 20.87 C-6), 141.21 (Ph C-10), 146.58 (Ph C-400), 153.19 (2C, Ph C-300þC-500),
(CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 49.63 158.05 (PhNHCO), 166.24 (PhCONH). DEPT C135 (DMSO-d6,
(pyrrolizine CH2-3), 56.63 (2C, 300-OCH3þ500-OCH3), 60.64 (400-OCH3), 125 MHz, d ppm): d 24.90 (pyrrolizine CH2-2), 25.70 (pyrrolizine
105.89 (2C, Ph CH-200þCH-600), 119.77 (2C, Ph CH-20þCH-60), 129.69 CH2-1), 49.61 (pyrrolizine CH2-3), 56.57 (2C, 300-OCH3þ500-OCH3),
(2C, Ph CH-30þCH-50). MS (EI): m/z (%) 475 ([M þ 1]þ, 2), 474 (Mþ, 60.63 (400-OCH3), 105.80 (2C, Ph CH-200þCH-600), 121.40 (2C, Ph CH-
9), 368 (34), 367 (37), 352 (6), 341 (4), 284 (6), 280 (1), 200 (11), 20þCH-60), 129.21 (2C, Ph CH-30þCH-50). MS (EI): m/z (%) 496
196 (11), 195 (100), 174 (2), 167 (6), 146 (2), 145 (1), 122 (4), 117 ([M þ 2]þ, 1), 494 (Mþ, 1), 480 (1), 450 (6), 436 (4), 400 (7), 396 (5),
(2), 107 (30), 106 (5), 92 (3), 78 (2), 77 (7). Anal. Calcd. for 387 (7), 372 (6), 352 (20), 340 (14), 339 (11), 329 (17), 328 (7), 327
C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.30; H, (9), 313 (13), 299 (12), 298 (15), 280 (4), 279 (11), 273 (21), 269
5.71; N, 11.99.
(15), 268 (48), 254 (20), 240 (26), 228 (30), 210 (40), 196 (23), 195
(31), 174 (100), 169 (31), 147 (29), 145 (17), 123 (31), 117 (29), 107
(44), 92 (36), 77 (14). Anal. Calcd. for C25H23ClN4O5 (494.93): C,
60.67; H, 4.68; N, 11.32. Found: C, 61.13; H, 4.87; N, 11.06.
2.1.2.3.
7-Cyano-N-(4-methoxyphenyl)-6-(3,4,5-trimethoxybenza-
mido)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (16c). The title
compound was prepared from the reaction of compound 14c
(0.90 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g,
2.1.2.5. N-(4-Bromophenyl)-7-cyano-6-(3,4,5-trimethoxybenzamido)
4.4 mmol) according to the general procedure B. Compound 16c -2,3-dihydro-1H-pyrrolizine-5-carboxamide (16e). The title com-
was obtained as a light buff solid product, m.p. 259–61 ꢁC, yield pound was prepared from the reaction of compound 14e (1.04 g,
69%. IRVmax/cmꢀ1 3196 (NHs), 3068, 3003 (aromatic C–H), 2963, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol)
2941, 2838 (aliphatic C–H) 2223 (CN), 1657, 1634 (COs), 1586, 1511 according to the general procedure B. Compound 16e was
(C═ C, C═ N), 1464, 1339, 1234 (C–N, C–O). 1H NMR (DMSO-d6, obtained as a light buff amorphous solid product, m.p. 286–8 ꢁC,
500 MHz, d ppm): d 2.39–2.44 (m, 2H, pyrrolizine CH2-2), 2.88 (t, yield 64%. IRVmax/cmꢀ1 3222 (NHs), 3060 (aromatic C–H), 2972,
2H, J¼ 7.0 Hz, pyrrolizine CH2-1), 3.71 (s, 3H, 40-OCH3), 3.72 (s, 3H, 2938, 2835 (aliphatic C–H) 2220 (CN), 1644 (COs), 1587, 1524
400-OCH3), 3.81 (s, 6H, 300-OCH3þ500-OCH3), 4.25 (t, 2H, J¼ 7.0 Hz, (C═ C, C═ N), 1414, 1388, 1296 (C–N, C–O). 1H NMR (DMSO-d6,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1319
500 MHz, d ppm): d 2.46–2.51 (m, 2H, pyrrolizine CH2-2), 3.04 (t, (m, 2H, indolizine CH2-7), 2.02–2.05 (m, 2H, indolizine CH2-6), 2.97
2H, J¼ 7.2 Hz, pyrrolizine CH2-1), 3.73 (s, 3H, 400-OCH3), 3.83 (s, 6H, (t, 2H, J¼ 7.3 Hz, indolizine CH2-8), 3.95 (s, 6H, 30-OCH3þ50-OCH3),
300-OCH3þ500-OCH3), 4.30 (t, 2H, J¼ 7.0 Hz, pyrrolizine CH2-3), 7.32 3.98 (s, 3H, 40-OCH3), 4.54 (t, 2H, J¼ 6.5 Hz, indolizine CH2-5), 7.12
(s, 2H, Ph CH-200þCH-600), 7.50 (d, 2H, J¼ 8.4 Hz, Ph CH-30þCH-50), (t, 1H, J¼ 7.3 Hz, Ph CH-400), 7.26 (m, 2H, Ph CH-300þCH-500), 7.32 (d,
7.57 (d, 2H, J¼ 8.6 Hz, Ph CH-20þCH-60), 9.91 (s, 1H, CONH), 10.29 2H, J¼ 7.6 Hz, Ph CH-200þCH-600), 7.69 (s, 2H, Ph CH-20þCH-60), 8.95
(s, 1H, CONH). 13C NMR (DMSO, 125 MHz, d ppm): d 24.90 (pyrroli- (s, 1H, N═CH), 10.96 (s, 1H, PhNHCO). 13C NMR (CDCl3, 125 MHz, d
zine CH2-2), 25.69 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), ppm): d 18.78 (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.02
56.56 (2C, 300-OCH3þ500-OCH3), 60.63 (400-OCH3), 84.86 (pyrrolizine (indolizine CH2-6), 46.96 (indolizine CH2-5), 56.42 (2C, 30-OCH3þ50-
C-7), 105.77 (2C, Ph CH-200þCH-600), 115.23 (CN), 115.73 (Ph C-40), OCH3), 61.18 (40-OCH3), 82.19 (indolizine C-1), 106.54 (2C, Ph CH-
118.67 (Ph C-100), 121.76 (2C, Ph CH-20þCH-60), 127.62 (pyrrolizine 20þCH-60), 106.73 (CN), 115.79 (indolizine C-2), 119.63 (Ph C-10),
C-5), 128.81 (pyrrolizine C-7a), 132.13 (2C, Ph CH-30þCH-50), 138.43 120.06 (2C, Ph CH-200þCH-600), 124.10 (Ph C-400), 128.72 (indolizine
(pyrrolizine C-6), 141.17 (Ph C-10), 146.61 (Ph C-400), 153.19 (2C, Ph C-3), 128.97 (2C, Ph CH-300þCH-500), 129.42 (indolizine C-8a), 138.35
C-300þC-500), 158.04 (PhNHCO), 166.23 (PhCONH). DEPT C135 (Ph C-100), 142.92 (Ph C-40), 153.73 (2C, Ph C-30þC-50), 158.80 (N═
(DMSO, 125 MHz, d ppm): d 24.90 (pyrrolizine CH2-2), 25.69 (pyrro- CH), 161.09 (PhNHCO). DEPT C135 (CDCl3, 125 MHz, d ppm): d 18.78
lizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.55 (2C, 300-OCH3þ500- (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.03 (indolizine CH2-
OCH3), 60.63 (400-OCH3), 105.75 (2C, Ph CH-200þCH-600), 121.75 (2C, 6), 46.97 (indolizine CH2-5), 56.41 (2C, 30-OCH3þ50-OCH3), 61.19
Ph CH-20þCH-60), 132.13 (2C, Ph CH-30þCH-50). MS (EI): m/z (%) (40-OCH3), 106.50 (2C, Ph CH-20þCH-60), 120.05 (2C, Ph CH-20þCH-
543 ([M þ 5]þ, 2), 541 ([M þ 3]þ, 3), 538 (Mþ, 3), 490 (31), 457 (12), 600), 124.02 (Ph C-400), 128.98 (2C, Ph CH-30þCH-500). MS (EI): m/z
422 (89), 368 (100), 341 (46), 318 (37), 296 (18), 200 (18), 175 (18), (%) 460 ([M þ 2]þ, 5), 458 (Mþ, 8), 422 (83), 396 (30), 382 (30), 356
121 (6), 100 (17), 73 (31). Anal. Calcd. for C25H23BrN4O5 (539.38): C, (47), 332 (100), 296 (15), 106 (13), 89 (26), 77 (12), 69 (31). Anal.
55.67; H, 4.30; N, 10.39. Found: C, 55.83; H, 3.91; N, 10.62.
Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. Found:
C, 67.88; H, 5.46; N, 12.53.
2.1.3. 1-Cyano-N-phenyl-2-((3,4,5-trimethoxybenzylidene)amino)-5,
6,7,8-tetrahydroindolizine-3-carboxamide (20)
2.1.4. 1-Cyano-N-phenyl-2-(3,4,5-trimethoxybenzamido)-5,6,7,8-tet-
A mixture of indolizine-3-carboxamides 19 (0.84 g, 3 mmol) and rahydroindolizine-3-carboxamide (21)
3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol), 0.5 mL glacial acetic A mixture of indolizine-3-carboxamides 19 (0.84 g, 3 mmol) and
acid in absolute ethanol (30 mL) was stirred under reflux for 5 h 3,4,5-trimethoxybenzoyl chloride (1 g, 4.4 mmol) in dry benzene
(Scheme 2). The solvent was then evaporated under reduced pres- (30 mL) was stirred for 48 h at room temperature (Scheme 2). The
sure. The solid obtained was collected and recrystallised from ace- reaction mixture was filtered, set aside. The precipitate obtained
tone–chloroform (1:1). Compound 20 was obtained as a yellow was recrystallised from ethanol–acetone (1:1). Compound 21 was
amorphous solid product, m.p. 247–9 ꢁC, yield 73%. IRVmax/cmꢀ1 obtained as a white solid product, m.p. 233–5 ꢁC, yield 65%.
3182 (NH), 3068 (aromatic C–H), 2938, 2875, 2824 (aliphatic C–H) IRVmax/cmꢀ1 3215, 3187 (NHs), 3061, 3040, 3015 (aromatic C–H),
2209 (CN), 1677 (COs), 1609, 1594, 1575 (C═C, C═N), 1481, 1330, 2960, 2944, 2839 (aliphatic C–H), 2221 (CN), 1648 (COs), 1587,
1230 (C–N, C–O). 1H NMR (CDCl3, 500 MHz, d ppm): d 1.91–1.95 1566 (C═ C, C═ N), 1317, 1235 (C–N, C–O). 1H NMR (DMSO-d6,
Scheme 2. Synthesis of compounds 20 and 21.

1320
A. M. SHAWKY ET AL.
500 MHz, d ppm): d 1.72–1.76 (m, 2H, indolizine CH2-7), 1.85–1.89 3 h, the supernatant was aspirated, and 100 lL of DMSO was
(m, 2H, indolizine CH2-6), 2.71 (t, 2H, J¼ 5.7 Hz, indolizine CH2-8),
added to each well. The optical density (OD) of the purple forma-
zan is proportional to the number of viable cells. When the
amount of formazan produced by treated cells is compared with
the amount of formazan produced by untreated control cells, the
strength of the drug in causing growth inhibition can be deter-
mined, through plotting growth curves of absorbance against
3.71 (s, 3H, 40-OCH3), 3.76 (s, 6H, 30-OCH3þ50-OCH3), 4.24 (t, 2H, J¼
5.6 Hz, indolizine CH2-5), 6.97 (d, 2H, J¼ 7.3 Hz, Ph CH-400), 7.23 (t,
2H, J¼ 7.7 Hz, Ph CH-300þCH-500), 7.37 (s, 1H, PhCONH), 7.44 (s, 2H,
Ph CH-20þCH-60), 7.58 (d, 2H, J¼ 7.9 Hz, Ph CH-200þCH-600), 10.92
(broad s, 1H, PhNHCO). 13C NMR (DMSO, 125 MHz, d ppm): d 19.15
(indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-
6), 45.90 (indolizine CH2-5), 56.21 (2C, 30-OCH3þ50-OCH3), 60.56
(40-OCH3), 88.27 (indolizine C-1), 105.69 (2C, Ph CH-20þCH-60),
116.52 (CN), 119.25 (2C, Ph CH-200þCH-600), 123.00 (Ph C-10), 125.71
(indolizine C-3), 128.80 (Ph CH-400), 129.10 (2C, Ph CH-300þCH-500),
129.38 (indolizine C-8a), 139.84 (indolizine C-2), 140.12 (Ph C-100),
140.20 (Ph C-40), 152.77 (2C, Ph C-30þC-50), 159.62 (PhNHCO),
166.29 (PhCONH). DEPT C135 (DMSO, 125 MHz, d ppm): d 19.15
(indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-
6), 45.90 (indolizine CH2-5), 56.21 (2C, 30-OCH3þ50-OCH3), 60.56
(40-OCH3), 105.68 (2C, Ph CH-20þCH-60), 119.25 (2C, Ph CH-200þCH-
600), 128.80 (Ph CH-400), 129.10 (2C, Ph CH-300þCH-500). MS (EI): m/z
(%) 475 ([M þ 1]þ, 3), 474 (Mþ, 10), 381 (82), 366 (6), 262 (4), 195
(64), 152 (6), 109 (20), 93 (57), 81 (71), 69 (98). Anal. Calcd. for
C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 66.21; H,
5.78; N, 11.66.
drug concentration, thus formulation concentration causing 50%
inhibition (IC50) compared to control cell growth (100%) were
determined. GraphPad Prism version 5.00 for Windows was used
for analysis (La Jolla, CA). Cytotoxicity of the new compounds was
calculated after treatment of cancer/normal cell lines for 72 h. The
absorbance of the reduced MTT was determined using a micro-
plate reader A570 nm.
2.2.2. Annexin V-FITC/PI apoptosis assay
The ability of compounds 16a,b,d to induce apoptotic changes in
MCF-7 cells was determined by Annexin V-FITC/PI staining accord-
ing to the previous report13,18,19. Additionally, the morphological
changes in MCF-7 cells were examined by microscope. Following
treatment with the test compounds at 0.1 mM for 24 h, the cells
were stained with annexin V and PI. The determination of nec-
rotic, early/late apoptotic changes were performed by flow cytom-
eter (FC500, Beckman Coulter, Miami, FL).
2.2. Biological evaluation
2.2.1. Evaluation of cytotoxic activity
2.2.3. Kinase inhibitory activity
2.2.1.1. Cell culture. MCF-7 (human breast adenocarcinoma),
A2780 (human ovarian cancer) and HCT116 (human colorectal car-
cinoma), and MRC-5 (normal foetal lung fibroblast) cell lines were
bought from the ATCC and were cultured in flasks which were
incubated at 37 ꢁC in an atmosphere of 5% CO2, 95% air, and
100% relative humidity, to maintain continuous logarithmic
growth. MCF-7, A2780, and HCT116 were maintained in RPMI-1640
media (100 U/mL penicillin, 100 mg/mL streptomycin, and 10% foe-
tal bovine serum, Gibco, Carlsbad, CA). MRC-5 cells were main-
tained in EMEM (100 U/mL penicillin, 100 mg/mL streptomycin, and
10% foetal bovine serum, Gibco, Carlsbad, CA). Doxorubicin (5 lg/
mL, for three months) added for RPMI-1640 media containing
MCF7 cells was used to develop doxorubicin-resistant MCF7/ADR
cells. All cancer and normal cell lines were cultured according to
our previous report13. Cells were used within 10–20 passages and
2.2.3.1. Kinase profiling test. Compounds 16a,b,d and imatinib
were evaluated for their inhibitory activities against 20 kinases at
a single concentration (10 lM). The test was performed using the
radiolabeled ATP determination method (KINEXUS Bioinformatics
Corporation, Vancouver, Canada). The test was performed follow-
ing the previous reports13,20
.
2.2.3.2. CDK-2 inhibitory assay. The ability of compounds 16a,b,d
to inhibit the activity of CDK-2 was determined using ADP-Glo
assay (Promega, Madison, WI). The assay was done according to
the manufacturer’s instructions and as described in the previ-
ous report21.
were checked for mycoplasma every 6 months, by measuring the 2.2.4. Cell cycle analysis
bioluminescence (Myco Alert sample detection kit; Lonza, Basel,
Switzerland) using the multiplate reader (Synergy HT, BioTek,
Winooski, VT).
Cell cycle perturbations of MCF-7 cells treated with compounds
16a,b,d at 0.1 mM concentration for 24 h was determined using
FC500 flow cytometer (FC500, Beckman Coulter, Miami, FL). The
cell cycle analysis was performed according to our previ-
ous report13.
2.2.1.2. MTT cell proliferation assay. The MTT assay was used to
measure the cytotoxicity and growth inhibitory activities and cyto-
toxicity of the new compounds according to our previous
report13,17. Cells from flasks of 70–80% confluency were separately
seeded in 96-well flat-bottom microculture plates (Nalgene-Nunc,
Thermo Fisher Scientific, Roskilde, Denmark) at a density of
3 ꢂ 103 cells (MCF-7, A2780, and MRC-5), 1 ꢂ 104 cells (HCT116), to
a volume of 180 lL/well of culture medium. A Neubauer haemo-
cytometer was used for cell counting. Cells were incubated at
37 ꢁC overnight to allow attachment to the wells. The final con-
centrations of each compound in wells were: 0–50 lM in 200 lL of
media (DMSO 0.1%). Lapatinib (Cayman, Ann Arbor, MI) was used
as a positive control at the same final concentrations. Medium
only (20 lL) was added to each control well, and each concentra-
tion was tested in triplicates (n ¼ 3). Following incubation for 72 h,
2.2.5. Tubulin polymerisation
2.2.5.1. Tubulin polymerisation assay. The effect of compounds
16a,b,d on tubulin polymerisation was measured in vitro using
tubulin polymerisation fluorescence-based assay (cytoskeleton, cat.
BK011P, https://www.cytoskeleton.com/bk011p). The assay was
performed following the previous report22 and according to the
manufacturer’s instructions. Briefly, 2 mg/mL porcine tubulin was
dissolved in buffer 1 (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA pH
6.9, 10 mM fluorescent reporter, 1 mM GTP, 15% glycerol) to a final
concentration of 10 mg/mL. Then tubulin solution was transferred
to a pre-warmed 96-well plate that contained the test compounds
16a,b,d (5 lM), paclitaxel (3 lM), CaCl2 (0.5 lM), and control buffer.
50 lL MTT was added into each well. Plates were incubated for The polymerisation of tubulin was monitored as fluorescence at

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1321
37 ꢁC for 60 min. Relative fluorescence units (RFU) were deter-
mined at 360 nm for excitation and at 460 nm for emission using
the Varioskan Flash spectral scanning multimode reader (Thermo
Fisher Scientific, Roskilde, Denmark) at a reading speed of 1
cycle/min.
The Schiff base 20 was also obtained from the condensation of
3,4,5-trimethoxybenzaldehyde and 2-aminoindolizine-3-carboxa-
mide 19 in ethanol. On the other hand, acylation of compound
19 with 3,4,5-trimethoxybenzoyl chloride yielded the benzamide
derivative 21. Spectral (IR, H NMR, 13C NMR, DEPT C135, and mass
spectra) and elemental analyses were performed to confirm the
chemical structure of compounds 20 and 21. The spectra of the
two compounds are also provided in Supplementary data (Figs.
2.2.5.2. Immunofluorescence staining. The MCF-7 cancer cells
plated on coverslips on six-well plate (1 ꢂ 105/well) were treated
with the indicated concentration of control, colchicine (3 lM), S6, S12, S49–S54, S96–S105, S125, and S141).
paclitaxel (3 lM), and the test compound 16b (5 lM) for 24 h as
previously reported23. After treatment, cells were rinsed twice with
3.2. Biological evaluation
PBS, fixed with 3.7% paraformaldehyde, and permeabilised with
0.1% Triton X-100. Cells then were blocked with 1% BSA in PBS
3.2.1. Cytotoxic activity
3.2.1.1. Evaluation of cytotoxic activity against cancer cell lines.
for 1 h before further incubation with anti-b-tubulin mouse mono-
clonal antibody overnight at 4 ꢁC (#86298, Cell Signaling, San
The cytotoxic activity of the new compounds (15a–e, 16a–e, 20,
and 21) was evaluated in vitro using MTT assay13. The MTT assay
depends on the reduction of the yellow tetrazole dye (MTT) to
the purple formazan by the active mitochondrial dehydrogenase
enzymes indicating the viability of the cells which can be meas-
ured as OD. The new compounds were evaluated against three
non-resistant cancer cell lines: MCF-7, and A2780 cell lines were
selected to compare cytotoxicity of the new compounds with the
previously reported results of compounds 9a,b13. Cytotoxicity of
the new compounds was also evaluated also against HCT116 cells
which lacks COX-2 protein33,34. Selection of this cell line was
based on the previous reports which revealed potent cytotoxic
activity of the five membered heterocyclic derivatives against
R
Francisco, CA). Cells were incubated with Alexa FluorV 488 second-
ary antibodies (Abcam, Cambridge, UK), after being washed with
PBS for 1 h in a darkroom. Cellular microtubules were observed
with the Nikon Eclipse Ti microscope (Minato-ku, Japan).
2.3. Molecular docking
The molecular docking simulation of the new compounds was car-
ried out by AutoDock 4.224. The crystal structure of CDK2 bound
to CAN508 (pdb code: 3TNW)25, EGFR bound to erlotinib (pdb
code: 1M17)26, Aurora A bound to VX6 (pdb code: 3E5A)27, and
tubulin bound to C-A428, were obtained from protein data bank
(http://www.rcsb.org/pdb). Ligand and protein files were prepared
according to the previous report13,29. Docking and grid parameter
files were prepared according to the previous reports30,31. The top
10 protein–ligand complexes were scored. The results including
binding modes, affinities, and interactions of the best-fitting con-
formations of the new compounds were visualised using
Discovery Studio Visualizer (v16.1.0.15350, Dassault Systems, San
Diego, CA)32.
HCT116 cells35,36
.
The results of the MTT assay (Table 1) revealed variable
degrees of cytotoxic activities for the new compounds with IC50
values in the range of 0.030–36.880 lM against the three cancer
cell lines. The results also revealed higher cytotoxic activities for
the benzamide derivatives (16a–e and 21) compared to their cor-
responding benzylidene analogues (15a–e and 20).
Compound 16b was the most potent in inhibiting the growth
of MCF-7 cells, while compounds 16c,d exhibited the highest
cytotoxic activity against HCT116 and A2780 cells, respectively. On
the other hand, lapatinib exhibited cytotoxic activity against the
three cancer cell lines with IC50 values in the range of
6.690–10.043 lM (Table 1).
The new compounds exhibited cytotoxic activities against
MCF-7 cells with IC50 values in the range 0.068–36.880 lM. In add-
ition, they inhibited the growth of A2780 cells with values in the
range 0.030–33.360 lM. Interestingly, the new compounds also
exhibited potent cytotoxic activities against HCT116 cells with IC50
values in the range of 00.032–19.835 lM. These results indicated
that COXs inhibition may not be essential for the cytotoxicity of
the compounds reported in this study.
3. Results and discussion
3.1. Chemistry
In this work, compound 11 was prepared from the reaction of
pyrrolidin-2-one 10 and malononitrile as previously reported13. On
the other hand, the acetanilides 13a–e were obtained from the
reaction of (un)substituted anilines 13a–e with chloroacetyl chlor-
ide according to the reported procedures13 (Scheme 1). The start-
ing materials 14a–e were prepared from the reaction of
compounds 11 and 13a–e in acetone13.
The Schiff bases 15a–e were obtained on refluxing compounds
14a–e with 3,4,5-trimethoxybenzaldehyde in absolute ethanol in
the presence of glacial acetic acid. On the other hand, the benza-
mide derivatives 16a–e were prepared from the reaction of com-
pounds 14a–e with 3,4,5-trimethoxybenzoyl chloride in dry
benzene (Scheme 1). Structural elucidation of the new compounds
was performed using both spectral and elemental analyses. Copies
of spectral data including IR, 1H NMR, 13C NMR, DEPT C135, and
mass spectra are provided in supplementary data (Figs. S1–S141).
In addition, compound 18 (Scheme 2) was obtained on the
treatment of piperidin-2-one 17 with dimethyl sulphate and malo-
nonitrile according to the reported procedures13. The starting
material, indolizine 19 was synthesised using the same reaction
conditions adopted for the synthesis of compound 14a with the
replacement of 2-(pyrrolidin-2-ylidene)malononitrile 11 by 2-
(piperidin-2-ylidene)malononitrile 18.
3.2.1.2. Evaluation of cytotoxic activity against doxorubicin-resist-
ant MCF-7/ADR cells. Encouraged by the high cytotoxic activity of
the new compounds against MCF-7 cells (Table 1), three of these
derivatives (16a,b,d) were also selected to evaluate their cytotox-
icity against doxorubicin-resistant MCF-7/ADR cells. The evaluation
was performed using MTT assay13. The results are presented in
Table 2. The results revealed cytotoxic activity for the three com-
pounds against MCF-7/ADR cells with IC50 in the range of
0.52–6.26 lM. Among the three derivatives, compound 16d with
the electron-withdrawing chloro atom exhibited the highest cyto-
toxic activity against MCF-7/ADR cells, while the methyl analogue
16b was the least active. These results suggested that electron-

1322
A. M. SHAWKY ET AL.
Table 1. Cytotoxicity (mM, IC50 SD) of compounds 15a–e, 16a–e, 20, and 21 against MCF-7, HCT116, and A2780 cancer cell lines in comparison to compounds 9a,b,
lapatinib, and colchicine.
IC50 (mM SD)a
Comp. no.
Linker
n
R
MCF-7
HCT116
A2780
Averageb
15a
15b
15c
15d
15e
20
16a
16b
–N═CH–
–N═CH–
–N═CH–
–N═CH–
–N═CH–
–N═CH–
–NHCO–
–NHCO–
–NHCO–
–NHCO–
–NHCO–
–NHCO–
–
1
1
1
1
1
2
1
1
1
1
1
2
–
–
–
–
H
CH3
OCH3
Cl
Br
H
36.880 4.400
35.860 4.440
0.616 0.010
21.750 3.180
0.1872 0.098
5.721 0.185
0.082 0.002
0.068 0.009
1.470 0.452
0.071 0.001
0.361 0.039
0.600 0.054
0.100 0.050
0.50 0.15
0.353 0.088
19.835 7.756
0.096 0.015
0.534 0.204
0.144 0.049
0.247 0.069
0.135 0.007
0.092 0.001
0.032 0.014
0.097 0.001
0.173 0.039
0.176 0.017
–
25.760 4.990
33.360 3.730
0.041 0.008
0.070 0.002
0.311 0.041
0.938 0.116
0.044 0.010
0.180 0.043
0.144 0.012
0.030 0.005
0.914 0.092
0.598 0.039
0.520 0.020
4.16 1.27
20.998
29.685
0.251
7.451
0.214
2.302
0.087
0.113
0.549
0.066
0.483
0.458
0.310
2.33
H
CH3
OCH3
Cl
Br
H
–
–
–
–
16c
16d
16e
21
9ac
9bc
–
–
–
–
Lapatinib
6.690 1.091
0.012 0.007
10.043 1.125
0.050 0.005
9.725 0.845
0.015 0.002
8.833
0.026
Colchicined
aIC50: concentration of test compound which reduce cellular growth to 50% after 72 h treatment, results represent mean IC50 value SD (n ¼ 3) of three independent
experiments.
bAverage of the three IC50 values against the three cancer cell lines.
cIC50 values quoted from our previous publication13
.
dIC50 values quoted from the previous publications37,38
.
Table 2. Cytotoxicity of compounds 16a, b, d against
MCF-7/ADR cells.
Table 3. Cytotoxicity of compounds 9a,b, 15a–e, 16a–e, 20, 21, and lapatinib
against normal MRC-5 cells.
Comp. no.
IC50 (mM SD)a
Selectivity indexb
IC50 (mM SD)a
16a
16b
16d
1.570 0.040
6.260 0.710
0.520 0.066
Comp. no.
MRC-5
MCF-7
HCT116
A2780
15a
15b
15c
15d
15e
20
16a
16b
16c
16d
16e
21
1.735 0.149
3.325 1.001
1.979 0.561
2.073 0.054
17.080 0.552
1.517 0.103
0.155 0.062
5.851 1.187
0.665 0.191
8.100 0.897
5.259 1.398
0.584 0.094
25.810 0.730c
9.00 0.13c
0.05
0.09
3.21
0.10
91.24
0.27
1.89
86.04
0.45
114.08
14.57
0.97
4.92
0.17
20.47
3.88
118.61
6.14
1.15
63.60
20.78
83.51
30.40
3.32
–
0.07
0.10
48.27
29.61
54.92
1.62
3.52
32.51
4.62
270.00
5.75
0.98
49.63
2.16
aIC50: concentration of test compound which reduce
cellular growth of MCF-7/ADR cells to 50% after 72 h
treatment, results represent mean IC50 SD, (n ¼ 3) of
three independent experiments.
withdrawing groups on the phenyl ring of scaffold A could be
used to enhance cytotoxic activity against MCF-7/ADR cells.
9a
9b
Lapatinib
258.10
18.00
2.04
–
1.36
3.2.1.3. Evaluation of cytotoxic selectivity. Selectivity towards can-
cerous cells is one of the important issues which must be consid-
ered during the discovery and development of new anticancer
agents. To assess the toxicity and selectivity of the new com-
pounds (15a–e, 16a–e, 20, and 21), their growth inhibitory activ-
ities against normal human foetal lung fibroblast MRC-5 cells were
evaluated using the MTT assay. The results expressed as IC50 val-
ues are presented in Table 3. The results revealed variable cyto-
13.660 2.900
1.40
aIC50 against MRC-5 cells after 72 h treatment with the test compound, results
represent mean IC50 SD (n ¼ 3).
bSelectively index (SI)¼IC50 value against normal MRC-5 cells/IC50 value against
cancer cell line.
cIC50 value quoted from our previous publication13
.
toxic activity of the new compounds against the normal MRC-5 against normal MRC-5 cells by their IC50 values against the corre-
cells (IC50¼0.155–17.080 lM), compared to 25.810 and 9.00 lM for sponding cancer cell line (Table 3).
compounds 9a,b, respectively13. However, compounds 15c,e, and
16a,b,d were more potent and/or more selective than compound 0.05–118.45) towards the three cancer cell lines than their benza-
9a. The selectivity of the new compounds towards each of the mide analogues 16a–e (SI ¼ 0.45–270). Among the new com-
three cancer cell lines was calculated by dividing their IC50 values pounds, compound 16d was the most selective towards MCF-7
The five Schiff bases 15a–e exhibited lower selectivity (SI ¼

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1323
Figure 4. SAR of cytotoxicity of the new compounds (15a–e, 16a–e, 20, and 21).
Table 4. Induction of apoptosis in MCF-7 cells treated with compounds 16a,b,d.
and A2780 cells, while compound 15e showed the highest select-
ivity to the HCT116 cancer cell line (Table 3).
% Cell numbera
Evaluation of cytotoxicity of the indolizine derivatives 20 and
21 against normal MRC-5 cells revealed IC50 values of 1.517 and
0.584 lM, respectively. The two compounds exhibited selectivity
indices in the range of 0.27–6.14 towards the three cancer cell
lines. Compound 20 was more cytotoxic towards the MRC-5 cells
than its pyrrolizine analogue 15a. On the other hand, the indoli-
zine 21 was less toxic to the normal cells compared to the pyrroli-
zine analogue 16a (Table 3).
Comp. no.
Live cells
Early apoptosis
Late apoptosis
Necrosis
Control
16a
16b
97.20 3.13
77.40 1.39
75.00 2.90
71.99 2.88
0.48 0.04
8.40 0.87
6.22 0.22
7.50 0.95
0.36 0.06
10.70 1.06
13.01 1.02
19.94 1.30
1.90 0.20
3.50 0.37
7.11 0.80
2.51 0.45
16d
aThe results were obtained on treatment of MCF-7 cells by compounds 16a,b,d
(0.1 lM) or vehicle control for 24 h (n¼ 3), values represent mean% SD of
three independent experiments.
3.2.1.4. Structure–activity relationship. The study of SAR of the
new compounds is represented in Figure 4. The Schiff base 15a
exhibited potent cytotoxic activity against HCT116 (IC50¼0.353 lM)
and very weak cytotoxicity against A2780 and MCF-7 cancer cell
lines with IC50 values of 25.76 and 36.88 lM, respectively. On the
other hand, replacement of the 3,4,5-trimethoxybenzylidene)amino
in compound 15a with 3,4,5-trimethoxybenzamide moiety
resulted in a significant increase in the cytotoxic activities against
the three cancer cell lines.
Replacement of the 3,4,5-trimethoxybenzylidene-amino moiety
in compound 15a with 3,4,5-trimethoxybenzamido moiety
resulted in a remarkable increase (2.6–585.4-fold) in cytotoxic
activities against the three cell lines. Expansion of the pyrrolidine
ring in compound 16a to piperidine ring resulted in an increase
in cytotoxic activity against the three cancer cell lines.
In conclusion, the 3,4,5-trimethoxybenzamido moiety and the
electron-withdrawing atoms on the phenyl ring at C-5 on the pyr-
rolizine scaffold are favoured for high cytotoxic outcomes.
The substituents on the phenyl ring showed variable effects on
cytotoxicity of the new compounds. In Schiff base derivatives
15a–e, substitution on para-position of the phenyl ring of com-
pound 15a with electron-donating (OCH3) or electron-withdrawing
(Cl/Br) atoms resulted in a significant increase (1.7–628.3-fold) in
cytotoxic activity against MCF-7 and A2780 cells, while substitu-
tion with CH3 group decreased cytotoxicity against both HCT116
and A2780 cancer cell lines (Figure 4).
3.2.2. Annexin V-FITC/PI apoptosis assay
The cytotoxic activity of our previously reported pyrrolizine-5-car-
boxamide 9a was mediated by the induction of apoptosis in MCF-
7 cells13,19. Accordingly, compounds 16a,b,d, the most active in
the MTT assay (Table 1) were selected to investigate their apop-
tosis-inducing activities. The cancer cells were treated with the
test compounds (0.1 lM) for 24 h. The induction of apoptosis in
MCF-7 cells by the selected compounds was investigated using
annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI)
staining assay. The assay procedures were the same as those
applied in the previous report13. The results are presented in
Table 4 and Figure 5. Compound 16a increased apoptosis (early
and late events) in MCF-7 cells to 19% compared to 1% in the
control. Moreover, compound 16b caused a significant increase of
MCF-7 apoptotic cells which was similar to the effect of com-
On the other hand, the study of SAR of the benzamide
derivatives 16a–e showed that substitution on the phenyl ring of
compound 16a with the electron-donating (OCH3) or electron-
withdrawing (Br) groups resulted in
a significant decrease
(3.3–20.8-fold) in the cytotoxicity against MCF-7 and A2780, while
substitution with CH3/Cl groups resulted in a very slight increase
in cytotoxic activity against both MCF-7 and HCT116 cell lines
(Figure 4).
Moreover, expansion of the pyrrolidine ring in compound 15a
to the piperidine ring (compound 20) resulted in 1.4–27.5-fold pound 16a. However, compound 16d was more potent in induc-
increase in cytotoxicity against the three cancer cell lines ing apoptosis in MCF-7 cells (26%) compared to compounds 16a
(Figure 4).
and 16b.

1324
A. M. SHAWKY ET AL.
Figure 5. Evaluation of apoptosis-inducing activity of compounds 16a,b,d in MCF-7 cells using annexin V FITC/PI staining assay for 24 h (n¼ 3). x-axis: annexin V, y-
axis: PI. (A) Vehicle control, (B) compound 16a (0.10 mM), (C) compound 16b (0.1 mM), (D) compound 16d (0.10 mM). Top left quarter: necrosis (PIþ/annexin V–); top
right quarter: late apoptosis (PIþ/annexin Vþ); bottom left quarter: living cells (PI–/annexin V–); bottom right: early apoptosis (PI–/annexin Vþ).
Table 5. Kinases inhibitory activity by compounds 9a, 16a,b,d and imatinib.
The test compounds also caused variable degrees of necrosis
in MCF-7 cells. Compounds 16a,d caused slight increases (3.50%
and 2.51%, respectively) in the necrotic events compared to by
the control (1.90%). On the other hand, compound 16b caused a
7.11% increase in necrotic events.
Compounds
Kinase
16a
16b
16d
Imatinib
9aa
ALK1
AMPK (A1/B1/G1)
–12%
0%
9%
–11%
–9%
1%
1%
–11%
51%
–21%
–15%
–12%
–11%
–13%
19%
–13%
–22%
–5%
–19%
2%
14%
18%
5%
–38%
–3%
13%
–2%
0%
–10%
13%
22%
–10%
2%
–1%
49%
–2%
17%
34%
4%
–5%
12%
26%
1%
In conclusion, the new compounds 16a,b,d induced higher ASK1
10%
–14%
–2%
53%
–19%
–7%
–17%
4%
Aurora A
BLK
BRAF
CDK2/cyclin A1
CK1 alpha 1
DYRK3
EGFR
EPHA1
FLT1
2%
apoptotic events in MCF-7 cells at lower concentration compared
to compound 9a (Table 4).
–8%
58%
–28%
–15%
–4%
–38%
–22%
21%
–9%
–18%
–14%
9%
7%
60%
–4%
6%
–25%
40%
25%
1%
3.2.3. Kinase inhibitory activity
3.2.3.1. Kinase profiling test. Compound 9a, the lead compound
in this study, was previously evaluated in a kinase profiling test to
5%
17%
–14%
–11%
23%
1%
22%
12%
1%
GRK1
1%
identify its mechanism of action13. The results revealed the ability GSK3 alpha
–3%
29%
–14%
33%
28%
3%
MSK1
of compound 9a to inhibit the activity of five oncogenic kinases
NEK1
including ALK1, CDK-2/cyclin A1, DYRK3, GSK3 alpha, and NEK1
p38 alpha
4%
–13%
–1%
–12%
–10%
9%
(inhibition%¼3–25%) (Table 5).
PDK1
PRKG1
–1%
In the current work, compounds 16a,b,d, the most active in
MTT assay were also selected for the kinase profiling test to inves-
tigate their potential mechanism of action. The three compounds
were tested at the same concentration (10 lM) against the same
20 kinases used in the previous report13. The test was performed
by KINEXUS Bioinformatics Corporation (Vancouver, Canada), fol-
lowing previous reports13,20. The results are presented in Table 5
and Figure 6.
SGK1
–19%
–13%
2%
3%
7%
The negative values indicate inhibition, while the positive values indicate the
activation in kinase activity.
aData quoted from our previous publication13
.
Treatment of CDK-2/cyclin A1 with compounds 16a,b,d
resulted in 19–28% inhibition in the activity of the kinase com-
pared to only 2% and 4% inhibition by imatinib and compound
9a, respectively. Compounds 16a,b also showed inhibitory activity
against EGFR with inhibition% of 30 and 11%, respectively.
Moreover, compound 16d exhibited 14% inhibition in the activity
of Aurora A kinase compared to 38% inhibition for imatinib.
The results revealed inhibition in the activities of 16 kinases by
compounds 16a,b,d with inhibition% in the range of 1–38%. The
activities of seven of these kinases were inhibited by the three
compounds (2–28%) (Table 5 and Figure 6).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1325
Figure 6. Inhibitory activity of compounds 9a, 16a,b,d, and imatinib against 20 protein kinases; negative values indicate inhibition in kinase activity, positive values
indicate the activation of the enzyme.
In addition, weak inhibition in the activities of ALK1 (11–19%),
BLK (2–11%), CK1 Alpha 1 (7–15%), and DYRK3 (4–17%) was also
observed following the treatment with compounds 16a,b,d. In
addition, compounds 16a,b displayed inhibitory activity against
EPHA1, MSK1, p38 Alpha, and SGK1 with inhibition% in the range
of 5–22% (Table 5). Although these inhibitions in the activities of
these kinases are weak, but they may also contribute to the over-
all cytotoxic activities of the three compounds.
The results of the kinase profiling test also showed significant
improvement in the inhibitory activities of compounds 16a,b,d
compared to compound 9a. This improvement was observed in
the number of oncogenic kinases (8–13 kinases) inhibited by the
new compared to only five kinases inhibited by compound 9a. In
addition, compounds 16a,b,d exhibited higher inhibitory activities
(1–38%) than those of compound 9a (3–25%) (Table 5 and
Figure 6). These results suggested that the aromatic fragment in
the side chain at C-6 of the pyrrolizine nucleus could have a bet-
ter impact on the inhibitory activities of the new pyrrolizines com-
pared to the aliphatic side chain in compound 9a.
Table 6. IC50 values of compounds 9a, 16a,b,d and
staurosporine against CDK-2.
Comp. no.
IC50 (lM)a SEM
16a
16b
16d
9a
0.086 0.016
0.265 0.021
0.113 0.017
1.270 0.066b
0.021 0.013
Staurosporine
aAverage of three determinations; IC50, concentration
which decrease CDK-2 activity to 50%.
bValue quoted from previous publication13
.
Moreover, compound 16d also exhibited potent inhibitory activity
against CDK-2 (IC50¼0.113 lM), while 16b with the electron-donat-
ing methyl group was the least active among the three benza-
mide derivatives (Table 6). These results suggested that for better
inhibitory activity of CDK-2, the phenyl ring in scaffold A should
be unsubstituted or substituted with electron-withdrawing
group(s) rather than the electron-donating (4-CH3) group.
3.2.4. Cell cycle analysis
Based on the results of the MTT assay (Table 1), compounds
16a,b,d were selected to investigate their effect on cell cycle
phases of MCF-7 cells. The cancer cells were treated with the test
compounds at 0.1 lM and vehicle (control) for 24 h according to
the previous report13. Cell cycle analysis was performed using
FC500 flow cytometer (FC500, Beckman Coulter, Miami, FL). The
revealed dramatic increases in the preG1 (11–14-fold) and G2/M
(5–7-fold) cell cycle phases at the expense of 5–10-fold decrease
of G1 events, all compared to control. On the other hand, the S
phase was the least affected with less than a twofold decrease.
Compound 16d showed the strongest preG1 (27.56%) and G2/M
3.2.3.2. CDK-2 inhibitory activity. The results of the kinase profiling
test (Table 5 and Figure 6) revealed weak to moderate inhibitory
activity against 7 kinases by compounds 16a,b,d. Among these kin-
ases, CDK-2 was inhibited by the three compounds with inhib-
ition% in the range 19–28%. To confirm the activity against CDK-2,
the three compounds were also evaluated for their CDK-2 inhibitory
activity using the ADP-Glo kinase assay kit (Promega, Madison, WI)
according to the previous reports21. The results expressed in the
IC50 values of the three compounds are presented in Table 6.
The results revealed higher inhibition in activity CDK-2 by com-
pounds 16a,b,d compared to compound 9a, which was matched
with the results of the kinase profiling test (Table 5). Among the (45.62%) cell cycle effects in MCF-7 cells (Table 7, Figure 7).
Cell cycle analysis of compound 9a (1 mM, 24 h) revealed block-
ring was the most potent as a CDK-2 inhibitor (IC50¼0.086 lM). ade of MCF-7 cells at the S phase with more than a threefold
three derivatives, compound 16a with the unsubstituted phenyl

1326
A. M. SHAWKY ET AL.
increase compared to the control (17.3–55.6%), at the expense of phenyl moiety aiming to obtain new cytotoxic agents that can
decreases in other cell cycle phases13. However, in the current interfere with tubulin polymerisation in cancer cells. Herein, com-
study, compounds 16a,b,d (0.1 mM) showed completely different
effects on cell cycle phases of MCF-7 cells at a much lower con-
centration compared to compound 9a. The three compounds
16a,b,d increased the preG1 and G2/M cell cycle phases in MCF-7
cells at the same time point (24 h) (Table 7).
pounds 16a,b,d, the most active in MTT assay were selected to
investigate their effects on the kinetics of the three phases of
tubulin polymerisation (I: nucleation, II: growth, and III: steady-
state equilibrium). In this assay, light scattering by microtubules
which is directly proportional to the concentration of the micro-
tubule polymer was used as a measure of tubulin polymerisation.
The study was performed using a fluorescence-based tubulin poly-
merisation assay kit (Cytoskeleton Inc., Denver, CO; Cat # BK011P)
following the manufacturer’s instructions (https://www.cytoskel-
eton.com/bk011p) and according to the previous report22.
Paclitaxel (3 lM) was used as a tubulin polymerisation enhan-
cer (positive control, enhancer), while calcium chloride (CaCl2,
0.5 lM) was used as a positive control (inhibitor) as per the manu-
facturer’s instructions (https://www.cytoskeleton.com/pdf-storage/
datasheets/bk011p.pdf). The vehicle was also used to evaluate the
polymerisation when tubulin is incubated in the absence of the
test compound. The effect of the selected compounds (5 lM) on
tubulin polymerisation was evaluated and compared with those of
the positive control. The results are represented in Figure 8.
3.2.5. Tubulin polymerisation
3.2.5.1. Tubulin polymerisation assay (kinetic study). In the current
study, the new compounds were designed bearing the trimethoxy
Table 7. Cell cycle effect of 16a,b,d on MCF-7 cells (24 h).
Cell cycle stage %
Comp. no.
preG1
G1
S
G2/M
Control
16a
16b
1.95 0.10
26.92 3.07
22.43 2.24
27.56 3.93
50.83 4.62
10.69 1.32
13.19 2.95
5.46 0.87
39.51 2.44
25.93 3.47
28.46 2.79
24.92 2.22
7.66 0.84
37.38 4.66
35.35 4.19
45.62 5.25
16d
Data shown in mean % SD (n ¼ 2), treatment for 24 h at 0.1 lM, experiment
was repeated 3ꢂ.
Figure 7. Cell cycle analysis of MCF-7 cells treated with (A) vehicle (control), (B) compound 16a (0.1 mM), (C) compound 16b (0.1 mM), and (D) compound 16d (0.1
mM) for 24 h. x-axis: DNA content, y-axis: % cell number; (E) bar chart showing the effect of vehicle (control) and compounds 16a,b,d (0.1 mM) on cell cycle stages of
MCF-7 cells after 24h treatment. Data shown in mean % SD (n ¼ 2) of three independent experiments, statistical differences, compared with control cells, were
ꢃ
ꢃꢃ
assessed by one-way ANOVA with the Tukey’s post hoc multiple comparison test (GraphPad Prism, La Jolla, CA). p< 0.05 and p< 0.01 were taken as significant.
Figure 8. Effect of compounds 16a,b,d on tubulin polymerisation: (A) tubulin polymerisation reactions of control, paclitaxel, CaCl2, and compounds 16a,b,d, I: nucle-
ation, II: growth, and III: steady-state equilibrium phases; (B) semi-quantitative analysis of the inhibition in tubulin polymerisation showing the AUC on treatment with
vehicle (control), paclitaxel, CaCl2, and compounds 16a,b,d.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1327
The results revealed that the tubulin polymerisation enhancer, compound 16b, the most active in tubulin polymerisation assay
paclitaxel at 3 lM eliminated the nucleation phase I and enhanced (Figure 8) was selected. The effect of compound 16b was eval-
the growth phase II. On the other hand, CaCl2 at 0.5 lM inhibited
tubulin polymerisation and reduced the final polymer mass
(Figure 8). The results also indicated moderate tubulin polymerisa-
tion inhibitory activity for compounds 16b,d compared to CaCl2.
The inhibitory effect of compound 16b was higher than com-
pound 16d. The effect of compound 16d on both nucleation and
growth phases was similar to those of CaCl2, while its effect on
the steady-state equilibrium phase was slightly lower than CaCl2
(Figure 8). On the other hand, compound 16a showed a similar
effect to that of paclitaxel on the nucleation phase. However, this
effect reached a plateau then started to decrease in the growth
phase. The inhibitory effect of compound 16a on tubulin polymer-
isation was continued in the steady-state phase.
In the current work, the tubulin polymerisation assay was used
as a qualitative assay to identify the potential mechanism of
action of the new compounds 16a,b,d. However, area under the
curve (AUC) indicates the total light scattered and corresponds to
the total mass of the polymerised tubulin (Figure 8(A)).
Accordingly, AUC was calculated for the tested compounds
16a,b,d and compared with those of the control, CaCl2, and pacli-
taxel (Figure 8(B)). The results showed a decrease in AUC of CaCl2
compared to the control indicating an inhibition in tubulin poly-
merisation and a decrease in the final polymer mass. On the other
hand, an increase in AUC was observed by paclitaxel relative to
uated in an immune staining study using confocal immunofluores-
cent microscopy. The test was performed according to the
previous report23. The cancer cells were incubated with the
vehicle (control) and compound 16b (5 lM) for 24 h. The nuclei of
the cancer cell stained blue with 40,6-diamidino-2-phenylindole
(DAPI), while microtubules were stained (green) with anti-b-tubulin
R
mouse monoclonal antibody and Alexa FluorV 488 secondary anti-
bodies (Abcam, Cambridge, UK).
The immunofluorescence images of the untreated/treated can-
cer cells were taken to visualise the effect of the test compounds
16b on the microtubule cytoskeleton. The results are represented
in Figure 9. The images of the control showed normal, clear slim,
and fibrous microtubules which wrap around the nuclei and
stretch through each cell. On the other hand, the cells treated
with compound 16b showed shrank and partially disorganised
microtubules which split partially into fragments. These results
indicated the ability of compound 16b to induce a disruption in
the formation of microtubules.
3.3. Molecular docking
In the current work, the molecular docking studies were per-
formed into protein kinases (PKs) and tubulin to understand the
mechanism of action of compounds 16a,b,d. The binding affin-
ities, modes, orientations, and interactions of the new compounds
into the relevant protein were investigated using AutoDock 4.224
and compared with those of the co-crystallised ligands. The results
of the docking studies were visualised by the Discovery studio
visualizer (Dassault Systems, San Diego, CA)32.
control indicating
a stabilisation in tubulin polymerisation.
Moreover, compounds 16a,b,d exhibited similar effects on tubulin
polymerisation like CaCl2. Among the three compounds, com-
pound 16b exhibited the highest reduction in the final polymer
mass. In addition, compound 16d showed a higher reduction in
the final polymer mass than compound 16a. Considering the
results of the three compounds, we can observe that compounds
16b,d have moderate inhibitory activity against tubulin polymer-
isation compared to the control and CaCl2, while the effect of
compound 16a was weak.
3.3.1. Docking study into protein kinases
To rationalise the improvement of kinase inhibitory activity of the
new compounds 16a,b,d compared to the lead compound 9a, a
molecular docking study of the new compounds into the active
3.2.5.2. Immunofluorescence staining. To assess the effect of the sites of CDK-2, EGFR, and Aurora A were performed. The crystal
new compounds on microtubule cytoskeleton in living cells, structure of CDK-2 (pdb code: 3TNW)25, EGFR (pdb code: 1M17)26,
Figure 9. Immunofluorescence confocal microscopy images assessing cellular microtubule networks of MCF-7 cells after treatment with vehicle (A), compound 16b (B)
for 24 h, nuclei were stained blue with 40,6-diamidino-2-phenylindole (DAPI), microtubules stained (green) with anti-b-tubulin mouse monoclonal antibody and Alexa
R
FluorV 488 secondary antibodies (Abcam, Cambridge, UK).

1328
A. M. SHAWKY ET AL.
Table 8. Docking results of compounds 16a,b,d into CDK-2 and EGFR in comparison to compounds 9a and the co-crystallised ligands.
Atoms in H-bonding
a
PK (pdb)
Ligand
16a
DGb
Kib
HBsc
5
In ligands
In tubulin
Lengthd (Å)
CDK-2 (3TNW)
–9.21
0.178 lM
CN
CONH
300-OCH3
400-OCH3
CONH
CN
NH of LEU83
NH of LEU83
NH3 of LYS89
NH3 of LYS89
C═O of ILE10
NH of LEU83
NH3 of LYS89
NH of LEU83
NH3 of LYS33
NH3 of LYS33
NH of ASP145
NH of LYS33
C═O of GLU51
C═O of GLU81
NH of LEU83
C═O of ASP145
OH of THR830
NH of ASP831
C═O of ASP831
NH3 of LYS721
NH3 of LYS721
NH of MET769
CYC773
2.08
2.61
2.10
1.95, 2.50
2.75
2.05
2.32
1.95
2.47
2.86
2.07
1.74
2.114
1.85
1.69
2.69
2.36
2.25
3.05
2.68
1.73
1.89
2.42
3.03
2.24
1.64
16b
16d
–7.91
–8.37
1.60 lM
3
3
CONH
CN
0.732 lM
300-OCH3
400-OCH3
CN
9a
–8.04
–6.47
1.290 mM
18.20 mM
1
5
CAN508e
OH
OH
NH2
Pyrazole N1
OH
CONH
CN
CONH
300-OCH3
400-OCH3
CN
EGFR (1M17)
16a
16b
–9.07
–8.60
223.89 nM
498.09 nM
3
3
16d
9a
–8.53
–8.51
555.92 nM
577.35 nM
1
2
CO
CONH
CONH
Pyrimidine N1
C═O of GLN767
NH of MET869
NH of MET769
Erlotinib
–7.39
3.84 mM
1
aBinding free energy (kcal/mol).
bInhibition constant.
cHBs, number of hydrogen bonds.
dLength in angstrom (Å).
eCAN508, -4-[(E)-(3,5-diamino-1H-pyrazol-4-yl)diazenyl)phenol.
and Aurora A (pdb code: 3E5A)27 was obtained from the protein orientations of the three compounds can account even partially
data bank (https://www.rcsb.org/). AutoDock (v.4.2) was used to for the difference in their inhibitory activity against CDK-2.
perform the docking study24. The study was performed following
Moreover, investigation of the binding interaction of com-
the previous reports29–31. Validation of the docking procedures into pound 16a into the active site of CDK-2 revealed the formation of
the selected kinases was also performed according to our previous five conventional hydrogen bonds with LEU83 and LYS89. One
report13. The results of the docking study of the new compounds carbon hydrogen bond was also observed between compound
and the co-crystallised ligands are presented in Table 8.
16a and HIS84. In addition, compound 16a displayed several
The results of the docking study of compounds 16a,b,d into the hydrophobic interactions with the key amino acids (ILE10, VAL18,
selected kinases revealed nice fitting into the active site of CDK-2 ALA31, LYS33, VAL64, PHE80, LEU134, and ALA144) in the ATP
and EGFR. These results were confirmed by the absence of any binding site of CDK-2. These binding interactions were similar to
steric clashes or unfavourable interactions with the amino acids in those reported for several CDK-2 inhibitors40. On the other hand,
the active sites of the two kinases. Moreover, compounds 16a,b,d each of compounds 16b,d displayed only three conventional
exhibited higher binding affinities towards CDK-2 and EGFR com- hydrogen bonds with amino acids in CDK-2. The fewer number of
pared to CAN508, and erlotinib, respectively (Table 8). The three conventional hydrogen bonds could also account for the lower
compounds 16a,b,d exhibited higher affinities (–7.91 to ꢀ9.21kcal/ inhibitory activities of compounds 16b,d compared to compound
mol) towards CDK-2 compared to ꢀ6.47 kcal/mol for CAN508 16a (Figure 10).
(IC50¼3.5 lM against CDK-2)39. Compound 16a showed the highest
Compounds 16a,b,d also displayed higher binding affinities
affinity towards CDK-2 followed by the chloro analogue 16d. This towards EGFR compared to erlotinib (Table 8). Among the three
order of binding affinities was in concordance with the inhibitory derivatives, compound 16a showed the highest affinity for EGFR
activities of the three compounds against CDK-2 (Table 6).
protein (DGb¼ ꢀ9.07 kcal/mol, Table 8). In addition, compound
To understand the differences in the inhibitory activity of the 16b displayed a binding affinity of ꢀ8.60 kcal/mol which was
three compounds against CDK-2 (Table 6), the binding modes and lower than the binding affinity of compound 16a. These results
interactions of compounds 16a,b,d were also investigated. The were matched with the inhibitory activities of the two compounds
results revealed that each of the two side chains in the three com- against EGFR (Table 5).
pounds can adopt an orientation that occupies the binding site of
CAN508 in CDK-2.
However, compound 16d displayed a good binding affinity
(DGb¼ ꢀ8.53 kcal/mol) although it did not show in vitro inhibitory
Investigation of the binding mode of compound 16a revealed activity against EGFR (Table 5). To explain this difference, the bind-
that the pyrrole ring together with the attached phenyl carboxa- ing interactions between the tested compounds (16a,b,d, and
mide side-chain adopted an orientation nearly superposing the erlotinib) and the amino acids in EGFR were investigated. The
position of CAN508 in CDK-2 (Figure 10). On the other hand, the results revealed that compound 16a exhibited three conventional
pyrrole ring and the trimethoxyphenyl moiety in compounds hydrogen bonds with THR830 and ASP831 (Figure 11). Compound
16b,d nearly superpose the positions of the imidazole and phenyl 16b also showed three conventional hydrogen bonds with LYS721
rings in CAN508, respectively. These differences in binding and MET769. However, investigation of the binding interactions of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1329
Figure 10. Binding modes/interactions of compounds 16a,b,d (shown as sticks, coloured by element) and CAN508 (coloured in yellow) into CDK-2 (pdb code: 3TNW):
(A) 3D binding mode of compound 16a overlaid with CAN508; (B) 3D binding mode of compound 16b overlaid with CAN508; (C) 3D binding mode of compound 16d
overlaid with CAN508; (D) 2D binding mode of compound 16a; (E) 2D binding mode of compound 16b; (F) 2D binding mode of compound 16d, all showing H-bond-
ing and hydrophobic interactions, hydrogen atoms were omitted for clarity.
Figure 11. Binding modes/interactions of compound 16a into EGFR (PDB code: 1M17): (A) 3D binding mode of compound 16a into the active site of EGFR, the co-
crystallised erlotinib shown as orange line, receptor shown as hydrogen bond surface, hydrogen atoms were omitted for clarity; (B) 2D binding mode of compound
16a into EGFR showing different types of interactions with amino acids in the active site of the protein, hydrogen atoms were omitted for clarity.
the best fit conformation of compound 16d into EGFR revealed 3.3.2. Docking study into tubulin
only one conventional hydrogen bond formed with CYC773 (Table In this part, another molecular docking study was performed into
8). These results indicated that the binding affinity of compound the binding site of CA-4/colchicine in tubulin protein to investi-
16d towards EGFR is due mainly to the hydrophobic interactions gate the binding mode/interactions of the new compounds correl-
with EGFR. The 2/3D binding modes of compounds 16b,d, and ate the obtained results with the inhibitory effects of these
erlotinib are provided in the Supplementary data (Figs. S142–144).
In addition, among the three derivatives 16a,b,d, only com-
pound 16d showed weak inhibitory activity against aurora A (14%
inhibition) in the kinase profiling test. The docking study of com-
pounds into the active site of aurora A (pdb code: 3E5A) revealed
the highest affinity for compound 16d, Supplementary data
(Table S3).
compounds on tubulin polymerisation (Figures 8 and 9). The crys-
tal structure of tubulin protein (pdb code: 5LYJ) co-crystallised
with CA-428 was obtained from the Protein Data Bank (https://
www.rcsb.org/structure/5lyj). The protein and ligand files were
prepared following the previous report29,41
.
To validate the docking procedures, the co-crystallised ligand
(CA-4) 1 was re-docked into the binding site of the co-crystallised

1330
A. M. SHAWKY ET AL.
Figure 12. Superposition of the re-docked/co-crystallised CA-4 1 into the binding site in tubulin (pdb code: 5LYJ): (A) 3D binding modes showing superposition of the
re-docked CA-4 (shown as sticks, coloured by element) over the co-crystallised ligand (CA-4, shown as sticks, coloured in blue) into CA-4 1 binding site in tubulin,
RMSD ¼ 0.9 Å; (B) 2D binding mode of CA-4 showing H-bonding and hydrophobic interactions with amino acids in tubulin, hydrogen atoms were omitted for clarity.
Table 9. Docking results of compounds 15a,b,d, 16a,b,d into tubulin protein (pdb code: 5LYJ) in comparison with the co-crystallised ligand 7BA (CA-4).
Atoms in H-bonding
a
Comp.
15a
DGb
Kib
HBsc
2
In ligands
In tubulin
Lengthd (Å)
–6.97
7.770 lM
4-CH3O
C═O
MeO groups
4-CH3O
C═O
MeO groups
–
3/4-CH3O
C═O
3/4-CH3O
C═O
7-CN
3/4-CH3O, CH2-3
C═O
3/4-CH3O, CH2-3
7-CN
3/4-CH3O, CH2-3
OH
CH3O groups
NH2 of ASN258
NH of ALA317
2.85
2.35
1.74–3.00
2.94
2.49
1.74, 2.17
–
2.03–2.74
2.08
1.83–2.26
2.22
2.46
2.07–2.61
1.49
2.56–3.04
2.62
2.43–2.80
2.18
ꢃ
3
2
ASN249, LEU248, ASN101, THR179
15b
15d
–6.35
–6.46
22.16 lM
18.41 lM
NH2 of ASN258
NH of ALA317
LEU248, ASN249
–
CYS241, ASN258
NH of ALA317
ꢃ
2
–
d
ꢃ
3
ꢃ
3
2
LEU248, ASN249, ASN101
NH2 of LYS352
16a
–8.80
347.84 nM
NH of ASP251
ꢃ
4
1
THR179, CYS241, GLY237, THR376, ALA316
NH2 of LYS352
CYS241, GLY237, THR376, ALA316, THR179
16b
16d
7BA
–10.97
–10.63
–8.72
9.06 nM
16.28 nM
407.88 nM
ꢃ
4
1
NH of ASP251
ꢃ
4
1
ASN101, GLY237, THR179
C═O of THR179
VAL238, VAL315, ASN350
ꢃ
3
1.93–2.75
7BA, compound 1 (CA-4); values with asterisks indicate the carbon hydrogen bonds, the underlined atoms are the atoms involved in the H-bonding interactions.
aBinding free energy (kcal/mol).
bInhibition constant.
cHBs, number of hydrogen bonds.
dLength in angstrom (Å).
ligand (7BA) in tubulin protein. The results revealed the superpos- also displayed higher binding free energies than their correspond-
ition of the re-docked CA-4 with the co-crystallised ligand with ing Schiff bases (15a–e and 20). These results were also matched
RMSD of 0.9 Å. Moreover, the re-docked CA-4 showed similar with the results of the cytotoxic activities of the two series
binding mode, orientation, and exhibited the same binding inter- (Table 1).
actions as those of the co-crystallised ligand (Figure 12).
To understand the reasons behind this noticeable difference in
Investigation of the binding interactions of CA-4 with the binding affinities of the two series, we have investigated their
amino acids in tubulin protein revealed one conventional hydro- binding modes/orientations into tubulin protein. The best-fit con-
gen bond with THR179, one pi-sulphur interaction with MET259, formations of the two series were overlaid with co-crystallised CA-
and three carbon hydrogen bonds with VAL238, VAL315, and 4 in Figure 13. Investigation of their binding modes revealed that
ASN350. In addition, several hydrophobic interactions with all the benzamide derivatives (16a–e and 21) exhibited binding
CYS241, ALA250, LEU255, ALA316, and LYS352 were observed orientations similar to that of CA-4. The two phenyl rings in each
(Figure 12).
of the six benzamide derivatives adopted orientations superposing
The results of the molecular docking study (Table 9) revealed a on the two phenyl rings of CA-4 with the trimethoxyphenyl moi-
nice fitting of the new compounds into the binding site of CA-4 eties extended towards the a/b interface (Figure 13).
in tubulin, where no steric clashes or unfavourable interactions
On the other hand, the Schiff base derivatives (15a–e and 20)
were observed. Moreover, the benzamide derivatives (16a–e and adopted completely different orientations from CA-4. The differ-
21) displayed higher binding affinity (DGb¼ ꢀ8.80 to ꢀ12.16 kcal/ ence in the binding orientations of the two series could account
mol) compared to ꢀ8.72 kcal/mol for CA-4, Supplementary data even partially for the difference in their binding affinities towards
(Table S4). Moreover, the benzamide derivatives (16a–e and 21) tubulin and their cytotoxic activities.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1331
Figure 13. An overlay of the best fit conformation of the new compounds with the co-crystallised CA-4 shown as sticks, coloured by element (pdb code: 5LYJ): (A)
compounds 15a–e and 20 (shown as blue lines) overlaid with the co-crystallised CA-4; (B) compounds 16a–e and 21 (shown as pink lines) overlaid with the co-crystal-
lised CA-4, receptor in the two figs shown as hydrogen bond surface, hydrogen atoms were omitted for clarity.
Figure 14. Binding modes/interactions of compounds 16a,b,d (shown as sticks, coloured by element) into CA-4 (shown as sticks, coloured in yellow) binding site in
tubulin (pdb code: 5LYJ): (A) 3D binding mode of compound 16a overlaid with the co-crystallised CA-4; (B) 3D binding mode of compound 16b overlaid with the co-
crystallised CA-4; (C) 3D binding mode of compound 16d overlaid with the co-crystallised CA-4; (D) 2D binding mode of compound 16a; (E) 2D binding mode of com-
pound 16b; (F) 2D binding mode of compound 16d, all showing H-bonding and hydrophobic interactions with amino acids in tubulin, hydrogen atoms were omitted
for clarity.
Among the new benzamide derivatives, compound 16b dis- co-crystallised CA-4, the trimethoxyphenyl moieties in the three
played the highest binding affinity towards tubulin. Moreover, the compounds were located deeply into the b-subunit showing sev-
chloro derivative 16d exhibited higher binding affinity than com- eral interactions with important amino acids such as THR179,
pound 16a. These results were quite matched with the results of VAL238, and/or CYS241, through carbon–hydrogen bonding and
hydrophobic interactions. Moreover, the pyrrolizine nucleus of
the three derivatives was located towards the a/b interface with
the double bond of the pyrrole ring (C2═C3) overlaid over the
tubulin polymerisation assay of the three compounds (Figure 8).
Furthermore, the binding orientations and interactions of the
benzamide derivatives 16a,b,d into tubulin protein were deeply
investigated. The results revealed that the three compounds double bond in CA-4. In addition, the two phenyl rings in the
adopted orientation like that of co-crystallised CA-4. In these three compounds were located on the same side of the pyrrole
orientations, the two phenyl rings of the three compounds ring adopting a cis-like conformation similar to the cis-conform-
superposed on the two phenyl rings of CA-4 (Figure 14). Like the ation of CA-4. These binding modes of compound 16a,b,d could

1332
A. M. SHAWKY ET AL.
account for their high binding affinities towards tubulin and
even partially for the high cytotoxic activities of benza-
mide derivative.
The docking study into the binding site of CA-4 in tubulin pro-
tein was also performed for the remaining compounds (15c,e,
16c,e, 20, and 21). The results of this study are provided in the
supplementary data (Table S4).
Funding
This work was funded by National Science, Technology, and
Innovation Plan (MAARIFAH), King Abdulaziz City for Science and
Technology (KACST), Kingdom of Saudi Arabia, grant number (12-
MED2304-10-R).
ORCID
4. Conclusions
Ahmed M. Gouda
http://orcid.org/0000-0003-4527-8885
In the present study, two new series of pyrrolizine derivatives
(15a–e and 16a–e) bearing 3,4,5-trimethoxyphenyl moiety were
designed, synthesised, and evaluated for their cytotoxic activities.
In addition, two new indolizines (20 and 21) were also synthesised
and for their cytotoxic activities. Structural confirmation of these
compounds was carried out by spectral (IR, 1H NMR, 13C NMR,
DEPT C135, and mass) and quantitative elemental analyses. The
results of the MTT assay revealed weak to potent cytotoxic activity
for the new compounds against three (MCF-7, HCT116, and
A2780) cancer cell lines with IC50 in the range of 0.03–36.88 lM.
Among the new derivatives, compounds 16a,b,d exhibited the
highest cytotoxicity against MCF-7 cells (IC50¼0.068–0.082 lM),
while compounds 16c,d were the most active against HCT116 and
A2780 cells, respectively. On the other hand, the new compounds
showed weak cytotoxicity against the normal MRC-5 cells
(IC50¼0.155–17.08 lM) compared to 13.66 lM for lapatinib.
Compounds 16a,b,d exhibited also cytotoxic activities against
doxorubicin-resistance MCF-7/ADR cells (IC50¼0.52–6.26 lM). The
study of SAR revealed higher cytotoxic activity and selectivity for
the benzamide derivatives (16a–e and 21) compared to their cor-
responding Schiff bases (15a–e and 20). The kinase profiling test
of compounds 16a,b,d revealed improvement in their inhibitory
activities against several kinases (inhibition%¼1–38%) compared
to compound 9a. Moreover, the three compounds exhibited
higher inhibitory activity against CDK-2 than compound 9a.
Mechanistic studies of compounds 16a,b,d also revealed weak to
moderate inhibitory activity against tubulin polymerisation. Cell
cycle analyses of MCF-7 cells treated with compounds 16a,b,d
revealed dramatic increases in the preG1 (11–14-fold) and G2/M
(5–7-fold) cell cycle phases at the expense of 5–10-fold decrease
of G1 events, all compared to the control. Compound 16d showed
the strongest preG1 (27.56%) and G2/M (45.62%) cell cycle effects.
The results of the docking study into tubulin/CDK-2/EGFR showed
higher binding affinities for compounds 16a,b,d compared to the
co-crystallised ligands (CAN508 and erlotinib). Taken together,
compounds 16a,b,d could serve as promising lead compounds for
the future development of new potent anticancer agents.
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA
Cancer J Clin 2019;69:7–34.
2. Housman G, Byler S, Heerboth S, et al. Drug resistance in
cancer: an overview. Cancers (Basel) 2014;6:1769–92.
3. Bayat Mokhtari R, Homayouni TS, Baluch N, et al.
Combination therapy in combating cancer. Oncotarget 2017;
8:38022–43.
4. Saputra EC, Huang L, Chen Y, Tucker-Kellogg L. Combination
therapy and the evolution of resistance: the theoretical mer-
its of synergism and antagonism in cancer. Cancer Res 2018;
78:2419–31.
5. Zheng W, Zhao Y, Luo Q, et al. Multi-targeted anticancer
agents. Curr Top Med Chem 2017;17:3084–98.
6. Singh H, Kinarivala N, Sharma S. Multi-targeting anticancer
agents: rational approaches, synthetic routes and structure
activity relationship. Anticancer Agents Med Chem 2019;19:
842–74.
7. Fu R-G, Sun Y, Sheng W-B, Liao D-F. Designing multi-tar-
geted agents: an emerging anticancer drug discovery para-
digm. Eur J Med Chem 2017;136:195–211.
8. Lu Y, Chen J, Xiao M, et al. An overview of tubulin inhibitors
that interact with the colchicine binding site. Pharm Res
2012;29:2943–71.
9. Li L, Jiang S, Li X, et al. Recent advances in trimethoxy-
phenyl (TMP) based tubulin inhibitors targeting the colchi-
cine binding site. Eur J Med Chem 2018;151:482–94.
10. Ducki S, Rennison D, Woo M, et al. Combretastatin-like chal-
cones as inhibitors of microtubule polymerization. Part 1:
synthesis and biological evaluation of antivascular activity.
Bioorg Med Chem 2009;17:7698–710.
11. Zhang X, Raghavan S, Ihnat M, et al. The design and discov-
ery of water soluble 4-substituted-2,6-dimethylfuro[2,3-d]pyr-
imidines as multitargeted receptor tyrosine kinase inhibitors
and microtubule targeting antitumor agents. Bioorg Med
Chem 2014;22:3753–72.
12. Zhang X, Kong Y, Zhang J, et al. Design, synthesis and bio-
logical evaluation of colchicine derivatives as novel tubulin
and histone deacetylase dual inhibitors. Eur J Med Chem
2015;95:127–35.
13. Shawky AM, Abourehab MAS, Abdalla AN, Gouda AM.
Optimization of pyrrolizine-based Schiff bases with 4-thiazo-
lidinone motif: design, synthesis and investigation of cyto-
toxicity and anti-inflammatory potency. Eur J Med Chem
2020;185:111780.
Acknowledgements
The authors would like to thank Dr. Hany Omar and Ms. Fatima
Hersi, Sharjah Institute for Medical Research, University of Sharjah,
United Arab Emirates for their kind help in the biological assays.
14. Attallah KM, Gouda AM, Ibrahim IT, Abouzeid L. Design, syn-
thesis, 99mTc labeling, and biological evaluation of a novel
pyrrolizine derivative as potential anti-inflammatory agent.
Radiochemistry 2017;59:630–8.
Disclosure statement
The authors declared that there is no conflict of interest and have
approved the article.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1333
15. Belal A, Gouda AM, Ahmed AS, Abdel Gawad NM. Synthesis 28. Gaspari R, Prota AE, Bargsten K, et al. Structural basis of cis-
of novel indolizine, diazepinoindolizine and pyrimidoindoli-
zine derivatives as potent and selective anticancer agents.
Res Chem Intermed 2015;41:9687–701.
and trans-combretastatin binding to tubulin. Chem 2017;2:
102–13.
29. Abdelbaset MS, Abdelrahman MH, Bukhari SNA, et al.
Design, synthesis, and biological evaluation of new series of
pyrrol-2(3H)-one and pyridazin-3(2H)-one derivatives as
tubulin polymerization inhibitors. Bioorg Chem 2021;107:
104522.
30. Almalki FA, Gouda AM, Bin AM, Almehmadi OM. Profens: a
comparative molecular docking study into cyclooxygenase-
1/2. Drug Invent Today 2019;11:480–7.
31. Gouda AM, Almalki FA. Carprofen: a theoretical mechanistic
study to investigate the impact of hydrophobic interactions
of alkyl groups on modulation of COX-1/2 binding selectiv-
ity. SN Appl Sci 2019;1:332.
32. BIOVIA. Dassault Systems, Discovery Studio Visualizer,
v16.1.0.15350. San Diego: Dassault Systems; 2016.
33. Sheng H, Shao J, Kirkland SC, et al. Inhibition of human
colon cancer cell growth by selective inhibition of cyclooxy-
genase-2. J Clin Invest 1997;99:2254–9.
34. Agarwal B, Swaroop P, Protiva P, Raj SV, et al. Cox-2 is
needed but not sufficient for apoptosis induced by Cox-2
selective inhibitors in colon cancer cells. Apoptosis 2003;8:
649–54.
35. Gomha SM, Riyadh SM, Mahmmoud EA, Elaasser MM.
Synthesis and anticancer activities of thiazoles, 1,3-thiazines,
and thiazolidine using chitosan-grafted-poly(vinylpyridine) as
basic catalyst. Heterocycles 2015;91:1227–43.
36. Gomha SM, Riyadh SM, Mahmmoud EA, Elaasser MM.
Synthesis and anticancer activity of arylazothiazoles and
1,3,4-thiadiazoles using chitosan-grafted-poly(4-vinylpyridine)
as a novel copolymer basic catalyst. Chem of Heterocycl
Compd 2015;51:1030–8.
16. Shawky AM, Ibrahim NA, Abourehab MAS, et al.
Pharmacophore-based virtual screening, synthesis, biological
evaluation, and molecular docking study of novel pyrroli-
zines bearing urea/thiourea moieties with potential cytotox-
icity and CDK inhibitory activities. J Enzyme Inhib Med
Chem 2021;36:15–33.
17. Gouda AM, Abdelazeem AH, Abdalla AN, Ahmed M.
Pyrrolizine-5-carboxamides: exploring the impact of various
substituents on anti-inflammatory and anticancer activities.
Acta Pharm 2018;68:251–73.
18. Marzouk AA, Abdel-Aziz SA, Abdelrahman KS, et al. Design
and synthesis of new 1,6-dihydropyrimidin-2-thio derivatives
targeting VEGFR-2: molecular docking and antiproliferative
evaluation. Bioorg Chem 2020;102:104090.
19. Gomaa HAM, El-Sherief HAM, Hussein S, et al. Novel 1,2,4-tri-
azole derivatives as apoptotic inducers targeting p53: syn-
thesis and antiproliferative activity. Bioorg Chem 2020;105:
104369.
20. Elsayed MSA, El-Araby ME, Serya RAT, et al. Structure-based
design and synthesis of novel pseudosaccharine derivatives
as antiproliferative agents and kinase inhibitors. Eur J Med
Chem 2013;61:122–31.
21. Wang Y, Chen Y, Cheng X, et al. Design, synthesis and bio-
logical evaluation of pyrimidine derivatives as novel CDK2
inhibitors that induce apoptosis and cell cycle arrest in
breast cancer cells. Bioorg Med Chem 2018;26:3491–501.
22. Sonawane V, Mohd Siddique MU, Jadav SS, et al. Cink4T, a
quinazolinone-based dual inhibitor of Cdk4 and tubulin
polymerization, identified via ligand-based virtual screening,
for efficient anticancer therapy. Eur J Med Chem 2019;165:
115–32.
ꢀ
37. Vilanova C, Dıaz-Oltra S, Murga J, Falomir E, et al. Design
23. Guan Q, Cong L, Wang Q, et al. Activated carbon/Brønsted
acid-promoted aerobic benzylic oxidation under “on-water”
condition: green and efficient synthesis of 3-benzoylquinox-
alinones as potent tubulin inhibitors. Eur J Med Chem 2020;
186:111894.
24. Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and
AutoDockTools4: automated docking with selective receptor
flexibility. J Comput Chem 2009;30:2785–91.
25. Baumli S, Hole AJ, Noble MEM, Endicott JA. The CDK9 C-
helix exhibits conformational plasticity that may explain the
selectivity of CAN508. ACS Chem Biol 2012;7:811–6.
26. Stamos J, Sliwkowski MX, Eigenbrot C. Structure of the epi-
dermal growth factor receptor kinase domain alone and in
complex with a 4-anilinoquinazoline inhibitor. J Biol Chem
2002;277:46265–72.
and synthesis of pironetin analogue/colchicine hybrids and
study of their cytotoxic activity and mechanisms of inter-
action with tubulin. J Med Chem 2014;57:10391–403.
38. Kumar A, Singh B, Sharma PR, et al. A novel microtubule
depolymerizing colchicine analogue triggers apoptosis and
autophagy in HCT-116 colon cancer cells. Cell Biochem
Funct 2016;34:69–81.
39. Krystof V, Cankar P, Frysova I, et al. 4-Arylazo-3,5-diamino-
1H-pyrazole CDK inhibitors: SAR study, crystal structure in
complex with CDK2, selectivity, and cellular effects. J Med
Chem 2006;49:6500–9.
40. Li Y, Zhang J, Gao W, et al. Insights on structural characteris-
tics and ligand binding mechanisms of CDK2. Int J Mol Sci
2015;16:9314–40.
41. Shawky AM, Abdalla AN, Ibrahim NA, et al. Discovery of new
pyrimidopyrrolizine/indolizine-based derivatives as P-glyco-
protein inhibitors: design, synthesis, cytotoxicity, and MDR
reversal activities. Eur J Med Chem 2021;218:113403.
27. Zhao B, Smallwood A, Yang J, et al. Modulation of kinase-
inhibitor interactions by auxiliary protein binding: crystallog-
raphy studies on Aurora A interactions with VX-680 and
with TPX2. Protein Sci 2008;17:1791–7.