Synthesis, molecular docking and antitumor activity of novel pyrrolizines with potential as EGFR-TK inhibitors

Article abstract of DOI:10.1016/j.bioorg.2015.02.006

A new series of pyrrolizine derivatives 4-8c were synthesized, their structures were confirmed by spectral and elemental analyses. Cytotoxic activity of these compounds was evaluated against breast (MCF7), colon (HCT116) and liver (HEPG2) cancer cell line

Full text of DOI:10.1016/j.bioorg.2015.02.006

Accepted Manuscript
Synthesis, molecular docking and antitumor activity of novel pyrrolizines with
potential as EGFR-TK inhibitors
Amany Belal
PII:
S0045-2068(15)00015-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.02.006
YBIOO 1792
Reference:
Bioorganic Chemistry
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Received Date:
14 November 2014
Please cite this article as: A. Belal, Synthesis, molecular docking and antitumor activity of novel pyrrolizines with
potential as EGFR-TK inhibitors, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.
2015.02.006
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Synthesis, molecular docking and antitumor activity of novel
pyrrolizines with potential as EGFR-TK inhibitors.
Amany Belal1,2,*
1Pharmaceutical Chemistry Department, College of Pharmacy, Taif University, Taif
21974, Kingdom of Saudi Arabia.
2Medicinal Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef 62514, Egypt.
* Correspondence:
Tel.: 0020822317950; Fax: 0020822317958; E-mail address: abilalmoh1@yahoo.com &
amanybilal2010@gmail.com
Keywords:
Pyrrolizine;
Pyrimidopyrrolizine;
Anticancer;
EGFR tyrosine kinases;
TK inhibitors.
1

Abstract
A new series of pyrrolizine derivatives 4-8c were synthesized, their structures were
confirmed by spectral and elemental analyses. Cytotoxic activity of these compounds was
evaluated against breast (MCF7), colon (HCT116) and liver (HEPG2) cancer cell lines
using sulphorhodamine-B (SRB) assay method. All the tested compounds showed highly
potent activity against MCF7 cell line with IC50 range equal 8-194 nM/ml and compound
8c was the best active one (IC50 = 8.6 nM/ml). 8b was the best active compound on both
HCT116 and HEPG-2 cancer cell lines; its IC50 is 26.5 and 12.3 nM/ml respectively.
Docking studies into ATP binding site of EGFR tyrosine kinase were performed to
predict their scores and mode of binding to amino acids, moreover, inhibitory activity of
these compounds against EGFR-TKs was evaluated; their inhibitory percent ranged from
40.4 to 97.6, compound 8c and 8b showed inhibitory activity at 97.6 and 88.4%
respectively.
2

1. Introduction
Pyrrolizine is a bicyclic system consists of two fused five member rings with one
nitrogen atom at the ring junction, 3H-pyrrolizine I (Fig. 1) is the parent compound of
this class [1]. The saturated perhydro pyrrolizine derivatives (pyrrolizidines) constitute
the main skeleton in several alkaloids [2], in addition, pyrrolizidine N-oxides proved to
have antitumor activity [3-5].
Insert fig. 1
Pyrrolizine analogues and their derivatives have attracted considerable interest in
medicinal chemistry; this nucleus was adopted as a scaffold in many compounds with
diverse biological activities as antitumor [6], anti-inflammatory [7], antiviral [8] and
antimicrobial [9].
The antitumor antibiotic clazamycin A and B are natural pyrrolizine derivatives
[10], moreover, phenyl pyrrolizinone derivative II (Fig. 2) showed a very fine cytotoxic
activity against human KB cells, its IC50 value was 70 nM [11]. The licofelone derivative
III showed two fold increase in activity over both 5-flurouracil and licofelone at MCF-7
and MDA-MB 231 carcinoma cells. Moreover, it was found to be as active as 5-FU and
two fold more active than licofelone at colon carcinoma cells HT-29 [12]. The acylated
pyrrolizine derivative IV (IPP) showed anticancer activity against different types of
tumors [13]. Moreover, the pyrimidopyrrolizine derivative V (Fig. 2) was reported to
inhibit the growth of Jensen's carcinoma 755 in rats by 30-40% [14]. Furthermore, our
previous study for exploring the antitumor activity of pyrroloazepine system (7 and 5
fused rings) was promising [15] and this encouraged us to further explore the pyrrolizine
system (5 and 5 rings) as well.
All these observations proved the importance of the pyrrolizine scaffold as a key
nucleus for the synthesis of promising antitumor drugs, this paid our attention to
synthesize a new pyrrolizines 4-8c and evaluate their antitumor activity against liver
(HEPG-2), breast (MCF-7) and colon cancer (HCT-116) cell lines.
The epidermal growth factor receptor (EGFR) is highly expressed in many types of
cancers especially breast, colon and bladder cancers [16]. Targeting this receptor
3

represents a good strategy for the design of new anticancer drugs [17]. Thus, molecular
docking studies for all the synthesized compounds 4-8c into ATP binding site of EGFR-
TKs was performed to investigate the ability of these novel derivatives to inhibit these
tumorogenic agents and explore their binding mode to EGFR-TKs. The synthesized
compounds were also evaluated for their inhibitory activity against EGFR-TK.
Insert fig. 2
2. Results and discussion
2.1. Chemistry
The present work includes preparation of new compounds 4-8c based on pyrrolizine
scaffold to evaluate their anticancer activity. Compound 1 was obtained according to the
previously reported method [18], heating compounds 1 and 2 under reflux for 20h in dry
acetone and in presence of dry potassium carbonate yielded a mixture of compounds 3
and 4 (1H-NMR spectrum of this mixture provided in the supplementary data), stirring of
1
this mixture in 1% NaOC2H5 at r.t. for 24 hours afforded compound 4 (scheme 1). H-
NMR spectrum of compound 4 (Supp. data) revealed characteristic signals for 3 CH2 of
pyrrolizine nucleus, aromatic protons signal at 7.31-7.50 ppm and a singlet at 5.39
corresponding to the NH2 (exchanged with D2O). Reaction of the obtained pyrrolizine
derivative 4 (scheme 2) with equimolar amount of acetyl chloride afforded the acylated
pyrrolizine derivative 5, which upon treatment with 1% sodium ethoxide at r.t. gave the
1
pyrimidopyrrolizine derivative 6, the H-NMR spectrum showed the appearance of
additional signal characterizing CH3 protons at 1.97 and 1.41 ppm respectively (Supp.
data). Chloro-acetylamino derivative 7 was obtained by reacting compound 4 with
1
chloroacetylchloride, H-NMR spectrum of compound 6 showed the appearance of a
singlet signal at 5.39 ppm for CH2Cl in addition to the other signals characterizing the
structure (Supp. data). Compound 6 was refluxed in absolute ethanol with excess amount
of different secondary amines to give compounds 8a-c (scheme 2), the 1H-NMR spectrum
showed the appearance of additional signals characterizing the added secondary amine in
addition to a singlet signal corresponding to CH2-CO. Mass spectrum and C13-NMR were
used also in structure confirmation (Supp. data and experimental section).
4

Insert scheme 1 and 2
2.2. Pharmacological screening
In this work cytotoxic activity of the newly synthesized pyrrolizines 4-8c were
evaluated against breast (MCF7), colon (HCT116) and liver (HEPG2) cancer cell lines,
using sulphorhodamine-B (SRB) assay method. IC50 was calculated and represented in
µM/ml (Table 1), moreover, percent of viability at different concentrations of the tested
compounds is presented in supplementary data (table 1-3). The tested compounds showed
a marked anticancer activity against all the tested cell lines, as for their activity against
MCF7 cell line; all compounds 4-8c showed great activity against breast cancer cells with
IC50 value in nanomolar range from 8.6 to 194 nM/ml, compound 8c was the best active
one its IC50 = 8.6 nM/ml, followed by compound 5 and 6 with IC50 values of 20 and 24
nM/ml respectively, the rest of the tested compounds are arranged in the following order
7 > 8a > 8b > 4 according to their IC50 values ( Table 1). Activity against colon cancer
cell line (HCT116) revealed that; compound 8b is the best active one with IC50 value
equals 26.5 nM/ml followed by compound 6 and 7 with IC50 value of 40 and 106 nM/ml
respectively, the other tested compounds showed IC50 values more than 200 nM/ml. All
the tested compounds showed potent activity against HEPG2 cancer cells except
compound 4 and 5 as their IC50 values exceeded 200 nM/ml, the active compounds
against HEPG2 cancer cells are 8b > 8c > 7 > 6 > 8a, their IC50 range is (12-112 nM/ml).
Further exploration for the mode of action of these pyrrolizines was performed through
docking the synthesized compounds into EGFR-TKs, moreover, the inhibitory properties
against these receptors were also evaluated, compound 8c was the most potent inhibitor
(97.6%) followed by compound 8b (88.4%) and the other compounds showed inhibitory
range from 40.4 to 62.8% (Table 2).
Insert table 1 and 2
2.3. Molecular docking
Epidermal growth factor receptor tyrosine kinases (EGFR-TKs) play a major role in
cell survival and signal transduction pathways, over expression of these kinases can led to
5

several types of human cancers, so, they are considered to be important targets in cancer
treatment [19-23].
Pyrrolizines represent an important scaffold for anticancer drugs [24], however, its
mechanistic studies are not sufficiently explored. In this work the synthesized compounds
were docked into ATP binding site of EGFR-TK to explore the ability of the novel
pyrrolizines to act as EGFR-TK inhibitors. AEE 788 was redocked into EGFR with a root
mean standard deviation (RMDS) = 1.2357, AEE 788 revealed score energy (S) = -17.17
kcal/mol and hydrogen bonding with Thr 854 through the N atom of piperidine moiety
(Fig. 3). All the docked compounds 4-8c showed good docking scores and also showed
hydrogen bonding with the receptor amino acids (Table 3), compounds 8b,c showed
better docking scores than the ligand itself, the best docking score was assigned for
compound 8c (-20.9 Kcal/mol), it showed a hydrogen bonding with Asp 855 in addition
to arene-cation interaction with Arg 841 amino acid (Fig. 5). Compound 8b (Fig. 4)
showed docking score (-17.8 Kcal/mol) better than 8a (-15.6 Kcal/mol) and both of them
revealed a hydrogen bonding with Met 793 through the nitrogen atom of the cyano group.
Compounds 5 and 6 also showed good docking scores and compound 6 revealed
hydrogen bonding with LYS 745 and ASP 855 amino acids (Table 3), the least docking
scores was assigned for compound 4 and 7. The synthesized compounds 4-8c were
evaluated for their inhibitory activity against EGFR-TK and the obtained results (Table 2)
proved molecular docking studies.
Insert table 3
Insert fig. 3a,b-4a,b-5a,b
Conclusion
From all the obtained data during this work we can conclude that i) The synthesized
pyrrolizine derivatives 4-8c showed cytotoxic activity against breast cancer cells MCF7
(all are active) greater than their cytotoxity against both liver cancer HEPG2 (five
compounds are active 6-8c) and colon cancer HCT116 (three compounds are active
6,7,8b). ii) Compound 8c (thiomorpholin-4-yl-acetamide derivative) is highly potent
against MCF7 (IC50 = 8.6 nM/ml), in addition to its great activity against HEPG2 (IC50 =
6

44 nM/ml). iii) Replacing thiomorpholine with morpholine revealed compound 8b which
is the most potent compound against HEPG2 and HCT116 cancer cell lines with IC50
values equal 12.3 and 26.5 nM/ml respectively, in addition to its activity against MCF7
(IC50 = 125 nM/ml), it can be considered as a broad spectrum cytotoxic compound
against the three tested cell lines. iv) Docking studies proved the ability of the
synthesized pyrrolizines to quick fit into the active site of EGFR-TK and can act as
promising inhibitors for these receptors, compounds 8a-c revealed the best score energy.
v) Compound 8c was the most potent inhibitor for EGFR-TK (97.6%) followed by
compound 8b (88.4%) and 8a (62.8%) these results confirmed the molecular docking
studies.
3. Experimental
3.1. Chemistry
Compound 1 was prepared according to the previously reported procedure [18].
Melting points were uncorrected; they were detected using Electrothermal Stuart 5MP3
digital melting point apparatus. Thin layer chromatography (TLC) was performed using
Kiesel gel 0.25 mm, 60 G F 254, Merck Silica gel plates and the running solvent system
was chloroform/methanol (9.5:0.5), ultraviolet light was used to detect the spots.
Elemental microanalyses were performed at the micro analytical center, Faculty of
Science, Cairo University, Mass spectra were detected on Fennigan MAT, SSQ 7000,
Mass spectrometer, (70 eV (EI)) at the micro analytical Center, Faculty of Science, Cairo
1
University. The H-NMR spectra were recorded in CDCl3 and DMSO-d6 on a Varian
Mercury spectrometer (400 MHz) and 13C-NMR (CDCl3) spectra were recorded at
100.62 MHz at the magnetic resonance unit at Beni-Suef University. Chemical shifts are
expressed in values (ppm) and tetramethylsilane (TMS) is the internal standard, addition
of D2O was used to confirm the exchangeable protons.
3.1.1. 2-Amino-3-benzoyl-6,7-dihydro-5H-pyrrolizine-1-carbonitrile 4.
To a mixture of 1 (0.13 g, 1 mmol) and 2 (0.2 g, 1 mmol) in dry acetone (20 ml), dry
potassium carbonate (0.5 g) was added and the reaction mixture was refluxed for 20
hours. The solvent was removed under vacuum, the obtained residue was washed with
7

water, dried then dissolved in 20 ml (1% NaOC2H5) at R.T. for 24 hours, filtered and
recrystallized from ethanol to give white solid, 0.2 g, 80% yield, mp 125-127oC. 1H-
NMR (CDCl3, 400MHz): δ 2.36 (m, 2H, CH2-6), 2.92 (t, 2H, CH2-7), 3.63 (t, 2H, CH2-
13
5), 5.39 (s, 2H, NH2 exchangeable with D2O), 7.31-7.50 (m, 5H, aromatic protons). C-
NMR(CDCl3) :25.2, 50.8, 52.1, 114, 114.6, 127.2, 128.3, 132.9, 140.1, 149.7, 150.9,
184.2. MS (EI): m/z (%): 250 (100), 251 (69). Anal. Calcd. For C15H13N3O (251.28): C,
71.70; H, 5.21; N, 16.72. Found: C, 71.81; H, 5.10; N, 16.43.
3.1.2. N-(3-Benzoyl-1-cyano-6,7-dihydro-5H-pyrrolizin-2-yl)-acetamide 5.
Compound 4 (0.25 g, 1 mmol) was dissolved in dioxan (20 ml) and acetyl chloride (2
mmol) was added, the reaction mixture was stirred at room temperature for 24 hours,
solvent was evaporated under vacuum. The obtained product was crystallized from
1
acetone. White solid, 0.2 g, 69% yield. mp 130-132oC. H-NMR (CDCl3, 400MHz): δ
1.97 (s, 3H, CH3), 2.48 (m, 2H, CH2-6), 3.05 (t, 2H, CH2-7), 3.92 (t, 2H, CH2-5), 7.46-
7.67 (m, 5H, aromatic protons), 8.67 (s, 1H, NH exchangeable with D2O). 13C-
NMR(CDCl3): 23.4, 25.2, 25.5, 50.4, 114.2, 119, 127.9, 128.1, 128.6, 132, 134.7, 138.7,
149.6, 168.5, 186.1. MS (EI): m/z (%): 293 (11), 250 (100). Anal. Calcd. For
C17H15N3O2 (293.32): C, 69.61; H, 5.15; N, 14.33. Found: C, 69.89; H, 5.04; N, 14.44.
3.1.3. 8-Benzoyl-6-methyl-2,3-dihydro-1H,5H-5,7,8a-triaza-cyclopenta[a]inden-4-one 6.
Compound 6 was prepared from compound 5 by dissolving ( 0.29 g, 1mmol) in 20
ml (1%NaOC2H5) at R.T. for 24 hours, white crystals were obtained, filtered and dried,
0.15 g, 50 % yield, mp 148-150oC. 1H-NMR (CDCl3, 400MHz): δ 1.41 (s, 3H, CH3), 2.37
(m, 2H, CH2-6), 2.93 (t, 2H, CH2-7), 3.75 (t, 2H, CH2-5), 5.38 (s, 1H, NH exchangeable
with D2O), 7.48-7.71 (m, 5H, aromatic protons). Anal. Calcd. For C17H15N3O2 (293.32):
C, 69.61; H, 5.15; N, 14.33. Found: C, 69.47; H, 5.13; N, 14.56.
3.1.4. N-(3-Benzoyl-1-cyano-6,7-dihydro-5H-pyrrolizin-2-yl)-2-chloro-acetamide 7.
To a solution of compound 4 (0.5 g, 2 mmol) in dioxan (20 ml) chloro acetyl
chloride (0.22 g, 2 mmol) was added drop wise, the reaction mixture was stirred at room
temperature for 12 hours, then evaporated under vacuum. The obtained white precipitate
1
was recrystallized from ethanol, 0.5 g, 77% yield, mp132- 134oC. H-NMR (DMSO-d6):
8

δ 2.05 (m, 2H, CH2-6), 3.03 (t, 2H, CH2-7), 3.75 (t, 2H, CH2-5), 5.39 (s, 2H, CH2-Cl),
7.44-8.14 (m, 5H, aromatic protons), 10 (s, 1H, NH exchangeable with D2O). MS (EI):
m/z (%): 327 (19.6), 105 (100). Anal. Calcd. For C17H14ClN3O2 (327.76): C, 62.30; H,
4.31; N, 12.82. Found: C, 61.98; H, 4.63; N, 12.32.
3.1.5. General procedure for the preparation of 8a-c.
Compound 7 (0.33 g, 1 mmol) was refluxed with the appropriate secondary amine (5
mmol) in absolute ethanol for 24 hours, the reaction mixture was cooled to room
temperature, the obtained white precipitate was filtered and recrystallized from ethanol.
3.1.5.1.
N-(3-Benzoyl-1-cyano-6,7-dihydro-5H-pyrrolizin-2-yl)-2-piperidin-1-yl-
acetamide 8a.
Compound 8a was prepared from compound 7 and piperidine. White solid, 0.19
g, 50% yield. mp 135-138oC. 1H-NMR (CDCl3, 400MHz): δ 1.36 (m, 2H, CH2-
piperidine), 1.49 (m, 4H, 2CH2-piperidine), 2.45 (m, 4H, 2CH2-piperidine), 2.52 (m, 2H,
CH2-6), 2.78 ( s, 2H, CH2-Cl), 3.11 (t, 2H, CH2-7), 4.08 (t, 2H, CH2-5), 7.41-7.65 (m,
5H, aromatic protons), 9.53 (s, 1H, NH exchangeable with D2O). MS (EI): m/z (%): 376
(5.19), 98 (100). Anal. Calcd. For C22H24N4O2 (376.45): C, 70.19; H, 6.43; N, 14.88.
Found: C, 70.49; H, 6.04; N, 14.44.
3.1.5.2.
N-(3-Benzoyl-1-cyano-6,7-dihydro-5H-pyrrolizin-2-yl)-2-morpholin-4-yl-
acetamide 8b.
Compound 8b was prepared from compound 7 and morpholine. White crystals,
1
0.18 g, 50 % yield, mp 138-140oC. H-NMR (DMSO-d6): δ 2.15 (m, 2H, CH2-6), 2.47
(m, 4H, 2CH2-morpholine), 3.07 (m, 4H, 2CH2-morpholine), 3.74 ( m, 4H, CH2-7, CH2-
Cl), 3.77 (t, 2H, CH2-5), 7.45-7.60 (m, 5H, aromatic protons), 9.69 (s, 1H, NH
exchangeable with D2O). MS (EI): m/z (%): 378 (2.2), 100 (100). Anal. Calcd. For
C21H22N4O3 (378.42): C, 66.65; H, 5.86; N, 14.81. Found: C, 66.47; H, 5.63; N, 14.56.
9

3.1.5.3.
N-(3-Benzoyl-1-cyano-6,7-dihydro-5H-pyrrolizin-2-yl)-2-thiomorpholin-4-yl-
acetamide 8c.
Compound 8c was prepared from compound 7 and thiomorpholine. Yellowish
1
crystals, 0.2 g, 50 % yield, mp 140-142oC. H-NMR (CDCl3, 400MHz): δ 2.48 (m, 2H,
CH2-6), 2.66 (m, 4H, 2CH2-thiomorpholine), 2.92 (m, 4H, 2CH2-thiomorpholine), 3 ( m,
4H, CH2-7, CH2-Cl), 4 (t, 2H, CH2-5), 7.53-7.66 (m, 5H, aromatic protons), 9.69 (s, 1H,
NH exchangeable with D2O). 13C-NMR(CDCl3): 25.1, 25.2, 25.6, 26.3, 50, 50.4, 114.2,
127.6, 127.9, 128.5, 128.7, 132, 132.07, 138.8, 138.9, 148.7, 185.5. MS (EI): m/z (%):
394 (2.6), 116 (100). Anal. Calcd. For C21H22N4O2S (394.49): C, 63.94; H, 5.62; N,
14.20. Found: C, 63.97; H, 5.63; N, 14.56.
3.2. Pharmacological studies
3.2.1. In vitro cytotoxic activity evaluation by SRB assay
Cytotoxic activity of the newly synthesized compounds was evaluated against
MCF7, HEPG2, HCT116 cancer cell lines using Sulphorhodamine-B (SRB) assay
method as previously reported by Skehan et al [25]. Antitumor activity evaluation was
performed at the Center for Genetic Engineering, Al-Azhar University, Cairo, Egypt.
Reagents and chemicals were purchased from Sigma Aldrich Chemical Company (St.
Louis, Mo, U.S.A.).The tested cell lines were obtained from the American Type Culture
Collection (ATCC, Minisota, U.S.A.) through the Tissue Culture Unit, The Egyptian
Organization for Biological Products and Vaccines (Vacsera, Egypt).
Cells were seeded for 24h in a 96 well microtiter plates at a concentration of 1000-
2000 cells/well, 100 µl/well, then cells were incubated for 48 h with various
concentrations ( 0, 6.25, 12.5, 25, 50, 100 µg/ml ) of the tested compounds, 3 wells were
used for each concentration, after incubation for 48h the cells were fixed with 10%
trichloroacetic acid 150 µl/well for 1 h at 4OC, washed by distilled water for 3 times.
Wells were stained for 10-30 min at r.t. with 0.4% SRB, dissolved in 1% acetic acid 70
µl/well. Washed with acetic acid 1% to remove unbound dye till colorless drainage
obtained.The plates were subjected to air drying, 24 h not exposed to UV. The dye was
solubilized with 150 µl/well of 10 mMTrise-EDTA (PH 7.4) for 5 min on a shaker at
10

1600 rpm. The optical density (OD) of each well was measured spectrophotometrically at
545 nm with an ELISA microplate reader. Survival curve was obtained by plotting the
percent of surviving cells against different concentrations of the tested compounds. The
IC50 values were calculated using sigmoidal concentration– response curve fitting models
(Sigmaplot software).
3.2.2. EGFR tyrosine kinase inhibitory activity
Kinase activity was determined using Kinase-Glo Plus luminescence kinase assay
kit, by quantitating the amount of ATP remaining in the solution of kinase reaction [26].
The luminescent signal is correlated with the amount present and it was inversely related
with kinase activity. The tested compounds were diluted to 100mM in 10% DMSO, then
5mL of the dilution was added to a 50mL reaction. All of the enzymatic reactions were
performed at 30°C for 40 min, 50mL of reaction mixture contains 10mM MgCl2, 40mM
Tris, pH 7.4, 0.1 mg/mL BSA, 0.2 mg/mL Poly (Glu, Tyr) substrate, 10mM ATP and
EGFR. Incubate the plate for 5 min at r. t. then add 50mL of Kinase-GloPlus
Luminescence kinase assay to each reaction. ADP-Glo assay kit is the protein kinase
assays used to determine IC50 values in which ADP generation was measured as it leads
to an increase in luminescence signal. The reaction mixture was incubated in a 96-well
plate at 30°C for 30 min, after the incubation period add 25mL of ADP-Glo reagent to
terminate the assay. Shake the 96-well plate for 30 min at ambient temperature then
incubate it, then add 50 mL of kinase detection reagent. Read the 96-well plate using the
ADP-Glo Luminescence reader. All the assay components were added to the blank
control except the substrate. By removing the blank control value you can obtain the
corrected activity for each protein kinase target.
3.3. Molecular docking
Docking studies were performed by downloading the pdb file : 2J6M [27], refined by
removing water molecules then protonated and the pocket was detected using Molecular
Operating Environment software 10.2008 (MOE) provided with chemical computing
group, Canada. 2J6M represent the EGFR-TK cocrystallized with AEE 788, verification
11

process was performed by redocking of the co crystallized ligand into the active site
using the default settings (Fig. 3). The synthesized pyrrolizines 2D was converted to 3D
by chemdraw, then protonated, subjected to energy minimization by Merck Molecular
force field (MMff 94x) and saved in an mdb data base file to be docked into the active
site of the receptor. Docking scores, hydrogen bonds and interacting groups are
represented in table 3.
Acknowledgments
This work was financially supported by Taif University, Taif, Kingdom of Saudi Arabia,
Grant No. 1-435-3287.
References
1- J. A. Joule, K. Mills (2010). Heterocyclic Chemistry (5th Edition). John Wiley & Sons.
2- A. Radominska-Pandya, Drug Metabolism Reviews, 2010, 42(1), 1–2.
Doi:10.3109/03602530903205450.
3- L. H. Patterson, Cancer Metastasis Rev. 1993, 12(2), 119-134.
Doi:10.1007/bf00689805.
4- L. H. Zalkow, J. A. Glinski, L. T. Gelbaum, T. J. Fleischmann, L. S. McGowan, M. M.
Gordon, J. med. chem. 1985, 28(6), 687-694. Doi: 10.1021/jm00383a001.
5- L. H. Zalkow, J. A. Glinski, L. T. Gelbaum, D. Moore, D. Melder, G. Powis, J. med.
chem. 1988, 31(8), 1520-1526. Doi: 10.1021/jm00403a008.
6- T.-L. Su, T.-C. Lee, R. Kakadiya. Eur. J. med. chem. Available online 18 September
2013. Doi: 10.1016/j.ejmech.2013.09.016.
7- C. A. Muller, J. K. Cheetham, T. H. Kuan and D. F. Power. Patent number: US
20130197051 A1, 1 Aug 2013. http://www.google.com.ar/patents/US20130197051 ( Last
accessed 2 Jan 2015).
8- C. C. J. Culvenor, J. A. Edgar, E. L. French, M. V. Jago, L. W. Smith,H. J.
Tweeddale. Patent number: US 3705905 A. Patented 12 Dec 1972.
http://www.google.com/patents/US3705905 (last accessed at 1 Jan 2015).
12

9- N. Sirisha and R. Raghunathan. ISRN Medicinal Chemistry, 2013, Article ID 492604,
1-8. Doi: 10.1155/2013/492604.
10- D. D. Buechter, and D. E. Thurston, J. Nat. Prod. 1987, 50(3), 360-367. Doi:
10.1021/np50051a004.
11- V. Perri, C. Rochais, J. S. O. Santos, R. Legay, T. Cresteil, P. Dallemagne, S. Rault,
Eur. J. Med. Chem. 2010, 45, 1146-1150. Doi:10.1016/j.ejmech.2009.12.021.
12- L. Wukun, Z. Jinpei, Z. Huibin, Q. Hai, Y. Jiahan, B. Kerstin, G. Ronald, Lett. Drug
Des. Discov. 2011, 8(10), 911-917. Doi: 10.2174/157018011797655223.
13- W. K. Anderson, J. S. New, P. F. Corey, Arzneim.-Forsch, 1980, 30(5), 765-767.
14- A. V. Kadushkin, I. N. Nesterova, T. V. Golovko, I. S. Nikolaeva, T. V. Pushkina, A.
N. Fomina, A. S. Sokolova, V. A. Chernov, V. G. Granik, Pharm. Chem. J. 1990,
24(12), 875-881. Doi: 10.1007/BF00766579.
15- A. Belal. Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8. Doi;
10.1002/ardp.201400004.
16- W. J. Gullick, Br. Med. Bull. 1991, 47(1), 87–98. pubmed/1863851.
17-Y. Yardern, A. Ullrich, Annu. Rev. Biochem. 1988, 57, 443-478.
18- A. Etienne, Y. Correia, Bull. Soc. Chim. Fr. 1969, 10, 3704–3712.
19- K.-W. Wu, P.-C. Chen, J. Wang, Y.-C. Sun, J Comput Aided Mol Des(2012)
26:1159–1169. Doi:10.1007/s10822-012-9606-6.
20 – Y. Cheng, W. Cui, Q. Chen, CH. Tung, M. Ji, F. Zhang, J Comput Aided Mol Des
(2011) 25:171–180. Doi: 10.1007/s10822-010-9408-7.
21- M.A. Olayioye, R.M. Neve, H.A. Lane, N.E. Hynes, EMBO J. 2000, 19, 3159-3167.
Doi: 10.1093/emboj/19.13.3159.
22- C. Fernandes, C. Oliveira, L. Gano, A. Bourkoula, I. Pirmettis, I. Santos, Bioorg.
Med. Chem. 2007, 15, 3974-3980. Doi:10.1016/j.bmc.2007.04.008.
13

23- H.-Q. Li, D.-D. Li, X. Lu, Y.-Y. Xu, H.-L. Zhu, Bioorg. Med. Chem. 2012, 20, 317-
323. Doi:10.1016/j.bmc.2011.10.085.
24- A. Belal, B. El-D. M. El-Gendy, Bioorg & Med Chem. 2014, 22, 46–53. Doi:
10.1016/j.bmc.2013.11.040.
25- Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren,
J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Nat. Cancer Inst. 1990, 82, 1107-1112.
Doi: 10.1093/jnci/82.13.1107.
26- D. Balzano, S. Santaguida, A. Musacchio, F. Villa, Chem. Biol. 2011, 18, 966–975.
Doi: 10.1016/j.chembiol.2011.04.013.
27-(http://www.rcsb.org/pdb/explore.do?structureId=2j6m. (Last accessed 18 oct. 2014).
14

Table 1: IC50 values for the newly synthesized pyrrolizine derivatives ( µM/ml ).
MCF7
HCT116
HEPG2
Comp.
No.
0.19415
0.02087
0.02400
0.05892
0.06298
0.12501
0.00862
0.481379
0.58563
0.040561
0.106048
0.20718
0.026496
0.31812
0.89068
0.263767
0.096117
0.052709
0.112743
0.012286
0.043859
4
5
6
7
8a
8b
8c
Table 2: EGFR tyrosine kinase inhibitory activity of pyrrolizine derivatives at 10µM.
Comp.
Percent
of inhibition
45.2
No.
4
58.24
54.27
40.4
5
6
7
8a
62.8
88.43
97.6
8b
8c
100
Erlotinib
15

Table 3: Docking scores, amino acid interactions and interacting groups of the docked
compounds into the active site of EGFR-TK.
Comp.
S
Interacting moieties
Amino acid
No.
4
(Kcal/mol)
-9.32
N of CN
Lys 745
-13.16
5
-11. 94
NH of pyrimidine
CO in pyrimidine
Asp 855
Lys 745
6
-9.72
-15.59
-17.77
-20.92
7
N of CN
N of CN
Met 793
Met 793
Asp 855
Arg 841
Thr 854
8a
8b
8c
N of thiomorpholine
Phenyl moiety
N-ethyl of piperidine
-17.17
AEE 788
16

I
Fig. 1. The parent compound (3H-pyrrolizine).
O
Cl
Ph
N
N
N
N
O
O
OH
N
II
III
OCONHCH(CH3)2
OCONHCH(CH3)2
H2N
N
N
N
N
Cl
Cl
COOC2H5
IV
V
Fig. 2. Antitumor active pyrrolizines and pyrimidopyrrolizines.
17

Fig 3. A) 2D interactions of AEE ligand with EGFR.
Fig 3. B) Docking predicted binding mode of AEE into the active site of EGFR.
18

Fig 4. A) 2D interactions of compound 8b with EGFR.
Fig 4. B) Docking predicted binding mode of compound 8b with the active site of EGFR.
19

Fig 5. A) 2D interactions of compound 8c with EGFR.
Fig 5. B) Docking predicted binding mode of compound 8c with the active site of EGFR.
20

NC
N
Br
CN
O
CN
CN
O
O
CN
NH2
NH2
CN
a
b
+
+
N
N
NH
O
1
2
4
3
4
Scheme 1: Synthesis of compound 4. Reagents and conditions: a) Dry acetone, K2CO3,
reflux 20h. b) 1% NaOC2H5, r.t. 24h.
CN
Cl
CN
R
CN
NH2
O
c
d
N
NH
N
N
O
N
H
4
O
O
O
a
CN
8a-c
7
O
N
N
H
8a : R =
8b : R =
8c: R =
N
N
N
5
O
O
b
S
O
H
N
N
N
6
O
Scheme 2 : Synthesis of compounds 5-8c. Reagents and conditions: a) Acetyl chloride,
dioxan, r.t. 24h. b) 1% NaOC2H5, r.t. 24h. c) Chloro acetyl chloride, dioxan, r.t. 12h. d)
Secondary amine, abs. ethanol, reflux 24h.
21

Graphical abstract
Highlights
 Design and synthesis of new pyrrolizines.
 Anticancer activity evaluation against MCF7, HEPG2 and HCT116.
 Docking studies into ATP binding site of EGFR-TK.
 Evaluating the new compounds as inhibitors for EGFR-TK.
22