Rational design, synthesis, and 2D-QSAR study of anti-oncological alkaloids against hepatoma and cervical carcinoma

Article abstract of DOI:10.1039/c4ra16663a

Antitumor active dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-diones 11-19 were regioselectively synthesized via azomethine ylide cycloaddition reactions with 3E,5E-1-alkyl-3,5-bis(arylmethylidene)-4-piperidones 3-7. Compounds 13, 14, and 16 reveal higher potency against the HeLa (cervical) tumor cell line than the standard reference cisplatin, while 11, and 12 seem more potent against the HepG2 (liver) carcinoma cell line relative to the standard reference doxorubicin hydrochloride as determined by in vitro Sulfo-Rhodamine-B bio-assay. 3D-Pharmacophores of the HeLa comprise five chemical features viz., two hydrogen bond acceptors, two hydrophobic centers and one positive ionizable center and HepG2 contains three chemical features viz., a hydrogen bond acceptor, a hydrophobic center and a positive ionizable center. These features of the tumor cell lines explain the variation of bioactivity relative to chemical structure. Statistically significant QSAR models describing the spiro-alkaloid bio-properties were obtained employing CODESSA-Pro software validating the observed pharmacological observations and identifying the most important parameters governing activity.

Full text of DOI:10.1039/c4ra16663a

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: A. Girgis, S. S.
Panda, M. N. Aziz, P. Steel, C. D. Hall and A. R. Katritzky, RSC Adv., 2015, DOI: 10.1039/C4RA16663A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 18
RSC Advances
View Article Online
DOI: 10.1039/C4RA16663A
RSC Advances
RSCPublishing
PAPER
Rational design, synthesis, and 2D-QSAR study of anti-
oncological alkaloids against hepatoma and cervical
carcinoma
Cite this: DOI: 10.1039/x0xx00000x
Adel S. Girgis,a Siva S. Panda,b Marian N. Aziz,a Peter J. Steel,c C. Dennis Hall*,b and
Alan R. Katritzkyb,d
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
Antitumor active dispiro[3
regioselectively synthesized via azomethine ylide cycloaddition reactions with
bis(arylmethylidene)ꢀ4ꢀpiperidones 3-7. Compounds 13 14, and 16 reveal higher potency against HeLa
H
ꢀindoleꢀ3,2'ꢀpyrrolidineꢀ3',3''ꢀpiperidine]ꢀ2(1
H
),4''ꢀdiones 11-19 were
www.rsc.org/
3
E,5Eꢀ1ꢀalkylꢀ3,5ꢀ
,
(cervical) tumor cell line than the standard reference cisplatin while, 11, and 12 seem more potent against
HepG2 (liver) carcinoma cell line relative to the standard reference doxorubicin hydrochloride as
determined by inꢀvitro SulfoꢀRhodamineꢀB bioꢀassay. 3DꢀPharmacophores of the HeLa comprise five
chemical features viz. two hydrogen bond acceptors, two hydrophobic centers and one positive ionizable
center and HepG2 contains three chemical features viz. a hydrogen bond acceptor, a hydrophobic center
and a positive ionizable center. These features of the tumor cell lines explain the variation of bioactivity
relative to chemical structure. Statistically significant QSAR models describing the spiroꢀalkaloid bioꢀ
properties were obtained employing CODESSAꢀPro software validating the observed pharmacological
observations and identifying the most important parameters governing activity.
Introduction
cancerous cells towards existing anticancer agents.
Therefore, a constant need to develop better alternatives to
Although there has been progress in diagnosing, treating and
managing cancer, the disease still results in the death of a
significant number of patients. It remains the second leading
cause of death worldwide after cardiovascular disease. The
worldwide cancer burden is expected to increase by as much
as 15 million new cases per year by 2020, according to the
World Health Organization, unless further preventive
measures are put into practice.1,2 Generally, cancers of the
breast, lung, colorectal, and prostate are the most frequent
types in developed countries and cancers of the stomach,
liver, oral cavity, and cervix the most frequent in developing
countries, although these patterns are changing, especially
due to population aging and life style changes.3,4. The
preliminary cancer treatment options usually remain a
combination of radiotherapy, surgery and chemotherapy.
Chemotherapy is considered one of the effective approaches
in suppressing tumor growth and eradication of tumors.
However, many patients undergoing chemotherapy suffer
from side effects such as nausea, vomiting, cachexia,
lethargy and poor oral intake.5 In spite of availability of a
large number of anticancer drugs, the development of new
chemotherapeutics is one of the most noteworthy challenges
due to nonꢀselectivity and emergence of resistance by
such problems is in demand.6
Recently we described regioselective synthesis of fluoroꢀ
substituted dispiroꢀoxindole and the structure was
investigated by Xꢀray and theoretical studies.7 We now
report full details of the synthesis of spiroꢀindoles within the
present work were bioꢀassayed for their antiꢀoncological
properties against human cervical (HeLa), and hepatoma
(HepG2) cancer cell lines, and the rationale for this is as
follows. Cervical carcinoma is the third most common
cancer and the fourth leading cause of cancerꢀrelated death
in women worldwide; every year, approximately 529,800
new cases are diagnosed, and approximately 275,000
women die from this disease.8,9 More than 80% of cervical
cancer cases occur in developing countries, while incidence
and mortality have substantially declined in developed
countries.10,11 Persistent infections with oncogenic types of
human papillomavirus (HPV) are the main risk factors for
cervical cancer development.12 From almost 160 HPV types
that have been characterized,13 close to 30 infect the
anogenital epithelium and 14 of them have been classified as
oncogenic types.14,15 The biological behavior of HPV
infections is influenced by viral cellular and host factors and
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

RSC Advances
Page 2 of 18
Paper
VRiewSCArtAicdlevOannlincees
DOI: 10.1039/C4RA16663A
varies in different lesions, even when the same viral type is
involved.16 Human cervical cancer cells can be categorized
according to HPV type as HeLa (HPVꢀ18+), MEꢀ180 (HPVꢀ
68+), SiHa (HPVꢀ16+), and SW756 (HPVꢀ18+) cells.17–19
Although several advances in screening, diagnostic and
treatment modalities have been made, the overall prognosis
of cervical cancer has not changed dramatically, and the
mortality rate still approaches 50%. The treatment of choice
of cervical cancer is represented by radiotherapy or surgery
for early stage disease and concurrent chemoradiation for
advanced stage patients.20 Concurrent chemotherapy and
radiotherapy (CCRT) is the standard of care for locally
advanced cervical cancer, able to achieve a 6% improvement
considered in the present work utilizing Discovery Studio
2.5 and Comprehensive Descriptors for Structural and
Statistical Analysis (CODESSAꢀPro) software. This allows
a better understanding of the observed pharmacological
activity and determines the most important structural
parameters controlling bioꢀactivity. These studies are also
used to validate the observed bioꢀdata.
Results and Discussion
Chemistry
3E,5Eꢀ1ꢀAlkylꢀ3,5ꢀbis(arylmethylidene)ꢀ4ꢀpiperidones
3-7, the starting agents for constructing the targeted spiroꢀ
alkaloids, were synthesized via baseꢀcatalyzed condensation
of 1ꢀalkylꢀ4ꢀpiperidones 1,2 with aromatic aldehydes
(Scheme 1). Due to the huge melting point difference (≈30
in a 5ꢀyear survival compared to radiotherapy alone.21–23
A
larger survival advantage occurs when adjuvant
chemotherapy is administered after CCRT.23–25 Cisplatin is
the drug of choice either alone or in combination with
topotecan.26 The combination of cisplatin with 5ꢀfluorouracil
has also been reported.27,28 However, severe sideꢀeffects like
boneꢀmarrow depression, neutropenia, thrombocytopenia
and anaemia due to haematological toxicity along with
°C) between the literature reported40
dichlorophenyl)methylidene]ꢀ1ꢀethylꢀ4ꢀpiperidones
3
E
,5Eꢀ3,5ꢀbis[(2,4ꢀ
and
3
our synthesized analogue, the structure of the compound was
investigated using a variety of spectroscopic techniques. The
IR spectrum of
3 reveals a strong stretching vibration band
neurotoxicity29
and
acquired
at
ν
= 1674 cmꢀ1 assignable to the α,βꢀunstaurated ketonic
nephrotoxicity
and
1
chemoresistance30 throughout the course of treatment have
limited the usage of cisplatin. Other reports describe the
severe renal toxicity and gastrointestinal side effects of
cisplatin that limits its application in clinic.31
function. The HꢀNMR spectrum of
3
exhibits the exocyclic
= 7.91
confirming the formation of a single geometrical isomer
(3 ,5 shows the olefinic
).41 The 13CꢀNMR spectrum of
methine carbon at = 136.1, and the carbonyl carbon at
186.3. A single crystal Xꢀray study of shows a half chair
form conformation for the piperidinyl ring. The exocyclic
olefinic bonds at ꢀ3 and ꢀ5 of the piperidinyl ring possess
E'ꢀconfigurations (Figure 1).
[3+2]ꢀCycloaddition of nonꢀstabilized azomethine ylides
(generated in situ via decarboxylative condensation of
isatins 8-9 with sarcosine 10) and 3 ,5Eꢀ1ꢀalkylꢀ3,5ꢀ
olefinic methine protons as a sharp singlet signal at
δ
E
E
3
δ
δ =
Liver malignancies including hepatocellular carcinoma,
cholangiocarcinoma and hepatoblastoma are jointly the fifth
most prevalent form of cancer and globally the third leading
cause of cancer related death, immediately after mortality
due to lung cancer and colon cancer.32 In addition, the liver
is a favorite site for metastasis of other cancers, in particular
colorectal cancer, esophageal cancer and pancreatic cancer.
The fiveꢀyear natural mortality rate for hepatocellular
carcinoma is more than 95%, and it affects more than
500,000 people worldwide per year.33 The major risk factors
for liver cancer are persistent infection with hepatitis B virus
(HBV) and hepatitis C virus (HCV), both of which increase
the risk of liver cancer some 20ꢀfold.34 Other wellꢀ
established liver cancer risk factors include cirrhosis,
aflatoxin exposure, heavy alcohol drinking, tobacco
smoking and some rare monogenic syndromes such as
3
C
C
E
,
E
bis(arylmethylidene)ꢀ4ꢀpiperidones 3-7 in refluxing ethanol
proceeds regioselectively to afford single products (silica gel
TLC) in good to excellent yields (62ꢀ98%) obeying the
reported procedure.42 The structure of the isolated products
was assigned as 1''ꢀalkylꢀ4'ꢀarylꢀ5''ꢀ(arylmethylidene)ꢀ1'ꢀ
methylꢀdispiro[3
Hꢀindoleꢀ3,2'ꢀpyrrolidineꢀ3',3''ꢀpiperidine]ꢀ
2(1
H
),4''ꢀdiones 11-19 based on their spectroscopic (IR, 1Hꢀ,
13CꢀNMR, 1H, 1HꢀCOSY, HSQC, HRMS) data and
elemental analysis (Figures S1ꢀS32 of supplementary
material). The reaction commences with nucleophilic attack
of the amino group of sarcosine 10 on the 3ꢀcarbonyl
function of isatin 8-9, followed by dehydration to form a
spiroꢀoxazalidinone system. This, expels carbon dioxide to
generate a reactive, nonꢀstabilized azomethine ylide, that
undergoes in situ 1,3ꢀdipolar cycloaddition to the exocyclic
olefinic linkage of piperidones 3-7 affording eventually
spiroꢀalkaloids 11-19 (Scheme 2).
hereditary
hemochromatosis
and
α
ꢀ1
antitrypsin
deficiency.35 Doxorubicin (also named as adriamycin, ADR)
has a broad antitumor effect as a typical DNA intercalating
agent, and is often used as the firstꢀline anticancer drug in
treatment of acute leukemia, malignant lymphoma, multiple
myeloma, breast cancer, osteosarcoma, soft tissue sarcoma
and liver cancer.36 The clinical application of doxorubicin is
limited by its toxicity to normal tissues and cells, especially
its cardiotoxicity.37 The only clinically approved
chemotherapy drug for advanced hepatocellular carcinoma
is sorafenib, which shows only modest efficacy, improving
survival of patients by just 3 months.38,39
3DꢀQSAR pharmacophoric generation and 2DꢀQSAR
(quantitative structureꢀactivity relationship) study is
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
methine HCꢀ4' is observed at
ꢀ3' ( ꢀ3'') and ꢀ3 (
respectively. The carbonyl carbons
= 176.3, 196.8, respectively. 1H, 1HꢀCOSY spectrum (Figure
S31 of supplementary material) and 1H,13Cꢀheteronuclear
single quantum coherence (HSQC) spectrum of compound
14 (Figures S32A, S32B of supplementary material) support
these interpretations.
δ
= 41.3 and the spiroꢀcarbons
O
O
C
C
C
C
ꢀ2') are exhibited at
δ
= 62.1, 76.2,
RCHO
base
R
R
Cꢀ2 and
Cꢀ4'' appear at δ
N
R'
R'
1-2
3-7
3 R = 2,4-Cl2C6H3, R' = Et
1 R' = Me
R' = Et
4
R = 4-FC6H4, R' = Et
5 R = 2-thienyl, R' = Et
2
A single crystal Xꢀray study of 14 (Figure 2), supports
the stereochemical structure. The indolyl as well as the 4ꢀ
fluorophenyl rings have planar configurations and the
6
R = 3-pyridinyl, R' = Me
7 R = 3-pyridinyl, R' = Et
Scheme 1 Synthetic route towards 3
piperidones 3-7
E,5Eꢀ1ꢀalkylꢀ3,5ꢀbis(arylmethylidene)ꢀ4ꢀ
exocyclic olefinic double bond has the Eꢀconfiguration. The
.
pyrrolidine ring has an envelope conformation with the flap
atom being the ring nitrogen which is out of the plane of the
remaining four atoms.
Figure 1 XꢀRay crystal structure of compound 3.
The IR spectrum of 11, representative of the family, exhibits
an indolyl amidic NH stretching vibration band at = 3285
cmꢀ1, and strong stretching vibration bands at
= 1701 and
1678 cmꢀ1 corresponding to the carbonyl of ketonic and
amidic functions, respectively. The methylene protons H2C
ν
ν
ꢀ
5', H2Cꢀ2'' and H2Cꢀ6'' of 11 are diastereotopic. The
methylene protons of the ethyl group attached at the
piperidinyl
Nꢀ1'' appear as a multiplet at δ = 1.93ꢀ2.11, due
to mutual coupling with each other and in turn with the
vicinal methyl protons (diastereotopic protons). The 13Cꢀ
NMR spectrum of 11 reveals the methylene carbons H2Cꢀ6'',
Figure 2 XꢀRay crystal structure of compound 14
.
H2Cꢀ5' and H2Cꢀ2'' at
δ
= 53.4, 57.0, 56.1, respectively. The
O
R
R
H
R
4'
O
N
R'
O
1'
N
CH3
O
H3C
CH2
O
O
4''
H3C
N+
O
N
X
O
R
ꢀCO2
H
X
2
NH
X
3-7
EtOH
ꢀH2O
N
1''
N
O
+
1
6''
H3C
OH
O
N
H
N
N
R'
H
10
H
8-9
11-19
X
8
X = H
9 X = Cl
11
R = 2,4ꢀCl2C6H3, R' = Et, X = H
3
R = 2,4ꢀCl2C6H3, R' = Et
12 R = 2,4ꢀCl2C6H3, R' = Et, X = Cl
13 R = 4ꢀFC6H4, R' = Et, X = H
14 R = 4ꢀFC6H4, R' = Et, X = Cl
15 R = 2ꢀthienyl,R' = Et, X = H
16 R = 2ꢀthienyl,R' = Et, X = Cl
17 R = 3ꢀpyridinyl, R' = Me, X = H
18 R = 3ꢀpyridinyl, R' = Et, X = H
19 R = 3ꢀpyridinyl, R' = Et, X = Cl
4 R = 4ꢀFC6H4, R' = Et
5 R = 2ꢀthienyl, R' = Et
6 R = 3ꢀpyridinyl, R' = Me
7 R = 3ꢀpyridinyl, R' = Et
Scheme 2 Synthetic route towards dispiro[3Hꢀindoleꢀ3,2'ꢀpyrrolidineꢀ3',3''ꢀpiperidines] 11-19.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

RSC Advances
Page 4 of 18
VRiewSCArtAicdlevOannlincees
Paper
DOI: 10.1039/C4RA16663A
observed antitumor properties was established via these
studies.
Antitumor properties
Antitumor properties of the synthesized dispiro[3Hꢀ
Computational chemistry
indoleꢀ3,2'ꢀpyrrolidineꢀ3',3''ꢀpiperidines]
11-19
were
screened against HeLa (cervical), and HepG2 (liver) human
tumor cell lines utilizing the reported inꢀvitro Sulfoꢀ
RhodamineꢀB standard method.43–49 The results in Table 1
(Figures S33, and S34 of supplementary material) show that,
all the synthesized compounds have considerable antitumor
activity against the tested cell lines with variable potencies.
3D-Pharmacophore modeling
The pharmacophore modeling technique has been widely
used in lead discovery and optimization as a key tool in
computer aided drug design. The 3Dꢀpharmacophore study
was performed using Discovery Studio 2.5 software
(Accelrys Inc., San Diego, CA, USA) which permits 3Dꢀ
pharmacophore generation, structural alignment, activity
prediction and 3Dꢀdatabase creation.45,50–52 A 3DꢀQSAR
pharmacophore protocol was used to generate predictive
pharmacophores via aligning different conformations in
which the molecules are likely to bind with the receptor. A
given hypothesis may be combined with known activity data
to create a 3Dꢀpharmacophore model that identifies overall
aspects of molecular structure governing activity. 3DꢀQSAR
pharmacophore was constructed using collections of
molecules with activities ranging over a number of orders of
magnitude. Pharmacophores explain the variability of
bioactivity with respect to the geometric localization of the
chemical features present in the molecules. The observed
HYPOGEN identifies a 3Dꢀarray of five chemical features
in the case of HeLa (cervical) and 3 features in case of
HepG2 (liver) tumor cell lines which are common to the bioꢀ
active set compounds 11-19 that are consistent with binding
to a proposed common receptor site.
Compounds 13, 14, and 16 reveal higher potency (IC50
4.87, 5.75, and 7.25 M, respectively) against HeLa
(cervical) cell line than the standard reference cisplatin (IC50
= 7.71
M) (clinically used against cervical carcinoma26).
On the other hand, compounds 11, and 12 seem more potent
(IC50 = 3.53, and 7.20 M, respectively) than doxorubicin
M) (clinically applicable agent
=
µ
µ
µ
hydrochloride (IC50 = 8.05
against liver carcinoma36).
µ
Structureꢀactivity relationships (SAR) based on the
observed antitumor activity data against HeLa (cervical
carcinoma) reveal that the nature of the substituent attached
to the phenyl group at
Cꢀ4' and consequently the exocyclic
olefinic linkage, seems to be a controlling factor governing
the antitumor properties. Substitution of this phenyl group
by fluorine atom enhances the observed antitumor properties
more than two chlorine atoms, as exhibited in pairs 11
(IC50 = 16.69, 4.87 M, respectively), and 12 14 (IC50
12.71, 5.75 M, respectively).
,
13
=
µ
,
µ
The five chemical features of the HeLa pharmacophore
are two hydrogen bond acceptors (HBAꢀ1, HBꢀ2), two
hydrophobic centers (Hꢀ1, Hꢀ2), and one positive ionizable
(PosIon) (Figure 3, Table 2 exhibits constraint distances and
angles between features of the generated 3Dꢀ
pharmacophore). On the other hand, the HepG2
pharmacophore contains three chemical features, a hydrogen
bond acceptor (HBA), a hydrophobic (H) and a positive
ionizable (PosIon) (Figure 4, Table 2). Table 3 exhibits fit
values and estimated/predicted activities of the synthesized
compounds 11-19 due to the generated 3Dꢀpharmacophore
models. Through the pharmacophore mapping study
(Figures S35, S36 of supplementary material) it has been
found that the major structural factors affecting the potency
of the synthesized compounds are related to their basic
skeleton. Additionally, most of the estimated activity as well
as the fit values derived from the generated pharmacophores
correlate with the experimentally observed potency. For
example, the most potent analogue 13 (R = 4ꢀFC6H4, R' =
SAR due to the observed antitumor activity data against
HepG2 (liver carcinoma) cell line describes a contrast
behavior than the aforementioned cervical cell line. 2,4ꢀ
Dichlorophenyl substituent at
Cꢀ4' and consequently the
exocyclic olefinic linkage, seems the best choice for
constructing antitumor active against hepatocellular
carcinoma compared with the 4ꢀfluorophenyl group as
exhibited in pairs 11, 13 (IC50 = 3.53, 10.90
respectively), and 12 14 (IC50 7.20, 12.50
respectively). Additionally, the 3ꢀpyridinyl group at
µ
µ
M,
M,
,
=
C
ꢀ4' and
consequently the exocyclic olefinic linkage, seem to
optimize antitumor activity against HepG2 (liver carcinoma)
when compared with the thienyl group as exhibited in pairs
15, 18 (IC50 = 18.42, 17.04
(IC50 = 17.34, 11.50 M, respectively). This can be
attributed to the ꢀdeficient heterocyclic properties of the 3ꢀ
µM, respectively), and 16, 19
µ
π
pyridinyl group compared with the electronꢀdonating
properties of thienyl group.
In order to better understand the observed antitumor
properties and determine the most important structural
parameters controlling bioꢀactivity, computational chemistry
studies were undertaken. Additionally, validation of the
Et, X = H; IC50 = 4.87
compounds, shows an estimated potency (IC50 = 5.75
ꢁ
M) among all the synthesized
M)
ꢁ
preserving its lead behavior and bioꢀpotency compared to
the standard reference used.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 5 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
Table 1. Antitumor properties of the synthesized compounds 11-19.
Entry
Compd.
R
R'
X
IC50, µg/ml (µM)a
HeLa
HepG2
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
2,4ꢀCl2C6H3
2,4ꢀCl2C6H3
4ꢀFC6H4
4ꢀFC6H4
2ꢀthienyl
2ꢀthienyl
3ꢀpyridinyl
3ꢀpyridinyl
3ꢀpyridinyl
ꢀꢀꢀ
Et
Et
Et
Et
Et
Et
Me
Et
Et
ꢀꢀꢀ
ꢀꢀꢀ
H
Cl
H
Cl
H
Cl
H
H
10.27 (16.69)
8.26 (12.71)
2.50 (4.87)
3.15 (5.75)
5.33 (10.89)
3.80 (7.25)
9.35 (20.08)
5.16 (10.76)
11.58 (22.53)
4.19 (7.22)
4.19 (7.71)
2.17 (3.53)
4.68 (7.20)
5.60 (10.90)
6.85 (12.50)
9.02 (18.42)
9.09 (17.34)
10.27 (22.06)
8.17 (17.04)
5.91 (11.50)
4.67 (8.05)
3.58 (11.89)
19
Cl
ꢀꢀꢀ
ꢀꢀꢀ
10
11
Doxorubicin hydrochloride
Cisplatin
ꢀꢀꢀ
aIC50 = concentration required to produce 50% inhibition of cell growth compared to control experimental.
Mapping of the HeLa 3Dꢀpharmacophore with
compound 13 (Figure S35 of supplementary material)
describes the correlation of the pyrrolidinyl nitrogen with
the positive ionizable feature. The same alignment was also
observed for compound 11 (R = 2,4ꢀCl2C6H3, R' = Et, X =
with the pharmacophoric hydrophobic feature, the
pyrrolidinyl nitrogen with the positive ionizable, and the
piperidinyl carbonyl is aligned with the hydrogen bond
acceptor feature. The potency difference of compounds 11
and 13 can be attributed to the slight difference in mode of
alignment and high hydrophobic properties of the 2,4ꢀ
dichlorophenyl group aligned with the hydrophobic feature
H; IC50 = 16.69, 15.00 ꢁM corresponding to the observed
and estimated potency, respectively). The high potency
difference between the observed/estimated activity of these
compounds (11, 13) explains the role of the substituent
than the corresponding pꢀfluorophenyl group aligned with
the same pharmacophoric feature. This observation is the
same mentioned for SAR rules governing HepG2 bioꢀdata.
The potency decrease of compounds 12 (R = 2,4ꢀCl2C6H3,
attached to the phenyl group linked at the
previously in SAR due to the observed bioꢀdata. The 2,4ꢀ
dichlorophenyl group attached to ꢀ4' position deactivates
the positive ionizable properties of the pyrrolidinyl nitrogen
much more than the ꢀfluorophenyl group (although the –I
Cꢀ4' as explained
C
R' = Et, X = Cl; IC50 = 7.20, 4.34
observed and estimated potency, respectively) and 14 (R =
4ꢀFC6H4, R' = Et, X = Cl; IC50 = 12.50, 13.55 ꢁM
ꢁM corresponding to the
p
effect of fluorine is higher than chlorine, the two chlorine
substituents combine to give a higher –I effect). The same
applies for compound 16 (R = 2ꢀthienyl, R' = Et, X = Cl;
corresponding to the observed and estimated potency,
respectively) “which exhibit typical alignment to
compounds 11, 13, respectively” compared to their similar
IC50 = 7.25, 10.42
estimated potency, respectively) when compared with
compound 19 (R = 3ꢀpyridinyl, R' = Et, X = Cl; IC50
22.53, 22.04 M corresponding to the observed and
ꢁ
M corresponding to the observed and
analogues 11 and 13 can be attributed to the effect of chloro
substitution attached to the indolyl group which decreases
the positive ionizable properties of the pyrrolidinyl nitrogen.
Mapping of the HepG2 3Dꢀpharmacophore with compound
=
ꢁ
estimated potency, respectively). This latter observation
correlates well with the electron donating properties of the
thienyl group (fiveꢀmembered heterocycle with one hetero
atom) strengthen the positive ionizable property of
pyrrolidinyl nitrogen upon compared with the effect of a 3ꢀ
pyridinyl group (πꢀdeficient heterocycle).
Mapping of the HepG2 3Dꢀpharmacophore with
compound 11 (R = 2,4ꢀCl2C6H3, R' = Et, X = H; IC50 = 3.53,
18 (R = 3ꢀpyridinyl, R' = Et, X = H; IC50 = 17.04, 18.90 ꢁM
corresponding to the observed and estimated potency,
respectively), describes the alignment of the 3ꢀpyridinyl
group attached to the exocyclic olefinic linkage with the
pharmacophoric hydrophobic, the pyrrolidinyl nitrogen with
the positive ionizable, and the piperidinyl carbonyl with the
hydrogen bond acceptor feature. A slightly modified
mapping is observed for compound 15 (R = 2ꢀthienyl, R' =
4.26
ꢁ
M corresponding to the observed and estimated
Et, X = H; IC50 = 18.42, 19.15 ꢁM corresponding to the
potency, respectively), the most potent analogue among all
the synthesized spiroꢀalkaloids (Figure S36 of
supplementary material), describes the alignment of the 2,4ꢀ
observed and estimated potency, respectively) where, of the
2ꢀthienyl group linked to the exocyclic olefinic linkage is
aligned with the pharmacophoric hydrophobic, the
pyrrolidinyl nitrogen with the positive ionizable, and the
indolyl carbonyl with the hydrogen bond acceptor feature.
The enhanced potency effect of compound 18 relative to
compound 15 can be attributed not only to the slight
difference in mode of alignment but also to the higher
hydrophobic properties of the 3ꢀpyridinyl group than the 2ꢀ
thienyl function. Another reason for the observed
pharmacological potency enhancement is extracted from the
differences in mode of alignment where, the hydrogen bond
dichlorophenyl group attached to the pyrrolidinyl
Cꢀ4' with
the pharmacophoric hydrophobic feature and the
pyrrolidinyl nitrogen with the pharmacophoric positive
ionizable while the piperidinyl carbonyl is aligned with the
hydrogen bond acceptor. A relatively similar mapping is
exhibited by compound 13 (R = 4ꢀFC6H4, R' = Et, X = H;
IC50 = 10.90, 11.51 ꢁM corresponding to the observed and
estimated potency, respectively), where the 4ꢀfluorophenyl
group attached to the exocyclic olefinic linkage is aligned
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

RSC Advances
Page 6 of 18
Paper
VRiewSCArtAicldevOanlnincees
DOI: 10.1039/C4RA16663A
acceptor property of the piperidinyl ketonic carbonyl is
higher than that of indolyl carbonyl. Meanwhile, potency
enhancement of compound 19 (R = 3ꢀpyridinyl, R' = Et, X =
acceptor feature. The same applies also for compound 16 (R
= 2ꢀthienyl, R' = Et, X = Cl; IC50 = 17.34, 17.59
ꢁM
corresponding to the observed and estimated potency,
respectively) that reveals a completely different mode of
alignment in the hypothesized pharmacophore. Where the
chlorine substituent of the indolyl group is aligned with the
pharmacophoric hydrophobic, the piperidinyl nitrogen with
the positive ionizable, and the indolyl carbonyl is aligned
with the hydrogen bond acceptor feature. This is could be
the reason for observed antitumor properties enhanced for
Cl; IC50 = 11.50, 12.26 ꢁM corresponding to the observed
and estimated potency, respectively) compared with its
similar analogue 18 is explained by the HepG2
pharmacophoric mapping of the former analogue in a
completely different mode of alignment where, the chlorine
substituent of the indolyl group is aligned with the
pharmacophoric hydrophobic, the piperidinyl nitrogen with
the positive ionizable, and the nitrogen of the 3ꢀpyridinyl
compound 16 relative to its similar analogue 15
.
group linked at the
Cꢀ4' is aligned with the hydrogen bond
Figure 3
(A) Constraint distances and (B) constraint angles of the generated 3Dꢀpharmacophore for the synthesized compounds 11-19 against HeLa (cervical) cell line
which contains two hydrogen bonding acceptor (HBAꢀ1, HBAꢀ2, green), two hydrophobics (Hꢀ1, Hꢀ2, light blue), and one positive ionizable (PosIon, red).
Figure 4
(A) Constraint distances and (B) constraint angles of the generated 3Dꢀpharmacophore for the synthesized compounds 11-19 against HepG2 (liver) cell line
which contains a hydrogen bonding acceptor (HBA, green), a hydrophobics (H, light blue), and a positive ionizable (PosIon, red).
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
Table 2 Constraint distances (Å) and angles (°) between features of the generated pharmacophores.
Cancer cell line
HeLa (cervical)
Constraint distances (Å)
(HBAꢀ1)‒(PosIon) = 3.146, (PosIon)‒(Hꢀ1) = 4.551,
(PosIon)‒(HBAꢀ2) = 4.494, (Hꢀ1)‒(HBAꢀ2) = 4.374,
(HBAꢀ2)‒(Hꢀ2) = 6.746; (Hꢀ2)‒(HBAꢀ1) = 4.581, (Hꢀ
1)‒(Hꢀ2) = 6.148
Constraint angles (°)
(HBAꢀ1)‒(Hꢀ1)‒(PosIon) = 39.22, (HBAꢀ1)‒(HBAꢀ
2)‒(PosIon) = 38.88, (HBAꢀ1)‒(Hꢀ1)‒(Hꢀ2) = 47.54, (HBAꢀ
1)‒(Hꢀ2)‒(PosIon) = 13.42
HepG2 (liver)
(H)‒(PosIon) = 8.239, (PosIon)‒(HBA) = 4.245,
(HBA)‒(H) = 7.672
(PosIon)‒(H)‒(HBA) = 30.68
Table 3. Best fit values and estimated/predicted activities for the synthesized compounds 11-19 mapped with the generated 3Dꢀpharmacophore models due to
HeLa (cervical) and HepG2 (liver) cancer cell lines.
Entry
Compd.
R
R'
X
HeLa (cervical) cell line
HepG2 (liver) cell line
Observed IC50,
Estimated IC50,
µM
Fit
Observed IC50,
Estimated IC50,
µM
Fit
value
5.75
5.74
5.31
5.24
5.09
5.13
5.04
5.10
5.29
µM
value
9.74
9.88
10.15
10.07
9.87
9.89
9.74
9.94
9.57
µM
3.53
7.20
10.90
12.50
18.42
17.34
22.06
17.04
11.50
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
19
2,4ꢀCl2C6H3
2,4ꢀCl2C6H3
4ꢀFC6H4
4ꢀFC6H4
2ꢀthienyl
Et
Et
Et
Et
Et
Et
Me
Et
Et
H
Cl
H
Cl
H
Cl
H
H
16.69
12.71
4.87
5.75
10.89
7.25
20.08
10.76
22.53
15.00
10.65
5.75
4.26
4.34
11.51
13.55
19.15
17.59
21.71
18.90
12.26
7.02
11.05
10.42
14.69
9.47
2ꢀthienyl
3ꢀpyridinyl
3ꢀpyridinyl
3ꢀpyridinyl
Cl
22.04
energy gradient was ≤0.01 kcal/mol. The RHF method was
used in spin pairing for the two semiꢀempirical tools.45,50,54,55
The resulting output files were exported to CODESSAꢀPro
that includes MOPAC capability for final geometry
optimization. CODESSAꢀPro software includes the
following: (a) a calculation engine for more than 500
descriptors and (b) an engine for the development of the
statistically most important linear and nonlinear QSAR
regression models. CODESSAꢀPro calculated 728 molecular
2D-QSAR study
Data set
The basic idea behind QSAR is to generate a relationship
between the chemical structure of an organic compound and
its physicoꢀchemical properties. Due to the limited
pharmacologically active data set mentioned in the present
study, external data points were considered. The external
data points are derived from spiroꢀalkaloids having the same
chemical scaffold (homogeneous/nonꢀdiverse data set
protocol) and their bioꢀproperties were determined by the
same standard technique adopted in the present study. The
QSAR study was undertaken using comprehensive
descriptors for structural and statistical analysis
(CODESSAꢀPro) software employing the synthesized
compounds of the present study 11,13,15-17,19 in addition
to compounds 20-44 which are recently reported by our
group53 forming 31 spiroꢀalkaloids used as a training set for
constructing QSAR models (Table 4). Compounds 12, 14,
and 18 (about one third of the synthesized analogues)
representing high, and low potent antitumor active agents,
were used as external data set for validating the attained
QSAR models (Table 5).
descriptors
including
constitutional,
topological,
geometrical, chargeꢀrelated, semiꢀempirical, molecularꢀtype,
atomicꢀtype and bondꢀtype descriptors for the exported 31
bioꢀactive spiroꢀalkaloids 11,13,15-17,19, and 20-44 which
were used as a training set in the present study. Different
mathematical transformations of the experimentally
observed property/activity (IC50,
ꢁ
M
which is the
concentration required to produce 50% inhibition of cell
growth compared to control experimental) against HeLa
(cervical) and HepG2 (liver) tumor cell lines of the training
set compounds were utilized for the present QSAR modeling
determination including property (IC50,
ꢁM), 1/property,
log(property) and 1/log(property) values in searching for the
best QSAR models.
QSAR modeling
Methodology
Best multiꢀlinear regression (BMLR) was utilized which is a
stepwise search for the best nꢀparameter regression
equations (where, n stands for the number of descriptors
used), based on the highest R2 (squared correlation
coefficient), R2cvOO (squared crossꢀvalidation “leave oneꢀ
out, LOO” coefficient), R2cvMO (squared crossꢀvalidation
“leave manyꢀout, LMO” coefficient), F (Fisher statistical
significance criteria) values, and s2 (standard deviation). The
QSAR models up to 3 and 4 descriptor models describing
Geometry of the compounds was optimized using molecular
mechanics force field (MM+) followed by the semiꢀ
empirical AM1 method implemented in the HyperChem 8.0
package. The structures were fully optimized without fixing
any parameters, thus bringing all geometric variables to their
equilibrium values. The energy minimization protocol
employed the PolakeꢀRibiere conjugated gradient algorithm.
Convergence to a local minimum was achieved when the
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

RSC Advances
Page 8 of 18
Paper
VRiewSCArtAicdlevOannlincees
DOI: 10.1039/C4RA16663A
bioꢀactivity of the antitumor active agents against HeLa
(cervical) and HepG2 (liver) cell line, respectively were
generated (obeying the thumb rule of 5:1, which is the ratio
between the data points and the number of QSAR descriptor
models). Statistical characteristics of the QSAR models are
presented in Tables 6, and 7. The established QSAR models
are statistically significant. The descriptors are sorted in
descending order of the respective values of the Student’s tꢀ
criterion, which is a widely accepted measure of statistical
significance of individual parameters in multiple linear
regressions. Figures 5, and 6 exhibit the QSAR multiꢀlinear
model plot of correlations representing the observed vs.
predicted IC50 values for HeLa and HepG2 tumor cell line
active agents, respectively. The scattered plots are uniformly
distributed, covering ranges, observed 0.688ꢀ1.387, 0.548ꢀ
1.375; predicted 0.723ꢀ1.416, 0.574ꢀ1.328 logarithmic units
for HeLa and HepG2 cell lines, respectively.
mentioned SAR rules governing bioꢀactivity concerning
type of substituent (halogen) attached to the phenyl group
linked at the
Cꢀ4' and consequently the exocyclic olefinic
linkage, affecting greatly the observed potency of the bioꢀ
active agent. The same phenomenon is also extracted from
the HeLa pharmacophoric hypothesis. The last descriptor of
the HeLa QSAR model (tꢀcriterion = 4.424), is HASAꢀ
2/SQRT(TMSA), which is also a charge related descriptor.
The areaꢀweighted surface charge of hydrogen bonding
acceptor atoms (HASA2) is determined by equation (2).56
ꢇꢈ
ꢊ
ꢉ
∑
HASA2
=
ꢈA
∈XHꢀacceptor .............................(2)
ꢂ
ꢋꢌꢋ
ꢃ
ꢉ
where, SA stands for solventꢀaccessible surface area of Hꢀ
bonding acceptor atoms, qA for partial charge on Hꢀbonding
acceptor atoms, and Stot
for total solventꢀaccessible
molecular surface area. This descriptor is considered the
second most effective parameter controlling the QSAR
model based on its coefficient (0.426). The present
descriptor also supported the attained SAR rules as
explained in the previous descriptor (FNSA2).
Molecular Descriptors
Molecular descriptors are the physicꢀchemical parameters
used to correlate chemical structure and property value
expressed as log(IC50). The descriptors were obtained based
on BMLR method. The descriptors controlling the bioꢀ
activity (property) by the established multiꢀlinear QSAR
models are presented in Tables 6, and 7 and are arranged,
based on their level of significance (tꢀcriterion).
HepGe (liver) tumor cell line
The attained HepG2 QSAR model exhibits 4 controlling
descriptors (Table 7) which are: min. (#HA, #HD) (Zefirov
PC), partial charged surface area for atom H, partial charged
surface area for atom O, and min. (>0.1) bond order for
atom C. The most important descript controlling the HepG2
QSAR model based on its tꢀcriterion (7.157) is minimum
(#HA, #HD) (Zefirov PC). This descriptor is a common for
both HeLa and HepG2 QSAR models explaining its
importance for the observed bioꢀproperties in variable tumor
cell lines. Although this descriptor is the most important one
governing the HepG2 QSAR model, its coefficient is the
minimum among all the other observed QSAR descriptor
(0.152). The second and the third important descriptors of
the HepG2 QSAR model are, partial charged surface area
for atom H, and partial charged surface area for atom O,
which are charge related descriptor. The partial positively or
negatively charged surface area is determined by equation
(3).56
HeLa (cervical) tumor cell line
The descriptors controlling bioꢀactivity of the synthesized
compounds against HeLa (cervical) tumor cell line
according to the attained 3 descriptor QSAR model (Table
6) are: min. (#HA, #HD) (MOPAC PC), FNSAꢀ2 fractional
PNSA (PNSAꢀ2/TMSA) (MOPAC PC), and HASAꢀ
2/SQRT(TMSA) (Zefirov PC) (all). The first descriptor
controlling the HeLa QSAR model is minimum (#HA,
#HD), which is a molecular type descriptor explaining the
capability of the bioꢀactive agent as hydrogen
donor/acceptor. Although this descriptor is considered the
most important one governing the attained QSAR model
based on its tꢀcriterion (9.200), its coefficient in the QSAR
is the minimum among all the other observed QSAR
descriptor (0.247). The second most important descriptor
controlling the HeLa QSAR model based on the tꢀcriterion
(5.546) is FNSAꢀ2 fractional PNSA (PNSAꢀ2/TMSA),
which is a charge related descriptor. The fractional total
charge weighted partial negative surface area (FNSA2) is
determined by equation (1).56
∑
ꢃ ꢍꢃA {δA< 0}.............................(3)
PNSA1
=
where, SA stands for positively or negatively charged
solventꢀaccessible atomic surface area. The partial charged
surface area for atom H descriptor participates in the HepG2
QSAR model with the greatest share among all the other
descriptors (coefficient = 83.943). However, the partial
charged surface area for atom O descriptor participated
negatively in the HepG2 QSAR model (coefficient = ꢀ
41.614), explaining that the higher of the latter descriptor
value, the lower of total log(IC50) observed, hence the higher
potency of the constructed agent against HepG2 tumor cell
line. This descriptor in particular can explain the potency of
ꢀꢁꢂꢃꢄ
FNSA2=
............................(1)
ꢅꢆꢂꢃ
where, PNSA2 stands for total charge weighted partial
negatively charged molecular surface area, and TMSA for
total molecular surface area. This descriptor has the highest
coefficient (0.596) among the other descriptors controlling
the HeLa QSAR model. This observation coincide with our
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 9 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
compounds 11 (R = 2,4ꢀCl2C6H3, R' = Et, X = H; IC50 = 3.53
M) relative to 13 (R = 4ꢀFC6H4, R' = Et, X = H; IC50
10.90 M). This can be correlated with the halogen
type/number substituted the phenyl group at ꢀ4' and
(IC50 range = 6.27ꢀ10.77
ꢁ
M) with relatively higher error
ꢁ
=
range (0.41ꢀ2.47) than the high potent analogues.
Additionally, the low potent analogues against HeLa cell
lines, compounds 11,17,19,36, and 40 (IC50 range = 11.20ꢀ
ꢁ
C
consequently the exocyclic olefinic linkage. The same
observation was mentioned in the SAR rules governing bioꢀ
activity and 3Dꢀpharmacophore modeling. The last
descriptor of the HepG2 QSAR model is min. (>0.1) bond
order for atom C, which is an atomic type descriptor.
24.36
range = 6.53ꢀ26.07
ꢁ
M) reveal higher deviated predicted potency (IC50
M) with error range = 1.64ꢀ5.99. From
ꢁ
all the above statistical observations, the attained HeLa
QSAR model can be considered a good predicative model
with a powerful ability to produce high potent HeLa
antitumor hits compared to those of mild or low potency.
Actually, this is an acceptable observation where most of the
training set compounds belong to the high potent HeLa
antitumor agents (20 compounds out of 31, i.e. two thirds of
the entire training set).
Validation of QSAR model
Internal validation
The reliability and statistical relevance of the QSAR models
are examined by internal and external validation procedures.
Internal validation is applied by the CODESSAꢀPro
technique employing both Leave One Out (LOO), which
involves developing a number of models with one example
omitted at a time, and Leave Many Out (LMO), which
involves developing a number of models with many data
points omitted at a time (up to 20% of the total data points).
The observed correlations due to the internal validation
techniques are R2cvOO = 0.738, 0.729; R2cvMO = 0.776,
0.741, for HeLa and HepG2 QSAR models, respectively.
Both of them are significantly correlated with the squared
correlation coefficient of the attained QSAR models (R2 =
0.815, 0.799 for HeLa and HepG2 QSAR models,
respectively). Standard deviation of the regressions (s2 =
0.008, 0.009 for HeLa and HepG2 QSAR models,
respectively) is also a measurable value for the attained
model together with the Fisher test value (F = 39.615,
25.768 for HeLa and HepG2 QSAR models, respectively)
that reflects the ratio of the variance explained by the model
and the variance due to their errors. A high value of Fꢀtest
compared with the s2 is a validation of the model. A
randomization test was also performed in the present study
which adds good support for the present QSAR models.
The predicted/estimated IC50 value of compound 13 (the
most potent synthesized analogue among all the training set
The predicted IC50 values (IC50 = 3.75ꢀ8.60
potent HepG2 analogues 11,20-24,26,28,29,31-35, and 37-
39 (IC50 3.53ꢀ7.49 M) relative to doxorubicin
hydrochloride (IC50 = 8.05 M standard reference used in
the present study “clinically used as liver antitumor agent”)
exhibit error range = 0.08ꢀ1.92. However, wilder error range
was observed for the mild HepG2 antitumor agents,
compounds 25,27, and 42-44 (IC50 = 8.73ꢀ9.37, 6.32ꢀ11.04
ꢁM) of the
=
ꢁ
ꢁ
ꢁ
M, corresponding to the observed and predicted values,
respectively, error range = 0.17ꢀ2.57) and low HepG2
antitumor agents, compounds 13,15-17,19,30,36,40, and 41
(IC50 = 10.90ꢀ23.71, 7.63ꢀ21.29 ꢁM, corresponding to the
observed and predicted values, respectively, error range =
0.37ꢀ5.31). This is an indication for the ability of utilization
of the attained HepG2 QSAR model for optimizing high
potent HepG2 (liver) antitumor agents than either mild or
low effective hits. The reason for this observation can be
attributed as mentioned in the former HeLa QSAR model,
due to participation of high number of potent agents
compared to mild and low potent analogues (14 out of 31
analogous of the training set i.e. 45%).
External validation
Compounds 12, 14, and 18 were used as an external test
set not only for validating the attained QSAR models but
also for examining their predicative ability. The test set
analogues experimentally exhibit high and low potency
against the tested cell lines. The variation in potency can
indicate the predication capabilities of the QSAR models.
Table 5 reveals the observed and predicted IC50 values of the
test set compounds. From the observed data, it has been
noticed that compound 14 which is considered a high potent
compounds) is 5.94
matched with the experimentally observed value (4.87
ꢁ
M based on the HeLa QSAR model,
M,
ꢁ
error “difference between observed and predicted values” =
1.07). All the other potent training set analogues
(compounds 16,20-29,31,33-35,38, and 42) relative to
cisplatin (standard reference clinically used against cervical
carcinoma, IC50 = 7.71 ꢁM) exhibit predicted IC50 values
matched with their experimentally observed potency (error
range = 0.06ꢀ1.12). Compounds 32, and 39 which are also
considered potent analogues against cervical carcimona
(IC50 = 5.55, 5.51 ꢁM corresponding to compounds 32, and
39, respectively) reveal relatively higher predicated potency
beyond the mentioned error range (predicted IC50 = 7.78,
7.84 ꢁM; error = 2.23, 2.33 corresponding to compounds 32,
and 39, respectively). The mild antitumor active agents
against HeLa cell line, compounds 15,30,37,41,43, and 44
agent against HeLa cell line (IC50 = 5.75
cisplatin, standard reference used, reveals a predicted IC50
5.64 M with minimum error value = 0.11. Compounds 12,
and 18 that considered low potent analogues (IC50 = 12.71,
ꢁM), relative to
=
ꢁ
10.76
23.70
ꢁ
ꢁ
M, respectively), afforded predicted IC50 = 8.99,
M, respectively (error values 3.72, 12.94,
=
respectively).
(IC50 range = 8.64ꢀ10.89
ꢁM), reveal predicted potency
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 9

RSC Advances
Page 10 of 18
View Article Online
DOI: 10.1039/C4RA16663A
RSC Advances
RSCPublishing
PAPER
Table 4 Observed and predicated values of training set compounds 11,13,15-17, and 19-44 according to the multiꢀlinear QSAR models.
Entry
Compd.
R
R'
X
HeLa (cervical) cell line
HepG2 (liver) cell line
Observed IC50,
Estimated
IC50, µM
12.26
5.94
10.48
7.86
26.07
20.89
5.92
5.41
6.30
5.72
5.28
5.90
5.43
5.95
5.71
6.21
7.09
6.71
7.78
7.68
7.17
6.54
6.53
6.27
6.94
7.84
20.24
8.18
Error
Observed
IC50, µM
3.53
10.90
18.42
17.34
22.06
11.50
7.46
5.66
6.03
6.26
5.73
8.89
5.43
8.73
5.77
5.82
14.18
7.32
5.46
6.15
6.68
6.68
13.67
5.91
6.95
Estimated
IC50, µM
3.75
Error
µM
16.69
4.87
10.89
7.25
20.08
22.53
6.21
5.92
6.74
5.08
4.96
5.78
5.20
6.51
5.15
5.44
8.64
6.65
5.55
6.96
6.45
7.22
11.20
8.74
6.10
5.51
24.36
8.94
6.86
9.65
9.88
1
2
3
4
5
6
7
8
11
13
15
16
17
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
2,4ꢀCl2C6H3
4ꢀFC6H4
2ꢀthienyl
Et
Et
Et
Et
Me
Et
Me
Me
Me
Me
Et
H
H
H
Cl
H
Cl
H
Cl
H
Cl
Cl
OMe
OMe
H
Cl
OMe
H
Cl
Cl
OMe
H
H
Cl
Cl
OMe
OMe
Cl
H
Cl
4.43
ꢀ1.07
0.41
ꢀ0.61
ꢀ5.99
1.64
0.29
0.51
0.44
ꢀ0.64
ꢀ0.32
ꢀ0.12
ꢀ0.23
0.56
ꢀ0.56
ꢀ0.77
1.55
ꢀ0.06
ꢀ2.23
ꢀ0.72
ꢀ0.72
0.68
ꢀ0.22
3.27
2.73
0.37
0.77
ꢀ4.82
ꢀ0.88
ꢀ0.50
ꢀ0.10
ꢀ0.08
ꢀ0.08
2.57
7.63
15.69
16.97
21.29
16.32
8.34
6.16
6.13
6.34
5.81
6.32
5.33
8.90
7.41
6.31
8.87
6.67
7.14
6.36
8.60
7.21
8.36
8.05
7.35
7.35
21.13
13.76
11.04
7.07
2ꢀthienyl
3ꢀpyridinyl
3ꢀpyridinyl
Ph
Ph
4ꢀClC6H4
4ꢀClC6H4
4ꢀClC6H4
4ꢀClC6H4
4ꢀClC6H4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Me
Et
0.10
4ꢀFC6H4
4ꢀFC6H4
4ꢀFC6H4
Me
Me
Me
Me
Me
Et
Me
Me
Et
Me
Et
Me
Et
ꢀ0.17
ꢀ1.64
ꢀ0.49
5.31
4ꢀH3CC6H4
4ꢀH3CC6H4
4ꢀH3CC6H4
4ꢀH3CC6H4
4ꢀH3COC6H4
4ꢀH3COC6H4
4ꢀH3COC6H4
4ꢀH3COC6H4
4ꢀH3COC6H4
4ꢀH3COC6H4
4ꢀMe2NC6H4
2ꢀthienyl
0.65
ꢀ1.68
ꢀ0.21
ꢀ1.92
ꢀ0.53
5.31
ꢀ2.14
ꢀ0.40
0.14
2.58
0.26
ꢀ1.75
1.33
ꢀ1.44
4.67
2.47
ꢀ0.84
ꢀ2.33
4.12
7.49
Me
Me
Me
Me
Me
23.71
14.02
9.29
8.40
9.37
0.76
2ꢀthienyl
2ꢀthienyl
5ꢀmethylꢀ2ꢀfuranyl
7.98
10.77
8.46
ꢀ1.12
ꢀ1.12
1.42
OMe
Cl
10.81
Table 5 Observed and predicated values of external test set compounds 12, 14, and 18 according to the multiꢀlinear QSAR models.
Entry
Compd.
R
R'
X
HeLa (cervical) cell line
HepG2 (liver) cell line
Observed IC50,
Estimated
IC50, µM
8.99
5.64
23.70
Error
Observed
IC50, µM
7.20
12.50
17.04
Estimated
IC50, µM
4.03
8.00
16.97
Error
µM
12.71
5.75
1
2
3
12
14
18
2,4ꢀCl2C6H3
4ꢀFC6H4
3ꢀpyridinyl
Et
Et
Et
Cl
Cl
H
3.72
0.11
ꢀ12.94
3.17
4.50
0.07
10.76
Table 6 Descriptor of the best multiꢀlinear QSAR model for the HeLa (cervical) tumor cell line active agents.
Entry
N=31, n=3, R2=0.815, R2cvOO=0.738, R2cvMO=0.776, F=39.615, s2=0.008
ID
0
D1
D2
D3
coefficient
0.141
0.247
0.596
0.426
s
t
Descriptor
Intercept
1
2
3
4
0.185
0.027
0.107
0.096
0.763
9.200
5.546
4.424
Min.(#HA, #HD) (MOPAC PC)
FNSAꢀ2 Fractional PNSA (PNSAꢀ2/TMSA) (MOPAC PC)
HASAꢀ2/SQRT(TMSA) (Zefirov PC) (all)
log(IC50) = 0.141 + (0.247 x D1) + (0.596 x D2) + (0.426 x D3)
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 10

Page 11 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
Table 7 Descriptor of the best multiꢀlinear QSAR model for the HepG2 (liver) tumor cell line active agents.
Entry
N=31, n=4, R2=0.799, R2cvOO=0.729, R2cvMO=0.741, F=25.768, s2=0.009
ID
0
D1
D2
D3
D4
coefficient
5.211
0.152
83.943
ꢀ41.614
ꢀ60.210
s
t
Descriptor
Intercept
Min.(#HA, #HD) (Zefirov PC)
Partial Charged Surface Area for atom H
Partial Charged Surface Area for atom O
Min. (>0.1) bond order for atom C
1
2
3
4
5
1.493
0.021
17.380
8.822
14.595
3.491
7.157
4.830
ꢀ4.717
ꢀ4.125
log(IC50) = 5.211 + (0.152 x D1) + (83.943 x D2) ‒ (41.614 x D3) – (60.210 x D4)
These observations give good sign for the predictive
capability of the attained HeLa QSAR model and support
the previous statement concerning its predictive power due
to the statistical values.
Predicted value for compound 12, IC50 = 4.03
correlated to its high potency against HepG2 cell line
(observed IC50 = 7.20 M). Although the error value due to
ꢁM, is
ꢁ
difference between the observed and predicted IC50 values is
considered a relatively high (error value = 3.17), the
observed potency of the analogue is still preserved relative
to doxorubicin hydrochloride, standard reference used (IC50
= 8.05
ꢁM). Compounds 14, and 18 which are considered
low potent HepG2 antitumor agents (observed IC50 = 12.50,
Figure 6 QSAR best multiꢀlinear model plot of correlations representing the
observed vs. predicted IC50 values for HepG2 (liver) tumor cell line active
agents (compounds 30, and 36 are outliers).
17.04
16.97
ꢁ
M, respectively) reveal predicted IC50 values = 8.00,
M, respectively (error values 4.50, 0.07,
ꢁ
=
respectively). The overall observations due to test set
predication values give a good indication for the predictive
power of the attained QSAR models for optimizing high
potent hits having spiroꢀalkaloid scaffold.
Conclusion
[3+2]ꢀCycloaddition reactions of nonꢀstabilized azomethine
ylide (generated in situ via decarboxylative condensation of
isatins 8,9 with sarcosine 10) and 3
bis(arylmethylidene)ꢀ4ꢀpiperidones 3-7 afforded 1''ꢀalkylꢀ4'ꢀ
arylꢀ5''ꢀ(arylmethylidene)ꢀ1'ꢀmethylꢀdispiro[3 ꢀindoleꢀ3,2'ꢀ
pyrrolidineꢀ3',3''ꢀpiperidine]ꢀ2(1 ),4''ꢀdiones 11-19 in
regioselective manner. Compounds 13 14, and 16 reveal
E,5Eꢀ1ꢀalkylꢀ3,5ꢀ
H
H
,
higher potency against HeLa (cervical) cell line than the
standard reference cisplatin, while compounds 11, and 12
seem more potent than doxorubicin hydrochloride (clinically
applicable agent against liver carcinoma) through inꢀvitro
SulfoꢀRhodamineꢀB bioꢀassay. 3Dꢀpharmacophore study
utilizing Discovery Studio 2.5 software explained the
antitumor variability of the tested compounds based on
chemical structural features. The HeLa pharmacophore
contains five chemical features; two hydrogen bond
acceptors, two hydrophobics, and one positive ionizable.
The HepG2 pharmacophore comprises three chemical
features; a hydrogen bond acceptor, a hydrophobic, and a
positive ionizable. 2DꢀQSAR study was undertaken utilizing
CODESSAꢀPro software in order to validate the antitumor
observed bioꢀdata and determine the most important
parameters controlling bioꢀactivity. Statistically significant
robust QSAR models describing the spiroꢀalkaloids bioꢀ
properties were obtained. External validation technique
Figure 5 QSAR best multiꢀlinear model plot of correlations representing the
observed vs. predicted IC50 values for HeLa (cervical) tumor cell line active
agents (compound 36 is an outlier).
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 11

RSC Advances
Page 12 of 18
Paper
VRiewSCArtAicdlevOannlincees
DOI: 10.1039/C4RA16663A
utilizing high and low potent synthesized agents, support the
predictive power of the attained QSAR models.
Homogeneity of the training set analogues (the same
chemical scaffold) may be the main factor for the success of
the QSAR models.
was evaporated till dryness. The separated solid upon
triturating the residue with methanol (5 ml) was collected
and crystallized from a suitable solvent.
4'-(2,4-Dichlorophenyl)-5''-[(2,4-
dichlorophenyl)methylidene]-1''-ethyl-1'-methyl-
dispiro[3
H
-indole-3,2'-pyrrolidine-3',3''-piperidine]-
Experimental
2(1 ),4''-dione (11). Obtained from reaction of
H
3
,
8
and 10
.
Melting points were determined on a capillary tube digital
Stuart SMP3 melting point apparatus. IR spectra (KBr) were
recorded on a JASCO 6100 FTꢀIR spectrophotometer. NMR
spectra were recorded on Mercury or Gemini NMR
spectrometers operating at 300 MHz for 1H (with TMS as an
internal standard) and 75 MHz for 13C, except 13CꢀNMR
spectra of compounds 17-19 which were recorded on a
Bruker Ascend 400/R (100 MHz) spectrometer. Highꢀ
resolution mass spectra (HRMS) were recorded on an
Agilent Technologies 6210 Time of Flight LC/MS
instrument operating in the ESI mode. The starting
compounds 4-7 were prepared according to the reported
procedures.57–59
Reaction time 12 h, pale yellow microcrystals from
n
ꢀ
butanol, mp 232ꢀ234 °C, yield 1.07 g (87%). IR: νmax/cmꢀ1
3285 (NH), 1701, 1678 (C=O), 1611, 1601. 1HꢀNMR
(DMSOꢀd6) (300 MHz):
NCH2CH3), 1.71 (d,
piperidinyl H2Cꢀ2''), 1.92 (s, 3H, pyrrolidinyl NCH3), 1.93ꢀ
2.11 (m, 2H, piperidinyl NCH2CH3), 2.91 (d, = 12.8 Hz,
1H, downfield H of piperidinyl H2Cꢀ2''), 3.10 (d, = 15.4
Hz, 1H, upfield H of piperidinyl H2Cꢀ6''), 3.28ꢀ3.40 (m, 2H,
downfield of piperidinyl H2Cꢀ6'' + upfield H of
pyrrolidinyl H2Cꢀ5), 3.77 (t, = 9.0 Hz, 1H, downfield H of
pyrrolidinyl H2Cꢀ5'), 4.89 (t, = 8.7 Hz, 1H, pyrrolidinyl
HCꢀ4'), 6.65 (d, = 7.6 Hz, 1H, arom. H), 6.82ꢀ6.84 (m, 2H,
arom. H), 7.04ꢀ7.08 (m, 1H, arom. H), 7.11 (d, = 8.3 Hz,
1H, arom. H), 7.34 (dd, = 8.4, 2.1 Hz, 1H, arom. H), 7.42
(s, 1H, olefinic CH), 7.47ꢀ7.51 (m, 2H, arom. H), 7.61 (d,
= 2.2 Hz, 1H, arom. H), 7.98 (d, = 8.5 Hz, 1H, arom. H),
10.57 (s, 1H, NH). 13CꢀNMR (DMSOꢀd6) (75 MHz):
11.0
(piperidinyl NCH2 H3), 33.9 (pyrrolidinyl N H3), 41.3
(pyrrolidinyl H ꢀ4'), 51.4 (piperidinyl N H2CH3), 53.4
(piperidinyl H2 ꢀ6''), 56.1 (piperidinyl H2 ꢀ2''), 57.0
(pyrrolidinyl H2 ꢀ5'), 62.1[spiro Cꢀ3' (Cꢀ3'')], 76.2 [spiro
3 ( ꢀ2')], 108.8, 121.2, 125.9, 127.1, 127.2, 128.4, 129.1,
131.2, 131.5, 132.0, 132.1, 134.1, 134.7, 134.8, 135.6,
135.8, 143.6 (arom. + olefinic ), 176.3 [indolyl =O (
2)], 196.8 [piperidinyl =O ( ꢀ4'')]. Anal. Calcd. for
δ
0.67 (t,
J = 7.1 Hz, 3H,
J
= 12.5 Hz, 1H, upfield H of
J
J
H
J
J
J
Synthesis
dichlorophenyl)methylidene]-1-ethyl-4-piperidone (3).
mixture of 1ꢀethylꢀ4ꢀpiperidone (0.64 ml, 5 mmol), and
of
3
E,5E-3,5-bis[(2,4-
J
A
J
2
J
2,4ꢀdichlorobenzaldehyde (1.75 g, 10 mmol) in 10 ml
methanol containing KOH (0.56 g, 10 mmol), was stirred at
room temperature (25 °C) for 3 h. The separated solid after
storing the reaction mixture overnight at room temperature,
was collected, washed with water, and crystallized from nꢀ
J
δ
C
C
C
C
C
C
butanol affording
3
as pale yellow microcrystals, mp 136ꢀ
C
Cꢀ
138 °C (lit. mp 100.4–103.7 °C)50, yield 1.95 g (88%). IR:
C
νmax/cmꢀ1 1674 (C=O), 1616, 1584. HꢀNMR (CDCl3) (300
1
MHz):
Hz, 2H, NCH2CH3), 3.64 (s, 4H, piperidinyl 2NCH2), 7.17
(d, = 8.3 Hz, 2H, arom. ꢀ6/6'), 7.29 (td, = 8.3, 3.4, 1.7
Hz, Hz, 2H, arom. ꢀ5/5'), 7.47 (t, = 1.7 Hz, 2H, arom.
3/3'), 7.91 (s, 2H, olefinic H’s). 13CꢀNMR (CDCl3) (75
MHz): 12.4 (piperidinyl NCH2 H3), 51.0 (piperidinyl
H2CH3), 54.0 [piperidinyl N H2 ( ꢀ2/6)], 127.1 (arom.
ꢀ5'), 130.1 (arom. ꢀ3'), 131.1 (arom. ꢀ6'), 132.2 (arom.
ꢀ4'), 133.2 (arom. ꢀ1'), 134.8 (arom. ꢀ2'), 135.5
H), 186.3 [piperidinyl
δ
0.99 (t,
J
= 7.2 Hz, 3H, NCH2CH3), 2.53 (q,
J
= 7.2
C
C
C
Cꢀ
C
C
J
H
J
C31H27Cl4N3O2 (615.39): C, 60.50; H, 4.42; N, 6.83. Found:
C, 60.89; H, 4.50; N, 6.46.
5-Chloro-4'-(2,4-dichlorophenyl)-5''-[(2,4-
dichlorophenyl)methylidene]-1''-ethyl-1'-methyl-
H
J
Hꢀ
δ
C
N
C
C
C
dispiro[3
H-indole-3,2'-pyrrolidine-3',3''-piperidine]-
C
C
C
C
2(1 ),4''-dione (12). Obtained from reaction of
H
3
,
9
and 10.
C
C
Reaction time 12 h, pale yellow microcrystals from
methanol, mp 203ꢀ204 °C, yield 0.81 g (62%). IR: νmax/cmꢀ1
3414 (NH), 1721, 1694 (C=O), 1607, 1585. 1HꢀNMR
(piperidinyl
Cꢀ3/5), 136.1 (olefinic C
Cꢀ4 (C=O)]. Anal. Calcd. for C21H17Cl4NO (441.19): C,
57.17; H, 3.88; N, 3.17. Found: C, 57.33; H, 3.56; N, 2.97.
Synthesis of 1''-alkyl-4'-aryl-5''-(arylmethylidene)-1'-
(DMSOꢀd6) (300 MHz):
piperidinyl NCH2CH3), 1.72 (d,
of piperidinyl H2Cꢀ2''), 1.94 (s, 3H, pyrrolidinyl NCH3),
1.98ꢀ2.13 (m, 2H, piperidinyl NCH2CH3), 2.95 (d, = 12.6
Hz, 1H, downfield H of piperidinyl H2Cꢀ2''), 3.15 (d, J =
δ
0.68 (t,
J = 7.1 Hz, 3H,
J
= 12.5 Hz, 1H, upfield H
methyl-dispiro[3
piperidine]-2(1
H
-indole-3,2'-pyrrolidine-3',3''-
H),4''-diones 11-19 (General procedure):
J
A mixture of equimolar amounts of the appropriate 1ꢀalkylꢀ
3,5ꢀbis(arylmethylidene)ꢀ4ꢀpiperidones 3-7 (2 mmol), the
corresponding isatins 8,9 and sarcosine 10 in absolute
ethanol (10 ml) was heated under reflux for the appropriate
time. The separated solid while refluxing was collected and
crystallized from suitable solvent affording compounds 11-
13,15,16, and 19. In the case of 14, the clear reaction
mixture was stored at room temperature overnight. The
separated solid was collected and crystallized from a
suitable solvent. In case of 17, and 18, the reaction mixture
13.6 Hz, 1H, upfield H of piperidinyl H2Cꢀ6''), 3.29ꢀ3.35 (m,
2H, downfield H of piperidinyl H2Cꢀ6'' + upfield H of
pyrrolidinyl H2Cꢀ5'), 3.75 (t,
pyrrolidinyl H2Cꢀ5'), 4.83 (t,
HCꢀ4'), 6.69 (d, = 8.3 Hz, 1H, arom. H), 6.78 (d,
Hz, 1H, arom. H), 7.16 (dd, = 8.2, 2.2 Hz, 1H, arom, H),
7.18 (s, 1H, arom. H), 7.37 (dd, = 8.4, 1.9 Hz, 1H, arom.
H), 7.43 (s, 1H, olefinic CH), 7.49ꢀ7.53 (m, 2H, arom. H),
7.63 (d, = 2.1 Hz, 1H, arom. H), 7.91 (d, = 8.5 Hz, 1H,
J
= 9.1 Hz, 1H, downfield H of
= 8.6 Hz, 1H, pyrrolidinyl
= 2.0
J
J
J
J
J
J
J
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 13 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
arom. H), 10.73 (s, 1H, NH). 13CꢀNMR (DMSOꢀd6) (75
MHz): 11.0 (piperidinyl NCH2CH3), 34.0 (pyrrolidinyl
H3), 41.6 (pyrrolidinyl ꢀ4'), 51.4 (piperidinyl
H2CH3), 53.3 (piperidinyl H2Cꢀ6''), 56.1 (piperidinyl
Hz, 1H, pyrrolidinyl HCꢀ4'), 6.59ꢀ7.37 (m, 12H, 11 arom. H
δ
+ olefinic CH), 10.55 (s, 1H, NH). 13CꢀNMR (DMSOꢀd6
)
N
N
H
C
C
H
C
(75 MHz):
(pyrrolidinyl
(piperidinyl NC
δ
N
10.9 (piperidinyl NCH2CH3),
H3), 44.2 (pyrrolidinyl ꢀ4'), 51.1
H2CH3), 53.2 (piperidinyl H2Cꢀ6''), 56.1
(piperidinyl H2Cꢀ2''), 56.7 (pyrrolidinyl H2Cꢀ5'), 64.5 [spiro
Cꢀ3' (Cꢀ3'')], 75.3 [spiro ꢀ3 ( ꢀ2')], 110.1, 114.9, 115.1,
34.1
C
HC
2Cꢀ2''), 57.0 (pyrrolidinyl H2Cꢀ5'), 62.7 [spiro Cꢀ3' (Cꢀ3'')],
ꢀ3 ( ꢀ2')], 110.4, 125.4, 126.7, 127.2, 127.3,
128.2, 128.5, 128.6, 129.2, 131.2, 131.5, 131.9, 132.2,
134.3, 134.5, 134.7, 135.5, 135.6, 142.5 (arom. + olefinic
), 176.0 [indolyl =O ( ꢀ2)], 196.8 [piperidinyl =O (
76.1 [spiro
C
C
C
C
C
115.5, 115.8, 124.8, 126.7, 128.4, 128.9, 130.8, 130.9,
131.0, 132.3, 132.4, 132.92, 132.94, 134.3, 134.31, 135.9,
C
C
C
C
Cꢀ
4'')]. Anal. Calcd. for C31H26Cl5N3O2 (649.84): C, 57.30; H,
4.03; N, 6.47. Found: C, 57.67; H, 3.77; N, 6.30.
1''-Ethyl-4'-(4-fluorophenyl)-5''-[(4-
142.4, 159.5, 160.5, 162.7, 163.8 (arom.
C
+ olefinic
C),
176.1 [indolyl
C
=O (
C
ꢀ2)], 198.0 [piperidinyl =O ( ꢀ4'')].
C
C
HRMS (ESI): m/z [M + H]+ calcd for C31H29ClF2N3O2:
548.1911, found: 548.1902.
fluorophenyl)methylidene]-1'-methyl-dispiro[3
3,2'-pyrrolidine-3',3''-piperidine]-2(1 ),4''-dione
Obtained from reaction of and 10. Reaction time 6 h,
H-indole-
H
(13).
1''-Ethyl-1'-methyl-4'-(2-thienyl)-5''-[(2-
4
,
8
thienyl)methylidene]-dispiro[3
3',3''-piperidine]-2(1 ),4''-dione (15). Obtained from
reaction of and 10. Reaction time 9 h, yellow
H-indole-3,2'-pyrrolidine-
colorless microcrystals from nꢀbutanol, mp 219ꢀ221 °C,
H
yield 1.01 g (98%). IR: νmax/cmꢀ1 3198 (NH), 1686 (C=O),
5, 8
1
1611, 1601. HꢀNMR (DMSOꢀd6) (300 MHz):
7.1 Hz, 3H, piperidinyl NCH2CH3), 1.63 (d,
δ
J
0.74 (t,
= 12.4 Hz,
J
=
microcrystals from N,Nꢀdimethylformamide, mp 236ꢀ237
°C, yield 0.84 g (86%). IR: νmax/cmꢀ1 3198 (NH), 1694, 1676
(C=O), 1614, 1576. 1HꢀNMR (DMSOꢀd6) (300 MHz):
1H, upfield H of piperidinyl H2Cꢀ2''), 1.94 (s, 3H,
pyrrolidinyl NCH3), 2.02ꢀ2.13 (m, 1H, upfield H of
piperidinyl NCH2CH3), 2.17ꢀ2.28 (m, 1H, downfield H of
δ
0.86 (t,
J
= 6.9 Hz, 3H, piperidinyl NCH2CH3), 1.86 (d,
J
=
12.3 Hz, 1H, upfield H of piperidinyl H2Cꢀ2''), 1.94 (s, 3H,
pyrrolidinyl NCH3), 2.12ꢀ2.19 (m, 1H, upfield H of
piperidinyl NCH2CH3), 2.34ꢀ2.42 (m, 1H, downfield H of
piperidinyl NCH2CH3), 2.90 (dd,
J = 14.9, 2.5 Hz, 1H,
upfield H of piperidinyl H2Cꢀ6''), 3.09ꢀ3.32 (m, 3H,
downfield H of piperidinyl H2Cꢀ2'' + downfield H of
piperidinyl H2Cꢀ6'' + upfield H of pyrrolidinyl H2Cꢀ5'), 3.75
piperidinyl NCH2CH3), 2.83 (d,
J = 15.1 Hz, 1H, upfield H
of piperidinyl H2Cꢀ6''), 3.23ꢀ3.33 (m, 3H, downfield H of
piperidinyl H2Cꢀ2'' + downfield H of piperidinyl H2Cꢀ6'' +
(t,
(dd,
13H, 12 arom. H + olefinic CH), 10.35 (s, 1H, NH).13Cꢀ
NMR (DMSOꢀd6 (75 MHz): 10.9 (piperidinyl
NCH2 H3), 34.0 (pyrrolidinyl N H3), 44.4 (pyrrolidinyl
ꢀ4'), 51.1 (piperidinyl N H2CH3), 53.2 (piperidinyl H2
6''), 56.0 (piperidinyl H2 ꢀ2''), 56.6 (pyrrolidinyl H2 ꢀ5'),
64.1 [spiro Cꢀ3' (Cꢀ3'')], 75.3 [spiro ꢀ3 ( ꢀ2')], 108.6,
J
= 9.6 Hz, 1H, downfield H of pyrrolidinyl H2Cꢀ5'), 4.61
J
= 10.7, 7.4 Hz, 1H, pyrrolidinyl HCꢀ4'), 6.56ꢀ7.36 (m,
upfield H of pyrrolidinyl H2Cꢀ5'), 3.77 (t,
downfield H of pyrrolidinyl H2Cꢀ5'), 4.87 (t,
pyrrolidinyl HCꢀ4'), 6.62 (d, = 7.8 Hz, 1H, arom. H), 6.70
(t, = 7.4 Hz, 1H, arom. H), 6.79ꢀ7.61 (m, 8H, 7 arom H +
olefinic CH), 7.81 (d, = 4.5 Hz, 1H, arom. H), 10.38 (s,
1H, NH). 13CꢀNMR (DMSOꢀd6
(75 MHz): 11.0
(piperidinyl NCH2 H3), 33.8 (pyrrolidinyl N H3), 40.2
(pyrrolidinyl H ꢀ4'), 51.4 (piperidinyl N H2CH3), 53.1
(piperidinyl H2 ꢀ6''), 54.8 (piperidinyl H2 ꢀ2''), 57.5
(pyrrolidinyl H2 ꢀ5'), 63.7 [spiro Cꢀ3' (Cꢀ3'')], 75.3 [spiro
3 ( ꢀ2')], 108.5, 120.4, 124.4, 126.0, 126.6, 126.9, 128.3,
128.8, 129.3, 132.2, 134.0, 137.6, 141.0, 143.6 (arom.
olefinic ), 176.3 [indolyl =O ( ꢀ2)], 196.5 [piperidinyl
=O (
ꢀ4'')]. HRMS (ESI): m/z [M + H]+ calcd for
J
= 9.7 Hz, 1H,
= 8.9 Hz, 1H,
J
)
δ
J
C
C
J
H
C
C
Cꢀ
J
C
C
)
δ
C
C
C
C
114.8, 115.1, 115.4, 115.6, 120.6, 126.7, 126.9, 128.5,
130.9, 131.0, 131.1, 131.12, 132.3, 132.4, 132.9, 134.6,
C
C
C
C
C
134.63, 135.5, 143.4, 159.5, 160.4, 162.7, 163.7 (arom.
olefinic ), 176.5 [indolyl =O ( ꢀ2)], 198.2 [piperidinyl
=O (
ꢀ4'')]. HRMS (ESI): m/z [M + H]+ calcd for
C
+
Cꢀ
C
C
C
C
C
C
C +
C31H29F2N3O2: 514.2301,found: 514.2326.
5-Chloro-1''-ethyl-4'-(4-fluorophenyl)-5''-[(4-
fluorophenyl)methylidene]-1'-methyl-dispiro[3
C
C
C
C
C
H
-indole-
),4''-dione (14).
and 10. Reaction time 10 h,
C27H27N3O2S2: 490.1617,found: 490.1640.
5-Chloro-1''-ethyl-1'-methyl-4'-(2-thienyl)-5''-[(2-
3,2'-pyrrolidine-3',3''-piperidine]-2(1
Obtained from reaction of
H
4
,
9
thienyl)methylidene]-dispiro[3
3',3''-piperidine]-2(1 ),4''-dione (16). Obtained from
reaction of and 10. Reaction time 11 h, pale yellow
microcrystals from
H-indole-3,2'-pyrrolidine-
pale yellow microcrystals from methanol, mp 210ꢀ212 °C,
H
yield 0.80 g (73%). IR: νmax/cmꢀ1 3157 (NH), 1701, 1678
5, 9
(C=O), 1599, 1585. 1HꢀNMR (DMSOꢀd6) (300 MHz):
δ
nꢀbutanol, mp 226ꢀ228 °C, yield 0.88 g
0.76 (t,
J
= 7.1 Hz, 3H, piperidinyl NCH2CH3), 1.64 (d,
J
=
(84%). IR: νmax/cmꢀ1 3183 (NH), 1695, 1672 (C=O), 1618,
1
12.5 Hz, 1H, upfield H of piperidinyl H2Cꢀ2''), 1.98 (s, 3H,
pyrrolidinyl NCH3), 2.06ꢀ2.14 (m, 1H, upfield H of
piperidinyl NCH2CH3), 2.18ꢀ2.30 (m, 1H, downfield H of
1574. HꢀNMR (DMSOꢀd6) (300 MHz):
Hz, 3H, piperidinyl NCH2CH3), 1.87 (d,
δ
J
0.86 (t,
= 12.5 Hz, 1H,
J = 7.2
upfield H of piperidinyl H2Cꢀ2''), 1.97 (s, 3H, pyrrolidinyl
piperidinyl NCH2CH3), 2.95 (d,
of piperidinyl H2Cꢀ6''), 3.10ꢀ3.34 (m, 3H, downfield H of
piperidinyl H2Cꢀ2'' + downfield H of piperidinyl H2Cꢀ6'' +
upfield H of pyrrolidinyl H2Cꢀ5'), 3.74 (t,
downfield H of pyrrolidinyl H2Cꢀ5'), 4.62 (dd,
J
= 13.9 Hz, 1H, upfield H
N
N
N
CH3), 2.14ꢀ2.23 (m, 1H, upfield
CH2CH3), 2.31ꢀ2.40 (m, 1H, downfield H of piperidinyl
CH2CH3), 2.85 (d, = 14.9 Hz, 1H, upfield H of
= 12.7 Hz, 1H, downfield H
H of piperidinyl
J
J
= 9.7 Hz, 1H,
= 10.7, 7.5
piperidinyl H2Cꢀ6''), 3.23 (d,
of piperidinyl H2Cꢀ2''), 3.28ꢀ3.41 (m, 2H, downfield H of
J
J
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 13

RSC Advances
Page 14 of 18
Paper
VRiewSCArtAicdlevOannlincees
DOI: 10.1039/C4RA16663A
piperidinyl H2Cꢀ6'' + upfield H of pyrrolidinyl H2Cꢀ5'), 3.74
(t, = 9.7 Hz, 1H, downfield H of pyrrolidinyl H2Cꢀ5'), 4.85
(dd, = 10.4, 7.4 Hz, 1H, pyrrolidinyl HCꢀ4'), 6.63 (d,
8.3 Hz, 1H, arom. H), 6.74 (d, = 2.0 Hz, 1H, arom. H),
6.96ꢀ7.61 (m, 7H, arom. H + olefinic CH), 7.85 (d, = 5.0
Hz, 1H, arom. H), 10.55 (s, 1H, NH). 13CꢀNMR (DMSOꢀd6
(75 MHz): 11.0 (piperidinyl NCH2CH3), 33.9 (pyrrolidinyl
H3), 34.7 (pyrrolidinyl ꢀ4'), 51.3 (piperidinyl
H2CH3), 53.1 (piperidinyl H2 ꢀ6''), 54.8 (piperidinyl
ꢀ2''), 57.7 (pyrrolidinyl H2 ꢀ5'), 64.0 [spiro Cꢀ3' (Cꢀ3'')],
75.3 [spiro ꢀ3 ( ꢀ2')], 109.9, 124.5, 124.7, 126.1, 126.6,
126.9, 128.4, 128.7, 128.9, 129.6, 132.3, 134.0, 137.5,
140.7, 142.5 (arom. + olefinic ), 175.9 [indolyl =O (
=O (
pyrrolidinyl H2Cꢀ5'), 3.78 (t,
J
= 9.8 Hz, 1H, downfield H of
J
pyrrolidinyl H2Cꢀ5'), 4.62 (dd,
pyrrolidinyl HCꢀ4'), 6.61 (d, = 7.8 Hz, 1H, arom. H), 6.88ꢀ
8.48 (m, 12H, 11 arom. H + olefinic CH), 10.41 (s, 1H,
NH). 13CꢀNMR (DMSOꢀd6) (100 MHz):
11.2 (piperidinyl
NCH2CH3), 34.4 (pyrrolidinyl N H3), 43.4 (pyrrolidinyl
ꢀ4'), 51.5 (piperidinyl N H2CH3), 53.5 (piperidinyl H2C
6''), 56.6 (piperidinyl H2 ꢀ2''), 56.8 (pyrrolidinyl H2 ꢀ5'),
64.6 [spiro Cꢀ3' (Cꢀ3'')], 75.9 [spiro ꢀ3 ( ꢀ2')], 109.3,
J = 9.9, 7.5 Hz, 1H,
J
J
=
J
J
J
δ
)
C
δ
H
C
C
ꢀ
N
N
H2
C
C
C
H
C
C
C
C
C
C
C
121.4, 124.0, 124.1, 126.8, 127.4, 129.3, 130.9, 133.6,
134.7, 135.6, 137.1, 137.4, 143.8, 148.4, 149.7, 150.7, 150.9
C
C
(arom.
C
+ olefinic ), 177.0 [indolyl
C
C=O (Cꢀ2)], 198.7
C
C
C
C
ꢀ
[piperidinyl
C
=O (Cꢀ4'')]. Anal. Calcd. for C29H29N5O2
2)], 196.6 [piperidinyl
H]+ calcd for C27H26ClN3O2S2: 524.1228,found: 524.1250.
C
C
ꢀ4'')]. HRMS (ESI): m/z [M +
(479.59): C, 72.63; H, 6.10; N, 14.60. Found: C, 72.86; H,
6.17; N, 14.78.
1',1''-dimethyl-4'-(3-pyridinyl)-5''-[(3-
5-Chloro-1''-ethyl-1'-methyl-4'-(3-pyridinyl)-5''-[(3-
pyridinyl)methylidene]-dispiro[3
H
-indole-3,2'-
),4''-dione
pyridinyl)methylidene]-dispiro[3
H
-indole-3,2'-
),4''-dione
pyrrolidine-3',3''-piperidine]-2(1
H
(17).
pyrrolidine-3',3''-piperidine]-2(1
H
(19).
Obtained from reaction of
6
,
8
and 10. Reaction time 12 h,
Obtained from reaction of 7, 9 and 10. Reaction time 11 h,
colorless microcrystals from toluene, mp 209ꢀ211 °C, yield
colorless microcrystals from ethanol, mp 244ꢀ246 °C, yield
0.75 g (81%). IR: νmax/cmꢀ1 3150 (NH), 1705, 1686 (C=O),
0.76 g (74%). IR: νmax/cmꢀ1 3408 (NH), 1709, 1686 (C=O),
1
1
1618, 1603. HꢀNMR (CDCl3) (300 MHz):
δ
1.71 (d,
J
=
1616, 1593. HꢀNMR (DMSOꢀd6) (300 MHz):
7.1 Hz, 3H, piperidinyl NCH2CH3), 1.72 (d,
δ
0.74 (t,
J =
12.6 Hz, 1H, upfield H of piperidinyl H2Cꢀ2''), 2.04 (s, 3H,
piperidinyl NCH3), 2.17 (s, 3H, pyrrolidinyl NCH3), 2.96
J = 12.3 Hz,
1H, upfield H of piperidinyl H2Cꢀ2''), 2.01 (s, 3H,
pyrrolidinyl NCH3), 2.09ꢀ2.26 (m, 2H, upfield H of
(dd,
3.23ꢀ3.38 (m, 3H, downfield H of piperidinyl H2Cꢀ2'' +
downfield of piperidinyl H2Cꢀ6'' + upfield H of
pyrrolidinyl H2Cꢀ5'), 3.92 (dd, = 10.8, 8.7 Hz, 1H,
downfield H of pyrrolidinyl H2Cꢀ5'), 4.79 (dd, = 10.8, 6.9
Hz, 1H, pyrrolidinyl HCꢀ4'), 6.65 (d, = 7.8 Hz, 1H, arom.
H), 6.95ꢀ8.51 (m, 12H, 10 arom. H + olefinic CH + NH),
8.59 (d,
= 1.8 Hz, 1H, arom. H). 13CꢀNMR (DMSOꢀd6
(100 MHz):
H3), 44.8 (pyrrolidinyl H
56.8 (pyrrolidinyl H2
[spiro Cꢀ3' (Cꢀ3'')], 75.4 [spiro
J = 14.7, 2.4 Hz, 1H, upfield H of piperidinyl H2Cꢀ6''),
piperidinyl
NCH2CH3 + downfield H of piperidinyl
H
N
CH2CH3), 3.05ꢀ3.34 (m, 4H, upfield H of piperidinyl H2Cꢀ
J
6'' + downfield H of piperidinyl H2Cꢀ2'' + downfield H of
piperidinyl H2Cꢀ6'' + upfield H of pyrrolidinyl H2Cꢀ5'), 3.77
J
J
(t,
(dd,
J
= 9.6 Hz, 1H, downfield H of pyrrolidinyl H2Cꢀ5'), 4.62
= 9.9, 7.5 Hz, 1H, pyrrolidinyl HCꢀ4'), 6.63 (d, = 8.1
J
J
J
)
Hz, 1H, arom. H), 6.80ꢀ8.51 (m, 11H, 10 arom. H + olefinic
CH), 10.59 (s, 1H, NH). 13CꢀNMR (DMSOꢀd6) (100 MHz):
δ
34.5 (pyrrolidinyl N
CH3), 43.2 (piperidinyl
N
C
Cꢀ4'), 56.3 (piperidinyl H2
Cꢀ6''),
δ
11.2 (piperidinyl NCH2
43.2 (pyrrolidinyl H ꢀ4'), 51.5 (piperidinyl N
(piperidinyl H2 ꢀ6''), 56.6 (piperidinyl H2
(pyrrolidinyl H2 ꢀ5'), 64.9 [spiro Cꢀ3' (Cꢀ3'')], 75.9 [spiro
3 ( ꢀ2')], 110.8, 124.0, 124.2, 125.5, 127.2, 129.0, 129.1,
130.7, 133.9, 134.4, 135.6, 137.2, 137.4, 142.7, 148.6,
150.0, 150.7, 150.9 (arom. + olefinic ), 176.7 [indolyl
=O ( ꢀ2)], 198.4 [piperidinyl =O ( ꢀ4'')]. Anal. Calcd.
C
H3), 34.5 (pyrrolidinyl N
H2CH3), 53.5
ꢀ2''), 56.9
CH3),
Cꢀ5'), 57.6 (piperidinyl H2
Cꢀ2''), 65.2
C
C
C
ꢀ3 ( ꢀ2')], 109.4, 121.4,
C
C
C
C
124.01, 124.04, 126.9, 127.4, 129.2, 130.8, 133.4, 134.6,
135.7, 137.1, 137.2, 143.6, 148.4, 149.7, 150.5, 150.8 (arom.
Cꢀ
C
C
+
olefinic
C), 177.1 [indolyl
C=O (Cꢀ2)], 198.3
[piperidinyl
C
=O ( ꢀ4'')]. Anal. Calcd. for C28H27N5O2
C
C
C
(465.56): C, 72.24; H, 5.85; N, 15.04. Found: C, 72.49; H,
5.82; N, 15.19.
1''-Ethyl-1'-methyl-4'-(3-pyridinyl)-5''-[(3-
C
C
C
C
for C29H28ClN5O2 (514.03): C, 67.76; H, 5.49; N, 13.62.
Found: C, 67.91; H, 5.56; N, 13.81.
pyridinyl)methylidene]-dispiro[3
H
-indole-3,2'-
),4''-dione
Single crystal X-ray
pyrrolidine-3',3''-piperidine]-2(1
H
(18).
Obtained from reaction of
7
,
8
and 10. Reaction time 12 h,
The Xꢀray single crystal diffraction data were collected at
120 K on an Agilent SuperNova instrument with focussed
microsource Cu Kα radiation (λ = 1.5418 Å) and ATLAS
CCD area detector. Details of the data collection conditions
and the parameters of the refinement are given in Table 8.
pale yellow microcrystals from toluene, mp 204ꢀ206 °C,
yield 0.84 g (88%). IR: νmax/cmꢀ1 3142 (NH), 1701, 1686
(C=O), 1618, 1597. 1HꢀNMR (DMSOꢀd6) (300 MHz):
δ
0.73 (t,
J
= 7.2 Hz, 3H, piperidinyl NCH2CH3), 1.73 (d,
J
=
12.0 Hz, 1H, upfield H of piperidinyl H2Cꢀ2''), 1.98 (s, 3H,
pyrrolidinyl NCH3), 2.03ꢀ2.13 (m, 1H, upfield H of
piperidinyl NCH2CH3), 2.17ꢀ2.25 (m, 1H, downfield H of
piperidinyl NCH2CH3), 3.00ꢀ3.32 (m, 4H, upfield H of
piperidinyl H2Cꢀ6'' + downfield H of piperidinyl H2Cꢀ2'' +
downfield
H of piperidinyl H2Cꢀ6'' + upfield H of
14 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 15 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
an atmosphere of 5% CO2. After 48 h, the cells were fixed,
washed and stained with SulfoꢀRhodamineꢀB (SRB) stain.
Excess stain was washed with acetic acid. The attached stain
was recovered with TrisꢀEDTA buffer. Cell survival and
drug activity were determined by measuring the color
intensity spectrophotometrically at 564 nm using an ELISA
microplate reader (Meter tech. Σ 960, USA). Data are
collected as mean values for experiments that were
performed in three replicates for each individual dose which
were measured by SRB assay. Control experiments did not
exhibit significant change compared to the DMSO vehicle.
Doxorubicin hydrochloride and cisplatin were used as
standard references during the present inꢀvitro bioactivity
screening assay. The percentage of cell survival was
calculated according to equation (4).
Table 8 Crystal data and structure refinement parameters for compounds
3 and 14.
Compound
3
14
Chemical formula
Mr
C21H17Cl4NO
441.16
C31H28ClF2N3O2
548.01
Crystal system, space
group
Monoclinic, C2/c
Monoclinic, P21/n
Temperature (K)
120
120
21.5694(4),
7.0397(1),
25.8262(3)
90.325(1)
3921.44(10)
8
11.97300 (13),
16.1952 (3), 14.09653
(16)
93.4721 (10)
2728.37 (6)
4
a, b, c (Å)
β (°)
V (Å3)
Z
Radiation type
ꢁ (mmꢀ1)
Crystal size (mm)
Cu Kα
5.577
0.22 x 0.18 x 0,14
SuperNova (Cu) Xꢀ
ray
Cu Kα
1.64
0.30 × 0.24 × 0.06
Diffractometer
SuperNova (Cu) Xꢀray
0.721, 0.922
Tmin, Tmax
0.373, 0.509
No. of measured,
independent and
observed [I> 2σ(I)]
reflections
ꢚ
ꢜ
Surviving fraction=ꢎꢏꢐꢑꢒꢓꢔ ꢕꢖꢗꢘꢑꢐꢙ ꢎ.ꢛ. ꢝꢞ ꢐꢟꢖꢓꢐꢖꢕ ꢒꢖꢔꢔꢘ ……(4)
ꢎ.ꢛ.ꢝꢞ ꢒꢝꢗꢐꢝꢔ ꢒꢖꢔꢔꢘ
21368, 3540, 3137
38016, 4913, 4248
The IC50 (concentration required to produce 50% inhibition
of cell growth compared to control experiment) was
determined using GraphꢀPad PRISM versionꢀ5 software.
Statistical calculations for determination of the mean and
standard error values were determined using SPSS 16
software. The observed antitumor properties are presented in
Table 1 (Figures S33, and S34 of supplementary material).
Rint
0.038
0.599
0.048
0.599
(sinθ/λ)max (Å−1)
R[F2> 2σ(F2)],
wR(F2), S
0.027, 0.075, 1.01
0.036, 0.096, 1.05
No. of reflections
No. of parameters
ꢁρmax, ꢁρmin (e Å−3)
3540
244
0.29, ꢀ0.22
4913
356
0.42, ꢀ0.23
The structures were solved using direct methods with
SHELXS60 and refined on F2 using all data by full–matrix
least square procedures with SHELXL–97.60 Multiscan
absorption corrections were done using SCALE3
ABSPACK. The non–hydrogen atoms were refined with
anisotropic displacement parameters. All hydrogen atoms
were included in calculated positions with isotropic
displacement parameters 1.2 times the isotropic equivalent
of their carrier atoms. Full crystallographic details of
Acknowledgements
This study was supported financially by the Science and
Technology Development Fund (STDF), Egypt, Grant No.
1357.
Notes and references
aPesticide Chemistry Department, National Research Centre, Dokki,
Cairo 12622, Egypt
compounds
3 and 14 have been deposited at the Cambridge
Crystallographic Data Centre (CCDC) as supplementary
publication numbers CCDC 1037561 and CCDC 1028899,
respectively.
bCenter for Heterocyclic Compounds, Department of Chemistry,
University of Florida, Gainesville, FL 32611ꢀ7200, USA. Eꢀmail:
charlesdennishall@gmail.com, tel: +1ꢀ352ꢀ392ꢀ0554
cChemistry Department, University of Canterbury, Christchurch, New
Zealand.
Antitumor activity screening
dDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589,
Saudi Arabia
Antitumor properties of the synthesized compounds 11-19
were screened by the National Cancer Institute, Cairo
University, Egypt, using the reported inꢀvitro Sulfoꢀ
RhodamineꢀB (SRB) standard technique adopting HeLa
(cervical) and HepG2 (liver) human tumor cell lines.43–49
Cells were seeded in 96ꢀwell microtiter plates at a
concentration of 5 x 104 – 105 cell/well in a fresh medium
and left for 24 h before treatment with the test compounds to
allow attachment of cells to the wall of the plate. The test
compounds were dissolved in dimethylsulfoxide (DMSO)
and diluted 1000ꢀfold in the assay. Different concentrations
of the compounds under test (0, 2.5, 6.25, 12.5, and 25
Electronic Supplementary Information (ESI) available: Copy of IR,
1H NMR, 13C NMR, HRMS/elemental analysis, HSQC and
pharmacological graphs of synthesized the compounds. See
DOI: 10.1039/b000000x/
1. H. Frankish, Lancet, 2003, 361, 1278.
2. D. Belpomme, P. Irigaray, A. J. Sasco, J. A. Newby, V.
Howard, R. Clapp, L. Hardell, Int. J. Oncol., 2007, 30,
1037‒1049.
3. C. Are, S. Rajaram, M. Are, H. Raj, B. O. Anderson, R. C.
Swamy, M. Vijayakumar, T. Song, M. Pandey, J. A. Edney, E.
L. Cazap, J. Surg. Oncol., 2013, 107, 221–226.
4. E. Carrasco, P. J. Álvarez, C. Melguizo, J. Prados, E. Álvarezꢀ
Manzaneda R. Chahboun, I. Messouri, M. I. Vázquezꢀ
ꢁ
g/ml) were added to the cell monolayer. Triplicate wells
were prepared for each individual dose. The monolayer cells
were incubated with the test compounds for 48 h at 37 °C, in
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 15

RSC Advances
Page 16 of 18
VRiewSCArtAicldevOanlnincees
Paper
DOI: 10.1039/C4RA16663A
Vázquez, A. Aránega, F. RodríguezꢀSerrano, Eur. J. Med.
Chem., 2014, 79, 1–12.
30. Z. H. Siddik, Oncogene, 2003, 22, 7265–7279.
31. X. Li, L. Ran, W. Fang, D. Wang, Biomedicine
Pharmacotherapy, 2014, 68, 291–297.
5. C. W. Mai, M. Yaeghoobi, N. AbdꢀRahman, Y. B. Kang, M.
R. Pichika, Eur. J. Med. Chem., 2014, 77, 378–387.
6. K. Singh, V. Verma, K. Yadav, V. Sreekanth, D. Kumar, A.
Bajaj, V. Kumar, Eur. J. Med. Chem., 2014, 87, 150‒158.
7. A. S. Girgis, S. S. Panda, E. S. M. Shalaby, A. F. Mabied, P.
J. Steel, C. D. Hall, A. R. Katritzky, RSC Adv., 2015, 5,
14780‒14787.
8. S. Nishio, K. Ushijima, T. Yamaguchi, Y. Sasajima, H. Tsuda,
T. Kasamatsu, M. Kage, M. Ono, M. Kuwano, T. Kamura,
Gynecol. Oncol., 2014, 132, 703–708.
9. A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, D.
Forman, CA Cancer J. Clin., 2011, 61, 69–90.
10. M. Abbas, K. Srivastava, M. Imran, M. Banerjee, Eur. J.
Obstet. Gynecol. Reprod. Biol., 2014, 176, 68–74.
11. R. Siegel, D. Naishadham, A. Jemal, CA Cancer J. Clin.,
2012, 62, 10–29.
32. A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M. J. Thun,
Cancer statistics, CA Cancer J. Clin., 2009, 59, 225–249.
33. Y. Bai, Y. Xue, X. Xie, T. Yu, Y. Zhu, Q. Ge, Z. Lu, Gene,
2014, 548, 91–100.
34. D. M. Parkin, Int. J. Cancer, 2006, 118, 3030‒3044.
35. H. B. ElꢀSerag, Gastroenterology, 2012, 142, 1264‒1273.
36. J. Yu, J. Z. Yang, S. W. Su, HeraldMed., 2013, 32, 174‒177.
37. T. Hirai, Y. Korogi, K. Ono, K. Maruoka, K. Harada, S.
Aridomi, M. Takahashi, Cardiovasc. Intervent. Radiol., 2001,
24, 176‒179.
38. A. R. de Lope, S. Tremosini, A. Forner, M. Reig, J. Bruix, J.
Hepatol., 2012, 56, S75‒S87.
39. J. M. Llovet, S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.
F. Blanc, A. C. de Oliveira, A. Santoro, J.ꢀL. Raoul, A.
Forner, M. Schwartz, C. Porta, S. Zeuzem, L. Bolondi, T. F.
Greten, P. R. Galle, J.ꢀF. Seitz, I. Borbath, D. Häussinger, T.
Giannaris, M. Shan, M. Moscovici, D. Voliotis, J. Bruix, N.
Engl. J. Med., 2008, 359, 378‒390.
12. J. M. Walboomers, M. V. Jacobs, M. M. Manos, F. X. Bosch,
J. A. Kummer, K. V. Shah, P. J. Snijders, J. Peto, C. J. Meijer,
N. Muñoz, J. Pathol., 1999, 189, 12–19.
13. R. D. Burk, A. Harari, Z. Chen, Virology, 2013, 445, 232–243.
14. V. Cogliano, R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El
Ghissassi, Lancet Oncol., 2005, 6, 204.
40. J. Wu, Y. Zhang, Y. Cai, J. Wang, B. Weng, Q. Tang, X.
Chen, Z. Pan, G. Liang, S. Yang, Bioorg. Med. Chem., 2013,
21, 3058–3065.
15. J. S. Smith, L. Lindsay, B. Hoots, J. Keys, S. Franceschi, R.
Winer, G. M. Clifford, Int. J. Cancer, 2007, 121, 621–632.
16. E. M. Burd, Clin. Microbiol. Rev., 2003, 16, 1–17.
17. S. Muthusami, D. S. Prabakaran, J.ꢀR. Yu, W.ꢀY. Park,
Cancer Lett., 2014, 351, 252–259.
18. C. P. Harris, X. L. Lu, G. Narayan, B. Singh, V. V. V. S.
Murty, Genes Chromosomes Cancer 36 (2003) 233–241.
19. L. Scotto, G. Narayan, S. V. Nadula, H. AriasꢀPulido, S.
Subramaniyam, A. Schneider, A. M. Kaufmann, J. D. Wright,
B. Pothuri, M. Mansukhani, V. V. Murty, Genes
Chromosomes Cancer, 2008, 47, 755–765.
41. A. S. Girgis, F. H. Osman, F. A. ElꢀSamahy, I. S. Ahmedꢀ
Farag, Chem. Pap. 60 (2006) 237‒242.
42. R. Grigg, S. Thianpatanagul, J. Chem. Soc., Chem. Commun.
(1984) 180‒181.
43. R. F. George, N. S. M. Ismail, J. Stawinski, A. S. Girgis, Eur.
J. Med. Chem., 2013, 68, 339–351.
44. A. M. Moustafa, A. S. Girgis, S. M. Shalaby, E. R. T. Tiekink,
Acta Crystallogr., Sect. E: Struct. Rep. Online, 2012, E68,
o2197–o2198.
45. A. S. Girgis, J. Stawinski, N. S. M. Ismail, H. Farag, Eur. J.
Med. Chem., 2012, 47, 312–322.
20. D. Lorusso, F. Petrelli, A. Coinu, F. Raspagliesi, S. Barni,
Gynecol. Oncol., 2014, 133, 117–123.
21. F. Landoni, E. Sartori, T. Maggino, P. Zola, V. Zanagnolo, S.
Cosio, F. Ferrari, E. Piovano, A. Gadducci, Gynecol. Oncol.,
2014, 132, 611–617.
22. C. Vale, J. F. Tierney, L. Stewart, M. Brady, K. Dinshaw, A.
Jakobsen, M. K. B. Parmar, G. Thomas, T. Trimble, D. S.
Alberts, H. Chen, S. Cikaric, P. J. Eifel, M. Garipagaoglu, H.
Keys, N. Kantardzic, P. Lal, R. Lanciano, F. Leborgne, V.
Lorvidhaya, H. Onishi, R. G. Pearcey, E. Pras, K. Roberts, P.
G. Rose, C. W. Whitney, J. Clin. Oncol., 2008, 26, 5802–
5812.
46. A. R. Katritzky, A. S. Girgis, S. Slavov, S. R. Tala, I.
StoyanovaꢀSlavova, Eur. J. Med. Chem., 2010, 45, 5183–
5199.
47. A. S. Girgis, Eur. J. Med. Chem., 2009, 44, 1257–1264.
48. A. S. Girgis, Eur. J. Med. Chem., 2009, 44, 91–100.
49. A. S. Girgis, H. M. Hosni, F. F. Barsoum, Bioorg. Med.
Chem., 2006, 14, 4466–4476.
50. A. S. Girgis, H. Farag, N. S. M. Ismail, R. F. George, Eur. J.
Med. Chem., 2011, 46, 4964‒4969.
51. A. S. Girgis, N. S. M. Ismail, H. Farag, Eur. J. Med. Chem.,
2011, 46, 2397‒2407.
52. A. S. Girgis, N. S. M. Ismail, H. Farag, W. I. ElꢀEraky, D. O.
Saleh, S. R. Tala, A. R. Katritzky, Eur. J. Med. Chem., 2010,
45, 4229‒4238.
23. L. A. RojasꢀEspaillat, P. G. Rose, Curr. Opin. Oncol., 2005,
17, 485–492.
24. A. DueñasꢀGonzález, J. J. Zarbá, F. Patel, J. C. Alcedo, S.
Beslija, L. Casanova, P. Pattaranutaporn, S. Hameed, J. M.
Blair, H. Barraclough, M. Orlando, J. Clin. Oncol., 2011, 29,
1678–1685.
53. A. S. Girgis, S. S. Panda, I. S. Ahmed Farag, A. M. Elꢀ
Shabiny, A. M. Moustafa, N. S. M. Ismail,G. G. Pillai,C. S.
Panda, C. D. Hall, A. R. Katritzky, Org. Biomol. Chem., 2015,
13, 1741‒1753.
25. A. Abe, H. Furumoto, M. Nishimura, M. Irahara, H. Ikushima,
Oncol. Lett., 2012, 3, 571–576.
26. R. Das, K. Bhattacharya, S. K. Samanta, B. C. Pal, C. Mandal,
Cancer Lett., 2014, 351, 81–90.
27. S. Schott, A. Brüning, Gynecol. Oncol., (in press)
28. J. F. De Los Santos, Jr. J. M. Straughn,
http://www.uptodate.com/contents/managementꢀofꢀlocallyꢀ
advancedcervicalꢀcancer
29. J. H. Maduro, E. Pras, P. H. Willemse, E. G. de Vries, Cancer
Treat. Rev., 2003, 29, 471–488.
54. A. D. Tiwari, S. S. Panda, A. S. Girgis,S. Sahu, R. F. George,
A. M. Srour, B. La Starza, A. M. Asiri, C. D. Hall, A. R.
Katritzky, Org. Biomol. Chem., 2014, 12, 7238‒7249.
55. I. S. Ahmed Farag, A. S. Girgis, A. M. Moustafa, B. E. M. Elꢀ
Gendy, A. F. Mabied, E. M. Shalaby, J. Mol. Struct., 2014,
1075, 327‒334.
56. CODESSAꢀPro Manual, pages 54,55,58,63
57. Z. Liu, J. L. MedinaꢀFranco, R. A. Houghten, M. A.
Giulianotti, Tetrahedron Lett., 2010, 51, 5003–5004.
16 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 17 of 18
RSC Advances
RSC Advances
View Article Online
Paper
DOI: 10.1039/C4RA16663A
58. F. A. M. AlꢀOmary, G. S. Hassan, S. M. ElꢀMessery, H. I. Elꢀ
60. G. M. Sheldrick, Acta Crystallogr., Sect. A, 2008, 64,
112‒122.
Subbagh, Eur. J. Med. Chem., 2012, 47, 65‒72.
59. S. Z. Vatsadze, M. A. Manaenkova, N. V. Sviridenkova, N. V.
Zyk, D. P. Krut´ko, A. V. Churakov, M. Yu. Antipin, J. A. K.
Howard, H. Lang, Russian Chem. Bull., International Ed.,
2006, 55, 1184‒1194.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 17

RSC Advances
Page 18 of 18
View Article Online
DOI: 10.1039/C4RA16663A
Graphical abstract
A series of novel substituted dispiro-oxindole were synthesized and screened for anti-
cancer properties. The anti-cancer data were validated by QSAR studies.