Substituted spirooxindole derivatives as potent anticancer agents through inhibition of phosphodiesterase 1

Article abstract of DOI:10.1039/c8ra02358a

Spirooxindole is a promising chemo therapeutic agent. Possible targets include cancers of the liver, prostate, lung, stomach, colon, and breast. Here, we demonstrate a one-pot three-component reaction via a [3 + 2] cycloaddition/ring contraction sequence of a dipolarophile (activated alkene) with in situ-generated azomethine ylide (1,3-dipoles) without the use of any catalyst. The reaction provides efficient access to synthetically useful and biologically important spirooxindoles in high yield (69-94%) with high diastereoselectivity. The synthesized compounds were subjected to cytotoxicity evaluation using colorectal cancer (HCT-116), hepatocellular carcinoma (HepG2), and prostate cancer (PC-3) cells. Compounds 4i, 4j, and 4k showed potent cytotoxic activity and high selectivity against HCT-116 cells when compared to cisplatin. Meanwhile compound 4d retained high cytotoxic activity and selectivity against HepG2 and PC-3 cells in comparison to cisplatin. The mechanism of compound 4d was further studied using phosphodiesterase 1 enzyme and showed 74.2% inhibitory activity. A possible binding mode for compound 4d to PDE-1 was investigated by molecular modeling using OpenEye software. Pose predictions for the active compounds were demonstrated by ROCS alignments. Compound 4d has a special geometry and differs from other active compounds.

Full text of DOI:10.1039/c8ra02358a

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Substituted spirooxindole derivatives as potent
anticancer agents through inhibition of
phosphodiesterase 1†
Cite this: RSC Adv., 2018, 8, 14335
ab
a
Hussien Mansur Ghawas,a
*
Assem Barakat,
Mohammad Shahidul Islam,
Abdullah Mohammed Al-Majid,a Fardous F. El-Senduny,
Yaseen A. M. M. Elshaiere and Hazem A. Ghabbourfg
Farid A. Badria,d
c
Spirooxindole is a promising chemo therapeutic agent. Possible targets include cancers of the liver,
prostate, lung, stomach, colon, and breast. Here, we demonstrate a one-pot three-component reaction
via a [3 + 2] cycloaddition/ring contraction sequence of a dipolarophile (activated alkene) with in situ-
generated azomethine ylide (1,3-dipoles) without the use of any catalyst. The reaction provides efficient
access to synthetically useful and biologically important spirooxindoles in high yield (69–94%) with high
diastereoselectivity. The synthesized compounds were subjected to cytotoxicity evaluation using
colorectal cancer (HCT-116), hepatocellular carcinoma (HepG2), and prostate cancer (PC-3) cells.
Compounds 4i, 4j, and 4k showed potent cytotoxic activity and high selectivity against HCT-116 cells
when compared to cisplatin. Meanwhile compound 4d retained high cytotoxic activity and selectivity
against HepG2 and PC-3 cells in comparison to cisplatin. The mechanism of compound 4d was further
studied using phosphodiesterase 1 enzyme and showed 74.2% inhibitory activity. A possible binding
mode for compound 4d to PDE-1 was investigated by molecular modeling using OpenEye software.
Pose predictions for the active compounds were demonstrated by ROCS alignments. Compound 4d has
a special geometry and differs from other active compounds.
Received 17th March 2018
Accepted 2nd April 2018
DOI: 10.1039/c8ra02358a
rsc.li/rsc-advances
guanosine monophosphate (cGMP). PDEs are 11 isoenzymes
(PDE1–PDE11) and their classication is based on their
1. Introduction
substrate, amino acid sequence, action or their distribution in
the body. These enzymes are important regulators of signal
transduction pathways regulating proliferation, apoptosis,
differentiation, vasodilation, vasoconstriction and inamma-
tion in cells. In breast and colon cancer cells, the increase in
intracellular concentrations of cAMP may induce apoptosis,
arrest growth, and reduce cell migration.2
The combined therapy of a multi-kinase inhibitor and a specic
phosphodiesterases (PDEs) inhibitor appears to be a good
therapy option for tumor treatment, as the tumor growth is
delayed and so the chance of survival is increased.1 Phospho-
diesterases (PDEs) are a ubiquitous family of enzymes that play
a role in regulating the intracellular level of the second
messengers cyclic adenosine monophosphate (cAMP) and cyclic
PDE-1 catalyzes the hydrolysis of the phosphodiester bond
between the catalytic tyrosine residue of topoisomerase I (TOP-
1) and DNA 30-phosphate during gene transcription.3 This
makes PDE-1 a rational anticancer target. PDE-1 inhibitors have
the potential to augment TOP-1 inhibitors as anticancer
agents.4
Although high intracellular levels of cAMP can effectively
inhibit the proliferation of cancer cells, compounds elevating
cAMP are not recommended for use as anti-cancer drugs
because of their high cytotoxicity.5–7 Limited numbers of PDE-1
inhibitors have been reported and there is still an unmet need
to discover novel PDE-1 inhibitors.8,9 Several studies have been
conducted via multidisciplinary international research groups
to develop more selective and effective potential anti-cancer
agents.10–17
aDepartment of Chemistry, College of Science, King Saud University, P. O. Box 2455,
Riyadh 11451, Saudi Arabia. E-mail: ambarakat@ksu.edu.sa; Fax: +966-11467-
5992; Tel: +966-11467-5901
bDepartment of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Ibrahimia, Alexandria 21321, Egypt
cDepartment of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt
dDepartment of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura
35516, Egypt
ePharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar
University, Assuit 71524, Egypt
fDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P. O. Box 2457, Riyadh 11451, Saudi Arabia
gDepartment of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura,
Mansoura 35516, Egypt
† Electronic supplementary information (ESI) available. CCDC 1583075 and
1818997. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c8ra02358a
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Spirooxindoles have unique structural features and a ubiq-
uitous class of biological activities, making them promising
candidates for new drug discovery.18 Over the past decade, this
class of compounds has enriched the repertoire of both oxin-
doles and other heterocyclic scaffolds and has attracted exten-
sive research efforts from synthetic and medical chemists
because of their unique chemopreventive properties.19–21 Two
2. Results and discussion
2.1. Synthesis of 4a–n
Anticancer compounds incorporating oxindoles were synthe-
sized using an efficient 1,3-dipolar cycloaddition reaction.17,31,32
The starting materials, a,b-unsaturated enone derivatives 1a–n
(Scheme 1), were synthesized by a condensation reaction of 3-
acetyl indole with substituted aryl aldehydes in the presence of
KOH in EtOH under reux. Then, the one-pot reaction of a,b-
unsaturated enone derivatives 1a–n with L-proline 2 and isatin 3
was carried out at 60 ꢀC in MeOH for 1.5–2.0 h to yield the nal
compound 4a–n, with 4 stereogenic centers in good to excellent
yield (69–94%). The molecular structure of the cycloadduct was
conrmed by nuclear magnetic resonance (NMR) spectroscopic
analysis. The reaction yielded the adduct 4a–n as a single
regioisomer. The structures of 4a–n and their derivatives were
deduced by 1H-NMR, 13C-NMR, mass spectrometry (MS),
infrared (IR) spectroscopy, elemental analysis, and X-ray crys-
tallography. The absolute congurations for the desired
compounds were assigned by X-ray, in addition to spectral
analysis studies. The aryl moiety and acylindole originating
from ring C were oriented as anti-conformation.
The proposed reaction mechanism of the three-component
reaction is shown in Scheme 2. It was assumed that azome-
thine ylide was formed exclusively, possibly due to the nucleo-
philic attack of proline into the active carbonyl of isatin with
conversion of the carbonyl group to alcohol. The resulting OH
will attack the carboxylic group in proline to form a lactone
functionality (intermediate I). In situ decarboxylation generates
the reactive azomethine ylide. The reaction of olen with azo-
methine ylide has four possibilities which will proceed regio-
selectively with path A exclusively and diastereoselectively with
path C to furnish the nal adduct (Table 1).34,35
examples
of
representative
spirooxindole-containing
compounds are NITD609 and MI-888 (Fig. 1), which are
currently in preclinical evaluation for the treatment of malaria
and human cancer, respectively.22,23 On the other hand, natu-
rally occurring spirocyclic oxindole alkaloids that could be iso-
lated, such as spirotryprostatins A and B, also show excellent
anticancer activities.24 Additionally, spirooxindole-containing
compounds have been reported to have antimycobacterial25 or
anti-inammatory26 activities, or can act as an acetylcholines-
terase (AChE) inhibitors.27,28
The importance of new anticancer agents originating from
these spirooxoindole architectures has stimulated our group
into designing and synthesizing a new series of spirooxoindoles
4a–n through some modications in the structure of reported
drugs, especially 6SJ29,30 (Fig. 1). These structural modications
include: (i) the substituted phenyl moiety tethered ring C was
replaced with 3-acyl indole. This replacement is expected to
increase the compound's potency, since this scaffold could
form extra hydrogen bonding and hydrophobic–hydrophobic
interactions. (ii) A substituted or non-substituted aryl group was
also installed on ring C. This aryl arm was expected to switch the
compound geometry and was suggested to be involved in extra
ligand–receptor interactions. Based on the aforementioned
information, and in continuation of our previous work,17,31–33 we
synthesized biologically important, highly substituted, and
functionalized spirooxindole derivatives, which are efficient
and powerful agents for the treatment of cancer.
Fig. 1 Chemical structures of reported anticancer spirooxindoles and the modified spirooxindole (4a–n).
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Scheme 1 The synthesis of the target spirooxindole derivatives 4a–n.
Scheme 2 Plausible approach for the target compounds 4a–n.
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Table 1 Synthesis of spirooxindole-pyrrolidine 4a–n
Entry
4a–n
Ar
Yield (%)
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
C6H5
p-MeC6H4
p-ClC6H4
2,4-Cl2C6H3
p-MeOC6H4
p-BrC6H4
84
92
80
74
86
77
84
78
85
72
76
94
89
69
p-FC6H4
m-FC6H4
9
m-MeC6H4
m-BrC6H4
p-CF3C6H4
2-Thiophene
2-Furan
10
11
12
13
14
3,4,5-Tri-MeOC6H2
of the tested compounds showed a better selectivity index
(SI: >1) over commonly used chemotherapeutic drug
2.2. Biological activity
a
Fourteen compounds were tested against three common cancer
cell lines, colorectal cancer (HCT-116), hepatocellular carci-
noma (HepG2), and prostate cancer (PC-3). Interestingly, most
(cisplatin, SI ¼ 0.3). On the other hand, compounds 4i, 4j, and
4k showed a remarkable cytotoxicity against HCT-116 with SI > 2
Table 2 The cytotoxic activity and selectivity of the synthesized compounds against a colorectal cancer cell line (HCT-116)a
VERO-B
(IC50, mM)
Entry
4a–n
HCT-116 (IC50, mM)
HCT-116 (IC50, mg mlꢁ1
)
SI*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Cisplatin
ND
ND
9.7
9.6
4.6
12.4
11
ND
26
22
9
ND
1.2
1.1
1
21 ꢂ 2
20 ꢂ 1.5
9 ꢂ 0.6
26 ꢂ 2
26
30
40
18
15
20
22
60
50
40
5
1
21 ꢂ 1.3
16 ꢂ 1
1.4
2.5
1.2
2.1
2.2
2.4
1.2
1.7
2
7.4
7
15 ꢂ 1.4
7 ꢂ 0.2
9 ꢂ 0.5
9 ꢂ 0.5
50 ꢂ 3.5
29 ꢂ 2
3.2
4.7
4.6
22.7
12.7
11.1
3.8
20 ꢂ 1.25
12.6 ꢂ 0.40
Positive control
0.4
a
SI*: selectivity index, ND: not determine.
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Table 3 The cytotoxic activity and selectivity of the synthesized compounds against hepatocellular carcinoma (HepG2) cellsa
VERO-B
(IC50, mM)
Entry
4a–n
HepG2 (IC50, mM)
HepG2 (IC50, mg mlꢁ1
)
SI*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Cisplatin
ND
ND
5.4
3.8
1
ND
26
22
9
ND
2.2
2.8
4.5
1.5
2.5
2
1.3
2.1
2.5
2.2
1.2
1.3
1.4
0.91
11.8 ꢂ 2
8 ꢂ 0.5
2 ꢂ 0.1
17.3 ꢂ 3
12 ꢂ 1.5
20 ꢂ 2
8.3
6.3
9.3
6.5
3.2
4.2
5.2
22.7
17.5
15.7
1.7
26
30
40
18
15
20
22
60
50
40
5
14 ꢂ 0.22
7 ꢂ 0.40
8 ꢂ 1
10 ꢂ 1.25
50 ꢂ 3
40 ꢂ 5
28 ꢂ 2
Positive control
5.5 ꢂ 1.5
a
SI*: selectivity index, ND: not determine.
and IC50 at 7, 9 and 9 mM, respectively, in comparison to 12.6 compound 4h (IC50 ¼ 15 mM). Moreover, the presence of two
mM for cisplatin, as presented in Table 2. chlorine atoms in compound 4d at positions C-2 and C-4 in the
Meanwhile, compound 4d showed a remarkable cytotoxicity aromatic ring greatly increased the anticancer activity (IC50 ¼ 2
against HepG2 with SI greater than 4 and IC50 at 2 mM versus 5.5 mM) in comparison to one chlorine at the para position in
mM and SI less than 1 for the standard drug cisplatin, as pre- compound 4c (IC50 ¼ 8 mM). Also, SAR showed that the addition
sented in Table 3. In addition, compounds 4c, 4i, and 4j pre- of triuoromethyl at position C-4 rather than methoxy as in
sented similar cytotoxicity against HepG2 with IC50 of 8, 7, and 8 compound 4e (IC50 ¼ 26 mM), bromine in compound 4f (IC50
¼
mM, respectively, with a better SI > 2 (Table 3 and Fig. 2).
21 mM) or uorine in compound 4g (IC50 ¼ 16 mM) improved the
Interestingly, compound 4d also showed superior cytotoxic cytotoxicity of compound 4k (IC50 ¼ 9 mM).
activity against prostate cancer cells at IC50 ¼ 2 mM (Table 4),
and its selectivity toward the cancer cells was greater than 4,
which makes it a promising anticancer candidate. Despite that,
2.4. Phosphodiesterase inhibitory study
compounds 4i, 4j, and 4k showed anticancer activity at a higher
IC50 (7, 7, and 9 mM, respectively) but their selectivity index was
still greater than 2 in comparison to the standard cisplatin (IC50
at 5 mM and SI ¼ 1) (Table 4).
Due to the increase in tumor resistance aer a refractory period,
there has been extensive research to discover new leads to
overcome the resistance and reduce the chemotherapeutic dose
in order to decrease the side effects on normal cells. One way is
to use phosphodiesterase 1 inhibitors in order to elevate the
level of cAMP. Compound 4d, which proved to be the most
active and selective anticancer compound among all the tested
2.3. Structural activity relationship (SAR)
The study of SAR showed that the meta substituted aromatic compounds, showed remarkable inhibitory activity against
ring with either methyl or bromine led to an increase in the phosphodiesterase enzyme (PD-1) at mM with 74.2%.
2
cytotoxicity of compounds 4i (IC50 ¼ 7 mM) and 4j (IC50 ¼ 9 mM), Compound 4d could be used in combination with other anti-
respectively, in comparison to the presence of a uorine atom in cancer drugs such as cisplatin for the treatment of solid tumors.
Fig. 2 Microscopic examination of the effect of compound 4i on the growth of hepatocellular carcinoma (HepG2).
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Table 4 The cytotoxic activity and selectivity of the synthesized compounds against prostate cancer cell linea
Entry
4a–n
PC-3 (IC50, mM)
PC-3 (IC50, mg mlꢁ1
)
VERO-B (IC50, mM)
SI*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Cisplatin
ND
ND
7.5
5.7
1
7.4
8.6
7.6
5.4
3.2
3.7
4.6
13
ND
26
22
9
ND
1.6
1.9
4.5
1.7
1.8
2.5
1.6
2.1
2.9
2.4
2.1
1.9
2.4
1
16.3 ꢂ 2
11.8 ꢂ 1.3
2 ꢂ 0.125
15.5 ꢂ 2
16.3 ꢂ 2.5
16.3 ꢂ 30
11.5 ꢂ 11.5
7 ꢂ 0.6
26
30
40
18
15
20
22
60
50
40
5
7 ꢂ 0.2
9 ꢂ 0.2
29 ꢂ 3
26 ꢂ 1.7
17 ꢂ 2
11.3
9.4
1.5
Positive control
5 ꢂ 0.45
a
SI*: selectivity index, ND: not determine.
It has been reported that the elevation of cAMP concentration in binding modes.
A library of substituted spirooxindole
the cells leads to the inhibition of survival pathways such as compounds was designed and energy minimized using
MAPK36 and antiapoptotic proteins like Bcl-2.37 Furthermore, MMFF94 force eld calculations for the catalytic domain of
a high level of cAMP could inhibit the interaction of the tumor PDE-1 which was obtained from the protein data bank (PDB
suppressor p53 protein with its regulator MDM-2.38
code: 1NOP)40 and was prepared for docking using OpenEye®
soware.41,42
Among all the compounds, compound 4d showed the best
consensus score of 19 by PDE-1 interaction. The pose and mode
for compound 4d bound to TDP1 are illustrated in Fig. 3. The
carbonyl group of the oxindole scaffold forms hydrogen
bonding (HB) interaction with the amino acid Thr 261. This
interaction is near the amino acid His 263. The other carbonyl
group that links the indole moiety with the remaining part of 4d
is also involved in HB interaction with the amino acid Ser 400.
The indole part is incorporated near the amino acid Lys 495.
The important pharmacophore 2,4-dichlorophenyl p-stacks
inside the active site near Ser 463.
2.5. Molecular docking and shape-matching studies
The crystallographic structure for PDE-1 illustrated that the
active site region contains pairs of highly conserved histidine
and lysine residues. The active site residues for catalysis are: His
263, Lys 265, His 493, and Lys 495.39 The docking of these
compounds with PDE-1 exhibited consensus scores and
binding modes which correlated with their biological activity as
anticancer agents. The target compounds were docked in the
active site of PDE-1 (PDB: 1NOP) in order to investigate their
Finally, the indole moiety of the compound is directed
toward the catalytic core of the enzyme through hydrophobic–
hydrophobic interactions with His 493.
Fig. 3 Visual representation of 4d docked with 1NOP showing two HB
interactions and hydrophobic–hydrophobic interactions as shown by Fig. 4 ROCS run for compound 4d with compounds 4k, 4j and 4i with
VIDA.
dissimilarity and not completely matched.
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In order to understand the diversity of scaffolds in the most
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active compounds, ROCS alignments for our compounds was
performed. ROCS is a fast shape comparison application. It uses
a smooth Gaussian function to represent the molecular
volume.43 ROCS is useful in pose prediction in the absence of
a protein structure.44 Compound 4d was selected as the query
molecule. Other target compounds were selected as the data-
base (dbase.t) le. The ROCS run for compound 4d with
compounds 4k, 4j and 4i represents a dissimilarity between 4d
and other compounds (Fig. 4).
For an explanation, compound 4d exhibited a unique
orientation in which the 2,4-dichlorophenyl moiety was located
axially to the pyrrolidine moiety. The indole part is perpendic-
ular to the oxindole moiety (Fig. 5). Superposition of
compounds 4j and 4k (Fig. 6) indicates the chemical similarity
between them as the m-Br phenyl and p-(CF3) phenyl govern the
molecular structure. The indole and oxindole moieties face each
other with a buckled shape. Compound 4i also adopts a buckled
shape in which both indole and oxindole moieties face each
other (Fig. 7) but it showed dissimilarities with compounds 4j
and 4k.
Fig. 6 Superposition for compounds 4j and 4k by ROCS exhibited
structural similarity.
3. Experimental
3.1. General procedure (GP1)
3.1.1. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-phenyl-
10,20,50,60,70,7a0'-hexahydrospiro[indoline-3,30-pyrrolizin]-2-
one (4a). Yield, 84%; mp 171 ꢀC; 1H-NMR (400 MHz, DMSO-d6)
d: 1.64–1.78 (m, 2H, CH2), 1.82–1.90 (m, 2H, CH2), 2.32–2.38 (m,
1H, CH2), 2.48–2.58 (m, 1H, CH2), 3.82–3.92 (m, 1H, CHN), 3.95
Enones 1a–p (0.5 mmol), isatin (74 mg, 0.5 mmol) and L-proline
(1.5 mmol) were dissolved in 20 ml of dry MeOH in a 50 ml
round-bottom ask. Then, the reaction mixture was heated for
1.5–2 h at 60–65 ꢀC. Aer the reaction was completed, as
monitored by thin-layer chromatography (TLC), the crude
material was subjected to column chromatography using ethyl
acetate/n-hexane (2 : 3), yielding compounds 4a–n.
Fig. 5 ROCS for 4d with specific geometry of the 2,4-dichlorophenyl Fig.
7
ROCS for compound 4i exhibited a buckled shape with
and indole moiety perpendicular to each other.
a different pose inside the 1NOP.
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(t, 1H, J ¼ 10.28 Hz, CHPh), 4.61 (d, 1H, J ¼ 11.76 Hz, CHCO), 6.92–7.11 (m, 4H, Ar–H), 7.34 (d, 2H, J ¼ 8.08 Hz, Ar–H), 7.37 (d,
6.53 (d, 1H, J ¼ 7.32 Hz, Ar–H), 6.90 (t, 1H, J ¼ 7.32 Hz, Ar–H), 1H, J ¼ 2.20 Hz, Ar–H), 7.57 (d, 1H, J ¼ 1.84 Hz, Ar–H), 7.78 (t,
6.92–7.04 (m, 2H, Ar–H), 7.08 (t, 1H, J ¼ 7.36 Hz, Ar–H), 7.32 (d, 2H, J ¼ 10.24 Hz, Ar–H), 7.96 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.33 (s,
1H, J ¼ 8.04 Hz, Ar–H), 7.36–7.47 (m, 4H, Ar–H), 7.78 (d, 1H, J ¼ 1H, NH), 11.86 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d:
8.04 Hz, Ar–H), 7.90 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.27 (s, 1H, NH), 26.9, 29.6, 47.2, 51.5, 63.1, 71.9, 73.4, 109.7, 111.8, 116.6, 121.1,
11.82 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 26.9, 29.7, 121.2, 121.7, 122.9, 125.0, 125.1, 127.4, 127.8, 128.8, 128.9,
47.3, 51.5, 63.5, 71.3, 73.4, 109.5, 111.9, 116.7, 119.6, 120.9, 129.0, 129.8, 131.7, 134.7, 136.4, 136.8, 141.8, 179.6, 188.9; IR
121.2, 121.6, 122.9, 125.1, 125.2, 127.8, 128.8, 129.9, 131.3, (KBr, cmꢁ1) nmax ¼ 3255, 2964, 2869, 1711.1619, 1619, 1520,
133.5, 136.3, 139.8, 141.6, 179.8, 189; IR (KBr, cmꢁ1) nmax
3386, 3248, 2958, 2867, 1716, 1618, 1520, 1470, 1422, 1243,
1137, 1153, 748, 698; [anal. calcd. for C29H25N3O2: C, 77.83; H, 4.63; N, 8.02]; LC/MS (ESI, m/z): 515.10 [M + H] for 515.12
¼
1470, 1425, 1335, 1243, 1136, 1110, 1046, 749; [anal. calcd. for
29H23Cl2N3O2: C, 67.45; H, 4.49; N, 8.14; found: C, 67.55; H,
C
5.63; N, 9.39; found: C, 77.75; H, 5.91; N, 9.49]; LC/MS (ESI, m/z):
447.20 [M + H] for 447.19 C29H25N3O2.
3.1.2. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(p-tolyl)-
10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-pyrrolizin]-2-one
C
29H23Cl2N3O2.
3.1.5. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(4-methox-
yphenyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
pyrrolizin]-2-one (4e). Yield (86%); yellow powder; mp: 122–
(4b). Yield (92%); orange powder; mp 174–176 ꢀC; 1H-NMR (400 124 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.64–1.76 (m, 2H, CH2),
MHz, DMSO-d6) d: 1.58–1.72 (m, 2H, CH2), 1.73–1.86 (m, 2H, 1.80–1.92 (m, 2H, CH2), 2.30–2.38 (m, 1H, CH2), 2.48–2.56 (m,
CH2), 2.13 (s, 3H, CH3), 2.25–2.34 (m, 1H, CH2), 2.42–2.52 (m, 1H, CH2), 3.52–3.58 (m, 1H, CH), 3.65 (s, 3H, OCH3), 3.82–3.86
1H, CH2), 3.78–3.83 (m, 1H, CH), 3.84 (t, 1H, J ¼ 12.08 Hz, CH), (m, 1H, CH), 3.89 (t, 1H, J ¼ 10.28 Hz, CH), 4.58 (d, 1H, J ¼
4.59 (d, 1H, J ¼ 11.00 Hz, CH), 6.51 (d, 1H, J ¼ 7.36 Hz, Ar–H), 11.00 Hz, CH), 6.53 (d, 1H, J ¼ 7.32 Hz, Ar–H), 6.83 (d, 2H, J ¼
6.88 (t, 1H, J ¼ 7.32 Hz, Ar–H), 6.92–7.05 (m, 6H, Ar–H), 7.27 (d, 8.80 Hz, Ar–H), 6.91 (t, 1H, J ¼ 7.36 Hz, Ar–H), 6.96–7.04 (m, 2H,
2H, J ¼ 8.80 Hz, Ar–H), 7.34 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.76 (d, Ar–H), 7.07 (t, 1H, J ¼ 7.32 Hz, Ar–H), 7.31–7.38 (m, 4H, Ar–H),
1H, J ¼ 8.04 Hz, Ar–H), 7.87 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.25 (s, 7.79 (d, 1H, J ¼ 8.04 Hz, Ar–H), 7.88 (d, 1H, J ¼ 2.92 Hz, Ar–H),
1H, NH), 11.78 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 10.25 (s, 1H, NH), 11.80 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-
20.6, 27.1, 30.1, 47.3, 52.0, 63.6, 71.6, 73.6, 109.6, 112.0, 117.0, d6) d: 27.0, 29.9, 47.3, 51.47, 54.9, 63.6, 71.5, 73.4, 109.4, 111.9,
121.0, 121.3, 121.7, 122.9, 125.2, 125.5, 127.4, 127.9, 128.8, 113.9, 116.9, 120.9, 121.3, 121.5, 122.9, 125.1, 125.4, 127.8,
129.1, 133.4, 135.7, 136.4, 137.3, 147.7, 180.1, 189.4; IR 128.5, 128.7, 132.1, 133.4, 136.3, 141.6, 157.9, 179.9, 189.3; IR
(KBr, cmꢁ1) nmax ¼ 3381, 3246, 2958, 2865, 1716, 1618, 1581, (KBr, cmꢁ1) nmax ¼ 3387, 3247, 2960, 2868, 1713, 1618, 1513,
1517, 1484, 1423, 1243, 1126, 749; [anal. calcd. for C30H27N3O2: 1469, 1437, 1244, 1178, 1138, 1034, 749; [anal. calcd. for
C, 78.07; H, 5.90; N, 9.10; found: C, 78.17; H, 5.82; N, 9.23]; LC/
MS (ESI, m/z): 461.20 [M + H] for 461.21 C30H27N3O2.
3.1.3. (20R,3S,7a0S)-10-(4-Chlorophenyl)-20-(1H-indole-3-
carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
C
30H27N3O3: C, 75.45; H, 5.70; N, 8.80; found: C, 75.31; H,
5.86; N, 9.03]; LC/MS (ESI, m/z): [477.20 [M + H] for 477.21
C
30H27N3O3.
3.1.6. (20R,3S,7a0S)-10-(4-Bromophenyl)-20-(1H-indole-3-
pyrrolizin]-2-one (4c). Yield (80%); beige powder; mp: 165– carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
167 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.60–1.74 (m, 2H, CH2), pyrrolizin]-2-one (4f). Yield (77%); yellow powder; mp: 101–
1.78–1.88 (m, 2H, CH2), 2.28–2.36 (m, 1H, CH2), 2.44–2.54 (m, 102 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.64–1.78 (m, 2H, CH2),
1H, CH2), 3.78–3.88 (m, 1H, CH), 3.94 (t, 1H, J ¼ 10.24 Hz, CH), 1.82–1.92 (m, 2H, CH2), 2.30–2.38 (m, 1H, CH2), 2.49–2.58 (m,
4.60 (d, 1H, J ¼ 11.72 Hz, CH), 6.52 (d, 1H, J ¼ 8.08 Hz, Ar–H), 1H, CH2), 3.82–3.90 (m, 1H, CH), 3.95 (t, 1H, J ¼ 10.28 Hz, CH),
6.89 (t, 1H, J ¼ 7.32 Hz, Ar–H), 6.94–7.02 (m, 2H, Ar–H), 7.06 (t, 4.64 (d, 1H, J ¼ 11.72 Hz, CH), 6.53 (d, 1H, J ¼ 7.36 Hz, Ar–H),
1H, J ¼ 7.32 Hz, Ar–H), 7.30 (t, 3H, J ¼ 7.32 Hz, Ar–H), 7.36 (d, 6.91 (t, 1H, J ¼ 8.04 Hz, Ar–H), 6.95–7.02 (m, 1H, Ar–H), 7.07 (t,
1H, J ¼ 7.36 Hz, Ar–H), 7.45 (d, 2H, J ¼ 8.76 Hz, Ar–H), 7.77 (d, 1H, J ¼ 7.32 Hz, Ar–H), 7.14 (t, 1H, J ¼ 7.36 Hz, Ar–H), 7.26 (t,
1H, J ¼ 7.32 Hz, Ar–H), 7.89 (d, 1H, J ¼ 3.68 Hz, Ar–H), 10.28 (s, 2H, J ¼ 7.36 Hz, Ar–H), 7.32 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.37 (d,
1H, NH), 11.82 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 1H, J ¼ 7.36 Hz, Ar–H), 7.42 (d, 2H, J ¼ 6.60 Hz, Ar–H), 7.78 (d,
27.0, 29.9, 47.36, 51.6, 63.6, 71.5, 73.5, 109.5, 112.0, 116.8, 121.0, 1H, J ¼ 8.04 Hz, Ar–H), 7.89 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.26 (s,
121.3, 121.7, 123.0, 125.2, 125.3, 127.9, 128.5, 128.9, 129.6, 1H, NH), 11.81 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d:
131.3, 131.6, 136.4, 139.4, 141.7, 180.0, 189.2; IR (KBr, cmꢁ1
nmax ¼ 3247, 2959, 2928, 2868, 1713, 1619, 1521, 1492, 1470, 121.2, 121.5, 122.8, 125.1, 125.4, 126.5, 127.6, 127.8, 128.4,
1424, 1334, 1243, 1138, 750, 532; [anal. calcd. for C29H24ClN3O2: 128.8, 133.4, 136.3, 140.4, 141.3, 179.9, 189.3; IR (KBr, cmꢁ1
)
26.9, 29.9, 46.7, 52.1, 63.4, 71.6, 73.4, 109.4, 111.9, 116.8, 120.9,
)
C, 72.27; H, 5.02; N, 8.72; found: C, 72.15; H, 5.13; N, 8.86]; LC/ nmax ¼ 3390, 3247, 2962, 2867, 1713, 1618, 1520, 1488, 1470,
MS (ESI, m/z): 481.21 [M + H] for 481.16 C29H24ClN3O2.
3.1.4. (20R,3S,7a0S)-10-(2,4-Dichlorophenyl)-20-(1H-indole-3-
carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
pyrrolizin]-2-one (4d). Yield, 74%; beige powder; mp: 149–
151 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.68–1.82 (m, 2H, CH2),
1420, 1330, 1243, 1138, 1009, 749; [anal. calcd. for
29H24BrN3O2: C, 66.17; H, 4.60; N, 7.98; found: C, 66.28; H,
4.51; N, 8.05]; LC/MS (ESI, m/z): 525.10 [M + H] for 525.11
C
C
29H24BrN3O2.
3.1.7. (20R,3S,7a0S)-10-(4-Fluorophenyl)-20-(1H-indole-3-
1.84–1.92 (m, 2H, CH2), 2.34–2.42 (m, 1H, CH2), 2.52–2.60 (m, carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
1H, CH2), 3.52–3.62 (m, 1H, CHN), 3.80–3.88 (m, 1H, CHPh), pyrrolizin]-2-one (4g). Yield (84%); orange powder; mp: 144–
4.79 (d, 1H, J ¼ 11.72 Hz, CHCO), 6.56 (d, 1H, J ¼ 7.36 Hz, Ar–H), 146 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.62–1.76 (m, 2H, CH2),
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1.80–1.92 (m, 2H, CH2), 2.30–2.38 (m, 1H, CH2), 2.48–2.56 (m,
1H, CH2), 3.80–3.98 (m, 2H, CH), 4.58 (d, 1H, J ¼ 10.24 Hz, CH),
6.53 (d, 1H, J ¼ 8.08 Hz, Ar–H), 6.81 (d, 1H, J ¼ 8.08 Hz, Ar–H),
6.90 (t, 1H, J ¼ 7.36 Hz, Ar–H), 6.96–7.11 (m, 4H, Ar–H), 7.28–
7.33 (m, 3H, Ar–H), 7.35 (d, 1H, J ¼ 6.60 Hz, Ar–H), 7.79 (d, 1H, J
¼ 8.04 Hz, Ar–H), 7.88 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.25 (s, 1H,
NH), 11.79 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 26.9,
30.64, 47.2, 51.3, 63.6, 71.4, 73.4, 109.4, 111.9, 114.3, 115.0,
115.3, 116.9, 120.9, 121.3, 121.5, 122.8, 125.1, 125.4, 127.8,
128.5, 128.7, 128.8, 129.5, 131.9, 133.3, 136.3, 141.6, 157.2,
179.9, 189.4; IR (KBr, cmꢁ1) nmax ¼ 3381, 3251, 2960, 2868, 1718,
1618, 1511, 1470, 1422, 1331, 1243, 1178, 1155, 1137, 1044, 861,
749; [anal. calcd. for C29H24FN3O2: C, 74.82; H, 5.20; N, 9.03;
found: C, 75.02; H, 5.17; N, 9.11]; LC/MS (ESI, m/z): 465.20 [M +
H] for 465.19 C29H24FN3O2.
3.1.8. (20R,3S,7a0S)-10-(3-Fluorophenyl)-20-(1H-indole-3-
carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
pyrrolizin]-2-one (4h). Yield (78%); yellow powder; mp: 149–
151 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.58–1.72 (m, 2H, CH2),
1.75–1.86 (m, 2H, CH2), 2.24–2.34 (m, 1H, CH2), 2.44–2.53 (m,
1H, CH2), 3.79–3.86 (m, 1H, CH), 3.94 (t, 1H, J ¼ 11.00 Hz, CH),
4.58 (d, 1H, J ¼ 11.72 Hz, CH), 6.48 (d, 1H, J ¼ 7.32 Hz, Ar–H),
6.85 (t, 1H, J ¼ 8.08 Hz, Ar–H), 6.91–6.98 (m, 3H, Ar–H), 7.02 (t,
1H, J ¼ 8.08 Hz, Ar–H), 7.20–7.26 (m, 4H, Ar–H), 7.33 (d, 1H, J ¼
7.32 Hz, Ar–H), 7.73 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.89 (d, 1H, J ¼
3.68 Hz, Ar–H), 10.22 (s, 1H, NH), 11.78 (s, 1H, NH); 13C-NMR
(100 MHz, DMSO-d6) d: 26.9, 29.7, 47.3, 51.8, 63.4, 71.3, 73.4,
109.5, 111.9, 113.3, 113.5, 114.4, 114.6, 116.7, 120.9, 121.2,
121.6, 122.9, 123.7, 125.1, 125.3, 127.8, 128.8, 130.3, 130.4,
133.7, 136.4, 141.7, 143.58, 161.0, 163.5, 179.8, 189.2; IR
(KBr, cmꢁ1) nmax ¼ 3388, 3249, 2962, 2867, 1717, 1617, 1587,
1519, 1469, 1424, 1332, 1241, 1142, 748; [anal. calcd. for
pyrrolizin]-2-one (4j). Yield (72%); yellow powder; mp: 110–
112 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.65–1.78 (m, 2H, CH2),
1.82–1.92 (m, 2H, CH2), 2.30–2.38 (m, 1H, CH2), 2.50–2.57 (m,
1H, CH2), 3.84–3.91 (m, 1H, CH), 3.97 (t, 1H, J ¼ 10.24 Hz, CH),
4.62 (d, 1H, J ¼ 11.72 Hz, CH), 6.52 (d, 1H, J ¼ 7.36 Hz, Ar–H),
6.89 (t, 1H, J ¼ 8.08 Hz, Ar–H), 7.00 (q, 2H, J ¼ 7.32 Hz, Ar–H),
7.07 (t, 1H, J ¼ 8.08 Hz, Ar–H), 7.24 (t, 2H, J ¼ 8.08 Hz, Ar–H),
7.13–7.39 (m, 3H, Ar–H), 7.47 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.65 (s,
1H, Ar–H), 7.77 (d, 1H, J ¼ 7.32 Hz, Ar–H), 7.92 (d, 1H, J ¼
2.96 Hz, Ar–H), 10.26 (s, 1H, NH), 11.82 (s, 1H, NH); 13C-NMR
(100 MHz, DMSO-d6) d: 26.8, 29.6, 47.3, 51.6, 63.5, 71.4, 73.4,
109.5, 111.9, 116.7, 120.9, 121.2, 121.6, 121.8, 122.9, 125.1,
125.2, 126.6, 127.8, 128.9, 129.5, 130.7, 130.8, 133.6, 136.3,
141.6, 143.3, 179.7, 189.1; IR (KBr, cmꢁ1) nmax ¼ 3403, 3253,
2958, 2866, 1715, 1619, 1520, 1470, 1424, 1332, 1241, 1134, 748;
[anal. calcd. for C29H24BrN3O2: C, 66.17; H, 4.60; N, 7.98; found:
C, 66.06; H, 4.49; N, 7.92]; LC/MS (ESI, m/z): 525.10 [M + H] for
525.11 C29H24BrN3O2.
3.1.11. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(4-[tri-
uoromethylphenyl)-10,20,50,60,70,7a0-hexahydrospiro[indo-
line-3,30-pyrrolizin]-2-one (4k). Yield (76%); yellow powder;
mp: 153–155 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.62–1.80 (m,
2H, CH2), 1.82–1.93 (m, 2H, CH2), 2.30–2.40 (m, 1H, CH2), 2.50–
2.58 (m, 1H, CH2), 3.86–3.94 (m, 1H, CH), 4.07 (t, 1H, J ¼
11.00 Hz, CH), 4.67 (t, 1H, J ¼ 11.76 Hz, CH), 6.53 (d, 1H, J ¼
7.36 Hz, Ar–H), 6.90 (t, 1H, J ¼ 7.32 Hz, Ar–H), 6.95–7.07 (m, 3H,
Ar–H), 7.33 (d, 1H, J ¼ 5.84 Hz, Ar–H), 7.39 (d, 1H, J ¼ 7.36 Hz,
Ar–H), 7.55–7.72 (m, 4H, Ar–H), 7.76 (d, 1H, J ¼ 8.08 Hz, Ar–H),
7.887791 (d, 1H, J ¼ 3.68 Hz, Ar–H), 10.28 (s, 1H, NH), 11.83 (s,
1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 26.9, 29.7, 47.2, 51.8,
63.4, 71.4, 73.4, 79.1, 109.4, 111.9, 116.6, 120.9, 121.2, 121.6,
122.9, 125.0, 125.2, 125.3, 127.5, 127.6, 128.6, 128.8, 133.6,
136.3, 141.6, 145.3, 179.7, 189.0; IR (KBr, cmꢁ1) nmax ¼ 3254,
2960, 2869, 1716, 1619, 1521, 1470, 1423, 1325, 1165, 1116,
1068, 1017, 7450; [anal. calcd. for C30H24F3N3O2: C, 69.89; H,
4.69; N, 8.15; found: C, 70.07; H, 4.82; N, 8.01]; LC/MS (ESI, m/z):
515.20 [M + H] for 151.18 C30H24F3N3O2.
C
29H24FN3O2: C, 74.82; H, 5.20; N, 9.03; found: C, 74.93; H,
5.09; N, 9.22]; LC/MS (ESI, m/z): 465.20 [M + H] for 465.19
C
29H24FN3O2.
3.1.9. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(m-tolyl)-
10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-pyrrolizin]-2-
one (4i). Yield mg (85%); yellow powder; mp: 141–143 C; H-
1
ꢀ
3.1.12. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(thiophen-
2-yl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-pyrrolizin]-2-
NMR (400 MHz, DMSO-d6) d: 1.62–1.72 (m, 2H, CH2), 1.76–
1.86 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.26–2.36 (m, 1H, CH2),
2.46–2.52 (m, 1H, CH2), 3.78–3.83 (m, 1H, CH)), 3.87 (t, 1H, J ¼
9.52 Hz, CH), 4.60 (d, 1H, J ¼ 11.00 Hz, CH), 6.49 (d, 1H, J ¼
7.32 Hz, Ar–H), 6.87 (t, 1H, J ¼ 8.04 Hz, Ar–H), 6.91–7.00 (m, 2H,
Ar–H), 7.03 (t, 1H, J ¼ 8.04 Hz, Ar–H), 7.10 (t, 2H, J ¼ 8.04 Hz, Ar–
H), 7.14–7.24 (m, 2H, Ar–H), 7.28 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.33
(d, 1H, J ¼ 7.32 Hz, Ar–H), 7.74 (d, 1H, J ¼ 8.08 Hz, Ar–H), 7.86
(d, 1H, J ¼ 2.92 Hz, Ar–H), 10.21 (s, 1H, NH), 11.76 (s, 1H, NH);
13C-NMR (100 MHz, DMSO-d6) d: 21.0, 26.9, 29.9, 47.2, 52.1,
63.4, 71.6, 73.4, 109.4, 111.9, 116.8, 120.9, 121.2, 121.5, 122.8,
124.6, 125.1, 125.4, 127.2, 127.8, 128.3, 128.4, 128.7, 133.4,
1
ꢀ
one (4l). Yield (94%); yellow powder; mp: 157–159 C; H-NMR
(400 MHz, DMSO-d6) d: 1.68–1.82 (m, 2H, CH2), 1.82–1.91 (m,
1H, CH2), 1.92–2.02 (m, 1H, CH2), 2.28–2.38 (m, 1H, CH2), 2.48–
2.56 (m, 1H, CH2), 3.88–3.98 (m, 1H, CH), 4.21 (t, 1H, J ¼
9.52 Hz, CH), 4.50 (d, 1H, J ¼ 11.76 Hz, CH), 6.52 (d, 1H, J ¼
7.32 Hz, Ar–H), 6.89–6.92 (m, 2H, Ar–H), 6.96–7.03 (m, 3H, Ar–
H), 7.11 (t, 1H, J ¼ 6.60 Hz, Ar–H), 7.28 (d, 1H, J ¼ 5.12 Hz, Ar–
H)), 7.32–7.35 (m, 2H, Ar–H), 7.82 (d, 1H, J ¼ 8.08 Hz, Ar–H),
7.85 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.27 (s, 1H, NH), 11.85 (s, 1H,
NH); 13C-NMR (100 MHz, DMSO-d6) d: 26.9, 30.0, 47.3, 64.6,
64.9, 71.5, 73.5, 109.5, 111.9, 116.8, 120.9, 121.3, 121.6, 122.9,
123.9, 124.4, 125.0, 125.1, 126.9, 127.8, 128.9, 133.4, 136.4,
141.6, 143.4, 179.7, 188.9; IR (KBr, cmꢁ1) nmax ¼ 3382, 3250,
2963, 2963, 2867, 1717, 1620, 1521, 1469, 1426, 1332, 1421,
1132, 749, 698; [anal. calcd. for C27H23N3O2S: C, 71.50; H,
5.11; N, 9.26; found: C, 71.37; H, 4.97; N, 9.04]; LC/MS (ESI, m/z):
453.10 [M + H] for 453.10 C27H23N3O2S.
136.3, 137.4, 140.3, 141.6, 180.1, 189.4; IR (KBr, cmꢁ1) nmax
¼
3382, 3248, 2957, 2865, 1716, 1618, 1521, 1470, 1422, 1331,
1243, 1152, 1111, 749; [anal. calcd. for C30H27N3O2: C, 78.07; H,
5.90; N, 9.10; found: C, 77.89; H, 6.03; N, 9.15]; LC/MS (ESI, m/z):
461.20 [M + H] for 461.21 C30H27N3O2.
3.1.10. (20R,3S,7a0S)-10-(3-Bromophenyl)-20-(1H-indole-3-
carbonyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
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3.1.13. (20R,3S,7a0S)-10-(Furan-2-yl)-20-(1H-indole-3-carbonyl)-
10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-pyrrolizin]-2-one
(4m). Yield (89%); beige powder; mp: 172–174 ꢀC; 1H-NMR (400
MHz, DMSO-d6) d: 1.72–1.96 (m, 3H, CH2), 2.00–2.10 (m, 1H,
CH2), 2.34–2.42 (m, 1H, CH2), 2.48–2.54 (m, 1H, CH2), 3.92–4.00
(m, 1H, CH), 4.09 (t, 1H, J ¼ 9.56 Hz, CH), 4.65 (d, 1H, J ¼
11.72 Hz, CH), 6.24 (d, 1H, J ¼ 2.88 Hz, Ar–H), 6.35 (t, 1H, J ¼
2.20 Hz, Ar–H), 6.57 (d, 1H, J ¼ 7.32 Hz, Ar–H), 6.95 (t, 1H, J ¼
7.32 Hz, Ar–H), 7.06–7.10 (m, 2H, Ar–H), 7.16 (t, 1H, J ¼ 7.32 Hz,
Ar–H), 7.35 (d, 1H, J ¼ 7.32 Hz, Ar–H), 7.42 (d, 1H, J ¼ 8.04 Hz,
Ar–H), 7.56 (d, 1H, J ¼ 1.48 Hz, Ar–H), 7.88 (d, 1H, J ¼ 8.04 Hz,
Ar–H), 7.94 (d, 1H, J ¼ 2.92 Hz, Ar–H), 10.32 (s, 1H, NH), 11.91
(s, 1H, NH); 13C-NMR (100 MHz, DMSO-d6) d: 26.9, 30.3, 45.4,
47.2, 61.2, 68.8, 73.2, 105.5, 109.5, 110.3, 111.9, 116.5, 120.9,
121.3, 121.6, 125.1, 125.2, 127.8, 128.8, 133.4, 136.8, 141.6,
141.9154.0, 179.6, 188.8; IR (KBr, cmꢁ1) nmax ¼ 3401, 3242, 2961,
2872, 1721, 1630, 1617, 1522, 1471, 1422, 1244, 1150, 1130,
1113, 1011, 751; [anal. calcd. for C27H23N3O3: C, 74.12; H,
5.30; N, 9.60; found: C, 73.98; H, 5.47; N, 9.51]; LC/MS (ESI, m/z):
437.20 [M + H] for 437.17 C27H23N3O3.
3.1.14. (20R,3S,7a0S)-20-(1H-Indole-3-carbonyl)-10-(3,4,5-tri-
methoxyphenyl)-10,20,50,60,70,7a0-hexahydrospiro[indoline-3,30-
pyrrolizin]-2-one (4n). Yield (69%); yellow powder; mp: 179–
181 ꢀC; 1H-NMR (400 MHz, DMSO-d6) d: 1.64–1.82 (m, 2H, CH2),
1.82–1.94 (m, 2H, CH2), 2.32–2.40 (m, 1H, CH2), 2.50–2.58 (m,
1H, CH2), 3.56 (s, 3H, OCH3), 3.70–3.72 (m, 1H, CH), 3.74 (s, 6H,
2xOCH3), 3.89–3.91 (m, 1H, CH), 4.64–4.67 (m, 1H, CH), 6.53 (d,
1H, J ¼ 8.08 Hz, Ar–H), 6.73 (s, 2H, Ar–H), 6.91 (t, 1H, J ¼
7.32 Hz, Ar–H), 6.96–7.05 (m, 2H, Ar–H), 7.08 (t, 1H, J ¼ 7.32 Hz,
Ar–H), 7.33 (d, 1H, J ¼ 8.04 Hz, Ar–H), 7.41 (d, 1H, J ¼ 7.36 Hz,
Ar–H), 7.79 (d, 1H, J ¼ 8.04 Hz, Ar–H), 7.99 (d, 1H, J ¼ 2.92 Hz,
Ar–H), 10.23 (s, 1H, NH), 11.80 (s, 1H, NH); 13C-NMR (100 MHz,
DMSO-d6) d: 26.9, 29.9, 47.3, 52.4, 55.8, 59.8, 63.2, 71.4, 73.6,
104.9, 109.4, 111.9, 116.9, 120.8, 121.2, 121.5, 122.8, 125.1,
125.4, 127.8, 128.7, 133.6, 136.0, 136.2, 136.4, 141.6, 152.8,
175.5, 189.4; IR (KBr, cmꢁ1) nmax ¼ 3256, 2937, 2870, 1720, 1619,
1590, 1510, 1468, 1427, 1332, 1243, 1187, 1154, 1126, 9997, 788,
743; [anal. calcd. for C32H31N3O5: C, 71.49; H, 5.81; N, 7.82;
found: C, 71.35; H, 5.92; N, 7.96]; LC/MS (ESI, m/z): 537.20 [M +
H] for 537.23 C32H31N3O5.
spirooxindole derivatives were solubilized in DMSO and stored
at ꢁ20 ꢀC. The viability of the cells was quantied using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),
which measures the activity of mitochondrial succinate dehy-
drogenase in viable cells.45,46
3.2.2. Cytotoxicity assay. The cells were seeded in 96-well
plates at a concentration of 5 ꢃ 104 cells per ml (100 ml per well).
A serial dilution of tested compounds or cisplatin was added
aer the cells were incubated overnight at 37 ꢀC and under 5%
CO2. DMSO was used as a negative control (0.1%). The cells
were incubated for 48 h. Aer that, 15 ml of MTT (5 mg mlꢁ1 in
PBS) was added to each well and incubated for another 4 h. The
formazan crystals were solubilized by 100 ml of acidied SDS
solution (10% SDS/0.01 N HCl in PBS). The absorbance was
measured aer 14 h of incubation at 37 ꢀC and under 5% CO2 at
570 nm by using a BioTek microplate reader. Each experiment
was repeated 3 times and a standard deviation was calculated
(SDꢂ). IC50 was calculated as the concentration that caused
50% inhibition of cell growth. The growth of the cells was
monitored and the images were acquired by Gx microscopes
(GXMGXD202 Inverted Microscope) at 10ꢃ magnication.
3.2.3. Selectivity index (SI) calculations. The selectivity
index was calculated with the following equation
ðIC50Þnormal
SI ¼
(1)
ðIC50Þcancer
Eqn (1): selectivity index (SI) equation: where IC50 normal ¼ the
concentration of the tested compound that killed 50% of
normal cells; IC50 cancer ¼ the concentration of the same tested
compound that killed 50% of cancer cells.
3.2.4. Phosphodiesterase I inhibition assay. A phosphodi-
esterase I inhibition assay was performed using snake venom
according to a previously reported method with minute varia-
tions. Briey, 33 mM Tris–HCl buffer of pH 8.8 (97 ml), 30 mM
magnesium acetate with an enzyme concentration of 0.000742
U wellꢁ1 and 0.33 mM bis-(p-nitrophenyl) phosphate (Sigma N-
3002, 60 ml) as substrate were taken. EDTA with an IC50 ꢂ SD of
274 ꢂ 0.007 mM was used as the positive control. Aer a pre-
incubation period of 30 min, the enzyme with the test
samples was observed spectrophotometrically for enzyme
activity on a microtiter plate reader at 37 ꢀC by following the rate
of change in OD minꢁ1 at 410 nm of the p-nitrophenol released
from p-nitrophenyl phosphate. All assays were processed in
triplicate.47
3.2.5. Docking studies. The docking studies were per-
formed using OpenEye Modelling soware. A virtual library of
spirooxindoles derivatives was used and their energies were
minimized using the MMFF94 force eld, followed by the
generation of multi-conformers using the OMEGA application.42
The whole library of minimized energy values was docked along
with the prepared PDE-1 (PDB ID: 1NOP)40–48 using the FRED
application to generate a physical property (DG) reecting the
predicted energy prole of the ligand-receptor complex. For
ROCS study, the most active compound was selected as the
query molecule. A library of compounds was adopted as the
database (t) le. The VIDA application49 was employed as
a visualization tool to show the poses of the ligands and the
3.2. Anticancer activity
3.2.1. Cell lines and drugs. The cytotoxic activity of the
compounds was tested against different mammalian cancer
cells, prostate carcinoma cells (PC-3), hepatocellular carcinoma
(HepG2) and colon cancer cells (HCT-116). African green
monkey kidney cells (Vero-B) were used as normal cells to study
the selectivity towards the cancer cells. The cell lines were ob-
tained from the American Type Culture Collection (ATCC). The
ꢀ
cells were cultivated at 37 C and 10% CO2 in DMEM (Lonza,
Germany) medium supplemented with 10% fetal bovine serum
(Lonza, Germany), 100 IU mlꢁ1 penicillin and 100 mg mlꢁ1
streptomycin
(Lonza,
Germany).
Cisplatin
(cis-dia-
mineplatinum(II) dichloride) was used as a positive control and
was obtained from Sigma-Aldrich®, then dissolved in 0.9%
saline and stored as an 8 mM stock solution at ꢁ20 ꢀC. The
14344 | RSC Adv., 2018, 8, 14335–14346
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4. Conclusions
In summary, inspired by synthesized spirooxindoles and
natural architectures, we have succeeded in generating potent
anticancer derivatives. The in vitro study revealed highly selec-
tive anticancer agents with a much better cytotoxic activity
against colorectal cancer (HCT-116), hepatocellular carcinoma
(HepG2), and prostate cancer (PC-3) when compared to the
commonly used chemotherapeutic cisplatin. In the phospho-
diesterase 1 enzyme inhibition studies, compound 4d proved to
exhibit a high cytotoxic activity against colorectal, prostate, and
liver cancers at IC50 ¼ 9, 2, and 2 mM with selectivity indices >1,
>4, and >4, respectively. Moreover, compound 4d showed the
best interaction with PDE-1 with a consensus score of 19. It
formed two HB interactions and also hydrophobic interactions.
The ROCS of this newly synthesized drug candidate adopted
a unique geometry unlike other derivatives. The aryl arm
controls the geometry of the compounds and in the case of 4d
the 2,4-dichlorophenyl moiety allowed the indole and oxoindole
moieties to occupy space perpendicularly. This unique char-
acter, high selectivity and promising activity against three
aggressive cancer cell lines of compound 4d make it a prom-
ising anticancer candidate. Therefore, this compound should
be considered a potential anti-cancer agent in combination with
widely used chemotherapeutic drugs to improve the response of
tumors. Currently, a more rigorous in vivo study is being
undertaken to disclose more preclinical information, such as
oral stability, bioavailability, and pharmacokinetics with the
anticipation of better activity and high safety margins.
Conflicts of interest
21 L. D. Quin and J. A. Tyrell, Fundamentals of heterocyclic
chemistry: importance in nature and in the synthesis of
pharmaceuticals, John Wiley & Sons, 2010.
There are no conicts to declare.
22 W. Wang and Y. Hu, Med. Res. Rev., 2012, 32, 1159–1196.
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24 S. Edmondson, S. J. Danishefsky, L. Sepp-Lorenzino and
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Acknowledgements
The authors would like to extend their sincere appreciation to
the Deanship of Scientic Research at King Saud University for
providing funding to the research group No. (RGP-257).
´
25 S. M. Rajesh, S. Perumal, J. C. Menendez, P. Yogeeswari and
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