Design, synthesis and cytotoxic activity of spiro(oxindole-3-3'-pyrrolidine) derivatives

Article abstract of DOI:10.2174/1570180814666170810120634

Background: Spiro[pyrrolidine-3,3'-oxindole] compounds are reported to be highly bioactive natural and synthetic products. Initially, spirooxindole alkaloids were isolated from plants of the Apocynaceae and Rubiaceae families, which were found to have a c

Full text of DOI:10.2174/1570180814666170810120634

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Letters in Drug Design & Discovery, 2018, 15, 37-45
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
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Design, Synthesis and Cytotoxic Activity of Spiro(oxindole-3-3'-
pyrrolidine) Derivatives
BENTHAM
S
CIENCE
Dilan Konyara,*, Cenk A. Andacb and Erdem Buyukbingola
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara-06100, Turkey;
bDepartment of Pharmaceutical Chemistry, School of Pharmacy, Istinye University, Istanbul-34010, Turkey
Abstract: Background: Spiro[pyrrolidine-3,3’-oxindole] compounds are reported to be highly
bioactive natural and synthetic products. Initially, spirooxindole alkaloids were isolated from plants
of the Apocynaceae and Rubiaceae families, which were found to have a common scaffold,
spiro[pyrrolidine-3,3’-oxindole], exhibiting anticancer activities., we specifically aimed at the syn-
thesis, characterization and anticancer activity of novel spiro[pyrrolidine-3,3' -oxindole] derivatives,
compounds 6a-c and 7.
Methods: The synthesis was initiated by Knovenegal condensation of indole-2-one with an appro-
priate benzaldehyde in presence of piperidine to afford compounds 3a-c. Compounds 6a-c were
A R T I C L E H I S T O R Yꢀ
synthesized by an asymmetric 1,3-dipolar cycloaddition between compounds 3a-c and (2S, 3R)-2,
3, 5, 6-tetrahydro-2, 3-diphenyl-1, 4-oxazin-6-one, which is an intermediate compound formed by
Received: February 06, 2017
the Schiff base reaction between 3-methyl-butanal and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl-
Revised: July 16, 2017
Accepted: July 24, 2017
1,4-oxazin-6-one, in presence of molecular sieves (4Å) under argon atmosphere. Compound 6a was
DOI:
then reacted with ethylamine-HCl in THF at room temperature to yield compound 7.
10.2174/1570180814666170810120634
Results: Cytotoxic effects of the compounds synthesized were determined on Huh7, MV, HCT116
and MCF7 cancer cell lines by the NCI-60 Sulforhodamine B Assay, using (S)-(+)-Camptothecin as
a positive control. In general, target compounds showed better cytotoxic activities against the
MCF7 and HCT116 cancer cell lines. It was found that compound 7 exhibited the most potent in-
hibitory activity with IC50 values of 4.8 µM, 3.9 µM, 14.9 µM and 8.2 µM against the MCF7,
HCT116, MV and Huh7 cell lines, respectively.
Conclusion: It was determined that compounds 6a&6b possess C6'(S)|C8'(R)|C9'(R) stereochemis-
try and compound 7 adopts C2'(S)|C4'(R)|C5'(R) stereochemistry. Cytotoxicity studies suggest that
compound 7 gave rise to the highest anticancer activity against MCF7, HCT116, and Huh7 cancer
cell lines.
Keywords: Oxindole, spiro pyrrolidino oxindole, 1,3-dipolar cycloaddition reaction, cytotoxic activity, cancer, drug design.
1. INTRODUCTION
features [6] as well as being inhibitors of the MDM2
(Mürine Double Minute)-p53 interaction [3]. Particularly,
spiro[pyrrolidine-3,3’-oxindole] compounds (Fig. 1) are re-
ported to be highly bioactive natural and synthetic products
[7]. Initially, spirooxindole alkaloids were isolated from
plants of the Apocynaceae and Rubiaceae families, which
were found to have a common scaffold, spiro[pyrrolidine-
3,3’-oxindole], exhibiting anticancer activities [8, 10]. For
instance, many natural spirooxindole alkaloids, such as Spi-
rotryprostatine A and B, horsifiline, elacomine etc. [9] ex-
hibit anticancer activity [9-11] as well as inhibitory poten-
tials for the p53-MDM2 interaction [10, 11] Synthesis of
spirooxindole derivatives fused with a five-membered het-
erocyclic ring including pyrrolidines, pyrrolines, pyrroles,
triazoles, and isoxazolidines were accomplished by the
Huisgen cycloaddition reaction, [12] a 1,3-dipolar cycloaddi-
Cancer has increasingly become a major cause of mortal-
ity throughout the world. 14.1 millions of cancer cases were
reported in 2012 [1]. Despite all advances in cancer diagno-
sis and therapy, the mortality rate remains to be still high.
Therefore, discovery of newer and safer anticancer agents
with extended-spectrum cytotoxicity against tumor cells be-
comes an urgent necessity [2]. It is known that spirooxindole
compounds have significant roles in drug design in terms of
antimicrobial [3], anti-HIV [4], antimalarial [5] and anticancer
*Address correspondence to this author at the Ankara University, Faculty of
Pharmacy, Department of Pharmaceutical Chemistry, Tandogan, Ankara-
06100, Turkey; Tel: +90 (312) 203 30 73; Fax: +90 (312) 213 10 81;
E-mail: konyar@pharmacy.ankara.edu.tr
1875-628X/18 $58.00+.00
©2018 Bentham Science Publishers

38 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Konyar et al.
Z
'
'
1
'/5
9
5'/
N
Y
8'/4'
6'/2'
X
O
N
H
Oxindole scaffold
O
O
O
O
N
N
O
O2N
O
NH
NH
6b
6a
HO
O
O
HN
O
Cl
N
O2N
N
O
O
NH
N
H
7
6c
Fig. (1). X, Y, and Z substituents constituting novel spiro[pyrrolidine-3,3′-oxindole] derivatives, namely compounds 6a-c and 7.
tion reaction. Here, we specifically aimed at the synthesis,
characterization and anticancer activity of novel
spiro[pyrrolidine-3,3' -oxindole] derivatives, compounds 6a-
c and 7 in Fig. (1), in which positions 6', 8' and 5' -9' on
compounds 6a-c and positions 2', 4' and 1' -5' on compound 7
are substituted with X, Y and Z. The novel spiro[pyrrolidine-
3,3’-oxindole] derivatives (6a-c) were synthesized by reac-
tion of the oxazinone and aldehyde with 3-benzyldene-1,3-
dihydro-indol-2-one derivatives in toluene were by 1,3-
dipolar cycloaddition reaction is shown in Scheme 1. The
stereochemistry of this class of compounds has a significant
influence on the binding affinities. Wang et al. suggested
that diastereomeric spirooxindole class of compounds had a
significant effect on binding affinities to MDM2; There is a
100-fold difference between binding affinity of the strongest
and the lowest potent stereoisomers. They have shown that
the binding affinities of the cis-cis isomers to MDM2 are
better than the trans-cis isomers. And also cis-cis isomer is
more effective than trans-cis isomer in p53 activation and
apoptosis induction, in vivo [13]. Chirality of the carbons at
positions 6', 8', 9' of compounds 6a-b and positions 2', 4', 5'
1
of compound 7 was determined by H-NMR and ROESY
NMR experiments. A previously reported spiro[pyrrolidine-
3,3'-oxindole] derivative, compound 6a [14], see Fig. (1),
was also synthesized, characterized, recrystallized for X-ray
crystallographic analysis and found to be the E-conformer
and used as a reference compound in biological activity stud-
ies [14]. Anticancer activities of compounds 6a-c and 7 were
determined on the Huh7 human liver, MCF7 human breast
and HCT116 human colon cancer cell lines.

Synthesis of Spiro(oxindole-3-3'-pyrrolidine) Derivatives
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 39
Ph
H
Ph
Ph
Ph
Ph
O
O
Ph
N
H
O
N
OHC
O
O
HN
4Ao mol.sieves,tol.
O
5
4
OH
Scheme 1. Proposed reaction mechanism pathway of compound 6a-c synthesis.
R1
O
O
Ph
Ph
CHO
N
H
,
CHO
4
Piperidine
+
O
O
R1
MeOH/80°C
N
H
N
4 A°mol.sieves, toluene/70°C
H
1
2
3a-c
3a (R1= -H)
3b (R1= -p-NO2)
3c (R1= -o-Cl)
HO
O
R1
Ph
2'
f
R1
3"
O
1'
3"
6"
O
2"
HN
a
3'
e
4"
5"
4'
4"
b
2"
d
N
c
1
Ph
H2N
9'
5'
2
5'
8'
N
2'
5"
6"
1'
1''
3
7'
1"
6'
5
4
3
1
4'
THF/r.t.
3'
3
1
O
2
O
2
N
H
N
H
6a-c
7
6a (R1= -H)
7 (R1= -p-NO2)
6b (R1= -p-NO2)
6c (R1= -o-Cl)
R1: -H, -p-NO2, -o-Cl
Scheme 2. Synthesis of 3-substituted-benzyldene-1,3-dihydro-indol-2-one (3a-c), 3',4'-diphenyl-8’-p-nitro-phenyl-6'-isobutyl-3',4',8,8'a-
tetrahydrospiro[3H-indol-3,7'(6'H)-[1H]pyrrolo[2,1-c][1,4]oxazine]-1',2(1H)-dione (6a-c) derivatives and 2'-(2-methylpropyl)-1,2-dihydro-
1'-[(1R,2S)-2-hydroksi-1,2-diphenylethyl]-N-ethyl-2-oxo-4'-p-nitrophenyl-spiro[3H-indol-3,3'-pyrrolidin]-5'-carboxamide (7). Reagents and
conditions: (a) Substituted benzaldehydes, piperidine, methanol, 80°C, 1.5 h, (b) (2S,3R)-2, 3, 5, 6-tetrahdro-2, 3-diphenyl-1, 4-oxazin-6-
one, 3-methyl-butyraldehyde, 4 A° mol.sieves, toluene, 70°C, (c) ethylamine-HCl, THF, r.t.
2. MATERIALS AND METHODS
2.1. Chemistry
(Merck), ether (Merck) were purchased in high purities and
freshly used.
Melting points were determined by a capillary melting
point apparatus (Electrothermal 9100, Essex, UK). Elemen-
tal analysis was performed by a Leco932 CHNS analyzer
(Leco, St. Joseph, MI, USA), which gave rise to elemental
compositions within 0.4% error-range of the calculated val-
2.1.1. Materials
2-Chloro benzaldehyde (Fluka), 4-nitro benzaldehyde
(Aldrich), benzaldehyde (Aldrich), (2S,3R)-2,3,5,6-tetra hy-
dro-2,3-phenyl-1,4-oxazin-6-one (Nantong Baihua Bio-
Pharmaceutical Co., Ltd.), oxindole (Aldrich), piperidine
(BDH Chemicals Ltd.), 3-methyl butiraldehyde (Aldrich),
4Aº molecular sieves (Sigma-Aldrich), petroleum ether
(Merck), acetone (Fluka), hexane (Sigma-Aldrich), ethyl
acetate (Sigma-Aldrich), methanol (Riedel de Haen), toluene
1
1
1
ues. H, H,1H-gCOSY, H,1H-ROESY, and gHSQC NMR
experiments were performed on a Varian Mercury-400 FT-
NMR spectrometer operating at 400 MHz (Varian Inc., Palo
Alto, CA, USA). All NMR spectra were acquired in CDCl3.
1H NMR FID data were collected between -2 and 14 ppm

40 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Konyar et al.
sieves (4Å) followed by stirring overnight at 70oC under
argon atmosphere. After completion of the reaction, the sol-
vent was evaporated in vacuo and the major crude product
was purified by silica gel column chromatography (hex-
anes/EtOAc, 5:1). Compound 6a (yield 16%), compound 6b
(yield 9%), and compound 6c (yield 5%).
with 32k data points. 1H,1H-gCOSY and 1H,1H-ROESY
NMR FID data were collected between -1 ppm and 9 ppm
with 400 data points in F1 dimension and 586 data points in
F2 dimension gHSQC NMR FID data were collected be-
tween -2 and 14 ppm with 960 data points in F2 (1H) dimen-
sion and between -10 and 180 ppm with 512 data points in
F1 (13C) dimension. All NMR FID data were post-processed
by Mestre-C(nD) v.1.1 (Mestre-C, Spain) and Varian NMR
software. All 1H NMR FID data were zero-filled to 32K data
points, linear-predicted and apodized with line-broadening
(3R,3'S,4'R,6'S,8'S,9'R)-3',4',8'-triphenyl-6'-(2-methylpropyl)-
3',4',8',9'-tetrahydro-1'H,6'H-spiro[2,3-dihydro-indole-3,
7'-pyrrolo[2,1-c][1,4]oxazine]-1',2(1H)-dione (Compound 6a)
1H-NMR (400 MHz, CDCl3) δ(ppm): 7.64 (1H, s, NH),
1
function before Fourier transformation. All H,1H-gCOSY
3
1
and H,1H-ROESY NMR FID data were zero-filled and lin-
7.34-6.94 (18H, m, HAr), 6.91 (1H, t, JHH= 8.0, 7.6 Hz, 6-
3
3
HAr), 6.56 (1H, d, JHH = 8.0 Hz, 7-HAr), 6.63 (1H, t, JHH
=
ear-predicted to 2K data points in F2 and F1 dimensions be-
fore Fourier transformation. In addition, line-predicted
ROESY FIDs were apodized with 90o Sine and 0.07 Hz
Gaussian functions before Fourier transformation in both
dimensions. Apodized ROESY FIDs were Fourier trans-
formed in inverse and quadrature modes along t2 dimension
and in inverse and quadrature modes using the hypercomplex
3
8.0, 7.2 Hz, 5-HAr), 6.23 (1H, d, JHH = 7.2 Hz, 4-HAr), 6.35
3
3
(1H, d, JHH = 4.0 Hz, 3'-H), 4.80 (1H, d, JHH = 4.4 Hz, 4'-
3
3
H), 4.39 (1H, d, JHH = 10.8 Hz, 9'-H), 4.25 (1H, d, JHH
=
3
10.8 Hz, 8'-H), 3.20 (1H, dd, JHH = 10.0, 4.0 Hz, 6′-H),
2.07-2.0 (1H, m, 6'H-iBu(-CH2)), 1.75-1.68 (1H, m, 6′H-
iBu(-CH2)), 1.05-1.04 (1H, m, 6'H-iBu(-CH)), 0.80 (3H, d,
3JHH = 6.8 Hz, 6'H-iBu(-CH3)), 0.47 (3H, d, JHH = 6.0 Hz,
3
1
protocol in t1 dimension. H NMR chemical shifts (δ) were
2
reported in ppm, 3JHH and JHH peak coupling constants were
6'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δ ]:
6.91/6.56 [6-HAr/7-HAr], 6.91/6.63 [6-HAr/5-HAr], 6.63/6.Η23
[5-HAr/4-HAr], 6.35/4.80 [3'-H/4'-H], 4.39/4.25 [8a′-H/8'-H],
3.20/2.07-2.0 [6'-Heq/CH2], 3.20/1.75-1.68 [6'-H/CH2], 2.07-
2.0/1.75-1.68 [CH2/CH2]; 13C-NMR (400 MHz, CDCl3)
δ(ppm): 129.8, 128.1, 127.7, 126.9, 125.7, 125.0, 122.0, 109.2,
83.7, 66.1, 60.5, 60.3, 56.0, 36.5, 24.9, 23.8, 21.6; MS (ESI)
m/z: 543 [M+H]+; Anal. (C36H34N2O3-0.5 H2O) requires C,
78.37; H, 6.40; N, 5.07; found C, 78.22, H, 6.62, N, 5.04.
reported in Hz, and peak splits were reported as s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). In NMR
assignments, ortho/meta/para aromatic hydrogens are de-
noted o/m/p respectively. Mass spectral data were acquired
on a Waters ZQ Micromass LC-MS spectrometer (Waters
Corporation, Milford, MA, USA) using the Electrospray
Ionization (ESI) method. The synthesis of compounds (6a-c, 7)
is schematically shown in Scheme 2.
(3R,3'S,4'R,6'S,8'S,9'R)-3',4'-diphenyl,8'--p-nitrophenyl-
6'-(2-methylpropyl)-3',4',8',9'-tetrahydro-1'H,6'H-spiro
[2,3-dihydro-indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-1',
2(1H)-dione (Compound 6b)
2.1.2. General Procedure for the Synthesis of 3-benzyldene-
1,3-dihydro-indol-2-one Derivatives (Compounds 3a-c)14
Piperidine (0.197 ml, 2 mmol) was added to a mixture of
oxindole (1 mmol) and the benzaldehyde derivative of inter-
est (1.1 mmol) in dry methanol. The reaction mixture was
then stirred under Argon atmosphere at 80°C for 1.5 hours.
The solvent of the reaction mixture was then evaporated to
yield the crude product, which was purified by silica gel col-
umn chromatography (hexanes/EtOAc, 2:1).
1H-NMR (400 MHz, CDCl3) δ(ppm): 7.88 (2H, d, JHH
=
3
8.4 Hz, 3''-HAr-5''-HAr), 7.35-7.16 (10H, m, HAr), 7.59 (1H, s,
NH), 7.10 (2H, d, 3JHH = 8.4 Hz, 2''-HAr-6''-HAr), 6.93 (1H, t,
3
3JHH = 8.0, 8.0 Hz, 6-HAr), 6.56 (1H, d, JHH = 8.0 Hz, 7-
3
HAr), 6.65 (1H, t, JHH = 7.6, 7.2 Hz, 5-HAr), 6.17 (1H, d,
3JHH = 7.6 Hz, 4-HAr), 6.31 (1H, d, 3JHH = 4.0 Hz, 3'-H), 4.80
(1H, d, 3JHH = 4.4 Hz, 4'-H), 4.32 (1H, d, 3JHH = 10.8 Hz, 9'-
3-Benzylidene-1,3-dihydro-indol-2-one (compound 3a)
Obtained as a yellow solid; M.P.: 164-166 °C (lit. M.P.: 175-
176 °C)11 ; MS(ESI) m/z 222 [M+H]+.
3
3
H), 4.28 (1H, d, JHH = 10.8 Hz, 8'-H), 3.16 (1H, dd, JHH
=
10.4, 3.2, 2.8 Hz, 6'-H), 2.15 (1H, ddd, 3JHH = 13.6, 10.4, 3.6
Hz, 6'H-iBu(-CH2)), 1.72 (1H, ddd, JHH = 13.6, 10.8, 3.2
3
3-(4'-Nitro)-benzylidene-1,3-dihydro-indol-2-one (com-
pound 3b) Obtained as a red solid; M.P.: 239-242 °C (lit.
M.P.: 245-250 °C)11; MS(ESI) m/z 267 [M+H]+.
Hz, 6'H-iBu(-CH2)), 1.06-1.02 (1H, m, 6'H-iBu(-CH)), 0.83
(3H, d, 3JHH = 6.4 Hz, 6'H-iBu(-CH3)), 0.48 (3H, d, J= 6.0 Hz,
6'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δΗ]:
7.88/7.10 [3''-HAr-5''-HAr /2''-HAr-6''-HAr], 6.93/6.56 [6-HAr/7-
HAr], 6.93/6.65 [6-HAr/5-HAr], 6.65/6.17 [5-HAr/4-HAr],
6.31/4.80 [3'-H/4'-H], 3.16/2.18-2.11 [6'-H/CH2], 3.16/1.75-
1.68 [6'-H/CH2], 2.18-2.11/1.75-1.68 [CH2/CH2]; MS(ESI)
m/z 588[M+H]+; Anal. (C36H33N3O5-1 H2O) requires C:
71.38; H: 5.82; N: 6.93; found C: 71.38, H: 5.68, N: 6.80.
3-(2'-Chloro)-benzylidene-1,3-dihydro-indol-2-one
(Compound 3c) Obtained as a red solid; M.P.: 145-148 °C
(lit. M.P.: 142 °C)11; MS (ESI) m/z 256 [M+H]+ (100%), 258
(M+H+2)+ (33%).
2.1.3. General Procedure for the Synthesis of the 3',4',8'-
triphenyl-6'-isobutyl-3',4',8,8'a-tetrahydrospiro[3H-indol-
3,7'(6'H)-[1H]pyrrolo[2,1c]-[1,4]oxazine]-1',2(1H)-dione
Derivatives (Compounds 6a-c)14
(3R,3'S,4'R,6'S,8'R,9'R)-3',4'-diphenyl,8'-o-chlorophenyl-
6'-(2-methylpropyl)-3',4',8',9'-tetrahydro-1'H,6'H-spiro
[2,3-dihydro-indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-1',
2(1H)-dione (Compound 6c)
1H-NMR (400 MHz, CDCl3) δ(ppm): 8.42 (1H, s, NH),
7.53 (1H, d, 3JHH = 7.6 Hz, 3''-HAr), 7.24-7.08 (13H, m, HAr),
7.05-6.98 (1H, m, 6-HAr), 6.76 (1H, d, 3JHH = 8.0 Hz, 7-HAr),
3-Benzylidene-1,3-dihydro-indol-2-one
compounds
3a/3b/3c, (0.475 mmol), 3-methyl-butyraldehyde (0.0509 ml,
0.475 mmol), and (2S,3R)-2,3,5,6-tetrahydro-2,3-diphenyl-
1,4-oxazine-6-one (0.1 g, 0.396 mmol) were dissolved in
toluene in the presence of 2 g of freshly activated molecular

Synthesis of Spiro(oxindole-3-3'-pyrrolidine) Derivatives
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 41
3
3
6.61 (1H, t, JHH = 8.0, 7.2 Hz, 5-HAr), 6.30 (1H, d, JHH
=
2.2. Determination of Anticancer Effects of Compounds
6a-c and 7 in Cell Culture
3
7.6 Hz, 4-HAr), 6.73 (1H, d, JHH = 3.6 Hz, 3'-H), 4.90 (1H,
3
3
d, JHH = 4.0 Hz, 4'-H), 4.78 (1H, d, JHH = 10.4 Hz, 9'-H),
Huh7 human hepatocellular carcinoma, MV (Mahlavu)
human hepatocellular carcinoma, MCF7 human breast carci-
noma and HCT116 human colon carcinoma cells were
grown in Dulbecco's Modified Eagles Medium (DMEM)
supplemented with 10% fetal bovine serum, 1% non-
essential amino acids, 1% penicillin and streptomycin
(GIBCO, Invitrogen) at 37°C under 5% CO2 atmosphere.
3
3
4.74 (1H, d, JHH = 10.8 Hz, 8'-H), 3.32 (1H, t, JHH = 7.2,
6.8 Hz, 6'-H), 1.92-1.85 (1H, m, 6'H-iBu(-CH2)), 1.49-1.42
(1H, m, 6'H-iBu(-CH2)), 1.02-0.96 (1H, m, 6'H-iBu(-CH)),
3
0.64 (3H, d, JHH = 6.4 Hz, 6'H-iBu(-CH3)), 0.47 (3H, d,
3JHH = 6.0 Hz, 6'H-iBu(-CH3)); COSY proton-proton corre-
lations [δΗ/δΗ]: 7.04-6.98/6.76 [6-HAr/7-HAr], 7.04-6.98/6.61
[6-HAr/5-HAr], 6.61/6.30 [5-HAr/4-HAr], 6.73/4.90 [3'-H/4'-
H], 4.78/4.74 [9'-H/8'-H], 3.32/1.92-1.85 [6'-H/CH2], 3.32/
1.49-1.44 [6'-H/CH2], 1.92-1.85/1.49-1.44 [CH2/CH2].
2.3. NCI-60 Sulforhodamine B Assay
Cells were plated in 96-well plates (2000-3000 cells/well
in 150µl/well) and grown for 24 hours at 37 °C. Grown cells
were then treated in duplicates with a series of dilutions (in
DMSO), ranging from 40µM to 2.5 µM, of the compounds
synthesized (6a-c and 7) as well as (S)-(+)-camptothecin (the
positive control). After 72 hours of incubation, cells were
fixed with 10% (v/v) trichloroacetic acid (MERCK) for an
hour and stained with sulforhodamine B (SRB) solution (50
µl of a 0.4% (m/v) of SRB in 1% acetic acid solution). For
the removal of unbound SRB, cells were washed out with
1% acetic acid and left for air-drying. SRB bound to protein
was solubilized in 10 mM Tris-base prior to absorbance
measurement (515 nm) using 96-well plate reader. Cells
treated with DMSO alone were used as the control in percent
inhibition and IC50 studies.
13C-NMR (400 MHz, CDCl3) δ(ppm): 129.5, 129.1, 127.9,
127.7, 126.2, 126.0, 124.9, 121.8, 109.3, 82.1, 69.5, 62.7,
61.5, 59.1, 53.4, 35.6, 29.6, 24.5, 23.8, 22.7, 22.2; MS(ESI)
m/z 577 [M+H]+ (100%), 579 [M+H+2]+ (40%); Anal.
(C36H33ClN2O3-0,25 C6H14-0,75 H2O) requires C: 73.57; H:
6.25; N: 4.57; found C: 73.41, H: 6.57, N: 4.47.
2.1.4. Synthesis of 2'-(2-methylpropyl)-1,2-dihydro-1'-
[(1R,2S)-2-hydroxy-1,2-diphenylethyl]-N-ethyl-2-oxo-4'-p-
nitrophenyl-spiro[3H-indol-3,3'-pyrrolidine]-5'-carboxamide
(2'S,3'S,4'S,5'R)-N-ethyl-1’-[(1R,2S)-2-hydroxy-1,2-
diphenylethyl]-2'-(2-methylpropyl)-4'-p-nitrophenyl-2(1H)-
oxo-spiro[2,3-dihydro-indole-3,3'-pyrrolidine-5'-
carboxamide (Compound 7)
Under nitrogen atmosphere, to a solution of compound 6
(0.587 g, 1 mmol) dissolved in 5 ml THF was added ethy-
lamine-HCl (0.164 g, 1.5mmol), followed by stirring over-
night. After brine was added into the reaction mixture, the
crude products were extracted with ethyl acetate for several
times. The combined organic phase was dried over anhy-
drous Na2SO4. The solvent was evaporated in vacuo and the
desired product was purified by silica gel column chroma-
tography (hexanes/acetone, 2:1, (yield: 45%) [14].
Percent inhibition values were determined by the follow-
ing equation [15]:
%inhibition=[{(average(ODtreatment)/average(ODDMSO))X10}]
(S)-(+)-Camptothecin (the positive control), compound
6a (the reference) and compounds 6b-c and 7 were tested for
their antitumor activities at their IC50 concentrations, given
in Table 1, against Huh7, MV, MCF7, HCT116 cancer cell
lines. Percent inhibition of the cells was determined using 5
dilutions of the compounds synthesized, with concentrations
ranging from 40 μM to 2.5 μM (in DMSO).
1H-NMR (400 MHz, CDCl3) δ(ppm): 7.53 (1H, s, indole-
3
NH), 7.52-7.22 (10H, m, HAr3), 7.75 (2H, d, JHH = 8.4 Hz,
3''-HAr-5''-HAr), 6.82 (2H, d, JHH = 7.6 Hz, 2''-HAr-6''-HAr),
3
6.99 (1H, t, JHH = 8.4, 7.6 Hz, 6-HAr), 6.65 (1H, s , amide(-
3. RESULTS AND DISCUSSION
NH)), 6.56 (1H, d, 3JHH = 8.0 Hz, 7-HAr), 6.15 (1H, d, J= 7.6
Hz, 4-HAr), 5.29 (1H, s, CH), 4.34 (1H, d, J= 8.8 Hz, 5'-H ),
The reference compounds (6a and the target compounds
6b-c and 7) were synthesized as described in the methods
section, following the steps shown in Scheme 2. The synthe-
sis was initiated by Knovenegal condensation of indole-2-
one in Scheme 2 with an appropriate benzaldehyde in the
presence of piperidine to afford compounds 3a-c [14]. Com-
pounds 6a-c were synthesized by an asymmetric 1,3-dipolar
cycloaddition between compounds 3a-c and (2S, 3R)-2, 3, 5,
6-tetrahydro-2, 3-diphenyl-1, 4-oxazin-6-one (compound 4
in Scheme 2), which is an intermediate compound formed by
the Schiff base reaction between 3-methyl-butanal (in
Scheme 1) and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl-
1,4-oxazin-6-one (compound 4 in Scheme 1), in the presence
of molecular sieves (4Å) under argon atmosphere. Com-
pound 6a was then reacted with ethylamine-HCl in THF at
room temperature to yield compound 7 [14]. After silica gel
chromatography separation, the major products for com-
pounds 6a (yield: 16%), 6b (yield: 9%), 6c (yield: 5%) and 7
(yield: 45%) were purified in the best possible highest yields
with more than 95% purity. All the compounds purified were
3
4.41 (1H, d, J= 6.0 Hz, CH), 3.88 (1H, d, JHH = 8.8 Hz, 4'-
H), 3.59 (1H, d, 3JHH = 9.2 Hz, 2'-H), 3.36-3.28 (1H, m, 5'H-
N-Et(-CH2)), 3.06-3.00 (1H, m, 5'H-N-Et(-CH2)), 2.56 (1H,
s, OH), 2.25 (1H, t, 2'H-iBu(-CH2)), 1.52 (1H, t, 2'H-iBu(-
CH2)), 1.10-1.08 (1H, m, 2'H-iBu(-CH), 0.99 (3H, t, 5'H-N-
3
Et(-CH3)), 0.82 (3H, d, JHH = 6.4 Hz, 2'H-iBu(-CH3)), 0.70
(3H, d, 3JHH = 6.0 Hz, 2'H-iBu(-CH3)); COSY proton-proton
correlations [δ /δΗ]: 7.75/6.82 [3''-HAr-5''-HAr /2''-HAr-6''-
HAr], 6.99/6.56 Η[6-HAr/7-HAr], 6.99/6.65 [6-HAr/5-HAr], 6.65/
6.15 [5-HAr/4-HAr], 6.65/3.36-3.28 [NH/CH2], 6.65/3.06-3.0
[NH/CH2], 5.29/4.41 [H2/H1], 5.28/2.56 [CH/OH], 4.34/
3.88 [5'H/4'H], 3.59/2.25 [2'-H/CH2], 3.59/1.52 [2'-H/CH2],
3.36-3.28/0.99 [CH2/CH3], 3.06-3.0/0.99 [CH2/CH3],
2.25/1.52 [CH2/CH2]; 13C-NMR (400 MHz, CDCl3) δ(ppm)
:
131.4, 129.7, 128.7, 128.2, 128.1, 126.9, 125.5, 122.9, 122.3,
109.5, 84.2, 70.5, 67.1, 65.8, 60.7, 60.0, 59.8, 58.1, 55.5,
40.6, 36.6, 34.2, 25.5, 23.7, 21.7, 14.3; MS(ESI) m/z
633[M+H]+ ; Anal.(C38H40N4O5-0,25H2O) requires C, 71.62;
H, 6.40; N, 8.79; found C, 71.67, H, 6.43, N, 8.45.

42 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Konyar et al.
3
Table 1. Experimental JHH (Hz) values and the correspond-
Table 2. ROESY NOE cross-peak intensities for compounds
6a, 6c and 7. NOE cross-peaks were referenced to
the prochiral geminal protons of –CH2- attached to
C6' of compounds 6a-c or C2' of compound 7.
ing HLA (Haasnoot, De Leeuw and Altona)16 dihe-
dral angles (HLAo) for the vicinal H8'-H9' proton pairs
of compounds 6a-c and the vicinal H4'-H5' proton
pair of compound 7. t stands for trans HLAo (~180o).
NOE Intensities
3JHH(Hz)/HLAo
-
-
6a
6c
7
-
6a
6b
6c
7
6'H-8'H
6'H-9'H
8'H-9'H
v.weak
weak
n.d.
n.d.
n.d.
2'H-4'H
2'H-5'H
4'H-5'H
v.weak
weak
n.d.
H8':H9'
H4':H5'
10.7/t
10.8/t
n.d.
n.a.
n.a.
n.a.
n.a.
9.1/t
medium
n.d.: not determined due to spectral overlap. n.a.: not applicable.
n.d.: not determined due to spectral overlap; weak: weak NOE intenstiy; v.weak: very
weak NOE intensity; medium: medium NOE intensity.
characterized by NMR, mass spectroscopy and elemental
analysis techniques, see the corresponding experimental section.
proton pairs, (Table 2). Stereochemistry determined for
compound 7 was directly correlated to compound 6b. As
In ESI mass spectroscopy studies, all compounds yielded
(M+H)+ positive ions with 100% relative abundances. Ele-
mental analysis results were found to be within the range of
calculated values with less than 0.4% deviations, proving the
chemical structure and purity of the compounds synthesized.
3
3
seen in Table 3, JH8'-H9' (compounds 6a & 6b) and JH4'-H5'
(compound 7) are within the range of ~9-12 Hz, Table 1,
giving rise to trans HLAo dihedral angles (~180o/t) [17],
which means that the C8'/C4' atom will have a different
stereoisomerism than the neighboring C9'/C5' atom. For
instance; a C8' atom with an R stereoisomerism would be
bound to a C9' atom with an R stereoisomerism or vice
versa. Stereoisomerism for the C6', C8' and C9' atoms of
compound 6c was not determined due to severe spectral
overlap of the H8' and H9' proton peaks. In Table 2, NOE
cross-peak intensities were correlated to proton-proton dis-
tances in three ranges: 1.8-2.8 Å for strong NOEs, 2.8-3.3
Åfor medium NOEs and 3.3-5.0 Å for weak NOEs [18].
Excluding homostereoisomeric possibilities with the C8'-C9'
(compounds 6a-c) and C4'-C5' (compound 7) atom pairs, 4
sets of stereoisomerism involving the C6'/C8'/C9' atoms of
compounds 6a & 6c and the C2'/C4'/C5' atoms of compound
7 were modeled and minimized by Discovery Studio v3.0
Visualizer (free trial version by Accelrys Software, San
Diego, CA, USA), for which dihedral angles and proton-
proton distances are listed in Table 3.
Proton chemical shifts listed in the experimental section
were determined by 1H and gCOSY NMR methods. 13C
chemical shifts, listed in the corresponding experimental
section, were determined by 13C and gHSQC NMR methods.
Stereoisomerism for the C6, C8' and C9' atoms of the pyr-
rolo[2,1-c][1,4]oxazine heterocyclic group in compounds 6a
and 6c, (Fig. 1), was studied by the application of the
Haasnoot, De Leeuwꢀ andꢀ Altona (HLA) Karplus equation
[16, 17] on 3JH8'-H9' coupling constants for the H8'-H9' vicinal
protons, see Table 1, as well as through-space NOE cross-
peak intensities determined for the H6'-H8', H6'-H9' and H8'-
H9' proton pairs by ROESY NMR. In terms of compound 7,
a hydrolyis product of compound 6b, stereoisomerism was
determined for the C4' and C5' atoms of the pyrrole group,
3
see Scheme 2, utilizing JH4'-H5', see Table 1, and NOE
cross-peak intensities for the H2'-H4', H2'-H5' and H4'-H5'
Table 3. Torsion (dihedral) angles and proton-proton through-space distances for modelled conformers of compounds 6a and 7
possessing possible different stereochemistries.
Compound 6a
Chirality
Torsion (o)
8'H-9'H
178/t
Distance (Å)
6'H:9'H
3.50
C6'
R
C8'
S
C9'
S
6'H:8'H
1.91
8'H:9'H
3.00
R
R
R
166/g+
3.74
1.86
3.09
S
S
S
-179/t
3.56
3.61
3.02
S
R
R
172/t
3.94
3.50
3.12
Compound 7
Chirality
Torsiono
4'H-5'H
176/t
Distance (Å)
2'H:5'H
3.52
C2'
R
C4'
S
C5'
S
2'H:4'H
1.93
4'H:5'H
3.04
R
R
R
173/g+
3.42
1.81
2.96
S
S
S
-176/t
3.47
3.75
3.05
S
R
R
172/t
3.84
3.57
3.14

Synthesis of Spiro(oxindole-3-3'-pyrrolidine) Derivatives
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 43
(A)
MCF7
100
90
80
70
60
50
40
30
20
10
00
7
6a
6c
CPT
20 mm
10 mM
5 mM
2.5 mM
40 mM
(B)
HCT116
100
90
80
70
60
50
40
30
20
10
00
6a
6c
7
CPT
20 mm
10 mM
5 mM 2.5 mM
40 mM
(C)
Huh7
100
80
60
40
20
0
6b
6c
7
6a
CPT
20 mm
10 mM
5 mM
2.5 mM
40 mM
Mahlavu
(D)
100
80
60
40
20
00
6b
6c
7
6a
CPT
20 mm
10 mM
5 mM 2.5 mM
40 mM
Fig. (2). Huh7, HCT116, MCF7 and Mahlavu cells were incubated at 72 hours. All molecules and controls were implemented to the cells in
dublicated with five different concentrations: 40, 20, 10, 5, and 2.5μM. After 72 h of incubation, SRB assays were generated and the IC50
values were calculated. mol. sieves, molecular sieves; tol, toluen; cycloadd., cycloaddition.
Looking at the computational dihedral angle (8'H-9'H)
and through-space proton-proton distances (6'H:8'H, 6'H:9'H
and 8'H:9'H) for compound 6a in Table 3, it is determined
that C6'(S)|C8'(R)|C9'(R) stereochemistry matches best the
experimental data for compound 6a with the corresponding
H8′-H9′ dihedral angle in Table 1 and the NOE cross-peak
intensities in Table 2, which conforms to the X-ray crystal-
lography data reported by Sebahar et al. [19], the spiro C7'
atom possesses S stereochemistry in asymmetric 1,3-dipolar
cycloaddition reaction products, therefore, so do compounds
6a-c &7.

44 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Konyar et al.
Table 4. IC50 values of camptothecin and compounds 6a-c & 7, determined on Huh7, MV (Mahlavu), MCF7 and HCT116 cancer
cell lines.
IC50 (μM)
Compound
MCF7
HCT116
MV
Huh7
Camptothecin
˂ 1
10.1
˂ 1
8.4
˂ 1
48.6
˂ 1
32.1
77.8
36.6
8.2
6a14
6b
6c
7
No inhibition
11.2
No inhibition
10.5
No inhibition
26.3
4.8
3.9
14.9
Computational dihedral angle (C8'H-C9'H) and through-
space proton-proton distances (6'H:8'H, 6'H:9'H and 8'H:9'H)
determined for the C2'(S)|C4'(R)|C5'(R) stereochemistry of
compound 7, listed in Table 3, points to the experimental
data obtained for compound 7, listed in Tables 1 and 2. As
compound 6b is the starting material for compound 7, the
pyrrole ring of compound 6b possesses identical stereochem-
istry as the pyrrolidine ring of compound 7, which are now
assigned as C6'(S)|C8'(R)|C9'(R) for compound 6b.
spectively. As compared to compound 6a [14], para-
nitrophenyl substitution in compound 6b knocked out cyto-
toxic activity against the MCF7, HCT116 and MV cell lines,
while a 2.4-fold decrease in cytotoxic activity was observed
against the Huh7 cell line. It is likely that p-nitrophenyl sub-
stitution increases repulsive electrostatic interactions at the
site of action. Ortho-chlorophenyl substitution in compound
c resulted in slight decrease in cytotoxic activity against the
MCF7, HCT116 and Huh7 cell lines, due possibly to C6'(R)
stereochemistry in compound 6c as compared to C6'(S)
stereochemistry in compound 6a.
Unfortunately, 8'H and 9'H proton peaks of compound 6c
severely overlap at 4.76 ppm, disabling determination of
6'H:8'H and 6'H:9'H through-space distances. Nevertheless, a
medium-to-weak NOE (~ 3.5 Å) and a strong NOE (< 3.5 Å)
for the 4'H:6'H and 4'H:[C6'-CH2-] proton pairs, respec-
tively, of the pyrrolo[2,1-c][1,4]oxazine heterocyclic ring
were detected by ROESY, which is a condition met only
when C6' adopts R stereochemistry that gives rise to
through-space distances of 3.4 Å and 2.65 Å for the 4'H:6'H
and 4'H:[C6'-CH2-] proton pairs, respectively. Otherwise, an
S stereochemistry with C6'(S) would give rise to distances of
2.88 Å (with a very strong NOE cross-peak) and 3.22 Å
(with a strong NOE cross-peak) for the 4'H:6'H and
4'H:[C6'-CH2-] proton pairs, respectively. This indeed makes
sense because a large ortho-substituent on a phenyl group,
such as chloride as in compound 6c (Fig. 1), is not sterically
allowed to accommodate on the same side of the 2-
methylpropyl group bound to C6'(S). Therefore, C6' of com-
pound 6c prefers to adopt R stereochemistry after the asym-
metric 1,3-cycloaddition reaction due to steric reasons.
Stereochemistry for C8' and C9' of compound 6c remains to
be unknown, except that C8' and C9' are homesteroisomeric.
Therefore we can explain with Cahn-Ingold-Prelog (CIP)
priority rule the stereochemistry at position C8' of compound
6c. Note that at position C8' of compound 6c a molecule with
the S assignment is produced because a Cl atom reverses the
Cahn-Ingold-Prelog (CIP) priority order [20].
CONCLUSION
In this study, novel spiro(oxindole-3-3'-pyrrolidine) de-
rivatives, compounds 6a-c & 7, were successfully synthe-
sized in good yields, characterized and tested for their anti-
cancer activities. It was determined that compounds 6a & 6b
possess C6'(S)|C8'(R)|C9'(R) stereochemistry and compound
7 adopts C2'(S)|C4'(R)|C5'(R) stereochemistry. Compound 6c
gains a C6'(R) stereochemistry while its C8'|C9' stereochem-
istry is unknown cytotoxicity studies suggest that compound
7, an amine hydrolysis product of compound 6b, gave rise to
the highest anticancer activity against MCF7, HCT116, and
Huh7 cancer cell lines, and therefore should be further stud-
ied as a potential drug candidate and as a lead compound for
its novel derivatives.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Cytotoxic effects of the compounds synthesized were
determined on Huh7, MV, HCT116 and MCF7 cancer cell
lines by the NCI-60 Sulforhodamine B Assay, using (S)-(+)-
Camptothecin as a positive control shown in Fig. (2). The
cell inhibition (IC50) results are given in Table 4. In general,
target compounds showed better cytotoxic activities against
the MCF7 and HCT116 cancer cell lines. It was found that
compound 7 exhibited the most potent inhibitory activity
with IC50 values of 4.8 µM, 3.9 µM, 14.9 µM and 8.2 µM
against the MCF7, HCT116, MV and Huh7 cell lines, re-
This study is funded by the Research Fund of Ankara
University (Grant No. 10B3336001). The authors acknowl-
edge Dr. Rengul Atalay at the Cancer System Biology
Laboratory, Graduate School of Informatics, Middle East
Technical University-Ankara, Turkey, and Deniz Cansen
Yildirim at the Department of Molecular Biology and
Genetics (DMBG), Faculty of Science, Bilkent University
(BU) at Ankara, Turkey, for implementing the cytotoxic
activity studies at the DMBG laboratories at BU-Ankara,

Synthesis of Spiro(oxindole-3-3'-pyrrolidine) Derivatives
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 45
logical evaluation and cellular target identification. Sci. Rep., 2016,
6, 322B.
Turkey. We also would like to thank the Instrumental
Analysis Facility in the School of Farmacy at Ankara
University-Ankara, Turkey for allowing us to use the NMR,
LC-MS and Elemental Analysis instruments therein.
[10]
[11]
Yu, B.; Yu, D.-Q.; Liu, H.-M. Spirooxindoles: Promising scaffolds
for anticancer agents. Eur. J. Med. Chem., 2015, 97, 673-698.
Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.; McEachern, D.; Shar-
gary, S.; Bernard, D.; Li, X.; Zhao, T. A potent small-molecule in-
hibitor of the MDM2-p53 interaction (MI-888) achieved complete
and durable tumor regression in mice. J. Med. Chem., 2013, 56(13),
5553-5561.
REFERENCES
[1]
Globacan Estimated Cancer Incidence. Mortality and prevalence
worldwide, 2012. [Available at http://globocan.iarc.fr/Default.aspx].
Nagarsenkar, A.; Prajapti, S.K.; Guggilapu, S.D.; Birineni, S.;
Kotapalli, S.S.; Ummanni, R.; Babu, B.N. Investigation of triazole-
linked indole and oxindole glycoconjugates as potential anticancer
agents: novel Akt/PKB signaling pathway inhibitors. Med. Chem.
Comm., 2016, 7(4), 646-653.
[12]
[13]
[14]
Vine, K.L.; Matesic, L.; Locke, J.M.; Ranson, M.; Skropeta, D.
Cytotoxic and anticancer activities of isatin and its derivatives: A
comprehensive review from 2000-2008. Anticancer Agents Med.
Chem., 2009, 9(4), 397-414.
Zhao, Y.; Liu, L.; Sun, W.; Lu, J.; McEachern, D.; Li, X.; Yu, S.;
Bernard, D.; Ochsenbein, P.; Ferey, V. Diastereomeric spirooxin-
doles as highly potent and efficacious MDM2 inhibitors. J. Am.
Chem. Soc., 2013, 135(19), 7223-7234.
Ding, K.; Wang, G.; Deschamps, J.R.; Parrish, D.A.; Wang, S.
Synthesis of spirooxindoles via asymmetric 1, 3-dipolar cycloaddi-
tion. Tetrahedron Lett., 2005, 46(35), 5949-5951.
Kerkvliet, G.J. Drug discovery screen adapts to changes. J. Natl.
Cancer Inst., 1990, 82(13), 1087-1088.
Haasnoot C.A.G.; De Leeuw F.A.A.M.; Altona C. The relationship
between proton-proton NMR coupling constants and substituent
electronegativities-I. Tetrahedron, 1980, 236, 2783-2793.
Andac, C.A.; Stringfellow, T.C.; Hornemann, U.; Noyanalpan, N.
NMR and amber analysis of the neamine pharmacophore for the
design of novel aminoglycoside antibiotics. Bioorg. Chem., 2011,
39(1), 28-41.
[2]
[3]
Zheng, G.-H.; Shen, J.-J.; Zhan, Y.-C.; Yi, H.; Xue, S.-T.; Wang,
Z.; Ji, X.-Y.; Li, Z.-R. Design, synthesis and in vitro and in vivo an-
titumour activity of 3-benzylideneindolin-2-one derivatives,
a
novel class of small-molecule inhibitors of the MDM2-p53 interac-
tion. Eur. J. Med. Chem., 2014, 81, 277-288.
[15]
[16]
[4]
[5]
Periyasami, G.; Raghunathan, R.; Surendiran, G.; Mathivanan, N.
Regioselective synthesis and antimicrobial screening of novel keto-
carbazolodispiropyrrolidine derivatives. Eur. J. Med. Chem., 2009,
44(3), 959-966.
Kumari, G.; Modi, M.; Gupta, S.K.; Singh, R.K. Rhodium (II)
acetate-catalyzed stereoselective synthesis, SAR and anti-HIV ac-
tivity of novel oxindoles bearing cyclopropane ring. Eur. J. Med.
Chem., 2011, 46(4), 1181-1188.
Prakash R.C.; Raja, S. Indolinones as promising scaffold as kinase
inhibitors: A review. Mini Rev. Med. Chem., 2012, 12(2), 98-119.
Das, S.K.; Bhattacharya, S.; Kundu, A. Rationalized design, syn-
thesis and pharmacological screening of amino acid linked spiro
pyrrolidino oxyindole analogs through environment friendly reac-
tion. J. Adv. Pharm. Technol. Res., 2013, 4(4), 198.
Marti, C.; Carreira, E.M. Construction of spiro [pyrrolidine3,
3′oxindoles] recent applications to the synthesis of oxindole alka-
loids. Eur. J. Org. Chem., 2003, 2003(12), 2209-2219.
Hati, S.; Tripathy, S.; Dutta, P.K.; Agarwal, R.; Srinivasan, R.;
Singh, A.; Singh, S.; Sen, S. Spiro [pyrrolidine-3, 3´-oxindole] as
potent anti-breast cancer compounds: Their design, synthesis, bio-
[17]
[18]
[6]
[7]
Andrews, P.R.; Chang, C.H.; Cooke, R.M.; Craik, D.J.; Edwards,
A.J.; Feeney, J.; Gerik, J.T.; Hodge, C.N.; Jaroszewski, J.W.;
Kessler, H.; King, G.F.; Konat, R.; Mackay, J.; Nicholson, L.K.;
Pavlopoulos, S.; Reid, D.G.; Searle, M.S.; Schmitt, W.; Sweeney,
P.J.; Wickham, G. NMR in drug design. CRC Press Inc: USA, 1996.
Sebahar, P.R.; Williams, R.M. The asymmetric total synthesis of
(+)-and (−)-spirotryprostatin B. J. Am. Chem. Soc., 2000, 122(23),
5666-5667.
[19]
[20]
[8]
[9]
Mata, P.; Lobo, A.M. Implementation of the Cahn-Ingold-Prelog
system for stereochemical perception in the LHASA program. J.
Chem. Inf. Comput. Sci., 1994, 34, 491-504.