Diversity-oriented synthesis of spiro-oxindole-based 2,5-dihydropyrroles via three-component cycloadditions and evaluation on their cytotoxicity

Article abstract of DOI:10.1039/c3ra40874d

The construction of a spiro-oxindole-based 2,5-dihydropyrrole scaffold with potential bioactivity has been established via an efficient three-component reaction of isatin, amino-ester and alkyne. This protocol represents the first 1,3-dipolar cycloaddition of electron-deficient alkynes with isatin-derived azomethine ylides, providing an easy access to spiro-oxindole-based 2,5-dihydropyrroles with structural diversity in high yields (up to 99%). In addition, the bioscreen of these new spiro-dihydropyrroles has led to the finding of sixteen compounds with promising cytotoxicity to MCF-7 cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra40874d

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Diversity-oriented synthesis of spiro-oxindole-based
2,5-dihydropyrroles via three-component
Cite this: DOI: 10.1039/c3ra40874d
cycloadditions and evaluation on their cytotoxicity3
Wei Tan,{a Xiao-Tong Zhu,{b Shu Zhang,*c Gui-Juan Xing,a Ren-Yi Zhua
and Feng Shi*a
The construction of a spiro-oxindole-based 2,5-dihydropyrrole scaffold with potential bioactivity has been
established via an efficient three-component reaction of isatin, amino-ester and alkyne. This protocol
represents the first 1,3-dipolar cycloaddition of electron-deficient alkynes with isatin-derived azomethine
ylides, providing an easy access to spiro-oxindole-based 2,5-dihydropyrroles with structural diversity in high
yields (up to 99%). In addition, the bioscreen of these new spiro-dihydropyrroles has led to the finding of
sixteen compounds with promising cytotoxicity to MCF-7 cells.
Received 20th February 2013,
Accepted 15th April 2013
DOI: 10.1039/c3ra40874d
www.rsc.org/advances
Introduction
The spiro-oxindole core is a privileged heterocyclic ring
system, which not only constitutes the core structural element
of pharmaceutically relevant natural products,1 but also is
featured in a large family of unnatural compounds exhibiting a
wide spectrum of important bioactivities such as antitumor,2
antimycobacterial,3 antimicrobial4 and antitubercular5 proper-
ties (Fig. 1).
On the other hand, the 2,5-dihydropyrrole skeleton also
exists in a variety of biologically significant compounds
possessing antioxidant,6 antimicrobial,7 anti-tumoral,8 and
anti-inflammatory9 activities (Fig. 2).
Considering the versatile bioactivities of the two structures
of spiro-oxindole and 2,5-dihydropyrrole, we hypothesize that
the integration of the two scaffolds into a spiro-oxindole-based
2,5-dihydropyrrole (in Scheme 1) may result in the discovery of
new drug candidates with unknown bioactivities. However,
hardly any approaches have been available to access this spiro-
architecture, yet.10 Therefore, the design of an efficient
protocol to access this novel target with structural diversity
Fig. 1 Biologically important molecules containing a spiro-oxindole core.
is highly desirable and valuable for medicinal chemistry and
drug discovery.
1,3-Dipolar cycloadditions of azomethine ylides to electron-
deficient olefins have offered a robust method to construct a
pyrrolidine skeleton.11 Inspired by this success, we envision
that the 1,3-dipolar cycloaddition of electron-deficient alkynes
aSchool of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou,
221116, China. E-mail: fshi@jsnu.edu.cn; Fax: +86(516) 83500065; Tel: +86(516)
83403165
bDepartment of Chemistry, School of Basic Education Sciences, Xuzhou Medical
College, Xuzhou, 221004, China
cClinical Research Center, the First Affiliated Hospital of Nanjing Medical University,
Nanjing, 210029, China. E-mail: zszqs@sina.com
Electronic supplementary information (ESI) available: Experimental details,
3
characterization, original NMR and HPLC spectra of product 4. CCDC 922100.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ra40874d
Fig. 2 Bioactive compounds containing a 2,5-dihydropyrrole core.
{ These two authors contributed equally to the work.
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Table 1 Optimization of reaction conditionsa
Entry B–H
Solvent
MS 1a : 2a : 3a Yield (%)b
c
1
2
3
4
5
6
7
8
PhCO2H
TsOH
CF3CO2H PhCH3
CF3CO2H 1,4-dioxane
CF3CO2H CH3CN
CF3CO2H EtOH
CF3CO2H ClCH2CH2Cl 3 Å 1.2 : 1 : 2
CF3CO2H ClCH2CH2Cl 4 Å 1.2 : 1 : 2
CF3CO2H ClCH2CH2Cl
PhCH3
PhCH3
3 Å 1.2 : 1 : 2
3 Å 1.2 : 1 : 2
3 Å 1.2 : 1 : 2
3 Å 1.2 : 1 : 2
3 Å 1.2 : 1 : 2
3 Å 1.2 : 1 : 2
—
—
Scheme 1 Design of the reaction to access
a spiro-oxindole-based 2,5-
c
dihydropyrrole scaffold.
16
45
33
c
—
48
42
to isatin-derived azomethine ylides will provide a straightfor-
ward entry to the spiro-oxindole-based 2,5-dihydropyrrole
scaffold (Scheme 1). Besides, the designed three-component
reactions of isatins, amino-esters and alkynes will facilitate the
diversity-oriented synthesis,12 which plays an important role in
offering a source of compounds for screening experiments
with the attempt to discover novel biologically active entities.13
However, electron-deficient alkynes have scarcely been
employed as dipolarophiles to react with azomethine ylides,14
especially with isatin-derived ones, which are more difficult to
form and have lower reactivity in subsequent cycloadditions
than their aldehyde-derived counterparts. Thus, the develop-
ment of 1,3-dipolar cycloadditions of electron-deficient
alkynes with isatin-derived azomethine ylides is of great
importance with regard to the chemistry of 1,3-dipolar
cycloadditions and the construction of the spiro-oxindole-
based 2,5-dihydropyrrole skeleton.
As a continuation of our efforts in developing new multi-
component reactions (MCRs) and synthesizing bioactive
heterocycles via MCRs,15 we herein report the efficient
diversity-oriented synthesis of novel spiro-oxindole-based 2,5-
dihydropyrroles (up to 99% yield, 36 examples) via an
unprecedented three-component 1,3-dipolar cycloaddition of
electron-deficient alkynes with isatin-derived azomethine
ylides. Additionally, the bioscreen of the products has led to
the finding of sixteen compounds with promising cytotoxicity
to MCF-7 cells.
d
c
9
—
1.2 : 1 : 2
—
56
73
70
10
11
12
CF3CO2H ClCH2CH2Cl 3 Å 1.2 : 1 : 8
CF3CO2H ClCH2CH2Cl 3 Å 1.2 : 1 : 12
CF3CO2H ClCH2CH2Cl 3 Å 1.2 : 1 : 20
a
Unless indicated otherwise, the reaction was carried out in 0.1
mmol scale in solvent (1 mL) with MS (100 mg) at 60 uC for 36 h.
Isolated yield. Trace product was observed. In the absence of
b
c
d
MS.
disclosed that 3 Å MS played an important role in controlling
the reactivity since trace product was observed in the absence
of MS (entry 9 vs. 7–8), which may be ascribed to the action of
3 Å MS in efficiently removing water molecules produced by
the condensation of 1a with 2a, thereby promoting the
generation of the isatine-derived azomethine ylide. Finally,
the mole ratio of 1a : 2a : 3a was fine tuned to improve the
yield (entries 10–12). To our delight, properly increasing the
stoichiometry of 3a rendered the reaction to proceed in a
much higher yield of 73% (entry 11).
With the optimal conditions in hand, the diversity-oriented
synthesis of spiro-oxindole-based 2,5-dihydropyrroles 4 was
then carried out by three-component reactions of various
isatins 1, diethyl 2-aminomalonate 2a and but-2-ynedioate 3
(Table 2). Firstly, the effect of N-substituents of isatin 1 on the
reaction was investigated. As shown in entries 1–7, this
protocol was amenable to a wide scope of isatins with different
types of N-substituents, including a hydrogen atom and alkyl,
phenyl or benzyl groups in high yields (64–99%). Obviously,
N-unsubstituted isatin 1b gave the highest yield of 99% among
different N-substituted isatins (entry 1). Basically, N-alkyl
substituted isatins gave a higher yield than N-phenyl and
N-benzyl substituted ones (entries 2–4 vs. 5–7). It seemed that
the size of the N-substituents also imposed some effect on the
reactivity. For instance, smaller N-substituents such as a
methyl group led to higher reactivity (entry 2), while larger
N-substituents as exemplified by a 1-naphthyl-methylene
group resulted in decreased reactivity (entry 7).
Results and discussion
The initial attempt to validate our hypothesis examined a
three-component reaction of N-benzylisatin 1a, diethyl 2-ami-
nomalonate 2a and dimethyl but-2-ynedioate 3a, which was
performed in the presence of 20 mol % Brønsted acid (B–H) in
toluene at 60 uC (Table 1).
However, a preliminary screening of the catalysts led to
disappointing results (Table 1, entries 1–3). Benzoic acid and
4-methylbenzenesulfonic acid (TsOH) could hardly catalyze
the reaction (entries 1–2), and trifluoroacetic acid could
catalyze the reaction only with a low yield of 16% (entry 3).
Subsequent screening of solvents revealed that 1,2-dichlor-
oethane was the most suitable for increasing the yield to 48%
(entry 7 vs. 3–6). Further evaluation of molecular sieves (MS)
The influence of substituents at the phenyl moiety of
isatins 1 on the reaction was studied next (entries 8–24).
Generally, the introduction of an electronically different
substituent at the C4, C5, C6 or C7 position of isatins was
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Table 2 Diversity-oriented synthesis of spiro-compounds 4a
Entry
4
R/R1 (1)
R2 (3)
Yield (%)b
1
2
3
4
5
6
7
8
4baa
4caa
4daa
4eaa
4faa
4aaa
4gaa
4haa
4iaa
H/H (1b)
Me/H (1c)
i-Pr/H (1d)
cyclopentyl/H (1e)
Ph/H (1f)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Me (3a)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
Et (3b)
99 (87)c
90
76
87
79
73
64
62
92
87
85
86
81
61
62
67
63
Fig. 3 The molecular structure of compound 4aaa.
delivering a quantitative yield of 99% to C7-substituted or
C5,C6-disubstituted ones (entries 19–21 vs. 22–24).
Bn/H (1a)
1-naphthyl-CH2/H (1g)
Me/4-Cl (1h)
Me/5-Me (1i)
Me/5-OMe (1j)
Me/5-Br (1k)
Me/5-Cl (1l)
Me/5-F (1m)
Bn/5-Me (1n)
Bn/5-OMe (1o)
Bn/5-Br (1p)
Bn/6-Br (1q)
Bn/7-Br (1r)
H/5-Me (1s)
H/5-OMe (1t)
H/5-F (1u)
Moreover, ethyl but-2-ynedioate 3b was also employed in
the reaction with some representative isatins to demonstrate
the applicability of this protocol and to provide a variety of
spiro-oxindole-based 2,5-dihydropyrroles 4 for further bioas-
says. As indicated in entries 25–36, ethyl but-2-ynedioate 3b
served as an appropriate substrate to furnish corresponding
spiro-compounds in high yields ranging from 65% to 99%.
Similarly, a wide range of isatins with different types of
N-substituents (entries 25–29) or with electronically different
substituents at various positions of the phenyl moiety (entries
30–36) could smoothly participate in the reactions to provide
the desired products with structural diversity.
In order to show the real difference of products with
quantitative yields, we further performed the reactions for
4baa, 4saa, 4taa, 4uaa and 4bab with a shorter reaction time of
16 h. The results indicated that products 4baa and 4saa were
easier to form than 4taa, 4uaa and 4bab in terms of yield
(entries 1 and 19 vs. 20–21 and 25, in parentheses).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
4jaa
4kaa
4laa
4maa
4naa
4oaa
4paa
4qaa
4raa
4saa
4taa
4uaa
4vaa
4waa
4xaa
4bab
4cab
4eab
4fab
4aab
4iab
4jab
4kab
4lab
4yab
4wab
4vab
79
99 (82)c
99 (63)c
99 (55)c
76
H/7-Br (1v)
H/7-CF3 (1w)
H/5,6-F2 (1x)
H/H (1b)
Me/H (1c)
cyclopentyl/H (1e)
Ph/H (1f)
58
52
99 (51)c
94
87
84
85
86
71
79
85
73
Bn/H (1a)
Me/5-Me (1i)
Me/5-OMe (1j)
Me/5-Br (1k)
Me/5-Cl (1l)
Me/6-Br (1y)
H/7-CF3 (1w)
H/7-Br (1v)
The structures of all these new spiro-compounds 4 were
unambiguously determined by IR, 1H NMR, 13C NMR and
HRMS (ESI ).16 Besides, the structure of compound 4aaa was
73
65
3
further confirmed by X-ray crystallographic analysis (Fig. 3).17
On the basis of our experimental results and previous
studies on the reaction mechanism,15b,c we proposed a
possible pathway and transition state of the reaction
(Scheme 2). Initially, the condensation of isatins 1 and diethyl
2-aminomalonate 2a in the presence of trifluoroacetic acid
a
Unless indicated otherwise, the reaction was carried out in 0.1
mmol scale in the presence of 20 mol% trifluoroacetic acid in 1,2-
dichloroethane (1 mL) with 3 Å MS (100 mg) at 60 uC for 36 h, and
b
c
the mole ratio of 1 : 2a : 3 was 1.2 : 1 : 12. Isolated yield. The
reaction time is 16 h.
tolerable to the reaction with good to excellent yields in most
cases. But in some cases, the position of the substituents
appeared to exert some impact on the reactivity. For N-methyl
isatins, there was no significant difference in reactivity when
varying the C5-substituent, and high yields of 81–92% were
obtained regardless of the electronic nature of the substituents
(entries 9–13). However, C4-substituted N-methyl isatin was
inferior to its C5-substituted counterpart in terms of yield
(entry 8 vs. 12). For N-benzyl isatins, C7-substituted isatin
delivered a higher yield of 79% than C5 or C6-substituted ones
(entry 18 vs. 14–17), while for N-unsubstituted isatins, C5-
substituted isatins exhibited a much superior reactivity of
Scheme 2 Possible reaction pathway and transition state.
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Table 3 Preliminary evaluation on the cytotoxicity of compounds 4a
Inhibition rate (%)
Entry
4
R/R1
R2 1 mg mL21 10 mg mL21 100 mg mL21
1
2
3
4
5
6
7
8
4baa H/H
Me 6.4 ¡ 1.9 12.7 ¡ 3.1 18.6 ¡ 3.4
b
4caa Me/H
4daa i-Pr/H
4aaa Bn/H
4naa Bn/5-Me
Me
—
5.2 ¡ 1.5
23.5 ¡ 3.3
Me 6.0 ¡ 2.0 10.0 ¡ 2.7 17.5 ¡ 2.0
b
b
b
b
b
Me
Me
—
—
—
—
—
12.2 ¡ 2.7
12.4 ¡ 2.6
13.1 ¡ 2.4
15.8 ¡ 3.0
11.0 ¡ 2.7
21.6 ¡ 2.9
Fig. 4 Compound 4bab inhibited cell viability and induced apoptosis in MCF-7
cells. (a) Effect of compound 4bab on MCF-7 cell viability. (b) The morphological
features of apoptosis were monitored by fluorescence microscopy after staining
with Hoechst 33 342. Scale bar: 50 mm. (c) The apoptosis rate was measured by
flow cytometry analysis after staining with Annexin V-FITC and PI. (d)
Compound 4bab (1, 10 and 100 mg mL21) induced MCF-7 apoptosis in a
concentration-dependent manner when incubated for 24 h. The data are
presented as mean ¡ SEM of four individual experiments. *P , 0.05; **P , 0.01,
compared with the control group.
4oaa Bn/5-OMe Me
7.8 ¡ 1.3
4paa Bn/5-Br
4raa Bn/7-Br
4saa H/5-Me
Me 5.2 ¡ 1.6 7.9 ¡ 1.9
b
Me
Me
—
—
5.6 ¡ 3.0
6.0 ¡ 2.1
b
9
10
11
12
13
14
15
16
17
4taa H/5-OMe Me 4.0 ¡ 1.2 10.6 ¡ 2.8 13.0 ¡ 2.9
b
b
b
b
4uaa H/5-F
4waa H/7-CF3
4bab H/H
4iab Me/5-Me
4jab Me/5-OMe Et 5.4 ¡ 2.9 9.7 ¡ 3.1
Me
Me
—
—
—
—
10.3 ¡ 2.6
13.3 ¡ 3.1
Et 6.3 ¡ 1.7 11.5 ¡ 2.9 28.0 3.3
Et
b
—
20.8 2.7
24.8 ¡ 2.6
20.7 ¡ 2.7
15.3 ¡ 2.7
b
4yab Me/6-Br
Et
—
—
9.6 ¡ 2.8
PCc
12.9 ¡ 2.5 35.3 ¡ 2.6
b
a
show any inhibition effect at this concentration. At
a
The cytotoxicity of the tested compounds was represented as
b
concentration of 10 mg mL21, five compounds exhibited
considerable cytotoxicity to MCF-7 cells (inhibition rate .
10%, entries 1, 3, 10, 13–14, column 6). Notably, compound
4iab showed the highest inhibition rate of 20.8% (entry 14,
column 6), which was much higher than the positive control.
At a concentration of 100 mg mL21, all sixteen compounds
displayed promising cytotoxicity to MCF-7 cells with inhibition
rates varying from 10.3% to 28.0%. Five compounds exhibited
remarkable cytotoxic activity (inhibition rate . 20%, entries 2,
9, 13–15, column 7). Among them, compound 4bab was the
most powerful to inhibit the growth of MCF-7 cells to 28.0%
(entry 13, column 7), which was comparable to the positive
control.
inhibition rate (mean ¡ S.D., n = 4) on MCF-7 cells. No inhibition
effect was observed. Doxorubicin hydrochloride was used as a
positive control (PC).
c
generated the corresponding azomethine ylides, which then
participated in 1,3-dipolar cycloadditions with but-2-ynedio-
ates 3 to afford products 4 via a sequential Michael addition
and Mannich-type cyclization rather than
a concerted
pathway.15b The trifluoroacetic acid served as a Brønsted
acid/Lewis base bifunctional catalyst to simultaneously acti-
vate both the isatin-derived azomethine ylides and but-2-
ynedioates by H-bonding interactions, thereby accelerating the
desired reaction to build up the structurally rigid spiro-
skeleton.
With the aim of gaining some insight into the possible
mechanism for the cytotoxicity of these compounds, we then
carried out an in-depth study on the cytotoxic mechanism of
representative compound 4bab. The effects of compound 4bab
on MCF-7 cell line viability were examined using the CCK8
assay. As shown in Fig. 4a, a typical dose-dependent inhibitory
effect on cell viability was discovered in MCF-7 cells that were
treated with different doses of compound 4bab (1, 10 and 100
mg mL21) for 24 h. To test whether the inhibition of cell growth
of compound 4bab was related to cell apoptosis, compound
4bab-induced MCF-7 cell apoptosis was determined using
Hoechst 33 324 staining. It was found that the percentage of
early apoptotic cells was markedly elevated in a dose-
dependent manner. As shown in Fig. 4b, untreated cells
exhibited regular and round-shaped nuclei. In contrast, the
condensation and fragmentation of nuclei, characteristic of
apoptotic cells, was evident in cells treated with different
concentrations of compound 4bab for 24 h. The results of flow
cytometry further confirmed the effects of compound 4bab on
In order to survey the possible bioactivity of these novel
spiro-oxindole-based 2,5-dihydropyrroles, all the synthesized
compounds 4 were subjected to a preliminary evaluation on
their in vitro cytotoxic activity, which was represented as the
inhibition rate of the tested compounds to mammary
carcinoma cell line MCF-7 at three concentrations of 1, 10
and 100 mg mL21. Among the tested compounds 4, sixteen
compounds exhibited an inhibition effect on MCF-7 cells at
different concentrations (Table 3). In detail, all sixteen
bioactive compounds and the positive control (PC) showed a
tendency of concentration-dependent cytotoxicity because
their inhibition rates were greatly enhanced with increasing
concentration. Although only six compounds slightly inhibited
the growth of MCF-7 cells at a concentration of 1 mg mL21
these results were better than the positive control (doxorubicin
hydrochloride), a powerful anticancer drug, which did not
,
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treated with MAPK inhibitors 1 h prior to compound 4bab
exposure. PD98059 is a potent, cell-permeable, and selective
inhibitor of MEK1, which results in inhibition of the
phosphorylation and activation of ERK1/2. SB203580 is a
pyridinyl imidazole compound which acts as a competitive
inhibitor of ATP binding on the p38 kinase, and thus serves as
a specific inhibitor of p38 MAPKs. SP600126 is a potent,
selective, reversible, and cell-permeable inhibitor of JNK. As
shown in Fig. 5b, PD98059, SB203580 and SP600125 alone
failed to affect the MCF-7 apoptosis. However, PD98059,
SB203580 and SP600125 significantly reduced compound
4bab-induced apoptosis of MCF-7. These results suggested
that compound 4bab induced MCF-7 apoptosis by the MAPK
pathway.
Fig. 5 Role of MAPKs in compound 4bab-induced apoptosis. (a) MCF-7 cells
were treated with 10 mg mL21 compound 4bab for the indicated time periods.
ERK1/2, p38 and JNK MAPKs and their phosphorylated forms were determined
by western blotting using specific antibodies. (b) Effects of MAPK inhibitors on
apoptosis induced by compound 4bab. MCF-7 cells were pretreated with 10
mM PD98059, SB203580 or SP600125 for 1 h and then exposed to 10 mg mL21
compound 4bab for 24 h (controls were not exposed to compound 4bab). After
the treatment, annexin V-PI double staining was used to analyze apoptotic cell
death. #P , 0.05, ##P , 0.01 vs. compound 4bab (10 mg mL21) treatment
groups. Data are presented as the mean ¡ SEM of three independent
experiments.
Conclusions
In summary, we have established the construction of a novel
spiro-oxindole-based 2,5-dihydropyrrole scaffold with poten-
tial bioactivity via efficient three-component reactions of
isatins, amino-ester and alkynes. In addition, this protocol
also represents the first 1,3-dipolar cycloaddition of electron-
deficient alkynes with isatin-derived azomethine ylides,
providing an easy access to spiro-oxindole-based 2,5-dihydro-
pyrroles with structural diversity. More importantly, the
bioscreen of these new spiro-dihydropyrroles has led to the
finding of sixteen compounds with promising cytotoxicity to
MCF-7 cells. This approach not only provides a valuable tool to
synthesize spiro-oxindole-based 2,5-dihydropyrroles with high
yields, atom-economy and operational simplicity, but also
greatly enriches the chemistry of 1,3-dipolar cycloadditions.
Significantly, the preliminary bioassay of these compounds
will cast light on their medicinal applications after further
structural modulation and biological studies.
MCF-7 cell apoptosis. Annexin-V binding and PI uptake were
detected in MCF-7 cells (Fig. 4c). The portion of annexin-V
positive cells increased from 2.8% in control cells to 11.5%,
24.8% and 42.5% in the cells treated with 1, 10 and 100 mg
mL21 of compound 4bab for 24 h, respectively (Fig. 4d).
The balance between survival and apoptosis signal path-
ways controls the cancer pathogenesis. Mitogen-activated
protein kinase (MAPK) signaling is a signaling pathway which
has received increasing attention as a target for cancer
prevention and treatment. MAPK includes JNK (c-Jun NH2-
terminal kinase), p38 MAPK (mitogen-activated protein
kinase) and ERK (extracellular signal-regulated kinase), lead-
ing to the activation of NF-kB, cell growth, and cell survival.19
An extracellular signal-regulated kinase (ERK)/MAPK pathway
has been demonstrated to be involved in the regulation of cell
apoptosis.20 P38 kinase has been demonstrated to be an
essential regulator of sustained G2 arrest.21 A previous study
has demonstrated that the duration and intensity of JNK
activation is associated with cell death.22 It has been reported
that MAPK is activated in various cancers and that the
activation of the MAPKs selectively phosphorylate cellular
targets, leading to regulation of gene expression and biological
events such as proliferation, differentiation, and apoptosis.23
To elucidate the mechanism of apoptosis that was induced by
compound 4bab, ERK1/2, p38 and JNK levels and their
phosphorylation states were measured. As shown in Fig. 5a,
phosphorylated ERK1/2 was significantly increased 60 min
after treatment of compound 4bab. Phosphorylated p38 and
JNK were increased 30 min after treatment of compound 4bab
and remained at a high level for up to 8 h. The total ERK 1/2,
p38 MAPK and JNK protein levels in the 4bab-treated group
were similar to the control group.
Experimental
General information
NMR spectra were measured respectively at 400 and 100 MHz
on a Brucker-400 MHz spectrometer. The solvent used for
NMR spectroscopy was CDCl3, using tetramethylsilane as the
internal reference. HRMS (Bio TOF Q) spectra were recorded
with an ESI resource on P-SIMS-Gly of Bruker Daltonics Inc.
Infrared spectra were recorded on a Nicolet MX-1E FT-IR
spectromter.
Analytical grade solvents for the column chromatography
and commercially available reagents were used as received. All
commercially available starting materials were used directly.
Substrates 1a, 1c–1r and 1y were synthesized according to the
literature methods.15c,18
General procedure for the synthesis of spiro-oxindole-based
2,5-dihydropyrroles 4
To examine the functional consequences of the ERK 1/2,
p38 MAPK and JNK phosphorylation changes, cells were pre-
A solution of isatins 1 (0.12 mmol), 2-aminomalonate 2a (0.1
mmol), trifluoroacetic acid (0.02 mmol), and 3 Å molecular
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sieves (100 mg) in 1,2-dichloroethane (0.5 mL) was stirred at rt
for 20 min. Then, this resultant mixture was added to but-2-
ynedioate 3 (1.2 mmol) and another 0.5 mL 1,2-dichlor-
oethane. The reaction mixture was stirred at 60 uC for 36 h.
Then the reaction mixture was filtered to remove the
molecular sieves, and the solid powder was washed with ethyl
acetate. The resultant solution was evaporated under the
reduced pressure, and the residue was purified through flash
column chromatography on silica gel to yield pure products 4.
(Flash column chromatography eluent, petroleum ether–ethyl
acetate = 4 : 1); reaction time = 36 h; yield: 87%; yellow sticky
oil; H NMR (400 MHz, CDCl3) d (ppm): 7.43 (dd, J = 7.4, 1.0
1
Hz, 1H), 7.27 (td, J = 7.8, 1.3 Hz, 1H), 7.03 (td, J = 7.6, 0.8 Hz,
1H), 6.88 (d, J = 7.9 Hz, 1H), 4.69 (t, J = 8.7 Hz, 1H), 4.40–4.29
(m, 4H), 3.82 (s, 3H), 3.76 (s, 1H), 3.50 (s, 3H), 2.08 (dt, J = 15.0,
7.4 Hz, 2H), 1.92 (d, J = 2.9 Hz, 4H), 1.69 (s, 2H), 1.35 (t, J = 7.0
Hz, 3H), 1.31 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.3, 168.7, 168.0, 162.6, 161.1, 142.6, 140.2, 139.7,
129.9, 128.9, 125.0, 122.8, 109.7, 79.9, 75.1, 63.0, 62.8, 53.0,
52.4, 52.2, 27.6, 27.3, 25.1, 25.1, 13.9, 13.8; IR (KBr): c 3334,
2998, 2954, 2859, 1742, 1673, 1607, 1443, 1307, 1239, 1157,
1127, 1035, 874, 735, 700 cm21; ESI FTMS exact mass calcd for
(C26H30N2O9 + Na)+ requires m/z 537.1844, found m/z 537.1833.
59,59-Diethyl 39,49-dimethyl 2-oxo-1-phenylspiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4faa). (Flash col-
umn chromatography eluent, petroleum ether–ethyl acetate =
4 : 1); reaction time = 36 h; yield: 79%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.54–7.48 (m, 3H), 7.46–7.37
(m, 3H), 7.24 (td, J = 7.8, 1.6 Hz, 1H), 7.10 (td, J = 7.6, 0.9 Hz,
1H), 6.79 (d, J = 7.8 Hz, 1H), 4.46–4.28 (m, 4H), 3.88 (s, 1H),
3.85 (s, 3H), 3.59 (s, 3H), 1.65 (s, 1H), 1.37 (t, J = 7.1 Hz, 3H),
1.30 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm):
174.0, 168.6, 167.9, 162.6, 161.3, 144.0, 139.9, 139.9, 134.3,
130.2, 129.6, 128.2, 126.3, 125.0, 123.7, 109.6, 80.1, 75.4, 63.1,
62.9, 52.5, 29.7, 13.9, 13.8; IR (KBr): c 3342, 2988, 2954, 2869,
1742, 1660, 1620, 1446, 1307, 1252, 1183, 1117, 1035, 939, 857,
Characterization of new compounds 4
59,59-Diethyl 39,49-dimethyl 2-oxospiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4baa). (Flash column
chromatography eluent, petroleum ether–acetone = 3 : 1);
reaction time = 36 h; yield: 99%; yellow solid; m.p. 145–147
uC; 1H NMR (400 MHz, CDCl3) d (ppm): 8.43 (s, 1H), 7.39 (d, J =
7.4 Hz, 1H), 7.26–7.20 (m, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.85 (d, J
= 7.8 Hz, 1H), 4.34 (dd, J = 14.1, 7.0 Hz, 4H), 3.84 (s, 3H), 3.75
(s, 1H), 3.55 (s, 3H), 1.34 (dt, J = 14.4, 7.1 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d (ppm): 176.5, 168.6, 168.0, 162.6, 161.3,
141.2, 140.0, 139.7, 130.3, 128.9, 125.0, 123.2, 110.3, 80.0, 75.5,
63.1, 62.8, 52.5, 13.92, 13.89; IR (KBr): c 3334, 2981, 2940, 1742,
1674, 1610, 1479, 1439, 1297, 1252, 1113, 1031, 871, 814, 709
cm21; ESI FTMS exact mass calcd for (C21H22N2O9 + H)+
requires m/z 447.1404, found m/z 447.1376.
59,59-Diethyl 39,49-dimethyl 1-methyl-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4caa). (Flash col-
789, 749, 693 cm21
; ESI FTMS exact mass calcd for
umn chromatography eluent, petroleum ether–acetone
=
(C27H26N2O9 + Na)+ requires m/z 545.1531, found m/z 545.1519.
59,59-Diethyl 39,49-dimethyl 1-benzyl-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4aaa). (Flash col-
umn chromatography eluent, petroleum ether–ethyl acetate =
4 : 1); reaction time = 36 h; yield: 73%; light yellow solid; m.p.
149–151 uC; 1H NMR (400 MHz, CDCl3) d (ppm): 7.45–7.36 (m,
3H), 7.36–7.23 (m, 3H), 7.20 (td, J = 7.8, 1.2 Hz, 1H), 7.02 (td, J =
7.6, 0.8 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 5.12 (d, J = 15.6 Hz,
1H), 4.66 (d, J = 15.6 Hz, 1H), 4.41–4.31 (m, 4H), 3.85 (s, 3H),
3.80 (s, 1H), 3.40 (s, 3H), 1.36 (t, J = 5.2 Hz, 3H), 1.33 (t, J = 5.2
Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.7, 168.6,
168.0, 162.6, 161.2, 143.2, 140.5, 139.6, 135.5, 130.2, 128.7,
128.6, 127.7, 124.7, 123.3, 109.3, 79.9, 75.0, 63.1, 62.9, 52.5,
52.3, 44.5, 13.9, 13.9; IR (KBr): c 3349, 2984, 2954, 2859, 1729,
1660, 1613, 1498, 1429, 1262, 1035, 926, 854, 809, 690, 598
cm21; ESI FTMS exact mass calcd for (C28H28N2O9 + H)+
requires m/z 537.1873, found m/z 537.1843.
59,59-Diethyl 39,49-dimethyl 1-(naphthalen-1-ylmethyl)-2-
oxospiro[indoline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate
(4gaa). (Flash column chromatography eluent, petroleum
ether–ethyl acetate = 4 : 1); reaction time = 36 h; yield: 64%;
yellow sticky oil; 1H NMR (400 MHz, CDCl3) d (ppm): 8.15 (d, J
= 8.4 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H),
7.59 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.56–7.37 (m, 4H), 7.15 (td, J
= 7.8, 1.2 Hz, 1H), 7.03 (td, J = 7.6, 0.7 Hz, 1H), 6.67 (d, J = 7.8
Hz, 1H), 5.57 (d, J = 16.3 Hz, 1H), 5.22 (d, J = 16.3 Hz, 1H), 4.38
(qd, J = 7.1, 1.7 Hz, 4H), 3.87 (s, 4H), 3.40 (s, 3H), 1.38 (t, J = 5.6
Hz, 3H), 1.34 (t, J = 5.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.9, 168.6, 167.9, 162.7, 161.2, 143.6, 140.7, 139.4,
133.8, 131.0, 130.3, 130.2, 128.8, 128.7, 128.3, 126.6, 125.9,
6 : 1); reaction time = 36 h; yield: 90%; light yellow solid;
m.p. 132–134 uC; 1H NMR (400 MHz, CDCl3) d (ppm): 7.41 (dd,
J = 7.4, 0.8 Hz, 1H), 7.31 (td, J = 7.8, 1.3 Hz, 1H), 7.06 (td, J = 7.6,
0.9 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 4.42–4.27 (m, 4H), 3.83 (s,
3H), 3.73 (s, 1H), 3.52 (s, 3H), 3.21 (s, 3H), 1.35 (t, J = 6.2 Hz,
3H), 1.31 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.3, 168.7, 168.0, 162.6, 161.1, 142.6, 140.2, 139.7,
129.9, 128.9, 125.0, 122.8, 109.7, 79.9, 75.1, 63.0, 53.0, 52.2,
27.3, 25.1, 13.9, 13.8; IR (KBr): c 3444, 3349, 2984, 2939, 2859,
1742, 1660, 1607, 1469, 1429, 1297, 1239, 1183, 1031, 871, 804,
749, 698 cm21; ESI FTMS exact mass calcd for (C22H24N2O9 +
Na)+ requires m/z 483.1374, found m/z 483.1353.
59,59-Diethyl 39,49-dimethyl 1-isopropyl-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4daa). (Flash col-
umn chromatography eluent, petroleum ether–ethyl acetate =
4 : 1); reaction time = 36 h; yield: 76%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.43 (dd, J = 7.4, 1.0 Hz, 1H),
7.27 (td, J = 7.8, 1.3 Hz, 1H), 7.02 (td, J = 7.6, 0.8 Hz, 1H), 6.95
(d, J = 7.9 Hz, 1H), 4.62–4.49 (m, 1H), 4.42–4.29 (m, 4H), 3.82
(s, 3H), 3.77 (s, 1H), 3.51 (s, 3H), 1.49 (d, J = 5.8 Hz, 3H), 1.47
(d, J = 5.7 Hz, 3H), 1.38–1.33 (m, 3H), 1.31 (d, J = 7.1 Hz, 3H);
13C NMR (100 MHz, CDCl3) d (ppm): 174.0, 168.7, 168.0, 162.6,
161.2, 142.7, 140.1, 139.7, 130.0, 129.0, 125.0, 122.7, 109.9,
79.9, 75.1, 63.0, 62.8, 52.4, 52.3, 44.5, 19.1, 13.9, 13.8; IR (KBr):
c 3458, 3339, 2964, 2926, 2852, 1729, 1660, 1607, 1453, 1443,
1289, 1252, 1117, 1035, 871, 772 cm21; ESI FTMS exact mass
calcd for (C24H28N2O9 + Na)+ requires m/z 511.1687, found m/z
511.1662.
59,59-Diethyl 39,49-dimethyl 1-cyclopentyl-2-oxospiro[indo-
line-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4eaa).
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125.3, 125.0, 124.7, 123.4, 123.0, 109.8, 80.0, 75.1, 63.2, 62.9,
52.6, 52.4, 42.7, 13.9, 13.8; IR (KBr): c 3461, 3334, 2994, 2923,
2854, 1729, 1660, 1620, 1501, 1469, 1361, 1294, 1249, 1183,
1117, 1031, 854, 817, 690 cm21; ESI FTMS exact mass calcd for
(C32H30N2O9 + Na)+ requires m/z 609.1844, found m/z 609.1836.
59,59-Diethyl 39,49-dimethyl 4-chloro-1-methyl-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4haa).
(Flash column chromatography eluent, petroleum ether–
acetone = 5 : 1); reaction time = 36 h; yield: 62%; light yellow
109.7, 80.1, 75.0, 63.2, 63.0, 52.6, 52.5, 26.7, 13.9, 13.8; IR
(KBr): c 3352, 2994, 2954, 2859, 1742, 1647, 1603, 1469, 1429,
1252, 1133, 1035, 939, 857, 749, 680 cm21; ESI FTMS exact
mass calcd for (C22H23BrN2O9 + Na)+ requires m/z 563.0462,
found m/z 563.0476.
59,59-Diethyl 39,49-dimethyl 5-chloro-1-methyl-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4laa).
(Flash column chromatography eluent, petroleum ether–
acetone = 5 : 1); reaction time = 36 h; yield: 86%; light yellow
solid; m.p. 128–130 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.43 (d, J = 2.1 Hz, 1H), 7.29 (dd, J = 8.3, 2.1 Hz, 1H), 6.74 (d, J =
8.3 Hz, 1H), 4.36 (m, 4H), 3.84 (s, 3H), 3.77 (s, 1H), 3.56 (s, 3H),
3.20 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d (ppm): 174.1, 168.4, 167.6, 162.5,
160.9, 142.5, 140.5, 138.7, 130.5, 130.1, 128.6, 125.3, 109.3,
80.1, 75.0, 63.2, 63.0, 52.6, 52.5, 26.8, 13.9, 13.8; IR (KBr): c
3334, 2954, 2844, 1729, 1660, 1620, 1456, 1294, 1252, 1180,
1113, 1088, 1048, 867, 806, 749, 693 cm21; ESI FTMS exact
mass calcd for (C22H23ClN2O9 + H)+ requires m/z 495.1170,
found m/z 495.1186.
59,59-Diethyl 39,49-dimethyl 5-fluoro-1-methyl-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4maa).
(Flash column chromatography eluent, petroleum ether–
acetone = 5 : 1); reaction time = 36 h; yield: 81%; light yellow
sticky oil; 1H NMR (400 MHz, CDCl3) d (ppm): 7.22 (dd, J = 7.5,
2.6 Hz, 1H), 7.01 (td, J = 8.8, 2.6 Hz, 1H), 6.74 (dd, J = 8.5, 4.0
Hz, 1H), 4.39–4.31 (m, 4H), 3.83 (s, 3H), 3.77 (s, 1H), 3.55 (s,
3H), 3.21 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H);
13C NMR (100 MHz, CDCl3) d (ppm): 174.3, 168.5, 167.7, 162.5,
161.0, 160.7, 158.3, 140.3, 139.9, 139.0, 130.4, 116.6, 116.3,
113.1, 112.9, 108.9, 108.8, 80.1, 75.3, 75.3, 63.1, 63.0, 52.5,
26.8, 13.9, 13.8; IR (KBr): c 3334, 2959, 2913, 2854, 1739, 1660,
1634, 1494, 1443, 1294, 1249, 1035, 864, 809, 693 cm21; ESI
FTMS exact mass calcd for (C22H23FN2O9 + H)+ requires m/z
479.1466, found m/z 479.1463.
1
sticky oil; H NMR (400 MHz, CDCl3) d (ppm): 7.27 (t, J = 8.0
Hz, 1H), 6.98 (dd, J = 8.2, 0.6 Hz, 1H), 6.72 (d, J = 7.4 Hz, 1H),
4.40–4.28 (m, 4H), 3.89 (s, 1H), 3.85 (s, 3H), 3.58 (s, 3H), 3.19
(s, 3H), 1.37–1.33 (m, 3H), 1.33–1.30 (m, 3H); 13C NMR (100
MHz, CDCl3) d (ppm): 173.7, 168.2, 167.6, 162.6, 161.5, 146.3,
141.6, 138.0, 131.8, 131.6, 124.0, 123.4, 106.8, 80.0, 75.2, 63.1,
62.8, 52.5, 26.8, 13.8; IR (KBr): c 3334, 2981, 2926, 2859, 1742,
1657, 1617, 1484, 1439, 1371, 1252, 1130, 1034, 871, 749, 693
cm21; ESI FTMS exact mass calcd for (C28H28N2O9 + H)+
requires m/z 495.1170, found m/z 495.1146.
59,59-Diethyl 39,49-dimethyl 1,5-dimethyl-2-oxospiro[indo-
line-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4iaa). (Flash
column chromatography eluent, petroleum ether–acetone =
5 : 1); reaction time = 36 h; yield: 92%; light yellow sticky oil;
1H NMR (400 MHz, CDCl3) d (ppm): 7.22 (s, 1H), 7.10 (d, J = 7.9
Hz, 1H), 6.69 (d, J = 7.9 Hz, 1H), 4.40–4.30 (m, 4H), 3.83 (s, 3H),
3.73 (s, 1H), 3.53 (s, 3H), 3.19 (s, 3H), 2.30 (s, 3H), 1.36 (t, J =
7.1 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H) ; 13C NMR (100 MHz, CDCl3)
d (ppm): 174.5, 168.7, 168.0, 162.7, 161.2, 141.6, 139.8, 139.8,
132.9, 130.6, 128.4, 125.4, 108.0, 80.0, 75.3, 63.1, 62.8, 52.5,
52.4, 26.6, 21.0, 13.9, 13.8; IR (KBr): c 3334, 2981, 2939, 2864,
1742, 1633, 1511, 1456, 1426, 1262, 1143, 1031, 871, 772, 693
cm21; ESI FTMS exact mass calcd for (C23H26N2O9 + H)+
requires m/z 475.1717, found m/z 475.1689.
59,59-Diethyl 39,49-dimethyl 5-methoxy-1-methyl-2-oxospir-
o[indoline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4jaa).
(Flash column chromatography eluent, petroleum ether–
acetone = 5 : 1); reaction time = 36 h; yield: 87%; light yellow
solid; m.p. 127–128 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.07 (d, J = 2.6 Hz, 1H), 6.83 (dd, J = 8.5, 2.6 Hz, 1H), 6.71 (d, J =
8.5 Hz, 1H), 4.39–4.31 (m, 4H), 3.83 (s, 3H), 3.77 (s, 3H), 3.75
(s, 1H), 3.54 (s, 3H), 3.19 (s, 3H), 1.38–1.33 (m, 3H), 1.31 (d, J =
7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.3, 168.6,
167.9, 162.6, 161.2, 156.5, 139.8, 139.7, 137.4, 129.8, 114.9,
111.7, 108.7, 80.0, 75.5, 63.0, 62.9, 55.8, 52.5, 52.4, 26.7, 13.9,
13.8; IR (KBr): c 3344, 2981, 2926, 2872, 1742, 1660, 1607, 1456,
1361, 1294, 1239, 1183, 1117, 1035, 871, 745, 693 cm21; ESI
FTMS exact mass calcd for (C23H26N2O10 + Na)+ requires m/z
513.1479, found m/z 513.1476.
59,59-Diethyl 39,49-dimethyl 5-bromo-1-methyl-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4kaa).
(Flash column chromatography eluent, petroleum ether–
acetone = 5 : 1); reaction time = 36 h; yield: 85%; light yellow
solid; m.p. 148–150 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.56 (d, J = 1.9 Hz, 1H), 7.44 (dd, J = 8.3, 2.0 Hz, 1H), 6.69 (d, J =
8.3 Hz, 1H), 4.41–4.30 (m, 4H), 3.84 (s, 3H), 3.76 (s, 1H), 3.57
(s, 3H), 3.20 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.32 (t, J = 7.1 Hz,
3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.0, 168.3, 167.6,
162.5, 160.9, 143.0, 140.5, 138.6, 133.1, 130.9, 128.0, 115.9,
59,59-Diethyl 39,49-dimethyl 1-benzyl-5-methyl-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4naa).
(Flash column chromatography eluent, petroleum ether–
acetone = 6 : 1); reaction time = 36 h; yield: 61%; light yellow
1
sticky oil; H NMR (400 MHz, CDCl3) d (ppm): 7.38 (d, J = 7.0
Hz, 2H), 7.34–7.28 (m, 2H), 7.28–7.21 (m, 2H), 6.99 (dd, J = 7.9,
0.9 Hz, 1H), 6.59 (d, J = 7.9 Hz, 1H), 5.10 (d, J = 15.6 Hz, 1H),
4.63 (d, J = 15.6 Hz, 1H), 4.37 (m, 4H), 3.85 (s, 3H), 3.80 (s, 1H),
3.41 (s, 3H), 2.27 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H), 1.33 (t, J = 7.1
Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.6, 168.6,
168.0, 162.7, 161.2, 140.8, 140.3, 139.7, 135.6, 133.0, 130.5,
128.7, 128.5, 127.7, 127.6, 125.4, 109.1, 79.9, 75.2, 63.1, 62.9,
52.5, 52.3, 44.5, 21.0, 13.9, 13.8; IR (KBr): c 3334, 2978, 2936,
2872, 1732, 1647, 1620, 1469, 1443, 1389, 1255, 1117, 1035,
933, 857, 745, 690 cm21; ESI FTMS exact mass calcd for
(C29H30N2O9 + H)+ requires m/z 551.2030, found m/z 551.2040.
59,59-Diethyl 39,49-dimethyl 1-benzyl-5-methoxy-2-oxospir-
o[indoline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4oaa).
(Flash column chromatography eluent, petroleum ether–
acetone = 6 : 1); reaction time = 36 h; yield: 62%; light yellow
solid; m.p. 145–147 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.41–7.35 (m, 2H), 7.35–7.27 (m, 3H), 7.07 (d, J = 2.6 Hz, 1H),
6.72 (dd, J = 8.5, 2.6 Hz, 1H), 6.60 (d, J = 8.5 Hz, 1H), 5.09 (d, J =
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15.5 Hz, 1H), 4.63 (d, J = 15.6 Hz, 1H), 4.37 (m, 4H), 3.84 (s,
3H), 3.82 (s, 1H), 3.74 (s, 3H), 3.42 (s, 3H), 1.36 (t, J = 6.0 Hz,
3H), 1.33 (t, J = 6.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.5, 168.6, 167.9, 162.6, 161.1, 156.5, 140.4, 139.6,
136.4, 135.6, 129.9, 128.7, 127.7, 114.9, 111.6, 109.9, 79.9, 75.4,
63.1, 62.9, 55.7, 52.5, 52.3, 44.6, 13.9, 13.9; IR (KBr): c 3444,
3334, 2984, 2923, 2859, 1729, 1610, 1498, 1469, 1371, 1296,
1252, 1170, 1120, 1018, 857, 745 cm21; ESI FTMS exact mass
calcd for (C29H30N2O10 + H)+ requires m/z 567.1979, found m/z
567.1973.
59,59-Diethyl 39,49-dimethyl 1-benzyl-5-bromo-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4paa).
(Flash column chromatography eluent, petroleum ether–
acetone = 6 : 1); reaction time = 36 h; yield: 67%; light yellow
solid; m.p. 118–120 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.56 (d, J = 2.0 Hz, 1H), 7.39–7.26 (m, 6H), 6.57 (d, J = 8.3 Hz,
1H), 5.09 (d, J = 15.6 Hz, 1H), 4.66 (d, J = 15.6 Hz, 1H), 4.37 (m,
4H), 3.86 (s, 3H), 3.83 (s, 1H), 3.46 (s, 3H), 1.39 (t, J = 7.1 Hz,
3H), 1.33 (t, J = 7.1 Hz, 3H) ; 13C NMR (100 MHz, CDCl3) d
(ppm): 174.2, 168.3, 167.6, 162.5, 160.8, 142.2, 141.1, 138.5,
135.0, 133.0, 131.0, 128.8, 128.0, 127.6, 116.0, 110.8, 80.1, 74.9,
63.3, 63.1, 62.8, 52.6, 44.6, 13.9, 13.8; IR (KBr): c 3458, 3346,
2988, 2923, 2862, 1739, 1657, 1607, 1484, 1364, 1297, 1235,
1103, 1028, 867, 817, 680 cm21; ESI FTMS exact mass calcd for
(C28H27BrN2O9 + Na)+ requires m/z 639.0776, found m/z
639.0770.
59,59-Diethyl 39,49-dimethyl 1-benzyl-6-bromo-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4qaa).
(Flash column chromatography eluent, petroleum ether–ethyl
acetate = 6 : 1); reaction time = 36 h; yield: 63%; light yellow
sticky oil; 1H NMR (400 MHz, CDCl3) d (ppm): 7.29 (dd, J = 7.1,
3.8 Hz, 3H), 7.26–7.17 (m, 3H), 7.08 (dd, J = 7.9, 1.6 Hz, 1H),
6.77 (d, J = 1.6 Hz, 1H), 5.01 (d, J = 15.6 Hz, 1H), 4.54 (d, J = 15.6
Hz, 1H), 4.34–4.22 (m, 4H), 3.76 (s, 3H), 3.68 (s, 1H), 3.35 (s,
3H), 1.26 (dd, J = 15.3, 7.2 Hz, 6H) ; 13C NMR (100 MHz, CDCl3)
d (ppm): 174.6, 168.5, 167.8, 162.5, 161.0, 144.5, 140.9, 138.8,
134.9, 128.9, 127.9, 127.7, 127.6, 126.3, 126.1, 123.9, 112.7,
79.9, 74.6, 63.2, 63.0, 52.6, 52.5, 44.6, 13.9, 13.8; IR (KBr): c
3334, 2981, 2954, 2859, 1742, 1660, 1617, 1484, 1429, 1294,
1252, 1113, 1035, 857, 817, 693 cm21; ESI FTMS exact mass
calcd for (C28H27BrN2O9 + Na)+ requires m/z 639.0776, found
m/z 639.0786.
59,59-Diethyl 39,49-dimethyl 5-methyl-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4saa). (Flash col-
umn chromatography eluent, petroleum ether–acetone
=
5 : 1); reaction time = 36 h; yield: 99%; yellow solid; m.p.
1
157–159 uC; H NMR (400 MHz, CDCl3) d (ppm): 8.28 (s, 1H),
7.20 (s, 1H), 7.08–7.00 (m, 1H), 6.74 (d, J = 7.9 Hz, 1H), 4.36 (m,
4H), 3.85 (s, 3H), 3.75 (s, 1H), 3.57 (s, 3H), 2.29 (s, 3H), 1.37 (t, J
= 7.1 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H) ; 13C NMR (100 MHz,
CDCl3) d (ppm): 176.5, 168.6, 168.0, 162.6, 161.3, 139.9, 139.8,
138.7, 132.8, 130.6, 128.9, 125.6, 110.0, 80.0, 75.6, 63.1, 62.8,
52.5, 52.5, 21.0, 13.9, 13.8; IR (KBr): c 3339, 2992, 2943, 2923,
2846, 1736, 1610, 1493, 1474, 1448, 1299, 1254, 1127, 1037,
864, 751 cm21; ESI FTMS exact mass calcd for (C22H24N2O9 +
Na)+ requires m/z 483.1374, found m/z 483.1364.
59,59-Diethyl 39,49-dimethyl 5-methoxy-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4taa). (Flash col-
umn chromatography eluent, petroleum ether–acetone
=
5 : 1); reaction time = 36 h; yield: 99%; light yellow solid;
1
m.p. 140–142 uC; H NMR (400 MHz, CDCl3) d (ppm): 8.21 (s,
1H), 7.03 (d, J = 2.0 Hz, 1H), 6.84–6.68 (m, 2H), 4.43–4.27 (m,
4H), 3.84 (s, 3H), 3.76 (s, 4H), 3.57 (s, 3H), 1.36 (t, J = 7.2 Hz,
3H), 1.32 (t, J = 7.2 Hz, 3H) ; 13C NMR (100 MHz, CDCl3) d
(ppm): 176.3, 168.6, 167.9, 162.6, 161.2, 156.3, 140.0, 139.6,
134.3, 130.2, 115.4, 111.6, 110.7, 80.0, 75.9, 63.1, 62.9, 55.7,
52.5, 52.5, 13.9, 13.8; IR (KBr): c 3321, 2984, 2929, 2859, 1742,
1660, 1607, 1469, 1443, 1371, 1294, 1239, 1183, 1143, 1103,
1035, 857, 779, 735 cm21; ESI FTMS exact mass calcd for
(C22H24N2O10 + H)+ requires m/z 477.1509, found m/z 477.1517.
59,59-Diethyl 39,49-dimethyl 5-fluoro-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4uaa). (Flash col-
umn chromatography eluent, petroleum ether–acetone
=
5 : 1); reaction time = 36 h; yield: 99%; light yellow solid;
1
m.p. 165–167 uC; H NMR (400 MHz, CDCl3) d (ppm): 8.42 (s,
1H), 7.18 (dd, J = 7.6, 2.6 Hz, 1H), 6.95 (td, J = 8.8, 2.6 Hz, 1H),
6.80 (dd, J = 8.5, 4.1 Hz, 1H), 4.35 (m, 4H), 3.85 (s, 3H), 3.80 (s,
1H), 3.58 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H);
13C NMR (100 MHz, CDCl3) d (ppm): 176.5, 168.4, 167.7, 162.4,
161.1, 160.5, 158.1, 140.4, 139.0, 137.1, 130.8, 116.8, 116.6,
113.2, 112.9, 111.1, 111.0, 80.0, 75.8, 63.2, 63.0, 52.6, 13.9,
13.8; IR (KBr): c 3468, 3349, 2971, 2939, 2862, 1742, 1670, 1607,
1484, 1443, 1347, 1294, 1249, 1183, 1130, 1035, 857, 755, 709
cm21; ESI FTMS exact mass calcd for (C21H21FN2O9 + H)+
requires m/z 465.1309, found m/z 465.1301.
59,59-Diethyl 39,49-dimethyl 1-benzyl-7-bromo-2-oxospiro[in-
doline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4raa).
(Flash column chromatography eluent, petroleum ether–ethyl
acetate = 6 : 1); reaction time = 36 h; yield: 79%; light yellow
solid; m.p. 134–137 uC; 1H NMR (400 MHz, CDCl3) d (ppm):
7.43–7.38 (m, 2H), 7.36–7.27 (m, 4H), 7.24 (s, 1H), 6.93 (dd, J =
8.1, 7.4 Hz, 1H), 5.47–5.25 (m, 2H), 4.40–4.31 (m, 4H), 3.84 (s,
3H), 3.80 (s, 1H), 3.51 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H), 1.31 (t, J =
7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 175.6, 168.5,
167.7, 162.5, 161.0, 141.0, 140.8, 138.9, 137.2, 136.1, 132.14,
128.4, 127.1, 126.8, 124.7, 124.1, 102.5, 79.9, 74.3, 63.2, 63.0,
52.6, 52.5, 45.2, 13.9, 13.8; IR (KBr): c 3334, 2968, 2872, 1729,
59,59-Diethyl 39,49-dimethyl 7-bromo-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4vaa). (Flash col-
umn chromatography eluent, petroleum ether–acetone
=
5 : 1); reaction time = 36 h; yield: 76%; light yellow solid;
m.p. 95–96 uC; 1H NMR (400 MHz, CDCl3) d (ppm): 7.70 (s,
1H), 7.37 (dd, J = 12.4, 4.1 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 4.41–
4.30 (m, 4H), 3.85 (s, 3H), 3.78 (s, 1H), 3.58 (s, 3H), 1.35 (t, J =
6.7 Hz, 3H), 1.30 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d (ppm): 174.8, 168.4, 167.6, 162.5, 160.9, 140.6, 140.4, 138.7,
132.8, 130.5, 124.5, 124.04, 103.0, 80.1, 76.5, 63.2, 63.0, 52.6,
13.9, 13.8; IR (KBr): c 3334, 2981, 2923, 2859, 1732, 1607, 1479,
1456, 1374, 1294, 1254, 1117, 1035, 857 cm21; ESI FTMS exact
mass calcd for (C21H21BrN2O9 + Na)+ requires m/z 549.0305,
found m/z 549.0320.
1660, 1607, 1474, 1443, 1307, 1239, 1173, 1031, 871, 749 cm21
;
ESI FTMS exact mass calcd for (C28H27BrN2O9 + Na)+ requires
m/z 639.0776, found m/z 639.0779.
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59,59-Diethyl 39,49-dimethyl 2-oxo-7-(trifluoromethyl)spir-
o[indoline-3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate
(4waa). (Flash column chromatography eluent, petroleum
ether–acetone = 6 : 1); reaction time = 36 h; yield: 58%; light
Tetraethyl 1-cyclopentyl-2-oxospiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4eab). (Flash column chroma-
tography eluent, petroleum ether–ethyl acetate
= 6 : 1);
reaction time = 36 h; yield: 87%; yellow sticky oil; 1H NMR
(400 MHz, CDCl3) d (ppm): 7.45 (dd, J = 7.4, 1.0 Hz, 1H), 7.27
(td, J = 7.8, 1.3 Hz, 1H), 7.03 (td, J = 7.6, 0.7 Hz, 1H), 6.88 (d, J =
7.9 Hz, 1H), 4.73–4.64 (m, 1H), 4.39–4.25 (m, 6H), 4.06–3.87
(m, 2H), 3.77 (s, 1H), 2.14–1.88 (m, 6H), 1.71–1.62 (m, 2H),
1.37–1.29 (m, 9H), 0.93 (t, J = 7.1 Hz, 3H) ; 13C NMR (100 MHz,
CDCl3) d (ppm): 174.4, 168.8, 168.2, 162.1, 160.7, 142.7, 140.3,
139.6, 129.8, 129.2, 125.1, 122.8, 109.6, 79.9, 75.1, 62.9, 62.8,
61.7, 61.3, 53.0, 27.7, 27.3, 25.2, 25.1, 13.9, 13.9, 13.8, 13.6; IR
(KBr): c 3484, 3344, 2981, 2939, 2859, 1739, 1663, 1607, 1456,
1439, 1294, 1249, 1130, 1038, 867, 749 cm21; ESI FTMS exact
mass calcd for (C28H34N2O9 + Na)+ requires m/z 565.2157,
found m/z 565.2131.
1
yellow sticky oil; H NMR (400 MHz, CDCl3) d (ppm): 7.87 (s,
1H), 7.62 (d, J = 7.4 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.14 (t, J =
7.8 Hz, 1H), 4.36 (dd, J = 7.0, 5.0 Hz, 4H), 3.85 (s, 3H), 3.80 (s,
1H), 3.56 (s, 3H), 1.34 (m, 6H); 13C NMR (100 MHz, CDCl3) d
(ppm): 175.3, 168.4, 167.6, 162.4, 160.7, 141.1, 138.5, 138.3,
131.0, 128.7, 126.9, 126.8, 125.0, 123.1, 122.3, 112.5, 112.2,
80.1, 74.2, 63.2, 63.0, 52.6, 52.5, 13.9, 13.8; IR (KBr): c 3349,
2981, 2949, 2859, 1739, 1660, 1607, 1498, 1446, 1299, 1235,
1130, 1031, 871, 814, 680 cm21; ESI FTMS exact mass calcd for
(C22H21F3N2O9
515.1286.
+
H)+ requires m/z 515.1277, found m/z
59,59-Diethyl 39,49-dimethyl 5,6-difluoro-2-oxospiro[indoline-
3,29-pyrrole]-39,49,59,59(19H)-tetracarboxylate (4xaa). (Flash col-
Tetraethyl 2-oxo-1-phenylspiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4fab). (Flash column chroma-
umn chromatography eluent, petroleum ether–acetone
=
tography eluent, petroleum ether–ethyl acetate
= 6 : 1);
5 : 1); reaction time = 36 h; yield: 52%; light yellow solid;
m.p. 175–177 uC; H NMR (400 MHz, CDCl3) d (ppm): 8.50 (s,
reaction time = 36 h; yield: 84%; yellow sticky oil; 1H NMR
(400 MHz, CDCl3) d (ppm): 7.56–7.34 (m, 6H), 7.23 (dd, J = 7.8,
1.2 Hz, 1H), 7.10 (td, J = 7.6, 0.7 Hz, 1H), 6.81 (d, J = 7.9 Hz,
1H), 4.43–4.21 (m, 6H), 4.12–3.94 (m, 2H), 3.88 (s, 1H), 1.42–
1.27 (m, 9H), 0.98 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d (ppm): 174.1, 168.8, 168.0, 162.1, 160.8, 144.0, 140.0,
139.8, 134.3, 130.1, 129.5, 128.4, 128.1, 126.2, 125.1, 123.7,
109.5, 80.1, 75.4, 63.0, 62.8, 61.8, 61.6, 13.9, 13.9, 13.9, 13.7; IR
(KBr): c 3458, 3336, 2981, 2951, 2861, 1739, 1663, 1608, 1484,
1439, 1307, 1257, 1183, 1133, 1048, 861, 821, 740, 710, 640
cm21; ESI FTMS exact mass calcd for (C29H30N2O9 + H)+
requires m/z 551.2030, found m/z 551.2030.
1
1H), 7.35–7.27 (m, 1H), 6.73 (dd, J = 9.7, 6.3 Hz, 1H), 4.35 (dt, J
= 12.1, 7.1 Hz, 4H), 3.85 (s, 3H), 3.78 (s, 1H), 3.61 (s, 3H), 1.37
(t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d (ppm): 176.5, 168.4, 167.7, 162.4, 161.0, 140.6, 138.5,
137.4, 124.7, 115.0, 114.7, 100.7, 100.5, 80.0, 75.3, 63.3, 63.1,
52.7, 52.6, 13.9, 13.8; IR (KBr): c 3344, 2954, 2841, 1729, 1660,
1607, 1484, 1443, 1265, 1209, 1143, 1601, 1031, 871, 807, 693,
598 cm21; ESI FTMS exact mass calcd for (C21H20F2N2O9 + Na)+
requires m/z 505.1029, found m/z505.1020.
Tetraethyl 2-oxospiro[indoline-3,29-pyrrole]-39,49,59,59(19H)-
tetracarboxylate (4bab). (Flash column chromatography elu-
ent, petroleum ether–acetone = 4 : 1); reaction time = 36 h;
yield: 99%; yellow sticky oil; 1H NMR (400 MHz, CDCl3) d
(ppm): 8.28 (s, 1H), 7.41 (d, J = 7.4 Hz, 1H), 7.23 (dd, J = 7.7, 1.2
Hz, 2H), 7.03 (td, J = 7.6, 0.9 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H),
4.49–4.17 (m, 6H), 4.11–3.83 (m, 2H), 3.76 (s, 1H), 1.37–1.30
(m, 9H), 0.98 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 176.5, 168.7, 168.1, 162.1, 160.8, 141.2, 139.9, 139.8,
130.2, 129.2, 125.1, 123.2, 110.1, 80.0, 75.5, 63.0, 62.8, 61.8,
61.6, 13.9, 13.9, 13.4; IR (KBr): c 3362, 3444, 2964, 2876, 1739,
1670, 1610, 1469, 1389, 1374, 1294, 1239, 1180, 1130, 1103,
Tetraethyl 1-benzyl-2-oxospiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4aab). (Flash column chro-
matography eluent, petroleum ether–ethyl acetate = 6 : 1);
reaction time = 36 h; yield: 85%; yellow sticky oil; 1H NMR (400
MHz, CDCl3) d 7.45–7.24 (m, 6H),7.19 (td, J = 7.8, 1.2 Hz, 1H)
7.02 (td, J = 7.6, 0.8 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 5.02 (d, J =
15.6 Hz, 1H), 4.74 (d, J = 15.6 Hz, 1H), 4.39–4.24 (m, 6H),4.01–
3.95 (m, 1H), 3.82–3.77 (m, 2H), 1.38–1.28 (m, 9H), 0.81 (t, J =
7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.8, 168.7,
168.1, 162.1, 160.7, 143.3, 140.2, 139.8, 135.5, 130.1, 128.8,
128.7, 127.7, 124.8, 123.3, 109.2, 79.9, 75.1, 63.0, 62.8, 61.8,
61.4, 44.5, 13.9, 13.9, 13.4; IR (KBr): c 3349, 2939, 2859, 1729,
1660, 1607, 1484, 1443, 1294, 1249, 1130, 1035, 857, 804, 680
cm21; ESI FTMS exact mass calcd for (C30H32N2O9 + Na)+
requires m/z 587.2000, found m/z 587.2009.
1035, 857, 749 cm21
; ESI FTMS exact mass calcd for
(C23H26N2O9 + Na)+ requires m/z 497.1531, found m/z 497.1531.
Tetraethyl 1-methyl-2-oxospiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4cab). (Flash column chroma-
tography eluent, petroleum ether–ethyl acetate
= 4 : 1);
reaction time = 36 h; yield: 94%; yellow sticky oil; 1H NMR
(400 MHz, CDCl3) d (ppm): 7.43 (d, J = 7.3 Hz, 1H), 7.31 (td, J =
7.7, 1.1 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H),
4.41–4.15 (m, 6H), 4.00–3.88 (m, 2H), 3.74 (s, 1H), 3.20 (s, 3H),
1.37–1.29 (m, 9H), 0.95 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d (ppm): 174.7, 168.7, 168.1, 162.1, 160.7, 144.0, 139.9,
139.8, 130.2, 128.7, 124.7, 123.3, 108.1, 79.9, 75.1, 63.0, 62.8,
61.7, 61.3, 29.6, 26.5, 13.9, 13.8, 13.5; IR (KBr): c 3334, 2994,
2954, 2859, 1739, 1660, 1484, 1429, 1262, 1035, 857, 804, 680,
598 cm21; ESI FTMS exact mass calcd for (C24H28N2O9 + Na)+
requires m/z 511.1687, found m/z 511.1687.
Tetraethyl 1,5-dimethyl-2-oxospiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4iab). (Flash column chroma-
tography eluent, petroleum ether–ethyl acetate
= 3 : 1);
reaction time = 36 h; yield: 86%; yellow sticky oil; 1H NMR
(400 MHz, CDCl3) d (ppm): 7.25 (m, 1H), 7.09 (m, 1H), 6.67 (d, J
= 7.9 Hz, 1H), 4.38–4.26 (m, 6H), 4.01–3.89 (m, 2H), 3.74 (s,
1H), 3.18 (s, 3H), 2.30 (s, 3H), 1.38–1.29 (m, 9H), 0.96 (t, J = 7.1
Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.6, 168.8,
168.1, 162.1, 160.7, 141.6, 140.0, 139.7, 132.9, 130.4, 128.6,
125.5, 107.9, 79.9, 75.3, 63.0, 62.8, 61.7, 61.3, 26.6, 21.0, 13.9,
13.8, 13.5; IR (KBr): c 3461, 3352, 2981, 2923, 2859, 1739, 1667,
1607, 1501, 1456, 1361, 1294, 1252, 1130, 1035, 939, 844, 762,
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693 cm21; ESI FTMS exact mass calcd for (C25H30N2O9 + Na)+
requires m/z 525.1844, found m/z 525.1840.
817, 698 cm21; ESI FTMS exact mass calcd for (C24H27BrN2O9 +
Na)+ requires m/z 589.0792, found m/z 589.0799.
Tetraethyl 5-methoxy-1-methyl-2-oxospiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4jab). (Flash column
Tetraethyl 2-oxo-7-(trifluoromethyl)spiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4wab). (Flash column
chromatography eluent, petroleum ether–ethyl acetate
=
chromatography eluent, petroleum ether–ethyl acetate =
3 : 1); reaction time = 36 h; yield: 71%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.09 (d, J = 2.6 Hz, 1H), 6.83
(dd, J = 8.5, 2.6 Hz, 1H), 6.70 (d, J = 8.5 Hz, 1H), 4.38–4.26 (m,
6H), 4.02–3.90 (m, 2H), 3.77 (s, 3H), 3.76 (s, 1H),3.17 (s, 3H),
1.37–1.29 (m, 9H), 0.97 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d (ppm): 174.4, 168.7, 168.0, 162.1, 160.6, 156.5, 139.9,
139.8, 137.4, 130.0, 114.9, 111.7, 108.6, 79.9, 75.5, 62.9, 62.8,
61.7, 61.4, 55.8, 26.6, 13.9, 13.9, 13.8, 13.5; IR (KBr): c 3334,
2991, 2926, 1742, 1660, 1607, 1456, 1374, 1252, 1198, 1127,
1035, 857, 762, 697 cm21; ESI FTMS exact mass calcd for
4 : 1); reaction time = 36 h; yield: 73%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.78 (s, 1H), 7.64 (d, J = 7.4
Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 4.38–
4.26 (m, 6H), 4.05–3.95 (m, 2H), 3.81 (s, 1H), 1.61 (s, 1H), 1.37–
1.29 (m, 9H), 0.98 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d (ppm): 175.4, 168.5, 167.7, 161.9, 160.2, 141.3, 138.5,
138.3, 131.2, 128.8, 126.8, 125.0, 123.1, 122.3, 112.4, 112.0,
80.0, 74.2, 63.1, 63.0, 62.7, 62.0, 61.7, 13.9, 13.8, 13.3; IR (KBr):
c 3331, 2926, 2852, 1732, 1663, 1617, 1469, 1449, 1361, 1294,
1252, 1184, 1130, 1031, 857, 789, 762 cm21; ESI FTMS exact
mass calcd for (C24H25F3N2O9 + Na)+ requires m/z 565.1404,
found m/z 565.1420.
(C25H30N2O10
541.1780.
+
Na)+ requires m/z 541.1793, found m/z
Tetraethyl 5-bromo-1-methyl-2-oxospiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4kab). (Flash column
Tetraethyl 7-bromo-2-oxospiro[indoline-3,29-pyrrole]-
39,49,59,59(19H)-tetracarboxylate (4vab). (Flash column chroma-
chromatography eluent, petroleum ether–ethyl acetate
=
tography eluent, petroleum ether–ethyl acetate
= 3 : 1);
3 : 1); reaction time = 36 h; yield: 79%; light yellow solid;
m.p. 128–130 uC; 1H NMR (400 MHz, CDCl3) d (ppm): 7.58 (d, J
= 2.0 Hz, 1H), 7.43 (dd, J = 8.3, 2.0 Hz, 1H), 6.68 (d, J = 8.3 Hz,
1H), 4.40–4.26 (m, 6H), 4.04–3.92 (m, 2H), 3.77 (s, 1H), 3.19 (s,
3H), 1.40–1.30 (m, 9H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (100
MHz, CDCl3) d (ppm): 174.2, 168.4, 167.8, 161.9, 160.4, 143.0,
140.5, 138.8, 132.9, 131.12, 128.0, 115.8, 109.6, 80.1, 75.0, 63.1,
63.0, 61.9, 61.6, 26.7, 13.9, 13.8, 13.5; IR (KBr): c 3349, 2994,
2958, 2872, 1742, 1660, 1633, 1488, 1443, 1265, 1035, 923, 814,
732, 690 cm21; ESI FTMS exact mass calcd for (C24H27BrN2O9 +
Na)+ requires m/z 589.0792, found m/z 589.0781.
reaction time = 36 h; yield: 65%; light yellow solid; m.p.
191–193 uC; H NMR (400 MHz, CDCl3) d (ppm): 7.58 (s, 1H),
1
7.39–7.37 (m, 2H), 6.95 (dd, J = 8.2, 7.5 Hz, 1H), 4.37–4.28 (m,
6H), 4.13–3.93 (m, 2H), 3.79 (s, 1H), 1.37–1.30 (m, 9H), 1.01 (t, J
= 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 174.9,
168.5, 167.8, 161.9, 160.3, 140.6, 140.4, 138.8, 132.7, 130.8,
124.5, 124.1, 102.9, 80.1, 76.5, 63.1, 62.9, 61.9, 61.7, 13.9, 13.9,
13.8, 13.4; IR (KBr): c 3349, 2968, 2913, 2859, 1739, 1647, 1610,
1494, 1443, 1297, 1249, 1130, 1035, 857, 804, 690 cm21; ESI
FTMS exact mass calcd for (C23H25BrN2O9 + Na)+ requires m/z
575.0636, found m/z 575.0639.
Tetraethyl 5-chloro-1-methyl-2-oxospiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4lab). (Flash column
Cytotoxic evaluation of compounds 4 to mammary carcinoma
cell line MCF-7
chromatography eluent, petroleum ether–ethyl acetate
=
3 : 1); reaction time = 36 h; yield: 85%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.45 (d, J = 2.1 Hz, 1H), 7.28
(dd, J = 8.3, 2.1 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H), 4.40–4.24 (m,
6H), 4.04–3.92 (m, 2H), 3.78 (s, 1H), 3.19 (s, 3H), 1.40–1.30 (m,
9H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.3, 168.4, 167.8, 161.9, 160.4, 142.5, 140.5, 138.8,
130.7, 130.0, 128.6, 125.3, 109.1, 80.1, 75.0, 63.1, 63.0, 61.9,
61.6, 29.6, 26.7, 13.9, 13.8, 13.5; IR (KBr): c 3459, 3334, 2981,
2929, 2862, 1725, 1660, 1607, 1501, 1473, 1361, 1284, 1252,
1120, 1031, 861, 819 cm21; ESI FTMS exact mass calcd for
(C24H27ClN2O9 + Na)+ requires m/z 545.1297, found m/z
545.1296.
The cytotoxicity of the tested compounds to MCF-7 cells was
assayed using the CCK8 (Cell Counting Kit-8) method. MCF-7
cells were seeded in 96-well plates at a density of 104 cells per
well with 200 mL complete culture medium. After adhesion for
24 h, the medium was changed to 1640 supplemented without
FBS and the compounds were added to the medium to final
concentrations ranging from 1 mg mL21 to 100 mg mL21. The
cells were then cultured for another 24 h. Cells that were not
exposed to compounds were used as controls and the wells to
which only culture medium was added served as blanks. At the
end of compound stimulation, the supernatant was removed,
and 100 mL 1640 medium containing 10 mL CCK8 was added to
each well for another 3 h at 37 uC. The culture plates were then
shaken for 10 min and the optical density (OD) values were
read at 450 nm. The inhibition rate was calculated according
Tetraethyl 6-bromo-1-methyl-2-oxospiro[indoline-3,29-pyr-
role]-39,49,59,59(19H)-tetracarboxylate (4yab). (Flash column
chromatography eluent, petroleum ether–ethyl acetate
=
5 : 1); reaction time = 36 h; yield: 73%; yellow sticky oil; 1H
NMR (400 MHz, CDCl3) d (ppm): 7.31 (d, J = 7.9 Hz, 1H), 7.19
(dd, J = 7.9, 1.6 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 4.38–4.26 (m,
6H), 4.03–3.92 (m, 2H), 3.72 (s, 1H), 3.18 (s, 3H), 1.36–1.29 (m,
9H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm): 174.5, 168.6, 167.9, 162.0, 160.5, 145.3, 140.4, 138.9,
127.8, 126.1, 123.9, 111.7, 80.0, 74.7, 63.1, 62.9, 61.8, 61.5,
29.6, 26.7, 13.9, 13.8, 13.6; IR (KBr): c 3349, 2981, 2926, 2872,
1745, 1660, 1617, 1498, 1456, 1357, 1289, 1249, 1031, 953, 871,
to the following formula: Inhibition rate = (OD450
2
control
OD450 sample)/OD450
6 100%, where OD stands for
control
optical density at 450 nm.
Morphological assessment and quantification of apoptotic
astrocytes
Hoechst staining. To quantify apoptotic cells, a MCF-7 cell
monolayer was fixed and stained with Hoechst 33324 (Sigma,
USA). The morphological features of apoptosis (cell shrinkage,
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Paper
H. Kakeya and H. Osada, Tetrahedron, 1996, 52, 12651; (d)
C. B. Cui, H. Kakeya and H. Osada, J. Antibiot., 1996, 49,
832.
2 (a) A. S. Girgis, Eur. J. Med. Chem., 2009, 44, 91; (b) V.
V. Kouznetsov, D. R. M. Arenas, F. Arvelo, J. S. B. Forero,
chromatin condensation, and fragmentation) were monitored
by fluorescence microscopy (Olympus BX 60, Japan). At least
400 cells from 12 randomly selected fields per dish were
counted, and each treatment was performed in triplicate.
Flow cytometry. MCF-7 cell apoptosis was estimated using
the Annexin-V Fluorescein (FITC) apoptosis detection kit
(Oncogene, USA) according to the kit instructions. The cell
samples were analyzed in a flow cytometry apparatus (Becton
˜
F. Sojo and A. Munoz, Lett. Drug Des. Discovery, 2010, 7,
632.
3 (a) S. U. Maheswari, K. Balamurugan, S. Perumal,
P. Yogeeswari and D. Sriram, Bioorg. Med. Chem. Lett.,
2010, 20, 7278; (b) R. Ranjith Kumar, S. Perumal,
P. Senthilkumar, P. Yogeeswari and D. Sriram, Eur. J.
Med. Chem., 2009, 44, 3821; (c) R. R. Kumar, S. Perumal,
P. Senthilkumar, P. Yogeeswari and D. Sriram, J. Med.
Chem., 2008, 51, 5731; (d) R. S. Kumar, S. M. Rajesh,
S. Perumal, D. Banerjee, P. Yogeeswari and D. Sriram, Eur.
J. Med. Chem., 2010, 45, 411.
4 (a) A. A. Raj, R. Raghunathan, M. R. SrideviKumari and
N. Raman, Bioorg. Med. Chem., 2003, 11, 407; (b)
G. Periyasami, R. Raghunathan, G. Surendiran and
N. Mathivanan, Bioorg. Med. Chem. Lett., 2008, 18, 2342;
(c) A. Thangamani, Eur. J. Med. Chem., 2010, 45, 6120.
5 (a) P. Prasanna, K. Balamurugan, S. Perumal, P. Yogeeswari
and D. Sriram, Eur. J. Med. Chem., 2010, 45, 5653; (b) S.
V. Karthikeyan, B. D. Bala, V. P. A. Raja, S. Perumal,
P. Yogeeswari and D. Sriram, Bioorg. Med. Chem. Lett., 2010,
20, 350.
6 M. C. Krishna, W. DeGraff, O. H. Hankovszky, C. P. Sar,
T. Kalai, J. Jeko, A. Russo, J. B. Mitchell and K. Hideg, J.
Med. Chem., 1998, 41, 3477.
7 G. Bhaskar, Y. Arun, C. Balachandran, C. Saikumar and P.
T. Perumal, Eur. J. Med. Chem., 2012, 51, 79.
8 W. K. Anderson and A. S. Milowsky, J. Med. Chem., 1987, 30,
2144.
9 J. K. Sugden and M. R. Tavakoli Saberi, Eur. J. Med. Chem.,
1979, 14, 189.
Dickinson FACSVantage SE, USA). Annexin
V binds to
phosphatidylserine that is translocated during apoptosis from
the inner to the outer leaflet of the plasma membrane. Live
cells with intact membranes are distinguished by their ability
to exclude propidium iodide (PI), which readily penetrates
dead or damaged cells. Dual analysis was introduced using a
quadrant dot plot, in which necrotic cells were identified as
single PI-positive, early apoptotic cells were annexin V-FITC-
positive only, and cells in late apoptosis were recognized as
double-positive for annexin V-FITC and PI. Cells that stained
negative for both annexin V-FITC and PI were classified as live
cells. Finally, the number of cells in each category was
expressed as a percentage of the total number of stained cells
counted.
Western blotting. Cells were washed twice with ice-cold PBS
and homogenized in 200 mL lysis buffer. After incubation for
20 min on ice, cell lysates were centrifuged (10 000 6 g for 10
min at 4 uC) and the protein concentration in the extracts was
determined by the Bradford assay.24 Proteins in cell extracts
(50 mg) were denatured with SDS sample buffer and separated
by 10% SDS-PAGE. Proteins were transferred to nitrocellulose
membranes using a Bio-Rad miniprotein-III wet transfer unit.
The membranes were incubated with 5% BSA dissolved in
TBST (pH 7.5, 10 mM Tris-HCl, 150 mM NaCl, and 0.1%
Tween-20) at room temperature for 1 h, washed three times
and incubated with different antibodies (JNK, phosphor-JNK,
ERK, phosphor-ERK p38 and phosphor-p38 at 1 : 1000) over-
night at 4 uC. The membranes were washed three times with
TBST buffer and incubated with the secondary antibody
(1 : 2000) for 1 h followed by four washings. Signal detection
was performed with an enhanced chemiluminescence kit.
Statistical analysis. All values were expressed as mean ¡
standard error of the mean (SEM). The significance of the
difference between the control and samples treated with
various drugs was determined by one-way ANOVA followed by
the post-hoc least significant difference (LSD) test. Differences
were considered significant at P , 0.05.
10 For the construction of a similar scaffold using radical
chemistry with limited substrate scope, see: (a) K. Jones, T.
C. T. Ho and J. Wilkinson, Tetrahedron Lett., 1995, 36, 6743;
(b) For reviews on synthesis of other spiro-oxindoles, see:
F. Zhou, Y.-L. Liu and J. Zhou, Adv. Synth. Catal., 2010, 352,
1381; (c) G. S. Singh and Z. Y. Desta, Chem. Rev., 2012, 112,
6104.
´
11 For reviews, see: (a) C. Najera and J. M. Sansano, Angew.
Chem., Int. Ed., 2005, 44, 6272; (b) L. M. Stanley and M.
P. Sibi, Chem. Rev., 2008, 108, 2887; (c) J. Adrio and J.
C. Carretero, Chem. Commun., 2011, 47, 6784; (d)
A. Moyano and R. Rios, Chem. Rev., 2011, 111, 4703; (e)
H. Pellissier, Tetrahedron, 2012, 68, 2197.
12 For reviews, see: (a) D. R. Spring, Org. Biomol. Chem., 2003,
1, 3867; (b) M. D. Burke and S. L. Schreiber, Angew. Chem.,
Int. Ed., 2004, 43, 46; (c) W. R. J. D. Galloway, A. Isidro-
Llobet and D. R. Spring, Nat. Commun., 2010, 1, 80; (d)
S. Dandapani and L. A. Marcaurelle, Curr. Opin. Chem.
Biol., 2010, 14, 362; (e) C. J. O’Connor, H. S. G. Beckman
and D. R. Spring, Chem. Soc. Rev., 2012, 41, 4444.
Acknowledgements
We are grateful for financial support from National Natural
Science Foundation of China (21002083 and 21232007) and
PAPD of Jiangsu Higher Education Institutions.
13 For some examples, see: (a) G. L. Thomas, R. J. Spandl, F.
G. Glansdorp, M. Welch, A. Bender, J. Cockfield, J.
A. Lindsay, C. Bryant, D. F. Brown, O. Loiseleur,
H. Rudyk, M. Ladlow and D. R. Spring, Angew. Chem., Int.
Ed., 2008, 47, 2808; (b) E. E. Wyatt, W. R. J. D. Galloway, G.
L. Thomas, M. Welch, O. Loiseleur, A. T. Plowright and D.
References
1 For reviews, see: (a) C. V. Galliford and K. A. Scheidt,
Angew. Chem., Int. Ed., 2007, 46, 8748; (b) C. Marti and E.
M. Carreira, Eur. J. Org. Chem., 2003, 2209; (c) C. B. Cui,
This journal is ß The Royal Society of Chemistry 2013
RSC Adv.

View Article Online
Paper
R. Spring, Chem. Commun., 2008, 4962; (c) V. Cerulli,
RSC Advances
16 See ESI for experimental details, characterization, original
3
NMR spectra of products 4.
L. Banfi, A. Basso, V. Rocca and R. Riva, Org. Biomol. Chem.,
2012, 10, 1255.
17 CCDC 922100. See ESI for details.
3
18 (a) B. M. Trost and Y. Zhang, J. Am. Chem. Soc., 2007, 129,
14548; (b) C. Marti and E. M. Carreira, J. Am. Chem. Soc.,
2005, 127, 11505; (c) Z. Liu, P. Gu, M. Shi, P. McDowell and
G. Li, Org. Lett., 2011, 13, 2314.
19 G. Pearson, F. Robinson, T. Beers Gibson, B. E. Xu,
M. Karandikar, K. Berman and M. H. Cobb, Endocr. Rev.,
2 0 0 1 , 2 2 , 1 5 3 , h t t p : / / w w w . j b c . o r g / c g i /
ijlink?linkType=ABST&journalCode=edrv&resid=22/2/153.
20 L. A. De Girolamo and E. E. Billett, J. Neurosci. Res., 2006,
83, 680.
21 Y. L. Hsu, P. L. Kuo, L. T. Lin and C. C. Lin, J. Pharmacol.
Exp. Ther., 2005, 313, 333.
22 D. N. Dhanasekaran and E. P. Reddy, Oncogene, 2008, 27,
6245.
23 (a) J. S. Sebolt-Leopold, Oncogene, 2000, 19, 6594; (b) J.
Y. Fang and B. C. Richardson, Lancet Oncol., 2005, 6, 322.
24 N. J. Kruger, Methods. Mol. Biol., 1994, 32, 9.
14 (a) E. Vedejs and J. W. Grissom, J. Am. Chem. Soc., 1988,
110, 3238; (b) E. Vedejs, S. Dax, G. R. Martinez and C.
K. McClure, J. Org. Chem., 1987, 52, 3470; (c) R. Huisgen
and K. Niklas, Heterocycles, 1984, 22, 21; (d) A. R. Katritzky,
W. K. Yeung, R. C. Patel and K. Burgess, Heterocycles, 1983,
´
20, 623; (e) J. Mancebo-Aracil, C. Najera and J. M. Sansano,
Org. Biomol. Chem., 2013, 11, 662.
15 (a) J. Yu, F. Shi and L.-Z. Gong, Acc. Chem. Res., 2011, 44,
1156; (b) F. Shi, S.-W. Luo, Z.-L. Tao, L. He, J. Yu, S.-J. Tu
and L.-Z. Gong, Org. Lett., 2011, 13, 4680; (c) F. Shi, Z.-
L. Tao, S.-W. Luo, S.-J. Tu and L.-Z. Gong, Chem. Eur. J.,
2012, 18, 6885; (d) F. Shi, G.-J. Xing, Z.-L. Tao, S.-W. Luo, S.-
J. Tu and L.-Z. Gong, J. Org. Chem., 2012, 77, 6970; (e)
F. Shi, G.-J. Xing, R.-Y. Zhu, W. Tan and S. Tu, Org. Lett.,
2013, 15, 128; (f) F. Shi, G.-J. Xing, W. Tan, R.-Y. Zhu and
S. Tu, Org. Biomol. Chem., 2013, 11, 1482.
RSC Adv.
This journal is ß The Royal Society of Chemistry 2013