5-Hydroxy-7-azaindolin-2-one, a novel hybrid of pyridinol and sunitinib: Design, synthesis and cytotoxicity against cancer cells

Article abstract of DOI:10.1039/c6ob00406g

Angiogenesis plays important roles in tumor growth and metastasis. Sunitinib (Sutent) is an antitumor agent targeting receptor tyrosine kinases which are involved in angiogenesis as well as cancer cell growth and survival. Using the pyridin-3-ol scaffold, which was previously reported as an excellent antioxidant and antiangiogenic platform, we have synthesized sunitinib mimics 6 by hybridizing bicyclic pyridinol 4 as a key scaffold and pyrrole-2-carbaldehydes 7 as side chains. Cytotoxicity assays showed that compounds 6 have comparable to better anticancer activity than sunitinib against five different cancer cell lines. In addition, compounds 6 showed even lower levels of cytotoxicity against normal cells, resulting in up to 26-fold better safety windows, than sunitinib. Signaling pathway-associated transcription factor reporter assay and western blot analyses revealed that apoptosis induction in MDA-MB-231 human breast cancer cells by 6F is mainly mediated through the p53 increase and down-regulation of phospho-signal transducer and activator of transcription 3 (STAT3) and its target gene products, cyclin D, Bcl-2, and survivin. The data strongly suggest that our hybrid compounds can provide a novel anticancer scaffold with improved and safer cytotoxicity profiles than sunitinib.

Full text of DOI:10.1039/c6ob00406g

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5-Hydroxy-7-azaindolin-2-one, a novel hybrid of
pyridinol and sunitinib: design, synthesis and
cytotoxicity against cancer cells†
Cite this: DOI: 10.1039/c6ob00406g
Sajita Shah,‡a Chaemin Lee,‡a Hyukjae Choi,a Jaya Gautam,a Hyeonjin Jang,a
Geum Jin Kim,a Yu-Jeong Lee,a Chhabi Lal Chaudhary,a Sang Won Park,b
Tae-gyu Nam,*b Jung-Ae Kim*a and Byeong-Seon Jeong*a
Angiogenesis plays important roles in tumor growth and metastasis. Sunitinib (Sutent®) is an antitumor
agent targeting receptor tyrosine kinases which are involved in angiogenesis as well as cancer cell growth
and survival. Using the pyridin-3-ol scaffold, which was previously reported as an excellent antioxidant
and antiangiogenic platform, we have synthesized sunitinib mimics 6 by hybridizing bicyclic pyridinol 4 as
a key scaffold and pyrrole-2-carbaldehydes 7 as side chains. Cytotoxicity assays showed that compounds
6 have comparable to better anticancer activity than sunitinib against five different cancer cell lines. In
addition, compounds 6 showed even lower levels of cytotoxicity against normal cells, resulting in up to
26-fold better safety windows, than sunitinib. Signaling pathway-associated transcription factor reporter
assay and western blot analyses revealed that apoptosis induction in MDA-MB-231 human breast cancer
cells by 6F is mainly mediated through the p53 increase and down-regulation of phospho-signal transdu-
cer and activator of transcription 3 (STAT3) and its target gene products, cyclin D, Bcl-2, and survivin. The
data strongly suggest that our hybrid compounds can provide a novel anticancer scaffold with improved
and safer cytotoxicity profiles than sunitinib.
Received 22nd February 2016,
Accepted 25th April 2016
DOI: 10.1039/c6ob00406g
www.rsc.org/obc
angiogenesis.8–15 One of the effective strategies is the discovery
of novel synthetic small molecules with potent antiangiogenic
Introduction
Angiogenesis, a vital process of new blood vessel formation, is activities, expecting that these molecules will prevent or slow
involved in growth, differentiation, and migration of endo- the growth of cancer.16–20 There are thirteen angiogenesis
thelial cells. It requires binding of chemical signals, such as inhibitors that are currently being used for cancer treatment in
vascular endothelial growth factor (VEGF) and platelet-derived humans. These agents fall into three categories; (1) mono-
growth factor (PDGF), to their receptors located on the surface clonal antibodies, such as bevacizumab, which directly bind to
of normal endothelial cells.1–5 Upon binding of angiogenic specific angiogenic signaling molecules extracellularly to
growth factors, the receptors activate receptor tyrosine kinases prevent their interactions with the corresponding receptors on
(RTKs) to relay signals within these cells that promote cell the surface of endothelial cells; (2) small molecule tyrosine
growth and survival.6,7
kinase inhibitors against multiple angiogenic factor receptors,
Because tumors, particularly solid tumors, cannot grow and such as sorafenib and sunitinib; and (3) inhibitors of the
spread without angiogenesis, tumor angiogenesis has been a mammalian target of rapamycin (mTOR), such as temsiroli-
target for anticancer drug development, and lots of efforts mus and everolimus. Among these three classes, the discovery
have been made to find efficient ways to block tumor-induced of small molecule RTK inhibitors has received a lot of atten-
tion from medicinal chemists in the past decade. Because
there is significant crosstalk between the growth factors
involved in vascularization,21 targeting only one RTK may not
aCollege of Pharmacy and Institute for Drug Research, Yeungnam University,
be an effective strategy due to the complexity of tumor angio-
genesis. Therefore, agents targeting a broad spectrum of RTKs
may be beneficial in the treatment of cancer angiogenesis.22
In addition to neovascularization, RTKs are also involved in
the regulation of critical processes in cancer cell growth and
survival. While strictly regulated in normal cells, RTKs in
Gyeongsan 38541, Republic of Korea. E-mail: jakim@yu.ac.kr, jeongb@ynu.ac.kr
bDepartment of Pharmacy and Institute of Pharmaceutical Science and Technology,
Hanyang University, Ansan, Gyeonggi-do 15588, Republic of Korea.
E-mail: tnam@hanyang.ac.kr
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6ob00406g
‡These authors contributed equally to this work.
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many cancer cells are constitutively activated by gain-of-func-
tion mutation, gene amplification, or overexpression.23,24 In
conjunction with angiogenesis signaling through RTKs in
endothelial cells, the aberrant signaling by oncogenic RTKs in
cancer cells has positioned RTK as an effective therapeutic
target against cancer by inhibiting growth and survival of both
cancer and endothelial cells.
Sunitinib has been used in its L-malic acid salt form in
humans since January 2006 for the treatment of two indi-
cations: advanced renal cell carcinoma (RCC) and imatinib-
resistant gastrointestinal stromal tumors (GIST). And an indi-
cation for pancreatic neuroendocrine tumors was added in
May 2011. Sunitinib inhibits cellular signaling by targeting
multiple RTKs including all receptors for VEGF and PDGF.25–30
The indolin-2-one (1) skeleton, shown in the sunitinib
structure, occupies an important position in the discovery of
new antitumor agents. Since sunitinib first appeared on the
market, intense research has been conducted on the structural
decoration and modification of this valuable skeleton 1.31–33 In
particular, the 3- and 5-positions of the indolin-2-one ring have
been the main targets for chemical modifications to increase
the antitumor activity. Most derivatization studies have started
from the core structure 2 ((Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)
methylene)indolin-2-one), and a large number of sunitinib ana-
logues were prepared by the combination of various substitu-
ents (i.e., A, G, and Z in structure 3) as shown in Fig. 1.34–46
We recently published a series of papers on the design and
preparation of pyridin-3-ol derivatives that showed excellent
antioxidant, antiangiogenic, and antitumor activities.47–52
Among the pyridinol derivatives, we paid special attention to
the bicyclic pyridinol 4 (4,6-dimethyl-5-hydroxy-7-azaindolin-2-
one)52 because of its similarities to the 5-fluoroindolin-2-one
moiety of sunitinib (Fig. 2). In our previous research, we found
that compound 4 has comparable antiangiogenic activity in
terms of 50% inhibitory dose (ID50 = 0.191 ng per CAM) to that
Fig. 2 Design of a novel hybrid scaffold.
of sunitinib malate (Sutent®, ID50 = 0.202 ng per CAM) as
measured by the quantitative chick embryo chorioallantoic
membrane (CAM) assay.52
We decided to explore the chemical space of the sunitinib
structure based on our pyridinol system. As the first stage of
our exploration, we designed a novel hybrid structure 6 by a
fusion of 4,6-dimethyl-5-hydroxy-7-azaindolin-2-one (4) (the
pyridinol part) and the (3,5-dimethylpyrrol-2-yl)methylene
moiety in 5 (the sunitinib part) as shown in Fig. 2. Surpris-
ingly, there is only one report on the modification of the C(5)-
position of 7-azaindolin-2-one as sunitinib analogues.46 In the
case of 5-halogenated-7-azaindolin-2-one (aza-3 in Fig. 1), it
was reported that 5-fluoro, 5-chloro and 5-bromo analogues of
sunitinib showed by and large comparable cytotoxicities rela-
tive to sunitinib depending on the nature of cancer cell lines.
With the identical side chain (Z group in aza-3), 5-chloro and
5-bromo analogues generally showed much better cytotoxici-
ties than the 5-fluoro ones against cancer cells, except when
Z = OEt where 5-fluoro and 5-chloro analogues were much
more cytotoxic than the 5-bromo ones.46 To our knowledge,
this study is the first report regarding 5-hydroxy-7-azaindolin-
2-one analogues of sunitinib. We here report the preparation
of the hybrid derivatives, their cytotoxicities against several
human cancer cell lines along with normal cell lines, and the
mechanism of action inducing apoptotic cancer cell death.
Results and discussion
Synthesis
Among the possible synthetic approaches for the novel com-
pounds 6, we adopted a convergent synthetic strategy for
efficient derivatization as shown in Scheme 1. We envisioned
that the target compounds 6 could be prepared by a nucleophi-
lic addition of the active methylene compound 4 to the alde-
hyde carbonyl group of 7 under the Knoevenagel condensation
reaction conditions. The bicyclic pyridinol 4 was prepared
from vitamin B6 (8) as we previously reported,52 and the 2-for-
mylpyrroles 7 were synthesized starting from tert-butyl aceto-
acetate (9) by the reported methods.25,36,38,40
Fig. 1 Sunitinib and its chemical modifications.
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ethanol gave the ethyl ester 14 with the benzyl protective group
removed. Under the typical azo coupling reaction conditions
with aniline, the phenyldiazenyl group was incorporated into
the C(6)-position of the pyridinol 14. The pyrrolidinone-fused
bicyclic pyridinol 4 was finally obtained by reductive cleavage
of the azo bond of 14 and subsequent intramolecular amida-
tion. The key intermediate 4 was prepared from vitamin B6 (8)
by a seven-step process in 35% overall yield.52
The preparation of the required 3,5-dimethyl-2-formyl-
pyrroles is shown in Scheme 3. First, the simplest analogue 17
was readily obtained from 3,5-dimethylpyrrole (16) by the Vils-
meier–Haack reaction. For more complex derivatives with car-
bonyl group substitution at the C(4)-position, well-established
Knorr pyrrole synthesis25,36,38,40 was applied for the C(4)-ester-
containing pyrroles. The synthesis commenced with nitrosa-
tion of tert-butyl acetoacetate (9) to give 2-oxime compound 18
in situ, and successive addition of zinc to this intermediate
converted the 2-oxime group to the amino group. The resulting
tert-butyl 2-aminoacetoacetate intermediate was then con-
densed with ethyl acetoacetate to produce 3,5-dimethylpyrrole
with tert-butyl ester and ethyl ester tethering at the 2- and
4-positions, respectively. Selective hydrolytic decarboxylation
Scheme 1 Overview of the convergent synthetic strategy for 6.
Scheme 2 shows the synthesis of 4,6-dimethyl-5-hydroxy-7-
azaindolin-2-one (4) from vitamin B6 (pyridoxine·HCl). Briefly,
selective cleavage of the C′(4)–OH bond in 8 with zinc dust in
acetic acid followed by methanolysis of the in situ-acetylated
C′(5)-acetate gave 4-deoxypyridoxine (10). After selective protec-
tion of the C(3)–OH with the benzyl group, the hydroxyl group
of the C′(5) position was converted into chloride, which was
then successively replaced with cyanide to afford a one carbon-
elongated compound 13. Ethanolysis of the nitrile under the
modified Pinner reaction conditions with sulfuric acid in
Scheme 2 Synthesis of 4,6-dimethyl-5-hydroxy-7-azaindolin-2-one
(4).
Scheme 3 Synthesis of 3,5-dimethyl-1H-pyrrole-2-carbaldehydes.
Yields are in parentheses.
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Scheme 4 Synthesis of novel pyridinol–sunitinib hybrid derivatives.
Yields are in parentheses.
Fig. 3 Determination of the alkene geometry of 6D by 1D NOE experi-
ment. (A) 1H-NMR spectrum (blue), NOE difference spectrum (red), (B)
expanded spectra (2.32–2.56 ppm), (C) NOE correlations in compound
6D.
of the tert-butyl ester group of 19, and the Vilsmeier–Haack
formylation at the C(2)-position gave the 2-formyl compound
21. The key compound for the introduction of various amides,
the carboxylic acid 22, was obtained through basic hydrolysis.
The pyrrole compounds 23–29 with various amido groups at
the C(4)-position were finally prepared by carbodiimide-
assisted amidation of the carboxylic acid 22.
HT-29 (colon cancer), A549 (lung cancer), and U937 (leukemia)
together with two normal cell lines, i.e. CHO-K1 (Chinese
hamster ovary cells) and HEK293 cells (Human embryonic
kidney cells). As shown in Table 1, compounds 6D, 6E, and 6F
showed almost comparable cytotoxicities (IC50) to sunitinib
against cancer cells. In particular, compound 6D, an exact
5-hydroxy-7-azaindolin-2-one version of sunitinib, was more
cytotoxic against MCF-7 and MDA-MB-231 (IC50: 6.09 and
5.31 μM, respectively) than sunitinib (IC50: 7.30 and 6.09 μM,
respectively), but not against HT-29, A549, and U937 cells,
demonstrating a certain level of selectivity of the 5-hydroxy-7-
azaindolin-2-one scaffold. Compound 6E showed similar cyto-
toxicities against cancer cells to 6F. Compound 6F showed the
best activity, better cytotoxicities against all five human cancer
cell lines (IC50: 3.21–6.90 μM) than sunitinib, whereas com-
pounds 6C, 6I and 6J were less cytotoxic against all five cancer
cell lines than sunitinib followed by least active compounds
6A, 6B, 6G, and 6H.
The final target compounds 6A–6J were readily obtained by
the Knoevenagel condensation reaction of 4,6-dimethyl-5-
hydroxy-7-azaindolin-2-one (4) with ten pyrrole-2-carbalde-
hydes (17, 21–29) in the presence of a secondary cyclic amine
(pyrrolidine or piperidine). All of the novel hybrid sunitinib
derivatives 6A–6J were well characterized by 1H-NMR,
13C-NMR, and MS (Scheme 4). The 1H NMR signals of 6D were
all assigned based on the 1D and 2D NMR data including
1H–1H COSY, HMQC, and HMBC spectra (see the ESI†).
The NOE enhancements were observed upon irradiation at
the vinylic proton signal in the 1D NOE experiment. In the
NOE difference spectrum of 6D, two singlet methyl signals
(Me2 and Me3 in Fig. 3) were enhanced by the irradiation of
the vinylic proton (δH 7.53) as shown in Fig. 3. The NOE
enhancements of the other proton signals except for the
solvent peaks were negligible or undetectable. The NOE corre-
lation between a vinylic proton and a methyl proton signal
(Me2) indicated that the double bond geometry of 6D is the Z
configuration (Fig. 3). Likewise, double bond geometry of the
other derivatives 6 synthesized in this study was speculated to
be the Z configuration.
In a report by Wang et al.,46 5-fluoro, 5-chloro, and 5-bromo
analogues (aza-3 compounds with Z = NH(CH2)2NEt2 in Fig. 1)
of 6D showed 1.2–1.3 times, 1.0–1.5 times, and 0.8–1.1 times
IC50’s relative to those of sunitinib against MCF-7, HT-20, and
A549 cells. Meanwhile, our 5-hydroxy analogue, 6D, revealed
0.8–4.3 times IC50’s relative to those of sunitinib against the
same cells, indicating that the 5-hydroxy group can slightly
Cytotoxicity
The cytotoxicity of all the synthesized compounds (6A–6J) and deteriorate the cytotoxicity of the 7-azaindolin-2-one scaffold.
sunitinib was screened in five different human cancer cell However, other 5-hydroxy compounds with different side
lines, i.e. MCF-7 (breast cancer), MDA-MB-231 (breast cancer), chains, such as 6F, showed much improved IC50’s compared to
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Table 1 Cytotoxicity of compounds (6A–6J) against five human cancer cell lines and two normal cell lines; IC50 values (μM)a
MCF-7
MDA-MB-231
HT-29
A549
U937
CHO-K1
HEK293
6A
6B
6C
6D
6E
6F
6G
6H
6I
31.62
39.81
14.79
6.09
8.70
6.90
34.67
26.91
12.64
10.23
7.30
10.23
24.54
19.49
5.31
8.12
3.52
43.65
14.79
14.49
16.21
6.09
16.62
22.88
26.24
20.89
15.17
4.47
43.35
16.62
57.54
36.12
4.89
13.86
18.19
19.95
19.95
13.24
6.08
50.11
12.07
30.08
26.30
6.98
33.88
12.88
14.79
8.01
14.79
3.21
52.48
9.77
>90
50.11
23.98
33.11
>90
51.28
43.35
58.88
14.79
>90
45.70
19.95
46.77
>90
40.73
>90
37.15
19.05
>90
21.37
7.30
6J
26.24
6.98
23.44
20.87
Sunitinib
a Data are shown as the mean of three independent experiments.
Table 2 Safety windows of compounds (6A–6J): IC50(CHO-K1)/IC50(cancer cells)|IC50(HEK293)/IC50(cancer cells)
MCF-7
MDA-MB-231
HT-29
A549
U937
6A
6B
6C
6D
6E
6F
6G
6H
6I
1.58|1.45
0.60|0.50
2.24|3.16
>14.78|>14.78
5.89|4.68
6.28|>13.04
1.70|1.07
0.55|0.71
>7.12|>7.12
2.29|2.09
2.86|1.00
4.90|4.47
0.98|0.81
1.70|2.40
>16.95|>16.95
6.32|5.02
12.32|>25.57
1.35|0.85
1.00|1.29
>6.21|>6.21
1.45|1.32
3.43|1.20
3.02|2.75
1.05|0.87
1.26|1.78
>4.31|>4.31
3.38|2.68
9.70|>20.13
1.36|0.86
0.89|1.15
>1.56|>1.56
0.65|0.59
4.27|1.49
3.62|3.30
1.32|1.10
1.66|2.34
>4.51|>4.51
3.87|3.08
7.13|14.80
1.18|0.74
1.23|1.58
>2.99|>2.99
0.89|0.81
2.99|1.05
1.48|1.35
1.86|1.55
2.24|3.16
>11.24|>11.24
3.47|2.75
13.50|28.04
1.12|0.71
1.51|1.95
1.00|1.00
0.89|0.81
2.99|1.05
6J
Sunitinib
sunitinib in all cancer cells, demonstrating the potential of the better safety window). In addition, they showed strikingly
scaffold. higher safety profiles in normal human renal cells (HEK) than
Interestingly, all the compounds in this study showed in normal murine cells (CHO), suggesting their potential as
higher IC50 values against normal cells than sunitinib anticancer agents against human cancers.
(Table 1), which led us to further investigate about their safety
profiles.
We have reported elsewhere that the antiangiogenic activity
of our core structure (4) was comparable to sunitinib in the
We calculated the safety window of compounds as a ratio of CAM assay.52 Therefore, the cytotoxicity of 6D, 6F and 6I
the IC50 value in normal cell lines (CHO-K1 or HEK293 cell against cancer cells may be largely attributed to the antiangio-
lines) relative to the IC50 value in cancer cell lines. As shown in
Table 2, compounds 6D and 6F showed a high level of safety
windows for both CHO-K1 and HEK293 cells while those of 6E
and 6I were slightly lower. For CHO-K1 cells, compound 6D
afforded as much as 4–5 times better safety windows than
Table 3 Inhibition of KDR kinase activity by compounds (6A–6J). The
concentration of all the tested compounds was 500 nM
sunitinib in MCF-7, MDA-MB-231 and U937 cells. The com-
pound showed <2-fold higher safety windows than sunitinib in
HT-29 and A549 cells. Compound 6F showed 3.5–4.5 times
better safety windows than sunitinib for MDA-MB-231 and
U937 cells whereas 2.2–2.4 times better for MCF-7, HT-29 and
A549 cells. In the meantime, for HEK293 cells, compound 6D
afforded as much as 10–14 times better safety windows than
sunitinib in MCF-7, MDA-MB-231 and U937 cells while 3–4
times higher safety windows in HT-29 and A549 cells. For
HEK293 cells, compound 6F showed 13–26 times better safety
windows than sunitinib for all cancer cells. These results
strongly indicate that our best compounds, 6D and 6F, are
KDR kinase activity
(% inhibition, mean SEM)
Compound
6A
6B
6C
6D
6E
6F
6G
6H
6I
22.2 3.3
26.1 2.0
66.8 2.0
78.1 0.8
70.5 2.2
13.2 2.4
49.0 3.4
23.1 2.9
35.8 3.2
34.9 2.5
96.5 0.2
6J
Sunitinib
Data are shown as mean
S.E.M. of at least three independent
potentially much safer agents than sunitinib (up to 26-fold experiments.
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Table 4 Effects of 6D, 6F, 6I and sunitinib on the cell cycle
Compound
Conc. (μM)
Sub-G1
G0/G1
S
G2/M
Control
Sunitinib
6D
—
10
1
3
10
1
3
10
1
3
10
18.54 0.29
41.76 1.50*
24.34 0.36*
36.98 1.29*
40.14 0.18*
30.87 0.71*
37.11 1.63*
41.77 0.74*
22.80 0.76
28.21 2.04*
37.32 0.94*
52.97 0.51
35.77 1.16*
48.28 0.61*
38.54 0.59*
42.35 2.03*
38.89 3.73*
38.93 1.10*
39.09 1.12*
48.61 0.45
46.12 1.14*
42.73 1.23*
9.06 0.18
19.85 0.46
15.00 0.30*
18.99 0.23
16.64 0.54*
12.99 1.42*
19.32 1.70
16.10 0.69*
15.35 0.53*
18.89 0.06*
18.44 0.72*
14.47 0.28*
7.60 0.15*
8.70 0.53
7.89 0.43
4.61 0.99*
7.45 1.07
8.14 0.27*
3.90 0.54*
9.98 1.05
7.48 0.41*
5.60 0.49*
6F
6I
The cell cycle was determined in cells treated with or without a compound in the presence of 10% fetal bovine serum for 48 h. *P < 0.05
compared to the vehicle-treated control.
genic activity of the core structure 4. However, the improved safety window. It is evident that some compounds have
selectivity in the cytotoxicity of the hybrid compounds, up to different primary target profiles from the others, although they
10-fold better safety windows, demonstrates the novelty of the all share the identical 5-hydroxy-7-azaindolin-2-one scaffold,
new core structure and hybridization strategy and is shedding and the selectivity is significantly dependent on the side
new light on the analogue design (Tables 2 and 3).
chain. This result strongly implies that our compounds are not
The key strategy of our hybridization is to use the 5-hydroxy- simple sunitinib-mimics but could be a novel kind of cytotoxic
7-azaindolin-2-one skeleton, which is well-characterized as an
antiangiogenic moiety.52 Since sunitinib has a certain degree
of toxicity to normal cells, we have adopted only a part of its
side chain. We anticipated that our hybrid compounds would
express both validated antiangiogenic activities from 5-hydroxy-
7-azaindolin-2-one and tuned drug-likeness from the side chain
of sunitinib. Indeed, 6F, for example, showed better IC50 values
than sunitinib against all the cancer cells used in this study
(Table 2). Other hybrid compounds also showed excellent cyto-
toxicity. Tables 2 and 3 clearly show that our hybrid compounds
are >10-fold less cytotoxic to normal cells (HEK and CHO cells)
than sunitinib, leading to up to 26-fold better safety windows.
This is obviously a top-priority benefit for anticancer com-
pounds and we believe that this is due to our hybridized struc-
ture. Non-selective cytotoxicity against cancer and normal cells
does not generate a very useful dataset to assess the potential of
anticancer compounds.
Inhibition of KDR by compounds 6A–6J
Sunitinib is a multi-kinase inhibitor, targeting many kinases
such as all types of VEGFR and PDGFR.25–30 In order to check
if our compounds target the same kinase as sunitinib, we
tested the inhibitory activity of our compounds against
VEGFR2, which is a representative and primary target of suniti-
nib, using the KDR assay system (Table 3). We expected that all
the compounds behave similarly to sunitinib due to the struc-
tural similarity. Sunitinib showed a very high level of inhi-
bition against KDR (96.5%) as expected. It was surprising that
only compound 6D, a direct 5-hydroxy-7-azaindolin-2-one ana-
logue of sunitinib, as well as 6C and 6E having similar or iden-
Fig. 4 Cell cycle analysis in sunitinib- and compound (6D, 6F, and 6I)-
treated MDA-MB-231 cells. The area parameter histogram was used to
determine the percentage of cells in sub-G1, G0/G1, S, and G2/M
tical side chains to sunitinib showed strong inhibition against
KDR. In contrast, non-similar side chain analogues 6A, 6B, 6F
and 6H hardly inhibit KDR (13.2–26.1%). Of weak inhibitors of
phases. The data for sub-G1 and each phase of the cell cycle are shown
KDR, compound 6F showed the best activity and the best in Table 4.
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platform which does not target KDR and possesses a fascinat- examined for the mechanism of action of the compounds. As
ing safety window.
shown in Table 4 and Fig. 4, treatment of MDA-MB-231 cells
with compounds 6D, 6F, and 6I for 48 hours significantly
increased the sub-G0/G1 fraction, an indicator of apoptosis. 6F
was the most effective, which is consistent with its superior
IC50 values.
Apoptotic cell death-inducing effects of the compounds
correlates with suppression of nuclear translocation of pSTAT3
With the striking side-chain dependence in the inhibition
against KDR, we also compared apoptosis-inducing effects of
the compounds by flow cytometric analysis of cells stained
with either propidium iodide (PI) alone for cell cycle analysis,
or FITC Annexin V in conjunction with PI for detection of the
translocation of phosphatidylserine from the inner to the
outer leaflet of the plasma membrane and the integrity of the
membrane. Because the highly invasive MDA-MB-231 cancer
cell line showed better sensitivity to the three compounds (6D,
6F, 6I) among the cancer cell lines tested, it was selected and
Similarly, compounds 6D, 6F, and 6I modestly increased
the number of cells that are FITC Annexin V positive (+) and PI
negative (−) (early apoptosis) relative to the control and dra-
matically increased cells that are FITC Annexin V (+) and PI (+)
(end stage apoptosis and death) in a concentration-dependent
manner (Fig. 5). Compared to sunitinib, 6F induced a similar
Table 5 Effects of compound 6F on 45-signal pathway-associated
transcription factor reporter activity in MDA-MB-231 cells
Transcription
factor
Reporter activity fold
Transcription factor
associated pathway
increasea (6F/control)
p53
E2F
HIF
SRE
AP1
Myc
NF-κB
SP1
STAT3
C/EBP
GATA
MEF2
VDR
TCF/LEF
ATF6
CRE
ERE
GRE
HSR
MTF1
GLI
IRF1
ISRE
KLF4
Nanog
Oct4
Pax6
Sox2
PR
RARE
RXR
SMAD
XRE
NFAT
LXR
AARE
AR
EGR1
HNF4
GAS
RBP-Jk
FOXO
PPAR
ERSE
ARE
2.2 0.1*
0.5 0.2*
0.4 0.1*
0.5 0.1*
0.3 0.1*
0.4 0.1*
0.4 0.1*
0.5 0.1*
0.2 0.1*
0.7 0.1*
0.7 0.1*
0.7 0.1*
0.8 0.1*
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.2
0.9 0.1
0.9 0.2
0.9 0.2
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.0 0.2
1.0 0.1
1.0 0.1
1.0 0.2
1.0 0.2
1.0 0.2
1.0 0.2
1.0 0.2
1.1 0.2
1.1 0.1
1.1 0.1
1.1 0.1
1.1 0.1
1.2 0.1
p53
E2F
HIF-1α
SRF/Elk-1
AP-1
c-Myc
NF-κB
SP1
STAT3
C/EBP
GATA
MEF2
VDR
TCF/LEF
ATF6
CREB
ER
GR
HSF-1
MTF-1
Gli
IRF1
STAT1/STAT2
KLF4
Nanog
Oct4
Pax6
Sox2
PR
RAR
RXR
Smad2/3/4
AhR
NFAT
LXR
ATF2/3/4
AR
EGR1
HNF4
STAT1
RBP-Jk
FOXO
PPAR
CBF/NFY/YY1
Nrf1/Nrf2
Fig. 5 Apoptotic cell death analysis in MDA-MB-231 cells treated with
either sunitinib or compounds (6D, 6F, and 6I). (A) Representative dot
plot of Annexin V (x-axis) versus PI (y-axis) analyses. (B) Percentage of
apoptotic cells (average of three independent experiments) populating
upper and lower right quadrants.
a Reporter activity was expressed as mean SEM from three indepen-
dent experiments. *P < 0.05 compared to the vehicle-treated control.
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level of apoptotic cell death at about 3-fold lower
concentration.
To identify relevant signaling molecules involved in the
action of compound 6F, we performed pathway-focused tran-
scription factor array assay using a Cignal 45-pathway finder
reporter array kit (Table 5). Compared to the vehicle-treated
control group, the DNA damage-associated p53 reporter
Fig. 6 Inhibitory effects of 6D, 6F, 6I, and sunitinib on STAT3 activation
(A and B) and NF-κB nuclear translocation (A and C). Protein extracted
from MDA-MB-231 cells treated with sunitinib and compounds (6D, 6F,
and 6I) were immunoblotted with anti p-STAT3, STAT3, and NF-κB anti-
bodies. β-Actin and lamin B were used for equal loading. The bands are
representative of three independent experiments, and bar graphs indi-
cate the mean SEM of relative densities. *P < 0.05 compared to the
vehicle-treated control group.
Fig. 7 Inhibitory effects of 6D, 6F, 6I, and sunitinib on the expressions
of p53 (A and B) and STAT3 target gene products (A and C). The bands
are representative of three independent experiments, and bar graphs
indicate the mean SEM of relative densities. *P < 0.05 compared to the
vehicle-treated control group.
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Western blotting clearly confirmed that the treatment of
MDA-MB-231 cells with 6F significantly inhibited phosphoryl-
ation of STAT3 both in the cytosol and nucleus and its nuclear
translocation in a concentration-dependent manner (Fig. 6A
and B), and such an inhibitory effect of 6F (10 μM) was better
than that of sunitinib, 6D or 6I. Nuclear translocation of NF-κB
was inhibited concentration-dependently by 6F (10 μM), simi-
larly to sunitinib, but less effectively than 6D (Fig. 6A and C).
The expression of p53 was highly increased by all the com-
pounds with the strongest effect by 6F (Fig. 7A and B). Like-
wise, 6F dramatically increased Bax (an apoptosis promoter),
which has the p53 binding site in its promoter region. Bcl-2
(an apoptosis inhibitor), which neutralizes the Bax action
through heterodimer formation, was down-regulated by 6F
(Fig. 7A and C). Such an increase in Bax/Bcl-2 ratio corre-
sponded to the induction of apoptosis by 6F. In addition,
cyclin D1 and survivin expressions were suppressed by all the
compounds, and the change was more effective in 6F-treated
cells than in sunitinib- or other compound-treated cells
(Fig. 7A and C).
We also investigated phosphorylation of phosphoinositide
3-kinase (PI3K), a downstream molecule of RTK and an
upstream signaling molecule of STAT3, and mitogen-activated
protein kinases (MAPKs). Compounds 6D, 6F, 6I, and sunitinib
inhibited phosphorylation of kinases such as PI3K, Akt, and
ERK, whereas JNK phosphorylation was not altered by any of
the compounds, including sunitinib (Fig. 8). Therefore,
although our compounds and sunitinib showed differential
inhibition profiles on KDR, it seems that they all share a
common downstream cascade of apoptosis pathways. In the
case of these kinase activation measurements, the inhibitory
effect of 6D was the strongest and better than that of
sunitinib.
Fig. 8 Inhibitory effects of 6D, 6F, 6I, and sunitinib on the phosphoryl-
ation of PI3K, Akt, and JNK. The bands are representative of three inde-
pendent experiments, and bar graphs indicate the mean
SEM of
relative densities. *P < 0.05 compared to the vehicle-treated control
groups.
Consistent with the cytotoxic activity, the apoptosis detec-
tion assay, signaling pathway array and western blot analyses
showed that compound 6F possesses better activity of apopto-
sis induction in MDA-MB-231 cells than sunitinib; this activity
activity was significantly increased, whereas E2F, Myc, NF-κB, was mainly mediated through the p53 increase and down-regu-
SP1, and STAT3 reporter activities were significantly sup- lation of pSTAT3 and its transcriptional activity.
pressed in 6F-treated cells (Table 5). The down-regulated tran-
scription factor signaling by 6F involved the pathways
associated with the cell cycle, MAPK, and STAT3. Among the
6F-induced changes, signal transducer and activator of tran-
Conclusions
scription 3 (STAT3) inhibition was the strongest. As sunitinib, We report here the synthesis and anticancer activity of suniti-
an antiangiogenic anticancer drug, is reported to suppress nib mimics 6. They were prepared by hybridizing bicyclic pyri-
STAT3 in renal cell carcinoma,53 the present results further dinol 4 as a key scaffold and pyrrole-2-carbaldehydes 7 as side
emphasize the antiangiogenic anticancer action of 6F.
chains. In the cytotoxicity assay using human cancer cell lines,
Because persistent activation of STAT3 is reported in a the hybrid compounds 6 showed comparable to better anti-
variety of cancer cells with worse prognoses54 and STAT3 regu- cancer activity than sunitinib. Their cytotoxicities against
lates target gene expressions related to the cell cycle (p53, normal cells were significantly lower than that of sunitinib,
cyclin B, cyclin D1), apoptosis (p53, Bcl-xL), and angiogenesis resulting in up to 26-fold better safety windows. In the KDR
(VEGF, HIF-1α), we confirmed these pathway-focused transcrip- kinase assay, the compounds showed very distinct inhibitory
tion factor array results by performing western blot analyses, activities depending on the side chains. The cytotoxicity and
focusing on the expression of phopho-STAT3 and its target apoptotic cell death data indicate that our hybrid compound
gene products, and compared the effects of 6F with those of 6F exerts the activity through apoptosis induction that is prin-
sunitinib and the other compounds, 6D and 6I.
cipally mediated via an increase in p53 level and down-regu-
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lation of STAT3 and its phosphorylation. 6F has anticancer
(Z)-Ethyl 5-((5-hydroxy-4,6-dimethyl-2-oxo-1H-pyrrolo[2,3-b]-
activity with improved and safer cytotoxicity profiles than suni- pyridin-3(2H)-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxy-
tinib against various cancer cell lines and normal cells, late (6B). To a mixture of 5-hydroxy-4,6-dimethyl-1H-pyrrolo
respectively. We have identified some 5-hydroxy-7-azaindolin- [2,3-b]pyridin-2(3H)-one (4) (43 mg, 0.242 mmol) and pyrroli-
2-one analogues which affect upstream signals in a totally dine (0.9 µL, 0.011 mmol) in ethanol (1.5 mL) was added ethyl
different manner from sunitinib, but show similar effects on 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylate (21) (52 mg,
downstream signals of apoptosis. Further study on the primary 0.266 mmol), and the resulting mixture was refluxed for
target of compound 6F which is a non-KDR inhibitor with the 24 hours. The mixture was cooled to room temperature and
best cytotoxicity and safety window should be followed in the the solids in the reaction mixture were collected by filtration.
future. Altogether, we suggest that pyridin-3-ol is a novel anti- The filter cake was washed with CH2Cl2, and dried to give 6B
cancer platform with controllable flexibility in inhibitory (56 mg, 65% yield) as an orange solid. Rf 0.43 (CHCl3 : MeOH =
activity against KDR through side chain modifications.
10 : 1). m.p. 316 °C (decomp.). 1H-NMR (DMSO-d6) δ 13.8 (s,
1H), 11.19 (br s, 1H), 8.25 (br s, 1H), 7.48 (s, 1H), 4.25–4.13 (m,
2H), 2.50–2.46 (m, 6H), 2.42 (s, 3H), 2.33 (s, 3H), 1.33–1.25 (m,
3H) ppm. 13C-NMR (DMSO-d6) δ 169.6, 164.4, 145.9, 144.8,
142.3, 139.7, 131.7, 129.2, 125.8, 125.0, 117.5, 114.8, 113.2,
59.2, 19.8, 14.6, 14.3, 12.4, 11.3 ppm. MS (ESI) m/z 356 [M +
Experimental
Synthesis
Unless noted otherwise, materials were purchased from com- H]+. HRMS (ESI) m/z calcd for C19H22N3O4 [M + H]+ 356.1610,
mercial suppliers and used without further purification. Air- found 356.1605.
or moisture-sensitive reactions were carried out under an inert
(Z)-5-((5-Hydroxy-4,6-dimethyl-2-oxo-1H-pyrrolo[2,3-b]pyridin-
gas atmosphere. The reaction progress was monitored by thin- 3(2H)-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid
layer-chromatography (TLC) using silica gel F254 plates. The (6C). To a mixture of 5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]
products were purified by flash column chromatography using pyridin-2(3H)-one (4) (50 mg, 0.281 mmol) and pyrrolidine
silica gel 60 (70–230 mesh) or by using the Biotage ‘Isolera (1.1 µL, 0.014 mmol) in ethanol (1 mL) was added 5-formyl-
One’ system with indicated solvents. Melting points were deter- 2,4-dimethyl-1H-pyrrole-3-carboxylic acid (22) (52 mg,
mined using a Fisher–Johns melting point apparatus and were 0.309 mmol), and the resulting mixture was refluxed for
not corrected. NMR spectra were obtained using a Bruker-250 3 hours. The mixture was cooled to room temperature and the
spectrometer 250 MHz for 1H-NMR, 1D NOE, 1H–1H COSY, solids in the reaction mixture were collected by filtration. The
and HMQC, and 62.5 MHz for 13C-NMR. The HMBC spectrum filter cake was washed with CH2Cl2, and dried to give 6C
of 6D was obtained by using a Jeol ECA-500 MHz spectrometer. (85 mg, 93% yield) as an orange solid. Rf 0.34 (CHCl3 : MeOH =
1
Chemical shifts (δ) were expressed in ppm using a solvent as 10 : 1). m.p. 296 °C (decomp.). H-NMR (DMSO-d6) δ 13.72 (s,
an internal standard and the coupling constant (J) in hertz. 1H), 12.08 (br s, 1H), 11.11 (s, 1H), 8.17 (br s, 1H), 7.49 (s, 1H),
Low-resolution mass spectra (LRMS) were obtained using an 2.52 (s, 3H), 2.47 (s, 3H), 2.42 (s, 3H), 2.33 (s, 3H) ppm.
Advion Expression CMS, and recorded in a positive ion mode 13C-NMR (DMSO-d6) δ 169.5, 165.9, 145.7, 144.7, 142.1, 139.7,
with an electrospray (ESI) source. High-resolution mass 131.9, 128.9, 125.6, 124.9, 117.0, 114.8, 113.9, 19.6, 14.3, 12.3,
spectra (HRMS) were obtained using a Thermo Scientific LTQ 11.2 ppm. MS (ESI) m/z 328 [M + H]+. HRMS (ESI) m/z calcd for
Orbitrap XL mass spectrometer, and recorded in positive ion C17H18N3O4 [M + H]+ 328.1297, found 328.1287.
mode with an electrospray (ESI) source.
(Z)-N-(2-(Diethylamino)ethyl)-5-((5-hydroxy-4,6-dimethyl-2-oxo-
(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-hydroxy-4,6- 1H-pyrrolo[2,3-b]pyridin-3(2H)-ylidene)methyl)-2,4-dimethyl-1H-
dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6A). To a mixture pyrrole-3-carboxamide (6D). To a mixture of 5-hydroxy-4,6-
of 5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (4) (25 mg,
(4) (162 mg, 0.908 mmol) and pyrrolidine (74 µL, 0.908 mmol) 0.140 mmol) and piperidine (28 µL, 0.281 mmol) in ethanol
in ethanol (2 mL) was added 3,5-dimethyl-1H-pyrrole-2-carbal- (1 mL) was added N-(2-(diethylamino)ethyl)-5-formyl-2,4-
dehyde (17) (123 mg, 0.988 mmol), and the resulting mixture dimethyl-1H-pyrrole-3-carboxamide (23) (53 mg, 0.196 mmol),
was refluxed for 40 hours. The mixture was cooled to room and the resulting mixture was refluxed for 20 hours. The
temperature and the solids in the reaction mixture were col- mixture was cooled to room temperature and the solids in the
lected by filtration. The filter cake was washed with CH2Cl2, reaction mixture were collected by filtration. The filter cake
and dried to give 6A (156 mg, 61% yield) as a brown solid. was washed with CH2Cl2, and dried to give 6D (46 mg, 77%
Rf 0.26 (CHCl3 : MeOH = 10 : 1). m.p. 260 °C (decomp.). yield) as an orange solid. Rf 0.31 (CHCl3 : MeOH = 7 : 1).
1H-NMR (DMSO-d6) δ 13.26 (s, 1H), 10.99 (s, 1H), 8.13 (s, 1H), m.p. 268 °C (decomp.). 1H-NMR (DMSO-d6) δ 13.50 (s, 1H),
7.43 (s, 1H), 6.00 (d, J = 2.2 Hz, 1H), 2.46 (s, 3H), 2.33 (s, 3H), 11.03 (s, 1H), 8.13 (br s, 1H), 7.42 (s, 1H), 7.36 (t, J = 5.2 Hz,
2.31 (s, 3H), 2.23 (s, 3H) ppm. 13C-NMR (DMSO-d6) δ 169.5, 1H), 3.30–3.21 (m, 2H), 2.50–2.37 (m, 12H), 2.30 (s, 6H), 0.95
145.4, 144.6, 141.9, 135.2, 131.5, 128.2, 126.3, 125.3, 115.4, (t, J = 7.0 Hz, 6H) ppm. 13C-NMR (DMSO-d6) δ 169.5, 164.6,
113.6, 112.4, 19.7, 13.5, 12.4, 11.4 ppm. MS (ESI) m/z 284 145.6, 144.7, 141.8, 135.2, 128.9, 128.7, 125.4, 125.1, 120.3,
[M + H]+. HRMS (ESI) m/z calcd for C16H18N3O2 [M + H]+ 116.0, 115.0, 51.6, 46.5 (2C), 36.9, 19.7, 13.2, 12.4, 11.8 (2C),
1
284.1394, found 284.1388.
10.5 ppm. H NMR (CD3OD) δ 7.53 (s, 1H), 3.51 (t, J = 7.0 Hz,
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2H), 2.76 (t, J = 7.0 Hz, 2H), 2.71 (q, J = 7.2 Hz, 4H), 2.52 (s, m.p. 327 °C (decomp.). 1H-NMR (DMSO-d6) δ 13.43 (s, 1H),
3H), 2.46 (s, 3H), 2.39 (s, 3H), 2.37 (s, 3H), 1.12 (t, J = 7.2 Hz, 11.09 (s, 1H), 8.18 (s, 1H), 7.43 (s, 1H), 3.51–3.40 (m, 2H),
6H) ppm. MS (ESI) m/z 426 [M + H]+. HRMS (ESI) m/z calcd for 3.27–3.18 (m, 2H), 2.46 (s, 3H), 2.33 (s, 3H), 2.29 (s, 3H), 2.19
C23H32N5O3 [M + H]+ 426.2505, found 426.2503.
(s, 3H), 1.92–1.74 (m, 4H) ppm. 13C-NMR (DMSO-d6) δ 169.5,
(Z)-5-((5-Hydroxy-4,6-dimethyl-2-oxo-1H-pyrrolo[2,3-b]pyridin- 165.2, 145.6, 144.7, 141.8, 132.6, 128.7, 127.7, 125.6, 125.2,
3(2H)-ylidene)methyl)-2,4-dimethyl-N-(2-(piperidin-1-yl)ethyl)- 122.1, 115.7, 115.1, 47.6, 45.2, 25.6, 24.1, 19.8, 12.6, 12.4,
1H-pyrrole-3-carboxamide (6E). To a mixture of 5-hydroxy-4,6- 10.2 ppm. MS (ESI) m/z 381 [M + H]+. HRMS (ESI) m/z calcd for
dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (4) (41 mg, C21H25N4O3 [M + H]+ 381.1927, found 381.1934.
0.229 mmol) and pyrrolidine (20 µL, 0.229 mmol) in ethanol
(Z)-3-((3,5-Dimethyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-yl)-
(2 mL) was added 5-formyl-2,4-dimethyl-N-(2-(piperidin-1-yl)- methylene)-5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-
ethyl)-1H-pyrrole-3-carboxamide (24) (70 mg, 0.252 mmol), 2(3H)-one (6H). To a mixture of 5-hydroxy-4,6-dimethyl-1H-
and the resulting mixture was refluxed for 40 hours. The pyrrolo[2,3-b]pyridin-2(3H)-one (4) (41 mg, 0.233 mmol) and
mixture was cooled to room temperature and the solids in the pyrrolidine (1.1 µL, 0.014 mmol) in ethanol (1 mL) was added
reaction mixture were collected by filtration. The filter cake 3,5-dimethyl-4-(piperidine-1-carbonyl)-1H-pyrrole-2-carbalde-
was washed with CH2Cl2, and dried to give 6E (73 mg, 73% hyde (27) (60 mg, 0.256 mmol), and the resulting mixture was
yield) as an orange solid. Rf 0.52 (CHCl3 : MeOH = 10 : 1). refluxed for 20 hours. The mixture was cooled to room temp-
m.p. 292 °C (decomp.). 1H-NMR DMSO-d6) δ 13.54 (s, 1H), erature and concentrated. The residue was purified by column
7.48–7.42 (m, 2H), 3.35–3.25 (m, 2H), 2.46 (s, 3H), 2.43 (s, 3H), chromatography (CH2Cl2 : MeOH = 20 : 1) to give 6H (47 mg,
2.40–2.24 (m, 12H), 1.55–1.27 (m, 6H) ppm. 13C-NMR (DMSO- 51% yield) as an orange solid. Rf 0.35 (CHCl3 : MeOH = 10 : 1).
d6) δ 169.4, 164.4, 145.6, 144.7, 141.8, 135.1, 128.8, 128.7, m.p. 305 °C (decomp.). 1H-NMR (DMSO-d6) δ 13.46 (s, 1H),
125.3, 125.0, 120.4, 116.0, 115.0, 57.5, 53.8 (2C), 36.1, 25.6 11.09 (s, 1H), 8.21 (br s, 1H), 7.43 (s, 1H), 3.60–3.40 (m, 4H),
(2C), 24.0, 19.6, 13.1, 12.3, 10.3 ppm. MS (ESI) m/z 438 [M + 2.46 (s, 3H), 2.33 (s, 3H), 2.27 (s, 3H), 2.17 (s, 3H), 1.65–1.36
H]+. HRMS (ESI) m/z calcd for C24H32N5O3 [M + H]+ 438.2505, (m, 6H) ppm. 13C-NMR (DMSO-d6) δ 169.5, 165.2, 145.7, 144.7,
found 438.2515.
141.8, 133.0, 128.8, 127.9, 125.6, 125.2, 120.3, 115.8, 115.1,
(Z)-N-Cyclohexyl-5-((5-hydroxy-4,6-dimethyl-2-oxo-1H-pyrrolo- 40.5 (2C), 24.1 (3C), 19.8, 12.4 (2C), 10.1 ppm. MS (ESI) m/z
[2,3-b]pyridin-3(2H)-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3- 395 [M + H]+. HRMS (ESI) m/z calcd for C22H27N4O3 [M + H]+
carboxamide (6F). To a mixture of 5-hydroxy-4,6-dimethyl-1H- 395.2083, found 395.2087.
pyrrolo[2,3-b]pyridin-2(3H)-one (4) (20 mg, 0.114 mmol) and
(Z)-3-((3,5-Dimethyl-4-(morpholine-4-carbonyl)-1H-pyrrol-2-yl)-
pyrrolidine (10.5 µL, 0.114 mmol) in ethanol (2.5 mL) was methylene)-5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-
added N-cyclohexyl-5-formyl-2,4-dimethyl-1H-pyrrole-3-carbox- 2(3H)-one (6I). To a mixture of 5-hydroxy-4,6-dimethyl-1H-
amide (25) (31 mg, 0.125 mmol), and the resulting mixture pyrrolo[2,3-b]pyridin-2(3H)-one (4) (58 mg, 0.326 mmol) and
was refluxed for 40 hours. The mixture was cooled to room pyrrolidine (1.5 µL, 0.018 mmol) in ethanol (1.5 mL) was
temperature and the solids in the reaction mixture were col- added
3,5-dimethyl-4-(morpholine-4-carbonyl)-1H-pyrrole-2-
lected by filtration. The filter cake was washed with CH2Cl2, carbaldehyde (28) (60 mg, 0.359 mmol), and the resulting
and dried to give 6F (36 mg, 78% yield) as an orange solid. mixture was refluxed for 4 hours. The mixture was cooled to
Rf 0.34 (CHCl3 : MeOH = 10 : 1). m.p. 362 °C (decomp.). room temperature and concentrated. The residue was purified
1H-NMR (DMSO-d6) δ 13.49 (s, 1H), 11.08 (br s, 1H), 8.21 (br s, by column chromatography (CH2Cl2 : MeOH = 20 : 1) to give 6I
1H), 7.55 (d, J = 8.0 Hz, 1H), 7.45 (s, 1H), 3.78–3.62 (m, 1H), (85 mg, 66% yield) as an orange solid. Rf 0.44 (CHCl3 : MeOH =
1
2.46 (s, 3H), 2.40 (s, 3H), 2.33 (s, 3H), 2.29 (s, 3H), 1.85–1.66 10 : 1). m.p. 319 °C (decomp.). H-NMR (DMSO-d6) δ 13.49 (s,
(m, 4H), 1.63–1.53 (m, 1H), 1.36–1.07 (m, 5H) ppm. 13C-NMR 1H), 11.10 (s, 1H), 8.19 (br s, 1H), 7.43 (s, 1H), 3.62–3.54 (m,
(DMSO-d6) δ 169.5, 163.9, 145.6, 144.7, 141.8, 134.8, 129.2, 4H), 3.53–3.39 (m, 4H), 2.46 (s, 3H), 2.33 (s, 3H), 2.29 (s, 3H),
128.7, 125.4, 125.2, 121.1, 115.8, 115.1, 47.7, 32.6 (2C), 25.3, 2.20 (s, 3H) ppm. 13C-NMR (DMSO-d6) δ 169.5, 165.5, 145.7,
24.8 (2C), 19.8, 13.2, 12.5, 10.4 ppm. MS (ESI) m/z 409 144.7, 141.9, 133.3, 128.8, 128.0, 125.7, 125.1, 119.3, 116.0,
[M + H]+. HRMS (ESI) m/z calcd for C23H29N4O3 [M + H]+ 115.0, 66.5 (2C), 40.5 (2C), 19.8, 12.5, 12.4, 10.1 ppm. MS (ESI)
409.2240, found 409.2236.
(Z)-3-((3,5-Dimethyl-4-(pyrrolidine-1-carbonyl)-1H-pyrrol-2-yl)- [M + H]+ 397.1876, found 397.1869.
m/z 397 [M + H]+. HRMS (ESI) m/z calcd for C21H25N4O4
methylene)-5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-
(Z)-3-((3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-
2(3H)-one (6G). To a mixture of 5-hydroxy-4,6-dimethyl-1H- pyrrol-2-yl)methylene)-5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]-
pyrrolo[2,3-b]pyridin-2(3H)-one (4) (44 mg, 0.248 mmol) and pyridin-2(3H)-one (6J). To a mixture of 5-hydroxy-4,6-dimethyl-
pyrrolidine (1.1 µL, 0.014 mmol) in ethanol (1 mL) was added 1H-pyrrolo[2,3-b]pyridin-2(3H)-one (4) (34 mg, 0.190 mmol)
3,5-dimethyl-4-(pyrrolidine-1-carbonyl)-1H-pyrrole-2-carbalde- and pyrrolidine (1.1 µL, 0.014 mmol) in ethanol (1 mL) was
hyde (26) (60 mg, 0.272 mmol), and the resulting mixture was added
3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-
refluxed for 18 hours. The mixture was cooled to room temp- pyrrole-2-carbaldehyde (29) (52 mg, 0.209 mmol), and the
erature and concentrated. The residue was purified by column resulting mixture was refluxed for 5 hours. The mixture was
chromatography (CH2Cl2 : MeOH = 15 : 1) to give 6G (48 mg, cooled to room temperature and the solids in the reaction
51% yield) as an orange solid. Rf 0.39 (CHCl3 : MeOH = 10 : 1). mixture were collected by filtration. The filter cake was washed
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with CH2Cl2, and dried to give 6J (57 mg, 73% yield) as an with PI solution containing RNase A and triton X-100 for
orange solid. Rf 0.23 (CHCl3 : MeOH = 10 : 1). m.p. 299 °C 40 min. A total of 20 000 events were analyzed by flow cytome-
(decomp.). 1H-NMR (DMSO-d6) δ 13.49 (s, 1H), 11.11 (s, 1H), try using an excitation wavelength set at 488 nm and emission
8.21 (br s, 1H), 7.45 (s, 1H), 3.49–3.35 (m, 4H), 2.47 (s, 3H), set at 575 nm.
2.33 (s, 3H), 2.31–2.27 (m, 7H), 2.21–2.17 (m, 6H) ppm.
Annexin V/PI staining for detection of apoptosis. FITC
13C-NMR (DMSO-d6) δ 169.5, 165.3, 145.7, 144.7, 141.9, 133.1, Annexin V apoptosis detection kit I (BD Biosciences, San Jose,
128.8, 128.0, 127.7, 125.6, 125.2, 119.8, 115.9, 64.2, 63.8, 55.2, CA, USA) was used according to the guidelines for the detec-
54.7, 45.7, 19.8, 12.4, 10.9, 10.1 ppm. MS (ESI) m/z 410 tion of apoptosis. In brief, after the cells were treated with
[M + H]+. HRMS (ESI) m/z calcd for C22H28N5O3 [M + H]+ compounds 6D, 6F, and 6I for an indicated time and concen-
410.2192, found 410.2205.
tration, they were trypsinized and washed with cold PBS two
times. Then, the cells were suspended in 1X binding buffer at
a concentration of 1 × 106 cells per mL. One hundred µL of cell
suspension was transferred to a culture tube and stained with
Cytotoxic effect and identification of apoptotic cell death
signaling
Cell lines and culture. Five human cancer cell lines, MCF-7 5 µL of FITC Annexin V and 5 µL of PI. The cells were further
(breast cancer cell line), MDA-MB-231 (breast cancer cell line), incubated for 15 min. at 25 °C, and 400 µL of the 1X binding
HT-29 (colon cancer cell line), A549 (lung cancer cell line), and buffer was added before analyzing the sample by flow cytome-
U937 (leukemia cell line), were obtained from American Type try (BD FACS VERSE).
Culture Collection (ATCC, USA). Two normal cell lines,
Cignal pathway reporter activity assay. The assay was per-
CHO-K1 (Chinese hamster ovary cell line) and HEK-293 formed using the Cignal 45-pathway reporter array kit
(human embryonic kidney cell line), were obtained from (QIAGEN Sciences, MA, USA) according to the manufacturer’s
Korean Cell Line Bank (KCLB). The cells were cultured in stan- protocol. Briefly, MDA-MB-231 cells were seeded in a 96-well
dard growth medium supplemented with 10% fetal bovine plate coated with the reporter assay constructs in the presence
serum (FBS), 1% penicillin/streptomycin and incubated at of Lipofectamine 2000 (Invitrogen Corporation, CA, USA) trans-
37 °C under a 5% CO2 atmosphere.
fection reagent. After 24 h transfection, the cells were treated
Cytotoxicity assay. The cytotoxicity of the compounds was with or without a drug for 18 hours. Then, the luciferase
assessed by measuring the cell viability decrease using the activity was measured using the Dual Luciferase Reporter assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit (Promega Corporation, WI, USA) on a Fluostar Optima
(MTT) staining method. Briefly, cells were seeded in a 96-well microplate reader (BMG Labtech GmbH, Offenberg, Germany).
plate (Falcon, USA) at a density of 2 × 104 cells per well. The
Western blotting. Protein extracts were loaded on 15% SDS
next day, cells were incubated with different concentrations of polyacrylamide electrophoresis gel, transferred to nitrocellu-
each compound for 24 hours. Then, 10 μL of MTT solution lose membranes, blocked with 5% skimmed milk, and probed
was added to the well and further incubated for 4 hours at with the respective antibodies. The membranes were washed
37 °C. At the end of the incubation, the media were removed, and exposed to horseradish peroxidase-conjugated secondary
and 200 μL of dimethyl sulfoxide (DMSO) was added into each antibodies. The intensity of the chemiluminescence was
well to solubilize the formazan crystals. Finally, the absor- measured using LAS-4000 after treating with enhanced chemi-
bance was measured at 540 nm using a microplate reader luminescence reagents.
(Molecular Devices, Versa MAX Sunnyvale, CA, USA).
Statistical analysis. Statistical significance was analyzed
KDR assay. KDR kinase enzyme system (Promega #V2681) using Student’s t-test or one-way ANOVA followed by the
and ADP-Glow™ kinase assay kit (Promega #V9101) were used Student–Newman–Keuls comparison method for the calcu-
to perform the KDR kinase assay. To a mixture consisting of lation of the differences between groups (GraphPad Prism 5.0
1.5 ng µL−1 of KDR enzyme, 0.2 µg µL−1 of poly(Glu4, Tyr1) software, San Diego, CA, USA). P values less than 0.05 were
and an indicated concentration of drugs, 50 µM of ATP (final considered statistically significant.
concentration) was added to start the reaction. The reaction
was carried out for 60 min at room temperature in a total
volume of 25 µL. Then, 25 µL of the ADP-Glow™ reagent was
added to the mixture with incubation for 40 min at room
Acknowledgements
temperature. Again after the addition of 50 µL of kinase detec- This work was supported by the Yeungnam University
tion reagent and incubation of the mixture at room tempera- Research Grant.
ture for 30 min, the luminescence was recorded using a
Fluostar optima microplate reader (BMG Labtech GmbH,
Offenburg, Germany).
Propidium iodide staining for cell cycle analysis. Propidium
Notes and references
iodide (PI) staining for cell distribution across the cell cycle
was performed with a BD FACSVERSE flow cytometer (BD Bio-
sciences, San Jose, CA). Drug-treated cells were trypsinized,
fixed with ethanol (70%) on ice for 15 min, and incubated
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