Synthesis and evaluation of novel 7-azaindazolyl-indolyl-maleimide derivatives as antitumor agents and protein kinase C inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.04.043

A series of novel 7-azaindazolyl-indolyl-maleimides were synthesized and evaluated for their antiproliferative activity in vitro against various human cancer cell lines and protein kinase C inhibitory activity. Compounds 8a-c, 8e and 14a were the most pro

Full text of DOI:10.1016/j.bmc.2009.04.043

Bioorganic & Medicinal Chemistry 17 (2009) 4763–4772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel 7-azaindazolyl-indolyl-maleimide
derivatives as antitumor agents and protein kinase C inhibitors
Qing Ye a, Ji Cao b, Xinglu Zhou b, Dan Lv a, Qiaojun He b, Bo Yang b, Yongzhou Hu a,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 7-azaindazolyl-indolyl-maleimides were synthesized and evaluated for their antiprolif-
erative activity in vitro against various human cancer cell lines and protein kinase C inhibitory activity.
Compounds 8a–c, 8e and 14a were the most promising compounds against K562, A549, ECA-109, KB and
SMMC-7721 cell lines in vitro. Compounds 9a–j showed moderate PKC inhibition. Further mechanism of
action studies revealed that the antiproliferative activity of compound 8b in KB cells might involve the
mitochondria-mediated apoptosis pathway.
Received 24 March 2009
Revised 20 April 2009
Accepted 21 April 2009
Available online 3 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
7-Azaindazolyl-indolyl-maleimides
Antiproliferative activity
PKC inhibition
1. Introduction
2. Results and discussion
Cancer is the second leading cause of death in the world. Cur-
rently, there are many different targets in cancer chemotherapy.
Among them, protein kinase C (PKC) is a suitable target in anti-
cancer drug design because its function is altered in some neopla-
sias, and this dysfunction is related to uncontrolled proliferation.
PKC inhibitors such as UCN-01, CGP 41251 and Enzastaurin are
in clinical trials for their potential role in cancer therapy
(Fig. 1).1–6 Bisindolylmaleimides (e.g., Ro 31-6233), were reported
to exhibit remarkable PKC inhibition.7–10 We reasoned that by
introducing nitrogen atoms into the indole ring, one might be able
to enhance the binding of the compounds to PKC. On the other
hand, SAR studies on bisindolylmaleimides indicated that attach-
ing hydrophilic substituent(s) on the appropriate position of the
compounds could enhance their inhibitory potency toward
PKC.11,12 This prompted us to design a series of novel bisindolyl-
maleimide analogues in an attempt to improve potency against
PKC and to find more potent anticancer compounds. In this paper,
we described the synthesis of the 7-azaindazolyl-indolyl-malei-
mides with a hydrophilic chain at N1-position of the 7-azaindaz-
ole ring, the evaluation of these compounds as anticancer agents
against various human cancer cell lines in vitro, and their PKC
inhibitory potency.
2.1. Chemistry
7-Azaindazolyl-indolyl-maleimides 9a–j, 10 and 14a–d were
prepared as shown in Scheme 1. Compound 3 was synthesized fol-
lowing the method described in the literature with minor modifi-
cation.13,14 Treatment of
3 with iodine in DMF afforded
compound 4,15 which was subsequently reacted with ethyl magne-
sium bromide and tributyltin chloride to generate 5.15 Stille cou-
pling of 5 with 616,17 in the presence of Pd(Ph3)2Cl2 succeeded to
obtain 7.15,18 Alkylation of 7 with different alkyl halides resulted
in 8a–j.15 Compounds 9a–j and 10 were prepared by treatment
of 8a–j or 7 with NH4OAc, respectively.19,20
Reaction of 7 with methylamine afforded 11, which was reacted
with 5 mol/L KOH in ethanol to generate 12.19–21 Compounds 13a–
d were obtained by treating 12 with different aryl amines in the
presence of catalytic amount of p-methylphenylsulfonic acid in
enthanol.22 Alkylation of 13a–d with 3-piperidinopropyl chloride
afforded 14a–d.15
2.2. Biological activity and molecular modeling
The prepared compounds and reference compound (Ro 31-
6233)10 were tested for their antiproliferative activity against
K562, A549, ECA-109, KB and SMMC-7721 cell lines by MTT assay
in vitro23 and PKC inhibitory activity using PepTagÒ Non-radioac-
tive Protein Kinase C Kit. The results are listed in Table 1.
* Corresponding author. Tel./fax: +86 571 88208460.
E-mail addresses: huyz@zju.edu.cn, huyz@zjuem.zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.043

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Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
Scheme 1. Synthetic route to compounds 9a–j, 10 and 14a–d. Reagents and conditions: (a) NH2NH2ÁH2O, EtOH, reflux; (b) CHMe2CH2CH2NO2, HBF4, CH3OH, 0–5 °C; (c) HPO2,
H2O, rt; (d) I2, KOH, DMF, rt; (e) EtMgBr, (Bu)3SnCl, THF, À5 to 0 °C; (f) Pd(Ph3)2Cl2, LiCl, toluene; (g) alkyl halides, K2CO3, DMF, 65–75 °C; (h) NH4OAc, 140 °C; (i) CH3NH2,
CH3OH, rt; (j) 5 mol/L KOH, EtOH, 30–35 °C; (k) aryl amines, PTS, EtOH; (l) 3-piperidinopropylchloride, K2CO3, DMF, 65–75 °C.
The data in Table 1 indicated that some compounds (i.e., 8a–e,
9c, 9e, 14a, 14d) exhibited moderate to higher antiproliferative
activity against tested cancer cells than the reference compound.
Among them, 8a–e and 14a exhibited more potent antiprolifera-
tive activity than 9a–j, suggesting that the presence of an aryl
group on the nitrogen of the maleimide ring was essential for anti-
proliferative activity. Moreover, when the aryl was unsubstituted
phenyl or 1-naphthyl, compounds (i.e., 8b, 14a) exerted better
activity against most tested cancer cells as compared to 14b–c.
The introduction of different hydrophilic side chains at the N1-po-
sition of the 7-azaindazole ring affected antiproliferative activity
remarkably. Compared with compound 7, the activity of 8a–c (R1
was dimethylamino, piperidinyl and imidazolyl, respectively) was
greatly improved while the activity of 8f and 8g (R1 was Pyrrolyl
and 1H-1,2,4-triazole-1-yl, respectively) was reduced. Introduction
of a 3-(piperidin-1-yl)propyl side chain at N1-position of 7-azain-
dazole ring increased the antiproliferative activity markedly as evi-
denced by comparing compounds 13a and 14a. The result
indicated that the alkaline-moieties at the end of the chain was
important for the activity. The length of alkyl linker also affected
the antiproliferative activity significantly. Comparison of the activ-
ity of 8c and 8e with 8d showed that compounds with a longer (3
or 4 carbon atoms) alkyl chain were more potent than compounds
with shorter alkyl chains. The same conclusion could be drawn
from the results of 9c–e.
and 14a–d showed less activity than 9a–j. This fact indicated that
a hydrogen bond donor in the maleimide ring was critical for main-
taining PKC inhibitory activity. Comparison of 10 with 9a–j
showed that compounds 9a–j with hydrophilic chain at 1-position
of 7-azaindazole ring possessed more potent inhibition. Com-
pounds 9c and 9e were about 10-fold more potent than 9b in
inhibiting PKC. It suggested that the length of the hydrophilic chain
attached to N1-position of the 7-azaindazole ring was a key feature
for enhancing inhibitory potency. However, the best inhibitory
activity, for the same length of alkyl chain, depended on the hydro-
philic group at the end of the chain (i.e., 9a–c). The result was not
consistent with the rank of their antiproliferative activity against
human cancer cell lines. This indicated that the tested compounds
might have other mechanism against human cancer cell lines be-
sides PKC inhibition.
The results prompted us to study other mechanism of the tested
compounds in detail. The antiproliferative mechanism of com-
pound 8b, a representative compound of this class, was further ex-
plored in KB cells.
KB cells were incubated with 10 lM and 20 lM 8b for 48 h, and
the effects of 8b on the morphology of KB cells were observed with
light microscope. As shown in Figure 2A, compound 8b exhibited
great antiproliferatvie activity against KB cells. In addition, the
apoptosis of KB cells was determined by Caspase-Glo 3/7 Kit and
Electrophoretic Analysis of DNA Fragmentation (Fig. 2B and
Fig. 2C).24 We found that compared with control cells, cells ex-
posed to 8b slightly increased caspase-3/7 activity, and resulted
Compared with the reference compound, compounds 9a–j
exhibited moderate inhibitory activity toward PKC, while 8a–g

Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
4765
Table 1
Antiproliferative and PKC inhibitory activity of the compounds 7, 8a–g, 9a–j, 10, 13a and 14a–da
b,c
d
Compound
R1
R2
n
IC50
(
lM)
IC50
(lM)
K562
A549
ECA-109
KB
SMMC-7721
PKC
Ro 31-6233
7
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
10
13a
14a
14b
14c
14d
17.46
3.26
1.70
3.79
5.51
26.43
5.03
36.56
>100
50.4
56.61
>100
13.01
20.55
1.56
43.06
21.65
>100
>100
>100
>100
>100
>100
2.38
>100
>100
>100
>100
>100
>100
>100
2.82
83.55
>100
5.84
12.31
1.44
53.78
>100
14.11
5.6
>100
>100
31.2
0.35 (0.55e)
>10
>10
>10
>10
>10
>10
>10
>10
0.72
0.65
0.90
9.10
0.86
1.10
2.20
5.95
1.86
3.15
>10
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
0
3
3
3
2
4
3
3
3
3
3
2
4
3
3
3
3
3
0
0
3
3
3
3
Me2N
1-Pipe
1-Imid
1-Imid
1-Imid
1-Pyrr
Tria
Me2N
1-Pipe
1-Imid
1-Imid
1-Imid
1-Pyrr
Tria
1-Mor
MePip
OH
H
H
1-Pipe
1-Pipe
1-Pipe
1-Pipe
3.05
6.8
34.77
>100
1.76
>100
>100
>100
7.67
>100
>100
29.16
>100
>100
>100
>100
>100
>100
>100
0.22
8.77
7.54
>100
47.30
>100
>100
>100
97.98
>100
>100
>100
>100
>100
>100
>100
>100
62.02
>100
13.44
>100
>100
43.95
>100
>100
>100
42.34
>100
>100
23.48
>100
>100
>100
>100
>100
46.11
>100
1.83
5.96
13.76
>100
31.75
21.56
>100
>100
46.73
85.78
>100
>100
3.18
H
H
1-Nap
1-Nap
ChlPh
2-Py
MetPh
>10
>10
>10
>10
1.80
>100
>100
28.68
>100
30.28
>100
7.30
3.34
>100
>100
1.74
>10
a
Abbreviations for R1 and R2: Imid, imidazolyl; Pipe, piperidinyl; Pyrr, pyrrolyl; Tria, 1H-1,2,4-triazole-1-yl; Mor, morpholinyl; MePip, 4-methyl-1-piperazinyl; Ph, phenyl;
Nap, naphthyl; ChlPh, 4-chlorophenyl; Py, pyridyl; MetPh, 4-methoxyphenyl.
b
c,d
e
The IC50 values represent the concentration which results in a 50% decrease in cell growth after 48 h incubation.
The IC50 values were the mean values of three repeated experiments.
From the literature.10
in a characteristic fragmentation of DNA, a common feature of
apoptotic cell death. DNA Fragmentation was later conducted in
apoptotic cells, compared with increasing caspase-3/7 activity.
aryl group containing compounds 8a–c, 8e and 14a were the most
promising compounds against the tested cancer cell lines. Com-
pounds 9a–j showed moderate PKC inhibition. Representative
compound 8b was used to investigate other antiproliferative
mechanism besides PKC inhibition in KB cells. Apoptosis pathway
contributed to the antiproliferative activity of compound 8b. The
results provided valuable information for the design of anticancer
agents and PKC inhibitors. Future progress on related series will
be reported in due course.
These theoretical events may explain that treating with 20 lM
8b, a severely high dose, could induce KB cells DNA Fragmentation.
In order to approach signal transduction inducing apoptosis, we
further detected JNK, Bax, Bcl-2, procaspase 9 and procaspase 3
protein expressions with Western Blotting methods (Fig. 3A). The
results suggested that compound 8b induced an increase of JNK
protein expression and an obvious decrease of Bcl-2, procaspase
9 and procaspase 3 protein expressions, but failed to exhibit any ef-
fect on Bax protein expression. The increase of Bax/Bcl-2 ratio,
which could induce the release of cytochrome c,25 was also ob-
served after treating with 8b in KB cells (Fig. 3B). Taken together,
all results suggested that mitochondria-mediated apoptosis path-
way was involved in 8b induced antiproliferative activity in KB
cells.
4. Experimental
4.1. Chemistry
All reactions were monitored by thin-layer chromatography
(TLC). All reagents were obtained from commercial sources and
used without further purification unless stated. Et2O, THF and ben-
zene were distilled from sodium–benzophenone. DMF was dis-
tilled from calcium hydride. Melting points were determined
with a BÜCHI Melting Point B-450 apparatus (Büchi Labortechnik,
Flawil, Switzerland). The 1H NMR spectra were recorded in DMSO-
d6 or CDCl3 on Bruker Avance DMX 400 at 400 MHz (chemical
shifts are expressed as d values relative to TMS as internal stan-
dard). ESI-MS were obtained on an Esquire-LC-00075 mass spec-
3. Conclusion
In conclusion, a series of novel 7-azaindazolyl-indolyl-malei-
mides were prepared. Some compounds showed moderate anti-
proliferative activity against human cancer cell lines including
K562, A549, ECA-109, KB and SMMC-7721. Among them, the N-

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Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
Figure 1. Structure of UCN 01, CGP 41251, Enzastaurin and Ro 31-6233.
trometer (Bruker, USA). Elemental analyses were performed by
ERBA-1110 analyzer (Carlo, Italy).
Na2SO4 and concentrated in vacuum to afford compound 4 (6.3 g,
87.5%) as a white solid, mp: 187–189 °C (Lit.26 188–190 °C).
4.2. 3-Iodo-1H-pyrazolo[3,4-b]pyridine (4)
4.3. 3-(Tributylstannyl)-1H-pyrazolo[3,4-b]pyridine (5)
Iodine (18.7 g, 73.6 mmol) was added to the solution of 3 (3.5 g,
29.4 mmol) in DMF (50 mL), followed by KOH (6.6 g, 118.0 mmol).
The reaction mixture was stirred at room temperature for 2 h. After
that, it was poured into brine (500 mL), and extracted with ethyl
acetate (3 Â 100 mL). The organic phase was combined, washed
with saturated NaHSO3 (50 mL) and brine (3 Â 300 mL), dried over
Under the protection of N2, compound 4 (8.0 g, 32.6 mmol) was
dissolved in THF (200 mL) and the solution was cooled down to
0 °C. Then ethyl magnesium bromide in Et2O (50 mL, 1.3 M) was
added dropwise. The mixture was stirred at 0 °C for 30 min, and
then a solution of (n-Bu)3SnCl (26.5 g, 82.2 mmol) in THF (20 mL)
was added dropwise. The mixture was stirred at 0 °C for 1 h then
Figure 2. The antiproliferative activity of compound 8b in KB cells: (A) compound 8b exhibited great antiproliferatvie activity against KB cells. KB cells were cultured with
10 M and 20 M compound 8b, respectively for 48 h, and then photographed by Leica DMI400B microscope; (B) compound 8b increased the activity of caspase-3/7. KB cells
were cultured with 10 M and 20 M compound 8b, respectively for 48 h, then followed the protocol of caspase-3/7 activity Kit; (C) compound 8b could obviously induced
DNA fragmentation in KB cells. KB cells were cultured with 10 M and 20 M compound 8b, respectively for 48 h, DNA in KB cells were extracted and loaded on agrose gel,
after electrophoresis, DNA ladder was photographed by Bio-Rad GD2000.
l
l
l
l
l
l

Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
4767
Figure 3. The apoptosis associated protein expression of KB cells treated with various concentrations of 8b: (A) the protein expression change in KB cells treated with 8b for
48 h. KB cells were cultured with 10 M and 20 M compound 8b, respectively for 48 h, then were lysed and subjected to Western Blotting using anti-JNK, anti-Bax, anti-Bcl-
l
l
2, anti-procaspase-9, anti-procaspase-3,and anti-tubulin antibodies; (B) compound 8b induced the decrease of Bcl-2 protein expression, and, the strong increase of Bax/Bcl-2
ratio in KB cells.
at room temperature for 1 h. After that, it was poured into brine
(400 mL), and extracted with ethyl acetate (3 Â 100 mL). The or-
ganic phase was combined, washed with brine (3 Â 100 mL), dried
over Na2SO4 and concentrated in vacuum. The residue was purified
by flash column chromatography on silica gel using petroleum
ether/ethyl acetate (6:1, v/v) as eluent to afford compound 5
(8.1 g, 60.9%) as a light yellow liquid, 1H NMR (CDCl3, d): 0.89
(9H, t, J = 7.2 Hz), 1.23–1.28 (6H, t, J = 7.2 Hz), 1.35–1.39 (6H, m),
1.60–1.64 (6H, m), 7.12–7.15 (1H, m), 8.07 (1H, dd, J = 1.6,
8.0 Hz), 8.62 (1H, dd, J = 1.6, 4.8 Hz), 12.71 (1H, br s). ESI-MS: m/
z = 409 [M+H]+. Anal. Calcd for C18H31N3Sn: C, 52.97; H, 7.66; N,
10.29. Found: C, 52.83; H, 7.64; N, 10.43.
Na2SO4 and concentrated in vacuum. The residue was purified by
flash column chromatography on silica gel using dichlorometh-
ane/methanol/ triethylamine (120:4:1, v/v/v) as eluent to afford
8a–i.
4.5.1. 3-(1-(3-(Dimethylamino)propyl)-1H-pyrazolo[3,4-
b]pyridine-3-yl)-4-(1-methyl-1H-indol-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (8a)
According to the general method, the reaction of compound 7
with 3-chloro-N,N-dimethylpropan-1-amine afforded compound
8a in 75.6% yield as a red solid, mp: 149–151 °C. 1H NMR (CDCl3,
d): 1.84–1.87 (2H, m), 2.14 (6H, s), 2.22 (2H, t, J = 7.2 Hz), 3.89
(3H, s), 4.48 (2H, t, J = 7.2 Hz), 6.33 (1H, d, J = 8.0 Hz), 6.78 (1H, t,
J = 8.0 Hz), 7.14–7.16 (2H, m), 7.31(1H, d, J = 8.0 Hz), 7.38–7.40
(1H, m), 7.49–7.53 (4H, m), 8.18 (1H, s), 8.25 (1H, d, J = 8.0 Hz),
8.56 (1H, d, J = 4.4 Hz). ESI-MS: m/z = 505 [M+H]+. Anal. Calcd for
C30H28N6O2: C, 71.41; H, 5.59; N, 16.66. Found: C, 71.23; H, 5.44;
N, 16.91.
4.4. 3-(1-Methyl-1H-indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-
3-yl)-1-phenyl-1H-pyrrole-2,5-dione (7)
Under the protection of N2, a mixture of compound 5 (2.0 g,
4.9 mmol), LiCl (0.48 g, 11.4 mmol), Pd(Ph3)2Cl2 (0.48 g,
0.75 mmol) and compound 6 (1.73 g, 5.14 mmol) in anhydrous tol-
uene (70 mL) was stirred at 100 °C for 10 h. After cooling, the mix-
ture was poured into water (500 mL) and extracted with ethyl
acetate (3 Â 100 mL). The organic phase was combined, washed
with brine (3 Â 100 mL), dried over Na2SO4 and concentrated in
vacuum. The residue was purified by flash column chromatogra-
phy on silica gel using dichloromethane/methanol (30:1, v/v) as
eluent to afford compound 7 (1.13 g, 55.0%) as a red solid, mp:
>250 °C. 1H NMR (DMSO-d6, d): 3.92 (3H, s), 6.33 (1H, d,
J = 8.0 Hz), 6.72 (1H, t, J = 8.0 Hz), 7.11 (1H, t, J = 8.0 Hz), 7.18–
7.22 (1H, m), 7.42–7.44 (1H, m), 7.49 (1H, d, J = 8.0 Hz), 7.51–
7.57 (4H, m), 8.18 (1H, dd, J = 1.6, 8.4 Hz), 8.25 (1H, s), 8.54 (1H,
dd, J = 1.6, 4.4 Hz), 14.02 (1H, s). ESI-MS: m/z = 420 [M+H]+. Anal.
Calcd for C25H17N5O2: C, 71.59; H, 4.09; N, 16.70. Found: C,
71.33; H, 4.14; N, 16.79.
4.5.2. 3-(1-Methyl-1H-indol-3-yl)-1-phenyl-4-(1-(3-(piperidin-
1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (8b)
According to the general method, the reaction of compound 7
with 1-(3-chloropropyl)piperidine afforded compound 8b in
73.6% yield as a red solid, mp: 158–161 °C. 1H NMR (CDCl3, d):
1.37–1.42 (2H, m), 1.51–1.55 (4H, m), 1.85–1.89 (2H, m), 2.23–
2.27 (6H, m), 3.89 (3H, s), 4.45 (2H, t, J = 7.2 Hz), 6.35 (1H, d,
J = 8.0 Hz), 6.78 (1H, t, J = 8.0 Hz), 7.11–7.18 (2H, m), 7.31 (1H, d,
J = 8.4 Hz), 7.38–7.42 (1H, m), 7.50–7.54 (4H, m), 8.19 (1H, s),
8.25 (1H, dd, J = 1.6, 8.0 Hz), 8.55 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS:
m/z = 545 [M+H]+. Anal. Calcd for C33H32N6O2: C, 72.77; H, 5.92;
N, 15.43. Found: C, 72.83; H, 5.96; N, 15.69.
4.5.3. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(1H-imidazol-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (8c)
4.5. General procedure for the synthesis of 8a–i
A mixture of compound 7 (0.24 mmol), K2CO3 (0.48 mmol) and
alkyl halides (0.36 mmol) in DMF (10 mL) was stirred at 65–75 °C
for 8 h. After cooling, the mixture was poured into water
(200 mL) and extracted with ethyl acetate (3 Â 50 mL). The organic
phase was combined, washed with brine (3 Â 100 mL), dried over
According to the general method, the reaction of compound 7
with 1-(3-chloropropyl)-1H-imidazole afforded compound 8c in
73.0% yield as a red solid, mp: 94–96 °C. 1H NMR (CDCl3, d):
2.04–2.07 (2H, m), 3.55 (2H, t, J = 6.8 Hz), 3.88 (3H, s), 4.37 (2H,
t, J = 6.4 Hz), 6.33 (1H, d, J = 8.0 Hz), 6.70–6.74 (2H, m), 6.98 (1H,

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Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
br s), 7.12 (1H, t, J = 8.4 Hz), 7.18–7.22 (1H, m), 7.24 (1H, br s), 7.30
(1H, d, J = 8.0 Hz), 7.39–7.41 (1H, m), 7.50–7.54 (4H, m), 8.12 (1H,
s), 8.38 (1H, dd, J = 1.6, 8.0 Hz), 8.59 (1H, dd, J = 1.6, 4.4 Hz). ESI-
MS: m/z = 528 [M+H]+. Anal. Calcd for C31H25N7O2: C, 70.57; H,
4.78; N, 18.58. Found: C, 70.33; H, 4.74; N, 18.79.
68.7% yield as a red solid, mp: 152–154 °C. 1H NMR (CDCl3, d):
1.88–1.90 (2H, m), 2.27–2.31 (6H, m), 3.62–3.66 (4H, m), 3.91
(3H, s), 4.48 (2H, t, J = 7.2 Hz), 6.32 (1H, d, J = 8.0 Hz), 6.77 (1H, t,
J = 8.0 Hz), 7.13–7.17 (2H, m), 7.32 (1H, d, J = 8.0 Hz), 7.38–7.40
(1H, m), 7.50–7.54 (4H, m), 8.18 (1H, s), 8.26 (1H, d, J = 8.4 Hz),
8.55 (1H, d, J = 4.4 Hz). ESI-MS: m/z = 547 [M+H]+. Anal. Calcd for
C32H30N6O3: C, 70.31; H, 5.53; N, 15.37. Found: C, 70.33; H, 5.44;
N, 15.59.
4.5.4. 3-(1-(2-(1H-Imidazol-1-yl)ethyl)-1H-pyrazolo[3,4-
b]pyridine-3-yl)-4-(1-methyl-1H-indol-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (8d)
According to the general method, the reaction of compound 7
with 1-(2-chloroethyl)-1H-imidazole afforded compound 8d in
71.2% yield as a red solid, mp: 145–148 °C. 1H NMR (CDCl3, d):
3.96 (3H, s), 4.55 (2H, t, J = 6.8 Hz), 4.89 (2H, t, J = 6.8 Hz), 6.32
(1H, d, J = 8.0 Hz), 6.73–6.75 (2H, m), 7.02 (1H, br s), 7.09–7.11
(1H, m), 7.16 (1H, t, J = 8.4 Hz), 7.36 (1H, d, J = 8.0 Hz), 7.41–7.43
(1H, m), 7.50–7.54 (4H, m), 8.15 (1H, dd, J = 1.6, 8.0 Hz), 8.18
(1H, s), 8.46 (1H, dd, J = 1.6, 4.4 Hz), 8.60 (1H, br s). ESI-MS: m/
z = 514 [M+H]+. Anal. Calcd for C30H23N7O2: C, 70.16; H, 4.51; N,
19.09. Found: C, 70.33; H, 4.44; N, 19.19.
4.5.9. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(4-methylpiperazin-
1-yl)propyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-
pyrrole-2,5-dione (8i)
According to the general method, the reaction of compound 14
with 1-(3-chloropropyl)-4-methylpiperazine afforded compound
8i in 70.5% yield as a red solid, mp: 195–198 °C. 1H NMR (CDCl3,
d): 1.82–1.85 (2H, m), 2.15–2.40 (13H, m), 3.90 (3H, s), 4.47 (2H,
t, J = 7.2 Hz), 6.33 (1H, d, J = 8.0 Hz), 6.77 (1H, t, J = 8.0 Hz), 7.13–
7.16 (2H, m), 7.31 (1H, d, J = 7.6 Hz), 7.38–7.39 (1H, m), 7.50–
7.52 (4H, m), 8.17 (1H, s), 8.27 (1H, d, J = 8.4 Hz), 8.50 (1H, d,
J = 4.4 Hz). ESI-MS: m/z = 560 [M+H]+. Anal. Calcd for C33H33N7O2:
C, 70.82; H, 5.94; N, 17.52. Found: C, 71.03; H, 5.84; N, 17.50.
4.5.5. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(4-(1H-imidazol-1-
yl)butyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (8e)
4.5.10. 3-(1-Methyl-1H-indol-3-yl)-1-phenyl-4-(1-(3-(tert-
butyldimethylsilyloxy)propyl)-1H-pyrazolo[3,4-b]pyridine-3-
yl)-1H-pyrrole-2,5-dione (8j)
According to the general method, the reaction of compound 7
with 1-(4-chlorobutyl)-1H-imidazole afforded compound 8e in
68.0% yield as a red solid, mp: 114–117 °C. 1H NMR (CDCl3, d):
1.63–1.65 (4H, m), 3.79 (2H, t, J = 6.4 Hz), 3.88 (3H, s), 4.44 (2H,
t, J = 6.4 Hz), 6.27 (1H, d, J = 8.0 Hz), 6.70 (1H, t, J = 8.0 Hz), 6.79
(1H, br s), 7.01 (1H, br s), 7.14–7.17 (2H, m), 7.32 (1H, d,
J = 8.0 Hz), 7.39–7.41 (2H, m), 7.50–7.54 (4H, m), 8.16 (1H, s),
8.30 (1H, d, J = 8.0 Hz), 8.57 (1H, d, J = 4.4 Hz). ESI-MS: m/z = 542
[M+H]+. Anal. Calcd for C32H27N7O2: C, 70.96; H, 5.02; N, 18.10.
Found: C, 70.83; H, 5.14; N, 18.29.
A
mixture of compound 7 (100.0 mg, 0.24 mmol), K2CO3
(86.2 mg, 0.48 mmol), (3-bromopropoxy) (tert-butyl)dimethylsi-
lane (91.1 mg, 0.36 mmol) in DMF (10 mL) was stirred at 60 °C
for 2 h. After cooling, the mixture was poured into water
(200 mL) and extracted with ethyl acetate (3 Â 50 mL). The organic
phase was combined and washed with brine (3 Â 100 mL), dried
over Na2SO4 and concentrated in vacuum. The residue was purified
by flash column chromatography on silica gel using dichlorometh-
ane/methanol (60:1, v/v) as eluent to afford 8j (97.5 mg, 68.6%) as a
red solid, mp: 110–113 °C. 1H NMR (CDCl3, d): 0.01 (6H, s), 0.88
(9H, s), 1.87–1.90 (2H, m), 3.52 (2H, t, J = 6.8 Hz), 3.92 (3H, s),
4.54 (2H, t, J = 7.2 Hz), 6.39 (1H, d, J = 8.0 Hz), 6.79 (1H, t,
J = 7.6 Hz), 7.16–7.18 (2H, m), 7.33 (1H, d, J = 8.0 Hz), 7.41–7.42
(1H, m), 7.51–7.55 (4H, m), 8.21(1H, s), 8.30 (1H, dd, J = 1.6,
8.0 Hz), 8.56 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS: m/z = 592 [M+H]+.
Anal. Calcd for C34H37N5O3Si: C, 69.01; H, 6.30; N, 11.83. Found:
c, 69.23; H, 6.17; N, 11.72.
4.5.6. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(1H-pyrrol-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (8f)
According to the general method, the reaction of compound 7
with 1-(3-chloropropyl)-1H-pyrrole afforded compound 8f in
77.0% yield as a red solid, mp: 85–88 °C. 1H NMR (CDCl3, d): 2.09
(2H, m), 3.60 (2H, t, J = 7.2 Hz), 3.89 (3H, s), 4.39 (2H, t,
J = 6.8 Hz), 6.07–6.09 (2H, m), 6.37 (1H, d, J = 8.0 Hz), 6.46–6.48
(2H, m), 6.74 (1H, t, J = 8.0 Hz), 7.14 (1H, t, J = 8.0 Hz), 7.18–7.20
(1H, m), 7.30 (1H, d, J = 8.0 Hz), 7.40–7.42 (1H, m), 7.50–7.54
(4H, m), 8.15 (1H, s), 8.35 (1H, dd, J = 1.6, 8.0 Hz), 8.58 (1H, dd,
J = 1.6, 4.4 Hz). ESI-MS: m/z = 527 [M+H]+. Anal. Calcd for
C32H26N6O2: C, 72.99; H, 4.98; N, 15.96. Found: C, 72.83; H, 4.94;
N, 15.89.
4.6. General procedure for the synthesis of 9a–j and 10
Under the protection of N2, compound 8a–j or 7 (0.057 mmol)
was heated with NH4OAc (57.0 mmol) for 6 h at 140 °C. The mix-
ture was cooled, poured into water (20 mL), adjusted to weak alka-
linity with Na2CO3 and extracted with ethyl acetate (3 Â 20 mL).
The organic layer was washed with brine (3 Â 50 mL), dried over
Na2SO4 and concentrated in vacuum. The residue was purified by
flash column chromatography on silica gel using dichlorometh-
ane/methanol/ triethylamine (120:4:1, v/v/v) as eluent to afford
9a–j and 10.
4.5.7. 3-(1-Methyl-1H-indol-3-yl)-1-phenyl-4-(1-(3-(1H-1,2,4-
triazol-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-
pyrrole-2,5-dione (8g)
According to the general method, the reaction of compound 7
with 1-(3-chloropropyl)-1H-1,2,4-triazole afforded compound 8g
in 69.4% yield as a red solid, mp: 163–165 °C. 1H NMR (CDCl3, d):
2.14–2.16 (2H, m), 3.87 (3H, s), 3.99 (2H, t, J = 6.8 Hz), 4.38 (2H,
t, J = 6.4 Hz), 6.39 (1H, d, J = 8.0 Hz), 6.74 (1H, t, J = 8.0 Hz), 7.06
(1H, t, J = 8.0 Hz), 7.26–7.28 (1H, m), 7.42–7.45 (2H, m), 7.53–
7.57 (4H, m), 7.97 (1H, s), 8.23–8.26 (2H, m), 8.29 (1H, br s), 8.59
(1H, dd, J = 1.6, 4.4 Hz). ESI-MS: m/z = 529 [M+H]+. Anal. Calcd for
C30H24N8O2: C, 68.17; H, 4.58; N, 21.20. Found: C, 68.33; H, 4.44;
N, 21.06.
4.6.1. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-
(dimethylamino)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-
pyrrole-2,5-dione (9a)
According to the general method, the reaction of compound 8a
with NH4OAc afforded compound 9a in 90.2% yield as a red solid,
mp: 192–194 °C. 1H NMR (CDCl3, d): 2.04–2.06 (2H, m), 2.30 (6H,
s), 2.49 (2H, t, J = 7.6 Hz), 3.87 (3H, s), 4.52 (2H, t, J = 6.8 Hz), 6.28
(1H, d, J = 8.4 Hz), 6.75 (1H, t, J = 8.0 Hz), 7.14–7.16 (2H, m), 7.30
(1H, d, J = 8.0 Hz), 8.11 (1H, s), 8.14 (1H, dd, J = 1.6, 8.0 Hz), 8.55
(1H, dd, J = 1.6, 4.0 Hz). ESI-MS: m/z = 429 [M+H]+. Anal. Calcd for
4.5.8. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-
dione (8h)
According to the general method, the reaction of compound 7
with 4-(3-chloropropyl)morpholine afforded compound 8h in

Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
4769
C24H24N6O2: C, 67.27; H, 5.65; N, 19.61. Found: C, 67.36; H, 5.60; N,
19.73.
8.0 Hz), 8.58 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS: m/z = 451 [M+H]+.
Anal. Calcd for C26H22N6O2: C, 69.32; H, 4.92; N, 18.66. Found: C,
69.46; H, 4.82; N, 18.49.
4.6.2. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(piperidin-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9b)
4.6.7. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(1H-1,2,4-triazol-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9g)
According to the general method, the reaction of compound 8b
with NH4OAc afforded compound 9b in 89.4% yield as a red solid,
mp: 108–111 °C. 1H NMR (CDCl3, d): 1.37–1.39 (2H, m), 1.52–
1.56 (4H, m), 1.90–1.93 (2H, m), 2.24–2.30 (6H, m), 3.86 (3H, s),
4.46 (2H, t, J = 7.2 Hz), 6.33 (1H, d, J = 8.0 Hz), 6.76 (1H, t,
J = 8.0 Hz), 7.12 (2H, m), 7.28 (1H, d, J = 8.0 Hz), 8.11 (1H, s), 8.15
(1H, dd, J =1.6, 8.4 Hz), 8.53 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS: m/
z = 469 [M+H]+. Anal. Calcd for C27H28N6O2: C, 69.21; H, 6.02; N,
17.94. Found: C, 69.03; H, 5.98; N, 18.03.
According to the general method, the reaction of compound 8g
with NH4OAc afforded compound 9g in 88.1% yield as a red solid,
mp: 237–239 °C. 1H NMR(CDCl3 + DMSO-d6, d): 2.08–2.10 (2H,
m), 3.63 (2H, t, J = 6.8 Hz), 3.77 (3H, s), 4.21 (2H, t, J = 6.4 Hz),
6.22 (1H, d, J = 8.0 Hz), 6.60 (1H, t, J = 8.0 Hz), 6.98 (1H, t,
J = 8.0 Hz), 7.07–7.08 (1H, m), 7.18 (1H, d, J = 8.0 Hz), 7.68 (1H, s),
7.78 (1H, s), 7.95 (1H, s), 8.16 (1H, dd, J = 1.6, 8.0 Hz), 8.46 (1H,
dd, J = 1.6, 4.4 Hz), 10.1 (1H, br s). ESI-MS: m/z = 453 [M+H]+. Anal.
Calcd for C24H20N8O2: C, 63.71; H, 4.46; N, 24.76. Found: C, 63.91;
H, 4.55; N, 24.59.
4.6.3. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(1H-imidazol-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9c)
4.6.8. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (9h)
According to the general method, the reaction of compound 8h
with NH4OAc afforded compound 9h in 93.6% yield as a red solid,
mp: 93–95 °C. 1H NMR (CDCl3, d): 1.86–1.88 (2H, m), 2.25–2.28
(6H, m), 3.62–3.65 (4H, m), 3.89 (3H, s), 4.49 (2H, t, J = 7.2 Hz),
6.27 (1H, d, J = 8.0 Hz), 6.74 (1H, t, J = 7.6 Hz), 7.12–7.14 (2H, m),
7.30 (1H, d, J = 8.4 Hz), 7.90 (1H, br s), 8.11 (1H, s), 8.16 (1H, dd,
J = 1.6, 8.0 Hz), 8.55 (1H, dd, J = 1.6, 4.0 Hz). ESI-MS: m/z = 471
[M+H]+. Anal. Calcd for C26H26N6O3: C, 66.37; H, 5.57; N, 17.86.
Found: C, 66.49; H, 5.51; N, 17.69.
According to the general method, the reaction of compound 8c
with NH4OAc afforded compound 9c in 92.2% yield as a red solid,
mp: 106–108 °C. 1H NMR (CDCl3, d): 2.02–2.04 (2H, m), 3.52 (2H,
t, J = 6.8 Hz), 3.82 (3H, s), 4.34 (2H, t, J = 6.4 Hz), 6.25 (1H, d,
J = 8.0 Hz), 6.66–6.68 (2H, m), 6.98 (1H, br s), 7.06 (1H, t,
J = 8.0 Hz), 7.17–7.19 (1H, m), 7.23–7.25 (2H, m), 8.04 (1H, s),
8.29 (1H, dd, J = 1.6, 8.0 Hz), 8.58 (1H, dd, J = 1.6, 4.4 Hz), 8.96
(1H, br s). ESI-MS: m/z = 452 [M+H]+. Anal. Calcd for C25H21N7O2:
C, 66.51; H, 4.69; N, 21.72. Found: C, 66.36; H, 4.75; N, 21.53.
4.6.4. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(2-(1H-imidazol-1-
yl)ethyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9d)
4.6.9. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(4-methylpiperazin-
1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9i)
According to the general method, the reaction of compound 8d
with NH4OAc afforded compound 9d in 88.3% yield as a red solid,
mp: 231–234 °C. 1H NMR (CDCl3, d): 3.91 (3H, s), 4.23 (2H, t,
J = 6.8 Hz), 4.76 (2H, t, J = 6.8 Hz), 6.29 (1H, d, J = 8.0 Hz), 6.76–
6.78 (2H, m), 6.95 (1H, br s), 7.10–7.11 (1H, m), 7.15 (1H, t,
J = 8.0 Hz), 7.29 (1H, br s), 7.33 (1H, d, J = 8.0 Hz), 8.07 (1H, s),
8.11–8.13 (2H, m), 8.50 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS: m/
z = 438 [M+H]+. Anal. Calcd for C24H19N7O2: C, 65.89.32; H, 4.38;
N, 22.41. Found: C, 65.66; H, 4.32; N, 22.58.
According to the general method, the reaction of compound 8i
with NH4OAc afforded compound 9i in 91.7% yield as a saffron yel-
low solid, mp: 102–104 °C. 1H NMR (CDCl3, d): 1.87–1.89 (2H, m),
2.25–2.60 (13H, m), 3.86 (3H, s), 4.47 (2H, t, J = 7.2 Hz), 6.30 (1H, d,
J = 7.6 Hz), 6.74 (1H, t, J = 8.0 Hz), 7.12–7.15 (2H, m), 7.29 (1H, d,
J = 8.0 Hz), 8.09 (1H, s), 8.16 (1H, dd, J = 1.6, 8.0 Hz), 8.53 (1H, dd,
J = 1.6, 4.4 Hz). ESI-MS: m/z = 484 [M+H]+. Anal. Calcd for
C27H29N7O2: C, 67.06; H, 6.04; N, 20.28. Found: C, 67.26; H, 5.98;
N, 20.03.
4.6.5. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(4-(1H-imidazol-1-
yl)butyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9e)
4.6.10. 3-(1-(3-Hydroxypropyl)-1H-pyrazolo[3,4-b]pyridine-3-
yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (9j)
According to the general method, the reaction of compound 8j
with NH4OAc afforded compound 9j in 49.0% as a red solid, mp:
119–121 °C. 1H NMR (CDCl3, d): 1.80–1.83 (2H, m), 3.32–3.35
(2H, m), 3.91 (3H, s), 4.46 (2H, t, J = 6.8 Hz), 4.53 (1H, t,
J = 5.2 Hz), 6.28 (1H, d, J = 8.0 Hz), 6.70 (1H, t, J = 8.0 Hz), 7.10
(1H, t, J = 8.4 Hz), 7.18–7.20 (1H, m), 7.46 (1H, d, J = 8.0 Hz), 8.11
(1H, s), 8.22 (1H, dd, J = 1.6, 8.0 Hz), 8.55 (1H, dd, J = 1.6, 4.0 Hz),
11.20 (1H, s). ESI-MS: m/z = 402 [M+H]+. Anal. Calcd for
C22H19N5O3: C, 65.83; H, 4.77; N, 17.45. Found: C, 65.69; H, 4.81;
N, 17.69.
According to the general method, the reaction of compound 8e
with NH4OAc afforded compound 9e in 87.0% as a red solid, mp:
93–95 °C. 1H NMR (CDCl3, d): 1.62–1.66 (4H, m), 3.78 (2H, t,
J = 6.4 Hz), 3.88 (3H, s), 4.44 (2H, t, J = 6.4 Hz), 6.21 (1H, d,
J = 8.0 Hz), 6.67 (1H, t, J = 7.6 Hz), 6.78 (1H, br s), 7.03 (1H, br s),
7.13–7.15 (2H, m), 7.30 (1H, d, J = 8.4 Hz), 7.37 (1H, br s), 8.10
(1H, s), 8.22 (1H, dd, J = 1.6, 8.0 Hz), 8.30 (1H, br s), 8.57 (1H, dd,
J = 1.6, 4.4 Hz). ESI-MS: m/z = 466 [M+H]+. Anal. Calcd for
C26H23N7O2: C, 67.08; H, 4.98; N, 21.06. Found: C, 67.19; H, 5.02;
N, 20.93.
4.6.6. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(1H-pyrrol-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-
dione (9f)
According to the general method, the reaction of compound 9f
with NH4OAc afforded compound 9f in 85.9% yield as a red solid,
mp: 195–198 °C. 1H NMR (CDCl3, d): 2.18–2.20 (2H, m), 3.62 (2H,
t, J = 7.2 Hz), 3.89 (3H, s), 4.40 (2H, t, J = 6.8 Hz), 6.07–6.09 (2H,
m), 6.30 (1H, d, J = 8.0 Hz), 6.49–6.51 (2H, m), 6.73 (1H, t,
J = 8.0 Hz), 7.12 (1H, t, J = 8.0 Hz), 7.20–7.21 (1H, m), 7.30 (1H, d,
J = 8.0 Hz), 7.69 (1H, br s), 8.10 (1H, s), 8.27 (1H, dd, J = 1.6,
4.6.11. 3-(1-Methyl-1H-indol-3-yl)-4-(1H-pyrazolo[3,4-
b]pyridin-3-yl)-1H-pyrrole-2,5-dione (10)
According to the general method, the reaction of compound 7
with NH4OAc produced compound 10 in 93.0% yield as a red solid,
mp: >250 °C. 1H NMR (DMSO-d6, d): 3.89 (3H, s), 6.25 (1H, d,
J = 8.0 Hz), 6.68 (1H, t, J = 8.0 Hz), 7.08 (1H, t, J = 8.0 Hz), 7.14–
7.17 (1H, m), 7.45 (1H, d, J = 8.4 Hz), 8.08 (1H, d, J = 8.0 Hz), 8.17
(1H, s), 8.52 (1H, d, J = 4.4 Hz), 11.20 (1H, s), 13.94 (1H, s). ESI-
MS: m/z = 334 [M+H]+. Anal. Calcd for C19H13N5O2: C, 66.47; H,
3.82; N, 20.40. Found: C, 66.23; H, 3. 38; N, 20.52.

4770
Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
4.7. 1-Methyl-3-(1-methyl-1H-indol-3-yl)-4-(1H-pyrazolo[3,4-
b]pyridine-3-yl)-1H-pyrrole-2,5-dione (11)
66.16; H, 3.55; Cl, 7.81; N, 15.43. Found: C, 66.23; H, 3.48; Cl,
7.61; N, 15.52.
32.0% Methylamine in methanol (16 mL) was added to com-
pound 7 (0.80 g, 1.9 mmol) and the mixture was stirred at room
temperature for 2 h. The solvent was removed in vacuum and the
residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol (30:1, v/v) as eluent to afford
compound 11 (0.58 g, 86.0%) as a red solid, mp: >250 °C. 1H NMR
4.9.3. 3-(1-Methyl-1H-indol-3-yl)-4-(1H-pyrazolo[3,4-
b]pyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrrole-2,5-dione (13c)
According to the general method, the reaction of 12 with pyri-
din-2-amine afforded compound 13c in 68.6% yield as a red solid,
mp: 233–235 °C. 1H NMR (DMSO-d6, d): 3.92 (3H, s), 6.35 (1H, d,
J = 8.0 Hz), 6.73 (1H, t, J = 8.0 Hz), 7.11 (1H, t, J = 8.0 Hz,), 7.15–
7.18 (1H, m), 7.49 (1H, d, J = 8.0 Hz), 7.52–7.54 (1H, m), 7.64 (1H,
t, J = 8.4 Hz), 8.06 (1H, td, J = 1.6, 8.0 Hz), 8.12 (1H, dd, J = 1.6,
8.0 Hz), 8.25 (1H, s), 8.54 (1H, dd, J = 1.6, 4.4 Hz), 8.66 (1H, dd,
J = 1.6, 4.8 Hz), 13.99 (1H, s). ESI-MS: m/z = 421 [M+H]+. Anal. Calcd
for C24H16N6O2: C, 68.56; H, 3.84; N, 19.99. Found: C, 68.73; H,
3.88; N, 19.82.
(DMSO-d6 ,
3.07 (3H, s), 3.90 (3H, s), 6.29 (1H, d, J = 8.0 Hz),
d):
6.69 (1H, t, J = 7.6 Hz), 7.08 (1H, t, J = 8.0 Hz), 7.13–7.16 (1H, m),
7.46 (1H, d, J = 8.0 Hz), 8.06 (1H, d, J = 8.0 Hz), 8.20 (1H, s), 8.52
(1H, dd, J = 1.2, 4.4 Hz), 13.97 (1H, s). ESI-MS: m/z = 358 [M+H]+.
Anal. Calcd for C20H15N5O2: C, 67.22; H, 4.23; N, 19.60. Found: C,
67.43; H, 4.38; N, 19.52.
4.8. 3-(1-Methyl-1H-indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridin-
3-yl)furan-2,5-dione (12)
4.9.4. 1-(4-Methoxyphenyl)-3-(1-methyl-1H-indol-3-yl)-4-(1H-
pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (13d)
According to the general method, the reaction of 12 with 4-
methoxyaniline afforded compound 13d in 71.3% yield as a red so-
lid, mp: >250 °C. 1H NMR (DMSO-d6, d): 3.82 (3H, s), 3.92 (3H, s),
6.36 (1H, d, J = 8.0 Hz), 6.72 (1H, t, J = 8.0 Hz), 7.08–7.11 (3H, m),
7.15–7.17 (1H, m), 7.43–7.46 (2H, m), 7.48 (1H, d, J = 8.0 Hz),
8.15 (1H, dd, J = 1.6, 8.0 Hz), 8.23 (1H, s), 8.44 (1H, dd, J = 1.6,
4.4 Hz), 13.99 (1H, s). ESI-MS: m/z = 450 [M+H]+. Anal. Calcd for
C26H19N5O3: C, 69.48; H, 4.26; N, 15.58. Found: C, 69.23; H, 4.38;
N, 15.42.
To a solution of compound 11 (0.62 g, 1.7 mmol) in ethanol
(50 mL), 5 M aqueous KOH (25 mL) was added. The mixture was
stirred at 25–30 °C for 5 h. Water (400 mL) was added to the solu-
tion and the mixture was acidified with 2 M HCl and extracted
with ethyl acetate (3 Â 80 mL). The combined organic layer was
washed with brine (3 Â 240 mL), dried over Na2SO4 and concen-
trated in vacuum. The residue was purified by flash column chro-
matography on silica gel using petroleum ether/ethyl acetate
(8:1, v/v) as eluent to afford compound 12 (0.44 g, 72.6%) as a saf-
fron yellow solid, mp: >250 °C. 1H NMR (DMSO-d6, d): 3.93 (3H, s),
6.34 (1H, d, J = 8.0 Hz), 6.77 (1H, t, J = 7.6 Hz), 7.14 (1H, t,
J = 8.0 Hz), 7.18–7.19 (1H, m), 7.51 (1H, d, J = 8.0 Hz), 8.15 (1H,
dd, J = 1.6, 8.0 Hz), 8.34 (1H, s), 8.56 (1H, dd, J = 1.6, 4.0 Hz),
14.20 (1H, s). ESI-MS: m/z = 345 [M+H]+. Anal. Calcd for
C19H12N4O3: C, 66.28; H, 3.51; N, 16.27. Found: C, 66.43; H, 3.48;
N, 16.39.
4.10. General procedure for the synthesis of 14a–d
A mixture of compounds 13a–d (0.1 mmol), K2CO3 (27.6 mg,
0.2 mmol), 1-(3-chloropropyl)piperidine (58.1 mg, 0.36 mmol) in
DMF (15 mL) was stirred at 70 °C for 8 h. After cooling, the mixture
was poured into water (300 mL) and extracted with ethyl acetate
(3 Â 50 mL). The organic phase was combined and washed with
brine (3 Â 150 mL), dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol/triethylamine (120:4:1, v/v/
v) as eluent to afford 14a–d.
4.9. General procedure for the synthesis of 13a–d
A mixture of 12 (80.0 mg, 0.23 mmol), arylamines (2.3 mmol)
and 4-methylbenzenesulfonic acid (8.0 mg, 0.046 mmol) in ethanol
(30 mL) was refluxed for 12 h. After that, the solvent was removed
in vacuum and the residue was purified by flash column chroma-
tography on silica gel using dichloromethane/methanol (30:1, v/
v) as eluent to afford 13a–d.
4.10.1. 3-(1-Methyl-1H-indol-3-yl)-1-(naphthalen-1-yl)-4-(1-
(3-(piperidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-
pyrrole-2,5-dione (14a)
According to the general method, the reaction of 13a with 1-(3-
chloropropyl)piperidine afforded compound 14a in 81.2% yield as a
red solid, mp: 161–163 °C. 1H NMR (CDCl3, d): 1.49–1.50 (2H, m),
1.83–1.87 (4H, m), 2.25–2.27 (2H, m), 2.63–2.67 (6H, m), 3.93
(3H, s), 4.54 (2H, t, J = 7.2 Hz), 6.39 (1H, d, J = 8.0 Hz), 6.85 (1H, t,
J = 8.0 Hz), 7.14–7.16 (1H, m), 7.21 (1H, t, J = 8.0 Hz), 7.37 (1H, d,
J = 8.0 Hz), 7.55–7.58 (3H, m), 7.63 (1H, t, J = 8.0 Hz), 7.79–7.81
(1H, m), 7.96–7.99 (2H, m), 8.24 (1H, s), 8.28 (1H, dd, J = 1.2,
8.0 Hz), 8.58 (1H, dd, J = 1.2, 4.4 Hz). ESI-MS: m/z = 595 [M+H]+.
Anal. Calcd for C37H34N6O2: C, 74.73; H, 5.76; N, 14.13. Found: C,
74.93; H, 5.68; N, 14.32.
4.9.1. 3-(1-Methyl-1H-indol-3-yl)-1-(naphthalen-1-yl)-4-(1H-
pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (13a)
According to the general method, the reaction of 12 with naph-
thalen-1-amine afforded compound 13a in 78.0% yield as a yellow
solid, mp: >250 °C. 1H NMR (DMSO-d6, d): 3.93 (3H, s), 6.40 (1H, d,
J = 8.0 Hz), 6.74 (1H, t, J = 8.4 Hz), 7.12 (1H, t, J = 8.0 Hz), 7.15–7.18
(1H, m), 7.50 (1H, d, J = 8.0 Hz), 7.60–7.64 (2H, m), 7.67–7.70 (2H,
m), 7.92 (1H, dd, J = 2.0, 8.0 Hz), 8.09–8.11 (2H, m), 8.16 (1H, dd,
J = 1.6, 8.0 Hz), 8.27 (1H, s), 8.54 (1H, dd, J = 1.6, 4.0 Hz), 14.20
(1H, br s). ESI-MS: m/z = 470 [M+H]+. Anal. Calcd for C29H19N5O2:
C, 74.19; H, 4.08; N, 14.92. Found: C, 74.33; H, 4.18; N, 15.02.
4.10.2. 1-(4-Chlorophenyl)-3-(1-methyl-1H-indol-3-yl)-4-(1-(3-
(piperidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-
pyrrole-2,5-dione (14b)
4.9.2. 1-(4-Chlorophenyl)-3-(1-methyl-1H-indol-3-yl)-4-(1H-
pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (13b)
According to the general method, the reaction of 13b with 1-(3-
chloropropyl)piperidine afforded compound 14b in 65.4% yield as a
red solid, mp: 135–137 °C. 1H NMR (CDCl3, d): 1.40–1.42 (2H, m),
1.50–1.53 (4H, m), 1.86–1.88 (2H, m), 2.23–2.27 (6H, m), 3.90
(3H, s), 4.46 (2H, t, J = 7.2 Hz), 6.34 (1H, d, J = 8.0 Hz), 6.77 (1H, t,
J = 8.0 Hz), 7.13–7.15 (2H, m), 7.32 (1H, d, J = 8.0 Hz), 7.46–7.49
(4H, m), 8.17 (1H, s), 8.21 (1H, dd, J = 1.6, 8.0 Hz), 8.55 (1H, dd,
J = 1.6, 4.4 Hz). ESI-MS: m/z = 580 [M+H]+. Anal. Calcd for
According to the general method, the reaction of 12 with 4-
chloroaniline afforded compound 13b in 71.4% yield as a red solid,
mp: >250 °C. 1H NMR (DMSO-d6, d): 3.92 (3H, s), 6.36 (1H, d,
J = 7.6 Hz), 6.73 (1H, t, J = 8.0 Hz), 7.11 (1H, t, J = 8.0 Hz), 7.15–
7.18 (1H, m), 7.48 (1H, d, J = 8.4 Hz), 7.60–7.64 (4H, m), 8.17 (1H,
d, J = 8.4 Hz), 8.25 (1H, s), 8.44 (1H, d, J = 4.0 Hz), 14.01 (1H, s).
ESI-MS: m/z = 455 [M+H]+. Anal. Calcd for C25H16ClN5O2: C,

Q. Ye et al. / Bioorg. Med. Chem. 17 (2009) 4763–4772
4771
C33H31ClN6O2: C, 68.44; H, 5.40; Cl, 6.12; N, 14.51. Found: C, 68.23;
H, 5.48; Cl, 6.21; N, 14.32.
chemiluminescence (ECL) Western Blotting detection reagents
(Amersham Biosciences, Piscataway, NJ).
4.10.3. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(3-(piperidin-1-
yl)propyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-1-(pyridin-2-yl)-1H-
pyrrole-2,5-dione (14c)
4.11.3. Electrophoretic analysis of DNA fragmentation
KB cells were lysed in 200.0 mL lysis buffer (10.0 mM EDTA;
50.0 mM Tris–HCl, pH 8.0; 0.5% sodium lauryl sulfate; 100.0 mg/
mL proteinase K) at 37 °C for 12 h, then incubated with RNase
(50.0 mg/ml) at 37 °C for an additional 1 h. After incubation, DNA
in the lysate was extracted with equal volume of phenol/chloro-
form/isoamyl alcohol (25:24:1), then with chloroform. DNA was
precipitated with two volumes of ethanol in the presence of
0.3 M sodium acetate. After centrifugation at 12,000g for 15 min,
the DNA pellets were washed with 70% ethanol, air-dried, and
resuspended in 20.0 mL TE (10.0 mM Tris–HCl and 1.0 mM EDTA,
pH 8.0). DNA was separated on 1.5% agarose gels containing
0.5 mg/ml ethidium bromide and photographed by Bio-Rad
GD2000 (Bio-Rad, Hercules, CA, USA).
According to the general method, the reaction of 13c with 1-(3-
chloropropyl)piperidine afforded compound 14c in 66.0% yield as a
red solid, mp: 138–141 °C. 1H NMR (CDCl3, d): 1.43–1.45 (2H, m),
1.65–1.69 (4H, m), 2.03–2.05 (2H, m), 2.40–2.44 (6H, m), 3.90
(3H, s), 4.48 (2H, t, J = 6.8 Hz), 6.35 (1H, d, J = 8.0 Hz), 6.78 (1H, t,
J = 8.0 Hz), 7.13–7.15 (2H, m), 7.34–7.35 (2H, m), 7.52 (1H, d,
J = 8.0 Hz), 7.90 (1H, t, J = 7.6 Hz), 8.21 (1H, s), 8.25 (1H, d,
J = 8.4 Hz), 8.54 (1H, d, J = 4.4 Hz), 8.71 (1H, d, J = 4.8 Hz). ESI-MS:
m/z = 546 [M+H]+. Anal. Calcd for C32H31N7O2: C, 70.44; H, 5.73;
N, 17.97. Found: C, 70.63; H, 5.68; N, 17.82.
4.10.4. 1-(4-Methoxyphenyl)-3-(1-methyl-1H-indol-3-yl)-4-(1-
(3-(piperidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-
pyrrole-2,5-dione (14d)
4.11.4. PepTag assay for nonradioactive detection of PKC
activity
According to the general method, the reaction of 13d with 1-(3-
chloropropyl)piperidine afforded compound 14d in 68.7% yield as a
red solid, mp: 165–168 °C. 1H NMR (CDCl3, d): 1.51–1.54 (2H, m),
1.81–1.84 (4H, m), 2.21–2.23 (2H, m), 2.56–2.60 (6H, m), 3.87
(3H, s), 3.90 (3H, s), 4.52 (2H, t, J = 6.4 Hz), 6.34 (1H, d,
J = 8.0 Hz), 6.80 (1H, t, J = 8.0 Hz), 7.03–7.05 (2H, m), 7.15–7.18
(2H, m), 7.34 (1H, d, J = 8.0 Hz), 7.38–7.39 (2H, m), 8.19 (1H, s),
8.24 (1H, dd, J = 1.6, 8.0 Hz), 8.57 (1H, dd, J = 1.6, 4.4 Hz). ESI-MS:
m/z = 575 [M+H]+. Anal. Calcd for C34H34N6O3: C, 71.06; H, 5.96;
N, 14.62. Found: C, 71.23; H, 5.88; N, 14.52.
The PepTag assay utilizes a brightly colored, florescent peptide
substrate that is highly specific to PKC (according to the manufac-
turer’s instructions (Promega). Phosphorylation by PKC changes
the net charge of the substrate from +1 to À1, thereby allowing
the phosphorylated and nonphosphorylated versions of the sub-
strate to be separated on an agarose (0.8%) gel. The phosphorylated
species migrates toward the positive electrode, while the nonphos-
phorylated substrate migrates toward the negative electrode. The
phosphorylated peptide in the band can then be visualized under
UV light. PKC recombination kinases were incubated with PKC
reaction mixture (25
lL) according to the manufacturer’s (Prome-
4.11. Pharmacology
ga) protocol at 30 °C for 30 min. The reactions were stopped by
placing the tubes in a boiling water bath. After adding 80% glycerol
4.11.1. Antiproliferative activity assay
(1 lL), the samples were loaded onto an agarose gel (0.8% agarose
The tumor cell lines (K562, A549, ECA-109, KB, SMMC-7721)
were obtained from Shanghai Institute of Pharmaceutical Industry.
The antiproliferative activity in vitro was measured using the
MTT assay. After treatment in 96-well plates, MTT solution
(5.0 mg/mL in RPIM-1640, Sigma, St Louis, MO, USA) was added
in 50 mM Tris–HCl, pH 8.0). The samples were separated on the
agarose gel in the same buffer at 100 V for 15 min, and the bands
were visualized under UV light.
Acknowledgments
(10.0
lL/well), and plates were incubated for a further 4 h at
37 °C. The purple formazan crystals were dissolved in 100.0
l
l
This study received financial support from the Nature Science
Foundation of Zhejiang Province (No. R20512), Zhejiang Provincial
Program For cultivation of High-level Innovative Health talents and
DMSO. After 5 min, the plates were read on an automated micro-
plate spectrophotometer (Bio-Tek Instruments, Winooski, VT,
USA) at 570 nm. Assays were performed in triplicate in three inde-
pendent experiments. The concentration required for 50% inhibi-
tion of cell viability (IC50) was calculated using the software
‘Dose-Effect Analysis with Microcomputers’. The tumor cell line
panel consisted of K562, A549, ECA-109, KB and SMMC-7721. In
all of these experiments, three replicate wells were used to deter-
mine each point.
Zhejiang
Province
‘New
Shoot’
Talents
Project
(No.
2007R40G2010042).
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