Synthesis and Biological Evaluation of 7-Azaisoindigo Derivatives

Article abstract of DOI:10.1002/ardp.200900268

A series of novel 7-azaisoindigo derivatives 3-14 were designed, synthesized, and structurally characterized by IR, ¹H-NMR, ¹³C-NMR, mass spectra, and elemental analyses. Their antiproliferative activities were evaluated in a hormone-independent prostate cancer cell line DU145. Among them, compounds 8, 9, 14 showed the highest activities. Our study also showed that compounds 7, 11, 12 exhibited higher inhibitory activities on CDK2/cyclin A than that of the positive control meisoindigo. Western blot analysis on DU145 cells treated with compounds 7 and 9 demonstrated that 7-azaisoindigo derivatives could decrease the level of CDK2 activity (phosphorylation) and the expression of cyclin D1, and increase the expression of endogenous cyclin-dependent inhibitor p27.

Full text of DOI:10.1002/ardp.200900268

1
60
Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
Full Paper
Synthesis and Biological Evaluation of 7-Azaisoindigo
Derivatives
1
2
3
3
1
1
Zhao-Hui Wang , Tao Wang , Shi-Ning Yao , Jing-cai Chen , Wei-Yi Hua , and Qi-Zheng Yao
1
School of Pharmacy, China Pharmaceutical University, Nanjing, P. R. China
Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing, P. R. China
Wuxi JC Pharmaceutical Technology Inc., Wuxi, P. R. China
2
3
A series of novel 7-azaisoindigo derivatives 3–14 were designed, synthesized, and structurally
1
13
characterized by IR, H-NMR, C-NMR, mass spectra, and elemental analyses. Their antiprolifera-
tive activities were evaluated in a hormone-independent prostate cancer cell line DU145. Among
them, compounds 8, 9, 14 showed the highest activities. Our study also showed that compounds
7, 11, 12 exhibited higher inhibitory activities on CDK2/cyclin A than that of the positive control
meisoindigo. Western blot analysis on DU145 cells treated with compounds 7 and 9 demon-
strated that 7-azaisoindigo derivatives could decrease the level of CDK2 activity (phosphoryla-
tion) and the expression of cyclin D1, and increase the expression of endogenous cyclin-depend-
ent inhibitor p27.
Keywords: Antitumor activity / 7-Azaisoindigo / CDK2/cyclin A assay / Synthesis /
Received: March 17, 2009; Accepted: November 23, 2009
DOI 10.1002/ardp.200900268
Introduction
Traditional medicines, in particular traditional Chinese
medicines (TCM), represent a valuable source of develop-
ing potential new antineoplastic drugs. One good exam-
ple is the successful development of the anti-chronic
myelocytic leukemic (CML) drug indirubin from the TCM
recipe Danggui Longhui Wan [1–4]. Indigo and its red iso-
Figure 1. Structures of indirubin I and methylisoindigotin II.
mer, indirubin I (Fig. 1), were isolated from Qingdai (an
active ingredient of the recipe) as active components
against CML [5–10] and have been approved for clinical to the catalytic site of the kinase. Indirubins are also
trials against chronic myelocytic and chronic granulo- strong inhibitors of an evolutionarily related kinase, gly-
cytic leukemia [11–14]. Indirubin and its derivatives have cogen synthase kinase-3b (GSK-3b) [16–18]. However,
clearly antiproliferative effects. Meijer and co-workers indirubin shows poor solubility, thus poor bioavailability
reported that indirubin derivatives selectively inhibit and gastrointestinal side effects. Developing new indiru-
cyclin-dependent kinases (CDKs), a family of key cell-cycle bin derivatives aimed at improving pharmacological
regulators [15], through competing with ATP for binding properties and reducing toxicities have been extensively
carried out by a group of Chinese scientists [19–21].
Consequently, methylisoindigotin II (Fig. 1), or abbre-
viated as meisoindigo, a bis-indole compound, showed a
higher antileukemic activity than indirubin and had
Correspondence: Qi-Zheng Yao, School of Pharmacy, China Pharma-
ceutical University, Nanjing, 210009, P. R. China.
E-mail: qz_yao@yahoo.com.cn
Fax: +86 25 866-34730
fewer clinical side effects [19, 22–25]. Meisoindigo
strongly inhibits DNA biosynthesis and microtubule
assembly in tumor cells [24, 26]. It also induces the leuke-
Abbreviation: rotational nuclear Overhauser effect spectroscopy (RO-
ESY)
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Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
7-Azaisoindigo Derivatives
161
Scheme 1. Synthetic route of 1-alkyl-59-halo-7-azaisoindigo derivatives.
Table 1. Synthesized 7-azaisoindigo derivatives 3–14.
Compound
R
X
Yield
%)
Compound
R
X
Yield
(%)
(
3
4
5
6
7
8
CH
3
F
72.6
80.2
72.8
71.8
74.1
78.6
9
10
11
12
13
14
i-C
3
H
5
7
Br
H
Cl
F
Br
H
79.7
78.5
73.8
74.9
81.4
72.8
n-C
i-C
4
H
9
H
Cl
Br
F
C
C
2
H
H
3
H
5
7
2
5
C
C
2
H
H
n-C
n-C
i-C H
3
7
4
H
H
9
2
5
4
9
n-C
4
H
9
Cl
mic cell differentiation by decreasing the c-myb expres- pound 2inthe presenceofsubstituted indolin-2-one deriv-
sion [27]. A recent study indicated that the anti-angiogen- atives produced the corresponding 1-alkyl-59-substituted-
esis effect of meisoindigo may also contribute to its anti- 7-azaisoindigos 3–14in 71–81% yield (Table 1).
leukemic effect [28].
Compounds 3–14 were structurally characterized by
1
13
A more recent study indicates that the presence of a their IR, H-NMR, C-NMR, ESI-MS spectra, and elemental
pyridine N-atom may reinforce the DNA- and/or enzyme- analysis. 7-Azaisoindigos might be formed by coupling
binding properties [29] of those nitrogenous aromatic an indole ring with a 7-azaindole ring at the 3,39-position
compounds. Therefore, we designed and synthesized a in (E)-configuration. Although there is no H-atom
series of 7-azaisoindigo derivatives 3–14, in which an attached to C-atoms of the double bond, we could eluci-
indole ring is coupled with a 7-azaindole ring at the 3,39- date their configuration through ROESY (Rotational
position, in order to get more potent and selective antitu- nuclear Overhauser Effect Spectroscopy) method.
mor agents. 7-Azaindole derivatives can inhibit the pro-
The ROESY results were applicable for the determina-
liferation of tumor cells [30, 31]. The 7-azaindole moiety tion of the E-/Z-configuration of compound 9 (Fig. 2). The
in the molecules may increase their antitumor activity. individual structures of the E- and Z-isomers were gener-
In this manuscript, we describe the methods to synthe- ated by the sybyl program. The calculated distances
size these compounds and the results of their activities between H-4 and H-49 was >0.5 nm for the E-isomer, which
on inhibitions of proliferation and CDK signaling in a was consistent with the result that no ROESY correlation
hormone-independent prostate cancer cell line.
was observed between H-4 and H-49, while for the Z-iso-
mer, the calculated distance between H-4 and H-49 was
0
.23 nm, which was considered to have a strong ROE cor-
relation.
Results and discussion
Synthesis
Antitumor activities
All 7-azaisoindigo derivatives 3–14 were obtained as The antitumor activities of compounds 3–14 were tested
described in the Scheme 1. The structures of compounds in cell line DU145 (hormone-independent prostatic carci-
3
–14 are indicated in Table 1.
The key intermediate, 1-alkyl-7-azaindole-2,3-dione 2, soindigo II as a positive control. The results expressed as
was prepared by alkylation of 7-azaindole and subsequent IC50 are summarized in Table 2.
noma) in vitro by the standard MTT method using mei-
oxidation with N-bromosuccinimide (NBS) in DMSO [32].
As shown in Table 2, most of the evaluated compounds
The required diversely substituted indolin-2-one deriv- showed potent inhibitory effects on the growth of DU145
atives were obtained commercially. Condensation of com- cells. Among them, compounds 8, 9, and 14 which pos-
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162
Z.-H. Wang et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
Table 3. Inhibition of the compounds 3–14 and II on CDK2/cylin
A.
Com-
pound
Inhibition IC50
Com-
Inhibition IC50
§
§
(%)
(lmol/L) pound (%)
(lmol/L)
&
3
4
5
6
7
8
9
12 € 1
12 € 2
12 € 1
13 € 1.5
75 € 5
11 € 0.5
11 € 1
ND
ND
ND
ND
76.0
ND
ND
10
11
12
13
14
16 € 3
78 € 7.5
81 € 9
11 € 1
11 € 0.6
ND
27.0
6.2
ND
ND
II
66 € 7
89.0
–5
§
Determined at a final concentration of 10 mol/L; & ND: not
determined.
Figure 2. ROESY (Rotational nuclear Overhauser effect spec-
troscopy) spectrum of compound 9.
Table 2. Antitumor activities of the compounds 3–14 and II.
Compound
IC50
Compound
IC50
(lmol/L)
(lmol/L)
3
4
5
6
7
8
43
14
10
14
17.5
9.0
9
10
11
12
13
14
8.3
10.5
14.0
20.7
15
DU145 cells were exposed to compounds 7 or 9 for 24 h. Cellular proteins were
extracted and immunoblotted using indicated antibodies where b-actin served as a
loading control.
9.1
Meisoindigo (II)
17.4
Figure 3. Western blotting analysis of protein levels of CDK2/
cyclin D1 and p27.
sess iPr or Bu residues at N(1), showed the strongest that an appropriate length of the alkyl chain at the N(1)-
growth inhibition on DU145 cells, while compounds 3 position (e.g. Et) and a F-atom at the 59-position were
and 12 containing a F-atom at the 59-position were less essential to the CDK2/cyclin A inhibition for these 7-aza-
potent than meisoindigo II. Other compounds showed isoindigo derivatives.
similar activities as the reference compound.
Effects on CDK2/cyclin D1 and p27 protein
Inhibitory effects on CDK2/cyclin A
expressions in DU145 cells
To evaluate whether these indirubin derivatives are also To further investigate whether 7-azaisoindigo derivatives
CDK inhibitors, we examined their activity on CDK2/ affect CDK activity in human tumor cells, we selected
cyclin A and meisoindigo II served as a positive control at CDK2/cyclin D1 kinase as a representative target. We
–
5
a final concentration of 10 mol/L. The results are listed found that the level of phosphorylated CDK2 kinase was
in Table 3. significantly reduced and expression of cyclin D1 was
We found that compounds 7, 11, 12 exhibited the markedly down-regulated in a concentration-dependent
strongest potency of inhibition of recombinant human manner when DU145 cells were exposed to compounds 7
CDK2/cyclin A kinase (percent inhibition >50%) at a final and 9 for 24 h. Interestingly, those compounds up-regu-
–
5
concentration of 10 mol/L in vitro, which was found to lated expression of endogenous CDK inhibitor p27 under
be more potent than meisoindigo. Our findings suggest the same experimental conditions (Fig. 3). The induction
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Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
7-Azaisoindigo Derivatives
163
of 7-azaisoindigo derivatives on expression of p27 is prob-
ably through their ability to activate the AhR receptor
pathway as was reported for indirubins previously [33].
These observations paralleled the growth inhibition of
the malignant cells.
in ethanol (15 mL), several drops of hydrochloric acid was added
until pH = 2–3. The mixture was stirred at 70–758C for 3 h. The
reaction mixture was then allowed to cool down. The precipitate
was formed, filtered, and washed by glacial acetic acid, water,
and ethanol respectively to obtain compounds 3–14. The com-
pounds were further purified by recrystallization in glacial ace-
tic acid to yield brownish red colored crystalline powders.
Conclusion
(
E)-3-(5-Fluoro-2-oxoindolin-3-ylidene)-1-methyl-1H-
pyrrolo[2,3-b]pyridin-2(3H)-one 3
In summary, we have synthesized a series of novel 7-
azaindigo derivatives 3–14. To evaluate the potential anti-
tumor activity of these compounds, we examined their
activities on inhibition of cell proliferation and CDK sig-
naling in DU145 human prostate cancer cells. We found
that most of the compounds showed a potent anticancer
activity. Among them, compounds 8, 9, and 14 displayed
a better activity than that of the positive control meisoin-
digo. Compounds 7, 11, and 12 significantly inhibited
the activity of CDK2/cyclin A which was found to be stron-
ger than that of the positive control agent meisoindigo
in DU145 cells. It was also found that these compounds
inhibited the activity of CDK2, down-regulated expres-
sion of cyclin D1, and up-regulated endogenous CDK
inhibitor p27. Based on the above observation, we found
that compound 11 was the most promising compound.
Thus, 7-azaisoindigo derivatives are novel anticancer
indirubin derivatives worth to be further investigated.
Yield: 72.6%; brownish red crystals; m.p.: 291–2938C; IR (KBr):
3
1
413, 3268, 3130, 1716, 1681, 1592, 1471, 1400, 1344, 1303,
1
255, 1101, 1052, 790, 605, 555; H-NMR (DMSO-d
), 6.85 (d, 1H, J = 8.76 Hz, 79-H), 7.11 (dd, 1H, J = 5.13, 7.89
Hz, 5-H), 7.26 (dd, 1H, J = 2.78, 8.76 Hz, 69-H), 8.27 (dd, 1H, J = 1.59,
6
) d: 3.63 (s, 3H,
N-CH
3
5
7
2
.13 Hz, 6-H), 9.00 (dd, 1H, J = 2.78 Hz, 49-H), 9.34 (dd, 1H, J = 1.59,
13
.89 Hz, 4-H), 11.01(s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
6
) d:
4.91 (NCH ), 110.08 (C79), 115.57 (C3a), 116.01 (C ), 117.99 (C39a),
3
5
119.36 (C ), 121.89 (C ), 130.78 (C ), 134.35 (C ), 136.42 (C ),
3
49
39
69
4
140.82 (C79a), 149.78 (C
6
), 156.05 (C59), 157.88 (C1a), 167.11 (C
2
),
+
1
68.73 (C29); ESI-MS m/z: 296.1 [M + H]. Anal. calcd. for
(295.27): C, 65.08; H, 3.41; N, 14.23. Found: C, 64.89;
H, 3.49; N, 14.37.
16 3 2
C H10FN O
(
E)-1-Butyl-3-(2-oxoindolin-3-ylidene)-1H-pyrrolo[2,3-b]
pyridin-2(3H)-one 4
Yield: 80.2%; brownish red crystals; m.p.: 215–2168C; IR (KBr):
3
430, 3137, 2958, 2485, 1708, 1637, 1461, 1326, 1211, 1103, 796,
1
750, 661, 597; H-NMR (DMSO-d
CH ), 1.67 (m, 2H, CH ), 3.82 (t, 2H, N-CH
-H), 7.02 (dt, 1H, J = 1.16, 8.08 Hz, 59-H), 7.10 (dd, 1H, J = 5.10, 7.86
Hz, 5-H), 7.38 (dt, 1H, J = 1.16, 7.68 Hz, 69-H), 8.25 (dd, 1H, J = 1.57,
6
) d: 0.92 (t, 3H, CH
3
), 1.33 (m, 2H,
2
2
2
), 6.88 (d, 1H, J = 7.68 Hz,
7
5
7
1
.10 Hz, 6-H), 9.12 (d, 1H, J = 8.08 Hz, 49-H), 9.33 (dd, 1H, J = 1.57,
Experimental
13
.86 Hz, 4-H), 11.02 (s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
3.49 (CH ), 19.54, 29.15 (CH ), 38.27 (NCH ), 109.70 (C79), 115.66
59), 117.89 (C3a), 121.27 (C ), 121.33 (C39a), 129.49 (C ), 129.70 (C49),
33.39 (C39), 135.14 (C69), 136.23 (C ), 144.67 (C79a), 149.45 (C ),
56.62 (C1a), 166.88 (C ), 168.80 (C29); ESI-MS m/z: 320.1 [M + H].
(319.3): C, 71.46; H, 5.37; N, 13.16.
Found: C, 71.20; H, 5.42; N, 12.82.
6
) d:
3
2
2
General
(
C
5
3
All melting points were measured on a Bꢀchi Melting Point B-
1
1
4
6
5
40 apparatus (Bꢀchi Labortechnik, Flawil, Switzerland) and
+
2
were uncorrected. Mass spectra (MS) were taken in ESI model on
17 3 2
Anal. calcd. for C19H N O
1
Agilent 1100 LC-MSD (Agilent, Palo Alto, CA, USA). Proton ( H)
nuclear magnetic resonance spectroscopy was performed using
Brucker ARX-300 (Bruker, Fꢁllanden, Switzerland), operating at
3
00 MHz with TMS as an internal standard. The chemical shifts
were reported in ppm (d) and coupling constants (J) values were
given in Hertz (Hz). Signal multiplicities were represented by: s
(
E)-3-(5-Chloro-2-oxoindolin-3-ylidene)-1-isopropyl-1H-
pyrrolo[2,3-b]pyridine-2(3H)-one 5
Yield: 72.8%; brownish red crystals; m.p.: 270–2718C; IR (KBr):
3
1
CH
1
(
singlet), d (doublet), dd (double doublet), t (triplet), dt (triple
1
3
triplet), m (multiplet). C-NMR (125 MHz) spectra were recorded
with Brucker AV-500 NMR spectrometer (Bruker). The IR spectra
were performed on a FTIR8400S (Shimadzu, Kyoto, Japan) in KBr
434, 3180, 1701, 1685, 1618, 1587, 1436, 1398, 1359, 1263,
1
099, 1002, 810, 765, 615; H-NMR (DMSO-d
6
) d: 1.51 (d, 6H, 2
3
), 4.78 (m, 1H, N-CH), 6.87 (d, 1H, J = 8.32 Hz, 79-H), 7.09 (dd,
–
1
pellets; the frequencies were expressed in cm . Elemental anal-
ysis was performed by an Elementar Vario EL III instrument (Her-
aeus GmbH, Hanau, Germany) for C, H, and N and the results
were within € 0.5% of the theoretical values. Unless noted other-
wise, all solvents and reagents were commercially available and
used without further purification.
H, J = 5.09, 7.90 Hz, 5-H), 7.45 (dd, 1H, J = 2.22, 8.32 Hz, 69-H), 8.27
(
(
dd, 1H, J = 1.52, 5.09 Hz, 6-H), 9.21 (d, 1H, J = 2.22 Hz, 49-H), 9.34
13
dd, 1H, J =1.52, 7.90 Hz, 4-H), 11.96 (s, 1H, N9-H); C-NMR (125
MHz, DMSO-d
6
) d: 19.00 (2 CH
59), 117.61 (C3a), 121.31 (C
33.40 (C39), 134.99 (C69), 136.26 (C
56.72 (C1a), 166.63 (C ), 168.90 (C29); ESI-MS m/z: 340.1 [M + H] (Cl
3
), 43.10 (NCH), 109.74 (C79), 115.71
(
C
5
), 121.37 (C39a), 129.76 (C
3
), 129.85 (C49),
),
1
1
4
), 144.71 (C79a), 149.31 (C
6
+
2
+
General procedure for the synthesis of compounds 3–14
To a solution of 1-alkyl-7-azaindole-2,3-dione (2.47 mmol) and
appropriately substituted indolin-2-one derivatives (2.47 mmol)
18 14 3 2
= 35), 342.1 [M + H] (Cl = 37). Anal. calcd. for C H ClN O
(339.08): C, 63.63; H, 4.15; N, 12.37. Found: C, 63.46; H, 4.21; N,
12.16.
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164
Z.-H. Wang et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
(
E)-3-(5-Bromo-2-oxoindolin-3-ylidene)-1-ethyl-1H-
135.26 (C69), 136.62 (C
66.60 (C ), 168.40 (C29); ESI-MS m/z: 382.1 [M – H] (Br = 79), 384.1
M – H] (Br = 81). Anal. calcd. for C18 (383.03): C, 56.27;
4 6
), 143.62 (C79a), 149.75 (C ), 156.99 (C1a),
–
1
[
2
pyrrolo[2,3-b]pyridin-2(3H)-one 6
Yield: 71.8%; brownish red crystals; m.p.: 293–2958C; IR (KBr):
3
1
–
3 2
H14BrN O
H, 3.67; N, 10.94. Found: C, 56.18; H, 3.73; N, 10.89.
434, 3197, 3131, 1693, 1618, 1589, 1452, 1359, 1315, 1241,
1
6 3
103, 896, 811, 611; H-NMR (DMSO-d ) d: 1.24 (t, 3H, CH ), 3.86
(
7
1
q, 2H, N-CH
2
), 6.83 (d, 1H, J = 8.29 Hz, 79-H), 7.11 (dd, 1H, J = 5.12,
(E)-1-Ethyl-3-(2-oxoindolin-3-ylidene)-1H-pyrrolo[2,3-b]
pyridin-2(3H)-one 10
Yield: 78.5%; brownish red crystals; m.p.: 253–2558C; IR (KBr):
.89 Hz, 5-H), 7.57 (dd, 1H, J = 2.06, 8.29 Hz, 69-H), 8.28 (dd, 1H, J =
.59, 5.12 Hz, 6-H), 9.33 (dd, 1H, J = 1.59, 7.89 Hz, 4-H), 9.37 (d, 1H,
1
3
J = 2.06 Hz, 49-H), 11.16 (s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
d: 13.51 (CH ), 39.57 (NCH ), 111.54 (C79), 112.89 (C59), 115.50 (C3a),
), 122.96 (C39a), 130.97 (C ), 131.72 (C49), 133.62 (C39),
35.33 (C69), 136.61 (C ), 143.65 (C79a), 149.91 (C ), 156.94 (C1a),
66.93 (C ), 168.41 (C29); ESI-MS m/z: 368.1 [M – H] (Br = 79), 370.1
(369.01): C, 55.15;
H, 3.27; N, 11.35. Found: C, 55.27; H, 3.33; N, 11.28.
6
)
3
1
3
446, 3141, 2518, 1710, 1637, 1616, 1511, 1483, 1461, 1324,
3
2
1
214, 1091, 1054, 941, 796, 754, 597; H-NMR (DMSO-d
6
) d: 1.24 (t,
1
1
1
17.95 (C
5
3
H, CH ), 3.86 (q, 2H, N-CH ), 6.87 (d, 1H, J = 7.24 Hz, 79-H), 7.00
3
2
4
6
(
7
6
dt, 1H, J = 8.01, 8.62 Hz, 59-H), 7.10 (dd, 1H, J = 5.10, 7.87 Hz, 5-H),
–
2
.39 (dt, 1H, J = 7.60, 8.79 Hz, 69-H), 8.26 (dd, 1H, J = 1.58, 5.10 Hz,
–
[
3 2
M – H] (Br = 81). Anal. calcd. for C17H12BrN O
-H), 9.13 (d, 1H, J = 8.01 Hz, 49-H), 9.34 (dd, 1H, J = 1.58, 7.87 Hz, 4-
13
H), 10.99 (s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
6
) d: 12.45 (CH
3
),
),
3
1
1
1
3.53 (NCH
21.46 (C39a), 129.68 (C
36.37 (C ), 144.75 (C79a), 149.42 (C
68.95 (C29); ESI-MS m/z: 292.0 [M + H]. Anal. calcd. for C17
2
), 109.58 (C79), 115.69 (C59), 117.65 (C3a), 121.37 (C
5
3
), 129.88 (C49), 133.38 (C39), 134.72 (C69),
), 156.89 (C1a), 166.68 (C ),
(
E)-1-Ethyl-3-(5-fluoro-2-oxoindolin-3-ylidene)-1H-
4
6
2
pyrrolo[2,3-b]pyridin-2(3H)-one 7
Yield: 74.1%; brownish red crystals; m.p.: 260–2628C; IR (KBr):
3
1
3
+
13 3 2
H N O
(
1
291.3): C, 70.09; H, 4.50; N, 14.42. Found: C, 69.98; H, 4.36; N,
4.60.
423, 3193, 3133, 1702, 1637, 1591, 1467, 1361, 1311, 1257,
201, 1103, 935, 811, 790, 765, 613; H-NMR (DMSO-d
1
6
) d: 1.24 (t,
H, CH ), 3.84 (q, 2H, N-CH ), 6.82 (d, 1H, J = 8.57 Hz, 79-H), 7.09
3
2
(
8
9
dd, 1H, J = 5.12, 7.88 Hz, 5-H), 7.23 (dd, 1H, J = 2.72, 8.57 Hz, 69-H),
(E)-3-(5-Chloro-2-oxoindolin-3-ylidene)-1-ethyl-1H-
pyrrolo[2,3-b]pyridin-2(3H)-one 11
Yield: 73.8%; brownish red crystals; m.p.: 269–2728C; IR (KBr):
.25 (dd, 1H, J = 1.57, 5.12 Hz, 6-H), 8.97 (d, 1H, J = 2.72 Hz, 49-H),
.33 (dd, 1H, J = 1.57, 7.88 Hz, 4-H), 10.99 (s, 1H, N9-H); C-NMR
13
(125 MHz, DMSO-d
6
) d: 12.43 (CH
15.48 (C3a), 116.04 (C ), 117.88 (C39a), 119.29 (C
30.65 (C39), 134.31 (C69), 136.56 (C
56.08 (C59), 156.89 (C1a), 166.60 (C
3
), 33.37 (NCH
2
), 110.04 (C79),
), 121.80 (C49),
3
1
415, 3195, 3133, 1695, 1618, 1589, 1454, 1359, 1315, 1259,
1
1
1
3
3
5
3
1
103, 811, 790, 763, 611, 559; H-NMR (DMSO-d
6
) d: 1.26 (t, 3H,
4
), 140.80 (C79a), 149.72 (C
), 168.69 (C29); ESI-MS m/z:
(309.09): C, 66.02; H,
.91; N, 13.59. Found: C, 65.95; H, 3.97; N, 13.27.
6
),
CH
3
), 3.86 (q, 2H, N-CH ), 6.88 (d, 1H, J = 8.34 Hz, 79-H), 7.11 (dd,
2
2
1
H, J = 5.11, 7.90 Hz, 5-H), 7.45 (dd, 1H, J = 2.13, 8.34 Hz, 69-H), 8.28
+
3 2
10.1 [M + H]. Anal. calcd. for C17H12FN O
(dd, 1H, J = 1.58, 5.11 Hz, 6-H), 9.24 (d, 1H, J = 2.13 Hz, 49-H), 9.34
(dd, 1H, J = 1.58, 7.90 Hz, 4-H), 11.16 (s, 1H, N9-H); C-NMR (125
13
MHz, DMSO-d
6
) d: 14.58 (CH
59), 118.13 (C3a), 122.48 (C
32.43 (C39), 133.72 (C69), 136.65 (C
56.92 (C1a), 166.87 (C ), 168.65 (C29); ESI-MS m/z: 326.1 [M + H] (Cl
35), 328.0 [M + H] (Cl = 37). Anal. calcd. for C17
3
), 35.47 (NCH
), 125.29 (C39a), 129.03 (C
), 143.37 (C79a), 149.98 (C
2
), 110.88 (C79), 115.71
), 130.98 (C49),
),
(
C
5
3
(
E)-1-Butyl-3-(5-chloro-2-oxoindolin-3-ylidene)-1H-
1
1
=
4
6
pyrrolo[2,3-b]pyridin-2(3H)-one 8
Yield: 78.6%; brownish red crystals; m.p.: 270–2738C; IR (KBr):
3
1
(
+
2
+
3 2
H12ClN O
432, 3310, 2956, 1704, 1618, 1589, 1456, 1400, 1384, 1313,
101, 979, 817, 595; H-NMR (DMSO-d
(
1
325.06): C, 62.06; H, 3.71; N, 12.90. Found: C, 61.94; H, 3.87; N,
2.78.
1
6
) d: 0.92 (t, 3H, CH
3
), 1.35
m, 2H, CH
2
), 1.66 (m, 2H, CH ), 3.82 (t, 2H, N-CH
2
2
), 6.88 (d, 1H, J =
8
2
1
1
2
1
1
1
.37 Hz, 79-H), 7.11 (dd, 1H, J = 5.10, 7.89 Hz, 5-H), 7.45 (dd, 1H, J =
.22, 8.37 Hz, 69-H), 8.27 (dd, 1H, J = 1.59, 5.10 Hz, 6-H), 9.22 (d,
H, J = 2.22 Hz, 49-H), 9.33 (dd, 1H, J = 1.59, 7.89 Hz, 4-H), 11.14 (s,
(E)-1-Butyl-3-(5-fluoro-2-oxoindolin-3-ylidene)-1H-
pyrrolo[2,3-b]pyridin-2(3H)-one 12
1
3
H, N9-H); C-NMR (125 MHz, DMSO-d
9.10 (CH ), 38.34 (NCH ), 110.95 (C79), 115.46 (C59), 118.03 (C3a),
22.45 (C ), 125.24 (C39a), 129.01 (C ), 130.91 (C49), 132.51 (C39),
33.78 (C69), 136.68 (C ), 143.31 (C79a), 149.95 (C ), 156.90 (C1a),
66.92 (C ), 168.48 (C29); ESI-MS m/z: 354.1 [M + H] (Cl = 35), 356.1
(353.8): C, 64.50; H,
.56; N, 11.88. Found: C, 63.88; H, 4.57; N, 11.37.
6
) d: 13.48 (CH
3
), 19.55,
Yield: 74.9%; brownish red crystals; m.p.: 260–2628C; IR (KBr):
3421, 3303, 3130, 2956, 1704, 1589, 1465, 1359, 1257, 1199,
2
2
1
5
3
1101, 815, 788, 597; H-NMR (DMSO-d ) d: 0.92 (t, 3H, CH ), 1.35
6
3
4
6
2 2 2
(m, 2H, CH ), 1.67 (m, 2H, CH ), 3.82 (t, 2H, N-CH ), 6.85 (d, 1H, J =
+
2
8.79 Hz, 79-H), 7.11 (dd, 1H, J = 5.13, 7.89 Hz, 5-H), 7.26 (dd, 1H, J =
2.78, 8.79 Hz, 69-H), 8.27 (dd, 1H, J = 1.59, 5.13 Hz, 6-H), 8.99 (d,
1H, J = 2.78 Hz, 49-H), 9.35 (dd, 1H, J = 1.59, 7.89 Hz, 4-H), 11.01 (s,
+
[
3 2
M + H] (Cl = 37). Anal. calcd. for C19H16ClN O
4
1
3
1
2
1
1
1
H, N9-H); C-NMR (125 MHz, DMSO-d
9.07 (CH ), 38.31 (NCH ), 110.24 (C79), 115.49 (C3a), 116.21 (C
18.05 (C39a), 119.50 (C ), 121.92 (C49), 130.79 (C39), 134.50 (C69),
36.64 (C ), 140.84 (C79a), 149.93 (C ), 156.98 (C59), 158.00 (C1a),
67.02 (C ), 168.86 (C29); ESI-MS m/z: 338.1 [M + H]. Anal. calcd. for
(337.30): C, 67.65; H, 4.78; N, 12.46. Found: C, 67.22;
H, 4.96; N, 12.44.
6
) d: 13.43 (CH
3
), 19.49,
2
2
5
),
(E)-3-(5-Bromo-2-oxoindolin-3-ylidene)-1-isopropyl-1H-
3
pyrrolo[2,3-b]pyridin-2(3H)-one 9
Yield: 79.7%; brownish red crystals; m.p.: 264–2658C; IR (KBr):
4
6
+
2
3475, 3415, 3197, 1718, 1697, 1664, 1616, 1583, 1438, 1307,
19 3 2
C H16FN O
1
6 3
1106, 783, 611, 541; H-NMR (DMSO-d ) d: 1.51 (d, 6H, 2 CH ), 4.78
(
m, 1H, N-CH), 6.82 (d, 1H, J = 8.31 Hz, 79-H), 7.08 (dd, 1H, J = 5.10,
7
1
1
1
1
.92 Hz, 5-H), 7.55 (dd, 1H, J = 2.04, 8.31 Hz, 69-H), 8.26 (dd, 1H, J =
(
E)-3-(5-Bromo-2-oxoindolin-3-ylidene)-1-butyl-1H-
.60, 5.10 Hz, 6-H), 9.31 (d, 1H, J = 2.04 Hz, 49-H), 9.33 (d, 1H, J =
1
3
.60 Hz, 4-H), 10.99 (s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
6
) d:
), 43.22 (NCH), 111.50 (C79), 112.90 (C59), 115.49 (C3a),
), 122.97 (C39a), 131.29 (C ), 131.75 (C49), 133.44 (C39),
pyrrolo[2,3-b]pyridin-2(3H)-one 13
Yield: 81.4%; brownish red crystals; m.p.: 255–2578C; IR (KBr):
3421, 3301, 2956, 1702, 1618, 1589, 1454, 1359, 1309, 1257,
8.99 (2 CH
17.71 (C
3
5
3
i
2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 343, 160–166
7-Azaisoindigo Derivatives
165
1
1
2
101, 815, 594; H-NMR (DMSO-d
6
) d: 0.92 (t, 3H, CH
3
), 1.34 (m,
Where T is the percent phosphorylation of the tested com-
pounds, and C is the percent phosphorylation of no inhibition
(control).
H, CH
2
), 1.67 (m, 2H, CH
2
), 3.81 (t, 2H, N-CH ), 6.83 (d, 1H, J = 8.33
2
Hz, 79-H), 7.11 (dd, 1H, J = 5.10 Hz, 5-H), 7.56 (dd, 1H, J = 2.04, 8.33
Hz, 69-H), 8.27 (dd, 1H, J = 1.58, 5.10 Hz, 6-H), 9.31 (d, 1H, J = 1.58
Hz, 4-H), 9.35 (d, 1H, J = 2.04 Hz, 49-H), 11.15 (s, 1H, N9-H); C-NMR
1
3
Determination of the expression of CDK2/Cyclin D1 and
p27 by Western blot
Androgen-independent prostate carcinoma cell line DU145 cells
were cultured in six-well plates in 10% FBS medium and treated
with different concentrations of compounds 7 and 9 for 24 h.
Cellular proteins were extracted in lysis buffer containing 150
mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
(
(
(
125 MHz, DMSO-d
NCH ), 111.49 (C79), 112.94 (C59), 115.47 (C3a), 118.03 (C
39a), 130.95 (C ), 131.73 (C49), 133.66 (C39), 135.30 (C69), 136.66 (C
43.68 (C79a), 149.96 (C ), 156.93 (C1a), 166.95 (C ), 168.38 (C29); ESI-
MS (m/z): 396.1 [M – H] (Br = 79), 398.1 [M – H] (Br = 81). Anal.
calcd. for C19 (397.20): C, 57.30; H, 4.05; N, 10.55.
Found: C, 57.22; H, 3.96; N, 10.44.
6
) d: 13.46 (CH
3
), 19.52, 29.09 (CH
), 122.96
),
2
), 40.00
2
5
C
3
4
1
6
2
–
–
H
3 2
16BrN O
1
0 lg/mL PMSF, 30 lg/mL aprotinin, and 50 Mm Tris-HCl (pH =
8
.0). The samples were then placed on ice for 10 min and centri-
(
E)-1-Isopropyl-3-(2-oxoindolin-3-ylidene)-1H-
fuged at 14 000 rpm for 30 min at 48C. Protein concentrations
were measured using a BCA protein assay kit (Pierce chemical,
Rockford, IL, USA). 100 lg of protein extracts were subjected to
SDS-PAGE using 8–15% (depending on the protein size to be ana-
lyzed) and electro-transferred to a Hybond-C nitrocellulose mem-
brane at 150 V for 1 h at 48C. The nitrocellulose membrane was
incubated overnight in a blocking solution containing 15%
pyrrolo[2,3-b]pyridin-2(3H)-one 14
Yield: 72.8%; m.p.: 228–2308C; IR (KBr): 3432, 3151, 1700, 1618,
1
6
1
438, 1400, 1384, 1336, 1103, 981; H-NMR (DMSO-d
6
) d: 1.51 (d,
H, 2 CH ), 4.78 (m, 1H, N-CH), 6.86 (d, 1H, J = 7.55 Hz, 79-H), 7.01
3
(
7
t, 1H, J = 8.04, 15.50 Hz, 69-H), 7.09 (dd, 1H, J = 5.09, 7.90 Hz, 5-H),
.39 (dd, 1H, J = 7.67, 8.64 Hz, 59-H), 8.25 (dd, 1H, J = 1.60, 5.09 Hz,
-H), 9.10 (d, 1H, J = 7.67 Hz, 49-H), 9.33 (dd, 1H, J = 1.60, 7.90 Hz, 4-
bovine skim milk in 10 mM PBS, followed by incubating for 1 h
with antiphospho-CDK2
6
Thr160
1
3
, anti-p27, anti-Cyclin-D1 (all from
H), 10.99 (s, 1H, N9-H); C-NMR (125 MHz, DMSO-d
CH
), 121.37 (C39a), 129.76 (C
36.26 (C ), 144.71 (C79a), 149.31 (C
68.90 (C29); ESI-MS m/z: 306.1 [M + H]. Anal. calcd. for C18
6
) d: 19.00 (2
Santa Cruz, Inc., Santa Cruz, CA, USA), or anti-b-actin (Sigma,
USA) antibody, respectively, and then with a second antibody.
After three washes with PBS containing 0.1% Tween 20, incu-
bated in ECL solution (NENTM Life Science Inc., Boston, MA, USA)
for 2 min, the protein signals in the membrane was detected by
an X-ray film.
3
), 43.10 (NCH), 109.74 (C79), 115.71 (C59), 117.61 (C3a), 121.31
), 129.85 (C49), 133.40 (C39), 134.99 (C69),
), 156.72 (C1a), 166.63 (C ),
(C
5
3
1
1
4
6
2
+
15 3 2
H N O
(305.3): C, 70.81; H, 4.95; N, 13.76. Found: C, 70.53; H, 5.04; N,
1
3.52.
The authors have declared no conflict of interest.
Biological testing
MTT assay
The MTT cell proliferation assay [34] was used to test the antitu- References
mor activity of synthesized compounds. The cells were seeded in
RPMI-1640 medium (100 lL) in a 96-well plate at a concentration
of 5000 cells per well. After culturing for 12 h at 378C and with
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final concentration of 5 lg/mL, and incubated for 4 h. The forma-
zane crystals were formed and dissolved in DMSO (100 lL) each
well. The optical density was measured at 570 nm with the refer-
ence wavelength 630 nm. All of the compounds were tested
thrice independently using the same DU145 cell line. IC50 (con-
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2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com