Synthesis of 1-substituted 3-pyridinylmethylidenylindolin-2-ones and 1-substituted 3-quinolinylmethylidenylindolin-2-ones as the enhancers of ATRA-induced differentiation in HL-60 cells

Article abstract of DOI:10.1016/j.bmc.2008.02.093

As part of our continuing search for potential differentiation agents, 1-benzyl-3-(4-pyridinylmethylidenyl)indolin-2-one (14) was selected as lead compound, and its new pyridinyl and quinolinyl analogs were synthesized and evaluated for differentiation-in

Full text of DOI:10.1016/j.bmc.2008.02.093

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4222–4232
Synthesis of 1-substituted 3-pyridinylmethylidenylindolin-
2-ones and 1-substituted 3-quinolinylmethylidenylindolin-2-ones
as the enhancers of ATRA-induced differentiation in HL-60 cells
Chi-Ying Hung,a Mei-Hua Hsu,b Li-Jiau Huang,b Chrong-Shiong Hwang,a
On Lee,a Chen-Yi Wu,a Chih-Hung Chena and Sheng-Chu Kuob,*
aBiomedical Engineering Center, Industrial Technology Research Institute, Hsinchu, Taiwan
bGraduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40421, Taiwan
Received 7 January 2008; revised 26 February 2008; accepted 27 February 2008
Available online 6 March 2008
Abstract—As part of our continuing search for potential differentiation agents, 1-benzyl-3-(4-pyridinylmethylidenyl)indolin-2-
one (14) was selected as lead compound, and its new pyridinyl and quinolinyl analogs were synthesized and evaluated for dif-
ferentiation-inducing activity toward HL-60 cells. Most of the tested compounds enhanced the ATRA-induced differentiation;
among them, 1-(1-phenylethyl)-3-(3-quinolinylmethylidenyl)indolin-2-one (25) was the most promising one. The two isomers,
25Z and 25E; consisting 25 were found to have similar differentiation activity. The combination of 25 with all trans retinoic
acid (ATRA) was found to induce complete differentiation of HL-60 cells and arrest the cells in the G0/G1 phase of the cell
cycle. Beside its excellent differentiation activity, 25 also exhibited relatively low cytotoxicity toward normal cells. Therefore,
compound 25 is recommended as a candidate for further development of novel enhancer of ATRA-induced differentiation in
HL-60 cells.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
chemotherapeutic agents, cytarabine (Ara-C) and ar-
senic trioxide (As2O3), were also used in combination
with ARTA to improve the APL treatment. But, Ara-
C and As2O3 are highly toxic in nature. Thus, the
search for novel differentiation enhancers that reduce
both the dosage and side effects of ATRA and induce
complete differentiation is an important task for
improving the treatment of APL.
Acute promyelocytic leukemia (APL) is a subtype of
acute myeloid leukemia (AML) characterized by the
presence of a fusion gene which alters the gene of reti-
noic acid receptor (RAR)-a and the gene of promyelo-
cytic leukemia (PML). Such fusion gene blocks the
maturation of hematopoietic stem cells and results in
APL. In 1988, the all trans retinoic acid (ATRA) was
used to induce leukemic cell differentiation and resulted
in almost complete remission in a high proportion of
patients with APL.1 But the effective dose of ATRA
in clinical use also led to undesirable side effect.2,3 Be-
sides, the remission in APL patients who had received
ATRA treatment lasted for only a few months before
the disease invariably recurred.4 The reason for recur-
rence is probably that ATRA induced incomplete dif-
During a massive screening in our laboratory for new
enhancers of ARTA-induced differentiation in HL-60
cells, 1-benzyl-3-(4-pyridinylmethylidenyl)indolin-2-one
(14) was identified as a new and potent enhancer for
ARTA-induced differentiation. The combined used of
2.5 lM of 14 with 5 nM of ATRA resulted in complete
differentiation (>90% NBT-positive) of HL-60 cells.
Moreover, the pyridine moiety of 14 makes it readily
convertible to water soluble HCl salt which can be used
readily in animal tests and clinical trials. Thus, 14 was
selected as a lead compound and its new pyridinyl and
quinolinyl analogs, which could be converted into HCl
salts, were synthesized and studied for cell differentia-
tion activity in the present work.
ferentiation
of
the
leukemia
cells.5
The
Keywords: Pyridinylidenylindolinone; Quinolinylidenylindolinone; In-
dolinone; ATRA; Differentiation; Leukemia; HL-60.
*
Corresponding author. Tel./fax: +886
sckuo@mail.cmu.edu.tw
4
22030760.; e-mail:
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.02.093

C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
4223
2. Results and discussion
analyses indicated that the reaction products 14–28 may
be a mixture of Z- and E-form isomers. Column chro-
matography of product 25, one of the most potent com-
pounds, yielded two configurational isomers, 25Z (mp
104.0–104.4 ꢁC) and 25E (mp 83.0–83.8 ꢁC) (as shown
in Fig. 1), with the same molecular formula
C26H20N2O. The two isomers could be recognized by
NOESY experiment and by the analysis of chemical
shifts. As shown in Figure 1, the Z-isomer (25Z) has a
NOESY correlation between H-4 (d 7.66) and H-90 (d
7.75), whereas the E-isomer (25E) exhibits a NOESY
correlation as follows: H-4 (d 7.60)/H-20 (d 9.25), H-40
(d 8.47). The chemical shifts of H-90, H-20 and H-40 in
2.1. Chemistry
The synthetic procedure of the target compounds 14–28
was illustrated in Scheme 1. Isatin (1) was first alkylated
with various arylalkyl halides (2–5) to afford the corre-
sponding 1-substituted isatins (6–9) which were then re-
duced with hydrazine hydrate to yield the 1-substituted
indolin-2-ones (10–13). Then the condensation of 10–
13 with various pyridinyl aldehydes or quinolinyl alde-
hydes, in the presence of piperidine, afforded the target
compounds 14–28. Results of TLC and NMR spectrum
O
O
(a)
(b)
O
ArCHBr
R
+
O
O
N
H
N
N
R
R
Ar
Ar
1
2-5
6-9
10-13
N
H
H
H
Ar'
O
N
Ar'CHO
(c)
O
O
N
N
N
CH3
CH3
R
Ar
14-28
25Z
25E
2
6
10
R= H
Ar=
3
7
11
R= CH3
Ar=
4
8
12
R= H
Ar=
5
9
13
R= H
Ar=
,
,
,
,
,
,
,
,
N
22
14
15
16
17
18
, R= H,
, R= H,
Ar=
, Ar'=
, Ar'=
, Ar'=
, R= H,
, R= H,
, R= H,
, R= H,
Ar=
Ar=
Ar=
Ar=
, Ar'=
, Ar'=
, Ar'=
, Ar'=
, Ar'=
N
N
23
24
Ar=
N
, R= CH3, Ar=
, R= CH3, Ar=
, R= CH3, Ar=
N
N
N
N
N
o
25
26
, Ar'=
, R= CH3, Ar=
, R= CH3, Ar=
, R= CH3, Ar=
, Ar'=
N
19
20
21
, Ar'=
, Ar'=
N
27
28
, R= H, Ar=
, R= H, Ar=
, Ar'=
, Ar'=
N
N
N
, R= H,
Ar=
, Ar'=
N
Scheme 1. Reagents and conditions: (a) tert-BuOK/THF; (b) NH2NH2ÆH2O; n (c) piperidine/MeoH.

4224
C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
N
H
H
2'
4'
4'
H
H
H
H
H
9'
N
9'
2
4
4
2'
3
3
5
6
5
6
O
O
2
N
N
7
7
H3C
H3C
25
Z
25E
Figure 1. Key NOESY (M) of 25Z and 25E.
Table 1. Chemical shifts of H-20, H-40 and H-90 in compounds 25Z
and 25E
with 5 nM of ATRA, in inducing the differentiation of
HL-60 cells. As shown in Table 2, the lead compound
(14) alone was inactive, but it significantly enhanced
the ATRA- induced cell differentiation at 1.0 lM.
Increasing the concentration of 14 to 2.5 lM resulted
in 94.2% cell differentiation, which is about 5 folds the
differentiation activity when 5 nM ATRA was used
alone (19.0%). During a series of systematic structural
modification, it was found that the replacement of pyri-
din-4-yl (14) with pyridin-3-yl (15) or pyridin-2-yl (16),
or to replace the pyridin-4-yl group with a pyridinyl
N-oxide (17), all resulted in slightly reduced activity.
However, the replacement of benzyl group of 14 with
1-phenylethyl group (18) significantly enhanced the dif-
ferentiation activity of ATRA at low concentration
(0.5 lM). On the contrary, higher concentration of 18
(2.0 lM) caused death of cell completely. Another
replacement of pyridin-4-yl group of 18 with pyridin-3-
yl (19) or pyridin-2-yl (20) also weakened its activity.
Based on the above structure–activity relationships, we
demonstrated that the pyridin-4-yl derivatives (14, 18)
exhibited more differentiation activity than both pyri-
din-3-yl-(15, 19) or pyridin-2-yl (16, 20) derivatives.
N
H
H
2'
4'
4'
H
H
H
H
4
H
N
9'
9'
4
2'
O
O
N
N
H3C
H3C
25
Z
25E
Compound
Chemical shift (d)
H-20
H-40
H-90
25Z
25E
9.87
9.25
9.33
8.47
7.75
8.05
both 25Z and 25E were documented in Table 1. As
shown, resonances of H-20 and H-40 of 25Z displayed
greater downfield shift than the corresponding protons
of 25E due to receiving the anisotropic effect from C-2
carbonyl group. Ascribing to the same reason, the signal
of H-90 of 25E located on the lower field than the corre-
sponding proton on the 25Z. Based on the above-men-
tioned result of NOESY experiment and chemical
shifts of H-90, H-20 and H-40 protons, 25Z was assigned
as Z-form of 1-(1-phenylethyl-3-(3-quinolinylmethylide-
nyl)indolin)-2-one, and 25E existed as its geometrical
isomer, E-form.
Alternatively, the pyridin-4-yl group of 14 was replaced
with quinolin-4-yl group (21) which resulted in lower
activity, or replaced with quinolin-3-yl (22) or quino-
lin-2-yl (23) which not only reduced activity but also
led to cell death at the concentration of 5 lM when com-
bined with 5 nM ATRA. Replacing simultaneously the
benzyl and pyridin-4-yl groups of 14 with 1-phenylethyl
and quinolin-4-yl groups, respectively, gave 24 with con-
siderable differentiation effect. However, at the concen-
tration of 5 lM, 24 led to cell death. During
subsequent modification, the quinolin-4-yl group of 24
was replaced with a quinolin-3-yl group to afford 25
which induced 33.9% cell differentiation at 2.5 lM,
and induced 94.5% cell differentiation, when combined
with 5 nM ATRA. We considered that 25 was the most
promising one among all tested compounds. The two
isomers (25Z and 25E), coexisting as 25, were separated
and evaluated, but no significant difference in their dif-
ferentiation activity was found. Next, the substitution
2.2. The effect of compounds 14–28 on the differentiation
of HL-60 cells
The nitroblue tetrazoliun reduction (NBT) cell differen-
tiation assay was used to measure the potency of com-
pounds 14–28, either used alone or in combination

C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
4225
Table 2. Cell differentiation and antiproliferation effect of compounds 14–28 against human leukemia HL-60 cells
N
H
H
H
Ar'
O
N
O
O
N
N
N
CH3
CH3
R
Ar
14 28
-
25
Concn (lM)
Z
25
E
Compounds
R1
H
Ar
Ar0
NBT (%)
MTT (%)
ꢀ5 nM ATRA +5 nM ATRA
ꢀ5 nM ATRA
0.7 0.3
+5 nM ATRA
19.0 1.2
Control
100.0 1.0
69.4 1.7
14
0.5
1.0
2.5
0.3 0.3
1.2 0.3
7.0 0.3#
38.2 5.6*
51.3 2.6*
94.2 4.1*
101.0 5.8
85.6 6.1#
24.4 5.1#
IC50 = 1.9 lM
61.4 3.2
48.1 2.9*
11.6 0.5*
N
15
16
H
H
1.0
2.5
5.0
0.7 0.3
0.8 0.3
2.7 0.3
21.7 3.3*
29.4 1.7*
74.3 3.4*
93.2 2.5#
75.9 3.3#
25.4 1.9#
IC50 = 3.8 lM
58.1 3.5*
48.8 3.8*
15.1 5.8*
N
1.0
2.5
5.0
0.7 0.3
0.7 0.3
3.4 1.4
22.7 5.5*
32.3 4.5*
76.0 3.1*
116.6 4.9
98.5 6.4
24.7 4.9#
IC50 = 3.6 lM
63.4 2.5*
46.3 1.5*
11.8 1.6*
N
N
17
18
19
H
1.0
2.5
5.0
1.6 1.2
3.2 0.6
11.5 1.9#
34.4 0.9*
67.2 3.8*
88.0 3.5*
90.0 3.8*
43.4 3.9#
22.0 0.9#
IC50 = 2.9 lM
58.7 14.0
30.9 1.1*
15.4 0.8*
O
CH3
CH3
0.5
1.0
2.0
1.0 0.5
0.8 0.6
Death
55.3 3.7*
72.5 2.6*
Death
66.7 2.7#
49.2 5.8#
Death
47.0 1.1*
42.1 1.0*
Death
N
1.0
2.5
5
0.8 0.3
2.5 0.5
19.2 2.1#
28.6 2.7*
41.9 2.0*
58.0 3.0#
88.0 0.4#
87.0 2.4#
19.6 5.5#
IC50 = 3.3 lM
63.7 1.0*
50.5 0.6*
9.0 1.3*
N
20
21
CH3
1.0
2.5
5.0
0.7 0.3
0.7 0.3
2.0 0.9
27.1 2.7*
32.3 4.6*
80.6 2.5*
90.0 1.9#
87.1 4.5#
55.7 0.8#
IC50 = 5.9 lM
73.7 2.2
58.9 3.3*
27.0 1.9*
N
H
1.0
5.0
0.8 0.3
2.1 0.3
4.7 0.3
21.5 0.3
37.6 3.4*
87.5 1.4*
85.2 7.7#
73.8 4.4#
51.2 0.6#
64.4 4.8
56.7 5.5*
35.7 1.6*
10.0
IC50 = 10.6 lM
N
22
H
1.0
2.5
5.0
0.7 0.3
0.8 0.3
23.6 0.9#
12.2 1.9
20.7 3.8*
Death
117.4 2.6
70.6 5.0#
10.9 2.8#
IC50 = 3.2 lM
70.2 3.9
48.4 1.5*
Death
N
(continued on next page)

4226
C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
Table 2 (continued)
Compounds R1
Ar
Ar0
Concn (lM)
NBT (%)
MTT (%)
ꢀ5 nM ATRA +5 nM ATRA ꢀ5 nM ATRA +5 nM ATRA
23
24
H
1.0
2.5
5.0
2.0 0.5
2.0 0.5
4.4 1.9
32.4 0.6*
53.7 0.3*
Death
82.7 3.7#
55.7 9.3#
12.2 1.7#
IC50 = 2.8 lM
63.7 0.6
33.7 3.3#
Death
N
CH3
1.0
2.5
5.0
1.2 0.7
20.8 2.4#
Death
30.3 2.0*
84.2 3.5*
Death
72.6 3.0#
31.0 2.6#
Death
61.3 2.7*
36.9 1.4*
Death
N
25
CH3
CH3
CH3
0.5
1.0
2.0
0.8 0.3
0.3 0.3
54.8 4.2*
79.3 2.3*
94.5 0.5*
89.5 5.1#
70.9 2.0#
24.7 4.2#
IC50 = 1.4 lM
45.1 2.0*
41.4 2.3*
20.4 0.6*
33.9 0.8#
N
N
N
N
25Z
25E
26
0.5
1.0
2.0
1.3 0.3
3.8 1.0
40.8 1.8#
53.1 4.2*
75.5 4.9*
93.7 3.2*
116.1 4.0
82.3 0.3#
20.3 2.9#
IC50 = 1.5 lM
72.8 6.4*
60.2 2.6*
32.2 2.0*
0.5
1.0
2.0
0.8 0.6
4.3 0.3
37.7 2.9#
52.1 2.3*
72.1 1.7*
90.2 2.6*
109.9 0.1
82.9 2.6#
25.5 0.8#
IC50 = 1.4 lM
71.0 2.5*
57.2 3.8*
29.0 1.3*
1.0
2.5
5.0
0.8 0.3
1.0 0.5
15.0 2.2#
30.0 1.5*
41.5 3.5*
75.6 0.4*
103.2 1.3
81.3 1.1#
18.5 3.2#
IC50 = 3.4 lM
62.2 1.6*
50.8 2.8*
15.7 0.7#
27
H
H
0.5
1.0
2.5
0.8 0.6
0.5 0.5
Death
16.2 2.8
24.0 2.0
Death
103.6 0.4
90.8 4.2
Death
71.3 4.9
59.4 4.1
Death
N
N
28
0.1
0.5
1.0
0.8 0.3
0.7 0.3
Death
46.8 4.4*
66.8 1.2*
Death
78.2 8.0#
59.6 1.4#
Death
39.2 3.8*
28.2 3.5*
10.1 0.9
HL-60 cells (4 · 104 cells/mL) were cultured with various concentrations of 14–28 for 72 h. After treatment, cells were harvested and examined by
NBT- and MTT-assay. Data are represented as means SD from four independent experiments.
* P < 0.001 compared with 5 nM ATRA used alone.
# P < 0.001 compared with vehicle control.
of the quinolin-3-yl group of 25 with quinolin-2-yl group
(26) resulted in attenuated activity. Finally, the benzyl
group of 14 was replaced with either a-naphthalinylm-
ethyl group to yield 27 with lowered activity, or replaced
with b-naphthalinylmethyl group to form 28, which at
concentration of 1 lM caused cell death.
ity was significantly enhanced by the addition of ATRA.
Comparing similar analogs, the pyridin-4-yl derivatives
(14, 18) demonstrated the best activity among pyridinyl
derivatives (14–20), and the quinolin-3-yl derivative of
25 (25Z, 25E) was found to be the most active among
quinolinyl derivatives (21–26).
2.3. The effect of compounds 14–28 on the proliferation of
HL-60 cells
2.4. Morphological change of HL-60 cells responding to
compound 25 alone or in combination with ATRA
The effect of compounds 14–28 on the proliferation of
HL-60 cells is summarized in Table 2. Most of the tested
compounds showed significant antiproliferative activity
with IC50 ranging between 0.5 and 5.0 lM. Their activ-
Comparing the morphological change of HL-60 cells
responding to 25 alone, or in combination with ATRA,
we found that the morphology of HL-60 cells changed
from round and smooth (Fig. 2A) before treatment into

C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
4227
Figure 2. Morphological changes in response to 25. HL-60 cells (4 · 104) were treated with vehicle control (A and A0), 2 lM 25 used alone (B and B0),
co-treated 2 lM 25 and 5 nM ATRA together (C and C0), 5 nM ATRA (D and D0) or 1 lM ATRA (E and E0) for 72 h (magnification 200·).
spindle-shape after treatment with 25 (Fig. 2B). Such
morphological change became more obvious (Fig. 2C)
when 25 was used in combination with 5 nM ATRA.
Similar morphological change was observed when
higher concentration of 1 lM ATRA (Fig. 2E) was used
alone.
2.5. The effect of compound 25 alone or in combination
with ATRA on the cell cycle distribution of HL-60 cells
The cellular DNA content was also measured by flow
cytometry, and the result in Figure 3 indicated that, fol-
lowing 72 h treatment with 25, the accumulation of cells
in the G0/G1 phase of cell cycle increased to 49.8%. The
combined use of 25 with 5 nM ATRA increased the G0/
G1 phase percentage further to 76.8%. Similar cell cycle
arrest in the G0/G1 phase was also observed when higher
concentration (1 lM) of ATRA was used alone. These
findings demonstrated that the combination of 25 with
5 nM ATRA arrested the HL-60 cells in the G0/G1
phase of cell cycle.
To confirm the above observation, Liu’s stain was also
applied to the cells which were then examined under
optical microscope. Again, the horseshoe morphology
was not seen in the nuclei of untreated cells (Fig. 2A0),
but was clearly seen in the nuclei of cells treated with
25 (Fig. 2B0). The population of horseshoe-shape nuclei
increased significantly when 25 was used in combination
with 5 nM ATRA (Fig. 2C0) or 1 lM ATRA used alone
(Fig. 2E0). The morphological changes of cells, un-
treated or treated with the combination of 25 and
5 nM ATRA, determined after application of Liu’s
stain, agreed well with the previous observation without
cell stain. These results indicated that the combined use
of 25 with 5 nM ATRA or higher concentration (1 lM)
of ATRA undoubtedly induced cell differentiation.
2.6. The effect of compound 25 alone or combined with
ATRA on the growth of human normal cells
The excellent differentiation activity of compound 25
prompted us to examine the cytotoxicity of 25 used
alone and combined with ATRA toward human normal
leukocytes in order to assess selectivity to HL-60 leuke-
mia cells. As shown in Figure 4, the IC50 value of 25 is
100
G0/G1
S
*
G0/G1
S
G2/M
140
compound 25
compound 25 + 5 nM ATRA
80
*
G2/M
120
*
60
#
100
#
*
*
*
*
40
80
60
40
20
0
20
#
#
&
&
0
0
5 nM
ATRA
1 μM
Vehicle
25
Both
*
*
Figure 3. Cell cycle distributions of 25 used alone or combined with
ATRA in HL-60 cells. HL-60 cells (4 · 104) were treated with vehicle
control, ATRA (5 nM or 1 lM), 2 lM 25 used alone, or co-treated
2 lM 25 and 5 nM ATRA together (both) for 72 h. After treatment,
cells were fixed and stained with PI and then the cell cycle distribution
was examined by flow cytometry. Data are represented as means SD
from four independent experiments. *P < 0.001 compared with the G0/
G1 phase of the vehicle control. #P < 0.001 compared with the S phase
of the vehicle control. &P < 0.001 compared with the G2/M phase of
the vehicle control.
Control
2
5
10
20
Conc. (μM) of compound 25
Figure 4. The cytotoxicity of 25 toward human normal leukocytes.
Human normal leukocytes (2 · 106) were treated with vehicle control,
25 used alone, or co-treated 25 and 5 nM ATRA together for 48 h.
After treatment, cells were harvested and examined using MTT assay.
Data are represented as means SD from four independent experi-
ments. *P < 0.001 compared with control.

4228
C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
14.0 lM which is about 10-fold higher than its cytotox-
icity against HL-60 cells (IC50 = 1.4 lM) (Table 2). We
then tested the cytotoxicity of the use of 25 in combina-
tion with 5 nM ATRA toward human normal leuko-
cytes and found that the concentration of compound
25 needed to accomplish 50% growth-inhibition of hu-
mane normal leukocytes to be 14.7 lM, which is about
30-fold higher than the concentration (0.5 lM) of 25
takes together with 5 nM ATRA, to accomplish 50%
growth-inhibition of HL-60 cells (Table 2). Such finding
indicated that the combined use of 25 with ATRA has
relatively low toxicity toward normal cells.
NMR (CDCl3): d 4.91 (s, 2H), 6.5–7.7 (m, 9H); HRMS:
m/z 237.0805 (calculated for C15H11NO2, 237.0790).
4.2.2. 1-(1-Phenylethyl)isatin (7).9 Isatin, PhMeCHBr
(3) and tert-BuOK in THF were allowed to react in
the same manner as described in the preparation of com-
pound 6 and the residue was purified by flash column
chromatography (EtOAc/n-hexane), recrystallized from
EtOAc/n-hexane to give 7 in 81% yield: mp 118–
1
120 ꢁC; H NMR (CDCl3): d 1.85 (d, 3H, J = 7.0 Hz),
5.77 (q, 1H, J = 7.0 Hz), 6.53 (d, 1H, J = 8.5 Hz), 7.01
(t, 1H, J = 7.5 Hz), 7.27–7.39 (m, 6H), 7.58 (dd, 1H,
J = 1.0, 7.5 Hz); HRMS: m/z 251.0926 (calculated for
C16H13NO2, 251.0946).
3. Conclusion
4.2.3. 1-(Naphthalen-1-ylmethyl)isatin (8). Isatin, 1-
(chloromethyl)naphthalene (4) and tert-BuOK in THF
were allowed to react in the same manner as described
in the preparation of compound 6 and the residue was
purified by flash column chromatography (EtOAc/n-
hexane), recrystallized from EtOAc/n-hexane to give 8
in 83% yield: mp 192–194 ꢁC; 1H NMR (CDCl3): d
5.40 (s, 2H), 6.72 (d, 1H, J = 8.5 Hz), 7.06 (t, 1H,
J = 8.0 Hz), 7.24–7.41 (m, 3H), 7.52–7.62 (m, 3H), 7.81
(dd, 1H, J = 2.0, 7.0 Hz), 7.88 (t, 1H, J = 8.0 Hz), 8.06
(d, 1H, J = 8.5 Hz); HRMS: m/z 287.0965 (calculated
for C19H13NO2, 287.0946).
A series of 1-substituted 3-pyridinylmethylidenylindolin-
2-ones and 1-substituted 3-quinolinylmethylidenylindo-
lin-2-ones were synthesized and evaluated for differenti-
ation-inducing activity toward HL-60 cells. Among the
target compounds, compound 25 was the most promis-
ing one. The combination of 25 with ATRA was found
to induce complete differentiation of HL-60 cells. Also,
25 showed relatively low cytotoxicity against normal
cells. Thus, compound 25 is recommended as a candi-
date for further development of novel enhancer of
ATRA-induced differentiation in HL-60 cells.
4.2.4. 1-(Naphthalen-2-ylmethyl)isatin (9). Isatin, 2-(bro-
momethyl)naphthalene (5) and tert-BuOK in THF were
allowed to react in the same manner as described in the
preparation of compound 6 and the residue was purified
by flash column chromatography (EtOAc/n-hexane),
recrystallized from EtOAc/n-hexane to give 9 in 75%
4. Experimental
4.1. Chemistry
4.1.1. General. All starting materials were commercially
available. Solvents and reagents were used without fur-
ther purification. Reactions were monitored either by
1
yield: mp 156 ꢁC (dec); H NMR (CDCl3): d 5.07 (s,
2H), 6.79 (d, 1H, J = 8.5 Hz), 7.06 (t, 1H, J = 7.5 Hz),
7.40–7.48 (m, 5H), 7.59 (d, 1H, J = 7.5 Hz), 7.77–7.82
(m, 2H), 8.14 (d, 1H, J = 8.5 Hz); HRMS: m/z
287.0935 (calculated for C19H13NO2, 287.0946).
TLC on silica gel plastic sheets (Kieselgel 60 F254
,
Merck) or by HPLC. Purification was performed by
flash chromatography using silica gel (particle size 63–
200 lm, Merck). NMR spectra were recorded on a Var-
ian 500-MHz spectrometer. Chemical shifts are reported
as ppm (d) relative to TMS as internal standard. The ra-
tio of E- to Z-isomer of compounds (14–28) was ana-
4.3. General procedure for reduction of N-aryl-oxindole
with NH2NH2ÆH2O (10–13)
4.3.1. 1-Benzyl-1,3-dihydroindol-2-one (10).10 A mixture
of N-benzylisatin (6, 0.5 g, 2.1 mmol) and NH2NH2Æ
H2O (12 mL) was refluxed for 30 min. The mixture
was extracted with ethyl acetate, washed with water,
dried over MgSO4 and evaporated. The residue was
purified by flash column chromatography (EtOAc/n-
hexane), and recrystallized from EtOAc/n-hexane to give
1
lyzed by H NMR.6,7 Mass spectra were recorded on a
JEOL JMS-SX102A spectrometer (HREI). IR spectra
were recorded on a Horiba FT-730 FT-IR spectrometer.
Melting points were determined on a Buchi B-540 appa-
ratus and are uncorrected.
4.2. General procedure of N-alkylation in base (6–9)
1
0.39 g of 10 in 84% yield: mp 80.3–81.0 ꢁC; H NMR
4.2.1. 1-Benzyl-isatin (6).8 To a solution of isatin (1,
5.0 g, 34.0 mmol) in THF (100 mL) was added tert-
BuOK (4.9 g, 43.8 mmol) at 0 ꢁC. After stirring for
30 min, PhCH2Br (2, 7.0 g, 40.8 mmol) was added drop-
wise over 20 min at 0 ꢁC. The reaction mixture was then
warmed to room temperature and stirred for additional
16 h, and then concentrated under reduced pressure.
The residue was dissolved in ethyl acetate, washed with
water, dried over MgSO4 and evaporated. The residue
was then purified by flash column chromatography
(EtOAc/n-hexane), and recrystallized from EtOAc/n-
hexane to give 5.1 g (63%) of 6: mp 126–127 ꢁC; 1H
(CDCl3): d 3.60 (s, 2H), 4.93 (s, 2H), 6.76 (d, 1H,
J = 7.5 Hz), 7.02 (t, 1H, J = 7.5 Hz), 7.18 (t, 1H,
J = 7.5 Hz), 7.22–7.37 (m, 6H); HRMS: m/z 223.0985
(calculated for C15H13NO, 223.0997).
4.3.2. 1-(1-Phenylethyl)indolin-2-one (11). 1-(1-Phenyl-
ethyl)isatin (7) and NH2NH2ÆH2O were allowed to react
in the same manner as described in the preparation of
compound 10 and the residue was purified by flash col-
umn chromatography (EtOAc/n-hexane), recrystallized
from EtOAc/n-hexane to give 11 in 88% yield: mp
1
63.9–64.8 ꢁC; H NMR (CDCl3): d 1.784 (d, 3H), 3.61

C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
4229
(s, 2H), 5.88 (q, 1H), 6.49 (d, 1H), 6.92–7.03 (m, 2H),
7.21–7.36 (m, 6H); HRMS: m/z 227.1168 (calculated for
C16H16NO, 237.1154).
to react in the same manner as described in the prepara-
tion of compound 14 and the residue was recrystallized
from EtOAc/i-propyl ether to give 16 in 81% yield: mp
46.0–46.4 ꢁC; IR (cmꢀ1) 3379, 3076, 1697; 1H NMR
(CDCl3): d 5.06 (s, 2H), 6.76 (d, 1H, J = 7.5 Hz), 7.08
(t, 1H, J = 7.5 Hz), 7.24–7.38 (m, 7H), 7.67 (d, 1H,
J = 7.5 Hz), 7.82–7.86 (m, 2H), 8.91 (d, 1H,
J = 4.5 Hz), 9.06 (d, 1H, J = 7.5 Hz); HRMS: m/z
312.1281 (calculated for C21H16N2O, 312.1263).
4.3.3. 1-(Naphthalen-1-ylmethyl)indolin-2-one (12). 1-
(Naphthalen-1-ylmethyl)-isatin (8) and NH2NH2ÆH2O
were allowed to react in the same manner as described
in the preparation of compound 10 and the residue
was purified by flash column chromatography (EtOAc/
n-hexane), recrystallized from EtOAc/n-hexane to give
1
12 in 85% yield: mp 153–154 ꢁC; H NMR (CDCl3): d
4.4.4. 1-Benzyl-3-(4-(N-oxy-pyridinylmethylidenyl))indo-
lin-2-one (17). 1-Benzyl-1,3-dihydroindol-2-one (10), 4-
pyridinecarboxaldehyde N-oxide and piperidine in
MeOH were allowed to react in the same manner as de-
scribed in the preparation of compound 14 and the res-
idue was recrystallized from EtOAc/i-propyl ether to
give 17 in 77% yield: mp 251 ꢁC (dec); IR (cmꢀ1) 3419,
3.68 (s, 2H), 5.39 (s, 2H), 6.66 (d, 1H, J = 8 Hz), 6.97
(t, 1H, J = 8 Hz), 7.09 (t, 1H, J = 7.5 Hz), 7.27–8.15
(m, 8H); HRMS: m/z 273.1133 (calculated for
C19H15NO, 273.1154).
4.3.4. 1-(Naphthalen-2-ylmethyl)indolin-2-one (13). 1-
(Naphthalen-2-ylmethyl)-isatin (9) and NH2NH2ÆH2O
were allowed to react in the same manner as described
in the preparation of compound 10 and the residue
was purified by flash column chromatography (EtOAc/
n-hexane), recrystallized from EtOAc/n-hexane to give
1
3082, 1682; H NMR (CDCl3): d 5.02 (s, 2H), 6.79 (d,
1H, J = 7.5 Hz), 7.09 (d, 1H, J = 7.5 Hz), 7.26–7.41
(m, 7H), 7.56 (d, 1H, J = 7.5 Hz), 8.23 (s, 1H), 8.38 (d,
2H, J = 6.0 Hz); HRMS: m/z 328.1207 (calculated for
C21H16N2O2, 328.1212).
1
13 in 86% yield as oil; H NMR (CDCl3): d 3.71 (s,
2H), 5.13 (s, 2H), 6.81 (d, 1H, J = 7.5 Hz), 7.04 (t, 1H,
J = 7.5 Hz), 7.18 (t, J = 7.5 Hz, 1H), 7.30 (d, 1H,
J = 7.5 Hz), 7.45–7.59 (m, 3H), 7.78–7.85 (m, 4H);
HRMS: m/z 273.1142 (calculated for C19H15NO,
273.1154).
4.4.5. 1-(1-Phenylethyl)-3-(4-pyridinylmethylidenyl)indo-
lin-2-one (18). To a stirred solution of 1-(1-phenyl-
ethyl)indolin-2-one (11, 3.0 g, 13.5 mmol) in MeOH
(30 mL) were added piperidine (50 mg) and 4-pyridine-
carboxaldehyde (1.6 g, 14.8 mmol) at 0 ꢁC. The mixture
was refluxed for 4 h, concentrated, extracted with ethyl
acetate, dried with MgSO4, and concentrated again.
The residue was purified by flash column chromatogra-
phy (EtOAc/n-hexane), and recrystallized from EtOAc/
n-hexane to give 3.0 g (73%) of 18: mp 70.1–70.3 ꢁC;
4.4. General procedure for condensation of indolin-2-one
with heteroaryl-aldehydes (14–28)
4.4.1. 1-Benzyl-3-(4-pyridinylmethylidenyl)indolin-2-one
(14). To a stirred solution of 1-benzyl-1,3-dihydroin-
dol-2-one (10, 3.2 g, 13.5 mmol) in MeOH (30 mL) were
added piperidine (50 mg) and 4-pyridinecarboxaldehyde
(1.6 g, 14.8 mmol) at 0 ꢁC. The mixture was heated un-
der reflux for 4 h, then extracted with ethyl acetate,
washed with water, dried over MgSO4 and evaporated.
The residue was purified by flash column chromatogra-
phy (ethyl acetate/n-hexane), and recrystallized from
EtOAc/n-hexane to give 14 in 53% yield: mp 60.0–
1
IR (cmꢀ1) 3396, 3008, 2973, 1711; H NMR (CDCl3):
d 1.91 (d, 3H, J = 7.0 Hz), 5.96 (q, 1H, J = 7.5 Hz),
6.56 (d, 1H, J = 8 Hz), 6.81 (t, 1H, J = 7.5 Hz), 7.09 (t,
1H, J = 7.5 Hz), 7.32 (t, 1H, J = 7.5 Hz), 7.37–7.46 (m,
5H), 7.53 (d, 2H, J = 6.0 Hz), 7.80 (s, 1H), 8.78 (d,
2H, J = 6.0 Hz); HRMS: m/z 326.1402 (calculated for
C22H18N2O, 326.1419).
4.4.6. 1-(1-Phenylethyl)-3-(3-pyridinylmethylidenyl)indo-
lin-2-one (19). 1-(1-Phenylethyl)indolin-2-one (11), 3-
pyridinecarboxaldehyde and piperidine in MeOH were
allowed to react in the same manner as described in
the preparation of compound 14 and the residue was
purified by flash column chromatography (EtOAc/n-
hexane), recrystallized from EtOAc-n-hexane to give
1
61.1 ꢁC; H NMR (CDCl3): d 4.74, 4.76 (2s, 2H), 6.51
(d, 1H), 6.62 (t, 1H), 6.78–7.39, 7.81 (m+d, 9H), 7.55
(s, 1H), 8.48–8.53 (m, 2H); HRMS: m/z 312.1245 (calcu-
lated for C21H16N2O, 312.1263).
4.4.2. 1-Benzyl-3-(3-pyridinylmethylidenyl)indolin-2-one
(15). 1-Benzyl-1,3-dihydro-indol-2-one (10), 3-pyridine-
carboxaldehyde and piperidine in MeOH were allowed
to react in the same manner as described in the prepara-
tion of compound 14 and the residue was recrystallized
from EtOAc/i-propyl ether to give 15 in 65% yield: mp
1
19 in 67% yield as oil; IR (cmꢀ1) 3419, 1702; H NMR
(CDCl3): d 1.88–1.92 (dd, 3H), 5.98 (q, 1H), 6.53–6.57
(m, 1H), 6.83 (t, 1H), 7.02–7.13 (m, 2H), 7.29–7.60
(m, 7H), 7.87–7.98 (m, 1H), 8.65–9.22 (m, 2H); HRMS:
m/z 326.1397 (calculated for C22H18N2O, 326.1419).
1
89.5–90.0 ꢁC; IR (cmꢀ1) 3420, 1697; H NMR (CDCl3):
d 5.02, 5.03 (2s, 2H, ArCH2-), 6.78 (d, 1H, J = 7.5 Hz),
6.89 (t, 1H, J = 7.5 Hz), 7.09 (t, 1H, J = 7.5 Hz), 7.07–
7.61 (m, 8H), 7.87, 7.98 (2s, 1H), 8.65–9.23 (m, 2H);
HRMS: m/z 312.1231 (calculated for C21H16N2O,
312.1263).
4.4.7. 1-(1-phenylethyl)-3-(2-pyridinylmethylidenyl)indo-
lin-2-one (20). 1-(1-Phenylethyl)indolin-2-one (11), 2-
pyridinecarboxaldehyde and piperidine in MeOH were
allowed to react in the same manner as described in
the preparation of compound 14 and the residue was
purified by flash column chromatography (EtOAc/n-
hexane), recrystallized from EtOAc-n-hexane to give
20 in 78% yield as oil; IR (cmꢀ1): 3425, 1700; 1H
NMR (CDCl3): d 1.92 (d, 3H, J = 7.5 Hz), 6.02
4.4.3. 1-Benzyl-3-(2-pyridinylmethylidenyl) indolin-2-one
(16). 1-Benzyl-1,3-dihydroindol-2-one (10), 2-pyridine-
carboxaldehyde and piperidine in MeOH were allowed

4230
C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
(q, 1H, J = 7.5 Hz), 6.55 (d, 1H, J = 7.5 Hz), 7.02 (t, 1H,
J = 7.5 Hz), 7.13 (t, 1H, J = 7.5 Hz), 7.29–7.45 (m, 5H),
7.67 (d, 1H, J = 8.0 Hz), 7.83 (t, 1H, J = 7.5 Hz), 7.86 (s,
1H), 8.90 (d, 2H, J = 5.0 Hz), 9.04 (d, 1H, J = 7.5 Hz);
HRMS: m/z 326.1431 (calculated for C22H18N2O,
326.1419).
J = 7.0 Hz), 7.68 (d, 1H, J = 4.5 Hz), 7.84 (t, 1H,
J = 7.0 Hz), 8.06 (d, 1H, J = 8.0 Hz), 8.26–8.28 (m,
2H), 9.04 (d, 1H, J = 4.5 Hz); HRMS: m/z 376.1617
(calculated for C26H20N2O, 376.1576).
4.4.12. 1-(1-Phenylethyl)-3-(3-quinolinylmethylidenyl)
indolin-2-one (25). 1-(1-Phenylethyl)indolin-2-one (11),
3-quinolinecarboxaldehyde and piperidine in MeOH
were allowed to react in the same manner as described
in the preparation of compound 14 and the residue was
recrystallized from MeOH to give 25: (isomer ratio, E/
4.4.8. 1-Benzyl-3-(4-quinolinylmethylidenyl)indolin-2-one
(21). 1-Benzyl-1,3-dihydroindol-2-one (10), 4-quinoline-
carboxaldehyde and piperidine in MeOH were allowed
to react in the same manner as described in the prepara-
tion of compound 14 and the residue was recrystallized
from MeOH to give 21 in 87% yield: mp 94.8–95.2 ꢁC;
1
Z = 3/1 by H NMR analysis) in 88% yield. The E-/Z-
form of 25 was separated by column chromatography
[CH2Cl2/MeOH (95:5)], and recrystallized from EtOAc/
n-hexane to give Z-isomer and E-isomer of 25. Z-isomer
(25Z): mp 104.0–104.4 ꢁC; IR (cmꢀ1) 3400, 3051, 1709;
1H NMR (CDCl3): d 1.92 (d, 3H, J = 7.0 Hz), 6.06 (q,
1H, J = 7.0 Hz), 6.55 (d, 1H, J = 7.5 Hz), 7.06 (t, 1H,
J = 7.5 Hz), 7.12 (t, 1H, J = 7.5 Hz), 7.32 (d, 1H,
J = 7.5 Hz), 7.39 (t, 2H, J = 7.5 Hz), 7.44 (d, 2H,
J = 7.5 Hz), 7.64 (t, 1H, J = 8.0 Hz), 7.66 (d, 1H, H4,
J = 8.8 Hz), 7.75 (s, 1H, H-90), 7.82 (t, 1H, J = 8.0 Hz),
8.05 (d, 1H, J = 7.5 Hz), 8.18 (d, 1H, J = 7.5 Hz), 9.33
1
IR (cmꢀ1) 3415, 3060, 2941, 1707; H NMR (CDCl3):
d 5.07 (s, 2H), 6.73–6.79 (m, 3H), 6.97 (d, 1H,
J = 7.5 Hz), 7.18 (t, 1H, J = 7.5 Hz), 7.30–7.43 (m,
4H), 7.63 (t, 1H, J = 7.5 Hz), 7.67 (d, 1H, J = 4.5 Hz),
7.84 (t, 1H, J = 7.5 Hz), 8.05 (d, 1H, J = 7.5 Hz), 8.26
(d, 1H, J = 8.5 Hz), 8.28 (s, 1H), 9.04 (d, 1H,
J = 7.5 Hz); HRMS: m/z 362.1477 (calculated for
C25H18N2O, 362.1419).
4.4.9. 1-Benzyl-3-(3-quinolinylmethylidenyl)indolin-2-one
(22). 1-Benzyl-1,3-dihydroindol-2-one (10), 3-quinoline-
carboxaldehyde and piperidine in MeOH were allowed
to react in the same manner as described in the prepara-
tion of compound 14 and the residue was recrystallized
from EtOAc/i-propyl ether to give 22 in 85% yield: mp
(s, 1H, H4 ), 9.87 (s, 1H, H2 ); 13C NMR (CDCl3): d
16.23, 48.79, 110.89, 119.45, 121.80, 124.21, 126.72,
127.04, 127.16, 127.42, 127.63, 128.40, 128.69, 128.91,
129.20, 129.35, 131.06, 132.52, 139.16, 139.37, 140.63,
147.76, 153.10, 166.06; E-isomer (25E): mp 83.0–
0
0
1
1
228.1–228.4 ꢁC; IR (cmꢀ1) 3429, 3060, 1685; H NMR
83.8 ꢁC; H NMR (CDCl3): d 1.94 (d, 3H, J = 7.0 Hz),
(CDCl3): d 5.07 (s, 2H), 6.80 (d, 1H, J = 7.5 Hz), 7.12
(t, 1H, J = 7.5 Hz), 7.25–7.40 (m, 7H), 7.62 (t, 1H,
J = 7.5 Hz), 7.67 (d, 1H, J = 7.5 Hz), 7.80 (t, 1H,
J = 7.5 Hz), 8.04 (d, 1H, J = 7.5 Hz), 8.16 (d, 1H,
J = 8.0 Hz), 9.30 (d, 1H, J = 2.0 Hz), 9.83 (d, 1H,
J = 2.0 Hz); HRMS: m/z 362.1432 (calculated for
C25H18N2O, 362.1419).
6.01 (q, 1H, J = 7.0 Hz), 6.58 (d, 1H, J = 7.5 Hz), 6.83
(t, 1H, J = 7.5 Hz), 7.10 (t, 1H, J = 7.5 Hz), 7.31–7.39
(m, 2H), 7.41 (t, 2H, J = 7.5 Hz), 7.46 (d, 2H,
J = 7.5 Hz), 7.60 (d, 1H, H4, J = 8.0 Hz), 7.68 (t, 1H,
J = 8.0 Hz), 7.85 (t, 1H, J = 8.0 Hz), 7.92 (d, 1H,
J = 8.0 Hz), 8.05 (s, 1H, H-90), 8.22 (d, 1H, J = 8.0 Hz),
8.47 (s, 1H, H4 ), 9.25 (s, 1H, H2 ); 13C NMR (CDCl3): d
16.35, 49.35, 111.19, 121.32, 121.77, 122.66, 126.71,
127.47, 127.49, 127.52, 128.22, 128.46, 128.72, 129.15,
129.55, 130.08, 130.67, 132.92, 136.36, 139.33, 142.64,
148.06, 150.44, 167.97; HRMS: m/z 376.1552 (calculated
for C26H20N2O, 376.1576).
0
0
4.4.10. 1-Benzyl-3-(2-quinolinylmethylidenyl)indolin-2-
one (23). 1-Benzyl-1,3-dihydroindol-2-one (10), 2-quino-
linecarboxaldehyde and piperidine in MeOH were al-
lowed to react in the same manner as described in the
preparation of compound 14 and the residue was recrys-
tallized from EtOAc/n-hexane to give 23 in 83% yield:
mp 266.8–267.2 ꢁC; IR (cmꢀ1) 3429, 3051, 1693; 1H
4.4.13. 1-(1-Phenylethyl)-3-(2-quinolinylmethylidenyl)
indolin-2-one (26). 1-(1-Phenylethyl)indolin-2-one (11),
2-quinolinecarboxaldehyde and piperidine in MeOH
were allowed to react in the same manner as described
in the preparation of compound 14 and the residue was
purified by flash column chromatography (EtOAc/n-hex-
ane), recrystallized from EtOAc/n-hexane to give 26 in
78% yield as oil; IR (cmꢀ1) 3416, 1703; 1H NMR (CDCl3):
d 1.92 (d, 3H, J = 7.5 Hz), 6.04 (q, 1H, J = 7.5 Hz), 7.05 (t,
1H, J = 7.5 Hz), 7.16 (t, 1H, J = 7.5 Hz), 7.31 (t, 2H,
J = 7.5 Hz), 7.39 (t, 2H, J = 7.5 Hz), 7.46 (d, 2H,
J = 8.0 Hz), 7.64 (t, 1H, J = 8.0 Hz), 7.74 (d, 1H,
J = 8.0 Hz), 7.83 (t, 1H, J = 8.0 Hz), 7.89 (d, 1H,
J = 8.0 Hz), 8.03 (s, 1H), 8.27–8.29 (m, 2H), 9.24 (d, 1H,
J = 7.5 Hz); HRMS: m/z 376.1552 (calculated for
C26H20N2O, 376.1576).
NMR (CDCl3):
d 5.03 (s, 2H), 6.78 (d, 1H,
J = 8.0 Hz), 7.11 (t, 1H, J = 8.0 Hz), 7.28–7.40 (m,
6H), 7.66 (t, 1H, J = 7.5 Hz), 7.75 (d, 1H, J = 8.0 Hz),
7.80–7.92 (m, 2H), 8.02 (s, 1H), 8.22 (d, 2H,
J = 8.0 Hz), 9.26 (d, 1H, J = 8.0 Hz); HRMS: m/z
362.1477 (calculated for C25H18N2O, 362.1422).
4.4.11. 1-(1-Phenylethyl)-3-(4-quinolinylmethylidenyl)
indolin-2-one (24). 1-(1-Phenylethyl)indolin-2-one (11),
4-quinolinecarboxaldehyde and piperidine in MeOH
were allowed to react in the same manner as described
in the preparation of compound 14 and the residue
was recrystallized from MeOH to give 24 in 90% yield:
mp 149.5–150.1 ꢁC; IR (cmꢀ1) 3396, 1710; 1H NMR
(CDCl3): d 1.95 (d, 3H, J = 7.0 Hz), 6.02 (q, 1H,
J = 7.0 Hz), 6.56 (d, 1H, J = 8.0 Hz), 6.68 (t, 1H,
J = 7.5 Hz), 6.96 (d, 1H, J = 7.5 Hz), 7.06 (t, 1H, J =
7.5 Hz), 7.33 (t, 1H, J = 7.5 Hz), 7.41 (t, 2H,
J = 7.5 Hz), 7.47 (d, 2H, J = 7.0 Hz), 7.63 (t, 1H,
4.4.14. 1-(1-Naphthylmethyl)-3-(4-pyridinylmethylidenyl)
indolin-2-one (27). 1-1-(Naphthalenylmethyl)indolin-2-
one (12), 4-pyridinecarboxaldehyde and piperidine in
MeOH were allowed to react in the same manner as

C.-Y. Hung et al. / Bioorg. Med. Chem. 16 (2008) 4222–4232
4231
described in the preparation of compound 14 and the
residue was purified by flash column chromatography
(EtOAc/n-hexane), recrystallized from EtOAc/n-hexane
buffered saline (PBS), and then 10 lL of MTT solution
(5 mg/mL) with 50 lL of cells suspension in HBSS was
added into each well of a 96-well plate and incubated
at 37 ꢁC in the dark for 2 h. Treatment of living cells
with MTT produces a dark blue formazan product,
whereas no such staining is observed in dead cells. The
formazan product was dissolved by adding 140 lL
DMSO and then the absorbance of the mixture was
measured on an ELISA reader at a best wavelength of
570 nm.
to give 27 in 89% yield: mp 102.3–102.7 ꢁC; IR (cmꢀ1
)
3417, 1711; 1H NMR (CDCl3): d 5.52 (s, 2H), 6.74
(d, 1H, J = 8.0 Hz), 6.89 (t, 1H, J = 7.5 Hz), 7.17 (t,
1H, J = 7.5 Hz), 7.35 (d, 1H, J = 7.0 Hz), 7.43 (t, 1H,
J = 7.5 Hz), 7.50 (d, 1H, J = 8.0 Hz), 7.56–7.60 (m,
3H), 7.65 (t, 1H, J = 7.5 Hz), 7.84 (d, 1H, J = 8.0 Hz),
7.87 (s, 1H), 7.94 (d, 1H, J = 8.0 Hz), 8.22 (d, 1H,
J = 8.0 Hz), 8.84 (d, 2H, J = 8.0 Hz); HRMS: m/z
362.1422 (calculated for C25H18N2O, 362.1419).
5.4. Cell cycle distribution analysis
4.4.15. 1-(2-Naphthylmethyl)-3-(4-pyridinylmethylidenyl)
indolin-2-one (28). 1-2-(Naphthalenylmethyl)indolin-2-
one (13), 4-pyridinecarboxaldehyde and piperidine in
MeOH were allowed to react in the same manner as de-
scribed in the preparation of compound 14 and the res-
idue was purified by flash column chromatography
(EtOAc/n-hexane), recrystallized from EtOAc/n-hexane
Cell cycle analysis by flow cytometry was performed as
described in the previous paper.13 HL-60 cells (4 · 104
cells/mL) were cultured with 2.5 lM compound 25 alone
or in combination with 5 nM ATRA for 72 h. After
treatment, the cells were collected, washed with cold
PBS, and fixed with 70% ice-cold ethanol at ꢀ20 ꢁC
overnight. Then the cells were centrifuged and resus-
pended in staining solution, containing 1% Triton X-
100 (Sigma), 0.1 mg/mL RNase (Sigma) and 4 lg/mL
phopidium iodide (Sigma), for 30 min at 37 ꢁC in the
dark and then analyzed on a fluorescence-activated cell
sorter flow cytometry (FACS-caliber, Becton Dickinson,
San Jose, CA, USA) and all the resulted histograms
were analyzed by ModFit Software.
to give 28 in 80% yield: mp 85.1–85.4 ꢁC; IR (cmꢀ1
)
1
3417, 3059, 3020, 1707; H NMR (CDCl3): d 5.20 (s,
2H), 6.82 (d, 1H, J = 8.0 Hz), 6.88 (t, 1H, J = 8.0 Hz),
7.20 (t, 1H, J = 8.0 Hz), 7.48–7.56 (m, 6H), 7.82–7.87
(m, 5H), 8.80 (d, 1H); HRMS: m/z 362.1417 (calculated
for C25H18N2O, 362.1419).
5. Bioassay
5.5. Statistic evaluation
5.1. Materials and methods
Values were expressed as means SD of three indepen-
dent experiments. Student’s t tests were used to assess
the statistical significance of the differences, with ‘P’ val-
ues of less than 0.05 being considered statistically
significant.
5.1.1. Cell culture and treatment. The human leukemia
cells HL-60 and normal leukocytes were maintained in
RPMI-1640 medium supplemented with 10% fetal
bovine serum (GIBCO/BRL), penicillin (100 U/mL)/
streptomycin (100 lg/mL) (GIBCO/BRL) and 1%
L-glutamine (GIBCO/BRL) at 37 ꢁC in a humidified
atmosphere containing 5% CO2. Logarithmically grow-
ing tumor cells were used for all experiments.
Acknowledgments
The authors thank the Ministry of economic affairs of
the Republic of China for financial support. The inves-
tigation was supported by research grant from the Na-
tional Science Council of the Republic of China
(NSC94-2323-B-039-001).
5.2. Cell differentiation assay
Cell differentiation effect was assessed using nitroblue
tetrazoliun reduction (NBT) assay.11 HL-60 cells
(4 · 104 cells/mL) were cultured with various concentra-
tions of 14–28 for 72 h. After treatment, the cells were
collected, washed with cold PBS, and then incubated
in 40 lL NBT solution containing 1 mg/mL NBT and
1 mg/mL phorbol phosphate 13-acetate (PMA) and
incubated at 37 ꢁC for 30 min. Then, cells were pelleted
and a hematocytometer was used to determine the pop-
ulation of NBT-positive cells among a total number of
at least 200 cells counted.
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