Indeno[1,2-c]isoquinolines as enhancing agents on all-trans retinoic acid-mediated differentiation of human myeloid leukemia cells

Article abstract of DOI:10.1016/j.bmc.2007.10.086

Induction of differentiation is a new and promising approach to cancer therapy, well illustrated by the treatment of acute myeloid leukemia with all-trans retinoic acid (ATRA). Using combination of ATRA and chemotherapy, adverse effects such as retinoic a

Full text of DOI:10.1016/j.bmc.2007.10.086

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1125–1132
Indeno[1,2-c]isoquinolines as enhancing agents on all-trans
retinoic acid-mediated differentiation
of human myeloid leukemia cells
Seung Hyun Kim,a Sang Mi Oh,b Ju Han Song,a Daeho Cho,c Quynh Manh Le,b
Suh–Hee Lee,b Won-Jea Chob and Tae Sung Kima,*
aSchool of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea
bCollege of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju, Republic of Korea
cDepartment of Life Sciences, Sookmyung Women’s University, Seoul, Republic of Korea
Received 11 September 2007; revised 25 October 2007; accepted 26 October 2007
Available online 30 October 2007
Abstract—Induction of differentiation is a new and promising approach to cancer therapy, well illustrated by the treatment of acute
myeloid leukemia with all-trans retinoic acid (ATRA). Using combination of ATRA and chemotherapy, adverse effects such as ret-
inoic acid syndrome have decreased, and long-term survival has improved. In this study, we demonstrated that the indeno[1,2-c]iso-
quinolines markedly enhanced differentiation of human myeloid leukemia HL-60 and NB4 cells when simultaneously combined with
a low dose of ATRA. Of the tested compounds, 6-(4-methoxybenzyl)-2,11-dimethyl-6H,11H-indeno[1,2-c]isoquinolin-5-one (IIQ-
16), an indeno[1,2-c]isoquinoline derivative, showed the highest differentiation-enhancing activity via a pathway involved with pro-
tein kinase C, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. The ability to enhance the differentiation potential
of ATRA by IIQ-16 may improve outcomes in the therapy of acute promyelocytic leukemia.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
pounds that have different mechanisms of action, such
as paclitaxel, curcumin, and silibinin.5–7
Leukemia can eventually be treated with agents that in-
duce terminal differentiation, presumably with less mor-
bidity than that produced by cyto-destructive agents.1
ATRA is able to induce a terminal differentiation of leu-
kemic cell lines, such as HL-60, NB4, and U937 cells,
and of short-term culture APL cells from humans.2
Moreover, ATRA is able to induce complete remission
(CR) in almost all patients with APL through in vivo
differentiation of APL blasts.3 Although ATRA can
bring about CR of APL, treatment with ATRA alone
showed severe side effects, including ATRA syndrome
and the induction of a secondary resistance to ATRA.4
ATRA syndrome combines fever, respiratory distress,
weight gain, pulmonary infiltrates, pericardial effusions,
and hypotension. Therefore, current attempts to over-
come this problem focus on the combination therapy
with non-toxic concentrations of ATRA and com-
The indenoisoquinolines are a class of cytotoxic mole-
cules that have been demonstrated to inhibit topoiso-
merase I enzyme by intercalating between DNA bases
at the enzyme’s cleavage site. This mechanism of action
is identical to the natural product camptothecin8 and its
clinically useful derivative topotecan.9 Various topoiso-
merase I inhibitors such as 10-hydroxycamptothecin and
camptothecin are known to induce and/or enhance the
differentiation of leukemia cells.10,11 Previously we syn-
thesized a series of indeno[1,2-c]isoquinoline derivatives,
which were demonstrated to exert inhibitory effects on
topoisomerase I activity.12
In this report, we investigated enhancing effects of the
indeno[1,2-c]isoquinoline derivatives on cellular differ-
entiation of human myeloid leukemia HL-60 cells, in
combinations of a low dose of ATRA. Human myeloid
leukemia HL-60 cell culture has been employed as an
excellent model system for studying cellular differentia-
tion in vitro. HL-60 cells are differentiated into a granu-
locytic lineage when treated with ATRA.2,13
Keywords: Indenoisoquinoline; Differentiation; Leukemia; All-trans
retinoic acid.
*
Corresponding author. Tel.: +82 2 3290 3416; fax: +82 2 3290
3921; e-mail: tskim@korea.ac.kr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.086

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S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
2. Results
specific inhibitors for extracellular signal-regulated ki-
nase (ERK) (PD 98059), c-Jun N-terminal kinase
(JNK) (SP 600125), PKC (protein kinase C) (GF
109203X, chelerythrine), and phosphatidylinositol 3-ki-
nase (PI3-K) (Wortmannin, LY 294002), in the presence
of indenoisoquinoline IIQ-16 alone or in combination
with 50 nM ATRA. Afterward, the degree of cellular
differentiation was assessed by nitroblue tetrazolium
reduction assay. Activation of PI3-K, PKC, and MAPK
has been known to be involved in the differentiation of
leukemia cells.15 As shown in Figure 4, inhibitors for
ERK, JNK, and PKC significantly suppressed HL-60 cell
differentiation treated with IIQ-16 in combination with
ATRA in a dose-dependent manner. In contrast, both
PI3-K inhibitors had no effects on HL-60 cell differentia-
tion enhanced by IIQ-16 in combination with ATRA.
In order to determine whether the indeno[1,2-c]isoquino-
line derivatives exerted any effects on the differentiation of
leukemia cells, HL-60 cells were seeded at a density of
2 · 105 cells/ml and treated with either medium alone,
or treated for 72 h with 200 nM of each of the inde-
no[1,2-c]isoquinoline derivatives, in the absence or pres-
ence of a low (nontoxic) dose of ATRA (50 nM). As
shown in Table 1, treatment with the indenoisoquinoline
derivatives induced little increase in the differentiation of
the HL-60 cells, by approximately 0.83–1.75%. Impor-
tantly, some of the indeno[1,2-c]isoquinoline derivatives
significantly enhanced cell differentiation when combined
with 50 nM ATRA, which by itself caused a relatively low
level of differentiation. Among the tested derivatives, 6-
(4-methoxybenzyl)-2,11-dimethyl-6H,11H-indeno[1,2-c]
isoquinolin-5-one (IIQ-16, Fig. 1B) profoundly potenti-
ated cell differentiation in a concentration-dependent
manner (Fig. 2A). Treatment of the cells with more than
100 nM of IIQ-16 also inhibited cell proliferation in a
dose-dependent manner (Fig. 2B). IIQ-16 also enhanced
cell differentiation and inhibited cell proliferation of hu-
man myeloid NB4 cells in a dose-dependent manner,
when combined with 50 nM ATRA (Fig. 2). For all treat-
ments, cell viability was in excess of 98% throughout the
incubation period, as shown by the results of the Trypan
blue exclusion assay (data not shown).
To further characterize involvement of PKC, ERK, and
JNK in the cell differentiation induced by ATRA and
IIQ-16, the protein levels of total PKC, pERK, and
pJNK were determined by Western blot analysis. As
shown in Figure 5, the levels of total PKC, pERK,
and pJNK were increased in the cells treated with IIQ-
16 and ATRA, compared with the levels in those treated
with single treatments of either IIQ-16 or ATRA. These
results demonstrate that indeno[1,2-c]isoquinolines
potentiate ATRA-induced HL-60 cell differentiation
via a PKC/ERK/JNK pathway.
To further determine the cell differentiation enhanced by
the indeno[1,2-c]isoquinoline derivatives, the morpho-
logic phenotypes and the expression of cell surface anti-
gens on HL-60 cells were analyzed. As shown in Figure
3A, Giemsa-stained undifferentiated control HL-60 cells
were predominantly myelocytes with round and regular
cell margins, and large nuclei, suggesting that the cells
were highly active in DNA synthesis and were rapidly
proliferating. The cells treated with 250 nM IIQ-16 or
50 nM ATRA alone exhibited relatively small changes
in cell morphology such as irregular cell margins. Com-
bined treatment of HL-60 cells with 50 nM ATRA plus
250 nM IIQ-16 resulted in significantly decreased cell
size, denser chromatin, and an increased cytoplasm to
nuclear ratio, which suggested less DNA synthesis. As
shown in Figure 3A, some cells showed a multilobed nu-
cleus, which is a sign of cell differentiation into a granu-
locytic lineage.
3. Discussion
In this study, we demonstrated that indeno[1,2-c]iso-
quinoline derivatives can potentiate ATRA-induced dif-
ferentiation in HL-60 promyelocytic leukemia cells,
which are widely used as a model system for studies of
differentiation. HL-60 cells were found to synergistically
differentiate into granulocytes when treated with the in-
deno[1,2-c]isoquinoline derivative IIQ-16 in combina-
tion with ATRA. Many previous studies have
uncovered some chemical combinations which exert
either an additive or a synergistic effect on the differen-
tiation of HL-60 cells. These combinations include reti-
noic acid with sodium butyrate, dimethylsulfoxide,
hexamethylene bisacetamide, or thalidomide.16
The mechanism by which the indeno[1,2-c]isoquinoline
derivative IIQ-16 potentiates ATRA-induced HL-60 cell
differentiation remains to be adequately clarified. ATRA
is believed to mediate biological responses, including cell
differentiation, as a consequence of their interaction
with nuclear receptors to regulate gene transcription
and with a putative cell membrane receptor to generate
rapid non-genomic effects including the opening of volt-
age-gated calcium and chloride channels,17 and the acti-
vation of PI3-K, PKC, and MAPK.15 In our study,
inhibitors of PKC, ERK, and JNK significantly inhib-
ited the enhancing effects on HL-60 cell differentiation
induced by IIQ-16 in combination with ATRA. This
finding strongly suggests that the potentiation of cell dif-
ferentiation by the indenoisoquinoline derivative in
combination with ATRA may occur via a PKC/ERK/
JNK-mediated signaling pathway.
Cytofluorometric analysis was also performed to deter-
mine the expression of specific surface antigens on HL-
60 cells. CD11b (Mac-1) is expressed on activated mono-
cytes, granulocytes, lymphocytes, and a subset of NK
cells. HL-60 leukemia cells express a cell surface marker,
CD11b, when differentiated into granulocytes by high
concentrations of ATRA.14 As shown in Figure 3B.
indenoisoquinoline IIQ-16 significantly increased the
number of CD11b-positive cells when combined with
50 nM ATRA, confirming that the indenoisoquinoline
potentiated ATRA-induced HL-60 cell differentiation.
To determine an action mechanism by which the indeno-
isoquinoline derivative potentiates ATRA-induced
HL-60 cell differentiation, HL-60 cells were treated with

S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
1127
Table 1. Effects of the indeno[1,2-c]isoquinoline derivatives on HL-60 cell differentiation
R1
R2
R3
Differentiation (%)
ATRA + IIQ
IIQ alone
Medium alone
ATRA alone
IIQ-1
0.83 0.38
—
1.50 0.87
—
27.00 2.75
28.33 13.99
–H
–H
–Me
IIQ-2
IIQ-3
IIQ-4
IIQ-5
IIQ-6
–H
–H
–H
–H
–H
–H
1.33 1.23
1.25 0.66
1.42 0.29
1.08 0.52
0.83 1.01
30.67 5.39
46.75 6.74**
38.58 0.88**
23.42 3.40
29.42 6.03
CH2
–Me
–Me
–OH
–OH
–Me
OMe
CH2
–Me
CH2
CH2
IIQ-7
–H
–OMe
0.92 0.14
38.92 9.00
OMe
OMe
OH
Me
IIQ-8
IIQ-9
IIQ-10
–H
–H
–H
1.17 0.14
1.75 0.43
1.58 0.80
37.83 3.06
33.08 6.03
38.83 6.03*
CH2
–OEt
–Me
–OCOCH3
CH2
CH2
OH
IIQ-11
–H
1.42 0.29
36.50 3.28*
OMe
OMe
(CH2)6 CH3
IIQ-12
IIQ-13
IIQ-14
IIQ-15
–H
–(CH2)6–CH3
0.83 0.14
1.25 0.66
1.25 0.25
1.75 0.50
40.75 6.19*
24.08 5.92
36.33 0.76*
58.75 6.95
CH2
–Me
–Me
–Me
–H
–Me
–H
OMe
OMe
CH2
–Me
–Me
IIQ-16
IIQ-17
IIQ-18
IIQ-19
IIQ-20
IIQ-21
IIQ-22
IIQ-23
IIQ-24
IIQ-25
IIQ-26
IIQ-27
IIQ-28
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–Me
1.25 1.00
0.83 0.29
1.33 0.14
1.17 0.72
1.42 0.14
1.42 0.38
0.92 0.58
1.33 0.63
1.25 0.43
1.00 0.25
1.92 0.29
0.92 0.14
1.50 0.66
76.08 10.16**
28.50 2.29
28.75 1.15
28.92 11.00
32.25 6.67
32.75 6.43
31.42 7.52
34.75 8.89
35.33 8.71
35.92 10.37
34.58 5.63
30.00 8.23
CH2
–OH
–OH
–OMe
–OMe
–OEt
–OEt
–OPr
–Me
OMe
OMe
OMe
CH2
–Me
CH2
–Me
CH2
–Me
–OPr
OMe
OMe
CH2
CH2
CH3
CH3
O
CH
–OBu
–OBu
–Me
OMe
CH2
–O(CH2)4CH3
–Me
28.42 7.73
(continued on next page)

1128
S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
Table 1 (continued)
R1
R2
R3
Differentiation (%)
IIQ alone
ATRA + IIQ
35.08 3.79*
IIQ-29
–Me
–O(CH2)4CH3
OMe
OMe
1.00 0.25
CH2
3
CH
IIQ-30
IIQ-31
–Me
–Me
–Me
1.50 0.87
1.17 0.52
33.00 5.17
32.17 6.33
(CH2)2
O
O
CH
CH
3
CH
CH33
(CH2)2 CH
CH2
HL-60 cells were treated for 72 h with medium or 50 nM ATRA alone, or with 50 nM ATRA in combination with 200 nM of the indeno[1,2-
c]isoquinoline derivatives. The cell differentiation was assessed by the NBT assay. Each value represents the mean SE mean (n = 3). *P < 0.05 and
**P < 0.01, relative to a group treated with ATRA alone.
In addition, the methyl substitution at the R1 and R2
positions of indeno[1,2-c]isoquinoline (IIQ-15, -16)
afforded higher enhancing effect on HL-60 cell differen-
tiation, and additional methoxybenzyl substitution at
the R3 position (IIQ-16) exhibited the most profound
synergistic effects on HL-60 cell differentiation when
combined with ATRA. This indicates that the substitu-
tion of indeno[1,2-c]isoquinoline at R1, R2, and R3 posi-
tions might affect the activity of signaling molecule(s)
involved in the enhancement of HL-60 cell differentia-
tion. Previously we reported that indeno[1,2-c]isoquino-
line and its analogs were demonstrated to inhibit
topoisomerase I activity.12 Among the synthesized com-
pounds, IIQ-22, IIQ-24, and IIQ-27 showed potent
topoisomerase I inhibition activity. In contrast, IIQ-16
exhibited weak inhibitory activity on topoisomerase I,
suggesting that the enhancing effects of the indeno[1,2-
c]isoquinoline derivatives on HL-60 cell differentiation
observed in this study were not correlated with the
inhibitory effects on topoisomerase I activity.
medium such that the final concentration of ethanol or
dimethylsulfoxide had no effect on the differentiation
and proliferation of HL-60 cells. All manipulations were
performed in subdued light. The cell lines HL-60 and
NB4 were maintained in RPMI-1640 medium supple-
mented with 10% fetal bovine serum (Gibco-BRL,
Grand Island, NY, USA).
4.2. Synthesis of indeno[1,2-c]isoquinoline derivatives
A number of indeno[1,2-c]isoquinolines with different
substituted groups at three positions (Fig. 1A) were syn-
thesized as previously described.12 The four compounds
2, IIQ-8, IIQ-11, and IIQ-12 were newly prepared as
shown in Scheme 1. The commercially available starting
lactone 1 was treated with PMBNH2 to give the indeno-
isoquinoline 2 in 95% yield. Indenoisoquinoline 2 was
reacted with MeMgBr or n-hexylMgBr to afford the cor-
responding alcohols IIQ-8 and IIQ-11, respectively, in
good yield. Dehydroxylation of IIQ-11 was performed
with TMSCl, NaI, and MeCN to provide the desired
IIQ-12 in 84% yield.
In conclusion, the indeno[1,2-c]isoquinoline derivative
IIQ-16 potentiates ATRA-induced HL-60 cell differenti-
ation via a PKC/ERK/JNK signaling pathway. These
findings imply that the indeno[1,2-c]isoquinoline deriva-
tives may prove useful in the treatment of leukemic
diseases.
4.3. General remarks
Melting points were determined by using the capillary
method on Electrothermal IA9200 digital melting point
apparatus and are uncorrected. Nuclear magnetic reso-
1
nance (NMR) data for H NMR were taken on Bruker
4. Experimental
4.1. Materials
AMX-R300 and are reported in ppm, downfield from
the peak of tetramethylsilane as an internal standard.
The data are reported as follows: chemical shift, number
of proton, multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet). IR spectra were recorded on
Nicolet 520P using KBr pellets. Mass spectra were ob-
tained on Platform II of Micromas applying the elec-
tron-impact (EI) method. Column chromatography
was performed on Merck silica gel 60 (70–230 mesh).
TLC was carried out using plates coated with silica gel
60 F254 purchased from Merck. Chemical reagents were
purchased from Aldrich Chemical Co. and used without
further purification. Solvents were distilled prior to use:
THF was distilled from sodium/benzophenone.
All-trans retinoic acid, phorbol 12-myristate 13-acetate
(PMA), 2-[4-morpholinyl]-8phenyl-1[4H]-benzopyran-
4-one (LY 294002), and 3-furo[4,3,2-de]indeno[4,5-h]-2-
benzopyran-3,6,9-trione (Wortmannin), ethanol, Giemsa
staining solution, methanol-free paraformaldehyde, and
all other reagents were purchased from the Sigma Chem-
ical Co. (St. Louis, MO, USA). Chelerythrine, 1-(5-iso-
quinolinesulfonyl)-2-methylpiperazine dihydrochloride
(H7), and 2-(20-amino-30-methoxyphenyl)-oxanaphtha-
len-4-one (PD 98059) were purchased from the Tocris
Cookson Ltd. (UK). SP 600125 was purchased from Cal-
biochem (San Diego, CA, USA). A stock solution of
1 mM ATRA was dissolved in dimethylsulfoxide. The
indenoisoquinoline derivatives were dissolved in dimeth-
ylsulfoxide to make a stock solution of 20 mM. The solu-
tions were diluted at least 1000-fold in the growth
4.3.1. 6-(4–Methoxybenzyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione (2). p-Methoxybenzyl amine (69 mg,
0.5 mmol) was added to a stirred solution of benz[d]inde-
no[1,2-b] pyran-5,11-dione (100 mg, 0.4 mmol) in CH2Cl2
(10 mL) at room temperature. The bright orange mixture

S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
1129
B
A
R2
Me
R1
Me
N
N
OMe
R3
O
O
Figure 1. The structures of indeno[1,2-c]isoquinolines (A) and IIQ-16 (B).
was stirred overnight and the solvent was removed in
vacuo to give the residue which was purified by column
chromatography on silica gel with Hexane/Ethyl acetate
(4:1) to afford the compound 2 (140 mg, 95%) as a reddish
solid. Mp 213–214 ꢁC. IR (cmꢀ1): 1700 (C@O), 1660
4.3.2. 11–Hydroxy-6-(4-methoxybenzyl)-11-methyl-6H,
11H-indeno[1,2-c]isoquinolin-5-one (IIQ-8). To a stirred
solution of 6-(4-methoxybenzyl)-6H-indeno[1,2-c] iso-
quinolin-5,11-dione 2 (100 mg, 0.27 mmol) in THF
(10 mL) at 0 ꢁC, CH3MgBr 1 M in butyl ether
(0.6 mL, 0.54 mmol) was added and the reaction mix-
ture was stirred while the temperature was increased to
room temperature. After additional stirring for 12 h,
the mixture was quenched by water and the reaction
mixture was filtered through Celite. The CH2Cl2 layer
was washed with brine, dried over anhydrous Na2SO4,
and concentrated to dryness. The residue was purified
by column chromatography on silica gel with n-hex-
ane/ethyl acetate (2:1) to yield IIQ-8 (82 mg, 79%) as a
yellow solid. Mp 172–177 ꢁC. IR (cmꢀ1): 3360 (OH),
1630 (amide). 1H NMR (CDCl3): d 8.22–6.75 (m,
12H), 5.55 (d, J = 16.4 Hz, 1H), 5.25 (d, J = 16.4 Hz,
1H), 3.71 (s, 3H), 1.84 (s, 3H). EIMS m/z (%): 383
(M+, 60%).
1
(amide). H NMR (CDCl3): d 8.76 (d, J = 8.3 Hz, 1H),
8.38 (d, J = 9.0 Hz, 1H), 7.79–6.86 (m, 10H), 5.74 (s,
2H), 3.77 (s, 3H). EIMS m/z (%): 367 (M+, 85%).
HL-60
IIQ-16
IIQ-16 + ATRA
A
NB4
IIQ-16
IIQ-16 + ATRA
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
*
4.3.3. 11–Hexyl-11-hydroxy-6-(4-methoxybenzyl)-6H, 11H-
indeno[1,2-c] isoquinolin-5-one (IIQ-11). The same pro-
cedure as described in the preparation of IIQ-8 was
used to give IIQ-11 (98%) as a yellow solid. Mp
M
0
25 50 100 200 250
M
0
25 50 100 200 250
1
145–146 ꢁC. IR (cmꢀ1): 3360 (OH), 1638 (amide). H
[IIQ-16] (nM)
[IIQ-16] (nM)
NMR (CDCl3): d 8.20 (d, J = 8.0 Hz, 1H), 7.72–6.67
(m, 11H), 5.54 (d, J = 16.5 Hz, 1H), 4.69 (m, 1H),
4.10 (m, 1H), 3.66 (s, 3H), 2.44–2.33 (m, 1H), 2.31–
2.03 (m, 1H), 1.03–0.97 (m, 6H), 0.69 (t, J = 6.9 Hz,
3H), 0.57–0.46 (m, 2H). EIMS m/z (%): 453 (M+,
72%).
B
HL-60
NB4
IIQ-16
IIQ-16
IIQ-16 + ATRA
IIQ-16 + ATRA
120
100
80
60
40
20
0
120
100
80
60
40
20
0
**
**
**
**
4.3.4. 11–Hexyl-6-(4-methoxybenzyl)-6H,11H-indeno[1,
2-c]isoquinolin-5-one (IIQ-12). To a mixture of Me3SiCl
(145 mg, 1.34 mmol), NaI (200 mg, 1.34 mmol) and
CH3CN (55 mg) was added a solution of 11–hexyl-11-
hydroxy-6-(4-methoxybenzyl)-6H,11H-indeno[1,2-c]iso-
quinolin-5-one IIQ-11 (101 mg, 0.22 mmol) in CH2Cl2
(2 mL). The mixture was stirred at room temperature
for overnight. The reaction mixture was diluted with
water and extracted with CH2Cl2. The organic layer
was washed with water, brine and dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the res-
idue was purified by column chromatography on silica
gel with hexane/ethyl acetate (4:1) to give the
compound IIQ-12 (81 mg, 84%) as a yellow solid. Mp
156–159 ꢁC. IR (cmꢀ1): 1642 (amide). 1H NMR
(CDCl3): d 8.54 (d, J = 8.0 Hz, 1H), 7.76–6.80 (m,
**
0
25 50 100 200 250
0
25 50 100 200 250
[IIQ-16] (nM)
[IIQ-16] (nM)
Figure 2. Effects of an indenoisoquinoline IIQ-16 on cell proliferation
and differentiation of human myeloid leukemia cells. Human myeloid
leukemia HL-60 and NB4 cells were, respectively, treated for 72 h with
ATRA (50 nM) or IIQ-16 (0–250 nM) alone, or with ATRA in
combination with various concentrations of IIQ-16 (0–250 nM). The
cellular differentiation was assessed by the NBT assay (A) and the cell
proliferationwas determinedbythe MTT assay (B). Eachvaluerepresents
the mean SE mean (n = 3). *P < 0.001, relative to an untreated group.
**P < 0.001, relative to a group treated with ATRA alone.

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S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
50 nM ATRA
250 nM IIQ-16
A
B
Medium
50 nM ATRA
250 nM IIQ-16
.1
1000
.1
1000
.1
1000
.1
1000
PE-anti CD11b
Figure 3. Morphologic and cytofluorometric analysis of HL-60 leukemia cells treated with ATRA alone or in combination with IIQ-16. (A) HL-60
cells were treated for 72 h with 50 nM ATRA or 250 nM IIQ-16, or with ATRA plus IIQ-16. The cells were assessed by morphologic analysis using
Giemsa stain. (B) The cells were assessed by cytofluorometric analysis using PE-conjugated anti-CD11 mAb (unshaded area), or isotype control mAb
(shaded area). The data are representative of three separate experiments.
11H), 5.81 (s, 2H), 4.13–4.10 (m, 1H), 3.73 (s, 3H), 2.28–
2.22 (m, 1H), 2.18–2.13 (m, 1H), 1.16–1.05 (m, 6H),
0.98–0.82 (m, 2H), 0.77 (t, J = 6.80 Hz, 3H). EIMS m/
z (%): 437 (M+, 92%).
lated as the percentage of live cells in the total cell
population. Cell proliferation was determined with the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
(MTT) assay. In brief, after each treatment, 10 ll of
MTT (5 mg/ml) was added to each well in 96-well plates.
After incubation for 4 h at 37 ꢁC, the crystals of viable
cells were dissolved with 100 ll of 0.04 N HCl in isopro-
panol. The absorbance of each well was then read at
540 nm using a kinetic microplate reader.
4.4. Determination of cell viability and proliferation
Cell viability was determined by the trypan blue exclu-
sion assay as previously described.18 Viability was calcu-
PD 98059
100
80
60
40
20
0
SP 600125
100
80
60
40
20
0
IIQ-16
ATRA
IIQ-16 + ATRA
0
10
20
0
5
10
[Inhibitor] (μM)
GF 109203X
Chelerythrin
Worthmannin
LY 294002
100
100
80
60
40
20
0
100
100
80
80
60
80
60
40
20
0
60
40
20
0
40
20
0
0
2.5
5
0
2.5
5
0
0.05
0.1
0
2.5
5
[Inhibitor] (μM)
[Inhibitor] (μM)
Figure 4. Effects of specific kinase inhibitors on HL-60 cell differentiation induced by 50 nM ATRA in combination with IIQ-16. HL-60 cells were
treated for 40 min with various concentrations of ERK inhibitor (PD 98059), JNK inhibitor (SP 600125), PKC inhibitors (GF 109203X,
chelerythrine), and PI3-K inhibitors (Wortmannin, LY 294002), followed by incubation with 50 nM ATRA or 250 nM IIQ-16, or with 50 nM ATRA
plus 250 nM IIQ-16. The cellular differentiation was assessed by the NBT reduction assay. Data are presented as percentage of differentiated cells
with means SE mean (n = 3).

S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
1131
A
B
Untreated
IIQ-16
4
3
2
1
0
ATRA
IIQ-16 + ATRA
Total PKC
pERK
pJNK
GAPDH
PKC
pERK
pJNK
Figure 5. Involvement of PKC, JNK, and ERK in HL-60 cell differentiation induced by ATRA and IIQ-16. HL-60 cells were incubated for 24 h with
50 nM ATRA or 250 nM IIQ-16, or with ATRA plus IIQ-16, or untreated. The protein levels were determined by Western blot analysis. The band
intensity of each of the treatment groups was determined via densitometric analysis and was expressed as fold of induction over untreated cells. The
experiment was repeated at least twice with similar results.
Me
O
O
HO
PMBNH2
CH2Cl2
95%
MeMgBr
THF
79%
N
O
N
OMe
OMe
O
O
O
IIQ-8
2
1
n-hexylMgBr
THF
98%
HO
TMSCl
NaI, CH3CN
84%
N
N
OMe
OMe
O
O
IIQ-11
IIQ-12
Scheme 1. Synthesis of indeno[1,2-c]isoquinoline derivatives.
4.5. Determination of cell differentiation
a cytospin centrifuge. The slides were fixed with metha-
nol and dried. The slides were stained with Giemsa
staining solution for 20 min and rinsed in deionized
water, air-dried, and observed under a microscope with
a camera. The stained cells were assessed for size, regu-
larity of the cell margin, and morphological characteris-
tics of the nuclei.
Cell differentiation was assessed by the nitroblue tetra-
zolium reduction assay, as previously described.19 This
assay is based on the ability of phagocytic cells to pro-
duce superoxide upon stimulation with PMA. For this
assay, 2 · 105 cells were harvested by centrifugation
and incubated with an equal volume of 1% NBT dis-
solved in PBS containing 200 ng/ml of freshly diluted
PMA at 37 ꢁC for 30 min in the dark. Cytospin slides
were prepared and were examined for blue-black nitro-
blue diformazan deposits, indicative of a PMA-stimu-
lated respiratory burst. At least 200 cells were assessed
for each experiment.
4.7. Immunofluorescent staining and cytofluorometric
measurements
Quantitative immunofluorescence measurements were
performed in an Epic XL flow cytofluorograph equipped
with a multi-parameter data acquisition and display sys-
tem. Briefly, a single-cell suspension was collected from
the various cultures and washed twice with ice-cold phos-
phate-buffered saline (PBS, pH 7.4). Afterward,
phytoerythrin (PE)-conjugated anti-human CD11b
monoclonal antibodies (Becton-Dickinson, San Jose,
4.6. Morphologic studies
Single-cell suspensions were prepared and 2 · 105 cells
were loaded into a cyto-funnel and spun at 500 rpm in

1132
S. H. Kim et al. / Bioorg. Med. Chem. 16 (2008) 1125–1132
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for 1 h. After incubation, the cells were washed with
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Cells were lysed in lysis buffer (50 mM Tris buffer, pH
7.5, containing 100 mM NaCl, 1% Nonidet P-40, 10%
glycerol, 1 mM EDTA, 1 mM NaF, 1 mM sodium
orthovanadate, 50 lg/ml leupeptin, 50 lg/ml aprotinin,
and 50 lg/ml phenylmethanesulfonyl fluoride) by incu-
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at 13,000 rpm at 4 ꢁC for 10 min. The proteins (15 lg) of
the supernatants were separated using a 10% sodium
dodecyl sulfate–polyacrylamide gel (SDS–PAGE) and
transferred to the nitrocellulose membrane. The blots
were probed with rabbit anti-human PKC, mouse anti-
pJNK, and mouse anti-pERK, washed, and exposed
to horseradish peroxidase-conjugated anti-mouse IgG2a
or rabbit IgG antibodies. Immunoreactive bands were
visualized by the enhanced chemiluminescence system
(Amersham, Buckinghamshire, UK).
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Acknowledgments
This study was supported by grants from the Korea
Health 21 R&D Project, Ministry of Health and Welfare
(01-PJ10-PG6-01GN16-0005) and the Seoul Research
and Business Program (10582) to T.S. Kim, and a grant
from the Korea Research Foundation (KRF-2004-
C00325) to W.-J. Cho.
17. (a) Norman, A. W.; Okamura, W. H.; Hammond, M. W.;
Bishop, J. E.; Dormanen, M. C.; Bouillon, R.; van
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Carson, M. C. Mol. Endocrinol. 1997, 11, 1518; (b)
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References and notes
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