Discovery and structure-activity relationship of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a new series of potent apoptosis inducers

Article abstract of DOI:10.1016/j.bmc.2007.09.046

We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors, with EC₅₀ values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine (6b) having EC₅₀ values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell growth inhibition assay, with GI₅₀ values of 16-42 nM for compound 6b.

Full text of DOI:10.1016/j.bmc.2007.09.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 222–231
Discovery and structure–activity relationship
of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines
as a new series of potent apoptosis inducers
Han-Zhong Zhang, Gisela Claassen, Candace Crogran-Grundy, Ben Tseng,
John Drewe and Sui Xiong Cai*
EpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 28 August 2007; accepted 28 September 2007
Available online 4 October 2007
Abstract—We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers
of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-
based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors,
with EC50 values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-
amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine
(6b) having EC50 values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell
growth inhibition assay, with GI50 values of 16–42 nM for compound 6b.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
at the P1 position of substrate for cleavage. Within the
caspase family, caspase-3 has been identified as one of
the key effector caspases that cleave multiple protein
substrates in cells, leading to irreversible cell death.6 In
our effort to discover and develop apoptosis inducers
as new anticancer agents, we have developed a cell-
based, high throughput screening technology, Apoptosis
Screening and AntiCancer Platform (ASAP), to identify
apoptosis inducers by measuring the activation of cas-
pase-3.7 One of the major advantages of this technology
is that by monitoring the activation of downstream cas-
pase-3 in a cell-based assay, we can discover compounds
that initiate the apoptosis signal pathway through a
known mechanism or known molecular targets, as well
as by novel mechanism or molecular targets.8
Apoptosis is a highly regulated process of cellular sui-
cide, and it plays a crucial role in normal cell develop-
ment and tissue homeostasis.1 Apoptosis enables
organisms to control their cell numbers and to eliminate
unneeded cells that may threaten their survival.2 It is
known that inappropriate apoptosis induction results
in excessive cell death, and could be the cause of degen-
erative diseases.3 Inadequate apoptosis, however, leads
to over proliferation of cells, and could result in cancer.4
It has been reported that the anti-tumor efficacy of sev-
eral chemotherapeutical agents is correlated to their
apoptosis inducing ability.5 Therefore, the identification
of apoptosis inducers represents an attractive approach
for the discovery and development of potential antican-
cer agents.
Applying this high throughput assay, we have discov-
ered and optimized several classes of small molecules
as novel apoptosis inducers. N-Phenyl nicotinamides,9
represented by 6-methyl-N-(4-ethoxy-2-nitro-phenyl)-
pyridine-3-carboxamide (1a), were discovered as a series
of potent apoptosis inducers that interact with tubulin.
4-Aryl-4H-chromenes, exemplified by 2-amino-3-cya-
no-7-dimethylamino-4-(3-bromo-4,5-dimethoxyphenyl)-
4H-chromene (1b),10 showed potent apoptosis inducing
activity and several of them demonstrated vascular
The mechanism of apoptosis involves a caspase cascade
that is activated sequentially. Caspases are a family of
cysteine proteases that require an asparatic acid residue
Keywords: Apoptosis inducers; HTS; N-Phenyl-1H-pyrazolo[3,4-b]qu-
inolin-4-amines; Caspases.
*
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@epicept.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.046

H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
223
fication of N-(4-acetylphenyl)-2,3-dihydro-1H-cyclo-
penta[b]quinolin-9-amine (2, Chart 1) as a potent
apoptosis inducer, and SAR study of 2 leading to the
discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-
amines as a series of potent apoptosis inducers.
OMe
MeO
Br
NO2
H
N
N
CN
O
OEt
O
N
O
1b
NH2
2. Results and discussion
2.1. Chemistry
1a
OH
Compounds 2, 6a, 6c, and 9a were purchased from a
commercial library. Compounds 6b, 6d–6g were pre-
pared as shown in Scheme 1. The key intermediate, 4-
Cl
O
CF3
N
O
O
O
O
S
chloro-1,3-dimethyl-1H-pyrazolo[3,4-b]quinoline
(5),
O
N
was prepared according to literature procedure.17 Reac-
tion of anthranilic acid with 4-methyleneoxetan-2-one in
CCl4, followed by treatment with acetic anhydride, pro-
OH
1c
1d
duced
2-(2-oxopropyl)-4H-benzo[d][1,3]oxazin-4-one
CF3
OMe
OMe
(3), which was reacted with methylhydrazine to produce
2-(1,3-dimethyl-1H-pyrazol-5-ylamino)benzoic acid (4).
Cyclization of 4 via treatment with POCl3 produced 5.
Reaction of 5 with a substituted aniline, such as 4-pro-
pionylbenzenamine, produced 1,3-dimethyl-N-(4-propi-
onylphenyl)-1H-pyrazolo[3,4-b]quinolin-4-amine (6b).
Acid 6h was prepared through hydrolysis of the corre-
sponding methyl ester 6f. Reduction of compound 6b
using sodium borohydride in methanol produced com-
pound 6i.
HN
N
O
N
N
N
H
N
1e
2
Chart 1.
disrupting activity (VDA) with anti-tumor efficacy in
several animal models.11 Gambogic acid (1c), isolated
from gamboge resin, was discovered as a fast apoptosis
inducer.12 Derivatives of gambogic acid with good
in vivo activity have been discovered and its molecular
target has been identified as transferrin receptor.13
3-Aryl-5-aryl-1,2,4-oxadiazoles, exemplified by 5-(3-
chlorothiophen-2-yl)-3-(4-trifluoromethylphenyl)-1,2,4-
oxadiazole (1d), were found to induce apoptosis
selectively in certain tumor types,14 and TIP47, an insu-
lin growth factor II (IGF II) receptor binding protein,
has been identified as its molecular target.15 In addition,
4-anilino-2-(2-pyridyl)pyrimidines such as 1e were re-
ported recently as a series of novel apoptosis inducers
that arrest cells at G2/M.16 Herein, we report the identi-
Other substituted 4-chloro-1H-pyrazolo[3,4-b]quino-
lines (8) were prepared following literature procedures18
as shown in Scheme 2. For example, reaction of 2-iodo-
benzoic acid with 1-methyl-1H-pyrazol-5-amine pro-
duced 2-(1-methyl-1H-pyrazol-5-ylamino)benzoic acid
(7c), which was cyclized by treatment with POCl3 to
produce 4-chloro-1-methyl-1H-pyrazolo[3,4-b]quinoline
(8c). Reaction of 8c with 4-acetylbenzenamine produced
N-(4-acetylphenyl)-1H-methyl-1H-pyrazolo[3,4-b]quin-
olin-4-amine (9c).
4-(4-Acetylphenoxy)-1,3-dimethyl-1H-pyrazolo[3,4-b]-
quinoline (10) was prepared via reaction of 5 with
40-hydroxyacetophenone. Similarly, reaction of 5 with
O
CO2H
1) CCl4
CH3NHNH2
O
+
O
NH2
2) Ac2O
N
O
CH2COCH3
3
R
R
CO2H
Cl
CH3
POCl3
CH3
N
HN
CH3
N
NH2
phenol
N
H
N
N
N
N
N
N
CH3
CH3
CH3
4
5
6
Scheme 1.

224
H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
R2
CO2H
R2
K2CO3
Cu
CO2H
I
N
+
H2N
N
N
R1
N
H
N
R1
R3
R3
7
R
R
H2N
Cl
R2
N
HN
R2
POCl3
N
N
N
R1
phenol
N
N
R1
R3
R3
8
9
Scheme 2.
4-methoxybenzenethiol produced 4-(4-methoxyphenyl-
thio)-1,3-dimethyl-1H-pyrazolo[3,4-b]quinoline 11 (Scheme
3). N-(4-Methoxycarbonylphenyl)-N,1,3-trimethyl-1H-
pyrazolo[3,4-b]quinolin-4-amine (12) was prepared via
reaction of 6f with MeI in DMF with sodium hydride
(Scheme 4).
the fluorogenic caspase-3 substrate N-(Ac-DEVD)-N0-
ethoxycarbonyl-R1107 in a caspase buffer and the sam-
ple was incubated at room temperature for 3 h. Using
a fluorescent plate reader, employing excitation at
485 nm and emission at 530 nm, the fluorescence of
cleaved product ethoxycarbonyl-R110 was measured,
thereby determining the level of caspase activation.
Compounds that induce apoptosis and activate caspases
produced a fluorescent signal that is higher than the
background (signal/background ratio). Compounds
found to give a ratio of >3 are considered active and re-
tested in triplet for confirmation. Compounds confirmed
to be active are then tested at several concentrations to
give a dose–response and the caspase activation activity
EC50 is calculated.
2.2. HTS assay
N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quino-
lin-9-amine (2) was identified as an inducer of apoptosis
using our cell- and caspase 3-based assays as described
previously.9 Briefly, human breast cancer T47D cells,
in a 384-well microtiter plate containing various concen-
trations of testing compounds, were incubated for 24 h
to induce apoptosis. To the treated cells was then added
Compound 2 was found to have an EC50 of 450 nM in
T47 breast cancer cell. It also demonstrated potent
activity on HCT116 colon cancer cell lines and
SNU398 liver cancer cell lines (Table 1). Encouraged
by these potent activities, a series of structurally related
compounds was purchased from commercial libraries
and tested, including 1,3-dimethyl-N-(4-acetylphenyl)-
1H-pyrazolo[3,4-b]quinolin-4-amine (6c) which was
found to have activity similar to that of compound 2.
Since replacement of the tricyclic cyclopenta[b]quinoline
by a pyrazolo[3,4-b]quinoline reduces Clog P by about 1
unit and introduces a basic pyrazolo ring, which could
improve solubility profile, we selected the pyrazolo[3,4-
b]quinoline system as the core scaffold to explore the
SAR including modification of various rings and the lin-
ker between top ring and the pyrazole[3,4-b]quinoline.
R
Cl
R
Q
N
CH3
N
CH3
N
110oC
+
N
N
N
CH3
CH3
QH
5
10, Q = O, R = COMe
11, Q = S, R = OMe
Scheme 3.
CO2Me
CO2Me
2.3. Structure–activity relationship (SAR) studies
MeI/NaH
DMF
The cell based caspase activation assay was used for the
testing of all the compounds related with 2 for SAR
studies. The caspase activation activity (EC50) of these
compounds in three cell lines, T47D breast cancer cells,
HCT116 colorectal cancer cells, and SNU398 liver can-
cer cells is summarized in Table 1. The SAR of substitu-
tion at the top phenyl ring was explored first. The para-
position was found to tolerate a wide variety of groups,
HN
N
N
N
N
N
6f
N
N
12
Scheme 4.

H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
Table 1. Caspase activation activity of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines
R1
225
X
N
R3
R2
N
N
R4
a
Compound
R1
X
R2
R3
R4
EC50 (lM)
T47D
HCT116
SNU398
2
0.45 0.023
1.01 0.058
0.071 0.007
0.52 0.017
0.44 0.037
>10
0.66 0.020
1.25 0.020
0.064 0.010
0.66 0.087
0.39 0.083
>10
0.44 0.06
0.48 0.069
6a
6b
6c
6d
6e
6f
4-MeO
4-EtOC
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
0.034 0.006
0.27 0.033
0.24 0.071
>10
4-MeOC
4-i-PrOC
4-NH2OC
4-MeO2C
3-MeO
H
H
H
H
0.54 0.017
7.26 0.47
>10
0.67 0.074
>10
>10
0.27 0.033
>10
>10
6g
6h
6i
H
4-HO2C
4-(1-HOPr)
4-MeO
H
H
0.14 0.010
>10
0.33 0.083
>10
0.053 0.012
>10
9a
9b
9c
9d
9e
9f
H
4-EtOC
H
Et
0.21 0.018
1.15 0.046
>10
0.19 0043
1.36 0.047
>10
0.15 0.019
0.73 0.073
>10
4-MeOC
4-MeOC
4-MeOC
4-MeOC
4-EtOC
H
Me
Me
Me
Me
Me
Me
Me
Me
H
i-Pr
Me
Me
Me
Me
Me
Me
6-Me
8-Me
8-NO2
H
>10
>10
>10
1.11 0.21
0.51 0.072
>10
0.94 0.37
0.48 0.081
>10
0.66 0.040
0.27 0.069
>10
9g
10
11
12
4-MeOC
4-MeO
4-MeO2C
S
NMe
H
5.47 0.19
5.21 0.63
6.67 0.18
6.08 0.91
4.99 0.073
1.33 0.015
H
a Data are mean of three or more experiments and are reported as means standard error of the mean (SEM).
such as acetyl, propionyl, methoxy carbonyl, and meth-
oxy. Compound 6b was found to be the most potent
compound in this series, which is about 7-fold more po-
tent than the original hit 2 in T47D cells. It is also inter-
esting to note that compound 6i with a 1-hydroxypropyl
at the para-position also showed potent activity. Intro-
duction of polar groups at the para-position of the phe-
nyl ring results in completely inactive compounds (6e
and 6h), suggesting a hydrophobic binding pocket at this
position. Compound 6g was about 7-fold less active
than 6a, suggesting substitution at the para-position is
preferred over meta-position.
than 6c. The 8-nitro analog 9g was about 7-fold less ac-
tive than 6b. These data indicate that substitution at the
8-position is relatively well tolerated. In comparison, the
6-Me analog 9e was not active up to 10 lM, indicating
that substitution at 6-position is not tolerated.
We also explored the replacement of the NH linker be-
tween the pyrazolo[3,4-b]quinoline scaffold and top phe-
nyl ring by other groups. Compound 10 with an O linker
was found to be inactive up to 10 lM, which is about 20-
fold less active than the corresponding NH analog 6c.
Compound 11, with a S linker, was 5-fold less active
than the corresponding NH analog 6a. The N-Me com-
pound 12 was 10-fold less active than the corresponding
NH analog 6f. Overall these data indicate that an NH
linker is highly preferred.
We then explored substitution at the 1- and 3-positions
of the pyrazolo ring. Compound 9a was inactive up to
10 lM, which was at least 10-fold less active than 6a,
indicating that an alkyl group at the 1-position of pyra-
zole ring is critical for activity. Compound 9b was 3-fold
less active than 6b, indicating that a small group such as
Me is preferred at the 1-position. Compound 9c was 2-
fold less active than the 3-Me analog 6c, and compound
9d with a 3-isopropyl group is inactive up to 10 lM,
indicating that a small substituent at the 3-position of
pyrazole is important for activity and a large group at
the 3-position is not tolerated.
The activities of these N-phenyl-1H-pyrazolo[3,4-b]-
quinolin-4-amines toward the human colorectal cancer
cells HCT116 and liver cancer cells SNU398 were
roughly parallel to their activity toward T47D cells.
SNU398 cells were slightly more sensitive than T47D
cells to these compounds as indicated by the about 2-
fold difference in EC50 values. HCT116 cells were about
as sensitive as T47D cells with similar EC50 values for
most of these compounds.
Substitutions at the 6- and 8-positions of the pyrazol-
o[3,4-b]quinoline also were explored. Compound 9f with
a Me group at the 8-position was about 2-fold less active
Selected compounds were also tested by the traditional
inhibition of cell proliferation (GI50) assay to confirm

226
H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
Table 2. Inhibition of cell proliferation activity of N-phenyl-1H-
pyrazolo[3,4-b]quinolin-4-amines
a
Compound
GI50 (lM)
T47D
HCT116
SNU398
2
0.56 0.042
0.026 0.004
>10
0.50 0.022
0.042 0.004
>10
0.17 0.001
0.016 0.001
>10
6b
9a
6i
0.59 0.122
0.93 0.033
0.14 0.013
a Data are mean of three or more experiments and are reported as
means standard error of the mean (SEM).
that the active compounds can inhibit tumor cell
growth. The growth inhibition assays in T47D,
HCT116, and SNU398 cells are run in a 96-well micro-
titer plate as described previously.9 The GI50 values were
summarized in Table 2. Compound 6b was highly active
with GI50 values of 16–42 nM in the three cell lines
tested. Compound 9a, which was inactive in the caspase
activation assay, also was inactive in the growth inhibi-
tion assay, confirming a good correlation between our
caspase activation assay and the traditional growth inhi-
bition assay.9
2.4. Additional characterization of compound 6b
The apoptosis-inducing activities of compound 6b were
further characterized by flow cytometry. T47D cells
were treated with 50 nM of compound 6b for 24 or
48 h, then stained with propidium iodide and analyzed
by flow cytometry. As shown in Figure 1a, control cells
have a normal cell cycle profile with most cells in the G1
phase. Cells treated with compound 6b for 24 h show a
shift of the population into G2/M phase (Fig. 1b), fol-
lowed by induction of apoptosis after 48-h treatment
(Fig. 1c). Similar results were found in several other cell
lines, including MDA MB435 (breast cancer), MX-1
(breast cancer), Jurkat (T-cell leukemia), and SNU398
(liver cancer) (data not shown).
Entry
Sub
G1
S
G2M
1a
1b
1c
0.9
4.3
67.8
43.5
11.0
13.6
7.6
17.3
44.0
57.0
12.0
18.8
Figure 1. Drug-induced apoptosis in T47D cells as measured by
flow cytometric analysis. The x-axis is the fluorescence intensity
and the y-axis is the number of cells with that fluorescence
intensity. (a) Control cells showing most of the cells in G1 phase
of the cell cycle. (b) Cells treated with 50 nM of compound 6b for
24 h showing most of the cells arrested in G2/M phase and
Since 6b arrested cells in G2/M followed by induction of
apoptosis, which is similar to many tubulin interacting
agents such as vinblastine and colchicine, we suspected
that 6b might interact with tubulin. In addition, a struc-
turally related N-(4-acetylphenyl)furo[2,3-b]quinoline-4-
amine (13, CIL-102, Chart 2) has been reported to bind
to tubulin and disrupt microtubule organization.19
Interestingly, when 6b was tested in a tubulin polymeri-
zation assay,16 no effects on tubulin polymerization were
observed up to 50 lM. Therefore, the molecular target
of 6b and related analogs remains to be identified.
reduction of cells in G1 and
S phases. (c) Cells treated with
50 nM of compound 6b for 48 h showing a progression from G2/
M to cells with sub-diploid DNA content, which are apoptotic
cells with fragmented nuclei.
O
3. Conclusion
HN
In conclusion, N-(3-acetylphenyl)-2,3-dihydro-1H-
cyclopenta[b]quinolin-9-amine (2) was identified as a po-
tent apoptosis inducer through our cell and caspase
based HTS assay. SAR study of 2 led to the identifica-
tion of a series of N-phenyl-1H-pyrazolo[3,4-b]quino-
lin-4-amines as potent apoptosis inducers. The top
N-phenyl group was found to tolerate various substitu-
O
N
13
Chart 2.

H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
227
tions, especially at the para-position. The NH linker be-
tween the top phenyl ring and pyrazolo[3,4-b]quinoline
was found to be important for activity, and its replace-
ment by NMe, O or S all led to decreased activity. A
Me group at the 1-position and a Me group at the 3-po-
sition were found to be important for activity. Substitu-
tion at the 8-position was found to be tolerated while
substitution at the 6-position led to inactive compound.
Through SAR study, compound 6b was identified as a
potent apoptosis inducer with an EC50 of 71 nM in
T47D cells, which was about 7-fold more potent than
hit 2. Compound 6b also is a potent inhibitor of cell pro-
liferation with a GI50 of 26 nM in T47D cells, which was
>10-fold more potent than 2. The ability of compound
6b to induce apoptosis was confirmed in a flow cytome-
try assay in T47D cells, which showed that treatment of
cells with 6b resulted in G2/M arrest, followed by apop-
tosis. However, unlike some of the classical G2/M arrest-
ing anticancer agents such as vinblastine which inhibit
tubulin polymerization, compound 6b was found to
have no effect in the tubulin polymerization assay, sug-
gesting that 6b induces apoptosis through a different
mechanism. Additional studies to identify the mecha-
nism of action of these compounds are in progress.
solids which was used for the next reaction without fur-
ther purification.
4.1.2. 2-(1,3-Dimethyl-1H-pyrazol-5-ylamino)benzoic acid
(4). To a solution of methylhydrazine (1.38 g, 30 mmol)
in water (6 mL) was added 3 (5.07 g, 25 mmol) with stir-
ring, then was added saturated aqueous sodium carbon-
ate (25 mL). The solution was stirred at room
temperature for 6 h. It was neutralized with 2 N HCl to
pH 5. The precipitates were filtered, washed with water,
and dried to give 4.7 g (79%) of 4 as solids which was used
for the next reaction without further purification.
4.1.3. 4-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-b]quinoline
(5). A mixture of 4 (2.37 g, 10 mmol) and POCl3 (6 ml)
was stirred at room temperature for 30 min and then
was heated to 100 ꢁC and stirred for 2 h. The solution
was cooled, poured into 100 mL of ice water, and ad-
justed to pH 4–5 with 2 N aqueous sodium hydroxide.
The precipitates were filtered, washed with water, dried,
and purified by flash chromatography (Hexane/ EtOAc
3:1) to give 1.8 g (78%) of 5 as solids. 1H NMR (CDCl3):
8.37 (d, J = 9.3 Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H), 7.77 (t,
J = 7.5 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 4.15 (s, 3H),
2.87 (s, 3H).
4. Experimental
4.1.4. 1,3-Dimethyl-N-(4-(methoxycarbonyl)phenyl)-1H-
pyrazolo[3,4-b]quinolin-4-amine (6f). A mixture of 5
(0.23 g, 1.0 mmol), methyl 4-aminobenzonate (0.30 g,
2.0 mmol), and phenol (1.5 g, 16 mmol) was stirred at
100 ꢁC for 4 h. To the mixture was added 2 N sodium
carbonate and the mixture was extracted with ethyl ace-
tate (3· 30 mL). The combined organic extracts were
washed with saline, dried, and concentrated to dryness,
and the residue was purified by column chromatography
(Hexane/EtOAc 3:1) to give 0.24 g (68%) of 6f as solids.
1H NMR (CDCl3): 8.08 (d, J = 8.4 Hz, 1H), 7.97 (d,
J = 8.7 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.73 (t,
J = 7.2 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 6.86 (d,
J = 8.7 Hz, 2H), 6.70 (bs, 1H), 4.16 (s, 3H), 3.88 (s,
3H), 2.46 (s, 3H). Anal. Calcd for C20H18N4O2Æ0.5
H2O: C, 69.35; H, 5.24; N, 16.17. Found: C, 69.46; H,
5.64; N, 16.30.
4.1. General methods and materials
Commercial-grade reagents and solvents were obtained
from Acros, Aldrich, or Lancaster and were used
without further purification except as indicated. All
reaction were stirred magnetically; moisture-sensitive
reactions were performed under argon in flame-dried
glassware. Thin-layer chromatography (TLC), usually
using ethyl acetate/hexane as the solvent system, was
used to monitor reactions. Solvents were removed by
rotary evaporation under reduced pressure; where
appropriate, the compounds were further dried using
1
a vacuum pump. The H NMR spectra were recorded
at 300 MHz. All samples were prepared as dilute solu-
tions in deuteriochloroform (CDCl3) with v/v 0.05%
tetramethylsilane (TMS). Chemical shifts are reported
in parts per million (ppm) downfield from TMS
(0.00 ppm) and J coupling constants are reported in
hertz. Elemental analyses were performed by Numega
Resonance Labs, Inc. (San Diego, CA). Human breast
cancer cells T47D and other cancer cell lines were ob-
tained from American Type Culture Collection
(Manassas, VA). Compounds 2, 6a, 6c, and 9a were
purchased from a commercial library (Aldrich Rare
chemicals) and the structures were confirmed by 1H
NMR and MS.
The following compounds were prepared from the cor-
responding substituted aniline and 5 by a procedure sim-
ilar to that described for the preparation of 6f.
4.1.5. 1,3-Dimethyl-N-(4-propionylphenyl)-1H-pyrazolo-
[3,4-b]quinolin-4-amine (6b). Yield: 35%, white solids.
1H NMR (CDCl3): 8.10 (d, J = 8.7 Hz, 1H), 7.92 (d,
J = 8.7 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.71 (t,
J = 7.2 Hz, 1H), 7.30 (t, J = 8.7 Hz, 1H), 6.85 (d,
J = 8.1 Hz, 2H), 6.69 (s, 1H), 4.15 (s, 3H), 2.95 (q,
J = 7.8 Hz, 2H), 2.45 (s, 3H), 1.25 (t, J = 7.8 Hz, 3H).
Anal. Calcd for C21H20N4O: C, 73.23; H: 5.85; N,
16.27. Found: C, 72.85; H, 5.84; N, 16.05.
4.1.1. 2-(2-Oxopropyl)-4H-benzo[d][1,3]oxazin-4-one (3).
To a solution of anthranilic acid (8.77 g, 64 mmol) in
CCl4 (100 mL) was added 4-methyleneoxetan-2-one
(5.38 g, 64 mmol) at 70 ꢁC, then the mixture was heated
at reflux for 20 min. To the mixture was added acetic
anhydride (6.53 g, 64 mmol) and the mixture was stirred
at 105 ꢁC for 1 h. The mixture was cooled and the prod-
uct was isolated by filtration to give 11.5 g (89%) of 3 as
4.1.6. 1,3-Dimethyl-N-(4-isobutyrylphenyl)-1H-pyrazolo-
[3,4-b]quinolin-4-amine (6d). Yield: 60%, white solids. 1H
NMR (CDCl3): 8.11 (d, J = 8.7 Hz, 1H), 7.97 (d,
J = 8.7 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.72 (m,
1H), 7.35 (m, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.73 (s,

228
H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
1H), 4.16 (s, 3H), 3.45 (q, J = 6.6 Hz, 2H), 2.48 (s, 3H),
1.19 (d, J = 6.6 Hz, 6H). Anal. Calcd for C22H22N4O: C,
73.72; H, 6.19; N, 15.63. Found: C, 73.09; H, 6.55; N,
15.16.
sodium sulfate and concentrated in vacuo to give 1.6 g
(66%) of crude 7f which was used for the next reaction
without further purification.
4.1.12.
4-Chloro-1,3,8-trimethyl-1H-pyrazolo[3,4-b]
4.1.7. N-(4-Carbamoylphenyl)-1,3-dimethyl-1H-pyrazolo-
[3,4-b]quinolin-4-amine (6e). Yield: 71%, white solids. 1H
NMR (CDCl3): 8.09 (d, J = 8.7 Hz, 1H), 7.97 (d,
J = 8.7 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.33 (t,
J = 8.4 Hz, 1H), 6.90 (t, J = 8.4 Hz, 2H), 6.69 (s, 1H),
5.69 (bs, 2H), 4.16 (s, 3H), 2.48 (s, 3H). Anal. Calcd
for C19H17N5O: C, 65.32; H, 5.48; N, 20.04. Found:
C, 65.60; H, 6.04; N, 19.74.
quinoline (8f). A mixture of 7f (1.4 g, 5.7 mmol) and
POCl3 (5 ml) was refluxed for 4 h. The solution was
cooled and diluted with ethyl acetate (40 mL), quenched
with 20 mL of ice water, and neutralized with saturated
potassium carbonate. The organic layer was separated,
dried, and evaporated, and the residue was purified by
flash chromatography (Hexane/EtOAc 3:1) to give
0.51 g (37%) of 8f as solids. H NMR (CDCl3): 8.20
(d, J = 9.0 Hz, 1H), 7.61 (d, J = 6.9 Hz, 1H), 7.26 (m,
1H), 4.15 (s, 3H), 2.86 (s, 3H), 2.83 (s, 3H).
1
4.1.8. N-(3-Methoxyphenyl)-1,3-dimethyl-1H-pyrazolo-
[3,4-b]quinolin-4-amine (6g). Yield 69%, white solids.
1H NMR (CDCl3): 8.02 (d, J = 8.7 Hz, 1H), 7.95 (d,
J = 8.7 Hz, 1H), 7.70 (t, J = 8.4 Hz, 1H), 7.26 (t,
J = 8.4 Hz, 1H), 7.16 (t, J = 8.4 Hz, 1H), 6.65 (s, 1H),
6.55–6.45 (m, 3H), 4.12 (s, 3H), 3.71 (s, 3H), 2.43 (s,
3H). Anal. Calcd for C19H18N4O): C, 71.68; H, 5.70;
N, 17.60. Found: C, 70.95; H, 5.97; N, 17.42.
4.1.13. N-(4-Acetylphenyl)-1,3,8-trimethyl-1H-pyrazolo[3, 4-b]
quinolin-4-amine (9f).
0.65 mmol), 1-(4-aminophenyl)ethanone
A
mixture of 8f (0.16 g,
(0.10 g,
0.72 mmol), and phenol (0.31 g, 3.3 mmol) was stirred
at 100 ꢁC for 4 h. To the mixture was added 2 N sodium
carbonate and the mixture was extracted with ethyl ace-
tate (3· 30 mL). The combined extracts were washed
with saline, dried, and concentrated to dryness, and
the residue was purified by column chromatography
(Hexane/EtOAc 3:1) to give 0.035 g (16%) of 9f as or-
ange solids. 1H NMR (CDCl3): 7.85 (d, J = 8.7 Hz,
2H), 7.84 (m, 1H), 7.58 (d, J = 6.9 Hz, 1H), 7.23 (m,
1H), 6.81 (d, J = 8.7 Hz, 2H), 6.65 (bs, 1H), 4.17 (s,
3H), 2.86 (s, 3H), 2.53 (s, 3H), 2.46 (s, 3H). Anal. Calcd
for C21H20N4OÆ1.2 H2O): C, 72.06; H, 6.45; N, 16.01.
Found: C, 72.38; H, 5.99; N, 15.89.
4.1.9. N-(4-Carboxyphenyl)-1,3-dimethyl-1H-pyrazolo-
[3,4-b]quinolin-4-amine (6h). To a mixture of 6f (80 mg,
0.23 mmol) in methanol (15 mL) was added 2 N NaOH
(1 mL). The mixture was refluxed for 4 h, then it was
concentrated. The residue was diluted with water
(5 mL), and the solution was washed with ethyl acetate
(10 mL). The aqueous solution was acidified to pH 5,
and it was extracted by ethyl acetate (3· 10 mL). The ex-
tracts were dried, concentrated to give 45 mg (59%) of
1
6 h as solids. H NMR (CD3OD): 8.20 (d, J = 9.0 Hz,
1H), 8.08 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 9.0 Hz, 2H),
7.79–7.74 (m, 1H), 7.42– 7.36 (m, 1H), 6.93 (d,
J = 9.0 Hz, 2H), 4.08 (s, 3H), 2.15(s, 3H). Anal. Calcd
for C19H16N4O2 Æ 0.3 H2O: C, 67.56; H, 4.95; N, 15.63.
Found: C, 67.65; H, 5.41; N, 16.44.
The following compounds 9b–9e and 9g were synthe-
sized from the corresponding substituted 2-iodobenzoic
acid, substituted 5-aminopyrazole and substituted ani-
line using procedures similar to that described for the
preparation of compounds 7f, 8f, and 9f.
4.1.10. N-(4-(1-Hydroxypropyl)phenyl)-1,3-dimethyl-1H-
pyrazolo[3,4-b]quinolin-4-amine (6i). To a solution of 6b
(103 mg, 0.30 mmol) in methanol (15 mL) was added so-
dium borohydrate (38 mg, 1.0 mmol). It was stirred at
room temperature for 2 h, diluted with water (5 mL),
quenched with 2 N HCl (3 mL), and then neutralized
to pH 8 by aqueous NaHCO3. The mixture was extracted
with EtOAc (3· 10 mL) and the extracts were dried and
concentrated to give 6i (100 mg, 96%) as white solids. 1H
NMR (CDCl3): 8.04 (d, J = 8.7 Hz, 1H), 7.93 (d,
J = 8.7 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.28–7.22 (m,
3H), 6.96 (d, J = 8.4 Hz, 2H), 6.69 (s, 1H), 4.57 (m,
1H), 4.12 (s, 3H), 2.38 (s, 3H), 1.81 (m, 2H), 0.91 (t,
J = 7.2 Hz, 3H). Anal. Calcd for C21H22N4O: C, 72.81;
H, 6.40; N, 15.63. Found: C, 73.51; H, 6.75; N, 15.78.
4.1.14. 2-(1-Ethyl-3-methylpyrazol-5-ylamino)benzoic acid
(7b). Yield: 97%.
4.1.15. 4-Chloro-1-ethyl-3-methyl-1H-pyrazolo[3,4-b]
quinoline (8b). Yield: 41%, solids. H NMR (CDCl3):
1
8.36 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.76
(m, 1H), 7.50 (m, 1H), 4.60 (q, J = 6.9 Hz, 2H), 2.88
(s, 3H), 1.55 (t, J = 6.9 Hz, 3H).
4.1.16. 1-Ethyl-3-methyl-N-(4-propionylphenyl)-1H-pyraz-
olo[3,4-b]quinolin-4-amine (9b). Yield: 67%, orange sol-
ids. H NMR (CDCl3): 8.07 (d, J = 7.8 Hz, 1H), 7.96
1
(d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.72 (m,
1H), 7.33 (m, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.70 (s,
1H), 4.60 (q, J = 7.5 Hz, 1H), 2.93 (q, J = 7.5 Hz, 1H),
1.58 (t, J = 7.8 Hz, 1H), 1.21 (t, J = 7.8 Hz, 1H). Anal.
Calcd for C22H22N4O.H2O: C, 70.19; H, 6.43; N,
14.88. Found: C, 70.70; H, 6.59; N, 14.06.
4.1.11. 2-(1,3-Dimethyl-1H-pyrazol-5-ylamino)-3-methyl-
benzoic acid (7f). A mixture of 2-iodo-3-methylbenzoic
acid (2.62 g, 10 mmol), 5-amino-1,3-dimethylpyrazole
(1.11 g, 10 mmol), potassium carbonate (1.38 g,
10 mmol), and Cu powder (0.32 g, 5.0 mmol) in water
(20 mL) was refluxed for 24 h and the solid was removed
by filtration. The aqueous solution was extracted with
ethyl acetate (50 mL). The extract was dried over
4.1.17. 2-(1-Methylpyrazol-5-ylamino)benzoic acid (7c).
Yield: 83%.
4.1.18. 4-Chloro-1-methyl-1H-pyrazolo[3,4-b]quinoline (8c).
Yield: 49%, solids. 1H NMR (CDCl3): 8.35 (d,

H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
229
J = 8.7 Hz, 1H), 8.32 (s, 1H), 8.12 (d, J = 8.7 Hz, 1H),
7.80 (t, J = 8.4 Hz, 1H), 7.55 (t, J = 8.4 Hz, 1H), 4.22
(s, 3H).
4.1.29. 4-(4-Acetylphenoxy)-1,3-dimethyl-1H-pyrazolo-
[3,4-b]quinoline (10). A mixture of 5 (0.12 g, 0.50 mmol)
and 40-hydroxyacetophenone (0.68 g, 5.0 mmol) was
heated at 110 ꢁC for 3 h. It was cooled to room temper-
ature and the mixture was purified by column chroma-
tography (Hexane/EtOAc 3:1) to give 0.095 g (57%) of
10 as white solids. 1H NMR (CDCl3): 8.11 (d,
J = 8.7 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.92 (m,
2H), 7.43 (t, J = 8.7 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H),
6.93 (m, 2H), 4.17 (s, 3H), 2.55 (s, 3H), 2.39 (s, 3H).
Anal. Calcd for C20H17N3O2: C, 71.33; H, 5.27; N,
12.48. Found: C, 71.61; H, 5.58; N, 12.70.
4.1.19. 1-Methyl-N-(4-acetylphenyl)-1H-pyrazolo[3,4-b]-
quinolin-4-amine (9c). Yield: 41%, white solids. 1H NMR
(CDCl3): 8.90 (d, J = 9.3 Hz, 2H), 8.03 (d, J = 8.4 Hz,
2H), 7.75 (t, J = 7.2 Hz, 1H), 7.53 (s, 1H), 7.40–7.30
(m, 3H), 4.17 (s, 3H), 2.63 (s, 3H). Anal. Calcd for
C19H16N4O: C, 72.14; H, 5.10; N, 17.71. Found: C,
71.75; H, 5.26; N, 17.47.
4.1.20. 2-(1-Methyl-3-isopropylpyrazol-5-ylamino)benzoic
acid (7d). Yield: 97%.
4.1.30. 4-(4-Methoxyphenylthio)-1,3-dimethyl-1H-pyraz-
A mixture of 5 (0.12 g,
olo[3,4-b]quinoline (11).
4.1.21. 4-Chloro-1-methyl-3-isopropyl-1H-pyrazolo[3,4
-b] quinoline (8d). Yield: 56%.
0.50 mmol) and 4-methoxybenzenethiol (0.42 g, 1.5
mmol) was heated at 110 ꢁC for 3 h. It was cooled to
room temperature and the mixture was purified by col-
umn chromatography (Hexane/EtOAc 3:1) to give
4.1.22. N-(4-Acetylphenyl)-1-methyl-3-isopropyl-1H-pyr-
azolo[3,4-b]quinolin-4-amine (9d). Yield: 32%, orange
solids. 1H NMR (CDCl3): 8.10 (d, J = 8.7 Hz, 1H),
7.92 (d, J = 8.7 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.71
(t, J = 7.2 Hz, 1H), 7.30 (t, J = 8.7 Hz, 1H), 6.80 (d,
J = 8.1 Hz, 2H), 6.69 (s, 1H), 4.18 (s, 3H), 3.30 (m,
1H), 2.55 (s, 3H), 1.58 (s, 3H), 1.35 (d, J = 7.2 Hz,
6H). Anal. Calcd for C22H22N4O: C, 73.72; H, 6.19;
N, 15.63. Found: C, 73.71; H, 5.97; N, 15.58.
1
0.068 g (41%) of 11 as solids. H NMR (CDCl3): 8.60
(d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.72 (t,
J = 8.1 Hz, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.04 (d,
J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.17 (s, 3H),
3.71 (s, 3H), 2.81 (s, 3H). Anal. Calcd for C19H17N3OS:
C, 68.03; H, 5.11; N, 12.53. Found: C, 68.30; H, 5.44; N,
12.56.
4.1.31. N-1,3-Trimethyl-N-(4-(methoxycarbonyl)phenyl)-
1H-pyrazolo[3,4-b]quinolin-4-amine (12). To a solution
of 6f (40 mg, 0.12 mmol) in DMF (2 mL) kept at 0 ꢁC
was added sodium hydride (4.0 mg, 0.17 mmol), fol-
lowed by dropwise addition of methyl iodide (98 mg,
0.69 mmol). It was stirred at room temperature for
0.5 h and was diluted with ethyl ester (30 mL). The solu-
tion was washed with water (3· 10 mL), dried, and con-
centrated to dryness, and the residue was purified by
column chromatography (Hexane/EtOAc 3:1) to give
4.1.23. 2-(1,3-Dimethyl-1H-pyrazol-5-ylamino)-5-methyl-
benzoic acid (7e). Yield: 60%.
4.1.24. 4-Chloro-1,3,6-trimethyl-1H-pyrazolo[3,4-b]quino
line (8e). Yield: 35%, solids. H NMR (CDCl3): 8.10 (s,
1
1H), 7.96 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H),
4.13 (s, 3H), 2.86 (s, 3H), 2.59 (s, 3H).
4.1.25. N-(4-Acetylphenyl)-1,3,6-trimethyl-1H-pyrazolo
[3,4-b]quinolin-4-amine (9e). Yield: 53%, white solids.
1H NMR (CDCl3): 8.02 (d, J = 8.4 Hz, 1H), 7.87 (d,
J = 8.7 Hz, 2H), 7.73 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H),
6.84 (d, J = 8.7 Hz, 2H), 6.62 (s, 1H), 4.15 (s, 3H),
2.54 (s, 3H), 2.48 (s, 3H), 2.44 (s, 3H). Anal. Calcd for
C21H20N4O.1.3H2O: C, 71.69; H, 6.47; N, 15.92. Found:
C, 71.81; H, 6.04; N, 15.64.
1
33 mg (80%) of 12 as solids. H NMR (CDCl3): 8.18
(d, J = 1.8 Hz, 1H), 8.05 (bs, 1H), 7.90 (d, J = 1.8 Hz,
1H), 7.73 (t, J = 8.1 Hz, 1H), 7.70 (bs, 1H), 7.38 (t,
J = 7.2 Hz, 1H), 7.05 (bs, 1H), 6.05 (bs, 1H), 4.20 (s,
3H), 3.85 (s, 3H), 3.55 (s, 3H), 2.31 (s, 3H). Anal. Calcd
for C21H20N4O2: C, 69.98; H, 5.59; N, 15.55. Found: C,
69.65; H, 5.56; N, 15.20.
4.1.26. 2-(1,3-Dimethyl-1H-pyrazol-5-ylamino)-3-nitroben-
zoic acid (7g). Yield: 54%.
4.2. Caspase activation assay (EC50)
T47D human breast cancer cells, HCT116 colon cancer
cells, and SNU398 liver cancer cells were grown accord-
ing to media component mixtures designated by Ameri-
can Type Culture Collection in RPMI-1640 +10% FCS
in a 5% CO2–95% humidity incubator at 37 ꢁC. Cells
were harvested using trypsin and washed at 600 g and
resuspended at 0.65 · 106 cells/mL into RPMI media
+10% FCS. An aliquot of 22.5 lL of cells was added
to a well of a 384-well microtiter plate containing
2.5 lL of 0.05–100 lM of test compound in RPMI-
1640 containing 25 mM HEPES media solution with
10% DMSO (0.005–10 lM final). An aliquot of
22.5 lL of cells was added to a well of a 384-well micro-
titer plate containing 2.5 lL of RPMI-1640 media solu-
tion with 10% DMSO and without test compound as the
control sample. The samples were then incubated at
4.1.27. 4-Chloro-1,3-dimethyl-8-nitro-1H-pyrazolo[3,4-b]
quinoline (8g). Yield: 52%, solids. H NMR (CDCl3):
8.59 (dd, J1 = 8.7 Hz, J2 = 1.2 Hz, 1H), 8.10 (dd,
J1 = 8.7 Hz, J2 = 1.2 Hz, 1H), 7.54 (m, 1H), 4.13 (s,
3H), 2.89 (s, 3H).
1
4.1.28. 1,3-Dimethyl-8-nitro-N-(4-propionylphenyl)-1H-
pyrazolo[3,4-b]quinolin-4-amine (9g). Yield: 57%, brown
solids. 1H NMR (CDCl3): 8.05 (dd, J1 = 8.7 Hz,
J2 = 1.5 Hz, 1H), 7.99 (dd, J1 = 8.7 Hz, J2 = 1.5 Hz,
1H), 7.89 (d, J = 9.0 Hz, 2H), 7.23 (m, 1H), 6.99 (s,
1H), 6.88 (d, J = 9.0 Hz, 2H), 4.08 (s, 3H), 2.93 (q,
J = 7.8 Hz, 2H), 2.48 (s, 3H), 1.25 (t, J = 7.8 Hz, 3H).
Anal. Calcd for C21H19N5O3: C, 64.77; H, 4.92; N,
17.98. Found: C, 65.26; H, 5.25; N, 17.30.

230
H.-Z. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 222–231
37ꢁC for 24 h in a 5% CO2–95% humidity incubator.
After incubation, the samples were removed from the
incubator and 25 lL of a solution containing 14 lM of
(Lmax ꢀ Lstart)]) = 0.5. The GI50 (lM) values for T47D,
HCT116, and SNU398 cells are summarized in Table 2.
N-(Ac-DEVD)-N0-ethoxycarbonyl-R110
fluorogenic
4.4. Measurement of apoptosis by flow cytometry
substrate in caspase buffer was added. The samples were
incubated at 37 ꢁC. Using a fluorescent plate reader
(Model Spectrafour Plus Tecan), an initial reading
(T = 0) was made approximately 1–2 min after addition
of the substrate solution employing excitation at 485 nm
and emission at 525 nm, to determine the background
fluorescence of the control sample. After the 3-h incuba-
tion, the samples were read for fluorescence as above
(T = 3 h).
Briefly, T47D cells were treated with 50 nM of com-
pound 6b and incubated for 24 h or 48 h at 37 ꢁC. Con-
trol cells were treated with the solvent (DMSO). After
the 24-h or 48-h incubation, cells were treated with pro-
pidium iodide and analyzed on a flow cytometer. All
flow cytometry analyses were performed on FACScali-
bur (Becton–Dickinson) using Cell Quest analysis soft-
ware. Fluorescence intensity is plotted on the x-axis
and the number of cells with that fluorescence intensity
is plotted on the y-axis. The sub-diploid amount of
DNA is indicative of apoptotic cells that have under-
gone DNA degradation or fragmentation.
4.2.1. Calculation. The relative fluorescence unit values
(RFU) were used to calculate the sample readings as fol-
lows. The activity of caspase activation was determined
by the ratio of the net RFU value for the test com-
pound to that of control samples. The EC50 (lM)
was determined by a sigmoidal dose–response calcula-
tion (XLFit3, IDBS), as the concentration of com-
pound that produces the 50% maximum response.
The caspase activation activity (EC50) in the three
cancer cell lines, T47D, HCT116, and SNU398 is sum-
marized in Table 1.
References and notes
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4.3. Cell growth inhibition assay (GI50)
Cells were grown and harvested as described above. An
aliquot of 45 lL of cells (4.4 · 104 cells/mL) was added
to a well of a 96-well microtiter plate, then 5 lL of
0.01–100 lM of test compound (0.001–10 lM final con-
centration) in RPMI-1640 media solution with 10%
DMSO was added. An aliquot of 45 lL of cells was
added to a well of a 96-well microtiter plate containing
5 lL of RPMI-1640 media solution with 10% DMSO
and without test compound as the control sample for
maximal cell proliferation (Lmax). The samples were
then incubated at 37 ꢁC for 48 h in a 5% CO2–95%
humidity incubator. After incubation, the samples were
removed from the incubator and 25 lL of CellTiter-Glo
reagent (Promega) was added. The samples were mixed
by agitation and incubated at room temperature for 10–
15 min. Plates were then read using a luminescent plate
reader (Model Spectrafluor Plus Tecan Instrument) to
give Ltest value.
Baseline for GI50 (dose for 50% inhibition of cell prolif-
eration) of initial cell numbers was determined by add-
ing an aliquot of 45 lL of cells and 5 lL of RPMI-
1640 media solution with 10% DMSO to wells of a 96-
well microtiter plate. The samples were then incubated
at 37 ꢁC for 0.5 h in a 5% CO2–95% humidity incubator.
After incubation, the samples were removed from the
incubator and 25 lL of CellTiter-Glo reagent (Promega)
was added. The samples were mixed by agitation and
incubated at room temperature for 10–15 min. Lumines-
cence was read as above to give Lstart value, defining
luminescence for initial cell number used as baseline in
GI50 determinations.
4.3.1. Calculation. GI50 (dose for 50% inhibition of cell
proliferation) is the concentration where [(Ltest ꢀ Lstart)/

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