Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure-activity relationships of the 4-aryl group

Article abstract of DOI:10.1021/jm049640t

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7- (dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G₂/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino) -4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)- 4H-chromene (4e), with an EC₅₀ of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI₅₀ values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI₅₀ value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MESSA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

Full text of DOI:10.1021/jm049640t

J. Med. Chem. 2004, 47, 6299-6310
6299
Discovery of 4-Aryl-4H-chromenes as a New Series of Apoptosis Inducers Using
a Cell- and Caspase-based High-Throughput Screening Assay. 1.
Structure-Activity Relationships of the 4-Aryl Group
William Kemnitzer,† John Drewe,† Songchun Jiang,† Hong Zhang,† Yan Wang,† Jianghong Zhao,† Shaojuan Jia,†
John Herich,† Denis Labreque,‡ Richard Storer,‡ Karen Meerovitch,‡ David Bouffard,‡ Rabindra Rej,‡
Real Denis,‡ Charles Blais,‡ Serge Lamothe,‡ Giorgio Attardo,‡ Henriette Gourdeau,‡ Ben Tseng,†
Shailaja Kasibhatla,† and Sui Xiong Cai*,†
Maxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, California 92121, and Shire Biochem Inc.,
275 Armand-Frappier Blvd., Laval, Quebec H7V 4A7, Canada
Received May 14, 2004
By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-
4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent
apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage,
as well as to arrest cells at the G2/M stage and to induce apoptosis as determined by the flow
cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-
activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was
obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxy-
phenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-
4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC50 of 19 and 11 nM in the caspase activation
assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-
4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with
GI50 values in the low nanomolar range for compound 1c. Significantly, compound 1c was found
to have a GI50 value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-
SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin
polymerization and to effectively inhibit the binding of colchicine to tubulin. These results
confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent
apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed
into future anticancer agents.
Introduction
Since many cancer cells exhibit abnormal inhibition
of apoptosis,3 we are interested in the discovery and
development of inducers of apoptosis as potential anti-
cancer agents. Toward this goal, we have developed a
cell-based high-throughput screening (HTS) system for
inducers of apoptosis8 using our novel fluorescent
caspase substrates.9,10 We have reported recently the
discovery and SAR studies of substituted N-phenylni-
cotinamides, exemplified by 6-methyl-N-(4-ethoxy-2-
nitrophenyl)pyridine-3-carboxamide (A),11 gambogic acid
(B),12 and substituted indole-2-carboxylic acid ben-
zylidenehydrazides, exemplified by 5-methyl-3-phenyl-
indole-2-carboxylic acid (4-methylbenzylidene)hydrazide
(C),13 as potent apoptosis inducers (Chart 1). Herein we
report the discovery and characterization of 2-amino-
3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylene-
dioxyphenyl)-4 H-chromene (1a) as a potent inducer of
apoptosis using our cell- and caspase-based apoptosis
HTS assay, as well as optimization of the screening hit
1a via SAR studies of the 4-aryl group.
Apoptosis, or programmed cell death, is the normal
process of cellular suicide that proceeds with charac-
teristic biochemical and cytological features, including
nuclear condensation and fragmentation. Apoptosis
enables organisms to control their cell numbers and to
eliminate unneeded cells that may threaten their sur-
vival.1 The correct balance between apoptosis and
inhibition of apoptosis is important in preserving tissue
homeostasis and organ morphogenesis.2 However, aber-
rations of this process underlie some pathological condi-
tions, with abnormal inhibition of apoptosis as one of
the hallmarks of cancer,3 while excessive apoptosis is
implicated in neurodegenerative disorders such as
Alzheimer’s disease.4 Apoptosis can be initiated by many
agents, including TNF-R and FAS ligand, as well as by
chemotherapy and radiation.5 The mechanism of apop-
tosis involves a cascade of initiator and effector caspases
that are activated sequentially. Caspases are a family
of cysteine proteases that require aspartic acid residues
at the P1 position of substrates for cleavage.6 Within the
caspase family, caspase-3, -6, and -7 have been identified
as key effector caspases that cleave multiple protein
substrates in cells, leading irreversibly to cell death.7
Results and Discussion
Chemistry. 2-Amino-3-cyano-7-(dimethylamino)-4-
(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene 1a
was obtained originally from a commercial compound
library. It was resynthesized by a one-pot reaction of
5-methoxypiperonal, 3-dimethylaminophenol, and ma-
lononitrile in good yield as a brown solid. Compounds
* Corresponding author. Tel: 858-202-4006. Fax: 858-202-4000.
E-mail: scai@maxim.com.
† Maxim Pharmaceuticals.
‡ Shire Biochem Inc.
10.1021/jm049640t CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/09/2004

6300 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Kemnitzer et al.
Chart 1
Scheme 4a
a Conditions: (a) malononitrile, EtOH, piperdine, rt, 12 h; (b)
3-(dimethylamino)phenol, EtOH, piperdine, reflux, 12 h.
Scheme 5a
Scheme 1a
a Conditions: (a) EtOH, piperidine, rt.
Scheme 2a
a Conditions: (a) molecular sieves, CH2Cl2, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone, rt, 1 h; (b) (1) Cu(I)Br-SMe2, THF, -78
°C, MeLi, 1 h; (2) 9, THF, -78 °C, 30 min.
a Conditions: (a) LAH, THF, -78 °C, 1 h, then rt, 20 h; (b)
MnO2, CH2Cl2, reflux, 18 h.
Scheme 3a
reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.
Compound 9 was then methylated using copper(I)
bromide-dimethyl sulfide and methyllithium to afford
compound 10.
HTS Assays. 2-Amino-3-cyano-7-(dimethylamino)-4-
(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene 1a
was identified as an inducer of apoptosis from a com-
mercial compound library using our cell-based apoptosis
induction HTS assay in HL60 B-cell promyelotic leu-
kemia cancer cells as described.8 Briefly, human HL60
cells, in a 96-well microtiter plate containing 10 µM of
test compound, were incubated for 3 h for the induction
of apoptosis. Caspase-3 fluorogenic substrate N-(Ac-
DEVD)-N′-ethoxycarbonyl-R11010 was then added to
cells, and the samples were mixed by agitation and
incubated at room temperature for 3 h. Using a fluo-
rescent plate reader, employing excitation at 485 nm
and emission at 525 nm, the fluorescence was measured
and the amount of caspase activation was determined.
Compounds that induce apoptosis and activate the
caspases yield a fluorescent signal higher than the
background (signal/background ratio). Compounds found
to give a ratio of >3 are considered active and retested
in triplicate for confirmation in both HL60 and T47D,
a solid human breast cancer tumor cell line. Compounds
confirmed to be active in both cell lines are then tested
at several concentrations to provide a dose response for
a Conditions: (a) (1) oxalyl chloride MnO2, CH2Cl2, 0 °C, DMF;
(2) CH3CN, THF, -78 °C, 7a, pyridine; (3) warm to -40 °C then
cooled to -78 °C; (4) LiAl(OtBu)3H, CuI.
1b-f, 1h-j, 2a-e, 3a-n, 4a-c, 4f, and 5a-c were
prepared in a manner similar to 1a from the corre-
sponding commercially available aldehydes by reaction
with 3-(dimethylamino)phenol and malononitrile (Scheme
1). For compounds 4d and 4e, the corresponding alde-
hydes (6 and 7) were synthesized starting from methyl
5-methyl-3-pyridinecarboxylate (Scheme 2) and 5-bro-
monicotinic acid (Scheme 3). Compound 1g was pre-
pared in two steps by the initial formation of 2-(3-bromo-
4-hydroxy-5-methoxybenzylidene)malononitrile (8) from
reaction of 3-bromo-4-hydroxy-5-methoxybenzaldehyde
and malononitrile (Scheme 4), followed by reaction of 8
with 3-(dimethylamino)phenol. Compound 10 was syn-
thesized in two steps starting from 1c (Scheme 5). First,
1c was oxidized to 4-(3-bromo-4,5-dimethoxyphenyl)-3-
cyano-7-(dimethylamino)-2-imino-2H-chromene (9) by

4-Aryl-4H-chromenes as Inducers of Apoptosis
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6301
Figure 2. Western blots of the time-course of drug-induced
poly(ADP)ribose polymerase (PARP) cleavage in Jurkat cells:
(a) DMSO-treated control cells; (b) cells treated with 1 µM of
staurosporine for 30 h; (c) cells treated with 2.5 µM of 1a for
5 h; (d) cells treated with 2.5 µM of 1a for 15 h; (e) cells treated
with 2.5 µM of 1a for 30 h.
Jurkat cells with the compound at a concentration of
2.5 µM for 5, 15, and 30 h. The cells were then lysed
and the proteins transferred to PVDF membrane. The
membranes were probed with a polyclonal antibody to
PARP, followed by goat-anti-rabbit HRP second anti-
body. Figure 2c shows that PARP cleavage can be
observed after cells were treated with 1a for 5 h. At 30
h, almost all the PARP have been cleaved (Figure 2e),
which is similar to what is observed with cells treated
with 1 µM of staurosporine (Figure 2b), a potent
apoptosis inducer for the same period of time.
The apoptosis-inducing activity of compound 1a was
also characterized by cell cycle analysis. T47D cells were
treated with 0.1 µM of compound 1a for 24 or 48 h,
stained with propidium iodide, and analyzed by flow
cytometry. An increase in the G2/M DNA content in cells
treated with compound 1a was observed, as shown in
Figure 3B,C. The subdiploid DNA content (apoptotic
sub-G1 area) of cells increased from 0.9% in the control
cells (Figure 3A) to 12% upon compound treatment for
48 h (Figure 3C), indicating the presence of apoptotic
cells that have undergone DNA degradation and nuclear
fragmentation. A similar level of apoptotic cells (10%)
is seen when T47D cells were treated with 0.1 µM of
paclitaxel.12 These results showed that treatment of
T47D cells by compound 1a for 48 h results in cell cycle
arrest in the G2/M phase, as well as induction of
apoptosis.
Structure-Activity Relationship (SAR) Studies.
The cell-based caspase HTS assay was used to test
analogues of compound 1a for SAR studies. A panel of
three different human cancer cell lines was used for
these experiments. Briefly, human breast cancer cell
line T47D, human nonsmall cell lung cancer cell line
H1299, and human colorectal cancer cell line DLD-1
were plated in 384-well microtiter plates containing
various concentration of test compound and incubated
at 37 °C for 24 h. After incubation, the samples were
treated with the N-(Ac-DEVD)-N-ethoxycarbonyl-R110
fluorogenic substrate. The caspase activation activity
(EC50) of compound 1a and its analogues in the three
cancer cell lines is summarized in Table 1.
Figure 1. Fluorescent micrographs of Jurkat cells treated
with drug and stained with a fluorescent DNA probe, Syto 16.
Figure 1A depicts control cells. Figure 1B depicts cells treated
with 0.1 µM of 1a for 24 h, showing shrunken and fragmented
nuclei.
the calculation of the caspase activation activity (EC50).
Compound 1a was found to induce apoptosis and
activate caspase in T47D cells with an EC50 value of
0.073 µM and a ratio of around 6 over untreated cells,
similar to that of substituted N-phenylnicotinamides11
and substituted indole-2-carboxylic acid benzylidene-
hydrazides.13
Characterization of Compound 1a. The ability of
compound 1a to induce apoptosis was confirmed in a
nuclear fragmentation assay, which is one of the hall-
marks of caspase-mediated apoptosis. Jurkat cells were
treated with 0.1 µM of compound 1a for 24 h followed
by staining with Syto 16 (Molecular Probes Inc, Eugene,
OR), a DNA stain that allows the visualization of
nuclear morphology. The compound-treated cells were
characterized by shrunken and fragmented nuclei with
condensed chromatin (Figure 1B). In contrast, the nuclei
of Jurkat cells treated with vehicle control (DMSO)
appeared to be normal with dispersed chromatin mod-
erately stained with Syto 16 (Figure 1A).
Table 1 shows that compound 1a has an EC50 for
caspase activation of 0.073, 0.093, and 0.083 µM in
T47D, H1299, and DLD-1 cells, respectively. By main-
taining the 2-amino, 3-cyano, and 7-dimethylamino
groups of 1a, the SAR at the 4-position was explored,
beginning with trisubstituted analogues. Replacing the
4,5-methylenedioxy ring by 4,5-dimethoxy groups gave
the trimethoxy-substituted analogue 1b, which was
slightly more potent than 1a in the T47D cells, sug-
gesting that the ring structure in the 4,5-position could
be replaced by two substituents. Replacement of the
The apoptosis-inducing activity of compound 1a was
also characterized by poly(ADP)ribose polymerase (PARP)
cleavage in Jurkat cells. PARP, an enzyme important
in genome surveillance, is one of the natural substrates
that is cleaved by caspases during apoptosis. The ability
of 1a to induce PARP cleavage was tested by treating

6302 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Kemnitzer et al.
2-methoxy group forces the phenyl ring into an unfavor-
able position. Compound 1j was >130-fold less active
than 1c, confirming that substitution in the 2-position
is not preferred.
The 3,5-dimethoxy analogue 2a is slightly more
potent than the 3,4,5-trimethoxy analogue 1b, indicat-
ing that the 4-methoxy group is not crucial for activity.
In comparison, the 3-bromo-5-methoxy analogue 2b is
slightly less potent than 1c, indicating that, when there
is a bromo group in the 3-position, the 4-methoxy group
does contribute to the potency. Compound 2b also was
slightly less potent than 2a, suggesting that for 3,5-
disubstituted analogues, an electron-donating group is
preferred over an electron-withdrawing group. This was
supported by the preparation of the 3,5-dichloro ana-
logue 2c, which was >3-fold less active than 2a. The
3,4-dimethoxy analogue 2d was >10-fold less potent
than 2a, further supporting the notion that substitution
in the 3,5-position is important for activity and substi-
tution in the 4-position contributes little to potency.
Similar to what has been observed in the trisubstituted
analogues, the 2,3-dimethoxy analogue 2e was >44-fold
less active than 2a, further confirming that substitution
in the 2-position is not preferred.
Monosubstituted analogues further highlight the
importance of substitution in the 3- and/or 5-positions
of the phenyl ring for activity. Monosubstitution at the
3-position of the phenyl ring with bromo (3a), methoxy
(3b), and cyano (3c) groups resulted in potent com-
pounds, which are only about 2-3-fold less potent than
the 3,5-dimethoxy analogue 2a and 3,4,5-trisubstituted
analogue 1c. The 3-fluoro (3d), 3-chloro (3e), and
3-methyl (3f) analogues also were found to be potent
compounds with potencies around 100 nM. The 3-hy-
droxy analogue 3g was >4-fold less potent than the
3-methoxy analogue 3b, suggesting that a hydrophobic
group is preferred in the 3-position. The 3-carboxylic
acid analogue 3h was >130-fold less potent than 3b,
indicating that a charged group is not tolerated in the
3-position. This dramatic reduction of activity of the
carboxylic acid analogue could be due to a combination
of low cell permeability and low affinity to its target.
Replacing the methoxy group in the 3-position with a
trifluoromethoxy group (3i) resulted in reduction of
potency by >6-fold compared to 3b, suggesting that
there might be a size-limited pocket in the 3-position.
The 3-benzyloxy (3j) and 3-dodecyloxy (3k) analogues
are >40- and >130-fold less potent than the 3-methoxy
analogue, further supporting the notion that there is a
size-limited pocket in the 3-position. The nonsubstituted
phenyl analogue (3l) was found to have a potency of 118
nM, which is only about 2-fold less active than the
3-methoxy analogue 3b. The 4-methoxy (3m) and 2-meth-
oxy (3n) analogues are >24- and > 4-fold less potent
than the 3-methoxy analogue 3b, and both are less
active than the nonsubstituted phenyl analogue 3l,
confirming that substitution in the 3-position is pre-
ferred and substitution in the 2- and 4-positions does
not contribute positively to the activity.
Figure 3. 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 0.1
µM of compound 1a for 24 h showing most of the cells arrested
in G2/M phase. (C) Cells treated with 0.1 µM of compound 1a
for 48 h showing a progression from G2/M to cells with
subdiploid DNA content, which are apoptotic cells with
fragmented nuclei.
methoxy group at the 3-position of 1b with a bromo
group (1c) resulted in 2-fold increase in potency, sug-
gesting that an electron-withdrawing group might be
preferred in the 3-position of these 3,4,5-trisubstituted
compounds. This was confirmed by the preparation of
the corresponding chloro (1d) and iodo (1e) analogues,
which are as active as the bromo analogue 1c. The 4,5-
methylenedioxy analogue 1f was 6-fold less active than
1c, suggesting that 4,5-dimethoxy groups are preferred
over the 4,5-methylenedioxy ring structure with an
electron withdrawing group in the 3-position. Replacing
the 4-methoxy group in 1c by a 4-hydroxy group (1g)
resulted in a 2-fold reduction of potency, suggesting that
the hydrophobic methoxy group is preferred over the
hydrophilic hydroxy group in the 4-position. Compounds
1h and 1i, with a methoxy group at the 2-position, are
>40-fold less active than the 3,4,5-trimethoxy analogue
1b, suggesting that there might be a space-limited
pocket around the 2-position, or due to steric effect, the
On the basis of the above SAR, we sought to replace
the phenyl group by a pyridyl group, which might
improve the aqueous solubility of the compound. The
3-pyridyl analogue 4a was almost 2-fold more potent
than the phenyl analogue 3l, suggesting that the

4-Aryl-4H-chromenes as Inducers of Apoptosis
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6303
Table 1. SAR of 4-Aryl-2-amino-3-cyano-7-(dimethylamino)-4H-chromene and Analogues in the Caspase Activation Assay
a Data are the mean of three or more experiments and are reported as mean ( standard error of the mean (SEM). NA ) not applicable.
nitrogen in the 3-pyridyl group contributes to activity.
In contract, the 2-pyridyl (4b) and 4-pyridyl (4c) ana-
logues were >4- and >3-fold less active than 3l, indicat-
ing that the nitrogen in either the 2- or the 4-position
contributes negatively to activity. Monosubstitution of
the 3-pyridyl analogue at the 5-position with a bromo
(4d) and a methyl (4e) group increased potency by >2-
and >6-fold, respectively, confirming the importance of
substitution in the 3-(5-) positions in both the phenyl
and the pyridyl ring system. The corresponding 3-meth-
yl-substituted 2-pyridyl analogue 4f was >20-fold less
potent than 4e, confirming the importance of the
position of the nitrogen in the pyridyl group. Therefore,
in the pyridyl series it is important to have the nitrogen
in the 3-position with a substitution at the 5-position.
This SAR corresponds to the 3,5-disubstitutions for the
phenyl series and suggests that the 3-pyridyl group is
a good replacement for the phenyl group.
The nonaromatic cyclohexyl analogue (5a) was >2.5-
fold less active than 3l, suggesting that a planar
structure of either a phenyl or pyridyl group is preferred
in the 4-position of the chromene structure. Extending
the nonsubstituted phenyl ring from the 4-position via
an ethyl linker (5b) resulted in >3.5-fold reduction in
potency relative to 3l, suggesting that the binding
pocket in the 4-position of chromene is size limited. This
is in agreement with the substituted phenyl series in
which a large group substituted in the 3-postion of the
phenyl group is not tolerated. Introduction of a bicyclic
quinolinyl group (5c) in the 4-position of chromene also

6304 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Kemnitzer et al.
Table 2. Comparison of Caspase Activation Activity and Inhibition of Cell Proliferation Activity of
4-Aryl-2-amino-3-cyano-7-(dimethylamino)-4H-chromenes
T47D
DLD-1
EC50a (µM)
GI50b (µM)
GI50/EC50
EC50a (µM)
GI50b (µM)
GI50/EC50
1c
2a
1b
3l
0.019 ( 0.004
0.023 ( 0.001
0.045 ( 0.003
0.118 ( 0.005
0.245 ( 0.045
0.290 ( 0.016
2.52 ( 0.12
0.092 ( 0.013
0.085 ( 0.010
0.11 ( 0.024
0.455 ( 0.027
0.605 ( 0.035
0.462 ( 0.092
8.31( 0.72
4.8
3.7
2.4
3.9
2.5
1.6
3.3
1.8
0.066 ( 0.014
0.077 ( 0.041
0.102 ( 0.014
0.169 ( 0.004
0.293 ( 0.034
0.343 ( 0.008
4.79 ( 0.07
0.070 ( 0.013
0.078 ( 0.013
0.29 ( 0.053
0.746 ( 0.074
0.735 ( 0.074
0.722 ( 0.077
1.1
1.0
2.8
4.4
1f
5a
3j
2.5
2.1
>10
9.89 ( 0.56
>2.1
2.4
1j
2.64 ( 0.22
4.68 ( 0.72
4.12 ( 0.09
a From Table 1. b Data are the mean of three experiments and are reported as mean ( standard error of the mean (SEM).
resulted in large drop of potency, confirming that there
is a size-limited pocket in the 4-position of chromene.
The 2-thiophenyl analogue 5d is about 2-fold less active
than the phenyl analogue 3l, and the 4-bromo-2-
thiophenyl analogue 5e is slightly more than 2-fold less
active than the 5-bromophenyl analogue 3a, suggesting
that a five-member ring heterocycle could partially
replace the six-member ring phenyl group.
The introduction of an additional methyl group in the
4-position of chromene of 1c resulted in the 4,4-disub-
stituted analogue 10, which is 178-fold less active than
its hydrogen-substituted analogue 1c. Structural model-
ing, comparing compounds 1c and 10 using ChemBats3D
(CambridgeSoft, Cambridge, MA), did not indicate that
the extra methyl group in the 4-position of 10 altered
the 3D orientation of the phenyl group relative to the
chromene ring. An alternate possibility is that the loss
of potency is simply an issue of pocket size and that the
methyl group is not tolerated within the binding site
because of unfavorable steric interaction, which is
consistent with the SAR found for the 2-position of the
phenol ring.
The activity of compounds 1c and 4e, two of the most
potent compounds in this series, is comparable to the
well-known antimitotic agent colchicine (Chart 1, com-
pound D). Compound 1c is about as potent as colchicine,
while compound 4e is slightly more potent than colchi-
cine in the caspase activation assay. Compounds 1c and
4e also are slightly more potent than vinblastin and
paclitaxel, two widely used anticancer drugs, in the
caspase activation assay. Interestingly, some structural
similarity between compound 1c and colchicine was
observed, including the preference for methoxy groups
in the phenyl ring,14 suggesting that compound 1c and
colchicine might interact with the same target.
The activities of these compounds toward the human
nonsmall cell lung cancer cell line H1299 and colorectal
cancer cell line DLD-1 was roughly parallel to their
activity toward T47D cells. H1299 cells were slightly
less sensitive (about 2-fold less sensitive as measured
by the EC50 value) to the compounds than T47D cells
in this assay. DLD-1 cells also were slightly less
sensitive (about 2-3-fold less as measured by the EC50
value) to the compounds than T47D cells.
a 96-well microtiter plate as described previously.11 The
GI50, along with the EC50 data and the ratio of GI50/
EC50, are summarized in Table 2.
Table 2 shows that compounds 1c, 2a, and 1b all are
potent inhibitors of tumor cells growth. Compound 1c
has a GI50 value of 0.092 µM and 0.070 µM in T47D
and DLD-1 cells, respectively. In general, the compound
that is more active in the apoptosis induction assay, as
measured by caspase activation, also is more potent in
the growth inhibition assay. For example, in T47 D cells,
compounds 1c is 132- and 90-fold more potent than 3j
in the caspase assay and growth inhibition assay,
respectively. In DLD-1 cells, compound 1c is 72- and
>140-fold more potent than 3j in the caspase assay and
growth inhibition assay, respectively. These data sug-
gest that generally there is a correlation between the
caspase activation activity and inhibition of cell prolif-
eration activity for these compounds. Similar to what
has been observed for the substituted N-phenylnico-
tinamides,11 these data indicate that the cell-based
caspase activation HTS assay not only is useful for the
identification of apoptosis inducers, but also can be used
for subsequent optimization and SAR studies of screen-
ing hits.
One of the serious problems of the currently used
cancer chemotherapeutic agents is multidrug resistance.
Many tumor cells overexpress the p-glycoprotein, a
plasma membrane transporter with a broad substrate
specificity that includes most of the conventional che-
motherapeutic drugs.15 This transporter efficiently re-
moves the drugs from the interior of the cell, resulting
in protection of the cancer cells from the toxic effects of
the drugs. Significantly, when compound 1c was tested
against the human uterus sarcoma MED-SA cells and
the multidrug resistant MED-SA/DX5 cells,11 it was
found that 1c is highly active with GI50 values of 2 nM
in both cells. In comparison, paclitaxel and vinblastin
are effective against MED-SA cells but at least 160- and
>20-fold less effective against MED-SA/DX5 cells, re-
spectively.11,16 This underscores a major potential ad-
vantage of this series of compoundssthey appear to be
effective against cells that overexpress the p-glyco-
proteinsand may therefore be useful in treating pa-
tients with cancers that are resistant to traditional
antitumor agents, including the taxanes.17
Selected compounds were also tested by the tradi-
tional inhibition of cell proliferation (GI50) assay to
confirm that the active compounds can inhibit tumor
cell growth, as well as to determine whether there is a
correlation between the activity from the caspase acti-
vation assay and the cell proliferation assay. The growth
inhibition assays in T47D and DLD-1 cells were run in
Since tubulin inhibitors are known to arrest cells in
the G2/M phase and some structural similarity between
compound 1c and colchicine was observed, we suspected
that this series of 4-arylchromenes might be inhibitors
of tubulin polymerization. The effect of compound 1c
on tubulin polymerization was measured using a cell-

4-Aryl-4H-chromenes as Inducers of Apoptosis
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6305
d6 sulfoxide (CD3SOCD3) with v/v 0.05% 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). Melting points were determined
on a glass capillary melting point apparatus. Human leukemia
cancer cells HL-60 and Jurkat, human breast cancer cells
T47D, human colon cancer cells DLD-1, human nonsmall cell
lung cancers H1299, human uterus sarcoma MES-SA cells, and
human uterus sarcoma doxorubicin-resistant MES-SA/DX5
cells were obtained from American Type Culture Collection
(Manassas, VA). Colchicine, vinblastin, and paclitaxel were
obtained from Sigma, and [3H]colchicine was from American
Radiolabeled Chemicals (St. Louis, MO). Tubulin was obtained
from Cytoskeleton (Boulder, CO). Compounds 1a, 5d, and 5e
were obtained from Chembridge (San Diego, CA) and their
free assay in which the amount of polymerized tubulin
in the presence and absence of drug is determined by
measuring the increase in absorbance at 340 nm.11
Compound 1a inhibits tubulin polymerization with an
IC50 value of 400 nM, a level of potency similar to
vinblastin (IC50 value of 300 nM), a well-known tubulin
inhibitor.11,16
To locate the binding site of 1c, the ability of 1c to
block the interaction of tubulin with [3H]colchicine was
measured. Compound 1c displaced [3H]colchicine with
an IC50 of 8 µM, suggesting that 1c binds at or close to
the colchicine site on â-tubulin.16 These data suggests
that compound 1c, and the 4-aryl-4H-chromene series
of compounds, most probably induce apoptosis through
the inhibition of tubulin polymerization.
1
structures were confirmed by H NMR.
5-Methyl-3-pyridinemethanol (6b). To a solution of
methyl 5-methyl-3-pyridinecarboxylate (6a, 5.02 g, 33.1 mmol)
in THF (10 mL) at -78 °C was added LiAlH4 (0.701 g, 17.5
mmol) in small portions over 5 min while the internal
temperature was maintained below -70 °C. The mixture was
stirred at -78°C for 1 h, and then the cold bath was removed.
The reaction mixture was stirred at room temperature for 6.5
h. TLC of the reaction mixture indicated the presence of
starting material, additional LiAlH4 (0.071 g, 1.7 mmol) was
added, and the mixture was stirred overnight. The reaction
mixture was quenched with water (5 mL), stirred at room
temperature for 0.5 h, and dried over MgSO4. The mixture was
filtered through a layer of Celite and the filter cake was
washed with ethyl acetate (100 mL). Evaporation of the solvent
gave compound 6b as a yellow oil (4.05 g, 99%): 1H NMR
(CDCl3) 8.40 (s, 1H), 8.37 (s, 1H), 7.31 (s, 1H), 4.71 (s, 2H),
2.35 (s, 3H).
5-Methyl-3-pyridinecarboxaldehyde (6c). A black mix-
ture of 6b (4.05 g, 32.9 mmol) and activated MnO2 (13.4 g,
131 mmol) in anhydrous dichloromethane (50 mL) was re-
fluxed for 18 h. The resulting black mixture was filtered
through a layer of Celite and the filter cake was washed with
dichloromethane (150 mL). The filtrate was concentrated in
vacuo to yield compound 6c as a yellow oil (3.23 g, 81%): 1H
NMR (CDCl3) 10.10 (s, 1H), 8.89 (s, 1H), 8.68 (s, 1H), 7.98 (s,
1H), 2.45 (s, 3H).
Conclusion
In conclusion, we have discovered a series of 4-aryl-
4H-chromenes as potent inducers of apoptosis using our
cell- and caspase-based HTS assay. The ability of
compound 1a, the screening hit, to induce apoptosis was
confirmed by nuclear fragmentation and PARP cleavage
assays in Jurkat cells, and flow cytometry assays in
T47D cells. SAR studies of the 4-position of the 4H-
chromenes showed that substituted phenyl and pyridyl
are the preferred group. These groups include 3,4,5-
trisubstituted phenyl, such as 3-bromo-4,5-dimethoxy-
phenyl (1c, MX58151),16 3-chloro-4,5-dimethoxyphenyl
(1d), and 3-iodo-4,5-dimethoxyphenyl (1e), as well as
5-substituted-3-pyridyl, such as 5-bromo-3-pyridyl (4d)
and 5-methyl-3-pyridyl (4e). Compounds 1c and 4e are
about as potent as or slightly more potent than colchi-
cine, vinblastin, and paclitaxel in the caspase activation
assay. Interestingly, introduction of an additional Me
group in the 4-position resulted in compound 10, which
is 178-fold less active than the corresponding 4-H
analogue 1c.
The 4-aryl-4H-chromenes were found to inhibit tu-
bulin polymerization, which might be the main mech-
anism of action of this series of apoptosis inducers.
Compound 1c retains activity in cells resistant toward
current antimitotic agents, taxanes and vinca alkaloids.
Therefore, they might have advantage for the treatment
of drug resistance cancers. Moreover, the 4-aryl-4H-
chromenes were found to disrupt preformed endothelial
cell capillary tubules, suggesting that they are likely to
work as tumor vasculature targeting agents,16 similar
to combretastatin A-4 phosphate (CA-4P) and ZD-
6126.18 Additional SAR studies and in vivo studies of
the 4-aryl-4H-chromenes series of apoptosis inducers are
in progress and will be reported in future publications.19
5-Bromo-3-pyridinecarboxaldehyde (7b). To a yellow
solution of oxalyl chloride (15.0 mL, 30.0 mmol, 2.0 M in
dichloromethane) and anhydrous dichloromethane (10 mL) at
0 °C was added anhydrous DMF (1.5 mL, 19 mmol). The
resulting white suspension was equilibrated to room temper-
ature and filtered and the white solid was collected. The white
solid (2.13 g, 16.6 mmol) was then added to a three-neck round-
bottom flask fitted with an internal thermometer and dissolved
in anhydrous CH3CN (25 mL) and anhydrous THF (55 mL).
The clear solution was cooled to -74 °C, and then 5-bromoni-
cotinic acid (7a, 0.672 g, 3.33 mmol) and anhydrous pyridine
(0.11 mL, 1.4 mmol) were added while the internal tempera-
ture was maintained below -70 °C. The reaction mixture was
warmed to -37 °C over 1 h and then cooled to -77 °C. The
reducing agent Li(tBuO)3AlH (6.65 mL, 6.65 mmol) was added
slowly to maintain the internal temperature below -71 °C and
was followed by the addition of copper iodide (0.050 g, 0.27
mmol). The mixture was stirred at -81 °C for 20 min and then
1 M HCl (20 mL) was added in small portions to maintain the
internal temperature below -67 °C. After the addition of 1 M
HCl, the mixture was equilibrated to room temperature,
diluted with ethyl acetate (150 mL), dried over Na2SO4, filtered
through Celite, and washed with additional ethyl acetate. The
organic filtrate was washed with water (2 × 20 mL), dried over
Na2SO4, filtered, and concentrated to yield compound 7b as a
brown solid (0.050 g, 8%): 1H NMR (CDCl3) 10.09 (s, 1H), 9.03
(s, 1H), 8.92 (s, 1H), 8.33 (s, 1H).
Experimental Sections
General Methods and Materials. Commercial-grade re-
agents and solvents were obtained from Acros, Aldrich, Lan-
caster, Salor, or TCI and were used without further purifica-
tion except as indicated. All reactions 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
1
compound was further dried using a vacuum pump. The H
2-Amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-
methylenedioxyphenyl)-4H-chromene (1a). To a mixture
of 3-(dimethylamino)phenol (0.76 g, 5.5 mmol), 5-methoxy-
piperonal (1.0 g, 5.5 mmol), and malononitrile (0.37 g, 5.5
NMR spectra were recorded at 300 MHz. All samples were
prepared as dilute solutions in either deuteriochloroform
(CDCl3) with v/v 0.05% tetramethylsilane (TMS) or dimethyl-

6306 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Kemnitzer et al.
mmol) in ethanol (20 mL) was added piperidine (1.2 mL, 11
mmol). The mixture was stirred at room temperature over-
night and then the solvent was evaporated. The resulting solid
was collected by filtration and washed with diethyl ether.
Recrystallization of the black solid (1:1, DMSO:water) yielded
(1.5 g, 74%) of compound 1a as a light brown solid: mp 168-
171 °C; 1H NMR (DMSO-d6) 6.85 (s, 1H), 6.82 (s, 2H), 6.52 (d,
J ) 1.5, 1H), 6.49-6.45 (m, 1H), 6.29 (d, J ) 1.5, 1H), 6.21 (d,
J ) 2.7, 1H), 5.94 (d, J ) 6.3, 2H), 4.54 (s, 1H), 3.81 (s, 3H),
2.86 (s, 6H).
The following compounds were prepared from the corre-
sponding substituted benzaldehyde, 3-(dimethylamino)phenol,
and malononitrile by a procedure similar to that described for
the preparation of compound 1a.
8.7, 1H), 6.45-6.42 (m, 1H), 6.30 (d, J ) 2.7, 1H), 4.64 (s, 1H),
4.63 (s, 2H), 2.94 (s, 6H). Anal. (C17H16N4O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-phenyl-4H-
chromene (3l). To a mixture of 3-dimethylaminophenol (0.129
g, 0.940 mmol), malononitrile (0.062 g, 0.94 mmol), and
benzaldehyde (96 µL, 0.94 mmol) in ethanol (5 mL) was added
piperidine (0.20 mL, 2.0 mmol). The white suspension was
stirred at room temperature for 2 h and the resulting precipi-
tate was collected by filtration, washed with methanol, and
then dried in vacuo to yield compound 3l as a white solid (0.202
g, 74%): mp 206-207 °C; 1H NMR (DMSO-d6) 7.32-7.27 (m,
2H), 7.22-7.14 (m, 3H), 6.82 (s, 2H), 6.78 (d, J ) 8.7, 1H),
6.45 (dd, J ) 8.7 and 2.4, 1H), 6.23 (d, J ) 2.4, 1H), 4.59 (s,
1H), 2.86 (s, 6H). Anal. (C18H17N3O) C, H, N.
The following compounds were prepared from the corre-
sponding substituted benzaldehyde, 3-(dimethylamino)phenol,
and malononitrile by a procedure similar to that described for
the preparation of compound 3l.
2-Amino-3-cyano-7-(dimethylamino)-4-(3,4,5-trimethoxy-
phenyl)-4H-chromene (1b): white solid (78%); mp 179-181
°C; 1H NMR (DMSO-d6) 6.90-6.84 (m, 3H), 6.48-6.46 (m, 3H),
6.23 (s, 1H), 4.57 (s, 1H), 3.72 (s, 6H), 3.62 (s, 3H), 2.87 (s,
6H). Anal. (C21H23N3O4) C, H. N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-iodo-4,5-
dimethoxyphenyl)-4H-chromene (1e): white solid (18%);
mp 176-177 °C; 1H NMR (DMSO-d6) 7.02 (d, J ) 1.8, 1H),
6.96 (s, 1H), 6.88 (s, 2H), 6.86 (d, J ) 8.7, 1H), 6.49-6.46 (m,
1H), 6.22 (d, J ) 2.4, 1H), 4.59 (s, 1H), 3.78 (s, 3H), 3.66 (s,
3H), 2.87 (s, 6H). Anal. (C20H20IN3O3) C, H, N.
2-Amino-4-(3-bromo-4,5-methylenedioxyphenyl)-3-cy-
ano-7-(dimethylamino)-4H-chromene (1f): brown solid
(56%); mp 232-234 °C (dec); 1H NMR (DMSO-d6) 6.87 (s, 2H),
6.84-6.80 (m, 2H), 6.67 (d, J ) 1.2, 1H), 6.50-6.46 (m, 1H),
6.22 (d, J ) 2.4, 1H), 6.08 (d, J ) 6.9, 2H), 4.58 (s, 1H), 2.87
(s, 6H). Anal. (C19H16BrN3O3) C, H, N.
2-Amino-3-cyano-4-(3,5-dimethoxyphenyl)-7-(dimethyl-
amino)-4H-chromene (2a): white solid (38%); mp 169-170
°C; 1H NMR (DMSO-d6) 6.85 (d, J ) 8.7, 1H), 6.81 (s, 2H),
6.46 (dd, J ) 8.7 and 2.7, 1H), 6.36-6.34 (m, 1H), 6.31 (d, J )
2.1, 2H), 6.22 (d, J ) 2.4, 1H), 4.52 (s, 1H), 3.70 (s, 6H), 2.86
(s, 6H). Anal. (C20H21N3O3) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-methylphe-
nyl)-4H-chromene (3f): white solid (24%); mp 174-175 °C;
1H NMR (DMSO-d6) 7.18 (t, J ) 7.5, 1H), 7.00 (d, J ) 7.2,
1H), 6.95 (d, J ) 2.1, 2H), 6.80 (s, 2H), 6.78 (d, J ) 8.7, 1H),
6.46-6.43 (m, 1H), 6.22 (d, J ) 2.4, 1H), 4.54 (s, 1H), 2.86 (s,
6H), 2.26 (s, 3H). Anal. (C19H19N3O) C, H, N.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-
(dimethylamino)-4H-chromene (1c): white solid (24%);
1
recrystallized from 5:1, hexanes:EtOAc; mp 200-202 °C; H
NMR (CDCl3) 6.89 (d, J ) 1.8, 1H), 6.79 (d, J ) 8.7, 1H), 6.72
(d, J ) 1.8, 1H), 6.46-6.43 (m, 1H), 6.28 (d, J ) 2.7, 1H), 4.58
(s, 1H), 4.57 (s, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 2.94 (s, 6H).
Anal. (C20H20BrN3O3) C, H, N.
2-Amino-4-(3-chloro-4,5-dimethoxyphenyl)-3-cyano-7-
(dimethylamino)-4H-chromene (1d): tan solid (57%); re-
crystallized from 5:1, hexanes:EtOAc; mp 190-192 °C; 1H
NMR (CDCl3) 6.82-6.69 (m, 3H), 6.48-6.44 (m, 1H), 6.30 (d,
J ) 2.7, 1H), 4.59 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 2.85 (s,
6H). Anal. (C20H20ClN3O3) C, H, N.
2-Amino-4-(3-bromo-5-methoxyphenyl)-3-cyano-7-(di-
methylamino)-4H-chromene (2b): white solid (50%); re-
crystallized from 5:1, hexanes:EtOAc; mp 210-212 °C; 1H
NMR (DMSO-d6) 6.99 (s, 1H), 6.91 (s, 2H), 6.85 (s, 1H), 6.84
(d, J ) 8.7, 1H), 6.77 (s, 1H), 6.49-6.46 (m, 1H), 6.24 (d, J )
3.0, 1H), 4.61 (s, 1H), 3.75 (s, 3H), 2.87 (s, 6H). Anal. (C19H18-
BrN3O2) C, H, N.
2-Amino-3-cyano-4-(3,5-dichlorophenyl)-7-(dimethyl-
amino)-4H-chromene (2c): yellow solid (24%); recrystallized
from 1:1, hexanes:dichloromethane; mp 212-214 °C; 1H NMR
(DMSO-d6) 7.47 (t, J ) 1.8, 1H), 7.20 (d, J ) 2.1, 2H), 6.98 (s,
2H), 6.83 (d, J ) 8.7, 1H), 6.49 (dd, J ) 8.9 and 2.6, 1H), 6.24
(d, J ) 2.4, 1H), 4.73 (s, 1H), 2.88 (s, 6H). Anal. (C18H15Cl2N3O)
C, H, N.
2-Amino-3-cyano-4-(3,4-dimethoxyphenyl)-7-(dimethyl-
amino)-4H-chromene (2d): white solid (23%); recrystallized
1
from 5:1, hexanes:EtOAc; mp 158-160 °C; H NMR (CDCl3)
6.81 (s, 1H), 6.79 (s, 1H), 6.75 (d, J ) 1.8, 1H), 6.69 (d, J )
1.8, 1H), 6.45-6.41 (m, 1H), 6.29 (d, J ) 3.0, 1H), 4.60 (s, 1H),
4.54 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 2.93 (s, 6H). Anal.
(C20H21N3O3*0.6H2O) C, H, N.
2-Amino-4-(3-benzyloxyphenyl)-3-cyano-7-(dimethyl-
amino)-4H-chromene (3j): white solid (76%); mp 197-198
1
2-Amino-4-(3-carboxyphenyl)-3-cyano-7-(dimethylami-
no)-4H-chromene (3h): yellow solid (80%); recrystallized
from 1:1:0.1, hexanes:EtOAc:MeOH; mp 204-206 °C; 1H NMR
(DMSO-d6) 7.72-7.68 (m, 1H), 7.64 (s, 1H), 7.29-7.25 (m, 2H),
6.84 (s, 2H), 6.76 (d, J ) 9.0, 1H), 6.45 (dd, J ) 8.9 and 2.6,
1H), 6.23 (d, J ) 2.7, 1H), 4.62 (s, 1H), 2.86 (s, 6H). Anal.
(C19H17N3O3) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-pyridyl)-4H-
chromene (4a): brown solid (93%); recrystallized from
1:1:0.1, hexanes:EtOAc:MeOH; mp 210-214 °C (dec); 1H NMR
(CDCl3) 8.49-8.47 (m, 2H), 7.52-7.49 (m, 1H), 7.26-7.22
(m, 1H), 6.75 (d, J ) 8.7, 1H), 6.45-6.41 (m, 1H), 6.30 (d, J )
2.4, 1H), 4.69 (s, 1H), 4.62 (s, 2H), 2.93 (s, 6H). Anal.
(C17H16N4O*0.3H2O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(2-pyridyl)-4H-
chromene (4b): brown solid (35%); recrystallized from 1:1,
hexanes:dichloromethane; mp 206-208 °C; 1H NMR (DMSO-
d6) 8.46 (dd, J ) 5.7 and 1.8, 1H), 7.73 (td, J ) 7.7 and 1.8,
1H), 7.23-7.20 (m, 2H), 6.88 (s, 2H), 6.85 (d, J ) 9.0, 1H),
6.44 (dd, J ) 8.9 and 2.6, 1H), 6.23 (d, J ) 3.0, 1H), 4.70 (s,
1H), 2.85 (s, 6H). Anal. (C17H16N4O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(4-pyridinyl)-
4H-chromene (4c): brown solid (47%); recrystallized from
1:1, hexanes:dichloromethane; mp 202-204 °C; 1H NMR
(CDCl3) 8.54-8.52 (m, 2H), 7.13-7.11 (m, 2H), 6.75 (d, J )
°C; H NMR (DMSO-d6) 7.46-7.33 (m, 5H), 7.22 (t, J ) 7.8,
1H), 6.87-6.79 (m, 5H), 6.75 (d, J ) 7.2, 1H), 6.45 (dd, J )
8.9 and 2.6, 1H), 6.22 (d, J ) 2.1, 1H), 5.04 (s, 2H), 4.56 (s,
1H), 2.86 (s, 6H). Anal. (C25H23N3O2) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-dodecyloxy-
phenyl)-4H-chromene (3k): white solid (54%); mp 94 °C;
1H NMR (DMSO-d6) 7.19 (t, J ) 7.8, 1H), 6.83-6.69 (m, 6H),
6.45 (dd, J ) 8.6 and 2.6, 1H), 6.22 (d, J ) 2.7, 1H), 4.55 (s,
1H), 3.90 (t, J ) 6.3, 2H), 2.86 (s, 6H), 1.70-1.65 (m, 2H),
1.38-1.24 (m, 18H), 0.85 (t, J ) 6.6, 3H). Anal. (C30H41N3O2)
C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(4-methoxyphe-
nyl)-4H-chromene (3m): yellow solid (80%); mp 174-176 °C;
1H NMR (CDCl3) 7.12-7.10 (m, 2H), 6.85-6.76 (m, 3H), 6.42
(dd, J ) 8.4 and 2.4, 1H), 6.28 (d, J ) 2.4, 1H), 4.60 (s, 1H),
4.51 (br s, 2H), 3.77 (s, 3H), 2.92 (s, 6H). Anal. (C19H19N3O2)
C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(2-methoxyphe-
nyl)-4H-chromene (3n): yellow solid (30%); mp 152-153 °C
1
(dec); H NMR (CDCl3) 7.20-7.14 (m, 1H), 7.00 (dd, J ) 7.5
and 1.8, 1H), 6.91-6.84 (m, 3H), 6.41 (dd, J ) 8.7 and 2.7,
1H), 6.28 (d, J ) 2.4, 1 H), 5.17 (s, 1H), 4.50 (br s, 2H), 3.85
(s, 3H), 2.91(s, 6H). Anal. (C19H19N3O2) C, H, N.
2-Amino-4-(5-bromo-3-pyridyl)-3-cyano-7-(dimethylami-
no)-4H-chromene (4d): brown solid (8%); mp 230-232 °C

4-Aryl-4H-chromenes as Inducers of Apoptosis
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6307
1
(dec); H NMR (DMSO-d6) 8.58 (t, J ) 2.3, 1H), 8.44 (d, J )
mmol), and 2,3,4-trimethoxybenzaldehyde (0.20 g, 1.0 mmol)
in ethanol (3.4 mL) was added piperidine (50 µL, 0.51 mmol).
The purple solution was stirred at room temperature for 24 h
and then the reaction solution was concentrated in vacuo. The
crude product was purified by flash column chromatography
(elution with EtOAc:hexanes, 2:1) to give compound 1h as a
yellow solid (0.20 g, 52%): mp 74-78 °C; 1H NMR (DMSO-d6)
6.73-6.71 (m, 5H), 6.45-6.41 (m, 1H), 6.22 (d, J ) 2.4, 1H),
4.74 (s, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.66 (s, 3H), 2.85 (s,
6H). Anal. (C21H23N3O4) C, H, N.
1.9, 1H), 7.75 (d, J ) 2.2, 1H), 7.01 (br s, 2H), 6.82 (d, J ) 8.8,
1H), 6.49 (d, J ) 8.8, 1H), 6.25 (s, 1H), 4.77 (s, 1H), 2.88 (s,
6H). Anal. (C17H15BrN4O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-py-
ridyl)-4H-chromene (4e): yellow solid (92%); mp 232-234
°C; 1H NMR (DMSO-d6) 8.26 (d, J ) 0.8, 2H), 7.29 (s, 1H),
6.90 (br s, 2H), 6.77 (d, J ) 8.5, 1H), 6.48-6.45 (m, 1H), 6.24
(d, J ) 2.2, 1H), 4.66 (s, 1H), 2.87 (s, 6H), 2.25 (s, 3H). Anal.
(C18H18N4O) C, H, N.
The following compounds were prepared from the corre-
sponding substituted benzaldehyde, 3-(dimethylamino)phenol,
and malononitrile by a procedure similar to that described for
the preparation of compound 1h.
2-Amino-3-cyano-7-(dimethylamino)-4-(6-methyl-2-py-
ridyl)-4H-chromene (4f): tan solid (66%); mp 212-214 °C;
1H NMR (CDCl3) 7.49 (t, J ) 7.5, 1H), 7.00-6.93 (m, 3H),
6.43-6.39 (m, 1H), 6.28 (s, 1H), 4.84 (s, 1H), 4.66 (s, 2H), 2.91
(s, 6H), 2.56 (s, 3H). Anal. (C18H18N4O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-quinolyl)-4H-
chromene (5c): yellow solid (69%); mp 224-226 °C; 1H NMR
(DMSO-d6) 8.71 (d, J ) 2.1, 1H), 8.12 (d, J ) 2.1, 1H), 7.99
(dd, J ) 7.7 and 4.0, 2H), 7.73 (td, J ) 7.7 and 1.5, 1H), 7.61
(t, J ) 7.5, 1H), 6.99 (s, 2H), 6.81 (d, J ) 8.7, 1H), 6.46 (dd, J
) 8.9 and 2.6, 1H), 6.28 (d, J ) 2.4, 1H), 4.91 (s, 1H), 2.87 (s,
6H). Anal. (C21H18N4O) C, H, N.
2-Amino-4-(2-bromo-4,5-dimethoxyphenyl)-3-cyano-7-
(dimethylamino)-4H-chromene (1j). To a purple suspension
of 3-(dimethylamino)phenol (0.250 g, 1.02 mmol), malononitrile
(0.067 g, 1.0 mmol), and 6-bromoveratraldehyde (0.140 g, 1.02
mmol) in ethanol (3.4 mL) was added piperidine (50 µL, 0.51
mmol). The brown suspension was stirred at room temperature
for 28 h, and the resulting precipitate was collected by
filtration, washed with ethanol, and purified by flash column
chromatography (elution with EtOAc:hexanes, 1:2) to give
compound 1j as a yellow solid (0.13 g, 29%): mp 94-98 °C;
1H NMR (CDCl3) 6.99 (s, 1H), 6.83 (d, J ) 8.5, 1H), 6.58 (s,
1H), 6.41 (dd, J ) 8.8 and 2.5, 1H), 6.27 (d, J ) 2.8, 1H), 5.23
(s, 1H), 4.57 (s, 2H), 3.86 (s, 3H), 3.75 (s, 3H), 2.93 (s, 6H).
Anal. (C20H20BrN3O3·0.3H2O) C, H, N.
The following compounds were prepared from the corre-
sponding substituted benzaldehyde, 3-(dimethylamino)phenol,
and malononitrile by a procedure similar to that described for
the preparation of compound 1j.
2-Amino-3-cyano-4-(2,3-dimethoxyphenyl)-7-(dimethyl-
amino)-4H-chromene (2e): white solid (58%); mp 72-74 °C;
1H NMR (DMSO-d6) 6.98 (t, J ) 7.2, 1H), 6.88 (d, J ) 7.5,
1H), 6.77 (s, 2H), 6.72 (d, J ) 8.4, 1H), 6.61 (d, J ) 7.2, 1H),
6.44-6.40 (m, 1H), 6.22 (d, J ) 1.8, 1H), 4.86 (s, 1H), 3.79 (s,
3H), 3.65 (s, 3H), 2.85 (s, 6H). Anal. (C20H21N3O3) C, N. H calcd,
6.02; found, 6.74.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-methoxyphe-
nyl)-4H-chromene (3b): yellow solid (19%); mp 161-162 °C;
1H NMR (CDCl3) 7.19 (t, J ) 0.6, 1H), 6.82-6.72 (m, 4H),
6.44-6.40 (m, 1H), 6.28 (d, J ) 2.7, 1H), 4.61 (s, 1H), 4.54 (s,
2H), 3.77 (s, 3H), 2.92 (s, 6H). Anal. (C19H19N3O2) C, H, N.
2-Amino-3-cyano-4-(3-cyanophenyl)-7-(dimethylamino)-
1
4H-chromene (3c): yellow solid (25%); mp 188-190 °C; H
NMR (DMSO-d6) 7.70 (d, J ) 7.5, 1H), 7.65 (s, 1H), 7.56-7.50
(m, 2H), 6.96 (s, 2H), 6.79 (d, J ) 8.4, 1H), 6.49-6.45 (m, 1H),
6.24 (d, J ) 2.4, 1H), 4.75 (s, 1H), 2.87 (s, 6H). Anal.
(C19H16N4O) C, H, N.
2-Amino-4-(3-chlorophenyl)-3-cyano-7-(dimethylamino)-
1
4H-chromene (3e): yellow solid (33%); mp 172-173 °C; H
NMR (DMSO-d6) 7.35 (t, J ) 7.5, 1H), 7.29-7.25 (m, 1H), 7.19
(t, J ) 1.8, 1H), 7.16-7.13 (m, 1H), 6.91 (s, 2H), 6.81 (d, J )
8.4, 1H), 6.49-6.45 (m, 1H), 6.23 (d, J ) 2.7, 1H), 4.66 (s, 1H),
2.87 (s, 6H). Anal. (C18H16ClN3O) C, H, N.
2-Amino-3-cyano-4-cyclohexyl-7-(dimethylamino)-4H-
chromene (5a): yellow solid (31%); mp 148-152 °C; 1H NMR
(DMSO-d6) 6.96 (d, J ) 8.4, 1H), 6.72 (s, 2H), 6.51 (dd, J )
8.7 and 2.4, 1H), 6.20 (d, J ) 2.4, 1H), 3.19 (d, J ) 3.3, 1H),
2.87 (s, 6H), 2.82 (s, 1H), 1.65-0.86 (m, 10H). Anal. (C18H23N3O)
C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(2-phenylethyl)-
1
4H-chromene (5b): yellow solid (17%); mp 140-142 °C; H
NMR (DMSO-d6) 7.36-7.08 (m, 6H), 6.75 (s, 2H), 6.56 (dd, J
) 8.4 and 2.4, 1H), 6.21 (d, J ) 2.4, 1H), 3.58 (t, J ) 6.9, 1H),
2.87 (s, 6H), 2.34-2.24 (m, 2H), 1.97-1.79 (m, 2H). Anal.
(C20H21N3O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(2,4,5-trimethoxy-
phenyl)-4H-chromene (1i). To a purple solution of 3-(dimeth-
ylamino)phenol (0.14 g, 1.0 mmol), malononitrile (0.067 g, 1.0
mmol), and 2,4,5-trimethoxybenzaldehyde (0.20 g, 1.0 mmol)
in ethanol (3.4 mL) was added piperidine (50 µL, 0.51 mmol).
The purple solution was heated at 50 °C for 10 h and the
resulting precipitate was collected by filtration, washed with
ethanol, and dried in vacuo to give compound 1i as a yellow
solid (0.17 g, 44%): mp 200-202 °C; 1H NMR (DMSO-d6) 6.80
(d, J ) 8.7, 1H), 6.71 (s, 2H), 6.68 (s, 1H), 6.55 (s, 1H), 6.44-
6.40 (m, 1H), 6.20 (d, J ) 2.4, 1H), 4.88 (s, 1H), 3.76 (d, J )
3.9, 6H), 3.59 (s, 3H), 2.84 (s, 6H). Anal. (C21H23N3O4) C, H,
N.
2-(3-Bromo-4-hydroxy-5-methoxybenzylidene)malono-
nitrile (8). To a mixture of 3-bromo-4-hydroxy-5-methoxy-
benzaldehyde (2.31 g, 10.0 mmol) and malononitrile (0.660 g,
10.0 mmol) in ethanol (20 mL) was added piperidine (0.50 mL,
0.50 mmol). The solution was stirred at room temperature
overnight and the resulting precipitate was collected by
filtration and dried to yield compound 8 (2.14 g, 77%) as a red
solid, which was used for the next reaction.
2-Amino-4-(3-bromo-4-hydroxy-5-methoxyphenyl)-3-
cyano-7-(dimethylamino)-4H-chromene (1g). To a mixture
of 8 (0.279 g, 1.00 mmol) and 3-(dimethylamino)phenol (0.137
g, 1.00 mmol) in ethanol (10 mL) was added piperidine (0.05
mL, 0.05 mmol) and the solution was refluxed overnight. The
solvent was removed in vacuo and the crude material was
purified by column chromatography (elution with EtOAc:
hexanes, 1:2) to give compound 1g (0.035 g, 8.4%) as a white
solid: mp 218-221 °C; 1H NMR (CDCl3) 6.88 (d, J ) 2.1, 1H),
6.78 (d, J ) 8.7, 1H), 6.67 (d, J ) 2.1, 1H), 6.44 (dd, J ) 2.4,
2-Amino-4-(3-bromophenyl)-3-cyano-7-(dimethylamino)-
1
4H-chromene (3a): yellow solid (62%); mp 180-182 °C; H
NMR (DMSO-d6) 7.43-7.39 (m, 1H), 7.33 (t, J ) 1.8, 1H), 7.29
(t, J ) 10.2, 1H), 7.20-7.17 (m, 1H), 6.92 (s, 2H), 6.81 (d, J )
8.4, 1H), 6.49-6.45 (m, 1H), 6.24 (d, J ) 2.7, 1H), 4.66 (s, 1H),
2.87 (s, 6H). Anal. (C18H16BrN3O2·0.2H2O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-fluorophenyl)-
1
4H-chromene (3d): white solid (38%); mp 157-158 °C; H
NMR (DMSO-d6) 7.39-7.31 (m, 1H), 7.06-6.95 (m, 3H), 6.89
(s, 2H), 6.82 (d, J ) 8.4, 1H), 6.47 (dd, J ) 8.7 and 2.4, 1H),
6.23 (d, J ) 2.1, 1H), 4.66 (s, 1H), 2.86 (s, 6H). Anal. (C18H16-
FN3O) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-hydroxyphe-
nyl)-4H-chromene (3g): white solid (25%); mp 176-178 °C;
1H NMR (DMSO-d6) 9.31 (s, 1H), 7.07 (t, J ) 7.8, 1H), 6.81-
6.79 (m, 3H), 6.64-6.55 (m, 3H), 6.48-6.44 (m, 1H), 6.22 (d,
J ) 2.4, 1H), 4.48 (s, 1H), 2.86 (s, 6H). Anal. (C18H17N3O2) C,
H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(3-trifluoro-
methoxyphenyl)-4H-chromene (3i): yellow solid (47%); mp
166-168 °C; 1H NMR (DMSO-d6) 7.45 (t, J ) 7.5, 1H), 7.21-
7.15 (m, 3H), 6.92 (s, 2H), 6.82 (d, J ) 8.7, 1H), 6.49-6.46 (m,
1H), 6.24 (d, J ) 2.7, 1H), 4.73 (s, 1H), 2.87 (s, 6H). Anal.
(C19H16F3N3O2) C, H, N.
2-Amino-3-cyano-7-(dimethylamino)-4-(2,3,4-trimethoxy-
phenyl)-4H-chromene (1h). To a purple solution of 3-(dimeth-
ylamino)phenol (0.14 g, 1.0 mmol), malononitrile (0.067 g, 1.0

6308 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Kemnitzer et al.
8.7, 1H), 6.28 (d, J ) 2.4, 1H), 5.81 (s, 1H), 4.55 (br s, 3H),
3.87 (s, 3H), 2.94 (s, 6H). Anal. (C19H18BrN3O3) C, H, N.
activity (EC50) in three cancer cell lines, T47D, H1299, and
DLD-1, are summarized in Table 1.
Morphological Assessment of Nuclear Fragmentation
of Apoptotic Cells. Jurkat cells, grown and harvested as
above, were treated with 0.1 µM of compound 1a for 24 h
followed by staining of the nucleus with Syto 16, a fluorescent
DNA dye. The cells were then observed under a fluorescent
microscope for chromosomal condensation and nuclear frag-
mentation (490 nm). The nuclei of Jurkat cells treated with
vehicle control (DMSO) are seen to be round with dispersed
chromatin that is moderately stained with Syto16 (Figure 1A).
In contrast, Jurkat cells treated with 0.1 µM of compound 1a
for 24 h have shrunken and fragmented nuclei (Figure 1B),
which is a hallmark of caspase-mediated apoptosis. These
results support the caspase activation assays, showing that
compound 1a can induce a key cellular marker of apoptosis.
Induction of PARP Cleavage in Jurkat Cells. The
ability of compound 1a to induce PARP cleavage was tested
by treating Jurkat cells with the compound at a concentration
of 2.5 µM for 5, 15, and 30 h. Control cultures were treated
with DMSO vehicle or staurosporine, a known apoptosis
inducer. At the end of the incubation period, the cells were
harvested by centrifugation, washed with PBS, and lysed in a
buffer containing 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a cocktail
of protease inhibitors (Complete Protease Inhibitor Tablets,
Roche Molecular Systems). The amount of protein in each
sample was measured by the method of Bradford using a
BioRad protein assay kit. Samples of lysate containing equiva-
lent amounts of protein were separated by SDS-PAGE and
transferred to PVDF membrane. The membranes were probed
with a polyclonal antibody to PARP (Enzyme Systems Prod-
ucts), followed by goat-anti-rabbit HRP second antibody.
Antibody binding was visualized by chemiluminescence (Pierce
SuperSignal). As illustrated in Figure 2, compound 1a induced
PARP cleavage starting at 15 h, and by 30 h virtually all of
PARP was converted into its cleaved form. These results
provide additional evidence that 1a induces caspase-mediated
apoptosis in cells.
Cell Cycle Analysis and Measurement of Apoptosis.
T47D cells were maintained and harvested as described above.
Cells (1 × 106) were treated with 0.1 µM of compound 1a for
24 or 48 h at 37 °C. As a control, cells were also incubated
with an equivalent amount of solvent (DMSO). Cells were
harvested at 1200 rpm and washed twice with 5 mM EDTA/
PBS. Cells were then resuspended in 300 µL of EDTA/PBS
and 700 µL of 100% ethanol, vortexed, and incubated at room
temperature for 1 h. Samples were centrifuged at 1200 rpm
for 5 min, and the supernatant was removed. A solution
containing 100 µg/mL of propidium iodide and 1 mg/mL of
RNAse A (fresh) was added to the samples and incubated for
1 h at room temperature. Samples were then transferred to
12 × 75 mm polystyrene tubes and analyzed on a flow
cytometer. All flow cytometry analyses were performed on a
FACScalibur (Becton Dickinson) using Cell Quest analysis
software. On the x-axis is plotted the fluorescence intensity
and on the y-axis is plotted the number of cells with that
fluorescence intensity. The T47D control cell population profile
is shown in Figure 3A with most of the cells in the G1 phase.
An increase in the G2/M DNA content of cells from 17% to
44% was observed when cells were treated with compound 1a
at 0.1 µM for 24 h (Figure 3B). After the cells were treated
with 0.1 µM of compound 1a for 48 h (Figure 3C), G2/M DNA
content of cells increased to 57%. Concurrently, the subdiploid
DNA content of cells (Figure 3C) increased from 0.9% to 12%.
The subdiploid amount of DNA (M1) is indicative of apoptotic
cells that have undergone DNA degradation and nuclear
fragmentation.
4-(3-Bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethyl-
amino)-2-imino-2H-chromene (9). To a solution of com-
pound 1c (2.00 g, 4.65 mmol) in dichloromethane (23 mL) was
added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.05 g, 4.65
mmol). The resulting green mixture was stirred at room
temperature for 5 h. The reaction mixture was then diluted
with ethyl acetate (250 mL), washed with sodium bicarbonate
saturated solution (2 × 50 mL) and brine (50 mL), dried over
sodium sulfate, and concentrated to yield 0.90 g (45%) of
compound 9 as an orange solid: 1H NMR (DMSO-d6) 8.33 (s,
1H), 7.28 (d, J ) 1.9, 1H), 7.21 (d, J ) 1.9, 1H), 6.87 (d, J )
9.1, 1H), 6.56 (dd, J ) 9.1 and 2.5, 1H), 6.40 (d, J ) 2.5, 1H),
3.84 (s, 3H), 3.82 (s, 3H), 3.03 (s, 6H).
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-
(dimethylamino)-4-methyl-4H-chromene (10). To a mix-
ture of copper bromide dimethyl sulfide (1.20 g, 5.84 mmol) in
anhydrous THF (16.7 mL) at -78 °C was slowly added
methyllithium (1.6 M in ether, 7.30 mL, 11.7 mmol). The
internal temperature of the reaction mixture was kept below
-72 °C throughout the addition process. To the mixture was
then added an orange solution of the compound 9 (0.50 g, 1.8
mmol) in anhydrous THF (20 mL) dropwise over 45 min while
the internal temperature was maintained below -70 °C. The
resulting orange mixture was stirred for 30 min at -78 °C
and then was warmed to 0 °C. It was then quenched with
ammonium chloride saturated solution (20 mL), extracted with
ethyl acetate (3 × 100 mL), washed with brine (25 mL), dried
over sodium sulfate, and concentrated. The residue was
purified by flash column chromatography (elution with EtOAc:
hexanes, 1:2) to give compound 10 as a yellow solid (0.11 g,
21%): mp 72-74 °C; 1H NMR (CDCl3) 6.86-6.76 (m, 3H), 6.45
(dd, J ) 8.7 and 2.6, 1H), 6.28 (d, J ) 2.6, 1H), 4.46 (br s, 2H),
3.84 (s, 3H), 3.80 (s, 3H), 2.93 (s, 6H), 1.87 (s, 3H). Anal.
(C21H22BrN3O3·0.5H2O) C, H, N.
Caspase Activation Assay (EC50). T47D, H1299, and
DLD-1 cells were grown according to media component mix-
tures designated by American Type Culture Collection in
RPMI-1640 +10% FCS in a 5% CO2-95% humidity incubator
at 37 °C. Cells were harvested using trypsin, washed at 600g,
and resuspended at 0.65 × 106 cells/mL into RPMI media +
10% FCS. An aliquot of 22.5 µL of cells was added to a well of
a 384-well microtiter plate containing 2.5 µL of 0.05-100 µM
of test compound in RPMI-1640 containing 25 mM HEPES
media solution with 10% DMSO (0.005 to 10 µM final). An
aliquot of 22.5 µL of cells was added to a well of a 384-well
microtiter plate containing 2.5 µL of RPMI-1640 media solu-
tion with 10% DMSO and without test compound as the control
sample. The samples were then incubated at 37 °C for 24 h in
a 5% CO2-95% humidity incubator. After incubation, the
samples were removed from the incubator and 25 µL of a
solution containing 14 µM of N-(Ac-DEVD)-N′-ethoxycarbonyl-
R110 fluorogenic substrate,10 20% sucrose, 20 mM DTT, 200
mM NaCl, 40 mM Na PIPES buffer pH 7.2, and 250 µg/mL
lysolecithin was added. The samples were incubated at room
temperature for 3 h. Using a fluorescent plate reader (Model
Spectrafluor 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 incubation, the samples were read for fluores-
cence as above (T ) 3 h).
Calculation. The relative fluorescence unit values (RFU)
were used to calculate the sample readings as follows: The
activity of caspase activation was determined by the ratio of
the net RFU value for the test compound to that of control
samples. Compound 1a was found to have a ratio (maximum
response) of 6.0. In comparison, colchicine and paclitaxel were
found to have a ratio of 7.5 and 6.4, respectively. The EC50
(µM) was determined by a sigmoidal dose-response calculation
(XLFit3, IDBS), as the concentration of compound that pro-
duces the 50% maximum response. The caspase activation
Cell Growth Inhibition Assays (GI50). Cells were grown
and harvested as described above. An aliquot of 45 µL of cells
(4.4 × 104 cells/mL) was added to a well of a 96-well microtiter
plate, then 5 µL of 0.01-100 µM of test compound (0.001 to
10 µM final concentration) in RPMI-1640 media solution with
10% DMSO was added. An aliquot of 45 µL of cells was added

4-Aryl-4H-chromenes as Inducers of Apoptosis
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6309
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283-292.
to a well of a 96-well microtiter plate containing 5 µL 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 µL 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 the give Ltest
value.
Baseline for GI50 (dose for 50% inhibition of cell prolifera-
tion) of initial cell numbers was determined by adding an
aliquot of 45 µL of cells and 5 µL 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 µL 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 the Lstart value, defining
luminescence for initial cell number used as baseline in GI50
determinations.
Calculation. GI50 (dose for 50% inhibition of cell prolifera-
tion) is the concentration where [(Ltest - Lstart)/(Lmax - Lstart)])
× 0.5. The GI50 (µM) values of compound 1b, 1e, 1f, 1g, 2b,
3h, 5a, and 5b in T47D and DLD-1 cells are summarized in
Table 2 in comparison with the caspase activation activity
(EC50).
Tubulin Polymerization Assay. Lyophilized tubulin (Cy-
toskeleton #ML113, 1 mg, MAP-rich) was assayed for the effect
of the test compound on tubulin polymerization according to
the recommended procedure of the manufacturer. To 1 µL of
each experimental compound (from a 100 x stock) in a 96-well
was added 99 µL of supplemented tubulin supernatant.
Incubation was done in a Molecular Devices plate reader at
37 °C, and absorbance readings at 340 nm were recorded every
minute for 1 h. The IC50 for tubulin inhibition was the
concentration found to decrease the initial rate of tubulin
polymerization by 50% as calculated with Prism 3.0.
Colchicine Binding Assay. A radioactive 96-well plate
tubulin-binding assay was used to measure the ability of drugs
to compete with [3H]colchicine on tubulin. Briefly, 5.5 µg/well
of biotin-labeled tubulin (Cytoskeleton, Denver, CO), diluted
in 80 mM PIPES pH 6.8 containing 1 mM EGTA, 1 mM MgCl2,
and 1 mM GTP, was coated into a FlashPlate streptavidin-
coated 96-well plate (Perkin-Elmer, Woodbridge, Ontario,
Canada) and incubated for 30 min at 37 °C. The wells were
then washed in a buffer of 80 PIPES pH 6.8, 1 mM EGTA,
and 1 mM MgCl2. A serial dilution of the compound to be tested
was made (10-4-10-9 M) in 80 mM PIPES, 1 mM EGTA, 1
mM MgCl2, and 1 mM GTP, and 0.36 µM of [3H]colchicine was
added to a final volume of 100 µL. The reaction mixture was
incubated at 37°C for 30 min, and the wells were then washed
in 80 mM PIPES pH 6.8, 1 mM EGTA, and 1 mM MgCl2. The
wells were allowed to dry and then 50 µL of Scintisafe Econo
1 (Fisher Scientific, St-Laurent, Quebec, Canada) was added
to each well and each was counted for radioactivity in a 1450
MicroBeta Trilux (Wallac, Turku, Finland). Each concentration
of the drugs was performed in triplicate and IC50’s were
generated using the PrismPad computer program (Graph-Pad
Software, Inc., San Diego, CA).
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