Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: Modifications of the 2- and 3-positions

Article abstract of DOI:10.1016/j.bmcl.2007.11.078

As a continuation of our efforts to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored modifications at the 2- and 3-positions. It was found that replacement of the 3-cyano group by an ester, including meth

Full text of DOI:10.1016/j.bmcl.2007.11.078

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 603–607
Discovery of 4-aryl-4H-chromenes as a new series
of apoptosis inducers using a cell- and caspase-based HTS assay.
Part 5: Modifications of the 2- and 3-positions
William Kemnitzer,a Songchun Jiang,a Yan Wang,a Shailaja Kasibhatla,a
Candace Crogan-Grundy,a Monica Bubenik,b Denis Labrecque,b Real Denis,b
Serge Lamothe,b Giorgio Attardo,b Henriette Gourdeau,b Ben Tseng,a
John Drewea and Sui Xiong Caia,*
aEpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
bShire Biochem Inc., 275 Armand-Frappier Boulevard, Laval, Que., Canada H7V 4A7
Received 27 October 2007; revised 17 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—As a continuation of our efforts to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer
agents, we explored modifications at the 2- and 3-positions. It was found that replacement of the 3-cyano group by an ester, includ-
ing methyl and ethyl ester, resulted in >200-fold reduction of activity. Conversion of the 2-amino group into an amide or urea
resulted in 4- to 10-fold drop of activity. Similarly, converting the 2-amino group into a hydrogen resulted in 4- to 10-fold reduction
of activity. Compound 3d was highly active with an EC50 value of 29 nM and a GI50 value of 6 nM in T47D cells. Importantly, the 2-
H analog 3d was found to be much more stable under acidic conditions compared to the 2-NH2 analog 3b, suggesting that 2-H
analogs might have better bioavailability than the 2-NH2 analogs.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Apoptosis is known to play a vital role in the elimina-
tion of damaged or excess cells. Defects in the apoptosis
pathways are one of the hallmarks of cancers that lead
to uncontrollable tumor cell growth, as well as tumor
resistance to chemotherapeutic treatment.1 The path-
ways of apoptosis involve a cascade of initiator and
effector caspases. Caspase-3 is known to be the main
executioner of apoptosis through cleavage of protein
substrates that leads to irreversible cell death.2 Since
many of the clinically useful cytotoxic agents are known
to induce apoptosis in cancer cells,3 we have been inter-
ested in the discovery and development of apoptosis
inducers as potential anticancer agents,4 and have there-
fore developed a cell-based high throughput-screening
assay for apoptosis inducers, using our proprietary fluo-
rescent caspase-3 substrate.5
new series of potent apoptosis inducers using our cell-
and caspase-based high-throughput screening (HTS)
assays. These compounds were found to be tubulin inhib-
itors, binding at or close to the binding site of colchicine,
possessing vascular targeting activity and showing high
activity in several anticancer animal models.6,7 From
our screening hit 2-amino-3-cyano-7-dimethylamino-4-
(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene
(1a), we have identified 2-amino-4-(3-bromo-4,5-dime-
thoxyphenyl)-3-cyano-7-dimethylamino-4H-chromene
(2a) as a lead compound.8 Additional SAR studies
showed that cyclization of the 7,8-positions9 into a ring
structure led to potent compounds, such as 2-amino-4-
(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7-dihydropyr-
rolo[2,3-h]chromene (3a).10 Introduction of a methyl
group at the 7-position of the pyrrolo ring led to highly
potent compounds such as 2-amino-4-(3-bromo-4,5-
dimethoxyphenyl)-3-cyano-4,7-dihydro-7-methylpyrrol-
o[2,3-h]chromene (3b)11 with EC50 values in the 2–3 nM
range (Chart 1).
We have recently reported the discovery and structure–
activity relationship (SAR) of 4-aryl-4H-chromenes as a
Keywords: Apoptosis inducers; Anticancer agents; HTS assay; SAR.
*
It has been reported that a series of structurally related
substituted 2-amino-3-cyano-4-phenyl-4H-naphtho[1,2-
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@epicept.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.078

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W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 603–607
OMe
OMe
O
MeO
Br
O
MeO
Br
OMe
CN
CN
CN
R
O
NH2
N
Me2N
O
2a
NH2
Me2N
O
NH2
1a
3a, R = H
3b, R = Me
NO2
CN
NO2
CN
O
O
N
O
NH2
O
4a
4b
Chart 1.
b]pyrans such as compound 4a blocked the synthesis
and not the activity of matrix-degrading metallopro-
teases, and thus might be useful for the prevention of
cartilage destruction associated with several degenera-
tive diseases of the articular joint such as rheumatoid
arthritis.12 Compound 4a was found to be unstable in
an acidic solution (50/50/0.1, v/v/v acetonitrile/water/
TFA), thus limiting its oral bioavailability. The 2-ami-
no group was found to be the cause of the instability of
4a and conversion of the amino group into a succinim-
ido group (4b) rendered the compound stable under
acidic conditions.13 Although most of the clinically
used tubulin interacting chemotherapeutics including
paclitaxel and doxetaxel are administered by iv injec-
tion, it is desirable to have orally active antitubulin
drugs. Currently there are several orally active tubulin
interacting agents in clinical development, including
ABT-75114 and indibulin,15 which are in phase II and
phase I clinical trials, respectively. We therefore ex-
plored the modification of the 2-amino group and the
3-cyano group, both as a continuation of our SAR
study as well as to improve stability of 4-aryl-4H-
chromenes under acidic conditions. We now wish to
report the SAR at the 2 and 3-positions of 4-aryl-
4H-chromenes.
The 2-succinimido 4H-chromene 2b was prepared via
reaction of amine 2a with succinyl chloride in pyridine.
Similarly, 2-ureido 2c was prepared via reaction of 2a
with phenyl isocyanate in pyridine (Scheme 1).
2-H analogs were prepared in a one-pot reaction via
diazotization of the amino group using t-butylnitrite
(t-BuNO2)16 in THF, followed by reduction of the dia-
zonium group using NaBH4, as shown for the prepara-
tion of 3c (Scheme 2).17 2-H analog 2d was prepared
OMe
MeO
Br
1) tBuNO2/THF, 2.5 h
2) NaBH4, overnight
3a
CN
H
40% yield
O
HN
3c
Scheme 2.
OMe
MeO
O
Br
ClCOCH2CH2COCl,
3 days, 10% yield
or PhNCO, 6 h,
2b, R =
N
23% yield
2a
CN
R
O
pyridine
O
2c, R = HN
NHPh
N
O
Scheme 1.

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 603–607
605
3d, with an EC50 of 29 nM, is still highly active but
about 10-fold less active than the corresponding 2-
NH2 analog 3b.
OMe
MeO
Br
MeI or EtBr,
Cs2CO3/THF,
2.5 days
We also explored the replacement of the 3-CN group by
an ester group. The 3-methyl ester 3f and 3-ethyl ester 3g
were found to have low activity with EC50 values of 1–
3 lM, which is >200-fold less active than the corre-
sponding 3-CN analog 3a. These data suggest that the
3-CN group is important for the activity and that there
might be a size limited pocket around the 3-position.
CN
H
3c
97% yield
R3
O
N
3d, R3 = Me
3e, R3 = Et
The activities of these compounds toward the colon can-
cer cells HCT116 and the hepatocellular carcinoma can-
cer cells SNU398 were roughly parallel to their activity
toward T47D cells. In general, both were slightly less
sensitive to these compounds than T47D cells in this as-
say as indicated by the slightly higher EC50 value.
Scheme 3.
from the corresponding amino compound 2a using a
similar procedure. 2-H analogs 3d–3e were prepared,
respectively, from reaction of 3c with iodomethane
and bromoethane in THF in the presence of Cs2CO3
(Scheme 3).18
Selected compounds were also tested by the traditional
inhibition of cell growth (GI50) assay to confirm that
the active compounds can inhibit tumor cell growth.
The growth inhibition assays in T47D, HCT116, and
SNU398 cells were run in a 96-well microtiter plate as
described previously.19 The GI50 are summarized in
Table 2. Compound 3d was found to be highly active
with GI50 values of 4–6 nM in the three cell lines tested.
Similar to what is observed in the caspase activation as-
say, compound 3g was found to have low activity in the
growth inhibition assay, suggesting a good correlation
between these two assays.17
The 3-ester 3f was prepared as shown in Scheme 4 by a
one-pot reaction of 3-bromo-4,5-dimethoxybenzalde-
hyde, 1H-indol-4-ol, and methyl 2-cyanoacetate in etha-
nol in the presence of piperidine, using a procedure
similar to the preparation of 3-cyano analogs such as
3a.8 Ester 3g was prepared similarly using ethyl 2-cya-
noacetate instead of methyl 2-cyanoacetate (Scheme 4).
The apoptosis inducing activity of these 4-aryl-4H-
chromenes was measured by our cell- and caspase-based
HTS assay19 in human breast cancer cells T47D, human
colon cancer cells HCT116, and hepatocellular carci-
noma cancer cells SNU398. The results are summarized
in Table 1. Starting from our lead compound 2a, we first
explored the effects of converting the 2-amino group
into an imide and urea group (Table 1). Succinimide
2b and urea 2c were 13- and 4-fold less active than 2a,
suggesting that the amino group might be important
for activity or the 2-position is size limited.
Since compound 3d was found to have good activity in
both the caspase activation assay and the growth inhibi-
tion assay, and the 2-NH2 group has been reported to
contribute to the instability of these compounds under
acidic conditions,13 we compared the stability of 2-H
analog 3d versus 2-NH2 analog 3b in acidic solution.
When a solution of the 2-amino compound 3b in
DMSO-d6 and D2O (v/v:8/2) with a drop of TFA (pH
1
4) was monitored by H NMR, it was found that after
the solution was stored at room temperature overnight,
all the NMR signals became multi-peaks, suggesting
that the compound undergoes rapid decomposition un-
der acidic conditions, similar to what had been reported
for structurally related compound 4b.13 In comparison,
a solution of the corresponding 2-H analog 3d in
We then explored the conversion of the 2-amino group
into a 2-H group. Compound 2d was about 4-fold less
active than 2a, suggesting that the 2-amino group is
important but not essential for the apoptosis inducing
activity of these compounds. Similarly, 2-H analogs
3c–3e were found to have good activity. Compound
OMe
MeO
Br
OMe
Ethanol,
piperidine,
over night
CO2R
MeO
Br
+
OH
+
HN
CO2R
NH2
CN
14-19% yield
CHO
O
HN
3f, R = Me
3g, R = Et
Scheme 4.

606
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 603–607
Table 1. SAR of 2- and 3-positions of 4-aryl-chromenes in the caspase activation assay
OMe
OMe
MeO
Br
MeO
Br
CN
R1
R2
R1
R3
N
O
O
N
2
3
R1
R2
R3
EC50 (lM)
a
Entry
T47D
HCT116
SNU398
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
NH2
Succinimido
NA
NA
NA
NA
CN
CN
CN
CN
CN
NA
NA
NA
NA
H
0.019 0.004b
0.25 0.01
0.009 0.0004d
0.42 0.03
0.019 0.0007d
0.19 0.03
NHCONHPh
H
0.068 0.001
0.065 0.002
0.005 0.0003c
0.002 0.0003d
0.047 0.005
0.029 0.002
0.070 0.014
1.4 0.5
0.084 0.008
0.093 0.013
0.014 0.002d
0.003 0.0004d
0.066 0.006
0.038 0.003
0.15 0.04
0.093 0.007
0.077 0.018
0.015 0.001d
0.002 0.0004d
0.052 0.003
0.034 0.004
0.087 0.017
1.0 0.4
NH2
NH2
H
Me
H
H
Me
Et
H
NH2
NH2
CO2Me
CO2Et
H
0.66 0.27
3.0 0.2
3g
H
3.2 0.3
2.2 0.2
a Data are means of three or more experiments and are reported as means standard error of the mean (SEM).
b Data from Ref. 8.
c Data from Ref. 10.
d Data from Ref. 11.
pounds in this series that are less active in the caspase
activation assay also were found to be less active in
the tubulin polymerization assay, confirming that tubu-
lin inhibition is the main mechanism of action of these
substituted chromenes.20
Table 2. Inhibition of cell growth of 4-aryl-chromenes
a
Compound
GI50 (lM)
T47D
HCT116
SNU398
2d
3c
3d
3g
0.066 0.012
0.073 0.011
0.006 0.001
3.3 0.4
0.051 0.003
0.053 0.005
0.006 0.001
4.3 0.5
0.017 0.001
0.034 0.014
0.004 0.001
1.6 0.1
In conclusion, we have explored the modification of the
2- and 3-positions of apoptosis inducing 4-aryl-4H-
chromenes as potential anticancer agents. It was found
that replacing the 3-CN group by an ester resulted in a
large drop of activity. Modification of the 2-NH2 group
into amide and urea resulted in 4- to 10-fold reduction
of activity. Converting the 2-NH2 group into a 2-H
group was found to be relatively well tolerated, with
only a 4- to 10-fold reduction of activity. Importantly,
the 2-H analog 3d was found to have good activity with
an EC50 value of 29 nM and a GI50 value of 6 nM in
T47D cells. Moreover, 3d has shown high stability under
acidic conditions in comparison to the 2-NH2 analog 3b,
confirming that the 2-NH2 group is responsible for the
instability of this class of compounds under acidic con-
ditions and suggesting that the 2-H analogs might have
improved oral bioavailability.
a Data are means of three or more experiments and are reported as
means standard error of the mean (SEM).
DMSO-d6/D2O/TFA under the same conditions was
found to be stable after 8 days, indicating that 3d with
a hydrogen at the 2-position was highly stable under
acidic conditions. Our results confirmed the early report
that the 2-amino group was responsible for the instabil-
ity of this class of compounds under acidic conditions
and suggested that the 2-des-amino analogs offer a
straightforward way to address the instability problems,
thus improving the bioavailability of this class of potent
apoptosis inducers.
We have reported that compound 2a and related com-
pounds are tubulin inhibitors that bind at or close to
the colchicine site of b-tubulin.6 Compounds 3c and 3d
were tested in the tubulin polymerization assay. Similar
to 2a, both compounds were found to inhibit tubulin
polymerization with IC50 values of less than 500 nM.
Therefore, converting the 2-amino group into a 2-H
group does not change the mechanism of action of these
compounds, which is induction of apoptosis through
inhibition of tubulin polymerization. In addition, com-
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stirred overnight (15 h). The THF solvent was evaporated
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1
solid (0.39 g, 40%): H NMR (CDCl3, 300 MHz) d 8.30 (bs,
1H), 7.45 (m, 1H), 7.24 (m, 1H), 7.13 (m, 1H), 6.96 (d,
J = 2.1 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 6.70 (d, J = 8.1 Hz,
1H), 6.67 (m, 1H), 4.81 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H).
18. Experimental procedure for the synthesis of 4-(3-bromo-
4,5-dimethoxy-phenyl)-3-cyano-4,7-dihydro-7-methyl-pyr-
ano[2,3-e]indole (3d). A mixture of 4-(3-bromo-4,5-dime-
thoxy-phenyl)-3-cyano-4,7-dihydro-pyrano[2,3-e]indole (51
mg, 0.12 mmol), iodomethane (23 lL, 0.37 mmol), and
cesium carbonate (120 mg, 0.37 mmol) in THF (2 mL) was
stirred at room temperature in a sealed tube for 2.5 days.
The reaction mixture was diluted with EtOAc (20 mL).
The mixture was filtered, and the solid was washed with
EtOAc (30 mL). The EtOAc solution was combined,
washed with brine (2· 5 mL), dried over MgSO4, and
evaporated to give an off-white solid. It was purified by
column chromatography (silica gel, EtOAc:hexanes/1:1) to
give 3d as an off-white solid (51 mg, 97%): 1H NMR
(CDCl3, 300 MHz) d 7.43 (m, 1H), 7.07 (d, J = 3.3 Hz,
1H), 7.05 (m, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.76 (d,
J = 2.4 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.58 (m, 1H),
4.81 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H).
19. Cai, S. X.; Nguyen, B.; Jia, S.; Herich, J.; Guastella, J.;
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17. Experimental procedure for the synthesis of 4-(3-bromo-
4,5-dimethoxy-phenyl)-3-cyano-4,7-dihydro-pyrano[2,3-
e]indole (3c). To a solution of 2-amino-4-(3-bromo-4,5-
20. For example, compound 3a, with an EC50 value of 5 nM
in the caspase activation assay, was found to have an IC50
value of 500 nM in the tubulin polymerization assay. In
comparison, the 4-phenyl analog of 3a, 2-amino-4-phenyl-
3-cyano-4,7-dihydro-pyrano[2,3-e]indole, with an EC50
value of 270 nM in the caspase activation assay, was
found to have an IC50 value of 3000 nM in the tubulin
polymerization assay.