Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2. Structure-activity relationships of the 7- and 5-, 6-, 8-positions

Article abstract of DOI:10.1016/j.bmcl.2005.07.066

As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the SAR of 4-aryl-4H-chromenes with modifications at the 7- and 5-, 6-, 8-positions. It was found that a small hydr

Full text of DOI:10.1016/j.bmcl.2005.07.066

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4745–4751
Discovery of 4-aryl-4H-chromenes as a new series
of apoptosis inducers using a cell- and caspase-based
high-throughput screening assay. 2. Structure–activity
relationships of the 7- and 5-, 6-, 8-positions
William Kemnitzer,a Shailaja Kasibhatla,a Songchun Jiang,a Hong Zhang,a
a
a
a
a
b
´
Jianghong Zhao, Shaojuan Jia, Lifen Xu, Candace Crogan-Grundy, Real Denis,
Nancy Barriault,b Louis Vaillancourt,b Sylvie Charron,b Jennifer Dodd,b
Giorgio Attardo,b Denis Labrecque,b Serge Lamothe,b Henriette Gourdeau,b
Ben Tseng,a John Drewea and Sui Xiong Caia,*
aMaxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
bShire Biochem Inc., 275 Armand-Frappier Blvd., Laval, Que., Canada H7V 4A7
Received 8 June 2005; revised 25 July 2005; accepted 25 July 2005
Available online 6 September 2005
Abstract—As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as novel anticancer
agents, we explored the SAR of 4-aryl-4H-chromenes with modifications at the 7- and 5-, 6-, 8-positions. It was found that a small
hydrophobic group, such as NMe2, NH2, NHEt, and OMe, is preferred at the 7-position. Di-substitution at either the 5,7-positions
or the 6,7-positions generally led to a large decrease in potency. Di-substitution at the 7,8-positions, in general, was found to result
in potent compounds. 7-NMe2, 7-NHEt, 7-OMe, and 7,8-di-NH2 analogs were found to have similar SAR for the 4-aryl group, and
several 7-substituted and 7,8-di-substituted analogs were found to have similar potencies as the lead compound MX58151 (2a) both
as caspase activators and inhibitors of cell proliferation.
Ó 2005 Elsevier Ltd. All rights reserved.
Apoptosis, or programmed cell death, is a well-controlled
process for the elimination of excessive cells that involves
a series of precisely regulated events.1 During apoptosis,
various characteristic changes occur within the cells,
including condensation of the nucleoplasm and cyto-
plasm, chromosomal DNA fragmentation, and the for-
mation of membrane-bound apoptotic bodies, which
are rapidly recognized and eliminated by adjacent cells.2
The mechanism of apoptosis involves a cascade of initia-
tor and effector caspases that are activated sequentially.3,4
Key effector caspases have been identified including cas-
pase-3, caspase-6, and caspase-7, that cleave multiple pro-
tein substrates in cells leading to irreversible cell death.5,6
It has been determined that many of the clinically useful
cytotoxic agents induce apoptosis in cancer cells.7,8 The
proapoptotic chemotherapeutic agents that target tubu-
lin (taxanes consisting of Taxol and Taxotere, and vinca
alkaloids consisting of vincristine, vinblastine, and vino-
relbine) are among the most successful and commonly
prescribed anticancer therapies. The emergence of
drug-resistant tumor cells, as well as dose-limiting neu-
rologic and bone marrow toxicity, however has limited
the use of tubulin targeting agents. The colchicine bind-
ing site, located on the monomeric unpolymerized a/b-
tubulin that is different from the binding sites of taxanes
and vinca alkaloids, represents another potential tubulin
target for the development of proapoptotic chemothera-
peutic agents. Recently, several tubulin inhibitors were
also shown to disrupt the tumor vasculature by targeting
endothelial cells, while sparing the normal vascula-
ture.9,10 Two such vascular targeting agents, combre-
tastatin A-4 phosphate prodrug (CA-4P) and ZD6126
(Chart 1), both destabilize tubulin by binding at the
Keywords: 4-Aryl-4H-chromenes; Apoptosis inducers; High-through-
put screening; Structure–activity relationships; Cell- and caspase-
based.
*
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@maxim.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.066

4746
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4745–4751
MeO
MeO
MeO
NHAc
MeO
OMe
OMe
OP(O)(ONa)2
OP(O)(ONa)2
OMe
ZD6126
(CA-4P)
OMe
O
O
MeO
Br
OMe
CN
CN
Me2N
O
2a
NH2
Me2N
O
NH2
1a
Chart 1.
Ar
CN
CN
CN
a
Ar-CHO
R
R
OH
O
NH2
Scheme 1. Reagents and conditions: (a) EtOH, piperidine, rt.
colchicine site and are currently undergoing clinical tri-
als.11,12 Compounds that induce apoptosis in cancer
cells by targeting the clinically validated tubulin/micro-
tubule system, while retaining activity in multi-drug-
resistant tumors, have the potential to offer new
treatment options in the field of oncology.13–15
our efforts to develop the apoptosis inducing 4-aryl-
4H-chromenes as potential anticancer therapeutics, we
now wish to report the SAR of 4-aryl-4H-chromenes
(1–5) with modifications at the 7- and 5-, 6-, 8-positions.
The substituted 4H-chromenes were generally prepared
by a one-pot reaction of the commercially available
substituted aryl aldehydes and phenols with malononit-
rile in the presence of a base, such as piperidine, in good
yield (Scheme 1), according to methods previously de-
scribed.18 For compound 3i, the appropriate starting
material, 5-methoxypyridine-3-carboxaldehyde (6c),
was synthesized in two steps in 16% overall yield. First,
3,5-dibromopyridine (6a) was converted to the 5-bromo-
3-methoxypyridine (6b) by refluxing with NaOMe, and
then the aldehyde was formed by treatment of 6b with
n-BuLi and DMF (Scheme 2).
We have reported the discovery, from our cell- and
caspase-based high-throughput screening (HTS) assay,
that 4-aryl-4H-chromenes are a new series of potent
apoptosis inducing agents possessing vascular target-
ing activity. These compounds were found to be tubu-
lin destabilizers, binding at or close to the binding site
of colchicine. They were also active in drug-resistant
cancer cell lines including the paclitaxel-resistant, mul-
ti-drug-resistant MES-SA/DX5 tumor cells, and were
found to be highly active in several anticancer animal
models.16,17 The 4-aryl-4H-chromenes, therefore, rep-
resent a promising series of novel apoptosis inducers
that could be developed into new therapeutic antican-
cer agents.
The one-pot reaction does not proceed well when the
starting phenol is substituted with electron-withdrawing
groups. Thus, compounds 2g and 2h could not be syn-
thesized directly via the one-pot reaction on account
We have recently reported the structure–activity
relationship (SAR) of the 4-aryl group of 4-aryl-4H-
chromenes. Starting from our screening hit, 2-amino-3-
cyano-7-dimethylamino-4-(3-methoxy-4,5-methylenedi-
oxyphenyl)-4H-chromene (1a), by maintaining the
2-amino, 3-cyano, and 7-dimethylamino groups of 1a,
we have identified 2-amino-4-(3-bromo-4,5-dimethoxy-
phenyl)-3-cyano-7-dimethylamino-4H-chromene (2a) as
a lead compound (Chart 1).18 As a continuation of
OMe
Br
OMe
N
N
N
a
b
CHO
Br
Br
6c
6a
6b
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, reflux, 3
days; (b) n-BuLi, DMF, À78 °C.

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4745–4751
4747
of the presence of electron-withdrawing halogens at the
meta-position of the phenol ring. We explored the prep-
aration of compounds 2g and 2h from the diazotization
of the 7-amino group of compound 2b. To avoid
interference with the diazotization process by the 2-ami-
no group, 2b was oxidized using 2,3-dichloro-5,6-dicy-
ano-1,4-benzoquinone to the imino-2H-chromene 7.
Diazotization of 7, followed by either bromination or
chlorination, introduced the desired electron-withdraw-
ing groups at the 7-position (8a and 8b). Reduction of
the imino-2H-chromenes by sodium borohydride then
yielded 2g and 2h in overall yields of 14 and 10%, respec-
tively (Scheme 3).
active than 1g, suggesting that a hydrophilic group is not
preferred at the 7-position.
We then explored substitution at the 7-position in com-
bination with substitution at either the 5-, 6-, or 8-posi-
tion (Table 1). The 5,7-dimethoxy analog 1i was not
active up to 10 lM and is >60-fold less active than 7-
methoxy analog 1g, indicating that a substituent at the
5-position is not tolerated. Interestingly, it has been ob-
served that an ortho-substituent at the 4-phenyl group
also resulted in a large reduction of activity.18 This is
probably because a substituent at the 5-position, as well
as an ortho-substituent at the 4-phenyl group, causes the
4-aryl group to rotate into an unfavorable position for
interaction with its target. The 6,7-dimethoxy analog
1j was also found to be inactive up to 10 lM and is
>60-fold less than the 7-methoxy analog 1g, indicating
that a substituent at the 6-position is not preferred.
The 6-methyl-7-ethylamino analog 1k was about 4-fold
less active than the 7-ethylamino analog 1c, suggesting
that a smaller group at the 6-position is more tolerated.
Compound 1l, with the 6- and 7-positions cyclized into a
methylenedioxy ring, thus limiting the size of the group
at the 6-position, was almost as potent as 1g, confirming
that a smaller group is more tolerated at the 6-position.
The 7-amino-8-methyl analog 1m was almost 4-fold
more potent than the 7-amino analog 1b, suggesting that
a substituent at the 8-position might be beneficial for
activity.
The apoptosis inducing activity of these 4-aryl-4H-
chromenes was measured by our cell- and caspase-based
HTS assay18 in two cell lines, human breast cancer cells
T47D and human non-small cell lung cancer cells
H1299, andthe results are given in Tables 1 and 2. Starting
from our screening hit 1a, by maintaining the 2-amino,
3-cyano, and 4-(3-methoxy-4,5-methylenedioxyphenyl)
groups, we first explored the effects of various substituents
at the 7-position (Table 1). The 7-amino analog 1b was
about 16-fold less potent than the 7-dimethylamino
analog 1a, suggesting that a slightly large and more
hydrophobic dimethylamino group at the 7-position is
important for the apoptosis inducing activity. The 7-eth-
ylamino analog 1c was about 3-fold more potent than 1b,
supporting the notion that a small and hydrophobic
group at the 7-position is important for activity. The 7-
diethylamino analog 1d was about 1.5-fold and 6-fold less
potent than 1c and 1a, respectively, suggesting that a large
group is not preferred at the 7-position. Compounds 1e
and 1f, with large phenylamino and N-morpholino
groups at the 7-position, were not active up to 10 lM
and are >100-fold less active than 1a, confirming that a
large group is not tolerated at the 7-position. The 7-meth-
oxy analog 1g had about half the activity of 1a, confirming
that a small and hydrophobic group is preferred at the 7-
position. The 7-hydroxy analog 1h was about 36-fold less
Based on these results, as well as our earlier results that
2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-
dimethylamino-4H-chromene (2a) is a lead compound
and 3-bromo-4,5-dimethoxyphenyl is one of the best
aryl groups at the 4-position in this series of analogs,18
we decided to concentrate our exploration efforts on
the combination of 3-bromo-4,5-dimethoxyphenyl at
the 4-position with different substituents at the 7-posi-
tion, as well as substituents at both 7- and 8-positions
(Table 1). Interestingly, 7-NH2 analog 2b was only
OMe
OMe
MeO
Br
MeO
Br
O
O
NC
NC
Cl
Cl
a
+
CN
CN
NH
H2N
O
NH2
H2N
O
2b
7
OMe
OMe
MeO
Br
MeO
Br
c
b
CN
CN
NH
X
O
NH2
X
O
2g, X = Br
2h, X = Cl
8a, X = Br
8b, X = Cl
t
Scheme 3. Reagents and conditions: (a) CH2Cl2/rt; (b) BuONO, CH3CN, CuBr2 or CuCl2, 0°C; (c) NaBH4, THF, rt.

4748
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4745–4751
Table 1. SAR of 4-(3-methoxy-4,5-methylenedioxyphenyl)- and 4-(3-bromo-4,5-dimethoxyphenyl)-2-amino-3-cyano-4H-chromenes in the caspase
activation assay
OMe
O
O
MeO
R5
Br
OMe
R5
R6
R7
CN
R6
R7
CN
O
NH2
O
NH2
R8
R8
2
1
Entry
R5
R6
R7
R8
EC50 (lM)a
T47D
H1299
1a
1b
1c
1d
1e
1f
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NMe2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
0.073 0.006b
1.2 0.03
0.33 0.06
0.48 0.06
>10
0.093 0.012b
1.7 0.09
0.40 0.04
0.53 0.01
>10
H
H
NHEt
NEt2
NHPh
Mopc
OMe
OH
H
H
H
>10
>10
1g
1h
1i
H
0.16 0.02
5.8 0.4
0.23 0.03
5.6 0.4
H
H
OMe
OMe
NHEt
>10
>10
>10
>10
1j
OMe
Me
1k
1l
1.1 0.03
0.21 0.01
0.31 0.01
0.019 0.004b
0.033 0.002
0.014 0.001
0.017 0.001
0.064 0.010
0.13 0.01
0.14 0.01
0.16 0.02
0.034 0.005
0.026 0.004
0.042 0.002
0.061 0.005
1.7 0.18
1.4 0.09
0.27 0.02
0.77 0.06
0.043 0.001b
0.067 0.005
0.027 0.002
0.034 0.002
0.092 0.004
0.21 0.01
0.28 0.02
0.29 0.02
0.081 0.014
0.053 0.007
0.087 0.008
0.18 0.034
2.5 0.28
OCH2O
1m
2a
2b
2c
2d
2e
2f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH2
NMe2
NH2
NHEt
OMe
OEt
OH
2g
2h
2i
Br
Cl
NH2
NH2
Me
NH2
Me
2j
2k
2l
Me
OH
OH
NH2
OH
2m
a Data are means of three or more experiments and are reported as mean standard error of the mean (SEM).
b Data from Ref. 18.
c Mop, N-morpholino.
slightly less active than 2a. This result contrasts with the
16-fold decrease in potency that was observed between
1a and 1b. In addition, the 7-ethylamino and 7-methoxy
analogs 2c and 2d were about as potent as 2a. These re-
sults suggest that the 3-bromo-4,5-dimethoxyphenyl at
the 4-position reduced the effects of substituents at the
7-position. Analog 2e, with a slightly larger 7-ethoxy
group, was about 4-fold less active than 2d, confirming
that a smaller group at the 7-position is preferred. The
7-hydroxy analog 2f was >7-fold less active than 2d,
confirming that a hydrophilic group is not preferred at
the 7-position. The 7-Br and 7-Cl analogs 2g and 2h
both were >7-fold less potent than 2d, indicating that
an electron-withdrawing group is less preferred than
the electron-donating group at the 7-position. The 7,8-
diamino analog 2i is a potent compound with an EC50
value of 34 nM, being only slightly less active than 2a.
The extra amino group at the 8-position is also expected
to increase the aqueous solubility of the compound. The
7-amino-8-methyl analog 2j and 7,8-dimethyl analog 2k
were also highly active with potency around 30–40 nM,
confirming that the presence of a substituent at the 8-po-
sition is not detrimental to activity. The 7-hydroxy-8-
amino analog 2l was about 2-fold more active than the
7-hydroxy analog 2f, confirming that a substituent at
the 8-position is beneficial for activity. The 7,8-dihy-
droxy analog 2m was about 20-fold less active than 2l,
and is the least potent 7,8-disubstituted analog pre-
pared, indicating that the hydrophilic hydroxy group is
not tolerated at the 8-position.
To determine whether modification of the 7-position
changes the SAR of the 4-aryl group, the 7-OMe,
7-NHEt, and the 7,8-diamino groups were selected to
explore different aryls at the 4-position (Table 2). For
the analogs with a 7-OMe group, similar to what was

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4745–4751
4749
Table 2. SAR of 7-methoxy-, 7-ethylamino-, and 7,8-diamino-4-aryl-2-amino-3-cyano-4H-chromenes in the caspase activation assay
R4
R4
R4
R5
R3
R5
R3
R5
R3
B
CN
CN
CN
MeO
O
NH2
EtHN
O
NH2
H2N
O
NH2
NH2
3
4
5
Entry
B
R3
R4
R5
EC50 (lM)a
T47D
H1299
3a
3b
3c
3d
3e
3f
C
C
C
C
C
C
C
N
N
C
C
C
C
C
C
C
C
C
C
C
C
OMe
OMe
OMe
Br
OMe
H
OMe
OMe
H
0.026 0.002
0.015 0.001
0.052 0.004
0.052 0.004
0.080 0.012
0.089 0.012
0.36 0.06
0.057 0.003
0.032 0.004
0.089 0.012
0.095 0.014
0.16 0.02
0.17 0.01
0.52 0.01
0.29 0.03
0.10 0.01
0.11 0.01
0.12 0.02
0.19 0.02
0.19 0.03
0.17 0.02
0.058 0.004
0.10 0.01
0.062 0.006
0.19 0.01
0.46 0.04
0.23 0.06
0.42 0.05
H
H
H
Cl
H
H
NO2
H
H
H
3g
3h
3i
H
H
H
H
H
0.17 0.02
OMe
OMe
OMe
OMe
Cl
H
H
0.047 0.009
0.049 0.006
0.055 0.010
0.11 0.01
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
OMe
H
OMe
OMe
H
H
H
H
0.12 0.01
0.11 0.01
NO2
Cl
H
H
OMe
OMe
OH
H
OMe
OMe
OMe
OMe
H
0.024 0.001
0.049 0.006
0.023 0.003
0.092 0.007
0.39 0.06
I
Br
OMe
CN
Br
H
H
H
0.15 0.01
0.39 0.05
5g
NO2
H
H
a Data are means of three or more experiments and are reported as mean standard error of the mean (SEM).
observed for analogs with a 7-NMe2 group,18 the 3,4,5-
trimethoxyphenyl analog 3a was only slightly less active
than 2d, the analog with the potent 3-bromo-4,5-dimeth-
oxyphenyl group at the 4-position. The 3,5-dimethoxy-
phenyl analog 3b and 3-substituted-phenyl analogs
3c–f all had good activity ranging from 15 to 89 nM.
The non-substituted phenyl analog 3g was much less ac-
tive, confirming the importance of a substituent at the
phenyl ring, especially at the 3-position. The 3-pyridyl
analog 3h was slightly more potent that the phenyl ana-
log 3g and the 5-methoxy-3-pyridyl analog 3i was about
as potent as the 3,4,5-trisubstituted-phenyl analog 3a,
confirming our previous observation that a 5-substitut-
ed-3-pyridyl has potency similar to that of a 3,4,5-trisub-
stituted-phenyl at the 4-position.18
analog 5b were about as potent as 2i, as expected from
our SAR studies in the 7-NMe2 series that the 3-bromo
group can be replaced by a chloro or iodo group.18
Analog 5c was about as potent as 2i, indicating that
3-bromo-4-hydroxy-5-methoxyphenyl group is a good
replacement for the 3-bromo-4,5-dimethoxyphenyl
group, similar to what was reported previously.18 More-
over, the hydroxy group in 5c might improve the solubil-
ity profile, as well as provide a useful handle for the
preparation of a phosphate prodrug, similar to what
has been successfully done in the case of combretastatin
A-4 phosphate prodrug.11 The 3,5-dimethoxyphenyl
analog 5d also had good activity. As expected, the
3-substituted analogs 5e–g were less active than the
3,4,5-trisubstituted analogs.
With an NHEt group at the 7-position, the 3,4,5-tri-
methoxyphenyl analog 4a and the 3,5-dimethoxyphenyl
analog 4b were slightly less potent than 2e, the one with
the potent 4-(3-bromo-4,5-dimethoxyphenyl) group.
Analogs 4c–e, with a 3-substituted-phenyl group at the
4-position, all had good activity with EC50 values of
around 100 nM, slightly less potent than the 3,4,5-tri-
substituted and 3,5-disubstituted analogs.
The activities of these compounds toward the human
non-small cell lung cancer cell line H1299 were roughly
parallel to their activity toward T47D cells. In general,
H1299 cells were slightly less sensitive (about 2-fold less
sensitive as measured by the EC50 value) to the com-
pounds than T47D cells in this assay.
Selected compounds were also tested by the traditional
inhibition of cell proliferation (GI50) assay to confirm
that the active compounds can inhibit tumor cell
growth. The growth inhibition assays in T47D and
In the 7,8-diamino series, the 3-chloro-4,5-dimethoxy-
phenyl analog 5a and the 3-iodo-4,5-dimethoxyphenyl

4750
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4745–4751
Table 3. Comparison of caspase activation activity and inhibition of cell proliferation activity of 4-aryl-2-amino-3-cyano-4H-chromenes
Entry
T47D
H1299
a
b
a
b
EC50 (lM)
GI50 (lM)
GI50/EC50
EC50 (lM)
GI50 (lM)
GI50/EC50
1b
1g
1h
1i
1.2 0.03
0.16 0.02
5.8 0.35
2.4 0.52
0.26 0.06
6.08 0.99
>10
2.1
1.6
1.0
1
1.7 0.08
0.23 0.025
5.63 0.38
>10
1.2 0.17
0.24 0.036
5.50 0.76
>10
0.74
1.0
0.98
1
>10
>10
1j
>10
1
>10
>10
1
1.1
1m
2a
2b
2d
2f
0.31 0.01
0.019 0.004
0.033 0.002
0.017 0.001
0.13 0.01
0.16 0.02
0.034 0.005
0.061 0.005
0.047 0.009
0.49 0.08
0.092 0.014
0.077 0.015
0.008 0.002
0.80 0.19
0.064 0.014
0.15 0.02
0.12 0.06
0.065 0.009
1.6
4.8
2.3
0.47
6.1
0.4
4.4
2.0
1.4
0.77 0.06
0.043 0.001
0.067 0.005
0.034 0.002
0.21 0.01
0.29 0.02
0.081 0.014
0.18 0.03
0.10 0.01
0.86 0.16
0.029 0.010
0.076 0.013
0.009 0.003
0.19 0.01
0.055 0.002
0.031 0.001
0.11 0.047
0.072 0.007
0.67
1.1
0.26
0.90
0.19
0.38
0.61
0.72
2h
2i
2l
3i
a From Table 1.
b Data are means of three experiments and are reported as mean standard error of the mean (SEM).
HA14-1 is not active up to 10 lM in our apoptosis
induction assay in several cell lines including T47D
and DLD-1.
O
N
O
O
Br
O
In conclusion, we have explored the SAR of the
apoptosis inducing 4-aryl-4H-chromenes as potential
anticancer agents via modifications at the 7- and 5-,
6-, 8-positions. It was found that small hydrophobic
groups, such as NMe2, OMe, or NHEt, are preferred
at the 7-position, while larger groups, such as NHPh, re-
sult in significant reduction of apoptotic activity. Inter-
estingly, disubstitution at the 7,8-positions, such as
7,8-diamino or 7,8-dimethyl, results in potent com-
pounds. Disubstitution at either the 5,7-positions or
the 6,7-positions generally results in a drastic decrease
in activity, with the exception of the cyclic 6,7-methylen-
edioxy analog that retains good potency. The 7-NMe2,
7-NHEt, 7-OMe, and 7,8-di-NH2 analogs were found
to have similar SAR for the 4-aryl group. Several 7-
substituted and 7,8-di-substituted analogs, such as 2b–
d, 2i–l, 3a–b, and 5a–c, were found to be as potent as
the lead compound 2a. In addition, some of these com-
pounds offer better potential for aqueous solubility, par-
ticularly 2i and 5a–c. Compound 5c also provides a
handle for the preparation of prodrugs.
O
NH2
HA14-1
Chart 2.
H1299 cells were run in a 96-well microtiter plate, as de-
scribed previously.18 The GI50, along with the EC50 data
and the ratio of GI50/EC50, are given in Table 3. Results
show that 2a, 2b, 2d, 2h, and 3i are the most potent
inhibitors of tumor cell growth among the compounds
tested. Compound 2d had a GI50 value of 0.008 and
0.009 lM in T47D and H1299 cells, respectively. The
GI50/EC50 ratio for most of these compounds was >1
or close to 1 both with T47D and H1299 cells, with a
few exceptions. For example, the GI50/EC50 ratio for
compounds 2d and 2h was less than 0.5 for both cell
lines, indicating that both compounds are more potent
in the growth inhibition assay than in the caspase assay.
Table 3 also shows that in general, the compound that is
more active in the apoptosis induction assay, as mea-
sured by caspase activation, is also more potent in the
growth inhibition assay.
References and notes
We have reported that compound 2a and related com-
pounds are tubulin inhibitors that bind at or are close
to the colchicine site of b-tubulin.18 Compounds 2b
and 2i were tested in the tubulin polymerization assay.
Both compounds were found to inhibit tubulin polymer-
ization with IC50 values less than 500 nM, similar to 2a.
Therefore, modification at the 7- and 8-positions does
not change the mechanism of action of these com-
pounds, that is induction of apoptosis through inhibi-
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