Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2. Structure-activity relationships of the 9-oxo-9H-fluorene ring

Article abstract of DOI:10.1016/j.bmcl.2009.11.025

As a continuation of our studies of apoptosis inducing 9-oxo-9H-fluorene-1-carboxamides as potential anticancer agents, we explored modification of the 9-oxo-9H-fluorene ring. SAR studies showed that most changes to the 9-oxo-9H-fluorene ring were not wel

Full text of DOI:10.1016/j.bmcl.2009.11.025

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1288–1292
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series
of apoptosis inducers using a cell- and caspase-based high-throughput
screening assay. 2. Structure–activity relationships of the
9-oxo-9H-fluorene ring
William Kemnitzer, Nilantha Sirisoma, Songchun Jiang, Shailaja Kasibhatla, Candace Crogan-Grundy,
*
Ben Tseng, John Drewe, Sui Xiong Cai
Epicept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation of our studies of apoptosis inducing 9-oxo-9H-fluorene-1-carboxamides as potential
anticancer agents, we explored modification of the 9-oxo-9H-fluorene ring. SAR studies showed that
most changes to the 9-oxo-9H-fluorene ring were not well tolerated, except the 9H-fluorene (2b) and
dibenzothiophene (2d) analogs, which were about twofold less active than the 9-oxo-9H-fluorene analog
2a. Significantly, introduction of substitutions at the 7-position of the 9-oxo-9H-fluorene ring led to com-
Received 12 October 2009
Revised 30 October 2009
Accepted 5 November 2009
Available online 12 November 2009
pounds 5a–5c with improved activity. Compound 5a was found to have EC50 values of 0.15–0.29 lM
Keywords:
against T47D, HCT116, and SNU398 cells, about fivefold more potent than the original lead 2a. As
opposed to the original lead compound 2a, compounds 5a–5b were active in a tubulin inhibition assay,
indicating a change of mechanism of action. The potent azido analog 5c could be utilized for target
identification.
Apoptosis inducers
Anticancer agents
SAR
Tubulin inhibition
Ó 2009 Published by Elsevier Ltd.
Apoptosis is a highly regulated process for the elimination of
excessive or damaged cells and plays an important role in normal
cell development and tissue homeostasis.1 It is well known that
inadequate apoptosis can lead to the over proliferation of cells
resulting in cancer, as well as cause resistance to cancer treat-
ment.2 The mechanism of apoptosis involves a cascade of initiator
and effector caspases that are activated sequentially. Among them,
caspase-3 has been identified as a key effector caspase that cleaves
multiple protein substrates in cells leading to cell death.3
Since it has been known that many clinically used anticancer
chemotherapeutic agents kill cancer cells at least partially through
induction of apoptosis,4 discovery of compounds that interact with
apoptosis regulators and promote or induce apoptosis could lead to
the development of novel anticancer agents.5,6 We therefore have
developed a cell- and caspase-based high-throughput screening
(HTS) technology called Anticancer Screening Apoptosis Program
(ASAP) using our novel fluorescent caspase-3 substrates for the dis-
covery of apoptosis inducers.7 The screening assay can be applied
to any cell type or cell line that expresses caspases. It can detect
any apoptosis inducer that triggers apoptosis at a point upstream
of caspase-3 activation by a caspase-mediated mechanism either
through known targets or novel targets.
Applying this assay, we have discovered and reported the struc-
ture–activity relationship (SAR) studies of several novel series of
apoptosis inducers, as well as the identification of novel molecular
targets.8 These include N-phenyl nicotinamides(1a),9 gambogic acid
(1b),10 4-aryl-4H-chromenes (1c),11 3-aryl-5-aryl-1,2,4-oxadiazoles
(1d),12 4-anilino-2-arylpyrimidines (1e),13 4-anilinoquinazolines
(1f),14 N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines (1g),15 N-
methyl-N-phenylnaphthalen-1-amines (1h),16 and 1-benzoyl-3-
cyanopyrrolo[1,2-a]quinolines (1i)17 (Chart 1). More recently, we
have reported the discovery and SAR study of N-aryl-9-oxo-9H-flu-
orene-1-carboxamides such as 2a as a novel series of apoptosis
inducers.18 SAR studies of the 1-carboxamide group showed that
substitution at the 2-position of the phenyl group are preferred for
apoptosis-inducing activity. Substitution at the 3- or 4-positions of
thephenylgroup, or replacement of thephenylgroupwith heterocy-
cles was not tolerated. Importantly, substitution at the 2-position of
the phenyl group with an N,N-dimethylamino, N,N-dimethylme-
thanamine or pyrazolyl group led to potent compounds with en-
hanced aqueous solubility properties.18 Herein, we wish to report
modification of the 9-oxo-9H-fluorene ring of N-aryl-9-oxo-9H-flu-
orene-1-carboxamides as apoptosis inducers.
Compounds 2b–2d, 2g–2i, and 5a (Tables 1 and 3) were pre-
pared in two steps using previously reported procedures18 starting
from the corresponding carboxylic acids (6a–6g), which was con-
verted to the carbonyl chlorides (7a–7g) by reaction with oxalyl
* Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202 4000.
E-mail address: scai@epicept.com (S.X. Cai).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.11.025

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1288–1292
OMe
1289
O
MeO
Br
OH
CF3
N
OMe
OMe
Cl
NO2
O
N
H
N
CF3
N
O
O
O
O
CN
N
O
N
N
H
S
O
N
O
O
1c
NH2
OEt
N
OH
1a
1b
1d
1e
OMe
OMe
CN
O
N
N
HN
N
O
N
O
N
H
N
N
N
N
Me
N
N
N
N
NMe2
1f
1g
1h
1i
2a
Chart 1.
chloride in DMF. Reaction of 7a–7g with 2-(1H-pyrazol-1-yl)ben-
zenamine produced the target compounds (Scheme 1). Among
the carboxylic acids, 9H-fluorene-1-carboxylic acid (6a), 6,7,8,9-
tetrahydro-5H-carbazole-1-carboxylic acid (6d), 2-benzoylbenzoic
acid (6e), and 2,3-dihydrobenzofuran-7-carboxylic acid (6f) were
obtained commercially. Dibenzofuran-1-carboxylic acid (6b) and
dibenzothiophene-1-carboxylic acid (6c) were prepared from lith-
iations of dibenzofuran and dibenzothiophene as reported previ-
ously,19 while 7-nitro-9-oxo-9H-fluorene-1-carboxylic acid (6g)
was prepared from nitration of 9-oxo-9H-fluorene-1-carboxylic
acid according to literature procedures.20,21 Compounds 4a–4d
were synthesized similarly from reaction of 7a with substituted
anilines (Scheme 2).
3b, with a hydroxyl group at the 9-position, was >3-fold less active
than the oxo analog 3a against T47D cells, and was not active in the
other two cell lines up to 10 lM, confirming the importance of the
9-oxo-9H-fluorene ring. Overall, except for the 9H-fluorene (2b)
and dibenzothiophene (2d) analogs, most of the modifications to
the 9-oxo-9H-fluorene ring led to inactive compounds.
Since the 9H-fluorene analog 2b showed reasonable activity, we
explored the synthesis of several analogs of 2b with different sub-
stitutions in the phenyl group (Table 2). The 2-methyl analog 4a
was slightly more active than 2b against T47D cells, but was not
active in HCT116 and SNU398 cells up to 10
and 4c, with an additional methyl group at the 3- or 4-position,
was not active up to 10 M in all three cell lines, confirmed our
lM. Compounds 4b
l
Compounds 2e and 2f were prepared from oxidation of diben-
zothiophene analog 2d using mCPBA (Scheme 3). Compound 3b
was synthesized from reduction of 3a using sodium borohydride
(Scheme 4). 7-Amino analog 5b was prepared from hydrogenation
of 7-nitro analog 5a. Diazotization of 5b followed by treatment
with sodium azide produced the azido analog 5c (Scheme 5). The
bromo analog 5d was prepared from bromination of 5a (Scheme 6).
The apoptosis-inducing activity of these N-aryl-9-oxo-9H-fluo-
rene-1-carboxamide analogs was measured by our cell- and cas-
pase-based HTS assay9 against three cell lines, T47D breast
cancer cells, HCT116 human colon cancer cells and SNU398 hepa-
tocellular carcinoma cancer cells, and the results are summarized
in Tables 1–3. Since we have found previously that a pyrazolyl
group at the 2-position of the phenyl group leads to potent com-
pounds with enhanced aqueous solubility properties,18 we decided
to maintain this group and explore the replacement of the 9-oxo-
9H-fluorene ring by other structures. 9H-Fluorene analog 2b was
about 2–3-folds less active than 2a, suggesting that the 9-oxo
group might contribute to activity. The dibenzofuran analog 2c
previous observation that except for the 2-position, substitution
at the other positions of the phenyl group is not preferred.18 Inter-
estingly, the 2,5-dimethyl analog 4d was about as active as 2b
against T47D cells but was not active in HCT116 and SNU398 cells
up to 10 lM.
To explore SAR further and prepare a reagent for potential tar-
get identification, we synthesized compounds with substitution at
the 7-position of the 9-oxo-9H-fluorene ring (Table 3). Interest-
ingly, the 7-nitro analog 5a was >5-fold more potent than 2a in
all three cell lines. Compound 5a was about 10-fold less active than
reference compounds vinblastine and paclitaxel and all were
broadly active against the three cell lines tested. The 7-amino ana-
log 5b and 7-azido analog 5c also were more active than 2a. The
azido analog 5c was synthesized as a potential photoaffinity label-
ing agent. The bromo analog 5d, prepared as an intermediate for
the preparation of a tritium labeled photoaffinity labeling agent,
also had good activity. Applying methods that we have successfully
used to identify the molecular targets of the 3-aryl-5-aryl-1,2,4-
oxadiazoles12 and 4-anilinoquinazolines14 series of apoptosis
inducers, treatment of 5d with tritium gas under hydrogenation
conditions would result in the replacement of the bromo by a tri-
tium, as well as reduction of the nitro group to an amino group. The
amino group can then be converted to an azido group, which
would give a radioactive version of 5c that could be used for target
identification.
was not active up to 10 lM, supporting the notion that the 9-oxo
group is important for activity. Interestingly, the dibenzothiophene
analog 2d, with the slightly larger thiophene ring compared to that
of the furan in 2c, was only about twofold less active than that of
2a and at least >5-fold more active than 2c. However, the corre-
sponding oxo analog 2e was not active up to 10 lM, while the di-
oxo analog 2f was active only in SNU398 cells. The loss of activity
for 2e suggests that the oxo group might have to be in a planar po-
sition to maintain potency. The 6,7,8,9-tetrahydro-5H-carbazole
analog 2g, benzophenone analog 2h and 2,3-dihydrobenzofuran
analog 2i were all inactive, indicating that the tricyclic and aro-
matic 9-oxo-9H-fluorene ring is important for activity. Compound
Selected compounds were also tested by the traditional cell
growth inhibition assay (GI50) to confirm that the active com-
pounds can inhibit tumor cell growth. The growth inhibition assays
in T47D, HCT116, and SNU398 cells were run in a 96-well microti-
ter plate according to previously described procedures9 and the
GI50 values are summarized in Table 4. The 7-nitro analog 5a was

1290
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1288–1292
Table 1
Table 2
Activity of analogs of N-aryl-9-oxo-9H-fluorene carboxamide with modified ring in
Activity of N-aryl-9H-fluorene-1-carboxamides in the caspase activation assay
the caspase activation assay
R
5
4
R
R
O
N
H
O
N
H
3
O
N
H
Me
N
N
Me
4
3
2
a
Compound
R
EC50 (lM)
a
Compound
R
EC50
(
lM)
T47D
HCT116
SNU398
T47D
HCT116
SNU398
1.1 0.1
4a
4b
4c
4d
H
1.7 0.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3-Me
4-Me
5-Me
2ab
3ab
1.4 0.3
1.3 0.1
2.4 0.2
a
Cells were treated with the test compounds for 48 h, data are the mean of three
O
or more experiments and are reported as mean standard error of the mean (SEM).
0.98 0.13
1.1 0.2
0.64 0.04
Table 3
O
Activity of 7-substituted N-aryl-9-oxo-9H-fluorene-1-carboxamides in the caspase
activation assay
2b
2c
2d
3.2 0.7
>10
6.2 1.3
>10
6.9 1.1
>10
R1
O
O
N
H
O
S
N
N
2.1 0.2
2.2 0.2
1.5 0.2
R
5
Compound
R
R1
EC50
(l
M)
a
2e
>10
>10
>10
S
T47D
HCT116
SNU398
O
5a
5b
5c
5d
H
H
H
Br
NAb
NA
NO2
NH2
N3
NO2
NA
0.29 0.03
0.97 0.03
0.47 0.04
1.3 0.05
0.032 0.006
0.037 0.003
0.22 0.03
0.37 0.15
0.29 0.03
1.5 0.03
0.036 0.009
0.018 0.001
0.15 0.02
0.15 0.01
0.15 0.02
1.4 0.1
0.026 0.008
0.009 0.001
2f
>10
>10
>10
>10
0.48 0.08
>10
S
O
O
Vinblastine
Paclitaxel
NA
2g
N
H
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
b
NA, not applied.
2h
2i
>10
>10
>10
>10
>10
>10
>10
O
a
b
Ar
Ar
COCl
Ar
CO2H
>10
O
N
H
O
N
6a-g
7a-g
N
3b
3.0 0.3
2b-d, 2g-i, 5a
OH
Scheme 1. Reagents and conditions: (a) oxalyl chloride, CH2Cl2, DMF, 0 °C; (b) 2-
(1H-pyrazol-1-yl)benzenamine, CH2Cl2, Et3N, 0 °C.
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
b
Data from Ref. 18.
a
found to have good activity in T47D, HCT116, and SNU398 cells,
with GI50 values of 0.11, 0.42, and 0.072 lM, respectively. Com-
R
pound 5a was more active than 2a, especially in SNU398 cells.
The 7-amino analog 5b also had good activity and was about as ac-
tive as that of 5a. Compounds 5a and 5b were about 10-fold less
active than the reference compounds vinblastine and paclitaxel
in the three cell lines.
O
4a-4d
N
H
O
Cl
7a
Scheme 2. Reagents and conditions: (a) aniline, CH2Cl2, Et3N, 0 °C.
The apoptosis-inducing activity of compound 5b was character-
ized by cell cycle analysis. T47D cells were treated with 2 lM of
compound 5b for 24 h or 48 h at 37 °C. Cells were then stained
with propidium iodide and analyzed by flow cytometry. Figure 1A
shows that control cells (treated with solvent DMSO) were mostly
in the G1 phase of the cell cycle. A large increase in G2/M content
(from 15% to 60%) was observed after 24 h of treatment (Fig. 1A
vs 1B). After 48 h of treatment, sub-G1 population increased to
50% with an equivalent decrease in G2/M content, indicating tran-
sition of the cells to apoptosis (Fig. 1C).

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1288–1292
1291
a
A
S
S
O
2d
O
O
+
O
N
O
N
H
H
N
N
N
N
2e
2f
Scheme 3. Reagents and conditions: (a) mCPBA, CH2Cl2, 0 °C.
a
O
OH
O
O
N
H
N
H
B
C
3a
3b
Scheme 4. Reagents: (a) NaBH4, MeOH.
R
a
O2N
O
O
O
O
N
H
N
H
N
N
N
N
5a
5b, R = NH2
5c, R = N3
b
Scheme 5. Reagents and conditions: (a) Pd/C, EtOH, EtOAc, H2(g), 40 psi, rt;
(b) (1) 1 N HCl, MeOH, 0 °C; (2) NaNO2, H2O, 0 °C, then NaN3.
O2N
a
5a
O
O
N
H
N
N
Entry
Sub
(%)
5
12
50
G1
(%)
68
11
10
S
(%)
10
14
10
G2M
(%)
15
60
24
Br
5d
1A
1B
1C
Scheme 6. Reagents: (a) Br2, acetic acid.
Figure 1. Drug-induced apoptosis in T47D cells as measured by flow cytometric
analysis. The x-axis is the fluorescence intensity and the y-axis is the number of
cells with that fluorescence intensity. (A) Control cells showing most of the cells in
Table 4
Inhibition of cell growth of N-aryl-9-oxo-9H-fluorene-1-carboxamides
Compound
GI50 (l
M)a
G1 phase of the cell cycle. (B) Cells treated with 2
showing a shift to G2/M. (C) Cells treated with 2
l
M of compound 5b for 24 h
lM of compound 5b for 48 h
T47D
HCT116
SNU398
showing a shift to sub-diploid DNA content indicative of apoptotic cells with
fragmented nuclei.
2ab
5a
5b
Vinblastine
Paclitaxel
0.16 0.05
0.11 0.05
0.15 0.03
0.007 0.001
0.026 0.003
1.1 0.2
0.42 0.19
0.51 0.04
0.010 0.003
0.060 0.007
0.78 0.22
0.072 0.011
0.084 0.014
0.005 0.001
0.061 0.005
tubulin inhibitors, inhibited polymerization fully at 0.5 lM, sug-
gesting that part of the apoptotic effect of compounds 5a and 5b
might be through inhibition of tubulin polymerization. This is dif-
ferent from the original hit 3a and lead compound 2a, which were
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
b
Data from Ref. 18.
not active in the tubulin assay up to 50 l
M,18 indicating a change
of mechanism of action with substitution at the 7-position. Similar
changes of mechanism of action due to simple structural variations
have been reported for other series of apoptosis inducers13,17 and
DNA minor groove binders.23
In conclusion, we have explored modifications of the 9-oxo-9H-
fluorene ring of the apoptosis inducing 9-oxo-9H-fluorene-1-car-
boxamides. SAR studies using the pyrazolyl group at the 2-position
of the phenyl ring of the 1-carboxamide revealed that most
changes to the 9-oxo-9H-fluorene ring were not well tolerated.
Since cell cycle analysis showed that treatment with 5b led to
G2/M arrest followed by induction of apoptosis, a characteristic
that is similar to tubulin inhibitors, we decided to test inhibition
of tubulin polymerization as a possible mechanism for this series
of compounds using previously reported procedure.22 Compounds
5a and 5b inhibited 50% of tubulin polymerization at 2 and 3
lM,
respectively, while vinblastine and colchicine, two known potent

1292
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1288–1292
11. (a) Kemnitzer, W.; Kasibhatla, S.; Jiang, S.; Zhang, H.; Wang, Y.; Zhao, J.; Jia, S.;
Herich, J.; Labreque, D.; Storer, R.; Meerovitch, K.; Bouffard, D.; Rej, R.; Denis,
R.; Blais, C.; Lamothe, S.; Attardo, G.; Gourdeau, H.; Tseng, B.; Drewe, J.; Cai, S.
X. J. Med. Chem. 2004, 47, 6299; (b) Gourdeau, H.; Leblond, L.; Hamelin, B.;
Desputeau, C.; Dong, K.; Kianicka, I.; Custeau, D.; Bourdeau, C.; Geerts, L.; Cai, S.
X.; Drewe, J.; Labrecque, D.; Kasibhatla, S.; Tseng, B. Mol. Cancer Ther. 2004, 3,
1375; (c) Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Crogan-Grundy, C.;
Labreque, D.; Bubenick, M.; Attardo, G.; Denis, R.; Lamothe, S.; Gourdeau, H.;
Tseng, B.; Kasibhatla, S.; Cai, S. X. J. Med. Chem. 2008, 51, 417.
12. (a) Zhang, H.-Z.; Kasibhatla, S.; Kuemmerle, J.; Kemnitzer, W.; Oliis-Mason, K.;
Qui, L.; Crogran-Grundy, C.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2005, 48,
5215; (b) Jessen, K.; English, N.; Wang, J.; Qui, L.; Brand, R.; Maliartchouk, S.;
Drewe, J.; Kuemmerle, J.; Zhang, H.-Z.; Gehlsen, K.; Tseng, B.; Cai, S. X.;
Kasibhatla, S. Mol. Cancer Ther. 2005, 4, 761.
The 9H-fluorene (2b) and dibenzothiophene (2d) analogs were
about twofold less active than the 9-oxo-9H-fluorene analog 2a.
Significantly, SAR studies of the 7-position of the 9-oxo-9H-fluo-
rene ring led to compounds 5a–5c, which were more potent than
2a. Compounds 5a and 5b were found to be active in the tubulin
inhibition assay, suggesting a change of mechanism of action from
that of the original lead 2a. Compound 5b in the future will be
evaluated for antitumor efficacy. Utilizing a radio labeled version
of the azido compound 5c, the proposed tubulin target will be val-
idated and secondary proapoptotic targets will be determined.
13. (a) Sirisoma, N.; Kasibhatla, S.; Nguyen, B.; Pervin, A.; Wang, Y.; Claassen, G.;
Tseng, B.; Drewe, J.; Cai, S. X. Bioorg. Med. Chem. 2006, 14, 7761; (b) Sirisoma,
N.; Pervin, A.; Nguyen, B.; Kasibhatla, S.; Tseng, B.; Drewe, J.; Cai, S. X. Bioorg.
Med. Chem. Lett. 2009, 19, 2305.
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Anderson, M.; Baichwal, V.; Mather, G. G.; Jessing, K.; Hussain, R.; Hoang, K.;
Pleiman, C. M.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2008, 51, 4771; (b)
Sirisoma, N.; Pervin, A.; Zhang, H.; Jiang, S.; Willardsen, A. J.; Anderson, M.;
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