Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: Structure-activity relationships of the 4-, 5-, 6-, 7- and 8-positions

Article abstract of DOI:10.1016/j.bmcl.2009.05.012

As a continuation of our efforts to discover and develop the apoptosis inducing 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as potential anticancer agents, we explored substitutions at the 4-, 5-, 6-, 7- and 8-positions of pyrrolo[1,2-a]quinoline. SAR studi

Full text of DOI:10.1016/j.bmcl.2009.05.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3481–3484
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series
of apoptosis inducers using a cell- and caspase-based high-throughput
screening assay. 2: Structure–activity relationships of the
4-, 5-, 6-, 7- and 8-positions
William Kemnitzer, Jared Kuemmerle, Songchun Jiang, Nilantha Sirisoma, Shailaja Kasibhatla,
*
Candace Crogan-Grundy, Ben Tseng, John Drewe, Sui Xiong Cai
EpiCept Corporation, Inc. 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 efforts to discover and develop the apoptosis inducing 1-benzoyl-3-cyanopyrrol-
o[1,2-a]quinolines as potential anticancer agents, we explored substitutions at the 4-, 5-, 6-, 7- and 8-
positions of pyrrolo[1,2-a]quinoline. SAR studies showed that substitution at the 6-position by a small
group such as Cl resulted in potent compounds. Substitutions at the 5- and 8-positions were tolerated
while substitutions at the 4- and 7-position led to inactive compounds. Several compounds, including
2c, 3a, 3b and 3f, were found to be highly active against human breast cancer cells T47D with EC50 values
Received 2 April 2009
Revised 1 May 2009
Accepted 4 May 2009
Available online 8 May 2009
Keywords:
Apoptosis inducers
Anticancer agents
HTS
of 0.053–0.080
carcinoma cancer cells SNU398 in the caspase activation assay. Compound 3f also was found to be highly
active with a GI50 value of 0.018 M against T47D cells in a growth inhibition assay.
Ó 2009 Elsevier Ltd. All rights reserved.
lM, but much less active against human colon cancer cells HCT116 and hepatocellular
l
SAR
Apoptosis is a highly regulated process of cellular suicide and
plays a key role for the maintenance of cell homeostasis.1 It is well
known that abnormal or excessive inhibition of apoptosis could re-
sult in uncontrolled tumor cell growth as well as tumor resistance
to chemotherapeutic treatment.2 The pathways of apoptosis in-
volve a cascade of sequentially activated initiator and effector
caspases.3 Caspase-3 is the main executioner in apoptosis and its
activation leads to the cleavage of many protein substrates and cell
death.4 Since many chemotherapeutics are known to induce apop-
tosis in cancer cells,5 promotion or activation of apoptosis via tar-
geting apoptosis regulators has been suggested as a promising
strategy for anticancer drug discovery.6
we have reported the discovery of 1-benzoyl-3-cyanopyrrolo[1,2-
a]quinolines (1f) as a potent apoptosis inducer. Structure–activity
relationship (SAR) study of the 1-benzoyl and 3-cyano groups
showed that substitution at the 4-position of the 1-benzoyl group
was important for activity, and replacing the 3-cyano by an ester or
ketone group led to inactive compounds.16 Herein we wish to re-
port the synthesis and SAR study of the 4-, 5-, 6-, 7- and 8-positions
of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as potent apoptosis
inducers.
1-Benzoyl-3-cyanopyrrolo[1,2-a]quinolines 2a–2e, 2h–2j, 3a–
3b and 3e–3f were synthesized in two steps from reaction of the
corresponding 2-bromoacetylphenones (4) with substituted quin-
olines (5) to give the quinolinium salts (6), followed by cyclization
with acrylonitrile in the presence of an oxidant, such as tetrapyrid-
inecobalt(II) dichromate (TPCD, Co(II)Py4(HCrO4)2) or manga-
Utilizing our proprietary fluorescent caspase-3 substrates,7 we
have developed a cell- and caspase-based HTS assays called Anti-
cancer Screening Apoptosis Program (ASAP) for the discovery of
apoptosis inducers as potential anticancer agents.8 Applying this
assay, we have reported the discovery of several series of novel
apoptosis inducers including 4-aryl-4H-chromenes (1a),9 gambo-
gic acid (1b),10 3-aryl-5-aryl-1,2,4-oxadiazoles (1c),11 4-anilino-2-
(2-pyridyl)pyrimidines (1d)12 and 4-anilinoquinazolines (1e)13
(Chart 1), as well as the identification of the molecular targets for
gambogic acid14 and 3-aryl-5-aryl-1,2,4-oxadiazoles.15 Recently,
nese(IV) oxide (MnO2) and
a base, as reported previously
(Scheme 1).16–18 Compounds 2f and 2g were prepared from reac-
tion of 1-benzoyl-3-cyano-6-hydroxypyrrolo[1,2-a]-quinoline
(2e) with N-(2-chloro-ethyl)-morpholine and 2-chloro-N,N-
dimethylethanamine in the presence of potassium carbonate and
potassium iodide, respectively (Scheme 2). Compounds 3c was
made from reaction of 6-chloro-3-cyano-1-(4-fluorobenzoyl)-pyr-
rolo[1,2-a]-quinoline (3a) with imidazole in the presence of potas-
sium carbonate (Scheme 3). Amino compounds 3d and 3g were
synthesized from reduction of the corresponding nitro compounds
* 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 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.012

3482
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3481–3484
O
OMe
OH
Br
MeO
Cl
O
CF3
N
O
O
O
O
S
CN
O
N
N
O
NH2
OH
1c
1a
1b
OMe
CF3
OMe
OMe
CN
N
N
N
N
O
N
N
N
N
H
1d
1e
1f
Chart 1.
R
R
Cl
O
Cl
O
Br
N
CN
Br
N
O
R
CN
N
O
CN
N
a
b
O
+
a
N
R1
R1
R1
2a-2e, 2h-2j,
3a-3b, 3e-3f
NH2
NO2
5
4
6
3b, 4-NO2
3e, 3-NO2
3d, 4-NH2
3g, 3-NH2
Scheme 1. Reagents and conditions: (a) CH3CN, 85 °C, 4–6 h; (b) acrylonitrile,
TPCD, DMF, Na2CO3, 95 °C, 5 h or acrylonitrile, MnO2, DMF, Et3N, 95 °C, 5 h.
Scheme 4. Reagents and conditions: (a) SnCl2, EtOH, 90 °C, 7 h.
OR
O
OH
O
Cl
O
Cl
O
CN
N
CN
N
CN
CN
N
N
a
a
OH
OMe
2f, R = CH2CH2(morpholino)
2g, R = CH2CH2NMe2
2e
3h
3f
Scheme 5. Reagents and conditions: (a) BBr3, CH2Cl2, À78 °C, then 10 °C, 1.5 h.
Scheme 2. Reagents and conditions: (a) RCl, acetone, K2CO3, KI, 60 °C, 2.5 days.
The apoptosis inducing activity of 1-benzoyl-3-cyanopyrrol-
o[1,2-a]quinolines was measured by our proprietary cell- and cas-
pase-based HTS assay as described previously19 in human breast
cancer cells T47D, human colon cancer cells HCT116 and hepato-
cellular carcinoma cancer cells SNU398, and the results are sum-
marized in Tables 1 and 2. The original hit 1f was a potent
compound against T47D cells and less active in HCT116 and
SNU398 cells and the SAR of the 1-benzoyl and 3-cyano groups
has been reported.16 Here we explored further the SAR of 1f by
maintaining the 1-benzoyl-3-cyanopyrrolo[1,2-a]quinoline struc-
ture, and introducing various substitutions at the 4-, 5-, 6-, 7-
and 8-positions of the pyrrolo[1,2-a]quinoline ring. Caspase activa-
tion data in Table 1 showed that compared with 1f, introduction of
a methyl group at the 4-position (2a) led to >70-fold reduction in
activity. A methyl group at the 5-position (2b) was somewhat tol-
erated with about sevenfold reduction in activity against T47D
cells, and became nonselective with similar activity in all the three
cell lines. The 6-chloro analog 2c was highly active against T47D
cells, and with lower activity in HCT116 and SNU398 cells, indicat-
Cl
O
Cl
O
CN
N
CN
N
a
N
F
N
3c
3a
Scheme 3. Reagents and conditions: (a) imidazole, DMF, K2CO3, 160 °C, 20 h.
3b and 3e using tin(II) chloride dihydrate (Scheme 4). The hydroxyl
compound 3h was prepared from demethylation of 3f using boron
tribromide (Scheme 5).

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3481–3484
3483
Table 1
Table 2
SAR of 4-, 5-, 6-, 7- and 8-positions of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines in
SAR of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines in the caspase activation assay
the caspase activation assay
R3
R3 R2
R1
R4
R5
CN
N
CN
N
O
O
R7
R6
Entry
R1
R2
R3
R4
R5
EC50 (l
M)a
Entry
R3
R6
R7
EC50
HCT116
(l
M)a
T47D
HCT116
SNU398
T47D
SNU398
1fb
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
Me
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
Cl
NO2
OH
ORc
ORd
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
0.078 0.002
5.8 0.2
0.52 0.09
0.083 0.018
>10
>10
>10
2.8 0.3
>10
>10
5.5 0.069
>10
0.31 0.01
1.9 0.02
>10
>10
>10
6.0 0.4
>10
>10
5.2 0.13
>10
0.22 0.02
>10
>10
>10
>10
4.9 0.1
>10
>10
5.0 0.1
3a
3b
Cl
Cl
F
NO2
H
H
0.053 0.017 >10
0.062 0.010 >10
>10
>10
N
N
3c
Cl
H
0.29 0.01
1.9 0.4
1.4 0.1
3d
3e
3f
Cl
Cl
Cl
Cl
Cl
H
NH2
H
H
H
H
H
NO2
0.15 0.02
0.15 0.01
3.8 0.3
>10
OMe 0.080 0.003 3.7 1.2
1.4 0.1
>10
Me
Cl
H
H
H
1.5 0.1
5.0 0.1
2.6 0.1
0.88 0.21
> 10
2j
0.28 0.03
>10
3g
NH2
OH
H
0.21 0.05
0.28 0.06
5.6 0.6
2.7 1.2
0.036 0.011 1.2 0.059
2.8 0.17 > 10
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).
3h
7ab
7bb
F
NO2
b
Data from Ref. 16.
R = CH2CH2(morpholino).
R = CH2CH2NMe2.
H
H
c
d
N
N
7cb
H
H
H
0.034 0.001 0.074 0.011 0.039 0.001
ing that a small group at the 6-position maintains both the potency
and selectivity of 1f. Interestingly, the 6-nitro analog 2d and 6-hy-
droxy analog 2e were not active in all the three cell lines up to
7db
7eb
H
H
NH2
H
0.17 0.022
OMe 0.17 0.014
0.24 0.044
1.7 0.42
0.032 0.006 0.036 0.009 0.026 0.008
0.037 0.003 0.018 0.001 0.009 0.001
0.20 0.036
1.6 0.51
Vinblastine NAc NA
NA
NA
10 lM. Compounds designed to improve aqueous solubility prop-
Paclitaxel
NA
NA
erties, were either inactive in all three cell lines (2-morpholinoeth-
oxy, 2f) or significantly less active (2-dimethylaminoethoxy, 2g),
indicating that the 6-position might be size limited. Moving to
the 7-position, introduction of a methyl or chloro group (2h and
2i) led to inactive compounds, indicating substitution at this posi-
tion is not tolerated. The 8-methyl analog 2j retained the selective
activity of 1f but was threefold less active against T47D cells, indi-
cating that a small substitution at the 8-position is tolerated.
We have found previously that substitution at the 1-benzoyl
group was important for activity and selectivity of 1f and related
compounds.16 To determine whether a chloro group at the 6-posi-
tion changes the SAR of the 1-benzoyl group, we prepared a group
of compounds with substitutions at the 3- and 4-positions of the
benzoyl group of the 6-chloro analog 2c (Table 2). Table 2 shows
that compounds with a small group, such as fluoro (3a) or nitro
group (3b) at the 4-position of the benzoyl group, were highly ac-
tive against T47D cells, and maintained good selectivity being inac-
tive in HCT116 and SNU398 cells. Analogs with either a relatively
large group imidazole (3c) or hydrophilic amino group (3d) at
the 4-position were 2–3-folds less active than 2c, and lost some
selectivity against HCT116 and SNU398 cells. The 3-nitro (3e), 3-
amino (3g) and 3-hydroxy (3h) analogs were 2–3-folds less active
than 2c, while the 3-methoxy analog 3f was about as active as 2c
with similar selectivity profiles. Overall, compounds 3a–3h were
all less active in HCT116 and SNU398 cells, showing that all the
compounds with a chloro group at 6-position of the pyrrolo[1,2-
a]quinoline ring have selectivity for T47D cells versus other cells.
For comparison, two well known tubulin interacting anticancer
agents vinblastine and paclitaxel were found to be active in all
the three cell lines (Table 2), suggesting that the selective com-
pounds may interact with different molecular targets from that
of vinblastine and paclitaxel.
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. 16.
NA, not applied.
c
Table 2 also includes data from several substituted analogs of 1f
for direct comparison.16 Compound 7a and the corresponding 6-
chloro analog 3a had similar activity profiles with high activity
against T47D cells and low activity in HCT116 and SNU398 cells.
Compounds 7e and 3f also had similar activity profiles, with high
activity against T47D cells and good selectivity. In contrast, the ni-
tro substituted compound 7b is >40 fold less active than 3b, indi-
cating that these two series of compounds do not have the exact
same SAR. Interestingly, compound 3c was >8-, 25- and 35-fold
less active than 7c against T47D, HCT116 and SNU398 cells, respec-
tively. These data indicated that an imidazolyl group at the 4-posi-
tion of the benzoyl group, which is preferred when there is no
substitution at the 4–8 positions, is not preferred with a chloro
group at the 6-position of the pyrrolo[1,2-a]quinoline ring. Simi-
larly, compound 3d was selective against T47D cells, while com-
pound 7d were broadly active against all three cell lines. These
data showed that a chloro group at the 6-position of the pyrrolo-
quinoline did change the SAR of the 1-benzoyl group.
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 as described previously.9 Compound 3f was found to be
highly active with a GI50 value of 0.018
and much less active in HCT116 and SNU398 cells, with GI50 values
of 8.6 and 4.8 M, respectively (Table 3). Compounds 3h also was
lM against T47D cells,
l

3484
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3481–3484
Table 3
3- and 4-positions of the benzoyl group of 2c were prepared and
found to maintain the high activity and selectivity of 2c. Through
SAR studies of the 4- to 8-positions of 1-benzoyl-3-cyanopyrrol-
o[1,2-a]quinolines, several potent compounds such as 2c, 3a, 3b
and 3f were identified, with low nanomolar potency against T47D
cells in the caspase activation assay.
Inhibition of cell growth of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines
Entry
GI50
HCT116
(l
M)a
T47D
SNU398
1fb
3f
0.070 0.015
0.018 0.001
0.16 0.05
0.041 0.008
0.007 0.001
0.026 0.003
5.8 0.13
8.6 1.6
5.9 0.2
0.065 0.015
0.010 0.003
0.060 0.007
2.0 0.50
4.8 0.2
2.6 0.4
0.059 0.009
0.005 0.001
0.061 0.005
3h
7cb
Vinblastine
Paclitaxel
References and notes
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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. 16.
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more active against T47D cells than HCT116 and SNU398 cells,
indicating that these 6-chloro substituted analogs maintain the
selectivity of the original hit 1f. In comparison, analog 7c as well
as the reference compounds vinblastine and paclitaxel, which were
active in all three cell lines in the caspase activation assay, also
were broadly active in the growth inhibition assay, indicating a
good correlation between caspase activation assay and growth
inhibition assay as has been observed for other series of apoptosis
inducers.8
We have previously found that compound 1f, which was active
against breast cancer cells but much less active against several
other cancer cell lines, arrested cells in G2/M followed by apoptosis
as characterized by cell cycle analysis, and was not active in the
tubulin polymerization assay.16 Compounds 2c, 3a and 3h, which
were selective against T47D cells, also were found to arrest T47D
cells in G2/M and induce apoptosis as assayed by cell cycle analy-
sis,9 and were inactive in a tubulin polymerization assay20 up to
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L.; Archer, S.; Ponce, A.; Sirisoma, N.; Jiang, S.; Zhang, H.-Z.; Gehlsen, K.; Cai, S.
X.; Green, D. R.; Tseng, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12095.
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50 lM. These data indicate that similar to 1f, compound 2c and re-
lated analogs are not tubulin inhibitors, and the mechanism of ac-
tion and molecular target for apoptosis induction remains to be
determined. In comparison, the nonselective compounds 2b, 7c
and 7d all inhibited tubulin polymerization, with IC50 values of less
than 5 lM.
In conclusion, we have explored the SAR of the 4-, 5-, 6-, 7- and
8-positions of the apoptosis inducing 1-benzoyl-3-cyanopyrrol-
o[1,2-a]quinolines. It was found that substitution at the 4- and 7-
positions by a small group such as methyl or chloro led to >70-folds
drop in activity against T47D cells compared to the corresponding
non-substituted analog 1f. Substitution at the 5- and 8-positions
by a methyl group were somewhat tolerated with 3–7-folds reduc-
tion in activity. Substitution at the 6-position by a smallchloro group
resulted in compound 2c that maintained the high activity and
selectivity of 1f. A group of compounds with substitutions at the
20. Barron, D. M.; Chatterjee, S. K.; Ravindra, R.; Roof, R.; Baloglu, E.; Kingston, D. G.
I.; Bane, S. Anal. Biochem. 2003, 315, 49.