Structure-activity studies of quinuclidinone analogs as anti-proliferative agents in lung cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2005.11.085

We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or with γ-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting analo

Full text of DOI:10.1016/j.bmcl.2005.11.085

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1156–1159
Structure–activity studies of quinuclidinone analogs as
anti-proliferative agents in lung cancer cell lines
a
a
a,b
a,
*
Ahmed Malki, Aravinda B. Pulipaka, Susan C. Evans and Stephen C. Bergmeier
a
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
b
Edison Biotechnology Institute, Ohio University, Athens, OH 45701, USA
Received 4 October 2005; revised 23 November 2005; accepted 28 November 2005
Available online 19 December 2005
Abstract—We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or
with c-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting
analogs significantly decreased cell viability in H1299 lung cancer cell lines. Two derivatives decreased cell proliferation in a
dose-dependent fashion alone or in the presence of c-radiation. Radiosensitization increased when derivative treatment preceded
radiation treatment for both derivatives. These preliminary studies show an evidence for both additive and synergistic cytotoxicity
for treatment of lung cancer by these novel quinuclidinone analogs.
Ó 2005 Elsevier Ltd. All rights reserved.
Tumors are complex collections of cells that respond to
therapies differently depending on the organ site and
type of cells. Chemotherapeutic agents are developed
through empirical screens to identify compounds or
molecules that kill cancer cells. Most cytotoxic agents
wide variety of cancers. Several examples of such small
molecules have been reported. The pyrimidine derivative
1 is reported to both stabilize wild-type p53, as well as to
8
–10
restore function to mutant p53.
2-Methoxyestradiol
1
1
(2) facilitates wild-type p53-mediated apoptosis. More
recently, a series of a-methylene ketones (e.g., 3) were
identified as having good anti-proliferative activity
1,2
used to treat cancer act by inducing apoptosis.
1
2
The combination of radiotherapy and chemotherapy is a
unique and effective therapy. The role of radiotherapy is
to control the primary tumor, while chemotherapy can
against p53 mutant cell types.
The quinuclidinone derivative 4 is also reported to in-
13,14
3
,4
be used to diminish distant metastases. Some chemo-
therapeutic drugs destroy tumor cells directly by their
own cytotoxic action but may also enhance the effects
of radiotherapy. Many studies show the advantages of
combining radiotherapy with chemotherapy, both in
terms of better local control of cancer and metastasis
duce apoptosis in a number of cell types.
This qui-
nuclidinone also restores the active conformation of
mutant p53. Original reports suggested 4 induced con-
formational change in p53. However, our study has
indicated that this compound decreases cell prolifera-
tion and induces apoptosis in a p53-independent
5
–7
prevention.
mechanism. The quinuclidinone
through the screening of a library of low molecular
4 was identified
1
4
Given the toxicity, development of resistance, and lack
of broad spectrum treatments, there is a continuing need
for the development of new chemotherapeutic agents for
the treatment of cancer. There has been an effort to
develop small molecules that restore function to mutant
p53, a tumor suppressor gene frequently mutated in a
weight molecules. Consequently, no structural activi-
ty studies have been reported for this intriguing
molecule.
The quinuclidine ring system itself is a common struc-
tural element of a number of pharmacologically active
small molecules, especially cholinergic ligands. In gener-
1
5,16
al, these ligands lack the 3-keto group seen in 4.
lack of a carbonyl group at C-3 has significant implica-
This
Keywords: Quinuclidinone; Anti-cancer; Radiation.
*
Corresponding author. Tel.: +1 740 517 8462; fax: +1 740 593 0148;
e-mail: bergmeis@ohio.edu
tions for the basicity of the tertiary amine. The pK of
a
the conjugate acid of quinuclidine is 11.4, while that of
0
960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.085

A. Malki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1156–1159
1157
1
7
the conjugate acid of quinuclidinone is only 7.5. These
combined differences may account for the differences in
activity between the two classes of compounds.
Given the need to develop new chemotherapeutic agents
with an improved safety profile, analogs of molecules
such as 4 should provide an excellent entry into novel
anti-cancer agents. These molecules should have a selec-
tive mode of action, they are structurally unique, and yet
have a great deal of known chemistry upon which to
prepare analogs.
We were particularly interested in molecule 4 as a start-
ing point for the synthesis of novel anti-cancer agents.
Given the simple structure of 4, a large number of ana-
logs could reasonably be proposed. In an initial series of
compounds, we chose to ask the following questions: (1)
is the diol necessary or could other functional groups
suffice, that is, is a H-bond acceptor or H-bond donor
Scheme 1. Reagents and conditions: (a) 34% aq HCHO, K
0 °C, 45%; (b) LiAlH (500 mol %), 0 °C, 60%; (c) AcCl, DMAP,
pyridine, 96%; (d) 8a, R =H, R =Ph, PhCHO, pTSA, D, 23%; 8b,
=R =CH (H C) C(OCH pTSA, D, 33%; 8c, =R
2 3
CO ,
5
4
1
2
R
1
2
3
,
3
2
3
)
2
,
R
1
2
=
(
or both) necessary for activity, and (2) is the carbonyl
needed, and how might that influence the pK of the
–CH (CH ) CH –, cyclohexanone, BF ÆOEt , HC(OEt) , 20%; 8d,
2
2 3
2
3
2
3
R
R
R
1
2
2
=H,
=CH
=3,4-Cl
R
2
=c-C
6
H
11
,
c-C
CH
PhCHO, BF
6
H
11CHO, p-TSA, D, 34%; 8e,
CHO, BF ÆOEt 31%; 8f,
36%; 8g,
, 21%.
R
R
1
=H,
=H,
a
amine.
2
CH
2
Ph, PhCH
2
2
3
2
,
1
2
Ph, 3,4-Cl
2
3
ÆOEt
2
,
1 2
R =H, R =
4
-(OMe)Ph, 4-(OMe)PhCHO, BF
3
ÆOEt
2
The commercially available quinuclidinone 5 was con-
verted to the diol 4 by two successive aldol condensa-
tions. With key diol in hand, three modifications to
this structure were carried out. The ketone was reduced
to the triol 6, using LiAlH . The diol was converted to
the diacetate 7 in excellent yield using excess acetyl
chloride. The diol was also converted to a series of
acetals by condensations with the appropriate carbonyl
compound. The yields for the acetal formation ranged
from 20 to 40%. Significantly, both the initial acetals
prepared showed similar or improved activities relative
to the parent compound 4. We thus chose to prepare
an additional set of acetals (8c–8f) using the same
1
8
As a control, we tested the starting quinuclidinone 5 in
2
1
the MTT assay. As expected, this compound showed
no activity. The initial lead compound 4 showed excel-
lent activity with less than 10% of the cells remaining
viable. Compound 6, in which the carbonyl group has
been reduced, showed complete loss of activity. There
are two possibilities to account for this observation.
The first is that the hydroxyl, while able to engage in
similar types of non-covalent contacts, is no longer in
an appropriate position. Alternatively, the lower basici-
ty of the quinuclidinones (as compared to quinuclidine)
is important for activity.
4
1
9
methods. Compounds 8a, 8d–8f appear to be a single
isomer at the anomeric carbon by H NMR (Fig. 1)
1
(
Scheme 1).
The diacetate 7 showed similar activity to the lead com-
pound 4, indicating that the free hydroxyls were not nec-
essary. As part of our study to determine the importance
of the hydroxyls, we initially prepared two acetals (8a,
Cell viability was measured via the methyl tetrazolium
MTT) bromide mitochondrial activity assay described
(
2
0
previously. The synthesized compounds decreased cell
survival and cell survival was further decreased if given
in conjunction with c-radiation (Fig. 2).
R =H, R =Ph, and 8b, R =R =CH ). Both these com-
1 2 1 2 3
pounds showed improved activity relative to the parent
diol 4. Based upon these results we prepared five addi-
tional acetals as analogs of the benzylidine (8a) and ace-
tonide (8b) derivatives. The acetal derived from
cyclohexanone (8c) showed decreased activity relative
to both the parent acetonide (8b) as well as the diol. This
may indicate a lack of tolerance to steric bulk around
the acetal ring. We prepared the acetal of cyclohexane
carboxaldehyde (8d) in order to determine if the aromat-
ic ring of 8a was essential. We observed a lowering of
activity relative to 8a. Compound 8e was prepared using
3-phenylpropionaldehyde. Our intention was to deter-
mine if the aromatic ring could be moved away from
the acetal and yet still retain activity, clearly this com-
pound is somewhat less potent than the benzylidine
8
8
a, but still as potent as the diol. Compounds 8f and
g were prepared to explore the effects of substitution
on the aromatic ring. The 3,4-dichloro derivative lost
some potency, but the 4-methoxy derivative lost almost
all activity.
Figure 1. Small molecules reported to restore the function to p53.

1158
A. Malki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1156–1159
Figure 2. Effect of quinuclidinone derivatives on cell viability of H1299 cells (null for p53) in the presence and absence of 4 Gy of c-radiation. Each
data point is an average of three independent experiments and expressed as M ± SD. All experiments were carried with a 4 mM concentration of
quinuclidinone analogs.
The compounds that showed the greatest effect on cell
viability, 8a and 8b, were chosen for further analysis.
Concentrations of 8a and 8b, ranging from 20 to
1000 lM, were prepared and dissolved in complete
Dulbecco’s modified essential media. H1299 cells that
have a deletion of the p53 gene were treated with the
compound alone, or in combination with 4 Gy of c-radi-
ation (IR) were assayed for cell survival. Compounds 8a
and 8b decreased the percent of cell survival in a dose-
dependent fashion both in the absence and presence of
c-radiation (Fig. 3). The GI50 for all compounds were
relatively similar. Compounds 8a and 8b alone had
GI s of 17.26 lM and 19.21 lM. In the presence of
5
0
c-radiation, the GI s decreased with compound
5
0
8
b + 4Gy being slightly more potent (4.74 lM) than
8
a + 4Gy (4.49 lM). Similar results were obtained from
dose–response curves of the same concentrations of
a and 8b in the presence of ultraviolet radiation
8
(
2
10 J/m ) (data not shown).
c-irradiation (IR) is a local treatment, focusing on a spe-
cific area of the body, while chemotherapy is systemic,
attacking cancer by treating the whole body. There con-
tinues to exist a need for new anti-cancer agents with im-
proved safety profiles, specifically those that have a
reduced liability for resistance and unwanted side effects.
In this study, we synthesized new acetals and di-acetates
that were potent in decreasing cell viability. These ana-
logs will enable us to examine important structural
determinants of small molecule macromolecular interac-
tions for anti-cancer activity. The discovery of a class of
highly selective and potent quinuclidinones will lead to
development of new anti-cancer drugs. Additional
experiments are required to determine the mechanism
of action of these new quinuclidinone derivatives as well
as to fully elucidate the structure–activity relationships
of this class of molecules.
References and notes
Figure 3. Dose–response curves of 8a and 8b in H1299 cells alone or in
the presence of 4 Gy c-radiation. Each data point is an average of three
independent experiments and expressed as M ± SD.
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and 100 lg/mL streptomycin at 37 °C in a 5% CO
2
atmosphere.
Cell proliferation (MTT assay): 4000–5000 cells/well in
100 lL of medium were seeded in a 96-well plate for 24 h
prior to drug treatment. The media were then changed to
media with analogs, and cells were treated with either c- or
UV-radiation. At the end of the incubation (24 h), 10 lL of
5 mg/mL MTT reagent (ATCC) was added to each well for
4 h. After incubation, 100 lL of detergent reagent was
added to each well to dissolve the formazan crystals. The
absorbance was determined at 570 nm. Assays were per-
formed in triplicate and standard error determined. GI50
values were obtained by averaging values generated from
non-linear regression analyses (Prism, GraphPad, San
Diego, CA) of individual concentration–response curves.