Novel substituted (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one and (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol derivatives as potent thermal sensitizing agents

Article abstract of DOI:10.1016/j.bmcl.2007.10.035

Use of ionizing radiation is essential for the management of many human cancers, and therapeutic hyperthermia has been identified as a potent radiosensitizer. Radiation therapy combined with adjuvant hyperthermia represents a potential tool to provide outstanding local-regional control for refractory disease. (Z)-(±)-2-(N-Benzylindol-3-ylmethylene)quinuclidin-3-ol (2) and (Z)-(±)-2-(N-benzenesulfonylindol-3-ylmethylene)quinuclidin-3-ol (4) were initially identified as potent thermal sensitizers that could lower the threshold needed for thermal sensitivity to radiation treatment. To define the structural requirements of the molecule that are essential for thermal sensitization, we have synthesized and evaluated a series of (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one (9), and (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol (10) analogs that incorporate a variety of substituents in both the indole and N-benzyl moieties. These systematic structure-activity relationship (SAR) studies were designed to further the development and optimization of potential clinically useful thermal sensitizing agents. The most potent analog was compound 10 (R¹ = H, R² = 4-Cl), which potently inhibited (93% inhibition at 50 μM) the growth of HT-29 cells after a 41 °C/2 h exposure.

Full text of DOI:10.1016/j.bmcl.2007.10.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6821–6824
Novel substituted (Z)-2-(N-benzylindol-3-ylmethylene)-
quinuclidin-3-one and (Z)-( )-2-(N-benzylindol-3-ylmethylene)-
quinuclidin-3-ol derivatives as potent thermal sensitizing agents
Vijayakumar N. Sonar,a Y. Thirupathi Reddy,a Konjeti R. Sekhar,b Soumya Sasi,b
Michael L. Freemanb and Peter A. Crooksa,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
bDepartment of Radiation Oncology/Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine,
Nashville, TN 37232, USA
Received 20 August 2007; revised 5 October 2007; accepted 8 October 2007
Available online 17 October 2007
Abstract—Use of ionizing radiation is essential for the management of many human cancers, and therapeutic hyperthermia has been
identified as a potent radiosensitizer. Radiation therapy combined with adjuvant hyperthermia represents a potential tool to provide
outstanding local-regional control for refractory disease. (Z)-( )-2-(N-Benzylindol-3-ylmethylene)quinuclidin-3-ol (2) and (Z)-( )-2-
(N-benzenesulfonylindol-3-ylmethylene)quinuclidin-3-ol (4) were initially identified as potent thermal sensitizers that could lower
the threshold needed for thermal sensitivity to radiation treatment. To define the structural requirements of the molecule that
are essential for thermal sensitization, we have synthesized and evaluated a series of (Z)-2-(N-benzylindol-3-ylmethylene)quinucli-
din-3-one (9), and (Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol (10) analogs that incorporate a variety of substituents in
both the indole and N-benzyl moieties. These systematic structure–activity relationship (SAR) studies were designed to further the
development and optimization of potential clinically useful thermal sensitizing agents. The most potent analog was compound 10
(R1 = H, R2 = 4-Cl), which potently inhibited (93% inhibition at 50 lM) the growth of HT-29 cells after a 41 °C/2 h exposure.
Ó 2007 Elsevier Ltd. All rights reserved.
Hyperthermia is one of the most potent radiosensitizers
identified to date.1 Over the last 30 years extensive
experimentation using cell and animal models has dem-
onstrated that hyperthermia can be an extremely effec-
tive radiation sensitizer, increasing the effectiveness of
the radiation by up to 5-fold.1,2 One important feature
is that thermal radiosensitization does not exhibit cell
type specificity, nor is it limited by hypoxic conditions,
suggesting that hyperthermia has the potential to over-
come significant radiation resistance. These studies have
been complemented by a recent meta analysis of 23 clin-
ical trials involving 1861 patients, which has shown that
radiation therapy administered with adjuvant hyperther-
mia yields highly significant (p < 0.0001) local-regional
control of aggressive tumors located in chest wall, cervi-
cal, bladder, and head and neck.3
Heat-mediated changes in protein conformation repre-
sent the underlying biophysical/biochemical mechanism
responsible for heat-mediated cytotoxicity.4 Westra
and Dewey5 were the first to hypothesize that cell death
following hyperthermic treatment was a consequence of
protein denaturation. Subsequent investigations have
validated their hypothesis.6–8 Thermal radiosensitization
is also a consequence of protein unfolding and aggrega-
tion,4 and, under most conditions, is directly related to
the degree of thermal sensitization.
However, for many cancers, suboptimal thermal dosing
limits radiosensitization.6 While temperatures of 41 °C
can be achieved clinically, administration of higher tem-
peratures can be challenging. In order to overcome this
problem we have undertaken a chemistry-driven ap-
proach to identify pharmacological agents that enhance
the degree of radiosensitization produced by a moderate
heat shock. The immediate goal was to synthesize small
molecules that could enhance a clinically achievable heat
shock but would not exhibit cytotoxicity at physiologi-
cal temperatures.
Keywords: Indolylquinuclidin-3-ones; Indolylquinuclidin-3-ols; Ther-
mal sensitization.
*
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257
7585; e-mail: pcrooks@email.uky.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.035

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V. N. Sonar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6821–6824
Previously, we have shown that structural mimics of
indomethacin produced thermal sensitization and ther-
mal radiosensitization of human HT-29 colon carci-
noma cells, MCF7 breast adenocarcinoma cells, and
H460 lung carcinoma cells at 41 °C. Biochemically, this
small molecule enhancement in thermal sensitization
activity induces a loss of mitochondrial membrane
potential, which is subsequently followed by mitotic
catastrophe. The most potent small molecule enhancers
identified were (Z)-2-(N-benzylindol-3-ylmethylene)quinu-
clidin-3-one,
(Z)-( )-2-(N-benzylindol-3-ylmethylene)
quinuclidin-3-ol, (Z)-2-(N-benzenesulfonylindol-3-ylmeth-
ylene)quinuclidin-3-one, and (Z)-( )-2-(N-benzenesulf-
onylindol-3-ylmethylene)quinuclidin-3-ol (1, 2, 3, and 4,
respectively)9 (Fig. 1). The indole nucleus played an
important role in the activity of these compounds, as lack
of an indole moiety produced inactive compounds. Ab-
sence of N-benzyl substitution also produced inactive or
less active compounds. Also, N-benzenesulfonyl
derivatives (3 and 4) were less potent than the N-benzyl
derivative (2).
These observations prompted us to undertake a more
detailed investigation of the structure–activity relation-
ships of various N-benzyl derivatives related to 1 and
2 (Fig. 1) that incorporate a variety of different substitu-
tions in the phenyl ring of the N-benzyl moiety and in
the indole moiety in these compounds. Thus, the present
study focuses on the synthesis and evaluation of thermal
sensitizing activity of various substituted (Z)-2-(N-ben-
zylindol-3-ylmethylene)quinuclidin-3-ones (9a–9l) and
(Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-
3-ols (10a–10l).
Scheme 1. Reagents and conditions: (a) Ac2O, Et3N, DCM, reflux; (b)
quinuclidin-3-one hydrochloride, THF, LDA, À78 °C; (c) 1 N NaOH
aq, reflux; (d) substituted benzyl halide, 50% NaOH aq, triethylben-
zylammonium chloride, DCM, rt; (e) NaBH4, methanol, rt.
The synthetic routes to the substituted (Z)-2-(N-benzy-
lindol-3-ylmethylene)quinuclidin-3-ones 9a–9l and the
(Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-
ols 10a–10l are illustrated in Scheme 1.
The appropriate 1-acetylindole-3-carboxaldehyde 6 was
prepared by N-acetylation of the corresponding indole-
3-carboxaldehyde 5 with acetic anhydride in the pres-
ence of triethylamine in dichloromethane under reflux.
Aldol condensation of 6 with quinuclidin-3-one hydro-
chloride in the presence of lithium diisopropylamide in
tetrahydrofuran at À78 °C afforded the (Z)-2-(N-acety-
lindol-3-ylmethylene)quinuclidin-3-one derivative, 7.11
Subsequent cleavage of the N-acetyl group by refluxing
with 1 N NaOH aqueous solution afforded the appropri-
ate (Z)-2-(1H-indol-3-ylmethylene)quinuclidin-3-one, 8.
The
substituted
(Z)-2-(N-benzylindol-3-ylmethyl-
ene)quinuclidin-3-one derivatives 9a–9l were prepared
in 85–90% yield by treating 8 with various substituted
benzyl halides under phase-transfer catalytic (PTC) con-
ditions using triethylbenzylammonium chloride and
50% w/v aqueous NaOH solution in dichloromethane.
Reduction of the above substituted (Z)-2-(N-benzylin-
dol-3-ylmethylene)quinuclidin-3-one derivatives 9a–9l
with NaBH4 in methanol afforded the corresponding
(Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-
3-ol derivatives 10a–10l in 80–85% yield. All the synthe-
1
sized compounds were characterized by H NMR and
13C NMR spectrometry and HR-MS analysis.10 The
geometry of the double bond in 3, 4, and 8 (R1 = H)
was established from X-ray crystallographic data.11,12
Figure 1. Chemical structures of the potent thermal radiosensitizing
agents 1–4.

V. N. Sonar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6821–6824
6823
Table 1. Relative survival of cultured HT-29 cells and the relative
potency of the substituted (Z)-2-(N-benzylindol-3-ylmethylene)quinu-
clidin-3-one analogs at 150 lM concentration
divided by initial number of cells heated, corrected for
plating efficiency. Survival following a 2 h, 41 °C heat
shock was not toxic: survival was not significantly differ-
ent from plating efficiency (p > 0.05 Student’s t test) and
was arbitrarily set at 1.0. The surviving fraction of cells
exposed to drug for 2 h at 41 °C was calculated from the
ratio of the number of colonies counted divided by the
initial number of cells treated, corrected for plating effi-
ciency and for survival following a 2h exposure to drug
at 37 °C. Cell survival following a 2h/37 °C exposure to
150 lM of compounds 9a–9l (Table 1) and compounds
10b, 10c, 10e, and 10h–10k (Table 2) was 70% or greater.
However, survival was significantly less than 70%
following a 2h/37 °C exposure to 150 lM of 10a, 10d,
10f, 10g, and 10l. Consequently these compounds were
evaluated at a concentration of 50 lM, a concentration
in which survival was 70% or more following a 2h/37 °C
exposure (Table 3).
Compound
Relative survivala
Relative
potencyb
of HT-29 cells at 41 °C
9a
9b
9c
1.00
1.00
0.98
1.00
1.00
0.56
0.71
0.86
1.00
0.81
0.96
0.75
0.50
1.00
1.00
1.00
1.00
1.00
1.00
1.79
1.41
1.00
1.00
1.00
1.00
1.00
2.00
1.00
9d
9e
9f
9g
9h
9i
9j
9k
9l
1
Vehicle
a Relative survival was calculated by correcting for vehicle effects and
effect of drugs at 37 °C.
Substituents in the indole moiety of (Z)-2-(N-benzylin-
dol-3-ylmethylene)quinuclidin-3-one analogs 9a–e and
9h–l did not increase thermal sensitivity compared to
vehicle control (p > 0.05 Student’s t test). However,
introduction of a 4-chloro or 4-fluoro substituent in
the N-benzyl moiety (compounds 9f and 9g) significantly
increased thermal sensitivity compared to solvent con-
trol (p < 0.05 Student’s t test).
b Relative potency is equal to the reciprocal value of relative survival
value and is statistically significant from vehicle control (p < 0.05
Student’s t test).
Table 2. Relative survival of cultured HT-29 cells and the relative
potency of the substituted (Z)-( )-2-(N-benzylindol-3-ylmethylene)
quinuclidin-3-ol analogs at 150 lM concentration
Relative survivala
Relative
potencyb
With regard to the (Z)-( )-2-(N-benzylindol-3-ylmethyl-
ene)quinuclidin-3-ol analogs, compounds 10b, 10h, 10j,
and 10k increased thermal sensitivity relative to vehicle
control (p < 0.05 Student’s t test) but were not as
effective as compound 2 (p > 0.05 Student’s t test) as
thermal sensitizers. (Z)-( )-2-(N-benzylindol-3-ylmeth-
ylene)quinuclidin-3-ol analogs 10a, 10d, 10f, 10g, and
10l were tested at a concentration of 50 lM and all
but 10l increased thermal sensitivity (p < 0.05 Student’s
t test). Analogs 10a, 10d, 10f, and 10g were significantly
more effective than compound 2 when tested at 50 lM
(p < 0.05 Student’s t test), 10f being the most potent.
Compound
of HT-29 cells at 41 °C
10a
10b
10c
10d
10e
10f
—
—
0.59
1.00
—
1.69
1.00
—
0.78
—
1.00
—
10g
10h
10i
—
—
0.61
0.90
0.52
0.58
—
1.64
1.00
1.92
1.72
—
10j
10k
10l
Based on the data from the HT-29 cell survival studies,
the following observation can be made. Substituted (Z)-
2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ones are
2
Vehicle
0.06
1.00
16.70
1.00
a Relative survival was calculated by correcting for vehicle effects and
effects of drugs at 37 °C.
b Relative potency is equal to the reciprocal value of relative survival
value, and is statistically significant from vehicle control (p < 0.05
Student’s t test).
Table 3. Relative survival of cultured HT-29 cells and the relative
potency of the substituted (Z)-( )-2-(N-benzylindol-3-ylmethylene)
quinuclidin-3-ol analogs at 50 lM concentration
Compound
Relative survivala
Relative
potencyb
Colony formation of tumor cells in culture was used to
assess potency.9 HT-29 cells growing exponentially in
T25 flasks (n = 4) were exposed to DMSO/drug for 2 h
with or without heat treatment (41 °C). Drug was then
washed off, and cells incubated in fresh medium for 14
days. Viable cells formed colonies (defined as greater
than 50 cells) that were stained with crystal violet and
counted. The plating efficiency was defined as the ratio
of the number of colonies counted divided by the initial
number of cells exposed to vehicle control (DMSO) for
2 h at 37 °C, and was typically P90%. The survival of
cells exposed to vehicle alone for 2 h at 41 °C was calcu-
lated from the ratio of the number of colonies counted
of HT-29 cells at 41 °C
10a
10d
10f
0.59
0.59
0.07
0.70
0.76
1.00
1.00
1.69
1.69
14.28
1.43
1.00
1.00
1.00
10g
10l
2
Vehicle
a Relative survival was calculated by correcting for vehicle effects and
effects of drugs at 37 °C.
b Relative potency is equal to the reciprocal value of relative survival
value and is statistically significant from vehicle control (p < 0.05
Student’s t test).

6824
V. N. Sonar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6821–6824
8. Nguyen, V. T.; Morange, M.; Bensaude, O. J. Biol. Chem.
1989, 264, 10487.
less effective sensitizers than the unsubstituted lead com-
pound, (Z)-( )-2-(N-benzylindol-3-ylmethylene)quinucli-
din-3-ol (2). Introducing a 4-chloro (but not a 4-fluoro)
substituent in the N-benzyl group afforded compound
10f, which exhibited the same magnitude of sensitization
as compound 2, but required only one-third the concen-
tration (50 vs 150 lM) to achieve this effect.
9. Sekhar, K. R.; Sonar, V. N.; Venkatraj, M.; Soumya, S.;
Laszlo, A.; Sawani, J.; Horikoshi, N.; Higashikubo, R.;
Bristow, R. G.; Borrelli, M. J.; Crooks, P. A.; Lepock, J.
R.; Roti Roti, J. L.; Freeman, M. L. Cancer Res. 2007, 67,
695.
10. Analytical data and yields for four of the most active
compounds: (9f): 1H NMR (300 MHz, CDCl3 d): 2.01 (m,
4H), 2.62 (p, J = 3.3 Hz, 1H), 2.98 (m, 2H), 3.18 (m, 2H),
5.35 (s, 2H), 7.03–7.29 (m, 7H), 7.46 (s, 1H), 7.87 (m, 1H),
8.36 (s, 1H); 13C NMR (75 MHz, CDCl3 d): 26.5, 40.6,
47.9, 50.5, 110.3, 110.8, 118.1, 119.4, 121.8, 123.0, 128.2,
129.6, 129.8, 133.8, 134.4, 135.2, 136.0, 140.5, 205.6; HR-
MS (EI+): m/z found: 376.1339, Calcd C23H21ClN2O (EI+)
In conclusion, a compound containing an N-benzylin-
dole nucleus linked to a quinuclidin-3-ol moiety via a
double bond with Z-geometry, and incorporating a lipo-
philic chloro substituent at the 4-position of the N-ben-
zyl group, exhibits potent thermal sensitizing properties.
The synthesis of such small molecules may afford prom-
ising tools of value in either clinical hyperthermia as
radiation sensitizers for the treatment of recurrent
tumors, or as enhancers of other cancer therapies that
may be augmented by hyperthermia, such as chemother-
apy and immunotherapy.
1
376.1337; yield: 89%: (10a): H NMR (300 MHz, CDCl3
d): 1.45 (m, 2H), 1.70 (m, 1H) 1.94 (m, 2H), 2.08 (m, 1H),
2.28 (s, 3H), 2.85 (m, 1H), 3.07 (m, 3H), 4.42 (br s, 1H),
5.30 (s, 2H), 6.59 (s, 1H), 6.98–7.21 (m, 7H), 7.70 (m, 1H),
8.10 (s, 1H); 13C NMR (75 MHz, CDCl3 d): 19.0, 21.8,
24.6, 30.6, 47.5, 48.2, 50.4, 71.2, 109.8, 113.6, 118.4, 119.8,
121.9, 126.6, 127.5, 128.2, 128.7, 134.4, 135.6, 136.6; HR-
MS (EI+): m/z found: 358.2038, Calcd C24H26N2O (EI+)
1
358.2039; yield: 82%: (10d): H NMR (300 MHz, CDCl3
Acknowledgment
d): 1.43 (m, 2H), 1.68 (m, 1H) 1.88 (m, 2H), 2.07 (m, 1H),
2.28 (s, 3H), 2.43 (s, 3H), 2.83 (m, 1H), 3.03 (m, 3H), 4.40
(br s, 1H), 5.26 (s, 2H), 6.56 (s, 1H), 6.93–7.08 (m, 6H),
7.50 (m, 1H), 8.04 (s, 1H); 13C NMR (75 MHz, CDCl3 d):
19.2, 21.3, 21.8, 25.3, 31.0, 47.3, 48.2, 50.3, 71.4, 109.7,
113.5, 118.3, 123.4, 126.5, 128.2, 129.0, 129.4, 130.0, 134.1,
134.7, 137.1; HR-MS (EI+): m/z found: 372.2193, Calcd
C25H28N2O (EI+) 372.2196; yield: 84%: (10f): 1H NMR
(300 MHz, CDCl3 d): 1.48 (m, 2H), 1.73 (m, 1H) 1.98 (m,
2H), 2.13 (m, 1H), 2.91 (s, 3H), 3.12 (m, 3H), 4.46 (br s,
1H), 5.31 (s, 2H), 6.66 (s, 1H), 7.03–7.29 (m, 7H), 7.69 (m,
1H), 8.18 (s, 1H); 13C NMR (75 MHz, CDCl3 d): 19.0,
21.8, 24.6, 30.6, 47.5, 48.2, 50.4, 71.2, 109.8, 113.6, 118.4,
119.8, 121.9, 126.6, 127.5, 128.2, 128.7, 134.4, 135.6, 136.6;
HR-MS (EI+): m/z found: 378.1492, Calcd C23H23ClN2O
(EI+) 378.1493; yield: 83%.
This research was supported by NIH/National Cancer
Institute Grant PO1 CA104457.
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