Syntheses and potential anti-prostate cancer activities of ionone-based chalcones

Article abstract of DOI:10.1016/j.bmcl.2008.12.089

We report the SAR studies of 43 ionone-based chalcones that demonstrate substantial in vitro anti-proliferative activities in LNCaP, MDA-PCa-2b, 22Rv1, C4-2B and PC-3 prostate cancer cell lines. Compound 25 with an IC₅₀ value of 0.74 μM in LNCa

Full text of DOI:10.1016/j.bmcl.2008.12.089

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1183–1186
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and potential anti-prostate cancer activities of ionone-based
chalcones
Jinming Zhou a,b,c, Guoyan Geng b,c, Gerald Batist a,b,c, Jian Hui Wu a,b,c,
*
a Department of Oncology, McGill University, Montreal, Canada
b Montreal Centre for Experimental Therapeutics in Cancer, Segal Cancer Center, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Canada
c Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, 3755 Cote-Ste-Catherine, Rd., Montreal, Que., Canada H3T 1E2
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the SAR studies of 43 ionone-based chalcones that demonstrate substantial in vitro anti-pro-
liferative activities in LNCaP, MDA-PCa-2b, 22Rv1, C4-2B and PC-3 prostate cancer cell lines. Compound
25 with an IC50 value of 0.74 lM in LNCaP cells potently antagonizes DHT-induced transactivation of the
wild type and the clinically relevant T877A, W741C and H874Y mutated androgen receptors, represent-
ing a novel chalcone as pan-antagonist of androgen receptor.
Received 24 September 2008
Revised 18 December 2008
Accepted 19 December 2008
Available online 25 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Androgen receptor
Mutation
Antiandrogen
Pan-antagonist
Ionone derivatives
Androgen receptor (AR) is a critical mediator of prostate can-
cer.1,2 Androgen ablation by chemical/surgical castration to sup-
press testicular androgen secretion and antiandrogens to
antagonize the action of residual androgen causes cancer regres-
sion. However, many men eventually failed this hormonal therapy
as initial response was almost always followed by a relapse to a
lethal hormone-refractory state, which is also referred to as castra-
tion-resistant prostate cancer (CRPC).3 Once prostate cancer be-
comes castration-resistant, further treatment is palliative and
patients eventually die of the disease. It has been established that
AR remains a critical target for therapeutic intervention in CRPC.2
The b-ionone is a phytochemical present in many fruits, vegeta-
bles and grains. It was found to exert anti-carcinogenic and antitu-
mor activities in melanoma4 and cancers of the colon,5 breast6 and
prostate.7 On the other hand, curcumin, the major pigment in the
dietary spice turmeric, was found to possess anti-inflammatory,
anti-oxidant, antiangiogenic and anticancer activities.8 Numerous
curcumin analogues have been synthesized and several of them
possess antiandrogenic activity.8,9 One widely used strategy for
structural modification in curcumin involves truncation of the cen-
tral conjugated b-diketone into a mono-carbonyl dienone, obtain-
ing better bioavailability.9 In our effort to develop natural
product-based anticancer agents, we have synthesized 43 io-
none-based chalcones by incorporating ionone and mono-carbonyl
dienone into one chemical entity and evaluated them for in vitro
cytotoxicity in a panel of prostate cancer cell lines, including
LNCaP, MDA-PCa-2b, 22Rv1, C4-2B and PC-3, as well as a nontu-
morigenic prostate epithelial cell line (RWPE-1). The most potent
compound has been evaluated for its anti-androgenic activity by
AR-dependent reporter assays.
Three series of ionone-based chalcones (compounds 1–43) were
synthesized by condensing commercial available substituted benz-
aldehyde with a-ionone, b-ionone and 4,5-epoxy-b-ionone, respec-
tively (Tables 1–3). Compounds 1, 2 and 12 which contain phenolic
hydroxyl were synthesized by acid-catalyzed Adol condensation.10
We found H2SO4-catalyzed reactions at room temperature gave 1,
2 and 12 in good yields, but AcOH-catalyzed reactions gave little
products. Chalcones 3–11 and 13–15 were obtained by facile Adol
condensation catalyzed by sodium hydroxide in ethanol (Scheme
1).11 Compounds 16–22 were obtained by Adol condensation cata-
lyzed by sodium hydroxide in water in the presence of cetyltrim-
ethyl ammonium bromide.12 Compounds 23–43 were
synthesized by Adol condensation catalyzed by sodium hydroxide
pellet in methanol. The epoxide ring opening of the 4,5-epoxy-b-
ionone during the reactions furnished 4-hydroxyl-a-ionone deriv-
atives 23–43 (Scheme 1), which was confirmed by the formation of
the 5,6-double bond as indicated by the proton NMR analyses.
Purification of the crude products were achieved by silica gel CC
(elutant: n-Hexane and EtOAc).
Cytotoxicity of chalcones 1–43 in a panel of prostate cancer cell
* Corresponding author. Tel.: +1 514 340 8222; fax: +1 514 340 8717.
E-mail address: jian.h.wu@mcgill.ca (J.H. Wu).
lines were evaluated by MTT assays.13 It should be noted that com-
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.089

1184
J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1183–1186
Table 1
Table 3
Cytotoxicity of b-ionone-based chalcones in LNCaP and PC-3 cells
Cytotoxicity of 4-hydroxy-b-ionone-based chalcones in five prostate cancer cell lines
and a normal prostate epithelial cell line (RWPE-1)
O
R2
R3
R4
O
R2
R3
R4
R6
R5
R6
a
Compound
Substituent
R4
Cytotoxicity, IC50
LNCaP PC-3
(lM)
R5
OH
R2
R3
R5
R6
Compound
Substituents
Cytotoxicity, IC50 (lM)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
OCH3
OH
OCH3
OC2H5
CF3
H
CF3
H
F
OH
OCH3
OCH3
OC2H5
H
CF3
H
H
H
F
H
OH
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
9.5
9.9
9.5
8.2
2.8
9.1
8.7
10.7
12.9
7.3
2.7
26.8
4.2
17.8
14.8
151.0
21.3
37.4
42.0
>50
7.7
12.0
7.2
R2 R3
R4 R5 R6 LNCaP PCa-2b 22Rv1 C4-2B PC-3 RWPE-1
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
CF3
H
H
CF3
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5.8
2.9
0.74
3.0
3.2
2.1
2.9
4.4
3.9
2.2
1.1
1.1
2.6
1.7
12.6
2.9
2.6
8.0
2.3
1.7
3.2
1.8
N.D.a 3.7 N.D.
1.4 3.5 0.73
N.D. 10.2 N.D.
15.5 N.D.
H
H
CF3
H
H
CF3
H
H
H
H
H
CF3
CF3
H
9.3
H
4.0
3.5
N.D. N.D.
1.9 N.D.
7.0 N.D.
22.1
18.7
25.8
10.0
> 50
18.9
35.0
47.8
CF3
H
2.2
3.3
5.6
7.5
3.2
2.4
N.D.
9
10
11
12
13
14
15
b-Ionone
N.D.
N.D.
N.D.
10.0
N.D.
N.D.
N.D.
N.D.
N.D.
19.0
N.D. 10.4 N.D.
N.D. 16.0 N.D.
4.0
7.3
H
H
H
H
H
F
F
H
H
H
NO2
NO2
NO2
CH3
H
F
H
6.2 N.D.
7.3 N.D.
3.0 N.D.
2.1 N.D.
4.6 N.D.
3.6 N.D.
8.8 N.D.
6.2 N.D.
7.2 1.9
CF3
CF3
CF3
H
F
H
H
CF3
H
H
F
H
H
CF3
H
N.D.
H
Ph
N.D. N.D.
F
3.8
2.0
8.9
4.4
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Inactiveb
F
H
a
F
H
H
CF3 4.9
IC50 is the concentration of compounds which causes a 50% inhibition as
H
H
H
H
H
H
CH3
CN
NO2
H
OCH3
CH(OEt)2
H
H
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
4.1
1.8
2.0
4.2
4.9
1.8
compared to the control (0.5% DMSO).
b
N.D. 2.2
Maximum tested concentration is 150 lM.
6.0
N.D.
N.D.
N.D.
1.9
4.1
5.8 1.9
N.D. 15.0 N.D.
N.D. N.D.
N.D. N.D.
6.5 N.D.
4.7 N.D.
H
Table 2
Cytotoxicity of
a
N.D., not determined.
a
-ionone-based chalcones in five prostate cancer cell lines
O
R2
R3
R4
O
O
R6
a) or b)
R5
R
+
RCHO
RCHO
1-15
Compound
Substituents
Cytotoxicity, IC50 (lM)
R2
R3
R4
R5
R6 LNCaP PCa
2b
22Rv1 C4-
2B
PC-
3
O
O
NaOH/H2O
+
R
16
17
18
19
20
21
22
CF3
H
H
F
F
H
H
H
CF3
H
H
H
H
H
CF3
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
22.6
1.0
4.7
1.6
1.7
N.D.a
4.4
7.0
8.3
3.0
18.4
2.9
3.3
9.4
3.5
11.7
4.4
5.2
6.0
4.7
4.0
6.2
19.0
5.2
7.9
4.8
3.1
2.9
5.9
16-22
O
O
NaOH/MeOH
+
RCHO
6
R
NO2
CH3
H
H
3.3
12.2
5.9
N.D.
4.7
15.2
5
23-43
4
O
a
N.D., not determined.
OH
Scheme 1. Preparation of ionone-based chalcones 1–43, where RCHO is a substi-
tuted benzaldehyde. Reagents and conditions: (a) H2SO4, MeOH, room temperature
(for compounds 1–2 and 12); and (b) NaOH, EtOH, room temperature (for
compounds 3–11 and 13–15).
pounds 3, 10 and 21 are known compounds, but their cytotoxicity
in prostate cancer cell lines have not been evaluated.12,14 The IC50
values were determined from cell survival curves and reported in
Tables 1–3.
The b-ionone-based chalcones 1–15 show considerable cytotox-
icity in LNCaP cell line and modest activity in PC-3 cells (Table 1).
Compound 5 and 11, both of which have electron-withdrawing
group at the meta position, are the two most potent b-ionone-
based chalcones. Moving trifluomethyl group (–CF3) from the meta
position to the para position or substituting –CF3 with –CH3 group
have substantially weakened the cytotoxicity in prostate cancer
cells (compare 5, 6 and 14). Substitution of meta-CF3 with meta flu-
oro (-F) decreases the cytotoxicity in LNCaP cells (compare 5 and
9). This indicates the electron-withdrawing –CF3 or –NO2 at the
meta position is critical for the cytotoxicity of b-ionone-based chal-
cones in LNCaP cell line. However, we found some b-ionone-based
chalcones are not stable enough. We have therefore focused the
studies on the other two series (Tables 2 and 3). Among the
a-ionone-based chalcones 16–22 (Table 2), compound 17 with a
meta-CF3 is the most potent compound in the series and replace-
ment of the meta-CF3 group with a meta-CH3 has severely reduced
its activity. Chalcones 19 and 20, which contain an ortho-F substi-
tution and the ortho-F and meta-CF3 double substitutions, respec-
tively, demonstrated dose-dependent cytotoxicity in LNCaP cells
with an IC50 value similar to that of compound 17. The importance
of an electron-withdrawing group at the meta position has also
been observed among the 4-hydroxy-b-ionone-based chalcones
23–43 (Table 3). In particular, compounds 25, 33, 34, 36, 39 and
40, which contain an electron-withdrawing group at the meta po-
sition, such as meta-CF3, –CN or –NO2, are much more potent than
compounds 23, 38 and 42, which contain –H, –CH3 and –OCH3 at
the meta position, respectively. Interestingly, compound 43 with

J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1183–1186
1185
a meta-CH(OEt)2 group, which has hydrogen bond acceptors and
possesses weak electron-withdrawing capability, shows potent
activity in LNCaP cells. This underscores the important role of the
hydrogen bond acceptors in the meta-CF3, –CN and –NO2 groups.
In addition, we have tested compounds 25, 39 and 40 in a nontu-
morigenic androgen-dependent prostate epithelial cell line
(RWPE-1) (Table 3). Compounds 25, 39 and 40 showed similar
cytotoxicity in both RWPE-1 and LNCaP cell lines.
Among the 43 chalcones, compound 25 demonstrated sub-
micromolar or low micromolar IC50 values in LNCaP, MDA-PCa-
2b, 22Rv1, C4-2B and PC-3 cell lines (Table 3). Both LNCaP and
MDA-PCa-2b cell lines are androgen-dependent and express mu-
tated ARs, with the T877A mutated AR in LNCaP and the T877A
and L701H double mutated AR in MDA-PCa-2b cells. The 22Rv1
cells express the H874Y mutated AR, and the growth of this cell
line is weakly stimulated by dihydrotestosterone (DHT). The C4-
2B cells are androgen-independent, and express the T877A mu-
tated AR. PC-3 cells, which lack endogenous AR, are androgen-
independent. Therefore, compound 25 has demonstrated potent
cytotoxicity in both androgen-dependent and androgen-indepen-
dent prostate cancer cells (Table 3).
To characterize antiandrogenic activity of chalcone 25, we
have investigated effect of 25 on the DHT-stimulated transacti-
vation of the wild type and the T877A, W741C and H874Y mu-
tated AR in transient transfection experiments, using PC-3 cells
(Fig. 1).15,16 We have included bicalutamide, an antiandrogen
used in the clinics, as a control. In consistent with previous stud-
ies,17 our reporter assays revealed that W741C mutation confers
resistance to bicalutamide (Fig. 1c). As shown in Figure 1, com-
pound 25 shows dose-dependent activity in suppressing 0.1 nM
DHT-induced transactivation of the T877A, W741C and H874Y
mutated ARs as well as modest antiandrogenic activity against
wild type AR. This indicates that chalcone 25 is a novel pan-anti-
androgen effective against the wild type and multiple mutated
ARs. Importantly, the AR W741C, T877A and H874Y mutations
have been characterized from patients with advanced prostate
cancer.18,19 The W741C and T877A mutations actually result in
paradoxical activation by bicalutamide17 and hydroxyfluta-
mide,18 respectively. Bicalutamide has been found to promote
tumor growth in a novel androgen-dependent prostate xenograft
model derived from a bicalutamide-treated patient.19 The T877A
mutant AR promotes prostate cancer cell growth and cell sur-
vival.20 Consequently, compound 25 represents a novel antian-
drogen that is simultaneously effective against multiple AR
mutants that confer resistance to antiandrogens currently used
in the clinics.
In summary, by condensing dietary ionones and substituted
benzaldhydes, 43 ionone-based chalcones have been synthesized
and evaluated in a panel of prostate cancer cell lines, including
LNCaP, MDA-PCa-2b, 22Rv1, C4-2B and PC-3. Among the com-
pounds synthesized, chalcone 25 shows the most potent
in vitro cytotoxic activities in prostate cancer cells. However,
compound 25 also shows potent cytotoxicity in an androgen-
dependent normal prostate cell line (RWPE-1). The AR-depen-
dent reporter assays revealed compound 25 is a novel antiandro-
gen for the wild type as well as the clinically relevant T877A,
W741C and H874Y AR mutants. In addition to its antiandrogenic
activity, the substantial cytotoxicity of 25 in AR-negative PC-3
cells indicates this lead compound is a multi-targeting agent.
The work to identify possible additional targets for 25 is in pro-
gress in our laboratory.
120
WT
a
100
80
60
40
20
0
DMSO
DHT
Bic-1.0 25-0.1 25-1.0
120
100
80
60
40
20
0
T877A
b
DMSO
DMSO
DMSO
DHT
Bic-1.0 25-0.1
25-1.0
c
d
W741C
200
150
100
50
Acknowledgments
We thank Dr Liang-Nian Song (Columbia University, NY, USA),
Dr. S. Srivastava (Uniformed Services University, USA), Dr. Osmu
Ogawa (Kyoto University, Kyoto, Japan) for providing AR express-
ing plasmids. This work was supported by the Canadian Institute
of Health Research (CIHR) via an operating grant to J.H.W. (Grant
No. MOP-74741). J.H.W. is a FRSQ investigator. The use of the com-
puter Linux cluster at Segal cancer centre for this work is
acknowledged.
0
DHT
Bic-1.0 25-0.1 25-1.0
H874Y
120
100
80
60
40
20
0
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.089.
DHT
Bic-1.0 25-0.1 25-1.0
Figure 1. Effect of bicalutamide (1.0
l
M) and compound 25 (0.1 and 1.0 lM) on
References and notes
DHT-induced transactivation of the wild type and the mutated ARs (a–d). PC-3 cells
were transiently transfected with pCMV-MMTV-Luc, Renilla null luciferase and AR
expressing plasmids. The cells were treated with 0.1 nM DHT with and without test
compound for 24 h. Relative luciferase activity was determined by dual luciferase
assay kit (Promega), standardized to Renilla luciferase control and normalized to
0.1 nM DHT without test compound (100%). Bic, bicalutamide; RLU, relative
luciferase unit.
1. Culig, Z.; Klocker, H.; Bartsch, G.; Hobisch, A. Endocr. Relat. Cancer 2002, 9, 155.
2. Balk, S. P. Urology 2002, 60, 132.
3. Taplin, M. E.; Ho, S. M. J. Clin. Endocrinol. Metab. 2001, 86, 3467.
4. Tatman, D.; Mo, H. B. Cancer Lett. 2002, 175, 129.
5. Janakiram, N. B.; Cooma, I.; Mohammed, A.; Steele, V. E.; Rao, C. V. Mol. Cancer
Ther. 2008, 7, 181.

1186
J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1183–1186
6. Duncan, R. E.; Lau, D.; El Sohemy, A.; Archer, M. C. Biochem. Pharmacol. 2004, 68,
1739.
7. Jones, S.; Mo, H. J. Nutr. 2005, 135, 3046S.
dissolved in isopropanol. The obsorbance was measured at 595 nm by a plate
reader (FLUOstar OPTIMA, BMG LabTech). All experiments were performed in
triplicate.
8. Shishodia, S.; Chaturvedi, M. M.; Aggarwal, B. B. Curr. Probl. Cancer 2007, 31,
243.
14. Gandhi, R. P.; Kumar, S.; Aryan, R. C.; Ishar, M. P. S. Synth. Commun. 1989, 19,
1759.
9. Mosley, C. A.; Liotta, D. C.; Snyder, J. P. Molecular Targets and Therapeutic Uses of
Curcumin in Health and Disease 2007, 595, 77.
10. Youssef, K. M.; El-Sherbeny, M. A.; El-Shafie, F. S.; Farag, H. A.; Al-Deeb, O. A.;
Awadalla, S. A. A. Arch. Pharm. (Weinheim) 2004, 337, 42.
11. Adams, B. K.; Ferstl, E. M.; Davis, M. C.; Herold, M.; Kurtkaya, S.; Camalier, R. F.;
Hollingshead, M. G.; Kaur, G.; Sausville, E. A.; Rickles, F. R.; Snyder, J. P.; Liotta,
D. C.; Shoji, M. Bioorg. Med. Chem. 2004, 12, 3871.
12. Chandra, N.; Pandey, S.; Ramesh; Suryawanshi, S. N.; Gupta, S. Eur. J. Med.
Chem. 2006, 41, 779.
13. Cell growth inhibition assay: LNCaP, 22Rv1, C4-2B and PC-3 cells were seeded at
a density of 6000–7000 cells per well in 96-well microtiter plates in RPMI 1640
supplemented with 10% FBS, 100 U/mL penicillin and 10 mg/mL streptomycin.
MDA-PCa-2b cells were seeded at the same density in medium BRFF-HPC1
(Athena Enzyme Systems, Baltimore, MD, USA) supplemented with 20% FBS.
RWPE-1 cells were seeded at the same density in keratinocyte serum-free
medium supplemented with 0.05 mg/mL bovine pituitary extract and 5 ng/mL
EGF (Cedarlane Laboratories, Burlington, ON, Canada). After overnight
incubation, the cells were treated with DMSO vehicle or test compounds at
various concentrations diluted from DMSO stock solution. The final DMSO
concentration is 0.5%. After 72 h of incubation, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) solution were added to each well and
incubated for another 2 h. The MTT formazan formed by viable cells was
15. Luciferase-reporter assay: PC-3 cells were seeded at a density of 60,000–70,000
cells per well in 24-well microtiter plates 24 h before transfection, and were
subsequently cotransfected with 100 ng of reporter MMTV-luciferase (pCMV-
MMTV-Luc), 10 ng of wild-type or mutant AR expressing plasmid (pCMV-AR-
wt, pCMV-AR-T877A, pCMV-W741C or pCMV-H874Y), and 10 ng of Renilla
null luciferase using LipofectamineTM 2000 reagent (Invitrogen) following
manufacturer’s protocol. Five hours after transfection, the medium was
changed to phenol red-free RPMI 1640 supplemented with 10% charcoal-
stripped FBS. After 16 h, the cells were treated with 0.1 nM DHT with and
without test compounds for 24 h. Luciferase activity was detected by dual
luciferase assay kit (Promega), standardized to the renilla luciferase control
and normalized to 0.1 nM DHT without test compound. All experiments were
performed in triplicate and repeated at least twice.
16. Song, L. N.; Gelmann, E. P. J. Biol. Chem. 2005, 280, 37853.
17. Hara, T.; Miyazaki, J.; Araki, H.; Yamaoka, M.; Kanzaki, N.; Kusaka, M.;
Miyamoto, M. Cancer Res. 2003, 63, 149.
18. Steketee, K.; Timmerman, L.; Ziel-van der Made, A.; Doesburg, P.; Brinkmann,
A. O.; Trapman, J. Int. J. Cancer 2002, 100, 309.
19. Yoshida, T.; Kinoshita, H.; Segawa, T.; Nakamura, E.; Inoue, T.; Shimizu, Y.;
Kamoto, T.; Ogawa, O. Cancer Res. 2005, 65, 9611.
20. Sun, C.; Shi, Y.; Xu, L. L.; Nageswararao, C.; Davis, L. D.; Segawa, T.; Dobi, A.;
McLeod, D. G.; Srivastava, S. Oncogene 2006, 25, 3905.