Antitumor agents 290. Design, synthesis, and biological evaluation of new LNCaP and PC-3 cytotoxic curcumin analogs conjugated with anti-androgens

Article abstract of DOI:10.1016/j.bmc.2012.05.011

In our continuing study of curcumin analogs as potential anti-prostate cancer drug candidates, 15 new curcumin analogs were designed, synthesized and evaluated for cytotoxicity against two human prostate cancer cell lines, androgen-dependent LNCaP and androgen-independent PC-3. Twelve analogs (5-12, 15, 16, 19, and 20) are conjugates of curcumin (1) or methyl curcumin (2) with a flutamide- or bicalutamide-like moiety. Two compounds (22 and 23) are C4-mono- and difluoro-substituted analogs of dimethyl curcumin (DMC, 21). Among the newly synthesized conjugates compound 15, a conjugate of 2 with a partial bicalutamide moiety, was more potent than bicalutamide alone and essentially equipotent with 1 and 2 against both prostate tumor cell lines with IC ₅₀ values of 41.8 μM (for LNCaP) and 39.1 μM (for PC-3). A cell morphology study revealed that the cytotoxicity of curcumin analogs or curcumin-anti-androgen conjugates detected from both prostate cancer cell lines might be due to the suppression of pseudopodia formation. A molecular intrinsic fluorescence experiment showed that 1 accumulated mainly in the nuclei, while conjugate 6 was distributed in the cytosol. At the tested conditions, anti-androgens suppressed pseudopodia formation in PC-3 cells, but not in LNCaP cells. The evidence suggests that distinguishable target proteins are involved, resulting in the different outcomes toward pseudopodia suppression.

Full text of DOI:10.1016/j.bmc.2012.05.011

Bioorganic & Medicinal Chemistry 20 (2012) 4020–4031
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitumor agents 290. Design, synthesis, and biological evaluation of new
LNCaP and PC-3 cytotoxic curcumin analogs conjugated with anti-androgens q
Qian Shi a,c, , Koji Wada a, Emika Ohkoshi a, Li Lin a, Rong Huang a, Susan L. Morris-Natschke a,
⇑
Masuo Goto b, Kuo-Hsiung Lee a,d,
⇑
a Natural Products Research Laboratories, UNC Eshelmen School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
b Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7090, USA
c AndroScience Corporation, 11175 Flintkote Ave., Suite F, San Diego, CA 92121, USA
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 February 2012
Revised 1 May 2012
Accepted 8 May 2012
Available online 15 May 2012
In our continuing study of curcumin analogs as potential anti-prostate cancer drug candidates, 15 new
curcumin analogs were designed, synthesized and evaluated for cytotoxicity against two human prostate
cancer cell lines, androgen-dependent LNCaP and androgen-independent PC-3. Twelve analogs (5–12, 15,
16, 19, and 20) are conjugates of curcumin (1) or methyl curcumin (2) with a flutamide- or bicalutamide-
like moiety. Two compounds (22 and 23) are C4-mono- and difluoro-substituted analogs of dimethyl cur-
cumin (DMC, 21). Among the newly synthesized conjugates compound 15, a conjugate of 2 with a partial
bicalutamide moiety, was more potent than bicalutamide alone and essentially equipotent with 1 and 2
Keywords:
Synthesis
Curcumin analogs
Conjugates
Cytotoxicity
against both prostate tumor cell lines with IC50 values of 41.8 lM (for LNCaP) and 39.1 lM (for PC-3). A
cell morphology study revealed that the cytotoxicity of curcumin analogs or curcumin–anti-androgen
conjugates detected from both prostate cancer cell lines might be due to the suppression of pseudopodia
formation. A molecular intrinsic fluorescence experiment showed that 1 accumulated mainly in the
nuclei, while conjugate 6 was distributed in the cytosol. At the tested conditions, anti-androgens sup-
pressed pseudopodia formation in PC-3 cells, but not in LNCaP cells. The evidence suggests that distin-
guishable target proteins are involved, resulting in the different outcomes toward pseudopodia
suppression.
Anti-prostate cancer
Morphology
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
nonsteroidal anti-androgens for several months.6,7 One proposed
mechanism for this syndrome is a mutation of the AR caused by
Statistically, prostate cancer is still ranked as the most common
cancer in American males (28%) and was the second leading cause of
cancer-related death among men in the United States (11%) in
2010.2 Constitutive activation of the androgen receptor (AR) by high
levels of androgens is presumed to be responsible for the progres-
sion of prostate cancer. Most presently available chemotherapeutic
agents are anti-androgens, including steroidal anti-androgens and
nonsteroidal anti-androgens. The steroidal anti-androgens, such
as cyproterone acetate, possess partial agonistic activity to the AR
and inter-activity with other hormonal systems, which can induce
many complications.3–5 The nonsteroidal anti-androgens, such as
flutamide and bicalutamide, have fewer side effects and were
thought to be pure AR antagonists. However, anti-androgen with-
drawal syndrome has been discovered in patients treated with
the long-term use of these nonsteroidal anti-androgens, so that
the nonsteroidal anti-androgens exhibit agonistic activity to the
mutant AR.8 Flutamide and bicalutamide (a racemic mixture)
(Fig. 1) are two well-known nonsteroidal anti-androgens, widely
used for the clinical treatment of prostate cancer.9,10 However,
induction of anti-androgen-withdrawal syndrome in patients has
been a considerable problem. Combination usage with a gonadotro-
pin-releasing hormone agonist, such as goserelin acetate or leupro-
lide acetate, is currently recommended and used in the clinic to
minimize anti-androgen withdrawal syndrome.11 To date, none of
the effective clinically available anti-androgens is able to kill pros-
tate cancer cells.
Curcumin (1) (Fig. 1), a major yellow pigment of Curcuma longa,
has been reported to have anti-prostate cancer activity in vitro and
in vivo.12–14 Although the mechanism of action is still unknown, it
has been associated with multiple proteins/signaling pathways,
such as NF-kB,15 STATs,16 AP-1,17 MAPK,18 and Akt,19 etc. More re-
cently, Ras has been reported to be a potential target protein re-
lated to the anticancer activity.20 However, the metabolic
q
Antitumor agents 290. For paper 289, see Ref. 1.
⇑
Corresponding authors. Tel.: +1 919 843 6325; fax: +1 919 966 3893 (Q.S.); tel.:
+1 919 962 0066; fax: +1 919 966 3893 (K.-H.L.).
E-mail addresses: qshi1@email.unc.edu (Q. Shi), khlee@unc.edu (K.-H. Lee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.011

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4021
( Fig. 2). The in vitro anti-prostate cancer activity of the newly
synthesized entities was examined and will be used to guide fur-
ther optimization of new conjugates. It is also known that an
enol-ketone linkage in 1 (and its analogs) is an important struc-
tural feature for its biological activities.25 In order to further con-
firm this interesting structure–activity phenomenon and the
effectiveness of both enol-ketone/diketone isomers on prostate
cancer, we designed and synthesized compounds 22 and 23, which
contain fluorine as an isostere of hydrogen at the C4 position.36
With the mono-fluoro substitution, we expect that 22 will be sta-
bilized as only the enol-ketone isomer, while the di-fluoro substi-
tuted 23 should retain the diketone form without tautomerization
to an enol-ketone isomer. We also determined the subcellular dis-
tribution of 1 and its conjugate 6 in PC-3 cells by capturing their
intrinsic fluorescence, and observed clear dissimilarity. From these
preliminary results, we hope to gain information not only on new
SAR but also regarding promising new anti-prostate cancer leads
and mechanism of action.
CF3
CF3
NO2
F
CN
O
O
R
S
N
H
N
H
O
O
OH
Flutamide, R = H
(R,S) Bicalutamide
Hydroxyflutamide, R = OH
O
OH
OCH3
OH
H3CO
HO
1
Curcumin
Figure 1. Structures of flutamide, hydroxyflutamide, bicalutamide, and curcumin.
instability of 1 leads to its poor bioavailability, which, in part, pre-
vents 1 from being used as an efficient therapeutic drug.21–23 Other
reports have identified curcumin analogs that were cytotoxic
against androgen-dependent LNCaP cells and androgen-indepen-
dent PC-3 cells.24–27 Some analogs demonstrated potent anti-AR
activity in LNCaP cells and PC-3 cells transfected with wild type
AR, and were even more potent than hydroxyflutamide, the active
metabolite of flutamide,28,29 although again, the exact mechanism
of action remains unclear.
2. Results and discussion
2.1. Chemistry
The starting material 2 was prepared according to a reported
synthetic method.37 As illustrated in Scheme 1, the conjugation
of 1 or 2 with flutamide-related analogs utilized 2-bromo-2-
methyl propanoic acid, which was reacted with 4-substituted-3-
trifluoromethylphenylamines (3a–e) in the presence of EDCI and
DMAP to give corresponding amides (4a–e) in 30–65% yields. Reac-
tion of these amides with 1 or 2 in the presence of anhydrous ce-
sium carbonate (sodium iodide was used as the catalyst for
making 5 and 10) gave the desired compounds 5–12 (2–10% yields)
(Scheme 1), along with the recovered starting materials (1 or 2)
(recovery rates: 39–92%). Various reaction conditions were tried
(such as different bases: Cs2CO3 or K2CO3; solvents: CH3CN, DMF,
acetone–DMF, CH2Cl2 or THF; reaction temperature: rt–80 °C and
reaction times: 4 h–overnight) to improve the yields of the target
compounds, but were not successful. The low reaction yields are
probably due to the low reactivity of 4a–4e, resulting from the ste-
ric hindrance of adjacent dimethyl groups. The synthesis of com-
pounds 15 and 16 (Scheme 2) began with the reaction of
methacryloyl chloride with 4-nitro-3-trifluoromethylphenylamine
(3a) or 4-cyano-3-trifluoromethylphenylamine (3b) to afford
N-(4-nitro-3-trifluoromethylphenyl)-methacrylamide (13a) or N-
(4-cyano-3-trifluoromethylphenyl)-methacrylamide (13b). The
resulting N-arylmethacrylamides were subsequently oxidized to
the corresponding racemic epoxides (14a and 14b) by using a com-
bination of hydrogen peroxide and trifluoroacetic anhydride.38
Epoxides 14a and 14b were further reacted with 2 in the presence
of sodium hydride through a ring-opening procedure to afford the
target conjugates 15 in 64% yield and 16 in 41% yield (Scheme 2).
Compounds 19 and 20 were synthesized in three steps beginning
from the reaction of vanillin in DMF with ethyl 4-bromobutyrate
in the presence of K2CO3 (for 17a) or with 11-bromoundercanoic
acid in the presence of NaI and Cs2CO3 (for 17b). The vanillin
It is also known that the conjugation of antitumor drugs with
other components such as antioxidants or other antitumor agents
has provided some advantages in improving the antitumor efficacy
and selectivity, while decreasing the systematic toxicity.30–32 In
our search for new anti-prostate cancer drugs with increased po-
tency and minimized adverse anti-androgen effects, we considered
conjugation of curcumin or its analogs with clinically used anti-
androgens, such as flutamide and bicalutamide, to be a possible ap-
proach to develop new anti-prostate cancer leads, in addition to
the conventional structural modification of the curcumin molecule
alone. Although 1 has been extensively studied recently in combi-
nations with different cancer therapeutic agents to treat cancers
through synergistic activity,33,34 the conjugation of 1 (or its analog)
with a cancer therapeutic agent has not been explored. Unlike the
principle of drug combination, the biological activity of a conjugate
basically results from a single molecule in which two or more ac-
tive components are tethered through covalent chemical bonds.
The mechanism of action of the conjugate, therefore, could be dif-
ferent from that of the combination forms. Ideally, the advance-
ment of the conjugation approach lies in ‘one molecule double or
multiple functions’.35 This field merits exploration, and in this
manner, we hope to develop new drug candidates potentially
inhibiting proliferation of prostate cancer cells and mitigating the
side effects of existing anti-androgens. Structurally, flutamide
and bicalutamide share
a N-(4-substituted-3-(trifluoromethyl)
phenyl)acetamido moiety, which may be an essential pharmaco-
phore for their anti-prostate cancer activity. New conjugates in
which curcumin (1) or methyl curcumin (2) were coupled with a
flutamide molecule (or N-arylmethacrylamide moiety of bicaluta-
mide) through various linkages were designed and synthesized
O
OH
CF3
OCH3
O
H3CO
R1O
R3
O
linker
N
H
R1 = H or CH3
R2
R2 = CH3 or OH
R3 = CN, NO2, F, Cl or CH3
Figure 2. Design of curcumin analogs conjugated with hydroxyflutamide or bicalutamide moiety.

4022
Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
O
O
CF3
R
EDCI, DMAP
anhy. Cs2CO3, NaI
OH
NH
Br
CF3
O
OH
Br
OCH3
OH
H3CO
R'O
H2N
R
4a-e
3a-e
R' = H, Curcumin (1)
R' = CH , Methylcurcumin (2)
3
O
OH
CF3
OCH3
O
H3CO
R'O
R
a
b
, R = NO2
O
, R = CN
c, R = F
5 - 12
7
N
H
d, R = Cl
e, R = CH3
8
9
10
11
12
5
6
NO2
H
R
CN
H
F
Cl
H
CH3 NO2
CN
Cl
R'
H
H
CH3 CH3
CH3
Scheme 1. Synthesis of conjugates 5–12.
O
O
CF3
O
CF3
H2O2, (CF3CO)2O
NH
R
NH
13a,b
R
Cl
O
CF3
H2N
R
a
b
, R = NO2
14a,b
, R = CN
3a,b
NaH, THF
O
OH
CF3
OCH3
O
R
H3CO
H3CO
O
O
OH
OCH3
OH
H3CO
H3CO
N
H
2
OH
15, R = NO2
16, R = CN
Scheme 2. Synthesis of conjugates 15 and 16.
OHC
OCH3
OH
OHC
OCH3
O
i)
17a,b
COOR
n
Vanillin
OH
O
O
OH
H3CO
H3CO
H3CO
H3CO
OCH3
O
ii), iii)
COOH
18a,b
n
O
OH
H3CO
H3CO
OCH3
iv)
H
N
CF3
CN
CF3
CN
O
n
H2N
19, n = 1
20, n = 8
O
19
20
17a
: i) Ethyl 4-bromobutyrate, K2CO3, DMF, ii) 1) B2O3, 2)
4) 1%HCl, iii) 10%HCl, THF, iv) EDCI, DMAP, CH2Cl2
(R = CH2CH3, n = 1), (nBuO)3B, 3) nBuNH2,
17b
: i) 4-Bromoundecanoic acid, Cs2CO3, NaI, DMF-dioxane, ii) 1) B2O3, 2)
(R = H, n = 8), (nBuO)3B,
3) nBuNH2, 4) 1%HCl, iv) EDCI, DMAP, CH2Cl2
Scheme 3. Synthesis of conjugates 19 and 20.
intermediates 17a and 17b were obtained in yields of 99% and 50%,
6-(3,4-dimethoxyphenyl)-4-hydroxyhexa-3,5-dien-2-one25 to af-
respectively (Scheme 3). Reaction of these intermediates with
ford the corresponding product 18a or 18b was achieved by

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4023
following a slightly modified literature procedure.39 Hydrolysis in
10% aq HCl followed by coupling with 4-amino-2-(trifluoro-
methyl)benzonitrile afforded the desired products 19 and 20.
Compound 22, a C4-mono-fluoro analog, was obtained by fluo-
rination of dimethyl curcumin (DMC, 21) with SelectFluor™ in
DMF. The yield of 22 was relatively low (7%) due to the difficulty
in controlling mono-substitution. Di-fluoro analog 23 was obtained
in the presence of sodium hydride as a major product in 31% yield
(Scheme 4).
Table 1
Cytotoxicity data for curcumin and curcumin analogs against LNCaP and PC-3 human
prostate cancer cell linesa
Compound
IC50 SE (lM)
LNCaP
PC-3
Curcumin (1)
Bicalutamide
Flutamide
2
36.4 2.4
74.0 5.9
81.8 2.1
47.0 3.2
>100
36.0 1.4
76.4 7.2
98.8 3.2
33.1 2.5
>100
5
6
7
8
55.0 4.1
54.8 2.5
>100
48.1 4.6
52.1 4.8
>100
2.2. Biological activity and SAR
9
>100
>100
>100
>100
41.8 3.9
73.0 4.2
96.3 8.3
>100
15.3 2.8
27.3 3.0
53.8 4.9
>100
>100
>100
>100
39.1 3.1
100.3 1.9
>100
The newly synthesized analogs were evaluated for cytotoxicity
against androgen-dependent LNCaP and androgen-independent
PC-3 human prostate cancer cell lines. The in vitro cytotoxicity bio-
assay was performed according to procedures described in litera-
ture.40 The test compounds were dissolved in DMSO as stock
solutions and stored in the dark at ꢀ20 °C. The final concentration
of DMSO was <0.5% (v/v) in all experiments. The human prostate
cancer cells were exposed to doses of the compounds for three
days. IC50 was defined as the concentration of test compound caus-
ing 50% inhibition of cell growth, as compared to the untreated
control. IC50 values were calculated by log—linear interpolation
of data points. Of the three marketed compounds, 1 was twice as
potent as the clinically used anti-androgens flutamide and bicalu-
tamide against both prostate cancer cell lines in vitro. The IC50 val-
10
11
12
15
16
19
20
21
22
23
>100
23.7 1.4
17.7 2.4
44.3 4.8
a
Cell proliferation was determined by SRB assay as described in ‘Section 4.13’.
Data are expressed as the means SE of at least three or more independent
experiments.
ues for 1 were 36.4 2.4 for LNCaP cell line and 36.0 1.4
lM for
was significantly reduced relative to the individual parent com-
pounds (1 or 2 and flutamide). Conjugates 6 and 7, which contain cy-
ano and fluoro groups, respectively, on the aniline ring in the
flutamide substructure, showed significant cytotoxicity against both
prostate cancer cell lines, while conjugates with nitro, chloro, and
methyl groups did not. The results again suggested that the substit-
uents on the flutamide substructure may be important for interac-
tion of the conjugate molecule with the target protein(s).
However, the curcuminoid portion of the molecule is also important,
as conjugate 11, which has the same cyano-flutamide substructure
found in 6, but is conjugated with 2 rather than 1, showed at least
twofold lower cytotoxicity against both LNCaP and PC-3 (IC50
PC-3 cell line compared with 81.8 2.1 M and 98.8 3.2
l
lM,
respectively, for flutamide and 74.0 5.9 and 76.4 7.2
respectively, for bicalutamide (Table 1).
Among the 12 conjugates, compound 15, a conjugate between 2
and N-[4-nitro-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methyl-
propanamide (hydroxyflutamide) exhibited the most potent cyto-
lM,
toxicity against both cell lines with IC50 values of 41.8 3.9
against LNCaP and 39.1 3.1 M against PC-3 proliferation. It
was essentially equipotent with its parent compound 2 (IC50
47.0 3.2 M against LNCaP and 33.1 2.5 M against PC-3) as
lM
l
l
l
well as 1 and almost twofold more potent than flutamide and bica-
lutamide (Table 1). Interestingly, 16, which had only a slight struc-
tural variation compared with 15 [the nitro group (NO2) on the
aniline ring of the hydroxyflutamide-related substructure of 15
was replaced with a cyano group (CN) in 16], was almost twofold
less active than 15 against both cancer cell lines (IC50
>100 lM) compared with 6 or 2, implying that the cytotoxic potency
depended on the substituents on both parts of the conjugate. Even
very slight structural deviations (e.g., NO2 vs CN in the flutamide
substructure and OCH3 vs OH in the curcuminoid part) could affect
the potency.
73.0 4.2 lM against LNCaP and 100.3 1.9 lM against PC-3). This
The identity of the linker between the curcuminoid phenoxy
oxygen and flutamide amido carbonyl also played a role in the
cytotoxic activity. As described above, conjugates 15 and 16 [lin-
ker = –CH2C(CH3)(OH)–] showed significant activity, while the cor-
responding 10 and 11 [linker = –C(CH3)2–] did not. In addition,
conjugates 19 and 20 with unbranched propyl and decyl linkers,
respectively, also did not show significant activity.
result suggests that substitutions in the flutamide portion of the
conjugate molecule can greatly impact the biological activity.
The curcuminoids 1 and 2 were also linked with several 4-substi-
tuted-3-trifluoromethylphenylanilines through an –O-C(CH3)2-CO–
amide bond generating compounds 5–9 and 10–12, respectively.
Surprisingly, except for 6 and 7, the anti-prostate cancer activity
O
OH
OCH3
OCH3
H3CO
H3CO
F
SelectFluor
O
OH
22
OCH3
OCH3
H3CO
H3CO
DMF
SelectFluor
O
O
OCH3
OCH3
H3CO
H3CO
21
NaH, THF
F
F
23
Scheme 4. Synthesis of conjugates 22 and 23.

4024
Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
The antiproliferation activities induced by the conjugates in-
PC-3
Bicalutamide
Flutamide
creased in a dose-dependent manner. Figures 3 and 4 show dose-
dependent curves of the conjugates 6 and 15 on viability of LNCaP
and PC-3 cells compared with bicalutamide and flutamide in RPMI-
1640 medium supplemented in 10% FBS, slightly modified from lit-
erature reported methods.41,42 Both conjugates demonstrated
more potent antiproliferation effects than the tested anti-
androgens.
Besides the 12 conjugates, two compounds, 22 and 23, were
synthesized from dimethyl curcumin 21 to investigate the role of
enol-ketone tautomerism in the anti-prostate cancer cytotoxicity.
Compound 21 was previously synthesized and reported to show
anti-prostate cancer activity.25,26 One of the important structural
features contributing to the activity is the extended unsaturated
system resulting from the enol-ketone bridge between the two
phenyl groups. The enol-ketone exists as a stable and predominate
tautomer in the equilibrium with its diketone form. Compound 22,
with mono-fluoro substitution at the C4 position, was designed to
maximize the enol-ketone form of 21, while compound 23, with a
di-fluoro replacement at C4, was designed to maintain the struc-
ture in the diketone form. Interestingly, compound 22 (IC50
100
80
60
40
20
0
Compound 6
Compound 15
0
20
40
60
80
100
Concentration (µM)
Figure 4. Dose–response curves of conjugates and anti-androgens in PC-3 cells.
Cells were cultured in RPMI-1640 medium supplemented with 10% FBS in the
presence of compound for 72 h. All data were calculated as the mean SE of
triplicate samples and are representative of at least three individual experiments.
27.3 3.0
most as potent as the parent compound 21 and twofold more cyto-
toxic than its di-fluoro analog 23 (IC50 53.8 4.9 M against LNCaP,
44.3 4.8 M against PC-3), supporting the postulate that the
lM against LNCaP, 17.7 2.4 lM against PC-3) was al-
l
l
enol-ketone tautomerism may play a certain role in inhibition of
prostate cancer cell growth in vitro. The physicochemical property
of the fluoro substitution may also affect the potency.
pseudopodia formation compared with the control. In contrast,
the androgen antagonist bicalutamide did not suppress the pseudo-
podia formation in LNCaP cells, even at a concentration as high as
Bicalutamide and flutamide are well known anti-androgens.
They inhibit prostate cancer cell growth through binding to the AR
on the prostate and preventing the binding of the natural ligand.
Although it has long been known that 1 and its analogs exhibit
anti-prostate cancer effects, the mechanism behind the biological
activity remains unclear. To identify the target points of cytotoxicity
from molecular cell biology level, morphologic changes generated
by active compounds were examined. LNCaP and PC-3 prostate tu-
mor cells were first treated with 1, 2, and two cytotoxic conjugates,
6 and 15, as well as the anti-androgen bicalutamide. After incuba-
tion with agents for 48 h, the cells were stained with DNA-specific
fluorescence dye DAPI, and the morphologic changes in the cells
were analyzed. For LNCaP cells, the observed cell morphology indi-
cated that 1, 2, and the conjugates strongly inhibited actin-based
100 lM (Fig. 5). These observations suggested that 1 and its analogs,
as well as conjugates, might share similar mechanism(s) of action in
LNCaP cytotoxicity by inhibiting F-actin formation, not only for the
production of pseudopodia, but also the dynamism of cytoskeletal
actin filaments. Interestingly, for PC-3 cells, all tested compounds,
including anti-androgen bicalutamide significantly suppressed
pseudopodia-like extension (Fig. 6). In addition, 1 and 2 induced nu-
clear enlargement, suggesting that nuclear division and cell cleav-
age were impaired in PC-3 cells, which might induce cell cycle
arrest at the G2-phase. In contrast to treatment with 1, multinucle-
ated cells were observed when cells were treated with conjugate 6
or 15 (Fig. 6), indicating that while 1 itself inhibited cell cycle pro-
gression,43 the conjugates induced irregular nuclear division result-
ing in production of polykaryotic cells that were subject to undergo
apoptosis. These findings led to the hypothesis that 1 and its analogs
may target the actin cytoskeletal dynamics, but it is still unclear
whether the irregular nuclear division induced by the conjugates
in PC-3 cells does or does not result from the suppression of F-actin
formation. Such morphologic changes were not observed in the cells
treated with the anti-androgen, and it is also notable that neither
flutamide nor bicalutamide showed significant cytotoxicity against
Bicalutamide
Flutamide
100
Compound 6
Compound 15
PC-3 cells within 48 h even at concentration of 100 lM (data not
shown), while suppression of pseudopodia-like formation was
observed.
80
60
40
20
0
The findings resulting from the current morphology study in
LNCaP and PC-3 prostate cancer cells clearly indicated that differ-
ent cellular molecules are targets of 1, its analogs, and conjugates,
as well as anti-androgens, though it is still unclear whether the cel-
lular defects distinguishable between PC-3 and LNCaP are caused
by expression of the AR. Because all test compounds in this study
displayed little or no cytotoxic selectivity towards LNCaP or PC-3
tumor cells, we have speculated that the displayed cytotoxicity
might not be associated completely or at all with androgen or
the AR. Nevertheless, because pseudopodia formation is highly re-
lated to cell migration and tumor metastasis,44 1 and its analogs,
including conjugates, may effectively inhibit prostate tumor
metastasis. The observed morphological change induced by the
anti-androgens in PC-3 cells may also lead to their marginal
0
20
40
60
80
100
Concentration (µM)
Figure 3. Dose–response curves of conjugates and anti-androgens in LNCaP cells.
Cells were cultured in RPMI-1640 medium supplemented with 10% FBS in the
presence of compound for 72 h. All data were calculated as the mean SE of
triplicate samples and are representative of at least three individual experiments.

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4025
Figure 5. Suppression of pseudopodia formation in LNCaP cells by curcumin analogs. Androgen-sensitive LNCaP cells were cultured for 48 h in RPMI-1640 medium
supplemented with 10% FBS in the presence of compound. Cells were fixed and stained with DAPI for DNA. Vigorous pseudopodia formation was seen in untreated (DMSO) or
100
lM bicalutamide-treated cells (arrows), while pseudopodia formation was limited in cells treated with 6 or 15, and completely impaired in cells treated with 1 or 2.
These observations implied that curcumin and its analogs strongly inhibited pseudopodia formation in LNCaP cells. Bar, 50
lm.
cytotoxicity in a certain degree. Cell invasion assays in the pres-
ence of 1-analogs are currently underway.
from 1-treated cells was accumulated in the cell nuclei, while for
6 treated cells, intrinsic fluorescence was detected only in the
cytosol. These findings demonstrated a different subcellular distri-
bution between 1 and the conjugate 6. Curcumin distributes and
accumulates mainly in the nuclear region, while the conjugate
accumulates mainly in the cytosol.
The structural uniqueness of 1 and its structural analogs results
in detectable molecular intrinsic fluorescence, which enables us to
detect the distribution of the drugs in the cells by capturing the
intrinsic fluorescence under a fluorescence microscope. Experi-
mentally, PC-3 cells were treated with 1 and conjugate 6, respec-
tively, for 1 h (this experiment was not performed in LNCaP cells
due to extremely slow growth), and as expected, the intrinsic green
fluorescence generated by treated compounds was observed in PC-
3 cells (Fig. 7). Interestingly, the intrinsic fluorescence captured
3. Conclusions
In this study, we designed and synthesized new 1-analogs,
including conjugates of 1 (or its analogs) and anti-androgens (such

4026
Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
Figure 6. Induction of nuclear fragmentation in PC-3 cells by curcumin analogs. Androgen-independent PC-3 cells were treated with compound as indicated for 48 h followed
by staining with DAPI for DNA. PC-3 cells treated with 1 or 2 often showed nuclear enlargement (black arrows), suggesting that PC-3 cells induced cell cycle arrest at G2/M.
Interestingly, when PC-3 cells were treated with 6 or 15, polykaryocytes (multinucleated cells) (arrow heads) were frequently observed. In addition, pseudopodia-like
extension (white arrow) was not seen in agent-treated cells. Bar, 50 lm.
as flutamide or a substructure of bicalutamide or modified mole-
cules). The preliminary SAR profile showed that conjugate 15
was equipotent to 1 and 2, and much more potent than flutamide
or bicalutamide. Two additional conjugates, 6 and 7, also showed
more cytotoxic potency than the clinically used anti-androgens flu-
tamide and bicalutamide against both androgen-dependent LNCaP
and androgen-independent PC-3 cell lines. These results implied
that appropriate accommodation of a large curcuminoid group on
the carbon adjacent to the amide carbonyl of flutamide or bicalu-
tamide may enhance the inhibition of prostate cancer cells prolif-
eration through different mechanism of action other than
targeting to the AR. Compound 22, a C4 mono-fluorinated curcumi-
noid, showed comparable potency to its parent compound 21 and
greater potency than its di-fluorinated analog 23. Morphological
and intrinsic fluorescence analysis indicated that 1, its analogs,
and conjugates may target the actin cytoskeletal dynamics in both
LNCaP and PC-3 cells. In PC-3 cells, 1 distributed and accumulated
mainly in the nuclei of the cells and inhibited cell cycle progression
by targeting the functional proteins in the nuclear region, while the
conjugates 6 and 15 accumulated mainly in the cytosol and in-
duced irregular nuclear division, which could lead to prokaryotic
cells and eventually apoptosis.

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4027
Figure 7. Subcellular distribution of curcumin (1) and conjugate 6 in PC-3 cells. PC-3 cells were briefly treated with 1 or 6. The intrinsic fluorescence of compound was
observed under the fluorescence microscope (left panels, green). DAPI was used for staining nuclei (center panels, red), and merged with intrinsic fluorescence (right panels).
The intrinsic fluorescence of 1 or 6 was detected and showed subcellular distribution of these compounds in PC-3 cells. Bar, 50 lm.
(s, 1H, aromatic-H), 8.06 (s, 1H, aromatic-H), ESI MS m/z 355.98
4. Experiments
4.1. Chemistry
(M+H)+.
4.2.3. 2-Bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
methylpropanamide (4b)
Melting points were determined on a Fisher–John melting point
apparatus and are uncorrected. Proton nuclear magnetic resonance
(1H NMR) and 13C NMR spectra were measured on Varian Gemini
300 or Inova 400 spectrometers with tetramethylsilane (TMS) as
the internal standard. Chemical shifts are reported in d (ppm).
Mass spectra (MS) were obtained on a Shimadzu LCMS-2010. Com-
biFlashÒ chromatographic system (Isco Companion) with a Grace
silica gel cartridge was used for general separation and purifica-
tion. Preparative thin layer chromatography (PTLC) on silica gel
plates (Kieselgel 60, F254, 1.50 mm) was also used for separation
and purification. Precoated silica gel plates (Kieselgel 60, F254,
0.25 mm) were used for thin layer chromatography (TLC) analysis.
Shimadzu LC-20AT prominence liquid chromatography was
Yield: 64%; white solid; mp 168–170 °C; 1H NMR (400 MHz,
CDCl3): d 1.90 (3H, s, CH3), 2.06 (3H, s, CH3), 7.82 (1H, d,
J = 8.6 Hz, Ar-H), 7.95 (1H, dt, J = 8.6, 2.1 Hz, Ar-H), 8.11 (1H, dd,
J = 6.6, 1.9 Hz, Ar-H), 8.80 (br, 1H, NH). ESI-MS (negative, m/z):
333.05 [MꢀH]ꢀ, 335.00 [M+2ꢀH]ꢀ.
4.2.4. 2-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-
methylpropanamide (4c)
Yield: 64%; white solid; mp 111–112 °C; 1H NMR (400 MHz,
CDCl3): d 1.89 (3H, s, CH3), 2.06 (3H, s, CH3), 7.20 (1H, t,
J = 9.3 Hz, Ar-H), 7.73–7.75 (1H, m, Ar-H), 7.84 (1H, td, J = 9.0,
2.7 Hz, Ar-H), 8.52 (br, 1H, NH). ESI-MS (negative, m/z): 325.95
[MꢀH]ꢀ, 328.00 [M+2ꢀH]+.
used for HPLC. Alltima 2.1 mm ꢁ 100 mm C18 3
lm was used as
HPLC column. All reagents and solvents were purchased from
Aldrich, Fisher, VWR, and other venders. Some chemicals were
used after purification, and others were used as purchased. Com-
pounds 1, 2, and 21 were synthesized by following literature
methods.25,26 All final compounds are >95% pure through HPLC
analysis.
4.2.5. 2-Bromo-N-(4-chloro-3-(trifluoromethyl)phenyl)-2-
methylpropanamide (4d)
Yield: 29%; white solid; mp 134–135 °C; 1H NMR (400 MHz,
CDCl3): d 2.05 (6H, s, CH3), 7.48 (1H, d, J = 8.8 Hz, Ar-H), 7.73 (1H,
dd, J = 8.8, 2.5 Hz, Ar-H), 7.91 (1H, d, J = 2.5 Hz, Ar-H), 8.54 (br,
1H, NH). ESI-MS (negative, m/z): 342.05 [MꢀH]ꢀ, 344.05
[M+2ꢀH]ꢀ, 345.95 [M+4ꢀH]ꢀ.
4.2. Synthesis of 2-bromo-2-methyl-N-(4-substituted-3-
(trifluoromethyl)-phenylpropanamides (4a–e)
4.2.6. 2-Bromo-N-(4-methyl-3-(trifluoromethyl)phenyl)-2-
methylpropanamide (4e)
4.2.1. General procedure
Yield: 49%; white solid; mp 125–126 °C; 1H NMR (400 MHz,
CDCl3): d 2.05 (6H, s, CH3), 2.46 (3H, s, Ar-CH3), 7.27 (1H, d,
J = 8.2 Hz, Ar-H), 7.64 (1H, dd, J = 8.2, 2.1 Hz, Ar-H), 7.78 (1H, d,
J = 2.1 Hz, Ar-H), 8.48 (br, 1H, NH). ESI-MS (negative, m/z):
322.15 [MꢀH]ꢀ, 324.00 [M+2ꢀH]ꢀ.
2-Bromo-2-methylpropionic acid (1.5 equiv), EDCI (1.5 or
2 equiv) and DMAP (1.5 or 2 equiv) were added to a solution of
4-substituted-3-(trifluoromethyl)aniline in CH2Cl2 (for 3a,b,c,e)
or pyridine (for 3d), and the mixture was stirred at 35 °C overnight
(60 °C, 4 h for pyridine as solvent) under N2. The mixture was di-
luted with CH2Cl2, washed with satd NH4Cl (aq), satd NaHCO3
(aq), and brine, then dried over anhydrous MgSO4. After removal
of the solvent in vacuo, the crude products were purified by silica
gel column chromatography eluting with n-hexane–EtOAc.
4.3. Synthesis of N-(4-substituted-3-(trifluoromethyl)phenyl)
pivalamide-curcumin or methylcurcumin analogs (5–12)
4.3.1. General procedure
Compounds 4a–4e (1.1, 1.5 or 3.0 equiv) and Cs2CO3 (1.5 equiv)
were added to a solution of 1 or 2 in CH3CN, and the mixture was
stirred at 60 °C for 3 h, 5 h or overnight under N2. The mixture was
quenched with 0.1 N HCl to pH 6 and extracted three times with
4.2.2. 2-Bromo-N-(4-nitro-3-(trifluoromethyl)phenyl)-2-
methylpropanamide (4a)
Yield: 12%, white solid powder; 1H NMR (300 MHz, CDCl3): d
2.06 (s, 3H, CH3), 2.07 (s, 3H, CH3), 7.98 (s, 1H, aromatic-H), 7.99

4028
Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
EtOAc. The combined organic layers were washed with H2O and
brine. After being dried over anhydrous Na2SO4, the solvent was re-
moved in vacuo. The crude products were purified by CombiFlashÒ
column chromatography or preparative-TLC eluting with n-hex-
ane–EtOAc.
4.3.7. N-(4-Nitro-3-(trifluoromethyl)phenyl)pivalamide-
methylcurcumin (10)
Yield: 3%; amorphous solid; 1H NMR (300 MHz, acetone-d6): d
8.55 (s, 1H), 8.32 (s, br, 1H), 8.18–8.20 (m, 1H), 7.66–7.61 (m,
2H), 7.46 (br s, 1H), 7.39–7.14 (m, 4H), 7.00–7.03 (m, 1H), 6.86–
6.73 (m, 2H), 6.03 (s, 1H), 3.98 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 1.57 (s, 6H, CH3); ESI MS m/z 657.3 (M+H)+.
4.3.2. N-(4-Nitro-3-(trifluoromethyl)phenyl)pivalamide-
curcumin (5)
Yield: 10%; amorphous solid; 1H NMR (300 MHz, DMSO-d6): d
8.49 (s, 1H), 8.36–8.32 (m 1H), 8.24–8.21 (m, 1H), 7.58–7.53 (m,
2H), 7.42 (br s, 1H), 7.32 (br s, 1H), 7.26–7.14 (m, 2H), 6.96–6.89
(m, 2H), 6.84–6.56 (m, 2H), 6.09 (s, 1H), 3.84 (s, 3H, OCH3), 3.81
(s, 3H, OCH3), 1.53 (s, 6H, CH3); ESI MS m/z 643.13 (M+H)+.
4.3.8. N-(4-Cyano-3-(trifluoromethyl)phenyl)pivalamide-
methylcurcumin (11)
Yield: 5% (starting with 0.13 mmol of methylcurcumin), amor-
phous solid; 1H NMR (400 MHz, CDCl3): d 1.79 (6H, s, CH3), 3.80,
3.93 and 3.94 (each 3H, s, OCH3), 4.96 (1H, s), 5.84 (1H, s, C@CH),
6.51 and 6.55 (each 1H, d, J = 16.0 Hz, CH@CH), 6.79 (1H, dd,
J = 8.4, 2.3 Hz, Ar-H0), 6.89 (1H, d, J = 8.4 Hz, Ar-H), 6.90 (1H, d,
J = 8.0 Hz, Ar-H), 6.96 (1H, d, J = 2.3 Hz, Ar-H0), 7.08 (1H, d,
J = 1.7 Hz, Ar-H), 7.10 (1H, s, Ar-H), 7.14 (1H, dd, J = 8.0, 1.7 Hz,
Ar-H), 7.15 (1H, dd, J = 8.2, 1.7 Hz, Ar-H), 7.59 and 7.63 (each 1H,
d, J = 15.8 Hz, CH@CH), 7.61 (1H, d, J = 8.6 Hz, Ar-H0). ESI-MS (posi-
tive, m/z): 637.30 [M+H]+.
4.3.3. N-(4-Cyano-3-(trifluoromethyl)phenyl)pivalamide-
curcumin (6)
Yield: 5% (starting with 0.26 mmol of curcumin), amorphous
solid; 1H NMR (400 MHz, CDCl3): d 1.58 (6H, s, CH3), 3.95 and
3.96 (each 3H, s, OCH3), 5.83 (1H, s, C@CH), 6.50 and 6.56 (each
1H, d, J = 15.8 Hz, CH@CH), 6.94 (1H, d, J = 8.2 Hz, Ar-H), 7.06
(1H, d, J = 1.7 Hz, Ar-H), 7.08 (1H, d, J = 8.0 Hz, Ar-H), 7.136 (1H,
dd, J = 8.2, 1.7 Hz, Ar-H), 7.141 (1H, d, J = 1.9 Hz, Ar-H), 7.17
(1H, dd, J = 8.4, 1.9 Hz, Ar-H), 7.61 and 7.62 (each 1H, d,
J = 15.8 Hz, CH@CH), 7.83 (1H, d, J = 8.4 Hz, Ar-H0), 8.03 (1H, dd,
J = 8.4, 2.1 Hz, Ar-H0), 8.09 (1H, d, J = 1.9 Hz, Ar-H0), 10.02 (1H,
s). ESI-MS (positive or negative, m/z): 623.50 [M+H]+, 621.30
[MꢀH]ꢀ.
4.3.9. N-(4-Chloro-3-(trifluoromethyl)phenyl)pivalamide-
methylcurcumin (12)
Yield: 3% (starting with 0.26 mmol of methylcurcumin), amor-
phous solid; 1H NMR (400 MHz, CDCl3): d 1.72 (6H, s, CH3), 3.78,
3.91 and 3.92 (each 3H, s, OCH3), 4.36 (1H, s), 5.82 (1H, s, C@CH),
6.49 and 6.53 (each 1H, d, J = 15.6 Hz, CH@CH), 6.75 (1H, dd,
J = 8.7, 2.7 Hz, Ar-H0), 6.87 (2H, d, J = 8.2 Hz, Ar-H), 6.98 (1H, d,
J = 2.7 Hz, Ar-H0), 7.067 (1H, s, Ar-H), 7.074 (1H, s, Ar-H0), 7.11
(1H, dd, J = 8.0, 1.7 Hz, Ar-H), 7.13 (1H, dd, J = 8.0, 1.7 Hz, Ar-H0),
7.27 (1H, d, J = 8.7 Hz, Ar-H0), 7.57 and 7.61 (each 1H, d,
J = 15.6 Hz, CH@CH). ESI-MS (positive, m/z): 646.15 [M+H]+.
4.3.4. N-(4-Fluoro-3-(trifluoromethyl)phenyl)pivalamide-
curcumin (7)
Yield: 6% (starting with 0.27 mmol of curcumin), amorphous so-
lid; 1H NMR (400 MHz, CDCl3): d 1.58 (6H, s, CH3), 3.95 (6H, s,
OCH3), 5.83 (1H, s, C@CH), 6.49 and 6.55 (each 1H, d, J = 15.8 Hz,
CH@CH), 6.94 (1H, d, J = 8.2 Hz, Ar-H), 7.05 (1H, s, Ar-H), 7.07
(1H, d, J = 8.6 Hz, Ar-H), 7.13 (1H, s, Ar-H), 7.16 (2H, d, J = 8.2 Hz,
Ar-H), 7.61 and 7.62 (each 1H, d, J = 15.8 Hz, CH@CH), 7.75 (1H,
d, J = 8.6 Hz, Ar-H0), 7.87 (1H, d, J = 8.6 Hz, Ar-H0), 7.90 (1H, s, Ar-
H0), 9.68 (1H, s). ESI-MS (positive or negative, m/z): 616.25
[M+H]+, 614.40 [MꢀH]ꢀ.
4.4. Synthesis of N-(4-substituted-3-(trifluoromethyl)phenyl)
methacrylamide (14a,b): N-(4-substituted-3-(trifluoromethyl)
phenyl)-2-hydroxy-2-methylpropanamide-methylcurcumin
(15–16)
4.4.1. General procedure
Methacryloyl chloride (1.2–3.0 equiv) was added dropwise to a
solution of 4-nitro-3-trifluoromethylphenylamine (3a) or 4-cyano-
3-trifluoromethylphenylamine (3b) in CH2Cl2 containing triethyl-
amine (1.2–3.0 equiv) at 0 °C. After stirring at room temperature
for 4–12 h with TLC monitoring, the reaction mixture was diluted
with CH2Cl2 and washed with H2O. The CH2Cl2 extract was then
dried over Na2SO4, concentrated, and purified by flash column
chromatography to afford the desired compounds.
4.3.5. N-(4-Chloro-3-(trifluoromethyl)phenyl)pivalamide-
curcumin (8)
Yield: 6% (starting with 0.33 mmol of curcumin), yellow solid;
mp 268–270 °C, 1H NMR (400 MHz, CDCl3): d 1.58 (6H, s, CH3),
3.95 and 3.96 (each 3H, s, OCH3), 5.83 (1H, s, C@CH), 6.50 and
6.55 (each 1H, d, J = 15.8 Hz, CH@CH), 6.94 (1H, d, J = 8.2 Hz, Ar-
H), 7.06 (1H, d, J = 1.9 Hz, Ar-H), 7.07 (1H, d, J = 8.2 Hz, Ar-H),
7.13 (1H, d, J = 1.9 Hz, Ar-H), 7.14 (1H, dd, J = 8.6, 1.9 Hz, Ar-H),
7.16 (1H, dd, J = 8.4 1.9 Hz, Ar-H), 7.49 (1H, d, J = 8.7 Hz, Ar-H0),
7.61 and 7.62 (1H, d, J = 15.8 Hz, CH@CH), 7.88 (1H, dd, J = 8.6,
2.5 Hz, Ar-H0), 7.96 (1H, d, J = 2.5 Hz, Ar-H0), 9.74 (1H, s). ESI-MS
(positive or negative, m/z): 632.10 [M+H]+, 634.25 [M+2+H]+,
630.35 [MꢀH]ꢀ, 632.35 [M+2ꢀH]ꢀ.
4.4.2. N-(4-Nitro-3-trifluoromethylphenyl)methacrylamide
(13a)
Yield: 41%; amorphous solid; 1H NMR (300 MHz, CDCl3): d 1.98
(s, 3H, CH3), 5.70 (s, 1H, ethylene-H), 5.79 (s, 1H, ethylene-H),
8.38–8.17 (m, 3H, aromatic-H); ESI MS m/z 274.2 (M+H)+.
4.3.6. N-(4-Methyl-3-(trifluoromethyl)phenyl)pivalamide-
curcumin (9)
4.4.3. N-(4-Cyano-3-trifluoromethylphenyl)methacrylamide
(13b)
Yield: 2% (starting with 0.27 mmol of 1), amorphous solid; 1H
NMR (400 MHz, CDCl3): d 1.58 (6H, s, CH3), 2.46 (3H, s, Ar-CH3),
3.80 and 3.95 (each 3H, s, OCH3), 5.85 (1H, s, C@CH), 6.56 and
6.57 (each 1H, d, J = 15.8 Hz, CH@CH), 6.77 (1H, dd, J = 8.4,
2.5 Hz, Ar-H), 6.88 (1H, d, J = 8.0 Hz, Ar-H), 6.97 (1H, d, J = 2.3 Hz,
Ar-H), 7.07 (1H, d, J = 8.2 Hz, Ar-H), 7.08 (1H, s, Ar-H), 7.15 (1H,
d, J = 8.0 Hz, Ar-H), 7.28 (1H, d, J = 8.7 Hz, Ar-H0), 7.61 and 7.63
(each 1H, d, J = 15.8 Hz, CH@CH), 7.80 (1H, d, J = 8.4 Hz, Ar-H0),
7.83 (1H, s, Ar-H0), 9.61 (1H, s). ESI-MS (positive or negative, m/
z): 612.25 [M+H]+, 610.15 [MꢀH]ꢀ.
Yield: 20%; amorphous solid; 1H NMR (300 MHz, CDCl3): d 2.08
(s, 3H, CH3), 5.60 (s, 1H, ethylene-H), 5.86 (s, 1H, ethylene-H),
8.05–7.78 (m, 3H, aromatic-H); ESI MS m/z 254.2 [M+H]+.
4.5. Synthesis of N-(4-substituted-3-(trifluoromethyl)phenyl)-
2-methyloxirane-2-carboxamide (14a,b)
4.5.1. General procedure
To a solution of N-arylmethacrylamides (13a or 13b, 1.7 mmol)
in CH2Cl2 (25 mL) was added 0.31 mL of hydrogen peroxide (30%,

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4029
w/w) followed by 1.25 mL of trifluoroacetic anhydride. After stir-
ring at rt for 2 h, the reaction was quenched by addition of sodium
bisulfide and extracted with CH2Cl2. The organic layer was washed
with satd NaHCO3 then brine, and dried over MgSO4. After removal
of the solvent by evaporation, the crude was purified through a
CombiFlashÒ chromatograph system to afford the desired products
as racemic epoxides.
9.8 mmol) were added and the mixture was stirred at 80 °C for
2 days under N2. The mixture was quenched with 0.1 N HCl to
pH 6 and extracted three times with EtOAc. The combined organic
layer was washed with H2O and brine, and then dried over anhy-
drous MgSO4, and the solvent was removed in vacuo. The crude
was purified by CombiFlashÒ column chromatography eluting with
n-hexane–EtOAc gradient to give the desired compound (1.7 g,
99%), white solid; mp 75–76 °C; 1H NMR (400 MHz, CDCl3): d
1.26 (3H, t, J = 7.2 Hz, OCH2CH3), 2.20 (2H, quintet, J = 6.8 Hz,
OCH2CH2CH2CO), 2.55 (2H, t, J = 7.2 Hz, OCH2CH2CH2CO), 3.92
(3H, s, OCH3), 4.15 (2H, q, J = 7.2 Hz, OCH2CH3), 4.17 (2H, t,
J = 6.4 Hz, OCH2CH2CH2CO), 6.99 (1H, d, J = 8.2 Hz, Ar-H), 7.41
(1H, d, J = 1.7 Hz, Ar-H), 7.44 (1H, dd, J = 8.2, 1.7 Hz, Ar-H), 9.85
(1H, s, CHO). ESI-MS (positive, m/z): 267.05 [M+H]+, 289.00
[M+Na]+.
4.5.2. N-(4-Nitro-3-(trifluoromethyl)phenyl)-2-methyloxirane-
2-carboxamide (14a)
Yield: 81%; pale-yellow powder; 1H NMR (300 MHz, CDCl3) d
1.70 (s, 3H, CH3). 3.04 (s, 2H, epoxide-H), 7.23 (s, 1H, NH), 7.82
(d, 1H, J = 8.7 Hz, aromatic-H), 8.17 (d, 1H, J = 8.7 Hz, aromatic-
H), 8.23 (s, 1H, aromatic-H); ESI MS m/z 291.7 [M+H]+.
4.5.3. N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-methyloxirane-
2-carboxamide (14b)
4.7.1. 4-(4-Formyl-2-methoxyphenoxy)undecanoic acid (17b)
Compound 17b was synthesized using a similar procedure to
that of making 17a with slight modification. To a solution of van-
illin (502 mg, 3.3 mmol) in DMF–dioxane (3:1, 20 mL), 11-bro-
moundecanoic acid (2.6 g, 9.9 mmol), NaI (2.5 g, 16.4 mmol) and
Cs2CO3 (2.1 g, 6.6 mmol) were added and the mixture was stirred
at 100 °C for 4 days under N2. The workup and purification steps
were as described above from 17a to give 17b (552 mg, 50%) as
an amorphous solid; 1H NMR (400 MHz, CDCl3): d 1.55–1.59 (2H,
m, CH2), 1.58–1.63 (12H, m, CH2), 1.88 (2H, quintet, J = 7.6 Hz,
CH2CH2CH2), 2.29 (2H, t, J = 7.6 Hz, CH2CH2CO), 3.93 (3H, s,
OCH3), 4.06 (2H, t, J = 6.6 Hz, OCH2CH2), 6.97 (1H, d, J = 8.0 Hz,
Ar-H), 7.41 (1H, s, Ar-H), 7.44 (1H, d, J = 8.4 Hz, Ar-H), 9.85 (1H,
s, CHO). ESI-MS (negative, m/z): 335.10 [MꢀH]ꢀ.
Yield: 70%; white powder; 1H NMR (300 MHz, CDCl3) d 1.68 (s,
3H, CH3), 3.01 (s, 2H, epoxide-H), 7.78 (d, 1H, J = 8.7 Hz, aromatic-
H),7.91 (dd, 1H, J = 2.4, 8.7 Hz, aromatic-H), 8.03 (d, J = 2.1 Hz, 1H,
aromatic-H), 8.42 (s, 1H, NH); ESI MS m/z 292.7 [M+Na]+.
4.6. Synthesis of N-(4-substituted-3-(trifluoromethyl)phenyl)-
2-hydroxy-2-methylpropanamide-methylcurcumin (15, 16)
4.6.1. General procedure
Methylcurcumin (2) (0.26 mmol in 3 mL of THF) was cooled to
0 °C. Sodium hydride (60%) (0.37 mmol) was added. After stirring
at rt for 5 min, 14a or 14b (0.27 mmol) was added. The resulting
mixture was heated to reflux for 24 h. The reaction mixture was di-
luted with CH2Cl2 and washed with water twice and extracted with
CH2Cl2. After purification through a CombiflashÒ system, the de-
sired product was obtained.
4.8. O-Butanoic acid substituted methylcurcumin (18a)
To a solution of 6-(3,4-dimethoxyphenyl)-4-hydroxyhexa-3,5-
dien-2-one25 (837 mg, 3.1 mmol) in EtOAc (8 mL) was added boric
anhydride (171 mg, 2.5 mmol). The solution was stirred at 70 °C for
1 h. To this solution were added 17a (1247 mg, 4.7 mmol) and trib-
utyl borate (1.3 mL, 4.8 mmol). The mixture was stirred for 0.5 h
and a solution of n-butylamine (0.46 mL, 4.7 mmol) in EtOAc
(4 mL) was added dropwise over 15 min at 85 °C. The stirring
was continued for 3 h at 100 °C. The mixture was then hydrolyzed
by adding 0.1 N HCl and stirred at 50 °C for 0.5 h. The organic layer
was separated, and the aqueous layer was extracted with EtOAc.
The combined organic layers were washed H2O and brine and dried
over anhydrous Na2SO4. After removal of solvent in vacuo, the
crude products were purified by CombiFlashÒ column chromatog-
raphy eluting with n-hexane–EtOAc gradient to give the ethyl ester
precursor of 18a (393 mg, 25%), amorphous solid, 1H NMR
(400 MHz, CDCl3): d 1.26 (3H, t, J = 7.2 Hz, OCH2CH3), 2.17 (2H,
quintet, J = 6.8 Hz, OCH2-CH2-CH2-CO), 2.54 (2H, t, J = 7.0 Hz,
OCH2-CH2-CH2-CO), 3.91, 3.92 and 3.93 (each 3H, s, OCH3), 4.11
(2H, t, J = 6.2 Hz, OCH2-CH2-CH2-CO), 4.15 (2H, q, J = 7.0 Hz,
OCH2CH3), 5.82 (1H, s, C@CH), 6.49 and 6.50 (each 1H, d,
J = 15.6 Hz, CH@CH), 6.878 (1H, d, J = 8.2 Hz, Ar-H), 6.883 (1H, d,
J = 8.4 Hz, Ar-H), 7.07 (2H, s, Ar-H), 7.13 (2H, t, J = 8.4 Hz, Ar-H),
7.59 and 7.60 (each 1H, d, J = 15.6 Hz, CH@CH). ESI-MS (positive
or negative, m/z): 497.20 [M+H]+, 495.10 [MꢀH]ꢀ.
4.6.2. N-(4-Nitro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methylpropanamido-methylcurcumin (15)
Yield: 64%; amorphous solid; 1H NMR (400 MHz, CDCl3): d 1.57
(s, 3H, CH3), 3.92–3.91 (m, 9H, OCH3), 3.96 (d, J = 9.6 Hz, 1H), 4.46
(d, J = 9.6 Hz, 1H), 5.81 (s, 1H), 6.49 (dd, J = 15.6, 1.2 Hz, 2H), 6.87
(d, J = 8.4 Hz, 1H), 7.06 (s, 2H), 7.13 (dd, J = 8.4, 1.6 Hz, 2H), 7.60
(d, J = 16.0 Hz, 1H), 7.62–7.54 (m, 1H), 7.95 (d, J = 8.4 Hz, 1H),
8.00–7.97 (m, 2H), 8.07 (dd, J = 8.8, 2.0 Hz, 1H), 9.30 (s, 1H,), 8.52
(s, 1H); 13C NMR (400 MHz, CDCl3): d 20.6 (CH3); 56.2 (OCH3 ꢁ 3),
75.1 (CH2), 84.9 [–C(OH)CH3CO], 101.6 (COCH@COH), 122.4 (CF3),
110.0, 111.0, 111.4, 116.2, 118.2, 122.1, 122.4, 123.4, 127.3 (aro-
matic CH), 120.5 (CHOHCH@CH), 139.7 (–CH@CH-Ph), 122.9 (COC
H@CH-Ph), 141.0 (COCH@CH-Ph), 128.1, 128.2, 130.6, 141.9,
143.4 (aromatic C), 149.0, 149.5, 150.0, 151.4 (aromatic C-OCH3);
173.5 (–C ONH–), 182.6 (enol COCHCOH), 184.2 (carbonyl C O-
CH@COH); ESI MS m/z 656.2 [M+H]+.
4.6.3. N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methylpropanamide-methylcurcumin (16)
Yield: 41%; amorphous solid; 1H NMR (300 MHz, acetone-d6): d
1.56 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 4.16 (d, J = 9.6 Hz, 1H), 4.42 (d, J = 9.6 Hz, 1H), 6.00 (s, 1H),
6.74 (d, J = 15.6 Hz, 2H), 7.02 (d, J = 15.9 Hz, 1H), 7.05 (d,
J = 15.9 Hz, 1H), 7.23 (t, J = 13.8, 7.2 Hz, 2H), 7.31 (d, J = 4.8 Hz,
2H), 7.59 (d, J = 15.9 Hz, 1H), 7.62 (d, J = 15.9 Hz, 1H), 8.06 (d,
J = 8.7 Hz, 1H), 8.31 (d, J = 8.7 Hz, 1H), 8.52 (s, 1H), 10.03 (s, 1H,);
ESI MS m/z 653.4 [M+H]+.
To a solution of resulting compound (393 mg, 0.79 mmol) in
THF (10 mL) was added 10% HCl (6 mL) and then the solution
was stirred at 70 °C overnight. To the mixture was added H2O
(20 mL) and extracted with EtOAc. The organic layer was washed
with H2O and brine and dried over anhydrous Na2SO4. After re-
moval of the solvent in vacuo, the crude was purified by Combi-
FlashÒ column chromatography eluting with n-hexane–EtOAc
gradient to afford 18a (263 mg, 71%), amorphous solid, 1H NMR
(400 MHz, CDCl3): d 2.19 (2H, quintet, J = 6.8 Hz, OCH2-CH2-CH2-
4.7. Ethyl 4-(4-formyl-2-methoxyphenoxy)butanoate (17a)
To a solution of vanillin (1.0 g, 6.6 mmol) in DMF (20 mL), ethyl
4-bromobutyrate (2.8 mL, 19.6 mmol) and K2CO3 (360 mg,

4030
Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
CO), 2.63 (2H, t, J = 7.0 Hz, OCH2-CH2-CH2-CO), 3.91, 3.93 and 3.94
(each 3H, s, OCH3), 4.13 (2H, t, J = 6.2 Hz, OCH2-CH2-CH2-CO), 5.82
(1H, s, C@CH), 6.50 (2H, d, J = 15.8 Hz, CH@CH), 6.89 (2H, d,
J = 8.4 Hz, Ar-H), 7.08 (2H, s, Ar-H), 7.14 (2H, t, J = 8.2 Hz, Ar-H),
7.60 and 7.61 (each 1H, d, J = 15.6 Hz, CH@CH). ESI-MS (positive
or negative, m/z): 469.10 [M+H]+, 467.15 [MꢀH]ꢀ.
J = 8.4, 1.8 Hz, 2H), 7.65 (d, J = 15.9 Hz, 2H),; ESI MS m/z 413.2
[MꢀH]ꢀ, 437.2 [M+Na]+.
4.11. 4,4-Difluoro substituted dimethylcurcumin (23)
A solution of dimethylcurcumin (21) (80 mg, 0.2 mmol) in THF
(5 mL) was added to a suspension of 10 mg of NaH (80% in mineral
oil) at 0 °C. The mixture was stirred at 0 °C for 0.5 h, then rt for 1 h.
A solution of SelectFluor™ (80 mg, 0.22 mmol) in DMF (2 mL) was
then added and the mixture was stirred for additional 8 h. After
additional 30 mg of SelectFluor™ in DMF (1 mL) was added, the
reaction mixture was continually stirred for 0.5 h. The solvent
was removed by distillation and the remained solid was purified
by flash column chromatography (eluting by n-hexane/
EtOAc = 6:1) and PTLC (n-hexane/EtOAc = 1:2) to give the desired
product 23 (27 mg, 31% yield) as yellow solid powder. 1H NMR
(300 MHz, CDCl3): d 3.92 (s, 12H, OCH3), 6.87 (d, J = 8.4 Hz, 2H),
6.96 (d, J = 15.6 Hz, 2H), 7.11 (d, J = 2.1 Hz, 2H), 7.21 (dd, J = 8.4,
2.1 Hz, 2H), 7.48 (d, J = 15.6 Hz, 2H); ESI MS m/z 433.2 [M+H]+,
455.1 [M+Na]+.
A similar procedure was used to prepare 18b.
4.8.1. O-Undecanoic acid substituted methylcurcumin (18b)
Yield: 25%; amorphous solid, 1H NMR (400 MHz, CDCl3): d 1.45–
146 (2H, m, CH2), 1.61–163 (12H, m, CH2), 1.86 (2H, quintet,
J = 7.6 Hz, CH2), 2.29 (2H, t, J = 7.6 Hz, CH2), 3.92, 3.93 and 3.94
(each 3H, s, OCH3), 4.05 (2H, t, J = 6.6 Hz, OCH2), 5.82 (1H, s, C@CH),
6.49 (1H, d, J = 15.8 Hz, CH@CH), 6.50 (1H, d, J = 15.6 Hz, CH@CH),
6.87 and 6.89 (each 1H, d, J = 8.4 Hz, Ar-H), 7.08 (2H, s, Ar-H), 7.13
(1H, d, J = 8.2 Hz, Ar-H), 7.15 (1H, d, J = 8.4 Hz, Ar-H), 7.61 (2H, d,
J = 15.8 Hz, CH@CH). ESI-MS (negative, m/z): 565.35 [MꢀH]ꢀ.
4.9. N-(4-Cyano-3-(trifluoromethyl)phenyl)butyramide-
methylcurcumin (19)
4-Amino-2-(trifluoromethyl)benzonitrile (97 mg, 0.52 mmol),
EDCI (100 mg, 0.52 mmol) and DMAP (63 mg, 0.52 mmol) were
added to a solution of 18a (120 mg, 0.26 mmol) in CH2Cl2 (8 mL),
and the mixture was stirred at 35 °C overnight under N2. The mix-
ture was diluted with CH2Cl2 and then washed with satd NH4Cl
(aq), satd NaHCO3 (aq), and brine, then dried over anhydrous
Na2SO4. After removal of the solvent in vacuo, the product was
purified by CombiFlashÒ column chromatography eluting with n-
hexane–EtOAc gradient to give the desired 19 (60 mg, 37%), amor-
phous solid, 1H NMR (400 MHz, CDCl3): d 2.27 (2H, quintet,
J = 6.8 Hz, OCH2-CH2-CH2-CO), 2.68 (2H, t, J = 7.0 Hz, OCH2-CH2-
CH2-CO), 3.84, 3.92 and 3.93 (each 3H, s, OCH3), 4.17 (2H, t,
J = 5.8 Hz, OCH2-CH2-CH2-CO), 5.82 (1H, s, C@CH), 6.49 and 6.50
(each 1H, d, J = 15.8 Hz, CH@CH), 6.88 and 6.90 (each 1H, d,
J = 8.4 Hz, Ar-H), 7.075 and 7.080 (each 1H, d, J = 2.1 Hz, Ar-H),
7.12 and 7.14 (each 1H, dd, J = 8.4, 1.7 Hz, Ar-H), 7.58 and 7.61
(each 1H, d, J = 15.8 Hz, CH@CH), 7.75 (1H, d, J = 8.4 Hz, Ar-H0),
7.95 (1H, s, Ar-H0), 7.97 (1H, dd, J = 8.4, 1.9 Hz, Ar-H0), 8.62 (1H, s,
NH). ESI-MS (positive, m/z): 637.40 [M+H]+.
4.12. Cell culture
PC-3 (ATCC CRL 1435) and LNCaP clone FGC (ATCC CRL 1740)
cell lines were obtained from Lineberger Comprehensive Cancer
Center (UNC-CH). PC-3 cells were cultured in RPMI-1640 medium
containing 25 mM HEPES and 2 mM
plemented with 10% heat-inactivated fetal bovine serum (Hy-
clone), 100 IU penicillin, 100 g/mL streptomycin, and 0.25 g/
mL amphotericin B (Mediatech). In addition, 1 mM sodium pyru-
vate (Mediatech) was added to the medium for LNCaP cells. Cells
were incubated in 5% CO2 and 95% air at 37 °C.
L-glutamine (Mediatech), sup-
l
l
4.13. Antiproliferative activity assay
Antiproliferative activity was determined by the sulforhoda-
mine B (SRB) colorimetric assay as previously described.45 In brief,
the cells (3–5 ꢁ 103 cells/well) were seeded in 96-well plates filled
with RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS) containing various concentrations of samples, and
incubated for 72 h. At the end of the exposure period, the attached
cells were fixed with cold 50% trichloroacetic acid for 30 min
followed by staining with 0.04% SRB (Sigma Chemical Co.)
for 30 min. The bound SRB was solubilized in 10 mM Tris-base
and the absorbance was measured at 515 nm on a Microplate
Reader ELx800 (Bio-Tek Instruments, Winooski, VT) with a Gen5
software. All results were representative of three or more
experiments.
4.9.1. N-(4-Cyano-3-(trifluoromethyl)phenyl)undecanamide-
methylcurcumin (20)
A similar procedure to that described above for 19 gave the de-
sired product 20 (46 mg, 15%), amorphous solid, 1H NMR
(400 MHz, CDCl3): d 1.43–1.45 (2H, m, CH2), 1.61–1.68 (10H, m,
CH2), 1.72–1.76 (2H, m, CH2), 1.83–1.86 (2H, m, CH2), 2.29 (2H, t,
J = 7.2 Hz, CH2CO), 3.91, 3.93 and 3.94 (each 3H, s, OCH3), 4.05
(2H, t, J = 6.4 Hz, OCH2), 5.83 (1H, s, C@CH), 6.49 and 6.50 (each
1H, d, J = 15.8 Hz, CH@CH), 6.88 (1H, d, J = 8.2 Hz, Ar-H), 6.89
(1H, d, J = 8.4 Hz, Ar-H), 7.08 (2H, s, Ar-H), 7.12 (1H, d, J = 8.9 Hz,
Ar-H), 7.14 (1H, d, J = 8.4 Hz, Ar-H), 7.61 (2H, d, J = 15.8 Hz,
CH@CH), 7.76 (1H, d, J = 8.4 Hz, Ar-H0), 7.96 (1H, d, J = 8.4 Hz, Ar-
H0), 8.00 (1H, s, Ar-H0). ESI-MS (positive, m/z): 735.50 [M+H]+.
4.14. Morphological analysis and intrinsic fluorescence of
compounds in cells
PC-3 and LNCaP tumor cells were maintained in RPMI-1640
medium supplemented with 10% FBS in 8-well chamber slides
(Lab-Tech) for 12 h prior to treatment with DMSO, 1 (30
(40 M), 6 (50 M), 15 (40 M), and flutamide (100 M), or bicalu-
tamide (100 M) for 48 h at 37 °C. For intrinsic fluorescence anal-
ysis, PC-3 cells were treated with 40 M compound for 1 h at 37 °C.
Cells were fixed with 4% paraformaldehyde in phosphate buffered
saline (PBS). Nuclei were labeled with 40,6-diamidino-2-phenylin-
dole (DAPI, Sigma), and cells were mounted with VECTASHIELD
(vector labs). Fluorescence labeled cells were observed using a Ni-
kon ECLLIPSE E400 fluorescence microscope and images were cap-
tured by a SPOT cooled digital camera controlled by the SPOT
Software (Diagnostic Instruments Inc.). Final images were prepared
using Adobe Photoshop ver 6.
lM), 2
4.10. 4-Fluoro substituted dimethylcurcumin (22)
l
l
l
l
l
Dimethylcurcumin (21) (198 mg, 0.5 mmol) in DMF (5 mL) was
added to SelectFluor™ (0.51 mmol). The mixture was stirred at rt
overnight under N2. The solvent was removed by distillation and
the remaining solid was dissolved in CH2Cl2 and washed with
water. After being dried over Na2SO4 the crude product was puri-
fied by flash column eluting by n-hexane/EtOAc = 6: 1 to afford
the desired product 22 as an orange powder in 7% yield. 1H NMR
(300 MHz, CDCl3): d 3.94–3.92 (m, 12H, OCH3) 6.88 (d, J = 8.4 Hz,
2H), 6.98 (dd, J = 15.9, 3.0 Hz, 2H), 7.12–7.11 (m, 2H), 7.21 (dd,
l

Q. Shi et al. / Bioorg. Med. Chem. 20 (2012) 4020–4031
4031
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Acknowledgments
This work was supported by National Cancer Institute Grant CA-
17625-32 awarded to K. H. Lee. This study was also supported in
part by the Taiwan Department of Health, China Medical Univer-
sity Hospital Cancer Research Center of Excellence (DOH100-TD-
C-111-005).
We would like to thank Drs. Ellen Weiss and Shoji Osawa (UNC-
CH) for offering their fluorescence microscope.
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