Antitumor Agents. 217.

Curcumin Analogues as Novel Androgen Receptor

Antagonists with Potential as Anti-Prostate Cancer Agents
Hi ronori Ohtsu,

Zhi yan Xi ao,

Junko I shi da,

Masahi ro Nagai ,

Hui -Kang Wang,

Hi deji I tokawa,

Chi ng-Yuan Su,

|
Charl es Shi h,
|
Tzuyi ng Chi ang,

Eugene Chang,

Yi Fen Lee,

Meng-Yi n Tsai ,

Chawnshang Chang,

and Kuo-Hsi ung Lee

,
*
Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7360, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 158-0098, J apan,
AndroScienceCorporation, 11175 FlintkoteAvenue, SuiteF, San Diego, California 92121, and GeorgeWhipple
Laboratory for Cancer Research, Departments of Pathology, Urology and Radiation Oncology, University of Rochester
Medical Center, 601 Elmwood Avenue, Box 626, Rochester, New York 14642
Received May 10, 2002
A number of curcumi n anal ogues were prepared and eval uated as potenti al androgen receptor
antagoni sts agai nst two human prostate cancer cel l l i nes, PC-3 and DU-145, i n the presence
of androgen receptor (AR) and androgen receptor coacti vator, ARA70. Compounds 4[5-hydroxy-
1,7-bi s(3,4-di methoxyphenyl )-1,4,6-heptatri en-3-one], 20[5-hydroxy-1,7-bi s[3-methoxy-4-(meth-
oxycarbonyl methoxy)phenyl ]-1,4,6-heptatri en-3-one], 22[7-(4-hydroxy-3-methoxyphenyl )-4-[3-
(4-hydroxy-3-methoxyphenyl )acryl oyl ]-5-oxohepta-4,6-di enoi c aci d ethyl ester], 23[7-(4-hydroxy-
3-methoxyphenyl )-4-[3-(4-hydroxy-3-methoxyphenyl )acryl oyl ]5-oxohepta-4,6-di enoi c aci d], and
39 [bi s(3,4-di methoxyphenyl )-1,3-propanedi one] showed potent anti androgeni c acti vi ti es and
were superi or to hydroxyfl utami de, whi ch i s the currentl y avai l abl e anti androgen for the
treatment of prostate cancer. Structure-acti vi ty rel ati onshi p (SAR) studi es i ndi cated that the
bi s(3,4-di methoxyphenyl ) moi eti es, the conjugated -di ketone moi ety, and the i ntramol ecul ar
symmetry of the mol ecul es seem to be i mportant factors rel ated to anti androgeni c acti vi ty.
The data further suggest that the copl anari ty of the -di ketone moi ety and the presence of a
strong hydrogen bond donor group were al so cruci al for the anti androgeni c acti vi ty, whi ch i s
consi stent wi th previ ous SAR resul ts for hydroxyfl utami de anal ogues. When the pharmacoph-
ori c el ements of di hydrotestosterone (DHT) and compound 4 are superposed, the resul ti ng
construct i mpl i es that the curcumi n anal ogues may functi on as a 17R-substi tuted DHT.
Compounds 4, 20, 22, 23, and 39have been i denti fi ed as a new cl ass of anti androgen agents,
and these compounds or thei r new syntheti c anal ogues coul d be devel oped i nto cl i ni cal tri al
candi dates to control androgen receptor-medi ated prostate cancer growth.
Introduction
The androgen receptor (AR) i s a member of a l arge
fami l y of l i gand-dependent transcri pti onal factors known
as the steroi d receptor superfami l y.
2,3
Androgens and
the AR pl ay an i mportant rol e i n the growth of prostate
cancer and normal prostate. Prostate cancer represents
the most common mal e mal i gnancy i n the Uni ted
States.
4
Recentl y, anti androgens such as hydroxyfl uta-
mi de (HF) i n combi nati on wi th surgi cal or medi cal
castrati on have been wi del y used for the treatment of
prostate cancer.
5
Both steroi dal and nonsteroi dal de-
ri vati ves are presentl y avai l abl e and have shown cl i ni cal
benefi t as chemotherapeuti c agents for prostate cancer
(Chart 1). The syntheti c steroi dal anti androgen cypros-
terone i s one of the fi rst anti androgens used cl i ni cal l y
i n Europe;
6
however, thi s steroi dal anti androgen showed
agoni sti c acti vi ty and overl appi ng effects wi th other
hormonal systems, i n addi ti on to a range of unpl easant
si de effects.
7
HF and bi cal utami de are nonsteroi dal
anti androgens, both of whi ch are thought to be pure
anti androgens wi thout agoni sti c acti vi ty. Bi cal utami de
has a l onger hal f-l i fe (6 days) and a hi gher bi ndi ng
affi ni ty to the AR than HF.
8,9
* To whom correspondence shoul d be addressed. Phone: (919)-962-
0066, Fax: (919)-966-3893. E-mai l : khl ee@unc.edu.

Anti tumor Agents. 217. For paper 216, see ref 1.

Uni versi ty of North Carol i na at Chapel Hi l l .

Hoshi Uni versi ty.

|
AndroSci ence Corporati on.

Uni versi ty of Rochester Medi cal Center.

Chart 1. Structures of Cyprosterone,
Hydroxyfl utami de, and Bi cal utami de
5037 J . Med. Chem. 2002, 45, 5037-5042
10.1021/jm020200g CCC: $22.00 2002 Ameri can Chemi cal Soci ety
Publ i shed on Web 10/12/2002
The agoni sti c acti vi ty of the anti androgen may resul t
i n anti androgen wi thdrawal syndrome.
10
A currentl y
accepted hypothesi s postul ates that mutati ons i n ARs
may account for why HF, the acti ve metabol i te of
fl utami de, can acti vate AR target genes and sti mul ate
prostate cancer growth.
10
The same mechani sm i s used
to expl ai n the fl utami de wi thdrawal syndrome, i n
whi ch pati ents who experi ence an i ncrease i n prostate-
speci fi c anti gen (PSA) whi l e taki ng fl utami de, have a
decrease i n PSA after wi thdrawal of treatment. I ndeed,
HF can acti vate AR target genes, such as PSA and
MMTV-LTR (a r epor ter gene whi ch expr esses l u-
ci ferase), i n the presence of ARA70, the fi rst i denti fi ed
AR coacti vator.
11
Because thi s syndrome often l eads to
the fai l ure of androgen-abl ati ve therapy, i t i s desi rabl e
to devel op better anti androgens wi thout agoni st acti vi ty.
The phenol i c di aryl heptanoi d curcumi n (1) i s the
major pi gment i n turmeri c. Curcumi n and i ts anal ogues
show potent anti oxi dant acti vi ty, anti i nfl ammatory
acti vi ty,
12
cytotoxi ci ty agai nst tumor cel l s,
13
and anti -
tumor-promoti ng acti vi ti es.
14,15
I n a previ ous paper, we
reported that two cycl i c di aryl heptanoi ds, 13-oxomyri -
canol and myri canone, exhi bi ted potent anti tumor-
promoti ng effects on DMBA-i ni ti ated and TPA-i nduced
mouse ski n carci nogenesi s.
16
Very recentl y, we have al so
eval uated the cytotoxi c effects of curcumi n anal ogues
and 1,3-di aryl -1,3-di ketopropane deri vati ves agai nst a
panel of human tumor cel l l i nes.
17
I n the present study,
we have prepared a number of curcumi n anal ogues and
eval uated thei r antagoni sti c acti vi ty agai nst the AR i n
the presence of ARA70, usi ng two human prostate
cancer cel l l i nes, PC-3 and DU-145. PC-3 cel l s are
androgen-i ndependent tumor cel l s that do not express
functi onal AR; DU-145 cel l s are androgen-i ndependent
tumor cel l s that express nei ther functi onal AR nor
endogenous ARA70.
Chemistry
Fi gures 1 and 2 show the structures of curcumi n
anal ogues and 1,3-di aryl -1,3-di ketopropane deri vati ves.
Curcumi n (1), demethoxycurcumi n (2), and bi sdemethox-
ycurcumi n (3) were obtai ned by col umn chromatography
(si l i ca gel , CHCl
3
-MeOH) of commerci al l y avai l abl e
curcumi n (Al dri ch), whi ch contai ned 2and 3as mi nor
components. Treatment of 1 wi th di azomethane gave
di methyl ated curcumi n (4) and monomethyl ated cur-
cumi n (9). Methyl ati on of 1wi th methyl i odi de and K
2
-
CO
3
furni shed the tri methyl ated deri vati ve 10, i n whi ch
a methyl group was al so i ntroduced at the C-4 posi ti on.
Compounds 5-8were synthesi zed by heati ng 1-4wi th
hi sti di ne hydrazi de, AcOH, and p-TsOH overni ght.
Hydrogenati on of 1wi th 10% Pd-C gave a mi xture of
11-13. Si mi l arl y, compounds 14-16 and 17, 18 were
obtai ned by hydrogenati on of 4 and 10, respecti vel y.
Heati ng 1 wi th methyl chl oroacetate, NaI , and K
2
CO
3
i n acetone furni shed a mi xture of mono(methoxycarbo-
Figure 1. Structures of Curcumi n Anal ogues 1-20.
5038 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 Ohtsu et al.
nyl methyl ) ether 19 and bi s(methoxycarbonyl methyl )
ether 20, whi ch were separated by preparati ve TLC
(PLC). Compounds 21-23were prepared from benzene
or vani l l i n and ethyl 4-acetyl -5-oxohexanoate as de-
scri bed previ ousl y i n the l i terature.
22
Compounds 21-
23 consti tute an unseparabl e mi xture of keto-enol
tautomeri c i somers. The syntheses of 24-38 were
descri bed i n our previ ous paper.
16,17
Compounds 39-
44were purchased from Al dri ch, I nc. (Mi l waukee, WI ).
Results and Discussion
Forty-four curcumi n deri vati ves (1-44) were tested
for antagoni sti c acti vi ty agai nst the AR usi ng two
di fferent human prostate cancer cel l s, PC-3 and DU-
145 (Fi gures 3A-C). The parental compound, curcumi n
(1), was i nacti ve i n al l cases. However, di methyl ated
curcumi n (4) showed si gni fi cant antagoni sti c acti vi ty
(reduci ng 70% of DHT-i nduced AR acti vi ty) when as-
sayed i n PC-3 cel l s transfected wi th wi l d-type AR
(wt.AR) and was more potent than HF (whi ch reduced
16%of DHT-i nduced AR acti vi ty, Fi gure 3A). Compound
4 al so showed the hi ghest antagoni st acti vi ty when
assayed i n DU-145 cel l s transfected wi th a mutant
LNCaP AR and ARA70 (showi ng a 45% reducti on i n
DHT-i nduced AR acti vi ty, Fi gure 3B), i ndi cati ng that
compound 4 i s an effecti ve antagoni st for both wt.AR
and mutant AR.
To determi ne the structural requi rements for AR
antagoni st acti vi ty i n thi s seri es of compounds, an SAR
study was conducted i n PC-3 and DU-145 cel l assay
systems. Compared wi th 4, monomethyl ated curcumi n
(9) l acks one O-methyl group at the para posi ti on on
one benzene ri ng and was si gni fi cantl y l ess acti ve than
4(Figure 3A). Thus, the bis(3,4-dimethoxyphenyl) groups
of 4 are i mportant to the acti vi ty. Compounds 14 and
15, whi ch were obtai ned by hydrogenati on of 4, were
as potent as HF wi th a 18% reducti on i n DHT-i nduced
AR acti vi ty but were consi derabl y l ess acti ve compared
to 4(Fi gure 3A). I ntroduci ng a methyl group at C-4 of
4 (10) resul ted i n decreased acti vi ty (Fi gure 3B). Con-
verti ng the -di ketone moi ety of 4to the correspondi ng
pyrazol e deri vati ve 8greatl y reduced the acti vi ty (data
not shown). Furthermore, 1,3-bi s(3,4-di methoxyphenyl )-
1,3-propandi one (39), whi ch contai ns the bi s-aryl groups
found i n 4, but l acks the conjugated doubl e bonds, was
l ess acti ve than 4 (Fi gure 3A and 3B), i ndi cati ng that
the conjugated doubl e bonds al so contri bute to the
acti vi ty of 4. These observati ons suggested that the bi s-
(3,4-di methoxyphenyl ) groups and the conjugated -di ke-
tone moi ety are cruci al for the acti vi ty.
Data i n Fi gure 3C shows a somewhat di fferent cel l
assay system where anti androgen acti vi ty was assayed
i n DU-145 cel l s transfected wi th wt.AR and ARA70.
Compounds 4, 20, 22, 23, and 39 showed comparabl e
or more potent anti androgen acti vi ty than HF i n thi s
assay system. Compounds 20 and 22 were al most
equi potent (54%and 53.8%reducti on, respecti vel y) and
were sl i ghtl y more acti ve than 4 (49.9%). Because
curcumi n (1) i tsel f was not acti ve, i ntroduci ng ei ther
methoxycarbonyl methyl groups at the phenol i c hy-
droxyl s (20) or an ethoxycarbonyl ethyl group at C-4 (22)
greatl y contri buted to the anti -AR acti vi ty i n DU-145
cel l s i n the presence of wt.AR and ARA70.
I n thi s study, we al so exami ned the anti androgen
acti vi ty of fl uorodi aryl heptanoi ds 24-29 and cycl i c
Figure 2. Structures of Curcumi n Anal ogues 21-44.
Novel Androgen Receptor Antagonists J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5039
di aryl heptanoi ds 30-38. Compounds 24-29have fl uo-
ri ne or tri fl uoromethyl substi tuents on both benzene
ri ngs but showed weak acti vi ty or were i nacti ve (data
not shown). Among the cycl i c di aryl heptanoi ds 30-38,
compound 30 was the most acti ve and was al most as
acti ve as HF (Fi gure 3A and 3C). The remai ni ng cycl i c
di aryl heptanoi ds showed weak antagoni sti c acti vi ty.
By cl osel y exami ni ng the structures and acti vi ti es of
the curcumi n anal ogues, addi ti onal structural features,
besi des the bi s(3,4-di methoxyphenyl ) groups and the
conjugated -di ketone moi ety, seem to be rel ated to the
anti androgeni c acti vi ty. The conjugated -di ketone moi -
ety i n the most acti ve compounds (4, 20, 22, 23, and
39) can be effecti vel y fi xed i nto a conformati on that
ensures copl anari ty. However, i n compounds 11-18, the
copl anari ty of the -di ketone moi ety i s l ost and the
anti androgeni c acti vi ty decreases dramati cal l y. Thi s
observati on i s consi stent wi th the correl ati on between
the anti androgeni c acti vi ty and the copl anari ty of the
-NH-CO-OH- moi ety i n HF anal ogues.
18
The hydro-
gen bond donor abi l i ty of the hydroxyl group i n the most
acti ve compounds i s enhanced by the conjugated system
(thi s abi l i ty wi l l be decreased by the formati on of
i ntramol ecul ar hydrogen bond wi th the carbonyl group;
however, the conjugated system makes the carbonyl
group i tsel f a very weak hydrogen bond acceptor), but
certai n compounds (e.g., 30-38) l acki ng such a hydroxyl
group are si gni fi cantl y l ess acti ve. The presence of a
strong hydrogen bond donor group i s al so consi stent
wi th the previ ous SAR for HF anal ogues.
18
I n addi ti on,
i ntramol ecul ar symmetry seems to be another i mpor-
tant factor rel ated to anti androgeni c acti vi ty.
I t was previ ousl y suggested that the steroi d A-ri ng
i s a pharmacophori c component necessary for mol ecul ar
recogni ti on between steroi d receptors and the l i gands,
whi l e the remai ni ng steroi d structure confers receptor
selectivity/specificity.
19,20
Additionally, the 17-hydroxyl-
3-one substructures have been consi dered essenti al for
effecti ve bi ndi ng of steroi ds to AR.
21
Usi ng these pre-
defi ned pharmacophori c el ements, we have superposed
DHT, the natural l y occurri ng l i gand of AR, wi th com-
pound 4(Fi gure 4) by al i gni ng the si x-membered ri ngs
(A-ri ng of DHTand a phenyl ri ng of 4) and the hydroxyl
groups. The resul ti ng superposi ti on suggests that the
curcumi n anal ogues may assume a conformati on si mi l ar
to that of 17R-substi tuted DHT i n order to exert thei r
anti androgeni c acti vi ty. The remai ni ng structural el e-
ments of compound 4 extend i nto the D-ri ng C17
vi ci ni ty, whi ch coi nci des wi th a steri cal l y favorabl e area
defi ned by a previ ous three-di mensi onal quanti tati ve
structure-acti vi ty rel ati onshi p (3D-QSAR) study usi ng
the comparati ve mol ecul ar fi el d anal ysi s (CoMFA)
techni que.
20
The consi stency between the SAR of cur-
cumi n anal ogues and known anti androgens i mpl i es that
thi s new cl ass of compounds may have a l i gand-
receptor recogni ti on pattern and i nteracti on mode si mi -
l ar to that of known anti androgens. Thi s knowl edge
woul d faci l i tate future mechani c i nvesti gati ons of cur-
cumi n anal ogues.
Figure 3. Suppressi on of DHT-medi ated MMTV (mouse memory tumor vi rus) transcri pti on AR acti vi ty by hydroxyfl utami de
(HF) and sel ected compounds. PC-3 and DU-145 cel l l i nes were seeded and cotransfected wi th reporter MMTV-l uci ferase (A and
C) or mutant AR expressi on pl asmi d (B) and ARA70 (B and C) usi ng SuperFect. Subsequentl y the transfected cel l s were harvested
and repl ated i n 10% charcoal -stri pped fetal bovi ne serum DMEM (Dul beccos Modi fi ed Eagl e Medi a) medi um. The cel l s were
then treated wi th dehydrotestosterone (DHT, 1 nM) and anti androgens (1 M) and harvested for detecti on of the l uci ferase acti vi ty.
The resul ts were averaged from two i ndependent experi ments.
Figure4. Superposi ti on of DHT and compound 4. DHT and
compound 4 are represented i n whi te and red, respecti vel y.
Both mol ecul es were constructed and opti mi zed wi th SYBYL
6.0. Compound 4was subjected to a geneti c al gori thm confor-
mati onal search. The l owest-energy conformer of 4 was
superposed to DHTby the si x-membered ri ngs (A-ri ng of DHT
and phenyl of 4) and the hydroxyl groups and was then fi el d-
fi t mi ni mi zed usi ng DHT as templ ate.
5040 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 Ohtsu et al.
Conclusion
I n concl usi on, we have prepared a number of cur-
cumi n anal ogues and eval uated thei r potenti al anti an-
drogen acti vi ty i n three di fferent assay condi ti ons usi ng
human prostate cancer cel l l i nes. Compounds 4showed
promi si ng anti androgen acti vi ti es i n al l assays. Com-
pounds 4, 20, 22, 23, and 39have been i denti fi ed as a
new cl ass of anti androgen agents. SAR studi es reveal ed
that bi s(3,4-di methoxyphenyl ) moi eti es, a conjugated
-di ketone, and an ethoxycarbonyl ethyl group at the C-4
posi ti on pl ay i mportant rol es i n the antagoni sti c acti v-
i ty. Further mechani sti c studi es and the devel opment
of new anti androgens are acti vel y underway i n our
l aboratory.
Experimental Section
Mel ti ng poi nts were determi ned on a Fi sher-Johns mel ti ng
apparatus and are uncorrected. Opti cal rotati ons were deter-
mi ned wi th a DI P-1000 pol ari meter.
1
H NMR spectra were
recorded on a Bruker AC-300 and JMN GX-500 spectrometer.
The chemi cal shi fts are presented i n terms of ppm wi th TMS
as the i nternal reference. MS spectra were recorded on
HP5989A and JMS D-300 i nstruments. El emental anal yses
were performed by Atl anti c Mi crol ab I nc., Norcross, GA, and
agreed wi th theoreti cal val ues to wi thi n (0.4%. New com-
pounds 9, 16, and 18 were homogeneous by HPLC anal yses
i n three di fferent sol vent systems. Compounds 1-3 were
obtai ned by col umn chromatography (si l i ca gel , CHCl
3-MeOH)
of commerci al l y avai l abl e curcumi n (Al dri ch), whi ch contai ned
2 and 3 as mi nor components. Compounds 39-44 were
purchased from Al dri ch, I nc (Mi l waukee, WI ).
Dimethylcurcumin (4). Curcumi n (1) i n Et
2O and MeOH
was treated wi th excess of di azomethane i n ether for 24 h.
The sol vents were removed i n vacuo, and the resi due was
puri fi ed by si l i ca gel col umn chromatography and PLC to yi el d
yel l ow needl es of 4(yi el d 19.8%); mp 129-130 C (MeOH) (l i t.
23
128-130 C);
1
H NMR (300 MHz, CDCl 3): 3.93 (12H, s,
OCH3 4), 5.82 (1H, s, 1-H), 6.48 (2H, d, 16 Hz), 6.88 (2H, d,
J ) 8 Hz), 7.08 (2H, bs), 7.15 (2H, bd), 7.61 (2H, J ) 16 Hz);
13
CNMR (300 MHz, CDCl 3): 55.9, 56.0, 101.3, 109.8, 111.1,
122.0, 122.6, 128.1, 140.4, 149.2, 151.0, 183.2.
Preparation of Pyrazole Derivative 8. To a sol uti on of
1-4 i n butanol and ethanol were added hi sti di ne hydrazi de
(1 equi v), aceti c aci d, and p-TsOH. The sol uti on was refl uxed
for 24 h, and then the sol vent was removed i n vacuo. The
resi due was puri fi ed by si l i ca gel col umn chromatography and
PLC.
Compound 8. Yel l ow powder (yi el d 17.5%), mp 166-168
C (MeOH);
1
H NMR (300 MHz, CDCl 3): 3.92 (6H, s, OCH3
2), 3.94 (6H, s, OCH3 2), 6.62 (1H, s, 1-H), 6.86 (2H, d, J
) 8 Hz), 6.93 (2H, d, J ) 16 Hz), 7.04 (2H, dd, J ) 8, 2 Hz),
7.06 (2H, bs), 7.05 (2H, d, J ) 16 Hz);
13
CNMR (300 MHz,
CDCl 3): 55.8, 55.9, 99.6, 108.6, 111.2, 115.8, 120.1, 129.7,
130.6, 149.1, 149.3; Anal . Cal cd (theoreti cal ) for C23H24N2O4
5
/4H2O: C, 66.57; H, 6.44; N, 6.75. Found: C, 66.44; H, 6.19;
N, 6.27.
Monomethylcurcumin (9). Curcumi n (1) i n MeOH was
treated wi th excess di azomethane i n Et2O for 24 h. After
removal of sol vents, the resi due was puri fi ed by si l i ca gel
col umn chromatography and PLC to yi el d a yel l ow amorphous
sol i d (yi el d 20%); mp 89-91C, [R]
D -3.6 (c ) 1.14, CHCl 3);
1
H NMR (300 MHz, CDCl 3): 3.93 (9H, s, OCH3 3) , 5.81
(1H, s, 1-H), 5.94 (1H, bs, OH), 6.49 (2H, bd, J ) 15 Hz), 6.93
(1H, d, J ) 8 Hz), 6.97 (1H, d, J ) 8 Hz), 7.10 (4H, m), 7.60
(2H, bd, J ) 15 Hz); EI MS m/z 382 (M
+
), HRFABMS 382.1396
(M + H
+
) (cal cd for C22H22O6: 382.1416).
Hydrogenation of 1, 4, and 10 (11-18). A sol uti on of
starti ng materi al i n EtOAc was shaken wi th 10%Pd-C under
H2 (45 psi ) overni ght usi ng a Parrs apparatus. The sol uti on
was fi l tered and concentrated i n vacuo to gi ve a resi due, whi ch
was puri fi ed by si l i ca gel col umn chromatography and PLC.
Tetrahydrocurcumin (11). Whi te powder, mp 92-93 C
(l i t.
23
95-96 C),
1
H NMR (300 MHz, CDCl 3): 2.53-2.58 (3H,
m), 2.78-2.88 (5H, m), 3.87 (6H, s, OCH3 2), 5.43 (1H, s,
1-H), 5.50 (2H, s, ArOH), 6.65 (2H, d, J ) 8 Hz), 6.69 (2H, s),
6.83 (2H, d, J ) 8 Hz);
13
C NMR (300 MHz, CDCl 3): 31.3,
40.4, 55.8, 99.8, 111.0, 114.3, 120.8, 132.6, 144.0, 146.4, 193.2.
Hexahydrocurcumin (12). Whi te powder, mp 87-88 C
(l i t.
23
78-80 C),
1
H NMR (300 MHz, CDCl 3): 1.60-1.81 (2H,
m), 2.53-2.97 (8H, m), 3.85 (6H, s, OCH3 2), 4.06 (1H, m,
2-H), 6.70 (4H, m), 6.80 (2H, d, J ) 8 Hz);
13
C NMR (300 MHz,
CDCl 3): 29.7, 31.7, 38.8, 45.8, 49.8, 56.3, 67.4, 111.5, 111.6,
114.8, 114.9, 121.2, 121.4, 133.0, 134.2, 144.2, 144.5, 146.9,
147.9, 211.9.
Octahydrocurcumin (13). Col orl ess oi l ,
1
H NMR (300
MHz, CDCl 3): 1.61 (2H, m), 1.75 (4H, m), 2.53-2.70 (4H,
m), 3.80 (6H, s, OCH3 2), 3.91 (2H, brs), 6.13 (2H, s, ArOH),
6.65 (2H, d, J ) 8 Hz), 6.69 (2H, bs) 6.82 (2H, bd, J ) 8 Hz),
13
C NMR (300 MHz, acetone-d6): 31.1, 39.8, 42.6, 35.6, 72.0,
111.0, 114.3, 120.6, 133.6, 143.6, 146.4.
Compound14. Whi te powder (yi el d 26.0%), mp 60-61 C,
1
H NMR (300 MHz, CDCl 3): 2.56 (3H, m), 2.86 (5H, m), 3.85
(12H, s, OCH3 4), 5.44 (1H, s, 1-H), 6.71 (4H, m), 6.78 (2H,
bd); Anal . Cal cd (theoreti cal ) for C23H28O6
1
/4H2O: C, 68.21;
H, 7.09. Found: C, 68.25; H, 7.06.
Compound15. Whi te powder (yi el d 20.0%), mp 94-95 C,
1
H NMR (300 MHz, CDCl 3): 1.65-1.80 (2H, m), 2.53-2.84
(8H, m), 3.85 (12H, s, OCH3 4), 4.05(1H, bs, 2-H), 6.68-
7.23 (4H, m), 6.79 (2H, bd), Anal . Cal cd (theoreti cal ) for
C
23H30O6
1
/4H2O: C, 67.88; H, 7.55. Found: C, 67.73; H, 7.49.
Compound 16. Col orl ess oi l (yi el d 4.2%), mp 60-61 C,
1
H NMR (300 MHz, CDCl 3): 1.55-1.65 (4H, m), 1.73-1.82
(3H, m), 2.60-2.72 (3H, m), 3.86 (6H, s, OCH3 2), 3.87 (8H,
bs, OCH3 2, 2,2-H), 6.72-6.78 (4H, m), 6.79 (2H, bd), 7.27-
(2H, s, OH 2), EI MS m/z: 404 (M
+
), HRFAB-MS m/z
404.219070 (M + H)
+
(cal cd for C23H32O6: 404.2198891).
Compound 17. Col orl ess oi l (yi el d 5.9%),
1
H NMR (300
MHz, CDCl 3): 1.10 (3H, d), 1.80 (1H, m), 2.43-2.82 (8H,
m), 3.86 (6H, s, OCH3 2), 3.87 (6H, s, OCH3 2), 3.94 (1H,
bs, 2-H),6.70-6.78 (6H, m), EI MS m/z 416 (M
+
).
Compound 18. Col orl ess oi l (yi el d 6.95%),
1
H NMR (300
MHz, CDCl 3): 0.95 (3H, d, 1-CH3), 1.52 (1H, m), 1.84 (2H,
m), 2.67 (6H, m), 3.83 (14H, bs, OCH3 4, 2, 2-H), 6.78 (6H,
m); EI MS m/z: 418 (M
+
), HRFAB-MS m/z 418.236618 (M +
H)
+
(cal cd for C24H34O6: 418.2355392).
Preparation of 19and 20. A mi xture of curcumi n (1, 100
mg, 0.81 mmol ) i n acetone (20 mL) wi th methyl chl oroacetate
(2 mL) and NaI (20 mg) was refl uxed wi th anhydrous potas-
si um carbonate (176 mg) for 24 h wi th sti rri ng. After fi l trati on
and removal of sol vent, the resi due was puri fi ed by si l i ca gel
col umn chromatography to yi el d the correspondi ng methyl
acetates 19 and 20.
Compound19:Yel l ow powder (yi el d 20.0%), mp 60-61 C,
mp 66-67 C, [R]
D -2.4 (c)2.08, CHCl 3);
1
H NMR (300 MHz,
acetone-d6): 3.73 (3H, s, COOCH3), 3.86 (6H, s, OCH3 2),
4.79 (2H, s, OCH2COO), 5.99 (1H, s, 1-H), 6.70 and 6.73 (both
1H, d, J ) 15.3 Hz), 6.88 (1H, d, J ) 8 Hz), 6.94 (1H, d, J )
8 Hz), 7.17 (2H, m), 7.33 (2H, m), 7.59 and 7.61 (both 1H, d,
J ) 15.3 Hz),
13
C NMR (300 MHz, CDCl 3): d 51.8, 55.9, 55.9,
65.9, 101.4, 111.2, 111.6, 114.3, 115.9, 121.8, 122.6, 123.0,
123.5, 127.6, 128.7, 129.8, 140.3, 141.3, 148.4, 149.8, 150.0,
150.4, 169.4, 183.4, 184.6; Anal. Calcd (theoretical) for C
24H24O8
3
/4H2O: C, 63.50; H, 5.66. Found: C, 63.53; H, 5.65.
Compound 20: Yel l ow powder (yi el d 20.0%), mp 141-142
C (MeOH), [R]D -0.29 (c) 5.86, CHCl 3);
1
H NMR (300 MHz,
CDCl 3): 3.80 (6H, s), 3.93 (6H, s), 4.73 (4H, s, OCH2COO
2), 5.82 (1H, s, 1-H), 6.50 (2H, d, J ) 16 Hz), 6.79 (2H, d, J )
8 Hz), 7.09 (4H, bs), 7.58 (2H, d, J ) 16 Hz),
13
C NMR (300
MHz, CDCl 3): 52.3, 56.0, 66.0, 101.4, 110.7, 113.6, 122.0,
122.7, 129.5, 140.1, 149.0, 149.7, 169.0, 183.1; Anal . Cal cd
(theoreti cal ) for C
27H28O10
1
/2H2O: C, 62.18; H, 5.60. Found:
C, 62.31; H, 5.57.
Compound 21: Yel l ow amorphous sol i d (yi el d 3.0%),
1
H
NMR (300 MHz, CDCl 3): 2.58 (2H, m), 2.95 (2H, m), 7.12
(2H, d, J ) 15 Hz), 7.40 (6H, m), 7.60 (4H, m), 7.81(2H, d, J
Novel Androgen Receptor Antagonists J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5041
) 15 Hz), 12.65(1H, bs); Anal . Cal cd (theoreti cal ) for
C22H20O4: C, 75.84, H, 5.79. Found: C, 75.56, H, 5.74.
Compound 22: Yel l ow amorphous sol i d (yi el d 25.0%),
Anal . Cal cd (theoreti cal ) for C26H28O8: C, 66.66, H, 6.02.
Found: C, 66.38, H, 6.16.
Compound 23: Yel l ow powder (yi el d 45.0%), mp 144-146
C (MeOH) [l i t.
22
71-73 C (CH2Cl 2)]; Anal . Cal cd (theoreti cal )
for C24H26O8
5
/2H2O: C, 59.87; H, 5.23. Found C, 59.94; H, 5.11.
The structures of 1-4, 10-13, 22, and 23 were confi rmed
by compari son of thei r physi cal spectral data wi th those
reported i n the l i terature.
22,23
Suppressionof DHT-MediatedTranscriptionActivity.
Cell Culture and Transfections. Human prostate cancer
DU145 and PC-3 cel l s were mai ntai ned i n Dul beccos mi ni -
mum essenti al medi um (DMEM) contai ni ng peni ci l l i n (25
uni ts/mL), streptomyci n (25 g/mL), and 10% fetal cal f serum
(FCS). For AR transacti vati on assay, PC-3 cel l s were trans-
fected wi th an AR expressi on pl asmi d and reporter gene.
Because of a l ow content of endogenous AR coacti vators, DU-
145 cel l s were transfected wi th expressi on pl asmi ds for AR
and ARA70, and reporter gene. The previ ousl y descri bed
condi ti ons were fol l owed wi th mi nor modi fi cati ons.
10
Trans-
fecti ons were performed usi ng the SuperFect ki t accordi ng to
manufacturers procedures (Qi agen, Chatsworth, CA). Bri efl y,
1 10
5
cel l s were pl ated on 35-mm di shes 24 h before
transfecti on, and then a reporter pl asmi d, MMTV-Luci ferase,
whi ch contai ns MMTV-LTR promoter and AR-bi ndi ng el e-
ment, was cotransfected wi th AR expressi on pl asmi d (wi l d type
or mutant), or pSG5ARA70. PRL-TK was used as an i nternal
control for transfecti on effi ci ency. The total amount of DNA
was adjusted to 3.0 g wi th pSG5 i n al l transcri pti onal
acti vati on assays. After a 2 h transfecti on, the medi um was
changed to DMEM-10%charcoal stri pped serum medi um, and
14-16 h l ater, the cel l s were treated wi th DHT, anti androgen,
or test compounds. After another 14-16 h, the cel l s were
harvested and tested for l uci ferase acti vi ty i n l uci ferase assays
(Promega, Dual Luci ferase Assay System, Madi son, WI ). Data
were expressed as rel ati ve l uci ferase acti vi ty as compared to
an i nternal l uci ferase posi ti ve control .
Acknowledgment. Thi s work was supported by
Nati onal Cancer I nsti tute Grant CA-17625 awarded to
K. H. Lee and DK-60905 to C. Chang.
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