Aza analogues of equol: Novel ligands for estrogen receptor β

Article abstract of DOI:10.1016/j.bmc.2007.05.071

3-Aryl-tetrahydroquinolines, aza analogues of equol, are synthesized and evaluated for their binding properties to the estrogen receptors ERα and ERβ. Several of these compounds exhibited binding selectivity for ER similar to that of genistein. Compounds

Full text of DOI:10.1016/j.bmc.2007.05.071

Bioorganic & Medicinal Chemistry 15 (2007) 5828–5836
Aza analogues of equol: Novel ligands for estrogen receptor b
Wuhong Chen,a,ꢀ Zhaohu Lin,b,ꢀ Mengmeng Ning,c,ꢀ Chunhao Yang,a,* Xueming Yan,a
Yuyuan Xie,a Xu Shena and Ming-Wei Wangc,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
bSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
cThe National Center for Drug Screening, 189 Guo Shou Jing Road, Shanghai 201203, China
Received 9 April 2007; revised 30 May 2007; accepted 31 May 2007
Available online 6 June 2007
Abstract—3-Aryl-tetrahydroquinolines, aza analogues of equol, are synthesized and evaluated for their binding properties to the
estrogen receptors ERa and ERb. Several of these compounds exhibited binding selectivity for ER similar to that of genistein. Com-
pounds 8c and 8d were found to have dual actions: antagonists for ERa and agonists for ERb in a yeast two-hybrid assay. These
compounds have no estrogenic effects on the uterus and bone in vivo.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
pocket, it is not surprising that 17b-estradiol displays a
similar affinity for both subtypes. A handful of com-
pounds that display some selectivity for ERb, including
genistein,7 DPN,8 ERB-041,9 and tetrahydrofluore-
none10 (Fig. 1), have been reported, but the desire of
developing more potent and selective ERb modulators
remains strong. In this paper, we describe the discovery
and characterization of a new class of ligands that are
selective for ERb.
The discovery of a second estrogen receptor, termed
ERb,1 has prompted intensive studies on respective
functions of the two estrogen receptor subtypes, ERa
and ERb. It is known that the distribution of these
two subtypes is different in human tissues. ERb is widely
expressed in the lung, brain,2 and prostate,3 but is not
the dominant subtype in the uterus and breast. These
observations suggest that estrogen receptors might be
tissue specific and ER subtype-selective ligands may pro-
duce different biological effects.4 ERa mediates many of
the well-documented activities elicited by estrogens in
mammary gland, bone, and uterine tissues. Elucidation
of the roles that ERb plays would reveal its therapeutic
value. For instance, an ERb selective agonist was shown
to exert potent action on experimental inflammation,
pointing to its potential to become a drug target.5
Equol, a key metabolite of daidzein, is a unique com-
pound which selectively mimics estrogen actions11 and
in the meanwhile antagonizes the effects of dihydrotes-
tosterone.12 Equol and dehydroequol were reported to
OH
OH
OH
O
CN
DPN
HO
O
HO
Although the two subtypes share about 56% sequence
homology, the ligand-binding pocket for ERb differs
from that of ERa by only two amino acids (ERa Leu
384 ! ERb Met336; ERa Met421 ! ERb ILe373).6
Considering this small change in the ligand-binding
Genistein
O
Br
HO
N
O
OH
HO
tetrahydrofluorenone
F
Keywords: 3-Phenyl-tetrahydroquinoline; ERb; Equol.
*
Corresponding authors. Tel./fax: +86 21 50806770 (C.Y.); e-mail:
chyang@mail.shcnc.ac.cn
Authors contributed equally to this article.
ERB-041
ꢀ
Figure 1. Selective estrogen receptor b ligands.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.071

W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
5829
possess modest selectivity for ERb (8- to 90-fold),13 and
the former could also inhibit bone loss in ovariecto-
mized mice.14 In order to study the structure-activity
relationship (SAR) of this class of compounds, we chose
tetrahydroquinoline as the scaffold because of its easy
modification. The different groups on nitrogen should
change the dominative conformations of the 3-aryl-tet-
rahydroquinolines and might promote selectivity for
ERb. Thus, a series of 7-hydroxy-3-phenyl-1,2,3,4-tetra-
hydroquinoline derivatives were prepared according to a
previously disclosed synthetic procedure.15
chloride or R-carbonyl chloride and Et3N in CH2Cl2 to
yield sulfonamide/amides 9 and subsequent deprotection
(BBr3) afforded 10. Finally, dehydrogenation of 5 with
palladium on activated charcoal at 270 ꢁC gave the aro-
matic ring 11 and deprotection with BBr3 in the same
way afforded 12.
The synthesis of 3-aryl-tetrahydroquinoline analogues
was accomplished using the synthetic transformations
shown in Scheme 2. The substituted trans-o-nitro-a-phe-
nyl-cinnamic acid was prepared by condensation of
4-methoxy-2-nitrobenzaldehyde with 4-methoxyphenyl-
acetic acid in the presence of acetic anhydride and
triethylamine under reflux, and catalytic hydrogenation
of the corresponding (E)-o-nitro-a-phenylcinnamic acid
over Pd/C afforded 3,4-dihydro-3-phenylcarbostyril 13
in good yield.
2. Results and discussion
2.1. Chemistry
The starting material 4-methoxy-2-nitrobenzaldehyde
was made from inexpensive and commercially available
4-methoxy-2-nitro-aniline according to the procedure
described in the literature.16 As shown in Scheme 1,
the 2-phenyl-3-(2-nitro-phenyl)-acrylonitrile derivative
3 was easily obtained by condensation of 4-methoxy-2-
nitrobenzaldehyde 1 with 4-methoxyphenylacetonitrile
2. Reduction of the acrylonitrile 3 was carried out with
NaBH4 in methanol and THF, thereby yielding propio-
nitrile 4. 3-Aryl-1,2,3,4-tetrahydro-quinoline 5 was di-
rectly obtained by Pd/C-catalyzed hydrogenation of 4.
Deprotection of the methoxy group of 5 was readily
achieved with BBr3 in CH2Cl2 to afford 6. Alkylation
of the nitrogen of 5 was effected with sodium borohy-
dride and neat carboxylic acids in THF to yield 7 and
removal of the methoxy protecting group was once
again achieved with BBr3 to afford N-alkyl tetrahydro-
quinolines 8. N-Sulfonylation/N-acylation of 5 was
readily accomplished by treating 5 with methanesulfonyl
The conversion of 13 to the corresponding thiolactam 15
(96%) was achieved by the Lawesson’s reagent17 in boil-
ing toluene. The thiolactam 15 reacted further with for-
mic anhydrazine in cyclohexanol to afford compound
17.18 In the preparation of compound 17, the reaction
should be carried out under nitrogen atmosphere and
low boiling point solvents should not be used. The meth-
oxy protecting group of 13, 15, 17 was removed using
BBr3 to unmask the phenol giving the desired analogues
14, 16, 18, respectively.
2.2. Biological activity
The compounds described above were primarily tested
for intrinsic activity in an ER binding assay with
[3H]17b-estradiol and full-length recombinant human
ERa and ERb proteins. First, these compounds were
tested at 10 and 1 lM, and their binding activities for
OMe
(c)
OMe
(b)
CHO
OMe
(a)
+
CN
NO2
NC
CN
NO2
MeO
MeO
NO2
MeO
1
2
3
4
OR
(g)
7
R=Me, R1=Me(a), Et(b), Pro(c),
cyclopropylmethyl(d)
8
R=H, R1=Me(a), Et(b), Pro(c),
cyclopropylmethyl(d)
RO
N
R1
OR
(e)
9
OR
R=Me,R 2=MeSO2(a), HCO(b),M eCO(c),
EtCO(d), cyclopropanecarbonyl(e),
4-(2-(piperidin-1-yl)ethoxy)benzoyl(f)
(g)
RO
N
10
R=H, R2=MeSO2(a), HCO(b),MeCO(c),
EtCO(d),cyclopropanecarbonyl(e),
4-(2-(piperidin-1-yl)ethoxy)benzoyl(f)
H
RO
N
R2
5
R=Me
R=H
(g)
OR
6
(
f
)
11 R=Me
12 R=H
(g)
RO
N
Scheme 1. Reagents and condition: (a) Na, CH3CH2OH; (b) NaBH4, THF-CH3OH; (c) H2, Pd/C, THF-CH3OH; (d) R2COOH, NaBH4, THF; (e)
R2COX, TEA, CH2Cl2; (f) Pd/C, heating under N2; (g) BBr3, CH2Cl2.

5830
W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
OMe
OMe
CHO
NO2
O2N
(a)
(b)
MeO
+
COOH
H
MeO
RO
COOH
OR
OR
(d)
OR
(c)
RO
N
RO
N
H
S
N
H
O
N
N
R=Me
17
13 R=Me
15 R=Me
16 R=H
(e)
(e)
(e)
R=H
14
18 R=H
Scheme 2. Reagents: (a) TEA, Ac2O; (b) H2, Pd/C, THF-CH3OH; (c) Lawesson’s reagent, THF; (d) HCONHNH2, Cyclohexanol; (e) BBr3, CH2Cl2.
Table 1. Binding property and selectivity for human ERa and ERb
ERa and ERb are shown in Figure 1. We found that all
the carbonyl amides of tetrahydroquinoline displayed
lower affinities to both ERs, and the hydrophobic prop-
erty may improve this feature. For example, N-alkyl
compounds of tetrahydroquinoline showed higher affin-
ities than amides, thioamide 16 also displayed higher
affinity than compound 14/18 because 16 is more hydro-
phobic. Then we examined the IC50 values of several ac-
tive compounds chosen from Figure 2 and the results are
summarized in Table 1. They generally exhibited poor
binding properties compared to the control compound,
and the IC50 of the most active compound 8b was at
least 12-fold less potent than genistein for ERb. Interest-
ingly, compound 10a with a methanesulfonyl group
showed an ERb binding selectivity close to that of
genistein.
Compound
ERa
IC50(lM)
ERb
IC50(lM)
fold Selectivity
for ERb
Genistein
8a
0.58
2.95
1.41
2.47
1.63
32.10
NA
0.0068
0.22
85.1
13.3
17.0
17.9
7.1
8b
8c
0.083
0.14
0.23
8d
10a
10e
16
0.52
2.95
61.5
—
NA
4.90
—
NA, not active.
We determined the EC50 values using the ERb agonist
genistein as a control.20 As shown in Tables 2 and 3,
all these compounds behaved as agonists on ERb; most
of them (except 8c and 8d) also displayed ERa agonism;
compounds 8c and 8d exhibited antagonist features on
ERa and significantly inhibited the effect induced by
To assess whether this class of compounds are of agonist
or antagonist nature, some of them were additionally
evaluated in a yeast two-hybrid assay. This system is
based on the ligand-dependent interaction between two
proteins, a hormone receptor and a co-activator, and
estrogenic activity is detected by a-galactosidase
activity.19
Table 2. In Yeast Binding Affinity for Selected ERb Ligands (1)a
Compound ERa EC50 (lM) ERb EC50 (lM) ERb
selectivityb
Binding activity for ERα
Genistein
6
0.81 0.06
>100
0.028 0.001
9.7 0.66
0.3 0.01
29
N/Ac
50
10 uM
1 uM
120
100
80
8a
8b
12
15.2 2.02
24.33 3.9
>100
0.58 0.02
37.5 1.52
41
N/A
60
40
a Dose-depedent effect of genistein and test compounds on ERa/b-
SRC-1 interactions as determined by the yeast two-hybrid assay.
b Selectivity is reported as EC50 (ERa)/ EC50 (ERb).
c N/A, selectivity could not be accurately determined under the assay
conditions used. Values are means SD from three independent
experiments.
20
0
-20
Geni
6
8a 8b 8c 8d 10a 10b 10c 10d 10e 10f 12 14 16 18
Binding activity for ERβ
10 uM
1 uM
120
100
80
Table 3. In Yeast Binding Affinity for Selected ERb Ligands (2)
60
a
Compound
ERa IC50 (lM)
ERb EC50 (lM)
40
20
8c
8d
2.65 0.49
5.57 0.30
4.75 0.27
6.89 0.74
0
Geni
6
8a 8b 8c 8d 10a 10b 10c 10d 10e 10f 12 14 16 18
-20
a IC50, the concentration of a test compound required to inhibit 50%
of the maximal a-galactosidase activity by
estradiol SEM.
1 nM of 17b-
Figure 2. Estrogen receptor binding characteristics of various 3-aryl-
tetrahydroquinoline analogues.

W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
5831
17b-estradiol (1 nM) (IC50 = 2.65 and 5.57 lM,
respectively).
apparatus. The MS(ESI) and HRMS (ESI) spectra were
obtained on an Finnigan MAT 95 mass spectrometer,
EI: 70 eV. The solvent was removed by rotary evapora-
tion under reduced pressure, and flash column
chromatography was performed on silica gel (200–300
mesh). Anhydrous solvents were obtained by distilling
from sodium wire.
The above compounds were next examined in vivo for
their abilities to affect uterine wet weight and bone min-
eral density. In contrast to raloxifene (control), the 10
compounds tested did not show any estrogenic effects
in ovariectomized mice including the ERa agonists
(Table 4). We presumed that the ERa agonists were eas-
ily transformed into hydrophilic metabolites that are less
active and eliminated quickly in vivo. The biotransfor-
mation of these compounds reduced estrogenic property
to a minimum.
4.2. Synthesis
4.2.1. Synthesis of (Z)-3-(4-methoxy-2-nitro-phenyl)-2-(4-
methoxy-phenyl)-acrylonitrile (3). A mixture of 4-meth-
oxy-2-nitrobenzaldehyde (1.38 g, 7.6 mmol) and
4-methoxyphenyl acetonitrile (1.12 g, 7.6 mmol) was
added to a solution of sodium (0.17 g, 7.6mmol) in abso-
lute ethanol (10 mL) while stirring. A few minutes later,
the mixture became warm, turned cloudy, and precipi-
tated. Stirring was continued for another 5 h. The mix-
ture was cooled and the product was separated by
filtration. The crude product was washed with distilled
water (20 mL), then with 95% ethanol (50 mL) to afford
a pure, bright yellow powder (1.82 g, 76.9%). Mp 143–
144 ꢁC. 1H NMR (300 MHz, CDCl3) d 3.86 (s, 3H),
3.94 (s, 3H), 6.97 (d, J = 8.8 Hz, 2H), 7.28 (m, 1H),
7.64 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 2.8 Hz, 1H),7.85
(s, 1H), 7.91 (d, J = 8.5 Hz, 1H). MS (EI): m/e (%) 310
(80M+), 293 (100), 149 (65), 122 (70).
3. Conclusion
In summary, we have identified 3-aryl-tetrahydroquino-
lines as a new class of ERb-selective ligands. Substitu-
tion at the nitrogen position, particularly with
methanesulfonyl group, is preferable for ERb selectivity.
Compounds 8c and 8d were found to be antagonists for
ERa and agonists for ERb in the yeast two-hybrid as-
say. And this series of compounds did not display any
observable estrogen-like activities in vivo.
4. Experimental
4.1. General
4.2.2. Synthesis of 3-(4-methoxy-2-nitro-phenyl)-2-(4-
methoxy-phenyl)-propionitrile (4). (Z)-3-(4-Methoxy-2-
nitro-phenyl)-2-(4-methoxy-phenyl)-acrylonitrile (1.55 g,
5 mmol) and sodium borohydride (0.28 g, 7.5 mmol)
were added to a mixture of THF (25 mL) and MeOH
(5 mL), stirred at ambient temperature for 4 h, and then
poured the mixture into cold water and neutralized with
1 mol Lꢀ1 HCl. The aqueous solution was extracted
with EtOAc. The combined organic phase was washed
successively with water, saturated NaHCO3, and brine
and then dried with anhydrous sodium sulfate. Removal
of the solvent afforded 3-(4-methoxy-2-nitro-phenyl)-2-
(4-methoxy-phenyl)-propionitrile (1.53 g, 98.2%), which
was used for the next step without further purification.
Mp 107-109 ꢁC. 1H NMR (300 MHz, CDCl3) d 3.15
(dd, J = 10.3 Hz, 13.5 Hz, 1H), 3.43 (dd, J = 5.2 Hz,
13.2 Hz, 1H), 3.82 (s, 3H), 3.88 (s, 3H), 4.28 (dd,
J = 5.3 Hz, 10.1 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 7.14
(dd, J = 2.9 Hz, 8.6 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H),
7.34 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 2.7 Hz, 1H). MS
(EI): m/e (%) 312 (7M+), 166 (100), 146 (85).
1
The H NMR spectra were recorded on Bruker AMX
400 MHz NMR spectrometer. The NMR data were re-
ported in parts per million relative to TMS or referenced
to the solvent in which they were run. Melting points
(uncorrected) were determined on a Buchi-510 capillary
Table 4. Effects on uterine wet weight and bone mineral density
Compound
Bone mineral
density (mg/cm3)
Uterine wet
weight (mg)
Raloxifene
608 34***
467 68*
455 53*
454 36*
439 28*
461 39*
461 19
587 67***
555 54***
475 48*
463 36*
451 28*
430 22**
483 22*
69.5 12.7***
31.4 5.8*
6
8a
31.8 4.0*
8b
8c
31.7 3.9*
28.3 3.3*
8d
30.8 12.2*
OVX+DW
Sham + DW
Raloxifene
10a
31.4 3.5
120.0 29.0***
62.8 12.2**
32.5 6.9*
4.2.3. Synthesis of 7-methoxy-3-(4-methoxy-phenyl)-
1,2,3,4-tetrahydro-quinoline (5). After several vacuum/
H2 cycles to remove air from the reaction flask, the stir-
red mixture of the 3-(4-methoxy-2-nitro-phenyl)-2-(4-
methoxy-phenyl)-propionitrile (1.62 g, 5.2 mmol), 10%
Pd/C (0.49 g, 30 wt% of the propanenitrile) in THF
(20 mL) and MeOH (5 mL) was hydrogenated at ordin-
ary pressure and at temperature (ca. 40 ꢁC) for 48 h. The
reaction mixture was filtered using a membrane filter
and filtrate was concentrated under reduced pressure.
The crude mixture was purified by flash silica gel column
chromatography, using petroleum ether/ethyl acetate
(V/V: 10/1) as eluent, thus obtaining the white crystal
10b
10c
36.4 10.4*
29.1 3.2*
33.1 4.8*
38.7 16.8*
36.8 11.4
129.1 16.7***
12
16
OVX+DW
Sham + DW
461
9
556 36***
Two months old ovariectomized Kunming mice were treated (sc) with
the test compounds at a dose of 4 lmol/kg qd for 4 weeks. Uterine wet
weight was measured on the day when mice were sacrificed (n = 5) per
group; mean SD.
* p > 0.05.
** p < 0.05.
*** p < 0.01 versus distilled water (DW) treated animals.

5832
W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
1
(0.85 g, 60.6%). Mp 146–147 ꢁC. H NMR (300 MHz,
CDCl3) d 2.92–2.97 (m, 2H), 3.26–3.31 (m, 2H), 3.59–
3.61 (m, 1H), 3.75 (s, 3H), 3.81 (s, 3H), 6.53 (dd,
J = 2.4 Hz, 8.3 Hz, 1H), 6.6 (d, J = 2.2 Hz, 1H), 6.89
(d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.5 Hz, 1H), 7.19 (d,
J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 33.9,
37.9, 48.4, 55.1, 55.2, 99.2, 102.9, 113.9, 114.0, 128.0,
130.0, 135.9, 144.7, 158.2, 158.9. MS (EI): m/e (%) 269
(100 M+), 148 (55).
J = 8.8 Hz, 2H). MS (EI): m/e (%) 323 (100M+), 149
(50), 121 (50).
4.2.5. General procedure for sulfonylation/acylation of the
tetrahydroquinoline nitrogen.
4.2.5.1. 1-Methanesulfonyl-7-methoxy-3-(4-methoxy-
phenyl)-1,2,3,4- tetrahydro-quinoline (9a). The THF
solution of the methanesulfonyl chloride (0.09 g,
0.825mmol) was added dropwise to a solution of 5
(0.21 g,
0.75 mmol),
triethylamine(0.11 mL,
4.2.4. General procedure for alkylation of the tetrahy-
droquinoline nitrogen.
0.825 mmol) in dry THF (10 mL) blanketed under nitro-
gen and cooled to 0 ꢁC. When the addition was com-
plete, the reaction mixture was stirred at room
temperature for 2 h. After removal of THF, the residue
was loaded on silica gel column and eluted with petro-
leum ether-ethyl acetate to afford the corresponding
product (0.13 g, 51.9%). Mp 135.5–136 ꢁC. 1H NMR
(300 MHz, CDCl3) d 2.83 (s, 3H), 2.87–3.20 (m, 3H),
3.50–3.60 (m, 1H), 4.22–4.30 (m, 1H), 3.80 (s, 3H),
3.81 (s, 3H), 6.68 (dd, J = 2.3 Hz, 8.2 Hz, 1H), 6.88 (d,
J = 8.8 Hz, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.17 (d,
J = 8.7 Hz, 2H), 7.40 (d, J = 2.4 Hz, 1H). MS (EI): m/e
(%) 347 (60M+), 214 (80), 121 (100).
4.2.4.1. 7-Methoxy-3-(4-methoxy-phenyl)-1-methyl-
1,2,3,4-tetrahydro- quinoline (7a). Compound 5 (0.27 g,
1 mmol) was dissolved in 10 mL of THF under N2,
chilled to 0 ꢁC, and 0.28 g (7.5 mmol) of NaBH4 added,
followed by slow addition of 3 mL of HCOOH. The
reaction mixture was allowed to warm to room temper-
ature and stirred overnight. The solvent was removed,
the residue was slurried in water and made basic with
1 mol Lꢀ1 NaOH, and the product was extracted into
EtOAc and chromatographed on a silica gel column.
Elution with petroleum ether/EtOAc (10/1) gave a white
solid (0.11 g, 38.1%). Mp 99–100 ꢁC. 1H NMR
(300 MHz, CDCl3) d 2.90–2.93 (m, 2H), 2.95 (s, 3H),
3.31 (d, J = 6.1 Hz, 2H), 3.14–3.20 (m, 1H), 3.80 (s,
3H), 3.81 (s, 3H), 6.30 (s, 2H), 6.88 (d, J = 8.6 Hz,
2H), 6.93 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 8.9 Hz, 2H).
MS (EI): m/e (%) 283 (100 M+), 162 (35).
4.2.5.2. 7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihydro-
2H-quinoline-1-carbaldehyde (9b). This compound was
prepared from compound 5 with formic acid and acetic
anhydride according to the similar procedure to 9a as a
1
solid in 72.3%. Mp 112–113 ꢁC. H NMR (300 MHz,
CDCl3) d 2.84–3.17 (m, 3H), 3.33–3.41 (m, 1H), 3.81
(s, 3H), 3.83 (s, 3H), 4.38–4.42 (m, 1H), 6.71 (dd,
J = 2.4 Hz, 8.5 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.88
(d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.3 Hz, 1H), 7.17 (d,
J = 8.7 Hz, 2H), 8.82 (s, 1H). MS (EI): m/e (%) 297
(100M+), 134 (70).
4.2.4.2.
1-Ethyl-7-methoxy-3-(4-methoxy-phenyl)-
1,2,3,4-tetrahydro-quinoline (7b). This compound was
prepared from compound 5 with acetic acid according
to the similar procedure to 7a as a solid in 88.2%. Mp
77–78 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.15 (t,
J = 7.2 Hz, 3H), 2.88–2.91 (m, 2H), 3.03–3.15 (m, 1H),
3.20–3.33 (m, 3H), 3.40–3.51 (m, 1H), 3.80 (s, 3H),
3.81 (s, 3H), 6.18 (dd, J = 2.6 Hz, 8.1 Hz, 1H), 6.23 (d,
J = 2.2 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 6.91 (d,
J = 8.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H). MS (EI): m/e
(%) 297 (100 M+), 282 (85), 121 (60).
4.2.5.3. 1-[7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-ethanone (9c). This compound was
prepared from compound 5 with acetyl chloride accord-
ing to the similar procedure to 9a as a solid in 96.5%.
1
Mp 102–103 ꢁC. H NMR (300 MHz, CDCl3) d 2.21
(s, 3H), 2.80–2.89 (m, 1H), 2.99–3.14 (m, 2H), 3.56–
3.64 (m, 1H), 3.79 (s, 3H), 3.81 (s, 3H), 4.11–4.22 (m,
1H), 6,71 (dd, J = 2.4 Hz, 8.5 Hz, 1H), 6.74 (d,
J = 2.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 7.08 (d,
J = 8.5 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H). MS (EI): m/e
(%) 311 (5M+), 149 (100).
4.2.4.3.
7-Methoxy-3-(4-methoxy-phenyl)-1-propyl-
1,2,3,4-tetrahydro-quinoline (7c). This compound was
prepared from compound 5 with propionic acid accord-
ing to the similar procedure to 7a as a solid in 87.4%.
Mp 84–85 ꢁC. H NMR (300 MHz, CDCl3) d 0.93 (t,
1
J = 7.3 Hz, 3H), 1.52–1.71 (m, 2H), 2.86–2.93 (m, 2H),
3.03–3.36 (m, 5H), 3.79 (s, 3H), 3.81 (s, 3H), 6.15–6.26
(m, 2H), 6.88 (d, J = 8.5 Hz, 2H), 6.89 (m, 1H), 7.16
(d, J = 8.7 Hz, 2H). MS (EI): m/e (%) 311 (50M+), 282
(100).
4.2.5.4. 1-[7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-propan-1-one (9d). This compound
was prepared from compound 5 with propionyl chloride
according to the similar procedure to 9a as a solid in
99%. Mp 89–90 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.14
(t, J = 7.4 Hz, 3H), 2.50 (q, J = 7.2 Hz, 2H), 2.80–2.89
(m, 1H), 2.99–3.18 (m, 2H), 3.57–3.62 (m, 1H), 3.80 (s,
3H), 3.81 (s, 3H), 4.11–4.20 (m, 1H), 6.71 (dd, J = 2.5
Hz, 8.5 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.86 (d,
J = 8.7 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 7.15 (d,
J = 8.6 Hz, 2H). MS (EI): m/e (%) 325 (100M+), 269 (100).
4.2.4.4. 1-Cyclopropylmethyl-7-methoxy-3-(4-meth-
oxy-phenyl)-1,2,3,4-tetrahydro-quinoline (7d). This com-
pound was prepared from compound
5
with
cyclopropanecarboxylic acid according to the similar
procedure to 7a with acetic acid as a solid in 36.2%.
Mp 97–98 ꢁC. 1H NMR (300 MHz, CDCl3) d 0.20–
0.25 (m, 2H), 0.52–0.55 (m, 2H), 0.92–0.94 (m, 1H),
2.91–3.07 (m, 3H), 3.08–3.20 (br, 1H), 3.41–3.45
(br, 3H), 3.80 (s, 3H), 3.81 (s, 3H), 6.20–6.46 (m, 2H),
6.89 (d, J = 8.8 Hz, 2H), 6.93 (m, 1H), 7.17 (d,
4.2.5.5.
Cyclopropyl-[7-methoxy-3-(4-methoxy-phe-
nyl)-3,4-dihydro-2H-quinolin-1-yl]-methanone (9e). This
compound was prepared from compound 5 with cyclo-

W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
5833
propanecarbonyl chloride according to the similar pro-
cedure to 9a as a solid in 93.5%. Mp 106–107 ꢁC. H
were removed and the residue was recrystallized with
ethanol to give 13 (1.32 g, 93%), Mp 182–183 ꢁC. H
1
1
NMR (300 MHz, CDCl3) d 0.80 (m, 2H), 1.12 (m,
2H), 2.00–2.09 (m, 1H), 2.80–2.91 (m, 1H), 2.99–3.18
(m, 2H), 3.57 (dd, J = 10.0 Hz, 12.7 Hz, 1H), 3.79 (s,
3H), 3.81 (s, 3H), 4.25 (dd, J = 0.9 Hz, 5.4 Hz, 1H),
6.70 (dd, J = 2.6 Hz, 8.3 Hz, 1H), 6.85 (d, J = 8.9 Hz,
2H), 7.05 (d, J = 2.5 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H),
7.15 (d, J = 8.8 Hz, 2H). MS (EI): m/e (%) 337
(95M+), 269 (100).
NMR (300 MHz, DMSO-d6) d 3.00–3.06 (m, 2H), 3.68
(s, 3H), 3.69 (s, 3H), 3.72–3.79 (m, 1H), 6.45–6.47 (m,
1H), 6.49–6.51 (m, 1H), 6.82 (d, J = 9.0 Hz, 2H), 7.05
(d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H). MS (EI):
m/e (%) 283 (100 M+), 149 (55), 57 (50).
4.2.8. 7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihydro-1H-
quinoline-2-thione (15).
A mixture of 13 (1.92 g,
6.8 mmol) and Lawesson’s reagent (2.06 g, 5.1 mmol)
was suspended in anhydrous toluene (25 mL) and then
refluxed under nitrogen atmosphere for 5 h. The solu-
tion was then cooled to room temperature and evapo-
rated. The residue was purified by flash
chromatography on silica gel (eluant CH2Cl2–MeOH,
50:1) providing 15 (1.95 g) in 96% yield as a pale yellow
4.2.5.6. [7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-
methanone (9f). This compound was prepared from
compound 5 with 4-(2-(piperidin-1-yl)ethoxy)benzoyl
chloride according to the similar procedure to 9a as a so-
lid in 96.8%. Mp 166–168 ꢁC. 1H NMR (300 MHz,
CDCl3) d 1.63–1.71 (m, 4H), 1.82–1.97 (m, 4H), 2.20–
2.37 (m, 2H), 2.71–2.84 (m, 2H), 2.91–3.02 (m, 1H),
3.07–3.25 (m, 2H), 3.54 (s, 3H), 3.71 (dd, J = 9.1 Hz,
12.6 Hz, 1H), 3.79 (s, 3H), 4.24 (dd, J = 4.7 Hz,
12.6 Hz, 1H), 4.54–4.60 (m, 2H), 6.45-6.46 (m, 1H),
6.63 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 6.79 (d, J = 8.8 Hz,
2H), 6.86 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H),
7.15 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.9 Hz, 2H). MS
(EI): m/e (%) 500 (10 M+), 98 (100).
1
solid: Mp 179–180 ꢁC. H NMR (300 MHz, CDCl3) d
3.00 (dd, J = 6.1 Hz, 16.1 Hz, 1H), 3.24 (dd,
J = 6.3 Hz, 15.9 Hz, 1H), 3.74 (s, 3H), 3.78 (s, 3H),
4.27 (t, J = 6.0 Hz, 1H), 6.44–6.47 (m, 1H), 6.60–6.62
(m, 1H), 6.78 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.2 Hz,
1H), 7.13 (d, J = 8.6 Hz, 2H). MS (EI): m/e (%) 299
(100M+).
4.2.9. 8-Methoxy-4-(4-methoxy-phenyl)-4,5-dihydro-1,2,4
triazolo[4,3-a]quinoline (17). A mixture of compound
15 (0.3 g, 1 mmol) and formic hydrazine 0.08 g (1.2
mmol) in 5 mL cyclohexanol was refluxed for 6 h under
a nitrogen atmosphere. After the solvent was removed
under reduced pressure, the residue was dissolved in
EtOAc and loaded on silica gel column and eluted with
CH2Cl2-MeOH (50:1) to afford a brown solid 0.28 g
4.2.6. Methoxy-3-(4-methoxy-phenyl)-quinoline (11). A
mixture of 5 (0.27 g, 1mmol) and palladium on activated
charcoal (10%) (54 mg) was heated at 270–280 ꢁC, under
nitrogen with stirring for 30 min. On cooling, methanol
was added and the reaction mixture was filtered through
Celite. The solvent was evaporated and the residue was
purified by flash silica gel column chromatography using
petroleum ether-ethyl acetate as eluent to afford white
solid (0.25 g, 96.2%). Mp 142–143 ꢁC. 1H NMR
(300 MHz, CDCl3) d 3.88 (s, 3H), 3.98 (s, 3H), 7.05
(d, J = 8.8 Hz, 2H), 7.22 (dd, J = 2.4 Hz, 8.9 Hz, 1H),
7.47 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.75
(d, J = 9.0 Hz, 1H), 8.20 (d, J = 2.3 Hz, 1H), 9.07 (d,
J = 2.3 Hz, 1H). MS (EI): m/e (%) 265 (100M+), 250
(60).
1
(90.2%), Mp 90–92.5 ꢁC. H NMR (300 MHz, CDCl3)
d 3.16–3.31 (m, 2H), 3.75 (s, 3H), 3.86 (s, 3H), 4.51 (t,
J = 7.1 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 6.82 (d,
J = 8.8, 2H), 6.96 (d, J = 2.5 Hz, 1H), 7.16 (d,
J = 8.5 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 8.71 (s, 1H).
MS (EI): m/e (%) 307 (5M+), 84 (100).
4.2.10. General procedure for protecting-group cleavage.
4.2.10.1.
3-(4-Hydroxy-phenyl)-1,2,3,4-tetrahydro-
quinolin-7-ol (6). The substrate 5 (0.20 g, 0.75 mmol)
was dissolved in CH2Cl2 (10mL) and cooled to ꢀ10 ꢁC
and BBr3 (0.75 g, 0.29mL, 3.0mmol) in CH2Cl2 was
added dropwise with stirring. As the solution of boron
tribromide was added, a white precipitate was formed.
The reaction mixture was allowed to attain room tem-
perature overnight with stirring, when a clear, brownish
yellow solution was obtained. The reaction mixture was
then hydrolyzed by careful shaking with 5 mL of water,
thus precipitating a white solid which was dissolved by
the addition of 1 mol Lꢀ1 sodium hydroxide, then neu-
tralized with dilute hydrochloric acid, extracted with
50 mL of ethyl acetate, and the organic layer was dried
over anhydrous magnesium sulfate. On removal of the
ethyl acetate under reduced pressure, a brownish yellow
oil remained which was purified by flash chromatogra-
phy on silica gel (eluant petroleum ether/ethyl acetate,
2:1) providing 6 (0.20 g) in 99% yield as a white solid.
Mp 228–229 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
2.61–2.83 (m, 3H), 3.0–3.07 (m, 1H), 3.15–3.21 (m,
1H), 5.87 (dd, J = 2.2 Hz, 7.8 Hz, 1H), 5.92 (d,
4.2.7. 7-Methoxy-3-(4-methoxy-phenyl)-3,4-dihydro-1H-
quinolin-2-one (13). A mixture of 1.81 g (10 mmol) of
4-methoxy-2-nitrobenzaldehyde, 2.41 g (14.5 mmol) of
4-methoxyphenylacetic acid, 5 mL (54 mmol) of acetic
anhydride, and 1 g (10 mmol) of triethylamine was re-
fluxed for 15 minutes in a 25-mL flask. The solution
was cooled to 90 ꢁC, and 5 mL of cold water was added
over a 5-min period at a rate that maintained the tem-
perature above 90 ꢁC. The solution was cooled to
20 ꢁC and the substituted trans-o-nitro-a-phenylcinnam-
ic acid precipitated in the form of light orange crystals.
It was separated by filtration and washed with 10 mL of
50% acetic acid and with water. After recrystallization
from 50 mL of ethanol, the dried acid was in the form
of yellow prisms weighing 2.39 g (71%) and melting at
205–206 ꢁC. The obtained (E)-3-(4-methoxy-2-nitro-
phenyl)-2-(4-methoxy-phenyl)-acrylic
acid
(1.65 g,
5mmol) was hydrogenated with 10% Pd/C (0.17 g) in
THF and MeOH at room temperature under atmo-
spheric pressure for 12 h. The catalyst and the solvent

5834
W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
J = 2.4 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.69 (d,
J = 8.4 Hz, 2H), 7.04 (d, J = 8.3 Hz, 2H). MS (EI): m/e
(%) 241 (100M+), 149 (60), 134 (65). HRMS (EI) calcd
for C15H15NO2, 241.1103; found, 241.1106.
4.2.10.7. 7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-2H-quinoline-1-carbaldehyde (10b). This compound
was prepared according to the similar procedure to 6
as a solid in 100%. Mp 238–239 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 2.78–2.95 (m, 2H), 3.28 (dd,
J = 10.2 Hz, 12.4 Hz, 1H), 4.01 (dd, J = 7.2 Hz,
14.2 Hz, 1H), 4.11 (dd, J = 3.7 Hz, 13.1 Hz, 1H), 6.55
(dd, J = 2.4 Hz, 8.1 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H),
6.78 (d, J = 2.3 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 7.10
(d, J = 8.7 Hz, 2H), 8.78 (s, 1H). MS (EI): m/e (%) 269
(100M+). HRMS (EI) calcd for C16H15NO3, 269.1052;
found, 269.1044.
4.2.10.2. 3-(4-Hydroxy-phenyl)-1-methyl-1,2,3,4-tetra-
hydro-quinolin-7-ol (8a). This compound was prepared
according to the similar procedure to 6 as a solid in
1
84.5%. Mp 203–204 ꢁC. H NMR (300 MHz, DMSO-
d6) d 2.70–2.73 (m, 2H), 2.78 (s, 3H), 2.93 (s, 1H),
3.11–3.15 (m, 2H), 5.98–6.00 (m, 2H), 6.68–6.70 (m,
3H), 7.05 (d, J = 8.0 Hz, 2H), 8.96 (s, 1H), 9.36 (s,
1H). MS (EI): m/e (%) 255 (100 M+), 148 (75). HRMS
(EI) calcd for C16H17NO2, 255.1259; found, 255.1262.
4.2.10.8. 1-[7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-ethanone (10c). This compound
was prepared according to the similar procedure to 6
as a solid in 77.0%. Mp 201–202 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 2.09 (s, 3H), 2.68–2.78 (m,
1H), 2.82-3.04 (m, 2H), 3.40–3.52 (m, 1H), 3.98–4.06
(m, 1H), 6.53 (dd, J = 2.4 Hz, 8.3 Hz, 1H), 6.70 (d,
J = 8.4 Hz, 2H), 6.78 (d, J = 2.3 Hz, 1H), 7.03 (d,
J = 8.5 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H). MS (EI): m/e
(%) 283 (100M+), 241 (65), 134 (55). HRMS (EI) calcd
for C17H17NO3, 283.1208; found, 283.1200.
4.2.10.3. 1-Ethyl-3-(4-hydroxy-phenyl)-1,2,3,4-tetra-
hydro-quinolin-7-ol (8b). This compound was prepared
according to the similar procedure to 6 as a solid in
93.6%. Mp 174–175 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 1.01 (t, J = 6.6 Hz, 3H), 2.64–2.78 (m, 2H), 3.09–
3.19 (m, 2H), 3.52–3.60 (m, 3H), 5.87–5.94 (m, 1H),
6.02 (s, 1H), 6.67–6.70 (m, 3H), 7.05 (d, J = 8.2 Hz,
2H), 8.91 (s, 1H), 9.36 (s, 1H). MS (EI): m/e (%) 269
(100 M+), 254 (95). Anal. calcd for C17H19NO2Æ0.25-
H2O: C, 74.56; H, 7.18; N. 5.11; found: C, 74.89; H,
7.10; N. 4.85.
4.2.10.9. 1-[7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-propan-1-one (10d). This com-
pound was prepared according to the similar
4.2.10.4. 3-(4-Hydroxy-phenyl)-1-propyl-1,2,3,4-tetra-
hydro-quinolin-7-ol (8c). This compound was prepared
according to the similar procedure to 6 as a solid in
1
procedure to 6 as a solid in 73%. Mp 204-205 ꢁC. H
NMR (300 MHz, DMSO-d6) d 0.94 (t, J = 7.4 Hz,
3H), 2.36–2.41 (m, 2H), 2.62–2.73 (m, 1H), 2.83–2.93
(m, 2H), 3.41–3.46 (m, 1H), 3.87–3.91 (m, 1H), 6.51
(dd, J = 2.4 Hz, 8.3 Hz, 1H), 6.67 (d, J = 8.5 Hz, 2H),
6.78 (d, J = 2.3 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.03
(d, J = 8.5 Hz, 2H). MS (EI): m/e (%) 297 (70 M+),
241 (100), 134 (45). HRMS (EI) calcd for C18H19NO3,
297.1365; found, 297.1369.
1
78.8%. Mp 144-145 ꢁC. H NMR (300 MHz, DMSO-
d6) d 0.84 (t, J = 7.4 Hz, 3H), 1.43–1.55 (m, 2H), 2.60–
2.78 (m, 2H), 2.80–2.90 (m, 1H), 2.99–3.09 (m, 1H),
3.14–3.25 (m, 3H), 5.88 (dd, J = 1.8 Hz, 7.6 Hz, 1H),
5.99 (d, J = 2.0 Hz, 1H), 6.64 (m, 1H), 6.67 (d,
J = 8.6 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H). MS (EI): m/e
(%) 283 (55 M+), 254 (100). HRMS (EI) calcd for
C18H21NO2, 283.1572; found, 283.1562.
4.2.10.10. Cyclopropyl-[7-hydroxy-3-(4-hydroxy-phe-
nyl)-3,4-dihydro-2H-quinolin-1-yl]-methanone (10e). This
compound was prepared according to the similar proce-
dure to 6 as a solid in 69%. Mp 267–268 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 0.76–0.85 (m, 4H), 1.97–2.01
(m, 1H), 2.62–2.78 (m, 1H), 2.86–2.93 (m, 2H), 3.41–
4.46 (m, 1H), 4.00–4.08 (m,1H), 6.55 (dd, J = 2.4 Hz,
8.2 Hz, 1H), 6.68 (d, J = 8.6 Hz, 2H), 6.91 (d,
J = 2.4 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 7.06 (d,
J = 8.5 Hz, 2H). MS (EI): m/e (%) 309 (40 M+), 241
(100), 84 (75). HRMS (EI) calcd for C19H19NO3,
309.1365; found, 309.1356.
4.2.10.5. 1-Cyclopropylmethyl-3-(4-hydroxy-phenyl)-
1,2,3,4-tetrahydro-quinolin-7-ol (8d). This compound
was prepared according to the similar procedure to 6
as a solid in 92.5%. Mp 124–125 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 0.17 (m, 2H), 0.41–0.43 (m,
2H), 0.75–0.80 (m, 1H), 2.60–2.75 (m, 2H), 2.75–2.89
(m, 2H), 3.24–3.29 (m, 3H), 5.93 (d, J = 8.3 Hz, 1H),
6.11 (s, 1H), 6.67–6.70 (m, 3H), 7.05 (d, J = 8.3 Hz,
2H), 8.92 (s, 1H), 9.36 (s, 1H). MS (EI): m/e (%) 295
(60M+), 149 (50), 115 (100). HRMS (EI) calcd for
C19H21NO2, 295.1572; found, 295.1568.
4.2.10.6.
3-(4-Hydroxy-phenyl)-1-methanesulfonyl-
4.2.10.11. [7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-2H-quinolin-1-yl]-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-
methanone (10f). This compound was prepared according
to the similar procedure to 6 as a solid in 80%. Mp 135-
136 ꢁC. 1H NMR (300 MHz, DMSO-d6) d 1.49–1.55 (m,
2H), 1.63–1.69 (m, 4H), 2.66–2.74 (m, 4H), 2.90–2.98 (m,
3H), 3.03–3.12 (m, 2H), 3.73 (dd, J = 8.0 Hz, 12.4 Hz,
1H), 4.06 (dd, J = 4.4 Hz, 12.7 Hz, 1H), 4.17 (t,
J = 5.6 Hz, 2H), 6.33–6.36 (m, 1H), 6.52 (dd,
J = 2.3 Hz, 8.1 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H), 6.88
(d, J = 8.7 Hz, 2H), 7.02–7.06 (m, 3H), 7.25 (d,
J = 8.7 Hz, 2H). MS (EI): m/e (%) 472 (15M+), 98
1,2,3,4-tetrahydro-quinolin-7-ol (10a). This compound
was prepared according to the similar procedure to 6
as a solid in 77.6%. Mp 231–232 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 2.69–2.89 (m, 2H), 2.98 (s,
3H), 3.36–3.50 (m, 2H), 3.92–4.05 (m, 1H), 6.51 (dd,
J = 2.5 Hz, 8.3 Hz, 1H), 6.72 (d, J = 8.5 Hz, 2H), 7.00
(d, J = 8.1 Hz, 1H), 7.12 (d, J = 2.6 Hz, 1H), 7.13 (d,
J = 8.6 Hz, 2H). MS (EI): m/e (%) 319 (5 M+), 200
(100), 121 (75). Anal. calcd for C16H17NO4SÆ0.5H2O:
C, 5, 5.52; N, 4.27; found: C, 58.38; H, 5.65; N,
4.178.52; H.

W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
5835
(100). HRMS (EI) calcd for C29H32N2O4, 472.2362;
found, 472.2357.
drop-out-agar plate without leucine and trypto-
phan(SD–TꢀLꢀ). The plates were incubated at 30 ꢁC
for 48 h for yeast growth. PCR was used to confirm
transformation with the target gene.
4.2.10.12. 3-(4-Hydroxy-phenyl)-quinolin-7-ol (12).
This compound was prepared according to the similar
1
procedure to 6 as a solid in 70%. Mp 185-186 ꢁC. H
4.4.2. a-Galactosidase activity assay. The quantitative
a-galactosidase activity assays were carried out by using
p-nitrophenyl a-D-galactopyranoside (PNP-a-Gal) as
the substrate according to the Clontech manual. The
corresponding yeast cells were cultured in SD minimal
medium (3 mL) without leucine and tryptophan (TꢀLꢀ)
(yeast nitrogen base without amino acids 6.7 g Lꢀ1, D-
(+)-glucose 20 g Lꢀ1, yeast synthetic drop-out medium
NMR (300 MHz, DMSO-d6) 6.94 (d, J = 8.8 Hz, 2H),
7.35 (d J = 8.3 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 8.03
(dd, J = 8.8 Hz, 21.7 Hz, 2H), 8.87 (s, 1H), 9.20 (dd,
J = 2.1 Hz, 16.0 Hz, 1H). MS (EI): m/e (%) 236
(25M+), 208 (65). HRMS (EI) calcd for C15H11NO2,
237.0790; found, 237.0784.
4.2.10.13. 7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-1H-quinolin-2-one (14). This compound was pre-
pared according to the similar procedure to 6 as a
solid in 100%. Mp > 300 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 2.91–3.10 (m, 2H), 3.57–3.62 (m, 1H),
6.30 (dd, J = 2.4 Hz, 8.0 Hz, 1H), 6.34 (d, J = 2.1 Hz,
1H), 6.65 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H),
6.97 (d, J = 8.6 Hz, 2H). MS (EI): m/e (%) 255 (100
M+), 122 (50). HRMS (EI) calcd for C15H13NO3,
255.0895; found, 255.0890.
supplement
without
leucine
and
tryptophan
1.54 g Lꢀ1) at 30 ꢁC with shaking (250 rpm). When the
OD600 of the cells had reached about 1.0, the cultures
were diluted 50 times with fresh TꢀLꢀ medium, and dif-
ferent concentrations of ligands (1 lL) or DMSO (1 lL;
as a blank) were then added to diluted cultures (999 lL).
After further incubation at 30 ꢁC for 16 h, each sample
(200 lL) was transferred to a 96-well plates (Corning
Costar 96-well flat-bottomed plate), and the OD600 va-
lue was measured by a Benchmark PlusTM microplate
spectrophotometer (Bio-Rad). the cells were centrifuged
at 3000 rpm for 30 min and 16 lL of supernatants was
transferred into a 96-well plate with fresh assay buffer
(48 lL; 1 volume of 100 mM PNP-a-Gal + 2 volumes
of 0.5 M sodium acetate, pH 4.5 per well). After a
further 60-min incubation at 30 ꢁC, the reaction was
terminated by the addition of stop solution (136 lL,
1 M Na2CO3) in each well, and the OD410 was
measured. One unit of a-galactosidase is defined as
the amount of enzyme that hydrolyzes 1 lmol
PNP-a-Gal to p-nitrophenol and D-galactose in 1 min
at 30 ꢁC in acetate buffer (pH 4.5).22 The a-galactosidase
activity was calculated according to the following
formula:
4.2.10.14. 7-Hydroxy-3-(4-hydroxy-phenyl)-3,4-dihy-
dro-1H-quinoline-2-thione (16). This compound was pre-
pared according to the similar procedure to 6 as a solid
1
in 100%. Mp 262-264 ꢁC. H NMR (300 MHz, DMSO-
d6) d 2.81 (dd, J = 4.1 Hz, 16.2 Hz, 1H), 3.08 (dd,
J = 6.3 Hz, 16.1 Hz, 1H), 4.11 (dd, J = 4.0 Hz, 5.9 Hz,
1H), 6.41 (dd, J = 2.4 Hz, 7.9 Hz, 1H), 6.57–6.59 (m,
2H), 6.60 (s, 1H), 6.90–6.94 (m, 3H). MS (EI): m/e (%)
271 (25 M+), 149 (30), 61 (100). HRMS (EI) calcd for
C15H13NO2S, 271.0667; found, 271.0658.
4.2.10.15. 4-(4-Hydroxy-phenyl)-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinolin-8-ol (18). This compound was
prepared according to the similar procedure to 6 as a so-
lid in 100%. Mp 205 ꢁC dec. 1H NMR (300 MHz,
DMSO-d6) d 3.05–3.18 (m, 2H), 4.44 (t, J = 6.8 Hz,
1H), 6.65 (d, J = 8.9 Hz, 2H), 6.68 (d, J = 2.2 Hz, 1H)
6.95 (d, J = 8.6 Hz, 2H), 7.16–7.19 (m, 2H), 9.24 (s,
1H). MS (EI): m/e (%) 278 (25Mꢀ1+), 210 (100). HRMS
(EI) calcd for C16H13N3O2, 279.1008; found, 279.0999.
a-Galactosidase
OD410 · Vf · 1000/[(n · b) · t · Vi · OD600
activity
[milliunits/(mL · cell)] =
]
t Elapsed time (min) of incubation.
Final volume of assay (200 lL).
Volume of culture medium supernatant added
(16 lL).
Vf
Vi
OD600 Optical density of overnight culture.
4.3. Ligand binding competition experiments
n · b
p-Nitrophenol molar absorbtivity b at 410 nm·
the light path (cm) = 10.5 (mL molꢀ1; Yeast
Protocols Handbook PT3024–1, Clontech).
The compounds were tested for intrinsic activity in an
ER binding assay with [3H]17b-estradiol and full-length
recombinant human ERa and ERb proteins according
to the procedure described in the literature.20
4.5. Bioassay on the OVX rat model
4.4. Yeast two-hybrid assay
The bone mineral density (BMD) of the tibial diaphysis
was measured by peripheral quantitative computed
tomography (pQCT) using an XCT Reaearch SA +
pQCT machine (Stratec Medizintechnik, Pforzheim,
Germany). The measurements were made with a
collimator opening of 0.2 mm on specimens embedded
in methylmethacrylate. One slice in the middiaphysis
of the tibias located 2.0 mm proximal from the
tibiofibular junction was measured. A voxel size of
0.09 mm and a threshold of 710 mg/cm3 were used
4.4.1. Yeast transformation and culture. The yeast strain
AH109 was obtained from Clontech (Palo Alto,CA),
and transformation was performed according to the lith-
ium acetate method.21 Briefly, 500 ng of plasmid DNA
was added to 50 lL of the competent cells and mixed
with 36 lL of 1 M lithium acetate, 240 lL of 50%
PEG3350, and 50 ng single-strain DNA at 30 ꢁC for
30 min followed by heat-shock (250 rpm) at 42 ꢁC for
30 min. The mixture was subsequently spread on a

5836
W. Chen et al. / Bioorg. Med. Chem. 15 (2007) 5828–5836
9. (a) Manas, E. S.; Unwalla, R. J.; Xu, Z. B.; Malamas, M.
S.; Miller, C. P.; Harris, H. A.; Hsiao, C.; Akopian, T.;
Hum, W. T.; Malakian, K.; Wolfrom, S.; Bapat, A.; Bhat,
R. A.; Stahl, M. L.; Somers, W. S.; Alvarez, J. C. J. Am.
Chem. Soc. 2004, 126, 15106–15119; (b) Malamas, M. S.;
Manas, E. S.; McDevitt, R. E.; Gunawan, I.; Xu, Z. B.;
Collini, M. D.; Miller, C. P.; Dinh, T.; Henderson, R. A.;
Keith, J. C.; Harris, H. A. J. Med. Chem. 2004, 47, 5021–
5040.
10. (a) Wilkening, R. R.; Ratcliffe, R. W.; Fried, A. K.; Meng,
D.; Sun, W.; Colwell, L.; Lambert, S.; Greenlee, M.;
Nilsson, S.; Thorsell, A.; Mojena, M.; Tudela, C.; Frisch,
K.; Chan, W.; Birzin, E. T.; Rohrerb, S. P.; Hammond,
M. L. Bioorg. Med. Chem. Lett. 2006, 16, 3896–3901; (b)
Wildonger, K. J.; Ratcliffe, R. W.; Mosley, R. T.;
Hammond, M. L.; Birzin, E. T.; Rohrer, S. P. Bioorg.
Med. Chem. Lett. 2006, 16, 4462–4466; (c) Parker, D. L.;
Meng, D.; Ratcliffe, R. W.; Wilkening, R. R.; Sperbeck,
D. M.; Greenlee, M. L.; Colwell, L. F.; Lambert, S.;
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for calculation of cortical BMD. Statistics were
computed using t-test.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (20502028 to C.H.Y.), Shanghai
Municipality Science and Technology Development
Fund (05ZR14141 to C.H.Y.), and the Ministry of Sci-
ence and Technology (2004CB518902 to M.-W.W.).
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