A new series of estrogen receptor modulators: Effect of alkyl substituents on receptor-binding affinity

Article abstract of DOI:10.1248/cpb.52.79

New types of selective estrogen receptor modulators (SERMs) were synthesized and evaluated for their binding affinity and biological effect on reproductive cells. A proposed lead structure (B) was derivatized to provide compounds 30 and 44, which showed g

Full text of DOI:10.1248/cpb.52.79

January 2004
Chem. Pharm. Bull. 52(1) 79—88 (2004)
79
A New Series of Estrogen Receptor Modulators: Effect of Alkyl
Substituents on Receptor-Binding Affinity
*
Atsushi KAMADA, Atsushi SASAKI, Noritaka KITAZAWA, Tadashi OKABE, Kazumasa NARA,
Shinichi HAMAOKA, Shin ARAKI, and Hiroaki HAGIWARA
Tsukuba Research Laboratories, Eisai Co., Ltd.; 5–1–3, Tokodai, Tsukuba, Ibaraki 300–2635, Japan.
Received September 5, 2003; accepted November 8, 2003
New types of selective estrogen receptor modulators (SERMs) were synthesized and evaluated for their
binding affinity and biological effect on reproductive cells. A proposed lead structure (B) was derivatized to pro-
vide compounds 30 and 44, which showed good estrogen-receptor binding affinity (Ki values: 6.3 and 10 nM, re-
spectively), as well as minimal impact on mammary and uterine carcinoma cells. Introduction of an alkyl group
in the core structure considerably enhanced receptor-binding affinity of the compounds tested. Synthesis and
structure–activity relationships of these compounds are described.
Key words estrogen; selective estrogen receptor modulator (SERM); receptor-binding affinity
The menopausal syndrome has a significant negative im- beneficial effects on osteoporosis and cardiovascular dis-
pact on the quality of life of many older women.1) A decline eases, and minimal effects on mammary and uterine tissues.
in estrogen production results in discomforts such as hot Following extensive investigation, novel types of non-
flushes, urogenital atrophy and mental disorders. Decreased steroidal, estrogen receptor partial agonists, termed as selec-
levels of circulating estrogen also elicit changes in the meta- tive estrogen receptor modulators (SERMs), have been iden-
bolic pathways of several organs. In the skeletal system, low tified.25—27) (Fig. 1)
levels of circulating estrogen result in the loss of essential
The common feature of SERM structures is the combina-
bone minerals, leading to osteoporosis and an increased inci- tion of a non-steroidal core and a basic side chain (Fig. 2,
dence of fracture.2) Estrogen also plays an important role in compound (A)). The core structure is comprised of an aro-
the maintenance of healthy blood-vessels and blood-lipid matic ring containing a phenolic-OH connected to another
profiles3,4); a reduction in estrogen levels has often been asso- aromatic ring by a spacer group. The basic side chain con-
ciated with an increased risk of cardiovascular disease. Addi- sists of an aryl moiety and an alkyl amine. On the basis of
tionally, a decline in mental function has been associated the structural features described above, development of
with a decrease in estrogen production.5—8)
SERMs as pharmaceutical products was initiated, and several
To alleviate these symptoms, estrogen has been adminis- compounds such as Raloxifene28,29) (on market, osteoporo-
tered to menopausal women to offset reduced estrogen pro- sis), Lasofoxifene30) (ph III, osteoporosis), Pipendoxifene31)
duction. Such hormone replacement therapy9—11) (HRT) has (ph III, osteoporosis) and Bazedoxifene31) (ph II, breast can-
been used for decades, and has relieved the suffering of mil- cer) have been identified as valuable tools or candidates for
lions of patients.12) However, the agonistic characteristics of treatment of menopausal symptoms.
HRT in mammary and uterine tissues have provoked con-
In our drug discovery program, we have pursued a novel
cerns relating to the increased risk of proliferation in estro- structural type of SERM compound. Subsequent to review of
gen-receptor-dependent breast and uterine cancers.13,14) the SAR of known SERMs, we deduced several types of sup-
Therefore, alternative methods of HRT have attracted the at- posed SERM compounds carrying novel combinations of
tention of the pharmaceutical industry.
core structures and side chains. Among them, we were inter-
In the 1960’s, Tamoxifen15,16) (Fig. 1) was discovered and ested in a compound (B) (Fig. 2). On comparison with exist-
subsequently marketed as an anti-breast cancer drug.17—19) ing SERMs, compound (B) contains the requisite topology
Interestingly, Tamoxifen exerts an antagonistic effect on for a SERM, but the side chain is attached in a different posi-
mammary tissues, but adversely acts as an agonist on other tion. It is well established that the topology of a side chain
tissues such as skeletal20,21) and cardiovascular systems.22—24) plays an important role in determination of the tissue-selec-
This unexpected discovery provided the impetus for pursuit tive agonist/antagonist profile in SERMs32,33); therefore we
of tissue-selective agonistic estrogen alternatives, which have reasoned that derivatization of compound (B) could result in
Fig. 1
To whom correspondence should be addressed. e-mail: a-kamada@hhc.eisai.co.jp
© 2004 Pharmaceutical Society of Japan

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Vol. 52, No. 1
the identification of a novel SERM with beneficial effects anhydride, and the produced triflate was subjected to
with respect to tissue selectivity. In order to evaluate the suit- Suzuki–Miyaura coupling followed by hydrogenolysis to pro-
ability of compound (B) as a new SERM candidate, synthesis vide 9. The attachment of an amine side chain and subse-
of the target compounds mentioned below was required to quent demethylation afforded 10.
permit biological testing. Here we report the synthesis and
the preliminary SAR results of our compounds.
Compound 16 was synthesized by the procedures given in
Chart 2. Aldehyde 12, derivatized from 11, was reacted with
Grignard reagent to give 13. Deoxygenation with silane fol-
lowed by hydrolytic deprotection afforded 14. Compound 14
Chemistry
The synthetic routes to compounds 6a, 6b and 10 are was subjected to copper-promoted arylation and subsequent
shown in Chart 1. debenzylation to produce 15, which was treated in a similar
Compound 1 was coupled with 3-methoxybenzoyl chlo- manner as described for 6a to afford 16.
ride via Fries rearrangement and subsequent selective
Chart 3 illustrates the synthetic routes to compounds 22,
monomethylation afforded 3a. According to a reported pro- 25a, 25b and 30. For the synthesis of 22, compound 18 was
cedure,34) 3a was treated with methyl chloroformate followed selectively demethylated using carbonyl-assisted demethyla-
by NaBH4 to provide the decarbonylated product 4a. Copper- tion promoted by CeCl3–NaI37) under mild conditions. De-
promoted phenolic-OH arylation35) of 4a with 4-tert-butyl- carbonylation of 19 using NaBH4–methyl chloroformate
dimethylsiloxyphenylboronic acid followed by desilylation gave complex mixtures. Alternatively, hydrogenolysis of 19
produced 5a. Introduction of an ethylpiperidine moiety on 5a was successfully performed to provide 20, which was con-
and subsequent demethylation furnished target compound verted to target compound 22 in a similar manner as de-
6a. Starting from 7,36) 6b was synthesized in a similar man- scribed for 6a. Syntheses of 25a and 25b were carried out
ner as described for 6a. For the synthesis of 10, the common following the procedures described for compound 10. For the
intermediate 4a was treated with trifluoromethanesulfonic synthesis of 30, introduction of a dimethyl moiety into 18
and subsequent application of the aforementioned procedures
were effectively conducted. In this case, NaBH4–methyl
chloroformate decarbonylation could be performed without
incurring significant problems.
Aniline type compounds 36a and 36c were synthesized as
shown in Chart 4. The Mizoroki–Heck reaction product 32
was hydrogenated to give aniline 33. Introduction of the side
chain moiety using amide formation followed by AlH3 reduc-
tion afforded 35. With or without the prior introduction of
methyl group on the aniline moiety of 35, deprotection was
effected utilizing EtSH–AlCl3 to provide target compounds
36a and 36c.
Fig. 2
Chart 1
Chart 2

January 2004
81
wald’s arylation39) permitted the smooth conversion of 38 to
39. CeCl3-promoted demethylation, NaBH4-reduction and
acid-promoted dehydration produced 41. Compound 41 was
Finally, compounds 44 and 51 were synthesized according
to Chart 5. Compound 3738) was subjected to intramolecular
Friedel–Crafts acylation to give 38. Application of Buch-
Chart 3
Chart 4
Chart 5

82
Vol. 52, No. 1
Antagonistic
Table 1. Binding Affinity and Effect on Reproductive Cells
hydrogenated to provide 42, which was converted to 44 in a
similar manner for 6a. For the synthesis of 51, compound
4540) was converted to 47, which was transformed to 51 in a
similar manner as described above.
ER binding MCF-7
Ki (nM) (%)
Ishikawa cell effect on MCF-7
(%)
(against 10 pM E2)
IC50 (nM)
Results and Discussion
Estradiol
Tamoxifen
Raloxifene
6a
6b
10
16
22
25a
25b
30
36a
36c
44
0.11 200 (1 nM)
300 (1 nM)
136 (100 nM)
102 (1 mM)
—
86.1
0.46
—
—
—
The synthesized compounds were tested for their ability to
140
130 (10 nM)
3
compete with H-estradiol for binding to the estrogen recep-
0.59 104 (1 mM)
tor.41) The compounds with a Ki value of double-figures or
lower were selected for further evaluation of their agonistic
activity on mammary and uterine tissues. Specifically, the se-
lected compounds were examined for their agonistic activity
in estrogen-receptor dependent proliferation of MCF-7 breast
adenocarcinoma cells.42) Estrogen-receptor dependent pro-
duction of alkali phosphatase (ALP) by Ishikawa cells43) was
used for measurement of the agonistic effect of the tested
compounds on uterine tissue. Additionally, the antagonistic
effect of tested compounds against estradiol (E2) was evalu-
ated. Tamoxifen and Raloxifene were used for comparative
purposes. Biological data is presented in Table 1. The princi-
pal aim of this study was to clarify whether compound (B)
was suitable for further derivatization studies directed toward
the identification of novel SERM compounds. Initially, opti-
mization of the spacer length was pursued.
190
—
—
—
—
—
—
—
—
Ͼ307
240
240
41
50
180
—
122 (100 nM) 122 (1 mM)
135 (100 nM) 128 (1 mM)
43.38
13.31
—
3.89
—
228.85
12.09
11.20
—
—
157 (100 nM)
—
6.3 111 (1 mM)
Ͼ235
—
31
10
50
143 (100 nM) 157 (1 mM)
125 (100 nM) 104 (1 mM)
104 (1 mM)
51
89 (1 mM)
Variation of the spacer length between A ring and B ring
influenced the binding affinity of the tested compounds to the
estrogen receptor. Compounds with a one-methylene spacer,
such as 6a and 6b, exhibited a significant decrease in binding
affinity. Carbon–oxygen exchange in the connecting section,
presented in compound 16, conferred no beneficial effect. In
contrast, introduction of a two-methylene carbon spacer im-
proved the receptor-binding affinity (RBA) of the compounds
tested. Thus, the RBA of compound 22 was over seven times
higher than that of compound 6b (Table 1, Fig. 3). A similar
relationship was noted between compound 10 and compound
25b; the former showed a decreased binding affinity when
Fig. 3
compared to the latter (Table 1). In addition, a predominance group, between A ring and B ring, for further optimization of
of two-carbon unit spacers is noticeable in known SERMs, so compound 22. Introduction of ring structures to drug-like
our synthetic effort was concentrated on production of com- substances has often had beneficial impact on their affinity to
pounds that contained two-methylene spacers between A ring biomolecules, so compounds 44 and 51 were synthesized
and B ring. Incidentally, compound 25a, which contained a with the aim of enhancing binding affinity to the estrogen re-
meta-alkylamine substituent, showed an improved binding ceptor (Fig. 3). As expected, compound 44 (obtained follow-
affinity over compound 25b, which contained a para-alkyl- ing introduction of the “ethyl” moiety) had an enhanced Ki
amine substituent. This phenomenon indicated that a subtle value of 10. However, compound 51 (obtained following in-
change in the topology of the side chain could significantly troduction of the “methyl” moiety) did not exhibit a distinct
affect the binding mode to the estrogen receptor.44) Accord- improvement in binding affinity. With this data, it was rea-
ingly, we set out to synthesize and evaluate derivatives con- soned that a certain level of “bulkiness” of an alkyl sub-
taining different side chain topologies.
stituent was required in the spacer group in order to enhance
The spacer between the core structure and the side chain receptor-binding affinity. This hypothesis led to the synthesis
(“Y” in Fig. 2) was another target of optimization. A of compound 30, which showed the strongest RBA among
biphenyl type (no spacer) compound 25b showed weak affin- our compounds (Fig. 3). To the best of our knowledge, there
ity to the estrogen receptor. Introduction of at least one atom is no explicit report, which states that the introduction of a
enhanced binding affinity to the estrogen receptor as shown dimethyl moiety to the spacer confers a considerable impact
in the case of compounds 22 and 36c. Interestingly, the pres- on the binding affinity of a SERM to the estrogen receptor.
ence of a hydrogen donor in the vicinity of the core structure At present, we have no rationale to explain this phenomenon,
lowered the RBA. As for Y, oxygen, nitrogen (aniline) and but we are sure that this discovery should facilitate the identi-
carbon could be candidates for further synthetic studies. In fication novel types of SERMs.
light of the RBA, there were no obvious differences between
them, so we selected an oxygen spacer for synthetic conve- Ishikawa cell assay indicated that the tested compounds ex-
nience. hibited weak agonistic activity. This fully exemplifies that
At this point, we turned our attention again to the spacer our synthetic compounds are true SERMs, which show mini-
The MCF-7 breast adenocarcinoma cell assay and

January 2004
83
successively 4-tert-butyldimethylsilyloxyphenylboronic acid (1.51 g,
4.0 mmol), copper acetate (727 mg, 4.0 mmol), powdered molecular shieves
4A (5 g) and triethylamine (2.02 g, 20.0 mmol). The resultant mixture was
stirred overnight at rt. The reaction mixture was filtered, and the filtrate was
absorbed on silica gel under reduced pressure. The gel was charged upon a
column of silica gel, and eluted with 10% AcOEt in hexane to give colorless
oil. The oily residue was dissolved in THF (10 ml), and to this was added 1 N
tetrabutylammonium fluoride in THF (4.0 ml, 4.0 mmol), and the resultant
mixture was stirred for 20 min at rt. The reaction mixture was evaporated
and partitioned between AcOEt and water. The organic layer was washed
with brine, dried over anhydrous magnesium sulfate and evaporated under
mal effect on mammary and uterine tissues. Antagonistic ef-
fect against estrogen-promoted cell growth was rather in ac-
cord with RBA of tested compounds.
Conclusion
In summary, new types of SERMs were identified, which
contain novel combinations of core structures and side
chains. The introduction of an alkyl moiety with “requisite
bulkiness” into the spacer between A ring and B ring consid-
erably enhances the affinity of those compounds to the estro- reduced pressure. The residue was chromatographed on silica gel (15—20%
1
AcOEt in hexane) to afford 5a (740 mg, 55%) as a colorless oil. H-NMR
(CDCl3): d (ppm) 3.68 (3H, s), 3.74 (3H, s), 3.92 (2H, s), 4.96 (1H, s), 6.33
(1H, d, Jϭ2.4 Hz), 6.55 (1H, dd, Jϭ2.4, 8.4 Hz), 6.69—6.83 (7H, m), 7.08
gen receptor. It was also evident that a hydrogen donor in the
spacer in the vicinity of the core structure confers a deleteri-
ous effect on the affinity to the estrogen receptor.
(1H, d, Jϭ8.4 Hz), 7.17 (1H, dd, Jϭ8.0, 8.0 Hz). HR-MS (ESI) m/z: Calcd
We are now conducting further research in this area, and
our results will be reported in future publications.
for C21H19O4: 335.1283 Found: 335.1319.
4-(3-Hydroxybenzyl)-3-[4-(2-piperidin-1-ylethoxy)phenoxy]phenol
(6a) To an ice-cooled, stirred solution of 5a (430 mg, 1.28 mmol) in N,N-
dimethylformamide (DMF) (4 ml) were added sodium hydride (118 mg,
Experimental
Melting points were measured using a Yanako melting-point apparatus 60% in mineral oil, 2.95 mmol), 1-(2-chloroethyl)piperidine hydrochloride
1
and are uncorrected. H-NMR spectra were recorded on a Varian Unity 400 (306 mg, 1.66 mmol). The resultant mixture was stirred overnight at rt. The
(400 MHz) spectrometer, and chemical shifts are expressed in ppm down- reaction mixture was quenched with water, partitioned between AcOEt and
field from tetramethylsilane (dϭ0) as an internal standard. The following water. The organic layer was washed with water and brine, dried over anhy-
abbreviations are used: sϭsinglet, dϭdoublet, tϭtriplet, qϭquartet, drous magnesium sulfate and evaporated under reduced pressure. The
mϭmultiplet, brϭbroad. 13C-NMR spectra were obtained on a Varian Unity residue was chromatographed on NH silica gel (5% AcOEt in hexane) to af-
400 (400 MHz) spectrometer. High-resolution mass spectra (HR-MS) were ford a pale yellow oil. To the residual oil was added pyridine hydrochloride
obtained on a Q-Tof Ultima global mass spectrometer. Elemental analysis (1.2 g), and the mixture was heated at 190 °C for 1 h 30 min. After cooling to
was performed with a Heraeus Elemental Analyzer CHN-O-RAPID. Materi- rt, the resultant solid was partitioned between THF and saturated sodium hy-
als were used as bought without any special purification. Silica gel (Kiesel- drogen carbonate solution, and the resultant mixture was extracted with
gel 60, Merck) was used for column chromatography, and silica gel (Kiesel- AcOEt. The organic layer was washed with brine, dried over anhydrous
gel 60 F254, layer thickness 0.25 mm, Merck) for analytical thin layer chro- magnesium sulfate and evaporated under reduced pressure. The residue was
matography (TLC).
chromatographed on NH silica gel (2% MeOH in AcOEt) to afford 6a
1
(2,4-Dihydroxyphenyl)-(3-methoxyphenyl)methanone (2) To an ice- (170 mg, 32%) as a colorless solid. mp 141—142 °C. H-NMR (CDCl3): d
cooled, stirred suspension of aluminum chloride (AlCl3) (14.0 g, 105 mmol) (ppm) 1.43 (2H, br s), 1.57—1.66 (4H, m), 2.52 (4H, br s), 2.71 (2H, t,
in dichloroethane (100 ml) were added 3-methoxybenzoyl chloride (18.8 g, Jϭ6.0 Hz), 3.85 (2H, s), 3.95 (2H, t, Jϭ6.0 Hz), 6.18 (1H, d, Jϭ2.8 Hz),
110 mol) and 2,4-dihydroxybenzene 1 (11.0 g, 100 mmol). The resultant 6.45 (1H, dd, Jϭ2.8, 8.4 Hz), 6.57—6.62 (4H, m), 6.67—6.71 (2H, m),
mixture was heated at 60 °C for 3 h, at 90 °C for 2 h 30 min, and at 70 °C
6.73—6.78 (1H, m), 7.00 (1H, d, Jϭ8.4 Hz), 7.06 (1H, dd, Jϭ7.6, 8.8 Hz).
overnight. After cooling, the reaction mixture was poured into ice-water, and 13C-NMR (DMSO-d6): d (ppm) 26.20, 35.21, 55.08, 58.61, 66.62, 105.06,
the resultant mixture was extracted with ethyl acetate (AcOEt)–tetrahydrofu- 105.13, 110.61, 110.70, 113.30, 113.38, 115.99, 116.08, 116.29, 119.84,
ran (THF) (2 : 1). The organic layer was dried over anhydrous magnesium 119.92, 120.81, 121.89, 129.76, 132.01, 143.39, 150.53, 155.27, 156.82,
sulfate and evaporated under reduced pressure. The residue was chro- 157.53, 157.91. Anal. Calcd for C26H29NO4: C, 74.44; H, 6.97; N, 3.34.
matographed on silica gel (30—70% AcOEt in hexane) to afford 2 (20.4 g, Found: C, 74.27; H, 7.06; N, 3.25. 6a was dissolved in THF and the solution
1
84%) as a pale yellow solid. mp 161—163 °C. H-NMR (CDCl3): d (ppm) was treated with 4 N solution of hydrogen chloride in AcOEt (4 N
3.79 (3H, s), 6.33—6.39 (2H, m), 7.10—7.18 (3H, m), 7.37 (1H, d,
Jϭ8.0 Hz), 7.43 (1H, dd, Jϭ8.0, 8.0 Hz), 10.76 (1H, br s), 12.19 (1H, s).
(2-Hydroxy-4-methoxyphenyl)-(3-methoxyphenyl)methanone (3a) To
a stirred solution of 2 (14.7 g, 60 mmol) in acetone (200 ml) were added
HCl–AcOEt) to give the corresponding hydrochloride (HCl) salt as an amor-
phous for biological evaluation.
(2-Hydroxy-4-methoxyphenyl)-(4-methoxyphenyl)methanone (3b) To
a stirred solution of 736) (9.68 g, 42.0 mmol) in acetone (200 ml) were added
potassium carbonate (12.4 g, 90 mmol) and iodomethane (9.37 g, 66 mmol). potassium carbonate (14.5 g, 105 mmol) and iodomethane (13.1 g,
The resultant mixture was stirred for 23 h at room temperature (rt), and then 92.4 mmol), and the resultant mixture was stirred for 12 h 30 min at rt. The
evaporated under reduced pressure. The residue was partitioned between reaction mixture was partitioned between AcOEt and water, and the organic
AcOEt and water, and the organic layer was washed with brine, dried over layer was washed with water, dried over anhydrous magnesium sulfate and
anhydrous magnesium sulfate and evaporated under reduced pressure. The evaporated under reduced pressure. The residue was chromatographed on
residue was chromatographed on silica gel (10% AcOEt in hexane) to afford silica gel (5—15% THF in hexane) to afford 3b (7.00 g, 64%) as a pale yel-
3a (10.3 g, 67%) as a pale yellow oil. 1H-NMR (CDCl3): d (ppm) 3.858 (3H, low solid. mp 111—113 °C. H-NMR (CDCl3): d (ppm) 3.87 (3H, s), 3.89
1
s), 3.864 (3H, s), 6.41 (1H, dd, Jϭ2.4, 8.8 Hz), 6.52 (1H, d, Jϭ2.4 Hz), 7.09 (3H, s), 6.42 (1H, dd, Jϭ2.4, 8.8 Hz), 6.52 (1H, d, Jϭ2.4 Hz), 6.96—7.01
(1H, dd, Jϭ2.4, 8.0 Hz), 7.15—7.22 (2H, m), 7.39 (1H, dd, Jϭ8.0, 8.0 Hz), (2H, m), 7.55 (1H, d, Jϭ8.8 Hz), 7.64—7.69 (2H, m), 12.68 (1H, s).
7.53 (1H, d, Jϭ8.8 Hz), 12.66 (1H, s).
5-Methoxy-2-(4-methoxybenzyl)phenol (4b) Starting from 3b (7.00 g,
5-Methoxy-2-(3-methoxybenzyl)phenol (4a) To an ice-cooled, stirred 27.1 mmol), 4b (5.02 g, 76%) was obtained as a colorless oil in a similar
1
solution of 3a (5.16 g, 20 mmol) in THF (100 ml) were added successively manner as described for 4a. H-NMR (CDCl3): d (ppm) 3.75 (3H, s), 3.77
triethylamine (3.04 g, 30 mmol) and methyl chloroformate (2.17 g, (3H, s), 3.86 (2H, s), 4.94 (1H, s), 6.38 (1H, d, Jϭ2.4 Hz), 6.45 (1H, dd,
23 mmol). The resultant mixture was stirred for 48 min at the same tempera- Jϭ2.4, 8.4 Hz), 6.80—6.86 (2H, m), 6.99 (1H, d, Jϭ8.4 Hz), 7.09—7.15
ture, and filtered. The filtrate was added to an ice-cooled, stirred solution of (2H, m).
sodium borohydride (3.03 g, 80 mmol) in water (50 ml), and the stirring was
4-[5-Methoxy-2-(4-methoxybenzyl)phenoxy]phenol (5b) Starting from
continued for 2 h 10 min. The reaction mixture was evaporated, partitioned 4b (977 mg, 4.0 mmol), 5b (544 mg, 40%) was obtained as a colorless oil in
between AcOEt and water, and the organic layer was washed with brine, a similar manner as described for 5a. 1H-NMR (CDCl3): d (ppm) 3.69 (3H,
dried over anhydrous magnesium sulfate and evaporated under reduced pres- s), 3.77 (3H, s), 3.89 (2H, s), 4.97 (1H, s), 6.33 (1H, d, Jϭ2.4 Hz), 6.55 (1H,
sure. The residue was chromatographed on silica gel (20% AcOEt in dd, Jϭ2.4, 8.4 Hz), 6.74—6.83 (6H, m), 7.06 (1H, d, Jϭ8.4 Hz), 7.10—7.15
1
hexane) to afford 4a (4.78 g, 98%) as a colorless oil. H-NMR (CDCl3): d (2H, m). HR-MS (ESI) m/z: Calcd for C21H19O4: 335.1283 Found: 335.1302.
(ppm) 3.75 (3H, s), 3.76 (3H, s), 3.90 (2H, s), 4.95 (1H, s), 6.38 (1H, d,
4-(4-Hydroxybenzyl)-3-[4-(2-piperidin-1-ylethoxy)phenoxy]phenol
Jϭ2.4 Hz), 6.45 (1H, dd, Jϭ2.4, 8.4 Hz), 6.72—6.82 (3H, m), 7.01 (1H, d, (6b) Starting from 5b (361 mg, 1.07 mmol), 6b (262 mg, 58%) was ob-
Jϭ8.4 Hz), 7.18—7.23 (1H, m).
tained as a colorless solid in a similar manner as described for 6a. mp 210—
4-[5-Methoxy-2-(3-methoxybenzyl)phenoxy]phenol (5a) To a stirred 212 °C. 1H-NMR (DMSO-d6): d (ppm) 1.30—1.39 (2H, m), 1.43—1.51
solution of 4a (977 mg, 4.0 mmol) in dichloromethane (50 ml) were added (4H, m), 2.40 (4H, br s), 2.61 (2H, t, Jϭ6.0 Hz), 3.69 (2H, s), 4.00 (2H, t,

84
Vol. 52, No. 1
Jϭ6.0 Hz), 6.09 (1H, d, Jϭ2.4 Hz), 6.40 (1H, dd, Jϭ2.4, 8.4 Hz), 6.59— (3H, s), 4.98—5.05 (4H, m), 6.00 (1H, s), 6.75 (1H, dd, Jϭ3.2, 8.8 Hz),
6.64 (m, 2H), 6.79—6.84 (m, 2H), 6.88—6.98 (5H, m). 13C-NMR (DMSO- 6.90—6.95 (3H, m), 7.00 (1H, d, Jϭ8.8 Hz), 7.27—7.44 (7H, m).
d6): d (ppm) 24.58, 26.21, 34.43, 55.08, 58.07, 66.63, 105.15, 105.23,
2-(4-Benzyloxybenzyl)-4-methoxyphenol (14) To an ice-cooled, stirred
110.62, 110.72, 115.63, 115.69, 116.29, 120.69, 122.75, 130.07, 131.87, solution of 13 (4.62 g, 12.1 mmol) in dichloromethane (50 ml) were added
132.05, 150.63, 155.21, 155.95, 156.63, 157.37. Anal. Calcd for C26H29NO4: successively trifluoroacetic acid (2.77 g, 24.3 mmol) and triethylsilane
C, 74.44; H, 6.97; N, 3.34. Found: C, 74.33; H, 7.25; N, 3.22. 6b was dis- (2.12 g, 18.2 mmol), and the resultant mixture was stirred for 54 min at the
solved in THF and the solution was treated with 4 N HCl–AcOEt to give the
corresponding HCl salt as an amorphous for biological evaluation.
Trifluoromethanesulfonic Acid 5-Methoxy-2-(3-methoxybenzyl)phenyl
same temperature. The reaction mixture was partitioned between AcOEt and
1 N sodium hydroxide solution, and the organic layer was washed with brine,
dried over anhydrous magnesium sulfate and evaporated under reduced pres-
Ester (8) To an ice-cooled, stirred solution of 4a (3.34 g, 13.7 mmol) and sure. The residue was dissolved in THF (40 ml), and to the solution were
pyridine (2.16 g, 27.3 mmol) in dichloromethane (50 ml) was added trifluo- added 4 N solution of HCl in 1,4-dioxane (10 ml) and five drops of water.
romethanesulfonic anhydride (4.63 g, 16.4 mmol) for 5 min, and the stirring After stirred overnight, the reaction mixture was evaporated under reduced
was continued for 20 min at the same temperature. The reaction mixture was pressure. The residue was chromatographed on silica gel (20—30% AcOEt
partitioned between dichloromethane and water, and the organic layer was in hexane) to afford 14 (2.70 g, 70%) as a pale yellow solid. mp 108—
dried over anhydrous magnesium sulfate and evaporated under reduced pres- 109 °C. 1H-NMR (CDCl3): d (ppm) 3.73 (3H, s), 3.89 (2H, s), 4.40 (1H, s),
sure. The residue was chromatographed on silica gel (5% AcOEt in hexane) 5.02 (2H, s), 6.63—6.73 (3H, m), 6.87—6.92 (2H, m), 7.10—7.15 (2H, m),
1
to afford 8 (3.90 g, 76%) as a yellow oil. H-NMR (CDCl3): d (ppm) 3.77 7.28—7.43 (5H, m).
(3H, s), 3.80 (3H, s), 3.97 (2H, s), 6.69—6.72 (1H, m), 6.74—6.79 (2H, m),
6.80—6.85 (2H, m), 7.09 (1H, d, Jϭ8.8 Hz), 7.21 (1H, dd, Jϭ8.0, 8.0 Hz).
4-[5-Methoxy-2-(4-methoxyphenoxy)benzyl]phenol (15) To a stirred
solution of 14 (481 mg, 1.50 mmol) in dichloromethane (30 ml) were added
5؅-Methoxy-2؅-(3-methoxybenzyl)biphenyl-4-ol (9) To a stirred solu- successively 4-methoxyphenylboronic acid (342 mg, 2.25 mmol), copper ac-
tion of 8 (376 mg, 1.00 mmol) in 1,4-dioxane (10 ml) under nitrogen atmo- etate (272 mg, 4.0 mmol), powdered molecular shieves 4A (3 g) and triethyl-
sphere were added 4-benzyloxyphenylboronic acid (251 mg, 1.10 mml), amine (759 mg, 7.50 mmol). The resultant mixture was stirred for 16 h at rt.
potassium carbonate (207 mg, 1.50 mmol) and tetrakis(triphenylphosphine)- The reaction mixture was filtered, and the filtrate was absorbed on silica gel
palladium (35 mg, 0.03 mmol), and the resultant mixture was heated at 80 °C under reduced pressure. The dried gel was charged upon a column of silica
for 4 h 40 min. The reaction mixture was cooled to rt and partitioned be- gel, and eluted with 5% AcOEt in hexane to give colorless oil. The oily
tween AcOEt and water. The organic layer was washed with brine, dried residue was dissolved in THF (15 ml), and the solution was charge with pal-
over anhydrous magnesium sulfate and evaporated under reduced pressure. ladium charcoal (Pd/C, 150 mg) and hydrogenolyzed under atmospheric
The residue was chromatographed on silica gel (5—6% AcOEt in hexane) to pressure for 20 h 30 min at rt. The reaction mixture was filtered, and the fil-
afford a colorless oil. The residual oil was dissolved in THF (10 ml) and the trate was evaporated under reduced pressure. The residue was chro-
solution was charged with palladium charcoal (Pd/C, 100 mg) and hy- matographed on silica gel (15—20% AcOEt in hexane) to afford 15
drogenolyzed under atmospheric pressure for 1 h at rt. The reaction mixture (398 mg, 79%) as colorless oil. 1H-NMR (CDCl3): d (ppm) 3.74 (3H, s),
was filtered, and the filtrate was evaporated under reduced pressure. The 3.78 (3H, s), 3.86 (2H, s), 4.91 (1H, s), 6.67—6.74 (4H, m), 6.77—6.84
residue was chromatographed on silica gel (30% AcOEt in hexane) to afford (5H, m), 7.03—7.08 (2H, m). HR-MS (ESI) m/z: Calcd for C21H19O4:
9 (167 mg, 52%) as a colorless oil. 1H-NMR (CDCl3): d (ppm) 3.72 (3H, s), 335.1283 Found: 335.1285.
3.80 (3H, s), 3.85 (2H, s), 5.16 (1H, s), 6.50—6.53 (m, 1H), 6.58 (1H, d,
Jϭ8.0 Hz), 6.68 (1H, dd, Jϭ2.4, 8.0 Hz), 6.77—6.85 (4H, m), 7.09—7.15
(4H, m). HR-MS (ESI) m/z: Calcd for C21H19O3: 319.1334 Found: 319.1360.
3-[4-(2-Cyclohexylethoxy)benzyl]-4-(4-hydroxyphenoxy)phenol (16)
Starting from 15 (290 mg, 0.86 mmol), 16 (235 mg, 65%) was obtained as a
pink solid in a similar manner as described for 6a. mp 204—212 °C (de-
6-(3-Hydroxybenzyl)-4؅-(2-piperidin-1-ylethoxy)biphenyl-3-ol (10) comp.) 1H-NMR (DMSO-d6): d (ppm) 1.30—1.38 (2H, m), 1.42—1.49 (4H,
Starting from 9 (154 mg, 0.48 mmol), 10 (70 mg, 44%) was obtained as a m), 2.39 (4H, br s), 2.60 (2H, t, Jϭ6.0 Hz), 3.70 (2H, s), 3.98 (2H, t,
1
colorless oil in a similar manner as described for 6a. H-NMR (CDCl3): d Jϭ6.0 Hz), 6.51—6.56 (2H, m), 6.61—6.70 (5H, m), 6.78—6.83 (2H, m),
(ppm) 1.45 (2H, br s), 1.58—1.68 (4H, m), 2.56 (4H, br s), 2.75 (2H, t, 7.02—7.07 (2H, m), 9.11 (2H, s). HR-MS (ESI) m/z: Calcd for C26H29N4O4:
Jϭ6.0 Hz), 3.75 (2H, s), 4.00 (2H, t, Jϭ6.0 Hz), 6.24 (1H, s), 6.51—6.61 420.2175 Found: 420.2179. 16 was dissolved in THF and the solution was
(4H, m), 6.62 (1H, d, Jϭ2.8 Hz), 6.76 (1H, dd, Jϭ2.8, 8.4 Hz), 6.85—6.90
treated with 4N HCl–AcOEt to give the corresponding HCl salt as an amor-
(2H, m), 7.03 (1H, dd, Jϭ8.0, 8.0 Hz), 7.08 (1H, d, Jϭ8.4 Hz). 13C-NMR phous for biological evaluation.
(CDCl3): d (ppm) 14.40, 21.29, 23.92, 25.00, 38.30, 54.94, 57.80, 60.75,
64.76, 113.32, 114.06, 115.04, 116.19, 117.41, 120.58, 129.54, 129.66,
1-(2,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)ethanone (18) To an
ice-cooled, stirred suspension of AlCl3 (7.58 g, 56.8 mmol) in dichloroethane
130.27, 132.01, 134.24, 143.08, 144.17, 154.77, 156.54, 157.45. HR-MS (100 ml) were added 4-methoxyphenylacetyl chloride (10.0 g, 54.2 mmol)
(ESI) m/z: Calcd for C26H30NO3: 404.2226 Found: 404.2189. 10 was dis- and 17 (8.23 g, 59.6 mmol). The resultant mixture was stirred at the same
solved in THF and the solution was treated with 4 N HCl–AcOEt to give the
corresponding HCl salt as an amorphous for biological evaluation.
temperature for 20 min, then at rt for 1 h. The reaction mixture was poured
into ice-water, and the resultant mixture was extracted with AcOEt. The or-
5-Methoxy-2-methoxymethoxybenzaldehyde (12) To a stirred solution ganic layer was washed with brine, dried over anhydrous magnesium sulfate
of 11 (25.2 g, 166 mmol) and N,N-diisopropylethylamine (42.8 g, 332 mmol) and evaporated under reduced pressure. The residue was chromatographed
in DMF (100 ml) and THF (100 ml) was added chloromethyl methyl ether
on silica gel (20—30% AcOEt in hexane) to afford 18 (8.02 g, 52%) as a
(20.0 g, 248 mmol), and the resultant mixture was stirred overnight at rt. The pale yellow solid. mp 76.5—78 °C. 1H-NMR (CDCl3): d (ppm) 3.78 (3H, s),
reaction mixture was partitioned between AcOEt and water, and the organic 3.84 (3H, s), 3.89 (3H, s), 4.21 (2H, s), 6.44 (1H, d, Jϭ2.4 Hz), 6.51 (1H,
layer was washed with water and brine, dried over anhydrous magnesium
dd, Jϭ2.4, 8.8 Hz), 6.81—6.86 (2H, m), 7.10—7.15 (2H, m), 7.78 (1H, d,
sulfate and evaporated under reduced pressure. The residue was chro- Jϭ8.8 Hz).
matographed on silica gel (10% AcOEt in hexane) to afford 12 (29.8 g, 92%)
1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (19)
1
as a pale yellow oil. H-NMR (CDCl3): d (ppm) 3.52 (3H, s), 3.81 (3H, s), To a stirred solution of 18 (2.76 g, 10.1 mmol) in acetonitrile (150 ml) were
5.24 (2H, s), 7.13—7.14 (1H, m), 7.16—7.20 (1H, m), 7.32—7.34 (1H, m),
10.47 (1H, s).
added sodium iodide (3.04 g, 20.2 mmol) and cerium chloride heptahydrate
(5.66 g, 15.2 mmol), and the resultant mixture was heated under reflux for
(4-Benzyloxyphenyl)-(5-methoxy-2-methoxymethoxyphenyl)methanol 20 h 30 min. An additional portion of sodium iodide (0.76 g, 5.1 mmol) and
(13) To stirred suspension of fine-ground magnesium (1.44 g, cerium chloride heptahydrate (1.89 g, 5.1 mmol) was added to the reaction
a
59.2 mmol) in THF (20 ml) was added dropwise a solution of 1-benzyloxy- mixture, and the reflux was continued for another 22 h 30 min. After cooling
4-bromobenzene (14.5 g, 55.1 mmol) in THF (80 ml) for 15 min. After the to rt, the reaction mixture was partitioned between AcOEt and water, and the
addition, the resultant mixture was heated at 60 °C for 1 h. After cooling in resultant mixture was filtered. The organic layer was washed with brine,
ice bath, a solution of 12 (9.81 g, 50.0 mol) in THF (50 ml) was added to the
dried over anhydrous magnesium sulfate and evaporated under reduced pres-
Grignard solution for 20 min and the resultant mixture was stirred for 50 min sure. The residue was chromatographed on silica gel (10—30% AcOEt in
at the same temperature. The reaction mixture was poured into saturated am- hexane) to afford 19 (2.46 g, 94%) as a colorless solid. mp 100—102 °C. 1H-
monium chloride solution-ice, and the resultant mixture was extracted with NMR (CDCl3): d (ppm) 3.79 (3H, s), 3.83 (s, 3H), 4.16 (s, 2H), 6.42 (1H, d,
AcOEt. The organic layer was washed with brine, dried over anhydrous Jϭ2.4 Hz), 6.44 (1H, dd, Jϭ2.4, 8.8 Hz), 6.85—6.90 (2H, m), 7.16—7.21
magnesium sulfate and evaporated under reduced pressure. The residue was (2H, m), 7.75 (1H, d, Jϭ8.8 Hz), 12.7 (1H, s).
chromatographed on silica gel (20—30% AcOEt in hexane) to afford 13
5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenol (20) A solution of 19
(10.2 g, 54%) as a yellow oil. 1H-NMR (CDCl3): d (ppm) 3.31 (3H, s), 3.75 (3.53 g, 13.0 mmol) in THF (40 ml) and acetic acid (10 ml) was hydro-

January 2004
85
genated over Pd/C (750 mg) under atmospheric pressure for 20 h at rt. An- C27H32NO3: 418.2382 Found: 418.2376.
other portion of Pd/C (500 mg) was added and the resultant mixture was hy-
1-(2,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)-2-methylpropan-1-one
drogenated under 4 atm for 8 h 30 min at rt. The reaction mixture was fil- (26) To a stirred solution of potassium tert-butoxide (7.97 g, 71.0 mmol)
tered and the filtrate was evaporated under reduced pressure. The residue and 18-crown-6 (626 mg, 2.37 mmol) in THF (200 ml) was added a solution
was chromatographed on silica gel (10—30% AcOEt in hexane) to afford 20
(2.74 g, 77%) as a colorless solid. mp 123—126 °C. 1H-NMR (CDCl3): d (50 ml), and the resultant mixture was stirred for 28 min at rt. The reaction
(ppm) 2.81 (4H, br s), 3.76 (3H, s), 3.79 (3H, s), 4.69 (1H, s), 6.35 (1H, d, mixture was filtered and the filtrate was absorbed on silica gel under reduced
Jϭ2.4 Hz), 6.43 (1H, dd, Jϭ2.4, 8.8 Hz), 6.80—6.85 (2H, m), 6.97 (1H, d, pressure. The gel was charged upon a column of silica gel, and eluted with
of 18 (6.78 g, 23.7 mmol) and iodomethane (13.4 g, 94.4 mmol) in THF
Jϭ8.8 Hz), 7.07—7.12 (2H, m).
4-{5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenoxy}phenol (21)
Starting from 20 (1.09 g, 4.2 mmol), 21 (644 mg, 44%) was obtained as a
15—25% AcOEt in hexane and the eluent was evaporated under reduced
pressure. To a stirred solution of the residue and iodomethane (8.40 g,
59.2 mmol) was added a solution of potassium tert-butoxide (6.64 g,
colorless solid in a similar manner as described for 5a. mp 78—80.5 °C. 1H- 59.2 mmol) in THF (60 ml) at once and the resultant mixture was stirred for
NMR (CDCl3): d (ppm) 2.79—2.89 (4H, m), 3.70 (3H, s), 3.78 (3H, s), 4.93 20 min at rt. The reaction mixture was filtered and the filtrate was absorbed
(1H, s), 6.34 (1H, d, Jϭ2.4 Hz), 6.55 (1H, dd, Jϭ2.4, 8.4 Hz), 6.76—6.86 on silica gel under reduced pressure. The gel was charged upon a column of
(6H, m), 7.03—7.08 (3H, m). HR-MS (ESI) m/z: Calcd for C22H21O4: silica gel, and eluted with 15—20% AcOEt in hexane and the eluent was
349.1440 Found: 349.1475.
evaporated under reduced pressure to afford 26 (6.22 g, 84%) as a colorless
4-[2-(4-Hydroxyphenyl)ethyl]-3-[4-(2-piperidin-1-ylethoxy)- solid. mp 85—88 °C. 1H-NMR (CDCl3): d (ppm) 1.52 (6H, s), 3.68 (3H, s),
phenoxy]phenol (22) Starting from 21 (519 mg, 1.48 mmol), 22 (111 mg, 3.74 (3H, s), 3.81 (3H, s), 6.19 (1H, dd, Jϭ2.0, 8.4 Hz), 6.35 (1H, d,
34%) was obtained as a colorless solid in a similar manner as described for Jϭ2.0 Hz), 6.42 (1H, d, Jϭ8.4 Hz), 6.82—6.90 (2H, m), 7.22—7.30 (2H,
6a. 22 was dissolved in THF and the solution was treated with 4 N
m).
1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)-2-methyl-
HCl–AcOEt to give the corresponding HCl salt as a colorless solid for ana-
lytical and biological evaluation. mp 169—170 °C. 1H-NMR (CD3OD): d propan-1-one (27) Starting from 26 (6.22 g, 19.8 mmol), 27 (4.86 g, 82%)
(ppm) 1.69 (2H, br s), 1.80—1.95 (4H, m), 2.65—2.77 (4H, m), 3.30 (4H,
br s), 3.53 (2H, t, Jϭ5.2 Hz), 4.33 (2H, t, Jϭ5.2 Hz), 6.20 (1H, d, Jϭ2.4 Hz), mp 86—89 °C. H-NMR (CDCl3): d (ppm) 1.60 (6H, s), 3.75 (3H, s), 3.78
6.46 (1H, dd, Jϭ2.4, 8.0 Hz), 6.58—6.64 (2H, m), 6.82—6.90 (4H, m), (3H, s), 6.06 (1H, dd, Jϭ2.4, 9.2 Hz), 6.36 (1H, d, Jϭ2.4 Hz), 6.83—6.88
6.94—7.02 (3H, m). 13C-NMR (CD3OD): d (ppm) 21.41, 22.89, 32.06, (2H, m), 7.08 (1H, d, Jϭ9.2 Hz), 7.13—7.18 (2H, m), 13.14 (1H, s).
36.06, 53.66, 56.04, 62.48, 105.77, 105.85, 110.28, 110.36, 114.72, 114.76, 5-Methoxy-2-[2-(4-methoxyphenyl)-2-methylpropyl]phenol (28) Start-
was obtained as a pale yellow solid in a similar manner as described for 19.
1
115.76, 119.12, 123.59, 129.22, 131.16, 133.10, 152.59, 153.55, 155.18, ing from 27 (2.94 g, 9.8 mmol), 28 (1.94 g, 69%) was obtained as a colorless
1
155.98, 156.67. HR-MS (ESI) m/z: Calcd for C27H32NO4: 434.2331 Found: solid in a similar manner as described for 4a. mp 101—102.5 °C. H-NMR
434.2325.
(CDCl3): d (ppm) 1.36 (6H, s), 2.76 (2H, s), 3.72 (3H, s), 3.80 (3H, s), 4.02
Trifluoromethanesulfonic Acid 5-Methoxy-2-[2-(4-methoxyphenyl)- (s, 1H), 6.25 (1H, d, Jϭ2.4 Hz), 6.36 (1H, dd, Jϭ2.4, 8.4 Hz), 6.74 (1H, d,
ethyl]phenyl Ester (23) Starting from 20 (2.67 g, 10.3 mmol), 23 (3.31 g, Jϭ8.4 Hz), 6.82—6.88 (2H, m), 7.20—7.26 (2H, m).
82%) was obtained as a yellow oil in a similar manner as described for 8.
1H-NMR (CDCl3): d (ppm) 2.79—2.92 (4H, m), 3.79 (3H, s), 3.80 (3H, s),
6.78—6.85 (4H, m), 7.06—7.15 (3H, m).
4-{5-Methoxy-2-[2-(4-methoxyphenyl)-2-methylpropyl]phenoxy}phe-
nol (29) Starting from 28 (1012 mg, 3.53 mmol), 29 (375 mg, 28%) was
obtained as a colorless oil in a similar manner as described for 5a. 1H-NMR
5؅-Methoxy-2؅-[2-(4-methoxyphenyl)ethyl]biphenyl-3-ol (24a) Start- (CDCl3): d (ppm) 1.33 (6H, s), 2.87 (2H, s), 3.66 (3H, s), 3.80 (3H, s), 4.80
ing from 23 (586 mg, 1.5 mmol), 24a (184 mg, 37%) was obtained as a col- (1H, s), 6.24 (1H, d, Jϭ2.4 Hz), 6.39 (1H, dd, Jϭ2.4, 8.4 Hz), 6.62 (1H, d,
orless oil in a similar manner as described for 9. 1H-NMR (CDCl3): d (ppm) Jϭ8.4 Hz), 6.76—6.84 (6H, m), 7.22—7.28 (2H, m). HR-MS (ESI) m/z:
2.61—2.67 (2H, m), 2.74—2.80 (2H, m), 3.76 (3H, s), 3.80 (3H, s), 5.09 Calcd for C24H25O4: 377.1753 Found: 377.1767.
(1H, br s), 6.59—6.62 (1H, m), 6.72—6.77 (3H, m), 6.80—6.88 (5H, m),
4-[2-(4-Hydroxyphenyl)-2-methylpropyl]-3-[4-(2-piperidin-1-
7.18 (1H, d, Jϭ8.4 Hz), 7.25 (1H, dd, Jϭ8.0, 8.0 Hz).
ylethoxy)phenoxy]phenol (30) Starting from 29 (201 mg, 0.53 mmol), 30
6-[2-(4-Hydroxyphenyl)ethyl]-3؅-(2-piperidin-1-yl-ethoxy)biphenyl-3- (119 mg, 49%) was obtained as a colorless oil in a similar manner as de-
ol (25a) Starting from 24a (184 mg, 0.55 mmol), 25a (122 mg, 53%) was scribed for 6a. 30 was dissolved in THF and the solution was treated with
obtained as a colorless oil in a similar manner as described for 6a. 25a was
dissolved in THF and the solution was treated with 4 N HCl–AcOEt to give
4 N HCl–AcOEt to give the corresponding HCl salt as a colorless solid for
analytical and biological evaluation. mp 213—214 °C. 1H-NMR (CD3OD): d
the corresponding HCl salt as a colorless solid for analytical and biological (ppm) 1.27 (6H, s), 1.56 (2H, br s), 1.89 (4H, br s), 2.77 (2H, s), 3.08 (2H,
1
evaluation. mp 213—215 °C. H-NMR (CD3OD): d (ppm) 1.54 (2H, br s), br s), 3.54 (2H, t, Jϭ4.8 Hz), 3.66 (2H, br s), 4.34 (2H, t, Jϭ4.8 Hz), 6.10
1.72—2.00 (4H, m), 2.49—2.56 (2H, m), 2.65—2.72 (2H, m), 3.05 (2H, (1H, d, Jϭ2.4 Hz), 6.29 (1H, dd, Jϭ2.4, 8.4 Hz), 6.54 (1H, d, Jϭ8.4 Hz),
br s), 3.52—3.64 (4H, m), 4.32—4.38 (2H, m), 6.54—6.60 (3H, m), 6.65—
6.62—6.69 (2H, m), 6.78—6.85 (2H, m), 6.95—7.01 (2H, m), 7.06—7.12
6.75 (3H, m), 6.86—6.91 (1H, m), 6.97—7.02 (1H, m), 7.09 (1H, d, (2H, m). 13C-NMR (CD3OD): d (ppm) 21.39, 22.86, 27.85, 38.49, 43.06,
Jϭ8.4 Hz), 7.35 (1H, dd, Jϭ8.0, 8.0 Hz). 13C-NMR (CD3OD): d (ppm) 53.65, 56.00, 62.46, 104.45, 104.53, 109.16, 109.25, 114.28, 115.73, 119.69,
21.35, 22.85, 34.82, 37.10, 53.68, 56.00, 61.92, 112.82, 114.37, 114.69,
120.51, 127.05, 132.92, 133.00, 140.52, 151.92, 153.65, 154.84, 156.57,
114.73, 115.47, 115.52, 116.15, 116.24, 122.73, 129.07, 129.18, 130.18, 156.93. Anal. Calcd for C29H36NO4Cl: C, 69.93; H, 7.29; N, 2.81. Found: C,
130.48, 132.97, 142.69, 144.04, 155.05. 155.14, 157.64. HR-MS (ESI) m/z: 69.65; H, 7.30; N, 2.74.
Calcd for C27H32NO3: 418.2382 Found: 418.2368.
4-Methoxy-1-[2-(4-methoxyphenyl)vinyl]-2-nitrobenzene (32) To a
5؅-Methoxy-2؅-[2-(4-methoxyphenyl)ethyl]biphenyl-4-ol (24b) Start- stirred solution of 31 (6.96 g, 30.0 mmol) in acetonitrile (60 ml) were added
ing from 23 (586 mg, 1.5 mmol), 24b (417 mg, 83%) was obtained as a col- 4-methoxystylene (4.23 g, 31.5 mmol), N,N-diisopropylethylamine (11.6 g,
orless solid in a similar manner as described for 9. mp 115—119 °C. 1H- 89.7 mmol), biphenyl-2-yl-di-tert-butylphosphane (537 mg, 1.80 mmol) and
NMR (CDCl3): d (ppm) 2.58—2.65 (2H, m), 2.74—2.79 (2H, m), 3.76 (3H, palladium acetate (404 mg, 1.80 mmol), and the resultant mixture was heated
s), 3.81 (3H, s), 5.11 (1H, s), 6.72—6.78 (2H, m), 6.81—6.90 (6H, m), at 80 °C for 11 h under nitrogen atmosphere. After cooling to rt, the reaction
7.13—7.20 (3H, m). HR-MS (ESI) m/z: Calcd for C22H21NO3: 333.1491 mixture was partitioned between AcOEt and water, and the organic layer
Found: 333.1528.
was washed with brine, dried over anhydrous magnesium sulfate and evapo-
6-[2-(4-Hydroxyphenyl)ethyl]-4؅-(2-piperidin-1-yl-ethoxy)biphenyl-3- rated under reduced pressure. The residue was chromatographed on silica
ol (25b) Starting from 24b (208 mg, 0.62 mmol), 25b (98 mg, 38%) was gel (10% AcOEt in hexane–10% AcOEt–10% THF in hexane) to afford 32
obtained as a colorless oil in a similar manner as described for 6a. 25b was (6.46 g, 76%) as a yellow solid. mp 108—110.5 °C. 1H-NMR (CDCl3): d
dissolved in THF and the solution was treated with 4 N HCl–AcOEt to give
(ppm) 3.84 (3H, s), 3.88 (3H, s), 6.88—6.93 (2H, m), 6.95 (1H, d,
the corresponding HCl salt as a colorless solid for analytical and biological Jϭ16.0 Hz), 7.14 (1H, dd, Jϭ2.8, 8.8 Hz), 7.41 (1H, d, Jϭ16.0 Hz), 7.42—
1
evaluation. mp 236—242 °C (decomp.). H-NMR (CD3OD): d (ppm) 1.70 7.48 (3H, m), 7.66 (1H, d, Jϭ8.8 Hz).
(2H, br s), 1.89 (4H, br s), 2.49—2.55 (2H, m), 2.65—2.71 (2H, m), 3.39
5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenylamine (33) A solu-
(4H, br s), 3.57 (2H, t, Jϭ4.8 Hz), 4.41 (2H, t, Jϭ4.8 Hz), 6.55—6.59 (3H, tion of 32 (1.40 g, 4.91 mmol) in THF (50 ml) was hydrogenated over Pd/C
m), 6.67—6.73 (3H, m), 7.02—7.08 (3H, m), 7.16—7.20 (2H, m). 13C- (400 mg) under atmospheric pressure for 20 h 50 min at rt. The reaction mix-
NMR (CD3OD): d (ppm) 21.46, 22.95, 34.91, 36.99, 53.74, 56.10, 62.04,
ture was filtered and the filtrate was evaporated under reduced pressure. The
110.40, 114.08, 114.69, 116.45, 116.52, 129.02, 130.29, 130.42, 132.98, residue was chromatographed on silica gel (10—20% AcOEt in hexane) to
136.03, 142.49, 155.01, 155.14, 156.93. HR-MS (ESI) m/z: Calcd for afford 33 (1.01 g, 80%) as a pink solid. mp 108—114 °C. 1H-NMR (DMSO-

86
Vol. 52, No. 1
d6): d (ppm) 2.54—2.60 (2H, m), 2.64—2.71 (2H, m), 3.61 (3H, s), 3.69
(3H, s), 4.84 (2H, s), 6.03 (1H, d, Jϭ2.4, 8.0 Hz), 6.19 (1H, d, Jϭ2.4 Hz),
6.75 (1H, d, Jϭ8.0 Hz), 6.78—6.86 (2H, m), 7.12—7.18 (2H, m).
(2H, s), 4.09 (2H, t, Jϭ6.0 Hz), 6.49 (1H, dd, Jϭ2.4, 8.4 Hz), 6.59 (1H, d,
Jϭ2.4 Hz), 6.61—6.66 (2H, m), 6.67—6.72 (2H, m), 6.84—6.89 (2H, m),
7.05 (1H, d, Jϭ8.4 Hz), 7.07—7.12 (3H, m). 13C-NMR (CDCl3): d (ppm)
24.06, 25.31, 32.53, 36.22, 42.14, 55.22, 57.92, 60.93, 65.47, 108.68,
110.89, 114.40, 115.54, 128.76, 129.52, 129.70, 130.62, 131.43, 134.09,
153.57, 154.41, 154.93, 157.59. HR-MS (ESI) m/z: Calcd for C29H37N2O3:
461.2804 Found: 461.2817. 36c was dissolved in THF and the solution was
treated with 4 N HCl–AcOEt to give the corresponding HCl salt as an amor-
phous for biological evaluation.
N-{5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenyl}-4-(2-piperidin-1-
ylethoxy)benzamide (34) To a stirred solution of 33 (643 mg, 2.50 mmol)
in 1,4-dioxane (20 ml) was added N,N-diisopropylethylamine (3 ml) and
4-(2-piperidin-1-ylethoxy)benzoyl chloride hydrochloride (989 mg, 3.25
mmol), and the resultant mixture was heated at 100 °C for 50 min. After
cooling to rt, the reaction mixture was evaporated, and the residue was parti-
tioned between AcOEt and saturated sodium hydrogen carbonate solution.
The organic layer was washed with brine, dried over anhydrous magnesium
sulfate and evaporated under reduced pressure. The residue was chro-
matographed on NH silica gel (50% AcOEt in hexane) to afford 34 (1.18 g,
97%) as a pale yellow solid. mp 103.5—104.5 °C. 1H-NMR (CDCl3): d
(ppm) 1.43—1.50 (2H, m), 1.58—1.67 (4H, m), 2.48—2.57 (4H, m), 2.78—
2.86 (6H, m), 3.76 (3H, s), 3.81 (3H, s), 4.17 (2H, t, Jϭ6.0 Hz), 6.74 (1H,
dd, Jϭ2.8, 8.4 Hz), 6.75—6.80 (2H, m), 6.90—6.98 (4H, m), 7.06 (1H, br s),
7.13 (1H, d, Jϭ 8.4 Hz), 7.47 (1H, d, Jϭ2.8 Hz), 7.53—7.58 (2H, m). HR-
MS (ESI) m/z: Calcd for C30H37N2O4: 489.2753 Found: 489.2788.
6,8-Dimethoxy-3,4-dihydro-2H-naphthalen-1-one (38) To
a stirred
suspension of 3738) (18.1 g, 80.7 mmol) in toluene (100 ml) was added oxalyl
chloride (15.4 g, 121 mmol), and the resultant mixture was stirred for 3 h
30 min at rt. The reaction mixture was evaporated under reduced pressure.
The residue was dissolved in dichloromethane (100 ml), and to this was
added a solution of stannic chloride in dichloromethane (1.0 M, 89.0 ml,
89.0 mmol) for 20 min with ice-cooling. After stirred for 2 h, the reaction
mixture was quenched with water and the resultant mixture was extracted
with dichloromethane. The organic layer was dried over anhydrous magne-
sium sulfate and evaporated under reduced pressure. The residue was chro-
matographed on silica gel (80% AcOEt in hexane–100% AcOEt) to afford
38 (16.0 g, 96%) as a pale yellow solid. mp 61—62 °C. 1H-NMR (CDCl3): d
(ppm) 1.98—2.06 (2H, m), 2.58 (2H, t, Jϭ6.0 Hz), 2.87 (2H, t, Jϭ6.0 Hz),
3.84 (3H, s), 3.88 (3H, s), 6.31—6.35 (2H, m).
{5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenyl}-[4-(2-piperidin-1-
ylethoxy)benzyl]amine (35) To an ice-cooled suspension of lithium alu-
minum hydride (607 mg, 16.0 mmol) and aluminum chloride (2.13 g,
16.0 mmol) in THF (100 ml) was added dropwise a solution of 34 (1.95 g,
4.00 mmol) in THF (20 ml), and the resultant mixture was stirred at the same
temperature for 3 h 7 min. The reaction mixture was diluted with THF and
quenched with 28% aqueous ammonia solution. The resultant suspension
was filtered off, and the filtrate was evaporated under reduced pressure. The
residue was chromatographed on NH silica gel (10—30% AcOEt in hexane)
6,8-Dimethoxy-2-(4-methoxyphenyl)-3,4-dihydro-2H-naphthalen-1-
one (39) To a stirred solution of 38 (11.9 g, 57.7 mmol) in THF (200 ml)
were added 4-bromoanisole (16.2 g, 86.6 mmol), sodium tert-butoxide
(11.1 g, 115 mmol), rac-BINAP (1.29 g, 2.07 mmol) and tris(dibenzylidene-
acetone)palladium (792 mg, 8.65 mmol), and the resultant mixture was
heated for 1 h 30 min at 75 °C under nitrogen atmosphere. After cooling to
rt, the reaction mixture was partitioned between AcOEt and water. The or-
ganic layer was washed with brine, dried over anhydrous magnesium sulfate
and evaporated under reduced pressure. The residue crystallized on stand-
ing, and the crude crystal was triturated in diethyl ether and filtered to give
39. The filtrate was evaporated under reduced pressure, and the residue was
chromatographed on silica gel (50—60% AcOEt in hexane) to give another
portion of 39. In total, 14.2 g (79%) of 39 were obtained as an off-white
solid. mp 115—117 °C. 1H-NMR (CDCl3): d (ppm) 2.22—2.36 (2H, m),
2.92—3.02 (2H, m), 3.69 (1H, dd, Jϭ5.6, 10.0 Hz), 3.78 (3H, s), 3.85 (6H,
s), 6.29—6.37 (2H, m), 6.80—6.86 (2H, m), 7.06—7.12 (2H, m).
1
to afford 35 (1.70 g, 90%) as a pale yellow oil. H-NMR (CDCl3): d (ppm)
1.40—1.48 (2H, m), 1.57—1.65 (4H, m), 2.47—2.55 (4H, m), 2.63—2.69
(2H, m), 2.77 (2H, t, Jϭ6.0 Hz), 2.81—2.87 (2H, m), 3.75 (3H, s), 3.78 (3H,
s), 4.10 (2H, t, Jϭ6.0 Hz), 4.18 (2H, s), 6.21—6.27 (2H, m), 6.77—6.82
(2H, m), 6.85—6.90 (2H, m), 6.95 (1H, d, Jϭ8.0 Hz), 7.02—7.08 (2H, m),
7.20—7.26 (2H, m). HR-MS (ESI) m/z: Calcd for C30H39N2O3: 475.2961
Found: 475.2954.
4-[2-(4-Hydroxyphenyl)ethyl]-3-[4-(2-piperidin-1-ylethoxy)benzyl-
amino]phenol (36a) To a stirred solution of 35 (474 mg, 1.00 mmol) in
dichloromethane (20 ml) was added AlCl3 (666 mg, 5.00 mmol) and
ethanethiol (0.37 ml, 5.00 mmol) and the resultant mixture was stirred for
27 min at rt. The reaction mixture was diluted with THF and quenched with
28% aqueous ammonia solution. The resultant suspension was filtered off,
and the filtrate was evaporated under reduced pressure. The residue was
chromatographed on NH silica gel (100% AcOEt–2% MeOH in AcOEt) to
8-Hydroxy-6-methoxy-2-(4-methoxyphenyl)-3,4-dihydro-2H-naph-
thalen-1-one (40) Starting from 39 (16.4 g, 52.5 mmol), 40 (8.68 g, 55%)
was obtained as a colorless solid in a similar manner as described for 19. mp
144.5—147 °C. 1H-NMR (CDCl3): d (ppm) 2.26—2.36 (2H, m), 2.90—2.98
(2H, m), 3.76 (1H, dd, Jϭ6.4, 9.2 Hz), 3.77 (3H, s), 3.83 (3H, s), 6.27—6.31
(2H, m), 6.86—6.91 (2H, m), 7.08—7.13 (2H, m), 12.88 (1H, s).
1
afford 36a (330 mg, 74%) as a pale yellow oil. H-NMR (CDCl3): d (ppm)
1.45 (2H, br s), 1.59—1.68 (4H, m), 2.60 (2H, br s), 2.64 (2H, t, Jϭ6.8 Hz),
2.73—2.81 (4H, m), 3.65 (1H, br s), 4.05—4.13 (2H, m), 6.09 (1H, d,
Jϭ2.4 Hz), 6.16 (1H, dd, Jϭ2.4, 8.0 Hz), 6.57—6.63 (2H, m), 6.74—6.79
(2H, m), 6.86—6.92 (3H, m), 7.06—7.10 (2H, m). 13C-NMR (CDCl3): d
(ppm) 24.06, 25.27, 32.96, 35.05, 47.88, 55.09, 57.86, 65.19, 99.06, 104.46,
114.81, 115.73, 118.12, 128.85, 129.64, 130.12, 131.59, 133.53, 147.06,
154.68, 155.86, 157.86. HR-MS (ESI) m/z: Calcd for C28H35N2O3: 447.2648
Found: 447.2638. 36a was dissolved in THF and the solution was treated
with 4 N HCl–AcOEt to give the corresponding HCl salt as an amorphous
for biological evaluation.
3-Methoxy-7-(4-methoxyphenyl)-5,6-dihydronaphthalen-1-ol (41) To
an ice-cooled, stirred suspension of lithium borohydride (1.10 g, 50.5 mmol)
in THF (60 ml) was added a solution of 40 (6.19 g, 20.7 mmol) in THF
(130 ml) for 10 min, and the resultant mixture was stirred for 20 min at the
same temperature. The reaction mixture was quenched with 2 N hydrochloric
acid solution and the resultant mixture was extracted with AcOEt. The or-
ganic layer was washed with brine, dried over anhydrous magnesium sulfate
and evaporated under reduced pressure. The residue was chromatographed
on silica gel (10—25% THF in hexane) to afford 41 (5.56 g, 95%) as a pur-
ple oil. 1H-NMR (CDCl3): d (ppm) 2.64—2.72 (2H, m), 2.84—2.92 (2H,
m), 3.78 (3H, s), 3.83 (3H, s), 4.97 (1H, br s), 6.23 (1H, d, Jϭ2.4 Hz), 6.37
(1H, d, Jϭ2.4 Hz), 6.88—6.94 (2H, m), 6.98 (1H, s), 7.46—7.52 (2H, m).
3-Methoxy-7-(4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-1-ol
(42) A solution of 41 (5.56 g, 19.7 mmol) in THF (50 ml) and MeOH
(50 ml) was hydrogenated over Pd/C (560 mg) under atmospheric pressure
for 12 h 40 min at rt. Another portion of Pd/C (280 mg) was added and the
resultant mixture was hydrogenated under atmospheric pressure for another
25 h 30 min at rt. The reaction mixture was filtered and the filtrate was evap-
orated under reduced pressure. The residue was chromatographed on silica
gel (10—20% THF in hexane) to afford 42 (4.67 g, 83%) as a colorless
{5-Methoxy-2-[2-(4-methoxyphenyl)ethyl]phenyl}methyl-[4-(2-
piperidin-1-ylethoxy)benzyl]amine (36b) To a stirred solution of 35
(712 mg, 1.50 mmol) in THF (30 ml) were added successively acetic acid
(675 mg, 11.25 mmol), 37% aqueous formaldehyde (0.27 ml, 3.38 mmol)
and sodium triacetoxyborohydride (699 mg, 3.30 mmol), and the resultant
mixture was stirred for 13 h 40 min at rt. The reaction mixture was parti-
tioned between AcOEt and 1 N sodium hydroxide solution, and the organic
layer was washed with brine, dried over anhydrous magnesium sulfate and
evaporated under reduced pressure. The residue was chromatographed on
NH silica gel (10—30% AcOEt in hexane) to afford 36b (733 mg, 73%) as a
pale yellow oil. 1H-NMR (CDCl3): d (ppm) 1.41—1.49 (2H, m), 1.57—1.65
(4H, m), 2.48—2.54 (4H, m), 2.53 (3H, s), 2.78 (2H, t, Jϭ6.0 Hz), 2.83—
2.90 (2H, m), 2.92—2.99 (2H, m), 3.79 (6H, s), 3.92 (2H, s), 4.10 (2H, t,
Jϭ6.0 Hz), 6.60 (1H, dd, Jϭ2.4, 8.8 Hz), 6.72 (1H, d, Jϭ2.4 Hz), 6.79—
6.87 (4H, m), 7.08—7.15 (3H, m), 7.23—7.28 (2H, m).
1
solid. mp 132—133.5 °C. H-NMR (CDCl3): d (ppm) 1.80—1.93 (1H, m),
2.02—2.11 (1H, m), 2.45—2.57 (1H, m), 2.80—2.98 (4H, m), 3.76 (3H, s),
3.80 (3H, s), 4.77 (1H, s), 6.25 (1H, d, Jϭ2.0 Hz), 6.29 (1H, d, Jϭ2.4 Hz),
6.83—6.89 (2H, m), 7.16—7.22 (2H, m).
4-[2-(4-Hydroxyphenyl)ethyl]-3-{methyl-[4-(2-piperidin-1-yl-
ethoxy)benzyl]amino}phenol (36c) Starting from 36b (471 mg, 0.97
mmol), 36c (410 mg, 92%) was obtained as a colorless oil in a similar man-
ner as described for 36a. H-NMR (CDCl3): d (ppm) 1.42—1.50 (2H, m),
1.62—1.70 (4H, m), 2.54 (3H, s), 2.61 (4H, br s), 2.76—2.89 (6H, m), 3.88
4-[3-Methoxy-7-(4-methoxyphenyl)-5,6,7,8-tetra-hydronaphthalen-1-
yloxy]phenol (43) Starting from 42 (569 mg, 2.0 mmol), 43 (362 mg,
48%) was obtained as a pale yellow oil in a similar manner as described for
5a. 1H-NMR (CDCl3): d (ppm) 1.82—1.95 (1H, m), 2.04—2.14 (1H, m),
2.58 (1H, dd, Jϭ11.2, 16.8 Hz), 2.84—3.01 (3H, m), 3.11 (1H, dd, Jϭ4.8,
1

January 2004
87
16.8 Hz), 3.71 (3H, s), 3.79 (3H, s), 6.20 (1H, d, Jϭ2.4 Hz), 6.43 (1H, d, Jϭ2.0 Hz), 6.56 (1H, d, Jϭ2.0 Hz), 6.75—6.80 (2H, m), 6.81—6.90 (4H,
Jϭ2.4 Hz), 6.74—6.80 (2H, m), 6.81—6.88 (4H, m), 7.17—7.22 (2H, m). m), 7.17—7.21 (2H, m). HR-MS (ESI) m/z: Calcd for C23H21O4: 361.1440
HR-MS (ESI) m/z: Calcd for C24H23O4: 375.1596 Found: 375.1615.
Found: 361.1469.
6-(4-Hydroxyphenyl)-4-[4-(2-piperidin-1-ylethoxy)phenoxy]-5,6,7,8-
2-(4-Hydroxyphenyl)-7-[4-(2-piperidin-1-ylethoxy)phenoxy]indan-5-ol
tetrahydronaphthalen-2-ol (44) Starting from 43 (189 mg, 0.50 mmol), (51) Starting from 50 (290 mg, 0.80 mmol), 51 (84 mg, 24%) was obtained
44 (104 mg, 45%) was obtained as a colorless solid in a similar manner as as a colorless solid in the same manner as described for 6a. 51 was dissolved
described for 6a. 44 was dissolved in THF and the solution was treated with
in THF and the solution was treated with 4 N HCl in AcOEt to give the cor-
4 N HCl–AcOEt to give the corresponding HCl salt as a colorless solid for responding HCl salt as a colorless solid for analytical and biological evalua-
1
analytical and biological evaluation. mp 197—198 °C. 1H-NMR (CD3OD): d tion. mp 203—205 °C. H-NMR (CD3OD): d (ppm) 1.68 (2H, br s), 1.82—
(ppm) 1.69 (2H, br s), 1.78—1.92 (5H, m), 1.96—2.05 (1H, m), 2.42 (1H, 1.95 (4H, m), 2.62 (1H, dd, Jϭ8.4, 15.6 Hz), 2.91 (1H, dd, Jϭ8.4, 15.6 Hz),
dd, Jϭ11.2, 16.4 Hz), 2.72—2.94 (4H, m), 3.20 (4H, br s), 3.44—3.50 (2H,
3.04 (1H, dd, Jϭ8.4, 15.6 Hz), 3.19 (1H, dd, Jϭ8.4, 15.6 Hz), 3.34 (4H,
m), 4.27—4.32 (2H, m), 6.07 (1H, d, Jϭ2.4 Hz), 6.36 (1H, d, Jϭ2.4 Hz), br s), 3.45—3.56 (3H, m), 4.32 (2H, t, Jϭ4.8 Hz), 6.13 (1H, d, Jϭ2.0 Hz),
6.67—6.72 (2H, m), 6.85—6.90 (2H, m), 6.94—6.99 (2H, m), 7.01—7.06
6.47 (1H, d, Jϭ2.0 Hz), 6.65—6.70 (2H, m), 6.88—6.93 (2H, m), 6.95—
(2H, m). 13C-NMR (DMSO-d6): d (ppm) 21.85, 23.01, 30.31, 30.44, 32.08, 7.00 (2H, m), 7.02—7.07 (2H, m). 13C-NMR (CD3OD): d (ppm) 21.38,
49.27, 53.30, 55.44, 63.35, 103.41, 103.47, 110.84, 110.93, 115.73, 116.60, 22.85, 37.37, 41.20, 44.94, 53.65, 56.01, 62.42, 103.62, 103.69, 106.45,
118.33, 120.01, 128.19, 137.52, 139.41, 151.49, 153.91, 156.02, 156.22, 106.54, 114.91, 114.88, 115.67, 119.22, 123.69, 127.60, 136.49, 146.87,
156.43. HR-MS (ESI) m/z: Calcd for C29H34NO4: 460.2488 Found: 152.15, 153.55, 153.68, 155.49, 157.73. Anal. Calcd for C28H32NO4Cl: C,
460.2489.
3-(3,5-Dimethoxyphenyl)-2-(4-methoxyphenyl)propionic acid (46) To
69.77; H, 6.69; N, 2.91. Found: C, 69.53; H, 6.76; N, 2.66.
Estrogen Receptor Binding Assay. Materials and Methods
an ice-cooled, stirred suspension of sodium hydride (2.40 g, 60% in mineral [2,4,6,7,16,17-3H] Estradiol, specific activity 143 Ci/mmol (TRK587) was
oil, 60.0 mmol) in DMF (40 ml) was added dropwise a solution of 4540) purchased from Amersham LIFE SCIENCE. Charcoal, dextran coated (C-
(7.47 g, 40.0 mmol) and methyl 4-methoxyphenylacetate (7.93 g, 44.0 mmol) 6197) and diethylstilbestrol (D-4628) were purchased from SIGMA. Bovine
in DMF (40 ml) for 6 min, and the resultant mixture was stirred for 30 min at uteri were obtained from a nearby slaughterhouse.
the same temperature. The reaction mixture was poured into 1 N hydrochlo-
Estrogen receptor binding assay was done according to the method of S.
ric acid solution-ice, and the resultant mixture was extracted with AcOEt. G. Korenman.41) Cytosol was obtained from a homogenate of bovine uterus
The organic layer was dried over anhydrous magnesium sulfate and evapo- by centrifugation at 105000ϫg for 30 min in 3 volumes of Buffer A (20 mM
rated under reduced pressure. The residue was chromatographed on silica
gel (10% AcOEt in hexane) to afford a pale yellow oil. To a stirred solution and 100 mM p-APMSF). 3H-Estradiol (10000 dpm) and competing com-
of the residual oil in THF (60 ml) and MeOH (40 ml) was added 5 N sodium pounds were mixed in a total volume of 80 ml Buffer A. Then 20 ml of uter-
hydroxide solution (15.0 ml, 60.0 mmol), and the resultant mixture was ine cytosol was added and incubated for 1 h at 23 °C. 100 ml of a charcoal,
stirred for 1 h at 60 °C. The reaction mixture was quenched with 5 N hy- dextran coated suspension (2% W/V) was added and mixed, incubated at
Tris/HCl, pH 7.5, containing 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol
drochloric acid solution (20.0 ml), and the resultant mixture was partitioned 23 °C for 10 min. The charcoal was then removed by centrifugation at
3
between AcOEt and water. The organic layer was dried over anhydrous mag- 2000ϫg for 10 min. The radioactivity of the bound H-Estradiol remaining
nesium sulfate and evaporated under reduced pressure to give a white solid. in 150 ml of the supernatant (total binding, dpm) was measured by liquid
The crude solid was triturated in tert-butyl methyl ether and filtered to afford
scintillation counter (LSC-6100, Aloka). Nonspecific binding (dpm) in the
supernatant was measured in the presence of 10 mM diethylstilbestrol. Spe-
46 (8.23 g, 65%) as a white solid. mp 119—120 °C. 1H-NMR (CDCl3): d
(ppm) 2.94 (1H, dd, Jϭ7.2, 14.0 Hz), 3.32 (1H, dd, Jϭ8.0, 14.0 Hz), 3.70 cific binding (dpm) was calculated by subtracting nonspecific binding (dpm)
(6H, s), 3.78 (3H, s), 3.75—3.84 (1H, m), 6.24—6.30 (3H, m), 6.82—6.87 from total binding (dpm). IC50 value (nM) was calculated as a concentration
3
(2H, m), 7.20—7.25 (2H, m).
of the compound, which shows 50% replacement of H-estradiol bound to
5,7-Dimethoxy-2-(4-methoxyphenyl)indan-1-one (47) To
a
stirred the estrogen receptors. Binding activity of the compound was shown as a Ki
suspension of 46 (7.68 g, 24.3 mmol) in toluene (150 ml) was added oxalyl value. The Ki value was obtained from the calculation; KiϭIC50/(1ϩ(C/Kd)).
chloride (4.62 g, 36.4 mmol), and the resultant mixture was stirred for 1 h C and Kd are concentration (nM) of 3H-Estradiol and dissociation constant
10 min at rt. The reaction mixture was evaporated under reduced pressure.
(nM) in the binding assay, respectively. The Kd value was calculated 0.3 nM
The residue was dissolved in dichloromethane (100 ml), and to this was by the Scatchard analysis (data not shown).
added a solution of stannic chloride in dichloromethane (1.0 M, 26.0 ml,
Effect on Reproductive Cells. Materials and Methods Cell Culture:
26.0 mmol) for 3 min with ice-cooling, and the stirring was continued for 1 h Response to estrogen was assessed by cell proliferation of MCF-7 cell, a
at the same temperature. The reaction mixture was quenched with water and human breast adenocarcinoma cell line, and expression of alkaline phos-
extracted with dichloromethane. The organic layer was dried over anhydrous phatese in Ishikawa cell, a human endometrial tumor cell line. MCF-7 cells
magnesium sulfate and filtered. The filtrate was absorbed on silica gel under and Ishikawa cells (subclone 3-H-12, No. 50, gifted by Dr. Nishida, Univer-
reduced pressure. The gel was charged upon a column of silica gel, and sity of Tsukuba, Japan) were maintained in minimum essential medium with
eluted with AcOEt–hexane (50—100%) and subsequently with THF to give Earles’s salts without phenol red (Invitrogen Corp., CA, U.S.A.) and 10%
47 (6.78 g, 94%) as a colorless solid. mp 121—123 °C. 1H-NMR (CDCl3): d fetal bovine serum, supplemented with 10% fetal bovine serum (FBS)
(ppm) 3.08 (1H, dd, Jϭ4.0, 17.2 Hz), 3.54 (1H, dd, Jϭ8.4, 17.2 Hz), 3.77
(3H, s), 3.79 (1H, dd, Jϭ4.0, 8.4 Hz), 3.90 (6H, s), 6.34 (1H, d, Jϭ1.2 Hz), vate (Invitrogen Corp., CA, U.S.A.), non-essential amino acids solution
6.52 (1H, d, Jϭ1.2 Hz), 6.80—6.86 (2H, m), 7.07—7.13 (2H, m). (1 : 100, Invitrogen Corp., CA, U.S.A.), and antibiotics–antimycotics
(V/V), 2 mM L-glutamine (Invitrogen Corp., CA, U.S.A.), 1 mM sodium pyru-
7-Hydroxy-5-methoxy-2-(4-methoxyphenyl)indan-1-one (48) Starting (1 : 100, penicillin G, streptomycin, and amphotericin B, Invitrogen Corp.,
from 47 (6.62 g, 22.1 mmol), 48 (2.81 g, 45%) was obtained as a brown solid CA, U.S.A.). The cells were maintained in 75-cm2 culture flasks at 37 °C in
in a similar manner as described for 19. mp 116—119 °C. 1H-NMR a 5% CO2 humidified incubator and were subcultured at a 1 : 20 to 40 ratio
(CDCl3): d (ppm) 3.12 (1H, dd, Jϭ4.0, 17.6 Hz), 3.58 (1H, dd, Jϭ8.0, once a week. The cells were used for assay before 100% confluence. The
17.6 Hz), 3.79 (s, 3H), 3.87 (s, 3H), 6.34 (1H, s), 6.51 (1H, s), 6.84—6.90 culture media were switched to assay media which was the same as mainte-
(2H, m), 7.08—7.13 (2H, m), 9.12 (1H, s).
6-Methoxy-2-(4-methoxyphenyl)indan-4-ol (49) Starting from 48 10% FBS, one day prior to plating. Stripped FBS was obtained by treating
(1.86 g, 6.5 mmol), 49 (1.04 g, 59%) was obtained as a colorless oil in a sim- the serum with dextran-coated charcoal to remove steroid hormones and was
nance medium except supplemented with 10% “stripped” FBS in place of
1
ilar manner as described for 20. H-NMR (CDCl3): d (ppm) 2.84 (1H, dd, used throughout all assays. The cells were removed from flasks using 0.05%
Jϭ8.0, 14.8 Hz), 3.00 (1H, dd, Jϭ8.4, 15.6 Hz), 3.23 (1H, dd, Jϭ8.0,
14.8 Hz), 3.27 (1H, dd, Jϭ8.4, 15.6 Hz), 3.60—3.72 (1H, m), 3.77 (3H, s),
Trypsin/0.53 mM EDTA (Invitrogen Corp., CA, U.S.A.). After neutralizing
of trypsin with assay media, the cells were pelleted and the trypsin/EDTA
3.80 (3H, s), 4.82 (1H, s), 6.26 (1H, d, Jϭ2.0 Hz), 6.43 (1H, d, Jϭ2.0 Hz), was removed by washing. The cells were re-suspended with assay medium.
6.82—6.88 (2H, m), 7.18—7.24 (2H, m). MCF-7 cells were plated on 96-well plate at 5000 cells/100 ml/well for cell
4-[6-Methoxy-2-(4-methoxyphenyl)indan-4-yloxy]phenol (50) Start- proliferation assay. Ishikawa cells were plated on 96-well plate at 20000
ing from 49 (780 mg, 2.89 mmol), 50 (379 mg, 38%) was obtained as a col- cells/100 ml/well for alkaline phosphatase assay. After 24-h incubation, the
orless solid in a similar manner as described for 5a. mp 113—113.5 °C. 1H- whole culture media were changed to 100 ml of fresh assay media. The test-
NMR (CDCl3): d (ppm) 2.81 (1H, dd, Jϭ8.4, 15.6 Hz), 3.02 (1H, dd, Jϭ8.4,
15.6 Hz), 3.21 (1H, dd, Jϭ8.4, 15.6 Hz), 3.33 (1H, dd, Jϭ8.4, 15.6 Hz),
3.58—3.68 (m, 1H), 3.73 (3H, s), 3.79 (3H, s), 4.71 (1H, br s), 6.26 (1H, d,
ing compounds were applied from 1 pM to 1 uM at final concentration. The
group of cells treated with 1 nM b-estradiol (Sigma, St. Louis, MO, U.S.A.)
was set up as positive control for each assay plate. For evaluation of antago-

88
Vol. 52, No. 1
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nistic effect against b-estradiol of testing compounds, 10 pM of b-estradiol
was applied at the same time.
Cell Proliferation Assay45) Cell proliferation of MCF-7 cells was as-
sessed by MTT reduction assay 3 d after compound application. Briefly, the
reaction was started by adding MTT (8 mg/ml, 10 ml, Sigma, St. Louis, MO,
U.S.A.) to each well and terminated by adding a solution containing 50% di-
methylformamide and 20% sodium dodecyl sulphate (pH 4.8). The amount
of formazan product was determined by measuring absorbance at 590 nm.
The MCF-7 proliferation score, which was calculated as ((sampleϪcon-
trol)/(1 nM b-estradiolϪcontrol)ϫ100ϩ100), was used as an index for com-
parison of potency of MCF-7 proliferation effect of compounds. IC50 value
(nM) was calculated as a concentration of the compound, which inhibits 50%
cell growth of 10 pM b-estradiol.
Alkaline Phosphatase Assay46) Alkaline phosphate activity in Ishikawa
cell was assessed by the method of Bessey–Lowry, using p-nitrophenylphos-
phate as substrate (Alkaline phospha B-test WAKO kit, Wako Pure Chemical
Industries, Ltd., Osaka). Briefly, The cultured cells were rinsed with Dulbec-
co’s phosphate-buffered saline and were incubated for 2 h in 37 °C CO2 incu-
bator with 100 ml of the 6.7 mM p-nitrophenylphosohate solution (6.7 mM).
The relative phosphatase activity was determined by measuring absorbance
at 405 nm. The relative alkaline phosphatase activity was expressed by using
“alkaline phosphatase (ALP) score” as index. ALP score was calculated as
((sampleϪcontrol)/(1 nM estradiolϪcontrol)ϫ200ϩ100).
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Acknowledgements We are especially grateful to Dr. Nishida (Univer-
sity of Tsukuba, Japan) for his generous gift of the Ishikawa cells. We are
grateful for the contributions of Physical Chemistry Department of Tsukuba
Research Laboratories for analytical support. We also acknowledge Dr. H.
Ogura and Dr. M. Nakagawa for helpful discussions.
31) Miller C. P., Collini M. D., Tran B. D., Harris H. A., Kharode Y. P.,
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