Practical synthesis and evaluation of the biological activities of 1α,25-dihydroxyvitamin D3 antagonists, 1α,25- dihydroxyvitamin D3-26,23-lactams. Designed on the basis of the helix 12-folding inhibition hypothesis

Article abstract of DOI:10.1021/jm050738x

A practical synthetic route to novel vitamin D antagonists of DLAM (1α,25-dihydroxyvitamin D₃-26,23-lactam) was developed from vitamin D₂ via the 1,3-dipolar cycloaddition reaction as a key step. Six DLAM derivatives (24 compounds) w

Full text of DOI:10.1021/jm050738x

2398
J. Med. Chem. 2006, 49, 2398-2406
Practical Synthesis and Evaluation of the Biological Activities of 1r,25-dihydroxyvitamin D3
Antagonists, 1r,25-dihydroxyvitamin D3-26,23-lactams. Designed on the Basis of the Helix
12-Folding Inhibition Hypothesis
Yusuke Nakano,† Yuko Kato,†,‡ Keisuke Imai,§ Eiji Ochiai,‡ Jun-ichi Namekawa,‡ Seiichi Ishizuka,‡ Kazuya Takenouchi,‡
Aya Tanatani,† Yuichi Hashimoto,† and Kazuo Nagasawa*,|
Institute of Molecular and Cellular Biosciences, UniVersity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, Teijin Institute for
Bio-medical Research, 4-3-2, Asahigaoka, Hino, Tokyo 191-8512, Japan, Drug DiscoVery Research, Astellas Pharma Inc., Miyukigaoka 21,
Tsukuba, Ibaraki 305-8585, Japan, and Department of Biotechnology and Life Science, Faculty of Technology, Tokyo UniVersity of Agriculture
and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan
ReceiVed July 29, 2005
A practical synthetic route to novel vitamin D antagonists of DLAM (1R,25-dihydroxyvitamin D3-26,23-
lactam) was developed from vitamin D2 via the 1,3-dipolar cycloaddition reaction as a key step. Six DLAM
derivatives (24 compounds) with a variety of nitrogen substituents and stereochemistries at C23 and C25
were synthesized. Among these new derivatives, (23S,25S)-DLAM isomers bound effectively to VDRs and
showed antagonistic activity in the HL-60 cell differentiation inhibition assay. The importance of the
substituent on the nitrogen of DLAMs for antagonistic activity was also suggested by computational docking
studies.
Introduction
1R,25-Dihydroxyvitamin D3 (1) (1,25-D3) (Figure 1), which
is a hormonally active form of vitamin D3, exhibits various
physiological actions, including the regulation of calcium
homeostasis, bone mineralization, proliferation and differentia-
tion of various types of cells, and immune modulation.1 Most
of these actions of 1,25-D3 are mediated by its specific vitamin
D receptor (VDR), which is a member of the nuclear receptor
(NR) superfamily.2 Like all NRs, the VDR has a DNA-binding
domain and a ligand-binding domain (LBD), which is formed
by 12 R-helices, containing an activation function 2 (AF-2)
domain.3 Among these helices, the carboxyl-terminal R-helix
(helix 12) plays an important role in the regulation of the
transcriptional activity of the receptor.4 The binding of the
ligands causes a conformational change within the LBD, that
is, the closure of helix 12 similar to that of a mouse trap.
Moreover, the LBD is involved in a variety of reversible
interactions with nuclear proteins, such as other NRs, coacti-
vators (CoAs), and corepressors (CoRs).5 The VDR favors
binding with a CoR in the absence of its ligands (apo form)
and acts as a transcriptional suppressor of the responsive genes.
In the apo form, helix 12 takes an open conformation. In
response to 1,25-D3, helix 12 is stabilized in the active closed
conformation (holo form), and CoA is recruited and binds to a
specific site located in helix 12, which results in the transcrip-
tional enhancement of the responsive genes.4b,6 These 1,25-D3-
modulated protein-protein interactions/dissociations are the
central molecular events of nuclear 1,25-D3 signaling. Therefore,
synthetic VDR ligands that inhibit helix 12 folding or the
optimal positioning of helix 12 in its agonistic conformation
should have the potential to act as antagonist.
So far, more than 3000 analogues of 1,25-D3 have been
synthesized as candidate ligands for VDRs, and most of them
Figure 1. Structures of vitamin D3 and its derivatives.
have been reported to show agonistic activity. However, the
1,25-D3 antagonists are expected to be effective for the treatment
of metabolic bone disease, represented by Paget’s disease,7 and/
or become tools for the elucidation of VDR function as well as
the mode of action of 1,25-D3. Only two types of ligands (except
our compounds, vide infra), the 25-carboxylic ester 2 (ZK168281)8
and 26,23-lactone 3 (TEI-9647)7g,9 have been reported as
antagonists so far (Figure 1). Compound 3 is an analogue of
* To whom correspondence should be addressed. Tel: +81-42-388-7295.
Fax: +81-42-388-7295. E-mail: knaga@cc.tuat.ac.jp.
† University of Tokyo.
‡ Teijin Institute for Bio-medical Research.
§ Astellas Pharma Inc..
| Tokyo University of Agriculture and Technology.
10.1021/jm050738x CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/30/2006

D3 Antagonists and D3-26,23-lactams
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2399
the natural metabolite compound 4, whereas compound 2 is a
derivative of the cyclopropyl-containing anti-psoriasis drug
calcipotriol (MC903).10 Recently, we have reported novel types
of vitamin D antagonist DLAMs 5 (DLAM-01) and 6 (DLAM-
1P),11 which were designed on the basis of the principle of
inhibition of folding of helix 12 in the NR.12 Among the new
derivatives, 6a ((23S,25S)-DLAM-1P) was found to competi-
tively bind to VDRs with 1,25-D3 and to inhibit the differentia-
tion of HL-60 cells induced by 1,25-D3. In this article, we
describe computer-assisted docking studies of 6a with VDRs
and report the results of the structure-activity relationship
studies of DLAM derivatives 7-11 and their novel and practical
synthetic method.
Figure 2. Docking structure of 6a with VDR-LBD in the presence
of helix-12.
Scheme 1. Synthesis of DLAM-01 (5) and 1P (6) by a
Convergent Route
Results and Discussion
Docking Studies of 6a with VDRs. We have recently
introduced a novel type of 1,25-D3 antagonists, DLAMs, which
have a lactam structure in the side chain.11 However, the
antagonistic activities of DLAMs is not potent, that is, even
the most active derivative among them, 6a ((23S,25S)-DLAM-
1P), possesses a 70-fold weaker antagonistic activity than that
of the known antagonist 3. As a precursor to further SAR studies
of DLAMs, we conducted a docking study of 6a with VDRs.
So far, six crystal structures of complexes consisting of
recombinant VDR-LBDs (∆165-215) and vitamin D3 deriva-
tives, that is, 1 (1,25-D3),13 20-epi-1,25-D3 (MC1288),14 26,-
27-dimethyl-24-homo-20-epi-22-oxa-1,25-D3 (KH1060),14 cal-
cipotriol,15 seocalcitol,15 and 19-nor-14-epi-23-yne-1,25-D3
(TX522),16 have been reported. They show practically the same
VDR-LBD and, therefore, validate the assumption that the new
DLAMs would also bind in the same way. We used the VDR-
LBD X-ray structure of the VDR-LBD/1 complex for the
docking studies with 6a.
complex, and the resulting substructure was used as a rigid
receptor model for docking with 6a. In this case, the strain
energy of 6a in the energy-minimized structure was decreased
to 10.0 kcal/mol, suggesting that there is indeed an unfavorable
interaction between 6a and helix 12. To examine this interaction,
helix 12 was brought back to the original position within VDR-
LBD (Figure 2), resulting in a conflict between the benzyl group
on the nitrogen atom of 6a and the Phe422 residue in helix 12.
This interaction could well be the cause of the antagonistic
activity of 6a, that is, the regulation/inhibition of the folding of
helix 12 and, therefore, the nature of the substituent on the
nitrogen was expected to affect the antagonistic activity of
DLAM. We therefore performed SAR studies focusing on the
substituents in the lactam moiety of DLAM.
First, computer-assisted reconstruction studies of the VDR-
LBD/1 complex were performed to ascertain the reliability of
the docking calculation. Namely, 1,25-D3 was deleted from the
reported X-ray structure of the VDR-LBD/1 complex (pdb
1DB1), and the remaining VDR-LBD structure was used as a
rigid receptor model. Energy minimization of the complex
structure with VDR-LBD and 1 was performed with the
CHARMm force field17 until a gradient convergence of less
than 0.01 kcal/mol Å was reached. The obtained docking
structure was consistent with the X-ray structure, supporting
the reliability of our calculation method. Compound 4 ((23S,-
26S)-1R,25-dihydroxyvitamin D3-26,23-lactone), which is struc-
turally related to 6a, was docked under the same conditions
and gave a docking structure similar to that of 1,25-D3 (1). Next,
we conducted docking studies of 6a with VDR-LBD on the
basis of the above results. The strain energy of 6a in the energy-
minimized structure of the complex with VDR-LBD was
calculated to be 29.4 kcal/mol, which is clearly higher than those
of 1,25-D3 (3.9 kcal/mol) and lactone 4 (11.6 kcal/mol). This
calculated value, which suggests the unfavorable/weak binding
of 6a to VDR compared to that of 1 and 4, is not consistent
with the moderate binding activity of 6a to VDR, that is, 6a
binds to VDR with an efficacy similar to that with 4.11 The
result suggests that the coformation of the VDR-LBD moiety
in the VDR-LBD/6a complex is different from that of the
VDR-LBD/1 and VDR-LBD/4 complexes. In fact, as men-
tioned above, compound 6a was initially designed as a ligand
that inhibits helix 12-folding.12 Hence, we next performed
docking to an artificial VDR-LBD template lacking the helix
12 moiety. The helix 12 moiety as well as the ligand molecule
was deleted from the X-ray structure of the VDR-LBD/1
Structure-Activity Relationship Studies on DLAM De-
rivatives. Synthesis of DLAM. We have previously reported
the synthesis of 5 (DLAM-01) and 6 (DLAM-1P) by means of
a convergent strategy as illustrated in Scheme 1.11 Though this
synthetic route was flexible enough to install various types of
A-ring moieties, the yields in the coupling reaction of the A-ring
and CD-ring synthons using the palladium catalyst18 and the
subsequent deprotection reaction were not consistent, and this
approach seemed unsuitable for preparing substantial amounts
of a range of DLAM derivatives for biological testing. Thus,
we decided to develop an alternative synthesis of DLAM for
the preparation of derivatives with various lactam substituents.
Compound 6 was synthesized directly from vitamin D2
(Scheme 2). Alcohol 12 was synthesized from vitamin D2 using
the Calverley method10 via the selective oxidation of the 1R
position19 and the oxidative degradation of the side chain. The
primary alcohol of 12 was reacted with p-toluenesulfonyl
chloride followed by sodium cyanide to give nitrile 14 in 95%
yield (2 steps). The nitrile group was reduced with DIBAL-H
to give aldehyde 15 in 96% yield. Reaction of aldehyde 15 with
N-benzylhydroxylamine gave nitrone 16, which was further
reacted with methyl methacrylate to give isoxazolidine 17 in
77% yield with four diastereomers at C23 and C25.20 The
reduction of the N-O bond of the isoxazolidine in the presence
21
of Mo(CO)6 gave lactam 18 in 55% yield. The TBS group

2400 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Scheme 2. Practical Synthesis of DLAM-1P (6) from VD2
Nakano et al.
a
a Reagents and Conditions: (a) TsCl, DMAP, CH2Cl2, 95%; (b) NaCN, DMSO 90 °C, 96%; (c) DIBAL-H, CH2Cl2, 0 °C, 96%; (d) BnNHOH-HCl,
Et3N, CH2Cl2; (e) methyl methacrylate, toluene, 90 °C, 77% (2 steps); (f) Mo(CO)6, NaBH4, CH3CN-H2O, 90 °C, 55%; (g) HF‚Py, THF, 0 °C; (h) HPLC
separation.
Table 1. VDR Binding Affinity and Antagonistic Activity of DLAM
Derivatives 6-11
VDR
antagonistic
activityb
(IC50, nM)
binding
DLAM
affinitya
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
2.74
0.25
0.18
0.25
8
0.51
0.34
0.19
0.68
0.19
0.09
0.03
5.24
0.3
700
NAc
NA
NA
207
NA
>2000
NA
2200
NA
NA
Figure 3. Structures of DLAMs 7-11. The suffixes a-d refer to the
stereoisomers at C23 and C25. a: (23S,25S), b: (23R,25R), c: (23S,-
25R), and d: (23R,25S).
NA
390
NA
9c
9d
0.09
0.1
NA
NA
was deprotected with HF-pyridine, and HPLC separation gave
(23S,25S)-6a, (23R,25R)-6b, (23S,25R)-6c, and (23R,25S)-6d
in 15, 15, 10, and 4% yields, respectively. The stereochemistries
of these compounds were determined by comparison with the
spectral data of authentic compounds prepared by the convergent
method.11,20 7 (DLAM-2P), 8 (DLAM-3P), 9 (DLAM-4P), 10
(DLAM-MPM), and 11 (DLAM-03I) were similarly synthesized
by changing the corresponding hydroxylamine (Figure 3).
Evaluation of the Biological Activities of DLAMs. The
relative binding affinity of DLAMs 6-11 to the VDR was
examined. The experiments were repeated at least three times.
The values differed from experiment to experiment, but the
results were basically reproducible (especially the order of
potency of the compounds). A competitive receptor binding
assay for the six types of compounds (24 compounds in total)
was performed using chick intestinal VDR,22 and the results
are summarized in Table 1. The DLAM derivatives with (23S,-
25S) stereochemistries showed higher VDR affinity than the
other corresponding diastereoisomers. Among them, 7a ((23S,-
25S)-DLAM-2P) exhibited the strongest binding affinity, which
was about 8% of that of 1 (for comparison, 3 and 4 show binding
affinities of about 12% and 8% of that of 1, respectively, under
the same conditions).23
10a
10b
10c
10d
11a
11b
11c
11d
2.2
0.3
660
NA
NA
NA
>1000
NA
0.09
0.1
1.52
0.14
0.14
0.11
NA
NA
a The potency of 1 is normalized to 100. b The antagonistic activity was
assessed in terms of IC50 for the differentiation of HL-60 cells induced by
10 nM of 1. c NA ) not an antagonist.
have aromatic groups on the lactam, scarcely induced HL-60
cell differentiation even at the high concentrations (>10-6 M)
(data not shown). However, 11a ((23S,25S)-DLAM-03I) induced
cell differentiation at a high concentration (>10-6 M) (data not
shown). Next, we investigated the antagonistic activity of
DLAMs, that is, the inhibitory activity of DLAMs on HL-60
cell differentiation induced by 10-8 M concentration of 1 was
tested using the NBT reducing-activity method (Table 1).
Among the six kinds of DLAM derivatives (24 compounds),
all of the isomers with (23S,25S) stereochemistries showed
antagonistic activity, and most of the other isomers did not.
Among the non-(23S,25S) stereoisomers, only 7c ((23S,25R)-
DLAM-2P) showed weak antagonistic activity. Compound 7a
((23S,25S)-DLAM-2P) showed the most potent antagonistic
activity, with an efficacy 3 times higher than that of 6a ((23S,-
25S)-DLAM-1P).26 The order of the antagonistic activity of
Next we examined the antagonistic activity of DLAMs 6-11
by the use of an HL-60 cell differentiation-inducing assay
system.24 First, the ability of DLAMs to induce HL-60 cell
differentiation, a typical agonistic activity of 1, was examined
by the NBT reducing-activity method.25 DLAMs 6-11, which

D3 Antagonists and D3-26,23-lactams
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2401
folding, which is a general feature of VDR activation; therefore,
these compounds can be expected to be species-nonspecific
VDR antagonists such as the ZK analogues (Figure 1, compound
2).
Conclusions
We have developed a practical synthetic route for DLAM
derivatives from vitamin D2 and used it to obtain new DLAM
derivatives with various substituents on the nitrogen atom of
the lactam moiety. Among these new derivatives, (23S,25S)-
DLAM isomers effectively bind to VDRs, and they also showed
antagonistic activity in the HL-60 cell differentiation inhibition
assay. The role of the substituent on the nitrogen of DLAMs
for antagonistic activity was investigated by computational
docking studies. Further SAR studies of DLAMs and investiga-
tions of the biological activities are in progress.
Experimental Section
Figure 4. Relationships of VDR binding affinity and antagonistic
activity of (23S,25S)-DLAM derivatives 6a-10a.
Synthesis of DLAMs. General Procedures. Flash chromatog-
raphy was performed on Silica gel 60 (spherical, particle size
0.040-0.100 mm; Kanto Kagaku). Optical rotations were measured
on a JASCO DIP polarimeter 370, using the sodium D line.
IR spectra were measured with a JASCO VALOR-III FT-IR
1
spectrophotometer. H and 13C NMR spectra were recorded on
JEOL JNM-ECP500 instrument. Mass spectra were recorded on
JEOL JMS-HX110 spectrometer with m-nitrobenzyl alcohol as the
matrix.
1R,3â-Bis-(tert-Butyldimethylsilyoxy)-9,10-secopregna-
5(Z),7(E),10(19)-triene-20(R)-methyl p-toluenesulfonate (13). To
a solution of alcohol 12 (575.8 mg, 1.00 mmol) in dichloromethane
(6 mL) was added DMAP (245 mg, 2.0 mmol) and p-toluenesulfo-
nyl chloride (248 mg, 1.3 mmol), and the mixture was stirred for
3 h at room temperature. To the reaction mixture was added H2O,
and the organic layer was extracted with dichloromethane. The
extracts were dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel chromatography (hexane/
ethyl acetate ) 10:1) to give 13 (697 mg, 0.96 mmol, 95%). [R]24
D
Figure 5. Transciptional assay of 6a with human and rat VDRs.
+44.06 (c 1.81, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.78
(d, J ) 8.3 Hz, 2H), 7.34 (d, J ) 8.3 Hz, 2H), 6.22 (d, J ) 11.0
Hz, 1H), 6.00 (d, J ) 11.2 Hz, 1H), 5.17 (d, J ) 1.5 Hz, 1H), 4.84
(d, J ) 2.2 Hz, 1H), 4.37 (dd, J ) 3.4, 6.3 Hz, 1H), 4.19 (m, 1H),
3.98 (dd, J ) 3.2, 9.3 Hz, 1H), 3.80 (dd, J ) 6.3, 9.3 Hz, 1H),
2.82 (d, J ) 12.4 Hz, 1H), 2.44 (m, 4H), 2.21 (dd, J ) 7.4, 13.1
Hz, 1H), 1.97-1.15 (m, 14H), 0.99 (d, J ) 6.6 Hz, 3H), 0.88
(s, 18H) 0.50 (s, 3H), 0.06 (m, 12H); 13C NMR (125 MHz, CDCl3)
δ 148.3, 144.5, 140.3, 135.3, 133.1, 129.7, 127.9, 123.0, 118.1,
111.2, 75.6, 72.0, 67.5, 55.9, 52.2, 46.0, 45.7, 40.2, 36.5, 28.7, 26.9,
25.8, 23.3, 22.1, 21.6, 17.0, 11.9, -4.7, -4.8, -5.1; m/z 729
(M + H+); HRMS: calcd for C41H69O5SSi2, 729.4404; found,
729.4384.
1R,3â-Bis-(tert-butyldimethylsilyoxy)-20(R)-cyanomethyl-9,10-
secopregna-5(Z),7(E),10(19)-triene (14). A mixture of 13 (618 mg,
0.85 mmol) and sodium cyanide (250 mg, 5.10 mmol) in DMSO
(10 mL) was stirred at 90 °C for 2 h. To the reaction mixture was
added H2O, and the organic layer was extracted with ethyl acetate.
The extracts were dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel chromatography
DLAMs was correlated with that of their binding affinity to
the VDR (Figure 4).
The effect of 6a on the transcriptional activation activity of
VDRs was examined using a luciferase reporter gene assay
system.9a The assay was conducted using COS-7 cells trans-
fected with human or rat VDR genes as well as 25(OH)-D3-
24-hydroxylase genes containing VDRE (vitamin D responsive
element) as a reporter gene.27 As shown in Figure 5, 6a ((23S,-
25S)-DLAM-1P) did not induce 25(OH)-D3-24-hydroxylase
gene expression in the human or rat VDR transfected system
even at a high concentration (>2 × 10-6 M), which is consistent
with the finding that 6a is ineffective as a VDR agonist. As
expected, 6a showed a dose-dependent inhibitory activity on
25(OH)-D3-24-hydroxylase gene expression induced by 10-8
M of 1 in both human and rat VDR transfected systems. These
results suggest that the (23S,25S)-DLAM derivatives inhibit the
activation of VDR at the transcriptional level irrespective of
the species difference of human and rat VDRs. It is noteworthy
that 3 (TEI-9647) shows agonistic activity toward rat VDRs at
the transcriptional level.28 These results suggest that the mo-
lecular mechanisms of VDR antagonistic activity elicited by
DLAMs and 3 are different. Compound 3 (TEI-9647) may elicit
its VDR antagonistic activity through alkylation of the cysteine
residue in the LBD of the human VDR.9f,28 The ineffectiveness
of 3 as an antagonist toward rat VDRs can be interpreted in
terms of the lack of corresponding cysteine residues in the LBDs
of rat VDRs. In contrast, DLAMs are expected to elicit their
VDR antagonistic activity through the inhibition of helix 12-
(hexane/ethyl acetate ) 50:1) to give 14 (496 mg, 0.85 mmol, 96%).
1
[R]24 +43.59 (c 3.37, CHCl3); H NMR (500 MHz, CDCl3) δ
D
6.23 (d, J ) 11.2 Hz, 1H), 6.02 (d, J ) 11.0 Hz, 1H), 5.18 (d, J )
1.5 Hz, 1H), 4.86 (d, J ) 2.2 Hz, 1H), 4.38 (dd, J ) 3.7, 6.6 Hz,
1H), 4.19 (m, 1H), 2.84 (d, J ) 12.0 Hz, 1H), 2.45 (d, J ) 10.0
Hz, 1H), 2.39-2.20 (m, 3H), 2.06-1.26 (m, 14H), 1.18 (d, J )
6.6 Hz, 3H), 0.88 (s, 18H) 0.56 (s, 3H), 0.07 (m, 12H); 13C NMR
(125 MHz, CDCl3) δ 148.3, 140.1, 135.5, 123.0, 119.0, 118.3,
111.2, 72.0, 67.5, 56.0, 55.1, 46.0, 45.7, 44.8, 40.2, 33.9, 29.7, 28.7,
27.5, 25.8, 24.8, 23.3, 22.0, 19.4, 12.0, -4.7, -4.8, -5.1; m/z 584
(M + H+); HRMS: calcd for C35H62NO2Si2, 584.4319; found,
584.4306.

2402 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Nakano et al.
1R,3â-Bis-(tert-butyldimethylsilyoxy)-20(R)-formylmethyl-
9,10-secopregna-5(Z),7(E),10(19)-triene (15). To a solution of 14
(563 mg, 0.96 mmol) in dichloromethane (10 mL) DIBAL-H (0.94
M in n-hexane, 1.23 mL, 1.16 mmol) was added at 0 °C and stirred
for 2 h. To the reaction mixture was added saturated aqueous NH4-
Cl (0.5 mL), and the resulting mixture was diluted with dichlo-
romethane. The mixture was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel
HF‚Py (0.9 mL) at 0 °C, and the mixture was stirred for 5 h. The
reaction mixture was diluted with ethyl acetate, and NaHCO3 (solid)
was added. The mixture was washed with H2O and brine, and the
organic layer was dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel chromatography
(chloroform/methanol ) 5:1) to give 6 as four diastereomer
mixtures. These mixtures were further purified by HPLC (PEGASIL
Silica 60-5 column, φ 20 × 250 mm, Senshu Pack, hexane/ethyl
acetate/IPA ) 51:45:4) to give 6a-d in 15% (2.3 mg, 4.3 µmol),
15% (2.3 mg, 4.3 µmol), 10% (1.5 mg, 2.8 µmol), and 4% (0.58
mg, 1.1 µmol, 3.8%) yields, respectively.
chromatography (hexane/ethyl acetate ) 100:1) to give 15 (540
1
mg, 0.92 mmol, 96%). [R]24 +19.66 (c 1.79, CHCl3); H NMR
D
(500 MHz, CDCl3) δ 9.75 (m, 1H), 6.24 (d, J ) 11.2 Hz, 1H),
6.03 (d, J ) 11.0 Hz, 1H), 5.19 (d, J ) 2.0 Hz, 1H), 4.87 (d, J )
2.4 Hz, 1H), 4.38 (dd, J ) 3.4, 6.3 Hz, 1H), 4.19 (m, 1H), 2.84 (d,
J ) 12.2 Hz, 1H), 2.49-2.45 (m, 2H), 2.25-1.27 (m, 16H), 1.03
Spectral Data for (23S, 25S)-DLAM-1P (6a). [R]24D +3.54 (c
0.20, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.33-7.22 (m, 5H),
6.37 (d, J ) 11.5 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.32 (s, 1H),
4.99 (s, 1H), 4.97 (d, J ) 11.1 Hz, 1H), 4.43 (m, 1H), 4.23 (m,
1H), 3.97 (d, J ) 12.0 Hz, 1H), 3.51 (m, 1H), 2.82 (d, J ) 12.4
Hz, 1H), 2.59 (d, J ) 10.3 Hz, 1H), 2.32 (dd, J ) 6.4, 13.7 Hz,
1H), 2.27 (dd, J ) 7.7, 13.7 Hz, 1H), 2.05-1.84 (m, 5 H), 1.66
(dd, J ) 5.1, 13.7 Hz, 1H), 1.68-1.17 (m, 12 H), 1.49 (s, 3H),
0.88 (m, 2H), 0.77 (d, J ) 6.0 Hz, 3H), 0.53 (s, 3H).
(d, J ) 6.6 Hz, 3H), 0.89 (s, 18H) 0.59 (s, 3H), 0.07 (m, 12H); 13
C
NMR (125 MHz, CDCl3) δ 203.3, 148.3, 140.4, 135.3, 123.0, 118.1,
111.2, 72.0, 67.5, 56.2, 50.8, 46.0, 45.8, 44.8, 40.4, 31.9, 28.8, 27.9,
25.8, 23.4, 22.1, 20.1, 12.0, -4.7, -4.8, -5.1; m/z 587 (M + H+);
HRMS: calcd for C35H63O3Si2, 587.4316; found, 587.4295.
1R,3â-Bis-(tert-butyldimethylsilyoxy)-20(R)-benzyliminomethyl-
9,10-secopregna-5(Z),7(E),10(19)-triene N-oxide (16). To a solu-
tion of aldehyde 15 (63 mg, 0.11 mmol) in dichloromethane (2
mL) was added N-benzylhydroxylamine hydrochloride (35 mg, 0.22
mmol) and triethylamine (60 µL, 0.43 mmol) at room temperature,
and the mixture was stirred for 2 h. The reaction mixture was cooled
to 0 °C and saturated aqueous NH4Cl was added. The organic layer
was extracted with dichloromethane, and the extracts were dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel chromatography (chloroform/methanol ) 20:
Spectral Data for (23R, 25R)-DLAM-1P (6b). [R]24 +13.73
D
1
(c 0.32, CHCl3); H NMR (500 MHz, CDCl3) δ 7.33-7.18 (m,
5H), 6.36 (d, J ) 11.1 Hz, 1H), 6.00 (d, J ) 11.1 Hz, 1H), 5.34 (s,
1H), 5.00 (s, 1H), 4.98 (d, J ) 15.0 Hz, 1H), 4.44 (m, 1H), 4.23
(m, 1H), 4.06 (d, J ) 15.4 Hz, 1H), 3.50 (m, 1H), 2.81 (d, J )
12.8 Hz, 1H), 2.60 (d, J ) 9.8 Hz, 1H), 2.48 (brs, 1H), 2.36 (dd,
J ) 7.7, 13.3 Hz, 1H), 2.31 (dd, J ) 6.4, 13.3 Hz, 1H), 2.04 (m,
1H), 1.92 (m, 3H), 1.77 (dd, J ) 5.1, 13.3 Hz, 1H), 1.66-1.20 (m,
11H), 1.50 (s, 3H), 1.03 (m, 2H), 0.88 (m, 1H), 0.86 (d, J ) 6.4
Hz, 3H), 0.48 (s, 3H).
1) to give 16 (78 mg, 0.11 mmol, 100%). [R]24 +39.84 (c 2.42,
D
1
Spectral Data for (23S, 25R)-DLAM-1P (6c). [R]24 +13.75
CHCl3); H NMR (500 MHz, CDCl3) δ 7.39-7.32 (m, 5H), 6.78
D
1
(brs, 1H), 6.22 (d, J ) 11.0 Hz, 1H), 6.00 (d, J ) 11.0 Hz, 1H),
5.17 (s, 1H), 4.94 (brs, 2H), 4.85 (m, 1H), 4.36 (m, 1H), 4.18 (m,
1H), 2.81 (d, J ) 11.2 Hz, 1H), 2.43 (d, J ) 12.8 Hz, 1H), 2.21
(m, 1H), 1.96-1.19 (m, 16H), 0.94-0.87 (m, 21H) 0.51 (s, 3H),
0.05 (s, 12H); 13C NMR (125 MHz, CDCl3) δ 148.1, 140.5, 135.1,
132.8, 129.2, 128.9, 123.0, 118.0, 111.2, 72.0, 69.1, 67.4, 56.5,
56.1, 46.0, 45.7, 44.7, 40.4, 34.3, 33.5, 28.7, 27.7, 25.8, 23.3, 22.0,
19.9, 11.9, -4.7, -4.9, -5.1; m/z 692 (M + H+); HRMS: calcd
for C42H70NO3Si2, 692.4894; found, 692.4920.
(c 0.155, CHCl3); H NMR (500 MHz, CDCl3) δ 7.34-7.18 (m,
5H), 6.36 (d, J ) 11.1 Hz, 1H), 6.00 (d, J ) 11.5 Hz, 1H), 5.32 (s,
1H), 5.00 (d, J ) 14.5 Hz, 1H), 4.99 (s, 1H), 4.43 (m, 1H), 4.23
(m, 1H), 4.06 (d, J ) 15.2 Hz, 1H), 3.27 (m, 1H), 2.82 (d, J )
12.4 Hz, 1H), 2.76 (brs, 1H), 2.60 (d, J ) 13.5 Hz, 1H), 2.33 (m,
1H), 2.21 (dd, J ) 6.4, 12.8 Hz, 1H), 2.19-1.95 (m, 5H), 1.73
(dd, J ) 8.1, 12.8 Hz, 1H), 1.68-1.20 (m, 12H), 1.35 (s, 3H),
0.88 (m, 4H), 0.76 (d, J ) 6.4 Hz, 3H), 0.51 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 176.8, 147.6, 142.6, 136.1, 133.2, 128.7,
127.9, 127.7, 124.8, 117.3, 111.9, 73.9, 70.8, 66.9, 56.9, 56.3, 51.3,
45.9, 45.2, 44.1, 42.8, 40.6, 40.5, 39.7, 32.8, 29.0, 27.9, 24.7, 23.5,
22.2, 18.6, 12.0; m/z 556 (M+Na+); HRMS: calcd for C34H47-
NO4Na, 556.3403; found, 556.3366.
1R,3â-Bis-(tert-butyldimethylsilyoxy)-20(R)-(2-benzyl-5-meth-
oxycarbonyl-5-methyl-isoxazolidine-3-yl)methyl-9,10-secopregna-
5(Z),7(E),10(19)-triene (17). A mixture of nitrone 16 (78 mg, 0.11
mmol) and methyl methacrylate (60 µL, 0.56 mmol) in toluene (4
mL) was heated at 90 °C for 2 h. The reaction mixture was
concentrated in vacuo. The residue was purified by silica gel
chromatography (hexane/ethyl acetate ) 10:1) to give 17 as four
Spectral Data for (23R, 25S)-DLAM-1P (6d). [R]24 -11.38
D
1
(c 0.058, CHCl3); H NMR (500 MHz, CDCl3) δ 7.33-7.14 (m,
5H), 6.36 (d, J ) 11.1 Hz, 1H), 6.00 (d, J ) 11.5 Hz, 1H), 5.33 (s,
1H), 5.01 (d, J ) 15.0 Hz, 1H), 4.99 (s, 1H), 4.43 (m, 1H), 4.23
(m, 1H), 4.06 (d, J ) 15.2 Hz, 1H), 3.28 (m, 1H), 2.81 (d, J )
12.8 Hz, 1H), 2.68 (brs, 1H), 2.60 (d, J ) 16.7 Hz, 1H), 2.32 (m,
1H), 2.27 (dd, J ) 6.4, 12.8 Hz, 1H), 2.17-1.92 (m, 5H), 1.87
(dd, J ) 8.1, 12.8 Hz, 1H), 1.75-1.22 (m, 11H), 1.39 (s, 3H),
1.06 (m, 2H), 0.87 (d, J ) 6.4 Hz, 3H), 0.48 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 177.0, 147.6, 142.6, 136.1, 133.1, 128.7,
127.7, 127.6, 124.9, 117.2, 111.8, 74.0, 70.8, 66.9, 57.3, 56.1, 52.9,
45.9, 45.2, 44.5, 42.9, 42.2, 40.5, 40.3, 34.5, 29.0, 27.7, 24.7, 23.4,
22.2, 20.2, 11.9; m/z 556 (M+Na+); HRMS: calcd for C34H47-
NO4Na, 556.3403; found, 556.3433.
1
diastereomer mixtures (68 mg, 0.086 mmol, 77%). H NMR (500
MHz, CDCl3) δ 7.40-7.31 (m), 6.23 (d, J ) 10.0 Hz), 6.01 (d, J
) 10.8 Hz), 5.18 (m), 4.86 (m), 4.38 (m), 4.19 (m), 4.13-3.88
(m), 3.76 (s), 3.13-1.26 (m), 0.89 (s), 0.69 (d, J ) 6.1 Hz), 0.54-
0.50 (m), 0.06 (s); m/z 793 (M + H+); HRMS: calcd for C47H78-
NO5Si2, 792.5419; found, 792.5408.
1R,3â-Bis-(tert-butyldimethylsilyoxy)-20(R)-(1-benzyl-3-hy-
droxy-3-methyl-pyrrolidin-2-one-5-yl)methyl-9,10-secopregna-
5(Z),7(E),10(19)-triene (18). To a solution of isoxsazolidine 17
(41 mg, 0.052 mmol) in CH3CN-H2O (7:1, 4 mL) was added Mo-
(CO)6 (74 mg, 0.28 mmol) and NaBH4 (1 mg, 0.026 mmol), and
the resulting mixture was stirred at 90 °C for 4 h. The reaction
mixture was filtered through a pad of Celite, and the filtrates were
concentrated in vacuo. The residue was purified by silica gel
chromatography (ethyl acetate) to give 18 as four diastereomer
mixtures (22 mg, 0.028 mmol, 55%). 1H NMR (500 MHz, CDCl3)
δ 7.38-7.20 (m), 6.23-6.21 (m), 6.02-5.99(m), 5.18 (s), 4.86 (m),
4.37 (m), 4.19 (m), 4.09 (d, J ) 15.0 Hz), 3.98 (d, J ) 15.0 Hz),
3.52 (m), 3.30 (m), 2.81 (m), 2.43 (m), 2.27-1.25 (m), 1.10-0.87
(m) 0.54-0.46 (m), 0.10-0.06 (m); m/z 763 (M + H+); HRMS:
calcd for C46H76O4Si2, 762.5313; found, 762.5348.
20(R)-(1-Phenylethyl-3-hydroxy-3-methyl-pyrrolidin-2-one-
5-yl)methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-di-
ol (7). DLAM-2P (7) was obtained from the corresponding nitrone,
which was prepared using aldehyde 15 and 2-phenylethylhydroxy-
lamine (19), as four diastereomer mixtures. These mixtures were
purified by HPLC (PEGASIL Silica 60-5 column, φ 20 × 250
mm, Senshu Pack, hexane/ethyl acetate/IPA ) 14:81:5) to give
7a-d in 26, 28, 8, and 11% yields, respectively.
Spectral Data for (23S, 25S)-DLAM-2P (7a). [R]24 +11.78
D
1
(c 0.52, CHCl3); H NMR (500 MHz, CDCl3) δ 7.31-7.21 (m,
20(R)-(1-Benzyl-3-hydroxy-3-methyl-pyrrolidin-2-one-5-yl)-
methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-diol (6).
To a solution of 18 (22 mg, 0.029 mmol) in THF (2 mL) was added
5H), 6.37 (d, J ) 11.1 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.32 (s,
1H), 4.99 (s, 1H), 4.43 (dd, J ) 4.3, 7.7 Hz, 1H), 4.23 (m, 1H),
3.79 (m, 1H), 3.47 (m, 1H), 3.19 (m, 1H), 2.90 (m, 1H), 2.85-

D3 Antagonists and D3-26,23-lactams
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2403
2.77 (m, 2H), 2.60 (d, J ) 9.8 Hz, 1H), 2.32 (dd, J ) 6.8, 13.3 Hz,
1H), 2.23 (dd, J ) 7.3, 13.3 Hz, 1H), 2.01-1.21 (m, 20H), 1.41
(s, 3H), 0.87 (d, J ) 5.6 Hz, 3H), 0.55 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 176.0, 147.7, 142.5, 138.8, 133.2, 128.8, 128.6,
126.6, 124.8, 117.3, 111.7, 74.0, 70.7, 66.9, 56.9, 56.3, 52.7, 45.9,
45.2, 42.9, 42.2, 40.5, 39.8, 39.5, 33.7, 33.1, 29.0, 28.0, 25.9, 23.5,
22.2, 18.6, 12.0; m/z 548 (M + H+); HRMS: calcd for C35H50-
NO4, 548.3740; found, 548.3758.
(m, 3H), 6.36 (d, J ) 11.1 Hz, 1H), 6.02 (d, J ) 11.5 Hz, 1H),
5.35 (s, 1H), 5.00 (s, 1H), 4.44 (m, 1H), 4.24 (m, 1H), 3.61 (m,
2H), 3.00 (m, 1H), 2.83 (m, 1H), 2.61 (m, 3H), 2.37 (dd, J ) 7.7,
13.3 Hz, 1H), 2.32 (dd, J ) 6.4, 13.3 Hz, 1H), 2.04-1.25 (m, 21H),
1.71 (dd, J ) 5.1, 13.3 Hz, 1H), 1.43 (s, 3H), 0.97 (d, J ) 6.8 Hz,
3H), 0.53 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.3, 147.6,
142.5, 141.2, 133.3, 128.4, 128.3, 126.0, 124.8, 117.3, 111.7, 74.1,
70.7, 66.9, 57.3, 56.2, 53.8, 45.9, 45.2, 42.8, 41.9, 41.0, 40.6, 40.4,
35.1, 33.1, 28.7, 25.8, 23.5, 22.3, 20.3, 12.0; m/z 562 (M + H+);
HRMS: calcd for C36H52NO4, 562.3896; found, 562.3926.
Spectral Data for (23R, 25R)-DLAM-2P (7b). [R]24 +32.12
D
1
(c 0.57, CHCl3); H NMR (500 MHz, CDCl3) δ 7.31-7.20 (m,
5H), 6.37 (d, J ) 11.1 Hz, 1H), 6.02 (d, J ) 11.1 Hz, 1H), 5.34 (s,
1H), 5.00 (s, 1H), 4.44 (dd, J ) 4.3, 7.7 Hz, 1H), 4.24 (m, 1H),
3.83 (m, 1H), 3.48 (m, 1H), 3.18 (m, 1H), 2.91 (m, 1H), 2.85-
2.76 (m, 2H), 2.60 (dd, J ) 3.0, 13.3 Hz, 1H), 2.53 (brs, 1H), 2.32
(dd, J ) 7.7, 13.3 Hz, 1H), 2.02-1.20 (m, 19H), 1.69 (dd, J )
5.1, 13.3 Hz, 1H), 1.49 (s, 3H), 0.96 (d, J ) 6.8 Hz, 3H), 0.56 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 176.0, 147.7, 142.5, 138.7,
133.3, 128.8, 128.6, 126.6, 124.8, 117.3, 111.8, 73.9, 70.8, 66.9,
57.4, 56.2, 53.9, 46.0, 45.2, 42.9, 42.8, 41.8, 41.1, 40.4, 35.1, 33.6,
29.0, 27.9, 25.9, 23.5, 22.3, 20.3, 12.0; m/z 548 (M + H+);
HRMS: calcd for C35H50NO4, 548.3740; found, 548.3704.
Spectral Data for (23S, 25R)-DLAM-3P (8c). [R]24 +56.21
D
(c 0.65, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.29 (m, 2H), 7.19
(m, 3H), 6.37 (d, J ) 11.1 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H),
5.33 (s, 1H), 4.99 (s, 1H), 4.43 (dd, J ) 4.3, 7.7 Hz, 1H), 4.23 (m,
1H), 3.63 (m, 1H), 3.48 (m, 1H), 3.01 (m, 1H), 2.83 (d, J ) 12.8
Hz, 1H), 2.60 (m, 3H), 2.31 (dd, J ) 6.4, 13.3 Hz, 1H), 2.23 (m,
1H), 2.04-1.24 (m, 22H), 1.33 (s, 3H), 0.95 (d, J ) 6.0 Hz, 3H),
0.55 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.6, 147.6, 142.5
141.2, 133.3, 128.5, 128.3, 126.1, 124.8, 117.3, 111.8, 73.8, 70.8,
66.9, 56.9, 56.3, 51.8, 45.9, 45.2, 42.8, 40.8, 40.5, 39.9, 39.8, 33.2,
32.8, 29.0, 27.9, 24.9, 23.5, 22.2, 18.7, 12.0; m/z 562 (M + H+);
HRMS: calcd for C36H52NO4, 562.3896; found, 562.3881.
Spectral Data for (23R, 25S)-DLAM-3P (8d). [R]24D +3.58 (c
Spectral Data for (23S, 25R)-DLAM-2P (7c). [R]24 +56.13
D
1
(c 0.16, CHCl3); H NMR (500 MHz, CDCl3) δ 7.30-7.19 (m,
1
5H), 6.37 (d, J ) 11.5 Hz, 1H), 6.02 (d, J ) 11.1 Hz, 1H), 5.33 (s,
1H), 4.99 (s, 1H), 4.44 (dd, J ) 4.3, 7.7 Hz, 1H), 4.23 (m, 1H),
3.97 (m, 1H), 3.26 (m, 1H), 3.19 (m, 1H), 2.90 (m, 1H), 2.83 (d,
J ) 13.3 Hz, 1H), 2.76 (m, 1H), 2.61 (d, J ) 13.3 Hz, 1H), 2.31
(dd, J ) 6.4, 13.3 Hz, 1H), 2.14 (dd, J ) 6.4, 12.8 Hz, 1H), 2.01-
1.13 (m, 19H), 1.62 (dd, J ) 8.1, 12.8 Hz, 1H), 1.25 (s, 3H), 0.89
(d, J ) 6.0 Hz, 3H), 0.54 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
176.6, 147.6, 142.5, 138.4, 133.2, 128.7, 128.6, 126.6, 124.8, 117.3,
111.8, 73.6, 70.8, 66.8, 56.9, 56.3, 51.8, 45.9, 45.2, 42.8, 40.9, 40.5,
39.9, 39.8, 33.7, 32.7, 29.0, 27.9, 24.7, 23.5, 22.2, 18.7, 12.0; m/z
548 (M + H+); HRMS: calcd for C35H50NO4, 548.3740; found,
548.3763.
0.24, CHCl3); H NMR (500 MHz, CDCl3) δ 7.29 (m, 2H), 7.18
(m, 3H), 6.38 (d, J ) 11.1 Hz, 1H), 6.03 (d, J ) 11.1 Hz, 1H),
5.35 (s, 1H), 5.01 (s, 1H), 4.44 (m, 1H), 4.24 (m, 1H), 3.66 (m,
1H), 3.43 (m, 1H), 3.02(m, 1H), 2.83 (d, J ) 12.8 Hz, 1H), 2.60
(m, 3H), 2.32 (dd, J ) 6.4, 13.3 Hz, 1H), 2.27 (dd, J ) 6.4, 12.8
Hz, 1H), 2.08-1.06 (m, 21H), 1.81 (dd, J ) 7.3, 12.8 Hz, 1H),
1.33 (s, 3H), 1.00 (d, J ) 6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 176.6, 147.7, 142.5, 141.3, 133.3, 128.5,
128.3, 126.1, 124.8, 117.3, 111.6, 73.9, 70.7, 66.9, 57.4, 56.2, 53.4,
45.9, 45.2, 42.9, 42.4, 40.8, 40.4, 40.3, 34.8, 33.3, 29.0, 27.9, 24.7,
23.5, 22.3, 20.4, 12.0; m/z 562 (M + H+); HRMS: calcd for C36H52-
NO4, 562.3896; found, 562.3892.
Spectral Data for (23R, 25S)-DLAM-2P (7d). [R]24D +1.81 (c
0.22, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.28-7.16 (m, 5H),
6.36 (d, J ) 11.5 Hz, 1H), 6.01 (d, J ) 11.5 Hz, 1H), 5.32 (s, 1H),
4.99 (s, 1H), 4.43 (m, 1H), 4.22 (m, 1H), 3.96 (m, 1H), 3.26 (m,
1H), 3.18 (m, 1H), 2.89 (m, 1H), 2.81 (m, 1H), 2.72 (m, 1H), 2.58
(m, 1H), 2.57-2.27 (m, 2H), 2.17 (dd, J ) 6.4, 12.4 Hz, 1H), 2.02-
1.13 (m, 18H), 1.75 (dd, J ) 8.1, 12.4 Hz, 1H), 1.23 (s, 3H), 0.96
(d, J ) 6.4 Hz, 3H), 0.55 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
176.6, 147.7, 142.6, 138.2, 133.2, 128.8, 128.6, 126.7, 124.9, 117.3,
111.8, 73.7, 70.8, 66.9, 57.4, 56.2, 53.4, 46.0, 45.2, 42.9, 42.4, 41.5,
40.8, 40.4, 34.8, 33.6, 29.0, 28.0, 24.6, 23.5, 22.3, 20.4, 12.0; m/z
548 (M + H+); HRMS: calcd for C35H50NO4, 548.3740; found,
548.3693.
20(R)-(1-Phenylbutyl-3-hydroxy-3-methyl-pyrrolidin-2-one-
5-yl)methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-di-
ol (9). DLAM-4P (9) was obtained from the corresponding nitrone,
which was prepared by using aldehyde 15 and 4-phenylbutylhy-
droxylamine (21), as four diastereomer mixtures. These mixtures
were purified by HPLC (PEGASIL Silica 60-5 column, φ 20 ×
250 mm, Senshu Pack, hexane/ethyl acetate/IPA ) 15:82:3) to give
9a-d in 17, 17, 12, and 10% yields, respectively.
Spectral Data for (23S, 25S)-DLAM-4P (9a). [R]24 +12.89
D
(c 0.94, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 2H), 7.17
(m, 3H), 6.37 (d, J ) 11.1 Hz, 1H), 6.02 (d, J ) 11.5 Hz, 1H),
5.33 (s, 1H), 4.99 (s, 1H), 4.43 (m, 1H), 4.23 (m, 1H), 3.64-3.56
(m, 2H), 2.96 (m, 1H),2.84 (d, J ) 12.8 Hz, 1H), 2.69-2.59 (m,
3H), 2.32 (dd, J ) 6.8, 14.5 Hz, 1H), 2.29 (m, 1H), 2.01-1.25 (m,
20(R)-(1-Phenylpropyl-3-hydroxy-3-methyl-pyrrolidin-2-one-
5-yl)methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-di-
ol (8). DLAM-3P (8) was obtained from the corresponding nitrone,
which was prepared by aldehyde 15 and 3-phenylpropylhydroxy-
lamine (20), as four diastereomer mixtures. These mixtures were
purified by HPLC (PEGASIL Silica 60-5 column, φ 20 × 250
mm, Senshu Pack, hexane/ethyl acetate/IPA ) 19:78:3) to give
8a-d in 21, 21, 14, and 11% yields, respectively.
23H), 1.60 (m, 1H), 1.42 (s, 3H), 0.92 (brs, 3H), 0.57 (s, 3H); 13
C
NMR (125 MHz, CDCl3) δ 175.9, 147.7, 142.5, 141.9, 133.2, 128.4,
128.3, 125.8, 124.8, 117.3, 111.7, 74.2, 70.7, 66.9, 56.9, 56.3, 52.0,
45.9, 45.2, 42.9, 40.5, 40.1, 39.8, 39.6, 35.3, 33.1, 29.0, 28.2, 28.0,
26.5, 25.9, 23.5, 22.2, 18.6, 12.0; m/z 576 (M + H+); HRMS: calcd
for C37H54NO4, 576.4053; found, 576.4082.
Spectral Data for (23R, 25R)-DLAM-4P (9b). [R]24 +24.27
D
Spectral Data for (23S, 25S)-DLAM-3P (8a). [R]24 +10.47
(c 1.05, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.26 (m, 2H), 7.16
(m, 3H), 6.37 (d, J ) 11.5 Hz, 1H), 6.03 (d, J ) 11.5 Hz, 1H),
5.34 (s, 1H), 5.00 (s, 1H), 4.44 (m, 1H), 4.23 (m, 1H), 3.63 (m,
1H), 3.58 (m, 1H), 3.00 (m, 1H), 2.96 (m, 1H), 2.83 (d, J ) 12.4
Hz, 1H), 2.62 (m, 3H), 2.36 (dd, J ) 7.7, 13.3 Hz, 1H), 2.32 (dd,
J ) 6.4, 13.3 Hz, 1H), 2.05-1.17 (m, 23H), 1.70 (dd, J ) 5.6,
13.3 Hz, 1H), 1.42 (s, 3H), 0.97 (d, J ) 6.4 Hz, 3H), 0.54 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 176.0, 147.7, 142.4, 141.9, 133.3,
128.4, 128.3, 125.8, 124.7, 117.3, 111.6, 74.1, 70.7, 66.8, 57.3,
56.1, 53.3, 45.9, 45.2, 42.9, 41.8, 40.8, 40.5, 40.4, 35.3, 35.0, 29.0,
28.3, 27.8, 26.3, 25.8, 23.4, 22.3, 20.3, 11.9; m/z 576 (M + H+);
HRMS: calcd for C37H54NO4, 576.4053; found, 576.4033.
D
(c 0.45, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.28 (m, 2H), 7.19
(m, 3H), 6.37 (d, J ) 11.1 Hz, 1H), 6.02 (d, J ) 11.1 Hz, 1H),
5.33 (s, 1H), 4.99 (s, 1H), 4.43 (dd, J ) 4.3, 7.7, Hz, 1H), 4.23
(m, 1H), 3.68 (m, 1H), 3.60 (m, 1H), 3.01 (m, 1H), 2.83 (d, J )
12.8 Hz, 1H), 2.63 (m, 3H), 2.32 (m, 1H), 2.29 (dd, J ) 7.3, 13.3
Hz, 1H), 2.07-1.25 (m, 21H), 1.60 (dd, J ) 5.6, 13.3 Hz, 1H),
1.43 (s, 3H), 0.95 (d, J ) 5.6 Hz, 3H), 0.57 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 176.1, 147.7, 142.4, 141.3, 133.3, 128.4,
128.3, 126.0, 124.8, 117.3, 111.7, 74.1, 70.7, 66.8, 56.9, 56.5, 52.2,
45.9, 45.2, 42.9, 40.5, 40.1, 39.9, 39.6, 33.1, 28.8, 28.0, 25.8, 23.5,
22.2, 18.7, 12.1; m/z 562 (M + H+); HRMS: calcd for C36H52-
NO4, 562.3896; found, 562.3869.
Spectral Data for (23S, 25R)-DLAM-4P (9c). [R]24 +44.34
D
Spectral Data for (23R, 25R)-DLAM-3P (8b). [R]24 +26.19
(c 0.61, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 2H), 7.17
(m, 3H), 6.37 (d, J ) 11.1 Hz, 1H), 6.02 (d, J ) 11.1 Hz, 1H),
D
(c 0.57, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 2H), 7.18

2404 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Nakano et al.
5.33 (s, 1H), 4.99 (s, 1H), 4.44 (m, 1H), 4.23 (m, 1H), 3.62 (m,
1H), 3.41 (m, 1H), 2.97 (m, 1H), 2.83 (d, J ) 12.5 Hz, 1H), 2.70-
2.57 (m, 3H), 2.31 (dd, J ) 6.4, 13.3 Hz, 1H), 2.22 (dd, J ) 6.4,
12.4 Hz, 1H), 2.01-1.20 (m, 25H), 1.68 (dd, J ) 7.7, 12.4 Hz,
1H), 1.33 (s, 3H), 0.91 (d, J ) 6.0 Hz, 3H), 0.55 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 176.7, 147.6, 142.5, 141.9, 133.3, 128.8,
128.4, 125.9, 124.8, 117.4, 111.9, 73.9, 70.8, 66.8, 56.9, 56.4, 51.6,
45.9, 45.2, 42.8, 40.8, 40.5, 39.9, 39.7, 35.2, 32.8, 29.0, 28.3, 27.9,
26.5, 24.9, 23.5, 22.2, 18.6, 12.0; m/z 576 (M + H+); HRMS: calcd
for C37H54NO4, 576.4053; found, 576.4015.
147.6, 142.6, 133.2, 129.2, 128.2, 124.8, 117.3, 114.1, 111.9, 74.0,
70.9, 66.9, 56.9, 56.3, 55.3, 51.0, 45.9, 45.3, 43.5, 42.8, 40.6, 40.5,
39.8, 32.9, 29.0, 27.9, 24.7, 23.5, 22.2, 18.7, 12.0; m/z 576 (M +
H+); HRMS: calcd for C35H50NO5 564.3689; found, 564.3718.
Spectral Data for (23R, 25S)-DLAM-MPM (10d). [R]24
D
1
+3.49 (c 0.53, CHCl3); H NMR (500 MHz, CDCl3) δ 7.08 (d, J
) 8.6 Hz, 2H), 6.85 (d, J ) 8.6 Hz, 2H), 6.36 (d, J ) 11.1 Hz,
1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.33 (s, 1H), 4.99 (s, 1H), 4.95 (d,
J ) 15.0 Hz, 1H), 4.44 (m 1H), 4.22 (m, 1H), 3.98 (d, J ) 15.0
Hz, 1H), 3.80 (s, 3H), 3.25 (m, 1H), 2.82 (d, J ) 12.4 Hz, 1H),
2.59 (d, J ) 14.1 Hz, 1H), 2.32 (m, 1H), 2.24 (dd, J ) 6.4, 12.4
Hz, 1H), 2.13-1.22 (m, 21H), 1.86 (dd, J ) 7.7, 12.4 Hz, 1H),
1.36 (s, 3H), 1.09 (m, 2H), 0.88 (d, J ) 6.5 Hz, 3H), 0.50 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 176.9, 159.1, 147.7, 142.6, 133.2,
129.0, 128.2, 124.9, 117.3, 114.1, 111.7, 74.0, 70.8, 66.9, 57.4,
56.2, 55.3, 52.9, 45.9, 45.3, 43.9, 42.9, 42.2, 40.5, 40.4, 38.8, 34.5,
29.0, 27.8, 24.7, 23.5, 22.3, 20.3, 11.9; m/z 576 (M + H+);
HRMS: calcd for C35H50NO5 564.3689; found, 564.3706.
20(R)-(1-(4-Isopropyl-3-hydroxy-3-methyl-pyrrolidin-2-one-
5-yl)methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-di-
ol (11). DLAM-03I (11) was obtained from the corresponding
nitrone, which was prepared by using aldehyde 15 and commercially
available isoprorylhydroxylamine hydrochloride (23), as four di-
astereomer mixtures. These mixtures were purified by HPLC (ODS-
AM, φ 30 × 250 mm, YMC-Pack, A ) 95% H2O/CH3CN, B )
0.5% H2O/40% MeOH/CH3CN; A:B ) 2:3) to give 11a-d in 4,
5, 5, and 6% yields, respectively.
Spectral Data for (23R, 25S)-DLAM-4P (9d). [R]24D -5.87 (c
1
0.41, CHCl3); H NMR (500 MHz, CDCl3) δ 7.28 (m, 2H), 7.17
(m, 3H), 6.38 (d, J ) 11.5 Hz, 1H), 6.03 (d, J ) 11.1 Hz, 1H),
5.34 (s, 1H), 5.01 (s, 1H), 4.44 (m, 1H), 4.23 (m, 1H), 3.68 (m,
1H), 3.37 (m, 1H), 2.98 (m, 1H), 2.83 (d, J ) 12.8 Hz, 1H), 2.68-
2.59 (m, 3H), 2.33 (m, 1H), 2.27 (dd, J ) 6.4, 12.4 Hz, 1H), 2.04-
1.25 (m, 23H), 1.81 (dd, J ) 7.7, 12.4 Hz, 1H), 1.33 (s, 3H), 0.98
(d, J ) 6.4 Hz, 3H), 0.55 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
176.6, 147.7, 142.6, 141.9, 133.2, 128.8, 128.4, 125.9, 124.8, 117.3,
111.7, 73.9, 70.8, 66.9, 57.4, 56.2, 53.1, 46.0, 45.2, 42.9, 42.4, 40.7,
40.4, 40.1, 35.3, 34.7, 29.0, 28.5, 27.9, 26.5, 24.8, 23.5, 22.3, 20.4,
11.9; m/z 576 (M + H+); HRMS: calcd for C37H54NO4, 576.4053;
found, 576.4066.
20(R)-(1-(4-Methoxybenzyl)-3-hydroxy-3-methyl-pyrrolidin-
2-one-5-yl)methyl-9,10-secopregna-5(Z),7(E),10(19)-triene-1R,3â-
diol (10). DLAM-MPM (10) was obtained from the corresponding
nitrone, which was prepared by using aldehyde 15 and 4-methox-
yphenylmethylhydroxylamine (22), as four diastereomer mixtures.
These mixtures were purified by HPLC (PEGASIL Silica 60-5
column, φ 20 × 250 mm, Senshu Pack, hexane:ethyl acetate:IPA
) 15:82:3) to give 10a-d in 17, 25, 14, and 14% yields,
respectively.
Spectral Data for (23S, 25S)-DLAM-03I (11a). 1H NMR (400
MHz, CDCl3) δ 6.38 (1H, d, J ) 11.5 Hz), 6.03 (1H, d, J ) 11.2
Hz), 5.33 (1H, s), 5.00 (1H, s), 4.44 (1H, br s), 4.24 (1H, br s),
4.02-3.92 (1H, m), 3.73-3.63 (1H, m), 2.87-2.80 (1H, m), 2.64-
2.21 (4H, m), 2.07-1.22 (25H, m), 1.00 (3H, d, J ) 5.9 Hz), 0.59
(3H, s); m/z 486 (M + H+); HRMS: calcd for C30H48NO4,
486.3583; found, 486.3531.
Spectral Data for (23S, 25S)-DLAM-MPM (10a). [R]24
D
-11.68 (c 0.68, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.15 (d, J
) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H), 6.36 (d, J ) 11.1 Hz,
1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.32 (s, 1H), 4.99 (s, 1H), 4.92 (d,
J ) 15.0 Hz, 1H), 4.43 (m 1H), 4.23 (m, 1H), 3.90 (d, J ) 15.0
Hz, 1H), 3.79 (s, 3H), 3.48 (m, 1H), 2.82 (d, J ) 12.4 Hz, 1H),
2.59 (d, J ) 13.7 Hz, 1H), 2.32 (m, 1H), 2.25 (dd, J ) 7.7, 13.3
Hz, 1H), 2.08-1.25(m, 14H), 1.65 (dd, J ) 5.1, 13.3 Hz, 1H),
1.48 (s, 3H), 0.89-0.84 (m, 5H), 0.79 (d, J ) 6.4 Hz, 3H), 0.53
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.1, 159.1, 147.7, 142.6,
133.2, 129.3, 127.9, 124.8, 117.3, 114.1, 111.7, 74.2, 70.7, 66.9,
56.8, 56.3, 55.3, 51.4, 45.9, 45.2, 43.7, 42.9, 40.4, 39.7, 39.3, 33.2,
29.0, 28.0, 26.1, 23.5, 22.2, 18.7, 12.0; m/z 564 (M + H+);
HRMS: calcd for C35H50NO5 564.3689; found, 564.3729.
Spectral Data for (23R, 25R)-DLAM-03I (11b).1H NMR (400
MHz, CDCl3) δ 6.38 (1H, d, J ) 11.2 Hz), 6.03 (1H, d, J ) 10.7
Hz), 5.33 (1H, s), 5.00 (1H, s), 4.44 (1H, br s), 4.24 (1H, br s),
3.95-3.84 (1H, m), 3.66-3.55 (1H, m), 2.88-2.80 (1H, m), 2.65-
2.26 (4H, m), 2.08-1.08 (25H, m), 1.02 (3H, d, J ) 6.3 Hz), 0.65
(3H, s); m/z 486 (M + H+); HRMS: calcd for C30H48NO4,
486.3583; found, 486.3541.
Spectral Data for (23S, 25R)-DLAM-03I (11c).1H NMR (400
MHz, CDCl3) δ 6.38 (1H, d, J ) 11.2 Hz), 6.02 (1H, d, J ) 11.5
Hz), 5.34 (1H, s), 5.00 (1H, s), 4.44 (1H, br s), 4.24 (1H, br s),
3.97-3.88 (1H, m), 3.60-3.49 (1H, m), 2.87-2.80 (1H, m), 2.63-
2.53 (2H, m), 2.32 (1H, dd, J ) 12.6, 6.7 Hz), 2.20 (1H, dd, J )
12.6, 6.7 Hz), 2.05-1.20 (25H, m), 0.99 (3H, d, J ) 6.3 Hz), 0.57
(3H, s); m/z 486 (M + H+); HRMS: calcd for C30H48NO4,
486.3583; found, 486.3572.
Spectral Data for (23R, 25R)-DLAM-MPM (10b). [R]24
D
+17.00 (c 1.10, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.12 (d, J
) 8.6 Hz, 2H), 6.85 (d, J ) 8.6 Hz, 2H), 6.36 (d, J ) 11.1 Hz,
1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.93 (d,
J ) 15.0 Hz, 1H), 4.44 (dd, J ) 4.3,7,7 Hz 1H), 4.23 (m, 1H),
3.97 (d, J ) 15.4 Hz, 1H), 3.79 (s, 3H), 3.47 (m, 1H), 2.82 (d, J
) 12.4 Hz, 1H), 2.59 (d, J ) 13.3 Hz, 1H), 2.34 (dd, J ) 7.7, 13.3
Hz, 1H), 2.32 (dd, J ) 6.4, 12.8 Hz, 1H), 2.08-1.89 (m, 5H),
1.76 (dd, J ) 5.1, 13.3 Hz, 1H), 1.69-0.99 (m, 14H), 1.49 (s,
3H), 0.87 (d, J ) 6.9 Hz, 3H), 0.50 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 176.4, 159.0, 147.7, 142.6, 133.2, 129.0, 127.9, 124.8,
117.3, 114.1, 111.7, 74.1, 70.8, 66.9, 57.3, 56.2, 55.3, 53.0 45.9,
45.2, 44.2, 42.9, 41.7, 40.7, 40.4, 35.0, 29.0, 27.8, 26.0, 23.5, 22.3,
20.1, 11.9; m/z 576 (M + H+); HRMS: calcd for C35H50NO5
564.3689; found, 564.3694.
Spectral Data for (23R, 25S)-DLAM-03I (11d).1H NMR (400
MHz, CDCl3) δ 6.38 (1H, d, J ) 11.2 Hz), 6.03 (1H, d, J ) 11.2
Hz), 5.33 (1H, s), 5.00 (1H, s), 4.43 (1H, br s), 4.23 (1H, br s),
3.89-3.80 (1H, m), 3.48-3.40 (1H, m), 2.87-2.80 (1H, m), 2.63-
2.50 (2H, m), 2.32 (1H, dd, J ) 12.7, 6.5 Hz), 2.25 (1H, dd, J )
12.7, 6.8 Hz), 2.10-1.15 (25H, m), 1.03 (3H, d, J ) 6.6 Hz), 0.57
(3H, s); m/z 486 (M + H+); HRMS: calcd for C30H48NO4,
486.3583; found, 486.3564.
Binding to the Vitamin D Receptor (VDR). Nuclear 1,25-D3
receptor protein was prepared from chick intestine at Teijin Pharma.
The VDR (0.2 mg) was dissolved in 1 mL of 25 mM phosphate
buffer (pH 7.4) containing 0.1 M KCl, 1 mM dithiothreitol, and
gelatin (1 mg). This solution was mixed with (26,27-methyl-3H)-
1,25-D3 (Amersham Biosciences Corp., 15 000 dpm, 180 Ci/mmol,
dissolved in 10 ml of ethanol) and various concentrations of a test
compound (dissolved in 40 mL ethanol) in a polypropylene tube
(Walter Sarstedt, 12 × 75 mm) and incubated for 60 min at 25 °C.
Then the mixture was cooled to 4 °C, and 40% poly(ethylene glycol)
6000 solution (1 mL) was added to each tube, and the tubes were
mixed vigorously and centrifuged at 2260g for 60 min at 4 °C.
The resulting pellet was dissolved in a scintillation cocktail (DuPont,
10 mL), and the radio activity was counted with a liquid scintillation
Spectral Data for (23S, 25R)-DLAM-MPM (10c). [R]24
D
+15.58 (c 0.46, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.11 (d, J
) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H), 6.36 (d, J ) 11.1 Hz,
1H), 6.01 (d, J ) 11.1 Hz, 1H), 5.33 (s, 1H), 4.99 (s, 1H), 4.94 (d,
J ) 14.5 Hz, 1H), 4.43 (dd J ) 4.3, 7.7 Hz, 1H), 4.23 (m, 1H),
3.90 (d, J ) 15.0 Hz, 1H), 3.79 (s, 3H), 3.26 (m, 1H), 2.82 (d, J
) 12.4 Hz, 1H), 2.59 (dd, J ) 3.0, 13.7 Hz, 1H), 2.31 (m, 1H),
2.19 (dd, J ) 6.4, 12.8 Hz, 1H), 2.02-1.14 (m, 20H), 1.71 (dd, J
) 8.1, 12.8 Hz, 1H), 1.33 (s, 3H), 0.88 (m, 1H), 0.78 (d, J ) 6.4
Hz, 3H), 0.52 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.7, 159.1,

D3 Antagonists and D3-26,23-lactams
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2405
(2) (a) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; Umesono, K.; Blumgerg, B.; Kastner, P.; Mark, M.; Chambon,
P.; Evans, R. M. The Nuclear Receptor Superfamily - The 2nd
Decade. Cell 1995, 83, 835-839. (b) Bjo¨rklund, S.; Almouzni, G.;
Davidson, I.; Nightingale, K. P. Global Transcription Regulators of
Eukaryotes. Cell 1999, 96, 759-767.
counter (Beckman, model LS6500). The relative binding affinity
of the test compounds for VDR was calculated from the concentra-
tion necessary to displace 50% of (26,27-methyl-3H)-1,25-D3 from
VDR. The relative affinity thus measured for 1,25-D3 was defined
as 100.
Assay for HL-60 Cell Differentiation. The human promyelo-
cytic leukemia cell line HL-60 was purchased from a cell bank
(Japanese Cancer Research Resources Bank, cell#: JCRB0085).
The HL-60 cells were cultured in RPMI-1640 (Life Technologies)
medium supplemented with 10% heat-inactivated fetal bovine serum
(FBS). The cell concentration at seeding was adjusted to 3 × 103
cells/mL, and the seeding volume was 1 mL/well. To assess the
vitamin D3-agonistic activity of test compounds, the HL-60 cells
were incubated in the presence or absence of 1,25-D3 (a positive
control) or a test compound (added to the culture in 1 mL of ethanol
solution) and incubated for 96 h at 37 °C in a humidified atmosphere
of 5% CO2/air without a medium change. To assess the vitamin
D3-antagonistic activity of test compounds, the HL-60 cells were
incubated with various concentrations of a test compound (added
to the culture in 1 mL of ethanol solution) in the presence of 1 ×
10-5 M 1,25-D3 (added to the culture in 1 mL of ethanol solution).
After incubation, the nitroblue tetrazolium (NBT)-reducing activity
of the HL-60 cells was measured. The HL-60 cells were collected
by centrifugation, washed with phosphate-buffered saline (PBS),
and re-suspended in the medium. To the cell suspension was added
NBT (Tokyo Kasei Kogyo) and 12-O-tetradecanoylphorbol-13-
acetate (TPA, Wako). The final concentrations of NBT and TPA
were 0.1% and 100 ng/mL, respectively. Then, the mixture was
incubated at 37 °C for 25 min, and the cells were collected by
centrifugation and re-suspended in PBS. Cytospin smears were
prepared, the counter-staining of the nuclei was done with a
Kemechrot solution, and the ratio of NBT-positive cells was counted
under a microscope.
Transactivation Assay. The COS-7 cells were maintained in
DMEM (Dulbecco’s modified Eagle’s medium), supplemented with
DCC-treated 10% FBS (JRH Bioscience). A VDR expression vector
was prepared by inserting a full-length human VDR gene into the
multicloning sites of the pTracer expression plasmid (Stratagene).
A reporter gene plasmid was prepared by inserting the promoter
region of the human 24-hydroxylase (Cyp24) gene containing two
sets of VDRE, which was obtained by polymerase chain reaction
(PCR), into pGL3-basic (Promega). The COS-7 cells (4 × 104 cells/
well) were cotransfected with the VDR expression vector (0.025
mg) and the reporter gene plasmid (0.25 mg) by using the
FuGENE6 (Roche) method. To standardize the transfection ef-
ficiency, pRL-TK (0.025 mg, Promega) was also cotransfected at
the same time. Transfection was continued for 4 h, and then the
medium was exchanged with a fresh a-MEM medium containing
10% FBS and various concentrations of a test compound. The
mixture was incubated for 24 h, and then the cells were washed
with Dulbecco’s phosphate-buffered saline (D-PBS). The luciferase
activity of the treated cells was measured by using the dual-
luciferase reporter assay system (Promega) and the fluoroskan ascent
FL (Thermo Labsystems) according to the protocol recommended
by the supplier.
(3) Moras, D.; Gronemeyer, H. The Nuclear Receptor Ligand-Binding
Domain: Structure and Function. Curr. Opin. Cell. Biol. 1998, 10,
384-391.
(4) (a) Brzozowski, A. M.; Pike, C. W.; Dauter, Z.; Hubbard, R. E.;
Bonn, T.; Engstro¨m, O.; O¨ hman, L.; Greene, G. L.; Gustafsson, J-A.;
Carlquist, M. Molecular Basis of Agonism and Antagonism in the
Oestrogen Receptor. Nature 1997, 389, 753-758. (b) Carlberg, C.
Molecular Basis of the Selective Activity of Vitamin D Analogues.
J. Cell. Biochem. 2003, 88, 274-281.
(5) Kliewer, S. A.; Umesono, K.; Mangelsdorf, D. J.; Evans, R. M.
Retinoid X-Receptor Interacts With Nuclear Receptors in Retinoic
Acid, Thyroid-Hormone and Vitamin-D3 Signaling. Nature 1992, 355,
446-449.
(6) (a) Carlberg, C. Ligand-Mediated Conformational Changes of the
VDR are Required for Gene Transactivation. J. Steroid Biochem.
Mol. Biol. 2004, 89-90, 227-232. (b) Christakos, S.; Dhawan, P.;
Liu, Y.; Peng, X.; Porta, A. New Insights into the Mechanisms of
Vitamin D Action. J. Cell. Biochem. 2003, 88, 695-705. (c)
Gonzalez, M. M.; Samenfeld, P.; Pera¨kyla¨, M.; Carlberg, C.
Corepressor Excess Shifts the Two-Side Chain Vitamin D Analogue
Gemini from an Agonist to an Inverse Agonist of the Vitamin D
Receptor. Mol. Endocrinol. 2003, 17, 2028-2038.
(7) (a) Menaa, C.; Barsony, J.; Reddy, S. V.; Cornish, J.; Cundy, T.;
Roodman, G. D. 1,25-Dihydroxyvitamin D3 Hypersensitivity of
Osteoclast Precursors from Patients with Paget’s Disease. J. Bone
Miner. Res. 2000, 15, 228-236. (b) Kurihara, N.; Reddy, S. V.;
Menaa, C.; Anderson, D.; Roodman, G. D. Osteoclasts Expressing
the Measles Virus Nucleocapsid Gene Display a Pagetic Phenotype.
J. Clin. InVest. 2000, 105, 607-614. (c) Leach, R. J.; Roodman, G.
D. Genetics of Endocrine Disease - The Genetics of Paget’s Disease
of the Bone. J. Clin. Endocrinol. Metab. 2001, 86, 24-28. (d) Reddy,
S. V.; Kurihara, N.; Menaa, C.; Landucci, G.; Forthal, D.; Koop, B.
A.; Windle, J. J.; Roodman, G. D. Osteoclasts Formed by Measles
Virus-Infected Osteoclast Precursors from hCD46 Transgenic Mice
Express Characteristics of Pagetic Osteoclasts. Endocrinology 2001,
142, 2898-2905. (e) Friedrichs, W. E.; Reddy, S. V.; Bruder, J. M.;
Cundy, T.; Cornish, J.; Singer, F. R.; Roodman, G. D. Sequence
Analysis of Measles Virus Nucleocapsid Transcripts in Patients with
Paget’s Disease. J. Bone Miner. Res. 2002, 17, 145-151. (f) Kurihara,
N.; Ishizuka, S.; Demulder, A.; Menaa, C.; Roodman, G. D. Paget’s
Disease - A VDR Coactivator Disease? J. Steroid Biochem. Mol.
Biol. 2004, 89-90, 321-325. (g) Ishizuka, S.; Kurihara, N.; Miura,
D.; Takenouchi, K.; Cornish, J.; Cundy, T.; Reddy, S. V.; Roodman,
G. D. Vitamin D Antagonist, TEI-9647, Inhibits Osteoclast Formation
Induced by 1R, 25-Dihydroxyvitamin D3 from Pagetic Bone Marrow
Cells. J. Steroid Biochem. Mol. Biol. 2004, 89-90, 331-334. (h)
Kurihara, N.; Reddy, S. V.; Araki, N.: Ishizuka, S.: Ozono, K.:
Cornish, J.: Cundy, T.: Singer, F. R.: Roodman, G. D. Role of
TAF (II)-17, a VDR Binding Protein, in the Increased Osteoclast
Formation in Paget’s Disease. J. Bone Miner. Res. 2004, 19, 1154-
1164. (i) Ishizuka, S.: Kurihara, N.: Reddy, S. V.: Cornish, J.:
Cundy, T.: Roodman, G. D. (23S)-25-Dehydro-1-alpha-hydroxyvi-
tamin D3-26, 23-Lactone, a Vitamin D Receptor Antagonist that
Inhibits Osteoclast Formation and Bone Resorption in Bone Marrow
Cultures from Patients with Paget’s Disease. Endocrinology 2005,
146, 2023-2030.
(8) (a) Herdick, M.; Steinmeyer, A.; Carlberg, C. Carboxylic Ester
Antagonists of 1R, 25-Dihydroxyvitamin D3 Show Cell-Specific
Actions. Chem. Biol. 2000, 7, 885-894. (b) Bury, Y.; Steinmeyer,
A.; Carlberg, C. Structure Activity Relationship of Carboxylic Ester
Antagonists of the Vitamin D3 Receptor. Mol. Pharmacol. 2000, 58,
1067-1074. (c) Toell, A.; Gonzalez, M. M.; Ruf, D.; Steinmeyer,
A.; Ishizuka, S.; Carlberg, C. Different Molecular Mechanisms of
Vitamin D3 Receptor Antagonists. Mol. Pharmacol. 2001, 59, 1478-
1485. (d) Va¨isa¨nen, S.; Pera¨kyla¨, M.; Ka¨rkka¨inen, A.; Steinmeyer,
A.; Carlberg, C. Critical Role of Helix 12 of the Vitamin D3 Receptor
for the Partial Agonism of Carboxylic Ester Antagonists. J. Mol. Biol.
2002, 315, 229-238.
(9) (a) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman,
A. W.; Ishizuka, S. Antagonistic Action of Novel 1R, 25-Dihydrox-
yvitamin D3-26,23-Lactone Analogues on Differentiation of Human
Leukemia Cells (HL-60) Induced by 1R,25-Dihydroxyvitamin D3.
J. Biol. Chem. 1999, 274, 16392-16399. (b) Ozono, K.; Saito, M.;
Miura, D.; Michigami, T.; Nakajima, S.; Ishizuka, S. Analysis of
the Molecular Mechanism for the Antagonistic Action of a Novel
1R,25-Dihydroxyvitamin D3 Analogue Toward Vitamin D Receptor
Function. J. Biol. Chem. 1999, 274, 32376-32381. (c) Bula, C. M.;
Acknowledgment. This work was supported by Grants-in-
Aid for Scientific Research from the ministry of Education,
Science, Sports and Culture, Japan and the funds from the
Mochida Memorial Foundation for Medicinal and Pharmaceuti-
cal Research and the TERUMO Life Science Foundation.
Supporting Information Available: Synthesis of hydroxy-
lamines 19-22 and1H NMR spectral data for 6-11. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Vitamin D Physiology, Molecular Biology, and Clinical, Applica-
tions. Holick, M. F., Ed.; Humana Press: Totowa, NJ, 1999. (b)
Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-Function-
Relationships in the Vitamin-D Endocrine System. Endocr. ReV.
1995, 16, 200-257.

2406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Nakano et al.
Bishop, J. E.; Ishizuka, S.; Norman, A. W. 25-Dehydro-1R-
Hydroxyvitamin D3-,26,23S-Lactone Antagonizes the Nuclear Vi-
tamin D Receptor by Mediating a Unique Noncovalent Conforma-
tional Change. Mol. Endcrinol. 2000, 11, 1788-1796. (d) Ishizuka,
S.; Miura, D.; Ozono, K.; Chokki, M.; Mimura, H.; Norman, A. W.
Antagonistic Actions in vivo of (23S)-25-Dehydro-1R-Hydroxyvi-
tamin D3,26,23-Lactone on Calcium Metabolism Induced by 1R,25-
Dihydroxyvitamin D3. Endcrinology 2001, 142, 59-67. (e) Toell,
A.; Gonzalez, M. M.; Ruf, D.; Steinmeyer, A.; Ishizuka, S.; Carlberg,
C. Different Molecular Mechanisms of Vitamin D3 Receptor
Antagonists. Mol. Pharmacol. 2001, 59, 1478-1485. (f) Takenouchi,
K.; Sogawa, R.; Manabe, K.; Saitoh, H.; Gao, Q.; Miura, D.; Ishizuka,
S. Synthesis and Structure-Activity Relationships of TEI-9647
Derivatives as Vitamin D3 Antagonists. J. Steroid Biochem. Mol. Biol.
2004, 89-90, 31-34. (g) Saito, S.; Masuda, M.; Matsunaga, T.; Saito,
H.; Anzai, M.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takimoto-
Kamimura, M.; Kittaka, A. 24,24-Dimethylvitamin D3-26,23-lactones
and their 2R-functionalized analogues as highly potent VDR antago-
nists. Tetrahedron 2004, 60, 7951-7961.
Minimization, and Dynamics Calculations. J. Comput. Chem. 1983,
4, 187-217.
(18) Trost, B. M.; Dumas, J.; Villa, M. New Strategies for the Synthesis
of VitaminD Metabolites via Pd-Catalyzed Reactions. J. Am. Chem.
Soc. 1992, 114, 9836-9845.
(19) Andrews, D. R.; Barton, D. H. R.; Hesse, R. H.; Pechet, M.
M.Synthesis of 25-Hydroxy- and 1R,25-Dihydroxy Vitamin D3 from
Vitamin D2 (Calciferol). J. Org. Chem. 1986, 51, 4819-4828.
(20) (a) Wovkulich, P. M.; Barcelos, F.; Batcho, A. D.; Sereno, J. F.;
Baggiolini, E. G.; Hennessy, B. M.; Uskokovic, M. R. Stereoselective
Total Synthesis of 1R,25S,26-Trihydroxycholecalciferol. Tetrahedron
1984, 40, 2283-2296. (b) Baggiolini, E. G.; Iacobelli, J. A.;
Hennessy, B. M.; Batcho, A. D.; Sereno, J. F.; Uskokovic, M. R.
Stereocontrolled Total Synthesis of 1R,25-Dihydroxycholecalciferol
and 1R,25-Dihydroxyergocalciferol. J. Org. Chem. 1986, 51, 3098-
3108.
(21) Cicchi, S.; Goti, A.; Brandi, A.; Guarna, A.; De Sarlo, F. 1,3-
Aminoalcohols by Reductive Cleavage of Isoxazolidines with Mo-
lybdenium Hexacarbonyl. Tetrahedron Lett. 1990, 31, 3351-3354.
(22) Ishizuka, S.; Bannai, K.; Naruchi, T.; Hashimoto, Y. Studies on the
Mechanism of Action of 1R,24-Dihydroxyvitamin D3 II. Specific
Binding of 1R,24-Dihydroxyvitamin D3 to Chick Intestinal Receptor.
Steroids 1981, 37, 33-43.
(23) Ishizuka, S.; Oshida, J.; Tsuruta, H.; Norman, A. W. The Stereo-
chemical Configuration of the Natural 1R,25-Dihydroxyvitamin D3-
26, 23-Lactone. Arch. Biochem. Biophys. 1985, 242, 82-89.
(24) Mangelsdorf, D. J.; Koeffler, H. P.; Donaldson, C. A.; Pike, J. W.;
Haussler, M. R. 1,25-Dihydroxyvitamin D3-Induced Differentiation
in a Human Promyelocytic Leukemia Cell Line (HL-60): Receptor-
Mediated Maturation to Macrophage-Like Cells. J. Cell Biol. 1984,
98, 391-398.
(25) Collins, S. J.; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C. Normal
Functional Characteristics of Cultured Human Promyelocytic Leu-
kemia Cells (HL-60) After Induction of Differentiation by Dimeth-
ylsulfoxide. J. Exp. Med. 1979, 149, 969-974.
(10) Calverley, M. J. Synthesis of MC-903, A Biologically-Active Vitamin
D Metabolite Analog. Tetrahedron 1987, 43, 4609-4619.
(11) (a) Kato, Y.; Hashimoto, Y.; Nagasawa, K. Novel Heteroatom-
Containing Vitamin D3 Analogs: Efficient Synthesis of 1R,25-
Dihydroxyvitamin D3-26,23-Lactam. Molecules 2003, 8, 488-499.
(b) Kato, Y.; Nakano, Y.; Sano, H.; Tanatani, A.; Kobayashi, H.;
Shimazawa, R.; Koshino, H.; Hashimoto, Y.; Nagasawa, K. Synthesis
of 1R,25-Dihydroxyvitamin D3-26,23-Lactams (DLAMs), a Novel
Series of 1R,25-Dihydroxyvitamin D3 Antagonist. Bioorg. Med.
Chem. Lett. 2004, 14, 2579-2583.
(12) Ishioka, T.; Tanatani, A.; Nagasawa, K.; Hashimoto, Y. Anti-
Androgens with Full Antagonistic Activity Toward Human Prostate
Tumor LNCaP Cells with Mutated Androgen Receptor. Bioorg. Med.
Chem. Lett. 2003, 13, 2655-2658.
(13) Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. The
Crystal Structure of the Nuclear Receptor for Vitamin D Bound to
its Natural Ligand. Mol. Cell 2000, 5, 173-179.
(26) 5a (DLAM-01) only showed a very weak binding affinity to the VDR
and antagonistic activity at a high concentration (>10-6 M).10b
(27) Chen, K.-S.; DeLuca, H. F. Cloning of the Human 1R,25-Dihydrox-
yvitamin D3 24-Hydroxylase Gene Promoter and Identification of 2
Vitamin Responsive Elements. Biochim. Biophys. Acta 1995, 1263,
1-9.
(28) (a) Pera¨kyla¨, M.; Molnar, F.; Carlberg, C. A Structural Basis for the
Species-Specific Antagonism of 26,23-Lactones on Vitamin D
Signaling. Chem. Biol. 2004, 11, 1147-1156. (b) Ochiai, E.; Miura,
D.; Eguchi, H.; Ohara, S.; Takenouchi, K.; Azuma, Y.; Kamimura,
T.; Norman, A. W.; Ishizuka, S. Molecular Mechanism of the Vitamin
D Antagonistic Actions of (23S)-25-Dehydro-1R-Hydroxyvitamin D3-
26,23-Lactone Depends on the Primary Structure of the Carboxyl-
Terminal Region of the Vitamin D Receptor. Mol. Endocrinol. 2005,
19, 1147-1157.
(14) Tocchini-Valentini, G.; Rochel, N.; Wurts, J. M.; Mitschler, A.;
Moras, D. Crystal Structures of the Vitamin D Receptor Complexed
to Superagonist 20-epi Ligands. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 5491-5496.
(15) Tocchini-Valentini, G.; Rochel, N.; Wurts, J. M.; Mitschler, A.;
Moras, D. Crystal Structures of the Vitamin D Nuclear Receptor
Liganded with the Vitamin D Side Chain Analogues Calcipotriol and
Seocalcitol, Receptor Agonists of Clinical Importance. Insights into
a Structural Basis for the Switching of Calcipotriol to a Receptor
Antagonist by Further Side Chain Modification. J. Med. Chem. 2004,
47, 1956-1961.
(16) Eelen, G.; Verlinden, L.; Rochel, N.; Claessens, F.; DeClercq, P.;
Vandewalle, M.; Tocchini-Valentini, G.; Moras, D. Superagonistic
Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained
by Vitamin D Receptor-Coactivator Interaction. Mol. Pharmacol.
2005, 67, 1566-1573.
(17) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;
Swaminathan, S.CHARMM: A Program for Macromolecular Energy,
JM050738X