Synthesis of 1alpha,25-dihydroxyvitamin D3-26,23-lactams (DLAMs), a novel series of 1 alpha,25-dihydroxyvitamin D3 antagonist.

Article abstract of DOI:10.1016/j.bmcl.2004.02.076

Novel vitamin D(3) analogs having a lactam structure in their side chains, 1 alpha,25-dihydroxyvitamin D(3)-26,23-lactams (DLAMs), were designed based on the principle of regulation of the folding of helix-12 in the vitamin D nuclear receptor (VDR). The n

Full text of DOI:10.1016/j.bmcl.2004.02.076

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2579–2583
Synthesis of 1a,25-dihydroxyvitamin D3-26,23-lactams (DLAMs),
a novel series of 1a,25-dihydroxyvitamin D3 antagonist
Yuko Kato,a Yusuke Nakano,a Hiroko Sano,a Aya Tanatani,a Hisayoshi Kobayashi,a
Rumiko Shimazawa,a Hiroyuki Koshino,b Yuichi Hashimotoa and Kazuo Nagasawaa,*
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bInstitute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received 22 January 2004; revised 20 February 2004; accepted 21 February 2004
Abstract—Novel vitamin D3 analogs having a lactam structure in their side chains, 1a,25-dihydroxyvitamin D3-26,23-lactams
(DLAMs), were designed based on the principle of regulation of the folding of helix-12 in the vitamin D nuclear receptor (VDR).
The new analogs were synthesized via 1,3-dipolar cycloaddition reaction and subsequent reduction of the isoxazolidine as key steps.
Among the analogs, (23S,25S)-DLAM-01 (4a) and (23S,25S)-DLAM-1P (5a) bind strongly to VDR. Moreover, these analogs were
found to inhibit the differentiation of HL-60 cells induced by 1a,25-dihydroxyvitamin D3.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
When VDR ligands inhibit the folding or induce the
mis-folding of helix-12, these compounds should be
antagonists, which are expected to be effective for
treatment of Pagetꢀs disease.5 However, only two types
of compounds, ZK168281 (2)6 and its analogs from
Schering and TEI-9647 (3)7 from Teijin, have so far been
reported as 1,25-D3 antagonists (Chart 1).
1a,25-Dihydroxyvitamin D3 (1,25-D3) (1), the hormon-
ally active form of vitamin D3, is an endogenous ligand
for the nuclear vitamin D receptor (VDR), which is a
member of the steroid/thyroid/androgen nuclear recep-
tor super-family, and plays critical roles in a variety of
biological activities, including regulation of calcium
homeostasis, bone mineralization and control of cellular
growth, differentiation, and apoptosis.1 The VDR con-
tains a DNA-binding domain (DBD), which is formed
by two zinc-finger motifs that are characteristic of the
nuclear receptor super-family, and a ligand-binding
domain (LBD), which consists of 11 a-helical structures,
containing a short trans-activation function 2 (AF-2)
domain.2 Among these helixes, helix-12, the last one,
plays an important role in the trans-activation activity.3
The agonistic signaling of 1 is induced through a con-
formational change of the LBD, that is, folding of helix-
12 to form a lid over the LBD pocket, which contains 1.4
Approximately 3000 analogs of 1 have been synthesized
as candidate ligands for VDR so far, and many of them
were reported to show agonistic activity.1b
OH
O
OH
OEt
H
H
H
H
HO
OH
HO
OH
1α,25-Dihydroxy
vitamin D3 (1)
ZK168281 (2)
OH
H
H
N
O
R
O
O
H
H
HO
OH
HO
OH
4: R = Me (DLAM-01)
5: R = Bn (DLAM-1P)
TEI-9647 (3)
Keywords: Vitamin D; Antagonist; Lactam.
* Corresponding author. Tel.: +81-358417848; fax: +81-358418495;
e-mail: nagasawa@iam.u-tokyo.ac.jp
Chart 1.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.076

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Y. Kato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2579–2583
We have recently succeeded in developing the nonanilide
type androgen antagonist on the basis of the principle of
inhibiting folding of the helix-12 of the nuclear andro-
gen receptor, a member of the nuclear receptor super-
family.8 Based upon this strategy, we decided to look for
a new type of VDR antagonist. In this paper, we
describe the design, synthesis and biological evaluations
of DLAMs 4 and 5 as novel antagonists having a lactam
moiety in their side chain. The former was designed as a
mis-folding inducer type antagonist, which was expected
to show partial antagonistic/agonistic activity, and the
latter was designed as a folding inhibitor type of
antagonist, which was expected to show full antagonistic
activity.
Lythgoe diol (7) and the aldehyde 8 (Scheme 1). The
aldehyde 8 was reacted with N-methylhydroxylamine to
give the nitrone 9, which was subsequently reacted with
methyl methacrylate to give the isoxazolidine 10 with
four possible diastereomers at C23 and C25.11a These
four diastereomers were separated with HPLC to give
10a–d in 26%, 28%, 10%, and 10% yields, respectively.
Their stereochemistries were determined by comparison
1
with the reported data of H NMR spectra and ½aꢀ
D
values by Uskokovic and co-workers.11 Each of the
diastereomers 10a–d was then carried on to the corre-
sponding CD-ring synthon 12a–d. Thus, the deprotec-
tion of the TBDMS ether of 10 with PTS, and reduction
of the N–O bond with hydrogen in the presence of
palladium on carbon gave the lactams 11 in 80% yield.
The secondary alcohol of 11 was oxidized with TPAP–
NMO,12 and the tertiary alcohol was protected as the
TMS ether with TMS-imidazole in 80% yield. Finally,
Wittig reaction was conducted with bromomethyl tri-
phenyl phosphonium bromide and LHMDS13 to afford
the vinyl bromides 12 in 25–71% yield.
2. Design and synthesis
At the outset of the design of the new 1,25-D3 analogs,
the lactam skeleton was chosen as a key component to
install in the side chain, since related analogs having a
lactone skeleton on their side chain are considered to be
promising structures for binding to the VDR.9 We thus
newly designed the DLAM series, in which a variety of
substituents can be installed on the nitrogen atom of the
lactam. These substituents were expected to sterically
influence the position of helix-12 so as to inhibit the
folding (e.g., having bulky group like benzyl group 5) or
the induce mis-folding (e.g., having less bulky group like
methyl group 4), which would result in VDR antago-
nistic activity. It had also aroused our interest that few
nitrogen-containing 1,25-D3 analogs with biological
activities have yet been reported.10
In contrast, the A-ring synthon 2014 was prepared from
the triol 13 derived from (S)-malic acid (Scheme 2).
Reaction of the triol 13 with p-anisaldehyde dimethyl-
acetal in the presence of a catalytic amount of cam-
phorsulfonic acid gave the six-membered benzylidene
alcohol 14 in 59% yield. After oxidation of the primary
alcohol of 14 under Swern conditions, the aldehyde
generated was reacted with the Wittig reagent to give 15
in 73% yield. The p-methoxybenzylidene acetal of 15
was selectively reduced with DIBAH at 0 °C to afford
the primary alcohol 16 in 70% yield. The oxidation of
the primary alcohol of 16 with Dess–Martin reagent15
followed by reaction with propargylmagnesium bromide
gave 17 in 67% yield as a 1:1 diastereomeric mixture.
The reaction of 17 with DDQ and subsequent purifica-
tion by silica gel column chromatography provided the
diol 18 and acetal 19 in 25% and 26% yields, respec-
The syntheses of DLAM-01 (4) and DLAM-1P (5) are
depicted in Schemes 1–3. The CD-ring synthon 12
was synthesized from vitamin D2 (6) via the Inhoffen–
O
N
OH
H
CHO
g
Me
H
H
h
a, b
c - f
Vitamin D2 (6)
H
H
H
HO
TBDMSO
TBDMSO
7
8
9
23
23
OTMS
CO2Me
i, j
OH
25
25
H
N
H
H
N
Me
O
N
k, l, m
O
Me
O
Me
H
H
H
TBDMSO
HO
Br
11a-d
10a; 23S, 25S
10b; 23R, 25R
10c; 23S, 25R
10d; 23R, 25S
12a; 23S, 25S
12b; 23R, 25R
12c; 23S, 25R
12d; 23R, 25S
Scheme 1. Reagents and conditions: (a) O3, CH2Cl2, ꢁ78 °C; (b) NaBH4, MeOH, 0 °C, 90% (two steps); (c) TsCl, DMAP, CH2Cl2, 0 °C; (d)
TBDMSOTf, 2,6-lutidine, CH2Cl2, 0 °C; (e) NaCN, DMSO, 90 °C; (f) DIBAL-H, CH2Cl2, 0 °C, 41% (four steps); (g) MeNHOHÆHCl, Et3N, CH2Cl2;
(h) methyl methacrylate, toluene, 110 °C, then HPLC separation, 10a (26%), 10b (28%), 10c (10%), 10d (10%); (i) p-TsOH, MeOH; (j) Pd/C, H2, 80%
(two steps); (k) TPAP, NMO, MS4A, CH2Cl2; (l) TMS-Im, CH2Cl2; (m) BrCH2PPh3ÆBr, LiHMDS, toluene, ꢁ78 °C, 12a (25%), 12b (42%), 12c
(49%), 12d (71%) (three steps).

Y. Kato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2579–2583
MP MP
2581
MP
OH
OH OH
O
O
O
O
a
O
d
b,c
HO
HO
13
16
14
MP = methyoxyphenyl
15
23
MP
OH
OH
25
H
N
OR OR
O
g, h
j, k
e, f
O
Me
H
17
18: R = H
i
20: R = TBS
MP
RO
OR
O
O
4a: 23S, 25S
4b: 23R, 25R
4c: 23S, 25R
4d: 23R, 25S
21a: R = TBS
4a : R =H
19
Scheme 2. Reagents and conditions: (a) p-anisaldehyde dimethylacetal, CSA, CH2Cl2, 59%; (b) (COCl)2, DMSO, Et3N, CH2Cl2, ꢁ78 °C; (c)
PPh3CH3Br, n-BuLi, THF, 0 °C, 73% (two steps); (d) DIBAH, CH2Cl2, 0 °C, 70%; (e) Dess–Martin reagent, CH2Cl2, 89%; (f) propargylmagnesium
bromide, Et2O, 0 °C, 67%; (g) DDQ, CH2Cl2; (h) silica gel, 18 (25%), 19 (26%); (i) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C, 99%; (j) 12a–d,
Pd2(dba)3CHCl3, Et3N, 100 °C, 21a (62%), 21b (90%), 21c (43%), 21d (53%); (k) HF-Py, THF, 4a (53%), 4b (73%), 4c (67%), 4d (98%).
O
N
taining 0.3 M KCl and 5 mM dithiothreitol just before
use. The receptor solution (500 lL, 0.35 mg protein) was
pre-incubated with 5 lL of ethanol solution of 1 or an
analog at various concentrations for 60 min at 25 °C.
Then, the receptor mixture was left to stand for 24 h
with 0.1 nM [3H]-1,25-D3 (Amersham) at 4 °C. The
bound and free [3H]-1,25-D3 were separated by treat-
ment with dextran-coated charcoal for 30 min at 4 °C
and centrifuged at 3000 rpm for 10 min. The radioac-
tivity of the supernatant (500 lL) with Atomlight
(PerkinElmer) was then counted. The relative binding
affinities of DLAM derivatives to VDR are summarized
in Table 1. The (23S,25S)-DLAM-01 (4a) and
(23S,25S)-DLAM-1P (5a) bound strongly to VDR,
while (23S,25R)-DLAM-01 (4c) and (23R,25S)-DLAM-
01 (4d) did not bind to VDR in the concentration range
examined.
Bn
H
a
8
H
TBDMSO
24
steps
(23S, 25S)-DLAM-1P (5a)
(23R, 25R)-DLAM-1P (5b)
Scheme 3. Reagents and conditions: (a) BnNHOHÆHCl, Et3N,
CH2Cl2.
tively. The diol 18 was reacted with TBSOTf to give 20
in 99% yield. The coupling reaction of 20 with 12a–d was
performed under palladium-catalyzed reaction condi-
tions14 to give 21a–d in 43–90% yield. The silyl ethers
were deprotected with HF-pyridine to give DLAM-01
(4a–d) in 53–98% yield. DLAM-1P (5) was also syn-
thesized similarly using N-benzylhydroxylamine
(Scheme 3). In this case, only (23S,25S)-5a and
(23R,25R)-5b were isolated after HPLC purification at
the final stage. These stereochemistries were determined
Next we investigated the ability of DLAMs to induce
HL-60 cell differentiation, a typical agonistic activity of
1,25-D3 (1), by way of the NBT reducing activity
method.18 Among the DLAM series, (23R,25R)-
DLAM-1P (5b) induced cell differentiation apparently at
a high concentration of 10ꢁ5 M. On the other hand, 4a
and 5a, which have strong affinity for the VDR, scarcely
by comparison with the H NMR spectra of 4a–d.16
1
Table 1. Relative binding affinities to VDR
Analog
IC50 (nM)
3. Results and discussion
1
0.5
4a
4b
4c
4d
5a
5b
10.2
50
The relative binding affinities of the DLAM 4a–d and
5a–b to the chick intestinal VDR were examined. A
competitive receptor binding assay for the synthesized
compounds was performed using VDR from chick
intestine as described previously.17 Chick intestinal 1,25-
D3 receptor was obtained from Yamasa Biochemical
and dissolved in 0.05 M phosphate buffer (pH 7.4) con-
>100
>100
1.9
30
IC50 values determined by in vitro competition binding to chick
intestinal VDR versus [3H]-1.

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Y. Kato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2579–2583
suggested to be important for antagonistic activity, and
further investigations of SAR studies are in progress.
120
100
80
60
40
20
0
Acknowledgements
The authors are grateful to Dr. K. Takenouchi and Dr.
S. Ishizuka (Teijin Pharma) for their generous supply of
TEI-9647. The authors also thank Ms. Kawachi and
Prof. H. Kagechika for their technical support on bio-
logical experiments and their helpful discussions. The
work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from the ministry
of Education, Science, Sports and Culture, Japan, and
the fund from the Mochida Memorial Foundation for
Medicinal and Pharmaceutical Research.
1 (10-8 M)
-7
-7
10-7 3 x 10 10-6
10-7 3 x 10 10-6
References and notes
5a (M)
3 (M)
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Figure 1. Effects of 5a on 1-induced HL-60 cell differentiation as
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1,25-D3 (1). To elicit the antagonistic activity, the ꢁSꢀ
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crucial, and the bulkiness of the substituent on nitrogen
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viding the third example of 1,25-D3 antagonist analogs.
The configuration of the lactam structure and the
bulkiness of the substituents on nitrogen of DLAM are

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(dd, J ¼ 4:7, 12.8 Hz, 1H), 2.60 (dd, J ¼ 2:1, 12.8 Hz, 1H),
2.31 (m, 1H), 2.27 (dd, J ¼ 7:7, 13.3 Hz, 1H), 2.10–0.87
(m, 23H), 0.77 (d, J ¼ 6:4 Hz, 3H), 0.53 (s, 3H); m=z 534
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534.3501. Data for 5b: 1H NMR (CDCl3, 500 MHz) d
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J ¼ 11:1 Hz, 1H), 5.34 (s, 1H), 5.00 (s, 1H), 4.98 (d,
J ¼ 15:0 Hz, 1H), 4.43 (m, 1H), 4.23 (m, 1H), 4.06 (d,
J ¼ 15:0 Hz, 1H), 3.50 (m, 1H), 2.81 (dd, J ¼ 3:4, 12.4 Hz,
1H), 2.60 (dd, J ¼ 2:6, 13.3 Hz, 1H), 2.37 (dd, J ¼ 7:7,
13.3 Hz, 1H), 2.32 (m, 1H), 2.04–0.99 (m, 23H), 0.86 (d,
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calcd for C34H48NO4 534.3583, found 534.3492.
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1
16. Spectra data for 5a: H NMR (CDCl3, 500 MHz) d 7.40–
7.14 (m, 5H), 6.37 (d, J ¼ 11:1 Hz, 1H), 6.01 (d,
J ¼ 11:5 Hz, 1H), 5.33 (s, 1H), 5.02 (s, 1H), 4.44 (m,
1H), 4.23 (m, 1H), 4.08–3.97 (m, 2H), 3.49 (m, 1H), 2.82