Dramatic Enhancement of Antagonistic Activity on Vitamin D Receptor: A Double Functionalization of 1α-Hydroxyvitamin D3 26,23-Lactones

Article abstract of DOI:10.1021/ol035922w

(Equation Presented) The synthesis of novel vitamin D receptor antagonists, 24-methyl-1α-hydroxyvitamin D₃ 26,23-lactones, is reported. We found that the biological activities of the vitamin D₃ lactones were affected by the structure of the lactone part. Furthermore, introduction of a 2α-methyl group into the 24-methylvitamin D₃ lactones dramatically enhanced their anti-vitamin D activity.

Full text of DOI:10.1021/ol035922w

ORGANIC
LETTERS
2
003
Vol. 5, No. 25
859-4862
Dramatic Enhancement of Antagonistic
Activity on Vitamin D Receptor: A
Double Functionalization of
4
1
r-Hydroxyvitamin D 26,23-Lactones
3
† ‡ ‡ †,§
Nozomi Saito, Hiroshi Saito, Miyuki Anzai, Akihiro Yoshida,
†
‡
‡
‡
Toshie Fujishima, Kazuya Takenouchi, Daishiro Miura, Seiichi Ishizuka,
Hiroaki Takayama,†,| and Atsushi Kittaka*,†
Faculty of Pharmaceutical Sciences, Teikyo UniVersity, Kanagawa 199-0195, Japan,
and Teijin Institute for Bio-Medical Research, Tokyo 191-8512, Japan
akittaka@pharm.teikyo-u.ac.jp
Received October 1, 2003
ABSTRACT
The synthesis of novel vitamin D receptor antagonists, 24-methyl-1r-hydroxyvitamin D
activities of the vitamin D lactones were affected by the structure of the lactone part. Furthermore, introduction of a 2r-methyl group into the
4-methylvitamin D lactones dramatically enhanced their anti-vitamin D activity.
3
26,23-lactones, is reported. We found that the biological
3
2
3
The seco-steroid hormone 1R,25-dihydroxyvitamin D
3
(1),
the C2R position of 1 to exhibit unique biological profiles
5
-7
which is the potent metabolite of vitamin D , regulates
3
with superagonistic activity on VDR.
In particular,
calcium and phosphorus homeostasis as well as cell dif-
ferentiation and proliferation of various types of tumor
introduction of the 2R-methyl (1a) showed 4-fold higher
1
,2
(3) Evans, R. M. Science 1988, 240, 889.
cells. Most of the biological responses of 1 are mediated
by its specific receptor, vitamin D receptor (VDR), which
is a member of the nuclear receptor superfamily and acts
as the ligand-dependent gene transcription factor with
coactivatiors.3 Recently, we reported some modification of
(
4) Takeyama, K.; Masuhiro, Y.; Fuse, H.; Endoh, H.; Murayama, A.;
Kitanaka, S.; Suzawa, M.; Yanagisawa, J.; Kato, S. Mol. Cell Biol. 1999,
9, 1049.
5) (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.; Miura, D.; Chokki,
1
(
M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8, 151. (b) Konno, K.;
Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.; Chokki, M.; Ishizuka, S.;
Yamaguchi, K.; Kan, Y.; Kurihara, M.; Miyata, N.; Smith, C.; DeLuca, H.
F.; Takayama, H. J. Med. Chem. 2000, 43, 4247. (c) Suhara, Y.; Nihei,
K.-i.; Tanigawa, H.; Fujishima, T.; Konno, K.; Nakagawa, K.; Okano, T.;
Takayama, H. Bioorg. Med. Chem. Lett. 2000, 10, 1129. (d) Suhara, Y.;
Nihei, K.-i.; Kurihara, M.; Kittaka, A.; Yamaguchi, K.; Fujishima, T.;
Konno, K.; Miyata, N.; Takayama, H. J. Org. Chem. 2001, 66, 8760. (e)
Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.; Kurihara, M.;
Takayama, H. Org. Lett. 2000, 2, 2619.
,4
†
Teikyo University.
Teijin Institute for Bio-Medical Research.
Present address: The Noguchi Institute and Japan Chemical Innovation
‡
§
Institute (JCII), Itabashi-ku, Tokyo 173-0003, Japan.
|
Present address: Japan Industry Research Center, Minato-ku, Tokyo
1
05-0001, Japan.
1) (a) Vitamin D; Feldman, D., Glorieux, F. H., Pike, J. W., Eds.;
(
Academic Press: New York, 1997. (b) Ettinger, R. A.; Deluca, H. F. AdV.
(6) For our account, see: Takayama, H.; Kittaka, A.; Fujishima, T.;
Suhara, Y. In Vitamin D Analogues in Cancer PreVention and Therapy,
Recent Results in Cancer Research 164; Reichrath, J., Friedrich, M., Tilgen,
W., Eds.; Springer-Verlag: Berlin, Heidelberg, 2003; pp 289-317.
Drug Res. 1996, 28, 269.
(2) Bouillon, R.; Okamura, W. H.; Norman, A W. Endocr. ReV. 1995,
1
6, 200.
1
0.1021/ol035922w CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/08/2003

sion in human osteosarcoma cells10b and in HL-60 cells10d
induced by 1. Moreover, TEI-9647 (2) shows antagonistic
action on the genomic-mediated calcium metabolism regu-
1
0e
lated by 1 in vivo. Recently, TEI-9647 (2) has received
considerable attention because of its possibility to be a
potential agent to inhibit the enhanced bone resorption in
1
3
patients with Paget’s disease, which is known as the most
flagrant example of disordered bone remodeling and the
second most common bone disease after osteoporosis in
1
3g
Anglo-Saxons.
The unique structures and unprecedented biological pro-
files of 2 and 3 prompted us to investigate the structure-
Figure 1.
3
activity relationships of the vitamin D lactones from the
standpoint of searching for more potential anti-vitamin D
molecules. In particular, we focused on the influence of the
structure including the stereochemistry of the lactone moiety
on the VDR binding affinity and antagonistic activity, and
we planned to introduce a methyl group into the C24 position
on the lactone ring of 2 and 3 (4-7) (Figure 3).
binding affinity to VDR relative to the natural hormone 1
5
a,b
(Figure 1).
Although more than 2000 analogues of 1 have been
8
synthesized over the past few decades, only two different
types of antagonists have been described. In 1999, studies
on the modification of the side-chain structure based on the
9
1
R,25-dihydroxyvitamin D
3
26,23-lactone metabolite de-
rived from 1 led to the discovery of the TEI-9647 (2) and
TEI-9648 (3) (Figure 2).1
0,11
3
Both vitamin D analogues 2
Figure 3.
Figure 2.
For the synthesis of 24-methylvitamin D lactones 4-7,
3
we utilized A-ring/CD-ring coupling methodology.14 First,
we synthesized the CD-ring precursors, which have the
and 3, which have an R-methylene-γ-butyrolactone part on
the side chain, are the first specific antagonists of VDR-
mediated genomic action of 1.12 Namely, 2 and 3 inhibit
2
3,24-syn lactone moiety, using Cr-mediated syn-selective
1
5
allylation (Scheme 1). According to Oshima’s protocol,
1
0a
differentiation of human leukemia cells (HL-60 cells), as
well as 25-hydroxyvitamin D 24-hydroxylase gene expres-
(
11) The other type of VDR antagonists of 25-carboxylic esters
3
ZK159222 and ZK168281, see: (a) Herdick, M.; Steinmeyer, A.; Carlberg,
C. J. Biol. Chem. 2000, 275, 16506. (b) Bury, Y.; Steinmeyer, A.; Carlberg,
C. Mol. Pharmacol. 2000, 58, 1067. (c) Herdick, M.; Steinmeyer, A.;
Carlberg, C. Chem. Biol. 2000, 7, 885. (d) Toell, A.; Gonzalez, M. M.;
Ruf, D.; Steinmeyer, A.; Ishizuka, S.; Carlberg, C. Mol. Pharmacol. 2001,
59, 1478. (e) V a¨ is a¨ nen, S.; Per a¨ kyl a¨ , M.; K a¨ rkk a¨ inen, J. I.; Steinmeyer,
A.; Carlberg, C. J. Mol. Biol. 2002, 315, 229.
(7) This concept was also good for 19-nor 1; see: Ono, K.; Yoshida,
A.; Saito, N.; Fujishima, T.; Honzawa, S.; Suhara, Y.; Kishimoto, S.;
Sugiura, T.; Waku, K.; Takayama, H.; Kittaka, A. J. Org. Chem. 2003, 68,
7
407.
8) For a review on synthesis of vitamin D3 analogues, see: Zhu, G. D.;
Okamura, W. H. Chem. ReV. 1995, 95, 1877. See also ref 2.
9) (a) Ishizuka, S.; Ishimoto, S.; Norman, A. W. Biochemistry 1984,
3, 1473. (b) Ishizuka, S.; Ohba, T.; Norman, A. W. In Vitamin D:
(
(12) Carlberg, C. J. Cell. Biochem. 2003, 88, 274.
(
(13) For recent reports on Paget’s bone disease, see: (a) Menaa, C.;
Barsony, J.; Reddy, S. V.; Cornish, J.; Cundy, T.; Roodman, G. D. J. Bone
Miner. Res. 2000, 15, 228. (b) Kurihara, N.; Reddy, S. V.; Menaa, C.;
Anderson, D.; Roodman, G. D. J. Clin. InVest. 2000, 105, 607. (c) Menaa,
C.; Reddy, S. V.; Kurihara, N.; Maeda, H.; Anderson, D. Cundy, T.; Cornish,
J.; Singer, F. R.; Bruder, J. M.; Roodman, G. D. J. Clin. InVest. 2000, 105,
1833. (d) Leach, R. J.; Singer, F. R.; Roodman, G. D. J. Clin. Endo. Metab.
2001, 86, 24. (e) Reddy, S. V.; Kurihara, N.; Menaa, C.; Landucci, G.;
Forthal, D.; Koop, B. A.; Windle, J. J.; Roodmann, G. D. Endocrinology
2001, 142, 2898. (f) Friedrichs, W. E.; Reddy, S. V.; Bruder, J. M.; Cundy,
T.; Cornish, J.; Singer, F. R.; Roodman, G. D. J. Bone Miner. Res. 2002,
17, 145. (g) Reddy, S. V.; Kurihara, N.; Menaa, C.; Roodman, G. D. ReV.
Endocr. Metab. Disord. 2001, 2, 195.
2
Molecular, Cellular and Chemical Endocrinology; Norman, A. W., Schaefer,
K., Grigoleit, H. G., von Herrath, D., Eds.; Walter de Gruyter: Berlin, 1988;
pp 143-144.
(10) (a) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman,
A. W.; Ishizuka, S. J. Biol. Chem. 1999, 274, 16392. (b) Ozono, K.; Saito,
M.; Miura, D.; Michigami, T.; Nakajima, S.; Ishizuka, S. J. Biol. Chem.
1
999, 274, 32376. (c) Miura, D.; Manabe, K.; Gao, Q.; Norman, A. W.;
Ishizuka, S. FEBS Lett. 1999, 460, 297. (d) Ishizuka, S.; Miura, D.; Eguchi,
H.; Ozono, K.; Chokki, M.; Kamimura, T.; Norman, A. W. Arch. Biochem.
Biophys. 2000, 380, 92. (e) Ishizuka, S.; Miura, D.; Ozono, K.; Chokki,
M.; Mimura, H.; Norman, A. W. Endocrinol. 2001, 142, 59. (f) Ishizuka,
S.; Miura, D.; Ozono, K.; Saito, M.; Eguchi, H.; Chokki, M.; Norman, A.
W. Steroids 2001, 66, 227.
(14) Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114,
9836.
4860
Org. Lett., Vol. 5, No. 25, 2003

Scheme 1
Scheme 3
3
Construction of the vitamin D triene skeleton was
accomplished by Pd-catalyzed alkenylative cyclization of
enyne 17 with the above CD-ring precursors (10, 11, 14, or
2
aldehyde 8, which was obtained from vitamin D , and allylic
1
6
bromide 9 were reacted with a low-valent Cr complex
prepared from CrCl and LiAlH to produce two lactone
1
6); then, acid-mediated deprotection provided the desired
3
4
2
4-methylvitamin D
3
-26,23-lactones 4-7 (Scheme 4).
derivatives 10 and 11, whose stereochemistries on the C23
and C24 positions (based on steroidal numbering) were
determined by NOE experiments and modified Mosher’s
1
7
method, in a total 95% yield with a ratio of 1:1.2.
Scheme 4
On the other hand, the CD-ring precursor having 23,24-
anti lactone 14 was synthesized from the 23,24-syn lactone
10 (Scheme 2). That is, DIBAL-H reduction of 10 followed
Scheme 2
The receptor binding affinities and antagonistic activities
of the vitamin D 26,23-lactones 4-7 were evaluated, and
3
both biological activities were markedly affected by the
structure of the lactone ring on the side-chain part (Table
1
). Binding affinity to the chick intestinal VDR was
by selective protection of the primary hydroxyl group gave
2. Inversion of the secondary hydroxyl group of 12 was
achieved by TPAP oxidation followed by LiAlH(OtBu)
reduction, and alcohol 13 was obtained in good yield. Finally,
deprotection of the pivaloyl group and subsequent oxidation
gave 23,24-anti lactone 14.
1
8
examined as described previously. The affinity of (23S)-
lactone analogues increased to be 2.4-fold (4) and 1.8-fold
1
3
(5) more potent than that of 2 by introducing the C24 methyl
group. In the case of (23R)-lactones, introducing the (24S)-
methyl unit (6) into 3 raised the affinity to 1.7 times higher
compared with that of 3, and the affinity of (24R)-analogue
Another syn lactone 11 was also transformed into ketone
(7) was almost the same as that of 3. Next, the antagonistic
15 through the same reaction steps. Treatment of 15 with
activities of 4-7 were assessed by the NBT-reduction
method in terms of 50% inhibitory concetration (IC50) for
DIBAL-H followed by oxidation produced the CD-ring
precursor having an anti lactone 16 (Scheme 3).
1
9
(
15) Okuda, Y.; Nakatsukasa, S.; Oshima, K.; Nozaki, H. Chem. Lett.
(18) (a) Eisman, J. A.; Hamstra, A. J.; Kream, B. E.; DeLuca H. F. Arch.
Biochem. Biophys. 1976, 176, 235. (b) Inaba, M.; DeLuca, H. F. Biochim.
Biophys. Acta 1989, 1010, 20.
(19) Collins, S. J.; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C. J.
Exp. Med. 1979, 149, 969.
1
985, 481.
(
(
16) Drewes, S. E.; Hoole, R. F. A. Synth. Commun. 1985, 1067.
17) Details of the determination of the stereochemistry are given in the
Supporting Information.
Org. Lett., Vol. 5, No. 25, 2003
4861

activities than those of 2, and synthesized the corresponding
2
R-methyl derivatives 4a and 5a from 10 and 16 with enyne
Table 1. Biological Activities of 24-Methylvitamin D
Lactones 4-7
3
2
0
1
7a, respectively (Scheme 5).
Biological evaluation disclosed that modification of the
antagonistic activitiyb
(IC50, nM)
A-ring with the 2R-methyl group dramatically enhanced the
antagonistic activities of 24-methylvitamin D lactones (Table
). Namely, (23S,24S)-2R,24-dimethyl analogue 4a showed
compd
VDR binding affinitya
3
1
100
12
29
22
7
2
TEI-9647 (2)
4
5
8.3
3.7
3.2
Table 2. Biological Activities of 2R,24-Dimethylvitamin D
Lactones 4a and 5a
3
TEI-9648 (3)
107.0
160.0
51.0
6
7
12
5
compd VDR binding affinitya antagonistic activity (IC50, nM)
b
a
The potency of 1 is normalized to 100 (see the Supporting Information).
Antagonistic activity was assessed in terms of 50% inhibitory concentration
IC50) for differentiation of HL-60 cells induced by 10 nM of 1.
2
4
5
12
63
23
8.3
0.19
0.13
b
a
a
(
a
The potency of 1 is normalized to 100 (see the Supporting Information).
b
Antagonistic activity was assessed in terms of 50% inhibitory concentration
differentiation of HL-60 cells induced by 1 (10 nM).
Introduction of a 24-methyl group into (23S)-lactone 2
increased the activity 2.2-fold (4) and 2.5-fold (5), respec-
tively. While the antagonistic activity of (23R)-lactone having
the (24S)-methyl group (6) was the same as that of 3,
(
IC50) for differentiation of HL-60 cells induced by 10 nM of 1.
ca. 5 times stronger VDR binding affinity and 38 times
higher antagonistic activity than 2. Although binding affinity
to the VDR of 5 was minimally affected by introducing the
(
3
23R,24R)-24-methylvitamin D lactone (7) showed three
2
R-methyl unit (5a), it is noteworthy that such modification
times higher anti-D activity than 3.
Next, we turned our attention to double functionalization
of vitamin D lactone analogues. That is, we planned to
3
considerably increased the anti-D activity to ca. 62-fold
stronger than that of 2.
In summary, we have succeeded in the development of
highly potent VDR antagonists based on the C24- and C2R-
functionalization of 1R-hydroxyvitamin D 26,23-lactones.
3
introduce a 2R-methyl substituent, which was one of our
positive motifs for VDR binding affinity,5a,b,6 into the new
3
24-methylvitamin D lactones. We expected a high increase
We expect that these analogues with high anti-D activity
would contribute to understanding the mechanisms involved
in the expression of antagonistic activity on VDR as well as
to finding the seeds of new medicines for treating Paget’s
bone disease. Further synthetic and biological studies are in
progress in our group.
in VDR binding affinity and striking improvement of the
antagonistic activity to VDR through such modifications. On
the basis of the above results, we focused on (23S)-lactone
analogues 4 and 5, which showed stronger antagonistic
Scheme 5
Acknowledgment. We thank Miss J. Shimode and Miss
M. Kitsukawa (Teikyo University) for spectroscopic mea-
surements. This study was supported by Grants-in-Aid from
the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Supporting Information Available: Experimental pro-
cedure and spectral data for the synthesis of 4-8, 10-16,
4
a, and 5a. Protocols and charts of vitamin D receptor
binding assay and assay for HL-60 cell differentiation to test
antagonistic activity. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL035922W
(20) Suhara, Y.; Kittaka, A.; Kishimoto, S.; Calverly, M. J.; Fujishima,
T.; Saito, N.; Sugiura, T.; Waku, K.; Takayama, H. Bioorg. Med. Chem.
Lett. 2002, 12, 3255.
4862
Org. Lett., Vol. 5, No. 25, 2003