Design and efficient synthesis of 2α-(ω-hydroxyalkoxy)- 1α,25-dihydroxyvitamin D3 analogues, including 2-epi-ED-71 and their 20-epimers with HL-60 cell differentiation activity

Article abstract of DOI:10.1021/jo0491051

A concise and efficient synthetic approach to 2α-(ω- hydroxyalkoxy)-1α,25-dihydroxyvitamin D₃ (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2α-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification" of 1 based on 4a-c with C20-epimerization, affording 2α-(ω-hydroxyalkoxy)-20-epi-1α, 25-dihydroxyvitamin D₃ (20-epi-4a-c). All three 2α-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.

Full text of DOI:10.1021/jo0491051

Design a n d Efficien t Syn th esis of
2r-(ω-Hyd r oxya lk oxy)-1r,25-d ih yd r oxyvita m in D3 An a logu es,
In clu d in g 2-epi-ED-71 a n d Th eir 20-Ep im er s w ith HL-60 Cell
Differ en tia tion Activity
Nozomi Saito,† Yoshitomo Suhara,†,| Masaaki Kurihara,‡ Toshie Fujishima,†,#
Shinobu Honzawa,† Hitoshi Takayanagi,† Toshiro Kozono,§ Masahiko Matsumoto,§
Masayuki Ohmori,§ Naoki Miyata,‡, Hiroaki Takayama,† and Atsushi Kittaka*,†
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University,
Sagamiko, Kanagawa 199-0195, J apan, National Institute of Health Sciences, Setagaya-ku,
Tokyo 158-8501, J apan, and Chugai Pharmaceutical Company, Ltd.,
Gotemba, Shizuoka 412-8513, J apan
akittaka@pharm.teikyo-u.ac.jp
Received May 27, 2004
A concise and efficient synthetic approach to 2R-(ω-hydroxyalkoxy)-1R,25-dihydroxyvitamin D3 (4a -
c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the
construction of the 2R-modified A-ring precursors (11a -c). It was found that the best ligand for
the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding
affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in
the induction of HL-60 cell differentiation for 4a -c was almost the same or weaker than that of 1
despite the strong binding affinity for the VDR. Next, we were interested in the “double modification”
of 1 based on 4a -c with C20-epimerization, affording 2R-(ω-hydroxyalkoxy)-20-epi-1R,25-dihy-
droxyvitamin D3 (20-epi-4a -c). All three 2R-substituted 20-epi analogues of 1 (20-epi-4a -c)
exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding
domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a -c
showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher
than that of the natural hormone 1.
In tr od u ction
distribution of VDR makes this hormone a potentially
useful therapeutic agent for certain cancers, skin dis-
eases, and immune disorders, and in fact, 1 and some
synthetic analogues of 1 are clinically used in the
treatment of bone diseases, secondary hyperparathyroid-
ism, and psoriasis.3 Therefore, it is interesting to design
and synthesize analogues of 1 with high VDR affinity in
terms of new drug development. To investigate the
structure-activity relationships of the natural hormone,
we systematically developed the A-ring-modified ana-
logues, such as 2-methyl-,6 2R-alkyl-, and 2R-(ω-hydroxy-
alkyl)-1R,25(OH)2D3 (Figure 1).7-10
The physiologically active metabolite of vitamin D3,
1R,25-dihydroxyvitamin D3 [1R,25(OH)2D3, 1], is the
nuclear hormone that regulates cellular growth, dif-
ferentiation, and apoptosis in addition to its classical role
in calcium homeostasis and bone mineralization.1-4 Most
of the biological effects of 1R,25(OH)2D3 are considered
to be mediated via binding to the specific intracellular
receptor, vitamin D receptor (VDR), which belongs to the
nuclear receptor superfamily acting as a ligand-depend-
ent transcription factor with coactivators.5 Ubiquitous
One of the striking results was that 2R-methyl-1R,25-
(OH)2D3 (2a ) showed a VDR binding potency that was
4-fold higher than that of 1.6 This simple A-ring modifi-
cation afforded for the first time an analogue, having the
natural side chain, with a VDR binding activity signifi-
cantly higher than that of the parent hormone 1; as a
* To whom correspondence should be addressed. Phone: (+81) 426-
85-3713. Fax: (+81) 426-85-3713.
† Teikyo University.
‡ National Institute of Health Sciences.
§ Chugai Pharmaceutical Co., Ltd.
| Present address: Kobe Pharmaceutical University, Higashinada-
ku, Kobe 658-8558, J apan.
#
Present address: Tokushima Bunri University, Shido, Kagawa
769-2193, J apan.
(5) (a) Evans, R. M. Science 1988, 240, 889-895. (b) Yanagisawa,
J .; Yanagi, Y.; Masuhiro, Y.; Suzawa, M.; Watanabe, M.; Kashiwagi,
K.; Toriyabe, T.; Kawabata, M.; Miyazono, K.; Kato, S. Science 1999,
283, 1317-1321.
(6) (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.; Miura, D.;
Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8, 151-
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Chokki, M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.;
Miyata, N.; Smith, C.; DeLuca, F. H.; Takayama, H. J . Med. Chem.
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Present address: Graduate School of Pharmaceutical Sciences,
Nagoya City University, Mizuho-ku, Nagoya 467-8603, J apan.
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10.1021/jo0491051 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/29/2004
J . Org. Chem. 2004, 69, 7463-7471
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Saito et al.
as a promising candidate for the treatment of osteoporo-
sis.3,12 Although ED-71 shows high calcemic activity and
a long half-life in plasma due to its strong affinity for
vitamin D binding protein (DBP, twice the affinity of 1R,-
25(OH)2D3),12,13 its binding affinity for bovine thymus
VDR is weaker than that of the natural hormone (13-
93%).12 We anticipated that 2R-(ω-hydroxyalkoxy)-1R,-
25(OH)2D3 compounds (4a -c), including 2-epi-ED-71,
could be better ligands for VDR and have potent vitamin
D3 activities.14 Furthermore, we were interested in a
structural cross talk in the vitamin D skeleton toward
biological activity, between the A-ring and the CD-ring
side chains through VDR binding, which would affect the
biological activity profile of 1R,25(OH)2D3.7d,15 Among the
various synthetic 1R,25(OH)2D3 analogues, the side-chain
structure of 20-epi-1 is especially noteworthy because 20-
epi-1 possesses a more potent activity in cell differentia-
tion and an immunosuppressive effect than the natural
hormone, despite a practically unchanged calcemic activ-
ity.16 The VDR binding potency of 20-epi-1 relative to that
of 1R,25(OH)2D3 is 4-5 times higher.16b,17 Thus, 20-epi
analogues of 4a -c were also synthesized based on Trost’s
convergent method,18 and VDR binding affinities and the
inducing effects of HL-60 cell differentiation of these
compounds were evaluated to understand the details of
the structure-activity relationships of 1R,25(OH)2D3
analogues.
F IGURE 1. Structures of 1R,25-dihydroxyvitamin D3 (1) (and
its 2R-substituted analogues 2-4), ED-71, and 20-epi-1 (and
its 2R-substituted analogues 20-epi-2a and 20-epi-4a -c).
Resu lts a n d Discu ssion
result, 2a shows a marked potency of transactivation of
target genes, induction of HL-60 cell differentiation, and
elevation of rat serum calcium concentration.11 Elonga-
tion of the 2R-alkyl chain, as in 2b-d , however, caused
a decrease in the agonistic activity for VDR.7a In regard
to the modification with the 2R-(ω-hydroxyalkyl) group,
it was found that 3c with the 2R-(3-hydroxypropyl) group
on 1 best fits the cavity of the ligand binding domain
(LBD) of VDR among the 2R-hydroxyalkyl series of 3 and
the binding activity of 3c is 3-fold higher than that of 1.7
On the other hand, Chugai Pharmaceutical Co. Ltd.
developed 2â-(3-hydroxypropoxy)-1R,25(OH)2D3 (ED-71)
Syn th esis. Convergent synthesis, in particular, Trost’s
A-ring/CD-ring connective strategy,18 seemed to be most
useful for synthesizing our target molecules. For the
synthesis of A-ring precursor enynes 11a -c, we chose
D-glucose as a chiral template for the desired stereo-
chemistry (1R,2R,3â; steroidal numbering) of the A-ring.
Methyl R-D-glucoside was converted to the known epoxide
5,19 and the regiospecific ring opening20 by an appropriate
alkanediol12a at C3 under basic conditions gave the
altrose configuration, in which the chiralities of C2, C3,
(12) (a) Miyamoto, K.; Murayama, E.; Ochi, K.; Watanabe, H.;
Kubodera, N. Chem. Pharm. Bull. 1993, 41, 1111-1113. (b) Tsugawa,
N.; Nakagawa, K.; Kurobe, M.; Ono, Y.; Kubodera, N.; Ozono, K.;
Okano, T. Biol. Pharm. Bull. 2000, 23, 66-71. (c) Ono, Y.; Watanabe,
H.; Shiraishi, A.; Takeda, S.; Higuchi, Y.; Sato, K.; Tsugawa, N.; Okano,
T.; Kobayashi, T.; Kubodera, N. Chem. Pharm. Bull. 1997, 45, 1626-
1630. (d) Ono, Y.; Watanabe, H.; Kawase, A.; Kubodera, N. Bioorg.
Med. Chem. Lett. 1994, 4, 1523-1526. For 19-nor and 2-(4-hydroxy-
butyl) analogues of ED-71, see: (e) Sicinski, R. R.; Perlman, K. L.;
DeLuca, H. F. J . Med. Chem. 1994, 37, 3730-3738. (f) Posner, G. H.;
J ohnson, N. J . Org. Chem. 1994, 59, 7855-7861.
(13) For the crystal structure of the DBP-25-hydroxyvitamin D3
complex, see: Verboven, C.; Rabijns, A.; De Maeyer, M.; Van Baelen,
H.; Bouillon, R.; De Ranter, C. Nat. Struct. Biol. 2002, 9, 131-136.
(14) We have reported a part of this work as a communication in
the following: Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.;
Kurihara, M.; Takayama, H. Org. Lett. 2000, 2, 2619-2622.
(15) Fujishima, T.; Liu, Z.-P.; Miura, D.; Chokki, M.; Ishizuka, S.;
Konno, K.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8, 2145-
2148.
(16) (a) Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.; Calverley,
M.; Hansen, K. Biochem. Pharmacol. 1991, 42, 1569-1575. (b) Dil-
worth, F. J .; Calverley, M. J .; Makin, H. L. J .; J ones, G. Biochem.
Pharmacol. 1994, 47, 987-993.
(17) Fujishima, T.; Konno, K.; Nakagawa, K.; Kurobe, M.; Okano,
T.; Takayama, H. Bioorg. Med. Chem. 2000, 8, 123-134.
(18) Trost, B. M.; Dumas, J .; Villa, M. J . Am. Chem. Soc. 1992, 114,
9836-9845.
(19) Wiggins, L. S. Methods Carbohydr. Chem. 1963, 2, 188-191.
(20) Beau, J .-M.; Aburaki, S.; Pougny, J .-R.; Sinay¨, P. J . Am. Chem.
Soc. 1983, 105, 621-622.
(7) (a) Suhara, Y.; Nihei, K.; Tanigawa, H.; Fujishima, T.; Konno,
K.; Nakagawa, K.; Okano, T.; Takayama, H. Bioorg. Med. Chem. Lett.
2000, 10, 1129-1132. (b) Suhara, Y.; Nihei, K.; Kurihara, M.; Kittaka,
A.; Yamaguchi, K.; Fujishima, T.; Konno, K.; Miyata, N.; Takayama,
H. J . Org. Chem. 2001, 66, 8760-8771. (c) Suhara, Y.; Kittaka, A.;
Kishimoto, S.; Calverley, M. J .; Fujishima, T.; Saito, N.; Sugiura, T.;
Waku, K.; Takayama, H. Bioorg. Med. Chem. Lett. 2002, 12, 3255-
3258. (d) Honzawa, S.; Suhara, Y.; Nihei, K.; Saito, N.; Kishimoto, S.;
Fujishima, T.; Kurihara, M.; Sugiura, T.; Waku, K.; Takayama, H.;
Kittaka, A. Bioorg. Med. Chem. Lett. 2003, 13, 3503-3506.
(8) Takayama, H.; Kittaka, A.; Fujishima, T.; Suhara, Y. In Vitamin
D Analogs in Cancer Prevention and Therapy; Reichrath, J ., Friedrich,
M., Tilgen, W., Eds.; Recent Results in Cancer Research 164; Springer-
Verlag: Berlin, 2003; pp 289-317.
(9) For 19-nor 1: (a) Yoshida, Y.; Ono, K.; Suhara, Y.; Saito, N.;
Takayama, H.; Kittaka, A. Synlett 2003, 1175-1179. (b) Ono, Y.;
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, 7407-7415.
(10) For VDR antagonists: (a) Saito, N.; Saito, H.; Anzai, M.;
Yoshida, A.; Fujishima, T.; Takenouchi, K.; Miura, D.; Ishizuka, S.;
Takayama, H.; Kittaka, A. Org. Lett. 2003, 5, 4859-4862. (b) Saito,
N.; Matsunaga, T.; Fujishima, T.; Anzai, M.; Saito, H.; Takenouchi,
K.; Miura, D.; Ishizuka, S.; Takayama, H.; Kittaka, A. Org. Biomol.
Chem. 2003, 1, 4396-4402. (c) Fujishima, T.; Kojima, Y.; Azumaya,
I.; Kittaka, A.; Takayama, H. Bioorg. Med. Chem. 2003, 11, 3621-
3631.
(11) Nakagawa, K.; Kurobe, M.; Ozono, K.; Konno, K.; Fujishima,
T.; Takayama, H.; Okano, T. Biochem. Pharmacol. 2000, 59, 691-702.
7464 J . Org. Chem., Vol. 69, No. 22, 2004

Design and Synthesis of 2-epi,20-epi-ED-71 Analogues
SCHEME 1. Syn th esis of th e A-Rin g P r ecu r sor sa
and C4 satisfy the 3â, 2R, and 1R stereochemistries of
the target molecules 4, respectively. Treatment of epoxide
5 with 1,2-ethanediol, 1,3-propanediol, or 1,4-butanediol
in the presence of KOtBu with heat followed by O-
silylation afforded protected methyl 3-O-(ω-hydroxy-
alkoxy)altropyranosides 6a -c in 81-90% yield. NBS
treatment21 of benzylidene acetals 6a -c gave bromides
7a -c in 75-91% yield. Previously, we exchanged the
resulting benzoyl group of 7 with the TBS group;14
however, the ester group is resistant to the later steps
being exposed under basic conditions, and it has made
the process three steps shorter. Reaction of bromides
7a -c with activated zinc powder and NaBH3CN provided
alcohols 8a -c in 70-86% yield.22 The diols were con-
verted to epoxides 9a -c through sulfonylation of the
primary alcohol followed by LiHMDS treatment in 60-
70% yield. Ethynylation of 9a -c using lithium TMS
acetylide in the presence of BF3‚OEt2 in THF and
subsequent solvolysis in K2CO3/MeOH supplied enynes
10a -c in 90-93% yield. Persilylation with TBSOTf/
2,6-lutidine afforded the desired protected enynes
11a -c for the palladium coupling in excellent yield
(Scheme 1).
The B-seco steroidal structure was constructed by
Trost’s palladium-catalyzed alkylative cyclization with
bromoolefin of the CD-ring counterpart (12),18 and sub-
sequent deprotection furnished the target 2R-(ω-hydroxy-
alkoxy)-1R,25(OH)2D3 in 32-66% yield in two steps
(Scheme 2). Next, each enyne was connected to the 20-
epi-CD-ring counterpart (14), which was synthesized
from vitamin D2 by our reported method,17 in the same
manner to yield 20-epi analogues (20-epi-4a -c) in 45-
57% yield (Scheme 3). All analogues were purified with
reversed-phase HPLC (recycled) for biological evalua-
tions.
Biologica l Eva lu a tion s. The VDR binding affinities
and potencies of induction of HL-60 cell differentiation
of the newly synthesized analogues (4a -c and 20-epi-
4a -c) are summarized in Table 1 in comparison with
those of the natural hormone 1 and 20-epi-1.16,17 In the
VDR binding assays23 using the bovine thymus VDR, 4a
and 4b showed a greater binding affinity for the VDR
(entries 2 and 3) and 4b reaches a peak in these three
analogues with the natural side chain (20R).
a
Reagents: (a) HOCH2(CH2)nCH2OH, KOtBu, 110 °C; (b) TBS-
Cl, Et3N, DMAP, CH2Cl2; (c) NBS, BaCO3, CCl4, reflux; (d) Zn
powder, NaBH3CN, 1-propanol/H2O (9/1), 95 °C; (e) 2,4,6-tri-
methylbenzenesulfonyl chloride, pyridine; (f) LiHMDS, THF, -78
to 0 °C; (g) TMSCCH, BuLi, BF3‚OEt2, THF, -78 °C to room
temperature; (h) K2CO3, MeOH; (i) TBSOTf, 2,6-lutidine, CH2Cl2,
0 °C.
SCHEME 2. Syn th esis of
2r-(ω-Hyd r oxya lk oxy)-1r,25(OH)2D3
a
Dock in g Stu d ies. We investigated a three-dimen-
sional structure of 2R-(3-hydroxypropoxy) analogue 4b
docking in the VDR ligand binding domain (LBD) based
on the crystal structure established by Moras et al.24 All
of the important hydrogen bonds, which make the ligand
(21) (a) Hanessian, S. Methods Carbohydr. Chem. 1972, 6, 183-189.
(b) Hanessian, S.; Plessas, N. R. J . Org. Chem. 1969, 34, 1035-1044.
(22) (a) Bernotas, R. C.; Pezzone, M. A.; Ganem, B. Carbohydr. Res.
1987, 167, 305-311. (b) Bernet, B.; Vasella, A. Helv. Chim. Acta 1979,
62, 1990-2016.
(23) (a) Imae, Y.; Manaka, A.; Yoshida, N.; Ishimi, Y.; Shinki, T.;
Abe, E.; Suda, T.; Konno, K.; Takayama, H.; Yamada, S. Biochim.
Biophys. Acta 1994, 1213, 302-308. (b) Fujishima, T.; Liu, Z.-P.;
Konno, K.; Nakagawa, K.; Okano, T.; Yamaguchi, K.; Takayama, H.
Bioorg. Med. Chem. 2001, 9, 525-535.
(24) Rochel, N.; Wultz, J . M.; Mitschler, A.; Klaholz, B.; Moras, D.
Mol. Cell 2000, 5, 173-179.
a
Reagents: (a) catalyst (Ph3P)4Pd, Et3N/toluene (1/1), reflux,
n ) 0 (75%), 1 (52%), and 2 (69%); (b) Bu4NF, THF, n ) 0 (78%),
1 (61%), and 2 (96%).
(25) Modeled structures were constructed by molecular dynamics
simulation and molecular mechanics energy minimization. The 32
amino acid residues were used for calculations. All calculations were
performed by MacroModel version 6.5.
(26) Kubodera, N.; Miyamoto, K.; Akiyama, M.; Matsumoto, M.;
Mori, T. Chem. Pharm. Bull. 1991, 39, 3221-3224.
anchor in the LBD, between 1R-OH and both Ser-237 and
Arg-274, 3â-OH and both Tyr-143 and Ser-278, and 25-
OH and both His-305 and His-397 can remain as in the
original X-ray structure, and an additional hydrogen
J . Org. Chem, Vol. 69, No. 22, 2004 7465

Saito et al.
SCHEME 3. Syn th esis of
2r-(ω-Hyd r oxya lk oxy)-20-epi-1r,25(OH)2D3
a
F IGURE 3. Modeled structure of 20-epi-4a (red) with Moras’
X-ray structure of the VDR-20-epi-1 (green) complex.28
explained by a possibly weaker stability of the ligand
(4a -c)-VDR with coactivator(s) complex, which switches
on the process in transactivation of the target genes.27
As noted in the Introduction, 20-epi-1, itself, exhibits
a VDR affinity 4-5 times stronger than that of 1;
however, introduction of the 2R-(3-hydroxypropoxy) group,
which strengthens the affinity for VDR in the case of
having the 20R natural side chain, causes an affinity
weaker than that of 20-epi-1, while these are still better
ligands if compared to 1. The docking study utilizing
Moras’ crystal structure of the 20-epi-1-VDR complex28
explains the different positions of the two hydroxyls
(1R,3â) on the A-ring of 20-epi-4a in the LBD of the VDR
from the originals that would be located at the ideal
positions for the binding of 20-epi-1 (Figure 3).25 Similar
results were obtained when we synthesized 2R-(ω-hy-
droxyalkyl)-20-epi-1R,25-dihydroxyvitamin D3 derivatives
and tested the VDR binding affinity.7d When the 2R
substituent was introduced, which also strengthens VDR
binding affinity in the case of having the natural side
chain (20R), into the 20-epi analogues of 1, the binding
affinity to VDR decreased, compared with that of 20-epi-
1.7d
a
Reagents: (a) see ref 17; (b) catalyst (Ph3P)4Pd, Et3N/toluene
(1/1), reflux; (c) HF/MeCN, n ) 0 (48%), 1 (57%), and 2 (45%) in
two steps.
TABLE 1. Rela tive Bin d in g Affin ity for Bovin e Th ym u s
VDR a n d HL-60 Cell Differ en tia tion Activitya
compound
VDR
HL-60
1
4a
4b
4c
100
120
180
40
100
100
70
40
20-epi-1
20-epi-4a
20-epi-4b
20-epi-4c
400
260
165
100
1810
5820
2120
2770
a
The potency of 1 is normalized to 100. The data are the mean
of three separate experiments26 (see the Supporting Information).
HL-60 cell differentiation activity26 was markedly high
with the 20-epi series compared to that of the natural
side-chain analogues 4a -c (Table 1).
Con clu sion s
We have developed an efficient synthetic route to the
novel biologically active 2R-(ω-hydroxyalkoxy)-1R,25-
(OH)2D3 complexes (4a -c) and their 20-epi counterparts
through the new A-ring precursors derived from D-
glucose. It was found that the VDR binding affinities of
4a , 4b, 20-epi-4a , and 20-epi-4b are stronger than that
F IGURE 2. Molecular modeling of 4b in the LBD of VDR.24,25
bonding network from the C2R terminal hydroxyl group
to Asp-144 and Tyr-236 would be properly formed (Figure
2).
However, the inducing effect of HL-60 cell differentia-
tion26 was not correlated to the binding affinity, and it
was between 70 and 100% of that of 1 despite the
stronger affinity for the VDR (Table 1). It could be
(27) Masuno, H.; Yamamoto, K.; Wang, X.; Choi, M.; Ooizumi, H.;
Shinki, T.; Yamada, S. J . Med. Chem. 2002, 45, 1825-1834.
(28) Tocchini-Valentini, G.; Rochel, N.; Wultz, J . M.; Mitschler, A.;
Moras, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5491-5496.
7466 J . Org. Chem., Vol. 69, No. 22, 2004

Design and Synthesis of 2-epi,20-epi-ED-71 Analogues
which was obtained from 5 (2.5 g, 9.5 mmol), KOtBu (3.5 g, 31
mmol), and 1,4-butanediol (45 mL), was dissolved in CH2Cl2
(19 mL). To the solution were added Et3N (6.6 mL, 47 mmol),
TBSCl (2.9 g, 19 mmol), and DMAP (116 mg, 0.95 mmol), and
the mixture was stirred at room temperature for 19 h. After
the usual workup, the crude product was purified by column
chromatography on silica gel (hexane/AcOEt ) 3/1) to give 6c
of the natural hormone. We investigated the potency of
inducing HL-60 cell differentiation and found the two 2R-
(ω-hydroxyalkoxy)-1R,25(OH)2D3 compounds (4b,c) ex-
hibit a rather lower effect, and all their 20-epi counter-
parts (20-epi-4a -c) showed higher potency. So far, in
many cases, 20-epi analogues of 1 are more potent in cell
growth and differentiation than the corresponding com-
pounds with the natural C20 stereochemistry, and 2R-
substituted 20-epi-4a -c analogues are no exception. We
propose that double modification on the 2R position and
the side chain would provide for the design and develop-
ment of new B-seco steroidal drugs for the treatment of
rickets, osteoporosis, psoriasis, certain cancers, and so
forth.2-4 These results would contribute to the under-
standing of the detail of structure-activity relationships
on the A-ring with variations of the CD-ring side chain.
Further biological testing is underway in our laboratories.
(3.9 g, 86% in two steps) as a colorless oil: [R]20 +60.7° (c
D
7.7, CHCl3); IR (neat) 3472 cm-1; H NMR (400 MHz, CDCl3)
1
δ 0.03 (s, 6H), 0.88 (s, 9H), 1.57-1.65 (m, 4H), 1.82 (d, J ) 5.8
Hz, 1H), 3.40 (s, 3H), 3.58-3.64 (m, 3H), 3.70-3.78 (m, 2H),
3.80 (t, J ) 3.1 Hz, 1H), 3.96 (dd, J ) 9.5, 3.1 Hz, 1H), 4.01
(dd, J ) 6.1, 3.1 Hz, 1H), 4.28-4.33 (m, 2H), 4.59 (s, 1H), 5.55
(s, 1H), 7.34-7.37 (m, 3H), 7.46-7.49 (m, 2H); 13C NMR (100
MHz, CDCl3) δ -5.0, 18.5, 26.1, 26.6, 29.6, 55.7, 58.7, 63.1,
69.4, 70.2, 71.7, 76.2, 77.2, 102.0, 102.3, 126.1, 128.1, 128.9,
137.6; EI-LRMS m/z 468 (M+). EI-HRMS calcd for C24H40O7-
Si 468.2543. Found 468.2540.
Meth yl 4-O-Ben zoyl-6-br om o-3-O-[2-{(ter t-bu tyldim eth -
ylsilyl)oxy}eth yl]-6-d eoxy-r-D-a ltr op yr a n osid e (7a ). To a
solution of 6a (1.9 g, 4.3 mmol) in CCl4 (44 mL) were added
BaCO3 (518 mg, 2.6 mmol) and NBS (817 g, 4.6 mmol) at room
temperature, and the mixture was refluxed for 1 h. After the
mixture was filtered, to the filtrate were added 10% Na2S2O3
aqueous solution and saturated NaHCO3 aqueous solution. The
aqueous layer was extracted with Et2O. The combined organic
layer was washed with saturated NaCl aqueous solution, dried
over Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt ) 5/1) to
Exp er im en ta l Section
Meth yl 4,6-O-Ben zylid en e-3-O-[2-{(ter t-bu tyld im eth -
ylsilyl)oxy}eth yl]-r-D-a ltr op yr a n osid e (6a ). To a suspen-
sion of 5 (1.5 g, 5.7 mmol) in ethylene glycol (25 mL) was added
KOtBu (2.1 g, 19 mmol), and the mixture was stirred at 110
°C for 24 h. The mixture was diluted with CH2Cl2, and the
organic layer was washed with saturated NH4Cl aqueous
solution, saturated NaCl aqueous solution, dried over Na2SO4,
and concentrated. To a solution of the crude product (1.9 g) in
CH2Cl2 (14 mL) were added TBSCl (1.1 g, 7.5 mmol), Et3N
(2.4 mL, 17 mmol), and DMAP (210 mg, 1.7 mmol) at 0 °C,
and the mixture was stirred at room temperature for 3 h. To
the mixture was added water, and the aqueous layer was
extracted with Et2O. The organic layer was washed with
saturated NaCl aqueous solution, dried over Na2SO4, and
concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 5/1-3/1) to give
give 7a (2.0 g, 86%) as a colorless oil: [R]25 +25.4° (c 1.0,
D
CHCl3); IR (neat) 3459, 1724, 1361, 1095 cm-1; 1H NMR (400
MHz, CDCl3) δ 0.02 (s, 3H), 0.03 (s, 3H), 0.86 (s, 9H), 3.03 (br
s, 1H), 3.50 (s, 3H), 3.55-3.65 (m, 3H), 3.68-3.83 (m, 4H),
3.99 (dd, J ) 8.2, 3.7 Hz, 1H), 4.32 (m, 1H), 4.71 (d, J ) 8.0
Hz, 1H), 5.47 (dd, J ) 6.4, 4.3 Hz, 1H), 7.45 (dd, J ) 7.8, 7.8
Hz, 2H), 7.58 (dd, J ) 7.8, 7.8 Hz, 1H), 8.05 (d, J ) 7.8 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ -5.2, 18.5, 26.0, 32.9, 56.0,
62.9, 69.6, 70.5, 70.6, 72.6, 77.9, 103.0, 128.3, 129.4, 129.7,
t
133.2, 165.6; EI-LRMS m/z 461 (M - Bu)+, 429, 308, 179,
6a (2.0 g, 81% in two steps) as a colorless oil: [R]25 +58.4° (c
D
105. EI-HRMS calcd for C18H26O779BrSi (M - tBu) 461.0631.
1
0.92, CHCl3); IR (neat) 3480, 1649, 1095 cm-1; H NMR (400
Found 461.0627.
MHz, CDCl3) δ 0.05 (s, 3H), 0.06 (s, 3H), 0.89 (s, 9H), 1.90 (d,
J ) 6.1 Hz, 1H), 3.39 (s, 3H), 3.65-3.90 (m, 5H), 3.92 (dd, J )
2.9, 2.9 Hz, 1H), 3.96 (dd, J ) 8.9, 2.9 Hz, 1H), 4.05 (ddd, J )
5.9, 2.9, 0.7 Hz, 1H), 4.25-4.35 (m, 2H), 4.59 (s, 1H), 5.55 (s,
1H), 7.32-7.40 (m, 3H), 7.45-7.52 (m, 2H); 13C NMR (100
MHz, CDCl3) δ -5.2, -5.1, 18.5, 26.0, 55.6, 58.5, 63.1, 69.4,
70.3, 73.2, 77.0, 77.1, 101.9, 102.2, 126.1, 128.1, 128.8, 137.5;
EI-LRMS m/z 440 (M+), 351, 305, 259, 121. EI-HRMS calcd
for C22H36O7Si 440.2231. Found 440.2227.
Meth yl 4-O-Ben zoyl-6-br om o-3-O-[3-{(ter t-bu tyldim eth -
ylsilyl)oxy}p r op yl]-6-d eoxy-r-D-a ltr op yr a n osid e (7b). In
a manner similar to that for the synthesis of 7a from 6a , a
crude product, which was obtained from 6b (3.6 g, 7.9 mmol),
NBS (1.5 g, 8.3 mmol), and BaCO3 (940 mg, 4.8 mmol) in CCl4
(79 mL), was purified by column chromatography on silica gel
(hexane/AcOEt ) 5/1) to give 7b (3.8 g, 91%) as a colorless oil:
[R]20D +46.0° (c 4.2, CHCl3); IR (neat) 1725 cm-1; 1H NMR (400
MHz, CDCl3) δ -0.04 (s, 3H), -0.03 (s, 3H), 0.83 (s, 9H), 1.67-
1.73 (m, 2H), 2.52 (br s, 1H), 3.50 (s, 3H), 3.55-3.70 (m, 6H),
3.73 (dd, J ) 7.3, 4.0 Hz, 1H), 3.97 (dd, J ) 7.3, 3.3 Hz, 1H),
4.35 (dt, J ) 3.7, 7.0 Hz, 1H), 4.70 (d, J ) 3.3 Hz, 1H), 5.45
(dd, J ) 7.0, 4.0 Hz, 1H), 7.43-7.47 (m, 2H), 7.58 (m, 1H),
8.03-8.08 (m, 2H); 13C NMR (100 MHz, CDCl3) δ -5.5, 18.2,
25.9, 32.7, 32.9, 55.9, 60.0, 67.9, 69.3, 69.8, 69.9, 76.9, 102.7,
Meth yl 4,6-O-Ben zylid en e-3-O-[3-{(ter t-bu tyld im eth -
ylsilyl)oxy}p r op yl]-r-D-a ltr op yr a n osid e (6b). In a manner
similar to that for the synthesis of 6a from 5, a crude product,
which was obtained from 5 (1.4 g, 5.1 mmol), KOtBu (1.9 g, 17
mmol), and 1,3-propanediol (25 mL), was dissolved in CH2Cl2
(20 mL). To the solution were added Et3N (2.1 mL, 15 mmol),
TBSCl (1.2 g, 7.6 mmol), and DMAP (63 mg, 0.51 mmol), and
the mixture was stirred at room temperature for 3 h. After
the usual workup, the crude product was purified by column
chromatography on silica gel (hexane/AcOEt ) 5/1-2/1) to give
t
128.5, 129.5, 129.8, 133.3, 165.7; EI-LRMS m/z 475 (M - -
Bu)+. EI-HRMS calcd for C19H2879BrO7Si (M - tBu)+ 475.0787.
Found 475.0786.
6b (2.1 g, 90% in two steps) as a colorless oil: [R]20 +68.8° (c
Meth yl 4-O-Ben zoyl-6-br om o-3-O-[4-{(ter t-bu tyldim eth -
ylsilyl)oxy}bu tyl]-6-d eoxy-r-D-a ltr op yr a n osid e (7c). In a
manner similar to that for the synthesis of 7a from 6a , a crude
product, which was obtained from 6c (1.0 g, 2.1 mmol), NBS
(460 mg, 2.6 mmol), and BaCO3 (240 mg, 1.2 mmol) in CCl4
(25 mL), was purified by silica gel column chromatography on
silica gel (hexane/AcOEt ) 5/1) and gave 7c (874 mg, 75%) as
D
3.9, CHCl3); IR (neat) 3463 cm-1; H NMR (400 MHz, CDCl3)
1
δ 0.02 (s, 3H), 0.03 (s, 3H), 0.87 (s, 9H), 1.78-1.82 (m, 2H),
1.93 (br s, 1H), 3.39 (s, 3H), 3.65-3.80 (m, 6H), 3.95 (dd, J )
8.8, 2.7 Hz, 1H), 3.99-4.00 (m, 1H), 4.26-4.33 (m, 2H), 4.89
(s, 1H), 5.55 (s, 1H), 7.34-7.37 (m, 3H), 7.47-7.49 (m, 2H);
13C NMR (100 MHz, CDCl3) δ -5.4, 18.3, 25.9, 33.3, 55.5, 58.6,
60.2, 68.5, 69.4, 70.0, 76.4, 76.9, 102.0, 102.3, 126.0, 126.2,
128.2, 129.0, 137.7; EI-LRMS m/z 454 (M+). EI-HRMS calcd
for C23H38O7Si 454.2387. Found 454.2387.
a colorless oil: [R]20 +36.0° (c 7.7, CHCl3); IR (neat) 3465,
D
1725 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.01 (s, 6H), 0.86 (s,
9H), 1.46-1.62 (m, 4H), 2.37 (d, J ) 3.7 Hz, 1H), 3.51 (s, 3H),
3.53-3.65 (m, 6H), 3.75 (dd, J ) 4.0, 7.3 Hz, 1H), 3.99 (dt, J
) 3.7, 7.3 Hz, 1H), 4.38 (dt, J ) 3.7, 6.7 Hz, 1H), 4.71 (d, J )
3.7 Hz, 1H), 5.47 (dd, J ) 6.7, 4.0 Hz, 1H), 7.45-7.48 (m, 2H),
Meth yl 4,6-O-Ben zylid en e-3-O-[4-{(ter t-bu tyld im eth -
ylsilyl)oxy}bu tyl]-r-D-a ltr op yr a n osid e (6c). In a manner
similar to that for the synthesis of 6a from 5, a crude product,
J . Org. Chem, Vol. 69, No. 22, 2004 7467

Saito et al.
7.60 (m, 1H), 8.04-8.07 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ -5.1, 18.3, 26.1, 26.4, 29.4, 32.9, 56.1, 62.8, 69.0, 69.9, 70.7,
76.9, 102.6, 128.4, 129.3, 129.7, 133.3, 165.5; EI-LRMS m/z
546 (M+). EI-HRMS calcd for C24H3979BrO7Si 546.1648. Found
546.1640.
tylenesulfonyl chloride (TmCl) (224 mg, 1.0 mmol) at 0 °C, and
the mixture was stirred at room temperature for 24 h. To the
mixture was added water, and the aqueous layer was extracted
with Et2O, washed with saturated NaCl aqueous solution,
dried over Na2SO4, and concentrated. The crude product was
dissolved in THF (9.3 mL). To the solution was added LiHMDS
(1.0 M solution in THF, 1.1 mL, 1.1 mmol) at -78 °C, and the
mixture was allowed to warm to 0 °C over 1 h. After the
mixture was stirred at 0 °C for 30 min, to the mixture was
added saturated NH4Cl aqueous solution. The aqueous layer
was extracted with Et2O. The organic layer was washed with
saturated NaCl aqueous solution, dried over Na2SO4, and
concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 20/1) to give
(2R,3S,4R)-4-Ben zoyloxy-3-[2-(ter t-bu tyld im eth ylsilyl-
oxy)eth oxy]h ex-5-en e-1,2-d iol (8a ). To a solution of 7a (2.8
g, 5.3 mmol) in 1-propanol/H2O (9/1, 53 mL) were added
activated Zn dust (24 g, 373 mmol) and NaBH3CN (3.7 g, 59
mmol) at 95 °C, and the mixture was stirred at the same
temperature for 1 h. After the mixture was filtered, to the
filtrate was added saturated NH4Cl aqueous solution, and the
aqueous layer was extracted with AcOEt. The organic layer
was washed with saturated NaCl aqueous solution, dried over
Na2SO4, and concentrated. The residue was purified by flash
column chromatography on silica gel (hexane/AcOEt ) 4/1-
9a (255 mg, 70% in two steps) as a colorless oil: [R]18 +33.0°
D
(c 1.1, CHCl3); IR (neat) 1725, 1647, 1603, 1269, 1109 cm-1
;
1H NMR (400 MHz, CDCl3) δ 0.05 (s, 6H), 0.88 (s, 9H), 2.65
(dd, J ) 4.9, 2.8 Hz, 1H), 2.77 (dd, J ) 4.6, 4.6 Hz, 1H), 3.12
(ddd, J ) 6.6, 3.8, 2.8 Hz, 1H), 3.29 (dd, J ) 6.6, 5.3 Hz, 1H),
3.65-3.85 (m, 4H), 5.31 (d, J ) 10.5 Hz, 1H), 5.40 (d, J ) 17.3
Hz, 1H), 5.68 (dd, J ) 6.1, 5.6 Hz, 1H), 6.05 (ddd, J ) 17.3,
10.5, 6.1 Hz, 1H), 7.45 (dd, J ) 7.8, 7.8 Hz, 2H), 7.58 (dd, J )
7.8, 7.8 Hz, 1H), 8.06 (d, J ) 7.8 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ -5.2, 18.4, 26.0, 43.6, 52.1, 62.7, 72.3, 74.8, 82.6,
118.1, 128.3, 129.5, 129.7, 132.8, 133.0, 165.0; EI-LRMS m/z
392 (M+), 376, 335, 305, 179, 105. EI-HRMS calcd for
2/1) to give 8a (1.5 g, 70%) as a colorless oil: [R]25 +1.47° (c
D
2.3, CHCl3); IR (neat) 3438, 1719, 1649, 1271, 1109 cm-1; H
1
NMR (400 MHz, CDCl3) δ 0.09 (s, 6H), 0.91 (s, 9H), 2.51 (m,
1H), 2.61 (br s, 1H), 3.62 (ddd, J ) 10.6, 8.4, 3.0 Hz, 1H), 3.68-
3.88 (m, 6H), 4.03 (ddd, J ) 10.6, 3.7, 3.2 Hz, 1H), 5.35 (d, J
) 10.6 Hz, 1H), 5.44 (d, J ) 17.2 Hz, 1H), 5.68 (dd, J ) 6.7,
3.9 Hz, 1H), 6.07 (ddd, J ) 17.2, 10.6, 6.7 Hz, 1H), 7.45 (dd, J
) 7.8, 7.8 Hz, 2H), 7.58 (dd, J ) 7.8, 7.8 Hz, 1H), 8.05 (d, J )
7.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ -5.2, 18.5, 26.0,
62.9, 63.4, 71.2, 74.4, 75.2, 82.5, 119.2, 128.3, 129.5, 132.4,
t
133.0, 165.2; EI-LRMS m/z 353 (M - Bu)+, 278, 219, 105.
C
21H32O5Si 392.2019. Found 392.2016.
t
EI-HRMS calcd for C17H25O6Si (M - Bu)+ 353.1420. Found
(3R,4R,5R)-3-Ben zoyloxy-4-[3-(ter t-bu tyld im eth ylsilyl-
353.1430.
oxy)p r op oxy]-5,6-ep oxyh ex-1-en e (9b). In a manner similar
to that for the synthesis of 9a from 8a , a crude product, which
was obtained from 8b (2.5 g, 5.8 mmol) and TmCl (1.5 g, 6.7
mmol) in pyridine (5.8 mL), was dissolved in THF (48 mL).
To the solution was added LiHMDS (1.0 M solution in THF,
7.2 mL, 7.2 mmol) at -78 °C, and the mixture was allowed to
warm to 0 °C over 1 h. After the usual workup, the crude
product was purified by flash column chromatography on silica
gel (hexane/AcOEt ) 30/1) to give 9b (1.4 g, 60% in two steps)
(2R,3S,4R)-4-Ben zoyloxy-3-[3-(ter t-bu tyld im eth ylsilyl-
oxy)p r op oxy]h ex-5-en e-1,2-d iol (8b). In a manner similar
to that for the synthesis of 8a from 7a , a crude product, which
was obtained from 7b (3.6 g, 6.8 mmol), activated zinc dust
(13 g, 203 mmol), and NaBH3CN (2.1 g, 34 mmol) in 1-pro-
panol/H2O (9/1, 68 mL), was purified by flash column chro-
matography on silica gel (hexane/AcOEt ) 3/1) to give 8b (2.5
g, 86%) as a colorless oil: [R]23D +26.1° (c 3.4, CHCl3); IR (neat)
3406, 1722, 1643, 1271, 1095 cm-1; 1H NMR (400 MHz, CDCl3)
δ 0.05 (s, 6H), 0.88 (s, 9H), 1.70-1.88 (m, 2H), 2.26 (br s, 1H),
3.05 (br s, 1H), 3.59-3.81 (m, 7H), 3.96 (dt, J ) 9.1, 5.7 Hz,
1H), 5.34 (ddd, J ) 10.6, 1.2, 1.2 Hz, 1H), 5.43 (ddd, J ) 17.3,
1.2, 1.2 Hz, 1H), 5.68 (dddd, J ) 6.3, 3.9, 1.2, 1.2 Hz, 1H),
6.05 (ddd, J ) 17.3, 10.6, 6.3 Hz, 1H), 7.45 (dd, J ) 7.9, 7.9
Hz, 2H), 7.58 (dd, J ) 7.8, 7.8 Hz, 1H), 8.05 (d, J ) 7.8 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ -5.2, -5.1, 18.4, 26.0, 33.0,
60.0, 63.7, 69.5, 71.1, 74.9, 81.0, 118.8, 128.3, 129.5, 129.8,
as a colorless oil: [R]19 +29.4° (c 4.8, CHCl3); IR (neat) 1724,
D
1651, 1601, 1269, 1109 cm-1 1H NMR (400 MHz, CDCl3) δ
;
0.03 (s, 6H), 0.87 (s, 9H), 1.80 (tt, J ) 6.4, 6.4 Hz, 2H), 2.62
(dd, J ) 4.9, 2.7 Hz, 1H), 2.76 (dd, J ) 4.6, 4.3 Hz, 1H), 3.10
(ddd, J ) 6.8, 3.9, 2.7 Hz, 1H), 3.18 (dd, J ) 6.8, 5.1 Hz, 1H),
3.60-3.75 (m, 3H), 3.83 (dt, J ) 9.5, 6.1 Hz, 1H), 5.31 (d, J )
10.7 Hz, 1H), 5.41 (d, J ) 17.3 Hz, 1H), 5.66 (dd, J ) 6.1, 5.1
Hz, 1H), 6.04 (ddd, J ) 17.3, 10.7, 5.1 Hz, 1H), 7.45 (dd, J )
7.8, 7.8 Hz, 2H), 7.58 (dd, J ) 7.8, 7.8 Hz, 1H), 8.05 (d, J )
7.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ -5.2, 18.3, 26.0,
33.2, 43.6, 52.2, 59.8, 67.5, 74.7, 82.4, 118.2, 128.2, 129.4, 129.8,
132.8, 132.9, 165.0; EI-LRMS m/z 406 (M+), 390, 349, 316,
179, 105. EI-HRMS calcd for C22H34O5Si 406.2175. Found
406.2177.
t
132.4, 133.1, 165.3; EI-LRMS m/z 367 (M - Bu)+, 293, 263,
t
233, 185, 105. EI-HRMS calcd for C18H27O6Si (M - Bu)+
367.1577. Found 367.1583.
(2R,3S,4R)-4-Ben zoyloxy-3-[4-(ter t-bu tyld im eth ylsilyl-
oxy)bu toxy]h ex-5-en e-1,2-d iol (8c). In a manner similar to
that for the synthesis of 8a from 7a , a crude product, which
was obtained from 7c (2.7 g, 4.8 mmol), activated zinc dust
(9.5 g, 145 mmol), and NaBH3CN (1.5 g, 24 mmol) in 1-pro-
panol/H2O (9/1, 48 mL), was purified by column chromatog-
raphy on silica gel (hexane/AcOEt ) 3/1) to give 8c (1.7 g, 81%)
(3R,4R,5R)-3-Ben zoyloxy-4-[4-(ter t-bu tyld im eth ylsilyl-
oxy)bu toxy]-5,6-ep oxyh ex-1-en e (9c). In a manner similar
to that for the synthesis of 9a from 8a , a crude product, which
was obtained from 8c (536 mg, 1.2 mmol) and TmCl (320 mg,
1.5 mmol) in pyridine (1.2 mL), was dissolved in THF (11 mL).
To the solution was added LiHMDS (1.0 M solution in THF,
1.2 mL, 1.2 mmol) at -78 °C, and the mixture was warmed to
0 °C over 1.5 h. After the usual workup, the crude product
was purified by flash column chromatography on silica gel
(hexane/AcOEt ) 25/1) to give 9c (328 mg, 64% in two steps)
as a colorless oil: [R]18 +21.4° (c 1.4, CHCl3); IR (neat) 3424,
D
1722, 1645, 1603, 1271, 1097 cm-1; 1H NMR (400 MHz, CDCl3)
δ 0.04 (s, 6H), 0.88 (s, 9H), 1.50-1.73 (m, 4H), 2.17 (m, 1H),
2.78 (d, J ) 4.9 Hz, 1H), 3.50-3.65 (m, 4H), 3.68-3.82 (m,
3H), 3.86 (ddd, J ) 9.0, 6.5, 6.5 Hz, 1H), 5.34 (d, J ) 10.6 Hz,
1H), 5.43 (d, J ) 17.2 Hz, 1H), 5.71 (dd, J ) 6.2, 4.4 Hz, 1H),
6.03 (ddd, J ) 17.2, 10.6, 6.4 Hz, 1H), 7.45 (dd, J ) 7.5, 7.5
Hz, 2H), 7.58 (dd, J ) 7.5, 7.5 Hz, 1H), 8.05 (d, J ) 7.5 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ -5.1, 18.5, 26.1, 26.7, 29.5,
62.8, 63.9, 70.9, 72.5, 74.8, 80.5, 118.8, 128.3, 129.6, 129.7,
as a colorless oil: [R]18 +31.4° (c 2.1, CHCl3); IR (neat) 1725,
D
1647, 1603, 1269, 1101 cm-1 1H NMR (400 MHz, CDCl3) δ
;
0.03 (s, 6H), 0.88 (s, 9H), 1.55-1.70 (m, 4H), 2.68 (dd, J ) 4.8,
2.7 Hz, 1H), 2.77 (dd, J ) 4.8, 3.9 Hz, 1H), 3.10 (ddd, J ) 6.9,
3.9, 2.7 Hz, 1H), 3.17 (dd, J ) 6.9, 5.1 Hz, 1H), 3.55-3.65 (m,
3H), 3.76 (dt, J ) 9.3, 5.9 Hz, 1H), 5.31 (ddd, J ) 10.6, 1.2,
1.2 Hz, 1H), 5.41 (ddd, J ) 17.2, 1.2, 1.2 Hz, 1H), 5.66 (dddd,
J ) 6.3, 5.1, 1.2, 1.2 Hz, 1H), 6.04 (ddd, J ) 17.2, 10.6, 6.3 Hz,
1H), 7.45 (dd, J ) 7.7, 7.7 Hz, 2H), 7.58 (dd, J ) 7.7, 7.7 Hz,
1H), 8.05 (d, J ) 7.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ
-5.1, 18.5, 26.1, 26.5, 29.5, 43.7, 52.4, 62.9, 71.0, 74.8, 82.4,
t
132.4, 133.1, 165.3; EI-LRMS m/z 381 (M - Bu)+, 259, 187,
179, 105. EI-HRMS calcd for C19H29O6Si (M - tBu)+ 381.1733.
Found 379.1726.
(3R,4R,5R)-3-Ben zoyloxy-4-[2-(ter t-bu tyld im eth ylsilyl-
oxy)eth oxy]-5,6-ep oxyh ex-1-en e (9a ). To a solution of 8a
(380 mg, 0.93 mmol) in pyridine (0.93 mL) was added mesi-
7468 J . Org. Chem., Vol. 69, No. 22, 2004

Design and Synthesis of 2-epi,20-epi-ED-71 Analogues
118.3, 128.3, 129.5, 129.8, 132.8, 133.0, 165.1; EI-LRMS m/z
420 (M+), 405, 363, 241, 217, 179, 105. EI-HRMS calcd for
1H), 3.38 (dd, J ) 4.8, 2.1 Hz, 1H), 3.55 (dt, J ) 9.1, 6.4 Hz,
1H), 3.63 (t, J ) 6.1 Hz, 2H), 3.75 (dt, J ) 9.1, 6.3 Hz, 1H),
5.62 (m, 1H), 4.48 (m, 1H), 5.27 (ddd, J ) 10.6, 1.6, 1.6 Hz,
1H), 5.43 (ddd, J ) 17.2, 1.6, 1.6 Hz, 1H), 5.93 (ddd, J ) 17.2,
10.6, 5.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ -5.12, 18.4,
23.8, 26.0, 26.6, 29.4, 62.8, 69.8, 70.3, 71.4, 72.4, 80.5, 80.7,
C
23H36O5Si 420.2232. Found 420.2235.
(3R,4S,5R)-4-[2-(ter t-Bu tyldim eth ylsilyloxy)eth oxy]oct-
1-en -7-yn e-3,5-d iol (10a ). To a solution of trimethylsilyl-
acetylene (0.35 mL, 2.5 mmol) in THF (2 mL) was added nBuLi
(1.5 M solution in hexane, 1.5 mL, 2.3 mmol) at -78 °C, and
the mixture was stirred at the same temperature for 30 min.
To the mixture were added a solution of 9a (384 mg, 0.98
mmol) in THF (6 mL) and BF3‚OEt2 (0.14 mL, 1.1 mmol) at
-78 °C, and the resulting mixture was warmed to 0 °C over 2
h. To the mixture was added saturated NH4Cl aqueous
solution, and the aqueous layer was extracted with AcOEt.
The organic layer was washed with saturated NaCl aqueous
solution, dried over Na2SO4, and concentrated. The residue was
dissolved in MeOH (3 mL). To the solution was added K2CO3
(406 mg, 2.9 mmol), and the mixture was stirred at room
temperature for 2 h. To the mixture was added H2O, and the
aqueous layer was extracted with AcOEt. The organic layer
was washed with saturated NaCl aqueous solution, dried over
Na2SO4, and concentrated. The residue was purified by flash
column chromatography on silica gel (hexane/AcOEt ) 4/1) to
t
116.2, 136.9; EI-LRMS m/z 285 (M - Bu)+, 211, 187, 147,
89. EI-HRMS calcd for C14H25O4Si (M - tBu)+ 285.1522.
Found 285.1525.
(3R,4S,5R)-3,5-Bis(ter t-bu tyldim eth ylsilyloxy)-4-[2-(ter t-
bu tyld im eth ylsilyloxy)eth oxy]oct-1-en -7-yn e (11a ). To a
solution of 10a (273 mg, 0.87 mmol) in CH2Cl2 (2.9 mL) were
added 2,6-lutidine (0.31 mL, 2.7 mmol) and TBSOTf (0.5 mL,
2.2 mmol) at 0 °C, and the mixture was stirred at the same
temperature for 3 h. To the mixture was added H2O, and the
aqueous layer was extracted with Et2O. The organic layer was
washed with saturated NaCl aqueous solution, dried over Na2-
SO4, and concentrated. The residue was purified by column
chromatography on silica gel (hexane/AcOEt ) 30/1) to give
11a (456 mg, 97%) as a colorless oil: [R]14 +0.24° (c 1.2,
D
CHCl3); IR (neat) 2124, 1647, 1255, 1098 cm-1; 1H NMR (400
MHz, CDCl3) δ 0.03 (s, 3H), 0.054 (s, 6H), 0.068 (s, 3H), 0.087
(s, 3H), 0.10 (s, 3H), 0.89 (s, 18H), 0.90 (s, 9H), 1.95 (t, J ) 2.7
Hz, 1H), 2.34 (ddd, J ) 16.8, 6.0, 2.7 Hz, 1H), 2.52 (ddd, J )
16.8, 5.4, 2.7 Hz, 1H), 3.40 (dd, J ) 5.3, 3.5 Hz, 1H), 3.61 (m,
1H), 3.71 (t, J ) 5.6 Hz, 2H), 3.78 (m, 1H), 3.90 (dd, J ) 5.6,
5.6 Hz, 1H), 4.32 (dddd, J ) 6.8, 3.7, 1.5, 1.2 Hz, 1H), 5.12
(ddd, J ) 10.3, 1.7, 1.2 Hz, 1H), 5.21 (ddd, J ) 17.3, 1.7, 1.5
Hz, 1H), 5.97 (ddd, J ) 17.3, 10.3, 6.8 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ -5.06, -5.00, -4.47, -4.06, -3.90, -3.87, 18.2,
18.3, 18.5, 24.1, 26.0, 26.1, 62.7, 69.9, 71.7, 73.8, 74.5, 82.1,
85.3, 115.9, 138.7; EI-LRMS m/z 542 (M+), 485, 371, 327, 233,
183, 171, 159. EI-HRMS calcd for C28H58O4Si3 542.3643.
Found 542.3654.
give 10a (285 mg, 93% in two steps) as a colorless oil: [R]18
D
-7.91° (c 1.1, CHCl3); IR (neat) 3438, 3314, 2122, 1645, 1255,
1103 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.09 (s, 6H), 0.91 (s,
9H), 2.02 (t, J ) 2.7 Hz, 1H), 2.46 (ddd, J ) 16.6, 5.9, 2.7 Hz,
1H), 2.55 (ddd, J ) 16.6, 7.1, 2.7 Hz, 1H), 3.18 (br d, J ) 3.9
Hz, 1H), 3.24 (d, J ) 5.4 Hz, 1H), 3.53 (dd, J ) 3.7, 3.7 Hz,
1H), 3.70-3.85 (m, 4H), 3.93 (m, 1H), 4.43 (m, 1H), 5.27 (d, J
) 10.5 Hz, 1H), 5.40 (d, J ) 17.0 Hz, 1H), 5.96 (ddd, J ) 17.0,
10.5, 6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ -5.2, -5.1,
18.5, 23.5, 26.0, 63.0, 69.9, 70.4, 72.7, 73.2, 80.6, 82.2, 116.6,
136.1; EI-LRMS m/z 257 (M - tBu)+, 239, 183, 171, 119. EI-
HRMS calcd for C12H21O4Si (M - tBu)+ 257.1210. Found
257.1197.
(3R,4S,5R)-3,5-Bis(ter t-bu tyldim eth ylsilyloxy)-4-[3-(ter t-
bu tyld im eth ylsilyloxy)p r op oxy]oct-1-en -7-yn e (11b). In
a manner similar to that for the synthesis of 11a from 10a , a
crude product, which was prepared from 10b (104 mg, 0.32
mmol), TBSOTf (0.18 mL, 0.78 mmol), and 2,6-lutidine (0.11
mL, 0.94 mmol) in CH2Cl2 (3 mL), was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 50/1) to give
(3R,4S,5R)-4-[3-(ter t-Bu tyld im eth ylsilyloxy)p r op oxy]-
oct-1-en -7-yn e-3,5-d iol (10b). In a manner similar to that
for the synthesis of 10a from 9a , a crude product, which was
obtained from 9b (1.2 g, 2.9 mmol), lithium trimethylsilyl-
acetylide [prepared from trimethylacetylene (1.1 mL, 7.4
mmol) and nBuLi (1.52 M solution in hexane, 4.4 mL, 6.7
mmol)], and BF3‚OEt2 (0.41 mL, 3.24 mmol) in THF (24 mL),
was treated with K2CO3 (1.2 g, 8.8 mmol) in MeOH (2.9 mL).
After the usual workup, the crude product was purified by
flash column chromatography on silica gel (hexane/AcOEt )
3/1) to give 10b (861 mg, 90% in two steps) as a colorless oil:
11b (177 mg, quantitative) as a colorless oil: [R]21 +0.3° (c
D
0.9, CHCl3); IR (neat) 2122 cm-1; H NMR (400 MHz, CDCl3)
1
δ 0.03 (s, 3H), 0.05 (s, 6H), 0.07 (s, 3H), 0.09 (s, 3H), 0.10 (s,
3H), 0.89 (s, 18H), 0.90 (s, 9H), 1.74-1.80 (m, 2H), 1.95 (t, J
) 2.6 Hz, 1H), 2.35 (ddd, J ) 16.9, 5.5, 2.6 Hz, 1H), 2.49 (ddd,
J ) 16.9, 5.5, 2.6 Hz, 1H), 3.35 (dd, J ) 5.5, 3.5 Hz, 1H), 3.60-
3.76 (m, 4H), 3.88 (dt, J ) 9.1, 5.5 Hz, 1H), 4.30 (dd, J ) 7.0,
3.5 Hz, 1H), 5.12 (br d, J ) 10.3 Hz, 1H), 5.20 (dt, J ) 17.2,
1.3 Hz, 1H), 5.95 (ddd, J ) 17.2, 10.3, 7.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ -5.1, -4.5, -4.1, -3.9, 18.2, 18.3, 18.5,
24.2, 26.0, 26.1, 33.7, 60.6, 69.5, 69.8, 71.6, 74.5, 82.1, 85.1,
115.8, 138.7; EI-LRMS m/z 499 (M - tBu)+. EI-HRMS calcd
[R]21 -10.5° (c 1.1, CHCl3); IR (neat) 3393, 2121, 1647, 1255,
D
1095 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.06 (s, 6H), 0.09 (s,
9H), 1.81 (tt, J ) 5.7, 5.7 Hz, 2H), 2.01 (t, J ) 2.6 Hz, 1H),
2.40-2.60 (m, 2H), 2.86 (d, J ) 5.5 Hz, 1H), 2.96 (d, J ) 5.5
Hz, 1H), 3.41 (dd, J ) 4.4, 2.4 Hz, 1H), 3.60-3.90 (m, 4H),
4.00 (m, 1H), 4.48 (m, 1H), 5.27 (d, J ) 10.5 Hz, 1H), 5.42 (d,
J ) 17.1 Hz, 1H), 5.95 (ddd, J ) 17.1, 10.5, 5.1 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ -5.2, 18.3, 23.6, 26.0, 33.0, 59.7,
68.0, 69.8, 70.3, 72.2, 80.6, 80.7, 116.1, 136.8; EI-LRMS m/z
t
for C25H51O4Si3 (M - Bu)+ 499.3096. Found 499.3074.
(3R,4S,5R)-3,5-Bis(ter t-bu tyldim eth ylsilyloxy)-4-[3-(ter t-
bu tyld im eth ylsilyloxy)bu toxy]oct-1-en -7-yn e (11c). In a
manner similar to that for the synthesis of 11a from 10a , a
crude product, which was obtained from 10c (615 mg, 1.8
mmol), TBSOTf (1.0 mL, 4.5 mmol), and 2,6-lutidine (0.65 mL,
5.6 mmol) in CH2Cl2 (3.6 mL), was purified by column
chromatography on silica gel (hexane/AcOEt ) 30/1) to give
11c (971 mg, 95%) as a colorless oil: [R]20D -2.1° (c 6.3, CHCl3);
IR (neat) 2122 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.03 (s, 3H),
0.04 (s, 6H), 0.07 (s, 3H), 0.08 (s, 3H), 0.10 (s, 3H), 0.89 (s,
18H), 0.90 (s, 9H), 1.24-1.28 (m, 2H), 1.57-1.60 (m, 2H), 1.95
(t, J ) 2.6 Hz, 1H), 2.35 (ddd, J ) 16.8, 5.5, 2.6 Hz, 1H), 2.49
(ddd, J ) 16.8, 5.5, 2.6 Hz, 1H), 3.36 (dd, J ) 5.5, 3.7 Hz, 1H),
3.55-3.69 (m, 4H), 3.86 (dt, J ) 9.1, 5.5 Hz, 1H), 4.30 (dd, J
) 3.7, 6.9 Hz, 1H), 5.12 (d, J ) 10.4 Hz, 1H), 5.20 (d, J ) 17.4
Hz, 1H), 5.96 (ddd, J ) 17.4, 10.4, 6.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ -5.1, -4.5, -4.1, -3.9, -3.8, 18.3, 18.4, 18.5,
24.2, 26.0, 26.1, 26.2, 26.8, 29.7, 63.2, 69.8, 71.7, 72.5, 74.6,
t
271 (M - Bu)+, 241, 185, 171, 133, 75. EI-HRMS calcd for
t
C
13H23O4Si (M - Bu)+ 271.1365. Found 271.1356.
(3R,4S,5R)-4-[4-(ter t-Bu tyldim eth ylsilyloxy)bu toxy]oct-
1-en -7-yn e-3,5-d iol (10c). In a manner similar to that for the
synthesis of 10a from 9a , a crude product, which was obtained
from 9c (855 mg, 2.0 mmol), lithium trimethylsilylacetylide
[prepared from trimethylacetylene (0.72 mL, 5.1 mmol) and
nBuLi (1.52 M solution in hexane, 3.1 mL, 4.7 mmol)], and BF3‚
OEt2 (0.28 mL, 32 mmol) in THF (24 mL), was treated with
K2CO3 (842 mg, 6.1 mmol) in MeOH (4 mL). After the usual
workup, the crude product was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 4/1) to give 10c
(647 mg, 93% in two steps) as a colorless oil: [R]18 -6.30° (c
D
0.98, CHCl3); IR (neat) 3314, 2122, 1645, 1255, 1095 cm-1; 1H
NMR (400 MHz, CDCl3) δ 0.05 (s, 6H), 0.89 (s, 9H), 1.55-
1.73 (m, 4H), 2.01 (t, J ) 2.6 Hz, 1H), 2.50 (dd, J ) 7.1, 2.6
Hz, 2H), 2.76 (br d, J ) 4.2 Hz, 1H), 2.93 (br d, J ) 5.8 Hz,
J . Org. Chem, Vol. 69, No. 22, 2004 7469

Saito et al.
t
82.1, 85.0, 115.8, 138.7; EI-LRMS m/z 513 (M - Bu)+. EI-
After the usual workup, the crude product was purified by
preparative TLC (10% MeOH in CH2Cl2) to yield 4c (15.1 mg,
66%, two steps from 11c). Further purification of 4c for
biological assays was conducted by reversed-phase recycle
HPLC (YMC-Pack ODS column, 20 × 150 mm, 9.9 mL/min,
CH3CN/H2O ) 6/4): [R]20D -22.1° (c 0.054, CHCl3); UV (MeOH)
HRMS calcd for C26H53O4Si3 (M - tBu)+ 513.3252. Found
513.3251.
2r-(3-Hyd r oxyeth oxy)-1r,25-d ih yd r oxyvita m in D3 (4a ).
To a solution of 12 (50 mg, 140 µmol) and 11a (50 mg, 92 µmol)
in toluene (1 mL) were added Et3N (1 mL) and Pd(PPh3)4 (32
mg, 28 µmol), and the mixture was stirred at 120 °C for 2 h.
The mixture was filtered through a silica gel pad. The filtrate
was concentrated, and the residue was purified by silica gel
column chromatography (hexane-hexane/AcOEt ) 95/5) to
give the coupling product 13a (57 mg, 75%) as a colorless oil,
which was used without any further purification. To the THF
(3 mL) solution of 13a (30 mg, 37 µmol) was added TBAF (1
M solution in THF, 0.18 mL, 0.18 mmol), and the mixture was
stirred at room temperature for 4 days. After the solution was
concentrated, the residue was purified by a preparative TLC
(10% MeOH in CH2Cl2) to give 4a (14 mg, 78%) as a white
powder. Further purification of 4a for biological assays was
conducted by reversed-phase recycle HPLC (YMC-Pack ODS
1
λmax 267 nm; H NMR (400 MHz, CDCl3) δ 0.54 (s, 3H), 0.93
(d, J ) 6.4 Hz, 3H), 1.21 (s, 6H), 1.25-2.00 (m, 25H), 2.23
(dd, J ) 13.3, 9.3 Hz, 1H), 2.68 (dd, J ) 13.3, 4.6 Hz, 1H),
2.83 (m, 1H), 3.35 (dd, J ) 7.6, 3.2 Hz, 1H), 3.61 (dt, J ) 9.5,
6.0 Hz, 1H), 3.68-3.77 (m, 4H), 4.05 (ddd, J ) 8.6, 7.6, 4.6
Hz, 1H), 4.42 (d, J ) 3.0 Hz, 1H), 5.10 (d, J ) 2.1 Hz, 1H),
5.39 (br s, 1H), 6.02 (d, J ) 11.3 Hz, 1H), 6.42 (d, J ) 11.3 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 12.1, 18.8, 20.8, 22.2, 23.5,
26.9, 27.6, 29.1, 29.2, 29.4, 29.7, 36.1, 36.4, 40.5, 40.8, 44.4,
45.9, 56.4, 56.5, 62.6, 68.2, 70.0, 71.1, 71.8, 84.6, 116.2, 117.1,
125.5, 131.5, 143.6, 144.2; EI-LRMS m/z 504 (M+), 486, 468.
EI-HRMS calcd for C31H52O5 504.3817. Found 504.3823.
20-ep i-2r-(2-H yd r oxye t h oxy)-1r,25-d ih yd r oxyvit a -
m in D3 (20-epi-4a ). To a solution of 14 (31 mg, 87 µmol) and
11a (71 mg, 0.13 mmol) in toluene (2 mL) were added Et3N (2
mL) and Pd(PPh3)4 (30 mg, 26 µmol), and the mixture was
stirred at 110 °C for 1.5 h. After the mixture was filtered
through a silica gel short column (hexane/AcOEt ) 10/1), the
filtrate was concentrated to give the crude product 20-epi-
vitamin D3 (45 mg). To a solution of the crude vitamin D3 in
MeCN (1 mL) was added HF/MeCN (1/9, 1 mL) at 0 °C, and
the mixture was stirred at room temperature for 1 h. To the
mixture was added saturated NaHCO3 aqueous solution at 0
°C, and the aqueous layer was extracted with AcOEt. The
organic layer was washed with saturated NaCl aqueous
solution, dried over Na2SO4, and concentrated. The residue was
purified by preparative TLC (AcOEt) to give 20-epi-4a (20 mg,
48% in two steps) as a colorless oil. Further purification of
20-epi-4a for biological assays was conducted by reversed-
phase recycle HPLC (YMC-Pack ODS column, 20 × 150 mm,
9.9 mL/min, CH3CN/H2O ) 6/4): [R]19D +12.4° (c 0.82, CHCl3);
UV (MeOH) λmax 266 nm; IR (neat) 3374, 1647, 1074 cm-1; 1H
NMR (400 MHz, CDCl3) δ 0.53 (s, 3H), 0.84 (d, J ) 6.4 Hz,
3H), 1.21 (s, 6H), 1.10-2.05 (m, 21H), 2.24 (dd, J ) 12.5, 11.5
Hz, 1H), 2.67 (dd, J ) 12.5, 4.6 Hz, 1H), 2.83 (m, 1H), 3.37
(dd, J ) 7.9, 3.0 Hz, 1H), 3.70 (m, 1H), 3.73-3.85 (m, 4H),
4.07 (m, 1H), 4.43 (d, J ) 2.9 Hz, 1H), 5.09 (d, J ) 1.5 Hz,
1H), 5.37 (d, J ) 1.5 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H), 6.42
(d, J ) 11.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 12.5, 18.7,
21.0, 22.2, 23.6, 27.4, 29.2, 29.3, 29.4, 35.5, 36.1, 40.5, 41.4,
44.4, 46.0, 56.2, 56.4, 61.9, 68.5, 71.1, 71.3, 72.5, 85.1, 116.6,
117.1, 125.3, 131.3, 143,3, 143.8; EI-LRMS m/z 476 (M+), 458,
440, 396, 378. EI-HRMS calcd for C29H48O5 476.3502. Found
476.3503.
column, 20 × 150 mm, 9.9 mL/min, CH3CN/H2O ) 6/4): [R]20
D
+59.1° (c 0.12, CHCl3); UV (MeOH) λmax 269 nm; 1H NMR (400
MHz, CDCl3/D2O) δ 0.54 (s, 3H), 0.93 (d, J ) 6.6 Hz, 3H), 1.21
(s, 6H), 1.25-1.80 (m, 14H), 1.83-1.89 (m, 1H), 1.96-2.01 (m,
2H), 2.23 (dd, J ) 13.0, 9.5 Hz, 1H), 2.67 (dd, J ) 13.0, 4.8
Hz, 1H), 2.83 (m, 1H), 3.38 (dd, J ) 8.1, 3.3 Hz, 1H), 3.72 (ddd,
J ) 9.5, 4.8, 2.7 Hz, 1H), 3.77-3.84 (m, 4H), 4.07 (ddd, J )
9.5, 7.9, 4.8 Hz, 1H), 4.43 (d, J ) 3.4 Hz, 1H), 5.10 (d, J ) 1.7
Hz, 1H), 5.38 (d, J ) 1.7 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H),
6.43 (d, J ) 11.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 12.1,
18.8, 20.8, 22.2, 23.5, 27.7, 29.1, 29.2, 29.4, 36.1, 36.4, 40.5,
41.2, 44.4, 45.9, 56.4, 56.6, 62.0, 68.6, 71.1, 71.4, 72.5, 85.0,
116.6, 117.1, 125.6, 131.4, 143.7, 144.1; EI-LRMS m/z 476
(M+), 458, 440. EI-HRMS calcd for C29H48O5 (M+) 476.3503.
Found 476.3527.
2r-(2-Hydr oxypr opoxy)-1r,25-dih ydr oxyvitam in D3 (4b).
In a manner similar to that for the synthesis of 4a from 11a
and 12, a crude product, which was obtained from 12 (219 mg,
0.39 mmol), 11b (130 mg, 0.36 mmol), and Pd(PPh3)4 (125.8
mg, 0.109 mmol) in toluene/Et3N (1/1, 10 mL), was purified
by silica gel column chromatography (hexane-hexane/AcOEt
) 95/5) to give the coupling product 13b (157 mg, 52%) as a
colorless oil, which was used without any further purification.
The coupling product 13b (157 mg, 0.19 mmol) was subjected
to desilylation by TBAF (1 M solution in THF, 0.94 mL, 0.94
mmol) in THF (3 mL) at room temperature for 4 days. After
the usual workup, the crude product was purified by prepara-
tive TLC (10% MeOH in CH2Cl2) to give 4b (56 mg, 61%) as a
white powder. Further purification of 4b for biological assays
was conducted by reversed-phase recycle HPLC (YMC-Pack
ODS column, 20 × 150 mm, 9.9 mL/min, CH3CN/H2O ) 6/4):
[R]25 +46.4° (c 0.55, CHCl3); UV (MeOH) λmax 267 nm; 1H
D
20-ep i-2r-(3-H yd r oxyp r op oxy)-1r,25-d ih yd r oxyvit a -
m in D3 (20-epi-4b). In a manner similar to that for the
synthesis of 20-epi-4a from 14 and 11a , a crude product, which
was obtained from 14 (22 mg, 60 µmol), 11b (51 mg, 91 µmol),
and Pd(PPh3)4 (21 mg, 18 µmol) in toluene/Et3N (1/1, 4 mL),
was dissolved in MeCN. To the solution was added HF/MeCN
(1/9, 1 mL) at 0 °C, and the mixture was stirred at room
temperature for 1.5 h. After the usual workup, the crude
product was purified by preparative TLC (AcOEt) to give 20-
epi-4b (17 mg, 57% in two steps) as a colorless oil. Further
purification of 20-epi-4b for biological assays was conducted
by reversed-phase recycle HPLC (YMC-Pack ODS column, 20
NMR (400 MHz, CDCl3) δ 0.54 (s, 3H), 0.93 (d, J ) 6.4 Hz,
3H), 1.21 (s, 6H), 1.25-2.10 (m, 23H), 2.24 (dd, J ) 13.4, 9.2
Hz, 1H), 2.69 (dd, J ) 13.4, 4.4 Hz, 1H), 2.82 (m, 1H), 3.38
(dd, J ) 7.5, 3.2 Hz, 1H), 3.75-3.91 (m, 5H), 4.05 (m, 1H),
4.44 (br d, J ) 2.8 Hz, 1H), 5.10 (d, J ) 1.8 Hz, 1H), 5.39 (br
s, 1H), 6.01 (d, J ) 11.3 Hz, 1H), 6.42 (d, J ) 11.3 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 12.1, 18.8, 20.8, 22.2, 23.5, 27.7,
29.1, 29.2, 29.4, 31.9, 36.1, 36.4, 40.5, 41.0, 44.4, 45.9, 56.4,
56.6, 61.2, 68.4, 68.5, 71.1, 71.9, 84.5, 116.1, 117.1, 125.5, 131.5,
143.6, 144.3; EI-LRMS m/z 490 (M+), 472, 454. EI-HRMS
calcd for C30H50O5 490.3660. Found 490.3638.
× 150 mm, 9.9 mL/min, CH3CN/H2O ) 6/4): [R]21 +11.0° (c
2r-(3-Hyd r oxybu toxy)-1r,25-d ih yd r oxyvita m in D3 (4c).
In a manner similar to that for the synthesis of 4a from 11a
and 12, a crude product, which was obtained from 11c (26 mg,
0.05 mmol), 12 (52 mg, 0.15 mmol), and Pd(PPh3)4 (16 mg, 14
µmol) in toluene/Et3N (1/3, 2.5 mL), was purified by prepara-
tive TLC (hexane/AcOEt ) 4/1) to give the coupling product
13c (26.5 mg, 69%) as a colorless oil, which was used without
any further purification. The coupling product 13c was
subjected to desilylation by TBAF (1 M solution in THF, 0.2
mL, 0.2 mmol) in THF (3 mL) at room temperature for 36 h.
D
1.31, CHCl3); UV (MeOH) λmax 267 nm; IR (neat) 3389, 1647,
1
1265, 1076 cm-1; H NMR (400 MHz, CDCl3) δ 0.53 (s, 3H),
0.84 (d, J ) 6.6 Hz, 3H), 1.20 (s, 6H), 1.10-1.73 (m, 15H),
1.75-1.90 (m, 4H), 1.90-2.00 (m, 2H), 2.22 (dd, J ) 13.4, 9.2
Hz, 1H), 2.64 (br s, 3H), 2.66 (dd, J ) 13.4, 4.8 Hz, 1H), 2.81
(m, 1H), 3.36 (dd, J ) 7.4, 3.3 Hz, 1H), 3.70-3.90 (m, 4H),
4.04 (m, 1H), 4.43 (br d, J ) 3.2 Hz, 1H), 5.08 (d, J ) 2.0 Hz,
1H), 5.37 (s, 1H), 6.01 (d, J ) 11.2 Hz, 1H), 6.40 (d, J ) 11.2
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 12.3, 18.5, 20.6, 22.1,
7470 J . Org. Chem., Vol. 69, No. 22, 2004

Design and Synthesis of 2-epi,20-epi-ED-71 Analogues
23.5, 27.3, 29.0, 29.2, 31.8, 35.4, 36.0, 40.4, 41.0, 44.3, 45.9,
56.1, 56.4, 61.1, 68.3, 68.4, 71.1, 71.9, 84.5, 116.1, 117.1, 125.4,
131.6, 143.4, 144.3; EI-LRMS m/z 490 (M+), 473, 472, 396,
267. EI-HRMS calcd for C30H50O5 490.3660. Found 490.3676.
20-ep i-2r-(2-H yd r oxyb u t oxy)-1r,25-d ih yd r oxyvit a -
m in D3 (20-epi-4c). In a manner similar to that for the
synthesis of 20-epi-4a , a crude product, which was obtained
from 14 (40 mg, 0.11 mmol), 11c (96 mg, 0.17 mmol), and Pd-
(PPh3)4 (39 mg, 34 µmol) in toluene/Et3N (1/1, 4 mL), was
dissolved in MeCN (1 mL). To the solution was added HF/
MeCN (1/9, 1 mL) at 0 °C, and the mixture was stirred at room
temperature for 2 h. After the usual workup, the crude product
was purified by preparative TLC (AcOEt) to give 20-epi-4c (25
mg, 45% in two steps) as a colorless oil. Further purification
of 20-epi-4c for biological assays was conducted by reversed-
phase recycle HPLC (YMC-Pack ODS column, 20 × 150 mm,
UV (MeOH) λmax 269 nm; IR (neat) 3376, 1645, 1078 cm-1; 1H
NMR (400 MHz, CDCl3) δ 0.53 (s, 3H), 0.84 (d, J ) 6.6 Hz,
3H), 1.10 (s, 6H), 1.08-2.05 (m, 25H), 2.23 (dd, J ) 13.4, 9.3
Hz, 1H), 2.50 (br s, 1H), 2.67 (dd, J ) 13.4, 4.5 Hz, 1H), 2.83
(m, 1H), 3.34 (dd, J ) 7.6, 3.1 Hz, 1H), 3.60 (dt, J ) 9.5, 5.8
Hz, 1H), 3.68 (t, J ) 5.7 Hz, 2H), 3.74 (dt, J ) 9.5, 5.9 Hz,
1H), 4.05 (ddd, J ) 8.8, 7.6, 4.6 Hz, 1H), 4.41 (br d, J ) 3.1
Hz, 1H), 5.09 (d, J ) 1.6 Hz, 1H), 5.38 (d, J ) 1.6 Hz, 1H),
6.02 (d, J ) 11.2 Hz, 1H), 6.41 (d, J ) 11.2 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 12.5, 18.7, 21.0, 22.2, 23.6, 27.0, 27.4, 29.2,
29.3, 29.4, 29.7, 35.5, 36.1, 40.5, 41.0, 44.4, 46.0, 56.2, 56.4,
62.3, 68.2, 69.9, 71.1, 71.8, 84.5, 116.1, 117.1, 125.3, 131.4,
143.2, 144.0; EI-LRMS m/z 504 (M+), 486, 396, 378. EI-
HRMS calcd for C31H52O5 504.3815. Found 504.3814.
Bin d in g Assa ys to th e Bovin e Th ym u s VDR. Bovine
thymus VDR was obtained from a commercial supplier, and
the affinity was evaluated according to the literature.23
Assa ys for In d u ction of HL-60 Cell Differ en tia tion . The
activity was estimated by superoxide anion production as
previously described.26 The superoxide radicals reduce the
cytochrome c, and the reduced cytochrome c is measured by
spectrophotometry at 550 nm.
Ack n ow led gm en t. We thank Ms. J unko Shimode
and Ms. Maroka Kitsukawa (Teikyo University) for the
spectroscopic measurements. This study was supported
by Grants-in-Aid from the Ministry of Education, Cul-
ture, Sports, Science and Technology, J apan and in part
by a grant (MF-16) from the Organization for Pharma-
ceutical Safety and Research. N.S. acknowledges Take-
da Science Foundation for support.
9.9 mL/min, CH3CN/H2O ) 6/4): [R]18 +8.1° (c 1.82, CHCl3);
D
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal details, 1H NMR and 13C NMR spectra for all new
compounds (6a -11a , 6b-11b, 6c-11c, 4a -c, 20-epi-4a -c),
charts of VDR binding assays of compounds 4a -c and 20-epi-
4a -c, and a chart for assays of induction of HL-60 cell
differentiation activity of compounds 4a -c and 20-epi-4a -c.
This material is available free of charge via the Internet at
http://pubs.acs.org.
J O0491051
J . Org. Chem, Vol. 69, No. 22, 2004 7471