Parallel synthesis of a vitamin D3 library in the solid-phase

Article abstract of DOI:10.1021/ja003976k

A highly efficient synthesis of the vitamin D₃ system on solid support is described. Two synthetic strategies for the solid-phase synthesis of vitamin D₃ were developed. One is for 11-hydroxy analogues, and the other is for most other synthetic analogues. In the latter strategy, the sulfonate-linked CD-ring 58 was initially immobilized on PS-DES resin to give solid-supported CD-ring 63 (Scheme 10). Similarly, solid-supported CD-ring 63 was prepared by attachment of the CD-ring 10 to the chlorosulfonate resin 64. The vitamin D₃ system was synthesized by Horner-Wadsworth-Emmons reaction of the A-ring phosphine oxide to a solid-supported CD-ring, followed by simultaneous introduction of the side chain and cleavage from resin with a Cu¹-catalyzed Grignard reagent. Parallel synthesis of the vitamin D₃ analogues was accomplished by a split and pool methodology utilizing radio frequency encoded combinatorial chemistry, and a manual parallel synthesizer for side chain diversification and deprotection. Additionally, we demonstrated the synthesis of various A-rings in a similar protocol for efficient preparation of building blocks.

Full text of DOI:10.1021/ja003976k

3716
J. Am. Chem. Soc. 2001, 123, 3716-3722
Parallel Synthesis of a Vitamin D3 Library in the Solid-Phase
Ichiro Hijikuro, Takayuki Doi, and Takashi Takahashi*
Contribution from the Department of Applied Chemistry, Graduate School of Science and Engineering,
Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
ReceiVed NoVember 15, 2000
Abstract: A highly efficient synthesis of the vitamin D3 system on solid support is described. Two synthetic
strategies for the solid-phase synthesis of vitamin D3 were developed. One is for 11-hydroxy analogues, and
the other is for most other synthetic analogues. In the latter strategy, the sulfonate-linked CD-ring 58 was
initially immobilized on PS-DES resin to give solid-supported CD-ring 63 (Scheme 10). Similarly, solid-
supported CD-ring 63 was prepared by attachment of the CD-ring 10 to the chlorosulfonate resin 64. The
vitamin D3 system was synthesized by Horner-Wadsworth-Emmons reaction of the A-ring phosphine oxide
to a solid-supported CD-ring, followed by simultaneous introduction of the side chain and cleavage from resin
with a CuI-catalyzed Grignard reagent. Parallel synthesis of the vitamin D3 analogues was accomplished by a
split and pool methodology utilizing radio frequency encoded combinatorial chemistry, and a manual parallel
synthesizer for side chain diversification and deprotection. Additionally, we demonstrated the synthesis of
various A-rings in a similar protocol for efficient preparation of building blocks.
Introduction
Scheme 1. Synthetic Study of the 11-Hydroxyvitamin D3
System on Solid-Support (XI ) OH, Strategy I)
1R,25-Dihydroxyvitamin D3 (1R,25-(OH)2D3, calcitriol) (8,
Scheme 2), the biologically active metabolite of vitamin D3, is
recognized as a steroid hormone.1 The natural hormone exhibits
a variety of physiological activities, such as regulation of
calcium and phosphorus metabolism, cell differentiation and
proliferation, and the immune system.2 Recently, to separate
these activities, especially cell differentiation and proliferation
activity from calcemic activity, and to enhance the specific
activities, a number of analogues have been synthesized by many
laboratories.3 Most of these have been modified in either the
A-ring or the side chain, while a few derivatives were altered
in the CD-ring, or in both the A-ring and the CD-ring side chain,
so-called “hybrid” analogues3c-e by Posner et al. These ana-
logues have been synthesized one by one, in various discrete
manners, many of which cannot be considered as consistently
convergent methods for analogue synthesis. In contrast, we have
(1) (a) Norman, A. W.; Bouillon, R.; Thomasset, M., Eds. Vitamin D:
Gene Regulation, Structure Function Analysis and Clinical Application;
Walter de Gruyter and Co.: Berlin, 1991. (b) Yamamoto, K.; Masuno, H.;
Choi, M.; Nakashima, K.; Taga, T.; Ooizumi, H.; Umesuno, K.; Scinska,
W.; VanHooke, J.; Deluca, H. F.; Yamada, S. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 1467.
(2) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. ReV. 1995,
16, 200.
recently reported the solid-phase synthesis of the 11-hydroxy-
vitamin D3 system in four steps by combining three components,
i.e. the A-ring, the CD-ring, and the side chain (in our
nomenclature, the CD-ring includes the carbons at the 20, 21,
and 22 positions, steroidal numbering, see 7 in Scheme 1).4 In
recent years, it has been proven that applications of solid-phase
chemistry, capable of generating combinatorial natural small
molecule libraries, to the drug discovery process is extremely
effective in terms of searching a wide range of analogues
rapidly.5 Therefore, it seems to be of great value to employ
(3) (a) Zhu, G.-D.; Okamura, W. H. Chem. ReV. 1995, 95, 1877. (b)
Ikekawa, N. Med. Res. ReV. 1987, 7, 333. For recent examples on the
synthesis of the vitamin D analogues, see: (c) Posner, G. H.; Wang, Q.;
Han, G.; Lee, J. K.; Crawford, K.; Zand, S.; Brem, H.; Peleg, S.; Dolan,
P.; Kensler, T. W. J. Med. Chem. 1999, 42, 3425. (d) Posner, G. H.; Lee,
J. K.; White, M. C.; Hutchings, R. H.; Dai, H.; Kachinski, J. L.; Dolan, P.;
Kensler, T. W. J. Org. Chem. 1997, 62, 3299. (e) Fujishima, T.; Konnno,
K.; Nakagawa, K.; Kurobe, M.; Okano, T.; Takayama, H. Bioorg. Med.
Chem. 2000, 8, 123. (f) Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca,
H. F. J. Med. Chem. 1998, 41, 4662. (g) Shimizu, M.; Iwasaki, Y.; Yamada,
S. Tetrahedron Lett. 1999, 40, 1697. (h) Mikami, K.; Osawa, A.; Isaka,
A.; Sawa, E.; Shimizu, M.; Terada, M.; Kubodera, N.; Nakagawa, K.;
Tsugawa, N.; Okano, T. Tetrahedron Lett. 1998, 39, 3359. (i) Courtney, L.
F.; Lange, M.; Uskokovic, M. R.; Wovkulich, P. M. Tetrahedron Lett. 1998,
39, 3363. (j) Oshida, J.; Okamoto, M.; Ishizuka, S.; Azumi, S. Bioorg. Med.
Chem. Lett. 1999, 9, 381. For a recent report for modification at the
6-position, see: (k) Addo, J. K.; Swamy, N.; Ray, R. Steroid 1999, 64,
273.
(4) Doi, T.; Hijikuro, I.; Takahashi, T. J. Am. Chem. Soc. 1999, 121,
6749.
(5) For reviews, see: (a) Dolle, R. E. J. Comb. Chem. 2000, 2, 383. (b)
Dolle, R. E.; Nelson, K. H., Jr. J. Comb. Chem. 1999, 1, 235. (c) Schreiber,
S. L. Science 2000, 287, 1964.
10.1021/ja003976k CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/31/2001

Parallel Synthesis of a Vitamin D3 Library in the Solid Phase
J. Am. Chem. Soc., Vol. 123, No. 16, 2001 3717
the A-ring and the CD-ring at the 8-position is accomplished
by Horner-Wadsworth-Emmons methodology.7 The Horner-
Wadsworth-Emmons methodology, pioneered by Lythgoe et
al.,7a is the most desirable as it could proceed with high chemo-
and stereoselectivity and in high yield, and the unreacted excess
A-ring moiety 2a could be recovered by simple purification after
quenching of the reaction with water. On the other hand, the
linkage between the CD-ring and the side chain is accomplished
by CuI-catalyzed Grignard reaction.8 The substitution reaction
of the C-22 sulfonyloxy group with the side chain via CuI-
catalyzed Grignard reagent 3, previously underutilized in vitamin
D3 synthesis,4,9 would be most desirable for direct coupling of
the side chain.10 Hence, it is important which component is
initially immobilized on a solid support. In solid-phase nucleo-
philic reactions, addition of excess nucleophiles to a solid-
supported electrophile is apparently desirable to complete the
reactions. Thus, attachment of the electrophilic CD-ring 1 to a
solid support is essential. The solid-supported CD-ring has two
electrophilic sites, which are required to be compatible during
the two carbon-carbon bond formations, without protecting
groups and/or functional group manipulation, which is undesir-
able for a practical combinatorial synthesis. On the basis of these
constraints, the Horner-Wadsworth-Emmons reaction of an
A-ring moiety to the polymer-supported 8-keto CD-ring is
performed prior to the alkylation of the sulfonyloxy group with
Grignard reagents to avoid Grignard addition at the 8-position
and facile epimerization at the 14-position adjacent to the
carbonyl group. Alkylation of a sulfonyloxy group at the 22-
position of the polymer-supported CD-ring with a Grignard
reagent would afford the vitamin D3 system, if this could be
performed at sufficiently low temperature (<30 °C) to avoid
isomerization of the preformed triene system. In our previous
investigation4 shown in Scheme 1 (strategy I in Figure 1), we
selected the hydroxy group at the 11-position as the loading
site of the CD-ring 4 on the solid support and PS-DES resin
(5).11 Although there was steric influence from not only the
substituent at the 11-position but also the polystyrene chain with
a four-carbon alkyl chain from the diethylsilyl group, we
anticipated that it would not affect the subsequent two steps,
i.e. (1) Horner-Wadsworth-Emmons reaction of A-ring moi-
eties 2a to the polymer-supported 8-keto CD-ring 6 and (2)
alkylation of the polymer-supported tosylate at the 22-position
with the Grignard reagent 3. Furthermore, PS-DES resin (5) is
appropriate as a polymer support because silyl protection has
proven to be effective in the synthesis of various vitamin D3
analogues and is readily cleaved even in the presence of the
unstable triene moiety. Finally, treatment of the polymer-
supported vitamin D3 with HF‚Py in THF would release the
desired vitamin D3 system 7 after aqueous workup and simple
Figure 1. Strategy of the vitamin D3 system on the solid-phase.
Scheme 2. Retrosynthesis of the Vitamin D3 System on a
Solid Support (Strategy II)
solid-phase chemistry to construct a combinatorial vitamin D3
library including analogues previously synthesized and to
investigate their structure-activity relationships.
In this article, we predominantly describe the improved solid-
phase synthesis of the vitamin D3 system (strategy II, Figure 1)
that is applicable to most synthetic analogues and the preparation
of its library utilizing radio frequency encoded combinatorial
(REC) chemistry,6 which is a useful technique for the identifica-
tion of members in a “split and pool” combinatorial library. In
addition, we wish to report an efficient synthesis of various
A-ring moieties by means of the Pd(0)-catalyzed intramolecular
Mizoroki-Heck reaction.
(7) (a) Lythgoe, B.; Moran, T. A.; Nambudiry, M. E. N.; Tideswell, J.;
Wright, P. W. J. Chem. Soc., Perkin Trans. 1 1978, 590. (b) Toh, H. T.;
Okamura, W. H. J. Org. Chem. 1983, 48, 1414. (c) Baggiolini, E. G.;
Iacobelli, J. A.; Hennessy, B. M.; Batcho, A. D.; Sereno, J. F.; Uskokovic,
M. R. J. Org. Chem. 1986, 51, 3098.
Solid-Phase Synthesis Strategy
(8) (a) Schlosser, M. Angew. Chem., Int. Ed. Engl. 1974, 13, 701. For a
review of Grignard reaction on the solid phase, see: (b) Franzen, R. G.
Tetrahedron 2000, 56, 685.
(9) Andrews, D. R.; Barton, D. H. R.; Hesse, R. H.; Pechet, M. M. J.
Org. Chem. 1986, 51, 4819.
(10) For another approach to formation of the side chain of the vitamin
D3 system, see: (a) Partrige, J. J.; Faber, S.; Uskokovic, M. R. HelV. Chim.
Acta 1974, 57, 764. (b) Lythgoe, B.; Roberts, D. A.; Waterhouse, I. J. Chem.
Soc., Perkin Trans. 1 1977, 2608. (c) Tachibana, Y.; Yokoyama, S.; Tsuji,
M. Bull. Chem. Soc. Jpn. 1989, 62, 2599. (d) Torneiro, M.; Fall, Y.; Castedo,
L.; Mourino, A. Tetrahedron Lett. 1992, 33, 105.
(11) (a) Hu, Y.; Porco, J. A., Jr.; Labadie, J. W.; Gooding, O. W.; Trost,
B. M. J. Org. Chem. 1998, 63, 4518. (b) Hu, Y.; Porco, J. A., Jr. Tetrahedron
Lett. 1998, 39, 2711. (c) Hu, Y.; Porco, J. A., Jr. Tetrahedron Lett. 1999,
40, 3289.
Consideration of the structure of the vitamin D3 system (see
8 in Scheme 2) readily led us to the assembly of the three
components: the A-ring, the CD-ring, and the side chain for
molecular diversification. Our strategy for the solid-phase syn-
thesis of vitamin D3 is shown in Figure 1. The linkage between
(6) (a) Nicolaou, K. C.; Winssinger, N.; Vourloumis, D.; Ohshima, T.;
Kim, S.; Pfefferkorn, J.; Xu, J.-Y.; Li, T. J. Am. Chem. Soc. 1998, 120,
10814. (b) Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor, J.; Winssinger,
N.; He, Y.; Ninkovic, S.; Sarabia, F.; Vallberg, H.; Roschangar, F.; King,
N. P.; Finlay, M. R. V.; Giannakakou, P.; Verdier-Pinard, P.; Hamel, E.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2097. (c) Xiao, X.-Y.; Parandoosh,
Z.; Nova, M. P. J. Org. Chem. 1997, 62, 6029.

3718 J. Am. Chem. Soc., Vol. 123, No. 16, 2001
Hijikuro et al.
Scheme 3. Retrosynthesis of Four Possible A-Ring
filtration through silica gel to remove silyl residues. Following
the above procedure, we performed the synthesis of twelve 11-
hydroxyvitamin D3 analogues.12 This strategy promises to
overcome the required two carbon-carbon bond-forming steps
on the solid phase, resulting in the consecutive coupling of the
CD-ring with the A-ring and side chain moieties. However, these
analogues are limited to “11-hydroxy”10d,13 analogues, and it is
also difficult to prepare various 11-hydroxy CD-rings. Therefore,
it is necessary to develop an alternative strategy to cover vitamin
D3 analogues beyond those encompassed by our earlier design.
To this end, as shown in Scheme 2 (strategy II in Figure 1),
attachment of the C-22 hydroxy group of the CD-ring 10 to a
sulfonate-linked resin 1114 would be acceptable in terms of not
requiring the C-11 hydroxy group. The solid-supported sul-
fonated CD-ring 9 would be available not only for CuI-catalyzed
Grignard reagents,4,9 but also other various nucleophiles, such
as lithium acetylide,10a R-lithiated sulfone,10c R-lithiated cyanide,10d
etc. As a consequence, we adopted the Horner-Wadsworth-
Emmons reaction of A-ring moiety 2a with 8-keto CD-ring 9,
followed by simultaneous alkylation and cleavage from the solid
support by CuI-catalyzed Grignard reaction with 3. Finally,
deprotection and simple purification of the crude cleavage
product would afford vitamin D3 analogues 8. In the present
strategy, there are many impurities in the cleavage solution,
therefore we should modify the workup procedure and purifica-
tion for parallel synthesis of a number of analogues.
Diastereomers of 1,25-(OH)2D3
as a synthon for the acyl anion of acrolein 22. The protected
cyanohydrins have been developed by Stork et al.19 and allowed
our group to accomplish the total syntheses of several natural
products.20 Chemoselective alkylation of the lithiated protected
cyanohydrin of acrolein 21 with (S)-epichlorohydrin (23)
(Scheme 4) followed by treatment with a catalytic amount of
copper(II) sulfate in methanol and water at 60 °C and shaking
with aqueous NaHCO3 gave crude enantiomerically pure
â-hydroxy ketone 19. The 1,3-anti diastereoselective reduction
Preparation of Various A-Ring Moieties
The modified CD-rings and the side chain moieties are readily
available from the Inhoffen-Lythgoe diol (46, Scheme 7
15
)
and bromo esters,16 respectively, whereas preparation of
the modified A-ring moieties3a,c-i is somewhat laborious. Thus,
generating a library of vitamin D3 analogues requires an efficient
synthesis of A-ring moieties via a similar protocol.
21
of the â-hydroxy ketone 19 with Me4NBH(OAc)3 in acetic
acid and acetonitrile followed by neutralization of the acetic
acid with aqueous NaOH afforded epoxy alcohol 2522 in 63%
yield from starting material 23. The 1,3-syn diastereoselective
reduction of 19 with Et3B-NaBH423 was also accomplished to
give diol 27,22 which potentially could be converted to the
corresponding phosphine oxide 12 (see Scheme 3).
THP protection of the hydroxy group of 25 gave epoxide
26, which was then transformed to acid 29a by alkylation with
potassium cyanide, TBS protection, reduction with DIBAL, and
oxidation. Treatment of acid 29a with carbonyl diimidazole,
followed by the addition of magnesium ethyl malonate, provided
the corresponding â-keto ester,24 which was converted to alcohol
15a by trapping the (Z)-enolate from the above â-keto ester
with Tf2NPh,25 and reduction of the ethyl ester. Palladium(0)-
catalyzed cyclization of 15a was carried out as previously
reported.4,25a,26 The reaction proceeded smoothly at room
One of our strategies, which could produce all possible A-ring
diastereomers of 1,25-(OH)2D3,17 is described in Scheme 3.
A-ring moieties 2 and 12-14 can be prepared from the
corresponding enol triflates 15-18 through Pd(0)-catalyzed
intramolecular Mizoroki-Heck reaction. The enol triflates 15-
18 can be synthesized by diastereoselective reductions and
various functional group transfomations of the â-hydroxy
ketones 19 and 20. Enantiomerically pure â-hydroxy ketones
1918 and 20 can be prepared by coupling of optically active
epichlorohydrins 23 and 24 with the protected cyanohydrin 21
(12) For the structures and spectral data of these analogues, see the
Supporting Information.
(13) For examples of C-11 substituted 1R,25-(OH)2D3, see: (a)
D’Halleweyn, C.; Van Haver, D.; Van der Eycken, J.; De Clercq, P.;
Vandewalle, M. Bioorg. Med. Chem. Lett. 1992, 2, 477. (b) Bouillon, R.;
Allewaert, K.; van Leeuwen, J. P. T. M.; Tan, B.-K.; Xiang, D. Z.; De
Clercq, P.; Vandewalle, M.; Pols, H. A. P.; Bos, M. P.; Van Baelen, H.;
Birkenha¨ger, J. C. J. Bio. Chem. 1992, 267, 3044. (c) Zhu, G.-D.; Van
Haver, D.; Jurriaans, H.; De Clercq, P. J. Tetrahedron 1994, 50, 7049. (d)
Kobayashi, N.; Higashi, T.; Shimada, K. J. Chem. Soc., Perkin Trans. 1
1994, 269.
(14) (a) Rueter, J. K.; Nortey, S. O.; Baxter, E. W.; Leo, G. C.; Reitz,
A. B. Tetrahedron Lett. 1998, 39, 975. (b) Baxter, E. W.; Rueter, J. K.;
Nortey, S. O.; Reitz, A. B. Tetrahedron Lett. 1998, 39, 979. (c) Zhong, H.
M.; Greco, M. N.; Maryanoff, B. E. J. Org. Chem. 1997, 62, 9326. (d)
Hunt, J. A.; Roush, W. R. J. Am. Chem. Soc. 1996, 118, 9998. (e) Takahashi,
T.; Tomida, S.; Inoue, H.; Doi, T. Synlett 1998, 1261. (f) Takahashi, T.;
Ebata, S.; Doi, T. Tetrahedron Lett. 1998, 39, 1369.
(19) Stork, G.; Moldnado, L. J. Am. Chem. Soc. 1971, 93, 5286.
(20) (a) Takahashi, T.; Kanda, Y.; Nemoto, H.; Kitamura, K.; Tsuji, J.
J. Org. Chem. 1986, 51, 3393. (b) Takahashi, T.; Shimizu, K.; Doi, T.;
Tsuji, J. J. Am. Chem. Soc. 1988, 110, 2674. (c) Takahashi, T.; Tsukamoto,
H.; Kurosaki, M.; Yamada, H. Synlett 1997, 1065.
(21) Evans, D. A.; Chapman, K. T.; Carreira, E. M. J. Am. Chem. Soc.
1988, 110, 3560.
(22) The stereochemistries of 27 and 28 were confirmed by 13C NMR
analysis of the gem-dimethyl group of the corresponding acetonides (δ 19.8,
30.0 from 27; δ 24.9, 25.2 from 28); see: Rychnovsky, S. D.; Skalitzky,
D. J. Tetrahedron Lett. 1990, 31, 945.
(15) (a) Inhoffen, H. H.; Quinkert, G.; Schuetz, S.; Friedrich, G.; Tober,
E. Chem. Ber. 1958, 91, 781. (b) Lythgoe, B.; Roberts, D. A.; Waterhouse,
I. J. Chem. Soc., Perkin Trans. 1 1977, 2608.
(16) Wilson, S. R.; Zhao, H.; Dewan, J. Bioorg. Med. Chem. Lett. 1993,
3, 341.
(17) For a study on a similar concept, see ref 3e.
(18) Trost, B. M.; Hanson, P. R. Tetrahedron Lett. 1994, 35, 8119.
(23) Chen, K.-M.; Gunderson, K. G.; Hardtmann, G. E.; Prasad, K.;
Repic, O.; Shapiro, M. J. Chem. Lett. 1987, 1923.
(24) Brooks, D. W.; Lu, L. D.-L.; Masamune, S. Angew. Chem., Int.
Ed. Engl. 1979, 18, 72.
(25) (a) Yokoyama, H.; Miyamoto, K.; Hirai, Y.; Takahashi, T. Synlett
1997, 187. For reviews of vinyl triflates, see: (b) Ritter, K. Synthesis 1993,
725. (c) Stang, P. J.; Hanack, M.; Subramanian, L. R. Synthtesis 1982, 85.

Parallel Synthesis of a Vitamin D3 Library in the Solid Phase
J. Am. Chem. Soc., Vol. 123, No. 16, 2001 3719
Scheme 5. Preparation of A-Ring Moiety 33
Scheme 4. Synthesis of A-Ring Moieties 2, and
1,3-syn-Diol 27 for A-Ring Moiety 12
Scheme 6. Synthesis of A-Ring Moieties 39, 41, and 44
temperature to furnish cyclized (Z)-diene 30a in 57% yield with
slight formation (<5%) of the corresponding (E)-diene via syn
Pd-H â-elimination. Dienyl alcohol 30a was chlorodehydroxy-
lated and then converted to the phosphine oxide 2a according
to the literature procedure.7b The phosphine oxide 2b, previously
synthesized by other groups,7c,27 was also prepared from 25 in
a similar manner.
THP-protected cyclic compound 30a is a key intermediate
that can lead to a variety of A-ring moieties by selective de-
protection at the C-1 or C-3 position. For example, deprotection
at the 3-position allowed for the subsequent etherification,
substitution, inversion, and reduction, etc. of the C-3 hydroxy
group. As one of these modifications, the etherified A-ring
moiety 33 was synthesized (Scheme 5). The allylic hydroxy
group of 30a was protected as its pivalate, which was converted
to alcohol 31 by desilylation with tetrabutylammonium fluoride.
The etherification of alcohol 31 with prenyl chloride in the
presence of tetrabutylammonium iodide provided ether 32.
Deprotection of the pivaloyl group and several transformations
(26) Takahashi, T.; Nakazawa, M. Synlett 1993, 37.
(27) (a) Posner, G. H.; Kinter, C. M. J. Org. Chem. 1990, 55, 3967. (b)
De Shrijver, J.; De Clercq, P. J. Tetrahedron Lett. 1993, 34, 4369. (c)
Kobayashi, S.; Shibata, J.; Shimada, M.; Ohno, M. Tetrahedron Lett. 1990,
31, 1577. (d) Stork, G.; Hutchinson, D.; Okabe, M.; Parker, D.; Ra, C.;
Ribereau, F.; Suzuki, T.; Zebovitz, T. Pure Appl. Chem. 1992, 64, 1809.
(e) Mascarenas, J. L.; Garcia, M.; Castedo, L.; Mourino, A. Tetrahedron
Lett. 1992, 33, 4365. (f) Nagasawa, K.; Ishihara, H.; Zako, Y.; Shimizu, I.
J. Org. Chem. 1993, 58, 2523. (g) Tazumi, K.; Ogasawara, K. J. Chem.
Soc., Chem. Commun. 1994, 1903. (h) Hatakeyama, S.; Numata, H.; Osanai,
K.; Takano, S. J. Org. Chem. 1989, 54, 3515. (i) Batty, D.; Crich, D. J.
Chem. Soc., Perkin Trans. 1 1991, 2894.
as shown in Scheme 5 gave phosphine oxide 33. Another
strategy is indicated in Scheme 6. We previously reported the
synthesis of the 2-hydroxy A-ring moiety 34,4,26 which can also
be a candidate to produce various A-ring phosphine oxides.
Michael addition to ethyl acrylate of intermediate 36, prepared
from 34 (Scheme 6), afforded the ester,28 which was reduced

3720 J. Am. Chem. Soc., Vol. 123, No. 16, 2001
Hijikuro et al.
Scheme 7. Initial Study of the Vitamin D3 Synthesis
(strategy II) on a Directly Sufonylchloride-Attached
Polystylene Resin
Scheme 8. Preparation of the Sulfonate-Linkers 54 and 55
proceeded successfully to give 49 in 52% isolated yield. The
solid-supported CD-ring 47 also provided 48 by Dess-Martin
oxidation33 or PDC oxidation.10d,34 However, the solid-bound
CD-ring 48 failed to couple with phosphine oxide 2a. We
attributed this failure to steric interferance by the polystyrene
chains. We decided to devise our own sulfonate linker to obtain
a solid-supported triene.
Preparation of Sulfonate-Linked CD-Ring Derivatives
The sulfonate linker is an attractive linker on solid-phase
synthesis as a traceless linker that can be displaced with various
nucleophiles.14 We designed the sulfonate linker 55 that has an
appropriate spacer with sufficient length to avoid steric interfer-
ence with the polystyrene chains of the solid support. As
illustrated in Scheme 8, Mitsunobu reaction of alcohol 51 with
methyl 4-hydroxybenzenesulfonate (52) provided 4-alkoxyben-
zenesulfonate 53. The sulfonate ester 53 was hydrolyzed with
lithium chloride in refluxing acetone to give the sulfonic acid
lithium salt, which was converted to the sulfonyl chloride 54
by PCl5 in DMF.14c,d THP-deprotection of 54 with CSA in EtOH
and THF provided hydroxy sulfonyl chloride 55 in 81% yield.35
Coupling of the sulfonyl chloride 54 with various CD-rings
afforded the corresponding sulfonate-linked CD-rings, three of
which were prepared as shown in Scheme 9. Sulfonylation of
54 with the CD-ring 10 prepared from 56 and subsequent
removal of the THP protecting group afforded the sulfonate-
linked CD-ring 58. The 11-methyl13a,b sulfonate-linked CD-ring
59 was also prepared from 57 (via route b) in the same manner.
The 20-epi3e,36 sulfonate-linked CD-ring 62 was synthesized as
follows. Treatment of aldehyde 60 with NaHCO3 in ethanol at
75 °C gave the 20-epimers whose ratio was 62:38 in favor of
the desired 20R-epimer.36 The mixture was reduced with NaBH4
to alcohols, which could be separated on silica gel to yield the
pure desired epimer 61 in 46% yield in two steps.3e Coupling
of epimer 61 with sulfonyl chloride 54 followed by deprotection
of THP and TBS, selective protection of the primary alcohol
by TBS, oxidation of the C-8 secondary alcohol, and TBS-
deprotection afforded the 22-epi sulfonate-linked CD-ring 62.
to alcohol 37. Alcohol 37 was transformed to the allylic chloride
with NCS and dimethyl sulfide.29 Under these conditions
approximately half of the isopropylidene group was removed
by a chloronium cation; therefore, the crude allylic chloride was
reprotected with 2,2-dimethoxy propane. TBS protection of the
primary hydroxy group afforded 38, which was converted to
phosphine oxide 39 as previously described. The A-ring synthon
44,30 an intermediate for the vitamin D3 analogue ED-71,31 was
assembled in a manner similar to the synthesis of 39.
The intermediate 36 was also converted to triene 40 by
treatment with DEAD and PPh3. Phosphine oxide 41 was readily
synthesized from 40. Unfortunately, phosphine oxide 41 did not
react with the CD-ring synthon 56 (Scheme 9) even at room
temperature, presumably because of the stabilization of the
intermediate carbanion that is highly delocalized due to the
internal olefin.
Initial Study of the Solid-Phase Vitamin D3 Synthesis
In our initial investigation of the solid-phase vitamin D3
synthesis (strategy II), we selected commercially available TsCl
resin (45, 1.58 mmol/g)32 as a solid support. It has chlorosulfonyl
groups attached to a polystyrene chain directly, as shown in
Scheme 7. Attachment of the CD-rings 46 and 10 was performed
according to the literature.32 Simultaneous alkylation and
cleavage from resin 47 by CuI-catalyzed Grignard reaction
Solid-Phase Synthesis of the Vitamin D3 System
(28) Kubodera, N.; Watanabe, H.; Kawanishi, T.; Matsumoto, M. Chem.
Pharm. Bull. 1992, 40, 1494.
The sulfonate-linked CD ring 58 was loaded onto chlorinated
PS-DES resin (5, 0.74 mmol/g)11 as shown in Scheme 10. The
loading yield of 63 was determined to be 95% by gravimetric
analysis. Subsequently, we developed another preparation of
63 by attachment of the CD-ring 10 with the sulfonyl chloride
(29) Corey, E. J.; Kim, C. U.; Takeda, M. Tetrahedron Lett. 1972, 4339.
(30) For a precedent report, see: Hatakeyama, S.; Iwabuchi, Y.; Irie,
H.; Kawase, A.; Kubodera, N. Bioorg. Med. Chem. Lett. 1997, 7, 2871.
(31) (a) See Supporting Information for our synthesis of ED-71. (b) Ono,
Y.; Kawase, A.; Watanabe, H.; Shiraishi, A.; Takeda, S.; Higuchi, Y.; Sato,
K.; Yamauchi, T.; Mikami, T.; Kato, M.; Tsugawa, N.; Okano, T.; Kubodera,
N. Bioorg. Med. Chem. 1998, 6, 2517. (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. (d) Okano, T.;
Tsugawa, N.; Masuda, S.; Takeuchi, A.; Kobayashi, T.; Takita, Y.; Nishii,
Y. Biochem. Biophys. Res. Commun. 1989, 163, 1444.
(33) (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(b) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899.
(34) Leyers, G. A.; Okamura, W. H. J. Am. Chem. Soc. 1982, 104, 6099.
(35) Alternative use of MeOH as a solvent resulted in affording 40%
methyl sulfonate byproduct with 45% desired product.
(32) This material and reaction conditions are available from Argonaut
Technologies. Home page: http://www.argotech.com.
(36) Calverley, M. J. Tetrahedron 1987, 43, 4609.

Parallel Synthesis of a Vitamin D3 Library in the Solid Phase
J. Am. Chem. Soc., Vol. 123, No. 16, 2001 3721
Scheme 9. Preparation of the Sulfonate-Linker Loaded
CD-Rings 58, 59, and 62
Scheme 10. Solid-Phase Synthesis of the Vitamin D3
System (66)a
resin 64 in the presence of DMAP in >90% yield, which is a
more general method for loading alcohols on a solid support
via a sulfonate-linker.14 It should be noted that use of DMAP
is essential for not only accelerating the reaction but also
ensuring attachment of the sulfonate-linked CD-ring.37 Sulfonyl
chloride resin 64 was prepared in one step by immobilizing
hydroxy sulfonyl chloride 55 to PS-DES resin in 83-87% yield,
practically without substitution of the chlorosulfonyl group.
Horner-Wadsworth-Emmons reaction of 63 with lithiated 67
in THF at -40 to -10 °C for 3 h yielded triene 65. Sequential
one-pot coupling of immobilized sulfonate 65 and cleavage from
the polymer-support by CuI-catalyzed Grignard reaction of 68
at room temperature for 6 h gave the crude alkylation product.
This crude protected product was immediately subjected to CSA
solution in methanol and water at room temperature for 6 h to
afford vitamin D3 analogue 66 in 47% yield from 63 (via route
a, b). These solid-phase reactions were monitored by 13C SR-
MAS (swollen resin-magic angle spinning) NMR38 and FT-
IR in comparison with the corresponding TES-protected com-
pounds prepared in solution. Thus, we have developed a solid-
phase synthesis of the vitamin D3 system, not requiring cleavage
from the resin for analysis.
Synthesis of the Vitamin D3 Library
On the basis of Scheme 10, we constructed a vitamin D3
combinatorial library utilizing Radiofreqency Encoded Com-
binatorial (REC) chemistry.6 Building blocks of a 72-member
vitamin D3 library are summarized in Figure 2. The 72
microreactors each containing ca. 25 mg of PS-DES resin (ca.
(37) In treatment with pyridine as a base, the sulfonate-CD ring was
cleaved from diethylsilyl resin by a generated pyridine hydrochloride. Use
of triethylamine did not complete the reaction within 12 h, and 2,6-lutidene
was not effective to do the reaction at all.
(38) Kobayashi, S.; Akiyama, R.; Furuta, T.; Moriwaki, M. Molecules
Online 1998, 2, 35.
Figure 2. Building blocks of a 72-member vitamin D3 library.

3722 J. Am. Chem. Soc., Vol. 123, No. 16, 2001
Hijikuro et al.
Figure 3. Flow chart of simultaneous coupling and cleavage from resin by Cu1-catalyzed Grignard reaction and deprotection in parallel using a
manual synthesizer.
18 µmol) were encoded and split into individual flasks (A, B,
C) according to radio frequency signals. After treatment of the
sulfonate-linked CD-rings with imidazole and recovery of excess
reagents individually, microreactors were pooled together for
washing and drying. These microreactors were decoded and
split, followed by coupling with A-rings in individual flasks
(1, 2, 3, 4). After recovery of the excess A-ring reagents
individually, microreactors were pooled together again for
washing and drying.
Conclusion
In the vitamin D field, we have described the first generation
of a combinatorial library via solid-phase chemistry. We have
developed two synthetic strategies for a three-component library
in only four steps including two nucleophilic additions. The latter
library was constructed in a split and pool methodology utilizing
REC chemistry.6 Our sulfonate linker as a traceless linker for
molecular diversification is effective for simultaneous alkylation
and cleavage from resin. We also demonstrated the workup and
purification in parallel using a manual parallel synthesizer for
efficient construction of a library. Additionally, preparation of
various A-ring moieties by a common protocol suggested the
desirability of such a parallel synthesis. These results may prove
useful for the general development of combinatorial libraries,
as well as that of the vitamin D3 system. Currently, biological
evaluation is in progress, and the structure-activity relationships
will be reported in due course.
In this parallel synthesis, sequential one-pot coupling and
cleavage from the resin with CuI-catalyzed Grignard reagent
and deprotection in solution were crucial. It is impractical to
line up many vessels on stirrers and work up the reaction mixture
in a conventional manner, i.e. via transfer of the mixture with
pipets and aqueous purification, and concentration on a rotary
evaporator. To overcome these difficulties, we utilized a manual
parallel synthesizer39 equipped with 20 vessels where agitation
and reaction temperature can be controlled, and that can be
connected to another vessel as shown in Figure 3. Side chain
diversification and deprotection in parallel was performed
according to the flowchart in Figure 3. In the Grignard
substitution with side chain IV, CuBr‚Me2S powder was placed
in vessels because steric interference with its neighbor diethyl
group prevented prompt formation of the cuprate. The filtrates
in vials were concentrated together on a centrifugal evaporator.
Finally, purification by GPC with chloroform for 30 min per
compound afforded a series of vitamin D3 analogues (2-9 mg)
in good purity.40 This sequence of manipulations was very
useful, especially when the cleavage product is a mixture and
is subject to further reaction. HRMS spectral data of all
Acknowledgment. This work was partly supported by a
Grant-in-Aid for JSPS Fellows (I.H., No. 10014) from the
Ministry of Education, Science, Sports, and Culture, Japan. We
gratefully acknowledge IRORI Quantum Microchemistry (U.S.A.)
for helpful advice and the supply of an AccuTag-100 instrument
and MicroKan reactors. We gratefully acknowledge Argonaut
Technologies (U.S.A.) for helpful advice.
Supporting Information Available: Experimental informa-
tion including spectral date of 10 “11-hydroxy” vitamin D3
analogues, 270 MHz 1H NMR and 67.8 MHz 13C NMR spectra,
100 MHz 13C SR-MAS NMR spectra of resins 63, 64, and 65,
and details on the preparation of ED-71 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
1
compounds and H,13C NMR spectral data of five compounds
selected randomly confirmed these structures.41
(39) Quest 210, Argonaut Technologies, San Carlos, CA 94070.
(40) On the way toward the 72-member library synthesis, we found that
addition of NaHCO3 powder (see operation vii in Figure 3) before
concentration of the reaction mixtures prevented decomposition of the
products.
JA003976K
(41) See Supporting Information.