Efficient synthesis of 2-modified 1α,25-dihydroxy-19-norvitamin D3 with Julia olefination: High potency in induction of differentiation on HL-60 cells

Article abstract of DOI:10.1021/jo034787y

Six novel 2-substituted analogues of 1α,25-dihydroxy-19-norvitamin D₃, 6a,b-8a,b, were efficiently synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared as 4-alkylated (3R,5R)-3,5-dihydroxycyclohex

Full text of DOI:10.1021/jo034787y

Efficien t Syn th esis of 2-Mod ified 1r,25-Dih yd r oxy-19-n or vita m in
D3 w ith J u lia Olefin a tion : High P oten cy in In d u ction of
Differ en tia tion on HL-60 Cells
Keiichiro Ono,† Akihiro Yoshida,†,‡ Nozomi Saito,† Toshie Fujishima,† Shinobu Honzawa,†
Yoshitomo Suhara,†,§ Seishi Kishimoto,| Takayuki Sugiura,| Keizo Waku,|
Hiroaki Takayama,† and Atsushi Kittaka*,†
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University,
Sagamiko, Kanagawa 199-0195, J apan, and Department of Hygienic Chemistry and Nutrition,
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, J apan
akittaka@pharm.teikyo-u.ac.jp
Received J une 9, 2003
Six novel 2-substituted analogues of 1R,25-dihydroxy-19-norvitamin D3, 6a ,b-8a ,b, were efficiently
synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared
as 4-alkylated (3R,5R)-3,5-dihydroxycyclohexanones 12-15 from (-)-quinic acid based on radical
allylation at the C4 position of methyl (-)-quinicate. The new type of the CD-ring coupling partner
23 was synthesized from 25-hydroxy Grundmann’s ketone 19 to apply to the modified J ulia
olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields,
including a deprotection step, were 47-62%. After the separation of the diastereomers based on
C2 stereochemistry, the structure (2R or 2â) was determined by 1H NMR experiments and compared
to DeLuca’s 2-methyl- and 2-ethyl-1R,25-dihydroxy-19-norvitamin D3. Thus, the synthesized 2R-
(3-hydroxypropyl)-1R,25-dihydroxy-19-norvitamin D3 (8a ) showed almost the same potency in
binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone 1, while its â-isomer
8b had only a 3% affinity. Both 2R-allyl- and 2R-propyl-1R,25-dihydroxy-19-norvitamin D3 (6a and
7a ) and their 2â-analogues (6b and 7b) possessed a weak affinity for the VDR. The strong VDR
ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1, and
interestingly, even the weaker ligand 8b showed a 6.7-fold higher potency in the cell differentiation
activity than that of 1.
In tr od u ction
prostate cancer, colon cancer, and breast cancer.4 The
stereochemistries of both hydroxyl groups at C1 and C3
of the A-ring are essential features for induction of cell
differentiation and apoptosis.5 We previously reported
that 2R-substituted analogues of 1 can exhibit enhanced
vitamin D receptor (VDR)6 agonist activities.7-11 On the
other hand, 19-nor A-ring analogues such as 1R,25-
dihydroxy-19-norvitamin D3 (5, Figure 1) were first
reported in 1990.12 This 19-nor analogue 5 showed a
selective activity profile, combining high potency in
inducing differentiation of malignant cells with very low
or no bone calcification activity.13 Further modifications
Recently, the A-ring modifications of 1R,25-dihydroxy-
vitamin D3 (1) have received much attention as potential
drugs for the treatment of osteoporosis,1 psoriasis,2
hereditary vitamin D-resistant rickets,3 and chemopre-
vention of cancer and cancer chemotherapy, especially
* To whom correspondence should be addressed. Phone: (+81)-426-
85-3715. Fax: (+81)-426-85-3714.
† Department of Pharmaceutical Chemistry, Faculty of Pharmaceu-
tical Sciences, Teikyo University.
‡ Present address: The Noguchi Institute and J apan Chemical
Innovation Institute (J CII), Itabashi-ku, Tokyo 173-0003, J apan.
§ Present address: Kobe Pharmaceutical University, Higashinada-
ku, Kobe 658-8558, J apan.
(4) (a) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. Rev.
1995, 16, 200-257. (b) Gross, C.; Peehl, D. M. In Vitamin D; Feldman,
D., Glorieux, F. H., Pike, J . W., Eds.; Academic Press: New York, 1997;
pp 1125-1139. (c) Brastitus, T. A.; Sitrin, M. Ibid. pp 1141-1154. (d)
Colston, K. Ibid. pp 1107-1123. (e) Morosetti, R.; Koeffler, H. P. Ibid.
pp 1155-1166.
(5) Nakagawa, K.; Kurobe, M.; Konno, K.; Fujishima, T.; Takayama,
H.; Okano, T. Biochem. Pharm. 2000, 60, 1937-1947.
(6) (a) Umezono, K.; Murakami, K. K.; Thompson, C. C.; Evans, R.
M. Cell 1991, 65, 1255-1266. (b) Evans, R. M. Science 1988, 240, 889-
895.
(7) 2-Methyl: (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.;
Miura, D.; Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998,
8, 151-156. (b) Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.;
Chokki, M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.;
Miyata, N.; Smith, C.; DeLuca, H. F.; Takayama, H. J . Med. Chem.
2000, 43, 4247-4265.
| Department of Hygienic Chemistry and Nutrition, Faculty of
Pharmaceutical Sciences, Teikyo University.
(1) ED-71: (a) Nishii, Y.; Okano, T. Steroids 2001, 66, 137-146. (b)
Kubodera, N.; Sato, K.; Nishii, Y. In Vitamin D; Feldman, D., Glorieux,
F. H., Pike, J . W., Eds.; Academic Press: New York, 1997; Chapter
63, pp 1071-1086.
(2) Yamada, S.; Shimizu, M.; Yamamoto, K. Med. Res. Rev. 2003,
23, 89-115.
(3) (a) Kittaka, A.; Kurihara, M.; Peleg, S.; Suhara, Y.; Takayama,
H. Chem. Pharm. Bull. 2003, 51, 357-358. (b) Gardezi, S. A.; Nguyen,
C.; Malloy, P. J .; Posner, G. H.; Feldman, D.; Peleg, S. J . Biol. Chem.
2001, 276, 29148-29156. (c) Swann, S. L.; Bergh, J . J .; Farach-Carson,
M. C.; Koh, J . T. Org. Lett. 2002, 4, 3863-3866. (d) Swann, S. L.; Bergh,
J .; Farach-Carson, M. C.; Ocasio, C. A.; Koh, J . T. J . Am. Chem. Soc.
2002, 124, 13795-13805.
10.1021/jo034787y CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/23/2003
J . Org. Chem. 2003, 68, 7407-7415
7407

Ono et al.
F IGURE 1. Structures of the natural hormone 1 and its 2R-substituted analogues (2-4),7-11 1R,25-dihydroxy-19-norvitamin D3
(5), and its 2-substituted analogues (6a ,b-8a ,b).
to the 19-nor A-ring were also studied, such as the
deletion of one of the hydroxyls14 and the introduction of
a 2R-methyl group.15,16 However, removal of the 10(19)-
methylene group reduces the VDR affinity due to its loss
of hydrophobic interaction with the ligand-binding do-
main (LBD) of the VDR. The binding affinity of 5 for the
porcine VDR has been reported as 30% of that of 1,17 and
in our present experiments for the bovine thymus VDR,
it was 17%. Recently, we synthesized the stable amide
analogues of 5, which contain a N5-C6 amide bond
(similar to the steroidal numbering) instead of the diene
system. However, these amide analogues showed almost
no affinity to the VDR.18
binding affinity for the VDR,8 we expected that 2R-(3-
hydroxypropyl)-1R,25-dihydroxy-19-norvitamin D3 (8)
might restore the binding affinity for the VDR up to the
level of the natural hormone. This type of compound
would show a unique biological activity profile of vitamin
D. DeLuca’s synthetic approach for 19-norvitamin D
analogues was based on the Horner-Wadsworth-
Emmons coupling reaction to construct the diene sys-
tem,15,19 which was developed by Lythgoe et al.20 How-
ever, utilization of this method for our synthetic goal was
found to be unsuitable, because a Horner-Wadsworth-
Emmons coupling between any of our 2-substituted
A-ring phosphine oxides (16-18) and the known 25-O-
protected 25-hydroxy Grundmann’s ketone 2021 was
unsuccessful. The CD-ring precursor could be elaborated
to incorporate the C6-C7 fragment, such as sulfone 23,
for the subsequent J ulia-type coupling with 2-substituted
A-ring ketones 12, 13, and 15.
Since we found that the introduction of the 2R-(3-
hydroxypropyl) group to 1 resulted in a 3-fold higher
(8) 2R-Alkyl and 2R-hydroxyalkyl: (a) Suhara, Y.; Nihei, K.; Kuri-
hara, M.; Kittaka, A.; Yamaguchi, K.; Fujishima, T.; Konno, K.; Miyata,
N.; Takayama, H. J . Org. Chem. 2001, 66, 8760-8771. (b) Suhara, Y.;
Nihei, K.; Tanigawa, H.; Fujishima, T.; Konno, K.; Nakagawa, K.;
Okano, T.; Takayama, H. Bioorg. Med. Chem. Lett. 2000, 10, 1129-
1132. (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, in press.
Resu lts a n d Discu ssion
Ch em istr y. Thioimidazolide 9,12b the A-ring precursor,
was allylated22 to afford methyl 4-allylquinicate 10 in
60% yield. Reduction of 10 with NaBH4 in EtOH and the
subsequent oxidative cleavage of the resulting vicinal diol
using NaIO4 gave ketone 12, which was the first 2-sub-
stituted A-ring precursor, in 82% yield in two steps.23
Hydrogenation of 12 on 10% Pd/C gave (R,R)-3,5-dihy-
droxy-4-propylcyclohexanone 13, the second 2-substituted
A-ring precursor, in 99% yield. When 10 was first
submitted to hydroboration, then reduced and cleaved
with periodate as before, 14 was produced in 68% overall
yield. Protection of the terminal hydroxyl group with
TBSCl resulted in a quantitative yield to afford 15, which
was the third 2-substituted A-ring precursor (Scheme 1).
Initially, these three ketones were converted to the
corresponding phosphine oxides 16-18 using the con-
ventional method,12b but all of the Horner-Wadsworth-
Emmons coupling reactions with 25-O-MOM-protected
Grundmann’s ketone 20 under basic conditions were
(9) 2R-Hydroxyalkoxy: (a) Kittaka, A.; Suhara, Y.; Takayanagi, H.;
Fujishima, T.; Kurihara, M.; Takayama, H. Org. Lett. 2000, 2, 2619-
2622. (b) Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.;
Kurihara, M.; Takayama, H. In Vitamin D Endocrine System: Struc-
tural, Biological, Genetic and Clinical Aspects; Norman, A. W.,
Bouillon, R., Thomasset, M., Eds.; University of California: Riverside,
2000; pp 49-52.
(10) Takayama, H.; Kittaka, A.; Fujishima, T.; Suhara, Y. J . Synth.
Org. Chem. J apan 2002, 60, 370-381.
(11) Takayama, H.; Kittaka, A.; Fujishima, T.; Suhara, Y. In
Vitamin D Analogs in Cancer Prevention and Therapy, Recent Results
in Cancer Research 164; Reichrath, J ., Friedrich, M., Tilgen, W., Eds.;
Springer-Verlag: Berlin Heidelberg, 2003; pp 289-317.
(12) (a) Perlman, K. L.; Sicinski, R. R.; Schnoes, H. K.; DeLuca, H.
F. Tetrahedron Lett. 1990, 31, 1823-1824. (b) Perlman, K. L.; Swenson,
R. E.; Paaren, H. E.; Schnoes, H. K.; DeLuca, H. F. Tetrahedron Lett.
1991, 32, 7663-7666.
(13) Sicinski, R. R.; Rotkiewics, P.; Kolinski A.; Sicinska, W.; Prahl,
J . M.; Smith, C. M.; DeLuca, H. F. J . Med. Chem. 2002, 45, 3366-
3380.
(14) (a) Mikami, K.; Osawa, A.; Isaka, A.; Terada, M.; Okano, T.
Tetrahedron Lett. 1998, 39, 3359-3362. (b) Okano, T.; Nakagawa, K.;
Kubodera, N.; Ozono, K.; Isaka, A.; Osawa, A.; Terada, M.; Mikami,
K. Chem. Biol. 2000, 7, 173-184.
(15) Sicinski, R. R.; Prahl, J . M.; Smith, C. M.; DeLuca, H. F. J .
Med. Chem. 1998, 41, 4662-4674.
(16) Mikami, K.; Koizumi Y.; Osawa, A.; Terada, M.; Takayama, H.;
Nakagawa, K.; Okano, T. Synlett 1999, 1899-1902.
(17) Zhou, X.; Zhu, G.-D.; Van Haver, D.; Vandewalle, M.; De Clercq,
P. J .; Verstuyf, A.; Bouillon, R. J . Med. Chem. 1999, 42, 3539-3556.
(18) Suhara, Y.; Kittaka, A.; Ono, K.; Kurihara, M.; Fujishima, T.;
Yoshida, A.; Takayama, H. Bioorg. Med. Chem. Lett. 2002, 12, 3533-
3536.
(19) Sicinski, R. R.; Perlman, K. L.; DeLuca, H. F. J . Med. Chem.
1994, 37, 3730-3738.
(20) Lythgoe, B. Chem. Soc. Rev. 1981, 449-475.
(21) (a) Kiegiel, J .; Wovkulich, P. M.; Uskokovic, M. R. Tetrahedron
Lett. 1991, 32, 6057-6060. (b) Sicinski, R. R.; DeLuca, H. F. Bioorg.
Med. Chem. Lett. 1995, 5, 159-162.
(22) Keck, G. E.; Enholm, E. J .; Yates, J . B.; Wiley, M. R. Tetrahe-
dron 1985, 41, 4079-4094.
(23) Reduction of 10 using DIBAL-H in toluene at rt gave diol 11
only in 55% yield.
7408 J . Org. Chem., Vol. 68, No. 19, 2003

Synthesis of 2-Modified 1R,25-Dihydroxy-19-norvitamin D3
SCHEME 1a
a
Reagents and conditions: (a) see ref 12b; (b) Bu3SnCH2CHdCH2, AIBN, benzene, reflux, 60%; (c) NaBH4, EtOH, 91%; (d) NaIO4,
MeOH-H2O (2:1), 90%; (e) H2, Pd/C, MeOH, 99%; (f) BH3‚THF, then 30% H2O2, 3 M NaOH; (g) NaBH4, EtOH; (h) NaIO4, MeOH-H2O
(2:1), 68% for three steps; (i) TBSCl, DMAP, Et3N, CH2Cl2, quant.
unsuccessful. It is known that in some cases this coupling
reaction results in a very low yield due to the structure
of the substituted A-ring phosphine oxide.13,24 Thus, we
changed the synthetic strategy, and we tried a modified
J ulia olefination recently reported by Hilpert and Wirz
for synthesizing retiferol (Ro 65-2299).25,26
Mo-catalyzed oxidation furnished sulfone 23 in 89% yield,
which was the substrate for the modified J ulia olefination
(Scheme 2).
Each ketone, 12, 13, and 15, and sulfone 23 were
coupled using LiHMDS in THF at -78 °C to give the
protected 2-substituted 19-norvitamin D3 as a diastereo-
isomeric mixture attributed to the C2 stereochemistry.
After partial purification through silica gel column chro-
matography, each mixture was treated with (+)-CSA in
MeOH to yield deprotected compounds in 47-62% yields.
Diastereomers were separated with reversed-phase HPLC
to afford the desired 2R- and 2â-substituted 19-norvita-
min D3 analogues 6a -8a and 6b-8b (Scheme 3).
Previously, conformational analysis on the A-ring of
2R- and 2â-methyl-1R,25-dihydroxy-19-norvitamin D3,15
and their ethyl counterparts,13 was reported in detail. The
conformational equilibrium of these 19-nor derivatives
The tertiary hydroxyl of ketone 19 was protected by a
MOM group, and the Horner-Wadsworth-Emmons
reaction using triethyl phosphonoacetate and sodium
hydride in THF gave the ester 21 (E/Z ) 92/8) in 98%
yield. Chemical shifts of the vinylic proton of the E- and
Z-isomers were 5.45 and 5.65 ppm, respectively. Trost
et al. assigned the (E,Z)-stereochemistry of the corre-
sponding bromoolefins using NMR: the vinylic proton of
the E-isomer (δ 5.63 ppm) appears in a higher field than
that of the Z-isomer (δ 5.93 ppm) due to the anisotropy
of the C-C single bond of the five-membered D-ring.27
(E)-Ethyl ester 21 was reduced to allylic alcohol 22 with
DIBAL-H in 99% yield, and sulfonation with 2-mercap-
tobenzothiazole under Mitsunobu conditions followed by
(26) Initially, we tried to connect ketone (A) with the two-carbon
elongated CD-ring phosphine oxide (24); however, only an elimination
product (B) was obtained under the basic conditions. The resemble
elimination reaction was reported in ref 25a. Ketone A was prepared
through simple acetylation of the corresponding alcohol in ref 19. Data
for B: 1H NMR (400 MHz, CDCl3) δ 0.048 (s, 3H), 0.053 (s, 3H), 0.85
(s, 9H), 2.14 (s, 3H), 2.60 (dd, J ) 3.2, 16.4 Hz, 1H), 2.71 (dd, J ) 6.0,
16.4 Hz, 1H), 4.44 (m, 1H), 5.59 (m, 1H), 6.10 (d, J ) 9.6 Hz, 1H), 6.62
(br d, J ) 9.6 Hz, 1H).
(24) Synthesis of 2,2-dimethyl-1R,25-dihydroxy-19-norvitamin D3:
Posner, G. H.; Woodard, B. T.; Crawford, K. R.; Peleg, S.; Brown, A.
J .; Dolan, P.; Kensler, T. W. Bioorg. Med. Chem. 2002, 10, 2353-2365.
(25) (a) Hilpert, H.; Wirz, B. Tetrahedron 2001, 57, 681-694. (b)
Baudin, J . B.; Hareau, G.; J ulia, S. A.; Ruel, O. Tetrahedron Lett. 1991,
32, 1175-1178.
(27) Trost, B. M.; Dumas, J .; Villa, M. J . Am. Chem. Soc. 1992, 114,
9836-9845.
J . Org. Chem, Vol. 68, No. 19, 2003 7409

Ono et al.
SCHEME 2a
a
Reagents and conditions: (a) MOMCl, (iPr)2NEt, CH2Cl2, quant; (b) (EtO)2P(O)CH2CO2Et, NaH, THF, 98% (E/Z ) 92/8); (c) DIBAL-
i
i
H, toluene, 99%; (d) 2-mercaptobenzothiazole, PrOCONdNCO2 Pr, Ph3P, THF, then cat. (NH4)6Mo7O24‚4H2O, 30%H2O2, EtOH, 89%.
SCHEME 3a
F IGURE 2. Preferred conformation of the 2-substituted 19-
norvitamin D3 analogues 6a -8a and 6b-8b. Left (6a -8a 2R-
equatorial chair conformation): NOE between H6 and H4R
was observed, 4.6% for R ) allyl (6a : J 3R,4â ) 9.6, J 1â,10R ) 4.1
Hz), 3.6% for R ) propyl (7a : J 3R,4â ) 9.2, J 1â,10R ) 4.7 Hz),
and 2.4% for R ) 3-hydroxypropyl (8a : J 3R,4â ) 9.6, J 1â,10R
)
4.3 Hz). Right (6b-8b 2â-equatorial chair conformation): NOE
between H6 and H4â was observed, 1.7% for R ) allyl (6b:
J 3R,4â ) 3.4, J 1â,10R ) 10.3 Hz), 4.2% for R ) propyl (7b: J 3R,4â
) 3.8, J 1â,10R ) 9.8 Hz), and 3.5% for R ) 3-hydroxypropyl
(8b: J 3R,4â ) 3.3, J 1â,10R ) 10.2 Hz).
TABLE 1. 1H NMR Ch em ica l Sh ifts of Rep r esen ta tive
P r oton s of 2-Meth yl, 2-Eth yl, a n d 2-Allyl An a logu es
a
Reagents and conditions: (a) LiHMDS, THF, -78 to 0 °C; (b)
2R-Mea 2R-Etb 2R-allyl 2â-Mea
2â-Etb
2â-allyl
(+)-CSA, MeOH, 47-62% in two steps, then reversed-phase HPLC
separation of each isomer.
1â-H
3R-H
4R-H
4â-H
10R-H
10â-H
7-H
3.96
3.61
2.60
2.13
2.80
2.22
5.82
6.37
2.80
0.54
0.94
1.13
4.14
3.64
2.60
4.08
3.70
2.61
3.51
3.90
3.54
4.09
3.64
4.02
2.40
2.43 2.34-2.42
is strongly shifted to one particular chair form (R-chair
or â-chair conformation), in which each alkyl group at
C2 preferentially takes its equatorial disposition, and the
1H NMR data and molecular calculations fully supported
the structures.13,15 Preferred conformations of the present
2-substituted 19-norvitamin D3 6a -8a and 6b-8b are
shown in Figure 2 based on the above discussion and the
1H NMR experiments including NOEs. The six new
analogues were put into two categories: one group had
NOE between H6 and H4R, while the other showed NOE
between H6 and H4â. The analogues belonging to the
former group exhibited a larger (axial-axial) J 3R,4â ) 9.2-
9.6 Hz and with a smaller J 1â,10R ) 4.1-4.7 Hz. On the
2.14 2.13-2.17 2.34 2.34-2.42
2.87 2.86
2.15 2.13-2.17 3.08
5.82
6.38
2.80
0.53
0.94
1.22
2.34
1.88-1.92
3.10
5.87
6.26
2.80
0.55
0.94
1.22
3.11
5.87
6.26
2.80
0.55
0.94
1.22
5.81
6.35
2.80
0.53
0.94
1.22
5.87
6.26
2.80
0.55
0.94
1.22
6-H
9â-H
18-H
21-H
26,27-H
a
b
Reference 15. Reference 13.
chemical shift of each proton can be seen in the 2R-series
vs 2â-series, respectively.
This C5-C6 J ulia-type olefination was found to be
applicable to the synthesis of 2-hydroxy-1R,25-dihydroxy-
19-norvitamin D3 (30), which was reported by DeLuca’s
group in 1994 and Yamada’s group in 2003.19,28 As shown
in Scheme 4, (-)-quinic acid was converted to 4,5-O-
isopropylidene lactone 26 in 80% yield. Reduction and
other hand, the later group showed a smaller J 3R,4â
)
3.3-3.8 Hz and a larger (axial-axial) J 1â,10R ) 9.8-10.3
Hz. In this manner, we were able to deduce the stereo-
chemistry of each isomer (Figure 2).
1
Moreover, the H NMR chemical shifts of representa-
tive protons of 2-methyl,15 2-ethyl,13 and 2-allyl analogues
are listed in Table 1, in which comparable correlation in
(28) Shimizu, M.; Iwasaki, Y.; Shibamoto, Y.; Sato, M.; DeLuca, H.
F.; Yamada, S. Bioorg. Med. Chem. Lett. 2003, 13, 809-812.
7410 J . Org. Chem., Vol. 68, No. 19, 2003

Synthesis of 2-Modified 1R,25-Dihydroxy-19-norvitamin D3
summarized in Table 2 (bovine thymus VDR).31 Data of
2R- and 2â-methyl, ethyl, and hydroxymethyl analogues
of 5 using the porcine intestinal VDR were reported.13,15
In the 2R-alkyl series, the 2R-propyl group in 7a
seemed to be too long to fit in the ligand-binding domain
(LBD) of the VDR. The 2R-allyl group in 6a may have
the same effect on binding. As a pure alkyl substituent
at the 2R-position, 2R-ethyl analogue of 5 showed high
binding affinity (83% of 1) for the porcine intestinal
VDR.13 From our experiments, the 2R-methyl analogue
of 1 was the strongest binder to the bovine thymus VDR
in the 2R-alkyl series.7,8 Introduction of the alkyl group
in the 2â-orientation (6b, 7b) decreased the affinity
(Table 2). However, 8a with the 3-hydroxypropyl group
at the 2R-position exhibited an equivalent binding affinity
to the natural hormone 1. The terminal hydroxyl group
of the 2R-(3-hydroxypropyl) substituent of 8a would form
an additional hydrogen bonding to Arg-274 or Asp-144
of the LBD to stabilize the ligand-VDR complex as in
the case of 2R-(3-hydroxypropyl) and 2R-(3-hydroxypro-
poxy) analogues of 1.8a,9a,11 On the other hand, the 2â-
isomer 8b was 33-fold less potent than 1. From our
experiments on the various 2-substituted analogues7-9
of 1 and DeLuca’s 2-substituted 19-nor analogues,13,15 it
can be said that 2R-isomers have a higher VDR affinity
than the corresponding 2â-isomers. Recently, Shimizu et
al. reported the synthesis of 2-hydroxyethoxy- and 2-(N,N-
diethylcarbamoyl)methoxy active 19-norvitamin D3, in
which the modified A-ring moiety was derived from
D-glucose. Interestingly, the 2â-hydroxyethoxy derivative
was more potent in the VDR binding than the 2R-
hydroxyethoxy counterpart.28
SCHEME 4a
a
Reagents and conditions: (a) 2,2-dimethoxypropane, (+)-CSA,
benzene, 80%; (b) NaBH4, EtOH; (c) NaIO4, CH2Cl2-H2O, 92% in
two steps; (d) TBSCl, Et3N, DMF, 95%; (e) 23, LiHMDS, THF,
-78 to 0 °C; (f) (+)-CSA, MeOH, 64% in two steps.
TABLE 2. Rela tive VDR Bin d in g Affin ity a n d HL-60
Cell Differ en tia tion Activity of 2-Su bstitu ted An a logu es
of 1r,25-Dih yd r oxy-19-n or vita m in D3
VDR binding
affinitya,b
HL-60 cell
compd
differentiationa,c
natural hormone (1)
19-nor (5)
2R-allyl (6a )
2R-propyl (7a )
2R-hydroxypropyl (8a )
2â-allyl (6b)
2â-propyl (7b)
2â-hydroxypropyl (8b)
100
17
3
100
27
54
84
3563
20
3
100
0.15
0.3
3
32
667
Next, we tested the ability of these new 19-nor ana-
logues to induce differentiation of HL-60 cells, which are
human promyelocytic leukemia cells, by the NBT reduc-
tion method.32 The results are summarized in Table 2.
The analogue 8a showed ca. 36-fold higher activity in
inducing cell differentiation, and 8b was 6.7-fold more
potent than 1, regardless of its low affinity for the VDR.
It is generally accepted that the affinity of a ligand for
the VDR and its cell-differentiating activity are not
always parallel, because the kinetics of VDR-ligand
complex formation to form a transcriptionally active horo-
form does not directly affect the kinetics in the binding
of a coactivator to the active VDR-ligand complex to
activate the target gene.33 The striking differential ability
of diastereomeric 8a and 8b to induce HL-60 cell dif-
ferentiation provides new insight into the structure-
activity relationships of vitamin D3 analogues, important
for the design of new, more potent drug candidates.
a
The potency of 1 was normalized to 100. b Bovine thymus VDR.
c Determined by an NBT assay; relative activity was calculated
at EC50. Data are the means of three separate experiments.
subsequent oxidative cleavage of the resulting vicinal diol
27 by sodium periodate gave cyclohexanone 28 in 92%
yield in two steps.29 TBS protection of the secondary
alcohol furnished a substrate 29 for the J ulia-type
coupling in 95% yield.30 Under the same basic conditions
as the previous modified J ulia olefination, O-protected
target compounds (a diastereoisomeric mixture) were
obtained. Deprotection using (+)-CSA in MeOH gave 30
in 64% yield in the last two steps (overall yield from (-)-
quinic acid was 45%).
VDR Bin d in g Affin ity a n d In d u ction of HL-60
Cell Differ en tia tion . Zhou et al. reported that the
binding affinity of 1R,25-dihydroxy-19-norvitamin D3 (5)
for the porcine intestinal VDR was 30% of that of the
natural hormone.17 In our experiments using the bovine
thymus VDR, it was 17% (Table 2). The introduction of
some kind of 2R-substituent, such as the methyl,7 3-hy-
droxypropyl,8 or 3-hydroxypropoxy group,9 to the natural
hormone 1 increases the binding affinity.10,11 We antici-
pated that the introduction of the 3-hydroxypropyl group
to 5 would increase the VDR affinity. The results are
Con clu sion
We efficiently synthesized three kinds of 2-substituted
active 19-norvitamin D3 analogues (6a ,b-8a ,b) in 11-
14 steps with 38-54% overall yields from (-)-quinic acid
(31) (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.
(32) Collins, S. J .; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C. J .
Exp. Med. 1979, 149, 969-974.
(33) Masuno, H.; Yamamoto, K.; Wang, X.; Choi, M.; Ooizumi, H.;
Shinki, T.; Yamada, S. J . Med. Chem. 2002, 45, 1825-1834.
(29) (a) Wang, Z.-X.; Miller, S. M.; Anderson, O. P.; Shi, Y. J . Org.
Chem. 1999, 64, 6443-6458. (b) Audia, J . E.; Boisvert, L.; Patten, A.
D.; Villalobos, A.; Danishefsky, S. J . J . Org. Chem. 1989, 54, 3738-
3740.
(30) (a) Izuhara, T.; Katoh, T. Tetrahedron Lett. 2000, 41, 7651-
7655. (b) Izuhara, T.; Katoh, T. Org. Lett. 2001, 3, 1653-1656.
J . Org. Chem, Vol. 68, No. 19, 2003 7411

Ono et al.
(400 MHz, CDCl3) major isomer: δ -0.02 (s, 3H), -0.01 (s,
3H), 0.00 (s, 6H), 0.80 (s, 9H), 0.82 (s, 9H), 1.15-1.36 (m, 2H),
1.45 (dd, J ) 2.2, 14.6 Hz, 1H), 1.52-1.68 (m, 2H), 1.84-1.99
(m, 1H), 2.08-2.09 (m, 1H), 2.45-2.49 (m, 1H), 3.23-3.31 (m,
2H), 4.10 (m, 1H), 4.38 (ddd, J ) 4.6, 5.1, 11.2 Hz, 1H), 4.48
(br s, 1H), 4.91 (m, 2H), 5.60-5.76 (m, 1H); minor isomer:
-0.01 (s, 9H), 0.03 (s, 3H), 0.05 (s, 3H), 0.84 (s, 9H), 3.89 (dt,
J ) 4.3, 10.4 Hz, 1H), 4.19-4.21 (m, 2H). 13C NMR (100 MHz,
CDCl3) major isomer: δ -5.05, -4.79, -4.77, -4.55, 17.89,
18.23, 25.79, 25.97, 28.94, 32.98, 38.81, 47.40, 65.05, 70.85,
71.78, 74.18, 116.08, 137.23; minor isomer: δ -4.59, -4.43,
-3.85, -3.68, 18.13, 18.21, 26.03, 30.82, 38.44, 44.37, 50.66,
67.30, 70.60, 70.74, 74.50, 115.75, 137.37. IR (neat): 3486,
3079, 2932, 2859, 1640, 1470, 1391, 1362, 1226, 1111, 1051,
911, 874, 833, 802, 777, 667 cm-1. EIMS: 430 (M+). HREIMS:
calcd for C22H46O4Si2 (M+) 430.2935, found 430.2939.
(3R,5R)-4-Allyl-3,5-bis[(ter t-bu tyld im eth ylsilyl)oxy]cy-
cloh exa n on e (12). To a solution of 11 (41 mg, 95 µmol) in
MeOH (2 mL) and H2O (1 mL) was added NaIO4 (31 mg, 0.14
mmol) at 0 °C. After being stirred at rt for 30 min, the reaction
mixture was diluted with AcOEt. Brine was added to the
mixture, the organic layer was separated, and the aqueous
layer was extracted with AcOEt. The organic layer and
extracts were combined and washed with brine, dried over
MgSO4, and concentrated in vacuo. Purification by silica gel
column chromatography (hexane/AcOEt ) 10:1) gave 34 mg
of 12 as a colorless oil (90%). [R]24D: -33.6 (c 1.23, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 0.05 (s, 12H), 0.86 (s, 9H), 0.88 (s,
9H), 1.7-1.9 (m, 1H), 2.2-2.3 (m, 1H), 2.3-2.4 (m, 2H), 2.43
(dd, J ) 3.6, 14.4 Hz, 1H), 2.49 (ddd, J ) 1.5, 5.7, 14.4 Hz,
1H), 2.64 (ddd, J ) 1.5, 4.8, 14.4 Hz, 1H), 4.0-4.1 (m, 1H),
4.2-4.3 (m, 1H), 5.08 (d, J ) 10.5 Hz, 1H), 5.09 (br d, J )
17.7 Hz, 1H), 5.81 (ddt, J ) 10.5, 17.7, 6.9 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ -4.7, -4.3, -4.0, 18.1, 25.9, 29.8, 30.9,
48.8, 49.2, 67.9, 69.6, 116.4, 136.9, 212.0. IR (neat): 2955, 2932,
2896, 2859, 2361, 1725, 1641, 1472, 1389, 1362, 1333, 1256,
1161, 1094, 980, 938, 889, 837, 777 cm-1. EIMS: 398 (M+).
HREIMS: calcd for C21H42O3Si2 (M+) 398.2673, found 398.2675.
and 25-hydroxy Grundmann’s ketone, utilizing the modi-
fied J ulia coupling reaction to connect between the C5
and C6 positions. This approach was a short and concise
way to introduce a three-carbon unit to the C2 position
of 19-norvitamin D3 (5). Further manipulation of the allyl
group of intermediate 12 can provide a shorter or longer
carbon functional group at C2. The 2R-(3-hydroxypropyl)
group in 8a contributed to a marked increase in both the
VDR binding affinity and potency in the induction of HL-
60 cell differentiation. As for the VDR binding affinity
of 8a , formation of the new hydrogen bonding between
the terminal hydroxyl group of the C2-substituent of 8a
and Arg-274 or Asp-144 would stabilize the ligand (8a )-
VDR complex. The 2â-counterpart showed 6.7-fold higher
activity in cell differentiation despite a lower affinity to
the VDR. The 2-propyl and 2-allyl groups were too long
as the alkyl substituents to form stable hydrophobic
contact with the LBD of the VDR, and analogues with
2-methyl and 2-ethyl groups were better binders to the
VDR.13,15
The present study implies that the 2R-(3-hydroxypro-
pyl) group in 8a can compensate for the loss of hydro-
phobic interaction of the 10(19)-exocyclic methylene
group in the LBD, which causes the low VDR affinity of
5. Further biological testing is underway in our labora-
tories.
Exp er im en ta l Section
Meth yl (1R,3R,4RS,5R)-4-Allyl-3,5-bis[(ter t-bu tyldim eth -
ylsilyl)oxy]-1-h yd r oxycycloh exa n eca r boxyla te (10). To a
solution of 912b (1.53 g, 2.81 mmol) in dry benzene (4 mL) was
added allyltributyltin (1.9 mL, 8.46 mmol) at rt. To the
resulting mixture was added the benzene solution (10 mL) of
ΑΙΒΝ (462 mg, 2.81 mmol) over 3 h at a refluxing temperature,
and the mixture was further refluxed for 3 h. After cooling,
the mixture was concentrated in vacuo, and purification by
silica gel column chromatography (hexane/AcOEt ) 10:1)
afforded 775 mg of 10 as a colorless oil (60%). This was an
inseparable mixture of isomers in a ratio of ca. 7:3 and was
used without further purification. 1H NMR (400 MHz, CDCl3)
major isomer: δ 0.06 (s, 6H), 0.07 (s, 6H), 0.88 (s, 9H), 0.89 (s,
9H), 1.24-1.37 (m, 1H), 1.50-1.80 (m, 3H), 1.93-1.99 (m, 2H),
2.09 (dd, J ) 2.4, 14.6 Hz, 1H), 2.59 (br d, J ) 14.6 Hz, 1H),
3.76 (s, 3H), 4.22 (m, 1H), 4.45 (ddd, J ) 4.6, 5.1, 11.2 Hz,
1H), 4.78 (s, 1H), 5.03 (d, J ) 9.8 Hz, 1H), 5.04 (d, J ) 17.1
Hz, 1H), 5.74 (m, 1H); minor isomer: 0.10 (s, 6H), 0.13 (s, 6H),
0.88 (s, 6H), 0.91 (s, 9H), 2.19 (dd, J ) 3.2, 12.9 Hz, 1H), 3.98
(dt, J ) 4.4, 10.3 Hz, 1H), 4.29 (m, 1H), 4.35 (s, 1H). 13C NMR
major isomer: -5.00, -4.92, -4.65, -4.47, 17.8, 18.2, 25.8,
25.9, 28.8, 33.5, 39.6, 46.9, 52.5, 64.4, 71.6, 76.4, 116.3, 137.0,
173.9; minor isomer: -4.81, -4.65, -3.95, -3.65, 17.7, 18.1,
25.96, 26.60, 27.9, 30.6, 39.0, 45.0, 49.9, 66.8, 70.2, 77.2, 115.9,
137.1. IR (neat): 3484, 2955, 2930, 2859, 1740, 1642, 1464,
1389, 1362, 1256, 1103, 876, 837, 777, 669 cm-1. EIMS: 401
(3R,5R)-3,5-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4-p r op yl-
cycloh exa n on e (13). To a solution of 12 (527 mg, 1.32 mmol)
in dry MeOH (15 mL) was added 10% Pd/C (50 mg), and the
mixture was stirred at rt in a hydrogen atmosphere (balloon)
for 15 h. The reaction mixture was filtered through a silica
gel pad, and the filtrate was concentrated in vacuo. Purifica-
tion by silica gel column chromatography (hexane/AcOEt )
5:1) afforded 524 mg of 13 as a colorless oil (99%). [R]24D: -29.5
1
(c 1.08, CHCl3). H NMR (400 MHz, CDCl3): δ 0.036 (s, 3H),
0.045 (s, 3H), 0.05 (s, 6H), 0.85 (s, 9H), 0.88 (s, 9H), 0.95 (t, J
) 7.2 Hz, 3H), 1.33 (m, 1H), 1.40-1.54 (m, 3H), 1.71 (ddt, J )
5.1, 8.0, 2.6 Hz, 1H), 2.31 (dd, J ) 8.0, 14.3 Hz, 1H), 2.43 (m,
2H), 2.62 (dd, J ) 4.7, 14.3 Hz, 1H), 4.02 (dt, J ) 4.7, 8.0 Hz,
1H), 4.31 (dt, J ) 2.8, 5.1 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ -4.9, -4.8, -4.7, -4.3, -4.2, 14.5, 18.1, 20.8, 25.8,
25.9, 28.7, 48.5, 48.8, 49.3, 68.2, 70.3, 207.7. IR (neat): 2957,
2932, 2896, 2859, 1725, 1464, 1362, 1335, 1256, 1171, 1088,
1005, 953, 920, 884, 835, 777, 666 cm-1. EIMS: 400 (M+).
HREIMS: calcd for C21H44O3Si2 (M+) 400.2829, found 400.2822.
(3R,5R)-3,5-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-4-(3-h y-
d r oxyp r op yl)cycloh exa n on e (14). To a solution of 10 (50
mg, 0.11 mmol) in dry THF (1 mL) was added BH3‚THF (1 M
solution in THF, 0.27 mL, 0.27 mmol) at 0 °C. After being
stirred at rt for 2 h, the reaction was quenched by the addition
of 3 M NaOH (1 mL) and 30% H2O2 (2 mL). After being stirred
at rt for an additional 2 h, the reaction mixture was poured
into saturated aqueous Na2SO3 and extracted with AcOEt. The
combined extracts were washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo. The residue was dissolved
in EtOH (1 mL), and NaBH4 (13 mg, 34 mmol) was added to
the solution at 0 °C. After being stirred at rt for 2 h, the
mixture was poured into saturated aqueous NH4Cl and
extracted with CH2Cl2. The organic extracts were combined
and washed with saturated aqueous NH4Cl and brine, dried
(M+
- -
tBu). HREIMS: calcd for C19H37O5Si2 (M+ tBu)
401.2180, found 401.2186.
(1R,3R,4RS,5R)-4-Allyl-3,5-bis[(ter t-bu tyldim eth ylsilyl)-
oxy]-1-(h yd r oxym eth yl)cycloh exa n -1-ol (11). To a solution
of 10 (1.52 g, 3.31 mmol) in EtOH (30 mL) was added NaBH4
(375 mg, 9.93 mmol) at 0 °C, and the mixture was stirred at
rt for 4 h. The reaction mixture was poured into saturated
aqueous NH4Cl and extracted with CH2Cl2. The combined
extracts were washed with saturated aqueous NH4Cl and
brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(hexane/AcOEt ) 5:1) to afford 1.29 g of 11 as a colorless oil
(91%). This was an inseparable mixture of isomers in a ratio
1
of ca. 7:3 and was used without further purification. H NMR
7412 J . Org. Chem., Vol. 68, No. 19, 2003

Synthesis of 2-Modified 1R,25-Dihydroxy-19-norvitamin D3
over MgSO4, and concentrated in vacuo. The residue contain-
ing vicinal diol was dissolved in MeOH (2 mL), and NaIO4 (70
mg, 327 mmol) in H2O (1 mL) was added to the solution at 0
°C. After being stirred at rt for 14 h, the reaction mixture was
diluted with AcOEt, and brine was added. The organic layer
was separated, and the aqueous layer was extracted with
AcOEt. The organic layer and extracts were combined and
washed with brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (hexane/AcOEt ) 2:1) to give 31 mg of 14 as a
colorless oil (68% in three steps). [R]24D: -39.4 (c 1.14, CHCl3).
1H NMR (400 MHz, CDCl3): δ 0.051 (s, 3H), 0.056 (s, 6H),
0.063 (s, 3H), 0.86 (s, 9H), 0.88 (s, 9H), 1.52-1.75 (m, 5H),
2.33 (dd, J ) 7.9, 14.4 Hz, 1H), 2.46 (m, 2H), 2.64 (dd, J )
4.6, 14.4 Hz, 1H), 3.69 (t, J ) 6.0 Hz, 2H), 4.04 (dt, J ) 4.6,
7.9 Hz, 1H), 4.33 (dt, J ) 2.9, 5.9 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ -4.8, -4.7, -4.3, -4.2, 18.1, 18.2, 22.7, 25.8, 25.9,
30.9, 48.7, 48.8, 49.2, 63.1, 68.3, 70.3, 207.5. IR (neat): 3426,
2955, 2930, 2894, 2859, 1719, 1472, 1362, 1256, 1096, 1061,
1271, 1254, 1146, 1096, 1042, 918, 866, 754 cm-1. EIMS: 394
(M+). HREIMS: calcd for C24H42O4 (M+) 394.3083, found
394.3091.
2-[(1R,2E,6R,7R)-7-[(R)-6-Met h oxym et h oxy-6-m et h yl-
h ep ta n -2-yl]-6-m eth ylbicyclo[4.3.0]n on a n -2-ylid en e]eth -
a n ol (22). To the (E/ Z)-mixture of 21 (751 mg, 1.90 mmol) in
toluene (14 mL) was added DIBAL-H (1.04 M solution in
toluene, 4.6 mL, 4.78 mmol) at -78 °C. After the mixture was
stirred at the same temperature for 1 h, the reaction was
quenched by adding saturated aqueous Na2SO4. The resulting
mixture was filtered through a pad of Celite, and the filtrate
was concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/AcOEt ) 5:1) to yield 666
1
mg of 22 as a colorless oil (99%). H NMR (400 MHz, CDCl3)
major isomer: δ 0.55 (s, 3H), 0.93 (d, J ) 6.4 Hz, 3H), 1.03 (m,
1H), 1.22 (s, 6H), 1.25-1.30 (m, 3H), 1.32-1.41 (m, 4H), 1.44-
1.56 (m, 5H), 1.65-1.68 (m, 3H), 1.84-2.05 (m, 3H), 2.63 (dd,
J ) 4.2, 12.2 Hz, 1H), 3.37 (s, 3H), 4.21 (d, J ) 7.2 Hz, 2H),
4.71 (s, 2H), 5.22 (t, J ) 7.0 Hz, 1H); minor isomer (selected):
δ 0.88 (d, J ) 6.4 Hz, 3H), 5.41 (t, J ) 7.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 12.0, 18.9, 20.7, 22.3, 23.6, 26.4, 26.5,
27.7, 28.8, 36.2, 36.5, 40.5, 42.3, 45.4, 55.1, 55.7, 56.7, 58.7,
76.4, 90.9, 119.1, 143.5. EIMS: 334 (M+ - H2O). HREIMS:
calcd for C22H38O2 (M+ - H2O) 334.2872, found 334.2876.
t
835, 775 cm-1. EIMS: 359 (M+ - Bu). HREIMS: calcd for
t
C
17H35O4Si2 (M+ - Bu) 359.2074, found 359.2078.
(3R,5R)-3,5-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4-[3-(ter t-
bu tyld im eth ylsilyloxy)p r op yl]cycloh exa n on e (15). To a
solution of 14 (204 mg, 0.49 mmol) in dry CH2Cl2 (5 mL) were
added Et3N (0.1 mL, 0.72 mmol), DMAP (6 mg, 50 µmol), and
TBSCl (88 mg, 0.58 mmol) at 0 °C. After being stirred at rt
for 2 h, the reaction mixture was poured into saturated
aqueous NH4Cl, and the organic layer was separated. The
aqueous layer was extracted with AcOEt. The combined
organic layer and extracts were washed with saturated aque-
ous NH4Cl and brine, dried over MgSO4, and concentrated in
vacuo. Purification by silica gel column chromatography (hex-
ane/AcOEt ) 10:1) afforded 259 mg of 15 as a colorless oil
(quant). [R]27D: -29.5 (c 1.38, CHCl3). 1H NMR (400 MHz,
CDCl3): δ 0.043 (s, 3H), 0.047 (s, 6H), 0.052 (s, 9H), 0.851 (s,
9H), 0.877 (s, 9H), 0.896 (s, 9H), 1.52-1.70 (m, 5H), 2.32 (dd,
J ) 7.9, 14.6 Hz, 1H), 2.44 (m, 2H), 2.63 (dd, J ) 4.6, 14.6 Hz,
1H), 3.64 (t, J ) 5.8 Hz, 2H), 4.03 (dt, J ) 4.6, 7.9 Hz, 1H),
4.32 (dt, J ) 2.4, 4.8 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ
-5.11, -5.08, -4.78, -4.64, -4.26, -4.20, 18.11, 18.13, 18.52,
23.10, 25.84, 25.93, 26.12, 31.16, 48.82, 48.87, 49.35, 63.52,
68.30, 70.31, 207.7. IR (neat): 2955, 2930, 2894, 2859, 1725,
1472, 1256, 1098, 909, 835, 775 cm-1. EIMS: 530 (M+).
HREIMS: calcd for C27H58O4Si3 (M+) 530.3643, found 530.3649.
(1R ,2E ,6R ,7R )-2-[2-(B e n zo t h ia zo le -2-s u lfo n y l)e t h -
ylid en e]-7-[(R)-6-m eth oxym eth oxy-6-m eth ylh ep ta n -2-yl]-
6-m eth ylbicyclo[4.3.0]n on a n e (23). To a solution of 2-mer-
captobenzothiazole (451 mg, 2.70 mmol) and PPh3 (707 mg,
2.70 mmol) in dry CH2Cl2 (5 mL) were added a solution of 22
(633 mg, 1.80 mmol) in dry CH2Cl2 (5 mL) and DIAD (0.52
mL, 1.80 mmol) at 0 °C. After being stirred at the same
temperature for 1 h, the mixture was concentrated. The
residue was dissolved in EtOH (10 mL), and to the solution
were added 30% H2O2 (1 mL) and (NH4)6Mo7O24‚4H2O (444
mg, 0.36 mmol) at 0 °C. After being stirred at rt for 2 h, the
mixture was poured into saturated aqueous Na2SO3 and was
extracted with AcOEt. The extracts were combined and
washed with brine, dried over MgSO4, and concentrated.
Purification by silica gel column chromatography (hexane/Et2O
1
) 3:1) gave 849 mg of sulfone 23 as a colorless oil (89%). H
NMR (400 MHz, CDCl3) major isomer: δ 0.26 (s, 3H), 0.86 (d,
J ) 6.4 Hz, 3H), 1.00 (m, 1H), 1.20 (s, 6H), 1.22-1.53 (m, 14H),
1.81-1.91 (m, 3H), 2.55 (br d, J ) 12.4 Hz, 1H), 3.36 (s, 3H),
4.21 (dd, J ) 6.8, 14.0 Hz, 1H), 4.43 (dd, J ) 8.8, 14.0 Hz,
1H), 4.70 (s, 2H), 5.02 (dd, J ) 6.8, 8.8 Hz, 1H), 7.58 (t, J )
8.0 Hz, 1H), 7.63 (t, J ) 8.0 Hz, 1H), 8.00 (d, J ) 8.0 Hz, 1H),
8.16 (d, J ) 8.0 Hz, 1H); minor isomer (selected): δ 0.78 (d, J
) 6.4 Hz, 3H), 1.21 (s, 6H), 3.37 (s, 3H), 4.71 (s, 2H). 13C NMR
(100 MHz, CDCl3): δ 11.7, 18.9, 20.6, 22.2, 23.2, 26.4, 26.5,
27.6, 29.1, 36.0, 36.4, 40.0, 42.3, 45.8, 53.9, 55.1, 56.0, 56.4,
76.3, 90.9, 104.0, 122.0, 125.2, 127.4, 127.7, 136.8, 151.9, 152.6,
165.7. IR (neat): 2948, 2874, 2363, 1657, 1599, 1567, 1472,
1424, 1381, 1327, 1240, 1148, 1090, 1040, 916, 853, 762, 731,
689, 639 cm-1. EIMS: 533 (M+). HREIMS: calcd for C29H43O4-
NS2 (M+) 533.2634, found 533.2637.
Eth yl [(1R,2E,6R,7R)-7-[(R)-6-Meth oxym eth oxy-6-m eth -
ylh ep ta n -2-yl]-6-m eth ylbicyclo[4.3.0]n on a n -2-ylid en e]a c-
eta te (21). To a suspension of NaH (675 mg, 20.3 mmol) in
THF (20 mL) was added (EtO)2P(O)CH2CO2Et (3.7 mL, 23.6
mmol) at 0 °C, and the mixture was stirred at rt for 17 h.
Ketone 20 (1.10 g, 3.38 mmol) was dissolved in THF (10 mL),
and the solution was added to the mixture above at 0 °C. After
being stirred at the same temperature for 14 h, the reaction
mixture was poured into saturated aqueous NH4Cl and
extracted with AcOEt. The combined extracts were washed
with saturated aqueous NH4Cl and brine, dried over MgSO4,
and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/AcOEt ) 8:1) gave 1.31 g of 21 as a
colorless oil (98%). This was an inseparable mixture of E/ Z-
isomers (E/ Z ) 92:8) and used without further purification.
1H NMR (400 MHz, CDCl3) major isomer (E): δ 0.57 (s, 3H),
0.93 (d, J ) 6.4 Hz, 3H), 1.04 (m, 1H), 1.21 (s, 6H), 1.28 (t, J
) 7.2 Hz, 3H), 1.31-1.46 (m, 7H), 1.48-1.55 (m, 3H), 1.60-
1.75 (m, 4H), 1.87-1.89 (m, 1H), 2.01 (br. d, J ) 15.6 Hz, 1H),
2.09 (dt, J ) 1.2, 10.0 Hz, 1H), 3.36 (s, 3H), 3.84 (br d, J )
14.4 Hz, 1H), 4.14 (dq, J ) 1.8, 7.2 Hz, 2H), 4.70 (s, 2H), 5.45
(s, 1H); minor isomer (Z) selected: δ 0.88 (d, J ) 6.4 Hz, 3H),
1.26 (s, 6H), 1.27 (t, J ) 7.2 Hz, 3H), 5.65 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 12.3, 14.5, 18.9, 20.7, 22.3, 24.0, 26.4,
26.5, 27.6, 29.7, 29.8, 36.1, 36.4, 40.2, 42.4, 47.2, 55.1, 56.8,
56.9, 59.5, 76.3, 91.0, 111.9, 163.1, 166.7. IR (neat): 2944, 2870,
2774, 2124, 2060, 1958, 1717, 1647, 1466, 1381, 1368, 1308,
(1R,2E,6R,7R)-7-[(R)-6-Meth oxym eth oxy-6-m eth ylh ep -
ta n -2-yl]-6-m eth yl-2-[2-(d ip h en ylp h osp h or yl)eth ylid en e]-
bicyclo[4.3.0]n on a n e (24). To a solution of 22 (133 mg, 0.38
mmol) in dry THF (3 mL) was added BuLi (1.58 M in hexane,
0.28 mL, 0.45 mmol) at 0 °C. After the mixture was stirred at
the same temperature for 5 min, freshly recrystallized TsCl
(86 mg, 0.45 mmol) was added at the same temperature. To
another flask charged with a solution of HPPh2 (0.13 mL, 0.76
mmol) in dry THF (1 mL) was added BuLi (1.58 M solution in
hexane, 0.48 mL, 0.76 mmol), and the resulting red solution
was added to the above tosylate solution at 0 °C. After being
stirred at the same temperature for 1 h, the reaction was
quenched by the addition of water. The solvents were evapo-
rated, and the residue was dissolved in CH2Cl2 (4 mL). To the
solution was added 30% H2O2 (2 mL) at 0 °C, and the mixture
was stirred at the same temperature for 30 min. The organic
layer was separated, and the aqueous layer was extracted with
J . Org. Chem, Vol. 68, No. 19, 2003 7413

Ono et al.
CH2Cl2. The combined organic layer was washed with aqueous
1 M Na2SO3, H2O, and brine, dried over MgSO4, and concen-
trated in vacuo. Purification by silica gel column chromatog-
raphy (hexane/AcOEt ) 1:1) afforded 178 mg of 24 as an
amorphous solid (88%). 1H NMR (400 MHz, CDCl3) major
isomer: δ 0.29 (s, 3H), 0.88 (d, J ) 6.4 Hz, 3H), 1.20 (s, 6H),
2.42 (br d, J ) 13.2 Hz, 1H), 3.04-3.27 (m, 2H), 3.356 (s, 3H),
4.69 (s, 2H), 5.00 (br dd, J ) 6.6, 14.2 Hz, 1H), 7.44-7.49 (m,
6H), 7.68-7.78 (m, 4H); minor isomer: δ 0.83 (d, J ) 6.8 Hz,
3H), 1.21 (s, 6H), 3.362 (s, 3H), 4.70 (s, 2H), 5.26 (m, 1H). 13C
NMR (100 MHz, CDCl3): δ 11.8, 14.9, 18.9, 20.7, 22.3, 23.2,
26.4, 26.5, 27.7, 36.1, 36.5, 40.4, 42.3, 49.7, 55.1, 56.5, 63.4,
76.4, 81.6, 90.9, 128.3, 128.4, 130.7, 130.8, 131.0, 131.1, 131.4,
131.5, 144.5. EIMS: 536 (M+). HREIMS: calcd for C34H49O3P
(M+) 536.3420, found 536.3409.
11.3 Hz, 1H, 7-H), 5.93 (dddd, J ) 7.3, 7.3, 10.2, 17.0 Hz, 1H,
-CH2CHdCH2), 6.26 (d, J ) 11.3 Hz, 1H, 6-H). 13C NMR (150
MHz, CDCl3): δ 12.0, 18.8, 20.8, 22.3, 23.5, 27.7, 29.0, 29.2,
29.4, 33.2, 36.1, 36.4, 37.7, 40.4, 44.0, 44.4, 45.8, 49.2, 56.3,
56.5, 68.8, 71.0, 71.1, 115.3, 116.5, 123.5, 131.1, 137.6, 143.1.
IR (neat): 3420, 2928, 2868, 1640 cm-1. EIMS: 444 (M+).
HREIMS: calcd for C29H48O3 (M+) 444.3603, found 444.3609.
(a R,1R,3R,7E)-2-P r op yl-9,10-seco-19-n or ch olest a -5,7-
d ien e-1,3,25-tr iol (7a ) a n d (a S,1R,3R,7E)-2-P r op yl-9,10-
seco-19-n or ch olesta -5,7-d ien e-1,3,25-tr iol (7b). These ana-
logues were obtained through the same procedure as described
above for the synthesis of 6a and 6b. A crude product, which
was obtained from 23 (74 mg, 139 µmol), 13 (43 mg, 107 µmol),
and LiHMDS (1 M solution in THF, 130 µL, 130 µmol), was
partially purified by silica gel column chromatography (hexane/
AcOEt ) 40:1) to give protected 19-norvitamin D3 (51 mg,
66%). To a solution of the crude 19-norvitamin D3 (46 mg, 64
µmol) in dry MeOH (2 mL) was added (+)-CSA (45 mg, 194
µmol) at 0 °C, and the mixture was stirred at rt for 22 h. The
usual workup and purification on silica gel column chroma-
tography (hexane/AcOEt ) 3:2) afforded 22.0 mg of 7 as a
mixture of isomers in a ratio of ca. 1:1 (51%). The isomers were
separated by reversed-phase HPLC (YMC-Pack ODS column,
20 × 150 mm, 9.9 mL/min) using CH3CN/H2O (90:10) as a
solvent system. Data for 7a (2R-propylated 5): tR ) 14.2 min.
[R]21D: +21.3 (c 0.36, CHCl3). UV (EtOH) λmax: 243.5, 252.0,
(aS,1R,3R,7E)-2-Allyl-9,10-seco-19-n or ch olesta-5,7-dien e-
1,3,25-t r iol (6a ) a n d (a R,1R,3R,7E)-2-Allyl-9,10-seco-19-
n or ch olesta -5,7-d ien e-1,3,25-tr iol (6b). To a solution of 23
(31.1 mg, 58.3 µmol) in dry THF (200 µL) was added LiHMDS
(1 M solution in THF, 55 µL, 55 µmol) at -78 °C. After the
mixture was stirred at the same temperature for 1 h, a solution
of 12 (17.1 mg, 42.9 µmol) in dry THF (200 µL) was added
dropwise to the mixture. After being stirred for 3 h, the
reaction mixture was poured into saturated aqueous NH4Cl
and extracted with Et2O. The combined organic layer was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatography
on silica gel (hexane/AcOEt ) 30:1) to yield crude protected
19-norvitamin D3 (18.0 mg, ca. 59%). To a solution of the crude
protected 19-norvitamin D3 (9 mg, 13 µmol) in dry MeOH (2
mL) was added (+)-CSA (10 mg, 23 µmol) at 0 °C. After being
stirred at rt for 19 h, the reaction mixture was diluted with
AcOEt. The resulting mixture was washed with saturated
aqueous NaHCO3 and brine, dried over Na2SO4, and concen-
trated. Purification by silica gel column chromatography
(hexane/AcOEt ) 3:2) afforded 4.0 mg of 6 as a mixture of
isomers in a ratio of ca. 1:1 (47%). The isomers were separated
by reversed-phase HPLC (YMC-Pack ODS column, 20 × 150
mm, 9.9 mL/min) using CH3CN/H2O (85:15) as a solvent
1
262.0 nm; λmin 247.5, 258.5 nm. H NMR (600 MHz, CDCl3):
δ 0.53 (s, 3H, 18-H3), 0.94 (d, J ) 6.6 Hz, 3H, 21-H3), 0.96 (t,
J ) 7.1 Hz, 3H, -CH2CH2CH3), 1.07 (m, 1H), 1.22 (s, 6H, 26-
H3 and 27-H3), 1.24-1.32 (m, 5H), 1.36-1.40 (m, 4H), 1.45-
1.56 (m, 7H), 1.60-1.68 (m, 4H), 1.89 (m, 1H), 1.99-2.01 (m,
2H), 2.12-2.17 (m, 2H), 2.60 (dd, J ) 4.5, 12.5 Hz, 1H, 4R-H),
2.80 (dd, J ) 4.7, 12.6 Hz, 1H, 9â-H), 2.84 (dd, J ) 4.7, 14.0
Hz, 1H, 10R-H), 3.64 (dt, J ) 4.5, 9.2 Hz, 1H, 3R-H), 4.09 (br
s, 1H, 1â-H), 5.82 (d, J ) 11.3 Hz, 1H, 7-H), 6.37 (d, J ) 11.3
Hz, 1H, 6-H). 13C NMR (150 MHz, CDCl3): δ 12.0, 14.4, 18.8,
20.4, 20.8, 22.2, 23.5, 27.6, 28.9, 29.2, 29.3, 29.4, 35.5, 36.1,
36.4, 40.5, 44.4, 45.2, 45.8, 48.9, 56.3, 56.5, 68.3, 71.1, 71.6,
115.2, 124.0, 131.3, 143.2. IR (neat): 3384, 2955, 2872, 1613
cm-1. EIMS: 446 (M+). HREIMS: calcd for C29H50O3 (M+)
446.3760, found 446.3763. Data for 7b (2â-propylated 5): tR
) 13.8 min. [R]21D: +15.2 (c 0.32, CHCl3). UV (EtOH): λmax
243.5, 252.0, 261.5 nm; λmin 247.0, 258.0 nm. 1H NMR (600
MHz, CDCl3): δ 0.55 (s, 3H, 18-H3), 0.94 (d, J ) 6.6 Hz, 3H,
21-H3), 0.95 (t, J ) 7.3 Hz, 3H, -CH2CH2CH3), 1.07 (m, 1H),
1.22 (s, 6H, 26-H3 and 27-H3), 1.24-1.33 (m, 5H), 1.37-1.53
(m, 11H), 1.66-1.67 (m, 3H), 1.88-1.91 (m, 2H), 1.99-2.02
(m, 2H), 2.35 (ddd, J ) 1.2, 3.8, 12.9 Hz, 1H, 4â-H), 2.40 (br d,
J ) 13.9 Hz, 1H, 4R-H), 2.80 (dd, J ) 4.7, 12.6 Hz, 1H, 9â-H),
3.10 (ddd, J ) 1.2, 4.4, 12.7 Hz, 1H, 10â-H), 3.54 (dt, J ) 4.4,
9.8 Hz, 1H, 1â-H), 4.05 (br s, 1H, 3R-H), 5.87 (d, J ) 11.3 Hz,
1H, 7-H), 6.26 (d, J ) 11.3 Hz, 1H, 6-H). 13C NMR (150 MHz,
CDCl3): δ 12.0, 14.4, 18.8, 20.2, 20.8, 22.3, 23.5, 27.7, 29.0,
29.2, 29.4, 29.6, 36.1, 36.4, 37.8, 40.5, 44.1, 44.4, 45.8, 49.1,
56.3, 56.5, 68.0, 71.1, 71.2, 115.3, 123.4, 131.3, 143.0. IR
(neat): 3382, 2942, 2872, 1613 cm-1. EIMS: 446 (M+). HRE-
IMS: calcd for C29H50O3 (M+) 446.3760, found 446.3757.
system. Data for 6a (2R-allylated 5): tR ) 16.6 min. [R]23
:
D
+22.3 (c 0.16, CHCl3). UV (EtOH): λmax 243.5, 252.0, 261.5
1
nm; λmin 247.5, 258.5 nm. H NMR (600 MHz, CDCl3): δ 0.53
(s, 3H, 18-H3), 0.94 (d, J ) 6.3 Hz, 3H, 21-H3), 1.06 (m, 1H),
1.22 (s, 6H, 26-H3 and 27-H3), 1.24-1.32 (m, 6H), 1.36-1.58
(m, 10H), 1.67 (m, 2H), 1.86-1.93 (m, 2H), 1.99-2.01 (m, 2H),
2.13-2.17 (m, 2H), 2.30 (ddd, J ) 6.0, 7.0, 14.2 Hz, 1H, -CH2-
CHdCH2), 2.50 (ddd, J ) 7.0, 8.4, 14.2 Hz, 1H, -CH2CHd
CH2), 2.61 (dd, J ) 4.7, 12.9 Hz, 1H, 4R-H), 2.80 (dd, J ) 4.4,
12.9 Hz, 1H, 9â-H), 2.86 (dd, J ) 4.1, 13.8 Hz, 1H, 10R-H),
3.70 (dt, J ) 4.7, 9.6 Hz, 1H, 3R-H), 4.08 (dt, J ) 4.1, 7.1 Hz,
1H, 1â-H), 5.07 (d, J ) 10.2 Hz, 1H, -CH2CHdCH2), 5.15 (d,
J ) 17.0 Hz, 1H, -CH2CHdCH2), 5.81 (d, J ) 11.3 Hz, 1H,
7-H), 5.92 (dddd, J ) 6.9, 6.9, 10.2, 17.0 Hz, 1H, -CH2CHd
CH2), 6.34 (d, J ) 11.3 Hz, 1H, 6-H). 13C NMR (150 MHz,
CDCl3): δ 12.0, 18.8, 20.8, 22.2, 23.4, 27.6, 28.9, 29.1, 29.3,
32.8, 35.5, 36.1, 36.4, 40.4, 44.4, 45.2, 45.8, 48.9, 56.3, 56.5,
68.7, 71.0, 71.2, 115.2, 116.5, 124.1, 131.2. IR (neat): 3353,
2936, 1644 cm-1. EIMS: 444 (M+). HREIMS: calcd for
29H48O3 (M+) 444.3603, found 444.3597. Data for 6b (2â-
(a R,1R,3R,7E)-2-(3-H yd r oxyp r op yl)-9,10-seco-19-n or -
ch olesta -5,7-d ien e-1,3,25-tr iol (8a ) a n d (a S,1R,3R,7E)-2-
(3-H yd r oxyp r op yl)-9,10-seco-19-n or ch olest a -5,7-d ien e-
1,3,25-tr iol (8b). These analogues were obtained through the
same procedure as described above for the synthesis of 6a and
6b. A crude product, which was obtained from 23 (28.8 mg,
54.0 µmol), 15 (21.8 mg, 41.1 µmol), and LiHMDS (1 M solution
in THF, 50 µL, 50 µmol), was partially purified by silica gel
column chromatography (hexane/AcOEt ) 30:1) to give pro-
tected 19-norvitamin D3 (30.5 mg). To a solution of the crude
19-norvitamin D3 (30.0 mg, 35.9 µmol) in dry MeOH (1 mL)
was added (+)-CSA (42 mg, 180 µmol) at 0 °C, and the mixture
was stirred at rt for 38 h. The usual workup and purification
on silica gel column chromatography (AcOEt/MeOH ) 10:1)
C
allylated 5): tR ) 16.0 min. [R]22D: +13.8 (c 0.13, CHCl3). UV
1
(EtOH): λmax 243.5, 252.0, 261.5 nm; λmin 247.0, 258.0 nm. H
NMR (600 MHz, CDCl3): δ 0.55 (s, 3H, 18-H3), 0.94 (d, J )
6.6 Hz, 3H, 21-H3), 1.07 (m, 1H), 1.22 (s, 6H, 26-H3 and 27-
H3), 1.24-1.33 (m, 5H), 1.37-1.57 (m, 11H), 1.66-1.70 (m,
2H), 1.88-1.92 (m, 2H), 1.99-2.02 (m, 2H), 2.28 (ddd, J ) 7.4,
7.7, 14.6 Hz, 1H, -CH2CHdCH2), 2.34 (ddd, J ) 1.4, 3.4, 14.0
Hz, 1H, 4â-H), 2.40 (br d, J ) 14.0 Hz, 1H,4R-H), 2.53 (ddd, J
) 6.7, 7.4, 14.6 Hz, 1H, -CH2CHdCH2), 2.80 (dd, J ) 4.2,
12.9 Hz, 1H, 9â-H), 3.11 (ddd, J ) 1.4, 4.7, 12.8 Hz, 1H, 10â-
H), 3.64 (dt, J ) 4.7, 10.3 Hz, 1H, 1â-H), 4.02 (ddd, J ) 3.4,
6.3, 6.9 Hz, 1H, 3R-H), 5.07 (d, J ) 10.2 Hz, 1H, -CH2CHd
CH2), 5.16 (d, J ) 17.0 Hz, 1H, -CH2CHdCH2), 5.87 (d, J )
7414 J . Org. Chem., Vol. 68, No. 19, 2003

Synthesis of 2-Modified 1R,25-Dihydroxy-19-norvitamin D3
afforded 11.8 mg of 8 as a mixture of isomers in a ratio of ca.
1:1 (62%). The isomers were separated by reversed-phase
HPLC (YMC-Pack ODS column, 20 × 150 mm, 9.9 mL/min)
using CH3CN/H2O (80:20) as a solvent system. Data for 8a
(2R-3-hydroxypropylated 5): tR ) 10.8 min. [R]22D: +11.4 (c
0.19, CHCl3). UV (EtOH): λmax 243.5, 252.0, 261.5 nm; λmin
247.5, 258.5 nm. 1H NMR (600 MHz, CDCl3): δ 0.53 (s, 3H,
18-H3), 0.94 (d, J ) 6.6 Hz, 3H, 21-H3), 1.07 (m, 1H), 1.22 (s,
6H, 26-H3 and 27-H3), 1.24-1.33 (m, 5H), 1.36-1.42 (m, 4H),
1.44-1.56 (m, 5H), 1.59-1.61 (m, 3H), 1.66-1.70 (m, 2H),
1.77-1.78 (m, 2H), 1.89 (m, 1H), 2.00-2.02 (m, 2H), 2.13-
2.18 (m, 2H), 2.60 (dd, J ) 4.7, 12.7 Hz, 1H, 4R-H), 2.80 (dd,
J ) 4.7, 12.6 Hz, 1H, 9â-H), 2.86 (dd, J ) 4.3, 13.9 Hz, 1H,
10R-H), 3.66 (dt, J ) 4.7, 9.6 Hz, 1H, 3R-H), 3.70-3.72 (m,
2H, -CH2CH2CH2OH), 4.10 (dt, J ) 4.3, 7.1 Hz, 1H, 1â-H),
5.82 (d, J ) 11.1 Hz, 1H, 7-H), 6.38 (d, J ) 11.1 Hz, 1H, 6-H).
13C NMR (150 MHz, CDCl3): δ 12.4, 18.8, 20.8, 22.2, 23.5, 27.7,
28.9, 29.2, 29.4, 30.2, 35.6, 36.1, 36.4(2C), 40.5, 44.4, 45.3, 45.8,
48.8, 56.3, 56.5, 63.0, 68.6, 71.1, 71.7, 115.2, 124.1, 131.1, 143.3.
IR (neat): 3362, 2924, 2855, 1635 cm-1. EIMS: 462 (M+).
HREIMS: calcd for C29H50O4 (M+) 462.3709, found 462.3712.
Bin d in g Assa ys to th e Bovin e Th ym u s VDR.31 Bovine
thymus 1R,25-dihydroxyvitamin D3 receptor was obtained from
Yamasa Biochemical (Chiba, J apan) and dissolved in 0.05 M
phosphate buffer (pH 7.4) containing 0.3 M KCl and 5 mM
dithiothreitol just before use. The receptor solution (500 µL,
0.23 mg protein) was preincubated with 50 µL of ethanol
solution of 1R,25-dihydroxyvitamin D3 or an analogue at
various concentrations for 60 min at 25 °C. Then, the receptor
mixture was left to stand overnight with 0.1 nM [3H]-1R,25-
dihydroxyvitamin D3 at 4 °C. The bound and free [3H]-1R,25-
dihydroxyvitamin D3 were separated by treatment with dextran-
coated charcoal for 30 min at 4 °C and centrifuged at 3000
rpm for 10 min. The radioactivity of the supernatant (500 µL)
with ACS-II (9.5 mL) (Amersham, UK) was then counted. This
experiment was repeated one more time for each analogue to
take the mean. The binding curves are shown in the Support-
ing Information. The relative potency of the analogues was
calculated from their concentration needed to displace 50% of
[3H]-1R,25-dihydroxyvitamin D3 from its receptor compared
with the activity of 1R,25-dihydroxyvitamin D3 (assigned a
100% value).
Data for 8b (2â-3-hydroxypropylated 5): tR ) 10.1 min. [R]21
:
D
+6.8 (c 0.16, CHCl3). UV (EtOH): λmax 243.5, 252.0, 261.5 nm.
λmin 247.0, 258.0 nm. 1H NMR (600 MHz, CDCl3): δ 0.55 (s,
3H, 18-H3), 0.94 (d, J ) 6.3 Hz, 3H, 21-H3), 1.07 (m, 1H), 1.22
(s, 6H, 26-H3 and 27-H3), 1.24-1.33 (m, 5H), 1.37-1.48 (m,
5H), 1.53-1.60 (m, 6H), 1.66-1.68 (m, 2H), 1.79-1.82 (m, 2H),
1.88-1.92 (m, 2H), 1.99-2.02 (m, 2H), 2.36 (ddd, J ) 1.5, 3.6,
13.7 Hz, 1H, 4â-H), 2.41 (br d, J ) 13.7 Hz, 1H, 4R-H), 2.80
(dd, J ) 4.7, 12.9 Hz, 1H, 9â-H), 3.10 (ddd, J ) 1.5, 4.8, 12.9
Hz, 1H, 10â-H), 3.56 (dt, J ) 4.8, 10.2 Hz, 1H, 1â-H), 3.69-
3.71 (m, 2H, -CH2CH2CH2OH), 4.05 (m, 1H, 3R-H), 5.87 (d, J
) 11.3 Hz, 1H, 7-H), 6.26 (d, J ) 11.3 Hz, 1H, 6-H). 13C NMR
(150 MHz, CDCl3): δ 12.0, 18.8, 20.8, 22.3, 23.5, 23.8, 27.7,
29.0, 29.2, 29.4, 29.9, 36.1, 36.4, 37.9, 40.4, 44.1, 44.4, 45.8,
49.0, 56.3, 56.5, 63.0, 68.3, 71.1, 71.2, 115.2, 123.5, 131.0, 143.2.
IR (neat): 3360, 2924, 2855, 1653 cm-1. EIMS: 462 (M+).
HREIMS: calcd for C29H50O4 (M+) 462.3709, found 462.3723.
(a RS,1R,3R,7E)-9,10-Seco-19-n or ch olest a -5,7-d ien e-
1,2,3,25-tetr a ol (30). To a solution of 23 (31.8 mg, 59.6 µmol)
in dry THF (200 µL) was added LiHMDS (1 M solution in THF,
55 µL, 55 µmol) at -78 °C in an argon atmosphere. After the
mixture was stirred at the same temperature for 1 h, a solution
of ketone 29 (11.0 mg, 36.6 µmol) in dry THF (200 µL) was
added dropwise. This was stirred for 3 h, the resulting mixture
was poured into saturated aqueous NH4Cl, and the organic
layer was separated. The aqueous layer was extracted with
Et2O. The combined organic layer was washed with brine,
dried over Na2SO4, and concentrated. Purification by silica gel
column chromatography (hexane/AcOEt ) 9:1) afforded crude
protected 2-hydroxy-19-norvitamin (22.7 mg) as a colorless oil,
which was used in the next step without further purification.
To a solution of crude protected 19-norvitamin (20.6 mg, 33.3
µmol) in dry MeOH (1 mL) was added (+)-CSA (35 mg, 168
mmol) at 0 °C in an argon atmosphere. After being stirred at
rt for 38 h, the reaction mixture was diluted with AcOEt. The
resulting mixture was washed with saturated aqueous
NaHCO3 and brine, dried over Na2SO4, and concentrated.
Purification by silica gel column chromatography (AcOEt/
MeOH ) 10:1) afforded 9.0 mg of 30 (diastereomer mixture)
as white powder in a ratio of ca. 1:1 (64%). 1H NMR (400 MHz,
CDCl3) â-isomer (selected): δ 0.55 (s, 3H), 0.94 (d, J ) 6.3 Hz,
3H), 1.22 (s, 6H), 2.62 (dd, J ) 4.0, 13.3 Hz, 1H), 2.80 (br d, J
) 12.7 Hz, 1H), 3.08 (dd, J ) 4.3, 13.1 Hz, 1H), 3.48-3.54 (m,
1H), 3.68 (m, 1H), 4.08 (m, 1H), 5.84 (d, J ) 11.0 Hz, 1H),
6.29 (d, J ) 11.0 Hz, 1H); R-isomer (selected): δ 2.90 (dd, J )
4.2, 14.4 Hz, 1H), 3.79 (m, 1H), 5.81 (d, J ) 11.2 Hz, 1H), 6.38
(d, J ) 11.2 Hz, 1H). These data are identical with those of
ref 19.
Assa ys for In d u ct ion of H L-60 Cell Differ en t ia t ion .
Activity of the analogues on differentiation of HL-60 cells was
estimated by nitroblue tetrazolium (NBT) reduction assay32
with some modifications. Human promyelocytic leukemia HL-
60 cells were grown at 37 °C in RPMI 1640 medium (Asahi
Technologlass Co., Chiba, J apan) supplemented with 10% fetal
bovine serum (Sigma, St. Louis, MO) in an atmosphere of 95%
air and 5% CO2. Cells were collected, suspended in 1.5 mL of
the culture medium containing various concentrations (0,
10-10-10-6 M) of the analogues at a density of ca. 5 × 105 cells/
mL, and then cultivated for 5 days. After the treatment, cells
were harvested and suspended in Tyrode’s solution (140 mM
NaCl, 2.7 mM KCl, 1.1 mM MgCl2, 1 mM CaCl2, 0.45 mM
NaH2PO4, 5.6 mM D-glucose, pH 7.4) containing 25 mM
HEPES and 0.05% NBT (Dojindo Laboratories, Kumamoto,
J apan). After preincubation at 37 °C for 7 min, phorbol 12-
myristate 13-acetate (4 µM, Sigma, St. Louis, MO) was added
to the cell suspension, and the suspension was incubated for
30 min at 37 °C. EDTA (10 mL) was added to stop the reaction,
and then the percentage of positive cells (blue-stained cells)
was determined using a hemocytometer. This experiment was
done for three times for each analogue. EC50 value was
estimated from the obtained dose-response curve (Supporting
Information). Relative differentiation activity was calculated
according to the following formula: relative differentiation
activity ) (EC50 of 1R,25-dihydroxyvitamin D3/EC50 of the
analogue) × 100.
Ack n ow led gm en t. We thank Ms. J unko Shimode
and Ms. Maroka Kitsukawa (Teikyo University) for
spectroscopic measurements. This study was supported
by a Grant-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology, J apan.
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 (10-15, 21-23, 6a ,b-8a ,b), charts of VDR binding
assays of compounds 6a ,b-8a ,b, and charts for assays of
induction of HL-60 cell differentiation activity of compounds
6a ,b-8a ,b. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O034787Y
J . Org. Chem, Vol. 68, No. 19, 2003 7415