Concise synthesis and biological activities of 2α-alkyl- and 2α-(ω-hydroxyalkyl)-20-epi-1α,25-dihydroxyvitamin D 3

Article abstract of DOI:10.1016/S0960-894X(03)00739-X

We found a concise route to the Trost A-ring precursor enyne for synthesizing 2α-alkylated 1α,25-dihydroxyvitamin D₃ (1) from D-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2α-substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2α-alkyl- and four analogues of 2α-(ω -hydroxyalkyl)-20-epi-1α,25-dihydroxyvitamin D₃ (5b-d and 6a-d) showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.

Full text of DOI:10.1016/S0960-894X(03)00739-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3503–3506
Concise Synthesis and Biological Activities of 2ꢀ-Alkyl- and
2ꢀ-(!-Hydroxyalkyl)-20-epi-1ꢀ,25-dihydroxyvitamin D3
Shinobu Honzawa,a Yoshitomo Suhara,a,y Ken-ichi Nihei,a Nozomi Saito,a
Seishi Kishimoto,b Toshie Fujishima,a Masaaki Kurihara,c Takayuki Sugiura,b
Keizo Waku,b Hiroaki Takayamaa and Atsushi Kittakaa,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko,
Kanagawa 199-0195, Japan
bDepartment of Hygienic Chemistry and Nutrition, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko,
Kanagawa 199-0195, Japan
cNational Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Received 22 May 2003; accepted 10 July 2003
Abstract—We found a concise route to the Trost A-ring precursor enyne for synthesizing 2a-alkylated 1a,25-dihydroxyvitamin D3
(1) from d-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2a-
substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2a-alkyl- and four analogues of 2a-
(o-hydroxyalkyl)-20-epi-1a,25-dihydroxyvitamin D3 (5b–d and 6a–d) showed higher binding affinity for vitamin D receptor (VDR)
and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.
# 2003 Elsevier Ltd. All rights reserved.
1a,25-Dihydroxyvitamin D3 (1), the hormonally active
form of vitamin D3, mediates intestinal calcium
absorption, and bone resorption and mineralization. In
addition, 1 has been found to exhibit a variety of biolo-
gical activities in many tissues and cells since the dis-
covery of the fact that 1 induces cell differentiation and
proliferation.1 This renewed interest has prompted
numerous efforts to synthesize a huge number of vita-
min D analogues in order to investigate biological roles
of this hormone and to develop potential therapeutic
agents.2,3
bovine thymus vitamin D receptor (VDR), elevation of
rat serum calcium concentration, and induction of HL-60
cell differentiation.4,5,6b
Previously, we synthesized A-ring modified analogues
2a–d and 3a–d, in which the 2a-alkyl or the 2a-hydro-
xyalkyl group was introduced to 1, to study the A-ring
conformation- and structure–activity relationships
(Fig. 1).4ꢀ6 The resulting analogues exhibited interesting
biological activities, in particular 2a-methyl and 2a-(3-
hydroxypropyl) analogues (2a and 3c) showed much
higher potency than 1 in terms of binding affinity for
*Corresponding author. Tel.: +81-426-85-3715; fax: +81-426-85-
3714; e-mail: akittaka@pharm.teikyo-u.ac.jp
yPresent address: Kobe Pharmaceutical University, Higashinada-ku,
Kobe 658-8558, Japan.
Figure 1. Structures of the natural hormone 1, its 2a-substituted ana-
logues 2–3; 20-epi-derivative 4, and its 2a-substituted analogues 5–6.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00739-X

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S. Honzawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3503–3506
For example, 2a-(3-hydroxypropyl) analogue 3c exhib-
ited 3-fold higher VDR binding affinity, approximately
500 times higher potency for elevation of rat serum cal-
cium concentration than those of 1.4b These remarkably
high activities are unique among vitamin D analogues
reported to date. On the other hand, structural modifi-
cations of the CD-ring side chain have been intensively
studied in the past.7 Among them, 20-epi-1a,25-dihy-
droxyvitamin D3 (4) is known as one of the typical
compounds exhibiting high cell differentiation activity
with relatively low calcemic effects.8ꢀ10 It is tempting to
speculate that modification at the 2a-position on the A-
ring may correlate to the structure of the CD-ring side
chain providing potential biological activity, which
would be different from its parent hormone. Indeed, we
synthesized all possible diastereomers of 2-methyl-1,25-
dihydroxyvitamin D3 with 20-epimerization, and found
2a-methyl-20-epi-1a,25-dihydroxyvitamin
D3
(5a)
exhibited exceptionally high potency in VDR binding,
transcriptional potency, and HL-60 cell differentiation
activity.11 We expect that combination of this 20-epi-
merization and 2a-substitution would lead to highly
promising analogues.12 We report here an improved
synthesis of the A-ring precursor enynes 8b–d for 2a-
alkylated 4 from d-glucose, utilizing highly active
Grignard reagents, and biological evaluation for VDR
binding affinity and HL-60 cell differentiation activity of
totally seven novel analogues of 2a-substituted 1a,25-
dihydroxyvitamin D3 with 20-epimerization of the CD-
ring moiety (5b–d, 6a–d).
Scheme 2. Reagents and conditions: (a) RMgCl, toluene, 11b (74%),
11c (75%), and 11d (83%); (b) NBS, BaCO3, CCl4, reflux, 12b (69%),
12c (85%), and 12d (68%); (c) Zn powder, NaBH3CN, 1-propanol–
H2O (10:1), 95 ꢁC, 13b (70%), 13c (75%), and 13d (67%); (d) (i) TmCl,
DMAP, CH2Cl2, (ii) LiHMDS, THF, ꢀ78 ꢁC to rt, 14b (57%), 14c
(76%), and 14d (72%) for two steps; (e) (i) TMSCCH, BuLi,
ꢁ
.
BF3 OEt2, THF, ꢀ78 C, (ii) K2CO3, MeOH, (iii) TBSOTf, 2,6-luti-
dine, 8b (72%), 8c (77%), and 8d (46%) for three steps.
n-butyl groups could be introduced in the same way. It
is important to note that the use of toluene is essential in
this step to realize shorter reaction time and higher
chemical yield as compared with the use of conventional
ethereal solvents such as diethyl ether and THF.15
Acetals 11b–d were treated with N-bromosuccinimide to
give bromo benzoates 12b–d. Bromides 12b–d reacted
with zinc in the presence of sodium cyanoborohydride
to give 1,2-diols 13b–d, which were converted to epox-
ides 14b–d through sulfonylation of the primary hydroxy
group, followed by base treatment. A-ring precursors 8b–
d were obtained by the reaction of 14b–d with lithium
acetylide, and protecting group manipulations.
The analogues were synthesized by employing the con-
vergent method of Trost and co-workers using palla-
dium-catalyzed coupling reaction of the A-ring synthon,
enyne, with the CD-ring portion.13 Scheme 1 outlines
the synthetic route to the target compounds 5b–d and
6a–d. The A-ring precursors 8b–d were synthesized in
eight steps from epoxide 10, which is readily available
from methyl a-d-glucoside (Scheme 2).14 The reaction
of ethyl magnesium chloride with epoxide 10 using tol-
uene as a reaction solvent gave epoxy ring opening
product 11b regioselectively in 74% yield. n-Propyl and
Enynes 9a–d were prepared by our reported method.4
Bromoolefin 7 with the 20-epi-CD-ring moiety was
synthesized from vitamin D2.16
Those synthons thus obtained were coupled using tet-
rakis(triphenylphosphine)palladium(0) as a catalyst fol-
lowed by deprotection of the silyl ethers with 1 M
TBAF in THF gave the desired compounds 5b–d17ꢀ19
and 6a–d.20ꢀ23 These target compounds were purified by
recycled reverse-phase HPLC for the biological eval-
uation.
We determined binding affinity of 5b–d and 6a–d to the
bovine thymus VDR.24 As we expected, the VDR affi-
nity of 20-epimers 5b–d and 6a–d were increased when
compared to that of the corresponding natural side
chain derivatives of 2b–d and 3a–d (Table 1). Especially,
the 2a-hydroxypropyl analogue 6c showed the highest
VDR binding affinity in this series, that is, 3.7-fold
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, TEA/toluene (3:1),
reflux; (b) 1 M TBAF in THF; 19–56% for two steps.

S. Honzawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3503–3506
3505
Table 1. Relative binding affinity of 2a-substituted-1 (2–3) and 2a-
substituted-20-epi-1 (5–6) for bovine thymus VDR and their potency
in induction of HL-60 cell differentiation
and 6a–d by NBT reduction method.25 As shown in
Table 1, 20-epimerization raised the induction of differ-
entiation activity of HL-60 cells markedly, it was more
than one order. Tendency to magnify the cell differ-
entiation activity was closely related to the VDR bind-
ing affinity,26 and 6c exhibited the highest potency in
this experiment. Although introduction of the 2a-sub-
stituent into the 20-epi analogue 4 did not cause very
dramatic alternation in the activity if compared with 5a,
it would be interesting to examine the other biological
activities in vivo. For example, ED-71, which has the
2b-(3-hydroxypropoxyl) group in 1, shows unique bio-
logical activity profiles; high calcemic activity along with a
long half-life in plasma, and the clinical trial of ED-71 as a
promising candidate for treatment of osteoporosis has
been conducted.27
Compd
Binding affinity
to VDRa
HL-60 cell
differentiationa,b
1
100
4005
404
100
2585
1064
444
2a
2b
2c
2d
3a
3b
3c
3d
4
5a
5b
5c
5d
6a
6b
6c
6d
204
84
74
104
204
704
864
3004
1204
40016
120011
150
119
33
2404
460
357116
968811
4200
1750
2300
570
In summary, we have synthesized seven new analogues
of 2a-alkyl- and 2a-(o-hydroxyalkyl)-20-epi-1a,25-
dihydroxyvitamin D3 and evaluated their VDR binding
affinity and HL-60 cell differentiation activity. These
double modifications on both the A-ring and the side
chain increased VDR binding affinity and potency in
induction of HL-60 cell differentiation compared to
those of the natural hormone 1. We consider that these
results provide important information for vitamin D
research with regard to the structure–activity relation-
ships of the A-ring and the side-chain moiety.28
190
320
370
183
4030
4900
3910
aThe potency of 1 is normalized to 100.
bData are the mean of three separate experiments.
higher than that of 1. Even 20-epimerization of 3a pro-
vided a better ligand (6a) in shape than the natural
hormone for the VDR.
Except 5a, however, simple 20-epimerization of the
parent steroid without 2a-(o-hydroxyalkyl) group (4)
provided the best results in VDR binding. The modeled
structure of 6c constructed by molecular dynamics cal-
culations (AMBER* force field) using MacroModel ver.
6.5. is shown in Figure 2. Although 6c (red) could form
an additional hydrogen bonding between the terminal
hydroxyl of the 2a-hydroxypropyl group and Arg 274 in
the ligand binding domain of the VDR, the positions of
the two hydroxyls (1a,3b) on the A-ring are slightly
different from the original positions in 4 (green).10 This
effect based on the 2a-substituent may decrease the
binding affinity to the VDR. Next, we evaluated the
HL-60 cell differentiation activity of the analogues 5b–d
Acknowledgements
We are grateful to Miss Junko Shimode and Miss
Maroka Kitsukawa for the spectroscopic measure-
ments. This work has been supported by Grants-in-Aid
from the Ministry of Education, Culture, Sports,
Science and Technology, Japan and in part by a grant
(MF-16) from the Organization for Pharmaceutical
Safety and Research.
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Figure 2. Modeled structure of 6c (red) with Moras X-ray structure of
the VDR-4 (green) complex.10

3506
S. Honzawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3503–3506
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0.53 (s, 3H), 0.85 (d, 3H, J=6.4 Hz), 0.92 (t, 3H, J=7.0 Hz),
1.21 (s, 6H), 2.24 (dd, 1H, J=8.6, 13.0 Hz), 2.66 (dd, 1H,
J=4.4, 13.2 Hz), 2.83 (m, 1H), 3.88 (ddd, 1H, J=4.4, 8.4, 8.4
Hz), 4.37 (d, 1H, J=3.2 Hz), 4.99 (d, 1H, J=1.8 Hz), 5.27 (d,
1H, J=1.8 Hz), 6.01 (d, 1H, J=11.2 Hz), 6.40 (d, 1H, J=11.2
Hz); HREIMS calcd for C31H52O3 (M+) 472.3916, found
472.3909.
20. Data for 6a: [a]2D7 +157.8 (c 0.03, CHCl3); UV (EtOH)
l
max 269 nm, lmin 226 nm; 1H NMR (400 MHz, CDCl3–D2O)
d 0.53 (s, 3H), 0.85 (d, 3H, J=6.6 Hz), 2.31 (m, 2H), 2.66 (dd,
1H, J=4.8, 13.2 Hz), 2.88 (m, 1H), 3.96 (dd, 1H, J=4.4, 11.4
Hz), 4.03 (m, 1H), 4.24 (m, 1H), 4.46 (d, 1H, J=2.9 Hz), 5.01
(d, 1H, J=2.2 Hz), 5.29 (d, 1H, J=2.2 Hz), 5.98 (d, 1H,
J=11.5 Hz), 6.45 (d, 1H, J=11.5 Hz); HREIMS calcd for
C28H44O3 (MꢀH2O)+ 428.3291, found 428.3316.
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Wurtz, J. M.; Mitschler, A.; Moras, D. Proc. Natl. Acad. Sci.
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21. Data for 6b: [a]2D7 +29.5 (c 0.007, CHCl3); UV (EtOH)
l
max 268 nm, lmin 227 nm; 1H NMR (400 MHz, CDCl3–D2O)
d 0.53 (s, 3H), 0.85 (d, 3H, J=6.4 Hz), 1.21 (s, 6H), 2.26 (dd,
1H, J=8.5, 13.4 Hz), 2.66 (dd, 1H, J=4.3, 13.4 Hz), 2.83 (m,
1H), 3.79 (m, 2H), 3.94 (m, 1H), 4.37 (bs, 1H), 5.02 (d, 1H,
J=1.8 Hz), 5.30 (bs, 1H), 6.01 (d, 1H, J=11.2 Hz), 6.40 (d,
1H, J=11.2 Hz); HREIMS calcd for C29H48O4 (M+)
460.3552, found 460.3532.
22. Data for 6c: [a]D27 +8.3 (c 0.01, CHCl3); UV (EtOH) lmax
267 nm, lmin 227 nm; 1H NMR (400 MHz, CDCl3–D2O) d
0.53 (s, 3H), 0.85 (d, 3H, J=6.6 Hz), 1.21 (s, 6H), 2.26 (dd,
1H, J=9.4, 13.5 Hz), 2.66 (dd, 1H, J=4.4, 13.5 Hz), 2.83 (m,
1H), 3.69 (m, 2H), 3.89 (dt, 1H, J=4.2, 8.4 Hz), 4.36 (d, 1H,
J=2.9 Hz), 4.99 (d, 1H, J=2.2 Hz), 5.27 (d, 1H, J=2.2 Hz),
6.00 (d, 1H, J=11.4 Hz), 6.40 (d, 1H, J=11.4 Hz); HREIMS
calcd for C30H50O4 (M+) 474.3709, found 474.3726.
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17. Data for 5b: [a]2D7 ꢀ911.9 (c 0.007, CHCl3); UV (EtOH)
23. Data for 6d: [a]2D7 ꢀ509.4 (c 0.06, CHCl3); UV (EtOH)
l
max 268 nm, lmin 227 nm; 1H NMR (400 MHz, CDCl3–D2O)
d 0.53 (s, 3H), 0.85 (d, 3H, J=6.6 Hz), 1.21 (s, 6H), 2.26 (dd,
1H, J=9.4, 13.5 Hz), 2.66 (dd, 1H, J=4.4, 13.5 Hz), 2.83 (m,
1H), 3.69 (m, 2H), 3.89 (dt, 1H, J=4.2, 8.4 Hz), 4.36 (d,
1H, J=2.9 Hz), 4.99 (d, 1H, J=2.2 Hz), 5.27 (d, 1H, J=2.2
Hz), 6.00 (d, 1H, J=11.4 Hz), 6.40 (d, 1H, J=11.4 Hz);
HREIMS calcd for C31H52O4 (MꢀH2O)+ 470.3760, found
470.3763.
24. Imae, Y.; Manaka, A.; Yoshida, N.; Ishimi, Y.; Shinki, T.;
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R. C. J. Exp. Med. 1979, 149, 969.
26. Discussion on biological activities and VDR binding affi-
nity, see: Masuno, H.; Yamamoto, K.; Wang, X.; Choi, M.;
Ooizumi, H.; Shinki, T.; Yamada, S. J. Med. Chem. 2002, 45,
1825.
l
max 268 nm, lmin 229 nm; 1H NMR (400 MHz, CDCl3–D2O)
d 0.53 (s, 3H), 0.85 (d, 3H, J=6.6 Hz), 1.21 (s, 6H), 2.20 (dd,
1H, J=8.5, 13.2 Hz), 2.66 (dd, 1H, J=4.1, 13.2 Hz), 2.83 (m,
1H), 3.89 (m, 1H), 4.34 (dd, 1H, J=4.5 Hz), 4.99 (d, 1H,
J=2.0 Hz), 5.27 (d, 1H, J=2.0 Hz), 6.00 (d, 1H, J=11.3 Hz),
6.40 (d, 1H, J=11.3 Hz); HREIMS calcd for C29H48O3 (M+)
444.3604, found 444.3594.
18. Data for 5c: [a]2D7 +459.7 (c 0.003, CHCl3); UV (EtOH)
l
max 268 nm, lmin 228 nm; 1H NMR (400 MHz, CDCl3–D2O)
d 0.53 (s, 3H), 0.85 (d, 3H, J=6.6 Hz), 1.21 (s, 6H), 2.26 (dd,
1H, J=8.4, 13.7 Hz), 2.66 (dd, 1H, J=4.2, 13.7 Hz), 2.83 (m,
1H), 3.92 (dt, 1H, J=4.2, 8.4 Hz), 4.34 (d, 1H, J=2.4 Hz),
4.93 (d, 1H, J=2.2 Hz), 5.18 (d, 1H, J=2.2 Hz), 6.05 (d, 1H,
J=11.2 Hz), 6.32 (d, 1H, J=11.2 Hz); HREIMS calcd for
C30H50O3 (M+) 458.3760, found 458.3765.
27. Kubodera, N.; Sato, K.; Nishii, Y. In Vitamin D;
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19. Data for 5d: [a]D22 +1.4 (c 0.33, CHCl3); UV (EtOH) lmax
268 nm, lmin 227 nm; 1H NMR (400 MHz, CDCl3–D2O) d