Systematic studies on synthesis, structural elucidation, and biological evaluation of A-ring diastereomers of 2-methyl-1α,25-dihydroxyvitamin D3 and 20-epi-2-methyl-1α,25-dihydroxyvitamin D3

Article abstract of DOI:10.1016/S0039-128X(00)00141-0

All possible A-ring diastereomers of 2-methyl-1α,25-dihydroxyvitamin D₃ (2) and 20-epi-2-methyl-1α,25-dihydroxyvitamin D₃ (3) were synthesized by palladium-catalyzed coupling reaction of A-ring 'enyne' synthons with CD-ring portions. The A-ring synthons were rationally synthesized via a novel and practical route, starting with methyl (R)-(+)- and (S)-(-)-3-hydroxy-2-methyl-propionate, in good yields. X-ray crystallographic analysis of 2α-methyl-1α,25-dihydroxyvitamin D₃ (2b) and conformational analysis of the A-ring of 2α-methyl-(2b) and 2β-methyl-1α,25-dihydroxyvitamin D₃ (2f) were carried out, and the results are described. All A-ring diastereomers (2 and 3), thus synthesized, were biologically evaluated both in vitro and in vivo. The biologic potency was highly dependent on the stereochemistry of the A-ring substituents. In particular, 2b showed 4-fold higher vitamin D receptor [VDR] binding activity than the natural hormone, and its 20-epimer (3b) exhibited exceptionally high activity, 12-fold more potent in VDR binding, 7-fold in calcium mobilization, and 590-fold in induction of human promyelocytic leukemia (HL-60) cell differentiation as compared with the natural hormone. Further, the 20-epi-2β-Me-1β, 3α(OH)₂ isomer (3g) had significant biologic potencies compared to the natural hormone despite having 1β-OH configuration. The transcriptional activities on human osteocalcin gene promoter, including VDRE in transfected mammalian cells, were also evaluated. Finally, there was a clear contrast between the effects of the 2-methyl group on the HL-60 cell differentiation- and apoptosis-inducing activities of 2 and 3. Copyright

Full text of DOI:10.1016/S0039-128X(00)00141-0

Steroids 66 (2001) 277–285
Systematic studies on synthesis, structural elucidation, and biological
evaluation of A-ring diastereomers of 2-methyl-1␣,25-dihydroxyvitamin
D3 and 20-epi-2-methyl-1␣,25-dihydroxyvitamin D3
Hiroaki Takayamaa,*, Katsuhiro Konnoa, Toshie Fujishimaa, Shojiro Makia, Zhaopeng Liua,
Daishiro Miurab, Manabu Chokkib, Seiichi Ishizukab, Connie Smithc, Hector F. DeLucac,
Kimie Nakagawad, Mayuko Kurobed, Toshio Okanod
aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
bInstitute for Bio–Medical Research, Teijin Ltd., Tokyo, Japan
cDepartment of Biochemistry, University of Wisconsin, WI, USA
dKobe Pharmaceutical University, Kobe, Japan
Abstract
All possible A-ring diastereomers of 2-methyl-1␣,25-dihydroxyvitamin D3 (2) and 20-epi-2-methyl-1␣,25-dihydroxyvitamin D3 (3) were
synthesized by palladium-catalyzed coupling reaction of A-ring ‘enyne’ synthons with CD-ring portions. The A-ring synthons were
rationally synthesized via a novel and practical route, starting with methyl (R)-(ϩ)- and (S)-(-)-3-hydroxy-2-methyl-propionate, in good
yields. X-ray crystallographic analysis of 2␣-methyl-1␣,25-dihydroxyvitamin D3 (2b) and conformational analysis of the A-ring of
2␣-methyl-(2b) and 2␤-methyl-1␣,25-dihydroxyvitamin D3 (2f) were carried out, and the results are described. All A-ring diastereomers
(2 and 3), thus synthesized, were biologically evaluated both in vitro and in vivo. The biologic potency was highly dependent on the
stereochemistry of the A-ring substituents. In particular, 2b showed 4-fold higher vitamin D receptor [VDR] binding activity than the natural
hormone, and its 20-epimer (3b) exhibited exceptionally high activity, 12-fold more potent in VDR binding, 7-fold in calcium mobilization,
and 590-fold in induction of human promyelocytic leukemia (HL-60) cell differentiation as compared with the natural hormone. Further,
the 20-epi–2␤-Me-1␤, 3␣(OH)2 isomer (3g) had significant biologic potencies compared to the natural hormone despite having 1␤-OH
configuration. The transcriptional activities on human osteocalcin gene promoter, including VDRE in transfected mammalian cells, were
also evaluated. Finally, there was a clear contrast between the effects of the 2-methyl group on the HL-60 cell differentiation- and
apoptosis-inducing activities of 2 and 3. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: 2-Methyl-1␣; 25-Dihydroxyvitamin D3; 20-epi-2-methyl-1␣; 25-Dihydroxyvitamin D3; Synthesis; A-ring conformation; VDR binding affinity;
HL-60 cell differentiation; Apoptosis
1. Introduction
tency or selective activity, whereas modification of the
A-ring in recent years has afforded other useful analogues,
such as ED-71 (Chugai group) [4], 19-nor analogues (De-
Luca group) [5] and 1-hydroxymethyl analogues (Posner
group) [6] that exhibit unique activity profiles. To investi-
gate the relationship between the structure of A-ring and the
various biologic functions of vitamin D, we synthesized
A-ring modified 1␣,25-(OH)2D3 analogs.
Our approach to the design and synthesis of A-ring modi-
fied analogues is based on the commonly accepted hypothesis
[7] that the A-ring conformation is in dynamic equilibrium
between two chair conformers, ␣-form and ␤-form in 1:1 ratio,
and that the ␤-form, in which 1␣-OH takes an equatorial
position, may be responsible for the biologic activity. We
The hormonally active form of vitamin D, 1␣,25-dihy-
droxyvitamin D3 [1␣,25(OH)2D3] (1), has a wide range of
biologic activities including cell differentiating and antipro-
liferative activities, in addition to its classic role in calcium
homeostasis [1], and these activities have been utilized to
develop therapeutic agents for cancer, psoriasis, and osteo-
porosis [2]. A majority of these agents [3] are altered in the
side-chain, providing many useful analogues with high po-
* Corresponding author. Tel.: ϩ81-426-85-3713; fax: ϩ81-426-85-
3713.
E-mail address: hi-takay@pharm.Teikyo-u.ac.jp (H. Takayama).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00141-0

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H. Takayama et al. / Steroids 66 (2001) 277–285
Scheme 1. Design and synthesis of A-ring diastereomers of 2-Me-1␣,25(OH)2-D3 (2) and 20-epi-2Me-1␣,25(OH)2-D3 (3).
expected that the introduction of a methyl group at the 2- or
4-position would shift the equilibrium, and resultant changes,
in conjunction with modification of the stereochemistry of the
two hydroxy groups, would cause changes in the spectrum of
biologic potency of various vitamin D functions. Therefore, we
intended to synthesize all possible A-ring diastereomers of
2-methyl-1␣,25-(OH)2D3, to evaluate their biologic activity in
detail, and to establish the relationship between the A-ring
structure and the potency in a wide variety of vitamin D
functions.
2. Design and synthesis
For the above reasons, we first synthesized all eight
possible diastereomers (2) of 2-methyl-1␣,25-1␣,25-
(OH)2D3, and confirmed that the potency of these com-
pounds varies depending on the configuration of the C-1 and
C-3 hydroxy groups, and the stereochemistry of the C-2-
methyl group. Furthermore, the remarkable effects of
2-methyl substitution on the potency prompted us to syn-
thesize all possible diastereomers (3) of 20-epi-2-methyl-
Scheme 2. Synthesis of the 2␣-methyl A-ring enynes.

H. Takayama et al. / Steroids 66 (2001) 277–285
279
Scheme 3. Synthesis of the 2␣-methyl A-ring enynes.
1␣,25-(OH)2D3, double modified in the A-ring and in the
side chain (Scheme 1).
esters [11]. The more polar (3R)-isomer was protected with
THP, followed by treatment with TBAF to furnish the
primary alcohol, which was then subjected Swern oxidation
to give the aldehyde in excellent yield. The reaction of the
aldehyde with Grignard reagent afforded a 1:1 mixture of
the diasteromeric allylalcohols. After removal of the THP,
the resulting mixture of enyne-diols, (2␣,1␣,3␤) and
(2␣,1␤,3␤) [the Greek letters denote the configurations at
C-2 (Me), C-1 (OH), and C-3 (OH), respectively] was
readily separable by silica gel column chromatography, and
the relative stereochemistry of the enyne-1,3-diol in each
isomer was determined by 13C-NMR analysis of the ace-
tonide [12] (Scheme 3).
The less polar (3S)-isomer was converted to the corre-
sponding enyne-diols (2␣,1␣,3␣), (2␣,1␤,3␣) via the same
procedure (Scheme 4). Thus, four diastereomers of 2␣-Me-
enyne-diol were synthesized.
The four diastereomers of 2␤-Me-enyne-diols [(2␤,1␣,3␤),
(2␤,1␤,3␣) and (2␤,1␣,3␣), (2␤,1␤,3␤)] were similarly syn-
thesized, stating with (2S)-propionic ester.
For the synthesis of analogues modified systematically
in both the A-ring and the side chain, we adapted the
convergent method developed by the Trost group ([8] and
references cited therein) employing palladium-catalyzed
coupling of the A-ring enyne synthon with the CD-ring
portion. Since the synthetic route to the precursor ‘enyne’
of the A-ring had not been fully established, we have
developed a novel and practical route to enynes for all
eight possible diastereomers of 2-methyl-1␣,25-(OH)2D3
[9,10].
The synthetic route as exemplified by the 2␣-Me-1␣-
OH–3␤-OH isomer is shown in Scheme 2. Commercially
available methyl (R)-(-)-3-hydroxy-2-methyl-propionate
was converted to the olefin in high yield in a usual manner,
and the latter, on treatment with peracid, afforded the ep-
oxide. The acetylene unit was introduced by reaction with
the epoxide to give a 1:1 mixture of the alcohols. These
isomers were readily separable by silica gel column chro-
matography, and the absolute configuration at C-3 of each
Finally, palladium-catalyzed coupling of the disilyl
ether of these enyne-diols (for example, 2␣,1␣,3␤ enyne-
1
isomer was determined by H-NMR analysis of its MTPA

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H. Takayama et al. / Steroids 66 (2001) 277–285
Scheme 4. The 2␤-methyl A-ring enynes.
diol), with the CD-ring portion, followed by deprotection
with CSA, gave all possible diastereomers of 20R- and
20S-2-Me-1,25-(OH)2D3 with the undoubted stereochem-
istries (2␣-Me-1␣,25-(OH)2D3) in good yields (Scheme
5).
3. Structural elucidation
An ORTEP drawing based on X-ray analysis of a fine
crystal of 2␣-methyl-1␣,25-(OH)2D3 (2b), containing water
of crystallization, is shown in Fig. 1. The results indicate
Scheme 5. Coupling of the A-ring enyne and the CD-ring portion.

H. Takayama et al. / Steroids 66 (2001) 277–285
281
tion-activity relationship of 2-substituted-19-nor-1␣,25-
(OH)2D3.
4. Biologic evaluation
The basic biologic activities [9,10] of the A-ring diaste-
reomers of 20-natural and 20-epi-2-Me-1␣,25-(OH)2D3
were evaluated with comparison with those of the natural
hormone, 1␣,25-(OH)2D3. The results are summarized in
Table 1.
The binding activity of 2-Me isomers to VDR of bovine
thymus was highly dependent on the stereochemistry of the
A-ring substituents and varied by as much as 104 -fold. The
1␣-isomers exhibited high affinity, whereas the 1␤-isomers
had virtually no affinity. It is noteworthy that 2␣-Me-1␣,25-
(OH)2-D3 (2b) showed 4-fold higher affinity than the natu-
ral hormone. This is the first example of an A-ring modified
analog with the same side chain as natural hormone, having
a significantly higher affinity than the natural hormone. This
isomer also exhibited 4-fold greater potency in elevation of
rat serum calcium concentration. The rank order of potency
in HL-60 cell differentiation was almost parallel to that of
VDR affinity (2b was twice as potent as the natural hor-
mone). In binding to DBP of calf serum, the 1␤-isomers
showed high affinity, whereas the 1␣-isomers had poor
affinity, in accordance with reported data. Furthermore, in
the 20-epi series, the potency also depended on the stereo-
chemistry of the A-ring substituents. Among them, 20-epi-
2␣-methyl-1␣,25-dihydroxyvitamin D3 (3b), doubly modi-
fied by 2-methyl substitution and 20-epimerization,
exhibited exceptionally high potency. It showed 12-fold
higher VDR affinity, 6.5-fold higher calcium mobilization
and 590 times higher potency in HL-60 cell differentiation,
as compared with the natural hormone, and is one of the
most potent analogues reported to date (comparable to KH-
1060). In binding to DBP, the 20-epi isomers had approx-
imately 300 times less affinity than the natural hormone. It
is noteworthy that the VDR affinity of 20-epi-2␤,1␣,3␤-
isomer (3f) was comparable to that of the natural hormone,
and that cell differentiation activity toward HL-60 cells was
higher in 3f and 20-epi-2␣,1␣,3␣-isomer (3a) in addition to
3b. It is also noteworthy that the 20-epi–2␤,1␤,3␣ isomer (3
g) had biologic activities [VDR [7], HL-60 [190], Ca [19]
compared to the natural hormone [100]] despite having
1␤-OH configuration.
Fig. 1. ORTEP drawing based on X-ray crystallographic analysis of 2␣-
Me-1␣,25-(OH)2D3.
that the A-ring exists in the chair ␤-form, in which C(1)-OH
takes equatorial and both C(2)-Me and C(3)-OH take axial
orientations. This conformation in the crystal, coupled with
a consideration of molecular packing, suggests that water in
the crystal is involved in hydrogen bonds with the C(1) and
C(3) hydroxyl groups [13].
To elucidate the A-ring conformation-activity (VDR af-
finity) relationships, the 1H-NMR spectra in CDCl3 solution
of the A-ring diastereomers thus synthesized were measured
1
and analyzed in detail. In our H-NMR analyses, the pro-
portions of ␣-form to ␤-form were calculated [14] from two
sets of coupling constants, diaxial C(1)-C(2)-H, and diaxial
C(2)-C(3)-H, in addition to diaxial C(3)-C(4)-H. In every
case, the A-ring is in equilibrium between chair ␣-form and
␤-form and the position of equilibrium depending on the
stereochemistry. Fig. 2 shows the results of conformational
analysis of 2␣-methyl- and 2␤-methyl-1␣,25-(OH)2D3
compared with the natural hormone. In 2␣-methyl-1␣,25-
(OH)2-D3, the ␣-form is predominant over the ␤-form (60:
40), whereas the ␤-form is predominant over the ␣-form
(75:25) in 2␤-methyl-1␣,25-(OH)2D3. Both ratios are sup-
ported by molecular mechanics calculation [15] with MM2
*(the energy differences of 2b and 2f are 0.95 kcal/mol and
0.93 kcal/mol, and the populations (␣-form/␤-form) are ca.
83/17 and 17/83 respectively). Recently, DeLuca’s group
[16] reported that axially oriented C(1)-OH group is re-
quired for bioactivity from a study on the A-ring conforma-
With regard to the in vivo activities (intestinal calcium
transport and bone calcium mobilization in a D-deficient
state), the 20-natural and 20-epi-2␣,1␣,3␤ isomers (2b and
3b) both had significantly higher potency than the natural
hormone, but no effect of 20-epimerization was observed in
this case. The results are shown in part in Table 2.
Finally, to clarify further biologic activity of the ana-
logues synthesized (2 and 3), their transcriptional activities
on human osteocalcin gene promoter, including VDRE in
transfected mammalian cells, were evaluated [17]. The rank

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H. Takayama et al. / Steroids 66 (2001) 277–285
Fig. 2. Conformations of 2␣-Me- and 2␤-Me-1␣,25-(OH)2D3 (2b and 2f).
Table 1
Basic biological activity of A-ring disastereomers of 2-methyl-1␣,25-(OH)2D3 (2) and their 20-epimers (3)
Compounds
VDR bindingc
DBP bindingd
HL-60 cell differentiatione
Ca mobilizationf
1␣,25-(OH)2-D3 (1)a
2␣, 1␣, 3␣ (2a)b
2␣, 1␣, 3␤ (2b)
2␣, 1␤, 3␣ (2c)
2␣, 1␤, 3␤ (2d)
100
4
400
Ͻ0.1
Ͻ0.1
100
45
68
1200
200
100
13
200
1.5
1
100
NTg
400
NT
NT
2␤, 1␣, 3␣ (2e)
2␤, 1␣, 3␤ (2f)
2␤, 1␤, 3␣ (2g)
2␤, 1␤, 3␤ (2h)
Ͻ0.3
13
0.8
21
79
1300
1000
1.5
10
3.0
1.5
NT
2
NT
NT
Ͻ0.1
20-epi-2␣, 1␣, 3␣ (3a)
20-epi-2␣, 1␣, 3␤ (3b)
20-epi-2␣, 1␤, 3␣ (3c)
20-epi-2␣, 1␤, 3␤ (3d)
17
1200
Ͻ0.1
Ͻ0.1
Ͻ0.3
Ͻ0.3
Ͻ0.3
Ͻ0.3
730
59 000
144
655
NT
NT
1
3
20-epi-2␤, 1␣, 3␣ (3e)
20-epi-2␤, 1␣, 3␤ (3f)
20-epi-2␤, 1␤, 3␣ (3g)
20-epi-2␤, 1␤, 3␤ (3h)
Ͻ0.1
160
7
Ͻ0.3
Ͻ0.3
Ͻ0.3
Ͻ0.3
6
2600
190
1
NT
115
19
Ͻ0.1
NT
a The activity of 1␣,25-(OH)2-D3 (1) is normalized to 100.
b The Greek letters denote the configuration of substituents.
c Bovine thymus.
d Calf serum.
e Cell differentiation was assessed in terms of NBT reductivity.
f Rat serum calcium level.
g Not tested.

H. Takayama et al. / Steroids 66 (2001) 277–285
283
Table 2
In vivo activity of intestinal calcium transport and bone calcium mobilization of 2-methyl-1␣,25-(OH)2-D3 and its 20-epimers
Stereoisomer
Amount
(pmol/d/7 days)
Intestinal calcium transport
(Serosa/Mucosa)
Bone calcium mobilization
(Serum Ca in mg/100 ml)
20-natural series
Exp. 1
D-Deficient
1␣,25-(OH)2D3
2␤, 1␣, 3␤ (2f)
0
260
260
500
3.8 Ϯ 0.28
5.0 Ϯ 0.20
4.3 Ϯ 0.27
4.3 Ϯ 0.16
4.1 Ϯ 0.26
5.5 Ϯ 0.12
4.1 Ϯ 0.2
5.0 Ϯ 0.09
Exp. 2
D-Deficient
1␣,25-(OH)2D3
2␣, 1␣, 3␤ (2b)
0
260
65
2.34 Ϯ 0.83
5.61 Ϯ 0.63
5.9 Ϯ 0.30
5.0 Ϯ 0.37
3.9 Ϯ 0.2
6.1 Ϯ 0.23
5.8 Ϯ 0.08
7.6 Ϯ 0.12
260
20-epi series
Exp. 3
D-Deficient
1␣,25-(OH)2D3
0
130
260
130
260
2.3 Ϯ 0.49
3.6 Ϯ 0.13
4.9 Ϯ 0.33
3.8 Ϯ 0.32
4.6 Ϯ 0.57
4.4 Ϯ 0.15
5.4 Ϯ 0.19
5.5 Ϯ 0.26
4.6 Ϯ 0.10
4.4 Ϯ 0.16
2␤, 1␣, 3␤ (3f)
Exp. 4
D-Deficient
1␣,25-(OH)2D3
2␣, 1␣, 3␤ (3b)
0
260
50
2.7 Ϯ 0.34
5.1 Ϯ 0.24
4.3 Ϯ 0.7
5.2 Ϯ 0.6
4.6 Ϯ 0.16
6.1 Ϯ 0.25
5.8 Ϯ 0.21
7.8 Ϯ 0.23
100
Fig. 3. Transcriptional activities of A-ring diastereomers of 2-methyl-1␣,25(OH)2-D3 (2) and their 20-epimers (3) on a human osteocalcin gene in human
osteosarcoma MG-63 cells.

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H. Takayama et al. / Steroids 66 (2001) 277–285
Fig. 4. Differentiation- or apoptosis-inducing effects of A-ring diastereomers of 2-methyl-1␣,25(OH)2D3 (2) on HL-60 cells.
Fig. 5. Differentiation- or apoptosis-inducing effects of A-ring diastereomers of 20-epi-2-methyl-1␣,25(OH)2D3 (3) on HL-60 cells.

H. Takayama et al. / Steroids 66 (2001) 277–285
285
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orders of their potencies corresponded to those of VDR
affinities and HL-60 differentiation activities. Fig. 3 shows
a typical transcriptional activity profile toward human os-
teocalcin gene in human osteosarcoma (MG-63) cells. The
results imply that the 2␣-Me group on the A-ring may be a
structure-specific motif for stimulating the VDR/VDRE-
mediated genomic action of 1␣,25-(OH)2D3.
Furthermore, the effects of the 2-methyl group of 2 and
3 on the activities for differentiation and apoptosis of HL-60
cells were examined [18]. Figs. 4 and 5 show a clear
contrast between the cell-differentiation and apoptosis-in-
ducing effects of 2-methyl-1␣,25(OH)2D3 (2) and 20-epi-
2-methyl-1␣,25(OH)2D3 (3). The 2␣-methyl isomers bear-
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differentiation, but failed to stimulate apoptosis. In contrast,
the 2␤-methyl isomers bearing 1␤- and 3␣-hydroxy groups
strongly stimulated apoptosis, but failed to induce differen-
tiation of HL-60 cells. These findings provide useful infor-
mation not only for studies on the structure-function rela-
tionship of 1␣,25-(OH)2D3 analogues but also for the
development of therapeutic agents for the treatment of leu-
kemia and cancers.
In conclusion, two sets of A-ring diastereomers of
2-methyl-1␣,25-(OH)2D3 and their 20-epimers were synthe-
sized, and proved to be very useful tools for studies on the
vitamin D biology and pharmacology.
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[18] Nakagawa K, Kurobe M, Konno K, Fujishima T, Takayama H,
Okano T. Structure specific control of differentiation and apoptosis
of human promyelocytic leukemia (HL-60) cells by A-ring diaste-
reomers of 2-methyl-1␣,25-dihydroxyvitamin D3 and its 20-epimer.
Biochem Pharmacol, In press.
This research was supported in part by Grants-In-Aid for
Scientific Research from the Ministry of Education, Sci-
ence, and Culture, JAPAN. The authors are grateful to Dr K.
Yamaguchi for X-ray analysis, and Dr M. Kurihara for the
calculation for conformational analysis.
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