New derivatives of 1α,25-dihydroxy-19-norvitamin D3 with two substituents at C-2: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmcl.2004.12.090

To examine the effect of 2,2-disubstitution on the biological activities of 19-norvitamin D analogs, novel 2,2-disubstituted-(20R)- and (20S)-1α,25-dihydroxy-19-norvitamin D₃ analogs were prepared and their biological activities were studied. A

Full text of DOI:10.1016/j.bmcl.2004.12.090

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1451–1455
New derivatives of 1a,25-dihydroxy-19-norvitamin D3 with
two substituents at C-2: synthesis and biological activity
Masato Shimizu,a,* Yukiko Iwasaki,b Mika Shimazaki,b Youhei Amano,b
Keiko Yamamoto,b Wolfgang Reischlc and Sachiko Yamadab
aLaboratory of Medicinal Chemistry, School of Biomedical Science, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
bInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku,
Tokyo 101-0062, Japan
cUniversity of Vienna, A-1090, Vienna, Austria
Received 29 November 2004; accepted 29 December 2004
Abstract—To examine the effect of 2,2-disubstitution on the biological activities of 19-norvitamin D analogs, novel 2,2-disubsti-
tuted-(20R)- and (20S)-1a,25-dihydroxy-19-norvitamin D3 analogs were prepared and their biological activities were studied. All
the synthesized analogs possessing hydrophobic 2a-substituents were more active than the corresponding 2b-isomers both in binding
to the vitamin D receptor and in activating gene transcription. The 2a-methyl-2b-hydroxy analog 9b was found to have markedly
higher transcriptional activity (32-fold) than the natural ligand 1a, although the two had the same binding affinity to the vitamin D
receptor. To our knowledge, this analog is among the most potent of 19-norvitamin D analogs.
Ó 2005 Elsevier Ltd. All rights reserved.
1a,25-Dihydroxyvitamin D3 (1a,25-(OH)2D3) 1a, in
addition to its well known crucial role in calcium and
phosphorus metabolism, bone mineralization, and the
prevention of rickets, regulates the growth, differentia-
tion, and function of several cell types, both normal
and malignant.1 It has also been shown to serve as a
modulator of the immune system. 1a,25-(OH)2D3 is a
secosteroid hormone that functions through a nuclear
receptor, the vitamin D receptor (VDR). This VDR is
a ligand-dependent transcription factor and is believed
to act by binding as a retinoid X receptor (RXR)/
VDR heterodimer to vitamin D-responsive elements
found in the promoter of target genes.1
of 19-norvitamin D has been shown to confer increased
affinity for the VDR, strong calcemic activity, as well as
high HL-60 differentiating activity.4–7 Recently the crys-
tal structures of the ligand binding domain (LBD) of the
rat VDR complexed with 2-substituted 19-norvitamin D
analogs have been disclosed,8 and in these complexes the
ligands are docked in the ligand binding pocket in a
manner similar to the natural hormone 1a in the human
VDR.9 We have developed a novel and efficient method
for preparation of the A-ring synthons of 19-norvitamin
D3 from D-glucose, and synthesized a number of 2-
substituted 19-norvitamin D3 analogs.10 From the struc-
ture–activity relationship (SAR) of the 2-functionalized
19-norvitamin D3 analogs, we showed that (20S)-1a,
25-dihydroxy-2b-hydroxyethoxy-19-norvitamin D3 had
the strongest binding affinity for the VDR (five times
more active than 1a) among the known 19-norvitamin
D analogs and significantly enhanced transcriptional
activity (30 times more active than 1a).11,12 In the course
of investigating the SAR of the 19-nor analogs, specifi-
cally the effect of the substituent at C-2 of the 19-norvi-
tamin D3 analog, we were interested to examine the
introduction of a second substituent at C-2 that might
have additional biologic effects due to ligand–protein
interactions among the substituents. Furthermore, the
1a,25-Dihydroxy-19-norvitamin D3 2a, which lacks the
10(19)-exomethylene of 1a, has low calcemic activity,
but still has the same cell differentiating ability as
1a.2,3 Recently, much attention has been focused on
the discovery of A-ring-modified 19-norvitamin D ana-
logs. Introduction of a substituent into the C-2 position
Keywords: 2,2-Disubustituted 19-norvitamian D; Synthesis; Vitamin D
receptor.
*
Corresponding author. Tel.: +81 03 5280 8117; fax: +81 03 5280
8005; e-mail: shimizu.mr@tmd.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.090

1452
M. Shimizu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1451–1455
O
OR
OR
limited number of 2,2-disubstituted 19-norvitamin D
analogs have been synthesized up to now.13 Here we re-
port the design, synthesis, biological activities, and
docking properties of a new class of (20R)- and (20S)-
19-norvitamin D analogs 3–12 with two different substit-
uents at the C-2 position (Fig. 1).
1
1) NaBH
2) BnBr
6
4
2
DMSO
1
4
(COCl)
5
2
TBSO
3
OTBS
OTBS
TBSO
3) aq. AcOH
OTMS
2
13
14: R =H; R =TMS 100%
1
1
1
2
15: R =Bn; R =TMS 85%
(sugar numbering)
2
16: R =Bn; R =H 80%
2
For synthesis of the target 2,2-disubstituted 19-norvit-
main D3 analogs 3–12, we used a Wittig–Horner cou-
pling approach14 involving the A-ring phosphine oxide
24 with the 25-hydroxy GrundmannÕs ketones possess-
ing a natural 20R- (25, 26) and an unnatural 20S- (20-
epi, 27) configuration (Fig. 2).
OBn
OBn
+
-
1) m-CPBA
MePPh Br
3
nBuLi
2) H /Pd-C
2
OTBS
OTBS
TBSO
TBSO
TBSO
TBSO
O
17 quant.
18 98%
O
OR
CH CO Me
1)
2
2
First, we prepared the A-ring synthon 24 with a
4,4-epoxy moiety, as outlined in Figure 2. The cyclohex-
anone derivative 13, bearing three silyl-protected hydr-
oxyl groups, was synthesized from D-glucose in 26%
yield.10 Reduction of 13 with NaBH4 yielded a 1-hydr-
oxy compound 14 (sugar numbering) as an approximate
2:1 diastereomeric mixture, which was treated with benz-
yl bromide to afford a benzyl ether 15. The trimethylsilyl
protecting group of 15 was selectively hydrolyzed by
treatment with a mixture of aqueous acetic acid in
THF to give a 4-hydroxy compound 16. Oxidation of
16 proceeded quantitatively under Swern conditions to
give the 4-keto derivatives 17 as a single compound.
The Wittig reaction of 17 with methyltriphenylphospho-
nium bromide afforded a 4-methylene product 18 in
excellent yield. Epoxidation of 18 with m-chloroperben-
zoic acid, followed by Pd/C-catalyzed hydrogenolysis of
the benzyl ether 19 proceeded quantitatively to provide
a spiro-oxirane 20 as an approximate 3:1 mixture of dia-
stereomers, which, upon Swern oxidation, gave a corre-
sponding 1-keto compound 21 as a single product.
DMSO
,
nBuLi
TMS
(COCl)
2
2) DIBAL-H
OTBS
OTBS
O
O
19: R=Bn 100%
20: R=H 100%
21 100%
R
P(O)Ph
2
i) TsCl
ii) Ph PLi
iii) H O
2
2
OTBS
TBSO
TBSO
OTBS
2
O
O
22: R=CO Me 95%
24 77%
2
23: R=CH OH 94%
2
OTES
OR
O
O
25: R=MOM; 26: R=TES
27
Figure 2. Synthetic scheme of the A-ring phosphine oxide with the
epoxy group.
Peterson olefination of 21 with methyl (trimethylsi-
lyl)acetate afforded an allylic ester 22 in an approximate
3:1 mixture of diastereomers due to the newly generated
double bond isomerism. The allylic ester 22 was reduced
with (i-Bu)2AlH to give an allylic alcohol 23. Transfor-
mation of 23 into the A-ring phosphine oxide 24 was
achieved in a one-pot process according to the proce-
dures described by DeLuca and co-workers.5
R'
R'
R'
19
5
R
4
2
3
1
HO
OH
HO
OH
HO
OH
O
O
1: R = CH
2
4
3
2: R = H
2
(2α-epoxide)
Protected 25-hydroxy GrundmannÕs ketones 25–27 with
a 20R- or unnatural 20S-configuration were prepared
starting from the readily available vitamin D3 or vitamin
D2 following the general procedures described previ-
ously (Fig. 2).15–18
(2β-epoxide)
(steroid numbering)
R'
R'
20
OH
OH
a: R' =
Ten 2,2-disubstituted 19-norvitamin D3 analogs 3a–12a
were successfully synthesized according to the sequence
shown in Figure 3. The key intermediates 28 (ca. 5:1)
and 29 (ca. 3:1) as a mixture of diastereomers were ob-
tained by condensation of the A-ring epoxide 24 with
the C/D-ring ketones 25 and 26, respectively. Deprotec-
tion of 28 with nBu4NF yielded the diastereomeric pairs
of 3a and 4a, together with fluorinated analogs 5a and
6a resulting from a nucleophilic ring-opening reaction
with fluoride anion. A methoxymethyl protected oxirane
b: R' =
HO
OH
HO
OH
OH
HO
R
R
5: R=CH F
6: R=CH F
2
2
2
7: R=CH OMe
8: R=CH OMe
2
9: R=Me
11: R=Et
10: R=Me
12: R=Et
Figure 1. Structures of the active vitamin D3 and its 19-norvitamin D
analogs.

M. Shimizu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1451–1455
1453
3a, 4a (60%) and 5a, 6a (8%)
7a, 8a (87%)
analog 9a was converted to a corresponding acetonide
33. In 33, an important correlation cross-peak was ob-
served between 2a-Me and 3a-H, but not between 1b-
H and 2a-Me. An NOE was also observed between
2a-Me and 10a-H. These correlations clearly demon-
strate the structure for 33 as indicated in Figure 3. In
the minor product 10a, a cross-peak was observed be-
tween 2b-Me and 1b-H or 4b-H or 10b-H, and the 2a-
configuration of the hydroxyl substituent was assigned.
These findings indicate that the major isomers of the
synthetic intermediates 28 and 29 have the 2b-epoxy
functionality.
24
25 or 26
nBuLi
nBu4NF
CSA
OR
OMOM
Me2Cu(CN)Li2
TBSO
OTBS
OH
HO
O
R
OH
28: R=TES 67%; 29: R=MOM 68%
31: R=Me (69% from 30)
32: R=Et (78% from 29)
It is well known that vitamin D analogs with the 20S-
configuration have enhanced VDR binding affinity with
respect to their 20R counterparts.21 We synthesized 2,2-
disubstituted 19-norvitamin D3 analogs 3b–12b with the
unnatural (20S)-side chain by analogous procedures as
described above (Fig. 3). Coupling of 24 with the pro-
tected (20S)-25-hydroxy C/D-ring ketone 27 yielded 34
as an approximate 3:1 isomeric mixture. Treatment of
34 with nBu4NF gave oxirane derivatives 3b and 4b, to-
gether with fluorinated products 5b and 6b, whereas
treatment of 34 with CSA in MeOH afforded the meth-
oxymethylated analogs 7b and 8b. LiAlH4 reduction of
3b and 4b, followed by hydrolysis, provided 9b and
10b (trace). Epoxide 34 was reacted with Me2Cu(CN)Li2
to yield 35, which was treated with CSA to afford 11b
and 12b.
nBu4NF
CSA
LiAlH4
9a, 10a (93%); 11a, 12a (99%)
OMOM
(CH3)2(OCH3)2
Me
4
10
1
H
OH
2
3
HO
OH
O
H
O
O
Me
Me
30 92%
33
7b, 8b (81%)
24
27 nBuLi
CSA
We evaluated the potencies of the 2,2-disubstituted 19-
norvitamin D3 analogs (3a,b–12a,b) to bind to the bo-
vine thymus VDR and to activate the gene in a transient
transcription assay in COS-7 cells, as described previ-
ously.22,23 Table 1 summarizes the results (Table 1). 2-
Methyl-2-hydroxy-19-norvitamin D3 analogs 9a and 9b
have higher transcriptional activities (Fig. 4), and the
largest difference in activity among the tested com-
pounds was found between the 2a-methyl compound
9a and the 2b-methyl isomer 10a. Other analogs 5a–
12a, except for the 2-methyl analogs, show comparable
efficacy, whereas the difference in activity between the
OTES
OTES
Me2Cu(CN)Li2
OTBS
OTBS
TBSO
TBSO
OH
O
35 88%
34 75%
nBu4NF
CSA
11b, 12b (81%)
3b, 4b (82%) and 5b, 6b (12%)
9b (67%); 10b (trace)
LiAlH4
3
2
1
0
Figure 3. Synthetic scheme of 2,2-disubstituted 19-norvitamin D3
analogs (3–12) with natural and unnatural C(20)-configuration.
29 was treated with camphor sulfonic acid (CSA) in
MeOH, yielding the 2-methoxymethylated analogs 7a
and 8a. The isomeric 2-methyl-19-nor analogs 9a and
10a were obtained by reductive ring cleavage of 30, fol-
lowed by removal of the methoxymethyl group of 31
with CSA. The nucleophilic oxirane cleavage reaction
of 29 with higher-order mixed organocuprate
19,20
1E-05 0.0001 0.001 0.01
0.1
1
10
100
1000
Me2Cu(CN)Li2
proceeded smoothly to afford a
Conc. (nM)
ring-opened product 32 in good yield, which after
deprotection with CSA, gave 11a and 12a. All C(2)-epi-
meric pairs (3a–12a) were separated by HPLC. The ster-
eochemistry at C-2 of 2,2-disubstituted 19-norvitamin D
analogs was assigned by phase-sensitive 2D NOESY
NMR analysis. The major 2-methyl-2-hydroxy-19-nor
Figure 4. Dose–response behavior of the most potent analog (9b) with
VDR in cellular reporter gene assays. Data represent the mean and
S.E. of at least three independent experiments for 1a,25-(OH)2D3
(triangle) or 9b (square). 1a,25-(OH)2D3: ED50 = 0.7 nM; 9b:
ED50 = 0.022 nM.

1454
M. Shimizu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1451–1455
Table 1. Relative VDR affinity and transcriptional activity of 2,2-
disubstituted 19-norvitamin D3 analogsa
phenyl ring of F150 contacts the newly introduced 2a-
methyl group in 9a, whereas hydrophilic R274 forms a
hydrogen bond with the 2b-hydroxyl moiety. Thus, the
A-ring is stabilized in the VDR LBP by both hydrophilic
and hydrophobic interactions. In isomer 10a with a 2a-
hydroxyl group, none of the hydrophobic and hydrogen
bonding interactions seen in 9a are observed.
Compounds
VDR affinity
Transcription
1a
3a
100
4
100
5c
4a
3b
3
50
4c
89c
18c
59b
14b
213b
46b
48b
8b
176b
39b
280c
6c
3200c
ND
67b
10b
92b
90b
4b
5a
20
2
0.02
In conclusion, we have described the synthesis and bio-
logical evaluation of novel 2,2-disubstituted 19-norvita-
min D3 analogs.26 2,2-Disubstituted analogs have
significant biological activities, in particular the 2a-
methyl-2b-hydroxy-19-nor analog 9b is characterized
by an extremely high ability to activate gene transcrip-
tion, and to our knowledge this is among the most po-
tent of 19-norvitamin D analogs. We expect that novel
2,2-disubstituted 19-norvitamin D3 analogs will have a
much broader spectrum of activities. Full details of the
synthesis and results of broad biological evaluation will
be reported in due course.
6a
5b
60
ND
6b
7a
4
0.1
8a
7b
20
8b
9a
10
30 (21d)
0.1 (2.6d)
100 (23d)
ND (18d)
2 (83e)
0.05 (1e)
5 (200e)
0.1 (17e)
10a
9b
10b
11a
12a
11b
12b
References and notes
ND: Not determined.
a Activities are presented as % effect of 1a.
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c Activity was assessed in terms of ED50
.
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two isomers is small. The replacement of hydrogen by
fluorine (CH3/CH2F transposition) causes markedly
reduced affinity for the VDR and transcriptional activ-
ity. These results suggest that the CH2F substituent
has a close steric relationship and biological similarity
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arrangement of two substituents at the C-2 position is
complementary to the amino acid residues of the LBP.
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26. The structures of all new compounds were confirmed
based on the 1H and 19F NMR, mass, and UV spectra.
The stereochemistry of the new compounds was estab-
lished from the 2D NOESY spectra.