Synthesis of 2α-propoxy-1α,25-dihydroxyvitamin D3 and comparison of its metabolism by human CYP24A1 and rat CYP24A1

Article abstract of DOI:10.1016/j.bmc.2009.05.032

A new vitamin D₃ analogue, 2α-propoxy-1α,25-dihydroxyvitamin D₃ (C3O1), was synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We studied the metabo

Full text of DOI:10.1016/j.bmc.2009.05.032

Bioorganic & Medicinal Chemistry 17 (2009) 4296–4301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2a-propoxy-1a,25-dihydroxyvitamin D3 and comparison
of its metabolism by human CYP24A1 and rat CYP24A1
Nozomi Saito a, , Yoshitomo Suhara a,à, Daisuke Abe b,§, Tatsuya Kusudo b,–, Miho Ohta c, Kaori Yasuda d,
Toshiyuki Sakaki d, , Shinobu Honzawa a,||, Toshie Fujishima a, , Atsushi Kittaka a,
*
*
a Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan
b Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
c Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, Osaka 559-0033, Japan
d Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Toyama 939-0398, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new vitamin D3 analogue, 2
from D-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling
method. We studied the metabolism of the new analogue by human CYP24A1 and rat CYP24A1 to learn of
species-based differences and found that the former has multiple metabolic pathways, but the latter has
only a single pathway.
a
-propoxy-1
a
,25-dihydroxyvitamin D3 (C3O1), was synthesized starting
Received 31 March 2009
Revised 11 May 2009
Accepted 12 May 2009
Available online 18 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D3 analogues
Metabolism
Human CYP24A1
Rat CYP24A1
1. Introduction
potentially useful therapeutic agent for certain cancers, skin dis-
eases, and immune disorders, and in fact, 1 and some synthetic
The physiologically active metabolite of vitamin D3, 1
a
,25-
analogues of 1 are used clinically in the treatment of bone diseases,
secondary hyperparathyroidism, and psoriasis.1 In order to investi-
gate the structure–activity relationship of the natural hormone, we
dihydroxyvitamin D3 (1), is a nuclear hormone that regulates cellu-
lar growth, differentiation, and apoptosis, in addition to its classical
role in calcium homeostasis and bone mineralization.1–3 Most of
the biological effects of 1 are considered to be mediated via binding
to the specific intracellular receptor, vitamin D receptor (VDR),
which belongs to the nuclear receptor superfamily and acts as a li-
gand-dependant transcription factor with coactivators.4 The ubiq-
uitous distribution of VDR in the body makes this hormone a
systematically synthesized A-ring modified analogues such as 2
methyl-, -alkyl- (2), -( -hydroxyalkyl)- (3), and -(
hydroxyalkoxy)-1
,25-dihydroxyvitamin D3 (4) (Fig. 1).5–10
In regard to modification with the 2 -(
it was found that the 2 -(3-hydroxypropyl) group on 1 best fits
the cavity of the ligand-binding domain (LBD) of VDR among the
a-
2a
2a
x
2a x
-
a
a
x-hydroxyalkyl) group,
a
2a-(x-hydroxyalkyl) analogues of 3, and the binding activity is
threefold higher than that of 1.7 Recently, an X-ray crystallographic
* Corresponding authors. Tel.: +81 766 56 7500x601; fax: +81 766 56 2498 (T.S.),
tel./fax: +81 42 685 3713 (A.K.).
E-mail addresses: tsakaki@pu-toyama.ac.jp (T. Sakaki), akittaka@pharm.teikyo-
u.ac.jp (A. Kittaka).
analysis showed the structure of the complex of the hVDR LBD
with 3c and 4b.11 The role of the 2
a-(3-hydroxypropyl) group in
3c was clarified: this moiety enters the water channel around the
A-ring moiety at the LBD of the VDR, and the terminal OH group
of 3c replaces one of the water molecules constituting the water
channel to maintain hydrogen bond networks, thus making the
complex up to threefold more stable than the VDR-1 complex. Chu-
gai Pharmaceutical Co. Ltd, developed 2b-(3-hydroxypropoxy)-
Present address: Faculty of Pharmaceutical Sciences, Hokkaido University,
Sapporo 060-0812, Japan.
à
Present address: Department of Environmental Sciences, Yokohama College of
Pharmacy, Yokohama 245-0066, Japan.
§
Present address: Process Engineering Section, Nagase ChemteX Corporation,
Kyoto, 620-0853, Japan.
–
Present address: Department of Biomedical Sciences, College of Life and Health
1a,25-dihydroxyvitamin D3 (ED-71, in Fig. 1) as a promising candi-
date for the treatment of osteoporosis in Japan.1,12 Although ED-71
shows high calcemic activity and a long half-life in plasma due to
its strong affinity for vitamin D binding protein (DBP, twice the
affinity of 1), its affinity for bovine thymus VDR is weaker than that
Sciences, Chubu University, Kasugai 487-8501, Japan.
||
Present address: Faculty of Pharmaceutical Sciences, Niigata University of
Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
Present address: Faculty of Pharmaceutical Sciences at Kagawa Campus, Toku-
shima-Bunri University, Kagawa 769-2193, Japan.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.032

N. Saito et al. / Bioorg. Med. Chem. 17 (2009) 4296–4301
4297
24
23
26
1
2
: R = H (1α,25-dihydroxyvitamin D3)
: R = (CH2)nCH3
OH
OH
H
H
C
D
2a: n = 0, 2b: n =1, 2c: n = 2, 2d: n = 3
: R = (CH2)nOH
H
H
3
7
6
3a
3b
3c
3d
: n = 1, : n = 2, : n = 3, : n = 4
3
A
4: R = OCH2(CH2)nCH2OH
4a 4b 4c
1
HO
HO
OH
OH
: n = 0, : n = 1, : n = 2
2
5 (C3O1)
R
: R = OCH2CH2CH3
O
OH ED-71
Figure 1. Structures of 1
a
,25-dihydroxyvitamin D3 (1), its 2
a
-substituted analogues (2–4), 2a-propoxy-1a,25-dihydroxyvitamin D3 (5, C3O1), and ED-71.
of the natural hormone (13–93%).12,13 It was found that one of our
synthetic analogues, 2 -(3-hydroxypropoxy)-1 ,25-dihydroxyvi-
tamin D3 (4b), that is, the 2 -epimer of ED-71, showed greater
affinity for VDR (180%) than did 1.8 We demonstrated that the bio-
logically active 4b was produced from the less active 2 -propoxy-
readily available from methyl a-D-glucoside, was chosen as the chi-
a
a
ral template.8,15 Regiospecific ring opening by 1-propanol at the C3
position established the altrose configuration of 7, in which the C2,
C3, and C4 asymmetric centers satisfy the corresponding desired
a
a
3b, 2a, and 1a stereochemistries of 5, respectively. Treatment of
1
a
,25-dihydroxyvitamin D3 (5, C3O1) through metabolic
x
-
the benzylidene acetal 7 with NBS gave the bromide 8. The reaction
of 8 with activated zinc powder in the presence of NaBH3CN gave
the alcohol 9. Sulfonylation of the primary hydroxy group and
LiHMDS treatment afforded the epoxide 11, into which was subse-
quently introduced the acetylene unit with the epoxide-ring open-
ing to give 12. Removal of the terminal TMS and benzoyl groups
under basic conditions afforded the enyne 13, and protection of
the secondary hydroxy groups with the TBS groups provided the
A-ring precursor 14.
Palladium-mediated alkylative cyclization with vinyl bromide
of the CD fragment 1516 from Grundmann’s ketone and subsequent
deprotection furnished C3O1 (5) (Scheme 2).
The affinity of C3O1 (5) for calf-thymus VDR was 64% as
compared with the affinity of the natural hormone 1. The metabo-
lism of C3O1 by human CYP27A1 produced O2C3 (4b), which
showed 180–204% binding affinity for the VDR.14 However, inacti-
vation of C3O1 by CYP24A1 also occurs in the body. This time we
focused on the metabolism of C3O1 by human CYP24A1 and rat
CPY24A1.
hydroxylation by CYP27A1.14 We report here the concise synthesis,
and the metabolism by human CYP24A1 and rat CYP24A1, of C3O1.
2. Results and discussion
In 2000, we reported that 2a-propyl-1a,25-dihydroxyvitamin
D3 (2c) showed strong calcemic activity in vivo, although its affin-
ity for VDR and HL-60 cell differentiation activity were weak
in vitro.7a We hypothesized that 2c was hydroxylated at the
x
-po-
sition of the 2
a-side chain of 2c through metabolism in the rat li-
ver, and consequently, inactive 2c was converted to active 3c,
which showed strong calcemic activity in vivo. We synthesized 5
to investigate whether this kind of metabolism actually occurs,
and proved that
5 was activated through hydroxylation by
CYP27A1 and 4b could be detected by LC–MS.14a
The route by which we synthesized 5 is shown in Scheme 1. To
create 1
a,2a,3b-stereochemistry on the A-ring of the target vita-
min D analogue (5), the known crystalline epoxide 6, which was
Scheme 1. Synthesis of the A-ring precursor 14 for C3O1.

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N. Saito et al. / Bioorg. Med. Chem. 17 (2009) 4296–4301
OH
H
1) Pd(PPh3)4
OH
H
NEt3, toluene
reflux
H
TBSO
OTBS +
2) TBAF, THF
50°C
H
O
14
15
5 (C3O1)
Br
HO
OH
O
Scheme 2. Trost coupling of the enyne 14 and bromoolefin 15.
As shown in Figure 3, the mass spectrum of M3 showed a
molecular ion at m/z 491 (M+H) and fragment ions at 473
(M+HꢀH2O), 455 (M+Hꢀ2H2O), 437 (M+Hꢀ3H2O), 413
(M+Hꢀ60ꢀH2O), 395 (M+Hꢀ60ꢀ2H2O), and 377 (M+Hꢀ60ꢀ
3. Metabolism of C3O1 by CYP24A1
reconstituted system containing adrenodoxin reductase
A
(ADR), adrenodoxin (ADX), and human CYP24A1 or rat CYP24A1
was examined for the metabolism of C3O1. Figure 2 shows HPLC
profiles of the substrate C3O1 and its metabolites produced by hu-
man CYP24A1 or rat CYP24A1. The rat CYP24A1-dependent metab-
olism of C3O1, giving three metabolites M2, M3, and M4, was quite
similar to that of 4b designated as O2C3 in our previous report.14a
However, in the human CYP24A1-dependent metabolism, we no-
ticed a clear difference between C3O1 and O2C3. M6 was a major
metabolite in the metabolism of C3O1, but M6 was not observed
in the metabolism of O2C3.14a Based on our previous studies, the
metabolites M1–M5 are assumed to be 23,26(OH)2–C3O1 (M1),
24-oxo-23(OH)–C3O1 (M2), a mixture of 23(OH)–C3O1 (M3) and
24(OH)–C3O1 (M3), 24-oxo-C3O1 (M4), and 25,26,27-trinor-24-
ene-C3O1 (M5), respectively.14a On the other hand, the metabolite
M6 is assumed to be 23-oxo-C3O1 based on the metabolism of
1b,25(OH)2-3-epi-D3.14b To confirm these assumptions, we col-
lected M3 and M6 produced by human CYP24A1, and subjected
them to mass spectrometric analysis.
3H2O). The fragment ion at m/z 413 indicates losses of a 2a-pro-
poxy group and a water molecule. In addition, fragment ions at
339 (M+Hꢀ60ꢀ74ꢀH2O), 321 (M+Hꢀ60ꢀ74ꢀ2H2O), and 303
(M+Hꢀ76ꢀ74ꢀ3H2O) which result after cleavage between C-23
and C-24 are characteristic of the 23-hydroxylated compound as
described previously.14a These results indicate that M3 contains
23(OH)–C3O1. However, based on the facts that the putative
metabolites of the C-24 oxidation pathway, 24-oxo-C3O1 (M4)
and 24-oxo-23(OH)–C3O1 (M2) were detected, it appeared that
M3 contained both 23(OH)–C3O1 and 24(OH)–C3O1. On the other
hand, the mass spectrum of M6 showed a molecular ion at m/z 489
(M+H) and fragment ions at 471 (M+HꢀH2O), 453 (M+Hꢀ2H2O),
411 (M+Hꢀ60ꢀH2O), 393 (M+Hꢀ60ꢀ2H2O). The fragment ion at
m/z 411 indicates losses of a 2a-propoxy group and a water mole-
cule. In addition, fragment ions at 353 (M+Hꢀ60ꢀ58ꢀH2O), 335
(M+Hꢀ60ꢀ58ꢀ2H2O) which result after cleavage between C-24
and C-25 are characteristic of the 23-oxo compound as described
previously.14b
Thus, human CYP24A1-dependent metabolism of C3O1 con-
tains a unique C-23 oxidation pathway from C3O1 to 23-oxo-
C3O1 (M6) via 23(OH)–C3O1 (M3), indicating that a significant
species-based difference in the CYP24A1-dependent metabolism
of C3O1 between humans and rats.14b
4. Conclusion
We synthesized a new 2a-modified vitamin D3 analogue (C3O1,
5) that showed moderate affinity for VDR, and then investigated
metabolic pathways for both human CYP24A1 and rat CPY24A1.
Human CYP24A1 showed multiple metabolic pathways, but rat
CPY24A1 has a single metabolic pathway. In previous report, we
proved that C3O1 was converted to active O2C3 (4b) by human
CYP27A1, and the catalytic efficiency of human CYP24A1 for
C3O1 and O2C3 was only 2% of the natural hormone 1.14a C3O1
would possess long half-life in vivo, and these results will help
us to develop C3O1 as a therapeutic agent.
5. Experimental
5.1. Synthesis
5.1.1. General
All manipulations were performed under an argon atmosphere
unless otherwise mentioned. All solvents and reagents were puri-
fied when necessary using standard procedures. Column chroma-
tography was performed on silica gel 60 N (Kanto Chemical Co.,
Figure 2. HPLC profiles of C3O1 and its metabolites with human CYP24A1 (A) and
rat CYP24A1 (B). After incubation with 4.0
lM of C3O1 for 60 min, the reaction
mixture was extracted, and analyzed by HPLC as described in Section 5.
Inc., 100–210 lm), and flash column chromatography was per-

N. Saito et al. / Bioorg. Med. Chem. 17 (2009) 4296–4301
4299
Figure 3. Mass spectra of the metabolites M3 (A) and M6 (B) produced by human CYP24A1. The metabolites were isolated by HPLC, and then analyzed by mass spectrometry
as described in Section 5.
formed on silica gel 60 (Merck, 40–63
l
m). NMR spectra were
NMR (400 MHz, CDCl3) d 0.91 (3H, t, J = 7.3 Hz), 1.62 (2H, m), 1.93
(1H, br d, J = 5.6 Hz), 3.40 (3H, s), 3.58 (1H, dt, J = 6.6, 9.5 Hz), 3.69
(1H, dt, J = 6.8, 9.5 Hz), 3.75 (1H, t, J = 9.8 Hz), 3.80 (1H, br t,
J = 2.9 Hz), 3.96 (1H, dd, J = 2.9, 9.5 Hz), 4.01 (1H, br dd, J = 2.9,
5.6 Hz), 4.28–4.36 (2H, m), 4.59 (1H, s), 5.56 (1H, s), 7.33–7.40
(3H, m), 7.45–7.52 (2H, m); 13C NMR (125 MHz, CDCl3) d 10.4,
23.1, 55.6, 58.6, 69.4, 70.2, 73.4, 76.1, 102.0, 102.3, 126.2, 128.2,
129.0, 137.7. IR (neat) 3462, 2963, 2934, 1456, 1383 cmꢀ1; EIMS
m/z 324 (M+); HREIMS Calcd for C17H24O6 (M+) 324.1573, found
324.1570.
measured on a JEOL AL-400 magnetic resonance spectrometer.
Infrared spectra were recorded on a JASCO FTIR-8000 spectrome-
ter. Mass spectra were measured on a JEOL JMX-SX 102 mass spec-
trometer. Specific optical rotations were measured on a JASCO DIP-
370 digital polarimeter.
5.1.2. Methyl 4,6-O-benzylidene-3-O-propyl-a-D-
altropyranoside (7)
To a stirred solution of epoxide 6 (3.40 g, 12.9 mmol) in 1-pro-
panol (70 mL) was added KOtBu (4.76 g, 42.5 mmol) at room tem-
perature. The solution was stirred at 110 °C for 3 days. The reaction
mixture was cooled to room temperature, and the solution was
partitioned between EtOAc (500 mL) and saturated aqueous NH4Cl
(200 mL). The organic layer was washed with saturated aqueous
NH4Cl, H2O, and brine (200 mL each), successively, and dried over
MgSO4. After filtration and concentration, the residue was purified
on silica gel column chromatography (10–33% EtOAc in hexane) to
5.1.3. Methyl 4-O-benzoyl-6-bromo-3-O-propyl-6-deoxy-a-D-
altropyranoside (8)
Benzylidene acetal 7 (2.10 g, 6.48 mmol) was dissolved in CCl4
(70 mL), and NBS (1.38 g, 7.78 mmol) and BaCO3 (1.28 g,
6.48 mmol) were added at room temperature. The mixture was
stirred at 80 °C for 35 min, and then cooled to room temperature.
The mixture was diluted with EtOAc (300 mL), and unsolved mate-
rials were filtered off with a Celite pad. The filtrate was washed
give 2.39 g of 7 as a colorless oil (57%). ½a D25
ꢁ
þ 89:5 (c 1.5, CHCl3). 1H

4300
N. Saito et al. / Bioorg. Med. Chem. 17 (2009) 4296–4301
with 1 M aqueous Na2S2O3, saturated aqueous NaHCO3, and brine
(100 mL each), successively. The organic layer was dried over
MgSO4, filtration and concentration followed by flash chromatog-
raphy on silica gel (20% EtOAc in hexane) gave 1.90 g of 8 as a col-
m/z 276 (M+); HREIMS Calcd for C16H20O4 (M+), 276.1362, found
276.1369.
5.1.6. (4R,5S,6R)-5-(Propoxy)oct-7-en-1-yn-4,6-diol (13)
orless oil (73%). ½a D25
ꢁ
þ 51:0 (c 1.1, CHCl3); 1H NMR (400 MHz,
To a solution of ethynyltrimethylsilane (0.17 mL, 1.20 mmol) in
THF (1 mL) was added a solution of BuLi in hexane (1.5 M, 0.69 mL,
1.04 mmol) at ꢀ78 °C, and the mixture was stirred at the same
temperature for 30 min. To the mixture were added a solution of
epoxide 11 (124.7 mg, 0.451 mmol) in THF (2.5 mL) and BF3ꢂOEt2
CDCl3) d 0.82 (3H, t, J = 7.4 Hz), 1.52 (2H, m), 2.49 (1H, br d,
J = 2.9 Hz), 3.47 (1H, dt, J = 6.6, 9.2 Hz), 3.51 (3H, s), 3.53 (1H, dt,
J = 6.6, 9.2 Hz), 3.60 (1H, dd, J = 6.8, 11.0 Hz), 3.64 (1H, dd, J = 3.9,
11.0), 3.76 (1H, dd, J = 3.9, 7.2 Hz), 3.99 (1H, br m), 4.38 (1H, dt,
J = 3.9, 6.8 Hz), 4.71 (1H, d, J = 3.4 Hz), 5.47 (1H, dd, J = 3.9,
6.8 Hz), 7.44–7.47 (2H, m), 7.57 (1H, m), 8.04–8.06 (2H, m); 13C
NMR (100 MHz, CDCl3) d 10.5, 23.1, 32.8, 56.1, 69.1, 69.8, 69.9,
72.5, 76.7, 102.6, 128.3, 129.3, 129.7, 133.2, 165.5; IR (neat)
3474, 1718, 1603, 1275, 1033 cmꢀ1; EIMS m/z 402 (M+); HREIMS
Calcd for C17H23O679Br (M+) 402.0678, found 402.0686.
(65
lL, 0.513 mmol) at ꢀ78 °C, and the resulting mixture was
warmed to room temperature over 2 h. After additional stirring
at room temperature for 1 h, the reaction was quenched by addi-
tion of saturated aqueous NH4Cl at 0 °C. The aqueous layer was ex-
tracted with EtOAc. The combined organic layer was washed with
brine, dried over Na2SO4, and concentrated. The residue was dis-
solved in MeOH (3 mL). To the MeOH solution was added K2CO3
(187.0 mg, 1.35 mmol) at 0 °C, and the mixture was stirred at room
temperature for 2 h. To the mixture was added saturated aqueous
NH4Cl at 0 °C, and the aqueous layer was extracted with EtOAc. The
combined organic layer was washed with brine, dried over Na2SO4
and concentrated. The residue was purified by flash column chro-
matography on silica gel (25% EtOAc in hexane) to give diol 13
5.1.4. (2R,3S,4R)-4-(Benzoyloxy)-3-(propoxy)hex-5-en-1,2-diol
(9)
Bromide 8 (1.27 g, 3.15 mmol) was dissolved in 1-propanol
(25 mL) and H2O (2.5 mL) was added. The solution was heated at
95 °C, and then Zn powder (6.11 g, 93.5 mmol) and NaBH3CN
(403.0 mg, 6.41 mmol) were added. The mixture was refluxed at
the same temperature for 20 min, and additional Zn powder
(4.10 g, 62.7 mmol) and NaBH3CN (398.0 mg, 6.33 mmol) were
added. After refluxing for 35 min, the reaction mixture was al-
lowed to cool to room temperature and filtered by a Celite pad.
The filtrate was concentrated in vacuo, and the residue was puri-
fied by a silica gel column chromatography (10% EtOAc in hexane)
(74.2 mg, 83% in two steps) as a colorless oil. ½a D20
ꢀ 0:74 (c 1.08,
ꢁ
CHCl3); 1H NMR (400 MHz, CDCl3) d 0.96 (3H, t, J = 7.4 Hz), 1.60–
1.69 (2H, m), 2.02 (1H, t, J = 2.7 Hz), 2.47 (1H, dd, J = 2.7,
16.7 Hz), 2.53 (1H, ddd, J = 1.2, 2.7, 16.7 Hz), 2.69 (1H, d,
J = 4.9 Hz), 2.90 (1H, d, J = 6.8 Hz), 3.39 (1H, dd, J = 2.2, 4.9 Hz),
3.49 and 3.70 (2H, each as dt, J = 6.6, 9.0 Hz), 4.05 (1H, dt, J = 2.2,
6.8 Hz), 4.50 (1H, br q, J = 4.9 Hz), 5.28 (1H, br d, J = 10.5 Hz),
5.44 (1H, br d, J = 17.1 Hz), 5.95 (1H, ddd, J = 4.9, 10.5, 17.1 Hz);
13C NMR (100 MHz, CDCl3) d 10.6, 23.2, 23.6, 69.8, 70.3, 72.3,
73.2, 80.4, 80.8, 116.2, 137.0; IR (neat) 3395, 2965, 2936, 2120,
1429, 1111, 1076, 930 cmꢀ1; EIMS m/z 198(M+); HREIMS calcd
for C11H18O3 (M+) 198.1256, found 198.1266.
to give 781.0 mg of 9 as a colorless oil (84%). ½a D20
þ 29:6 (c 1.38,
ꢁ
CHCl3); 1H NMR (400 MHz, CDCl3) d 0.94 (3H, t, J = 7.1 Hz), 1.62
(2H, tq, J = 7.1 Hz), 3.50 (1H, dt, J = 6.6, 9.1 Hz), 3.60 (1H, t,
J = 4.7 Hz), 3.74–3.82 (4H, m), 5.34 (1H, dt, J = 1.4, 10.4 Hz), 5.43
(1H, dt, J = 1.4, 17.3 Hz), 5.71–5.73 (1H, m), 6.05 (1H, ddd, J = 6.3,
10.4, 17.3 Hz), 7.26–7.47 (2H, m), 7.57–7.60 (1H, m), 8.05–8.07
(2H, m); 13C NMR (100 MHz, CDCl3) d 10.6, 23.2, 63.8, 71.0, 74.2,
74.8, 80.4, 118.1, 128.3 (2C), 129.5 (2C), 129.8, 132.5, 133.1,
165.4; IR (neat) 3432, 2963, 2936, 2878, 1719, 1647, 1271, 1111,
713 cmꢀ1; EIMS m/z 276 (M+ꢀH2O); HREIMS calcd for C16H20O4
(M+ꢀH2O) 276.1362, found 276.1369.
5.1.7. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-4-
(propoxy)oct-1-en-7-yne (14)
To a cooled (0 °C) and stirred solution of the diol 13 (300 mg,
1.52 mmol) in CH2Cl2 (20 mL) were added 2,6-lutidine (811 lL,
7.58 mmol) and TBSOTf (1.04 mL, 4.55 mmol). After stirring at
0 °C for 1 h, the solution was diluted with EtOAc (50 mL), and then
washed with water and brine (30 mL each). The organic layer was
separated and dried over MgSO4. Filtration and concentration fol-
lowed by flash chromatography on silica gel (10% EtOAc in hexane)
5.1.5. (3R,4S,5R)-3-(Benzoyloxy)-4-(propoxy)-5,6-epoxyhex-1-
ene (11)
To a solution of diol 9 (220.2 mg, 0.748 mmol) in pyridine
(0.75 mL) was added 2,4,6-trimethylbenzenesulfonyl chloride
(TmCl, 180.0 mg, 0.823 mmol) at 0 °C, and the mixture was stirred
at room temperature for 14 h. To the mixture was added water at
0 °C, and the aqueous layer was extracted with Et2O. The combined
organic layer was washed with brine, dried over Na2SO4 and con-
centrated. The residue was dissolved in THF (7.5 mL). To the solu-
tion was added a solution of LiHMDS in THF (1.0 M, 0.82 mL,
0.82 mmol) at ꢀ78 °C, and the resulting mixture was warmed to
0 °C over 1 h. To the mixture was added brine at 0 °C, and the aque-
ous layer was extracted with EtOAc. The combined organic layer
was washed with brine, dried over Na2SO4 and concentrated. The
residue was purified by flash column chromatography on silica
gel (5% EtOAc in hexane) to give epoxide 11 (148.8 mg, 72% in
gave 580 mg of 14 as a colorless oil (90%). ½a D20
ꢁ
0:070 (c 1.43,
CHCl3); 1H NMR (400 MHz, CDCl3) d 0.03, 0.07, 0.09, and 0.10
(12H, each as s), 0.90 (3H, t, J = 7.4 Hz), 0.89 and 0.90 (18H, each
as s), 1.56 (2H, m), 1.95 (1H, t, J = 2.7 Hz), 2.36 (1H, ddd, J = 2.7,
5.6, 16.8 Hz), 2.50 (1H, ddd, J = 2.7, 5.6, 16.8 Hz), 3.36 (1H, dd,
J = 3.7, 5.6 Hz), 3.52 (1H, dt, J = 6.6, 8.9 Hz), 3.62 (1H, dt, J = 7.0,
8.9 Hz), 3.87 (1H, q, J = 5.6 Hz), 4.31 (1H, dd, J = 3.7, 6.8 Hz), 5.13
(1H, dt, J = 1.2, 10.3 Hz), 5.21 (1H, dt, J = 1.2, 17.3 Hz), 5.97 (1H,
ddd, J = 6.8, 10.3, 17.3 Hz); 13C NMR (100 MHz, CDCl3) d ꢀ4.6,
ꢀ4.2, ꢀ4.1, ꢀ4.0, 10.7, 18.1, 18.2, 23.4, 24.2, 25.9 (3C), 26.0 (3C),
69.8, 71.7, 74.3, 74.5, 82.1, 84.9, 115.8, 138.8; IR (neat) 2957,
2932, 2888, 2859, 1472, 1254, 1094, 835, 777 cmꢀ1; EIMS m/z
two steps) as a colorless oil. ½a D20
ꢁ
þ 47:8 (c 0.48, CHCl3); 1H NMR
369 (Mꢀ Bu)+; HREIMS Calcd for C19H37O3Si2 (Mꢀ Bu)+ 369.2281,
t
t
(400 MHz, CDCl3) d 0.93 (3H, t, J = 7.4 Hz), 1.61 (2H, m), 2.63 (1H,
dd, J = 2.7, 4.9 Hz), 2.78 (1H, dd, J = 4.2, 4.9 Hz), 3.11 (1H, ddd,
J = 2.7, 4.2, 6.8 Hz), 3.19 (1H, dd, J = 5.1, 6.8 Hz), 3.55 and 3.71
(2H, each as dt, J = 6.9, 9.5 Hz), 5.32 (1H, dt, J = 1.2, 10.5 Hz), 5.42
(1H, dt, J = 1.2, 17.2 Hz), 5.67 (1H, ddd, J = 1.2, 5.1, 6.4 Hz), 6.06
(1H, ddd, J = 6.4, 10.5, 17.2 Hz), 7.44–7.49 (2H, m), 7.51–7.56 (1H,
m), 8.05–8.08 (2H, m); 13C NMR (100 MHz, CDCl3) d 10.5, 23.0,
43.5, 52.2, 72.6, 74.6, 82.2, 118.1, 128.3, 129.4, 129.8, 132.8,
133.0, 165.1; IR (neat) 2969, 2940, 1726, 1176, 1109 cmꢀ1; EIMS
found 369.2275.
5.1.8. 2
The silyl ether 14 (40 mg, 93.7
(40 mg, 112 mol) were dissolved in TEA/toluene (3:1, 2.0 mL),
and tetrakis(triphenylphosphine)palladium(0) (8.0 mg, 7.72 mol)
a
-Propoxy-1
a
,25-dihydroxyvitamin D3 (C3O1, 5)
lmol) and vinyl bromide 15
l
l
was added. After stirring at room temperature for 15 min, the
resulting yellow solution was refluxed for 2 h. The reaction mix-
ture was filtered through a pad of silica gel. Concentration followed

N. Saito et al. / Bioorg. Med. Chem. 17 (2009) 4296–4301
4301
by preparative thin-layer chromatography on silica gel (20% EtOAc
in hexane) gave a crude coupling product as a white solid, which
was used in the next step without further purification.
has been supported in part by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (to N.S.,
Y.S., T.F., and S.H.) and by Grant-in-Aid from Japan Society for the
Promotion of Science (to A.K.).
To a cold (0 °C) and stirred solution of the crude protected vita-
min D in THF (2.0 mL) was added 1 M TBAF in THF (1 mL, 1 mmol).
The reaction mixture was stirred at 50 °C for 1 h. The solution was
diluted with EtOAc (20 mL), and the solution was washed with H2O
and brine (2.0 mL each). The aqueous layer was extracted with
EtOAc (6 ꢃ 2.0 mL), and the combined organic layer was dried over
Na2SO4. Filtration and concentration followed by preparative thin-
layer chromatography on silica gel (20% MeOH in CH2Cl2) gave
28 mg of 5 as a white powder (42% in two steps). Further purifica-
tion for biological evaluation was conducted by using reversed-
phase recycle HPLC (YMC-Pack ODS column, 20 ꢃ 150 mm,
References and notes
1. In Vitamin D, 2nd ed.; Feldman, D., Pike, J. W., Glorieux, F. H., Eds.; New vitamin
D analogues are described in chapters 80–88; Elsevier Academic: New York,
2005.
2. (a) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocrinol. Rev. 1996, 16, 200;
(b) Brown, A. J.; Slatopolsky, E. Mol. Aspect Med. 2008, 29, 433; (c) Laverny, G.;
Penna, G.; Uskokovic, M.; Marczak, S.; Maehr, H.; Jankowski, P.; Ceailles, C.;
Vouros, P.; Smith, B.; Robinson, M.; Reddy, G. S.; Adorini, L. J. Med. Chem. 2009,
52, 2204.
3. (a) Ettinger, R. A.; DeLuca, H. F. Adv. Drug Res. 1996, 28, 269; (b) DeLuca, H. F.
Nutr. Rev. 2008, 66, S73.
4. (a) Evans, R. M. Science 1988, 240, 889; (b) Yanagisawa, J.; Yanagi, Y.; Masuhiro,
Y.; Suzawa, M.; Watanabe, M.; Kashiwagi, K.; Toriyabe, T.; Kawabata, M.;
Miyazono, K.; Kato, S. Science 1999, 283, 1317.
5. For reviews, see: (a) Saito, N.; Honzawa, S.; Kittaka, A. Curr. Top. Med. Chem.
2006, 6, 1273; (b) Kittaka, A. Yakugaku Zasshi (in Japanese) 2008, 128, 1235;
(c) Takayama, H.; Kittaka, A.; Fujishima, T.; Suhara, Y.. In Reichrath, J.,
Friedrich, M., Tilgen, W., Eds.; Vitamin D Analogs in Cancer Prevention and
Therapy, Recent Results in Cancer Research 164; Springer: Berlin Heidelberg,
2003; p 289.
6. (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.; Miura, D.; Chokki, M.;
Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8, 151; (b) Konno, K.; Fujishima, T.;
Maki, S.; Liu, Z.-P.; Miura, D.; Chokki, M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.;
Kurihara, M.; Miyata, N.; Smith, C.; DeLuca, F. H.; Takayama, H. J. Med. Chem.
2000, 43, 4247.
7. (a) Suhara, Y.; Nihei, K.; Tanigawa, H.; Fujishima, T.; Konno, K.; Nakagawa,
K.; Okano, T.; Takayama, H. Bioorg. Med. Chem. Lett. 2000, 10, 1129; (b)
Suhara, Y.; Nihei, K.; Kurihara, M.; Kittaka, A.; Yamaguchi, K.; Fujishima, T.;
Konno, K.; Miyata, N.; Takayama, H. J. Org. Chem. 2001, 66, 8760; (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; (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, 13, 3503.
8. (a) Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.; Kurihara, M.;
Takayama, H. Org. Lett. 2000, 2, 2619; (b) Saito, N.; Suhara, Y.; Kurihara, M.;
Fujishima, T.; Honzawa, S.; Takayanagi, H.; Kozono, T.; Matsumoto, M.; Ohmori,
M.; Miyata, N.; Takayama, H.; Kittaka, A. J. Org. Chem. 2004, 69, 7463.
9.0 mL/min, CH3CN/H2O = 85:15). ½a D20
þ 56:1 (c 1.21, CHCl3); UV
ꢁ
(EtOH) kmax 267.0 nm, kmin 228.4 nm; 1H NMR (400 MHz, CDCl3)
d 0.54 (3H, s), 0.93 (3H, d, J = 5.9 Hz), 0.96 (3H, t, J = 7.0 Hz), 1.05
(1H, m), 1.21 (6H, s), 1.24–1.72 (17H, m), 1.89 (1H, m), 1.92–2.02
(2H, m), 2.17–2.32 (3H, m), 2.69 (1H, dd, J = 4.4, 13.4 Hz), 2.83
(1H, m), 3.34 (1H, dd, J = 3.3, 7.4 Hz), 3.50 (1H, dt, J =9.3, 6.6 Hz),
3.63 (1H, dt, J = 6.7, 9.3 Hz), 4.05 (1H, ddd, J = 4.6, 7.7, 8.6 Hz),
4.41 (1H, d, J = 2.9 Hz), 5.10 (1H, d, J = 1.3 Hz), 5.40 (1H, d,
J = 1.3 Hz), 6.03 (1H, d, J = 11.2 Hz), 6.42 (1H, d, J = 11.2 Hz); 13C
NMR (100 MHz, CDCl3) d 10.6, 12.1, 18.8, 20.8, 22.2, 23.2, 23.5,
27.7, 29.1, 29.2, 29.4, 36.1, 36.4, 40.5, 40.7, 44.4, 45.9, 56.4, 56.6,
68.3, 71.1, 71.7, 71.8, 84.3, 116.3, 117.2, 125.5, 131.6, 143.5,
144.3; EIMS m/z 474 (M+); HREIMS calcd for C30H50O4 (M+)
474.3710, found 474.3701.
5.2. Metabolism
The activity of CYP24A1 towards C3O1 was measured using the
membrane fraction prepared from the recombinant Escherichia coli
cells expressing human CYP24A1 or rat CYP24A1.14a The reconsti-
tuted system contains 0.02
1.0 M of adrenodoxin (ADX), 0.1
reductase (ADR), 4.0 M of C3O1, 0.5 mM of NADPH, 100 mM
l
M of human CYP24A1 or rat CYP24A1,
9. For our 2a-substituted 19-norvitamin D3 analogues, see: Ono, K.; Yoshida, A.;
l
lM of NADPH–adrenodoxin
Saito, N.; Fujishima, T.; Honzawa, S.; Suhara, Y.; Kishimoto, S.; Sugiura, T.;
Waku, K.; Takayama, H.; Kittaka, A. J. Org. Chem. 2003, 68, 7407.
l
Tris–HCl (pH 7.4) and 1 mM EDTA. The reaction was initiated by
addition of NADPH. After 60 min of incubation at 37 °C, the reac-
tion mixture was extracted with 4 vol. of CHCl3–CH3OH (3:1).
The organic phase was recovered and dried up in vacuo. The resul-
tant residue was dissolved in CH3CN and applied to HPLC under the
10. For VDR antagonists, see: (a) Saito, N.; Kittaka, A. ChemBioChem. 2006, 7, 1478;
(b) Saito, N.; Matsunaga, T.; Saito, H.; Anzai, M.; Takenouchi, K.; Miura, D.;
Namekawa, J.; Ishizuka, S.; Kittaka, A. J. Med. Chem. 2006, 49, 7063; (c)
Fujishima, T.; Kojima, Y.; Azumaya, I.; Kittaka, A.; Takayama, H. Bioorg. Med.
Chem. 2003, 11, 3621.
11. Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.; Takayama, H.; Rochel, N.;
Moras, D. J. Med. Chem. 2006, 49, 5199.
following conditions: column, YMC-Pack ODS-AM (5 lm)
12. (a) Miyamoto, K.; Murayama, E.; Ochi, K.; Watanabe, H.; Kubodera, N. Chem.
Pharm. Bull. 1993, 41, 1111; (b) Matsumoto, T.; Miki, T.; Hagino, H.; Sugimoto,
T.; Okamoto, S.; Hirata, T.; Yanagisawa, Y.; Hayashi, Y.; Fukunaga, M.; Shiraki,
M.; Nakamura, T. J. Clin. Endocrinol. Metab. 2005, 90, 5031; (c) Tsugawa, N.;
Nakagawa, K.; Kurobe, M.; Ono, Y.; Kubodera, N.; Ozono, K.; Okano, T. Biol.
Pharm. Bull. 2000, 23, 66; (d) Ono, Y.; Watanabe, H.; Shiraishi, A.; Takeda, S.;
Higuchi, Y.; Sato, K.; Tsugawa, N.; Okano, T.; Kobayashi, T.; Kubodera, N. Chem.
Pharm. Bull. 1997, 45, 1626; (e) Ono, Y.; Watanabe, H.; Kawase, A.; Kubodera, N.
Bioorg. Med. Chem. Lett. 1994, 4, 1523; For 19-nor and 2-(4-hydroxybutyl)
analogs of ED-71, see: (f) Sicinsky, R. F.; Perlman, K. L.; DeLuca, H. F. J. Med.
Chem. 1994, 37, 3730; (g) Posner, G. H.; Johnson, N. J. Org. Chem. 1994, 59, 7855.
(4.6 mm ꢃ 300 mm) (YMC Co., Kyoto, Japan); UV detection,
265 nm; flow-rate, 1.0 mL minꢀ1; column temperature, 40 °C; mo-
bile phase, CH3CN: a linear gradient of 20–100% CH3CN aqueous
solution per 25 min and 100% CH3CN for 12 min.
5.2.1. LC–MS analysis of the metabolites
The metabolite M3 and M6 produced by human CYP24A1 was
isolated from HPLC effluents, and subjected to mass spectrometric
analysis using a Finnegan Mat TSQ-70 with atmospheric pressure
chemical ionization, positive mode, as described previously.14
The conditions of LC were described below: column, reverse phase
Synthesis and biological activity of 2b-butoxy-1
a,25-dihydroxyvitamin D3
have been described in Refs. 12c,d..
13. For the crystal structure of DBP–25-hydroxyvitamin D3 complex, see:
Verboven, C.; Rabijns, A.; De Maeyer, M.; Van Baelen, H.; Bouillon, R.; De
Ranter, C. Nat. Struct. Biol. 2002, 9, 131.
14. (a) Abe, D.; Sakaki, T.; Kusudo, T.; Kittaka, A.; Saito, N.; Suhara, Y.; Fujishima, T.;
Takayama, H.; Hamamoto, H.; Kamakura, M.; Ohta, M.; Inouye, K. Drug Metab.
Dispos. 2005, 33, 778; (b) Kusudo, T.; Sakaki, T.; Abe, D.; Fujishima, T.; Kittaka,
A.; Takayama, H.; Hatakeyama, H.; Ohta, M.; Inouye, K. Biochem. Biophys. Res.
Commun. 2004, 321, 774.
ODS column (
l
Bondapak C18, 5
l
m, Waters) (6 mm ꢃ 150 mm);
mobile phase, 80% CH3OH aqueous solution per 25 min; flow-rate,
1.0 mL minꢀ1; UV detection, 265 nm.
Acknowledgments
15. Wiggins, L. S. Methods Carbohydr. Chem. 1963, 2, 188.
16. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114, 9836.
We are grateful to Ms. Junko Shimode and Ms. Akiko Tonoki
(Teikyo University) for the spectroscopic measurements. This work