Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues having a spiro-oxetane fused at the C2 position in the A-ring

Article abstract of DOI:10.1016/j.bmc.2013.06.032

Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D₃ (3a-d) bearing a spiro-oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d), respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a beneficial effect on the VDR affinity.

Full text of DOI:10.1016/j.bmc.2013.06.032

Bioorganic & Medicinal Chemistry 21 (2013) 5209–5217
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues
having a spiro-oxetane fused at the C2 position in the A-ring
⇑
Toshie Fujishima , Takato Nozaki, Tsutomu Suenaga
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Sanuki, Kagawa 769-2193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D3 (3a–d) bearing a spiro-
oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent
manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d),
respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary
biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that
the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a
beneficial effect on the VDR affinity.
Received 26 May 2013
Revised 11 June 2013
Accepted 12 June 2013
Available online 20 June 2013
Keywords:
Vitamin
Hormone
Nuclear receptor
Oxetane
Ó 2013 Elsevier Ltd. All rights reserved.
Steroid
1. Introduction
complex and the transcriptional machinery, can be influenced by
modifications to the ligands, particularly to the A-ring of 1.
The hormonally active form of vitamin D3, 1
a
,25-dihydroxyvi-
The binding mode of 1 to the receptor, as well as the binding
modes of several other active VDR ligands, have been solved by
X-ray crystallography, with the results indicating that all three of
the hydroxy groups in 1, as well as in the active ligands, were
recognised by the same two amino acid residues via hydrogen
bonding interactions.2 The X-ray crystal structure of the VDR com-
plexed with 1 also revealed the presence of a hydrophilic region in
the vicinity of the A-ring that was capable of accommodating bulky
substituents at the C2 position of the A-ring as in the seco-ste-
roids.2c Several groups, including our own, found a series of impor-
tant A-ring analogues with elevated affinities for VDR via the
introduction of a substituent with hydrogen bonding capability.3
In addition, it has been suggested that this cavity consists of a sleek
hydrophobic area surrounded by lipophilic amino acid residues
such as Phe-150, Leu-233 and Tyr-236, in the close vicinity of the
C2 position in the A-ring.3d In this regard, we generated a series
of the C2-methyl analogues (2a–c)3a–c with unique activity pro-
tamin D3 (1: Fig. 1), exhibits a broad range of biological activities,
including cell differentiation-inducing, anti-proliferative and apop-
tosis-stimulating activities in many normal and malignant cells, in
addition to its classical role in calcium-phosphorus homeostasis.1
The vitamin D receptor (VDR) is generally considered to be the ma-
jor molecular target of vitamin D3 and its active metabolites. The
VDR itself belongs to the nuclear receptor super-family, and be-
haves as a ligand-inducible transcriptional factor. Further insights
into the structure–function relationships of vitamin D and its
metabolites are needed to allow for our understanding of these
beneficial effects on cells in disease states to be developed into
therapeutic agents against cancer, psoriasis and immune disorders,
as well as developing a deeper understanding of the subtype-free
VDR and how it could be used to deliver a variety of different
actions.
The binding of the seco-steroid hormone 1 to the ligand-binding
domain (LBD) of VDR triggers a conformational change in the
trans-activation domain of the receptor (AF-2) that initiates an en-
tire sequence of reactions resulting in a genomic response.1 The
specific interactions of modified ligands with the LBD have been
the subject of considerable levels of attention from chemists, be-
cause the interface provided by the VDR for the binding of tran-
scriptional co-activators, as well as the overall stability of the
files, including 2,2-dimethyl-1a,25-dihydroxyvitamin D3 (2c:
Fig. 1),3e bearing substituents that could access this hydrophobic
area.
Four-membered cyclic ethers, which are otherwise known as
oxetanes, can act as surrogates not only for geminal dimethyl
groups, but also for ‘deeper’ carbonyl groups with high hydrogen
bonding avidity.4 The nominal analogy of oxetanes to carbonyl
groups has been demonstrated, and the former can be beneficial
when a larger volume occupancy and deeper oxygen placement
are required in the receptor pocket.4b The substitution of a geminal
⇑
Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181.
E-mail address: tofu@kph.bunri-u.ac.jp (T. Fujishima).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.032

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T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
conducted according to the convergent method pioneered by Trost
et al.5 The A-ring enyne precursors (13a,b) were synthesised from
pentaerythritol (4), according to the route depicted in the scheme
(Scheme 1). The protection of one of the four hydroxy groups in
pentaerythritol (4) with a p-methoxybenzyl (PMB) group gave
the triol 5 in 75% yield.6 Conversion of 5 to the mono-tosylate 6,
followed by treatment with sodium hydride, provided the key oxe-
tane intermediate 7 in excellent yield.7 Oxidation of the primary
alcohol in 7 using the Swern procedure gave the corresponding
aldehyde 8 in 99% yield. Subsequent treatment of the aldehyde
with vinylmagnesium bromide afforded an enantiomeric mixture
of the allylic alcohols ( )-9 in 81% yield. The protection of the sec-
ondary alcohol in ( )-9 with a tert-butyldimethylsilyl (TBS) ether
was achieved under standard conditions using TBS chloride (TBSCl)
and imidazole to give ( )-10 in 96% yield. Subsequent deprotection
of the PMB group with 2,3-dichloro-5,6-dicyano-p-benzoquinone
(DDQ) gave the primary alcohol ( )-11 in 95% yield without affect-
ing the oxetane structure. The primary alcohol ( )-11 was then
oxidised using the Swern procedure to afford the corresponding
aldehyde in 93% yield, which was reacted with allenylmagnesium
bromide to give a diastereomeric mixture of homopropargyl alco-
hols in 95% yield, as an approximately 45:55 mixture of the diaste-
reomers in favour of the 1,3-syn-diols. Following the removal of the
TBS protective group with tetra-n-butylammonium fluoride
(TBAF), it became possible to separate the resulting mixture of
the requisite A-ring enyne synthons ( )-12a and ( )-12b by silica
gel column chromatography. The relative stereochemistries of
the 1,3-diols in the different enantiomeric mixtures were
determined by 13C NMR analysis of the corresponding acetonides
(Scheme 2).8 A variety of different conditions were evaluated for
the protection of the diols, including TBSCl-imidazole, TBS
trifluoromethanesulfonate (triflate)-2,6-lutidine, and N-methyl-N-
(tert-butyldimethylsilyl)trifluoroacetamide.9 Unfortunately, how-
ever, these methods failed to provide the desired products in good
yield. The protection of the diols in ( )-12a and ( )-12b as the
corresponding TBS groups was ultimately achieved using tert-
butyldimethylsilyloxy N-phenylbenzimidate (TBS-BEZA)10 in the
presence of catalytic pyridinium trifluoromethanesulfonate
(triflate) to afford the requisite A-ring precursors ( )-13a and
( )-13b, respectively, in 80% yield in both cases.
25
H
H
OH
OH
H
H
3
1
2
HO
OH
HO
OH
R2 R1
O
1: R1 = R2 = H
2a: R1 = CH3, R2 = H
2b: R1 = H, R2 = CH3
2c: R1 = R2 = CH3
3a
Figure 1. Chemical structures of 1a,25-dihydroxyvitamin D3 (1), the C2-methyl-
introduced vitamin D3 (2a–c) and novel vitamin D analogue (3a) having a spiro-
oxetane fused at the C2 position in the A-ring.
dimethyl group with an oxetane should lead to an increase in
aqueous solubility, as well as a reduction in the rate of metabolic
degradation in the majority of cases,4b and changes of this type
could be advantageous for VDR ligands. In view of our results with
the C2-methyl analogues, it was envisaged that the incorporation
of a spiro-oxetane at the C2 position in the A-ring of 1 would be
of interest to generate novel ligands capable of better filling the
hydrophilic cavity. Herein, we describe the synthesis of all four
possible A-ring stereoisomers incorporating a spiro-oxetane fused
at the C2 position (3a–d) as novel vitamin D analogues capable
of filling the hydrophilic cavity with an oxetane, combined with
beneficial effects of the C2-methyl substituent.
2. Results and discussion
2.1. Synthesis
The syntheses of these novel seco-steroid analogues bearing a
spiro-cyclic oxetane fused at the C2 position in the A-ring were
HO
HO
OH
PMBO
HO
OH
PMBO
TsO
OH
a
The coupling reactions of the protected A-ring enyne precursors
( )-13a and ( )-13b with the CD-ring portion 155 in the presence of
tetrakis(triphenylphosphine)palladium, followed by deprotection
with TBAF, proceeded smoothly to afford the novel seco-steroids
3a,b and 3c,d, respectively (Scheme 3). The presence of a spiro-
oxetane structure in the four A-ring stereoisomers (3a–d) was
b
OH
OH
OH
4
5
6
PMBO
PMBO
PMBO
c
d
e
OH
OR
O
O
O
O
O
a
( )-12a
(less polar)
( )-14a
Δδ = 0.6
7
8
9: R = H
10: R = TBS
f
O
O
δ 24.3
δ 24.9
HO
g
d, h, i
OTBS
O
RO
j
OR
RO
j
OR
a
O
O
12a: R = H
13a: R = TBS
O
( )-12b
(more polar)
( )-14b
11
12b: R = H
13b: R = TBS
O
O
Δδ = 10.4
δ 19.2
δ 29.6
Scheme 1. Reagents: (a) NaH; PMBCl, TBAI/DMF, 75%; (b) TsCl/pyridine, 63%; (c)
NaH/THF, 90%; (d) oxalyl chloride, DMSO, Et3N/CH2Cl2, 99% from 8, 93% from 11; (e)
vinylmagnesium bromide/toluene, 81%; (f) TBSCl, imidazole/CH2Cl2, 96%; (g) DDQ/
CH2Cl2, 95%; (h) allenylmagnesium bromide/ether, 95%; (i) TBAF/THF, 96%; (j) TBS-
BEZA, pyridinium triflate/CH2Cl2, 80% from 12a,b.
Scheme 2. Determination of relative stereochemistry of the 1,3-diols by 13C NMR
analysis. Reagents: (a) 2,2-dimethoxypropane, CSA, 82% from ( )-12a, 62% from ( )-
12b.

T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
5211
H
H
OH
OH
H
H
( )-13a
a, b
HO
OH
HO
OH
3b
3a
H
O
O
OH
H
H
H
OH
OH
15
Br
( )-13b
H
H
a, b
HO
OH
HO
OH
3d
3c
O
O
Scheme 3. Coupling reactions of the A-ring enynes with the CD-ring portion. Reagents: (a) (Ph3P)4Pd, Et3N/toluene; (b) TBAF/THF, 16–20% (two steps).
confirmed by 1H NMR analysis. The NMR spectra of the stereoiso-
Table 1
Vitamin D receptor binding affinities of the four A-ring stereoisomeric spiro-oxetane
mers showed four distinctive doublets with geminal coupling
constants in the range of 6.1–6.5 Hz, indicating the presence of a
slightly restricted four-membered ring. In the case of 3a, the vicinal
analogues (3a–d)a
Compounds
VDR affinityb
coupling constant of 6.9 Hz between the H(3
a and H(4b protons
1
2a
2b
2c
100
400c
13c
suggested that the introduction of the spiro-oxetane had not had
a significant effect on the conformational preference of the A-ring,
based on a comparison with the parent compound 1 and its 2,2-
dimethyl counterpart 2c.11 The absolute stereochemistry of the
1,3-diols in the A-ring was determined using the bis-MTPA meth-
ods that we developed previously (Fig. 2).12 The four stereoiso-
meric spiro-oxetane analogues were purified using a recycling
HPLC method to afford pure specimens for biological evaluation.
3d
3a (1R, 3R)
3b (1S, 3S)
3c (1R, 3S)
3d (1S, 3R)
5
0.001
0.04
0.002
a
b
c
The potency of 1a,25-dihydroxyvitamin D3 (1) was normalised as 100.
Bovine thymus vitamin D receptor.
Ref. 3a.
Ref. 3e.
d
2.2. Vitamin D receptor (VDR) binding affinity
The affinities of the novel compounds for the VDR were evalu-
ated using bovine thymus VDR. Table 1 provides a summary of
the relative VDR binding affinities of the spiro-oxetane analogues
(3a–d) in comparison with the natural hormone 1, the potency of
which was normalised to 100 by definition, as well as the
possessed the 1a- and 3b-hydroxy groups in the natural configura-
tion, showed the highest level of affinity toward the VDR. Further-
more, the replacement of the C2-gem-dimethyl group of 2c with
the oxetane in this case resulted in a slight increase in the affinity
toward the VDR. The other stereoisomers (3b–d) showed a reduced
affinity to the VDR relative to the parent A-ring stereoisomers of
the natural hormone 1. It is noteworthy that isomer 3d bearing
2a
-methyl-(2a)3a 2b-methyl-(2b)3a and 2,2-dimethyl analogues
(2c).3e Of the four stereoisomers (3a–d), isomer 3a, which
+ 0.103
- 0.025
- 0.041
+ 0.032
H
H
H
H
OH
OH
OH
OH
+ 0.057
+ 0.112
- 0.075
- 0.173
- 0.084
- 0.236
+ 0.073
+ 0.196
+ 0.107
- 0.104
- 0.115
- 0.053
+ 0.243
+ 0.034
+ 0.094
- 0.203
+ 0.150
- 0.071
- 0.191
+ 0.054
- 0.213
+ 0.193
- 0.007
+ 0.132
- 0.171
+ 0.544
- 0.430
+ 0.084
MTPAO
OMTPA
MTPAO
OMTPA
MTPAO
OMTPA
MTPAO
OMTPA
O
O
3a
3b
O
O
3c
3d
Figure 2. Determination of absolute configuration at the C1 and C3 positions of 3a–d by 1H NMR analysis of their bis-MTPA esters.12

5212
T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
the 1b- and 3b-hydroxy groups, which showed the weakest level of
binding to the VDR of the four A-ring stereoisomers,3b showed a
recognisable level of affinity. The introduction of an oxetane at
the C2 position as in 3d, particularly the oxygen atom, could there-
fore provide some degree of compensation for the unfavourable
interactions provided by the 1b-hydroxy group.
129.6, 159.4; HRMS (ESI+) m/z calcd for C13H20O5Na [M+Na]+
279.1203, found 279.1217.
4.3. 3-Hydroxy-2-hydroxymethyl-2-[(4-
methoxybenzyloxy)methyl]propyl 4-methylbenzenesulfonate
(6)
To a stirred solution of 5 (6.67 g, 26.0 mmol) in dry pyridine
(70 mL) was added p-toluenesulfonyl chloride (5.45 g, 28.6 mmol)
under an atmosphere of argon at 0 °C, and the resulting mixture
was stirred at room temperature for 2 h. After the addition of water
(25 mL) to the reaction mixture at 0 °C, the solvent was removed
under the reduced pressure to give a residue, which was dissolved
with CH2Cl2 (400 mL) before being washed with brine (70 mL). The
organic layer was dried over sodium sulfate and filtered. Evapora-
tion of the filtrate afforded a residue, from which 6 (6.72 g) was
separated by silica gel column chromatography (ethyl acetate/n-
hexane/methanol = 10:5:1) as a colourless oil in 63% yield.
Compound 6: 1H NMR (400 MHz, TMS, CDCl3) d 2.30 (2H, t,
J = 6.0 Hz), 2.45 (3H, s), 3.42 (2H, s), 3.63 (4H, d, J = 6.0 Hz), 3.81
(3H, s), 4.13 (2H, s), 4.37 (2H, s), 6.86 (2H, d, J = 8.4 Hz), 7.16 (2H,
d, J = 8.4 Hz), 7.34 (2H, d, J = 8.4 Hz), 7.78 (2H, d, J = 8.4 Hz); 13C
NMR (100 MHz, TMS, CDCl3) d 21.5, 44.9, 55.1, 62.9, 69.1, 69.7,
73.2, 113.7, 127.8, 129.0, 129.5, 129.8, 132.3, 144.9, 159.2, 145.1,
159.4; HRMS (ESI+) m/z calcd for C20H26NaO7S [M+Na]+ 433.1291,
found 433.1242.
3. Conclusions
In conclusion, we have designed and synthesised four novel
A-ring analogues of active vitamin D3 bearing an oxetane fused
at the C2 position. A convergent synthetic method using palladium
catalysis allowed us to reach the novel seco-steroids bearing a un-
ique spiro-oxetane structure (3a–d). The enyne precursors (13a,b)
required for the construction of the A-ring were prepared from
pentaerythritol according to an eleven-step procedure in excellent
overall yield. The introduction of a spiro-oxetane into the A-ring
resulted in an increase of the polarity and solubility properties of
the products compared with their geminal dimethyl counterparts.
Our concise synthetic route from pentaerythritol to the enyne pre-
cursors (13a,b) provided an effective demonstration that the oxe-
tane structure is considerably robust and tolerant of a variety of
synthetic conditions, which may be of additional importance to
any future work conducted in this area. Preliminary biological
evaluation of the novel analogues using bovine thymus VDR sug-
gested that the incorporation of a spiro-oxetane at the C2 position
instead of a gem-dimethyl group had a beneficial effect on the VDR
affinity.
4.4. 3-[(4-Methoxybenzyloxy)methyl]-oxetan-3-ylmethanol (7)
To a solution of 6 (4.50 g, 11.0 mmol) in dry THF (45 mL) was
added sodium hydride (660 mg, 60% in mineral oil, 16.5 mmol) in
a potion-wise manner with stirring under an atmosphere of argon
at 0 °C, and the resulting mixture was stirred for 16 h at room tem-
perature. After the addition of water (30 mL) to the reaction mix-
ture at 0 °C, the whole was extracted with ethyl acetate
(200 mL Â 3). The combined organic layer was washed with brine,
dried over sodium sulfate and filtered. Evaporation of the filtrate
afforded a residue, from which 7 (2.36 g) was separated by silica
gel column chromatography (ethyl acetate/n-hexane = 2:1) as a
colourless oil in 90% yield.
4. Experimental section
4.1. General information
NMR spectra were recorded on a Bruker AVANCE-400, a Bruker
AVANCE-700, or a JEOL ECX-400 spectrometer. The chemical shifts
have been expressed in ppm relative to tetramethylsilane (TMS).
Mass spectra and electrosprey ionisation high-resolution mass
spectra (ESI-HRMS) were recorded on a Bruker micrOTOF. Ultravi-
olet spectra were recorded with a Jasco V-660 spectrophotometer.
Recycling preparative HPLC was performed on a Shimadzu CBM-
20A equipped with an LC-6AD pump and an SPD-M20A diode array
detector.
Compound 7: 1H NMR (400 MHz, TMS, CDCl3) d 2.30 (1H, t,
J = 5.6 Hz), 3.78 (2H, s), 3.81 (3H, s), 3.93 (2H, d, J = 5.6 Hz), 4.41
(2H, d, J = 6.0 Hz), 4.48 (2H, d, J = 6.0 Hz), 4.49 (2H, s), 6.90 (2H,
d, J = 8.8 Hz), 7.22 (2H, d, J = 8.8 Hz); 13C NMR (100 MHz, TMS,
CDCl3) d 43.9, 55.2, 66.3, 73.3, 73.4, 76.5, 113.8, 129.3, 129.6,
159.4; HRMS (ESI+) m/z calcd for C13H18O4Na [M+Na]+, 261.1097,
found 261.1080.
4.2. 2-Hydroxymethyl-2-(4-methoxybenzyloxy)methyl-1,3-
propanediol (5)
4.5. 3-[(4-Methoxybenzyloxy)methyl]-oxetane-3-carbaldehyde
(8)
To a solution of pentaerythritol 4 (3.40 g, 25.0 mmol) and
tetra-n-buthylammonium iodide (TBAI) (250 mg, 0.68 mmol) in
dry N,N-dimethylformamide (DMF) (37.5 mL) was added sodium
hydride (405 mg, 60% in mineral oil, 28.1 mmol) in a potion-wise
manner with stirring under an atmosphere of argon at 0 °C. Upon
completion of the addition, the stirring was continued for 1 h at
A solution of oxalyl chloride (0.42 mL, 0.61 g, 4.8 mmol) in CH2-
Cl2 (5 mL) was added to a stirred solution of dimethylsulfoxide
(DMSO) (0.68 mL, 9.7 mmol) in dry CH2Cl2 (5 mL) under an atmo-
sphere of argon at À78 °C, and the mixture was stirred at the same
temperature for 30 min. The resulting mixture was transferred to a
solution of 7 (500 mg, 2.1 mmol) in CH2Cl2 (10 mL), and the reac-
tion mixture was stirred at À78 °C for 40 min. Subsequently, neat
triethylamine (2.6 mL, 18.5 mmol) was added to the reaction mix-
ture followed by stirring for 50 min, while the temperature was
elevated from À78 to 0 °C. The reaction was quenched by the addi-
tion of water (5 mL) and the whole was extracted with ether
(100 mL Â 3). The combined organic layer was washed with brine
(5 mL), dried over sodium sulfate and filtered. Evaporation of the
filtrate afforded a residue, from which 8 (490 mg) was separated
by silica gel column chromatography (ethyl acetate/n-hex-
ane = 1:2) as a colourless oil in 99% yield.
room temperature.
A solution of p-methoxybenzyl chloride
(PMBCl) (1.06 g, 6.76 mmol) in dry DMF (1.25 mL) was then added
to the reaction mixture with stirring at 0 °C, and the resulting mix-
ture was stirred overnight at room temperature. After the addition
of methanol (3.0 mL) to the reaction mixture at 0 °C, the solvent
was removed under the reduced pressure to give a residue, from
which 5 (1.30 g) was separated by silica gel column chromatogra-
phy (ethyl acetate/n-hexane/methanol = 10:5:1) as a colourless so-
lid in 75% yield.
Compound 5: 1H NMR (400 MHz, TMS, CDCl3) d 2.53 (3H, t,
J = 5.6 Hz), 3.48 (2H, s), 3.71 (6H, d, J = 5.6 Hz), 3.80 (3H, s), 4.44
(2H, s), 6.88 (2H, d, J = 8.8 Hz), 7.22 (2H, d, J = 8.8 Hz); 13C NMR
(100 MHz, TMS, CDCl3) d 44.9, 55.3, 65.0, 72.0, 73.5, 113.9, 129.3,
Compound 8: 1H NMR (400 MHz, TMS, CDCl3) d 3.80 (3H, s), 3.89
(2H, s), 4.50 (2H, d, J = 8.0 Hz), 4.52 (2H, s), 4.79 (2H, d, J = 8.0 Hz),

T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
5213
6.87 (2H, d, J = 8.8 Hz), 7.22 (2H, d, J = 8.8 Hz), 9.85 (1H, s); 13C
NMR (100 MHz, TMS, CDCl3) d 53.1, 53.3, 69.6, 73.26, 73.31,
113.9, 129.4, 159.4, 199.9; HRMS (ESI+) m/z calcd for C13H16O4Na
[M+Na]+ 259.0940, found 259.0946.
chromatography (ethyl acetate/n-hexane = 1:3) as a colourless oil
in 95% yield.
Compound 11: 1H NMR (400 MHz, TMS, CDCl3) d 0.07 (3H, s),
0.12 (3H, s), 2.79 (1H, dd, J = 8.0, 2.8 Hz), 3.93 (1H, dd, J = 11.1,
8.0 Hz), 4.02 (1H, ddd, J = 11.1, 2.7, 1.0 Hz), 4.19 (1H, d,
J = 6.5 Hz), 4.37 (1H, d, J = 6.5 Hz), 4.57 (1H, dd, J = 6.1, 1.0 Hz),
4.64 (1H, d, J = 6.2 Hz), 4.66 (1H, br d, J = 7.0 Hz), 5.26 (1H, ddd,
J = 10.4, 1.4, 1.2 Hz), 5.32 (1H, dt, J = 17.2, 1.4 Hz), 5.84 (1H, ddd,
J = 17.2, 10.4, 7.0 Hz); 13C NMR (100 MHz, TMS, CDCl3) d À5.1,
À4.1, 18.0, 25.7, 46.9, 65.5, 74.9, 77.6, 79.2, 117.7, 136.1; HRMS
(ESI+) m/z calcd for C13H26NaO3Si [M+Na]+ 281.1543, found
281.1543.
4.6. 1-{3-(4-Methoxybenzyloxy)methyl-oxetan-3-yl}prop-2-en-
1-ol (9)
A solution of vinylmagnesium bromide (14% in THF, 0.29 mL,
0.29 mmol) was added to
a stirred solution of 8 (64 mg,
0.27 mmol) in dry toluene (5 mL) at À78 °C in a drop-wise manner
under an atmosphere of argon. After having been stirred for 35 min
at the same temperature, the reaction was quenched by the addi-
tion of saturated aqueous NH4Cl. The resultant mixture was ex-
tracted with ethyl acetate (30 mL Â 3), and the combined organic
layer was washed with brine, dried over magnesium sulfate and fil-
tered. Evaporation of the filtrate afforded a residue, from which 9
(58 mg) was separated by silica gel column chromatography (ethyl
acetate/n-hexane = 1:2) as a colourless oil in 81% yield.
4.9. (1RS)-{3-[1-(RS)-Hydroxyprop-2-en-1-yl]oxetan-3-yl}but-3-
yn-1-ol (12a:1,3-anti-diol) and (1SR)-{3-[1-(RS)-hydroxyprop-2-
en-1-yl]oxetan-3-yl}but-3-yn-1-ol (12b:1,3-syn-diol)
A solution of oxalyl chloride (0.84 mL, 1.24 g, 9.80 mmol) in
CH2Cl2 (15 mL) was added to a solution of DMSO (1.39 mL,
19.6 mmol) in dry CH2Cl2 (15 mL) under an atmosphere of argon
at À78 °C, and the mixture was stirred at the same temperature
for 30 min. The resulting mixture was transferred to a solution of
11 (1.10 g, 4.26 mmol) in dry CH2Cl2 (30 mL), and the reaction mix-
ture was stirred at À78 °C for 40 min. Subsequently, neat triethyl-
amine (5.2 mL, 37.5 mmol) was added to the reaction mixture
followed by stirring for 1 h, while the temperature was elevated
from À78–0 °C. The reaction was quenched by the addition of
water (15 mL) and the whole was extracted with ether
(80 mL Â 3). The combined organic layer was washed with brine,
dried over sodium sulfate and filtered. Evaporation of the filtrate
afforded a residue, from which the desired aldehyde (1.03 g) was
separated by silica gel column chromatography (ethyl acetate/n-
hexane = 1:9) as a colourless oil in 93% yield.
3-[1-(tert-Butyldimethylsilyloxy)prop-2-en-1-yl]oxetane-3-
carbaldehydee1H NMR (400 MHz, TMS, CDCl3) d 0.07 (3H, s), 0.10
(3H, s), 0.88 (9H, s), 4.51 (1H, d, J = 6.5 Hz), 4.58 (1H, d, J = 6.3 Hz),
4.67 (1H, br d, J = 7.0 Hz), 4.731 (1H, d, J = 6.3 Hz), 4.734 (1H, d,
J = 6.5 Hz), 5.28 (1H, ddd, J = 10.4, 1.2, 1.1 Hz), 5.35 (1H, dt,
J = 17.1, 1.3 Hz), 5.84 (1H, ddd, J = 17.1, 10.4, 7.0 Hz), 9.88 (1H, s);
13C NMR (100 MHz, TMS, CDCl3) d À5.1, À4.0, 18.0, 25.6, 57.0,
72.1, 73.7, 75.3, 118.4, 135.6, 200.6; HRMS (ESI+) m/z calcd for C13-
H26NaO3Si [M+Na]+ 281.1543, found 281.1552.
To a stirred solution of the aldehyde (1.03 g, 4.02 mmol) dis-
solved in dry ether (40 mL) was added a solution of allenylmagne-
sium bromide (ca. 2 M in ether, 12 mL, 24 mmol) at À78 °C under
an atmosphere of argon. After having been stirred for 30 min at
the same temperature, the reaction was quenched by the addition
of saturated aqueous NH4Cl (20 mL). The resultant mixture was ex-
tracted with ethyl acetate (50 mL Â 3), and the combined organic
layer was washed with brine, dried over magnesium sulfate and fil-
tered. Evaporation of the filtrate afforded a residue, from which a
diastereomeric mixture of homopropargyl alchohols (1.13 g) was
obtained by silica gel column chromatography (ethyl acetate/n-hex-
ane = 1:5) as a colourless oil in 95% yield. The ratio between the min-
or and major isomers was deduced to be 45:55 by 1H NMR analysis
using the protons at 4.18 ppm and 3.98 ppm, respectively.
Compound 9: 1H NMR (400 MHz, TMS, CDCl3) d 3.78 (1H, d,
J = 8.8 Hz), 3.81 (3H, s), 3.82 (1H, d, J = 8.8 Hz), 4.25 (1H, d,
J = 6.4 Hz), 4.47 (2H, s), 4.50 (3H, complex), 4.68 (1H, d,
J = 6.2 Hz), 5.25 (1H, dt, J = 10.5, 1.5 Hz), 5.37 (1H, dt, J = 17.1,
1.5 Hz), 5.87 (1H, ddd, J = 17.1, 10.5, 6.0 Hz), 6.89 (2H, m), 7.23
(2H, m); 13C NMR (100 MHz, TMS, CDCl3) d 45.8, 55.3, 73.2, 73.5,
75.2, 76.3, 77.4, 113.9, 117.1, 129.3, 129.5, 136.3, 159.5; HRMS
(ESI+) m/z calcd for C15H20O4Na [M + Na]+ 287.1254, found
287.1254.
4.7. 3-{[1-(tert-Butyldimethylsilyl)oxy]prop-2-enyl}-3-[(4-
methoxybenzyl)oxy]methyl-oxetane (10)
To a stirred solution of 9 (1.60 g, 6.1 mmol) and imidazole
(1.24 g, 18.2 mmol) in dry CH2Cl2 (60 mL) was added tert-butyldi-
methylsilyl chloride (TBSCl) (1.82 g, 12.1 mmol) at 0 °C under an
atmosphere of argon, and the resulting mixture was stirred at
room temperature for 9 h. Additional charges of imidazole
(1.24 g, 18.2 mmol) and TBSCl (1.82 g, 12.1 mmol) were then made
to the reaction, and the resulting mixture was stirred at room tem-
perature for 13 h. The reaction mixture was then diluted with ethyl
acetate (300 mL) and was washed successively with water (30 mL),
and brine (30 mL). The organic layer was dried over sodium sulfate,
filtered and concentrated. The residue was purified by silica gel
column chromatography (ethyl acetate/n-hexane = 1:2) to give 10
(2.20 g) as a colourless oil in 96% yield.
Compound 10: 1H NMR (400 MHz, TMS, CDCl3) d 0.03 (3H, s),
0.06 (3H, s), 0.88 (9H, s), 3.55 (1H, d, J = 9.2 Hz), 3.71 (1H, d,
J = 9.2 Hz), 3.81 (3H, s), 4.32 (1H, d, J = 5.8 Hz), 4.35 (1H, br d,
J = 7.4 Hz), 4.42 (1H, d, J = 5.9 Hz), 4.44 (1H, d, J = 11.7 Hz), 4.50
(1H, d, J = 11.7 Hz), 4.54 (1H, d, J = 5.8 Hz), 4.59 (1H, d, J = 5.9 Hz),
5.17 (1H, ddd, J = 10.3, 1.7, 0.9 Hz), 5.24 (1H, ddd, J = 17.2, 1.7,
1.2 Hz), 5.82 (1H, ddd, J = 17.2, 10.3, 7.4 Hz), 6.88 (2H, m), 7.26
(2H, m); 13C NMR (100 MHz, TMS, CDCl3) d À5.0, À3.9, 18.1, 25.8,
47.6, 55.3, 71.2, 73.1, 74.4, 75.1, 75.3, 113.8, 117.1, 129.3, 130.3,
137.2, 159.2; HRMS (ESI+) m/z calcd for C21H34NaO4Si [M+Na]+
401.2119, found 401.2074.
4.8. 3-{[1-(tert-Butyldimethylsilyl)oxy]prop-2-enyl}-3-
hydroxymethyl-oxetane (11)
A mixture of alcohols: 1H NMR (400 MHz, TMS, CDCl3) (minor
isomer) d 0.10 (3H, s), 0.14 (3H, s), 0.88 (9H, s), 2.06 (1H, t,
J = 2.6 Hz), 2.68 (2H, dd, J = 6.7, 2.7 Hz), 3.73 (1H, d, J = 3.3 Hz),
4.18 (1H, dt, J = 6.7, 3.3 Hz), 4.42 (1H, d, J = 6.7 Hz), 4.48 (1H, d,
J = 6.4 Hz), 4.67 (1H, d, J = 6.4 Hz), 4.68 (1H, d, J = 6.7 Hz), 4.69
(1H, br d, J = 7.4 Hz), 5.33 (1H, ddd, J = 10.4, 1.2, 1.0 Hz), 5.36 (1H,
dt, J = 17.2, 1.2 Hz), 5.94 (1H, ddd, J = 17.2, 10.4, 7.4 Hz); (major iso-
mer) d 0.07 (3H, s), 0.11 (3H, s), 0.90 (9H, s), 2.07 (1H, t, J = 2.6 Hz),
2.50 (1H, ddd, J = 16.9, 8.7, 2.7 Hz), 2.64 (1H, d, J = 4.7 Hz), 2.70 (1H,
ddd, J = 16.9, 4.1, 2.7 Hz), 3.98 (1H, dt, J = 8.8, 4.4 Hz), 4.40 (1H, d,
J = 7.0 Hz), 4.46 (1H, d, J = 6.4 Hz), 4.53 (1H, d, J = 7.0 Hz), 4.56
To a stirred solution of 10 (160 mg, 0.42 mmol) in a mixture of
CH2Cl2 (5 mL) and water (0.5 mL) was added 2,3-dichloro-5,6-dicy-
ano-p-benzoquinone (DDQ) (143 mg, 0.63 mmol) at 0 °C, and the
resulting mixture was stirred at room temperature for 50 min.
The reaction mixture was then diluted with ether (50 mL), and
was washed successively with saturated aqueous NaHCO3
(5 mL Â 2), and then with brine (5 mL). The organic layer was dried
over magnesium sulfate, filtered and concentrated to afford a res-
idue, from which 11 (102 mg) was separated by silica gel column

5214
T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
(1H, br d, J = 7.2 Hz), 4.63 (1H, d, J = 6.3 Hz), 5.26 (1H, ddd, J = 10.4,
1.4, 1.2 Hz), 5.33 (1H, dt, J = 17.1, 1.4 Hz), 5.89 (1H, ddd, J = 17.1,
10.4, 7.2 Hz).
atmosphere of argon, and the resulting mixture was stirred at
the same temperature for 1.5 h. Water (1.5 mL) was then added
to the reaction mixture, and the whole was extracted with ether
(10 mL Â 3). The combined organic layer was washed with brine
(5 mL), dried over sodium sulfate, and filtered. The filtrate was con-
centrated under the reduced pressure to give a residue, from which
13b (204 mg) was separated by silica gel column chromatography
(ethyl acetate/n-hexane = 1:49) as a colourless oil in 80% yield.
Compound 13b: 1H NMR (400 MHz, TMS, CDCl3) d 0.06 (3H, s),
0.09 (3H, s), 0.10 (3H, s), 0.14 (3H, s), 0.90 (9H, s), 0.93 (9H, s),
1.93 (1H, t, J = 2.7 Hz), 2.45 (1H, ddd, J = 17.2, 4.8, 2.7 Hz), 2.52
(1H, ddd, J = 17.2, 5.1, 2.8 Hz), 4.06 (1H, t, J = 4.9 Hz), 4.33 (1H, d,
J = 6.1 Hz), 4.44 (1H, br d, J = 7.1 Hz), 4.46 (1H, d, J = 5.9 Hz), 4.62
(1H, d, J = 6.0 Hz), 4.80 (1H, d, J = 6.1 Hz), 5.26 (1H, br d,
J = 10.3 Hz), 5.31 (1H, br d, J = 17.1 Hz), 6.03 (1H, ddd, J = 17.1,
10.3, 7.5 Hz); 13C NMR (100 MHz, TMS, CDCl3) d À4.8, À4.0, À3.6,
18.2, 23.8, 25.8, 52.0, 70.6, 70.8, 73.6, 74.2, 74.9, 81.3, 117.4,
137.5; HRMS (ESI+) m/z calcd for C22H42NaO3Si2 [M+Na]+
433.2565, found 433.2521.
A solution of tetra-n-buthylammonium fluoride (TBAF) (1.0 M
in THF, 1.4 mL, 1.4 mmol) was added to a stirred solution of the
diastereomeric mixture as described above (340 mg, 1.2 mmol) in
THF (5 mL) at 0 °C in a drop-wise manner, and the resulting mix-
ture was stirred for 1 h at room temperature. Brine was then added
to the reaction mixture and the whole was extracted with ethyl
acetate (30 mL Â 3). The combined organic layer was washed with
brine (10 mL), dried over sodium sulfate, and filtered. Evaporation
of the filtrate afforded a residue, from which 12a (89 mg, 42%, less
polar) and 12b (124 mg, 57%, more polar) were obtained by silica
gel column chromatography (ethyl acetate/n-hexane = 1:3) both
as colourless oils in a combined yield of 99%.
Compound 12a: 1H NMR (400 MHz, TMS, CDCl3) d 2.07 (1H, t,
J = 2.8 Hz), 2.60 (1H, ddd, J = 16.8, 6.1, 2.7 Hz), 2.70 (1H, ddd,
J = 16.8, 7.9, 2.8 Hz), 2.71 (1H, complex), 3.12 (1H, br d,
J = 6.0 Hz), 4.29 (1H, dt, J = 7.8, 6.1 Hz), 4.42 (1H, d, J = 6.9 Hz),
4.59 (2H, m), 4.61 (1H, d, J = 7.0 Hz), 4.63 (1H, m), 5.42 (1H, dt,
J = 10.4, 1.3 Hz), 5.48 (1H, dt, J = 17.1, 1.3 Hz), 6.20 (1H, ddd,
J = 17.1, 10.4, 6.4 Hz); 13C NMR (100 MHz, TMS, CDCl3) d 23.1,
48.3, 71.2, 73.6, 74.8, 75.3, 75.7, 80.1, 118.6, 135.9; HRMS (ESI+)
m/z calcd for C10H14NaO3 [M+Na]+ 205.0835, found 205.0816.
Compound 12b: 1H NMR (400 MHz, TMS, CDCl3) d 2.09 (1H, t,
J = 2.6 Hz), 2.15 (1H, d, J = 3.5 Hz), 2.61 (1H, ddd, J = 16.9, 8.2,
2.6 Hz), 2.62 (1H, d, J = 4.1 Hz), 2.69 (1H, ddd, J = 16.9, 4.3,
2.6 Hz), 4.10 (1H, dt, J = 8.4, 4.3 Hz), 4.49 (1H, d, J = 6.6 Hz), 4.53
(1H, m), 4.57 (1H, d, J = 6.6 Hz), 4.63 (1H, d, J = 6.6 Hz), 4.66 (1H,
d, J = 6.6 Hz), 5.37 (1H, dt, J = 10.4, 1.3 Hz), 5.45 (1H, dt, J = 17.1,
1.3 Hz), 6.17 (1H, ddd, J = 17.1, 10.4, 6.6 Hz); 13C NMR (100 MHz,
TMS, CDCl3) d 23.6, 49.4, 71.3, 71.5, 73.2, 74.6, 75.1, 80.8, 118.2,
136.6; HRMS (ESI+) m/z calcd for C10H14NaO3 [M+Na]+ 205.0835,
found 205.0821.
4.12. General procedure for synocedure for synthesis of
acetonides
To a stirred solution of the 1,3-diols (12a,b:10–28 mg) in dime-
thoxypropane (1–1.5 mL) was added camphorsulfonic acid (CSA)
(0.2 equiv) at room temperature under an atmosphere of argon,
and the resulting mixture was stirred for 0.5–1.5 h. Evaporation
of the solvent, followed by purification by silica gel column chro-
matography (ethyl acetate/n-hexane = 1:4) gave the corresponding
acetonides (14a,b) as colourless oils in 62–82% yields. The syn-1,3-
diol required an extended reaction time to complete the reaction
with a slightly reduced yield.
Compound 14a: 1H NMR (400 MHz, TMS, CDCl3) d 1.36 (3H, s),
1.37 (3H, s), 2.08 (1H, t, J = 2.6 Hz), 2.64 (1H, ddd, J = 16.9, 7.9,
2.6 Hz), 2.81 (1H, ddd, J = 16.9, 4.7, 2.6 Hz), 3.91 (1H, dd, J = 7.9,
4.7 Hz), 4.25 (1H, br d, J = 5.9 Hz), 4.29 (1H, d, J = 7.3 Hz), 4.34
(1H, d, J = 6.8 Hz), 4.63 (1H, d, J = 6.8 Hz), 4.73 (1H, d, J = 7.2 Hz),
5.43 (1H, ddd, J = 10.6, 1.7, 1.4 Hz), 5.48 (1H, dt, J = 17.2, 1.6 Hz),
6.17 (1H, ddd, J = 17.2, 10.6, 6.0 Hz); 13C NMR (100 MHz, TMS,
CDCl3) d 21.2, 24.3, 24.9, 47.6, 70.3, 71.0, 73.3, 74.1, 75.2, 80.5,
101.2, 118.6, 133.1.
4.10. 3-{[1-(RS)-(tert-Butyldimethylsilyl)oxy]but-3-ynyl}-3-{[1-
(RS)-(tert-butyldimethylsilyl)oxy]prop-2-enyl}oxetane (13a)
To a stirred solution of 12a (66 mg, 0.36 mmol) and pyridinium
trifluoromethanesulfonate (67 mg, 0.29 mmol) in dry THF (5 mL)
was added tert-butyldimethylsilyloxy N-phenylbenzimidate (TBS-
BEZA) (677 mg, 2.17 mmol) at À20 °C under an atmosphere of ar-
gon, and the resulting mixture was stirred at the same temperature
for 2 h. Water (1 mL) was added to the reaction mixture, and the
whole was extracted with ether (10 mL Â 3). The combined organic
layer was washed with brine (5 mL), dried over sodium sulfate, and
filtered. The filtrate was concentrated under the reduced pressure
to give a residue, from which 13a (119 mg) was separated by silica
gel column chromatography (ethyl acetate/n-hexane = 1:99) as a
colourless oil in 80% yield.
Compound 14b: 1H NMR (400 MHz, TMS, CDCl3) d 1.37 (3H, s),
1.49 (3H, s), 2.05 (1H, t, J = 2.7 Hz), 2.64 (1H, ddd, J = 17.2, 7.7,
2.7 Hz), 3.01 (1H, ddd, J = 17.2, 4.1, 2.7 Hz), 4.00 (1H, dd, J = 7.7,
4.2 Hz), 4.23 (1H, br d, J = 6.4 Hz), 4.32 (1H, d, J = 7.0 Hz), 4.46
(1H, J = 6.9 Hz), 4.49 (1H, d, J = 7.2 Hz), 4.58 (1H, d, J = 7.2 Hz),
5.489 (1H, dt, J = 10.1, 1.5 Hz), 5.490 (1H, dt, J = 17.5, 1.5 Hz),
6.22 (1H, ddd, J = 17.5, 10.1, 6.3 Hz); 13C NMR (100 MHz, TMS,
CDCl3) d 19.2, 21.2, 29.6, 43.9, 69.8, 70.9, 72.1, 72.5, 74.7, 81.4,
99.4, 120.0, 133.3.
Compound 13a: 1H NMR (400 MHz, TMS, CDCl3) d 0.04 (3H, s),
0.06 (3H, s), 0.070 (3H, s), 0.072 (3H, s), 0.90 (9H, s), 0.92 (9H, s),
1.99 (1H, t, J = 2.7 Hz), 2.59 (2H, dd, J = 7.2, 2.7 Hz), 3.73 (1H, d,
J = 10.1 Hz), 4.00 (1H, d, J = 10.1 Hz), 4.27 (1H, br d, J = 7.8 Hz),
4.30 (1H, d, J = 5.7 Hz), 4.56 (1H, d, J = 5.8 Hz), 4.88 (1H, t,
J = 7.2 Hz), 5.17 (1H, ddd, J = 10.3, 1.7, 0.7 Hz), 5.22 (1H, ddd,
J = 17.2, 1.7, 1.0 Hz), 5.83 (1H, ddd, J = 17.2, 10.3, 7.7 Hz); 13C
NMR (100 MHz, TMS, CDCl3) d À5.6, À4.9, À3.9, 18.1, 22.1, 25.8,
50.5, 61.6, 70.3, 70.6, 72.2, 74.1, 80.2, 81.7, 117.2, 137.7; HRMS
(ESI+) m/z calcd for C22H42NaO3Si2 [M+Na]+ 433.2565, found
433.2524.
4.13. (5Z,7E)-(1R,3R)-2,2-(Methyleneoxy)methano-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (3a) and (5Z,7E)-(1S,3S)-
2,2-(methyleneoxy)methano-9,10-seco-5,7,10(19)-
cholestatriene-1,3,25-triol (3b)
To a stirred solution of the A-ring enyne precursor 13a (50 mg,
0.12 mmol) and the CD-ring portion 15 (52 mg, 0.15 mmol) in a
mixture of toluene (6.3 mL) and triethylamine (2.2 mL) was added
tetrakis(triphenylphosphine)palladium (70 mg, 0.06 mmol) at
room temperature under an atmosphere of argon. After having
been heated at reflux for 40 min, the reaction mixture was diluted
with ether and filtered through a small pad of silica gel. Evapora-
tion of the filtrate gave a residue, which was purified by silica gel
column chromatography (ethyl acetate/n-hexane = 1:19, then
1:1) to give a mixture of protected vitamins (82 mg). The crude
vitamins dissolved in THF (2 mL) were treated with TBAF (1.0 M
4.11. 3-{[1-(SR)-(tert-Butyldimethylsilyl)oxy]but-3-ynyl}-3-{[1-
(RS)-(tert-butyldimethylsilyl)oxy]prop-2-enyl}oxetane (13b)
To a stirred solution of 12b (114 mg, 0.63 mmol) and pyridini-
um trifluoromethanesulfonate (143 mg, 0.63 mmol) in dry THF
(7 mL) was added TBS-BEZA (1.56 g, 5.00 mmol) at 0 °C under an

T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
5215
in THF, 0.85 mL, 0.85 mmol) at room temperature for 16 h. Brine
was added to the mixture and the whole was extracted with ethyl
acetate. The organic layer was dried over sodium sulfate, filtered
and concentrated. The residue was purified by silica gel column
chromatography (ethyl acetate/n-hexane = 1:2, then 6:1), followed
by silica gel preparative TLC (ethyl acetate/n-hexane = 6:1) to give
3a,b (9.0 mg) in 16% yield (two steps). Separation and further puri-
fication of 3a (less polar) and 3b (more polar) were conducted on a
recycling HPLC (COSMOSILÒ Cholester column, 10 Â 250 mm,
5 mL minÀ1, methanol/water = 75:25).
(1H, d, J = 6.5 Hz), 5.05 (1H, d, J = 1.5 Hz), 5.37 (1H, d, J = 1.5 Hz),
5.99 (1H, d, J = 11.3 Hz), 6.47 (1H, d, J = 11.3 Hz); 13C NMR
(175 MHz, TMS, CDCl3) d 12.1, 18.8, 20.8, 22.2, 23.4, 27.6, 29.1,
29.2, 29.3, 36.1, 36.4, 40.5, 41.3, 44.4, 45.9, 47.4, 56.3, 56.5, 71.1,
73.4, 76.1, 79.5, 116.1, 116.7, 126.6, 129.4, 143.9; HRMS (ESI+) m/
z calcd for C29H46NaO4 [M+Na]+ 481.3288, found 481.3286.
Compound 3d: UV (EtOH) kmax 264 nm, kmin 227 nm; 1H NMR
(400 MHz, TMS, CDCl3) d 0.55 (3H, s), 0.94 (3H, d, J = 6.4 Hz), 1.21
(6H, s), 2.39 (1H, m), 2.45 (1H, dd, J = 14.4, 3.6 Hz), 2.83 (1H, dd,
J = 11.9, 4.0 Hz), 4.29 (1H, d, J = 6.4 Hz), 4.33 (1H, d, J = 6.5 Hz),
4.35 (1H, m), 4.62 (1H, br s), 4.72 (1H, d, J = 6.5 Hz), 4.76 (1H, d,
J = 6.5 Hz), 5.08 (1H, d, J = 1.9 Hz), 5.39 (1H, d, J = 1.8 Hz), 6.04
(1H, d, J = 11.3 Hz), 6.48 (1H, d, J = 11.5 Hz); 13C NMR (100 MHz,
TMS, CDCl3) d 11.9, 18.8, 20.8, 22.3, 23.7, 27.6, 29.15, 29.23, 29.4,
36.1, 36.4, 40.4, 41.3, 44.4, 46.0, 47.3, 56.3, 56.5, 71.1, 73.6, 76.3,
79.9, 116.4, 116.6, 126.7, 129.5, 143.98, 144.03; HRMS (ESI+) m/z
calcd for C29H46NaO4 [M+Na]+ 481.3288, found 481.3269.
Compound 3a: UV (EtOH) kmax 266 nm, kmin 227 nm; 1H NMR
(400 MHz, TMS, CDCl3) d 0.53 (3H, s), 0.93 (3H, d, J = 6.4 Hz), 1.21
(6H, s), 1.98 (1H, m), 2.19 (1H, dd, J = 14.0, 6.9 Hz), 2.46 (1H, dd,
J = 13.9, 3.5 Hz), 2.80 (1H, dd, J = 11.6, 3.5 Hz), 4.31 (1H, dd,
J = 6.7, 3.9 Hz), 4.46 (1H, d, J = 6.3 Hz), 4.49 (1H, br s), 4.52 (1H, d,
J = 6.1 Hz), 4.60 (1H, d, J = 6.3 Hz), 4.67 (1H, d, J = 6.1 Hz), 5.05
(1H, s), 5.44 (1H, s), 5.98 (1H, d, J = 11.3 Hz), 6.36 (1H, d,
J = 11.2 Hz); 13C NMR (100 MHz, TMS, CDCl3) d 12.0, 18.8, 20.8,
22.2, 23.6, 27.6, 29.1, 29.2, 29.3, 36.1, 36.4, 40.4, 41.2, 44.4, 45.9,
50.0, 56.3, 56.5, 70.3, 71.1, 74.1, 74.2, 74.8, 114.0, 116.7, 125.2,
131.8, 143.8, 144.6; HRMS (ESI+) m/z calcd for C29H46NaO4
[M+Na]+ 481.3288, found 481.3277.
4.15. General procedure for synthesis of bis-MTPA esters
A solution of the vitamins (from 0.3 to 0.5 mg) described above
dissolved in dry CH2Cl2 was treated with DMAP (26 equiv) and (R)-
or (S)-MTPACl (16 equiv) at room temperature under an atmo-
sphere of argon. The reaction mixture was purified by preparative
TLC (ethyl acetate/n-hexane = 1:2) without pretreatment to afford
the corresponding bis-(S)-MTPA or bis-(R)-MTPA ester.
Compound 3b: UV (EtOH) kmax 266 nm, kmin 227 nm; 1H NMR
(400 MHz, TMS, CDCl3) d 0.54 (3H, s), 0.94 (1H, d, J = 6.4 Hz), 1.22
(6H, s), 1.99 (1H, m), 2.17 (1H, dd, J = 13.4, 7.9 Hz), 2.48 (1H, dd,
J = 13.9, 4.0 Hz), 2.81 (1H, dd, J = 12.4, 4.3 Hz), 4.27 (1H, m), 4.46
(1H, d, J = 6.2 Hz), 4.54 (1H, d, J = 6.4 Hz), 4.56 (1H, br s), 4.60
(1H, d, J = 6.3 Hz), 4.70 (1H, d, J = 6.2 Hz), 5.07 (1H, s), 5.44 (1H,
s), 5.98 (1H, d, J = 11.3 Hz), 6.38 (1H, d, J = 11.3 Hz); 13C NMR
(175 MHz, TMS, CDCl3) d 12.0, 18.8, 20.8, 22.3, 23.6, 27.6, 29.1,
29.2, 29.4, 36.1, 36.4, 40.4, 41.4, 44.4, 46.0, 49.9, 56.3, 56.5, 69.7,
71.1, 74.4, 75.3, 114.8, 116.6, 125.4, 131.5, 144.0, 144.2; HRMS
(ESI+) m/z calcd for C29H46NaO4 [M+Na]+ 481.3288, found
481.3279.
4.15.1. Bis-(S)-MTPA ester of 3a
1H NMR (400 MHz, TMS, CDCl3) d 0.441 (3H, s), 0.931 (3H, d,
J = 6.6 Hz), 1.216 (6H, s), 2.243 (1H, m), 2.737 (1H, dd, J = 13.2,
4.7 Hz), 2.802 (1H, m), 3.472 (3H, s), 3.542 (3H, s), 3.985 (1H, d,
J = 6.7 Hz), 4.073 (1H, d, J = 6.7 Hz), 4.199 (1H, d, J = 6.4 Hz), 4.608
(1H, d, J = 6.5 Hz), 5.181 (1H, dd, J = 10.3, 4.5 Hz), 5.298 (1H, s),
5.679 (1H,s), 5.892 (1H, d, J = 11.8 Hz), 5.916 (1H, s), 6.470 (1H,
d, J = 11.4 Hz), 7.345–7.716 (10H, m); HRMS (ESI+) m/z calcd for
4.14. (5Z,7E)-(1R,3S)-2,2-(Methyleneoxy)methano-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (3c) and (5Z,7E)-(1S,3R)-
2,2-(methyleneoxy)methano-9,10-seco-5,7,10(19)-
C
49H60F6NaO8 [M+Na]+ 913.4085, found 913.4079.
4.15.2. Bis-(R)-MTPA ester of 3a
cholestatriene-1,3,25-triol (3d)
1H NMR (400 MHz, TMS, CDCl3) d 0.338 (3H, s), 0.931 (1H, d,
J = 6.4 Hz), 1.225 (6H, s), 2.159 (1H, m), 2.587 (1H, m), 2.745 (1H,
m), 3.434 (3H, s), 3.598 (3H, s), 4.268 (1H, d, J = 6.6 Hz), 4.331
(1H, d, J = 6.7 Hz), 4.391 (1H, d, J = 6.7 Hz), 4.617 (1H, d,
J = 6.9 Hz) 5.204 (1H, s), 5.338 (1H, dd, J = 10.8, 5.8 Hz), 5.547
(1H, s), 5.785 (1H, d, J = 11.2 Hz), 5.971 (1H, s), 6.358 (1H, d,
J = 10.9 Hz), 7.344–7.591 (10H, m); HRMS (ESI+) m/z calcd for C49-
H60F6NaO8 [M+Na]+ 913.4085, found 913.4045.
To a stirred solution of the A-ring enyne precursor 13b (55 mg,
0.13 mmol) and the CD-ring portion 15 (57 mg, 0.16 mmol) in a
mixture of toluene (6.8 mL) and triethylamine (2.4 mL) was added
tetrakis(triphenylphosphine)palladium (46 mg, 0.04 mmol) at
room temperature under an atmosphere of argon. After having
been heated at reflux for 40 min, the reaction mixture was diluted
with ether, and filtered through a small pad of silica gel. Evapora-
tion of the filtrate gave a residue, which was purified by silica gel
column chromatography (ethyl acetate/n-hexane = 1:10), followed
by silica gel preparative TLC (ethyl acetate/n-hexane = 1:2), to give
a mixture of protected vitamins (56 mg). The vitamins were then
dissolved in THF (0.8 mL) before being treated with TBAF (1.0 M
in THF, 0.24 mL, 0.24 mmol) at room temperature for 1.5 h. Brine
was added to the mixture and the whole was extracted with ethyl
acetate. The organic layer was dried over sodium sulfate, filtered
and concentrated. The residue was purified by silica gel column
chromatography (ethyl acetate/n-hexane = 1:4, then 2:1), followed
by silica gel preparative TLC (ethyl acetate/n-hexane = 5:1) to give
3c,d (12 mg) in 20% yield (two steps). Separation and further puri-
fication of 3c (more polar) and 3d (less polar) were conducted on a
recycling HPLC (COSMOSILÒ Cholester column, 10 Â 250 mm,
5 mL minÀ1, methanol/water = 75:25).
4.15.3. Bis-(S)-MTPA ester of 3b
1H NMR (400 MHz, TMS, CDCl3) d 0.511 (3H, s), 0.934 (3H, d,
J = 6.4 Hz), 1.218 (6H, s), 2.163 (1H, m), 2.555 (1H, dd, J = 13.5,
4.4 Hz), 2.730 (1H, m), 3.458 (3H, s), 3.589 (3H, s), 4.265 (1H, d,
J = 6.7 Hz), 4.365 (1H, d, J = 6.7 Hz), 4.406 (1H, d, J = 6.7 Hz), 4.617
(1H, d, J = 6.7 Hz), 5.226 (1H, s), 5.339 (1H, dd, J = 10.5, 5.0 Hz),
5.547 (1H, s), 5.847 (1H, d, J = 11.6 Hz), 5.907 (1H, s), 6.316 (1H,
d, J = 10.8 Hz), 7.331–7.569 (10H, m); HRMS (ESI+) m/z calcd for
C
49H60F6NaO8 [M+Na]+ 913.4085, found 913.4039.
4.15.4. Bis-(R)-MTPA ester of 3b
1H NMR (400 MHz, TMS, CDCl3) d 0.536 (3H, s), 0.940 (3H, d,
J = 6.4 Hz), 1.216 (6H, s), 2.234 (1H, m), 2.746 (1H, dd, J = 13.1,
4.5 Hz), 2.805 (1H, m), 3.461 (3H, s), 3.555 (3H, s), 4.082 (1H, d,
J = 6.8 Hz), 4.172 (1H, d, J = 6.7 Hz), 4.215 (1H, d, J = 6.5 Hz), 4.632
(1H, d, J = 6.4 Hz), 5.216 (1H, dd, J = 10.5, 4.6 Hz), 5.347 (1H, s),
5.718 (1H, s), 5.951 (1H, d, J = 11.6 Hz), 5.966 (1H, s), 6.489 (1H,
d, J = 11.6 Hz), 7.295–7.562 (10H, m); HRMS (ESI+) m/z calcd for
Compound 3c: UV (EtOH) kmax 264 nm, kmin 228 nm; 1H NMR
(400 MHz, TMS, CDCl3) d 0.53 (3H, s), 0.93 (3H, d, J = 6.4 Hz), 1.21
(6H, s), 1.99 (1H, m), 2.39 (1H, m), 2.44 (1H, dd, J = 14.5, 4.0 Hz),
2.83 (1H, dd, J = 12.4, 4.0 Hz), 4.30 (1H, m), 4.31 (1H, d, J = 6.3 Hz),
4.35 (1H, d, J = 6.4 Hz), 4.58 (1H, br s), 4.71 (1H, d, J = 6.4 Hz), 4.76
C
49H60F6NaO8 [M+Na]+ 913.4085, found 913.4042.

5216
T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
4.15.5. Bis-(S)-MTPA ester of 3c
Acknowledgements
1H NMR (400 MHz, TMS, CDCl3) d 0.430 (3H, s), 0.942 (3H, d,
J = 6.4 Hz), 1.218 (6H, s), 1.85 (1H, m), 1.93–2.03 (2H, m), 2.302
(1H, dd, J = 14.4, 7.0 Hz), 2.480 (1H, dd, J = 14.4, 4.0 Hz), 2.667
(1H, m), 3.216 (3H, s), 3.307 (3H, s), 4.203 (1H, d, J = 6.6 Hz),
4.321 (1H, d, J = 6.8 Hz), 4.342 (1H, d, J = 6.8 Hz), 4.393 (1H, d,
J = 6.7 Hz), 5.138 (1H, s), 5.430 (1H, dd, J = 6.8, 4.2 Hz), 5.477 (1H,
s), 5.694 (1H, s), 5.767 (1H, d, J = 11.4 Hz), 6.150 (1H, d,
J = 11.4 Hz), 7.387–7.568 (10H, m); HRMS (ESI+) m/z calcd for C49-
H60F6NaO8 [M+Na]+ 913.4085, found 913.4090.
This work was supported in part by a Grant-in-Aid for scientific
research (Grant number 23590142) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology in Japan.
Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra for the
novel compounds 5–11, 12a,b, 13a,b, 14a,b and 3a–d) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2013.06.032.
4.15.6. Bis-(R)-MTPA ester of 3c
1H NMR (400 MHz, TMS, CDCl3) d 0.471 (3H, s), 0.939 (3H, d,
J = 6.3 Hz), 1.228 (6H, s), 2.248 (1H, m), 2.693 (1H, dd, J = 13.2,
4.9 Hz), 2.752 (1H, dd, J = 12.2, 3.5 Hz), 3.552 (3H, s), 3.647 (3H,
s), 4.232 (1H, d, J = 6.9 Hz), 4.276 (1H, d, J = 6.8 Hz), 4.479 (1H, d,
J = 6.4 Hz), 4.523 (1H, d, J = 6.4 Hz), 4.895 (1H, s), 4.933 (1H, s),
5.122 (1H, dd, J = 10.6, 4.8 Hz), 5.524 (1H, s), 5.820 (1H, d,
J = 11.2 Hz), 6.390 (1H, d, J = 10.9 Hz), 7.382–7.716 (10H, m); HRMS
(ESI+) m/z calcd for C49H60F6NaO8 [M+Na]+ 913.4085, found
913.4083.
References and notes
1. (a)Vitamin D; Feldman, D., Wesley Pike, J., Adam, J. S., Eds., 3rd ed.; Elsevier
Academic Press: Burlington, 2011; (b) Bikle, D. J. Clin. Endocrinol. Metab. 2009,
96, 26; (c) Haussler, M. R.; Whitfield, G. K.; Kaneko, I.; Haussler, C. A.; Hsieh, D.;
Hsieh, J.-C.; Jurutka, P. W. Calcif. Tissue Int. 2013, 92, 77; (d) Norman, A. W.
Endocrinology 2006, 147, 5542; (e) Plum, L. A.; DeLuca, H. F. Nat. Rev. Drug Disc.
2010, 9, 941.
2. (a) Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. Mol. Cell 2000, 5,
173; (b) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mitschler, A.; Moras, D.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5491; (c) Hourai, S.; Fujishima, T.; Kittaka,
A.; Suhara, Y.; Takayama, H.; Rochel, N.; Moras, D. J. Med. Chem. 2006, 49,
5199; (d) Eelen, G.; Valle, N.; Sato, Y.; Rochel, N.; Verlinden, L.; De Clercq, P.;
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1029, 15.
3. (a) 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, H. F.;
Takayama, H. J. Med. Chem. 2000, 43, 4247; (b) Fujishima, T.; Konno, K.;
Nakagawa, K.; Kurobe, M.; Okano, T.; Takayama, H. Bioorg. Med. Chem. 2000, 8,
123; (c) Fujishima, T.; Liu, Z.-P.; Konno, K.; Nakagawa, K.; Okano, T.;
Yamaguchi, K.; Takayama, H. Bioorg. Med. Chem. 2001, 9, 525; (d) Fujishima,
T.; Konno, K.; Nakagawa, K.; Tanaka, M.; Okano, T.; Kurihara, M.; Miyata, N.;
Takayama, H. Chem. Biol. 2001, 1011, 8; (e) Fujishima, T.; Kittaka, A.; Yamaoka,
K.; Takeyama, K.; Kato, S.; Takayama, H. Org. Biomol. Chem. 1863, 2003, 1; (f)
Fujishima, T.; Tsutsumi, R.; Negishi, Y.; Fujii, S.; Takayama, H.; Kittaka, A.;
Kurihara, M. Anticancer Res. 2006, 24, 2633; (g) Kittaka, A.; Suhara, Y.;
Takayanagi, H.; Fujishima, T.; Kurihara, M.; Takayama, H. Org. Lett. 2000, 2,
2619; (h) Suhara, Y.; Nihei, K.; Kurihara, M.; Kittaka, A.; Yamaguchi, K.;
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(h) Ono, Y.; Watanabe, H.; Shiraishi, A.; Tanaka, S.; Higuchi, Y.; Sato, K.;
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K.; Okano, T. Biol. Pharm. Bull. 2000, 23, 66.
4.15.7. Bis-(S)-MTPA ester of 3d
1H NMR (400 MHz, TMS, CDCl3) d 0.552 (3H, s), 0.944 (3H, d,
J = 6.4 Hz), 1.223 (6H, s), 2.271 (1H, dd, J = 11.6, 9.9 Hz), 2.682
(1H, dd, J = 13.5, 4.8 Hz), 2.758 (1H, m), 3.481 (3H, s), 3.603 (3H,
s), 4.242 (1H, d, J = 6.9 Hz), 4.280 (1H, d, J = 6.7 Hz), 4.462 (1H, d,
J = 6.4 Hz), 4.492 (1H, d, J = 6.3 Hz), 4.901 (1H, s), 4.962 (1H, s),
5.174 (1H, dd, J = 9.9, 4.5 Hz), 5.558 (1H, s), 5.812 (1H, d,
J = 11.2 Hz), 6.386 (1H, d, J = 11.1 Hz), 7.379–7.421 (10H, m); HRMS
(ESI+) m/z calcd for C49H60F6NaO8 [M+Na]+ 913.4085, found
913.4085.
4.15.8. Bis-(R)-MTPA ester of 3d
1H NMR (400 MHz, TMS, CDCl3) d 0.520 (3H, s), 0.935 (3H, d,
J = 6.3 Hz), 1.218 (6H, s), 2.278 (1H, dd, J = 14.0, 7.9 Hz), 2.489
(1H, dd, J = 13,9, 4.4 Hz), 2.685 (1H, m), 3.292 (3H, s), 3.361 (3H,
s), 4.265 (1H, d, J = 6.6 Hz), 4.324 (1H, d, J = 6.7 Hz), 4.376 (1H, d,
J = 6.6 Hz), 4.391 (1H, d, J = 6.3 Hz), 5.104 (1H, s), 5.388 (1H, dd,
J = 7.4, 4.1 Hz), 5.405 (1H, s), 5.682 (1H, s), 5.778 (1H, d,
J = 10.9 Hz), 6.190 (1H, d, J = 11.5 Hz), 7.348–7.571 (10H, m); HRMS
(ESI+) m/z calcd for C49H60F6NaO8 [M+Na]+ 913.4085, found
913.4122.
4. (a) Wuitschik, G.; Rogers-Evans, M.; Buckl, A.; Bernasconi, M.; Märki, M.; Godel,
T.; Fischer, H.; Wagner, B.; Parrilla, I.; Schuler, F.; Schneider, J.; Alker, A.;
Schweizer, W. B.; Müller, K.; Carreira, E. M. Angew. Chem., Int. Ed. 2008, 47,
4512; (b) Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.;
Schuler, F.; Rogers-Evans, M.; Müller, K. J. Med. Chem. 2010, 53, 3227; (c)
Burkhard, J. A.; Guérot, C.; Knust, H.; Roger-Evans, M.; Carreira, E. M. Org. Lett.
1944, 2010, 12.
4.16. Competitive vitamin D receptor (VDR) binding assay
5. (a) Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114, 9836; (b) Trost,
B. M.; Dumas, J. J. Am. Chem. Soc. 1924, 1992, 114.
6. For procedures concerning monobenzylation of pentaerythrithol, see: (a)
Lehmann, J.; Weitzel, U. P. Carbohydr. Res. 1996, 294, 65; (b) Brand, M.; Lainé,
C.; Réthoré, G.; Laurent, I.; Neven, C.; Lemiègre, L.; Benvegnu, T. J. Org. Chem.
2007, 72, 8262.
7. For intra-molecular substitution to produce an oxetane, see: (a) Behrendt, J. M.;
Bala, K.; Golding, P.; Hailes, H. C. Tetrahedron Lett. 2005, 46, 643; (b) Jiang, Z.-H.;
Budzynski, W. A.; Skeels, L. A.; Kranz, M. J.; Koganty, R. R. Tetrahedron 2002, 58,
8833.
8. (a) Rychnovsky, S. D.; Skalitzky, J. Tetrahedron Lett. 1990, 31, 945; (b)
Rychnovsky, S. D.; Rogers, B.; Yang, G. J. Org. Chem. 1993, 58, 3511.
9. For N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and the successful
application of the above reagent, see: (a) Mawhinney, T. P.; Madson, M. A. J.
Org. Chem. 1982, 47, 3336; (b) Boger, D. L.; Kim, S. H.; Mori, Y.; Weng, J.-H.;
Rogel, O.; Castle, S. L.; McAtee, J. J. J. Am. Chem. Soc. 1862, 2001, 123; (c) Pearson,
A. J.; Ciurea, D. V.; Velankar, A. Tetrahedron Lett. 1922, 2008, 49.
10. Misaki, T.; Kurihara, M.; Tanabe, Y. Chem. Commun. 2001, 2478.
11. The A-ring conformational equilibria of 1 and its analogues can be estimated
by 1H NMR spectra using the vicinal coupling constants between protons at the
C3 and C4 positions, which is confirmed based upon the following references.
The bovine thymus VDR receptor was obtained from Yamasa
Biochemical (Chiba, Japan) and dissolved in 0.05 M phosphate
buffer (pH 7.4) containing 0.3 M KCl and 5 mM dithiothreitol
immediately prior to use. The receptor solution (500
pre-mixed with 50 of an ethanol solution of
dihydroxyvitamin D3 or an analogue at various concentrations
for 60 min at 25 °C, prior to the before addition of [3H]-1
,25-
dihydroxyvitamin D3 (50 L). The receptor mixture was then left
to stand overnight with 0.1 nM [3H]-1
,25-dihydroxyvitamin D3
at 4 °C. The bound and free [3H]-1
,25-dihydroxyvitamin D3 were
separated by treatment with a dextran-coated charcoal (Norit SX-
II) suspension (200 L) for 30 min at 4 °C, followed by centrifuga-
l
L) was
l
L
1a,25-
a
l
a
a
l
tion at 3000 rpm for 10 min. The supernatant (500 lL) was mixed
with Insta-GelÒ Plus (9.5 mL) (PerkinElmer, USA) and the radioac-
tivity was counted. The relative potency of the analogues was cal-
culated from the concentration required to displace 50% of the
The above vicinal coupling constant of 3a (J3 –4b = 6.9 Hz) was in good
a
accordance with that of
1 (J3 –4b = 6.4 Hz), and with that of 2c (J3
a
a
–
[3H]-1
the activity of 1
100 by definition.
a
,25-dihydroxyvitamin D3 from the receptor compared with
4b = 6.6 Hz): (a) Eguchi, T.; Ikekawa, N. Bioorg. Chem. 1990, 18, 19; (b) Anet, F.
A. L. J. Am. Chem. Soc. 1962, 84, 1053.
a,25-dihydroxyvitamin D3, which was assigned as
12. The modified Mosher’s method that used a simple derivation of secondary
alcohols to their methoxy-trifluoromethyl-phenylacetic acid (MTPA) esters

T. Fujishima et al. / Bioorg. Med. Chem. 21 (2013) 5209–5217
5217
was shown to be applicable to determine the absolute configuration of 1,3-
diols by their bis-MTPA ester products. In the cases of the bis-MTPA esters of
1,3-syn-diols (3c, d), one of the protons at the C4 position that was syn to the
MTPA ester at the C3 position resulted in an opposite sign value. All the other
positions showed no irregular values: Konno, K.; Fujishima, T.; Liu, Z.-P.;
Takayama, H. Chirality2002, 14, 72.