Synthesis and complete structure determination of a sperm-activating and -attracting factor isolated from the ascidian ascidia sydneiensis

Article abstract of DOI:10.1021/acs.jnatprod.7b01052

For the complete structure elucidation of an endogenous sperm-activating and -attracting factor isolated from eggs of the ascidian Ascidia sydneiensis (Assydn-SAAF), its two possible diastereomers with respect to C-25 were synthesized. Starting from ergosterol, the characteristic steroid backbone was constructed by using an intramolecular pinacol coupling reaction and stereoselective reduction of a hydroxy ketone as key steps, and the side chain was introduced by Julia-Kocienski olefination. Comparison of the NMR data of the two diastereomers with those of the natural product led to the elucidation of the absolute configuration as 25S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.

Full text of DOI:10.1021/acs.jnatprod.7b01052

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Synthesis and Complete Structure Determination of a Sperm-
Activating and -Attracting Factor Isolated from the Ascidian Ascidia
sydneiensis
†
‡
†
⊥
‡
†
Tomohiro Watanabe, Hajime Shibata, Makoto Ebine, Hiroshi Tsuchikawa, Nobuaki Matsumori,
‡
§
†
†
†
Michio Murata, Manabu Yoshida, Masaaki Morisawa, Shu Lin, Kosei Yamauchi, Ken Sakai,
,†
and Tohru Oishi*
†
Faculty and Graduate School of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
‡
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, 1024 Koajiro, Misaki, Miura, Kanagawa
§
2
38-0225, Japan
⊥
Tokyo Kasei Gakuin University, 2600 Aihara, Machida, Tokyo 194-0292, Japan
*
S Supporting Information
ABSTRACT: For the complete structure elucidation of an endogenous
sperm-activating and -attracting factor isolated from eggs of the ascidian
Ascidia sydneiensis (Assydn-SAAF), its two possible diastereomers with
respect to C-25 were synthesized. Starting from ergosterol, the character-
istic steroid backbone was constructed by using an intramolecular pinacol
coupling reaction and stereoselective reduction of a hydroxy ketone as key
steps, and the side chain was introduced by Julia−Kocienski olefination.
Comparison of the NMR data of the two diastereomers with those of the
natural product led to the elucidation of the absolute configuration as 25S;
thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.
perm activation and chemotaxis, which ubiquitously occur
addition, Ciinte-SAAF was proved to be active toward C.
savignyi, another species in the same genus, indicating that there
is tolerance on the species specificity. On the other hand,
S
in animals and plants, play an important role in
2b
1
fertilization. Several nonpeptide small molecules are known
to act as chemoattractants in fertilization. We have previously
isolated the sperm-activating and -attracting factor (SAAF)
from the eggs of the ascidian Ciona intestinalis (Ciinte-SAAF,
Assydn-SAAF (Figure 1) was isolated from the eggs of the
3
ascidian Ascidia sydneiensis. The molecular structure of Assydn-
SAAF was determined by NMR and ESI/TOF-MS analysis
using approximately 2.6 μg of sample to be 3,7,8,26-
2
Figure 1). Ciinte-SAAF was the first example of a single agent
3
tetrahydroxycholest-22-ene-3,26-disulfate, while the configu-
concomitantly inducing both sperm activation and attraction. In
rations at C-20 and C-25 remained unknown. Although the
structure closely resembles that of Ciinte-SAAF, it was found
that Ciinte-SAAF did not activate the sperm of A. sydneiensis,
3
,4
suggesting that there is high genus specificity. Although the
mechanisms underlying the sperm activation/attraction as well
as genus specificity are not fully elucidated, unambiguous
structure determination of Assydn-SAAF is necessary for further
investigations. Herein we report the synthesis and complete
structure determination of Assydn-SAAF based on the chemical
synthesis of plausible diastereomers and comparison of the
NMR data.
RESULTS AND DISCUSSION
■
From the biosynthetic point of view, we assumed that the
absolute configuration at C-20 of Assydn-SAAF (1) would be
Figure 1. Structure of Ciinte-SAAF and proposed structure of Assydn-
SAAF.
Received: December 24, 2017
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
identical with that of Ciinte-SAAF, as shown in Figure 2, and
and compare the NMR data with those of the natural product.
Although the structure of the steroid core was determined by
NMR analysis, it was difficult to elucidate the configuration of
the nonprotonated carbon at C-8. Therefore, prior to the
synthesis of 2a and 2b, we planned to prepare model
compound 3a corresponding to the steroid core and its C-8-
supportive NOE correlations between H-12a−H -21 together
3
1
epimer 3b (Figure 2) and compare the H NMR data with
those of the natural product.
Olefin 8, the key intermediate of 3a and 3b, was synthesized
from ergosterol as shown in Scheme 1. The diene moiety on
the B-ring was protected via hetero-Diels−Alder reaction using
phthalhydrazide in the presence of Pb(OAc) to give
4
5
cycloadduct 4 in 93% yield. Inversion of the secondary
alcohol at C-3 was carried out via Parikh−Doering oxidation
(
96%) followed by reduction with L-Selectride (lithium tri-sec-
butylborohydride) to afford α-alcohol 5 as a single isomer
66%). The C-22/C-23 double bond was selectively cleaved by
(
6
ozonolysis and following hydride reduction gave the primary
alcohol (71%). Removal of the phthalazide moiety with
LiAlH4 liberated the diene on the B-ring to afford 6. Birch
5
7
reduction of diene 6 proceeded regio- and stereoselectively to
furnish the 5α-cholest-7-ene skeleton. The primary hydroxy
group of the diol was selectively protected as pivaloate 7 in 76%
yield for three steps. The remaining secondary alcohol was
protected as a p-methoxybenzyl (PMB) ether under acidic
8
conditions to provide olefin 8 in 83% yield.
Having the key intermediate in hand, we moved on to the
synthesis of 3a and 3b as shown in Scheme 2. It is known that
OsO -mediated dihydroxylation of 5α-cholest-7-ene and its
4
9
derivatives provides α-diol selectively. Thus, dihydroxylation of
hindered olefin 8 with OsO was carried out in the presence of
4
10
1
,4-diazabicyclo[2.2.2]octane (DABCO), and the reaction
proceeded stereoslectively to afford the desired 7α,8α-diol 3b
in 80% yield as a single diastereomer. Because initial attempts
to prepare 3a directly from 8 via epoxidation/ring-opening
sequence were unsuccessful, oxidative cleavage of the C-7/C-8
Figure 2. Proposed structure of Assydn-SAAF (1), two possible
diastereomers with respect to C-25, (25S)-2a and (25R)-2b, and
model compounds 3a and 3b.
11,12
double bond followed by intramolecular pinacol coupling
3
with H -18−H-20 were observed. To determine the complete
of the resulting ketoaldehyde was examined. Although
ozonolysis of olefin 8 was unsuccessful and gave a complex
mixture, and attempts of oxidative cleavage of diol 3b with
3
structure of Assydn-SAAF, we planned to synthesize both
diastereomers with respect to C-25, (25S)-2a and (25R)-2b,
a
Scheme 1. Synthesis of Olefin 8 from Ergosterol
a
Reagents and conditions: (a) phthalhydrazide, Pb(OAc) , CH Cl , 0 °C to rt, 4 h, 93%; (b) SO ·pyridine, dimethylsulfoxide (DMSO), Et N,
4
2
2
3
3
CH Cl , rt, 2.5 h, 96%; (c) L-Selectride, tetrahydrofuran (THF), −78 °C, 30 min, 66%; (d) O , pyridine, CH Cl , −60 °C, 20 min; NaBH , MeOH,
2
2
3
2
2
4
−
60 °C to rt, 2 h, 71%; (e) LiAlH , THF, 80 °C, 1.5 h; (f) Li, NH , tert-amyl alcohol, THF, −78 °C, 1.5 h; (g) PvCl, pyridine, CH Cl , 0 °C, 2 h,
4
3
2
2
7
6% (3 steps); (h) p-methoxybenzyl trichloroacetimidate (PMBTCA), La(OTf) , toluene, 0 °C, 40 min, 83%.
3
B
DOI: 10.1021/acs.jnatprod.7b01052
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Article
a
Scheme 2. Synthesis of 3a−3d from 8
a
Reagents and conditions: (a) K OsO ·2H O, K Fe(CN) , K CO , MeSO NH , DABCO, t-BuOH/H O/CH Cl , rt, 36 h, 80%; (b) Pb(OAc) ,
2
4
2
3
6
2
3
2
2
2
2
2
4
CH Cl , 0 °C, 35 min, 92%; (c) SmI , MeOH, THF, 0 °C, 45 min, 85%; (d) SO ·pyridine, DMSO, Et N, CH Cl , rt, 13 h, 85%; (e) NaBH(OAc) ,
2
2
2
3
3
2
2
3
i-Pr NEt, THF, 0 °C to rt, 15 h, 93%; (f) DIBALH, CH Cl , −78 °C to rt, 2 h, 91%; (g) SO ·pyridine, DMSO, Et N, CH Cl , rt, 12 h, 76%; (h)
2
2
2
3
3
2
2
NaBH , THF, MeOH, 0 °C, 1.5 h, 89%.
4
Figure 3. Plausible transition states of the intramolecular pinacol coupling of 9.
NaIO resulted in no reaction, treatment of diol 3b with
Therefore, inversion of configuration at C-7 was examined via
an oxidation/reduction sequence. Parikh−Doering oxidation of
diol 3c gave hydroxy ketone 10 in 85% yield, and reduction of
10 with NaBH(OAc) in the presence of i-Pr NEt resulted in
4
Pb(OAc) proceeded smoothly to afford ketoaldehyde 9 in
4
92% yield. Then, intramolecular pinacol coupling of 9 was
carried out by treating with SmI in the presence of MeOH as a
2
3
2
proton source to give 7β,8β-diol 3c, the C-7 epimer of 3a, in
the formation of diol 3a in 93% yield as a single isomer,
85% yield as a single diastereomer. In the absence of MeOH,
presumably due to neighboring participation of the C-8
the yield of 3c decreased to 52% with concomitant formation of
byproducts. The structure of diol 3c was confirmed by NOE
experiments. The stereochemical outcome can be explained as
hydroxy group. It is noteworthy that addition of i-Pr NEt is
2
important to avoid the formation of the 8-deoxy byproduct.
The absolute configuration of 3a was confirmed by X-ray
crystallographic analysis of the corresponding primary alcohol
11, derived from 3a by removing the pivaloyol group with
diisobutylalminum hydride (DIBALH). The ORTEP drawing
of 11 is depicted in Figure 4.
Then, we examined conversion of 3b into 3d, the remaining
one of the four diastereomers at C-7 and C-8, via inversion of
the secondary alcohol. Parikh−Doering oxidation of 3b gave
hydroxy ketone 12 in 76% yield, and successive reduction with
shown in Figure 3. The SmI -mediated intramolecular pinacol
2
coupling is thought to proceed via a chelation transition state
bridging oxygen atoms at C-7 and C-8 with the samarium ion.
Among the possible chelation transition states TS1−TS3, TS1,
giving compound 3c, appears to be most favored compared
with TS2 and TS3, in which severe steric repulsions exist.
Although the stereochemistry at C-8 was successfully
controlled, that at C-7 was opposite that of Assydn-SAAF.
C
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Journal of Natural Products
Article
for 3b−d. Thus, the configuration of the vic-diol moiety of
Assydn-SAAF was confirmed to be 7α-OH/8β-OH as reported.
Having confirmed the configuration at C-8, we moved on to
the synthesis of (25S)-2a and (25R)-2b from 3a (Scheme 3).
The secondary alcohol at C-7 of 3a was protected as a
triethylsilyl (TES) ether (92%), the pivaloyl group was
removed by reduction with DIBALH (99%), and oxidation of
the resulting primary alcohol furnished aldehyde 13 (84%).
1
3,14
Then, Julia−Kocienski olefination
of aldehyde 13 was
examined by using sulfone (S)-14a, which was prepared from
known alcohol (S)-16a derived from (S)-2-methyl-1,4-
1
5
Figure 4. Structure of 11 in the crystalline state.
butanediol (Scheme 4), via protection of the primary alcohol
as a PMB ether (99%) and molybdate-H O -catalyzed
NaBH provided 7β,8α-diol 3d as the sole product, presumably
2
2
4
due to attack of hydride on the less hindered side. Thus, we
have established the synthetic route to construct the steroid
core of Assydn-SAAF including all possible diastereomers at C-7
and C-8.
With all four diastereomers 3a−d in hand, H NMR coupling
patterns of the H-7 proton were compared with that of Assydn-
oxidation of the corresponding tetrazol-5-yl thioether (89%).
As shown in Table 1, a solution of aldehyde 13 in
tetrahydrofuran (THF) was added to a solution of sulfone
(S)-14a and potassium bis(trimethylsilyl)amide (KHMDS) in
toluene, but resulted in the formation of a complex mixture
(entry 1). Next, KHMDS was added to a premixed solution of
aldehyde 13 and sulfone 14a in THF to afford 15a in 42% yield
as an inseparable E/Z mixture in a 2:1 ratio (entry 2). When
the base was changed to lithium bis(trimethylsilyl)amide
1
SAAF as shown in Figure 5. The H-7 proton signal of the
(
LHMDS), the yield was improved to 77%, but the E/Z ratio
decreased to 1:1 (entry 3). Addition of Hexamethylphosphoric
triamide (HMPA) as a cosolvent improved the E/Z ratio to 5:1,
but the yield decreased to 28% (entry 4). When KHMDS was
used in THF/HMPA, 15a was obtained in 50% yield in a 7:1
ratio. Finally, the E/Z ratio was improved to 11:1 by using a
THF solution of KHMDS to afford 15a in 51% yield (entry 6).
Then, the PMB groups of 15a were removed with 2,3-dichloro-
5
,6-dicyano-1,4-benzoquinone (DDQ) in 86% yield. In this
1
reaction, addition of aqueous NaHCO was necessary to avoid
Figure 5. Comparison of H NMR peak shapes of the H-7 proton
3
between natural 1 and synthetic 3a−d. 1: 500 MHz in D O. 3a−d:
the removal of the TES group. The resulting triol was
converted to the 3,26-disulfate ester by treatment with a SO3·
pyridine complex. After removal of the TES group with HF·
pyridine, the undesired Z-isomer was separated by preparative
TLC. Finally, treatment with ion-exchange resin (Amberlite IR-
2
6
00 MHz in CDCl .
3
natural product exhibited a characteristic splitting pattern
3
2c,d
reported as a broad triplet, J < 3 Hz. Among the synthetic
120B) afforded (25S)-2a in 48% yield for two steps. On the
specimens 3a−d, only 3a showed a similar peak shape to the
natural product, while larger J values than 3 Hz were observed
other hand, (25R)-2b was synthesized in an analogous
sequence from aldehyde 13 and sulfone (R)-14b, prepared
a
Scheme 3. Synthesis of Assydn-SAAF 2a and Its C-25-Epimer 2b
a
Reagents and conditions: (a) TESOTf, 2,6-lutidine, CH Cl , 0 °C to rt, 1.5 h, 92%; (b) DIBALH, CH Cl , −78 °C to rt, 1 h, 99%; (c) SO ·
2
2
2
2
3
pyridine, DMSO, Et N, CH Cl , 0 °C to rt, 7 h, 84%; (d) KHMDS, THF/HMPA, −78 °C to rt, 2 h, 51%, E/Z = 11:1 for (25S)-15a, 50%, E/Z = 6:1
for (25R)-15b; (e) DDQ, CH Cl , saturated NaHCO (aq), 0 °C, 20 min, 86% for (25S), 41% for (25R); (f) SO ·pyridine, pyridine, rt, 1 h; (g) HF·
pyridine, MeOH, 0 °C to rt, 7 h, then Amberlite IR-120B (Na ), MeOH, 48% in two steps for (25S)-2a, 72% in two steps for (25R)-2b.
3
2
2
2
2
3
3
+
D
DOI: 10.1021/acs.jnatprod.7b01052
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Article
a
Scheme 4. Synthesis of Sulfone 14a and 14b
a
Reagents and conditions: (a) PTSH, diisopropyl azodicarboxylate (DIAD), PPh , THF, −78 °C to rt, 4 h, 25%; (b) PMBTCA, La(OTf) , toluene,
3
3
rt, 23 h, 99%; (c) (NH ) Mo O ·4H O, H O , EtOH, rt, 5.5 h, 89%; (d) LiAlH , THF, −78 °C to rt, 17.5 h, 98%; (e) PTSH, DIAD, PPh , THF,
−
4
6
7
24
2
2
2
4
3
78 °C to rt, 7.5 h, 22%; (f) PMBTCA, La(OTf) , toluene, rt, 14 h, 90%; (g) (NH ) Mo O ·4H O, H O , EtOH, rt, 6 h, 91%.
3 4 6 7 24 2 2 2
Table 1. Optimization of Julia−Kocienski Reaction Using 13 and 14a
entry
base
solvent
yield/%
ratio/E:Z
a
1
KHMDS in toluene
KHMDS in toluene
LHMDS in THF
LHMDS in THF
KHMDS in toluene
KHMDS in THF
THF
THF
THF
0
b
2
42
77
28
50
51
2:1
1:1
b
3
b
4
THF/HMPA (4:1)
THF/HMPA (4:1)
THF/HMPA (4:1)
5:1
b
5
7:1
b
6
11:1
a
b
Aldehyde 13 was added to the mixture of sulfone 14a and base. Base was added to the mixture of aldehyde 13 and sulfone 14a.
from (R)-2-methylsuccinic acid via alcohol (R)-16b (Scheme
product revealed that the structure of Assydn-SAAF is
(3R,7R,8S,20R,25S)-3,7,8,26-tetrahydroxycholest-22-ene-3,26-
disulfate. The pure synthetic specimen was also used to confirm
the sperm-activating and -attracting activity. Further studies for
identification of the receptor of SAAF as well as the relevant
signal transduction pathway(s) are in progress.
4
).
With both diastereomers (25S)-2a and (25R)-2b in hand,
1
13
their H and C NMR chemical shifts were compared with
those of the natural product (Table 2). The chemical shifts of
(
25S)-2a are identical with those of Assydn-SAAF (1), while
1
there are noticeable differences in the H NMR chemical shifts
of (25R)-2b at the H -24 to H -26 positions. Therefore, the
2
2
EXPERIMENTAL SECTION
■
configuration at C-25 was determined to be S, and that at C-20
was also confirmed. Thus, complete structure of Assydn-SAAF
was determined to be (3R,7R,8S,20R,25S)-3,7,8,26-tetrahydrox-
ycholest-22-ene-3,26-disulfate, and the first synthesis of Ascidia-
SAAF was achieved.
General Experimental Procedures. Melting points were
measured using a Yanaco MP-J3 micro melting point apparatus
without correction. Optical rotations were recorded on a JASCO P-
1
010 polarimeter. IR spectra were recorded on a JASCO FT/IR-400.
1
13
H and C NMR spectra were measured on JEOL JNM-ECA-600 or
Biological activities of the synthetic specimens were
JNM-ECS400 instruments. Chemical shifts were reported in ppm from
tetramethylsilane (TMS) with reference to internal residual solvent
2
a
evaluated based on the methods previously reported.
Synthetic Ascidia-SAAF 2a elicited sperm-activating and
attracting activity against the sperm of the ascidian A.
sydneiensis at 5.0 μM. It is noteworthy that the C-25-epimer
1
[ H NMR: CHCl3 (7.26), C HD5 (7.16), acetone-d5 (2.05),
6
1
3
-
CD
acetone-d
b, NMR spectra were measured in D O with referring the chemical
2
HOD (3.31); C NMR: CDCl
3 6 6
(77.16), C D (128.06),
(29.84), CD OD (49.00)]. For compounds 1, 2a, and
6
3
2c
2
2
b also elicited comparable activity to 2a.
2
1
13
shifts to trace MeOH (3.34 ppm for H NMR and 49.5 ppm for
C
NMR). High-resolution ESI mass spectra (HRESIMS) were measured
on a Bruker microTOFfocus spectrometer. All reactions sensitive to air
or moisture were performed under an argon atmosphere with dry
glassware unless otherwise noted. The anhydrous solvents, CH Cl ,
THF, and toluene were purchased from Kanto Chemical Co. Inc. or
Wako Pure Chemical Industries Ltd. and used without further
dehydration unless otherwise stated. 2,6-Lutidine, triethylsilyl
trifluoromethanesulfonate (TESOTf), and HMPA were distilled
before use. Amberlite IR-120B was ion-exchanged with 1 M aqueous
CONCLUSION
■
In conclusion, we have developed the stereodivergent synthetic
route to construct the steroid core of Assydn-SAAF including all
possible diastereomers at C-7 and C-8 via an intramolecular
pinacol coupling reaction and stereoselective reduction of a
hydroxy ketone as key steps, and syntheses of (25S)- and
25R)-Assydn-SAAF were achieved via a Julia−Kocienski
reaction to introduce the side chain. Comparison of NMR
data of the synthetic specimens with those of the natural
2
2
(
NaOH, then washed with H O to neutrality, followed by conditioning
2
with MeOH. Powdered Sm metal was purchased from Nippon
E
DOI: 10.1021/acs.jnatprod.7b01052
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Article
Table 2. 13C and H NMR Chemical Shifts of 1, 2a, and 2b in D O
1
a
2
b
SAAF (1)
(25S)-2a
(25R)-2b
c
c
c
c
position
1
δC
δH
δC
ΔδC
δH
ΔδH
δC
ΔδC
δH
ΔδH
33.3
1.55
33.3
0.0
1.54
1.20
1.84
1.81
4.66
1.59
1.55
1.90
1.90
1.18
3.56
−0.01
0.02
0.00
0.01
0.01
−0.01
0.00
0.00
0.00
0.00
0.00
33.4
0.1
1.56
1.18
1.84
1.80
4.65
1.60
1.56
1.90
1.90
1.18
3.56
0.01
0.00
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
1
.18
1.84
.80
2
26.5
26.5
0.0
26.6
0.1
1
3
4
78.5
18.0
4.65
1.60
78.5
18.0
0.0
0.0
78.4
18.0
−0.1
0.0
1
.55
5
6
32.6
31.5
1.90
1.90
32.6
31.5
0.0
0.0
32.6
31.5
0.0
0.0
1
.18
7
8
9
72.0
n.d.
3.56
72.1
77.4
50.2
36.0
32.9
40.8
0.1
72.0
77.4
50.2
36.0
32.8
40.8
0.0
50.2
36.0
32.9
40.7
1.25
0.0
0.0
0.0
0.1
1.24
−0.01
0.0
0.0
1.26
0.01
10
11
12
1.59
1.99
1.57
2.00
1.21
−0.02
0.01
−0.1
0.1
1.60
2.00
1.22
0.01
0.01
0.01
1
.21
0.00
1
1
1
3
4
5
43.2
53.9
18.6
43.2
53.9
18.7
0.0
0.0
0.1
43.2
53.9
18.7
0.0
0.0
0.1
1.54
1.47
1.55
1.47
1.37
1.73
1.28
1.15
0.90
0.90
2.07
0.99
5.39
5.39
2.02
1.94
1.87
3.95
3.83
0.93
0.01
0.00
−0.01
0.00
0.00
0.01
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.01
1.56
1.48
1.37
1.73
1.28
1.15
0.90
0.90
2.06
0.99
5.39
5.39
2.08
1.86
1.89
3.92
3.85
0.92
0.02
0.01
1
.38
1.73
.28
−0.01
0.00
16
28.4
28.4
0.0
28.4
0.0
1
0.00
17
18
19
20
21
22
23
24
56.6
13.5
11.5
39.7
20.3
140.5
125.5
36.0
1.14
0.90
0.90
2.07
0.98
5.39
5.39
2.02
56.6
13.5
11.5
39.9
20.3
140.5
125.3
36.0
0.0
0.0
0.0
0.2
0.0
0.0
56.6
13.6
11.5
39.9
20.4
140.4
125.4
36.0
0.0
0.1
0.01
0.00
0.0
0.00
0.2
−0.01
0.01
0.1
−0.1
−0.1
0.0
0.00
−0.2
0.0
0.00
0.06
1
.93
−0.07
0.02
2
2
5
6
33.5
74.1
1.87
3.95
33.5
74.1
0.0
0.0
33.5
74.2
0.0
0.1
−0.03
0.02
3
.83
27
16.4
0.92
16.4
13
0.0
16.2
−0.2
b
0.00
a
1
Residual MeOH was adjusted to 49.5 ppm for C NMR (150 MHz) and 3.34 ppm for H NMR (600 MHz). Data of 1 are adopted from the
literature (ref 3). Δδ = δ(synthetic 2a or 2b) − δ(natural 1).
c
Yttrium Co., Ltd. All other chemicals were obtained from local
vendors and used as supplied unless otherwise stated. Thin-layer
chromatography (TLC) was performed on Merck silica gel 60 F254
precoated plates (0.25 mm thickness) for the reaction analyses.
Column chromatography was performed using Kanto silica gel 60N
Na SO , filtered, and concentrated under reduced pressure. The
2 4
residue was purified by column chromatography (silica gel, hexane/
EtOAc = 10/1 → 1/3) to give diene 4 (12.4 g, 22.3 mmol, 93%) as a
yellow foam: [α]22 −137 (c 1.05, CHCl ); R = 0.32 (hexane/EtOAc
D
3
f
= 2/1); IR (neat) 3441, 2955, 2871, 1678, 1601, 1460, 1381, 1311,
−1
1
(
spherical, neutral, 100−210 μm). Flash column chromatography was
1180, 1071, 1043, 1027, 974, 755, 725, 697 cm ; H NMR (600
performed on Kanto silica gel 60N (spherical, neutral, 40−50 μm). For
reversed-phase preparative TLC (RP-pTLC), Merck silica gel 60 RP-
MHz, CDCl ) δ 8.17−8.13 (m, 1H), 8.13−8.08 (m, 1H), 7.72−7.65
3
(m, 2H), 6.65 (d, J = 8.3 Hz, 1H), 6.27 (d, J = 8.3 Hz, 1H), 5.22 (dd, J
= 15.6, 7.6 Hz, 1H), 5.15 (dd, J = 15.6, 8.4 Hz, 1H), 4.00−3.92 (m,
2H), 3.73−3.65 (m, 1H), 2.13−2.06 (m, 2H), 2.05−1.96 (m, 2H),
1.88−1.81 (m, 2H), 1.78−1.64 (m, 3H), 1.62−1.56 (m, 1H), 1.50−
1.24 (m, 8H), 1.03 (s, 3H), 1.02 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9
Hz, 3H), 0.84 (s, 3H), 0.83 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz,
3H); the hydroxy proton was not observed probably due to the rapid
1
8 F254S precoated plates (0.25 mm thickness) were used. X-ray
crystallographic analysis was performed on a Bruker SMART APEX
CCD-detector diffractometer.
Synthetic Procedures. (22E)-5α,8α-(1,4-Dioxo-1,2,3,4-tetrahy-
drophthalazine-2,3-diyl)ergosta-6,22-dien-3β-ol (4). Phthalhydra-
zide (16.4 g, 101 mmol), Pb(OAc) (22.3 g, 40.2 mmol), and acetic
4
13
acid (4.4 mL, 77 mmol) were added to a solution of ergosterol (10.0 g,
exchange with contaminating H O; C NMR (150 MHz, CDCl ) δ
2
3
2
4.0 mmol) in dry CH Cl (267 mL) at 0 °C. The reaction mixture
161.9, 159.8, 138.2, 135.4, 132.9 (2C), 132.3, 130.7, 130.0, 129.1,
127.1, 127.0, 68.6, 67.7, 67.1, 56.7, 50.5, 49.0, 44.3, 42.8, 40.7, 40.0,
39.4, 35.6, 35.0, 33.2, 29.3, 28.3, 24.7, 21.9, 21.0, 20.0, 19.8, 18.7, 17.6,
2
2
was stirred at 0 °C for 3 h and then at room temperature (rt) for 1 h.
The reaction mixture was cooled to 0 °C and quenched with
powdered Al O (41.3 g, 405 mmol). After stirring at rt for 30 min, the
+
2
3
13.4; HRESIMS m/z 579.3554 [M + Na] (calcd for C H N O Na,
36 48
2
3
resultant mixture was filtered through a pad of Celite. The filtrate was
extracted with EtOAc, and the organic layer was washed with saturated
579.3557).
(22E)-5α,8α-(1,4-Dioxo-1,2,3,4-tetrahydrophthalazine-2,3-diyl)-
aqueous NaHCO and saturated aqueous NaCl, dried over anhydrous
ergosta-6,22-dien-3-one (S1). DMSO (7.0 mL, 99 mmol), Et N (7.0
3
3
F
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
mL, 50 mmol), and SO ·pyridine (4.74 g, 28.9 mmol) were added to a
(dd, J = 11.7, 7.6 Hz, 1H), 3.68−3.63 (brd, J = 10.0 Hz, 1H), 3.39−
3.34 (m, 1H), 2.83 (s, 1H), 2.26 (dd, J = 16.5, 3.4 Hz, 1H), 2.17 (dd, J
= 13.1, 4.8 Hz, 1H), 2.02 (ddd, J = 13.1, 3.4, 3.4 Hz, 1H), 2.00−1.94
(m, 1H), 1.92−1.87 (m, 1H), 1.84 (ddd, J = 13.1, 13.1, 3.4 Hz, 1H),
1.65−1.28 (m, 11H), 1.06 (d, J = 6.2 Hz, 3H), 0.99 (s, 3H), 0.86 (s,
3
solution of alcohol 4 (5.53 g, 9.93 mmol) in dry CH Cl (50 mL) at 0
2
2
°
C. After stirring at rt for 3 h, the reaction was quenched with
saturated aqueous NH Cl at 0 °C and extracted with Et O. The
4
2
organic layer was washed with saturated aqueous NaCl, dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure.
3H); 13C NMR (150 MHz, CDCl ) δ 165.0, 159.8, 139.0, 132.97,
2
4
3
The residue was purified by column chromatography (silica gel,
132.89, 130.8, 130.3, 128.8, 127.0, 126.9, 68.2, 67.8, 65.8, 64.6, 53.5,
hexane/EtOAc = 4/1 → 1/2) to give ketone S1 (5.27 g, 9.50 mmol,
50.8, 48.3, 44.5, 41.5, 39.3, 38.5, 32.3, 31.9, 27.9, 27.3, 24.4, 21.9, 18.5,
16.8, 13.2; HRESIMS m/z 513.2720 [M + Na] (calcd for
6%) as a yellow foam: [α]22D −25.4 (c 1.08, THF); R = 0.53
+
9
f
(
hexane/EtOAc = 1/3); IR (neat) 2955, 2870, 1717, 1635, 1603,
C H N O Na, 513.2724).
30
38
2
4
−
1
1
464, 1386, 1330, 1264, 1221, 1188, 1066, 1026, 975, 772, 694 cm ;
(20S)-3α-Hydroxy-20-methyl-5α-pregn-7-en-21-yl Pivalate (7).
1
H NMR (600 MHz, CDCl ) δ 8.17−8.14 (m, 2H), 7.72−7.67 (m,
LiAlH (233 mg, 6.13 mmol) was added to a solution of diol S2
3
4
2
H), 6.59 (d, J = 8.4 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H), 5.26 (dd, J =
5.6, 7.2 Hz, 1H), 5.18 (dd, J = 15.6, 7.2 Hz, 1H), 4.02 (d, J = 18.0 Hz,
H), 3.47 (dd, J = 12.0, 6.0 Hz, 1H), 2.80 (d, J = 18.0 Hz, 1H), 2.61−
.57 (m, 1H), 2.48−2.41 (m, 1H), 2.15−1.32 (m, 15H), 1.10 (s, 3H),
.04 (d, J = 6.0 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.86 (s, 3H), 0.85
(792 mg, 1.61 mmol) in dry THF (23 mL) at 0 °C. After stirring at 80
°C for 2 h, the reaction mixture was cooled to 0 °C and treated
1
1
2
1
sequentially with H
O (0.75 mL). The resultant solution was diluted with Et
2
O (0.25 mL), 2 M aqueous NaOH (0.50 mL), and
2
H
O and
2
stirred for 10 min. The cake was filtered through a pad of Celite, and
the filtrate was concentrated under reduced pressure. The crude diene
6 (594 mg) was used in the next reaction without further purification:
13
(
d, J = 6.6 Hz, 3H), 0.83 (d, J = 7.2 Hz, 3H); C NMR (150 MHz,
CDCl ) δ 204.6, 159.9, 156.9, 135.7, 135.3, 133.2, 132.9, 132.5, 131.6,
3
1
4
1
30.9, 129.2, 127.2, 127.1, 70.2, 67.5, 56.0, 51.1, 50.6, 44.8, 44.6, 42.9,
1.5, 39.9, 38.6, 36.2, 34.3, 33.2, 27.7, 24.3, 23.8, 21.3, 20.1, 19.8, 17.9,
7.6, 13.5; HRESIMS m/z 577.3408 [M + Na] (calcd for
R = 0.48 (hexane/EtOAc = 1/2); HRESIMS m/z 353.2461 [M +
f
+
Na] (calcd for C22H O Na, 353.2451).
34 2
+
A solution of crude diene 6 (594 mg) and tert-amyl alcohol (1.05
mL, 9.66 mmol) in dry THF (20 mL + 10 + 4 mL rinse) was added to
C H N O Na, 577.3401).
3
6
46
2
3
(
22E)-5α,8α-(1,4-Dioxo-1,2,3,4-tetrahydrophthalazine-2,3-diyl)-
a stirred deep-blue solution of Li (113 mg, 16.3 mmol) in liquid NH
3
ergosta-6,22-dien-3α-ol (5). L-Selectride (1.0 M in THF, 0.235 mL,
(20 mL) at −78 °C. After stirring at −78 °C for 1 h, the reaction
0
.235 mmol) was added to a solution of ketone S1 (99.5 mg, 0.179
mmol) in dry THF (1.6 mL) at −78 °C. After stirring at −78 °C for
0 min, the reaction was quenched with 2 M aqueous NaOH (0.36
mL, 0.72 mmol) and H O (30% in H O, 51 μL, 0.45 mmol). The
mixture was quenched with solid NH Cl (8.65 g, 162 mmol), and the
4
mixture was stirred at rt for 2 h. After addition of saturated aqueous
3
NH Cl and H O, the reaction mixture was extracted with THF. The
4
2
organic layer was washed with saturated aqueous NaCl, dried over
2
2
2
resultant solution was stirred at rt for 20 min and extracted with Et O.
anhydrous Na SO , filtered, and concentrated under reduced pressure.
2
2
4
The organic layer was washed with saturated aqueous NaCl, dried over
The crude olefin S3 (683 mg) was used in the next reaction without
further purification.
anhydrous Na SO , filtered, and concentrated under reduced pressure.
2
4
The residue was purified by flash column chromatography (silica gel,
Pivaloyl chloride (0.48 mL, 3.9 mmol) was added to a solution of
crude olefin S3 (683 mg) in pyridine (11 mL) and dry CH Cl (8.0
hexane/EtOAc = 5/1 → 1/1) to give olefin 5 (65.7 mg, 0.118 mmol,
2
2
6
6%) as a yellow foam: [α]24D +271 (c 1.06, CHCl ); R = 0.58
mL) at 0 °C. After stirring at 0 °C for 2 h, the reaction mixture was
3
f
(
hexane/EtOAc = 1/2); IR (neat) 3384, 2956, 2870, 1653, 1586,
quenched with saturated aqueous NaHCO and extracted with EtOAc.
3
−1 1
1
457, 1370, 1220, 1159, 1068, 971, 873, 772 cm ; H NMR (600
The organic layer was washed with saturated aqueous KHSO and
4
MHz, CDCl ) δ 8.14−8.08 (m, 2H), 7.69−7.63 (m, 2H), 6.63 (d, J =
saturated aqueous NaCl, dried over anhydrous Na SO , filtered, and
3
2
4
8
5
1
3
1
.3 Hz, 1H), 6.27 (d, J = 8.3 Hz, 1H), 5.22 (dd, J = 15.1, 7.6 Hz, 1H),
.15 (dd, J = 15.1, 8.3 Hz, 1H), 4.31 (d, J = 16.5 Hz, 1H), 4.13 (brs,
H), 4.00 (dd, J = 11.7, 6.9 Hz, 1H), 2.85 (s, 1H), 2.26 (dd, J = 16.5,
.4 Hz, 1H), 2.16 (dd, J = 13.1, 4.8 Hz, 1H), 2.07−1.97 (m, 2H),
.95−1.80 (m, 4H), 1.66−1.57 (m, 2H), 1.50−1.28 (m, 8H), 1.02 (d, J
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, hexane/EtOAc = 1/0 → 1/1)
to give pivalate ester 7 (508 mg, 1.22 mmol, 76% for the three steps)
as a colorless solid: [α]23 +8.80 (c 1.04, CHCl ); mp 140−141 °C; R
D
3
f
= 0.43 (hexane/EtOAc = 3/1); IR (neat) 3278, 2936, 2873, 1730,
1479, 1445, 1397, 1383, 1366, 1284, 1160, 1029, 1007, 979, 943, 905,
=
6.2 Hz, 3H), 0.99 (s, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.84 (s, 3H),
.83 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H); 13C NMR (150
MHz, CDCl ) δ 165.0, 159.8, 138.9, 135.4, 132.9, 132.8, 132.3, 130.8,
−1
1
0
848, 794, 770 cm ; H NMR (600 MHz, CDCl ) δ 5.19−5.16 (m,
3
1H), 4.08−4.04 (m, 2H), 3.77 (dd, J = 11.0, 7.6 Hz, 1H), 2.03−1.99
(m, 1H) 1.90−1.79 (m, 2H), 1.79−1.49 (m, 12H), 1.49−1.39 (m,
3
1
4
1
30.3, 129.0, 127.0, 126.9, 68.3, 65.8, 64.6, 56.7, 50.9, 48.6, 44.2, 42.8,
1.5, 40.0, 39.3, 33.2, 32.3, 31.9, 28.3, 27.9, 24.4, 21.9, 21.0, 20.0, 19.8,
8.6, 17.6, 13.3; HRESIMS m/z 579.3557 [M + Na]+ (calcd for
4H), 1.39−1.22 (m, 3H), 1.20 (s, 9H), 1.02 (d, J = 6.9 Hz, 3H) 0.78
13
(s, 3H), 0.56 (s, 3H); C NMR (150 MHz, CDCl ) δ 178.8, 139.2,
3
C H N O Na 579.3557).
118.0, 69.4, 66.6, 54.9, 53.0, 49.6, 43.7, 39.6, 39.0, 36.4, 35.7, 34.87,
3
6
48
2
3
(
20S)-5α,8α-(1,4-Dioxo-1,2,3,4-tetrahydrophthalazine-2,3-diyl)-
34.78, 32.1, 29.7, 28.9, 27.5, 27.4 (3C), 23.1, 21.4, 17.4, 12.2, 12.1;
+
2
0-methylpregn-6-ene-3α,21-diol (S2). O was bubbled through a
HRESIMS m/z 439.3178 [M + Na] (calcd for C H O Na,
3
27 44
3
solution of olefin 5 (701 mg, 1.26 mmol) and pyridine (0.84 mL, 10
mmol) in dry CH Cl (32 mL) at −60 °C for 20 min. O was then
439.3183).
2
2
2
(20S)-3α-(4-Methoxybenzyl)oxy-20-methyl-5α-pregn-7-en-21-yl
Pivalate (8). La(OTf) (42.3 mg, 0.0722 mmol) was added to a
bubbled through the solution for 10 min to remove excess O . NaBH
3
4
3
(
383 mg, 10.1 mmol) and MeOH (30 mL) were added to the solution.
solution of 4-methoxybenzyl-2,2,2-trichloroacetimidate (300 mg, 1.06
mmol) and alcohol 7 (298 mg, 0.714 mmol) in dry toluene (7.2 mL)
at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was
After stirring at −60 °C for 1.5 h, the reaction mixture was warmed to
rt and stirred for 40 min. The reaction was quenched with saturated
aqueous NH Cl at 0 °C. The mixture was extracted with Et O, and the
quenched with saturated aqueous NaHCO and then extracted with
4
2
3
combined organic layer was washed with saturated aqueous KHSO4,
EtOAc. The organic layer was washed with saturated aqueous NaCl,
saturated aqueous NaHCO , and saturated aqueous NaCl. The organic
dried over anhydrous Na SO , filtered, and concentrated under
reduced pressure. The residue was purified by flash column
3
2
4
layer was dried over anhydrous Na SO , filtered, and concentrated
2
4
under reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 2/1 → 1/5) to give diol
chromatography (silica gel, hexane/EtOAc = 1/0 → 1/1) to give
2
2
olefin 8 (322 mg, 0.600 mmol, 83%) as a colorless foam: [α] +16.4
D
S2 (438 mg, 0.893 mmol, 71%) as a yellow powder: [α]28 −14.7 (c
(c 1.23, CHCl ); R = 0.45 (hexane/EtOAc = 7/1); IR (neat) 2933,
D
3
f
1
2
1
8
.45, CHCl ); R = 0.20 (hexane/EtOAc = 1/2); IR (neat) 3451, 2947,
2872, 1723, 1613, 1586, 1513, 1463, 1397, 1362, 1283, 1246, 1161,
3
f
−1 1
874, 1634, 1600, 1461, 1438, 1391, 1317, 1267, 1217, 1170, 1101,
1072, 1036, 977, 942, 823, 771 cm ; H NMR (600 MHz, CDCl ) δ
3
−1 1
067, 1040, 1004, 975, 931, 906 cm ; H NMR (600 MHz, CDCl ) δ
7.26 (d, J = 7.8 Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H), 5.19−5.16 (m, 1H),
4.41 (s, 2H), 4.07 (dd, J = 10.8, 3.0 Hz, 1H), 3.81−3.76 (m, 4H), 3.65
(brs, 1H), 2.03−1.98 (m, 1H), 1.90−1.75 (m, 5H), 1.75−1.64 (m,
3
.15−8.07 (m, 2H), 7.71−7.64 (m, 2H), 6.65 (d, J = 8.3 Hz, 1H), 6.27
(
d, J = 8.3 Hz, 1H), 4.31 (brd, J = 15.8 Hz, 1H), 4.14 (brs, 1H), 4.01
G
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
4
=
H), 1.64−1.40 (m, 6H), 1.40−1.22 (m, 5H), 1.21 (s, 9H), 1.03 (d, J
aqueous NaCl, dried over anhydrous Na SO , filtered, and
2
4
13
7.2 Hz, 3H), 0.79 (s, 3H), 0.57 (s, 3H); C NMR (150 MHz,
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, hexane/EtOAc = 7/1 → 1/1)
CDCl ) δ 178.7, 159.0, 139.3, 131.6, 128.9 (2C), 118.0, 113.8 (2C),
3
2
4
D
7
3
2.9, 69.4, 69.2, 55.3, 54.9, 52.9, 49.5, 43.6, 39.6, 39.0, 36.4, 35.2, 34.7,
2.7, 32.6, 29.7, 27.5, 27.4 (3C), 25.8, 23.1, 21.3, 17.4, 12.4, 12.0;
to give diol 3c (456 mg, 0.799 mmol, 85%) as a colorless solid: [α]
+20.1 (c 1.03, CHCl ); mp 163−164 °C; R = 0.31 (hexane/EtOAc =
3
f
+
HRESIMS m/z 559.3783 [M + Na] (calcd for C H O Na,
5
3/1); IR (neat) 3533, 2933, 2870, 1725, 1613, 1513, 1458, 1397, 1362,
3
5
52
4
−1 1
59.3758).
1286, 1247, 1169, 1057, 1036, 977, 946, 825, 774, 756 cm ; H NMR
(
20S)-7α,8α-Dihydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-5α-
(600 MHz, CDCl ) δ 7.26 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz,
3
pregnan-21-yl Pivalate (3b). A solution of olefin 8 (51.0 mg, 95.1
μmol) in CH Cl (0.05 mL + 0.05 mL rinse) was added to a stirred
mixture of K OsO ·2H O (1.9 mg, 5.2 μmol), DABCO (1.6 mg, 14
μmol), K Fe(CN) (91.3 mg, 0.277 mmol), K CO (39.6 mg, 0.287
2
H), 4.43 (d, J = 11.4 Hz, 1H), 4.39 (d, J = 11.4 Hz, 1H), 4.04 (dd, J =
2
2
10.2, 3.6 Hz, 2H), 3.80 (s, 3H), 3.76 (dd, J = 10.8, 7.2 Hz, 1H), 3.63−
3.59 (m, 1H), 3.47 (dd, J = 11.4, 4.8 Hz, 1H), 2.06 (br, 1H), 2.02 (dt, J
= 12.4, 3.4 Hz, 1H), 1.86−1.76 (m, 2H), 1.76−1.67 (m, 2H), 1.67−
1.49 (m, 6H), 1.49−1.40 (m, 3H), 1.40−1.32 (m, 1H), 1.32−1.27 (m,
2
4
2
3
6
2
3
mmol), and MeSO NH (26.6 mg, 0.280 mmol) in t-BuOH/H O
2
2
2
(
1:1, v/v, 0.94 mL) at 0 °C. After stirring at rt for 36 h, the reaction
1
H), 1.27−1.22 (m, 1H), 1.22−1.15 (m, 2H), 1.20 (s, 9H), 1.10 (ddd,
mixture was quenched with solid Na S O at 0 °C and stirred at rt for
1
layer was washed with saturated aqueous NaCl, dried over anhydrous
Na SO , filtered, and concentrated under reduced pressure. The
2
2
3
J = 10.3, 9.6, 9.6 Hz, 1H), 0.99 (d, J = 6.6 Hz, 3H), 0.97 (s, 3H), 0.94
(s, 3H), 0.90 (dd, J = 13.1, 2.8 Hz, 1H); C NMR (150 MHz, CDCl3)
0 h. The reaction mixture was extracted with Et O, and the organic
13
2
δ 178.9, 159.1, 131.5, 129.1 (2C), 113.9 (2C), 76.2, 76.1, 72.9, 69.5,
69.4, 58.7, 55.6, 55.4, 52.7, 44.3, 41.1, 39.1, 37.4, 35.7, 35.5, 34.3, 33.7,
2
4
residue was purified by flash column chromatography (silica gel,
3
5
2.6, 27.8, 27.4 (3C), 25.3, 22.7, 18.0, 17.1, 13.8, 11.6; HRESIMS m/z
hexane/EtOAc = 3/1 → 1/3 with addition of 1 v/v % Et N) to give
+
3
93.3808 [M + Na] (calcd for C H O Na, 593.3813).
diol 3b (42.5 mg, 74.5 μmol, 80%) as a colorless solid: [α]2 −14.7 (c
3
35 54
6
D
(
20S)-8β-Hydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-7-oxo-
1
.56, CHCl ); mp 157−158 °C; R = 0.17 (hexane/EtOAc = 3/1); IR
3
f
5α-pregnan-21-yl Pivalate (10). DMSO (0.15 mL, 2.1 mmol), Et N
3
(
neat) 3274, 2934, 2853, 1726, 1613, 1512, 1480, 1459, 1397, 1362,
(
0.15 mL, 1.1 mmol), and SO ·pyridine (103 mg, 0.627 mmol) were
3
−1 1
1
284, 1245, 1151, 1075, 1037, 973, 949, 827, 771 cm ; H NMR (600
added to a solution of diol 3c (69.9 mg, 0.122 mmol) in dry CH Cl
2
2
MHz, CDCl ) δ 7.26 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H),
3
(1.2 mL) at 0 °C. After stirring at rt for 13 h, the reaction mixture was
4
1
3
.42 (d, J = 12.4 Hz, 1H), 4.41 (d, J = 12.4 Hz, 1H), 4.14−4.09 (m,
H), 4.04 (dd, J = 10.8, 3.6 Hz, 1H), 3.82−3.77 (m, 1H), 3,79 (s, 3H),
.61 (brs, 1H), 2.96−2.88 (m, 1H), 2.13−2.03 (m, 1H), 1.99−1.92
quenched with saturated aqueous NH Cl at 0 °C and extracted with
4
Et O. The organic layer was washed with saturated aqueous KHSO
2
4
and saturated aqueous NaCl, dried over anhydrous Na SO , filtered,
2
4
(
m, 2H), 1.84−1.66 (m, 6H), 1.60−1.53 (m, 4H), 1.52−1.42 (m, 3H),
.37−1.13 (m, 6H), 1.20 (s, 9H), 1.01 (d, J = 6.0 Hz, 3H), 0.85 (s,
and concentrated under reduced pressure. The residue was purified by
column chromatography (silica gel, hexane/EtOAc = 10/1 → 1/1) to
give hydroxy ketone 10 (59.1 mg, 0.104 mmol, 85%) as a colorless
1
3
1
5
2
13
H), 0.79 (s, 3H); C NMR (150 MHz, CDCl ) δ 178.8, 159.0,
3
31.7, 129.0 (2C), 113.8 (2C), 78.5, 72.6, 70.4, 69.3 (2C), 61.7, 55.7,
5.4, 53.7, 43.3, 40.3, 39.1, 38.5, 36.5, 36.2, 36.0, 32.3, 31.0, 27.4 (3C),
24
foam: [α] −17.0 (c 1.17, CHCl ); R = 0.35 (hexane/EtOAc = 3/
D
3
f
1
1
); IR (neat) 3502, 2934, 1712, 1612, 1513, 1462, 1397, 1362, 1340,
285, 1247, 1168, 1063, 1036, 975, 946, 824, 754, 666 cm ; H NMR
7.1, 25.8, 23.9, 22.9, 17.3, 15.0, 12.1; HRESIMS m/z 593.3801 [M +
−1 1
+
Na] (calcd for C H O Na, 593.3813).
35
54
6
(
600 MHz, CDCl ) δ 7.22 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz,
3
(
5R,20S)-3α-(4-Methoxybenzyl)oxy-20-methyl-7,8-dioxo-7,8-
2
3
2
4
1
3
H), 4.37 (s, 2H), 4.05 (dd, J = 10.2, 3.0 Hz, 1H), 3.78 (s, 3H), 3.78−
.74 (m, 1H), 3.62 (brs, 1H), 3.04 (dd, J = 14.4, 12.0 Hz, 1H), 2.11−
.04 (m, 1H), 2.02−1.93 (m, 2H), 1.93−1.85 (m, 1H), 1.85−1.75 (m,
H), 1.74−1.66 (m, 1H), 1.66−1.51 (m, 5H), 1.49 (brd, J = 13.1 Hz,
H), 1.39−1.31 (m, 1H), 1.30−1.08 (m, 5H), 1.19 (s, 9H), 1.15 (s,
seco-5α-pregnan-21-yl Pivalate (9). Pb(OAc) (1.29 g, 2.33 mmol)
was added to a solution of diol 3b (838 mg, 1.47 mmol) in dry CH Cl2
4
2
(
15 mL) at 0 °C. After stirring at rt for 35 min, the reaction mixture
was quenched with H O at 0 °C and filtered through a pad of Celite.
The filtrate was extracted with Et O, and the organic layer was washed
with saturated aqueous NaHCO and saturated aqueous NaCl, dried
over anhydrous Na SO , filtered, and concentrated under reduced
2
2
13
H), 0.99 (d, J = 6.0 Hz, 3H), 0.94 (s, 3H); C NMR (150 MHz,
3
CDCl ) δ 211.7, 178.7, 159.0, 131.2, 128.9 (2C), 113.8 (2C), 81.4,
3
2
4
7
3
2.7, 69.5, 69.2, 58.9, 55.3, 52.4, 52.1, 43.3, 43.1, 41.7, 39.9, 39.0, 36.9,
pressure. The residue was purified by flash column chromatography
5.3, 33.9, 32.8, 27.3 (4C), 25.3, 19.9, 17.8, 17.1, 14.0, 11.5; HRESIMS
(
silica gel, hexane/EtOAc = 2/1 → 0/1) to give ketoaldehyde 9 (773
+
24
m/z 591.3656 [M + Na] (calcd for C H O Na, 591.3656).
mg, 1.36 mmol, 92%) as a colorless foam: [α]
CHCl ); R = 0.49 (hexane/EtOAc = 2/1); IR (neat) 2958, 2873,
+2.7 (c 0.82,
35 52
6
D
(
20S)-7α,8β-Dihydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-5α-
3
f
pregnan-21-yl Pivalate (3a). NaBH(OAc) (827 mg, 3.12 mmol) was
3
1
9
3
4
723, 1613, 1513, 1458, 1397, 1364, 1285, 1247, 1159, 1079, 1035,
76, 937, 823, 754 cm ; H NMR (600 MHz, CDCl ) δ 9.69 (dd, J =
.4, 1.4 Hz, 1H), 7.25 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H),
.41 (s, 2H), 4.04 (dd, J = 10.8, 3.6 Hz, 1H), 3.81−3.76 (m, 1H), 3.79
−1
1
added to a solution of hydroxy ketone 10 (59.1 mg, 0.104 mmol) and
3
i-Pr NEt (2.5 mL, 14 mmol) in dry THF (5.2 mL) at 0 °C. After
2
stirring at rt for 15 h, the reaction mixture was quenched with
saturated aqueous NH Cl at 0 °C and extracted with EtOAc. The
4
(
s, 3H), 3.53 (brs, 1H), 2.66 (brs, 1H), 2.47−2.37 (m, 3H), 2.17−2.12
organic layer was washed with saturated aqueous NaCl, dried over
(
m, 1H), 2.09−1.99 (m, 2H), 1.92−1.82 (m, 2H), 1.82−1.67 (m, 4H),
anhydrous Na SO , filtered, and concentrated under reduced pressure.
1
1
2
6
2
5
.67−1.58 (m, 4H), 1.54−1.36 (m, 4H), 1.20 (s, 9H), 1.03 (s, 3H),
2
4
.02 (d, J = 7.6 Hz, 3H), 0.61 (s, 3H); 13C NMR (150 MHz, CDCl ) δ
The residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 10/1 → 1/3) to give diol 3a (55.3 mg, 0.0969 mmol,
3
10.7, 203.1, 178.7, 159.1, 131.2, 129.1 (2C), 113.9 (2C), 72.1, 69.5,
9.0, 63.1, 55.4 (2C), 53.5 (2C), 50.6, 44.3, 39.3, 39.0, 36.9, 35.7, 32.1,
7.5, 27.3 (3C), 26.2 (2C), 25.3, 19.3, 17.8, 17.3, 12.4; HRESIMS m/z
9
3%) as a colorless foam: [α]22 +6.82 (c 1.11, CHCl ); R = 0.12
D 3 f
(hexane/EtOAc = 3/1); IR (neat) 3525, 2936, 1714, 1613, 1513,
+
1458, 1397, 1362, 1287, 1246, 1169, 1124, 1065, 1034, 971, 943, 897,
91.3659 [M + Na] (calcd for C H O Na, 591.3656).
35
52
6
−1 1
8
21, 756 cm ; H NMR (600 MHz, CDCl ) δ 7.27 (d, J = 8.4 Hz,
3
(
20S)-7β,8β-Dihydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-5α-
pregnan-21-yl Pivalate (3c). Diiodomethane (1.30 mL, 16.2 mmol)
was added dropwise to a vigorously stirred mixture of Sm metal (3.01
g, 20.0 mmol, treated in a glovebox) and dry THF (200 mL) at 0 °C.
The mixture was vigorously stirred at 0 °C for 10 min and then at rt
2H), 6.87 (d, J = 8.4 Hz, 2H), 4.45 (d, J = 11.4 Hz, 1H), 4.40 (d, J =
11.4 Hz, 1H), 4.06 (dd, J = 10.8, 3.0 Hz, 1H), 3.80 (s, 3H), 3.75 (dd, J
= 10.2, 7.2 Hz, 1H), 3.63 (brs, 1H), 3.48 (brs, 1H), 2.07−2.02 (m,
1H), 1.98 (dt, J = 13.2, 3.6 Hz, 1H), 1.92 (td, J = 13.2, 1.8 Hz, 1H),
1.91−1.84 (m, 1H), 1.80 (brd, J = 13.8 Hz, 1H), 1.75−1.68 (m, 2H),
1.68−1.54 (m, 4H), 1.48−1.42 (m, 3H), 1.41−1.21 (m, 8H), 1.20 (s,
9H), 1.13 (dt, J = 14.4, 2.8 Hz, 1H), 0.99 (d, J = 6.0 Hz, 3H), 0.95 (s,
for 3 h to give a deep-blue suspension of SmI (ca. 0.08 M in THF).
2
This suspension (47.0 mL, 3.76 mmol) was added to a solution of
ketoaldehyde 9 (537 mg, 0.945 mmol) in freshly distilled THF (47
mL) and MeOH (0.26 mL) at 0 °C. After stirring at 0 °C for 45 min,
6H); 13C NMR (150 MHz, CDCl ) δ 178.9, 159.1, 131.7, 129.1 (2C),
3
the reaction was quenched with saturated aqueous NH Cl and
113.9 (2C), 76.5, 73.1, 72.6, 69.5, 69.3, 55.4, 53.4, 53.2, 49.8, 43.2,
40.4, 39.1, 36.4, 35.5, 33.5, 32.5 (2C), 32.4, 27.4 (3C), 27.2, 25.4, 18.7,
4
extracted with Et O. The organic layer was washed with saturated
2
H
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
+
1
7.8, 17.0, 13.7, 11.5; HRESIMS m/z 593.3806 [M + Na] (calcd for
1H), 3.89 (brd, J = 8.3 Hz, 1H), 3.79 (s, 3H), 3.78 (dd, J = 10.3, 6.9
Hz, 1H), 3.63 (brs, 1H), 2.69−2.61 (m, 1H), 2.04 (ddd, J = 14.4, 8.3,
6.2 Hz, 1H), 1.94 (ddd, J = 12.4, 3.4, 3.4 Hz, 1H), 1.89−1.81 (m, 1H),
1.80−1.73 (m, 3H), 1.72−1.65 (m, 2H), 1.61 (dd, J = 13.8, 6.9 Hz,
1H), 1.58−1.50 (m, 4H), 1.47−1.34 (m, 4H), 1.34−1.20 (m, 4H),
1.20 (s, 9H), 1.17−1.09 (m, 1H), 1.03 (s, 3H), 1.01 (d, J = 6.2 Hz,
C H O Na, 593.3813).
3
5
54
6
(
20S)-3α-(4-Methoxybenzyl)oxy-20-methyl-5α-pregnane-
α,8β,21-triol (11). DIBALH (1.0 M in toluene, 0.80 mL, 0.80 mmol)
was added to a solution of pivalate 3a (55.3 mg, 0.0969 mmol) in dry
CH Cl (1.0 mL) at −78 °C. After stirring at −78 °C for 1 h, the
reaction mixture was warmed to rt. After stirring at rt for 1 h, the
reaction mixture was quenched with MeOH at 0 °C. After addition of
Et O and saturated aqueous Na /K tartrate, the mixture was stirred at
rt for 1 h and then extracted with EtOAc. The organic layer was
washed with saturated aqueous NaCl, dried over anhydrous Na SO ,
7
2
2
3H), 0.90 (s, 3H); 13C NMR (150 MHz, CDCl ) δ 178.9, 159.0,
3
131.7, 129.0 (2C), 113.8 (2C), 76.7, 73.0, 69.30, 69.25, 69.17, 62.8,
55.4, 55.2, 54.4, 43.4, 40.0, 39.1, 37.2, 36.9, 36.1, 35.0, 32.6, 30.3, 27.4
(3C), 26.9, 25.9, 23.9, 20.6, 17.1, 14.0, 13.5; HRESIMS m/z 593.3820
+
+
2
+
2
4
[M + Na] (calcd for C H O Na, 593.3813).
35 54
6
filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (silica gel, hexane/EtOAc =
(20S)-8β-Hydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-7α-
(triethylsilyl)oxy-5α-pregnan-21-yl Pivalate (S4). TESOTf (0.16 mL,
0.71 mmol) was added to a solution of diol 3a (199 mg, 0.348 mmol)
and 2,6-lutidine (0.16 mL, 1.4 mmol) in dry CH Cl (3.5 mL) at 0 °C.
2
/1 → 1/3) to give triol 11 (42.7 mg, 0.0877 mmol, 91%) as a
23
colorless solid: [α] +7.51 (c 1.42, CHCl ); mp 188−189 °C; R =
D
3
f
2
2
0
1
7
2
1
.22 (hexane/EtOAc = 1/1); IR (neat) 3416, 2934, 1612, 1586, 1512,
443, 1361, 1302, 1246, 1171, 1145, 1125, 1032, 990, 968, 896, 822,
After stirring at rt for 1.5 h, the reaction mixture was quenched with
saturated aqueous NaHCO at 0 °C and extracted with EtOAc. The
3
−1 1
55, 666 cm ; H NMR (600 MHz, CDCl ) δ 7.26 (d, J = 8.4 Hz,
organic layer was washed with saturated aqueous NaCl, dried over
3
H), 6.87 (d, J = 8.4 Hz, 2H), 4.45 (d, J = 11.4 Hz, 1H), 4.39 (d, J =
1.4 Hz, 1H), 3.80 (s, 3H), 3.63−3.59 (m, 2H), 3.46 (brs, 1H), 3.33
anhydrous Na SO , filtered, and concentrated under reduced pressure.
2
4
The residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 30/1 → 10/1) to give silyl ether S4 (220 mg, 0.321
mmol, 92%) as a colorless foam: [α]23 +2.21 (c 1.00, CHCl ); R =
(
dd, J = 10.2, 7.2 Hz, 1H), 2.07−2.01 (m, 1H), 1.98 (dt, J = 12.4, 3.4
Hz, 1H), 1.90 (td, J = 13.8, 2.8 Hz, 1H), 1.89−1.83 (m, 1H), 1.80
D
3
f
(
brd, J = 15.1 Hz, 1H), 1.77−1.49 (m, 8H), 1.49−1.40 (m, 3H), 1.38−
0.57 (hexane/EtOAc = 4/1); IR (neat) 3527, 2937, 2843, 1727, 1614,
1513, 1458, 1397, 1363, 1285, 1246, 1168, 1129, 1086, 1036, 1015,
1
0
1
.15 (m, 7H), 1.12 (dt, J = 13.8, 2.8 Hz, 1H), 1.01 (d, J = 7.2 Hz, 3H),
.94 (s, 3H), 0.93 (s, 3H); 13C NMR (150 MHz, CDCl ) δ 159.1,
974, 946, 824, 792, 772, 741 cm ; H NMR (600 MHz, CDCl ) δ
−1 1
3
3
31.6, 129.1 (2C), 113.9 (2C), 76.5, 73.1, 72.5, 69.5, 67.9, 55.4, 53.1
7.26 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 4.44 (d, J = 11.4 Hz,
1H), 4.37 (d, J = 11.4 Hz, 1H), 4.00 (dd, J = 10.2, 2.4 Hz, 1H), 3.82
(dd, J = 10.8, 7.2 Hz, 1H), 3.80 (s, 3H), 3.60 (brs, 1H), 3.48 (brs, 1H),
2.21 (brt, J = 13.1 Hz, 1H), 1.96 (dt, J = 12.4, 2.8 Hz, 1H), 1.86−1.66
(m, 5H), 1.65−1.56 (m, 2H), 1.56−1.49 (m, 2H), 1.46−1.40 (m, 2H),
1.40−1.23 (m, 5H), 1.23−1.16 (m, 2H), 1.20 (s, 9H), 1.08 (dt, J =
14.4, 2.8 Hz, 1H), 1.01−0.99 (m, 4H), 0.934 (t, J = 8.3 Hz, 9H), 0.930
(
1
2C), 49.8, 43.2, 40.4, 38.3, 36.4, 33.5, 32.5, 32.42, 32.38, 27.3, 25.3,
8.6, 17.7, 16.6, 13.7, 11.5; HRESIMS m/z 509.3239 [M + Na] (calcd
+
for C H O Na, 509.3237).
30
46
5
(
20S)-8α-Hydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-7-oxo-
5
α-pregnan-21-yl Pivalate (12). i-Pr NEt (0.063 mL, 0.36 mmol) and
2
SO ·pyridine (28.6 mg, 0.179 mmol) were added to a solution of diol
3
13
3
b (20.5 mg, 0.0359 mmol) in dry CH Cl /DMSO (4:1, v/v, 0.90
(s, 3H), 0.92 (s, 3H), 0.65−0.54 (m, 6H); C NMR (150 MHz,
2
2
mL) at 0 °C. After stirring at rt for 12 h, the reaction mixture was
quenched with water at 0 °C and extracted with EtOAc. The organic
layer was washed with water and saturated aqueous NaCl, dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure.
CDCl ) δ 178.8, 158.9, 131.9, 128.7 (2C), 113.7 (2C), 77.2, 73.2, 73.1,
3
69.4, 69.2, 55.4, 53.4, 52.8, 49.3, 42.9, 40.5, 39.1, 36.4, 35.4, 33.6, 32.7,
32.0, 31.9, 27.3 (3C), 27.0, 26.1, 18.6, 17.7, 17.2, 13.7, 11.6, 7.2 (3C),
+
2
4
5.5 (3C); HRESIMS m/z 707.4700 [M + Na] (calcd for
The residue was purified by column chromatography (silica gel,
C H O SiNa, 707.4677).
41
68
6
hexane/EtOAc = 9/1 → 4/1 with 1% v/v Et N) to give hydroxy
(20S)-3α-(4-Methoxybenzyl)oxy-20-methyl-7α-(triethylsilyl)oxy-
3
29
ketone 12 (15.5 mg, 0.0273 mmol, 76%) as a colorless foam: [α]
5α-pregnane-8β,21-diol (S5). DIBALH (1.0 M in toluene, 1.7 mL, 1.7
mmol) was added to a solution of silyl ether S4 (136 mg, 0.199 mmol)
in dry CH Cl (2.0 mL) at −78 °C. After stirring at −78 °C for 40
D
−
14.3 (c 0.77, benzene); R = 0.31 (EtOAc/hexane = 1/3); IR (neat)
f
3
1
342, 2969, 2934, 2912, 2868, 1726, 1712, 1612, 1513, 1285, 1244,
2
2
−1 1
167, 1088, 1036 cm ; H NMR (600 MHz, C D ) δ 7.31 (d, J = 8.3
min, the reaction mixture was warmed to rt. After stirring at rt for 20
6
6
Hz, 2H), 6.86 (d, J = 8.9 Hz, 2H), 4.36 (d, J = 11.7 Hz, 1H), 4.33 (d, J
11.7 Hz, 1H), 4.08 (dd, J = 11.0, 3.4 Hz, 1H), 3.87 (dd, J = 11.0, 6.9
Hz, 1H), 3.59−3.52 (m, 1H), 3.48−3.45 (m, 1H), 3.33 (s, 3H), 2.59
dd, J = 17.2, 6.9 Hz, 1H), 2.27−2.17 (m, 1H), 1.80 (dd, J = 17.2, 11.7
min, the reaction mixture was quenched with MeOH at 0 °C. After
+
+
=
addition of Et O and saturated aqueous Na /K tartrate, the reaction
2
mixture was stirred for 1 h and then extracted with EtOAc. The
organic layer was washed with saturated aqueous NaCl, dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure.
(
Hz, 1H), 1.79−1.68 (m, 4H), 1.67−1.61 (m, 1H), 1.62−1.56 (m, 1H),
2
4
1
9
6
.55−1.44 (m, 2H), 1.43−1.36 (m, 2H), 1.34−1.23 (m, 4H), 1.22 (s,
The residue was purified by flash column chromatography (silica gel,
H), 1.16 (dd, J = 13.1, 7.6 Hz, 1H), 1.10−1.01 (m, 2H), 1.00 (d, J =
hexane/EtOAc = 7/1 → 1/1) to give diol S5 (119 mg, 0.198 mmol,
13
26
.9 Hz, 3H), 0.95−0.88 (m, 1H), 0.88 (s, 3H), 0.64 (s, 3H); C NMR
99%) as a colorless foam: [α]
+5.44 (c 1.02, CHCl ); R = 0.16
D 3 f
(
150 MHz, C D ) δ 210.0, 177.8, 159.7, 132.0, 129.2 (2C), 114.1
(hexane/EtOAc = 4/1); IR (neat) 3430, 2935, 2873, 1613, 1513,
1463, 1362, 1301, 1246, 1220, 1170, 1149, 1086, 1037, 973, 943, 825,
6
6
(
2C), 78.8, 72.5, 69.7, 69.2, 59.3, 58.8, 54.8, 53.7, 44.2, 43.1, 39.9, 39.0,
−1 1
3
1
5
6.5, 36.2, 35.5, 32.5, 32.4, 27.5 (3C), 27.4, 26.0, 22.9, 19.9, 17.4, 14.4,
772, 742, 675 cm ; H NMR (600 MHz, CDCl ) δ 7.26 (d, J = 8.3
3
+
3.7; HRESIMS m/z 591.3650 [M + Na] (calcd for C H O Na,
Hz, 2H), 6.86 (d, J = 8.3 Hz, 2H), 4.44 (d, J = 11.7 Hz, 1H), 4.37 (d, J
= 11.7 Hz, 1H), 3.80 (s, 3H), 3.63 (dd, J = 10.3, 3.4 Hz, 1H), 3.59
(brt, J = 2.8 Hz, 1H), 3.48 (brs, 1H), 3.34 (dd, J = 10.3, 7.2 Hz, 1H),
2.20 (tt, J = 13.1, 2.8 Hz, 1H), 1.96 (dt, J = 12.4, 3.4 Hz, 1H), 1.86−
1.69 (m, 4H), 1.66−1.49 (m, 6H), 1.46−1.40 (m, 2H), 1.40−1.22 (m,
6H), 1.18 (td, J = 12.4, 3.4 Hz, 1H), 1.12 (ddd, J = 10.3, 9.6, 9.6 Hz,
1H), 1.08 (dt, J = 13.8, 2.8 Hz, 1H), 1.02 (d, J = 6.2 Hz, 3H), 0.94 (t, J
35
52
6
91.3656).
(
20S)-7β,8α-Dihydroxy-3α-(4-methoxybenzyl)oxy-20-methyl-5α-
pregnan-21-yl Pivalate (3d). NaBH (15.5 mg, 0.410 mmol) was
4
added to a solution of hydroxy ketone 12 (15.5 mg, 0.0273 mmol) in
dry THF/MeOH (4:1, v/v, 1.0 mL) at 0 °C. After stirring at 0 °C for
1
.5 h, the reaction mixture was quenched with saturated aqueous
1
3
NH Cl and extracted with EtOAc. The organic layer was washed with
= 8.3 Hz, 9H), 0.93 (s, 3H), 0.92 (s, 3H), 0.65−0.54 (m, 6H); C
4
water and saturated aqueous NaCl, dried over anhydrous Na SO ,
NMR (150 MHz, CDCl ) δ 158.9, 131.9, 128.7 (2C), 113.7 (2C),
2
4
3
filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (silica gel, hexane/EtOAc =
77.3, 73.2, 73.1, 69.2, 68.0, 55.4, 53.2, 52.7, 49.3, 43.0, 40.5, 38.5, 36.4,
33.7, 32.7, 32.0, 31.9, 27.2, 26.1, 18.7, 17.7, 16.6, 13.7, 11.6, 7.2 (3C),
5.5 (3C); HRESIMS m/z 623.4099 [M + Na] (calcd for
+
9
8
/1 → 3/2 with 1% v/v Et N) to give diol 3d (13.9 mg, 0.0244 mmol,
3
29
9%) as a colorless solid: [α]
+5.8 (c 0.69, CHCl ); R = 0.18
C H O SiNa, 623.4102).
D
3
f
36 60
5
(
1
2
hexane/EtOAc = 3/1); IR (neat) 3333, 2918, 1710, 1513, 1236,
(20S)-8β-Hydroxy-3α-(4-methoxybenzyl)oxy-7α-(triethylsilyl)oxy-
5α-pregnane-20-carbaldehyde (13). DMSO (55 μL, 0.78 mmol),
Et N (55 μL, 0.39 mmol), and SO ·pyridine (37.5 mg, 0.229 mmol)
−1 1
165, 1052 cm ; H NMR (600 MHz, CDCl ) δ 7.25 (d, J = 8.9 Hz,
3
H), 6.85 (d, J = 8.9 Hz, 2H), 4.41 (s, 2H), 4.04 (dd, J = 11.0, 3.4 Hz,
3
3
I
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
were added to a solution of diol S5 (29.8 mg, 0.0496 mmol) in dry
CH Cl (0.50 mL) at 0 °C. After stirring at rt for 7 h, the reaction
0.0534 mmol) in H O (30% in H O, 0.60 mL) was added to a
2
2
2
solution of sulfide S6a (205 mg, 0.534 mmol) in EtOH (0.76 mL) at
rt. After stirring at rt for 5.5 h, the reaction mixture was extracted with
EtOAc. The organic layer was washed with saturated aqueous NaCl,
dried over anhydrous Na SO , filtered, and concentrated under
2
2
mixture was quenched with saturated aqueous NH Cl at 0 °C and
4
extracted with Et O. The organic layer was washed with saturated
2
aqueous KHSO and saturated aqueous NaCl, dried over anhydrous
4
2
4
Na SO , filtered, and concentrated under reduced pressure. The
reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 7/1 → 3/1) to give
2
4
residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 20/1 → 7/1) to give aldehyde 13 (24.8 mg, 0.0414
mmol, 84%) as a colorless foam: [α]22 +4.67 (c 1.08, CHCl ); R =
2
4
sulfone 14a (198 mg, 0.475 mmol, 89%) as a colorless foam: [α]
D
−6.90 (c 1.01, CHCl ); R = 0.54 (hexane/EtOAc = 2/1); IR (neat)
D
3
f
3
f
0
1
9
9
2
3
1
.57 (hexane/EtOAc = 3/1); IR (neat) 3545, 2936, 2873, 1722, 1613,
513, 1457, 1362, 1301, 1246, 1170, 1148, 1128, 1086, 1037, 1011,
3354, 2959, 1730, 1611, 1513, 1497, 1462, 1340, 1302, 1247, 1174,
−
1 1
1151, 1091, 1034, 918, 824, 770, 763, 689 cm ; H NMR (600 MHz,
CDCl
−
1 1
77, 943, 824, 791, 772, 742 cm ; H NMR (600 MHz, CDCl ) δ
3
) δ 7.68 (d, J = 7.2 Hz, 2H), 7.65−7.57 (m, 3H), 7.24 (d, J = 9.0
3
.56 (d, J = 2.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz,
H), 4.45 (d, J = 11.4 Hz, 1H), 4.37 (d, J = 11.4 Hz, 1H), 3.80 (s, 3H),
.60 (brs, 1H), 3.48 (brs, 1H), 2.36−2.30 (m, 1H), 2.25−2.17 (m,
H), 1.93 (dt, J = 12.4, 2.8 Hz, 1H), 1.87−1.79 (m, 2H), 1.79−1.73
Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 4.42 (s, 2H), 3.84−3.73 (m, 5H),
3.37 (dd, J = 9.6, 4.8 Hz, 1H), 3.26 (dd, J = 9.6, 7.2 Hz, 1H), 2.10−
2.02 (m, 1H), 2.00−1.92 (m, 1H), 1.90−1.82 (m, 1H), 0.98 (d, J = 7.2
Hz, 3H); 13C NMR (150 MHz, CDCl
) δ 159.4, 153.6, 133.2, 131.6,
3
(
1
3
m, 2H), 1.66−1.53 (m, 4H), 1.47−1.20 (m, 9H), 1.11−1.06 (m, 1H),
130.3, 129.8 (2C), 129.4 (2C), 125.2 (2C), 114.0 (2C), 74.5, 73.0,
+
.10 (d, J = 6.6 Hz, 3H), 0.95 (s, 3H), 0.94 (t, J = 7.6 Hz, 9H), 0.93 (s,
H), 0.65−0.55 (m, 6H); the hydroxy proton was not observed
55.4, 54.5, 32.7, 26.4, 17.0; HRESIMS m/z 439.1411 [M + Na] (calcd
for C20H N O SNa, 439.1410).
24 4 4
13
probably due to the rapid exchange with contaminating H O;
C
(25S)-3α,26-Bis(4-methoxybenzyl)oxy-7α-(triethylsilyl)oxy-5α-
cholest-22-en-8β-ol (15a). Aldehyde 13 (71.9 mg, 0.120 mmol) and
sulfone 14a (150 mg, 0.360 mmol) were dissolved in freshly distilled
THF (4.1 mL) and HMPA (1.2 mL) and cooled to −78 °C. A freshly
prepared solution of KHMDS (0.5 M in freshly distilled THF, 0.72
mL, 0.36 mmol) was added to the above solution, and the mixture was
stirred at −78 °C for 1 h. After stirring at rt for 1 h, the reaction
mixture was cooled to 0 °C and quenched with saturated aqueous
2
NMR (150 MHz, CDCl ) δ 205.3, 158.9, 131.9, 128.7 (2C), 113.7
3
(
2C), 77.2, 73.3, 73.0, 69.2, 55.4, 52.4, 51.6, 49.4, 49.3, 43.5, 40.4, 36.4,
3
5
3.7, 32.6, 32.0, 31.9, 26.6, 26.1, 19.0, 17.7, 14.0, 13.3, 11.7, 7.2 (3C),
.5 (3C); HRESIMS m/z 621.3944 [M + Na] (calcd for
+
C H O SiNa, 621.3946).
3
6
58
5
(
S)-2-Methyl-4-((1-phenyl-1H-tetrazol-5-yl)thio)butan-1-ol
1
(
16a). PPh (269 mg, 1.02 mmol) and 1-phenyl-1H-tetrazole-5-thiol
3
(
PTSH) (179 mg, 1.00 mmol) were added to a solution of (S)-(−)-2-
NH
combined organic layer was washed with saturated aqueous NaCl,
dried over anhydrous Na SO , filtered, and concentrated under
4
Cl. The resultant mixture was extracted with Et O, and the
2
methyl-1,4-butanediol (110 mg, 1.03 mmol) in dry THF (5.4 mL) at
rt. The solution was cooled to −78 °C, and Diisopropyl
azodicarboxylate (DIAD) (1.9 M in toluene, 0.56 mL, 1.06 mmol)
was added. After stirring at −78 °C for 1 h, the reaction mixture was
warmed to −20 °C over 1 h. After stirring at −20 °C for 2 h, the
reaction mixture was concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 5/1 → 1/3) to give sulfide 16a (65.7 mg, 0.249
mmol, 25%) as a colorless oil: [α]24 −8.02 (c 1.08, CHCl ); R = 0.55
2
4
reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 24/1 → 4/1) to give
olefin 15a (E/Z = 11:1, 48.2 mg, 0.0611 mmol, 51%) as a colorless
foam: [α]25
−15.0 (c 1.39, CHCl ); R = 0.48 (hexane/EtOAc = 5/
D
3 f
1); IR (neat) 3566, 2935, 2872, 1717, 1613, 1586, 1513, 1457, 1362,
1301, 1246, 1207, 1171, 1149, 1086, 1038, 1012, 974, 945, 908, 822,
792, 741, 724, 677 cm ; H NMR of E-isomer (600 MHz, CDCl ) δ
−1
1
D
3
f
3
(
hexane/EtOAc = 1/2); IR (neat) 3415, 2930, 2873, 1720, 1597,
7.28−7.24 (m, 4H), 6.89−6.84 (m, 4H), 5.29−5.17 (m, 2H), 4.45 (d, J
= 11.7 Hz, 1H), 4.42 (s, 2H), 4.37 (d, J = 11.7 Hz, 1H), 3.80 (s, 6H),
3.59 (brs, 1H), 3.48 (brs, 1H), 3.29 (dd, J = 8.9, 6.2 Hz, 1H), 3.21 (dd,
J = 8.9, 6.2 Hz, 1H), 2.23−2.18 (tt, J = 13.1, 2.8 Hz, 1H), 2.10−2.04
(m, 1H), 2.01−1.95 (m, 1H), 1.94 (dt, J = 12.4, 3.4 Hz, 1H), 1.86−
1.70 (m, 5H), 1.68−1.50 (m, 5H), 1.50−1.41 (m, 3H), 1.41−1.27 (m,
3H), 1.25−1.14 (m, 3H), 1.11−1.04 (m, 2H), 0.97 (d, J = 6.9 Hz,
3H), 0.94 (t, J = 7.6 Hz, 9H), 0.93 (s, 3H), 0.91 (s, 3H), 0.89 (d, J =
1
6
499, 1461, 1387, 1279, 1243, 1174, 1075, 1041, 1014, 983, 912, 762,
−1 1
93 cm ; H NMR (600 MHz, CDCl ) δ 7.57−7.52 (m, 5H), 3.60
3
(
(
1
dd, J = 11.0, 5.5 Hz, 1H), 3.53 (dd, J = 11.0, 6.9 Hz, 1H), 3.48−3.38
m, 2H), 2.14 (br, 1H, OH), 2.01−1.94 (m, 1H), 1.87−1.79 (m, 1H),
13
.78−1.71 (m, 1H), 0.97 (d, J = 7.2 Hz, 3H); C NMR (150 MHz,
CDCl ) δ 154.7, 133.8, 130.3, 129.9 (2C), 124.0 (2C), 67.4, 35.0, 33.0,
3
+
3
0.9, 16.5; HRESIMS m/z 287.0937 [M + Na] (calcd for
13
C H N OSNa, 287.0937).
6.9 Hz, 3H), 0.65−0.54 (m, 6H); C NMR of E-isomer (150 MHz,
1
2
16
4
(
S)-5-((4-((4-Methoxybenzyl)oxy)-3-methylbutyl)thio)-1-phenyl-
CDCl ) δ 159.2, 158.9, 138.7, 131.9, 131.1, 129.3 (2C), 128.7 (2C),
3
1
H-tetrazole (S6a). La(OTf) (14.3 mg, 0.0244 mmol) was added to a
125.4, 113.9 (2C), 113.7 (2C), 77.4, 75.3, 73.2, 73.1, 72.8, 69.2, 56.5,
55.4 (2C), 53.0, 49.3, 42.8, 40.5, 39.9, 36.7, 36.4, 34.0, 33.7, 32.7, 32.0,
3
mixture of 4-methoxybenzyl-2,2,2-trichloroacetimidate (108 mg, 0.380
mmol) and sulfide 16a (65.7 mg, 0.249 mmol) in dry toluene (2.5
31.9, 28.2, 26.1, 20.6, 18.6, 17.7, 16.9, 13.8, 11.6, 7.2 (3C), 5.6 (3C);
+
mL) at rt. After stirring at rt for 19 h, La(OTf) (30.1 mg, 0.0514
HRESIMS m/z 811.5303 [M + Na] (calcd for C49
H
76
O
6
SiNa,
3
mmol) was added to the reaction mixture. After stirring at rt for 4 h,
the reaction mixture was quenched with saturated aqueous NaHCO3
at 0 °C and extracted with EtOAc. The organic layer was washed with
saturated aqueous NaCl, dried over anhydrous Na SO , filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, hexane/EtOAc = 10/1 → 3/
811.5303).
(25S)-7α-(Triethylsilyl)oxy-5α-cholest-22-ene-3α,8β,26-triol (S7a).
DDQ (57.5 mg, 0.246 mmol) was added to a mixture of olefin 15a
(48.2 mg, 0.0611 mmol, E/Z = 11:1) in dry CH
saturated aqueous NaHCO (0.12 mL) at 0 °C. After stirring at rt for
20 min, the reaction mixture was cooled to 0 °C and quenched with
saturated aqueous NaHCO and saturated aqueous Na 3. After
extraction with EtOAc, the combined organic layer was washed with
O and saturated aqueous NaCl, dried over anhydrous Na SO
Cl (1.2 mL) and
2
4
2
2
3
1
) to give sulfide S6a (94.9 mg, 0.247 mmol, 99%) as a colorless foam:
3
S O
2 2
24
[
(
α] −1.27 (c 1.01, CHCl ); R = 0.49 (hexane/EtOAc = 2/1); IR
D
3
f
neat) 2956, 1726, 1611, 1513, 1499, 1462, 1387, 1302, 1246, 1174,
H
2
2
,
4
−1
1
1
089, 1035, 1015, 915, 824, 760, 694 cm ; H NMR (600 MHz,
filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (silica gel, hexane/EtOAc =
10/1 → 1/1) to give triol S7a (28.9 mg, 0.0526 mmol, 86%, E/Z =
CDCl ) δ 7.59−7.51 (m, 5H), 7.24 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4
3
Hz, 2H), 4.42 (s, 2H), 3.79 (s, 3H), 3.50−3.44 (m, 1H), 3.41−3.35
11:1) as a colorless foam: [α]26
−28.2 (c 1.44, CHCl ); R = 0.10
(
m, 1H), 3.34−3.27 (m, 2H), 2.00−1.90 (m, 2H), 1.72−1.65 (m, 1H),
D
3 f
0
1
7
.98 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl ) δ 159.3, 154.6,
(hexane/EtOAc = 5/1); IR (neat) 3394, 2935, 2873, 1716, 1508,
3
33.9, 130.6, 130.2, 129.9 (2C), 129.3 (2C), 124.0 (2C), 113.9 (2C),
1457, 1414, 1372, 1231, 1216, 1146, 1087, 1002, 974, 944, 902, 868,
−
1 1
4.9, 72.9, 55.4, 33.3, 33.0, 31.4, 16.9; HRESIMS m/z 407.1506 [M +
841 cm ; H NMR (600 MHz, CDCl ) δ 5.34−5.22 (m, 2H), 4.02
3
+
Na] (calcd for C H N O SNa, 407.1512).
(brt, J = 2.5 Hz, 1H), 3.51 (dd, J = 10.3, 6.2 Hz, 1H), 3.48 (brs, 1H),
3.43 (dd, J = 10.3, 6.2 Hz, 1H), 2.17−2.10 (m, 1H), 2.07−1.97 (m,
2H), 1.95 (dt, J = 13.1, 3.4 Hz, 1H), 1.91−1.85 (m, 1H), 1.84 (td, J =
20
24
4
2
(
S)-5-((4-((4-Methoxybenzyl)oxy)-3-methylbutyl)sulfonyl)-1-phe-
nyl-1H-tetrazole (14a). A solution of (NH ) Mo O ·4H O (66.2 mg,
4
6
7
24
2
J
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
3.6, 2.1 Hz, 1H), 1.77−1.67 (m, 3H), 1.66−1.50 (m, 6H), 1.50−1.42
reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 1/1 → 0/1) to give
(
2
6
m, 2H), 1.35−1.12 (m, 8H), 1.11−1.06 (m, 1H), 1.05 (dt, J = 13.8,
.8 Hz, 1H), 0.98 (d, J = 6.4 Hz, 3H), 0.97 (t, J = 7.9 Hz, 9H), 0.92 (s,
diol (932 mg, 8.95 mmol, 98%) as a colorless oil: [α]26 +22.1 (c 1.05,
D
13
H), 0.90 (d, J = 6.9 Hz, 3H), 0.64−0.55 (m, 6H); C NMR (150
CHCl ); R = 0.30 (hexane/EtOAc = 0/1); IR (neat) 3318, 2928,
3
f
−1 1
MHz, CDCl ) δ 138.8, 125.5, 77.3, 73.2, 68.2, 66.7, 56.5, 53.0, 49.4,
2359, 1458, 1220, 1036, 836, 773, 671 cm ; H NMR (600 MHz,
3
4
1
5
2.8, 40.5, 39.9, 36.6, 36.5, 36.2, 35.7, 33.1, 32.5, 31.5, 28.4, 28.2, 20.6,
CDCl ) δ 3.77−3.73 (m, 1H), 3.65−3.61 (m, 1H), 3.54 (dd, J = 10.2,
3
8.6, 17.7, 16.6, 13.8, 11.3, 7.2 (3C), 5.5 (3C); HRESIMS m/z
4.2 Hz, 1H), 3.40 (dd, J = 10.2, 7.2 Hz, 1H), 3.35 (brs, 2H), 1.83−1.75
+
13
71.4152 [M + Na] (calcd for C H O SiNa, 571.4153).
(m, 1H), 1.63−1.52 (m, 2H), 0.91 (d, J = 7.2 Hz, 3H); C NMR (150
33
60
4
Dipyridinium (25S)-8β-Hydroxy-7α-(triethylsilyl)oxy-5α-cholest-
MHz, CDCl ) δ 68.3, 61.1, 37.7, 34.3, 17.4; HRESIMS m/z 127.0714
3
+
2
2-ene-3α,26-diyl Disulfate (S8a). SO ·pyridine (11.1 mg, 0.0676
[M + Na] (calcd for C H O Na, 127.0730).
3
5
12
2
mmol) was added to a solution of triol S7a (3.8 mg, 6.9 μmol, E/Z =
(R)-2-Methyl-4-((1-phenyl-1H-tetrazol-5-yl)thio)butan-1-ol (16b).
5
1
:1) in freshly distilled pyridine (0.28 mL) at rt. After stirring at rt for
PPh (1.77 g, 6.75 mmol) and PTSH (1.20 g, 6.75 mmol) were added
3
h, the solvent was removed under reduced pressure. Et O was then
to a solution of (R)-(+)-2-methyl-1,4-butanediol (740 mg, 7.11 mmol)
in dry THF (18 mL) at rt. The resultant solution was cooled to −78
°C, and DIAD (1.9 M in toluene, 3.8 mL, 7.2 mmol) was added to the
mixture. After stirring at −78 °C for 1 h, the reaction mixture was
warmed to −20 °C over 2 h. After stirring at −20 °C for 2 h, the
mixture was warmed to rt over 2 h. After stirring at rt for 30 min, the
reaction mixture was concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 5/1 → 1/3) to give sulfide 16b (392 mg, 1.48 mmol,
22%) as a colorless oil: [α]26 +8.95 (c 1.02, CHCl ); R = 0.57
2
added to the residue and decanted to remove excess SO ·pyridine. The
precipitates were dissolved in CHCl , and insoluble impurities were
removed by decantation. The supernatant was concentrated under
reduced pressure. Crude disulfate S8a (6.0 mg) thus obtained was
3
3
used in the next reaction without further purification: R = 0.27
f
1
(
4
5
CHCl /MeOH = 5/2); H NMR (600 MHz, CDCl ) δ 8.92 (d, J =
.8 Hz, 4H), 8.33 (t, J = 7.2 Hz, 2H), 7.87 (t, J = 7.2 Hz, 4H), 5.31−
.21 (m, 2H), 4.75 (brs, 1H), 3.98 (dd, J = 9.6, 6.6 Hz, 1H), 3.90 (dd,
3
3
J = 9.6, 6.6 Hz, 1H), 3.45 (brs, 1H), 2.22−2.11 (m, 2H), 2.05−1.96
D
3
f
(
m, 2H), 1.96−1.85 (m, 2H), 1.85−1.77 (m, 2H), 1.74−1.54 (m, 5H),
(hexane/EtOAc = 1/2); IR (neat) 3396, 2929, 2874, 1597, 1499,
−1
1
1
0
.54−1.41 (m, 4H), 1.35−1.23 (m, 3H), 1.23−1.16 (m, 2H), 1.16−
.01 (m, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.94−0.88 (m, 18H), 0.62−
.50 (m, 6H). The two pyridinium protons were not observed
1461, 1387, 1279, 1243, 1075, 1041, 1015, 984, 771, 763, 693 cm ;
1
H NMR (600 MHz, CDCl ) δ 7.59−7.51 (m, 5H), 3.61 (dd, J = 11.4,
3
6
.6 Hz, 1H), 3.54 (dd, J = 11.4, 6.6 Hz, 1H), 3.48−3.39 (m, 2H), 2.01
probably due to the rapid exchange with contaminating H O.
2
(br, OH), 2.01−1.95 (m, 1H), 1.87−1.80 (m, 1H), 1.78−1.72 (m,
1
1
H), 0.98 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl ) δ 154.7,
Disodium (22E,25S)-7α,8β-Dihydroxy-5α-cholest-22-ene-3α,26-
diyl Disulfate (2a). HF·pyridine (70% HF in pyridine, 7 μL, 0.3
mmol) was added to a solution of crude disulfate S8a (6.0 mg) in
MeOH (0.14 mL) at 0 °C. After stirring at rt for 7 h, the reaction
mixture was quenched with Et N at 0 °C and concentrated under
reduced pressure. Purification by RP-pTLC (MeOH/H O = 2/1)
afforded ammonium salt (4.6 mg) as a colorless foams. Undesired Z-
isomer was separated and removed at this point. A solution of the
above ammonium salt (4.6 mg) in MeOH was passed through a
column packed with Amberlite IR-120B cation exchange resin (Na
form) pre-equilibrated with 1 M aqueous NaOH, H O, and MeOH.
3
33.8, 130.3, 129.9 (2C), 124.0 (2C), 67.4, 35.0, 33.1, 31.0, 16.5;
+
HRESIMS m/z 287.0938 [M + Na] (calcd for C H N OSNa,
12
16
4
2
87.0937).
3
(R)-5-((4-((4-Methoxybenzyl)oxy)-3-methylbutyl)thio)-1-phenyl-
2
1H-tetrazole (S6b). La(OTf) (87.4 mg, 0.149 mmol) was added to a
3
solution of 4-methoxybenzyl-2,2,2-trichloroacetimidate (616 mg, 2.18
mmol) and sulfide 16b (385 mg, 1.45 mmol) in dry toluene (14 mL)
at rt. After stirring at rt for 14 h, the reaction mixture was quenched
+
with saturated aqueous NaHCO at 0 °C and then extracted with
3
2
EtOAc. The organic layer was washed with saturated aqueous NaCl,
The column was eluted with MeOH, and the eluate was concentrated
dried over anhydrous Na SO , filtered, and concentrated under
2
4
under reduced pressure to obtain (25S)-SAAF 2a (2.1 mg, 3.3 μmol,
reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 10/1 → 5/1) to give
4
8% for the three steps) as a colorless foam: [α]26D −7.6 (c 0.23,
2
6
D
MeOH); IR (neat) 3676, 2937, 2923, 2865, 2844, 1716, 1698, 1653,
sulfide S6b (499 mg, 1.30 mmol, 90%) as a colorless foam: [α]
−1 1
1
558, 1507, 1473, 1373, 1213, 1055, 1033, 1012, 908 cm ; H NMR
+1.41 (c 1.05, CHCl ); R = 0.52 (hexane/EtOAc = 2/1); IR (neat)
3 f
(
600 MHz, D O) [chemical shifts were referred to trace MeOH (3.34
2932, 1730, 1612, 1513, 1499, 1462, 1387, 1302, 1246, 1174, 1089,
2
1
13
−1
1
ppm for3 H NMR, and 49.5 ppm for C NMR) as originally
reported ] δ 5.42−5.36 (m, 2H), 4.66 (brs, 1H), 3.95 (dd, J = 9.6, 6.0
Hz, 1H), 3.83 (dd, J = 9.6, 6.0 Hz, 1H), 3.56 (brs, 1H), 2.10−2.06 (m,
1034, 1015, 914, 822, 774, 762, 693 cm ; H NMR (600 MHz,
CDCl ) δ 7.58−7.51 (m, 5H), 7.24 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0
3
Hz, 2H), 4.42 (s, 2H), 3.80 (s, 3H), 3.50−3.45 (m, 1H), 3.41−3.36
(m, 1H), 3.33−3.28 (m, 2H), 1.99−1.90 (m, 2H), 1.71−1.65 (m, 1H),
1
1
1
H), 2.04−1.99 (m, 2H), 1.96−1.94 (m, 1H), 1.92−1.79 (m, 5H),
.76−1.70 (m, 1H), 1.63−1.53 (m, 6H), 1.50−1.45 (m, 1H), 1.40−
.33 (m, 1H), 1.32−1.12 (m, 6H), 0.99 (d, J = 6.2 Hz, 3H), 0.93 (d, J
0.98 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl ) δ 159.3, 154.6,
3
133.9, 130.7, 130.2, 129.9 (2C), 129.3 (2C), 124.0 (2C), 113.9 (2C),
=
6.9 Hz, 3H), 0.901 (s, 3H), 0.895 (s, 3H). The two hydroxy protons
75.0, 72.9, 55.4, 33.4, 33.0, 31.5, 17.0; HRESIMS m/z 407.1534 [M +
13
+
were not observed due to H/D exchange with the solvent; C NMR
Na] (calcd for C H N O SNa, 407.1512).
20
24
4
2
a
(
150 MHz, D O) δ 140.5, 125.3, 78.5, 77.4, 74.1, 72.1, 56.6, 53.9,
(R)-5-((4-((4-Methoxybenzyl)oxy)-3-methylbutyl)sulfonyl)-1-phe-
2
5
2
0.2, 43.2, 40.8, 39.9, 36.02, 35.99, 33.5, 33.3, 32.9, 32.6, 31.5, 28.4,
6.5, 20.3, 18.7, 18.0, 16.4, 13.5, 11.5; chemical shifts were referred to
nyl-1H-tetrazole (14b). A solution of (NH ) Mo O ·4H O (65.4 mg,
4
6
7
24
2
a
0.0529 mmol) in H O (30% in H O, 0.59 mL) was added to a
2 2 2
13
trace MeOH (3.34 ppm for 1H NMR, and 49.5 ppm for C NMR) as
originally reported; HRESIMS m/z 296.1184 [M − 2Na] (calcd for
C H O S , 296.1182).
solution of sulfide S6b (204 mg, 0.529 mmol) in EtOH (0.76 mL) at
rt. After stirring at rt for 6 h, the reaction mixture was extracted with
EtOAc. The organic layer was washed with saturated aqueous NaCl,
dried over anhydrous Na SO , filtered, and concentrated under
3
2−
2
7
44 10 2
(
R)-(+)-2-Methyl-1,4-butanediol. LiAlH (1.13 g, 29.8 mmol) was
4
2
4
added to a solution of (R)-(+)-methylsuccinic acid (1.20 g, 9.11
mmol) in dry THF (30 mL) at −78 °C. After stirring at rt for 5 h, the
reduced pressure. The residue was purified by flash column
chromatography (silica gel, hexane/EtOAc = 7/1 → 3/1) to give
2
7
reaction mixture was cooled to 0 °C and treated with LiAlH (700 mg,
sulfone 14b (201 mg, 0.482 mmol, 91%) as a colorless foam: [α]
4
D
1
8.4 mmol). After stirring at rt for 1 h, the reaction mixture was
+6.83 (c 1.01, CHCl ); R = 0.54 (hexane/EtOAc = 2/1); IR (neat)
3 f
warmed to 60 °C. After stirring at 60 °C for 11.5 h, the reaction
2958, 1731, 1612, 1513, 1498, 1463, 1340, 1302, 1247, 1175, 1152,
1092, 1034, 822, 783, 768, 760, 689 cm ; H NMR (600 MHz,
−1
1
mixture was cooled to 0 °C and treated sequentially with H O (2.0
2
mL), 3 M aqueous NaOH (2.5 mL), and H O (6.0 mL). The resultant
CDCl ) δ 7.66 (d, J = 7.8 Hz, 2H), 7.61−7.56 (m, 3H), 7.24 (d, J = 7.8
2
3
mixture was diluted with Et O and stirred for 15 min. The cake was
Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H), 4.42 (s, 2H), 3.82−3.73 (m, 5H),
3.37 (dd, J = 9.6, 4.8 Hz, 1H), 3.26 (dd, J = 9.6, 4.8 Hz, 1H), 2.09−
2.03 (m, 1H), 1.98−1.93 (m, 1H), 1.88−1.81 (m, 1H), 0.97 (d, J = 7.2
2
filtered through a pad of Celite, and the filtrate was extracted with
THF. The organic layer was washed with saturated aqueous NaCl,
dried over anhydrous Na SO , filtered, and concentrated under
Hz, 3H); 13C NMR (150 MHz, CDCl ) δ 159.2, 153.5, 133.1, 131.5,
2
4
3
K
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
5
30.3, 129.7 (2C), 129.3 (2C), 125.2 (2C), 113.9 (2C), 74.4, 72.8,
5.3, 54.4, 32.5, 26.3, 16.8; HRESIMS m/z 439.1423 [M + Na] (calcd
(CHCl /MeOH = 5/2); H NMR (600 MHz, CD OD) δ 8.90 (brs,
3 3
+
4H), 8.72−8.66 (m, 2H), 8.17−8.11 (m, 4H), 5.31−5.20 (m, 2H),
4.61 (brs, 1H), 3.89−3.84 (m, 1H), 3.82−3.76 (m, 1H), 3.44 (brs,
1H), 2.25−2.07 (m, 2H), 2.07−1.78 (m, 6H), 1.76−1.58 (m, 5H),
.58−1.35 (m, 5H), 1.35−1.02 (m, 6H), 1.02−0.91 (m, 21H), 0.65−
.60 (m, 6H); three protons (the pyridinium and the hydroxy
protons) were not observed probably due to the H/D exchange with
the solvent.
Disodium (22E,25R)-7α,8β-Dihydroxy-5α-cholest-22-ene-3α,26-
diyl Disulfate (2b). HF·pyridine (70% HF in pyridine, 20 μL, 0.76
mmol) was added to a solution of crude disulfate S8b (17.1 mg) in
MeOH (0.20 mL) at 0 °C. After stirring at rt for 17.5 h, the reaction
was quenched with Et N at 0 °C and concentrated under reduced
pressure. Purification by RP-pTLC (MeOH/H O = 2/1) afforded an
ammonium salt of desilylated product (6.4 mg) as a colorless foam.
Undesired Z-isomer was separated and removed at this point. A
solution of the above ammonium salt (6.4 mg) in MeOH was passed
through a column packed with Amberlite IR-120B cation exchange
resin (Na form) pre-equilibrated with 1 M aqueous NaOH, H O, and
MeOH. The column was eluted with MeOH, and the eluate was
concentrated under reduced pressure to obtain (25R)-SAAF 2b (4.7
for C H N O SNa, 439.1410).
20
24
4
4
(
25R)-3α,26-Bis(4-methoxybenzyl)oxy-7α-(triethylsilyl)oxy-5α-
cholest-22-en-8β-ol (15b). A mixture of aldehyde 13 (15.9 mg, 0.0265
mmol) and sulfone 14b (34.2 mg, 0.0821 mmol) was dissolved in
freshly distilled THF (0.90 mL) and HMPA (0.27 mL) and cooled to
1
0
−
78 °C. A solution of KHMDS (0.5 M in freshly distilled THF, 0.16
mL, 0.080 mmol) was added to the above mixture and stirred at −78
C for 1 h. The reaction mixture was warmed to rt and stirred for 1 h.
°
The reaction mixture was cooled to 0 °C and quenched with saturated
aqueous NH Cl. After extraction with Et O, the combined organic
4
2
layers were washed with saturated aqueous NaCl, dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (silica gel,
3
2
4
2
hexane/EtOAc = 24/1 → 7/1) to give olefin 15b (E/Z ≈ 6:1, 10.5
mg, 0.0133 mmol, 50%) as a colorless foam: [α]25 −14.7 (c 0.53,
D
CHCl ); R = 0.46 (hexane/EtOAc = 5/1); IR (neat) 3566, 2930,
3
f
2
1
6
4
1
1
6
1
3
3
6
872, 1731, 1613, 1586, 1513, 1457, 1443, 1372, 1362, 1301, 1246,
220, 1171, 1149, 1087, 1038, 1012, 973, 945, 908, 822, 772, 744, 724,
73 cm ; H NMR of E-isomer (600 MHz, CDCl ) δ 7.28−7.24 (m,
H), 6.89−6.84 (m, 4H), 5.28−5.15 (m, 2H), 4.45 (d, J = 11.7 Hz,
H), 4.42 (s, 2H), 4.37 (d, J = 11.7 Hz, 1H), 3.80 (s, 6H), 3.59 (brs,
H), 3.48 (brs, 1H), 3.28 (dd, J = 8.9, 6.2 Hz, 1H), 3.21 (dd, J = 8.9,
.2 Hz, 1H), 2.20 (tt, J = 13.1, 2.8 Hz, 1H), 2.13−2.05 (m, 1H), 2.03−
.91 (m, 2H), 1.86−1.70 (m, 5H), 1.68−1.50 (m, 5H), 1.50−1.41 (m,
H), 1.41−1.24 (m, 6H), 1.12−1.04 (m, 2H), 0.96 (d, J = 6.2 Hz,
H), 0.94 (t, J = 8.3 Hz, 9H), 0.93 (s, 3H), 0.91 (s, 3H), 0.89 (d, J =
+
2
−1
1
3
2
3
D
mg, 7.4 μmol, 72% for the three steps) as a colorless foam: [α] −4.1
c 0.24, MeOH); IR (neat) 3445, 2939, 2871, 1716, 1647, 1558, 1541,
507, 1456, 1373, 1227, 1055, 1033, 1011 cm ; H NMR (600 MHz,
(
1
−1 1
1
D O) [chemical shifts were referred to trace MeOH (3.34 ppm for H
2
13
3
NMR and 49.5 ppm for C NMR) as originally reported ] δ 5.42−
5
.36 (m, 2H), 4.65 (brs, 1H), 3.92 (dd, J = 9.6, 5.4 Hz, 1H), 3.85 (dd,
13
J = 9.6, 5.4 Hz, 1H), 3.56 (brs, 1H), 2.09−2.06 (m, 2H), 2.01−1.99
(m, 1H), 1.94−1.79 (m, 6H), 1.76−1.70 (m, 1H), 1.62−1.55 (m, 6H),
1.51−1.46 (m, 1H), 1.40−1.33 (m, 1H), 1.28−1.13 (m, 6H), 0.99 (d, J
= 6.6 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 0.904 (s, 3H), 0.900 (s, 3H);
two hydroxy protons were not observed probably due to the H/D
.6 Hz, 3H), 0.65−0.54 (m, 6H); C NMR of E-isomer (150 MHz,
C D ) δ 159.7, 159.5, 138.9, 132.3, 131.6, 129.3 (2C), 128.9 (2C),
6
6
1
5
3
26.1, 114.1 (2C), 114.0 (2C), 77.2, 75.2, 73.9, 73.6, 73.0, 69.7, 57.0,
4.8 (2C), 53.5, 49.9, 43.2, 41.0, 40.4, 37.1, 36.8, 34.5, 34.2, 33.1, 32.9,
2.4, 28.8, 26.1, 21.0, 18.8, 18.2, 17.2, 14.0, 12.0, 7.4 (3C), 5.9 (3C);
+
13
a
HRESIMS m/z 811.5326 [M + Na] (calcd for C H O SiNa,
exchange with the solvent; C NMR (150 MHz, D O) δ 140.4,
49
76
6
2
8
11.5303).
25R)-7α-(Triethylsilyl)oxy-5α-cholest-22-ene-3α,8β,26-triol
S7b). DDQ (17.5 mg, 0.0748 mmol) was added to a solution of olefin
125.4, 78.4, 77.4, 74.2, 72.0, 56.6, 53.9, 50.2, 43.2, 40.8, 39.9, 36.0
(2C), 33.5, 33.4, 32.8, 32.6, 31.5, 28.4, 26.6, 20.4, 18.7, 18.0, 16.2, 13.6,
(
2
−
(
1
1
1.5; HRESIMS m/z 296.1193 [M − 2Na] (calcd for C H O S ,
a
2
7
44 10 2
5b (19.6 mg, 0.0248 mmol, E/Z ≈ 6:1) in dry CH Cl (0.50 mL) and
2
2
296.1182). Chemical shifts were referred to trace MeOH (3.34 ppm
3
pH 7 buffer (0.10 mL) at 0 °C. After stirring at rt for 20 min, the
reaction was quenched with saturated aqueous NaHCO and saturated
aqueous Na S O at 0 °C and then extracted with EtOAc. The organic
layer was washed with H O and saturated aqueous NaCl, dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (silica gel,
hexane/EtOAc = 14/1 → 1/1) to give triol S7b (5.6 mg, 0.010 mmol,
1
13
for H NMR, and 49.5 ppm for C NMR) as originally reported.
3
X-ray Crystallographic Analysis of 11. A colorless crystal of 11 was
obtained from a mixture of CH Cl /n-hexane. Mo Kα radiation (λ =
2
2
3
2
2
2
0
.710 73 Å) was used for X-ray diffraction data collection (Supporting
2
4
Information Table S1 for details): C H O , M = 486.67,
3
0
46
5
orthorhombic, space group P2 2 2 , a = 6.7944(14) Å, b =
1
1 1
1
2
0
3.003(3) Å, c = 29.832(6) Å, α = 90°, β = 90°, γ = 90°, V =
25
4
=
1%, E/Z ≈ 6:1) as a colorless foam: [α] −52.2 (c 0.28, CHCl ); R
3
3
−1
D
3
f
635.6(9) Å , Z = 4, ρ = 1.226 g/m , μ = 0.081 mm , crystal size
calc
0.12 (hexane/EtOAc = 5/1); IR (neat) 3405, 2935, 2873, 1717,
3
.10 × 0.08 × 0.05 mm , F(000) = 1064, 15 400 reflections collected,
1
7
457, 1414, 1372, 1219, 1146, 1087, 1002, 974, 944, 902, 868, 841,
91 cm ; H NMR (600 MHz, CDCl ) δ 5.33−5.23 (m, 2H), 4.02
−1
1
6024 independent reflections (Rint = 0.0512), R
1
= 0.0459 [I > 2σ(I)],
3
wR = 0.0757 [I > 2σ(I)], R = 0.0751 (all data), wR = 0.0848 (all
2
1
2
(
brs, 1H), 3.50 (dd, J = 10.8, 6.0 Hz, 1H), 3.48 (brs, 1H), 3.44 (dd, J =
0.8, 6.0 Hz, 1H), 2.14 (tt, J = 13.1, 2.8 Hz, 1H), 2.06 (dt, J = 13.4, 6.2
data), goodness of fit = 1.030, Flack parameter = 0.8(9). The crystal
structure was solved with SHELXS-97 and refined using SHELXL-97
with the assistance of KENX. Crystallographic data for 11 have been
deposited with the Cambridge Crystallographic Data Centre
1
Hz, 1H), 2.04−1.98 (m, 1H), 1.95 (dt, J = 12.4, 2.8 Hz, 1H), 1.89−
1
1
6
3
7
3
.80 (m, 2H), 1.78−1.43 (m, 12H), 1.33 (dd, J = 13.1, 3.4 Hz, 1H),
.30−1.15 (m, 5H), 1.14−1.30 (m, 2H), 1.02 (brs, 1H), 0.98 (d, J =
.2 Hz, 3H), 0.96 (t, J = 8.3 Hz, 9H), 0.91 (s, 6H), 0.90 (d, J = 6.9 Hz,
H), 0.65−0.55 (m, 6H); C NMR (150 MHz, C D ) δ 138.8, 126.2,
7.2, 73.9, 67.8, 66.3, 56.9, 53.5, 49.9, 43.2, 41.0, 40.4, 36.9, 36.8, 36.5,
(deposition number 1813183). Copies of the data can be obtained,
13
free of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: + 44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
6
6
5.9, 33.5, 33.0, 31.7, 29.1, 28.7, 20.9, 18.8, 18.2, 16.7, 13.9, 11.7, 7.4
+
(
3C), 5.8 (3C); HRESIMS m/z 571.4167 [M + Na] (calcd for
C H O SiNa, 571.4153).
3
3
60
4
ASSOCIATED CONTENT
■
Dipyridinium (25R)-8β-Hydroxy-7α-(triethylsilyl)oxy-5α-cholest-
2
2-ene-3α,26-diyl Disulfate (S8b). SO ·pyridine (17.0 mg, 0.104
*
S
Supporting Information
3
mmol) was added to a solution of triol S7b (5.6 mg, 0.010 mmol, E/Z
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b01052.
≈
6:1) in freshly distilled pyridine (0.40 mL) at rt. After stirring at rt
for 2 h, the solvent was removed under reduced pressure. Et O was
2
added to the residue and decanted to remove excess SO ·pyridine. The
precipitates were dissolved in CHCl , and insoluble impurities were
removed by decantation. The supernatant was concentrated under
reduced pressure. The crude disulfate S8b (17.1 mg, E/Z ≈ 6:1) was
used in the next reaction without further purification: R = 0.20
3
1H and 13C NMR spectra of new synthetic compounds;
3
details about X-ray crystallographic analysis of 11 (PDF)
X-ray crystallographic data for 11 (CIF)
f
L
DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
AUTHOR INFORMATION
Corresponding Author
Tel: +81 92 802 4176. E-mail: oishi@chem.kyushu-univ.jp.
■
*
ORCID
Nobuaki Matsumori: 0000-0003-0495-2044
Tohru Oishi: 0000-0002-3057-9354
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was partly supported with grants from JST ERATO
Lipid Active Structure.
■
REFERENCES
■
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DOI: 10.1021/acs.jnatprod.7b01052
J. Nat. Prod. XXXX, XXX, XXX−XXX