Synthesis of the antiproliferative agent hippuristanol and its analogues from hydrocortisone via Hg(II)-catalyzed spiroketalization: Structure-activity relationship

Article abstract of DOI:10.1021/jm401799j

An efficient synthesis of hippuristanol (1), a marine-derived highly potent antiproliferative steroidal natural product, and nine closely related analogues has been accomplished from the commercially available hydrocortisone utilizing Hg(II)-catalyzed spi

Full text of DOI:10.1021/jm401799j

Article
pubs.acs.org/jmc
Synthesis of the Antiproliferative Agent Hippuristanol and Its
Analogues from Hydrocortisone via Hg(II)-Catalyzed
Spiroketalization: Structure−Activity Relationship
Ragam Somaiah,† Kontham Ravindar,† Regina Cencic,‡ Jerry Pelletier,‡ and Pierre Deslongchamps*,†
†Dep
Medecine, Ville de Queb
́
artement de Chimie, Faculte
́
des Sciences et de Gen
ec, G1V 0A6, Canada
́ ́
ie, Pavillon Alexandre-Vachon, Universite Laval, 1045, Avenue de la
́
́
ec, Queb
́
‡Department of Biochemistry and Oncology, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, H3G 1Y6,
Canada
S
* Supporting Information
ABSTRACT: An efficient synthesis of hippuristanol (1), a
marine-derived highly potent antiproliferative steroidal natural
product, and nine closely related analogues has been
accomplished from the commercially available hydrocortisone
utilizing Hg(II)-catalyzed spiroketalization of 3-alkyne-1,7-diol
motif as a key strategy. This practical synthetic sequence
furnished 1 in 11% overall yield from hydrocortisone in 15
linear steps. Modifications to the parent molecule 1
encompassed changing the functional groups on rings A and
E. Each analogue was screened for their effects on inhibition of
cap-dependent translation, and the assay results were used to
establish structure−activity relationships. These results suggest
that the stereochemistry and all substituents of spiroketal portion (rings E and F) and C3-α and C11-β hydroxyl functional
groups on rings A and C, respectively, are critical for the inhibitory activity of natural product 1.
structurally close analogues of spiroketals appendage (E and F
rings, Scheme 1) including structure−activity relationship
(SAR) studies, starting from hecogenin acetate via 11-
ketotigogenin, employing our own protocol of Hg(II)-catalyzed
INTRODUCTION
■
Hippuristanol (1), a polyoxygenated marine-derived steroidal
natural product, was isolated from the Gorgonian Isis hippuris.1
We have previously identified hippuristanol as a highly potent
candidate for the selective inhibition of eukaryotic initiation
factor (elF)4A-RNA binding activity that can be used to
distinguish between elF4A-dependent and independent modes
of translation initiation in vitro and in vivo.2 Eukaryotic protein
synthesis is regulated at the level of initiation; here, a 40s
ribosomal subunit and associated factors (43s preinitiation
complex) are recruited to an mRNA by four initiation factors,
namely, the eIF4F complex, which comprises the cap-binding
protein, eIF4E, a large scaffolding protein, eIF4G, and an RNA
helicase, elF4A. Eukaryotic initiation factor (elF)4A is the
prototypical member of the DEAD-box family of RNA
helicases, and it is required to unwind local secondary structure
proximal to the 5′ m7GpppN cap structure. Compound 1 binds
to eIFA and inhibits its RNA binding activity, thus inhibiting
translation initiation.2 Because of the selective translation
inhibition activity of 1 and its effects on cellular proliferation, it
is considered a promising lead for the development of
anticancer chemotherapeutic agents.
Scheme 1. Retrosynthetic Analysis of Hippuristanol (1) and
Analogues 2−10
Inspired by fascinating structural features and promise as an
anticancer and antiviral lead structure and in combination with
lowest natural abundance, we have recently disclosed synthetic
routes for 1 (in 5.54% overall yield in 12 steps) and its
Received: November 21, 2013
Published: March 3, 2014
© 2014 American Chemical Society
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cascade spiroketalization of semiprotected 3-alkyne-1,7-diol
motifs and Suarez cyclizations.3 Yu and co-workers reported the
synthesis of 1 and some interesting analogues in order to verify
the effect of size, stereochemical aspects, and different
substitution patterns of spiroketal appendage (E and F rings,
Scheme 1) in relation to the biological activity.4 We and Yu
previously have concluded that the hydroxyl group at C20, the
“R” configuration at C22 spiroketal carbon, the three methyl
groups on ring F, and the size (5/5) of the spiroketal portion
were crucial for the inhibitory activity of 1 (Figure 1).3,4
alkyne-1,7-diol motif along with several unique transformations.
Modifications to the parent molecule 1 included removal or
changing the substituents and functional groups of rings A, C,
and E. Structure−activity relationships of all analogues were
established based on the screening results.
RESULTS AND DISCUSSION
■
In the retrosynthetic analysis, as described in Scheme 1, we
envisioned the synthesis of 1 and its analogues (2−10) from
the advanced suitable semiprotected 3-alkyne-1,7-diol inter-
mediate A, via Hg(II)-catalyzed cascade spiroketalization.
Hydrocortisone (11) was considered the best choice of
commercially available and cost-effective starting material to
produce intermediate A, via β-hydroxy ketone B, as it contains
the favorable steric rigidity along with suitably positioned
functional groups.
Synthesis of Hippuristanol (1) and 22-epi-Hippurista-
nol (2). As described in retrosynthetic analysis in Scheme 1, all
analogues of natural product 1 were to be synthesized from the
advanced suitable semiprotected 3-alkyne-1,7-diol intermediate
A, via Hg(II)-catalyzed cascade spiroketalization, which could
be readily prepared from the versatile enone intermediate 19.
Thus, our synthetic efforts began from 11 as shown in Scheme
2. Thus, natural product 11 was transformed into its bis-
methyleneketal derivative 12 using paraformaldehyde in
CH2Cl2, H2O, and HCl, in good yield of 85%. Reduction of
enone 12 under Birch conditions5 (lithium, liquid NH3, t-
BuOH, THF, −33 °C) furnished C3-β-hydroxyl product
(steroid numbering) 13 with trans ring juncture in excellent
yield of 92%. Selective inversion of C3-β-hydroxyl group of diol
13 under Mitsunobu conditions6 (DIAD, TPP, BzOH, THF)
and subsequent LiAlH4 reduction of benzoate 14 in anhydrous
THF provided 3α-11β-diol 15 in 89% and 92% yields,
Figure 1. Hippuristanol and its analogues.
In this study we aimed to develop a practical synthetic route
for natural product 1 and understand further the importance of
the functional groups/substituents on rings A, C, and E for
inhibitory activity as well as to identify analogues with
enhanced biological activity. Herein, we report an efficient
synthetic route for 1 and nine closely related new series of
analogues, starting from commercially available and cost-
effective hydrocortisone4 employing our own protocol of
Hg(II)-catalyzed cascade spiroketalization of semiprotected 3-
Scheme 2. Synthesis of Enone 19
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Scheme 3. Synthesis of Hippuristanol (1)
respectively.3 Then diol 15 was protected as its dibenzyl ether
16 using standard conditions (NaH, PhCH2Br, TBAI, THF,
reflux). Deprotection7 of bis-methylene ketal group in
compound 16 using 48% aqueous HF in THF cleanly furnished
corresponding dihydroxy ketone 17 in 78% yield. Dihydroxy
ketone 17 was converted to bisacetoxy ketone 18 using
standard reaction conditions (Ac2O, PTSA, toluene), and then
pyrolytic elimination8 of the 17α-acetoxy group of 18 was
carried out to obtain α,β-unsaturated 21-acetoxy ketone 19 in
85% yield (Scheme 2).
was subjected to bromohydroxylation using N-bromoacetamide
(NBA)11 in aqueous acetone/THF to provide the 16β-hydroxy-
17α-bromide 21 in 70% yield, which on subsequent treatment
with n-Bu3SnH and triethylborane (Et3B) in anhydrous CH2Cl2
resulted in debrominated product 22 in 95% yield. This
synthetic sequence provided hydroxy ketone 22 in 15% overall
yield in 11 steps from commercially available natural product
11, which was compared with our previous synthesis in 3%
overall yield in 21 steps starting from hecogenin acetate.3
Addition of lithiated alkyne coupling partner 23 (which was
prepared using our own method)3 onto 16β-hydroxyketone 22
provided exclusively the expected Cram’s product 24 in 85%
yield. Hg(II)-catalyzed cascade spiroketalization3 of semi-
protected alkynediol 24 provided the desired spiroketal 25 in
excellent yield of 90%, which on debenzylation with lithium in
liquid ammonia resulted in analogue 2 as a single diastereomer
(22S) in 94% yield. Analogue 2 was converted to natural
product 1 using pyridine p-toluenesulfonate (PPTS) mediated
epimerization in CHCl3 at room temperature in 85% yield
(brsm, based on recovered starting material).3 1H, 13C, and
remaining analytical data of the synthetic material were in good
accordance with those of the natural product as well as with the
reported data.2−4
Then we turned our attention to establish suitable reaction
conditions to access the required α,β-unsaturated methyl
ketone 20 from α,β-unsaturated-21-acetoxy enone 19. Thus,
we first explored zinc in acetic acid mediated reductive
cleavage9 of the 21-acetoxy group in compound 19 to give
20. We obtained only 50% yield which could be due to
overreduction of C20-keto group. To overcome this problem,
we adopted a two-step procedure for this conversion. Thus, we
first treated compound 19 with methylmagnesium bromide in
anhydrous THF to acquire the corresponding diol, which was
10
subsequently subjected to silica gel supported NaIO4
mediated cleavage in anhydrous CH2Cl2 to obtain the required
enone intermediate 20 in good yield of 70%, for two steps
(Scheme 3).
Having key enone intermediate 20 in hand, we adopted our
own reported3 synthetic sequence to furnish the natural
product 1 as illustrated in Scheme 3. Accordingly, enone 20
With the above promising results, we then decided to expand
our research toward the synthesis of various analogues of
natural product 1 using the chemistry similar to that employed
for the synthesis of 1 as shown in Scheme 2. Enone 19 was
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Scheme 4. Synthesis of 21-Methylhippuristanol (3) and Its 22-Epimer 4
Scheme 5. Synthesis of 21-Hydroxy-22-epi-hippuristanol (5)
used as a versatile building block for the synthesis of analogues
related to E ring (3−8), whereas synthetic compound 1 was
used as a precursor for analogues related to A and C rings (9
and 10) (Figure 1).
Synthesis of Analogues 3 and 4. Synthesis of 21-
methylhippuristanol 3 (hippuristanol numbering) and its 22-
epimer 4 was started from enone 19. Addition of ethyl-
magnesium bromide to acetoxyenone 19 and subsequent silica
supported NaIO4 cleavage provided ethyl vinyl ketone 26.
Bromohydroxylation of 26 using N-bromoacetamide in
aqueous acetone and THF gave the corresponding bromohy-
drin 27 in 75% yield. n-Bu3SnH and Et3B mediated
debromination of 27 gave the required β-hydroxy ketone 28
in 93% yield. Lithiated alkyne 23 addition onto hydroxy ketone
28 furnished exclusively the desired Cram’s product 29 in
excellent yield of 90%. As was observed above, exposure of
semiprotected 3-alkyn-1,7-diol 29 to Hg(OTf)2 in aqueous
acetonitrile and acetic acid at ambient temperature cleanly
provided the spiroketal, which upon debenzylation with lithium
in liquid ammonia resulted in analogue 4 as a single
diastereomer. Epimerization of spiroketal 4 at C22 using
PPTS in anhydrous CHCl3 at room temperature furnished
analogue 3 in 80% yield (brsm). Here slow epimerization was
observed compared to the conversion of 2 into 1 (Scheme 4).
Synthesis of Analogue 5. In order to verify the effect of a
more polar hydroxyl group in the vicinity of spiroketal segment
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Scheme 6. Synthesis of Analogues 6−8
Scheme 7. Synthesis of Analogues 9 and 10
on inhibitory activity and the relative orientation of the
spiroketal unit, we planned to prepare the 21-hydroxy
derivative of 1 (hippuristanol numbering) from acetate
intermediate 19 which has all the skeletal requirements.
Hence, hydrolysis of acetate 19 using K2CO3 in MeOH−
THF and subsequent benzyl protection using Ag2O and benzyl
bromide in anhydrous Et2O 12 resulted in benzyl ether 30.
Compound 30 on exposure to N-bromoacetamide (NBA) in
aqueous acetone/THF underwent bromohydroxylation to give
16β-hydroxy-17α-bromide 31 in moderate yield, which on
treatment with Bu3SnH/Et3B in anhydrous CH2Cl2 resulted in
debrominated product 32. The addition of lithiated alkyne 23
onto β-hydroxy ketone 32 cleanly furnished the propargyl
alcohol 33 in excellent yield of 82%. As was observed in prior
experiments, exposure of semiprotected 3-alkyn-1,7-diol 33 to
Hg(OTf)2 in aqueous acetonitrile at room temperature cleanly
furnished the spiroketal 34, which on debenzylation using
lithium in liquid ammonia resulted in the 22S spiroketal
analogue 5 as a single diastereomer in a cascade manner. The
stereochemistry of 5 was assigned on the basis of the analogy of
the data (1H and 13C spectra) and from previous similar work
(vide infra). PPTS mediated C22-epimerization of 5 was not
observed, which could be due to the more favorable
intramolecular hydrogen bonding between spiroketal oxygen
(ring F) and α-oriented primary C20-hydroxymethylene group
of the spiroketal 5 (Scheme 5).
Synthesis of Analogues 6, 7, and 8. Silica-supported
NaIO4 cleavage of glycol 5 in anhydrous CH2Cl2 cleanly
furnished the desired ketone analogue 8 in 85% yield, which
was used as a precursor for the synthesis of analogues 20-
desmethylhippuristanol (6) and 20-desmethyl-22-epi-hippuris-
tanol (7). Therefore, NaBH4/EtOH reduction of ketone 8 at
room temperature resulted in analogue 7 as single diaster-
1
eomer, which was confirmed by the comparison of H (δ16H
=
4.42) and 13C NMR (δC22 = 113.0) data with data of analogue
2 and natural product 1. PPTS mediated epimerization at
spiroketal carbon of analogue 7 at room temperature afforded
analogue 6 in 86% yield (brsm). Initial observation of the Rf
value on TLC and lowering of the δ value of C22 spirocarbon
(δ 112.7 ppm) in 13C NMR and δ at 4.15 ppm in H NMR
1
confirmed inversion of the stereochemistry at spiroketal carbon
(Scheme 6).
Synthesis of Analogues 9 and 10. Synthetic compound 1
was used as a precursor for the synthesis of analogues 9 and 10
as illustrated in Scheme 7. Oxidation of 1 using pyridinium
dichromate (PDC) in anhydrous pyridine furnished 3,11-
diketone in 75% yield. NaBH4 mediated selective C3-ketone
reduction followed by C11-ketone reduction using LiAlH4 in
anhydrous THF afforded the desired 3β,11β-diol analogue 9
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1
Table 1. Correlation of the H and 13C NMR Data and the [α]D Values between the Diastereomeric Analogues
compd with the “R” stereochemistry at C22 (hippuristanol numbering)
compd with the “S” stereochemistry at C22 (hippuristanol numbering)
compd
δH‑16 (ppm)
δC‑22 (ppm)
[α]2D0
compd
δH‑16 (ppm)
δC‑22 (ppm)
[α]2D0
1 (hippuristanol)
4.15
3.94
4.15
4.01
4.28
115.2
115.5
112.7
115.0
115.3
+31.0
+10.3
+43.5
+38.5
+28.5
2 (22-epi-hippuristanol)
4.33
4.14
4.42
4.15
4.88
118.6
118.4
113.0
117.5
109.0
−45.2
−15.4
−55.3
−23.5
−20.5
3
4
7
5
8
6
9
10
with complete substrate controlled stereoselection.13 3β-
Hydroxyl group of 9 was selectively converted to the 3α-
phthalimide 35 using Mitsunobu conditions (phthalimide,
diisopropyl azodicarboxylate (DIAD) and triphenylphosphine)
and then converted to the corresponding 3α-amine analogue
10 by treating with hydrazine hydrate in absolute ethanol at
reflux temperature.14
C3-α-hydroxyl group with β-OH or α-NH2 functional group
resulted in the less active analogue 9 or 10, respectively. These
results show that the C3 α-OH (hippuristanol numbering) can
greatly influence the activity of the molecule 1 (Figure 2).
Structural Correlations between C22 Diastereomers
of Analogues. Structural and stereochemical elucidation of 1
and 2 was unambiguously reported by Higa and co-workers
using extensive spectroscopic analysis and X-ray diffraction
studies.1 We and Yu have successfully assigned the stereo-
chemistry of spiroketal appendage (E/F rings) of several
analogues of natural product 1 on the basis of the correlation of
Rf values, specific rotation, and 1H NMR (δH‑16) and 13C NMR
(δC‑22) data with data reported for 1 and its 22-epimer 2.3,4
Using the above analogy, herein we assigned the stereo-
chemistry of spiroketal appendage (E and F rings) of analogues
3−10. Analogues 3 and 6, having the same configuration as 1 at
C22 (R), have their δH‑16 and δC‑22 chemical shifts at lower
values of 3.94, 4.15 ppm and 115.5, 112.7 ppm, while their C-
22 epimers (S) 4 and 7 have those signals at higher values of
4.14, 4.42 ppm and 118.4, 113.0 ppm, respectively, and this was
attributed to the data reported for 1 and 2. Analogues 9 and 10
have their δC‑22 values 115 and 115.3, respectively, and are very
close to the compound 1 data. Analogues 5 and 8 have δC‑22
values of 117.5 and 109, respectively, which are very close to
the observed compound 2 data. Analogues 3, 6, 9, and 10 (“R”
stereochemistry at spiroketal carbon) have shown diagnostic
specific rotation values compared to those of 4, 5, 7, and 8 (“S”
stereochemistry at spiroketal carbon) (Table 1).
Figure 2. Evaluation of analogues of 1 as inhibitors of eukaryotic
translation initiation. (A) Schematic representation of bicistronic FF/
HCV/Ren construct used to evaluate the inhibitory potential of
analogues of natural product 1 in in vitro translations. (B) Effect of
analogues of 1 on cap-dependent (FF) in vitro translations. Cap-
independent (HCV-Ren) translation serves as internal standard.
Anisomycin (Aniso) is included as control compound inhibiting cap-
dependent as well as cap-independent eukaryotic translation.
Compounds were tested at a final concentration of 10 μM. The
activity relative to DMSO is presented. Results are the average of
duplicates with the error of the mean shown.
Biological Activity of Analogues 3−10. Earlier it was
found that analogues possessing “S” stereochemistry at
spiroketal carbon (C22) had less inhibitory activity compared
to their C22 “‘R” diastereomers.1−4 The similar stereochemical
requirement at spiroketal carbon is observed in the new series
of analogues 3−10. For analogue 4 with a methyl group
extension at C21 and analogue 7 with the lack of C21 methyl
group and in combination with C22, “S” stereochemistry
showed less activity compared to their epimers (C22, “R”) 3
and 6, respectively. Hence, we conclude that “R” stereo-
chemistry at the spiroketal carbon (C22) and C21 methyl
group is crucial for the inhibitory activity. Methyl group
extension onto the C21 carbon in analogue 3 resulted in a
decrease in the activity compared to natural product 1.
Analogue 5 with polar hydroxyl functionality extension at
C21 together with 22S stereochemistry showed less activity
compared to 1 and 2. Analogue 6 that lacks the methyl group at
C20 is also less active, which suggests that the C20 methyl
substitution is also essential for the activity. C20 ketone
analogue 8, possessing “S” stereochemistry at spiroketal carbon,
which lacks the methyl and the hydroxyl groups at C20,
displayed low activity compared to 1 and 2. Replacement of the
CONCLUSION
■
We have developed an efficient synthetic route featuring
Hg(II)-catalyzed cascade spiroketalization using the semi-
protected 3-alkyne-1,7-diol motif 24 to produce natural product
1 and nine closely related analogues 2−10. This synthetic
sequence produced 1 in 11% overall yield from commercially
available 11, which was compared with Yu’s synthesis in 3.6%
overall yield in 16 steps and our own previously reported
synthetic route in 5.54% overall yield in 12 steps starting from
11-ketotigogenin. These results suggest that the “R” stereo-
chemistry at spiroketal carbon (C22) and all substituents of 5,5-
spiroketal portion (rings E and F) and C3-α and C11-β
hydroxyl functional groups on ring A and C, respectively, are
critical for the inhibitory activity of 1. Further study on the
structure−activity relationships of 1 and its analogues is under
progress to increase the bioavailability while maintaining or
even increasing the inhibitory activity and will be reported in
due course.
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added to disperse the newly formed reagent followed by a slow
addition of a solution of enone 12 (14.95 g, 37.0 mmol) in anhydrous
THF (100 mL) and t-BuOH (10 mL). A cautious addition is needed
here to ensure a regular and smooth ammonia reflux. The reaction
mixture was then stirred at −33 °C for 3 h. NH4Cl powder was
carefully and slowly added at −33 °C to quench the excess of
unreacted lithium, and ammonia was allowed to evaporate under a
stream of air with stirring for an additional 4 h. The resulting mixture
was diluted with water, extracted with EtOAc, and washed with brine.
The organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. Purification by silica gel column chromatog-
raphy (60% EtOAc/hexanes) provided the 3β,11β-diol 13 (14.02 g,
92%) as a white solid. Mp 238−243 °C; [α]2D0 −50.4 (c 0.8, CHCl3);
1H NMR (CDCl3, 300 MHz) δ 5.22 (s, 1H), 5.02 (s, 2H), 5.03 (s,
1H), 4.37 (m, 1H), 3.97 (q, 2H, J = 9.1 Hz), 3.58 (m, 1H), 2.10−0.7
(m, 22H), 1.07 (s, 3H), 1.05 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ
109.9, 98.4, 92.8, 91.4, 70.3, 69.0, 66.3, 58.7, 53.0, 45.7, 40.0, 39.8,
37.1, 35.2, 32.2, 31.8, 31.2, 30.7, 28.6, 27.8, 23.6, 15.3, 14.2; IR (NaCl)
cm−1 3615, 3494, 3010, 2929, 1235. HRMS (ESI) m/z: [M]+ calcd for
C23H36O6 408.2512; found 408.2507 0.0012.
EXPERIMENTAL SECTION
■
In Vitro Transcriptions and Translations. The bicistronic
mRNA construct FF/HCV/Ren (Novac et al.) was linearized with
BamHI, followed by in vitro transcription using SP6 RNA polymerase.
The resulting mRNA was in vitro translated at a concentration of 4
ng/μL in rabbit reticulocyte lysate (RRL) from Promega according to
the instructions of the manufacturer. In vitro translations were
performed in the presence of 0.5% DMSO or 10 μM of the indicated
compounds. Firefly (FF) and renilla (Ren) luciferase activities were
measured on a Berthold Lumat LB 9507 luminometer, and values
obtained were normalized against vehicle control, which was set at 1.
General Procedures. All reactions were performed under argon
atmosphere with oven (80 °C) or flame-dried glassware. Tetrahy-
drofuran (THF) and diethyl ether (Et2O) were distilled from sodium
benzophenone under argon atmosphere immediately prior to use.
Dichloromethane, toluene, N,N-dimethylformamide, and acetonitrile
were freshly distilled over calcium hydride under argon atmosphere.
For the NMR spectra assignments, the following abbreviations were
used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet, ABq, AB
quartet; br, broad. Chemical shifts are reported in values relative to the
1
solvent used (CHCl3: 7.26 ppm for H NMR and 77.0 ppm for 13C
(3R,5S,8S,9S,10S,11S,13S,14S)-11-Hydroxy-10,13-dimethyl-
hexadecahydrodispiro[cyclopenta[a]phenanthrene-17,4′-
[1,3]dioxolane-5′,4″-[1,3]dioxolan]-3-yl Benzoate (14). 3β,11β-
Diol 13 (9.87 g, 24.2 mmol) was weighed in a flame-dried round-
bottom flask and dissolved in anhydrous THF (359 mL) under argon
atmosphere. Benzoic acid (BzOH, 4.83 g, 39.9 mmol) and
triphenylphosphine (TPP, 15.52 g, 59.2 mmol) were added to the
reaction mixture at 0 °C. After 5 min, diissopropylazodicarboxylate
(DIAD, 11.69 mL, 59.4 mmol) was added at 0 °C. The mixture was
allowed to stir at room temperature for 3 h. Then the reaction mixture
was concentrated as such (without further workup) and subjected to
silica gel column chromatography (20% EtOAc/hexanes) to afford the
3α-benzoate 14 (11 g, 89%) as a white foamy solid. [α]2D0 −54.4 (c
NMR) as internal standard. Optical rotations were measured at the
sodium D line (589 nm) with a 1.00 dm path length cell. Infrared
spectra were recorded as neat liquid films, and only the most
significant absorption bands are reported (in cm−1). Experimental
procedures for all the new compounds and known compounds without
published experimental procedures are described below. Compounds
that are not presented in the main text (manuscript) are numbered
starting from S1. The HPLC purities of all analogues to be tested were
determined with a Shimadzu Prominence apparatus (Kyoto, Japan)
equipped with a Shimadzu LCMS-2020 mass spectrometer, an APCI
(atmospheric pressure chemical ionization) probe, and an Alltima HP
C18 analytical reversed-phase column (250 mm × 4,6 mm, 5 μm) and
with a solvent gradient of MeOH/water. All final compounds showed
a purity of ≥95% (96.4−99.5%) except for compounds 3, 4, 5, and 6
(88.0−94.4%). The IUPAC nomenclature was used in the
Experimental Section, and the names of steroid derivatives were
generated using ACD/Laboratories (Chemist’s version) software.
(8S,9S,10R,11S,13S,14S)-11-Hydroxy-10,13-dimethyl-
1,6,7,8,9,10,11,12,13,14,15,16-dodecahydrodispiro-
[cyclopenta[a]phenanthrene-17,4′-[1,3]dioxolane-5′,4″-[1,3]-
dioxolan]-3(2H)-one (12). To a solution of water (450 mL) and
concentrated HCl (450 mL) was added paraformaldehyde (150 g),
and the mixture was stirred for 4 h at room temperature. Then
hydrocortisone 11 (15 g, 41.4 mmol) and CH2Cl2 (750 mL) were
added and stirred for an additional 4 h at room temperature. The
mixture was placed overnight (without stirring) and extracted with
CH2Cl2 (3 × 100 mL). Organic layers were washed with brine, dried
over anhydrous Na2SO4, concentrated under reduced pressure.
Purification by silica gel column chromatography (40% EtOAc/
hexanes) afforded bis-methylene ketal 12 (14.17 g, 85%) as a white
1
0.58, CHCl3); H NMR (CDCl3, 300 MHz) δ 8.04−8.01 (m, 2H),
7.56−7.40 (m, 3H), 5.20 (m, 1H), 5.19 (s, 1H), 5.03 (s, 1H), 5.02 (s,
1H), 5.01 (s, 1H), 4.40 (m, 1H), 3.96 (q, 2H, J = 9.2 Hz), 2.10−0.8
(m, 21H), 1.07 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 165.8, 132.7,
131.0, 129.5, 129.4, 128.3, 109.9, 94.8, 91.9, 91.4, 70.5, 70.3, 68.2, 57.9,
53.4, 45.8, 41.2, 40.2, 36.0, 32.9, 32.4, 32.1, 31.3, 30.7, 27.7, 25.9, 23.6,
15.3, 15.2, 14.5. HRMS (ESI) m/z: [M]+ calcd for C30H40O7
512.2774; found 512.2781 0.0015.
(3R,5S,8S,9S,10S,11S,13S,14S)-10,13-Dimethyl-
hexadecahydrodispiro[cyclopenta[a]phenanthrene-17,4′-
[1,3]dioxolane-5′,4″-[1,3]dioxolane]-3,11-diol (15). 3α-Benzoate
14 (11.0 g, 21.5 mmol) was placed in a flame-dried two-necked round-
bottom flask under argon. Anhydrous THF (27.5 mL) was added, and
the system was cooled to 0 °C. LiAlH4 solution (1.18 M in THF, 109
mL, 0.128 mmol) was added slowly and cautiously at 0 °C. After 30
min of stirring at 0 °C the mixture was slowly allowed to reflux for 4 h.
The reaction mixture was then cooled to 0 °C and quenched with a
minimum amount of cold water (10 mL) cautiously and very slowly.
The reaction mixture was diluted with EtOAc (50 mL) and stirred for
2.5 h at room temperature and then filtered on Celite using EtOAc
and CH2Cl2. The filtrate was dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. Purification of the crude
product by silica gel column chromatography (50% EtOAc/hexanes)
afforded 3α,11β-diol 15 (8.1 g, 92%) as a light yellow viscous liquid.
[α]2D0 −65.9 (c 0.57, CHCl3); 1H NMR (CDCl3, 300 MHz) δ 5.21 (s,
1H), 5.04 (s, 2H), 5.02 (s, 1H), 4.41 (m, 1H), 4.03 (m, 1H), 3.98 (q,
2H, J = 9.1 Hz), 2.15−0.8 (m, 22H), 1.07 (s, 3H), 1.03 (s, 3H); 13C
NMR (CDCl3, 75 MHz) δ 109.9, 94.8, 91.8, 91.4, 70.3, 68.3, 66.3,
57.8, 53.2, 45.8, 40.1, 39.8, 36.2, 35.2, 32.2, 31.9, 31.3, 30.8, 28.6, 27.9,
23.7, 15.3, 14.3; IR (NaCl) cm−1 3615.5, 3494, 3011, 2929, 1455,
1235, 1083. HRMS (ESI) m/z: [M]+ calcd for C23H36O6 408.2512;
found 408.2519 0.0012.
solid. [α]2D0 +24.5 (c 0.75, CHCl3); H NMR (CDCl3, 300 MHz) δ
1
5.64 (s, 1H), 5.19 (s, 1H), 5.01 (s, 1H), 5.0 (s, 1H), 4.99 (s, 1H), 4.38
(m, 1H), 3.96 (q, 2H, J = 9.2 Hz), 2.55−0.9 (m, 18H), 1.42 (s, 3H),
1.09 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 199.6, 172.4, 122.2,
109.9, 94.8, 91.6, 91.4, 70.2, 68,5, 56.1, 52.6, 45.6, 40.6, 39.2, 34.9,
33.8, 32.5, 32.0, 31.3, 30.9, 23.7, 21.0, 15.2; IR (NaCl) cm−1 3615.3,
3490.4, 3010, 2990, 1725, 1675, 1631.5, 1452, 1232. HRMS (ESI) m/
z: [M]+ calcd for C23H32O6 404.2199; found 404.2202 0.0012.
(3S,5S,8S,9S,10S,11S,13S,14S)-10,13-Dimethylhexadeca-
hydrodispiro[cyclopenta[a]phenanthrene-17,4′-[1,3]-
dioxolane-5′,4″-[1,3]dioxolane]-3,11-diol (13). A flame-dried
two-neck round-bottom flask was topped with a dry ice condenser.
The system was flushed with argon. The condenser was filled with a
dry ice/acetone mixture. Ammonia was condensed using a −78 °C
trap, The hexane washed lithium metal (8.42 g) was added cautiously
through the side neck of the round-bottom flask. The mixture was
stirred at −33 °C until no further lithium was seen, and when bronze
globules (golden liquid) started to appear, the system was allowed to
equilibrate to the refluxing temperature (−33 °C). THF (10 mL) was
(3R,5S,8S,9S,10S,11S,13S,14S)-3,11-Bis(benzyloxy)-10,13-
dimethylhexadecahydrodispiro[cyclopenta[a]phenanthrene-
17,4′-[1,3]dioxolane-5′,4″-[1,3]dioxolane] (16). To the 0 °C
cooled suspension of NaH (60% dispersion in mineral oil, 2.62 g, 65.5
mmol) in anhydrous THF (15 mL) under argon atmosphere was
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added diol 15 (4.80 g, 11.7 mmol) in anhydrous THF (87 mL), and
the mixture was stirred for 30 min at 0 °C and for 30 min at reflux
temperature. Then it was cooled again to 0 °C, and to it was added
TBAI (2.07 g, 5.6 mmol) followed by benzyl bromide (10.7 mL, 90.3
mmol). The reaction mixture was refluxed for 36 h and then cautiously
quenched by adding cold water (dropwise) at 0 °C. Extraction was
with Et2O (3 × 100 mL), and washing was with brine solution. The
organic fractions were dried over anhydrous Na2SO4, filtered using
sintered funnel, and solvents were removed under vacuum. Purification
of the crude product by silica gel column chromatography (6%
EtOAc/hexanes) afforded the dibenzyl ether 16 (5.57 g, 85%) as a
concentrated under reduced pressure. Purification by silica gel column
chromatography (15% EtOAc/hexanes) afforded the monoacetoxy
enone 19 (1.63g, 85%) as a viscous liquid. Rf = 0.5 (SiO2, 20% EtOAc/
hexanes); 1H NMR (CDCl3, 400 MHz) δ 7.39−7.21 (m, 10H), 6.75−
6.71 (m, 1H) 5.0−4.87 (ABq, 2H, J = 16.0 Hz), 4.55−4.43 (q, 2H, J =
12.4 Hz), 4.78−4.18 (ABq, 2H, J = 11.3 Hz), 4.0−3.94 (m, 1H),
3.65−3.60 (m, 1H), 3.01−2.94 (dd, 1H, J = 14.4, 1.9 Hz), 2.42−2.34
(m, 1H), 2.19 (s, 3H), 2.17−0.85 (m, 16 h), 1.17 (s, 3H), 1.03(s, 3H);
13C NMR (CDCl3, 100 MHz) δ 190.7, 170.6, 153.1, 144.2, 139.6,
139.2, 128.5, 128.3, 127.9, 127.5, 127.2, 75.6, 73.37, 70.3, 69.8, 65.81,
59.57, 58.0, 46.6, 40.9, 36.5, 36.1, 32.9, 32.5, 32.4, 30.87, 28.1, 25.4,
20.8, 17.8, 14.8; IR (NaCl) cm−1 2928, 2856, 1746, 1682, 1460, 1200.
HRMS (ESI) m/z: [M]+ calcd for C37H46O5 570.3345; found
570.3322 0.0012.
white foamy solid. [α]D20 −17.1 (c 0.68, CHCl3); H NMR (CDCl3,
1
300 MHz) δ 7.38−7.26 (m, 10 H), 5.22 (s, 1H), 5.10 (s, 1H), 5.09
(1s, 1H), 5.08 (s, 1H), 4.67 (d, 1H, J = 11.5 Hz), 4.0 (q, 2H, J = 9.2
Hz), 4.03 (m, 1H), 4.0 (s, 2H), 3.64 (m, 1H), 2.45 (d, 1H, J = 12.1
Hz), 2.2−0.8 (m, 19H), 1.07 (s, 3H), 1.02 (s, 3H); 13C NMR (CDCl3,
75 MHz) δ 139.5, 139.3, 128.3, 128.2, 128.1, 127.6, 127.4, 127.3,
127.0, 110.1, 95.0, 94.9, 91.9, 91.5, 75.7, 73.2, 73.1, 72.8, 70.3, 69.6,
58.1, 53.8, 53.6, 46.1, 40.5, 36.1, 32.7, 32.5, 37.3, 31.5, 31.3, 28.1, 27.9,
25.4, 23.8, 23.7, 22.7, 14.3, 14.2; IR (NaCl) cm−1 3011, 2930, 2875,
1700.2, 1453.8, 1356.3. HRMS (ESI) m/z: [M]+ calcd for C37H48O6
588.3451; found 588.3456 0.0017.
(3α,5α,11β)-3,11-Bis(benzyloxy)pregn-16-en-20-one (20). To
the methylmagnesium bromide solution (3.0 M in THF, 2.92 mL, 8.76
mmol) in anhydrous THF (10 mL) was added monoacetoxy enone 19
(1.0 g, 1.75 mmol) in anhydrous THF (10 mL) at −10 °C (ice and
salt mixture bath) under inert atmosphere. The mixture was stirred at
0 °C for 1 h and for an additional 1.5 h at room temperature. Then the
reaction was quenched with saturated aqueous NH4Cl (10 mL)
solution cautiously in dropwise fashion at 0 °C. The resulting mixture
was acidified with 3 N aqueous HCl (pH 6.0), extracted with EtOAc
(3 × 50 mL), washed with brine solution, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
diol was used as such in the next step. Accordingly, the crude diol was
dissolved in anhydrous CH2Cl2 and then treated with silica gel
supported NaIO4 (1.8 g) at room temperature under argon
atmosphere. After completion of the reaction, the contents were
filtered with sintered funnel and washed with CH2Cl2. Filtrate was
concentrated under reduced pressure and the crude enone was
subjected to silica gel column chromatography (10% EtOAc/hexanes)
to afford the enone 20 (629 mg, 70% yield for two steps) as a white
(3α,5α,11β)-3,11-Bis(benzyloxy)-17,21-dihydroxypregnan-
20-one (17). To the solution of bismethylene ketal 16 (1.74 g, 2.9
mmol) in THF (15 mL) was added dropwise 48% HF (80 mL) in a
plastic round-bottom flask at room temperature. The mixture was
stirred for 6 h at room temperature. Then the contents were poured
slowly into saturated aqueous NaHCO3 solution, extracted with
CH2Cl2 (3 × 100 mL), and washed with water and brine solution.
Drying over anhydrous Na2SO4 and concentration under reduced
pressure gave the crude product. Purification by silica gel column
chromatography (40% EtOAc/hexanes) afforded the dihydroxy ketone
17 (1.25 g, 78%) as a white solid. [α]2D0 +62.8 (c 0.65, CHCl3); H
1
solid. [α]2D0 +95.6 (c 0.7, CHCl3); H NMR (CDCl3, 400 MHz) δ
1
NMR (CDCl3, 300 MHz) δ 7.41−7.26 (m, 10H), 4.73 (s, 1H), 4.67
(d, 1H, J = 19.8 Hz), 4.58 (d, 1H, J = 11.4 Hz), 4.50 (q, 2H, J = 12
Hz), 4.32 (d, 1H, J = 19.8 Hz), 4.25 (d, 1H, J = 11.4 Hz), 4.05 (m,
1H), 3.64 (m, 1H), 2.67 (m, 1H), 2.1−0.8 (m, 20H), 1.0 (s, 3H), 0.90
(s, 3H); 13C NMR (CDCl3, 75 MHz) δ 212.5, 139.3, 138.7, 128.3,
128.2, 127.5, 127.4, 127.3, 127.1, 89.0, 75.3, 73.2, 70.3, 69.6, 67.4, 57.9,
52.6, 48.4, 40.4, 36.1, 34.2, 32.7, 32.6, 32.5, 31.8, 31.5, 28.0, 23.8, 16.8,
14.6; IR (NaCl) cm−1 3608.3, 3489.6, 3011, 2928, 2858, 1660, 1357,
1278. HRMS (ESI) m/z: [M]+ calcd for C35H46O5 546.3345; found
546.3334 0.0016.
7.41−7.23 (m, 10H), 6.71−6.69 (m, 1H), 4.82−4.21 (ABq, 2H, J =
11.7 Hz), 4.50 (q, 2H, J = 12.1 Hz), 4.0−3.94 (m, 1H), 3.67−3.60 (m,
1H), 3.0−2.98 (dd, 1H, J = 14.0, 1.9 Hz), 2.38−2.30 (m, 1H), 2.27 (s,
3H), 2.13−0.82 (m, 17H), 1.16 (s, 3H), 1.0 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 197.0, 156.3, 144.4, 139.5, 139.2, 128.3, 128.1,
127.7, 127.4, 127.3, 126.9, 73.2, 70.2, 69.6, 59.4, 58.4, 45.8, 40.8, 36.4,
36.2, 32.7, 32.4, 32.3, 30.7, 27.9, 27.1, 25.3, 17.5, 14.7; IR (NaCl) cm−1
2922, 2862.5, 1665.3, 1589.3, 1454, 1359, 1060. HRMS (ESI) m/z:
[M]+ calcd for C35H44O3 512.3290, found: 512.3291 0.0015.
Enone 20 Using Zn-AcOH. To monoacetoxy 19 (1.0 g, 1.75
mmol) in AcOH (5 mL) and H2O (1 mL) was added zinc dust (6.0 g)
at 0 °C. The reaction was monitored by TLC. After completion of the
reaction, the contents were filtered with sintered funnel and washed
with MeOH. Filtrate was concentrated under reduced pressure. The
crude enone was purified by silica gel column chromatography (8%
EtOAc/hexanes) to obtain the enone 20 (449 mg, 50% yield) as a
white solid. Rf = 0.7 (SiO2, 20% EtOAc/hexanes); [α]2D0 +96.0 (c 0.5,
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.40−7.22 (m, 10H), 6.71−
6.66 (m, 1H), 4.82−4.20 (ABq, 2H, J = 11.7 Hz), 4.50 (q, 2H, J = 12.1
Hz), 4.0−3.95 (m, 1H), 3.65−3.60 (m, 1H), 3.0−2.97 (dd, 1H, J =
14.4, 2.3 Hz), 2.38−2.30 (m,1H), 2.26 (s, 3H), 2.12−0.93 (m, 17H),
1.15 (s, 3H), 1.0 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 197.0,
158.5, 144.6, 139.6, 139.4, 128.5, 128.2, 127.9, 127.6, 127.4, 127.1,
75.8, 73.4, 70.3, 69.8, 59.5, 58.5, 46.0, 41.0, 36.5, 32.9, 32.5, 32.48,
32.45, 30.9, 28.1, 27.3, 25.5, 17.7, 14.9.
(3α,5α,11β,16β)-3,11-Bis(benzyloxy)-17-bromo-16-hydroxy-
pregnan-20-one (21). Enone 20 (252 mg, 0.49 mmol) was dissolved
in acetone (6 mL), THF (3 mL) and H2O (1.5 mL), and to the
mixture was added NBA (N-bromoacetamide, 203.7 mg, 1.47 mmol)
at room temperature. The mixture was stirred for 18 h in the dark, and
additional NBA (135.7 mg, 0.98 mmol) was added at room
temperature. The stirring continued for an additional 10 h at room
temperature, and the mixture was diluted with EtOAc (30 mL). The
organic layer was washed with saturated Na2SO3 solution and brine
solution. The sample was dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
(3α,5α,11β)-3,11-Bis(benzyloxy)-20-oxopregnane-17,21-diyl
Diacetate (18). To a solution of dihydroxy ketone 17 (1.96 g, 3.59
mmol) in toluene (37.3 mL) were added acetic anhydride (20.8 mL)
and PTSA monohydrate (679 mg, 3.59 mmol) at room temperature,
and the mixture was stirred at the same temperature for 24 h. After
completion of the reaction, water was added and extracted with
dichloromethane, and washing was with saturated aqueous NaHCO3
solution. Drying over anhydrous Na2SO4, concentration under
reduced pressure, and purification by silica gel column chromatog-
raphy (15% EtOAc/hexanes) afforded the bisacetoxy ketone 18 (2.01,
1
90.6%). H NMR (CDCl3, 400 MHz) δ 7.40−7.21 (m, 10 H), 4.99−
4.18 (ABq, 2H, J = 16.7 Hz), 4.70−4.45 (m, 4H),4.07−4.02 (m, 1H),
3.65−3.60 (m, 1H), 2.83−2.74 (m, 1H), 2.30−2.24 (m, 1H), 2.17 (s,
3H), 2.11 (s, 3H), 1.96−0.87 (m, 18H), 0.98 (s, 3H), 092 (s, 3H); 13C
NMR (CDCl3, 100 MHz) δ 199.7, 171.2, 170.5, 139.6, 138.0, 128.5,
128.3, 128.1, 129.9, 127.6, 127.5, 127.3, 95.0, 73.4, 73.0, 71.89, 70.4,
69.8, 58.0, 54.7, 53.3, 47.8, 40.6, 36.3, 32.7, 31.7, 30.8, 28.1, 25.5, 23.9,
21.5, 20.8, 15.5, 14.7; IR (NaCl) cm−1 2927.3, 2863.6, 1736.4. HRMS
(ESI) m/z: [M]+ calcd for C39H50O7 630.3556; found 630.3569
0.0019.
(3α,5α,11β)-3,11-Bis(benzyloxy)-20-oxopregn-16-en-21-yl
Acetate (19). To a solution of bisacetoxy ketone 18 (2.05g, 3.25
mmol) in toluene (20 mL) and DMSO (10 mL) was added potassium
acetate (1.26g, 12.89 mmol) at room temperature, and the mixture was
stirred at 120 °C for 6 h. Contents were cooled to room temperature,
water was added and extracted with EtOAc (3 × 100 mL). The sample
was washed with brine solution, dried over anhydrous Na2SO4, and
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by silica gel column chromatography (10% EtOAc/hexanes) to get the
washed with hexanes) was added cautiously through the side neck of
the round-bottom flak. The contents were stirred at −33 °C until no
further lithium was seen. When bronze globules (golden liquid) started
to appear, the system was allowed to equilibrate to the refluxing
temperature (−33 °C). THF (1.5 mL) was added to disperse the
newly formed reagent, and then to the mixture was added slowly a
solution of dibenzyl ether 25 (84 mg, 0.130 mmol). A cautious
addition is needed to ensure a regular and smooth ammonia reflux.
The reaction mixture was then stirred at −33 °C for 2 h, and to the
mixture was carefully and slowly added NH4Cl powder at −33 °C to
quench the excess of lithium. Ammonia was allowed to evaporate
under a stream of air. To the residue was added H2O (5 mL), and
extraction was with EtOAc (5 mL). The combined extracts were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography (15%
acetone/hexanes) afforded 22-epi-hippuristanol (2) (56.4 mg, 94%) as
α-bromo ketone 21 (208 mg, 70%) as a viscous liquid. [α]2D0 +17.4 (c
1
0.35, CHCl3); H NMR (CDCl3, 300 MHz) δ 7.36−7.25 (m, 10H),
4.95 (dd, 1H, J = 4.1 Hz), 4.68−4.30 (q, 2H, J = 11.4 Hz), 4.49 (q, 2H,
J = 11.2 Hz), 4.04 (m, 1H), 3.62 (m, 1H), 2.39 (s, 3H), 2.9−1.0 (m,
19 H), 1.37 (s, 3H), 1.0 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 204.1,
139.4, 138.7, 128.3, 128.2, 127.6, 127.4, 127.3, 85.5, 82.0, 75.5, 73.1,
70.7, 69.7, 57.8, 50.6, 45.7, 40.4, 37.9, 36.1, 34.3, 32.7, 32.5, 32.2, 31.8,
28.5, 27.8, 25.4, 15.8, 14.6; IR (NaCl) cm−1 3605, 3517, 2940.9,
2859.1, 1702, 1693.2, 1454, 1359, 1270. HRMS (ESI) m/z: [M −
OBr]+ calcd for C35H45O4Br 513.3368; found 513.3365 0.0015.
(3α,5α,11β,16β)-3,11-Bis(benzyloxy)-16-hydroxypregnan-
20-one (22). To a solution of bromide 21 (150 mg, 0.24 mmol) in
anhydrous CH2Cl2 (3 mL) was added Bu3SnH (0.651 mL, 2.46
mmol) followed by Et3B (1 M in hexane, 0.24 mL, 0.48 mmol) and air
(with empty syringe, 1.5 mL) under inert atmosphere. The mixture
was allowed to stir for 45 min at room temperature and then
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography (30% EtOAc/hexanes)
to afford the hydroxy ketone 22 (123.6 mg, 95%) as a white solid.
[α]2D0 +52.0 (c 0.5, CHCl3); 1H NMR (C6D6 + CCl4 (1:9), 400 MHz)
δ 7.11−6.96 (m, 10H), 4.43−3.39 (q, 2H, J = 11.3 Hz), 4.25−4.19 (m,
2H), 3.96−3.92 (m, 1H), 3.74−3.69 (m, 1H), 3.39−3.34 (m, 1H),
2.32−2.25 (m, 1H), 2.04−1.95 (m, 1H), 1.87 (s, 3H), 1.81−0.58 (m,
18H), 0.97 (s, 3H), 0.79 (s, 3H); 13C NMR (C6D6 + CCl4 (1:9), 100
MHz) δ 210.0, 139.3, 138.4, 128.0,128.1, 127.9, 127.6, 127.5, 127.4,
127.2, 127.1, 127.0, 75.0, 73.0, 71.6, 70.5, 69.6, 67.0, 58.7, 55.7, 43.3,
40.4, 40.3, 36.7, 36.2, 32.7, 32.6, 32.4, 31.8, 31.38, 28.0, 25.6, 16.4,
14.7; IR (NaCl) cm−1 3606.3, 3495.1, 2930.1, 2854.5, 1713.1, 1681.3,
1450, 1363.1. HRMS (ESI) m/z: [M]+ calcd for C35H46O4 530.3396;
found 530.3408 0.0016.
a white solid. [α]2D0 −45.2 (c 1.0, CHCl3/CH3OH (1:1)); H NMR
1
(CDCl3 + 2% CD3OD, 400 MHz) δ 4.37−4.28 (m, 1H),4.17 (br s,
1H), 3.98 (br s, 1H), 2.17−2.08 (m,1H), 2.08−2.0 (m, 1H), 1.99−
0.74 (m, 19H), 1.20 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.25 (s, 3H),
1.23 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H), 0.89 (d, 3H, J = 7.0 Hz),
0.70−0.63 (dd, 1H, J = 10.93, 3.1 Hz); 13C NMR (CDCl3 + 2%
CD3OD, 100 MHz) δ 118.6, 84.5, 82.3, 79.4, 67.8, 66.5, 58.5, 58.1,
48.3, 42.1, 41.0, 39.9, 36.3, 35.2, 32.5, 31.7, 31.6, 30.3, 28.9, 28.4, 28.0,
27.1, 22.9, 19.1, 14.0, 13.9. HRMS (ESI) m/z: [M]+ calcd for
C28H46O5 462.3345; found 462.3348. LRMS for C28H47O5 [M + H]+
= 463.45. HPLC purity of 96.4% (retention time = 15.8)
(3α,5α,11β,17ξ,22R,24S)-24-Methyl-22,25-epoxyfurostan-
3,11,20-triol (1). 22-epi-Hippuristanol (2) (63 mg, 0.135 mmol) was
dissolved in CHCl3 (2 mL) in a single neck round-bottom flask, and to
this solution was added pyridine p-toluenesulfonate (PPTS, 5.3 mg,
0.021 mmol) at room temperature. After the mixture was stirred for 12
h at room temperature, the solvent was removed under reduced
pressure. Chromatography of the residue (without further workup) on
silica gel (15% acetone/hexanes) afforded hippuristanol (1) (18.4 mg,
85% brsm (based on recovered starting material of 42%)) as a white
solid. [α]2D0 +31.0 (c 0.5, CCl4); 1H NMR (400 MHz, 9:1 CCl4/C6D6)
δ 4.05−3.96 (m, 2H), 3.73−3.68 (m, 1H), 2.69 (s, 1H), 2.15−2.07 (m,
1H), 1.98−1.91 (m, 1H), 1.80−0.55 (m, 18H), 1.15 (s, 3H), 1.05 (s,
3H), 1.0 (s, 3H), 1.0 (s, 3H), 1.0 (s, 3H), 0.81(s, 3H), 0.79 (s, 3H),
0.53−0.46 (m, 1H); 13C NMR (75 MHz, 9:1 CCl4/C6D6) δ 115.2,
84.1, 80.0, 79.2, 68.0, 66.4, 66.0, 58.6, 57.6, 50.6, 42.5, 42.1, 41.1, 40.0,
36.0, 34.4, 34.4, 33.1, 31.7, 30.6, 29.4, 29.1, 28.4, 23.6, 18.9, 15.4, 14.3.
HRMS (ESI) m/z: [M + Na]+ calcd for C28H46O5Na 485.3238; found
485.3234. LRMS for C28H47O5 [M + H]+ = 463.45. HPLC purity of
99.5% (retention time = 17.0).
Dihydroxyalkyne 24. To the known3 terminal alkyne 23 (150 mg,
1.2 mmol) in anhydrous THF (2.5 mL) under argon was added n-
butyllithium solution (1.6 M solution in hexane, 0.481 mL, 0.706
mmol) at −78 °C. The resultant mixture was stirred at −78 °C for 1.5
h, followed by the addition of hydroxy ketone 22 (135 mg, 0.253
mmol) in anhydrous THF (2.0 mL). The resultant mixture was stirred
at −78 °C for 4 h. Then the reaction was quenched with saturated
aqueous NH4Cl solution at −78 °C. The mixture was warmed to 0 °C,
diluted with water, extracted with EtOAc, dried over anhydrous
Na2SO4, filtered, and concentrated under vacuum. Purification by flash
column chromatography (silica gel, gradient elution with 4−20%
EtOAc/hexanes) afforded the coupled product 24 (157 mg, 85%) as a
viscous liquid. Here, we obtained the product as a mixture of
diastereomers; hence, analytical data were not provided.
(3α,5α,11β,17ξ,22S,24S)-3,11-Bis(benzyloxy)-24-methyl-
22,25-epoxyfurostan-20-ol (25). To a stirred mixture of dihydrox-
yalkyne 24 (117 mg, 0.159 mmol) and H2O (14.5 mg, 0.799 mmol) in
CH3CN (2 mL) was added Hg(OTf)2 (16 mg, 0.0319 mmol) at room
temperature, and the mixture was stirred for 10 min at the same
temperature. Reaction was quenched by the addition of Et3N (65 μL).
The resulting mixture was diluted with Et2O (5 mL), extracted with
Et2O, and the combined extracts were dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The crude product
was subjected to silica gel column chromatography (elution with 12%
EtOAc/hexanes) to afford the spiroketal 25 (93 mg, 90%) as a white
solid. 1H NMR (C6D6 + CCl4 (1:9), 400 MHz) δ 7.13−7.0 (m, 10H),
4.47−3.91 (ABq, 2H, J = 10.93 Hz), 4.25−4.14 (m, 3H), 3.63 (s br,
1H), 3.35 (br s, 1H), 2.31−2.21 (m, 2H), 1.89−1.68 (m,2H), 1.65−
0.65 (m,19 H), 1.45 (d, 1H, J = 7.8 Hz), 1.23 (s, 3H), 1.0 (s, 3H), 1.0
(s,3H), 0.75 (s, 3H), 0.73 (s, 3H), 0.58−0.49 (dd, 1H, J = 11.32, 3.12
Hz). 13C NMR (C6D6 + CCl4 (1:9), 100 MHz) δ 139.4, 138.5, 128.1,
128.0, 127.9, 127.7, 127.4, 127.18, 127.15, 127.0, 119.7, 84.3, 82.4,
79.7, 74.7, 73.0, 70.5, 69.5, 66.7, 58.9, 58.2, 44.8, 42.7, 40.3, 36.1, 32.7,
32.59, 32.65, 31.6, 30.6, 31.0, 29.6, 28.3, 25.6, 23.0, 20.1, 14.6.
(3α,5α,11β,17ξ,22S,24S)-24-Methyl-22,25-epoxyfurostan-
3,11,20-triol (2). A flame-dried two-neck round-bottom flask was
topped with a dry ice condenser, and the system was flushed with
argon. The condenser was filled with a dry ice/acetone mixture.
Ammonia was condensed, and the lithium metal (16 mg, 2.611 mmol,
1-[(3R,5S,8S,9S,10S,11S,13S,14S)-3,11-Bis(benzyloxy)-10,13-
dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl]propan-1-one (26). To a
stirred solution of ethylmagnesium bromide (3.0 M in THF, 1.61
mL, 4.8 mmol) in anhydrous THF (5 mL) was added acetoxy enone
19 (300 mg, 0.48 mmol) in anhydrous THF (5 mL) at −10 °C (ice
and salt mixture bath) under argon atmosphere. The mixture was
stirred at 0 °C for 1 h and for an additional 1.5 h at room temperature,
then quenched with saturated aqueous NH4Cl (10 mL) solution
cautiously in dropwise fashion at 0 °C. The resulting mixture was
acidified with 3 N aqueous HCl (pH 6.0), extracted with EtOAc (3 ×
30 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. This crude diol was used as such
in the next step without further purification. Accordingly, the crude
diol (176 mg, 0.29 mmol) was dissolved in anhydrous CH2Cl2 (10
mL) and then treated with silica gel supported NaIO4 (629 mg) at
room temperature under argon atmosphere. After completion of the
reaction, the contents were filtered with sintered funnel and washed
with CH2Cl2. Filtrate was concentrated under reduced pressure and
the crude enone was subjected to silica gel column chromatography
(8% EtOAc/hexanes) to afford the desired enone 26 (141 mg, 85%
yield) as a white solid. Rf = 0.7 (SiO2, 20% EtOAc/hexanes); 1H NMR
(CDCl3, 400 MHz) δ 7.39−7.2 (m, 10 H), 6.72−6.63 (m, 1H), 4.81−
4.19 (ABq, 2H, J = 11.32 Hz), 4.49 (q, 2H, J = 12.1 Hz), 4.0−3.94 (m,
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1H), 3.65−3.59 (m, 1H), 3.07−2.99 (m, 1H), 2.7−2.56 (m, 2H),
2.37−0.93 (m,17 H), 1.15 (s, 3H), 1.08 (t, 3H, J = 7.02 Hz), 1.03 (s,
3H); 13C NMR (CDCl3, 100 MHz) δ 199.8, 155.7, 142.7, 139.4,
139.1, 128.2, 128.0, 127.7, 127.3, 127.2, 126.9, 75.5, 73.1, 70.0, 69.5,
59.3, 58.2, 45.9, 40.7, 36.3, 36.2, 32.7, 32.3, 32.2, 32.1, 32.0, 30.7, 27.9,
25.2, 17.5, 14.6, 8.2. HRMS (ESI) m/z: [M + H]+ calcd for C36 H47 O3
527.352; found 527.3512.
mL), and the resulting mixture was diluted with Et2O (5 mL) and
water (5 mL) and extracted with Et2O (3 × 10). The combined
extracts were dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified by silica gel
column chromatography (elution with 10% EtOAc/hexanes) to afford
the corresponding spiroketal (104 mg, 85%) which was used directly
in next step.
A flame-dried two-neck round-bottom flask was topped with a dry
ice condenser, and the system was flushed with argon. The condenser
was filled with a dry ice/acetone mixture. Ammonia was condensed,
and the lithium metal (9.4 mg, 1.58 mmol, washed with hexanes) was
added cautiously through the side neck of the round-bottom flak. The
contents were stirred at −33 °C until no further lithium was seen.
When bronze globules (golden liquid) started to appear, the system
was allowed to equilibrate to the refluxing temperature (−33 °C).
THF (1.5 mL) was added to disperse the newly formed reagent, and
then to the mixture was added slowly a solution of the above dibenzyl
ether (104 mg, 0.15 mmol). A cautious addition is needed to ensure a
regular and smooth ammonia reflux. The reaction mixture was then
stirred at −33 °C for 2 h, and to the mixture was carefully and slowly
added NH4Cl powder at −33 °C to quench the excess of lithium.
Ammonia was allowed to evaporate under a stream of air. To the
residue was added H2O (5 mL), and extraction was with EtOAc (5
mL). The combined extracts were dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. Purification by silica
gel column chromatography (20% acetone/hexanes) afforded 21-
methyl-22-epi-hippuristanol 4 (71 mg, 94%) as a colorless solid. Rf =
1-[(3R,5S,8S,9S,10S,11S,13S,14S,16S,17S)-3,11-Bis-
(benzyloxy)-17-bromo-16-hydroxy-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl]propan-
1-one (27). Enone 26 (141 mg, 0.26 mmol) was dissolved in acetone
(6 mL), THF (3 mL), and H2O (1.5 mL). Then to the mixture was
added NBA (N-bromoacetamide, 110.7 mg, 0.80 mmol) at room
temperature. The mixture was stirred for 6 h in the dark at room
temperature. Then the mixture was diluted with EtOAc (20 mL). The
organic layer was washed with saturated Na2SO3 solution and brine
solution, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified by silica gel
column chromatography (10% EtOAc/hexanes) to give the α-bromo
ketone 27 (125 mg, 75%) as a viscous liquid. Rf = 0.6 (SiO2, 20%
EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 7.39−7.24 (m,
10H), 5.0−4.95 (m, 1H), 4.66−4.32 (ABq, 2H, J = 11.7 Hz), 4.51−
4.48 (m, 2H), 4.47−4.44 (m, 1H), 4.07−4.02 (m, 1H), 3.66−3.62 (m,
1H), 3.05−2.97 (m, 1H), 2.48−2.34 (m, 2H), 2.05−0.84 (m, 17 H),
1.37 (s, 3H), 1.16 (t, 3H, J = 7.0 Hz), 1.02 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 207.1, 139.3, 138.6, 128.5, 128.9, 128.2, 127.6, 127.3,
127.2, 127.2, 85.8, 82.0, 75.6, 73.1, 70.7, 69.6, 57.7, 50.6, 45.6, 40.3,
38.0, 36.1, 34.2, 33.6, 32.6, 32.4, 32.1, 31.7, 27.8, 25.4, 15.8, 14.5, 8.5.
1-[(3R,5S,8S,9S,10S,11S,13S,14S,16S,17R)-3,11-Bis-
(benzyloxy)-16-hydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl]propan-1-one (28). To a sol-
ution of bromide 27 (125 mg, 0.20 mmol) in anhydrous CH2Cl2 (5
mL) were added Bu3SnH (0.53 mL, 2.0 mmol) followed by Et3B (1 M
in hexane, 0.32 mL, 0.32 mmol) and air (with empty syringe, 5 mL)
under inert atmosphere. The mixture was allowed to stir for 45 min at
room temperature and concentrated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
(20% EtOAc/hexanes) to afford the desired hydroxy ketone 28 (101.5
mg, 93% yield) as a white solid. Rf = 0.5 (SiO2, 30% EtOAc/hexanes).
1H NMR (C6D6 + CCl4, 400 MHz) δ 7.14−6.95 (m, 10H), 4.42−3.98
(ABq, 2H, J = 11.3 Hz), 4.26−4.18 (m, 3H), 3.74−3.70 (m, 1H),
3.39−3.34 (m, 1H), 2.27−1.96 (m, 3H), 1.87−0.61 (m, 14H), 0.96 (s,
3H), 0.84 (t, 3H, J = 7.41 Hz), 0.79 (s, 3H); 13C NMR (CDCl3, 100
MHz) δ 212.4, 139.1, 138.2, 127.7, 127.4, 127.6, 127.4, 127.3, 127.2,
126.96, 126.91, 126.8, 74.9, 72.7, 71.5, 70.4, 69.3, 65.8, 58.4, 55.4, 43.4,
40.2, 37.5, 35.9, 32.4, 32.3, 32.2, 31.1, 27.8, 26.6, 25.4, 16.3, 14.5; IR
(NaCl) cm−1 3606.3, 3495.1, 2930.1, 2854.5, 1713.1, 1681.3, 1450,
1363.1. HRMS (ESI) m/z: [M + H]+ calcd for C36H49O4 545.3625;
found 545.3622.
(2S,2′R,4S,4a′S,4b′S,5′S,6a′S,7′R,9a′S,10a′S,10b′S,12a′S)-7′-
Ethyl-4,4a′,5,5,6a′-pentamethylicosahydro-3H-spiro[furan-
2,8′-naphtho[2′,1′:4,5]indeno[2,1-b]furan]-2′,5′,7′-triol (4). To
the known terminal alkyne 23 (109 mg, 0.18 mmol) in anhydrous
THF (2.5 mL) under argon was added n-butyllithium solution (1.6 M
solution in hexane, 0.29 mL, 0.46 mmol) at −78 °C. The resultant
mixture was stirred at −78 °C for 1.5 h, followed by the addition of
hydroxy ketone 28 (101.5 mg, 0.18 mmol) in anhydrous THF (2 mL).
The resultant mixture was stirred at −78 °C for 6 h. Then the reaction
was quenched with saturated aqueous NH4Cl solution at −78 °C. The
mixture was warmed to 0 °C, diluted with water, extracted with EtOAc
(3 × 10 mL), dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (gradient elution with 5−15% EtOAc/
hexane) to afford the desired coupled product 29 (139 mg, 90%) as
a mixture of diastereomers and was used as such in the next reaction.
To a stirred mixture of dihydroxyalkyne 29 (139 mg, 0.18 mmol) and
H2O (16.8 mg, 0.93 mmol) in CH3CN (2.0 mL) was added Hg(OTf)2
(18.7 mg, 0.037 mmol) at room temperature. Then 0.1 mL of acetic
acid was added and the mixture was stirred for 10 min at the same
temperature. Reaction was quenched by the addition of Et3N (0.5
0.45 (SiO2, 30% acetone/hexanes); [α]2D0−15.4 (c 0.25, CH2Cl2); H
1
NMR (C6D6 + CCl4, 400 MHz) δ 4.19−4.09 (m, 1H), 4.04−3.98 (m,
1H), 3.74−3.67 (m, 1H), 2.13−2.01 (m, 1H), 1.85−0.65 (m, 21H),
1.08 (s, 3H), 1.06 (s, 3H), 0.79 (s, 3H); 13C NMR (C6D6 + CCl4, 100
MHz) δ 118.4, 84.8, 83.2, 78.6, 67.4, 65.5, 64.0, 58.5, 57.8, 48.8, 41.6,
40.3, 39.4, 36.0, 35.4, 32.3, 31.4, 31.2, 30.7, 30.0, 29.3, 28.6, 27.8, 23.2,
19.34, 13.83, 14.04, 8.6. HRMS (ESI) m/z: [M + H]+ calcd for
C29H49O5 477.3575; found 477.3571. LRMS for C29H49O5 [M + H] =
477.45. HPLC purity of 94.4% (retention time = 17.2).
(2R,2′R,4S,4a′S,4b′S,5′S,6a′S,7′R,9a′S,10a′S,10b′S,12a′S)-7′-
ethyl-4,4a′,5,5,6a′-Pentamethylicosahydro-3H-spiro[furan-
2,8′-naphtho[2′,1′:4,5]indeno[2,1-b]furan]-2′,5′,7′-triol (3). 21-
Methyl-22-epi-hippuristanol 4 (71 mg, 0.14 mmol) was dissolved in
CHCl3 (2 mL), and to this solution was added pyridine p-
toluenesulfonate (PPTS, 5.6 mg, 0.02 mmol) at room temperature.
The mixture was stirred for 12 h. The solvent was removed under
reduced pressure. The crude residue was purified (without further
workup) by silica gel column chromatography using 15% acetone/
hexanes to afford the desired 21-methylhippuristanol 3 (28.0 mg, 80%
brsm (based on recovered starting material of 40%)) as a colorless
liquid. Rf = 0.5 (SiO2, 30% acetone/hexanes); [α]2D0 +10.3 (c 0.25,
CH2Cl2); 1H NMR (400 MHz, 9:1 CCl4/C6D6) δ 4.05−3.89 (m, 2H),
3.71 (br s, 1H), 2.69 (s, 1H), 2.16−2.03 (m, 1H), 1.89 (dd, 1H, J =
13.6 Hz), 1.80−0.58 (m, 27H), 1.15 (s, 3H), 1.09 (s, 3H), 1.01 (s,
3H), 1.0 (s, 3H), 0.81 (s, 3H); 13C NMR (100 MHz, CCl4/C6D6
(9:1)) δ 115.5, 83.4, 81.2, 80.0, 67.5, 65.6, 60.7, 58.2, 57.1, 49.7, 41.85,
41.83, 40.3, 39.6, 36.2, 35.4, 33.8, 32.5, 31.2, 30.0, 28.6, 28.3, 27.9,
22.9, 18.3, 14.8, 13.7, 8.4. HRMS (ESI) m/z: [M + H]+ calcd for
C29H49O5 477.3575; found 477.3575. LRMS for C29H49O5 [M + H] =
477.45. HPLC purity of 94.1% (retention time = 18.0).
(3α,5α,11β)-3,11-Bis(benzyloxy)-21-hydroxypregn-16-en-
20-one (S1). To a stirred solution of ketoacetate 19 (500 mg, 0.87
mmol) in THF (5 mL), MeOH (5 mL), and H2O (2 drops) was
added K2CO3 (120 mg, 0.87 mmol) at room temperature, and the
reaction was monitored by TLC. After completion of the reaction,
solvent was removed under reduced pressure and the reaction mixture
was diluted with EtOAc (20 mL) and washed with H2O (2 × 20 mL)
and brine. The sample was dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by flash chromatography (15% EtOAc/hexanes) to provide the
corresponding alcohol S1 (324 mg, 70%) as a colorless solid. Rf = 0.46
(30% EtOAc/hexanes). 1H NMR (CDCl3, 400 MHz) δ 7.39−7.23 (m,
10 H), 6.75−6.71 (m, 1H), 4.81−4.23 (ABq, 2H, J = 11.32 Hz), 4.55−
2520
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4.47 (ABq, 2H, J = 12.24 Hz), 4.45−4.35 (ABq, 2H, J = 16.13 Hz),
4.01−3.97 (m, 1H), 3.65−3.62 (m, 1H), 2.97 (dd, 1H, J = 14.44, 1.95
Hz), 2.43−2.33 (m, 1H), 2.19−1.98 (m, 2H), 1.88−1.76 (m, 2H),
1.62−0.91 (m, 14H), 1.19 (s, 3H), 1.03 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 196.7, 152.6, 144.8, 139.6, 139.2, 128.5, 128.3, 127.8,
127.5, 127.4, 127.2, 75.7, 73.3, 70.4, 69.8, 65.2, 59.61, 58.2, 46.5, 40.9,
36.5, 36.3, 32.8, 32.5, 32.4, 30.8, 28.0, 25.5, 17.9, 14.8. HRMS (ESI)
m/z: [M + H]+ calcd for C35 H45O4 529.3312; found 529.3306.
(3α,5α,11β)-3,11,21-Tris(benzyloxy)pregn-16-en-20-one
(30). To a stirred solution of alcohol S1 (324 mg, 0.61 mmol) in dry
diethyl ether (10 mL) under argon at room temperature was added
silver oxide (284 mg, 1.22 mmol) followed by benzyl bromide (0.087
mL, 0.73 mmol), and the mixture was allowed to stir for 48 h at room
temperature. After completion of the reaction, the mixture was filtered
through Celite pad and washed with Et2O (20 mL). The original
filtrate and Et2O washings were combined and evaporated under
reduced pressure. The crude product was purified by silica gel column
chromatography (5% EtOAc/hexanes) to afford the benzyl ether 30
(303 mg, 80%) as a colorless foamy solid. Rf = 0.80 (SiO2, 20%
solution (1.6 M solution in hexane, 0.27 mL, 0.44 mmol) at −78 °C.
The resultant mixture was stirred at −78 °C for 1.5 h, followed by the
addition of hydroxy ketone 32 (113 mg, 0.17 mmol) in anhydrous
THF (2 mL). The resulting mixture was stirred at −78 °C for 6 h.
Then the reaction was quenched with saturated aqueous NH4Cl
solution at −78 °C. The mixture was warmed to 0 °C, diluted with
water, extracted with EtOAc (3 × 10 mL), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product was purified by flash column chromatography (gradient
elution with 5−15% EtOAc/hexane) to afford the desired coupled
product 33 (121 mg, 82%) as an inseparable mixture of diastereomers
(Rf = 0.6 (SiO2, 30% EtOAc/hexanes)). This was used in the next
reaction without further analysis. To a stirred mixture of
dihydroxyalkyne 33 (121 mg, 0.14 mmol) and H2O (0.013 g, 0.72
mmol) in CH3CN (2.0 mL) was added Hg(OTf)2 (14 mg, 0.029
mmol) at room temperature, and the mixture was stirred for 10 min at
the same temperature. Reaction was quenched by the addition of Et3N
(90 μL). The resulting mixture was diluted with Et2O (5 mL),
extracted with Et2O, and the combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The crude product 34 (87 mg, 80%) was used as such for the next
step. A flame-dried two-neck round-bottom flask was topped with a
dry ice condenser, and the system was flushed with argon. The
condenser was filled with a dry ice/acetone mixture. Ammonia was
condensed, and the lithium metal (13.9 mg, 2.32 mmol, washed with
hexanes) was added cautiously through the side neck of the round-
bottom flask. The contents were stirred at −33 °C until no further
lithium was seen. When bronze globules (golden liquid) started to
appear, the system was allowed to equilibrate to the refluxing
temperature −33 °C. THF (1.0 mL) was added to disperse the newly
formed reagent, and then to the mixture was added slowly a THF
solution of 34 (87 mg, 0.11 mmol). A cautious addition is needed to
ensure a regular and smooth ammonia reflux. The reaction mixture
was then stirred at −33 °C for 2 h, and to the mixture was carefully
and slowly added NH4Cl powder at −33 °C to quench the excess of
lithium. Ammonia was allowed to evaporate under a stream of air. To
the residue was added H2O (10 mL), and extraction was with EtOAc
(3 × 10 mL). The combined extracts were dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure.
Purification by silica gel column chromatography (15% acetone/
hexanes) afforded C-21-hydroxy-22-epi-hippuristanol (5) (47.8 mg,
86%) as a white solid. Rf = 0.5 (SiO2, 30% acetone/hexanes); [α]D20
1
EtOAc/hexanes); H NMR (CDCl3, 500 MHz) δ 7.42−7.25 (m, 15
H), 6.74−6.70 (m, 1H), 4.82−4.23 (ABq, 2H, J = 11.24 Hz), 4.67−
4.34 (m, 4H), 4.02−3.97 (m, 1H), 3.66−3.62 (m, 1H), 3.08−3.01 (dd,
1H, J = 14.42, 1.95 Hz), 2.39−2.31 (m, 1H), 2.14−2.0 (m, 2H), 1.88−
1.79 (m, 2H), 1.68−0.96 (m, 14H), 1.19 (s, 3H), 1.03 (s, 3H); 13C
NMR (CDCl3, 125 MHz) δ 194.9, 153.4, 143.6, 139.4, 139.1, 137.5,
128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.3, 127.2, 126.9, 75.5, 73.2,
73.1, 72.5, 70.15, 69.6, 59.3, 57.9, 46.4, 40.7, 36.3, 36.1, 32.7, 32.6,
32.3, 32.2, 30.6, 27.9, 25.3, 17.6, 14.6. HRMS (ESI) m/z: [M +
H]+calcd for C42 H51O4 619.3782; found 619.3779.
(3α,5α,11β,16β)-3,11,21-Tris(benzyloxy)-17-bromo-16-hy-
droxypregnan-20-one (31). To a stirred solution of enone 30 (303
mg, 0.48 mmol) in acetone (8 mL), THF (2 mL), and H2O (4 mL)
was added NBA (N-bromoacetamide) (202 mg, 1.46 mmol) at room
temperature. The mixture was stirred for 5 h at room temperature.
The mixture was diluted with EtOAc (20 mL). The organic layer was
washed with saturated Na2SO3 solution and brine solution, dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatography
(10% EtOAc/hexanes) to get the desired hydroxy bromide 31 (140
mg, 40%) as a viscous liquid. Rf = 0.5 (SiO2, 20% EtOAc/hexanes); 1H
NMR (CDCl3, 400 MHz) δ 7.41−7.20 (m, 15 H), 5.05−4.99 (m,
1H), 4.74−4.22 (ABq, 2H, J = 11.71 Hz), 4.67−4.21 (m, 5H), 3.99
(m, 1H), 3.62 (m, 1H), 2.49−2.32 (m, 2H), 2.05−0.83 (m, 18H), 1.38
(s, 3H), 0.99 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 200.8, 139.3,
138.7, 136.5, 128.6, 128.2, 128.13, 128.11, 127.6, 127.3, 127.2, 127.1,
85.3, 81.4, 75.0, 73.6, 73.1, 73.0, 70.3, 69.6, 57.6, 50.4, 46.5, 40.3, 37.4,
36.0, 34.3, 32.5, 32.59, 32.50, 31.5, 27.5, 25.3, 16.2, 14.5. HRMS (ESI)
m/z: [M + NH4]+calcd for C42H55BrNO5 733.3292; found 733.3289.
(3α,5α,11β,16β)-3,11,21-Tris(benzyloxy)-16-hydroxypre-
gnan-20-one (32). To a stirred solution of hydroxy bromide 31 (140
mg, 0.19 mmol) in anhydrous CH2Cl2 (5 mL) was added Bu3SnH
(0.51 mL, 1.9 mmol) followed by Et3B (1 M in hexane, 0.31 mL, 0.31
mmol) and air (with empty syringe, 3 mL) under inert atmosphere.
The mixture was allowed to stir for 45 min at room temperature, then
directly concentrated under reduced pressure. The resulting residue
was purified by silica gel column chromatography (20% EtOAc/
hexanes) to afford the desired hydroxy ketone 32 (113 mg, 91%) as a
white solid. Rf = 0.4 (SiO2, 30% EtOAc/hexanes); [α]2D0 +38.0 (c 0.3,
1
−23.5 (c 1.0, acetone); H NMR (C6D6 + CCl4 (1:9), 400 MHz) δ
4.19−411 (m, 1H), 4.03 (br s, 1H), 3.75 (br s, 1H), 3.41 (d, 1H, J =
11.71 Hz), 3.19 (d, 1H, J = 11.32 Hz), 2.61−2.55 (br s, 1H), 2.24 (s,
1H), 2.14−2.04 (m, 1H), 1.93−0.60 (m, 29H), 1.83 (s, 3H), 1.09 (s,
3H), 0.98 (s, 3H); 13C NMR (C6D6+CCl4 (1:9), 100 MHz) δ 117.5,
83.1, 83.0, 79.5, 68.1, 67.3, 67.1, 63.4, 58.4, 58.1, 48.4, 41.6, 40.2, 39.6,
38.6, 36.1, 35.4, 32.3, 31.4, 30.4, 29.3, 28.5, 27.8, 23.4, 18.0, 14.0, 13.8.
HRMS (ESI) m/z: [M + H]+ calcd for C28H47O6 479.3367; found
479.3383. LRMS for C28H47O6 [M + H] 479.45. HPLC purity of 88.0
(retention time = 15.1).
(2S,2′R,4S,4a′S,4b′S,5′S,6a′S,9a′S,10a′S,10b′S,12a′S)-2′,5′-
Dihydroxy-4,4a′,5,5,6a′-pentamethyloctadecahydro-3H-spiro-
[furan-2,8′-naphtho[2′,1′:4,5]indeno[2,1-b]furan]-7′(1′H)-one
(8). To a vigorously stirred suspension of silica gel supported NaIO4
reagent (0.198 g) in CH2Cl2 (5 mL) was added a solution of vicinal
diol 5 (47.8 mg, 0.09 mmol) in anhydrous CH2Cl2 (10 mL). The
reaction was monitored by TLC until disappearance of the diol. After
completion of the reaction, the mixture was filtered through sintered
glass funnel and silica gel and thoroughly washed with CH2Cl2 (3 × 15
mL). The filtrate was concentrated under reduced pressure and
purified by silica gel column chromatography (10% acetone/hexanes)
to afford the ketone 8 (37.9 mg, 85%) as a white solid. Rf = 0.7 (SiO2,
30% acetone/hexanes). [α]2D0 −20.5 (c 1.0, acetone); 1H NMR (C6D6
+ CCl4 (1:9), 400 MHz) δ 4.92−4.85 (m, 1H), 4.39−4.34 (m, 1H),
4.08−4.04 (m, 1H), 2.35 (d, 1H, J = 7.0 Hz), 2.31−0.80 (m, 24H),
1.31 (s, 3H), 1.03 (s, 3H), 1.02 (s, 3H), 0.98 (d, 3H, J = 6.6 Hz); 13C
NMR (CDCl3, 100 MHz) δ 211.4, 109.0, 86.1, 79.5, 67.7, 66.3, 59.7,
58.0, 57.9, 47.8, 43.3, 41.7, 39.8, 38.9, 36.3, 35.2, 33.2, 32.3, 31.7, 31.0,
1
CH2Cl2); H NMR (C6D6 + CCl4 (1:9), 400 MHz) δ 7.13−6.95 (m,
15 H), 4.40−3.91 (ABq, 2H, J = 11.32 Hz), 4.34−3.63 (m, 8H), 3.35
(br s, 1H), 3.27 (d, 1H, J = 3.51 Hz), 2.19−2.10 (m, 2H), 2.06−1.96
(m, 1H), 1.80−0.58 (m, 16H), 0.99 (s, 3H), 0.77 (s, 3H); 13C NMR
(C6D6 + CCl4 (1:9), 100 MHz) δ 209.1, 139.1, 138.3, 136.9, 128.1,
127.8, 127.7, 127.45, 127.41, 127.2, 126.9, 126.8, 75.7, 74.6, 72.7, 71.6,
70.1, 69.3, 63.0, 58.3, 55.3, 43.4, 40.1, 39.4, 37.0, 35.9, 32.43, 32.41,
32.3, 31.0, 27.69, 26.65, 16.82, 14.50. HRMS (ESI) m/z: [M + H]+
calcd for C42H53O5 637.3888; found 637.3903.
(3α,5α,11β,17ξ,22S,24S)-24-Methyl-22,25-epoxyfurostan-
3,11,20,21-tetrol (5). To the alkyne 23 (104 mg, 0.53 mmol) in
anhydrous THF (2.5 mL) under argon was added n-butyllithium
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Article
28.6, 28.2, 27.7, 22.3, 17.4, 14.1, 13.9. HRMS (ESI) m/z: [(M + H) +
(−H2O)]+ calcd for C27H41O4 429.2999; found 429.2997. LRMS for
C27H41O4 [M + H − H2O] = 429.40. HPLC purity of 99.4%
(retention time = 16.2).
quenched with a minimum amount of cold water (5 mL) cautiously.
The reaction mixture was diluted with EtOAc (5 mL) and stirred for
2.5 h at room temperature and then filtered using Celite-cintered
funnel. The filtrate was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. Purification of the crude
product by silica gel column chromatography (30% acetone/hexanes)
afforded 3-β,11-β diol 9 (38.0 mg, 75% yield) as a colorless solid. Rf =
(2S,2′R,4S,4a′S,4b′S,5′S,6a′S,7′R,9a′S,10a′S,10b′S,12a′S)-
4,4a′,5,5,6a′-Pentamethylicosahydro-3H-spiro[furan-2,8′-
naphtho[2′,1′:4,5]indeno[2,1-b]furan]-2′,5′,7′-triol (7). To a
stirred solution of ketone 8 (37.9 mg, 0.084) in anhydrous ethanol
(4.0 mL) was added NaBH4 (6.4 mg, 0.16 mmol) at 0 °C, and the
reaction mixture was allowed to stir at ambient temperature for 1 h.
The resulting solution was poured into ice cold water, and to it was
added Et2O. Then the sample was extracted with Et2O (3 × 10 mL),
dried over anhydrous Na2SO4, filtered using sintered funnel, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (20% acetone/hexanes) to give the
desired 20-desmethyl-22-epi-hippuristanol (7) (34.2 mg, 90%) as a
colorless solid. Rf = 0.5 (SiO2, 30% acetone/hexanes); [α]2D0 −55.3 (c
0.4 (SiO2, 30% Acetone/Hexanes). [α]2D0 +38.5 (c 1.0, acetone). H
1
NMR (C6D6 +CCl4 (1:9), 400 MHz) δ 4.01−3.91 (m, 2H), 3.25−3.13
(m, 1H), 2.71−2.68 (br s, 1H), 2.15−2.07 (m, 1H), 1.93 (dd, 1H, J =
14.0, 2.3 Hz), 1.79−0.29 (m, 20H), 1.16 (s, 3H), 1.06 (s, 3H), 1.02 (s,
3H), 1.01 (s, 3H), 0.83 (s, 3H), 0.80 (d, 3H, J = 7.02 Hz); 13C NMR
(C6D6 + CCl4, 100 MHz) δ 115.0, 83.8, 79.9, 78.8, 70.4, 67.8, 66.0,
58.3, 57.0, 50.0, 45.79, 42.0, 41.5, 40.6, 36.3, 35.6, 34.0, 32.6, 31.4,
31.2, 30.1, 29.0, 28.5, 28.0, 23.0, 18.3, 14.9, 14.0; IR (NaCl) cm−1
3615.5, 3494, 3011, 2929, 1455, 1235, 1083. HRMS (ESI) m/z: [M +
H]+ calcd for C28H47O5 463.3418; found 463.3433. LRMS for
C28H47O5 [M + H]+ = 463.45. HPLC purity of 98.8% (retention time
= 15.6).
1
0.25, CHCl3); H NMR (CDCl3 + 2% CD3OD, 400 MHz) δ 4.45−
4.37 (m, 2H), 4.24−4.18 (br s, 1H), 3.97−3.91 (br s, 1H), 2.23−0.79
(m, 22H), 1.20 (s, 3H), 1.19 (s, 3H), 0.95 (s, 3H), 0.92 (s, 3H), 0.88
(d, 3H, J = 7.0 Hz); 13C NMR (CDCl3 + 2% CD3OD, 100 MHz) δ
113.0, 79.3, 76.4, 76.1, 63.8, 62.0, 55.8, 54.5, 54.1, 44.0, 37.5, 37.1,
35.8, 35.5, 32.1, 31.1, 28.8, 28.3, 27.6, 26.5, 24.5, 24.3, 23.8, 19.0, 14.0,
2-[(3α,5α,11β,17ξ,22R,24S)-11,20-Dihydroxy-24-methyl-
22,25-epoxyfurostan-3-yl]-1H-isoindole-1,3(2H)-dione (35). To
a stirred solution of 3β,11β-hydroxyhippuristanol (9) (38.0 mg, 0.08
mmol) in dry THF (5 mL) was added TPP (22.4 mg, 0.08 mmol) and
phthalimide (12.0 mg, 0.08 mmol) at 20 °C under nitrogen
atmosphere. The resulting suspension was cooled to 5 °C and stirred
for 10 min, and then DIAD (0.017 mL, 0.09 mmol) in THF (0.1 mL)
was added slowly to the reaction mixture, which was slowly allowed to
room temperature and was stirred for 48 h at room temperature.
Methanol was added to the reaction mixture, and the resulting slurry
was stirred for 1 h. The solid was filtered off and solid was washed with
dichloromethane and combined filtrates were evaporated to give the
phthalimide 35 (48.6 mg, 65% yield). Rf = 0.6 (SiO2, 30% acetone/
10.07, 10.0. HRMS (ESI) m/z: [M
+
H]+ calcd for
C27H45O5449.3262; found 449.3252. LRMS for C27H45O5 [M + H]+
= 449.40. HPLC purity of 96.5% (retention time = 13.4)
(2R,2′R,4S,4a′S,4b′S,5′S,6a′S,7′R,9a′S,10a′S,10b′S,12a′S)-
4,4a′,5,5,6a′-Pentamethylicosahydro-3H-spiro[furan-2,8′-
naphtho[2′,1′:4,5]indeno[2,1-b]furan]-2′,5′,7′-triol (6). 20-Des-
methyl-22-epi-hippuristanol (7) (34.2 mg, 0.076 mmol) was dissolved
in CHCl3 (1.0 mL) in a single-neck round-bottom flask, and to this
solution was added pyridine p-toluenesulfonate (PPTS, 2.8 mg, 0.01
mmol) at room temperature. After the mixture was stirred for 12 h at
room temperature, the solvent was removed under reduced pressure.
The crude residue (without further workup) was purified by silica gel
column chromatography (18% acetone/hexanes) to afford the 20-
desmethylhippuristanol (6) (15.3 mg, 85% brsm (based on recovered
starting material of 40%)) as a white solid. Rf = 0.6 (SiO2, 30%
1
hexanes). H NMR (C6D6 + CCl4, 400 MHz) δ 7.53−7.48 (m, 2H),
7.29−7.26 (m, 2H), 4.31−4.24 (br s, 1H), 4.08−3.97 (m, 2H), 2.69−
2.66 (br s, 1H), 2.15−2.07 (m, 1H), 1.92 (dd, 1H, J = 14..0, 2.3 Hz),
1.79−0.47 (m, 19H), 1.18 (s, 3H), 1.07 (s, 3H), 1.02 (s, 3H), 1.01 (s,
3H), 0.89 (s, 3H), 0.80 (d, 3H, J = 7.02 Hz); 13C NMR (C6D6 + CCl4
(1:9), 100 MHz) δ 167.9, 132.8, 132.2, 122.4, 115.0, 83.8, 80.0, 78.9,
67.5, 65.9, 58.1, 56.8, 50.0, 47.1, 42.0, 41.5, 41.1, 40.6, 38.7, 35.3, 33.9,
33.6, 32.3, 32.0, 30.0, 28.5, 27.9, 23.0, 21.4, 18.3, 14.9, 14.4.
acetone/hexanes); [α]D20 +43.5 (c 0.2, CH2Cl2); H NMR (400 MHz,
1
CD2Cl2) δ 4.29−4.20 (m, 2H), 4.15 (dd, 1H, J = 17.17, 8.19 Hz),
3.99−3.94 (br s, 1H), 2.51 (d, 1H, J = 8.1 Hz), 2.16 (d, 1H, J = 5.46
Hz), 2.13−0.73 (m, 20H), 1.25 (s, 3H), 1.22 (s, 3H), 1.14 (s, 3H), 1.0
(s, 3H), 0.92 (d, 3H, J = 6.63 Hz); 13C NMR (100 MHz, CD2Cl2) δ
112.7, 84.6, 80.0, 78.5, 67.9, 66.1, 58.8, 58.1, 57.8, 49.0, 46.4, 42.0,
41.6, 39.8, 36.2, 35.3, 33.8, 32.4, 31.6, 30.2, 28.6, 27.9, 27.4, 22.2, 18.6,
13.8, 13.7. HRMS (ESI) m/z: [M + H]+ calcd for C27H45O5 449.3262;
found 449.3252. LRMS for C27H45O5 [M + H]+ = 449.40. HPLC
purity of 92.5% (retention time = 15.8).
(3β,5α,11β,17ξ,22R,24S)-24-Methyl-22,25-epoxyfurostan-
3,11,20-triol (9). Hippuristanol (1) (100 mg, 0.2 mmol) was
dissolved in pyridine (5.0 mL). PDC (325 mg, 0.86 mmol) was added
at 0 °C under argon atmosphere. The reaction mixture was warmed to
room temperature and stirred for 4 h at room temperature. After
completion of the reaction, contents were poured into water, then
extracted with ether, and the combined organic layers were dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure. The residue was purified by flash chromatography (20%
acetone/hexanes) to afford a mixture of C3-mono ketone and 3,11-
diketone, which was used as such in the next reaction. To a stirred
solution of above mixture of ketones (63.0 mg, 0.13 mmol) in MeOH
(2 mL) was added NaBH4 (51.9 mg, 1.3 mmol) at 0 °C. After 10 min
the reaction mixture was warmed to reflux temperature for 4 h and
then cooled to 0 °C. Brine solution was added to the reaction mixture
and warmed to room temperature and extracted with EtOAc (3 × 10
mL), dried over anhydrous Na2SO4, filtered using sintered funnel, and
concentrated under reduced pressure. The crude residue was purified
by a short pad of silica gel and further treated with LiAlH4 solution
(1.18 M in THF, 0.93 mL, 1.09 mmol) at 0 °C in THF under argon
atmosphere. The mixture was stirred for 30 min at 0 °C and slowly
allowed to reflux for 4 h. The reaction mixture was cooled to 0 °C and
(3α,5α,11β,17ξ,22R,24S)-3-Amino-24-methyl-22,25-epoxy-
furostan-11,20-diol (10). To a stirred solution of phthalimide 35
(48.6 mg, 0.08 mmol) in dry EtOH (2 mL) was added hydrazine
monohydrate (0.48 mL, 9.9 mmol) at room temperature under
nitrogen atmosphere. The resulting suspension was heated to reflux for
24 h. The mixture was allowed to cool to 0 °C, and the solid was
filtered off. The solid was washed with dichloromethane and combined
filtrates were dried with anhydrous Na2SO4 and evaporated to give the
crude residue. The crude residue was purified by Al2O3 (neutral)
column chromatography (15% MeOH−CH2Cl2) to afford the desired
amine 10 (30 mg, 80% yield). Rf = 0.3 (SiO2, 20% MeOH/CH2Cl2) as
a white solid. [α]D20 +28.5 (c 0.25, CH2Cl2); H NMR (CD2Cl2, 500
1
MHz) δ 4.32−4.24 (m, 2H), 3.36−3.28 (br s, 1H), 3.16−3.07 (br s,
1H), 2.41−2.34 (m, 2H), 2.18−2.10 (m, 1H), 2.06−0.82 (m, 18H),
1.36 (s, 3H), 1.30 (s, 3H), 1.23 (s, 3H), 1.20 (s, 3H), 1.04 (s, 3H),
0.99 (d, 3H, J = 7.1 Hz); 13C NMR (CD2Cl2, 125 MHz) δ 115.3, 84.3,
79.9, 79.1, 68.0, 66.1, 57.8, 57.0, 49.74, 46.4, 42.1, 41.9, 40.8, 39.5,
36.4, 33.9, 33.7, 32.5, 31.2, 30.2, 28.9, 28.1, 28.0, 22.7, 21.6, 18.4, 14.4,
14.0; IR (NaCl) cm−1 2920.35, 1455, 1260, 1018.1, 799.57. HRMS
(ESI) m/z: [M + H]+ calcd for C28H48NO4 462.3578; found 462.3591.
LRMS for C28H48NO4 [M + H]+ = 462.45. HPLC purity of 96%
(retention time = 4.8).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C spectra for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Article
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AUTHOR INFORMATION
Corresponding Author
*Phone: 418-603-3753. E-mail: pierre.deslongchamps@chm.
ulaval.ca.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Natural Sciences and Engineering Research
Council of Canada (NSERCC) and Canadian Institutes of
Health Research (CIHR) for funding. We also thank the
Organic Synthesis Service at the CHU of Quebec Research
Center (http://pfchem.crchudequebec.ca) for performing
purity analyses of compounds.
ABBREVIATIONS USED
■
RNA, ribonucleic acid; eIF, eukaryotic initiation factor; mRNA,
messenger ribonucleic acid; DEAD, aspartic acid−glutamic
acid−alanine−aspartic acid; G, glycine; N, asparagine; SAR,
structure−activity relationship; THF, tetrahydrofuran; DIAD,
diisopropyl azodicarboxylate; TPP, triphenylphosphine; TBAI,
tetrabutylammonium iodide; PTSA, p-toluenesulfonic acid;
NBA, N-bromoacetamide; PPTS, pyridinium p-toluenesulfo-
nate; Rf, retention factor (in chromatography); TLC, thin-layer
chromatography; NMR, nuclear magnetic resonance; PDC,
pyridinium dichromate; FF, firefly; Ren, renilla; DMSO,
dimethyl sulfoxide; HPLC, high-performance liquid chroma-
tography; high-pressure liquid chromatography
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