Total synthesis and biological activity of the proposed structure of phaeosphaeride A

Article abstract of DOI:10.1021/jo301662e

The total synthesis of the structure assigned to the natural product phaeosphaeride A 1a was accomplished. The key steps involve the addition of vinyllithium reagent 7 to the acetonide-protected aldehyde 8 to access the carbon backbone of 1a, the introduction of the methoxylamino group followed by intramolecular hetero-Michael cyclization, and methanol elimination to form the dihydropyran ring. In this study, both enantiomers of 1a were synthesized and tested for biological activity. Preliminary results showed that (6R,7R,8R)-1a and (6S,7S,8S)-1a inhibit STAT3-dependent transcriptional activity in a dose-dependent manner and exhibit antiproliferative properties in breast (MDA-MB-231) and pancreatic (PANC-1) cancer cells.

Full text of DOI:10.1021/jo301662e

Article
pubs.acs.org/joc
Total Synthesis and Biological Activity of the Proposed Structure of
Phaeosphaeride A
Anthoula Chatzimpaloglou,† Maria P. Yavropoulou,‡ Karien E. Rooij,§ Ralf Biedermann,∥ Uwe Mueller,⊥
Stefan Kaskel,∥ and Vasiliki Sarli*,†
†Faculty of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece
‡Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, Netherlands
§Department of Radiology, Leiden University Medical Center, Leiden, Netherlands
∥Department of Inorganic Chemistry, Dresden University of Technology, Mommenstr. 6, 01069 Dresden, Germany
⊥Department BESSY-MX Group, F-12, Helmholtz Zentrum Berlin fur Materialien und Energie, Albert-Einstein-Str. 15, 12489 Berlin,
̈
Germany
S
* Supporting Information
ABSTRACT: The total synthesis of the structure assigned to
the natural product phaeosphaeride A 1a was accomplished.
The key steps involve the addition of vinyllithium reagent 7 to
the acetonide-protected aldehyde 8 to access the carbon
backbone of 1a, the introduction of the methoxylamino group
followed by intramolecular hetero-Michael cyclization, and
methanol elimination to form the dihydropyran ring. In this
study, both enantiomers of 1a were synthesized and tested for
biological activity. Preliminary results showed that (6R,7R,8R)-
1a and (6S,7S,8S)-1a inhibit STAT3-dependent transcriptional
activity in a dose-dependent manner and exhibit antiprolifer-
ative properties in breast (MDA-MB-231) and pancreatic
(PANC-1) cancer cells.
these efforts, the design of potent and cell-permeable STAT3
inhibitors remains a highly challenging task. Currently, there are
no STAT3 inhibitors on the market, although some have
entered clinical trials for treatment of solid tumors and
lymphomas.
In 2006, Clardy and co-workers isolated phaeosphaeride A
and its stereoisomer, phaeosphaeride B, from the endophytic
fungus FA39 (Phaeosphaeria avenaria).11 Phaeosphaeride A was
reported to selectively inhibit STAT3/DNA binding with an
IC50 of 0.61 mM and exhibit promising cell growth inhibition in
STAT3-dependent U266 multiple myeloma cells with an IC50
of 6.7 μM. Interestingly, phaeosphaeride B was inactive against
STAT3.
Phaeosphaerides A and B are structurally related to the
fungal metabolites curvupallide A,12 the spirocyclic lactams
spirostaphylotrichins/triticones,13 and the antibiotic benesudon
(Scheme 1).14 Phaeosphaerides contain a dihydroxylated
dihydropyran ring fused to a five-membered cyclic O-methyl
hydroxamate with an exocyclic C3−C14 double bond. The
interesting structural features coupled with their significant
biological activity have made them attractive synthetic targets.
Clardy and co-workers proposed structure 1a for phaeosphaer-
INTRODUCTION
■
The signal transducer and activator of transcription factors
(STATs) regulate the expression of genes that mediate many
physiological processes including cell growth, survival, differ-
entiation, and motility.1 Upon stimulation, STATs become
transcriptionally active by phosphorylation on specific tyrosine
residues, which leads to dimer formation, nuclear translocation,
and gene transcription.2 Among the STAT family members,
STAT3 and STAT5 proteins are believed to contribute to the
pathogenesis of a variety of human solid tumors and blood
malignancies.3 The possible downstream targets on which
STAT3 promotes oncogenesis may be through the tran-
scription of proliferation and antiapoptosis-associated genes,4
such as BCL-2, BCL-XL, IL-17, IL-23, MCL1, survivin,5 and
tumor angiogenesis (VEGF, HIF-1).6 Importantly, blocking
STAT3 signaling in tumor cells by a dominant negative form of
STAT3, antisense approaches, or siRNAs has been shown to
induce apoptosis, inhibit cell proliferation, suppress angio-
genesis, and stimulate immune responses.7 Therefore, small
molecule inhibitors of STAT3 signaling are of great therapeutic
potential. So far, a number of STAT3 inhibitors have been
identified by rational design or through the screening of
chemical libraries. Examples include peptidomimetics,8 small
molecules (STA21, stattic, S3I-201, STX-0119),9 and natural
products (curcumin, galiellalactone, cucurbitacins).10 Despite
Received: August 17, 2012
Published: October 10, 2012
© 2012 American Chemical Society
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Scheme 1. Originally Proposed Structure of Phaeosphaeride
A, Its Isomers, and Three Structurally Related Natural
Products
Scheme 3. Synthesis of Aldehyde 8
(triphenylphosphoranylidene) propionate 10 to give the E-
ester in high isomeric purity (>97%). Wittig reagent 10 was
prepared from methyl bromopropionate as described in the
literature.16 Consecutively, olefin 11 was dihydroxylated with
OsO4 and N-methylmorpholine-N-oxide (NMO) in THF to
stereoselectively give the diol 12 in 90% yield as a white solid.
After protection of the vicinal diol system in the form of an
acetonide, the ester group was reduced by DIBALH, and the
alcohol was successfully oxidized with the SO3·py complex in
DMSO and Et3N to aldehyde 8 in 91% yield.
With aldehyde 8 in hand, we focused our attention to the key
reaction of lithiated tetronate 7 to 8. Tetronate derivative 15
was prepared from methyl tetronate in a three-step sequence
according to Yoshii et al.17 Deprotonation of 15 with LDA in
THF at −78 °C as described by Yoshii18 and addition of the
resulting vinyllithium species to aldehyde 8 gave intermediate 6
as a single stereoisomer in 53% yield along with partial recovery
of starting materials. The anti-selective addition to aldehyde 8
may be explained by using the Felkin−Ahn transition state
shown below (Scheme 4).19
ide A and structure 1d for phaeosphaeride B, on the basis of
two-dimentional NMR data and mass spectrometric analysis.
However, both the present work and the recent publication of
Tamura suggest an incorrect assignment of the reported
structure of phaeosphaeride A.15
Herein, we report our investigation leading to the stereo-
selective synthesis of the proposed structure of phaeosphaeride
A 1a and, additionally, the asymmetric synthesis of both
enantiomers of 1a that have been achieved by applying the
Sharpless asymmetric dihydroxylation reaction. Preliminary
biological evaluation of the synthetic phaeosphaerides revealed
potent STAT3 inhibitors that exhibit antiproliferative activity in
human breast and pancreatic cancer cells.
Scheme 4. Synthesis of Tetronate 6
RESULTS AND DISCUSSION
■
According to our retrosynthetic strategy, the carbon backbone
of 1a was envisioned to arise from the addition of vinyllithium
reagent 7 to the acetonide-protected aldehyde 8 (Scheme 2).
Scheme 2. Retrosynthetic Analysis of Compound 1a
The following steps involved the introduction of the
methoxylamino group and the subsequent formation of the
dihydropyran ring. Reaction of tetronate 6 with MeONH2·HCl,
using LiHMDS as a base in THF at −78 °C, easily afforded
derivatives 16 in high yield (95%).20 After acetonide
deprotection of 16 with TFA/H2O, the cyclization of the
resulting triol was investigated. Initial attempts were focused on
the acid-catalyzed ring closure of 18 to provide the desired
dihydropyrans 20 through a transition state of the 6-endotrig
type.21
Upon treatment of 18 with p-TsOH in toluene at 60 °C or
TFA in dichloromethane, the hetero-Michael reaction took
place, but a complicated reaction mixture was obtained
including intermediate compounds 19 and products of acid-
catalyzed alcohol dehydration. On the other hand, treatment of
Introduction of the methoxylamino group, followed by
acetonide deprotection, was anticipated to provide precursor
5, ideally functionalized for the preparation of the dihydropyran
ring. Finally, the target molecule was planned to arise from an
intramolecular hetero-Michael 6-endo-trig cyclization followed
by an elimination of methanol.
The synthesis of aldehyde 8 was first investigated (Scheme
3). Hexanal was treated with the stabilized ylid methyl
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Scheme 5. Completion of the Synthesis of 1a
18 with TBAF in THF gave rise to the dihydropyrans 20 at
room temperature. The final transformation necessary to
complete the total synthesis of 1a was the regioselective
dehydration of 20. Rewardingly, exposure of intermediates 20
to 1 equiv of p-TsOH in toluene produced 1a in 87% yield
(over two steps) (Scheme 5).
correlation between H-9 and OH-6 as well as OH-7 suggests
that these protons are positioned on the same side of the ring.
These findings are in agreement with the published data for the
proposed structure of phaeosphaeride A by Tamura’s group.15
Synthesis of (6R,7R,8R)-1a and (6S,7S,8S)-1a. The
synthesis of (6R,7R,8R)-1a and (6S,7S,8S)-1a was also
successful by using the Sharpless asymmetric dihydroxylation
of unsaturated ester 11, providing sufficient quantities of
material for biological evaluation of both enantiomers.
Compound 11 was asymmetrically oxidized with commercial
AD-mix-β reagent to form (2S,3R)-12 in enantioexcess ≥91%
ee and 90% yield or with AD-mix-a reagent to give (2R,3S)-12
in enantioexcess ≥82% ee and 94% yield (Scheme 6).22 The
enantiomeric excess of (2S,3R)-12 and (2R,3S)-12 was
indirectly determined by conversion of both enantiomers to
The stereochemistry of C-6 of 1a was determined by NOESY
experiments (Figure 1). However, the spectral data of synthetic
1
the corresponding (S)-mandelate esters and H NMR analysis
of the resulting diastereomeric mixture. It should be noted that
Tamura et al. reported the asymmetric dihydroxylation of ethyl
(E)-2-methyl-oct-2-enoate under similar conditions (AD-mix-β,
MeSO2NH2 in t-BuOH, H2O), but with higher ee values (98%
ee, 99% yield).15 Intermediates (2S,3R)-12 and (2R,3S)-12
were then advanced through the previously described steps to
complete the synthesis of (6R,7R,8R)-1a and (6S,7S,8S)-1a.
In addition, we were able to determine the crystal structure
of synthetic (6R,7R,8R)-1a by synchrotron radiation, which
confirmed our NMR structural and stereochemical assignments
(Figure 2).23
Figure 1. Selected NOESY correlations of compound 1a.
1a did not match with the data reported for the natural
phaeosphaeride A, leading to the conclusion that the structure
of 1a assigned for phaeosphaeride A is incorrect. Detailed
structural analysis using 2D-NMR and NOESY fully supported
our assignment of synthetic 1a as the proposed structure of
phaeoshaeride A. For example, H-6 correlated with H-8, and
both of them correlated with H-15. Furthermore, the
Scheme 6
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Figure 2. Crystal structure of (6R,7R,8R)-1a.
Biological Evaluation of (6R,7R,8R)-1a and (6S,7S,8S)-
1a. Inhibition of STAT3-Dependent Transcriptional Activity.
Compounds (6R,7R,8R)-1a and (6S,7S,8S)-1a were examined
for their effects in rat preosteoblastic cells (UMR106), and
(6S,7S,8S)-1a was further tested in breast (MDA-MB-231) and
pancreatic (PANC-1) cancer cells. In all cases, cells were
transiently transfected with STAT3-specific luciferase reporter
construct, and STAT-3 phosphorylation was induced by human
oncostatin M (hOSM) at a concentration of 10 ng/mL for 6
h.24 Oncostatin M belongs to the family of gp130-signaling
cytokines, which upon binding to the IL-6/GP130 receptors
induce receptor dimerization, activation of the associated Jak
kinases, and phosphorylation of the SH2-containing STAT-
S.25−27Activation of STAT3 signaling cascade after treatment
with hOSM has been previously reported for UMR106, PANC-
1, and MDA-MB-231 cells.27−29 Results from the luciferase
assay showed both enantiomers to significantly reduce STAT3-
dependent transcriptional activity in UMR106 cells with similar
potency in a dose-dependent manner. The maximum dose
tested was 20 μM (Figure 3a). Compound (6S,7S,8S)-1a
displayed a comparable inhibitory effect in PANC-1 and MDA-
MB-231 cancer cells, showing higher potency in the former cell
line (Figure 3b).
To determine whether the ability of the enantiomers to
suppress hOSM-induced STAT3 activity in rat preosteoblastic
cells was due to inhibition of cell proliferation, a cell viability
assay (MTS) was performed using the same time points and
inhibitor concentrations as in the transcriptional activity
experiments. No effect on cell proliferation was observed
after 6 h incubation, indicating that the compounds were able
to reduce STAT3 activity without affecting the survival of the
treated cells (Figure 4).
Inhibition of Cell Proliferation in PANC-1 and MDA-MB-
231 Cancer Cells. Constitutive activation of STAT3 has been
associated with invasion, survival, and growth of a number of
cancers.30 Therefore, we examined the growth inhibitory
activities of (6R,7R,8R)-1a and (6S,7S,8S)-1a in PANC-1 and
MDA-MB-231 cells that express high levels of STAT3. A
luminescence cell viability assay (Cell Titer Glo) was used to
Figure 3. STAT3-dependent transcriptional activity. Cells were
transiently transfected with a STAT3-specific luciferase reporter
construct, followed by treatment with 10 ng/mL of hOSM and
different concentrations of the inhibitors for 6 h. Results are reported
relative to cells treated with 10 ng/mL of hOSM alone (**:p < 0.001).
generate dose−response curves and evaluate cell viability
following 72 h of treatment with different concentrations of
(6R,7R,8R)-1a and (6S,7S,8S)-1a.
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breast (MDA-MB-231) cancer cells in the low micromolar
range. On the basis of our experience and considering the fact
that the original isolation spectra of phaeosphaeride A indicate
a NOESY correlation between H-6 and H-8, the most probable
structure for phaeosphaeride A is 1c or its enantiomer. Further
investigation leading to the assignment of the natural
phaeosphaeride A structure and detailed studies on the
mechanism of phaeosphaeride-induced STAT3 inhibition are
in progress and will be reported in due course.
Figure 4. Cell toxicity of STAT3 inhibitors in rat preosteoblastic cells.
Results are reported relative to untreated cells.
EXPERIMENTAL SECTION
■
General Experimental Details. All reactions were carried out
under an atmosphere of Ar unless otherwise specified. Commercial
reagents of high purity were purchased and used without further
purification, unless otherwise noted. Reactions were monitored by
TLC and using UV light as a visualizing agent and aqueous ceric
sulfate/phosphomolybdic acid, ethanolic p-anisaldehyde solution,
potassium permanganate solution, and heat as developing agents.
The 1H and 13C NMR spectra were recorded at 300 and 75 MHz, and
tetramethylsilane was used as an internal standard. Chemical shifts are
Gemcitabine, a chemotherapeutic agent for pancreatic and
breast cancer, shown in a recent study to be more beneficial on
pancreatic cancer patients compared to conventional medi-
cations,31 has been used as a positive control. Both compounds
decreased significantly malignant cell proliferation in a dose-
dependent manner, in the low micromolar range (Figure 5),
consistently with their capacity to inhibit STAT3 signaling.
1
indicated in δ values (ppm) from internal reference peaks (TMS H
0.00; CDCl3 1H 7.26, 13C 77.00; DMSO-d6 1H 2.50, 13C 39.51).
Melting points (m.p.) are uncorrected. High-resolution mass spectra
(HRMS) were recorded on a mass spectrometer at a 4000 V emitter
voltage.
(E)-Methyl 2-Methyloct-2-enoate, 11. To a solution of hexanal
(10 g, 100 mmol) in THF (400 mL) was added methyl
(triphenylphosphoranylidene) propionate (104 g, 300 mmol) at
room temperature. Then, the reaction mixture was stirred for 8 h at
the reflux temperature. The solvent was removed under reduced
pressure. After the addition of Et2O (80 mL), the mixture was filtered
and concentrated. The crude product was purified by flash
chromatography (eluent; hexane/ethyl acetate = 20/1) to afford
(E)-methyl 2-methyloct-2-enoate (13.61 g, 80%) as yellow oil: 1H
NMR (300 MHz, CDCl3) δ 0.89 (t, J = 6.0 Hz, 3H), 1.26−1.33 (m,
4H), 1.39−1.46 (m, 2H), 1.83 (s, 3H), 2.17 (dd, J = 7.5, 14.5 Hz, 2H),
3.74 (s, 3H), 6.77 (dt, J = 9.0, 6.0 Hz, 1H); 13C NMR (75 MHz,
CDCl3) 12.2, 13.8, 22.4, 28.1, 28.5, 31.4, 51.5, 127.2, 142.7, 168.6; FT-
IR 1647, 1717, 2858, 2928, 2955; HRMS (ESI-TOF) m/z for
C10H19O2 [M + H]+ calcd 171.1379, found 171.1380.
Methyl 2,3-Dihydroxy-2-methyloctanoate, ( )-12. To a
solution of 3 g (17.6 mmol) of olefin 11 in 180 mL of THF were
added 6.2 g (52.9 mmol) of N-methylmorpholine N-oxide and 1.7 mL
of a 2.5% by weight solution of OsO4 in tert-butanol, and the mixture
was stirred for 24 h. Aqueous solution of sodium bisulfite (100 mL)
was then added, and the resulting mixture was stirred for 30 min. The
aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the
combined organic layers were dried with anhydrous MgSO4. The
solvent was removed under reduced pressure. Purification by flash
chromatography (eluent; hexane/ethyl acetate = 1/1) afforded the diol
12 (3.22 g, 15.8 mmol) in 90% yield as a white solid: mp = 40−42 °C;
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 6.0 Hz, 3H), 1.26−1.44
(m, 6H), 1.35 (s, 3H), 1.55−1.60 (m, 2H), 1.85 (br, 1H), 3.34 (br,
1H), 3.70 (d, J = 9.6 Hz, 1H), 3.81 (s, 3H); 13C NMR (75 MHz,
CDCl3) 13.9, 21.6, 22.5, 25.5, 30.2, 31.7, 52.9, 75.5, 77.5, 176.7; FT-IR
1735, 2858, 2954, 3448; HRMS (ESI-TOF) m/z for C10H21O4 [M +
H]+ calcd 205.1434, found 205.1435.
Figure 5. Cell toxicity of STAT3 inhibitors in cancer cells compared to
chemotherapy drug gemcitabine. Results are reported relative to
untreated cells (**:p < 0.001 compared to untreated cells).
(2S,3R)-Methyl 2,3-Dihydroxy-2-methyloctanoate, (+)-12. To
a stirring solution of AD-mix-β (20.9 g, 1.4 g/mmol) in 58 mL of t-
BuOH/H2O (1:1) was added methanesulfonamide (2.86 g, 30.0
mmol, 2.00 equiv). The solution was stirred well for 15 min until one
phase was present. The reaction mixture was cooled to 0 °C, and a
solution of ester 11 (2.55 g, 15 mmol, 1.00 equiv) in 58 mL of t-
BuOH/H2O (1:1) was added. The reaction mixture was warmed to
room temperature over 2 h and stirred overnight. The mixture was
quenched with a saturated solution of Na2SO3 and stirred for 1 h. The
aqueous layer was extracted with CH2Cl2, and the combined organic
extracts were dried over MgSO4, filtered, and concentrated under
CONCLUSIONS
■
In summary, we have synthesized the originally proposed
structure of phaeosphaeride A 1a in a highly diastereoselective
manner from readily available starting materials. Both
enantiomers of 1a were synthesized and evaluated for their
ability to inhibit STAT3-dependent transcriptional activity. Our
results demonstrate that (6R,7R,8R)-1a and (6S,7S,8S)-1a
decrease the growth of pancreatic (PANC-1) and human
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reduced pressure. The product was purified by flash chromatography
3.76 (s, 3H), 4.17 (t, J = 6.0 Hz, 1H); 13C NMR (75 MHz, CDCl3)
13.9, 19.7, 22.4, 25.4, 26.3, 28.2, 29.5, 31.7, 52.3, 80.2, 82.5, 108.7,
173.7; FT-IR 1736, 2936, 2954, 2989; HRMS (ESI-TOF) m/z for
C13H25O4 [M + H]+ calcd 245.1747, found 245.1742.
(4S,5R)-Methyl 2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carbox-
ylate (+)-S-3. The synthesis was carried out using the same procedure
used for the synthesis of ( )-S-3, starting from (+)-12. [α]D20 +4.38 (c
= 3.33 g/mL, CHCl3).
(4R,5S)-Methyl 2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carbox-
ylate (−)-S-3. The synthesis was carried out using the same procedure
used for the synthesis of ( )-S-3, starting from (−)-12. [α]D20 −4.0 (c
= 3.00 g/mL, CHCl3).
on silica gel (eluent; hexane/ethyl acetate = 5/1) to give 2.76 g of diol
23
(+)-12 as a white solid (90%, ee > 91%). [α]D +36.2 (c= 0.714 g/
mL, CHCl31). The enantiomeric purity was determined by comparative
analysis of H NMR spectra of the corresponding mandelate ester.
(2S,3R)-Methyl 3-((S)-2-Acetoxy-2-phenylacetoxy)-2-hy-
droxy-2-methyloctanoate, S-1. To a solution of diol (+)-12 (35
mg, 0.171 mmol) in 2.2 mL of CH2Cl2 were added (S)-(+)-α-
acetoxyphenylacetic acid (35 mg, 0.18 mmol), dicyclohexylcarbodii-
mide (39 mg, 0.188 mmol), and a catalytic amount of DMAP (≈3 mg)
successively. After 45 min, the CH2Cl2 was removed, and the residue
was diluted with ether and washed with water. The aqueous layer was
extracted with ether, and the combined extracts were dried over
Na2SO4, filtered, and concentrated. The residue was purified by flash
chromatography on silica gel (eluent; hexane/ethyl acetate = 2/1) to
2,2,4-Trimethyl-5-pentyl-1,3-dioxolan-4-yl Methanol, ( )-S-
4. To a solution of ester ( )-S-3 (2 g, 8.19 mmol) in CH2Cl2 (45 mL)
was added a solution of DIBALH (1 M in dichloromethane, 24.6 mL,
24.6 mmol) at 0 °C. After the reaction mixture had been stirred for 30
min at the same temperature, methanol was added. The mixture was
allowed to warm to room temperature. Then, saturated aqueous
potassium sodium tartrate was added to the solution. The mixture was
extracted with CH2Cl2; the organic layer was washed with water and
brine and dried over MgSO4; and the solvent was evaporated. The
residue was purified by flash chromatography (eluent; hexane/ethyl
acetate = 5/1) to give 1.68 g of alcohol ( )-S-4 as colorless oil in 95%
1
give 50 mg of (S)-mandelate ester as a white solid (77%): H NMR
(300 MHz, CDCl3) δ 0.74 (t, J = 7.2 Hz, 3H), 0.85−1.26 (m, 6H),
1.35 (s, 3H), 1.50 (m, 2H), 2.17 (s, 3H), 3.31 (br, 1H), 3.72 (s, 3H),
5.14 (dd, J = 4.7, 7.5 Hz, 1H), 5.81 (s, 1H), 7.34−7.38 (m, 3H), 7.44−
7.47 (m, 2H); 13C NMR (75 MHz, CDCl3) 13.7, 20.5, 22.0, 22.1, 24.4,
28.1, 31.4, 53.1, 74.4, 76.1, 78.2, 127.6, 128.6, 129.2, 133.6, 168.1,
169.9, 175.2; HRMS (ESI-TOF) m/z for C20H28O7Na [M + Na]+
calcd 403.1727, found 403.1729.
1
yield. ( )-S-4: H NMR (300 MHz, CDCl3) δ 0.86 (t, J = 6.0 Hz,
(2R,3S)-Methyl 2,3-Dihydroxy-2-methyloctanoate, (−)-12.
To a stirring solution of AD-mix-α (25 g, 1.4 g/mmol) in 68 mL of
t-BuOH/H2O (1:1) was added methanesulfonamide (3.34 g, 35.2
mmol, 2.00 equiv). The solution was stirred well for 15 min until one
phase was present. The reaction mixture was cooled to 0 °C, and a
solution of ester 11 (3 g, 17.6 mmol, 1.00 equiv) in 35 mL of t-BuOH/
H2O (1:1) was added. The reaction mixture was warmed to room
temperature over 2 h and stirred overnight. The mixture was quenched
with a saturated solution of Na2SO3 and stirred for 1 h. The aqueous
layer was extracted with CH2Cl2, and the combined organic extracts
were dried over MgSO4, filtered, and concentrated under reduced
pressure. The product was purified by flash chromatography on silica
gel (eluent; hexane/ethyl acetate = 5/1) to give 3.38 g of (2R,3S)-
methyl 2,3-dihydroxy-2-methyloctanoate as a white solid (94%, ee ≥
3H), 1.01 (s, 3H), 1.29−1.33 (m, 6H), 1.30 (s, 3H), 1.41 (s, 3H),
1.49−1.54 (m, 2H), 2.26 (br, 1H), 3.35 (dd, J = 9.0, 11.7 Hz, 1H),
3.49 (dd, J = 3.9, 11.7 Hz, 1H), 3.98 (dd, J = 3.0, 9.0 Hz, 1H); 13C
NMR (75 MHz, CDCl3) 13.9, 18.6, 22.4, 26.5, 26.7, 28.6, 29.4, 31.9,
65.6, 78.0, 82.6, 106.9; FT-IR 2861, 2934, 2986, 3472; HRMS (ESI-
TOF) m/z for C12H24O3Na [M + Na]+ calcd 239.1618, found
239.1610.
((4R,5R)-2,2,4-Trimethyl-5-pentyl-1,3-dioxolan-4-yl)methanol,
(+)-S-4. The synthesis was carried out using the same procedure used
20
for the synthesis of ( )-S-4, starting from (+)-S-3. [α]D +8.67 (c =
2.814 g/mL, CHCl3).
((4S,5S)-2,2,4-Trimethyl-5-pentyl-1,3-dioxolan-4-yl)methanol,
(−)-S-4. The synthesis was carried out using the same procedure used
20
for the synthesis of ( )-S-4, starting from (−)-S-3. [α]D −7.5 (c =
23
82%). [α]D −30.7 (c = 0.724 g/mL, CHCl3). The enantiomeric
1.385 g/mL, CHCl3).
1
purity was determined by comparative analysis of H NMR spectra of
2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carbaldehyde,
)-8. Alcohol ( )-S-4 (1 g, 4.62 mmol) was dissolved in
the corresponding mandelate ester.
(
(2R,3S)-Methyl 3-((S)-2-Acetoxy-2-phenylacetoxy)-2-hy-
droxy-2-methyloctanoate, S-2. To a solution of (2R,3S)-methyl
2,3-dihydroxy-2-methyloctanoate (20 mg, 0.097 mmol) in 1.3 mL of
CH2Cl2 were added (S)-(+)-α-acetoxyphenylacetic acid (20 mg, 0.102
mmol), dicyclohexylcarbodiimide (22 mg, 0.106 mmol), and a catalytic
amount of DMAP (≈1.7 mg) successively. After 45 min, the CH2Cl2
was removed, and the residue was diluted with ether and washed with
water. The aqueous layer was extracted with ether, and the combined
extracts were dried over Na2SO4, filtered, and concentrated. The
residue was purified by flash chromatography on silica gel (eluent;
hexane/ethyl acetate = 2/1) to give 30 mg of (S)-mandelate ester as a
white solid (81%): 1H NMR (300 MHz, CDCl3) δ 0.84 (t, J = 6.5 Hz,
3H), 1.08−1.35 (m, 6H), 1.23 (s, 3H), 1.65 (m, 2H), 2.18 (s, 3H),
3.14 (s, 1H), 3.35 (s, 3H), 5.11 (t, J = 6.5 Hz, 1H), 5.84 (s, 1H), 7.34−
7.40 (m, 3H), 7.45−7.49 (m, 2H); 13C NMR (100 MHz, CDCl3) 13.8,
20.5, 22.2, 22.3, 24.8, 28.4, 31.5, 52.5, 74.6, 75.9, 78.2, 127.5, 128.6,
129.1, 133.6, 168.1, 170.1, 174.8; HRMS (ESI-TOF) m/z for
C20H28O7Na [M + Na]+ calcd 403.1727, found 403.1737.
Methyl 2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carboxy-
late, ( )-S-3. To a stirring solution of diol ( )-12 (2 g, 9.79
mmol) in 20 mL of CH2Cl2 and dimethoxypropane (1.48 mL, 11.75
mmol) was added CSA (100 mg) at 0 °C. The mixture was stirred at
room temperature overnight. Then, the mixture was quenched with a
saturated solution of NaHCO3. The aqueous layer was extracted with
CH2Cl2, and the combined organic extracts were dried over MgSO4,
filtered, and concentrated under reduced pressure. The product was
purified by flash chromatography on silica gel (eluent; hexane/ethyl
acetate = 5/1) to give 2.25 g of compound ( )-S-3 (94%). ( )-S-3:
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 6.0 Hz, 3H), 1.32 (s, 3H),
dichloromethane (40 mL). Triethylamine (3.9 mL) was added, and
the mixture was cooled to 0 °C. A solution of SO3-pyridine (2.94 g,
18.49 mmol) in anhydrous dimethylsulfoxide (7.4 mL) was added, and
the reaction mixture was stirred at 0 °C for 1 h and then at room
temperature overnight. The mixture was extracted with CH2Cl2, and
the organic layer was washed with water and brine and dried over
MgSO4. The solvent was evaporated, and the residue was purified by
flash chromatography (eluent; hexane/ethyl acetate = 10/1) to afford
901 mg of aldehyde in 91% yield. ( )-8: 1H NMR (300 MHz, CDCl3)
δ 0.89 (t, J = 6.0 Hz, 3H), 1.17 (s, 3H), 1.30−1.35 (m, 6H), 1.42 (s,
3H), 1.43 (m, 1H), 1.45 (s, 3H), 1.56 (m, 1H), 4.01 (dd, J = 3.3, 8.8
Hz, 1H), 9.59 (s, 1H); 13C NMR (75 MHz, CDCl3) 13.8, 15.9, 22.3,
26.0, 26.2, 28.2, 29.0, 31.6, 77.4, 85.6, 109.1, 201.6; HRMS (ESI-TOF)
m/z for C12H23O3 [M + H]+ calcd 215.1642, found 215.1642.
(4S,5R)-2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carbaldehyde,
(+)-8, and (4R,5S)-2,2,4-Trimethyl-5-pentyl-1,3-dioxolane-4-carbal-
dehyde, (−)-8. The synthesis was carried out using the same
procedure used for the synthesis of ( )-8, starting from (+)-S-4 and
(−)-S-4, respectively.
3-(Hydroxy-2,2,4-(trimethyl-5-pentyl-1,3-dioxolan-4-yl)-
methyl)-4-methoxy-5-methylenefuran-2(5H)-one, ( )-6. Diiso-
propylamine (0.5 mL, 3.66 mmol) was dissolved in THF (2.2 mL) and
cooled to −78 °C in a dry ice/acetone bath for 15 min. To this
solution was n-BuLi (2.3 mL, 1.6 M in hexanes) dropwise over 2 min,
and the deprotonation was allowed to proceed for 1 h. Tetronate (420
mg, 3.33 mmol) dissolved in a mixture of THF (2.8 mL) was added
dropwise to the LDA solution over 6 min during which the solution
turned lemon yellow. The reaction was stirred for exactly 5 min after
the addition was complete during which the color of the reaction
darkened to light brown. Aldehyde ( )-8 (724 mg, 3.38 mmol)
1.32−1.34 (m, 6H), 1.39 (s, 3H), 1.46 (s, 3H), 1.49−1.59 (m, 2H),
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dissolved in THF (3.5 mL) was added slowly to the lithiated tetronate
over 5 min, and the reaction was allowed to proceed for 2 h. The
reaction was quenched at −78 °C with a saturated aqueous NH4Cl
solution (30 mL) and allowed to reach room temperature. The
mixture was extracted with AcOEt (20 mL), and the combined organic
extracts were dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash chromatography
(eluent; hexane/ethyl acetate = 7/1) to afford 432 mg (1.27 mmol,
53% yield) of ( )-6 accompanied with recovered starting materials
[213 mg of aldehyde (1 mmol) and 76 mg of ester (0.6 mmol)].
extracted with ethyl acetate. The organic layers were dried over
MgSO4 and filtered, and the solvent was removed under reduced
pressure. The residue was purified by flash chromatography (eluent;
ethyl acetate). NMR spectra showed that the product was
contaminated with a significant amount of TBAF. The mixture was
used to the next step without further purification. ( )-20: HRMS
+
(ESI-TOF) m/z for C15H26NO6 [M + H] calcd 316.1755, found
316.1755.
Compounds ( )-20 (0.043 mmol) were dissolved in toluene (1
mL). Then p-TSA monohydrate (10 mg, 0.052 mmol) was added, and
the mixture was stirred at 60 °C for 30 min (control by TLC). The
reaction was allowed to cool to room temperature and treated with
0.05 mL of Et3N. The mixture was extracted with CH2Cl2, and the
organic layer was washed with water and brine and dried over MgSO4.
The solvent was removed under reduced pressure, and the residue was
purified by silica gel chromatography (eluent; hexane/ethyl acetate =
1/1) to afford 11 mg of compound ( )-1a as a white solid in 87%
yield over two steps. ( )-1a: mp = 136−138 °C; 1H NMR (300 MHz,
CDCl3) δ 0.91 (t, J = 6.6 Hz, 3H), 1.29 (s, 3H), 1.26−1.55 (m, 5H),
1.65−1.83 (m, 2H), 1.99 (m, 1H), 2.74 (s, 1H), 3.24 (s, 1H), 3.89
(1H, overlapping with OMe), 3.91 (s, 3H), 4.36 (s, 1H), 5.03 (d, J =
1.5 Hz, 1H), 5.09 (d, J = 1.5 Hz, 1H);13C NMR (75 MHz, CDCl3)
13.9, 22.5, 22.6, 25.5, 27.4, 31.5, 64.4, 65.7, 69.0, 85.1, 92.8, 103.0,
136.4, 156.6, 166.8; 1H NMR (300 MHz, DMSO-d6) δ 0.87 (t, J = 6.5
Hz, 3H), 1.09 (s, 3H), 1.24−1.37 (m, 5H), 1.53 (m, 1H), 1.67−1.84
(m, 2H), 3.78 (s, 3H), 3.99 (1H, overlap with H-8), 4.03 (d, J = 6.9
Hz, overlap with H-6), 4.49 (s, 1H), 4.95 (d, J = 1.5 Hz, 1H), 4.96 (d,
J = 1.5 Hz, 1H), 5.15 (d, J = 7.2 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6) 13.8, 21.9, 22.6, 25.1, 26.9, 31.0, 63.6, 64.6, 68.7, 84.9, 90.7,
104.9, 136.6, 156.3, 165.8; FT-IR 1087, 1433, 1638, 1703, 2855, 2920,
2954, 3446; LC-ESI MS 320.29; HRMS (ESI-TOF) m/z for
C15H24NO5 [M + H]+ calcd 298.1649, found 298.1648.
1
( )-6: H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 6.0 Hz, 3H), 1.14
(s, 3H), 1.16−1.39 (m, 5H), 1.32 (s, 3H), 1.41 (s, 3H), 1.43−1.69 (m,
3H), 3.91 (dd, J = 9.6, 1.8 Hz, 1H), 4.24 (1H, overlapping CH and
OCH signals), 4.27 (s, 3H), 4.78 (d, J = 9.9 Hz, 1H), 5.11 (d, J = 2.7
Hz, 1H), 5.13 (d, J = 2.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) 13.9,
16.6, 22.5, 26.6, 27.0, 28.7, 30.3, 31.8, 60.1, 72.3, 83.0, 84.3, 93.7,
103.1, 107.4, 149.4, 162.5, 170.7; FT-IR:1623, 1751, 2860, 2933, 2956,
2983, 3474; HRMS (ESI-TOF) m/z for C18H29O6 [M + H]+ calcd
341.1959, found 341.1960.
3-((R)-Hydroxy((4R,5R)-2,2,4-trimethyl-5-pentyl-1,3-dioxolan-4-
yl)methyl)-4-methoxy-5-methylenefuran-2(5H)-one, (+)-6. The syn-
thesis was carried out using the same procedure used for the synthesis
20
of ( )-6, starting from (+)-8. [α]D +7.2 (c = 1.461g/mL, CHCl3).
3-((S)-Hydroxy((4S,5S)-2,2,4-trimethyl-5-pentyl-1,3-dioxolan-4-
yl)methyl)-4-methoxy-5-methylenefuran-2(5H)-one, (−)-6. The syn-
thesis was carried out using the same procedure used for the synthesis
20
of ( )-6, starting from (−)-8. [α]D −7.0 (c = 2.00 g/mL, CHCl3).
5-Hydroxy-3-(hydroxy(2,2,4-trimethyl-5-pentyl-1,3-dioxo-
lan-4-yl)methyl)-1,4-dimethoxy-5-methyl-1H-pyrrol-2(5H)-
one, ( )-16. A stirred suspension of O-Me-hydroxylamine hydro-
chloride (37 mg, 0.44 mmol, 1.5 equiv) in dry THF (4 mL) at −78 °C
and under argon atmosphere was treated with a 1 M solution of
LiHMDS (2.7 mmol, 2.7 mL). After 10 min, a solution of the ester
( )-6 (100 mg, 0.29 mmol, 1 equiv) in a minimum amount of dry
THF was added. After 2 h stirring at −78 °C, the reaction was
quenched with a saturated aqueous solution of NH4Cl, warmed to
room temperature, and extracted with AcOEt. The combined organic
layers were dried, filtered, and concentrated under reduced pressure.
The residue was purified by flash chromatography (eluent; hexane/
ethyl acetate = 1/1) to give 107 mg of compound (95%) ( )-16: FT-
IR 1646, 2863, 2933, 3448; HRMS (ESI-TOF) m/z for C19H34NO7
[M + H]+ calcd 388.233, found 388.2337.
(6R,7R,8R)-1a. The synthesis was carried out using the same
procedure used for the synthesis of ( )-1a, starting from (+)-18.
20
[α]D +78.7 (c = 0.128 g/mL, CH2Cl2).
(6S,7S,8S)-1a. The synthesis was carried out using the same
procedure used for the synthesis of ( )-1a, starting from (−)-18.
20
[α]D −75.8 (c = 0.441 g/mL, CH2Cl2).
Cell Lines. Human breast cancer cell line MDA-MB-231 was
purchased from ATCC. The human pancreatic carcinoma cell line
PANC-1 and rat osteosarcoma cells (UMR106) were a gift from Dr.
H. W. Verspaget, Department of Gastroenterology, Leiden University
Medical Center, The Netherlands. All cell lines were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Breda,
The Netherlands), containing 100 U/mL of penicillin (Invitrogen),
100 U/mL of streptomycin (Invitrogen), and 10% fetal bovine serum
(FBS; Cambrex, Belgium) in a humidified atmosphere of 5% CO2 at
37 °C.
5-Hydroxy-3-((R)-hydroxy((4R,5R)-2,2,4-trimethyl-5-pentyl-1,3-di-
oxolan-4-yl)methyl)-1,4-dimethoxy-5-methyl-1H-pyrrol-2(5H)one,
(+)-16, and 5-Hydroxy-3-((S)-hydroxy((4S,5S)-2,2,4-trimethyl-5-pen-
tyl-1,3-dioxolan-4-yl)methyl)-1,4-dimethoxy-5-methyl-1H-pyrrol-
2(5H)-one, (−)-16. The synthesis was carried out using the same
procedure used for the synthesis of ( )-16, starting from (+)-6 and
(−)-6, respectively.
5-Hydroxy-1,4-dimethoxy-5-methyl-3-(1,2,3-trihydroxy-2-
methyloctyl)-1H-pyrrol-2(5H)-one, ( )-18. A mixture of trifluoro-
acetic acid and water (3 mL of a 1:1 vol/vol solution) at 0 °C was
added to the acetonide derivative ( )-16 (62 mg, 0.16 mmol). The
reaction mixture was stirred at 0 °C for 1 h and was then decanted into
aqueous NaHCO3 and extracted with CH2Cl2. The combined organic
phases were dried over MgSO4, filtered, and concentrated. The residue
was purified by flash chromatography (eluent; ethyl acetate) to afford
50 mg of derivatives ( )-18 in 90% yield. ( )-18: FT-IR 1646, 1685,
2858, 2929, 3421; HRMS (ESI-TOF) m/z for C16H30NO7 [M + H]+
calcd 348.2017, found 348.2009.
5-Hydroxy-1,4-dimethoxy-5-methyl-3-((1R,2S,3R)-1,2,3-trihy-
droxy-2-methyloctyl)-1H-pyrrol-2(5H)-one, (+)-18, and 5-Hydroxy-
1,4-dimethoxy-5-methyl-3-((1S,2R,3S)-1,2,3-trihydroxy-2-methyloc-
tyl)-1H-pyrrol-2(5H)-one, (−)-18. The synthesis was carried out using
the same procedure used for the synthesis of ( )-18, starting from
(+)-16 and (−)-16, respectively.
Proposed Structure of Phaeosphaeride A, ( )-1a. Com-
pounds ( )-18 (15 mg, 0.043 mmol) were dissolved in THF (1 mL),
and TBAF (0.065 mmol, 0.1 M solution in THF) was added. The
mixture was stirred at room temperature for 12 h. After the addition of
saturated NaHCO3 solution (2 mL), the reaction mixture was
STAT3 Inhibitors. All STAT3 inhibitors were dissolved in sterile
DMSO to make a 10 mM stock solution. Aliquots of the stock solution
were stored at −20 °C.
STAT3-Dependent Transcriptional Luciferase Activity. Cells were
seeded at a density of 20 000 cells/well in 96-well plates. The second
day, the cells were transiently cotransfected with 20 ng of the STAT3-
specific luciferase reporter construct25 and 1.25 ng of CMV-renilla
construct using FuGENE HD transfection reagent (Promega Benelux
BV, Leiden, The Netherlands), according to the manufacturer’s
protocol. Cells were treated with 10 ng/mL of Oncostatin M and a
dose range of the synthesized compounds the next day. After 6 h,
luciferase assays were performed using the Dual Luciferase Reporter
Assay system (Promega), according to the manufacturer’s protocol.
Luciferase activity was measured using a luminescence counter. Firefly
luciferase activity was corrected for renilla luciferase activity.
Cell Viability Assays. To test the potential cytotoxicity of the
synthesized molecules, UMR106 cells were seeded at a density of 19
000 cells/well in 96-well plates. To simulate the transfection
conditions, cells were treated with a dose range of the enantiomers
(6R,7R,8R)-1a and (6S,7S,8S)-1a on the third day after seeding. A
tetrazolium-based (MTS) assay (CellTiter 96 AQueous, Promega) was
performed 6 h after stimulation, according to the manufacturer’s
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Article
protocol. The reagent was added directly to the assay wells at the
recommended ratio of 20 μL of reagent to 100 μL of culture medium.
Cells were then incubated for approximately 30 min at 37 °C, and
absorbance was measured at 490 nm in a microplate reader.
Human breast (MDA-MB-231) and pancreatic (PANC-1) cancer
cell lines were seeded in opaque-walled 96-well plates, compatible with
the luminometer used, at a density of 5000 cells/well. Different
concentrations of (6R,7R,8R)-1a and (6S,7S,8S)-1a or gemcitabine
(100 nM) were added in triplicate to the plates in the presence of 10%
FBS. Cells were incubated at 37 °C for a period of 72 h, and the
luminescent cell viability assay (CellTiter-Glo, Promega) was
performed according to the manufacturer’s protocol. After 72 h,
cells were treated with a volume of CellTiter-Glo Reagent equal to the
volume of cell culture medium present in each well and incubated for
approximately 10 min at room temperature to stabilize the
luminescent signal. Luminescence was then recorded using a
luminescence counter.
(8) Coleman, D. R.; Ren, Z.; Mandal, P. K.; Cameron, A. G.; Dyer, G.
A.; Muranjan, S.; Campbell, M.; Chen, X.; McMurray, J. S. J. Med.
Chem. 2005, 48, 6661−6670.
(9) (a) Schust, J.; Sperl, B.; Hollis, A.; Mayer, T. U.; Berg, T. Chem.
Biol. 2006, 13, 1235−1242. (b) Song, H.; Wang, R.; Wang, S.; Lin, J.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4700−4705. (c) Siddiquee, K.;
Zhang, S.; Guida, W. C.; Blaskovich, M. A.; Greedy, B.; Lawrence, H.
R.; Yip, M. L.; Jove, R.; McLaughlin, M. M.; Lawrence, N. J.; Sebti, S.
M.; Turkson, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7391−7396.
(d) Matsuno, K.; Masuda, Y.; Uehara, Y.; Sato, H.; Muroya, A.;
Takahashi, O.; Yokotagawa, T.; Furuya, T.; Okawara, T.; Otsuka, M.;
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(12) Abraham, W. R.; Meyer, H.; Abate, D. Tetrahedron 1995, 51,
4947−4952.
ASSOCIATED CONTENT
■
S
* Supporting Information
Characterization data of the described compounds and X-ray
crystal data for compound (6R,7R,8R)-1a. This material is
available free of charge via the Internet at http://pubs.acs.org.
(13) Sugawara, F.; Takahashi, N.; Strobel, G. A.; Strobel, S. A.; Lu, H.
S. M.; Clardy, J. J. Am. Chem. Soc. 1988, 110, 4086−4087.
(14) Thines, E.; Arendholz, W. R.; Anke, H.; Sterner, O. J. Antibiot.
1997, 50, 13−17.
AUTHOR INFORMATION
■
(15) Kobayashi, K.; Okamoto, I.; Morita, N.; Kiyotani, T.; Tamura,
O. Org. Biomol. Chem. 2011, 9, 5825−5832.
Corresponding Author
*E-mail: sarli@chem.auth.gr.
(16) Handa, M.; Scheidt, K. A.; Bossart, M.; Zheng, N.; Roush, W. R.
J. Org. Chem. 2008, 73, 1031−1035.
Notes
The authors declare no competing financial interest.
(17) Takeda, K.; Yano, S.; Sato, M.; Yoshii, E. J. Org. Chem. 1987, 52,
4135−4137.
(18) For examples in the synthesis of tetronate derivatives with
lithiated tetronate 7, see: (a) Hori, K.; Hikage, N.; Inagaki, A.; Mori,
S.; Nomura, K.; Yoshii, E. J. Org. Chem. 1992, 57, 2888−2902.
(b) Zapf, C. W.; Harrison, B. A.; Drahl, C.; Sorensen, E. J. Angew.
Chem., Int. Ed. 2005, 44, 6533−6537. (c) Snider, B. B.; Zou, Y. Org.
Lett. 2005, 7, 4939−4941. (d) Niu, D.; Hoye, T. R. Org. Lett. 2012, 14,
828−831. (e) Couladouros, E. A.; Bouzas, E. A.; Magos, A. D.
Tetrahedron 2006, 62, 5272−5279.
ACKNOWLEDGMENTS
■
The authors are grateful to Professor Jon Clardy (Harvard
Medical School) for providing an authentic sample of
(−)-Phaeosphaeride A. The authors thank Ineke Oudshoorn
(Dept. of Radiology, LUMC) for technical assistance, Dr. Hein
Verspaget (Dept. of Gastroenterology, LUMC) for the PANC-
1 cell line, and Professor Clemens Lowik (Dept of Radiology,
LUMC) for critical reading of the manuscript.
̈
(19) Cherest, M.; Felkin, H.; Prudent, N. Tetrahedron Lett. 1968, 18,
2199−2204.
(20) Gissot, A.; Volonterio, A.; Zanda, M. J. Org. Chem. 2005, 70,
6925−6928.
(21) Baldwin, J. E.; Cutting, J.; Dupont, W.; Kruse, L.; Silberman, L.;
Thomas, R. C. Chem. Soc. Chem. Commun. 1976, 736−738.
(22) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev.
1994, 94, 2483−2547.
(23) Crystal data for 1a (293 K): C15H23NO5; fw 297.34;
monoclinic; space group P21; a = 10.213(2) Å, b = 5.204(1) Å, c =
15.962(3) Å, β = 100.28(3)°; V = 834.7(3) Å3; Z = 2; Dcalcd = 1.183
mg/m3, R1 = 0.0778 [I > 2σ(I)], wR2 = 0.2191 (all data); GOF =
1.045. For details, see the Supporting Information.
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