Stereospecific total syntheses of proteasome inhibitors omuralide and lactacystin

Article abstract of DOI:10.1021/jo201453x

Omuralide, a transformation product of the microbial metabolite lactacystin, was the first molecule discovered as a specific inhibitor of the proteasome and is unique in that it specifically inhibits the proteolytic activity of the 20S subunit of the prot

Full text of DOI:10.1021/jo201453x

ARTICLE
pubs.acs.org/joc
Stereospecific Total Syntheses of Proteasome Inhibitors Omuralide
and Lactacystin
Wenxin Gu† and Richard B. Silverman*
Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and
Drug Discovery, Northwestern University, Evanston, Illinois 60208-3113, United States
S Supporting Information
b
ABSTRACT: Omuralide, a transformation product of the
microbial metabolite lactacystin, was the first molecule discov-
ered as a specific inhibitor of the proteasome and is unique in
that it specifically inhibits the proteolytic activity of the 20S
subunit of the proteasome without inhibiting any other protease
activities of the cell. The total syntheses of omuralide and (+)-
lactacystin are reported. An important key intermediate is
synthesized at an early stage, which allows analogues of these two natural products to be made readily.
’ INTRODUCTION
Omuralide (2; also called clasto-lactacystin-β-lactone), a trans-
formation product of the microbial metabolite lactacystin,6 was
originally isolated by Omura and co-workers from cultures of a
terrestrial Streptomyces sp.7 Omuralide, remarkable because it was
the first molecule discovered to be a truly specific inhibitor of the
proteasome, is unique in that it specifically inhibits the proteolytic
activity of the 20S subunit of the proteasome without inhibiting
any other protease activities of the cell.8 This is an important
attribute, which can be utilized in future potential drug design.
Stimulated by the unusual structure of lactacystin, its remark-
able biological activity, and the scarcity of natural material,
several research groups undertook total syntheses.9À15 The first
total synthesis of lactacystin in 19929 made lactacystin (and
radiolabeled lactacystin) available and was instrumental in re-
search, which demonstrated that the biological activity of lacta-
cystin results from its potent, highly selective, and irreversible
inhibition of proteasome-mediated peptidase activity.4 Syntheses
of omuralide (2) also have been reported.13,15,16 Conclusive
evidence was obtained that lactacystin is converted in vivo to the
equipotent β-lactone omuralide (2), which is the actual agent
that acts by acylation of the amino terminal threonine residue of a
proteasome subunit. This result was confirmed by X-ray crystal-
lographic studies at 2.4 Å resolution.17À19
The proteasome, a high-molecular-weight, multicatalytic pro-
tease complex responsible for most nonlysosomal intracellular
protein degradation, is emerging as an important target for cancer
chemotherapy, because small-molecule inhibitors of its catalytic
activity induce apoptosis in both in vitro and in vivo models of
humanmalignanciesandareprovingtohaveefficacy in early clinical
trials.1 A role for the proteasome in programmed cell death was
uncovered using small-molecular-weight, cell-permeable inhibitors,
which induce apoptosis in a variety of tumor-derived cell lines.2
Given the importance of the proteasome to normal cellular home-
ostasis, however, it is likely that inhibitors induce programmed cell
death by affecting many apoptosis-associated pathways. Eukaryotic
intracellular protein degradation occurs predominantly through the
ubiquitin proteasome pathway (UPP) composed of the Ub-con-
jugating system and the 26S proteasome.3
Lactacystin (1), a microbial natural product that inhibits cell
proliferation and induces neurite outgrowth in a murine neuro-
blastoma cell line, has become a widely used reagent in functional
studies of the proteasome.4 The proteasome is composed of a
20S catalytic core particle and additional subunits that are
thought to be involved in the recognition and unfolding of
ubiquitinated proteins; the composite structure has a sedimenta-
tion coefficient of 26S. Lactacystin binds to certain catalytic
subunits of the 20S proteasome and inhibits the three best
characterized peptidase activities of the proteasome, two irrever-
sibly and all at different rates.5
Many analogues of 1 were synthesized to discover the most
potent agent.20,21 There is an absolute requirement for the
β-lactone ring, and the stereochemical fidelity is dictated by that
of the natural product. Furthermore, methylation of the y-lactam
nitrogen abolishes activity. The one region of the molecule
that supported chemical modification was the C4 alkyl group.
Replacement of the methyl group at C7 with short aliphatic
chains enhanced the potency of the lactone inhibitor. The
best activities were recorded for ethyl, n-propyl, isopropyl, and
Received: July 18, 2011
Published: September 14, 2011
r
2011 American Chemical Society
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Figure 1. Retrosynthetic plan for lactacystin and omuralide.
Scheme 1. Synthesis of 8
Scheme 2. Synthesis of Key Intermediate 12
n-butyl, all possessing 2À3 times the potency of the correspond-
ing (+)-lactacystin-β-lactone.
5. In our hands, the acylation of 4 in a two-phase reaction
medium of saturated aqueous NaHCO3 in methylene chloride
gave the best results and made the workup easier than that
reported. A Dieckmann condensation followed by reduction
generated alcohol 7 and a minor amount of the epimer, which
was removed by recrystallization to generate the desired isomer
in a 60% overall yield for the two steps. Protection of the
β-hydroxyl group of alcohol 7 also was explored. The TBS ether,
benzyl ether, and acetate were attempted, all of which, however,
resulted in production of an unsaturated lactam side product as a
result of in situ elimination of the protected alcohol of 7. This
suggests that cis substitution at the β position of this bicyclic
lactam is unstable and favors elimination. Diol 8 was obtained in a
quantitative yield by reaction of 7 in a mixture of propane-1,3-
dithiol and acidic trifluoroethanol.
From the view of medicinal chemistry, an ideal synthesis of a
target molecule is one in which a stable key intermediate can
be used to efficiently assemble a diverse library of analogues of
the target on a large scale. Here we report a synthesis of
lactacystin (1) and omuralide (2) via an important intermediate
(3, Figure 1), leading to the efficient preparation of analogues in
which the moieties of 3 can readily be replaced by other groups.
’ RESULTS AND DISCUSSION
With L-serine methyl ester as the starting material, β-hydro-
xylated lactam 8 was prepared in five steps with very high
stereoselectivity by the elegant method of Maloney and co-
workers for diastereoselectively controlled hydroxylated
lactams22 (Scheme 1). Thus, serine-derived oxazolidine 4 was
acylated with ethyl malonyl chloride to give the N-acyl derivative
Key intermediate 12 was prepared as shown in Scheme 2.
The two hydroxyl groups of 8 were protected as TBS ethers
by treatment with TBS chloride in DMF with imidazole as base.
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Scheme 3. Synthesis of 17
the lactam, and only a trace amount of the O-methylated enolate
was observed. It was previously reported that Boc protection of
a lactam amide nitrogen assisted in stabilization of the α-anion
by formation of a cyclic transition state with the lactam carbonyl
group and the lithium cation.10
Therefore, the PMB group of 15 was exchanged for a Boc
protecting group (19) in two steps (Scheme 4). The TBS ether
was removed with TBAF in acetic acid; the acetic acid is
important, as decomposition was observed when only TBAF
was utilized. Methylation of 20 was performed by the method of
Donohoe et al.11 with LDA and HMPA, generating 21 in 60%
yield with very high stereoselectivity. After Boc deprotection and
hydrolysis of the methyl ester and the acetic ester of 22, the
β-lactone was formed by the reported coupling procedure
using BOPCl,18a yielding omuralide (2) in a total of 19 steps.
Further treatment of 2 with N-acetyl L-cysteine by the reported
procedure18a gave (+)-lactacystin (1).
Figure 2. X-ray crystal structure of 16.
The lactam of the resulting bis-TBS ether 9 was protected as its
PMB ether (10). Initially, Boc was used as the protecting group
for 9; however, in the next step the Boc group migrated from the
lactam to the adjacent hydroxyl group after its TBS ether was
removed. The TBS ether at the primary alcohol in 10 was then
regioselectively deprotected. After we screened various acidic
and fluoride reagent conditions, TFA/H2O/CH2Cl2 (9/1/1)
was found to be the most efficient conditions to prepare alcohol
11, which was DessÀMartin periodinane oxidized to aldehyde
12, the key intermediate.
Next, the two hydrophobic subunits of the β-hydroxy lactam
skeleton were installed (Scheme 3). Aldehyde 12 was treated
with 2-propenyl Grignard reagent and trimethylchlorosilane,
as was reported earlier for addition to aldehydes with high
stereoslectivity,18a giving only one stereoisomer of adduct 13.
A six-membered-ring transition state, with Mg2+ coordinating to
the carbonyl groups of the ester and aldehyde, was proposed to
account for the stereoselectivity of the addition.18a Hydrogena-
tion of 13 followed by acylation and deprotection of the TBS
ether of 15 gave 16; the absolute stereochemistry of 16 was
confirmed by X-ray crystallography (Figure 2). Unfortunately,
various bases, including LDA and potassium and sodium HMDS,
followed by methyl iodide, failed to methylate the α-position of
’ CONCLUSION
We have completed total stereospecific syntheses of omuralide
(2) and (+)-lactacystin (1) in 3% (19 steps) and 1.5% (20 steps)
overall yields, respectively. Key intermediate 12 was prepared
at an early stage of the synthesis; then the two alkyl substituents
were introduced at a later stage. The introduction of these
substituents permits analogous modifications of these natural
products. The PMB protecting group was found to be important
for the introduction of the isopropyl substituent, and the Boc
protecting group was critical for the installation of the methyl
group.
’ EXPERIMENTAL SECTION
(2R,3S)-Methyl 3-(tert-Butyldimethylsilyloxy)-2-((tert-butyl-
dimethylsilyloxy)methyl)-5-oxopyrrolidine-2-carboxylate (9).
To a solution of 8 (5 g, 26.43 mmol) in DMF (200 mL) was added
TBSCl (8.76 g, 58.15 mmol) followed by imidazole (7.20 g, 106 mmol)
at room temperature. The reaction mixture was stirred overnight. The
reaction was quenched by the addition of saturated aqueous NH4Cl
solution and then was extracted with EtOAc (3 Â 500 mL). The
combined organic phases were washed with brine, dried with Na2SO4,
and concentrated. The residue was purified by flash column
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Scheme 4. Final Steps in the Synthesis of Omularide (2) and Lactacystin (1)
chromatography (hexanes/EtOAc 100/0 to 0/100) to afford 9 (10.4 g,
94%) as a colorless oil: [α]25D = +31.19° (c 1.0, CHCl3); 1H NMR (500
MHz, CDCl3) δ 6.30 (s, 1H), 4.30 (dd, J = 6.4, 3.6 Hz, 1H), 4.00 (d, J =
9.8 Hz, 1H), 3.70 (s, 3H), 3.61 (d, J = 9.8 Hz, 1H), 2.60 (dd, J = 16.8,
6.5 Hz, 1H), 2.31 (dd, J = 16.8, 3.6 Hz, 1H), 0.83 (s, 9H), 0.82 (s, 9H),
0.03 (s, 3H), 0.02 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H); 13C NMR (126
MHz, CDCl3) δ 174.9, 170.0, 73.7, 71.2, 66.4, 52.4, 40.6, 26.3, 26.1, 25.9,
25.8, 25.7, 25.6, 25.4, 25.2, 18.3, 17.9, À4.3, À4.7, À5.1, À5.5, À5.6,
À6.0; HRMS (ESI) calcd for [(C19H39NO5Si2) + H]+ 418.2421, found
418.2424.
with Na2SO4, and concentrated. The residue was purified by flash
column chromatography (hexanes/EtOAc 100/0 to 0/100) to afford 11
(5.8 g, 73%) as a colorless oil, which gradually changed to a white solid:
[α]25D = +10.99° (c 1.6, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27
(d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 4.73 (d, J = 15.0 Hz, 1H),
4.49 (t, J = 8.2 Hz, 1H), 4.09 (d, J = 15.0 Hz, 1H), 3.95 À 3.71 (m, 4H),
3.55 (s, 3H), 2.65 (ddd, J = 51.3, 16.5, 8.2 Hz, 1H), 0.84 (s, 9H), 0.05
(d, J = 4.8 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 174.3, 170.4, 159.4,
130.0, 129.3, 114.3, 77.5, 77.2, 77.0, 75.1, 68.8, 61.4, 55.4, 52.2, 44.2,
39.0, 25.6, 17.9; HRMS (ESI) calcd for [(C21H33NO6Si) + H]+
424.2153, found 424.2155.
(2R,3S)-Methyl 3-(tert-Butyldimethylsilyloxy)-2-((tert-butyldi-
methylsilyloxy)methyl)-1-(4-methoxybenzyl)-5-oxopyrroli-
dine-2-carboxylate (10). NaH (60% in mineral oil, 1.05 g, 26.33 mmol)
was prewashed with hexanes and then was suspended in DMF (300 mL).
To the mixture was carefully added a solution of 9 (10 g, 23.94 mmol) in
DMF (100 mL) at 0 °C. The resulting mixture was stirred for 1 h before
PMBCl (4.5 g, 3.90 mL, 28.73 mmol) was added dropwise, followed by
TBAI (1.77 g, 4.79 mmol). The reaction mixture was stirred at room
temperature overnight and then was quenched by adding saturated aqueous
NH4Cl solution and extracted with EtOAc (3 Â 500 mL). The combined
organic phases were washed with brine (30 mL), dried with Na2SO4, and
concentrated. The residue was purified by flash column chromatography
(hexanes/EtOAc 100/0 to 0/100) to afford 10 (10.6 g, 82%) as a colorless
oil: [α]25D = +55.82° (c 0.8, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.19
(d, J = 8.5 Hz, 2 H), 6.78 (d, J = 8.5 Hz, 2 H), 4.94 (d, J = 15 Hz, 1 H), 4.56
(t, J = 8.5 Hz, 1 H), 4.98 (dd, J = 12, 50 Hz, 1H), 3.84 (d, J = 12 Hz, 1H),
3.77 (s, 3H), 3.27 (s, 3H), 2.63 (d, J = 8.5 Hz, 1H), 0.89 (s, 9H), 0.81 (s,
9H), 0.10 (s, 6H), 0.09 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 173.9,
170.1, 159.1, 130.6, 128.5, 113.7, 95.0, 73.8, 68.4, 59.0, 55.5, 51.6, 43.5, 39.1,
26.0, 25.9, 25.7, 25.6, 18.3, 17.9, À5.0, À5.4, À5.8; HRMS (ESI) calcd for
[(C27H47NO6Si2) + H]+ 538.3013, found 538.3020.
(2S,3S)-Methyl 3-(tert-Butyldimethylsilyloxy)-2-formyl-1-(4-
methoxybenzyl)-5-oxopyrrolidine-2-carboxylate (12). To a
solution of 11 (5.0 g, 11.80 mmol) in CH2Cl2 (500 mL) was added
DessÀMartin periodinane (3.75 g, 8.85 mmol) in several portions at
0 °C. The resulting mixture was stirred at room temperature for 2 h.
Then an aqueous solution of Na2S2SO3 was added very carefully to
quench the reaction. To the resulting mixture was added saturated
aqueous NaHCO3 solution, which was extracted with CH2Cl2 (3 Â
500 mL). The combined organic phases were washed with brine
(50 mL), dried with Na2SO4, and concentrated. The residue was
purified by flash column chromatography (hexanes/EtOAc 100/0 to
0/100) to afford 12 (4.4 g, 88%): [α]25 = +56.99° (c 1.0, CHCl3);
D
1H NMR (500 MHz, CDCl3) δ 9.76 (s, 1H), 7.02 (d, J = 8.2 Hz, 2H),
6.73 (d, J = 8.1 Hz, 2H), 4.65 (d, J = 14.6 Hz, 1H), 4.46 (t, J = 7.1 Hz,
1H), 4.31 (d, J = 14.6 Hz, 1H), 3.69 (s, 3H), 3.47 (s, 3H), 2.58 (ddd, J =
23.1, 16.4, 7.2 Hz, 2H), 0.76 (s, 9H), À0.04 (d, J = 8.1 Hz, 6H); 13C
NMR (126 MHz, CDCl3) δ 172.6, 167.6, 159.4, 131.0, 127.2, 113.8,
77.8, 77.5, 77.2, 77.0, 70.7, 55.2, 52.3, 44.4, 39.0, 25.8, 25.4, 25.3, 24.9,
17.7; HRMS (ESI) calcd for [(C21H31NO6Si) + H]+ 422.1999, found
422.1997.
(2R,3S)-Methyl 3-(tert-Butyldimethylsilyloxy)-2-(hydroxy-
methyl)-1-(4-methoxybenzyl)-5-oxopyrrolidine-2-carboxy-
late (11). To a stirred solution of TFA/H2O (9/1, 500 mL) was added
a solution of 10 (10 g, 18.59 mmol) in CH2Cl2 (50 mL) at room
temperature. The reaction was monitored by TLC until the starting
material was consumed. Most of the solvent and TFA was evaporated,
and the crude product was extracted with EtOAc (3 Â 500 mL).
The combined organic phases were washed with brine (30 mL), dried
(2S,3S)-Methyl 3-(tert-Butyldimethylsilyloxy)-2-((S)-1-hy-
droxy-2-methylallyl)-1-(4-methoxybenzyl)-5-oxopyrrolidine-
2-carboxylate (13). To a stirred solution of aldehyde 12 (4.0 g,
9.49 mmol) in THF (500 mL) at À40 °C was added TMSCl (5.15 g,
6.0 mL, 47.5 mmol). Isopropenylmagnesium bromide (0.5 M in THF,
57 mL, 28.47 mmol) was then added dropwise at À40 °C. After the
mixture was stirred for 2 h at À40 °C, the reaction was quenched with
saturated aqueous NH4Cl at that temperature and the mixture stirred
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for an additional 0.5 h. The resulting mixture was extracted with EtOAc
(3 Â 500 mL). The combined organic phases were washed with brine
(30 mL), dried with Na2SO4, and concentrated. The residue was purified
by flash column chromatography (hexanes/EtOAc 100/0 to 0/100) to
2H), 5.54 (d, J = 5.8 Hz, 1H), 4.79 (d, J = 15.3 Hz, 1H), 4.68 (dd, J = 7.5,
4.8 Hz, 1H), 4.33 (d, J = 15.3 Hz, 1H), 3.79 (s, 3H), 3.48 (s, 3H), 2.93
(dd, J = 17.7, 7.7 Hz, 1H), 2.62 (dd, J = 17.7, 4.6 Hz, 1H), 2.13 (s, 3H),
1.89 (td, J = 13.1, 6.5 Hz, 1H), 0.97 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.6
Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 174.5, 170.0, 159.0, 129.7,
128.7, 113.9, 75.8, 69.3, 55.4, 52.3, 46.1, 38.6, 28.9, 21.1; HRMS (ESI)
calcd for [(C20H27NO7) + H]+ 394.1842, found 394.1850.
(2R,3S)-Methyl 2-((S)-1-Acetoxy-2-methylpropyl)-3-(tert-
butyldimethylsilyloxy)-5-oxopyrrolidine-2-carboxylate (18).
To a solution of 15 (1.4 g, 2.76 mmol) in CH3CN/H2O (1/1, 100 mL),
was added ceric ammonium nitrate (4 g, 7.30 mmol) at room tempera-
ture. The resulting solution was stirred until the TLC showed no more
starting material. The solution was extracted with EtOAc (3 Â 300 mL),
and the combined organic phases were washed with brine (30 mL), dried
withNa2SO4, andconcentrated. Theresidue was purified by flash column
chromatography (hexanes/EtOAc 100/0 to 0/100) to afford 18 (1.04 g,
97%) as a colorless oil: [α]25D = +16.00° (c 0.5, CHCl3); 1H NMR (499
MHz, CDCl3) δ 7.74 (s, 1H), 5.36 (d, J = 5.6 Hz, 1H), 4.23 (s, 1H), 3.68
(s, 3H), 2.61 À 2.35 (m, 1H), 2.10 (d, J = 17.0 Hz, 1H), 2.03 (s, 3H), 1.80
(m, 1H), 0.80 (d, J = 4.6 Hz, 6H), 0.75 (s, 9H), À0.04 (s, 6H); 13C NMR
(126 MHz, CDCl3) δ 176.4, 170.3, 169.3, 78.9, 75.5, 73.0, 52.4, 41.2,
29.9, 25.5, 20.9, 19.7, 18.6, 17.7, À5.0, À5.3; HRMS (ESI) calcd for
[(C18H33NO6Si) + H]+ 388.2155, found 388.2162.
(2R,3S)-1-tert-Butyl 2-Methyl 2-((S)-1-Acetoxy-2-methyl-
propyl)-3-(tert-butyldimethylsilyloxy)-5-oxopyrrolidine-1,2-
dicarboxylate (19). To a solution of 18 (900 mg, 2.32 mmol) in THF
(20 mL) was added Boc2O (760 mg, 3.48 mmol) at room temperature.
Then Et3N (470 mg, 647 μL, 4.64 mmol) was added dropwise, followed
by DMAP (57 mg, 0.46 mmol). The reaction mixture was then refluxed
overnight under N2. The solvent was evaporated, and saturated NH4Cl
solution (50 mL) was added. The mixture was extracted with EtOAc
(3 Â 200 mL). The combined organic phases were washed with brine
(30 mL), dried with Na2SO4, and concentrated. The residue was
purified by flash column chromatography (hexanes/EtOAc 100/0 to
0/100) to afford 19 (950 mg, 83%) as a colorless oil: [α]25D = À41.82°
(c 0.6, CHCl3); 1H NMR (499 MHz, CDCl3) δ 5.85 (d, J = 3.6 Hz, 1H),
4.72 (dd, J = 8.3, 6.2 Hz, 1H), 3.60 (s, 3H), 2.83 (dd, J = 18.1, 8.6 Hz,
1H), 2.56 (dd, J = 18.1, 5.9 Hz, 1H), 2.05 (s, 3H), 1.71 (m, 1H), 1.41 (s,
9H), 0.86 (d, J = 6.8 Hz, 6H), 0.77 (s, 9H), 0.04 (s, 3H), À0.00 (s, 3H);
13C NMR (126 MHz, CDCl3) δ 172.1, 169.8, 168.8, 148.4, 74.9, 74.2,
65.3, 52.1, 41.3, 29.2, 27.7, 25.4, 21.6, 21.2, 17.6, 17.3, À4.2, À5.3; HRMS
(ESI) calcd for [(C23H41NO8Si) + Na]+ 510.2499, found 510.2502.
(2R,3S)-1-tert-Butyl 2-Methyl 2-((S)-1-Acetoxy-2-methyl-
propyl)-3-hydroxy-5-oxopyrrolidine-1,2-dicarboxylate (20).
To a solution of TBAF (400 mg, 453 μL, 1.54 mmol) in THF (5 mL)
was added AcOH (92 mg, 87 μL, 1.54 mmol) at room temperature.
Then a solution of 19 (500 mg, 1.03 mmol) in THF (5 mL) was added
dropwise to the reaction mixture. The resulting reaction mixture was
stirred overnight. The solution was added to saturated NH4Cl solution
(50 mL) and extracted with EtOAc (3 Â 100 mL). The combined
organic phases were washed with brine (30 mL), dried with Na2SO4, and
concentrated. The residue was purified by flash column chromatography
afford 13 (3.8 g, 87%) as a colorless glass: [α]25 = +50.00° (c 0.9,
D
CHCl3); 1H NMR (499 MHz, CDCl3) δ 7.17 (d, J = 8.1 Hz, 2H), 6.71 (d,
J = 8.1 Hz, 2H), 5.02 (d, J = 51.3 Hz, 2H), 4.78 (d, J = 15.1 Hz, 1H), 4.67
(d, J = 4.7 Hz, 1H), 4.63 À 4.55 (m, 1H), 4.08 (d, J = 5.0 Hz, 1H), 4.01 (d,
J = 14.9Hz, 1H), 3.66 (s, 3H), 3.20 (s, 3H), 2.74 (dd, J= 16.7, 7.5 Hz, 1H),
2.33 (dd, J = 16.8, 4.6 Hz, 1H), 1.70 (s, 3H), 0.74 (s, 9H), À0.03 (s, 6H);
13C NMR (126 MHz, CDCl3) δ 174.1, 171.2, 158.4, 142.7, 129.7, 129.0,
116.6, 113.2, 76.5, 76.3, 69.6, 55.0, 51.4, 45.4, 40.2, 25.3, 20.4, 17.5, À4.7,
À5.4; HRMS (ESI) calcd for [(C24H37NO6Si) + H]+ 464.2444, found
464.2446.
(2S,3S)-Methyl 3-(tert-butyldimethylsilyloxy)-2-((S)-1-hy-
droxy-2-methylpropyl)-1-(4-methoxybenzyl)-5-oxopyrroli-
dine-2-carboxylate (14). To a stirred solution of 13 (3.0 g, 6.47
mmol) in EtOH (50 mL) in a single-necked flask was added Pd/C (10%,
300 mg) very carefully at room temperature. The flask was evacuated
and connected to a hydrogen balloon through a three-way stopcock
adaptor. The solution was evacuated under vacuum for 3 min with
stirring, and was filled with H2. This cycle was repeated three times.
Then the reaction was stirred overnight under H2. The reaction mixture
was filtered through an EtOAc prewashed Celite layer to give a clear
filtrate. The Celite layer was was washed with EtOAc again. The filtrate
was evaporated to give 14 (3.0 g, 99%) as a clear colorless oil: [α]25
=
D
+21.63° (c 0.8, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.21 (d, J = 8.5
Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H), 4.73 (dd, J = 8.2, 6.0 Hz, 1H), 4.67 (d,
J = 14.9 Hz, 1H), 4.08 (dd, J = 4.7, 3.3 Hz, 1H), 4.04 (d, J = 14.8 Hz, 1H),
3.73 (s, 3H), 3.41 (d, J = 4.8 Hz, 1H), 3.27 (s, 3H), 2.76 (dd, J = 17.1, 8.4
Hz, 1H), 2.43 (dd, J = 17.1, 5.9 Hz, 1H), 1.78 À 1.60 (m, 1H), 0.92 (t, J =
7.1 Hz, 6H), 0.78 (s, 9H), 0.02 (d, J = 4.7 Hz, 6H); 13C NMR (126 MHz,
CDCl3) δ 174.5, 172.5, 158.9, 130.4, 128.8, 113.6, 76.8, 76.2, 68.2, 55.3,
51.7, 45.4, 40.0, 28.7, 25.5, 25.4, 22.4, 17.7, 17.4, À4.6, À5.2; HRMS
(ESI) calcd for [(C24H39NO6Si) + H]+ 466.2632, found 466.2634.
(2S,3S)-Methyl 2-((S)-1-Acetoxy-2-methylpropyl)-3-(tert-
butyldimethylsilyloxy)-1-(4-methoxybenzyl)-5-oxopyrroli-
dine-2-carboxylate (15). To a solution of 14 (2.4 g, 5.15 mmol) in
pyridine (20 mL) at 0 °C was added Ac2O (789 mg, 729 μL, 7.73 mmol),
followed by DMAP (126 mg, 1.03 mmol). The reaction mixture was
stirred overnight at room temperature, and the solvent was evaporated.
To the resulting residue was added saturated NH4Cl solution, and the
aqueous phase was extracted with EtOAc (3 Â 300 mL). The combined
organic phases were washed with brine (30 mL), dried with Na2SO4, and
concentrated. The residue was purified by flash column chromatography
(hexanes/EtOAc 100/0 to 0/100) to afford 15 (2.36 g, 90%) as a
1
colorless oil: [α]25 = +2.31° (c 1.0, CHCl3); H NMR (500 MHz,
D
CDCl3) δ 7.25 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 5.49 (d, J =
5.5 Hz, 1H), 4.67 (d, J = 15.5 Hz, 1H), 4.56 (dd, J = 7.0, 3.5 Hz, 1H), 4.35
(d, J = 15.5 Hz, 1H), 3.77 (s, 3H), 3.47 (s, 3H), 2.71 (dd, J = 17.0, 7.0 Hz,
1H), 2.41 (dd, J = 17.0, 3.5 Hz, 1H), 2.09 (s, 3H), 1.89 (m, 1H), 0.94 (d,
J = 7.0 Hz, 3H), 0.84 (d, J = 7.0 Hz, 3H), 0.85 (s, 9 H), 0.08 (s, 3 H), 0.06
(s, 3 H); 13C NMR (126 MHz, CDCl3) δ 174.6, 170.4, 169.0, 158.8,
129.2, 129.1, 113.7, 77.0, 70.0, 55.4, 51.9, 46.6, 40.6, 29.3, 25.6, 21.6,
21.2, 19.0, 17.8, À4.3, À5.1; HRMS (ESI) calcd for [(C26H41NO7Si) +
H]+ 508.2762, found 508.2752.
(hexanes/EtOAc 100/0 to 0/100) to afford 20 (295 mg, 77%) as a
1
colorless oil: [α]25 = +31.19° (c 1.0, CHCl3); H NMR (500 MHz,
D
CDCl3) δ 5.91 (d, J = 3.6 Hz, 1H), 4.89 (td, J = 8.8, 3.7 Hz, 1H), 3.75 (s,
3H), 2.90 (dd, J = 17.9, 9.0 Hz, 1H), 2.76 (dd, J = 17.9, 8.7 Hz, 1H), 2.22
(t, J = 8.2 Hz, 1H), 2.16 (s, 3H), 1.84 (m, 1H), 1.50 (s, 9H), 0.97 (d, J =
6.9 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
172.1, 169.7, 169.7, 148.6, 84.8, 75.4, 74.2, 65.5, 52.6, 38.4, 29.2, 27.8,
21.5, 21.1, 17.3; HRMS (ESI) calcd for [(C17H27NO8) + Na]+
396.1634, found 396.1638.
(2R,3S)-Methyl2-((S)-1-Acetoxy-2-methylpropyl)-3-hydroxy-
1-(4-methoxybenzyl)-5-oxopyrrolidine-2-carboxylate (16).
15 (2.0 g, 3.94 mmol) was added to a solution of TFA/H2O (10/1,
50 mL), and the resulting reaction mixture was heated to reflux for 3 h.
Most of the solvent was evaporated, and the crude product was purified
directly by flash column chromatography (hexanes/EtOAc 100/0 to
0/100) to afford 19 (1.4 g, 90%): [α]25D = À11.81° (c 0.5, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 7.23 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz,
(2R,3S,4R)-Methyl 2-((S)-1-Acetoxy-2-methylpropyl)-3-
hydroxy-4-methyl-5-oxopyrrolidine-2-carboxylate (21). To a
solution of diisopropylamine (52 mg, 72 μL, 0.52 mmol) in THF (5 mL)
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The Journal of Organic Chemistry
ARTICLE
was added butyllithium in hexane (200 μL of 2.5 M, 0.52 mmol) at 0 °C.
After being stirred for 10 min, the solution was cooled to À78 °C and
stirred for 10 min. Iodomethane (487 mg, 213 μL, 3.43 mmol) was
added, followed by the dropwise addition of a solution of 20 (160 mg,
0.43 mmol) and HMPA (0.5 mL) in THF (3 mL). After this mixture
was stirred at À78 °C for 1 h, additional HMPA (0.5 mL) was added.
The reaction mixture was stirred at À78 °C for 3 h and quenched with
saturated NH4Cl (20 mL). After the mixture was poured into water
(30 mL), the products were extracted with EtOAc (3 Â 50 mL). The
combined organic phases were washed with brine (30 mL), dried with
Na2SO4, and concentrated. The residue was purified by flash column
chromatography (hexanes/EtOAc 100/0 to 0/100) to afford 21 (100
mg, 60%) as a colorless oil: [α]25D = À16.76° (c 0.5, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 5.89 (d, J = 3.8 Hz, 1H), 4.89 (dd, J = 9.8, 5.6 Hz,
1H), 3.73 (s, 3H), 2.91 (dq, J = 15.4, 7.7 Hz, 1H), 2.16 (s, 3H), 2.00
(d, J = 5.6 Hz, 1H), 1.92 À 1.73 (m, 1H), 1.51 (s, 9H), 1.32 (d, J =
7.7 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 176.2, 170.3, 169.8, 149.0, 84.9, 75.4, 72.4, 67.1,
52.7, 40.9, 29.4, 28.0, 21.8, 21.3, 17.6, 10.7; HRMS (ESI) calcd for
[(C18H29NO8) + Na]+ 410.1791, found 410.1789.
pyridine) δ 9.94 (s, 1H), 8.79 (d, J = 8.0 Hz, 1H), 5.41 (dd, J = 13.0, 6.5
Hz, 2H), 5.34 (d, J = 7.1 Hz, 1H), 4.59 (d, J = 7.0 Hz, 1H), 4.06 (dd, J =
13.5, 4.8 Hz, 1H), 3.85 (dd, J = 13.5, 6.7 Hz, 1H), 3.54 À 3.41 (m, 1H),
2.24 (dd, J = 13.5, 6.8 Hz, 1H), 2.02 (s, 3H), 1.56 (d, J = 7.6 Hz, 3H),
1.25 (d, J = 6.6 Hz, 3H), 1.17 (d, J = 6.8 Hz, 3H).
’ ASSOCIATED CONTENT
Supporting Information. Figures giving 1H and 13C
S
b
1
NMR spectra of all new compounds and H NMR spectra of
all known compounds and a CIF file giving crystallographic data
for 16. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: Agman@chem.northwestern.edu. Tel: 847-491-5653.
Present Addresses
†Vertex Pharmaceuticals, Inc. 130 Waverly Street, Cambridge,
MA 02139.
Omuralide (2). Trifluoroacetic acid (5 mL) was added to a solution
of 21 (50 mg, 0.13 mmol) in CH2Cl2 (5 mL) at 4 °C. The reaction
mixture was stirred at room temperature for another 2 h and concen-
trated. The crude product was purified by flash column chromatography
(hexanes/EtOAc 100/0 to 0/100) to afford 22 (30 mg, 81%): [α]25
=
’ ACKNOWLEDGMENT
D
À18.25° (c 0.4, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H),
5.53 (d, J = 6.3 Hz, 1H), 4.32 (d, J = 5.4 Hz, 1H), 3.88 (s, 3H), 2.88 À
2.53 (m, 1H), 2.12 (s, 3H), 1.91 (dt, J = 13.2, 6.6 Hz, 1H), 1.26 (s, 1H),
1.18 (d, J = 7.3 Hz, 3H), 0.92 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 180.8, 171.4, 170.5, 78.5, 76.0, 75.5, 53.5, 41.3, 30.5, 21.1,
19.6, 18.4, 8.2; HRMS (ESI) calcd for [(C13H21NO6) + Na]+ 310.1267,
found 310.1274.
We are grateful to the National Institutes of Health (Grant R41
CA116971) for financial support of this research. We thank
Dr. Charlotte Stern (Northwestern University) for help with the
X-ray data.
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