Proteasome inhibition by a totally synthetic β-lactam related to salinosporamide A and omuralide

Article abstract of DOI:10.1021/ja056284a

A new and effective proteasome inhibitor, β-lactam 3, has been accessed enantioselectively by multistep synthesis from the readily prepared intermediates 7 and 8 which were joined by a [2 + 2]-cycloaddition reaction to form the spiro β-lactam 9 stereosele

Full text of DOI:10.1021/ja056284a

Published on Web 10/14/2005
Proteasome Inhibition by a Totally Synthetic â-Lactam Related to
Salinosporamide A and Omuralide
Philip C. Hogan and E. J. Corey*
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received September 12, 2005; E-mail: corey@chemistry.harvard.edu
Salinosporamide A (1) and omuralide (2) are potent naturally
derived substances which inhibit proteasome function with very
high selectivity.1-3 Proteasome inhibition offers considerable
promise in the therapy of a number of types of cancer and is already
used for multiple myeloma.4 Several routes have been developed
for the syntheses of 1 and 2.1,5 One potential problem with the use
of 1 or 2 as therapeutic agents is their short half-life in solution at
pH 7 or in serum (estimated as 5-10 min). Because of this potential
shortcoming, we have developed a synthesis of the â-lactam 3,
which is expected to be much more stable than the corresponding
â-lactone (or 1 and 2).
The pathway of the synthesis is outlined in Scheme 1. The known
alcohol 46a was converted to oxazolidinone 56b (93% yield) by
successive acylation with phenyl chloroformate and pyridine in
dichloromethane and reduction of the azide function with concomi-
tant cyclization. N-Protection of 5 with tert-butylpyrocarbonate-
4-(dimethylamino)pyridine (DMAP)-Et3N afforded 6. Reductive
cleavage of the benzyl ester subunit in 6 provided the carboxylic
acid 7.
the â-lactam 9 in 43% yield. Noteworthy in this transformation is
the use of an imine derived from (S)-(-)-1-(4-methoxyphenyl)-
ethylamine. The corresponding imine derived from 4-methoxyben-
zylamine led to â-lactam but in lower yields (∼12%). Cleavage of
the oxazolidinone ring in 9 was accomplished by exposure to Cs2-
CO3-MeOH to afford the alcohol 10 in high yield (94%).9
Oxidation of the 2-trimethylsilylfuran subunit in 10 by peroxyacetic
acid gave the butenolide 11.10 Catalytic reduction of 11 provided
the butyrolactone 12 in 91% yield, the remainder of the material
being the corresponding diastereomer. Cleavage of the t-butoxy-
carboxyl group of 12 was accomplished with trimethylsilyl tri-
fluoromethanesulfonate and 2,6-lutidine in dichloromethane at 23
°C. Any remaining trimethylsilyl trifluoromethanesulfonate was
quenched by addition of methanol and triethylamine to the reaction
mixture. Fluoride treatment during workup provided the desired
butyrolactam 13 in 86% yield. The chlorine atom was introduced
by selective reaction of the primary hydroxyl of 13 with dichlo-
rotriphenylphosphorane and pyridine in acetonitrile at 23 °C to give
14 in 80% yield. Oxidative cleavage of the (S)-1-(-)-(4-methoxy-
phenyl)ethylamide protecting group in 14 with ceric ammonium
nitrate (CAN) cleanly afforded the â-lactam 3 in 89% yield. We
were pleased that 3 is completely stable at pH 7 and 23 °C for 24
h.
Addition of a solution of 7 to a solution of 1-chloro-N,N-2-
trimethylpropenylamine7 led to smooth formation of the corre-
sponding acid chloride. Rapid addition of the acid chloride to a
mixture of the imine 88 and triethylamine gave stereoselectively
Scheme 1
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C O M M U N I C A T I O N S
that for 1 or 2, the slow rate is more than compensated for by the
greatly increased aqueous stability of 3 under physiological
conditions.
It seems reasonable that the pathway of proteasome inhibition
by the â-lactam 3 follows that of omuralide and salinosporamide
A, that is, acylation of a catalytically active threonine of a
proteolytic â-subunit. It is likely also that this acylation is rendered
irreversible by ring closure involving the chloroethyl group as an
electrophile, as appears to be the case for salinosporamide A,5b since
treatment of 3 with methanolic base afforded the bicyclic pyrrolidine
15. This fact, the observation of proteasome inhibition in vitro, and
the indefinite stability in neutral aqueous solution suggest that 3 is
a worthy candidate for further biological evaluation.
Figure 1.
Acknowledgment. We thank Drs. Lawrence Dick and Chris-
topher Tsu (Millennium Pharmaceuticals, Inc.) for advice on
determining 20S proteasome inhibition.
Supporting Information Available: Experimental synthetic pro-
cedures as well as physical and spectral data for compounds 3 and
5-15, experimental procedures for incubation of 20S proteasome with
3, and determination of inactivation rates. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 2. Plot of ln(fluorescence/time) versus time during proteasome
inactivation experiments. The slope of these lines are equivalent to kobs for
the inactivation of 20S proteasome by the â-lactam 3. The half-lives of
inactivation of 20S proteasome by 3 are approximately 58 and 165 min at
concentrations of 3 at 30 and 10 µM, respectively.
References
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Determination of the inhibitory activity of 3 against the 20S
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reported by McCormack and Dick et al.11 Incubation of 20S
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inactivation was determined by addition of Suc-LLVY-AMC to a
2 mL aliquot of the incubation experiment and monitoring the rate
of production of free AMC by fluorescence spectrophotometry. A
plot of ln(fluorescence/time) versus time provided a linear decay
the slope of which gave kobs for proteasome inactivation (Figure
2). The rate of proteasome inactivation was proportional to the
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and 30 µM time course experiments gave values for kobs/[3] as 7
and 6.7 M-1 s-1, respectively. To monitor background inactivation
of 20S proteasome,13 each of these of experiments was run side by
side with a control experiment identical in all respects except for
the exclusion of 3. In the control experiment correlating to 30 µM
of 3, after 3 h, ∼30% of initial proteasome activity was lost. In the
control experiment correlating to 10 µM of 3, after 9.5 h, ∼50%
of the initial proteasome activity remained. In each the correspond-
ing experiments containing 3, less than 10% of the initial protea-
some activity remained at these respective time points. Although
the rate of proteasome inhibition by 3 is considerably slower than
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(12) Obtained from Boston Biochem; Suc ) succinyl; AMC ) aminometh-
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(13) In vitro proteasome preparations are much less stable than the native in
vivo proteins.
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