Aspartic protease inhibitors: Expedient synthesis of 2-substituted statines

Article abstract of DOI:10.1021/ol016331d

(Equation presented) General stereocontrolled synthesis of all four (2,3)-stereoisomers of 2-substituted statines is described. The 2,3-syn and 2,3-anti isomers were synthesized via β-ketoester reduction and aldol reactions, respectively. Peptides contain

Full text of DOI:10.1021/ol016331d

ORGANIC
LETTERS
2001
Vol. 3, No. 17
2725-2728
Aspartic Protease Inhibitors: Expedient
Synthesis of 2-Substituted Statines
Jeremy M. Travins,† Matthew G. Bursavich,† Daniel F. Veber,‡ and
,†
Daniel H. Rich*
Department of Chemistry and School of Pharmacy, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53706, and GlaxoSmithKline, 709 Sweedland Road,
King of Prussia, PennsylVania 19406
dhrich@facstaff.wisc.edu
Received June 22, 2001
ABSTRACT
General stereocontrolled synthesis of all four (2,3)-stereoisomers of 2-substituted statines is described. The 2,3-syn and 2,3-anti isomers were
synthesized via â-ketoester reduction and aldol reactions, respectively. Peptides containing 2-substituted statines inhibit porcine pepsin with
nanomolar IC50 values.
Aspartic proteases play a crucial role in the onset or
proliferation of many diseases including AIDS (HIV pro-
tease), hypertension (renin), malaria (plasmepsin), and
Alzheimer’s disease (â-secretase; BACE), and much work
has been done to find both peptidomimetic and non-
peptidomimetic inhibitors of these enzymes. A key structural
element in many of these inhibitors is a hydroxyl or
hydroxyl-like subunit that binds to the two catalytic aspartates
in the enzyme active site. This structural feature was
discovered in the peptide natural product pepstatin,1 which
contains two copies of the unnatural amino acid statine
(Figure 1), a unit proposed to mimic the transition state for
amide bond hydrolysis.2 On the basis of this principle, a
number of transition-state analogues have been developed
and incorporated into peptides.3
These molecules replace the dipeptide subunit that contains
the scissile amide bond (P1-P1′ residues). Although statine
peptides are good aspartic protease inhibitors, there are two
obvious differences between a statine and a dipeptide: statine
is one backbone atom shorter than a dipeptide and it lacks a
P1′ side chain. The idea that functionalization of the statine
C2 position would restore the P1′ side chain to yield improved
or more selective aspartic protease inhibitors was explored
by Veber et al.7 Since then, 2-substituted statines have not
The most common transition-state isosteres utilized in
peptide-like aspartic protease inhibitors are the “hydroxy-
ethylenes”,4 “hydroxyethylamines”,5 and statines6 (Figure 1).
† University of Wisconsin-Madison.
‡ GlaxoSmithKline.
(1) Umezawa, H.; Aoyagi, T.; Morishima, H.; Matsazuki, M.; Hamada,
M.; Takeguchi, T. J. Antibiot. 1972, 25, 251.
(2) (a) Marshall, G. R. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1976, 35,
2494. (b) Marciniszyn, J., Jr.; Hartsuck, J. A.; Tang, J. J. Biol. Chem. 1976,
251, 7088.
Figure 1. Transition-state isostere dipeptide mimetics.
10.1021/ol016331d CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/03/2001

been investigated as aspartic protease inhibitors. However,
since 2-substituted statines are components of bleomycin8
and the dolastatins,9 a number of syntheses specific to these
natural products have been developed. The 2,3-syn stereo-
isomers have been accessed via a number of chiral auxiliary
mediated reactions,10 whereas the synthesis of the 2,3-anti
stereoisomers has been less explored.11 Herein, we describe
a general and expedient synthesis of four C2,C3 diastereo-
mers of 2-substituted statines, their incorporation into pep-
tides, and porcine pepsin inhibition data.
diastereoselectivity corresponds to the diastereomeric ratio
of the starting â-ketoester, with the 2,3-syn stereochemistry
arising via intramolecular hydride delivery in the proposed
zinc borohydride-â-ketoester six-membered chelate.14 The
individual diastereomers were easily purified using standard
silica gel flash chromatography. Removal of the Boc group
or the benzyl ester allows for peptide coupling the N- or
C-terminus, respectively.15
The (2,3)-anti stereoisomers were more difficult to obtain.
Initial attempts involved alkylation of N,N-dibenzyl statine
ethyl ester, using methodology similar to that used for the
synthesis of dolastatins.8 However, poor yields were obtained
(e25%) even when reactive alkylating agents such as allyl
bromide were used. The low yields, limited availability of
reactive alkylating agents, and number of steps compelled
us to develop a new route.
The synthesis of the (2,3)-syn-2-iBu-statines began with
the synthesis of â-ketoester 1 (Scheme 1),12 obtained via
Scheme 1
The (2,3)-anti stereoisomers were synthesized (Scheme 2)
Scheme 2
coupling of Boc-leucine acyl-imidazole with the enolate of
benzylisocaproate. While this synthesis of â-ketoester 1
offers a quick entry to the â-ketoester, the product is difficult
to purify from the side products.13 Fortunately, reduction of
crude 1 with ethereal zinc borohydride was chemoselective
for the â-ketoester, yielding the desired (2S,3S,4S)- and
(2R,3R,4S)-alcohols 2 and 3 in a ratio of 1:1.4. The
(3) (a) Rich, D. H. Peptidase Inhibitors. In ComprehensiVe Medicinal
Chemistry. The Rational Design, Mechanistic Study and Therapeutic
Application of Chemical Compounds; Hansch, C., Sammes, P. G., Taylor,
J. B., Eds.; Pergamon Press: New York; 1990; Vol. 2, pp 391-441. (b)
Rich, D. H. Med. Res. ReV. 1993, 13, 327.
(4) (a) Szelke, M.; Jones, D. M.; Hallett, A. European Patent Application
EP 45665, 1982; Chem. Abstr. 1982, 97, 39405p. (b) Szelke, M.; Jones, D.
M.; Atrash, B.; Hallett, A.; Leckie, B. J. Proc. Am. Pept. Symp. 8th 1983,
579. (c) Holladay, M. W.; Rich, D. H. Tetrahedron Lett. 1983, 24, 4401.
(d) Greenlee, W. J. Med. Res. ReV. 1990, 10, 173.
as separable diastereomers by employing aldol methodology
developed by Heathcock and co-workers.16 Deprotonation
of 2,6-dimethylphenyl isocaproate yielded the lithium E-
enolate, which reacted with Boc-leucinal to give the pair of
2,3-anti products 4a and 5a via the Zimmerman-Traxler
transition state.17 Limiting the amount of LDA in the reaction
proved to be critical as an excess caused epimerization of
the C2 carbon. Separation of the diastereomers18 from each
other was facile; however, each product was contaminated
with recovered Boc-leucinal. Saponification of the aryl ester
took place via the methyl ester by employing 2 N NaOH in
methanol, yielding the Boc-2-iBu-statines 4b and 5b ready
for peptide coupling. Alkylation of the acid with cesium
(5) (a) Gordon, E. M.; Godfrey, J. D.; Pluscec, J.; Von Langen, D.;
Natarajan, S. Biochem. Biophys. Res. Commun. 1985, 126, 419. (b) Rich,
D. H.; Green, J.; Toth, M. V.; Marshall, G. R.; Kent, S. B. H. J. Med.
Chem. 1990, 33, 1285.
(6) Rich, D. H. J. Med. Chem. 1985, 28, 263.
(7) Veber, D. F.; Bock, M. G.; Brady; S. F.; Ulm, E. H.; Cochran, D.
W.; Smith, G. H.; LaMont, B. I.; DiPardo, R. M.; Poe, Freidinger, R. M.;
Evans, B. E.; Boger, J. S. Trans. Biochem. Soc. 1984, 12, 956.
(8) (a) Narita, M.; Otsuka, M.; Kobayashi, S.; Ohno, M. Tetrahedron
Lett. 1982, 23, 525. (b) DiPardo, R. M.; Bock, M. G. Tetrahedron Lett.
1983, 24, 4805.
(9) (a) Tomioka, K.; Kanai, M.; Koga, K. Tetrahedron Lett. 1991, 32,
2395. (b) Shioiri, T.; Hayashi, K.; Hamada, Y. Tetrahedron 1993, 49, 1913.
(10) (a) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Boger,
J. S., Freidinger, R. M.; Veber, D. F. Chem. Commun. 1985, 109. (b) Rivero,
R. A.; Greenlee, W. J. Tetrahedron Lett. 1991, 32, 2453.
(11) Hayashi, K.; Hamada, Y.; Shioiri, T. Tetrahedron Lett. 1991, 32,
7287.
(14) (a) Nakata, T.; Oishi, T. Tetrahedron Lett. 1980, 21, 1641. (b) Oishi,
T.; Nakata, T. J. Synth. Org. Jpn. 1981, 39, 633.
(15) Removal of the Boc-group followed by cyclization to the γ-lactam
and 1H NMR analysis provided absolute stereochemical assignment for each
diastereomer based on the L-leucine starting material. See Supporting
Information.
(16) Heathcock, C. H.; Pirrung, M. C.; Montgomery, S. H.; Lampe, J.
Tetrahedron 1981, 37, 4081.
(12) Paris, M.; Fehrentz, J. J.; Heitz, A.; Martinez, J. Tetrahedron Lett.
1998, 39, 1569.
(13) Boc-Leu-OBn was a major identified impurity, along with unidenti-
fied products of benzyl ester self-condensation.
(17) Zimmerman, H. E.; Traxler, M. D. J. Am. Chem. Soc. 1957, 79,
1920.
(18) Absolute stereochemistry was again assigned via 1H NMR analysis
of the corresponding lactams. See Supporting Information.
2726
Org. Lett., Vol. 3, No. 17, 2001

Scheme 3
carbonate and benzyl bromide yielded the benzyl esters 4c
and 5c.
porcine pepsin by about 2 orders of magnitude.22 Further-
more, replacement of the N-Iva unit with N-acetyl also
weakens binding. Consequently, we predicted that control
peptide Ac-V-K-Sta-Ala-OMe would inhibit porcine pepsin
with a low µM IC50 value. This unoptimized peptide
sequence was chosen so that deleterious or improved binding
contributions would be readily observable. When tested
against porcine pepsin (Table 1) using a previously described
Each of the four Boc-2-iBu-Sta-OBn diastereomers, 2, 3,
4c, and 5c, was incorporated into a peptide of the sequence
Ac-Val-Lys-(2-iBuSta)-Ala-OMe (Scheme 3). Initially we
chose this amino acid sequence to target â-amyloid convert-
ing enzyme (BACE; â-secretase).19 The sequence is a hybrid
of the normal Alzheimer precursor protein (APP) cleavage
site (VKM-DAE) and the APP Swedish mutant (VNL-
DAE)20 and an unexpectedly effective inhibitor of BACE
(EVN-StaV-AEF).21 The peptide synthesis began with the
removal of the 2-iBu-statine N-Boc group, followed by
coupling with Fmoc-Lys(Nꢀ-Boc)-OH to give pseudotripep-
tide 6. The N-Fmoc group was cleaved, and the resulting
amine was coupled with Fmoc-Val-OH to afford 7. Removal
of the N-Fmoc group, acetylation of the resulting amine, and
subsequent hydrogenolysis of the benzyl ester yielded peptide
acid 9. Coupling of 9 with an amino acid ester, followed by
treatment of the resultant peptide with TFA, yielded the
functional 2-iBu-statine-containing peptides 11a-d and 12
in 40-50% overall yields.
Table 1. Activities of statine and 2-iBu-Statine peptides
against porcine pepsin
To estimate the contribution to binding provided by the
additional 2-isobutyl C2-substituent, the analogous statine-
containing peptide, Ac-V-K-Sta-Ala-OMe (13), was synthe-
sized. A series of lysine-substituted, statine-containing
peptides previously synthesized here demonstrated that lysine
in the P2 position of the peptide diminished binding to
(19) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E.
A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Leoloff, R.; Luo, Y.;
Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.;
Curran, E.; Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers, G.;
Citron, M. Science 1999, 286, 735.
(20) Citron, M.; Oltersdorf, T.; Haass, C.; McConlogue, L.; Hung, A.
Y.; Seubert, P.; Vigo-Pelfrey, C.; Lieberburg, I.; Selkoe, D. J. Nature 1992,
360, 672.
(21) Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello,
R.; Davis, D.; Doan, M.; Dovey, H. V.; Frigon, N.; Hong, J.; Jacobsen-
Croak, K.; Jewett, N.; Keim, P.; Knops, J.; Lieberburg, I.; Power, M.; Tan,
H.; Tatsuno, G.; Tung, J.; Schenk, D.; Seubert, P.; Suomensaari, S. M.;
Wang, S.; Walker, D.; Zhao, J.; McConlogue, L.; John, V. Nature 1999,
402, 537.
fluorometric assay,23 Ac-V-K-Sta-Ala-OMe gave an IC50 of
2 µM, in good agreement with prediction. The diastereomeric
(22) Kuzmic, P.; Sun, C. Q.; Zhao, Z. C.; Rich, D. H. Tetrahedron 1991,
47, 2519.
Org. Lett., Vol. 3, No. 17, 2001
2727

2-iBu-Sta-containing peptides also inhibited porcine pepsin
in the 0.1-10 µM range. However, none of these peptides
showed inhibition of BACE at concentrations of 10 µM.
The inhibition data demonstrate that the S-hydroxyl 2-i-
Bu-Sta peptides are potent inhibitors of porcine pepsin, with
activities in the 100 nM range (Table 1). As expected for
peptides of this length, the 2-iBu-Sta peptides containing the
R-hydroxyl group are weaker inhibitors, having low micro-
molar potencies. Similar to the observed inhibition of renin
by 2-substituted statine peptides,6 the (2R,3S,4S)-2-iBu-Sta
peptides are 3-4 times more active against porcine pepsin
than the (2S,3S,4S)-diastereomer. The preference for the 2R
stereochemistry can be rationalized on the basis of the
positions of the statine-C2 pro-R and pro-S protons in the
X-ray structures of statine peptides bound to porcine pepsin.
Most importantly, the (3S)-2-iBu-Sta peptides 11a and 12
are at least 10-fold more active than the statine peptide 13
for inhibition of porcine pepsin. Further optimization is
expected to provide improved inhibitors against this and other
aspartic peptidases.
Figure 2. Functionalization of the C2 side chain.
These strategies are being applied to synthesize other
2-substituted statines, as illustrated by 2-allylstatine (Figure
2). The allyl side chain permits entry to a variety of
derivatives designed to mimic Asn, Gln, Glu, and Arg side
chains. Oxidative cleavage of the allyl side chain using
RuCl3/NaIO4 gave the Leu-Asp dipeptide mimics 14a and
14b in good yields. Alternatively, hydroboration-oxidation
of the alkene can provide the terminal alcohol, which can
be converted into a number of functional groups. Elaboration
of the allyl side chain into the Arg side chain was recently
accomplished in the synthesis of a Phe-Arg hydroxyethylene
isostere, utilizing a similar substrate.24 On the basis of the
inhibitory activity of the 2-iBu-Sta-containing peptides
against aspartic proteases, these molecules may offer new
templates for the design of selective aspartic protease
inhibitors.
Acknowledgment. This work was supported in part by
grants from GlaxoSmithKline and from the National Insti-
tutes of Health (GM50113). We thank Dr. Thomas Meek
for his support and encouragement, Lindsay J. Howett (GSK)
for providing BACE assay data, and Dr. Martha Vestling
for obtaining MS data.
Supporting Information Available: Detailed experi-
mental procedures for synthesis and characterization of
representative compounds and absolute stereochemical as-
1
signments via H NMR analysis. This material is available
(23) (a) Flentke, G. R.; Glinski, J.; Satyshur, K.; Rich, D. H. Protein
Expression Purif. 1999, 16, 213. (b) Peranteau, A. G.; Kuzmic, P.; Angell,
Y.; Rich, D. H. Anal. Biochem. 1995, 227, 242.
free of charge via the Internet at http://pubs.acs.org.
(24) Brewer, M.; Rich, D. H. Org Lett. 2001, 3, 945.
OL016331D
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