Synthesis and evaluation of keto-glutamine analogues as inhibitors of hepatitis A virus 3C proteinase.

Article abstract of DOI:10.1021/jo0157831

Hepatitis A virus (HAV) 3C enzyme is a picornaviral cysteine proteinase involved in the processing of the initially synthesized viral polyprotein and is therefore important for viral maturation and infectivity. Although it is a cysteine proteinase, this enzyme has a topology similar to those of the chymotrypsin-like serine proteinases. Since the enzyme recognizes peptide substrates with a glutamine residue at the P(1) site, a number of ketone-containing glutamine compounds analogous to nanomolar inhibitors of cathepsin K were synthesized and tested for inhibition against HAV 3C proteinase. In addition, a 3-azetidinone scaffold was incorporated into the glutamine fragment but gave only modest inhibition. However, introduction of a phthalhydrazido group alpha to the ketone moiety gave significantly better inhibitors with IC(50) values ranging from 13 to 164 microM, presumably due to the effect of intramolecular hydrogen bonding to the ketone. In addition, the tetrapeptide phthalhydrazide 24 was found to be a competitive reversible inhibitor (K(i) = 9 x 10(-6) M) and also showed no loss of inhibitory potency in the presence of dithiothreitol.

Full text of DOI:10.1021/jo0157831

VOLUME 67, NUMBER 10
MAY 17, 2002
© Copyright 2002 by the American Chemical Society
Articles
Syn th esis a n d Eva lu a tion of Keto-Glu ta m in e An a logu es a s
In h ibitor s of Hep a titis A Vir u s 3C P r otein a se
Yeeman K. Ramtohul,† Michael N. G. J ames,‡,| and J ohn C. Vederas*,†,§
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2, and Department
of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
john.vederas@ualberta.ca
Received May 22, 2001
Hepatitis A virus (HAV) 3C enzyme is a picornaviral cysteine proteinase involved in the processing
of the initially synthesized viral polyprotein and is therefore important for viral maturation and
infectivity. Although it is a cysteine proteinase, this enzyme has a topology similar to those of the
chymotrypsin-like serine proteinases. Since the enzyme recognizes peptide substrates with a
glutamine residue at the P1 site, a number of ketone-containing glutamine compounds analogous
to nanomolar inhibitors of cathepsin K were synthesized and tested for inhibition against HAV 3C
proteinase. In addition, a 3-azetidinone scaffold was incorporated into the glutamine fragment but
gave only modest inhibition. However, introduction of a phthalhydrazido group R to the ketone
moiety gave significantly better inhibitors with IC50 values ranging from 13 to 164 µM, presumably
due to the effect of intramolecular hydrogen bonding to the ketone. In addition, the tetrapeptide
phthalhydrazide 24 was found to be a competitive reversible inhibitor (Ki ) 9 × 10-6 M) and also
showed no loss of inhibitory potency in the presence of dithiothreitol.
In tr od u ction
family, which contains more than 200 known members
including other pathogens such as the human rhinovirus
(HRV), foot and mouth disease virus, poliovirus (PV), and
encephalomyocarditis (EMCV).2 HAV is the only known
member of the genus hepatovirus and causes an acute
form of infectious hepatitis.2 Occasional outbreaks of
hepatitis A still occur in the developed world, and these
are usually attributed to contaminated food and drinking
water.3 Picornaviruses possess a small positive single-
stranded RNA genome whose translation in the host cells
produces a single ∼250 kDa polyprotein. In HAV, the 3C
Cysteine proteinases are among the most attractive
targets for chemotherapy because of their role in the
pathogenesis of many diseases, including osteoporosis,
arthritis, cancer, viral, and parasitic infections.1 The
hepatitis A virus (HAV) belongs to the picornavirus
* To whom correspondence should be addressed. Tel (780) 492 5475.
FAX (780) 492 8231.
† Department of Chemistry, University of Alberta.
‡ Department of Biochemistry, University of Alberta.
§ Canada Research Chair in Bioorganic and Medicinal Chemistry.
| Canada Research Chair in Protein Structure and Function.
(1) For reviews on cysteine proteinases, see: (a) Otto, H.-H.;
Schirmeister, T. Chem. Rev. 1997, 97, 133-171. (b) Leung, D.;
Abbenante, G.; Fairlie, D. P.; J . Med. Chem. 2000, 43, 305-341. (c)
See also the Symposium-in-Print on cysteine protease inhibitors:
Thompson, S. K. Bioorg. Med. Chem. 1999, 7, 571.
(2) Hollinger, F. B.; Ticehurst, J . R. In Fields Virology; Fields, B.
N.; Knipe, D. M.; Howley, P. M.; Channock, R. M.; Melnick, J . L.;
Monath, T. P.; Roizmann, B. E.; Strauss, S. E., Eds., Lippincott-Raven
Publishers: Philadelphia, 1996.
(3) Pebody, R. G.; Leino, T.; Ruuutu, P.; Kinnunen, L.; Davidkin,
I.; Nohynek, H.; Leinikki, P. Epidemiol. Infect. 1998, 120, 55-59.
10.1021/jo0157831 CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/08/2001

3170 J . Org. Chem., Vol. 67, No. 10, 2002
Ramtohul et al.
proteinase is responsible for the co- and posttranslational
processing of this viral polyprotein into structural and
nonstructural components and is therefore essential for
viral maturation and infectivity.4,5 In addition, 3C pro-
teinases have a unique active site geometry4a which
makes these enzymes highly specific in their choice of
substrate. The cleavage sites of the 3C proteinases, which
are absent in noninfected mammalian cells, have a high
degree of conserved residues6,7 and therefore a specific
inhibitor of the 3C enzyme could potentially inhibit a
large number of serotypes.
Crystal structures of HAV 3C,8 HRV 3C,9 and PV 3C10
proteinases have been reported, and structurally these
enzymes are distinct from the papain family in that they
have a two-domain â-barrel fold that is characteristic of
the chymotrypsin-like serine proteinases.4,6 An active site
cysteine (Cys-172) of the HAV 3C proteinase acts as the
nucleophile with assistance from a histidine residue (His-
44), which behaves as a general acid-base catalyst, with
the formation of the tetrahedral intermediate being
promoted by an electrophilic oxyanion hole.4 The enzyme
typically binds four to five residues (P5 to P1, Figure 1)
preceding the scissile peptide bond and two to three
residues (P1′ to P3′) following the site of cleavage.11 In
addition, hydrogen bonding between the carbonyl oxygen
side chain of the P1 glutamine of the substrate (or peptidic
inhibitor) and His-191 at the S1 subsite is one of the key
recognition events.4,6 For an octapeptide substrate (Ac-
ELRTQSFS-NH2) mimicking the 2B/2C junction of the
viral polyprotein precursor with glutamine at the P1
position (Figure 1), the kcat is about 1.8 s-1 with a Km of
2.1 mM at pH 7.5.6 In search of potential therapeutic
leads, enzyme inhibition studies on HAV 3C proteinase
have employed a variety of compounds, including peptide
aldehydes,12 peptide fluoromethyl ketones,13 iodoacet-
amides,14 azapeptides,15 azodicarboxamides,16 and â-lac-
tones.17
F igu r e 1. A preferred cleavage site of HAV 3C proteinase
showing the sequence at the 2B-2C junction of the viral 250
kDa precursor protein with rationale for inhibition by keto-
glutamine derivatives and structures of cathepsin K inhibitors
1 and 2.
(4) (a) Bergmann, E. M.; J ames, M. N. G. In Proteases as Targets
for Therapy; Von der Helm, K., Korant, B. Eds.; Springer: Heiderlberg,
1999. (b) Gorbalenya, A. E.; Snijder, E. J . Perspect. Drug Discov. Des.
1996, 6, 64-86.
(5) Rueckert, R. R. In Fields Virology; Fields, B. N.; Knipe, D. M.;
Howley, P. M.; Channock, R. M.; Melnick, J . L.; Monath, T. P.;
Roizmann, B. E.; Strauss, S. E.; Eds.; Lippincott-Raven Publishers:
Philadelphia, 1996.
Recently, 1,3 diamino ketone 118 and R-alkoxy ketone
219 were reported as potent and selective inhibitors of
cathepsin K, a cysteine proteinase of the papain super-
family involved in the process of bone resorption (Figure
(6) Malcolm, B. A. Protein Sci. 1995, 4, 1439-1445.
(7) Krausslich, H. G.; Wimmer, E. Annu. Rev. Biochem. 1988, 57,
701-754. (b) Rasnick, D. Perspect. in Drug Discov. Des. 1996, 6, 47-
63.
(8) (a) Bergmann, E. M.; Mosimann, S. C.; Chernaia, M. M.;
Malcolm, B. A.; J ames, M. N. G. J . Virol. 1997, 71, 2436-2448. (b)
For crystal structure of HAV 3C with N-iodoacetyl-Val-Phe-NH2, see:
Bergmann, E. M.; Cherney, M. M.; McKendrick, J .; Frormann, S.; Luo,
C.; Malcolm, B. A.; Vederas, J . C.; J ames, M. N. G. Virology 1999, 265,
153-163.
(9) (a) Matthews, D. A.; Smith, W. W.; Ferre, R. A.; Condon, B.;
Budahazi, G.; Sisson, W.; Villafranca, J . E.; J anson, C. A.; McElroy,
H. E.; Gribskov, C. L.; Worland, S. Cell 1994, 77, 761-771. (b)
Matthews, D. A.; Dragovich, P. S.; Webber, S. E.; Fuhrman, S. A.;
Patick, A. K.; Zalman, L. S.; Hendrickson, T. F.; Love, R. A.; Prins, T.
J .; Marakovits, J . T.; Zhou, R.; Tikhe, J .; Ford, C. E.; Meador, J . W.;
Ferre, R. A.; Brown, E. L.; Binford, S. L.; Brothers, M. A.; DeLisle; D.
M.; Worland, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11000-11007.
(10) Mosimann, S. C.; Cherney, M. M.; Sia, S.; Plotch, S.; J ames,
M. N. G. J . Mol. Biol. 1997, 273, 1032-1047.
(11) Using the notation of Schechter and Berger. Schechter, I.;
Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157-162.
(12) Malcolm, B. A.; Lowe, C.; Shechosky, S.; Mckay, R. T.; Yang,
C. C.; Shah, V. J .; Simon, R. J .; Vederas, J . C.; Santi, D. V. Biochemistry
1995, 34, 8172-8179.
(13) Morris, T. S.; Frormann, S.; Shechosky, S.; Lowe, C.; Lall, M.
S.; Gauss-Mu¨ller, V.; Purcell, R. H., Emerson, S. U.; Vederas, J . C.;
Malcolm, B. A. Bioorg. Med. Chem. 1997, 5, 797-807.
(14) McKendrick, J . E.; Frormann, S.; Luo, C.; Semchuck, P.;
Vederas, J . C.; Malcolm, B. A. Int. J . Mass. Spectrom. 1998, 176, 113-
124.
(15) Huang, Y.; Malcolm B. A.; Vederas, J . C. Bioorg. Med. Chem.
1999, 7, 607-619.
(16) Hill, R. D.; Vederas, J . C. J . Org. Chem. 1999, 64, 9538-9546.
(17) Lall, M. S.; Karvellas, C.; Vederas, J . C. Org. Lett. 1999, 1, 803-
806.
(18) (a) Yamashita, D. S.; Smith, W. W.; Zhao, B.; J anson, C. A.;
Tomaszek, T. A.; Bossard M. J .; Levy, M. A.; Oh H.-J .; Carr, T. J .;
Thompson, S. K.; Ijames, C. F.; Carr, S. A.; McQueney, M.; D’Alesio,
K. J .; Amegadzie, B. Y.; Hanning, C. R.; Abdel-Meguid, S.; DesJ ardais,
R. L.; Gleason, J . G.; Veber, D. F. J . Am. Chem. Soc. 1997, 119, 11351-
11352. (b) Marquis, R. W.; Yamashita, D. S.; Ru, Y.; LoCastro, S. M.;
Oh, H.-J .; Erhard, K. F.; DesJ ardais, R. L.; Head, M. S.; Smith, W.
W.; Zhao, B.; J anson, C. A.; Abdel-Meguid, S. S.; Tomaszek, T. A.; Levy,
M. A.; Veber, D. F. J . Med. Chem. 1998, 41, 3563-3567.
(19) Marquis, R. W.; Ru, Y.; Yamashita, D. S.; Oh H.-J .; Yen, J .;
Thompson, S. K.; Carr, T. J .; Levy, M. A.; Tomaszek, T. A.; Ijames, C.
F.; Smith, W. W.; Zhao, B.; J anson, C. A.; Abdel-Meguid, S. S.; D′Alesio,
K. J .; McQueney, M. S.; Veber, D. F. Bioorg. Med. Chem. 1999, 7, 581-
588.

Inhibitors of Hepatitis A Virus 3C Proteinase
J . Org. Chem., Vol. 67, No. 10, 2002 3171
Sch em e 1
1). Both ketone 1 and 2 are nanomolar inhibitors with
Ki,app of 22 nM and IC50 of 3.7 nM, respectively, and were
found to form a hemi-thioketal bond between the ketone
carbonyl and the active site cysteine.18,19 In addition,
these compounds contain a ketone moiety which has a
reduced electrophilicity compared to their aldehyde
counterparts and can therefore react more specifically
with the proteinase as opposed to a variety of thiols
present in the mamalian cells. We therefore chose to
investigate a series of analogues containing this ketone
functionality as potential inhibitors of HAV 3C protein-
ase. Although HAV 3C proteinase has a preference for a
glutamine residue at the P1 site, it has been shown that
replacement of the glutamine with N,N-dimethylglutamine
has no significant effect on substrate recognition or
cleavage.20 Hence, for the ease of synthesis and to hinder
any interaction of the side-chain glutamine primary
amide with the ketone moiety, we chose to synthesize
1,3-diamino ketone (e.g., 11), R-heteroatom ketone (e.g.,
17), and cyclic ketone (e.g., 25) analogues bearing the
N,N-dimethylglutamine motif at the P1 site and a side-
chain phenyl moiety mimicking the P2′ phenylalanine.
Although these compounds lack the functionality that
mimics the P1′ amino acid, which is normally a serine
residue, previous studies show that this residue does not
contribute significantly to recognition of the substrate as
compared to P4 (Leu), P1 (Gln), and P2′ (Phe).6
glutamic acid R-benzyl ester 3 with dimethylamine using
ethyl chloroformate gives 4 in 85% yield.13 Removal of
the benzyl group by catalytic hydrogenation affords Boc-
N,N-dimethylglutamine 5 which can be subsequently
converted to the thioester 6 using a procedure similar to
that described by Fukuyama et al.21 Conversion of
thioester 6 to the aldehyde 7 proceeds in good yield by
reduction with triethylsilane in the presence of a catalytic
amount of palladium on carbon.21 Reaction of 7 with
potassium cyanide generates cyanohydrin 8 in a 1:1
diastereomeric mixture.22 Reduction of 8 over PtO2 at 50
psi is sluggish and affords the amino alcohol 9 in 43%
yield. Coupling of 9 to 4-phenylbutyric acid or benzyl
chloroformate followed by Dess-Martin periodinane23
oxidation proceeds readily to generate the desired com-
pounds 11 and 13, respectively. Although the R-center
bearing the glutamine side chain could potentially be
prone to epimerization, this was not observed with 11,
13, or other aminoketones described below.
Assay of 11 and 13 for inhibition against HAV 3C
proteinase employed an overexpressed C24S mutant in
which the nonessential surface cysteine is replaced with
serine and which displays catalytic parameters indistin-
guishable from the wild-type proteinase.24a The enzyme
activity is monitored using a fluorometric assay at an
enzyme concentration of approximately 0.1 µM and
(21) Fukuyama, T.; Lin, S. C.; Li, L. J . Am. Chem. Soc. 1990, 112,
7050-7051.
Resu lts a n d Discu ssion
(22) Greenlee, W. J .; Springer, J . P.; Patchett, A. A. J . Med. Chem.
1989, 32, 165-170.
Compounds 11 and 13 were selected as initial targets
based on their structural similarity to 1 and 2, potent
inhibitors of cathepsin K.18,19 They were synthesized as
outlined in Scheme 1. Coupling of the Boc-protected
(23) (a) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155-
4156. (b) Dess, D. B.; Martin, J . C. J . Am. Chem. Soc. 1991, 113, 7277-
7287.
(24) (a) Malcolm, B. A.; Chin, S. M.; J ewell, D. A.; Stratton-Thomas,
J . R.; Thudium, K. B.; Rosenberg, S. Biochemistry 1992, 31, 3358-
3363. (b) Wang, Q. M.; J ohnson, R. B.; Cohen, J . D.; Voy, G. T.;
Richardson, J . M.; J unghein, L. N. Antiviral Chem. Chemother. 1997,
8, 303-310.
(20) J ewell, D. A.; Swietnicki, W.; Dunn, B. N.; Malcolm, B. A.
Biochemistry 1992, 31, 7862-7869.

3172 J . Org. Chem., Vol. 67, No. 10, 2002
Ramtohul et al.
Ta ble 1
Sch em e 2
compounds
IC50 values,a µM
% inhibitiona,b
11
13
16
17
18
20
21
23
24
25
26
28
no inhibition
20 ( 1
>500
20 ( 2
89
146
164
15 ( 7
20 ( 5
13
358
258
a
0.1 µM HAV 3C, 10 µM Dabcyl-GLRTQSFS-Edans, 2 mM
EDTA, 0.1 mg/mL BSA, 100 mM KH2PO4/K2HPO4 at pH 7.5, 15
min preincubation of the enzyme with inhibitor. The IC50 values
reported are within (10% error. bInhibition at [I] ) 100 µM.
Dabcyl-GLRTQSFS-Edans as the substrate. The enzyme
is incubated with the appropriate inhibitor for 15 min,
and reaction is initiated by the addition of substrate.
Disappointingly, compound 11 shows no inhibition, and
13 gives poor inhibition of 20 ( 1% at a concentration of
100 µM (Table 1). This contrasts the nanomolar inhibition
achieved by the structurally related 1,3-diamino ketone
1 against mammalian cysteine proteinase, cathepsin
K.18,19 Although it might initially seem that the Boc group
in 11 and 13 is a poor mimic of the peptide chain in
substrates and interferes with enzyme recognition, the
work described below shows that this is not the primary
cause of weak binding.
The marked contrast in potency of 11 and 13 with HAV
3C proteinase, as compared to 1 and 2 with cathepsin
K,18,19 indicates the importance of factors beyond simple
accommodation of the side chain(s) in the active site and
electrophilicity of the keto group.25 Although certain more
electrophilic N-acylated R-amino aldehydes (“peptide
aldehydes”) such as Ac-Leu-Ala-Ala-(N,N-dimethyl)-
glutaminal are very potent slow-binding inhibitors of
HAV 3C proteinase (Ki* of 42 nM),12 their potential use
as therapeutic agents is limited by poor bioavailability
due to the reactive nature of the aldehyde moiety. The
corresponding N-acetyl-(N,N-dimethyl)glutaminal, which
is missing the P4-P2 residues, is a weak competitive
inhibitor with Ki ) 2.5 mM.12 Hence, in addition to
replacement of peptidic residues, the goals of the current
work included adjustment of the electrophilicity of the
reactive carbonyl to enhance metabolic stability and
maintain selective inhibition of the target enzyme. To
further examine the effect of the R-heteroatom substitu-
ent and its electron-withdrawing capability, compounds
16 and 17 were synthesized and tested (Scheme 2).
Following the literature procedure of Morris et al.,13 acid
5 can be converted to the R-diazoketone 14 by formation
of the mixed anhydride with ethyl chloroformate followed
by trapping with diazomethane. Treatment of 14 with
aqueous HBr provides the bromomethyl ketone 15 which
is subsequently converted to the phthalimidomethyl
ketone 16 or phenoxymethyl ketone 17 by nucleophilic
displacement with potassium phthalimide or sodium
phenoxide, respectively. However, testing with the HAV
Another approach to enhancing the electrophilicity of
the target carbonyl group in an inhibitor involves in-
tramolecular hydrogen bonding to the doubly bonded
oxygen, such as in compound 18, which has the phthal-
hydrazido N-H proton available for such an interaction
in a six-membered chelate. Such an idea has been
successfully applied to the design of R-hydroxy keto-
methylene dipeptide inhibitors of angiotensin converting
enzyme.26 This should also increase the conformational
rigidity of the molecule, thereby reducing the entropic
cost for tight binding to the target protein.27 Nucleophilic
displacement of sodium phthalhydrazide on bromomethyl
ketone 15 affords the desired compound 18 as well as
the dimer 19 in a 2:1 ratio (Scheme 3). Testing of 18
against HAV 3C proteinase gives reversible inhibition
with an IC50 of 89 µM. This contrasts strongly with the
poor inhibition shown by 16 (IC50 > 500 µM), which has
related functionality but lacks the analogous hydrogen
bond.28 Following this result, it seemed that analogues
of 18 with a nitro group on the phenyl ring could increase
the acidity of the N-H proton and thereby enhance the
electrophilicity of the ketone moiety. Reaction of bromo-
methyl ketone 15 with sodium 3-nitrophthalhydrazide
affords 20 and 21 (Scheme 3).29 However, incubation of
(26) (a) Kim, B. H.; Soo, G. C.; Hu, T.-K. J . Org. Chem. 1999, 64,
5036-5041. (b) Kim, B. H.; Ryu, E. J .; Chung, Y. J . Bioorg. Med. Chem.
Lett. 1994, 4, 2799. (c) Kim, B. H.; Chung, Y. J .; Ryu, E. R. Tetrahedron
Lett. 1993, 34, 8465-8468.
(27) (a) Ding, J .; Fraser, M. E.; Meyer, J . H.; Bartlett, P. A.; J ames,
M. N. G. J . Am. Chem. Soc. 1998, 120, 4610-4621. (b) Meyer, J . H.;
Bartlett, P. A. J . Am. Chem. Soc. 1998, 120, 4600-4609. (c) Smith,
W. W.; Bartlett, P. A. J . Am. Chem. Soc. 1998, 120, 4622-4628. (d)
Khan, A. R.; Parrish, J . C.; Fraser, M. E.; Smith, W. W.; Bartlett, P.
A.; J ames, M. N. G. Biochemistry 1998, 37, 16839-16845.
(28) Examination of the ketone carbonyl IR absorption band of
compounds 16 (1745 cm-1) and 18 (1735 cm-1) indicates a lower
absorption frequency for 18 possibly due to the effect of intramolecular
hydrogen bonding to the ketone moiety, see: (a) Silverstein, R. M.;
Webster, F. X. in Spectrometric Identification of Organic Compounds,
6th ed.; J ohn Wiley & Sons: New York, 1998; p 96. (b) Pimentel, G. C.;
McClellan, A. L. The Hydrogen Bond; W. H. Freeman and Co.: San
Francisco, 1960; Chapter 5.
3C enzyme indicates only weak inhibition, with IC50
>
500 µM for 16 and 20 ( 2% inhibition at a concentration
of 100 µM for 17 (Table 1).
(25) Mjalli, A. M. M.; Chapman, K. T.; MacCoss, M.; Thornberry,
N. A.; Peterson, E. P. Bioorg. Med. Chem. Lett. 1994, 4, 1965-1968.

Inhibitors of Hepatitis A Virus 3C Proteinase
J . Org. Chem., Vol. 67, No. 10, 2002 3173
Sch em e 3
Sch em e 5
inhibition of HAV 3C (15 ( 7% at 100 µM) (Table 1).
Thus, removal of the aromatic ring coupled with in-
creased flexibility and change of the preferred conforma-
tion of the hydrazido functionality32 drastically reduces
binding to the enzyme.
Although a major objective of this work was removal
of peptidic linkages from potential inhibitors of the 3C
cysteine proteinase, it is desirable to estimate the relative
contribution to binding of such residues by attachment
of the successful “warhead” motifs. In the case of (N,N-
dimethyl)glutaminal, an analogue with an appropriate
tripeptide (N-Ac-Leu-Ala-Ala) attached to the N-terminus
is about 9000 times more effective than the simple
N-acetyl derivative.12 Hence 18 was coupled to the
tripeptide Ac-Leu-Ala-Ala-OH to test whether this would
drastically influence molecular recognition of the inhibi-
tor and whether the Boc group is detrimental to binding.
The tripeptide fragment Ac-Leu-Ala-Ala is a reasonable
substitute for the Leu-Arg-Thr sequence in substrate
analogues as it has been shown that neither arginine nor
threonine side chains contribute significantly to bind-
ing.20 This peptide sequence also simplifies the synthesis
by eliminating side chain deprotection steps. Cleavage
of the Boc protecting group of 18 with trifluoroacetic acid
followed by coupling with Ac-Leu-Ala-Ala-OH using
HBTU proceeds smoothly to afford the desired tetrapep-
tide 24 in 70% yield over two steps (Scheme 5). Com-
pound 24 is a competitive reversible inhibitor of the HAV
3C enzyme with a Ki of 9 µM.33 Furthermore, the
inhibitory activity of 24 is not affected in the presence of
a 100-fold excess of dithiothreitol (DTT), indicating that
this type of phthalhydrazido system could be specific
inhibitor of the HAV 3C proteinase which would not react
inadvertently with ubiquitous thiols (e.g., glutathione).
Although the peptide residues in 24 do enhance the
inhibition by about a factor of 7 relative to 18, the effect
is not nearly so dramatic as with the N-acylated R-amino
aldehyde, (N,N-dimethyl)glutaminal. Thus, 18 represents
an interesting lead structure that could be further
modified to generate drug candidates for picornaviral
cysteine proteinases.
Sch em e 4
20 and 21 with the HAV 3C proteinase does not show
improved inhibition and gives IC50 values of 146 and 164
µM, respectively (Table 1). Possibly the nitro group has
unfavorable steric or electronic interactions in the region
of the enzyme active site which binds the P2′ phenyl-
alanine residue in substrate analogues.8b To examine the
motif further, compound 23 was prepared as it has the
hydrazido N-H for intramolecular hydrogen bond forma-
tion but is missing the ring system of 18, 20, and 21
(Scheme 4). Conversion of 14 to the hydroxymethyl
ketone 22 proceeds by treatment with 1 N HCl.30 This is
then coupled to the dimethyl hydrazodicarboxylate group
via a Mitsunobu-type31 reaction to give 23 in low yield
(20%). Enzymatic testing of 23 shows weak reversible
Another approach to enhancing electrophilicity and
reactivity of R-amino ketones is the introduction of a rigid
3-azetidinone scaffold at the P1 site. Thus reaction of
R-diazoketone 14 with a catalytic amount of rhodium(II)
(29) Isomeric compounds 20 and 21 were characterized by using
HMQC and HMBC NMR techniques, e.g., the key correlation in the
HMBC spectrum of 20 is between the phthalhydrazido N-H proton
(δH 11.04 ppm) and the aromatic C-NO2 (δC 156 ppm) quaternary
carbon.
(30) Pirrung, M. C.; Rowley, E. G.; Holmes, C. P. J . Org. Chem. 1993,
53, 5683-5689.
(31) Dow, R. L.; Kelly, R. C.; Schletter, I.; Wierenga, W. Synth.
Commun. 1981, 11, 43-53.
(32) Harris, J . M.; Bolessa, E. A.; Vederas, J . C. J . Chem. Soc.,
Perkin Trans. 1 1995, 1951-1959.
(33) Segel, I. H. In Enzyme Kinetics, J ohn Wiley & Sons: New York,
1975; pp 100-125.

3174 J . Org. Chem., Vol. 67, No. 10, 2002
Ramtohul et al.
Sch em e 6
Su m m a r y
In conclusion, the present work provides access to
several electrophilic keto-glutamine analogues which
may allow development of potent inhibitors of 3C cysteine
proteinases as therapeutic agents for picornaviral infec-
tions. Interestingly, analogues of inhibitors of the mam-
malian cysteine proteinase, cathepsin K, display rela-
tively weak inhibition with HAV 3C proteinase. However,
phthalhydrazido analogues 18 and 24 show good inhibi-
tion, possibly due to the effect of intramolecular hydrogen
bonding to the ketone moiety and restricted conforma-
tional mobility. The absence of peptidic bonds in 18
makes it an especially promising structure for further
modification. It is also likely that such keto-glutamine
derivatives may be potent inhibitors of other picornaviral
cysteine proteinases, such as HRV 3C, as most of these
enzymes require a glutamine residue at the P1 site in
the substrate. Additional studies with modified analogues
and other 3C proteinases will be reported in the future.
Exp er im en ta l Section
Gen er a l Meth od s a n d En zym e Assa ys. Most general
procedures and instrumentation have been previously de-
scribed.35 Peptide fragment Ac-Leu-Ala-Ala-OH was prepared
using standard solution-phase chemistry with 2-(1H-benzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) as the coupling reagent.36 HPLC purification was
performed on a Rainin HPLC system (Waters C18 Bondapak,
25 × 100 mm, 15 mL/min flow rate, linear gradient of
acetonitrile in 0.1% TFA/H2O). Recombinant C24S HAV 3C
proteinase was expressed in Escherichia coli and purified as
reported previously.24a Purity of the enzyme was greater than
90% as determined by SDS-PAGE analysis.24a Proteinase
acetate dimer34 generates a carbenoid which inserts
intramolecularly into the N-H bond to afford 25 along
with 26 as a side product in a ratio of 3:1 (Scheme 6).
Urethane 26 presumably forms by addition of the car-
bonyl oxygen atom of the Boc protecting group to the
carbene followed by loss of the tert-butyl group. Testing
of 25 with the HAV 3C enzyme gives moderate inhibition
with an IC50 of 358 µM, and the urethane 26 displays 20
( 5% inhibition at a concentration of 100 µM (Table 1).
Thus the azetidinone motif in 25 appears inherently less
effective compared to the hydrogen-bonding phthal-
hydrazide motif in 18. To examine the influence of
additional peptidic residues with this four-membered ring
system, the tripeptide Ac-Leu-Ala-Ala was attached to
25. To prevent unwanted side reactions, the ketone
functionality of 25 is reduced prior to coupling. Thus
reaction of 25 with sodium borohydride followed by
treatment with trifluoroacetic acid affords the amino
alcohol 27. This is coupled to Ac-Leu-Ala-Ala-OH as
described previously and subsequently oxidized with
Dess-Martin periodinane23 to afford the target 28 in 71%
yield. Incubation of 28 with the HAV 3C proteinase
displays only a slight enhancement of reversible inhibi-
tion (IC50 ) 258 µM) relative to 25 despite the presence
of the P4-P1 peptide backbone. Although the reversible
binding of the azetidinones to the target enzyme is
significantly greater than that of an optimized octapep-
tide substrate (Km of 2.1 mM), this cyclic ketone motif
appears to be less promising for HAV 3C inhibitor
development than the phthalhydrazido system. Never-
theless, it may prove promising for other types of cysteine
proteinases of therapeutic interest.
concentrations were determined spectrophotometrically ꢀ280
)
1.2 mg/mL. Enzyme was dialyzed against reaction buffer to
remove dithiothreitol (DTT) immediately prior to use and its
activity monitored by a fluorometric assay similar to one
described for HRV 3C proteinase.24b Assays were done at 30
°C in a solution containing 100 mM KH2PO4/K2HPO4 at pH
7.5, 2 mM EDTA, 0.1 mg/mL bovine serum albumin (BSA),
10 µM fluorogenic substrate Dabcyl-GLRTQSFS-Edans
(Bachem), 0.1 µM HAV 3C proteinase, and 1% DMF. Enzyme
was incubated with the appropriate inhibitor for 15 min and
reaction initiated by the addition of substrate. Increase in
fluorescence (λex 336 nm, λem 472 nm) was continuously
monitored using a Shimadzu RF5301 spectrofluorometer. For
proteinase inhibition studies, the initial 3 min of the reaction
were used for calculation purposes. Inhibitor stock solutions
were prepared at 10 mM in DMF and serial dilutions made in
DMF. At least five different inhibitor concentrations were
examined along with a control sample containing no inhibitor
under the conditions described above. The HAV 3C proteinase
activity in the presence of the specified inhibitor was expressed
as a percentage of that obtained from the respective control
samples. For inhibitors displaying dose-dependent inhibition
of the proteinase activity, IC50 values were determined from
plots of the relative proteinase activity versus the log of
inhibitor concentration. IC50 values were not determined for
compounds showing weak inhibition. The competitive inhibi-
tion constant (Ki) for compound 24 was determined from the
Dixon plot.33 The sensitivity of inhibitor 24 to DTT was
evaluated using reactions similar to those described above, but
with the addition of up to 5 mM molar concentrations of DTT
to the inhibitor-containing mixture followed by the addition
of enzyme.
(35) (a) Harris, J . M.; Bolessa, E. A.; Mendonca, A. J .; Feng, S.-C.;
Vederas, J . C. J . Chem. Soc., Perkin Trans. 1 1995, 1945-1950. (b)
Merkler, D. J .; Glufke, U.; Ritenour-Rodgers, K. J .; Baumgart, L. E.;
DeBlassio, J . L.; Merkler, K. A.; Vederas, J . C. J . Am. Chem. Soc. 1999,
121, 4904-4905.
(36) Bodansky, M. Peptide Chemistry; Springer, New York, 1988,
pp 55-146, and references therein.
(34) (a) Sengupta, S.; Das, D. Synth. Commun. 1998, 3, 403-408.
(b) For a review on organic synthesis with R-diazocarbonyl compounds
see: Ye, T.; McKervey, M. A. Chem. Rev. 1994, 94, 1091-1160.

Inhibitors of Hepatitis A Virus 3C Proteinase
J . Org. Chem., Vol. 67, No. 10, 2002 3175
(4S)-N,N-Dim et h yl-4-(ter t-b u t yloxyca r b on yla m in o)-
p en ta n oic Acid Ben zyl Ester (4). This compound was
prepared from a modified procedure of Matsoukas et al.37 To
a stirred solution of Boc-Glu-OBn (5.00 g, 14.82 mmol) in dry
dichloromethane (70 mL) at 0 °C was added triethylamine
(2.27 mL, 16.30 mmol) followed by ethyl chloroformate over a
period of 5 min. After stirring at 0 °C for 30 min, solid
dimethylamine hydrochloride (1.33 g, 16.83 mmol) and more
triethylamine (2.60 mL, 18.51 mmol) were added. The reaction
mixture was stirred at 0 °C for a further 2 h and then allowed
to stir overnight at room temperature. The solvent was
removed in vacuo and the residue diluted with water (60 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic
layers were washed with 1 N HCl (20 mL), dried over MgSO4,
and concentrated under reduced pressure. Recrystallization
of the crude product from dichloromethane/hexane gave white
further 2 h at 0 °C and then filtered through Celite and washed
successively with more DMF, water, and acetone. The filtrate
was evaporated in vacuo and purified by column chromatog-
raphy (SiO2, 5% MeOH/EtOAc) to yield a colorless oil (3.31 g,
91%); [R]26D +23.2° (c 1.0, CHCl3); IR (CHCl3 cast) 3295, 2878,
1733, 1707, 1634, 1505 cm-1 1H NMR (300 MHz, CDCl3) δ
;
1.40 (s, 9H), 1.92-2.00 (m, 1H), 2.12-2.25 (m, 1H), 2.30-2.51
(m, 2H), 2.92 (s, 3H), 2.98 (s, 3H), 4.10-4.19 (m, 1H), 5.60 (br
s, 1H), 9.48 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 24.2, 28.3,
28.7, 35.6, 37.2, 59.5, 79.9, 157.7, 171.5, 200.0; HRMS (EI)
calcd for C12H22N2O4 (M+) 258.1579, found 258.1578. Anal.
Calcd for C12H22N2O4: C, 55.80, H, 8.58, N, 10.84. Found C,
55.87, H, 8.69, N, 10.89.
(4S,5RS)-N,N-Dim eth yl-4-(ter t-bu tyloxycar bon ylam in o)-
5-cya n o-5-h yd r oxyp en ta n a m id e (8). Aldehyde 7 (1.32 g,
5.13 mmol) was dissolved in MeOH (60 mL), and to this
solution was added AcOH (0.44 mL, 7.70 mmol) followed by
potassium cyanide (0.5 g, 7.70 mmol). The reaction mixture
was stirred at room temperature for 18 h and then concen-
trated in vacuo. The residue was slurried with EtOAc (20 mL)
and filtered. The filtrate was concentrated and the crude
product purified by column chromatography (SiO2, 10% MeOH/
EtOAc) to afford a white solid which was recrystallized from
dichloromethane/hexane (1.03 g, 70%); mp 114-115 °C; IR
(CHCl3 cast) 3307, 2350, 1709, 1627 cm-1; 1H NMR (300 MHz,
CDCl3) (1:1 mixture of diasteromers, data for one compound)
δ 1.42 (s, 9H), 1.94-2.10 (m, 2H), 2.36-2.52 (m, 2H), 2.92 (s,
3H), 2.98 (s, 3H), 3.79-3.95 (m, 1H), 4.54 (d, J ) 9.0 Hz, 1H),
5.43 (d, J ) 7.4 Hz, 1H), 5.80-6.00 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 25.0, 36.0, 37.3, 38.3, 39.3, 39.6, 53.7, 54.6, 64.2, 65.2,
80.4, 80.6, 118.5, 118.7, 154.0, 156.7, 172.8; HRMS (EI) calcd
for C16H17N2O3 (M+) 285.1239, found 285.1231. Anal. Calcd for
needles (4.61 g, 85%); mp 97-98 °C, lit.37 mp 99-100 °C; [R]26
D
-25.8° (c 1.0, EtOH), lit.37 [R]20D -27.5° (c 1.0, MeOH); 1H NMR
(300 MHz, CDCl3) δ 1.40 (s, 9H), 1.92-2.02 (m, 1H), 2.11-
2.22 (m, 1H), 2.25-2.40 (m, 2H), 2.85 (s, 3H), 2.88 (s, 3H),
4.22-4.38 (m, 1H), 5.12 (d, J ) 13.2 Hz, 1H), 5.18 (d, J ) 12.9
Hz, 1H), 5.42 (br d, J ) 5.4 Hz, 1H), 7.25-7.29 (m, 5H); HRMS
(EI) calcd for C19H28N2O5 (M+) 364.1998, found 364.1999.
(4S)-N,N-Dim et h yl-4-(ter t-b u t yloxyca r b on yla m in o)-
p en ta n oic Acid (5). Glutamine 4 (2.95 g, 8.10 mmol) was
dissolved in MeOH (25 mL) in the presence of 10% Pd/C (300
mg, 10% w/w). The suspension was stirred under a hydrogen
atmosphere until the uptake of hydrogen ceased (approxi-
mately 6 h). Filtration through Celite, followed by removal of
the solvent in vacuo, afforded the title compound which was
recrystallized from dichloromethane/hexane to give white
crystalline solid (2.22 g, 90%); mp 121-122 °C, lit.38 mp 124-
125 °C; [R]26 +3.5° (c 1.0, EtOH); lit.38 [R]20 +2.2° (c 1.0,
C
16H17N2O3: C, 54.72, H, 8.12, N, 14.73. Found C, 54.44, H,
D
D
1
8.24, N, 14.42.
EtOH); H NMR (300 MHz, CDCl3) δ 1.40 (s, 9H), 1.95-2.04
(m, 1H), 2.21-2.29 (m, 1H), 2.42-2.82 (m, 1H), 2.70-2.84 (m,
1H), 2.95 (s, 3H), 3.05 (s, 3H), 4.15 (q, J ) 5.6 Hz, 1H), 5.65
(d, J ) 5.6 Hz, 1H); HRMS (EI) calcd for C12H22N2O5 (M+)
274.1529, found 274.1533.
(4S,5RS)-N,N-Dim eth yl-4-(ter t-bu tyloxycar bon ylam in o)-
6-a m in o-5-h yd r oxyh exa n a m id e Aceta te Sa lt (9). To a
solution of cyanohydrin 8 (55 mg, 0.19 mmol) in propan-2-ol
(10 mL) and AcOH (22 µL, 0.38 mmol) was added PtO2 (10
mg, 0.05 mmol). The mixture was agitated under a hydrogen
atmosphere at 50 psi for 24 h after which it was filtered
through Celite and washed through with more propan-2-ol.
Evaporation of the solvent in vacuo and purification of the
crude product by column chromatography (SiO2; CHCl3/MeOH/
AcOH 90:9:1) provided the title compound as a light brown
foam (28.9 mg, 43%). IR (CHCl3 cast) 3400, 2924, 1705, 1633
(4S)-N,N-Dim et h yl-4-ter t-b u t yloxyca r b on yla m in o-4-
(eth a n esu lfa n ylca r bon yl)bu tyr a m id e (6). This was pre-
pared by an adaptation of the Malcolm et al. procedure.12
A
solution of acid 5 (5.00 g, 18.25 mmol) in dry CH2Cl2 at 0 °C
was treated with triethylamine (17.98 mL, 129.56 mmol)
followed by ethyl chloroformate (6.29 mL, 65.69 mmol) over a
period of 15 min. Ethanethiol (5.53 mL, 74.82 mmol) and
DMAP (22.3 mg, 1.82 mmol) were added, and the reaction
mixture was stirred for a further 2 h at 0 °C. The reaction
was quenched by the addition of glacial acetic acid (5 mL),
and the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in dichloromethane (50
mL) and washed subsequently with 5% aq NaHCO3 (25 mL),
5% aq citric acid (25 mL), and brine (25 mL). The organic layer
was dried over MgSO4 and evaporated in vacuo. Recrystalli-
zation of the crude product from dichloromethane/hexane
furnished the desired product as an off-white crystalline solid
(4.49 g, 78%); mp 122-123 °C; [R]26D -14.6° (c 1.0, CHCl3); IR
(CHCl3 cast) 3214, 2979, 1710, 1674, 1618, 1539 cm-1; 1H NMR
(300 MHz, CDCl3) δ 1.22 (t, J ) 7.5 Hz, 3H), 1.42 (s, 9H), 1.95-
2.00 (m, 2H), 2.30-2.50 (m, 2H), 2.84 (q, J ) 7.5 Hz, 2H), 2.95
(s, 3H), 3.00 (s, 3H), 4.22-4.37 (m, 1H), 5.70 (d, J ) 7.5 Hz,
1H); 13C NMR (75 MHz, CDCl3) δ 27.5, 28.4, 29.4, 35.7, 37.2,
60.6, 80.0, 155.6, 172.1, 201.8; HRMS (EI) calcd for C14H26N2-
SO4 (M+) 318.1613, found 318.1604. Anal. Calcd for C14H26N2-
SO4: C, 52.81, H, 8.23, N, 8.79. Found C, 52.51, H, 8.37, N,
8.61.
cm-1 1H NMR (300 MHz, CDCl3) (1:1 mixture of diastereo-
;
mers, data for one compound) δ 1.42 (s, 9H), 1.74-2.08 (m,
5H), 2.38-2.50 (m, 2H), 2.92 (s, 3H), 2.98 (s, 3H), 3.00-3.15
(m, 0.5H), 3.21-3.32 (m, 0.5H), 3.42-3.68 (m, 1H), 3.92-4.05
(m, 0.5H), 4.08-4.19 (m, 0.5H), 5.72 (d, J ) 7.5 Hz, 1H), 6.05
(d, J ) 7.5 Hz, 1H), 7.00-7.40 (m, 3H); 13C NMR (75 MHz,
CDCl3) δ 27.9, 28.5, 29.5, 29.7, 35.8, 37.4, 43.1, 51.9, 69.3, 70.3,
75.8, 79.3, 156.7, 173.3, 173.5; HRMS (ES) calcd for C13H28N3O4
(M - CH3CO2+) 290.2079, found 290.2080. Anal. Calcd for
C
15H31N3O6: C, 51.56, H, 8.94, N, 12.03. Found C, 51.22, H,
8.63, N, 12.21.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
h yd r oxy-6-(4-p h en yl-bu tyr yla m in o)h exa n a m id e (10). A
solution of 4-phenylbutyric acid (56.8 mg, 0.35 mmol) in
dichloromethane (10 mL) at 0 °C was treated with triethyl-
amine (52 µL, 0.38 mmol) followed by dropwise addition of
ethyl chloroformate (33.1 µL, 0.35 mmol). The mixture was
stirred for a further 20 min and subsequently treated with
the amino alcohol 9 (110 mg, 0.32 mmol) and triethylamine
(52 µL, 0.38 mmol). The reaction mixture was allowed to warm
to room temperature over 3 h and quenched with saturated
aqueous NaHCO3 (10 mL) and extracted with EtOAc (3 × 5
mL). The combined organic layers were dried over MgSO4, and
evaporation of the solvent in vacuo followed by purification of
the crude product by column chromatography (SiO2, 1%
MeOH/EtOAc) afforded the desired compound as a colorless
oil (84.3 mg, 61%); IR (CHCl3 cast) 3323, 2929, 1694, 1633,
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxop en ta n a m id e (7). To a solution of thioester 6 (4.49 g,
14.15 mmol) in dry DMF (100 mL) at 0 °C was added
triethylsilane (13.53 mL, 84.90 mmol) followed by 10% Pd/C
(450 mg, 10% w/w). The reaction mixture was stirred for a
(37) Matsoukas, J .; Theodoropoulos, D. Org. Magn. Reson. 1979, 12,
393-395.
(38) Stahl, G. L.; Smith, C. W.; Walter, R.; Tsgenidis, T.; Stavropou-
los, G. J . Med. Chem. 1980, 2, 213-217.
1497 cm-1 1H NMR (300 MHz, CD2Cl2) (2:1 mixture of
;
diasteromers, data for major compound) δ 1.40 (s, 9H), 1.65-
2.10 (m, 4H), 2.15-2.50 (m, 4H), 2.65 (m, 2H), 2.90 (s, 3H),

3176 J . Org. Chem., Vol. 67, No. 10, 2002
Ramtohul et al.
2.96 (s, 3H), 3.10-3.30 (m, 1H), 4.40-4.65 (m, 3H), 4.35 (br s,
0.3H), 4.55 (br s, 0.7H), 5.15 (d, J ) 9.1 Hz, 0.7H), 5.50 (d, J
) 7.2 Hz, 0.3H), 6.58-6.02 (m, 0.7H), 6.84-6.87 (m, 0.3H),
7.10-7.35 (m, 5H); 13C NMR (75 MHz, CD2Cl2) δ 27.1, 27.6,
28.5, 29.7, 29.9, 35.6, 35.8, 36.1, 37.4, 42.9, 53.0, 71.4, 74.4,
79.5, 126.2, 128.6, 128.8, 142.3, 157.0, 173.3, 173.5, 173.7,
174.9; HRMS (ES) calcd for C23H37N3O5Na (MNa+) 458.2631,
found 458.2631.
mmol). The reaction mixture was stirred for 30 min, and the
mixture was filtered into an ethereal solution of diazomethane
(200 mL, ca. 91 mmol) at 0 °C. After stirring for a further 4 h,
the solvent was evaporated and the crude product recrystal-
lized from dichloromethane/hexane to give an off-white solid
(4.35 g, 80%); mp 112-113 °C, lit.13 mp 112-113 °C; [R]26
D
-1.3° (c 1.1, CHCl3), lit.13 [R]26D -1.7° (c 1.1, CHCl3); 1H NMR
(300 MHz, CDCl3) δ 1.40 (s, 9H), 1.75-1.90 (m, 1H), 2.02-
2.20 (m, 1H), 2.38 (dt, J ) 16.5, 6.0 Hz, 1H), 2.48 (dt, J )
16.5, 6.0 Hz, 1H), 2.90 (s, 3H), 2.98 (s, 3H), 4.11-4.19 (m, 1H),
5.55 (br s, 1H), 5.40 (d, J ) 5.8 Hz,1H); HRMS (ES) calcd for
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-(4-p h en ylbu tyr yla m in o)h exa n a m id e (11). To a solu-
tion of alcohol 10 (80 mg, 18.5 mmol) in dichloromethane (5
mL) at room temperature was added Dess-Martin periodinane
(199 mg, 46.2 mmol). The resulting mixture was stirred for 4
h after which it was quenched with 1.3 N NaOH (5 mL) and
stirred for a further 15 min. The mixture was diluted with
water (10 mL) and extracted with EtOAc (3 × 5 mL), and the
organic layers were dried over MgSO4. The solvent was
removed in vacuo, and the residual oil was purified by column
chromatography (SiO2, 5% MeOH/EtOAc) to provide the title
C
13H22N4O4Na (MNa+) 321.1539, found 321.1533.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-6-
br om o-5-oxoh exa n a m id e (15). This known compound was
prepared by a modification of the Morris13 procedure. A
solution of the diazo-ketone 14 (4.09 g, 13.7 mmol) in THF
(150 mL) at 0 °C was treated dropwise with aq 48% HBr (2.43
mL, 14.4 mmol). The reaction mixture was then stirred at 0
°C until the evolution of gas ceased and quenched with
saturated aqueous NaHCO3 (20 mL) and the solvent removed
in vacuo. The residue was diluted with H2O (50 mL) and
extracted with EtOAc (3 × 30 mL). The combined organic
layers were dried over MgSO4 and then concentrated under
reduced pressure. Purification of the crude product by column
chromatography (SiO2, 1:1 CHCl3/EtOAc) afforded the desired
product which was recrystallized from dichloromethane/hexane
to give a light yellow solid (743 mg, 81%); mp 59-60 °C, lit.13
compound as a colorless oil (61 mg, 76%); [R]26 +2.1° (c 1.4,
D
CHCl3); IR (CHCl3 cast) 3312, 2927, 1701, 1636, 1497 cm-1
;
1H NMR (300 MHz, CD2Cl2) δ 1.40 (s, 9H), 1.80-2.00 (m, 3H),
2.00-2.18 (m, 1H), 2.22 (t, J ) 8.2 Hz, 2H), 2.28-2.50 (m, 2H),
2.85 (t, J ) 7.3 Hz, 2H), 2.90 (s, 3H), 2.98 (s, 3H), 4.18-4.32
(m, 3H), 5.94-5.98 (m, 1H), 6.38-6.44 (m, 1H), 7.10-7.35 (m,
5H); 13C NMR (75 MHz, CD2Cl2) δ 26.5, 27.6, 28.4, 29.2, 35.5,
35.7, 35.8, 37.4, 47.2, 58.8, 80.1, 126.2, 128.7, 128.8, 142.2,
149.6, 156.1, 172.5, 173.3, 206.1; HRMS (ES) calcd for
mp 61 °C; [R]26 +21.4° (c 1.3, CHCl3); 1H NMR (300 MHz,
D
C
23H35N3O5Na (MNa+) 456.2474, found 456.2472.
CDCl3) δ 1.40 (s, 9H), 1.90-2.04 (m, 1H), 2.10-2.25 (m, 1H),
2.36 (dt, J ) 18.1, 5.7 Hz, 1H), 2.49 (dt, J ) 10.4, 5.9 Hz, 1H),
2.92 (s, 3H), 2.98 (s, 3H), 4.12 (d, J ) 13.5 Hz, 1H), 4.19 (d, J
) 13.5 Hz, 1H), 4.45-4.57 (m, 1H), 5.62-5.74 (m, 1H); HRMS
(ES) calcd for C13H24N2O479Br (MH+) 351.0914, found 351.0914.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-(N-p h th a lim id o)h exa n a m id e (16). To a solution of
bromoketone 15 (400 mg, 1.14 mmol) in DMF (10 mL) was
added potassium phthalimide (253 mg, 1.37 mmol). The
reaction mixture was heated at 60 °C for 6 h after which the
solvent was removed in vacuo and the residue diluted with
H2O (20 mL) and extracted with EtOAc (3 × 10 mL). The
combined organic layers were dried over MgSO4, and the
solvent was evaporated under reduced pressure. Purification
of the crude product by column chromatography (SiO2, 5%
MeOH/EtOAc) furnished the desired product which was
recrystallized from dichloromethane/diethyl ether to give a
(4S,5RS)-N,N-Dim eth yl-4-(ter t-bu tyloxycar bon ylam in o)-
6-(ben zyloxycar bon ylam in o)-5-h ydr oxyh exan am ide (12).
A solution of amino alcohol 9 (76.5 mg, 0.22 mmol) in H2O/
THF (2:1, 6 mL) was treated with NaHCO3 (64.4 mg, 0.77
mmol). After gas evolution ceased, benzyl chloroformate (34
µL, 0.24 mmol) was added, and stirring was continued
overnight at room temperature. The reaction mixture was
diluted with water (5 mL) and extracted with EtOAc (3 × 5
mL), and the combined organic layers were dried over MgSO4.
Evaporation of the solvent followed by purification of the crude
product by column chromatography (SiO2, 10% MeOH/EtOAc)
furnished the title compound as a colorless oil (79 mg, 85%);
1
IR (CHCl3 cast) 3332, 2932, 1706, 1651, 1632, 1575 cm-1; H
NMR (300 MHz, CDCl3) (1:1 mixture of diasteromers) δ 1.41
(s, 9H), 1.80-2.10 (m, 2H), 2.20-2.50 (m, 2H), 2.92 (s, 3H),
2.98 (s, 3H), 3.00-3.15 (m, 1H), 3.35-3.40 (m, 1H), 3.55-3.64
(m, 2H), 5.05 (d, J ) 8.1 Hz, 1H), 5.12 (d, J ) 7.3 Hz, 1H),
5.28-5.30 (m, 1H), 5.60-5.64 (m, 1H), 5.92 (br s, 1H), 7.25-
7.40 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 25.2, 26.5, 28.4,
29.6, 33.8, 35.8, 37.3, 37.3, 43.7, 52.5, 53.2, 66.7, 66.9, 70.5,
74.0, 79.6, 128.1, 128.5, 136.6, 136.7, 156.6, 156.9, 173.2;
HRMS (ES) calcd for C21H33N3O6Na (MNa+) 446.2267, found
446.2263.
white powder (351 mg, 74%); mp 127-128 °C; [R]26 -5.0° (c
D
1.0, CHCl3); IR (CHCl3 cast) 3289, 2930, 1745, 1720, 1623, 1524
cm-1 1H NMR (300 MHz, CDCl3) δ 1.42 (s, 9H), 1.92-2.05
;
(m, 1H), 2.21-2.30 (m, 1H), 2.36 (dt, J ) 16.5, 7.0 Hz, 1H),
2.51 (dt, J ) 15.7, 7.0 Hz), 2.94 (s, 3H), 2.98 (s, 3H), 4.39-
4.48 (m, 1H), 4.65 (d, J ) 18.4 Hz, 1H), 4.77 (d, J ) 18.1 Hz,
1H), 5.72-5.82 (m, 1H), 7.64-7.72 (m, 2H), 7.79-7.85 (m, 2H);
13C NMR (75 MHz, CDCl3) δ 26.6, 28.3, 28.8, 44.3, 44.6, 57.7,
80.1, 123.5, 132.2, 134.1, 155.8, 167.6, 172.2, 202.4; HRMS (ES)
calcd for C21H27N3O6Na (MNa+) 440.1798 found, 440.1794.
Anal. Calcd for C21H27N3O6: C, 60.42 H, 6.52, N, 10.07. Found
C, 60.68, H, 6.56, N, 10.08.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-6-
(ben zyloxyca r bon yla m in o)-5-oxoh exa n a m id e (13). This
was prepared by employing the same procedure as that
described for the preparation of compound 11. A solution of
alcohol 12 (68 mg, 0.16 mmol) in dichloromethane (4 mL) was
treated with Dess-Martin periodinane (103 mg, 0.24 mmol),
and the reaction mixture was stirred for 2 h. Purification by
column chromatography (SiO2, 5% MeOH/EtOAc) gave the title
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-p h en oxyh exa n a m id e (17). A solution of phenol (147
mg, 0.16 mmol) in DMF (5 mL) was treated with sodium
hydride (3.9 mg, 0.16 mmol) at room temperature. After 15
min, the phenoxide solution was cooled to 0 °C, and the
bromoketone 15 (50 mg, 0.14 mmol) was added in one portion.
The reaction mixture was stirred for a further 1 h at 0 °C and
then allowed to warm to room temperature over 3 h. The
reaction mixture was quenched with saturated aqueous NH4-
Cl (5 mL) and the solvent removed in vacuo. The residue
obtained was diluted with H2O (10 mL) and extracted with
EtOAc (3 × 10 mL). The combined organic layers were dried
over MgSO4, and evaporation of the solvent followed by
purification by column chromatography (SiO2, 30% hexane/
EtOAc) afforded the desired product as a colorless oil (361 mg,
69%); [R]26D +24.2° (c 0.6, CHCl3); IR (CHCl3 cast) 3304, 1745,
1703, 1634, 1599 cm-1; 1H NMR (300 MHz, CD2Cl2) δ 1.42 (s,
9H), 1.98 (m, 1H), 2.12-2.25 (m, 1H), 2.40 (dt, J ) 6.9, 5.9
product as a colorless oil (48.8 mg, 72%); [R]26 -2.6° (c 1.3,
D
CHCl3); IR (CHCl3 cast) 3318, 1712, 1635, 1507 cm-1; 1H NMR
(300 MHz, CD2Cl2) δ 1.41 (s, 9H), 1.82-1.98 (m, 1H), 2.01-
2.20 (m, 1H), 2.20-2.50 (m, 2H), 2.92 (s, 3H), 2.98 (s, 3H),
4.15-4.40 (m, 3H), 5.11 (s, 2H), 5.54 (br s, 1H), 5.90 (br s, 1H),
7.20-7.40 (m, 5H); 13C NMR (75 MHz, CD2Cl2) δ 26.4, 28.4,
29.2, 35.2, 48.7, 58.3, 67.1, 80.1, 128.3, 128.4, 128.8, 129.0,
129.1, 137.2, 156.1, 156.6, 172.5, 206.1; HRMS (ES) calcd for
C
21H31N3O6Na (MNa+) 444.2110, found 444.2114.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-d ia zoh exa n a m id e (14). This known compound was
prepared using the same literature procedure as that described
by Morris et al.13 To a cooled 0 °C solution of acid 5 (5.0 g,
18.2 mmol) in THF (120 mL) was added triethylamine (2.79
mL, 20 mmol) followed by ethyl chloroformate (1.94 mL, 20

Inhibitors of Hepatitis A Virus 3C Proteinase
J . Org. Chem., Vol. 67, No. 10, 2002 3177
Hz, 1H), 2.49 (dt, J ) 16.7, 6.1 Hz, 1H), 2.90 (s, 3H), 2.98 (s,
3H), 4.38-4.44 (1H, m), 4.85 (d, J ) 17.2 Hz, 1H), 4.92 (d, J
) 17.1 Hz, 1H), 5.95 (br d, J ) 5.3 Hz, 1H), 6.90 (d, J ) 6.6
28.3, 28.7, 35.9, 37.5, 56.9, 69.1, 80.3, 115.9, 127.4, 130.0, 130.5,
132.5, 146.0, 146.3, 155.7, 158.1, 173.0, 202.5; HRMS (ES)
calcd for C21H27N5O8Na (MNa+) 500.1757, found 500.1763.
Hz, 1H), 7.00 (t, J ) 7.3 Hz, 2H), 7.30 (t, J ) 7.5 Hz, 2H); 13
C
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-6-
h yd r oxy-5-oxoh exa n a m id e (22). Diazoketone 14 (2.5 g, 8.39
mmol) was dissolved in THF (50 mL) and cooled to 0 °C. After
5 min, 1 N HCl (21 mL, 16.8 mmol) was added dropwise until
evolution of gas ceased. The reaction mixture was stirred for
5 h at 0 °C after which it was carefully quenched with
saturated aqueous NaHCO3 (10 mL). The solvent was removed
in vacuo and the residue diluted with H2O (30 mL) and
extracted with EtOAc (3 × 15 mL). The combined organic
layers were dried over MgSO4, and evaporation of the solvent
followed by purification by column chromatography (SiO2, 5%
MeOH/EtOAc) furnished the desired product as a white solid
(1.93 g, 80%) after recrystallization from dichloromethane/
NMR (75 MHz, CD2Cl2) δ 26.2, 28.2, 29.2, 35.5, 37.2, 57.4, 71.2,
80.0, 114.8, 121.7, 129.7, 158.1, 172.3, 205.7; HRMS (ES) cacld
for C19H29N2O5 (MH+) 365.2076, found 365.2075.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-(2,3-dih ydr oph th alazin e-1,4-dion e)h exan am ide (18)
a n d 2,3-Bis[(4S)-N,N-d im eth yl-4-(ter t-bu tyloxyca r bon yl-
a m in o)-5-oxo-6-m eth ylh exa n a m id e]-2,3-d ih yd r op h th a la -
zin e-1,4-d ion e (19). To a suspension of sodium phthal-
hydrazide (58 mg, 0.31 mmol) in DMF was added the
bromoketone 15 (100 mg, 0.28 mmol) in small portions over 1
h. After stirring for 5 h at room temperature, the solvent was
removed in vacuo and the residue diluted with H2O (10 mL)
and extracted with EtOAc (3 × 10 mL). The combined organic
layers were dried over MgSO4, and evaporation of the solvent
followed by purification by column chromatography (SiO2, with
a gradient elution of 20% EtOAc/hexane to 100% EtOAc)
yielded 18 as a white powder (42 mg, 52%) after recrystalli-
zation from dichloromethane/diethyl ether and dimer 19 as
an oil (59 mg, 29%).
hexane; mp 118-119 °C; [R]26 +21.0° (c 1.0, CHCl3); IR
D
(microscope) 3277, 3011, 1720, 1697, 1604, 1518 cm-1; 1H NMR
(300 MHz, CDCl3) δ 1.40 (s, 9H), 1.89-2.00 (m, 1H), 2.08-
2.41 (m, 2H), 2.28-2.54 (m, 3H), 2.92 (s, 3H), 2.98 (s, 3H),
4.30-4.39 (m, 1H), 4.38 (d, J ) 17.3 Hz, 1H), 4.43 (d, J ) 19.1
Hz, 1H), 5.85 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.6,
28.3, 28.9, 35.8, 37.2, 56.8, 66.5, 80.1, 155.8, 172.2, 209.9;
HRMS (EI) calcd for C13H24N2O5 (M+) 288.1685, found 288.1683.
Da ta for 18: mp 153-154 °C; [R]26 -8.2° (c 1.0, MeOH);
D
IR (microscope) 3273, 1735, 1702, 1650, 1618 cm-1; H NMR
1
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-(1,2-dim eth oxycar bon ylh ydr azin o)h exan am ide (23).
To a solution of triphenylphosphine (182 mg, 0.69 mmol) in
THF at -78 °C was added dimethyl azodicarboxylate (95 µL,
0.86 mmol) over 5 min. After 30 min of stirring at -78 °C,
hydroxyketone 22 (100 mg, 0.35 mmol) was added, and the
reaction mixture was slowly allowed to warm to room-
temperature overnight. The solvent was removed in vacuo and
the residue purified by column chromatography (SiO2, gradient
elution of 3:1 hexane/EtOAc to 1:1 hexane/EtOAc) to afford
(300 MHz, CDCl3) δ 1.40 (s, 9H), 2.00-2.40 (m, 2H), 2.42-
2.58 (m, 2H), 2.92 (s, 3H), 2.98 (s, 3H), 4.42-4.57 (m, 1H), 5.08
(d, J ) 16.7 Hz, 1H), 5.18 (d, J ) 16.7 Hz, 1H), 5.80 (d, J )
5.8 Hz, 1H), 7.70-7.85 (m, 2H), 8.05 (d, J ) 7.3 Hz, 1H), 8.40
(d, J ) 6.7 Hz, 1H), 10.70 (br s, 1H); 13C NMR (75 MHz, CDCl3)
δ 26.7, 28.4, 28.7, 35.8, 37.4, 57.1, 68.6, 80.2, 123.9, 124.7,
127.0, 129.0, 132.2, 133.5, 149.6, 155.8, 159.9, 172.4, 203.8;
HRMS (ES) calcd for C21H29N4O6 (MH+) 433.2087, found
433.2083. Anal. Calcd for C21H28N4O6: C, 58.32 H, 6.53, N,
12.96. Found C, 58.03, H, 6.49, N, 12.69.
23 as a pale yellow oil (29.6 mg, 20%); [R]26 +3.8° (c 0.5,
Da ta for d im er 19: [R]26D -20.8° (c 1.8, CHCl3); IR (CHCl3
D
CHCl3); IR (CH2Cl2) 3280, 2958, 1740, 1712, 1631, 1506 cm-1
;
1
cast) 3288, 1740, 1706, 1632, 1592 cm-1; H NMR (300 MHz,
1H NMR (300 MHz, CDCl3) (6:1 mixture of conformers) δ 1.40
(s, 9H), 1.89-2.01 (m, 1H), 2.05-2.20 (m, 1H), 2.35 (dt, J )
16.5, 6.3 Hz, 1H), 2.49 (dt, J ) 16.5, 6.6 Hz, 1H), 2.92 (s, 3H),
2.98 (s, 3H), 3.72-3.90 (m, 6H), 4.28-4.37 (br m, 1H), 4.86 (d,
J ) 17.3 Hz, 1H), 4.95 (d, J ) 17.3 Hz, 1H), 5.75 (d, J ) 17.3
Hz, 1H);13C NMR (75 MHz, CDCl3) δ 26.4, 28.3, 28.9, 35.8,
37.3, 55.3, 59.9, 69.3, 155.4, 162.4, 172.4, 203.0; HRMS (ES)
calcd for C17H30N4O8Na (MNa+) 441.1961, found 441.1953.
CD2Cl2) δ 1.42 (s, 18H), 1.82-2.02 (m, 2H), 2.20-2.38 (m, 2H),
2.40-2.55 (m, 4H), 2.96 (s, 6H), 3.00 (s, 6H), 4.20-4.41 (m,
2H), 4.92-5.21(m, 4H), 6.28 (br d, J ) 2.2 Hz, 1H), 6.47 (br d,
J ) 1.9 Hz, 1H), 7.82 (dt, J ) 14.2, 6.6 Hz, 2H), 8.06 (d, J )
7.2 Hz, 1H), 8.32 (d, J ) 7.2 Hz, 1H); 13C NMR (75 MHz, CD2-
Cl2) δ 26.6, 27.2, 28.4, 29.3, 29.8, 35.8, 35.9, 37.4, 37.5, 57.2,
58.1, 58.6, 68.7, 80.0, 80.1, 124.0, 124.8, 127.4, 129.1, 132.7,
133.6, 149.2, 156.3, 159.2, 172.8, 204.2, 204.4; HRMS (ES)
calcd for C34H51N6O10 (MH+) 703.3666, found 703.3663.
(4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r bon yla m in o)-5-
oxo-6-(5-n itr o-2,3-d ih yd r o-p h th a la zin e-1,4-d ion e)h exa n -
a m id e (20) a n d (4S)-N,N-Dim eth yl-4-(ter t-bu tyloxyca r -
b on yla m in o)-5-oxo-6-(8-n it r o-2,3-d ih yd r op h t h a la zin e-
1,4-d ion e)h exa n a m id e (21). A similar procedure was em-
ployed as that described for the preparation of 18. Sodium
3-nitrophthalhydrazide (72 mg, 0.31 mmol) was added to the
bromo-ketone 15 (100 mg, 0.28 mmol) in DMF (10 mL) over 1
h and allowed to stir for 5 h. Purification by column chroma-
tography (SiO2, with a gradient of 20% EtOAc/hexane to 100%
EtOAc) furnished compounds 20 (46 mg, 34%) and 21 (22 mg,
16%) as light yellow foams.
(4S)-N,N-Dim eth yl-4-(a cetyl-L-leu cyl-L-a la n yl-L-a la n yl)-
a m in o-5-oxo-6-(2,3-d ih yd r o-p h t h a la zin e-1,4-d ion e)h ex-
a n a m id e (24). Trifluroacetic acid (2 mL) was added to a
solution of 18 (65 mg, 0.12 mmol) in dichloromethane (2 mL)
at 0 °C. After 1.5 h, the reaction mixture was concentrated in
vacuo and the residue triturated with diethyl ether to yield
the trifuoroacetate salt as
a light brown foam (60 mg,
quantitative). The salt obtained was used in the next step
without any further purification. To a solution of Ac-Leu-Ala-
Ala-OH (64 mg, 0.15 mmol) in DMF (8 mL) at room temper-
ature was added DIPEA (51 µM, 0.29 mmol) followed by HBTU
(59 mg, 0.15 mmol). The mixture was stirred for 30 min after
which it was treated with a solution of the trifluoroacetate salt
(60 mg, 0.13 mmol) in DMF (2 mL). After 6 h of stirring, the
solvent was removed in vacuo and the crude product purified
by HPLC (linear gradient elution over 20 min of 0 to 40% of
acetonitrile in 0.1% TFA/H2O, tR 16.6 min) to afford the title
product as a white powder after lyophilization (59 mg, 70%);
Da ta for isom er 20: [R]26 +2.1° (c 2.8, CHCl3); IR
D
(microscope) 3224, 1739, 1671, 1623, 1601 cm-1; 1H NMR (300
MHz, CDCl3) δ 1.42 (s, 9H), 1.98-2.12 (m, 1H), 2.24-2.60 (m,
3H), 2.92 (s, 3H), 2.98 (s, 3H), 4.40-4.51 (m, 1H), 5.09 (d, J )
16.8 Hz, 1H), 5.23 (d, J ) 16.6 Hz, 1H), 5.78-5.80 (m, 1H),
7.72 (d, J ) 7.0 Hz, 1H), 7.92 (t, J ) 7.9 Hz, 1H), 8.23 (d, J )
7.3 Hz, 1H), 11.04 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.4,
28.3, 28.7, 35.9, 37.5, 57.2, 69.0, 80.3, 119.6, 125.7, 126.0, 126.7,
134.1, 148.6, 148.9, 156.0, 156.4, 173.0, 203.4; HRMS (ES)
calcd for C21H27N5O8Na (MNa+) 500.1757, found 500.1752.
m.p 88-90 °C; [R]26 -60.0° (c 0.3, CHCl3); IR (CHCl3, cast)
D
3279, 2958, 1740, 1653, 1600, 1540 cm-1; 1H NMR (300 MHz,
CD3OD) (1:1 mixture of conformers) δ 0.88 (d, J ) 5.7 Hz, 3H),
0.92 (d, J ) 6.4 Hz, 3H) 1.28-1.44 (m, 6H), 1.45-1.70 (m, 3H),
1.85-2.08 (m, 4H), 2.24-2.58 (m, 3H), 2.92 (s, 3H), 3.05 (s,
3H), 3.10-3.20 (q, J ) 7.2 Hz, 1H), 3.20-4.40 (m, 2H), 4.60
(dt, J ) 9.6, 4.9 Hz, 1H), 5.15 (d, J ) 16.9 Hz, 1H), 5.23 (d, J
) 12.2 Hz, 1H), 7.93 (dt, J ) 14.9, 7.5 Hz, 2H), 8.12 (d, J )
7.8 Hz, 1H), 8.32 (d, J ) 6.7 Hz, 1H); 13C NMR (125 MHz,
CD3OD) δ 17.5, 17.7, 21.8, 22.0, 23.3, 23.4, 25.9, 26.6, 26.8,
30.1, 35.8, 37.7, 41.7, 50.7, 50.9, 51.2, 53.3, 54.0, 57.2, 69.9,
125.0, 126.0, 127.4, 129.9, 133.5, 134.9, 151.4, 161.7, 174.6,
Da ta for isom er 21: [R]26 +158.8° (c 0.6, CHCl3); IR
D
(CHCl3 cast) 3183, 1741, 1672, 1630, 1599 cm-1; 1H NMR (300
MHz, CDCl3) δ 1.42 (s, 9H), 1.98-2.17 (m, 1H), 2.22-2.38 (m,
2H), 2.42-2.60 (m, 1H), 2.92 (s, 3H), 2.98 (s, 3H), 4.40-4.52
(m, 1H), 4.92 (d, J ) 16.5 Hz, 1H), 5.12 (d, J ) 16.7 Hz, 1H),
5.61 (d, J ) 4.6 Hz, 1H), 7.81-7.92 (m, 2H), 8.58 (dd, J ) 14.2,
1.9 Hz, 1H), 11.04 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.8,

3178 J . Org. Chem., Vol. 67, No. 10, 2002
Ramtohul et al.
174.9, 175.3, 175.5, 204.5; HRMS (ES) calcd for C30H43N7O8-
Na (MNa+) 652.3071, found 652.3068.
CN) δ 23.0, 29.9, 30.1, 35.9, 37.6, 54.2, 65.4, 68.3, 174.2; HRMS
(ES) calcd for C8H17N2O2 (M-CF3CO2+) 173.1290, found
173.1286.
(2S)-2-(N,N-Dim eth ylpr opan am id-3-yl)-1-(ter t-bu tyloxy-
ca r bon yl)a zetid in -3-on e (25) a n d (4S)-4-(N,N-Dim eth yl-
p r op a n a m id -3-yl)-[1,3]-oxa zin a n e-2,5-d ion e (26). A solu-
tion of diazoketone 14 (800 mg, 2.7 mmol) in benzene (50 mL)
was added dropwise over 2 h to a refluxing solution of
rhodium(II) acetate dimer (2.22 mg, 0.027 mmol) in benzene
(50 mL). The mixture was heated under reflux for a further 1
h at which time the color changed from green to pink. The
solvent was evaporated and the residue diluted with EtOAc
(30 mL) and filtered through a pad of Celite. Concentration of
the filtrate followed by purification of the crude product by
column chromatography (SiO2, 5% MeOH/EtOAc) afforded 25
(378 mg, 52%) and 26 (99 mg, 17%) as pale yellow oils.
(2S )-2-(N ,N -Dim e t h yl-p r op a n a m id -3-yl)-1-(a ce t yl-L-
leu cyl-L-a la n yl-L-a la n yl)a zet id in -3-on e (28). This com-
pound was prepared using the same coupling procedure as that
described for the preparation of 24. Ac-Leu-Ala-Ala-OH (95
mg, 0.30 mmol) in DMF (8 mL) at room temperature was
treated with DIPEA (126 µM, 0.72 mmol) followed by HBTU
(120 mg, 0.32 mmol). The trifluoroacetate salt 27 (95 mg, 0.33
mmol) in DMF (2 mL) was added. After 6 h of stirring, the
solvent was removed in vacuo and the crude product purified
by HPLC (linear gradient elution over 30 min of 5 to 50%
acetonitrile in 0.1% TFA/H2O, tR 13.9 min.) to afford the
peptidyl alcohol as an oil (87 mg, 58%); IR (CHCl3, cast) 3286,
2986, 2852, 1632, 1537 cm-1; 1H NMR (300 MHz, CD3OD) (3:2
mixture of diastereomers and conformers) δ 0.92 (d, J ) 4.0
Hz, 3H), 0.95 (d, J ) 6.5 Hz, 3H), 1.21-1.29 (m, 6H), 1.52-
1.60 (m, 2H), 1.61-1.78 (m, 2H), 1.98 (s, 3H), 2.00 (s, 3H), 2.10
(dt, J ) 14.7, 7.3 Hz, 1H), 2.22-2.40 (m, 1H), 2.48-2.60 (m,
2H), 2.94 (s, 3H), 3.05 (s, 3H), 3.65 (ddd, J ) 25.9, 10.5, 4.4
Hz, 1H), 3.98-4.10 (m, 1H), 4.22-4.42 (m, 4H), 4.52-4.70 (m,
2H); HRMS (ES) calcd for C22H39N5O6Na (MNa+) 492.2798,
found 492.2791. The alcohol was oxidized using a similar
procedure as that described for the preparation of 11. Dess-
Martin periodinane (206 mg, 0.48 mmol) was added to a
solution of the alcohol (75 mg, 0.16 mmol) in DMF (5 mL).
After stirring for 6 h at room temperature, the solvent was
removed in vacuo and the crude product purified by HPLC
(linear gradient elution over 30 min of 5 to 50% acetonitrile
in 0.1% TFA/H2O, tR 16.9 min) to afford the title product as a
white powder after lyophilization (53 mg, 71%); m.p 56-58
Da ta for 25: [R]26 +28.7° (c 1.2, CHCl3); IR (CHCl3 cast)
D
2927, 1822, 1701, 1640, 1456 cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.40 (s, 9H), 2.13 (q, J ) 7.1 Hz, 2H), 2.34-2.55 (m, 2H),
2.90 (s, 3H), 2.96 (s, 3H), 4.50 (dd, J ) 16.8, 4.2 Hz, 1H), 4.65
(d, J ) 16.6 Hz, 1H), 4.95 (dt, J ) 9.3, 4.2 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 25.9, 28.2, 28.4, 35.4, 37.1, 69.1, 80.9, 82.1,
156.5, 171.5, 200.1; HRMS (EI) calcd for C13H22N2O4 (M+)
270.1579, found 270.1586.
Da ta for 26: [R]26 -2.4 (c 0.4, MeOH); IR (CHCl3 cast)
D
3268, 2927, 1715, 1625, 1503 cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.98-2.11 (m, 1H), 2.18-2.32 (m, 1H), 2.46-2.52 (m, 2H),
2.92 (s, 3H), 2.98 (s, 3H), 3.92 (dd, J ) 5.2, 0.9 Hz, 1H), 4.55
(d, J ) 17.5 Hz, 1H), 4.61 (d, J ) 17.5 Hz, 1H), 6.95 (br s, 1H);
13C NMR (125 MHz, CDCl3) δ 25.2, 29.4, 35.8, 37.8, 59.2, 71.6,
154.7, 172.0, 203.4; HRMS (ES) calcd for C9H14N2O4Na (MNa+)
237.0851, found 237.0854.
(2S)-2-(N,N-Dim eth ylp r op a n a m id -3-yl)-3-h yd r oxya ze-
tid in e Tr iflu or oa ceta te sa lt (27). To a solution of ketone
25 (258 mg, 0.95 mmol) in EtOH (10 mL) at 0 °C was added
NaBH4 (180 mg, 4.77 mmol). The mixture was stirred for 4 h
after which it was quenched with saturated aqueous citric acid
(2 mL). The solvent was evaporated and the residue obtained
was diluted with water (15 mL). The aqueous layer was
extracted with EtOAc (3 × 5 mL), and the combined organic
layers were dried over MgSO4. Evaporation of the solvent
followed by purification of the crude product by column
chromatography (SiO2, 5% MeOH/EtOAc) furnished the alco-
hol as a colorless oil (187 mg, 72%); IR (CHCl3 cast) 3356, 2917,
°C; [R]26 -29.5° (c 0.2, CHCl3); IR (CHCl3, cast) 3286, 2958,
D
1826, 1779, 1641, 1539 cm-1; 1H NMR (300 MHz, CD3CN) (1:1
mixture of conformers) δ 0.87 (d, J ) 6.5 Hz, 3H), 0.92 (d, J )
6.5 Hz, 3H), 1.20-1.32 (m, 6H), 1.50 (t, J ) 7.0 Hz, 2H), 1.58-
1.72 (dq, J ) 8.2, 5.5 Hz, 1H), 1.98 (s, 3H), 2.05-2.12 (m, 2H),
2.45-2.62 (m, 2H), 2.88 (s, 3H), 2.98 (s, 3H), 4.15-4.44 (m,
3H), 4.65-5.12 (m, 3H), 7.05-7.25 (m, 3H); 13C NMR (75 MHz,
CD3CN) δ 17.9, 21.9, 22.1, 22.8, 23.0, 23.3, 25.5, 25.9, 29.3,
35.7, 37.5, 40.9, 41.3, 48.0, 49.8, 53.7, 71.1, 82.7, 173.2, 173.7,
200.6; HRMS (ES) calcd for C22H37N5O6Na (MNa+) 490.2642,
found 490.2642.
1685, 1629, 1455 cm-1 1H NMR (300 MHz, CDCl3) (1:1
;
mixture of diastereomers) δ 1.40 (s, 9H), 1.94-2.05 (m, 1H),
2.20-2.42 (m, 2H), 2.50-2.60 (m, 1H), 2.92 (s, 3H), 3.00 (s,
3H), 3.60 (dd, J ) 10.4, 3.7 Hz, 1H), 3.95-4.12 (m, 2H), 4.38-
4.48 (m, 1H), 5.20 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 21.6,
22.0, 28.1, 28.4, 30.5, 35.7, 37.3, 56.5, 62.9, 67.8, 79.3, 156.8,
173.5; HRMS (ES) calcd for C13H24N2O4Na (MNa+) 295.1634,
found 295.1633. The Boc protecting group was cleaved using
a similar procedure as that described for the preparation of
24. Trifluroacetic acid (2 mL) was added to a solution of the
alcohol (127 mg, 0.47 mmol) in dichloromethane (2 mL) at 0
°C. After 1.5 h, the reaction mixture was concentrated in vacuo,
and the trifluoroacetate salt 27 was obtained as an oil (120
mg, 90%). This was used in the next step without any further
purification. An analytical sample was purified by HPLC
(linear gradient elution over 15 min. of 5 to 60% acetonitrile
in 0.1% TFA/H2O, tR 4.3 min.) IR (CHCl3 cast) 3286, 2924,
Ack n ow led gm en t. The authors thank Drs. Bruce
Malcolm (Shering Plough), David Wishart, Scott Watson
(Faculty of Pharmacy, University of Alberta), Ernst
Bergmann, and Craig Garen (Biochemistry Department,
University of Alberta) for enzyme preparation and
helpful suggestions. This work was supported by the
U.S. National Institutes of Health (Grant AI 38249), the
Natural Sciences and Engineering Research Council of
Canada, and the Alberta Heritage Foundation for Medi-
cal Research (Scholarship to Y. K. R.). The viral particles
are pictures of mengovirus (PDB 2MEV) from Science
(Luo, H. et al. Science 1987, 235, 182): GRASP virus
image by Dr. J ean-Yves Sgro, University of Wisconsin
at Madison.
1632, 1537, 1461 cm-1 1H NMR (300 MHz, CD3OD) (2:1
;
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of selected compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
mixture of diastereomers) δ 2.09 (m, 2H), 2.40-2.69 (m, 2H),
2.92 (s, 3H), 3.02 (s, 3H), 3.75 (dd, J ) 11.3, 4.7 Hz, 1H), 4.18
(dd, J ) 11.0, 6.5 Hz, 1H), 4.43 (dd, J ) 13.0, 6.0 Hz, 1H),
4.64 (ddd, J ) 11.2, 6.6, 4.8 Hz, 1H);13C NMR (125 MHz, CD3-
J O0157831