Discovery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-3- [2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-6, 6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a selective, potent, orally bioavailable hepatitis C virus NS3 protease inhibitor: A potential therapeutic agent for the treatment of hepatitis C infection

Article abstract of DOI:10.1021/jm060325b

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-α or polyethylene glycol (PEG)-interferon-α alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.

Full text of DOI:10.1021/jm060325b

6074
J. Med. Chem. 2006, 49, 6074-6086
Discovery of (1R,5S)-N-[3-Amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-
3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-
6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a Selective, Potent,
Orally Bioavailable Hepatitis C Virus NS3 Protease Inhibitor: A Potential Therapeutic Agent
for the Treatment of Hepatitis C Infection
Srikanth Venkatraman,* Ste´phane L. Bogen, Ashok Arasappan, Frank Bennett, Kevin Chen, Edwin Jao, Yi-Tsung Liu,
Raymond Lovey, Siska Hendrata, Yuhua Huang, Weidong Pan, Tejal Parekh, Patrick Pinto, Veljko Popov, Russel Pike,
Sumei Ruan, Bama Santhanam, Bancha Vibulbhan, Wanli Wu, Weiying Yang, Jianshe Kong, Xiang Liang, Jesse Wong,
Rong Liu, Nancy Butkiewicz, Robert Chase, Andrea Hart, Sony Agrawal, Paul Ingravallo, John Pichardo, Rong Kong,
Bahige Baroudy, Bruce Malcolm, Zhuyan Guo, Andrew Prongay, Vincent Madison, Lisa Broske, Xiaoming Cui,
Kuo-Chi Cheng, Yunsheng Hsieh, Jean-Marc Brisson, Danial Prelusky, Walter Korfmacher, Ronald White,
Susan Bogdanowich-Knipp, Anastasia Pavlovsky, Prudence Bradley, Anil K. Saksena, Ashit Ganguly, John Piwinski,
Viyyoor Girijavallabhan, and F. George Njoroge
Schering Plough Research Institute, K-15, 2015 Galloping Hill Road, Kenilworth New Jersey 07033
ReceiVed March 21, 2006
Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and
hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only
therapeutic regimens are subcutaneous interferon-R or polyethylene glycol (PEG)-interferon-R alone or in
combination with oral ribavirin. Although combination therapy is reasonably successful with the majority
of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only
about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery
of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been
advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described.
X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.
Introduction
replication. This has been a field of intense investigation by
various groups worldwide.6 BILN-2061 and VX-950 are two
novel protease inhibitors that have been advanced into clinical
studies in humans and demonstrated to be efficacious.7 The
X-ray crystal structure of the enzyme reveals a featureless,
shallow, highly solvent exposed active site located near the
surface in a cleft between two â-barrel subdomains.8 Charac-
teristic residues of the catalytic triad, histidine-57, and aspartic
acid-81 are located in the N-terminal, whereas serine-139 forms
part of the C-terminal subdomain. Cysteine is conserved in the
P1 position of the natural substrate of NS3 protease in all three
trans-cleavage sites and is replaced by threonine in the cis-
cleavage event. The P1′ is a small hydrophobic amino acid, either
a serine or alanine. The P2, P3, and P4 sites are hydrophobic
amino acids, and P5 and P6 usually contain acidic amino acids
such as aspartic acid or glutamic acid.
An estimated 170 million people worldwide are infected with
hepatitis C virus (HCV), making it an impending public threat
that leads to liver cirrhosis, carcinoma, or liver failure.1 The
slow progression of the disease in combination with mild
symptoms has made early detection difficult. Pegylated R-in-
terferon alone or in combination with ribavirin is the preferred
treatment for HCV viral infection.2 Although 80% of genotype-
2-infected patients respond initially, only 40% of genotype-1-
infected patients show sustained response to interferon treatment.
Lack of effective methods to treat genotype-1 HCV infections
and patients relapsing from interferon therapy necessitate
discovery of new drugs. Significant efforts are now directed
toward development of therapies that target key enzymes vital
to HCV replication and maturation.3
Hepatitis C virus is a positive strand RNA with a single open
frame of ∼9600 nucleotides. It encodes a single polypeptide of
3000 amino acids that is post-translationally modified to produce
mature virions.4 The single polypeptide contains all the structural
and nonstructural proteins: C-E1-E2-P7-NS2-NS3-NS4A-NS4B-
NS5A-NS5B. NS3, a trypsin-like serine protease, catalyzes cis
cleavage of the NS3-NS4A junction, followed by trans cleavage
of the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B to
produce functional proteins.5 The central role played by NS3
protease in the development of mature hepatitis C virus makes
it an excellent target for drug discovery. Development of small-
molecule inhibitors for this enzyme would potentially arrest the
processing of the aforementioned polyprotein required for viral
Strategy and Initial Leads
We initially screened numerous compound libraries in an
effort to identify a potential lead candidate that could be
appropriately modified into a “druglike” entity. Our initial efforts
failed to generate potential leads; thus, we resorted to structure-
based drug design. We envisioned trapping the catalytic serine
with conventional electrophiles such as aldehydes, ketones,
trifluoromethyl ketones, and ketoamides. Most of these traps
did not impart desirable activity with the inhibitors evaluated.
A ketoamide containing undecapeptide 1 that spanned from P6
to P5′ (Figure 1), a mixture of diasteromers at P1, had an
excellent activity of Ki* ) 1.9 nM in the inhibition of NS3
protease and thus became our starting point for drug
development.
* To whom correspondence should be addressed. Phone: 908-740-3758.
Fax: 908-740-7152. E-mail: Srikanth.Venkatraman@spcorp.com.
10.1021/jm060325b CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/07/2006

Carboxamide for Treatment of Hepatitus C
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6075
Figure 1. Truncation of lead undecapeptide 1 to P3-P2′ derived pentapeptide inhibitors.
ylcyclopropylproline motif at the P2 position resulted in
Inhibitor 1, though an excellent inhibitor of HCV NS3
protease, lacked selectivity against human neutrophil elastase
(HNE), a structurally related serine protease. Moreover, with
such a large number of peptidic bonds in the molecule, the
compound was bound to have poor pharmacokinetics (PK). It
was therefore necessary to modify this compound by incorporat-
ing appropriate properties that would render it druglike.
Replacement of C-terminal tetrapeptide methionine-serine-
tyrosine-serine from P2′-P5′ with -NH2 yielded truncated
compound 2 with a Ki* ) 0.043 µM. Although this truncation
resulted in a 25-fold loss in potency in comparison to 1, the
molecular weight of the resultant inhibitor had been drastically
reduced by half. Side chain modification and introduction of
various amino acids at P sites of inhibitor 1 pointed to
cyclohexylglycine as an excellent P3 and to leucine as a desirable
P2 residue that provided potent inhibitors. Parallel results from
our laboratories also showed that introduction of phenylglycine
amide at P2′ yielded P3 truncated inhibitors of type 3, which
were equipotent to inhibitor 2.9 Although P2 leucine containing
inhibitors of type 3 were very potent in an enzyme inhibition
assay, they lacked activity in cellular assay.10 However, P2
proline containing inhibitors displayed good enzyme binding
and moderate cellular potency in a replicon-based assay.11
Comparing inhibitors 2 and 3, we envisioned introducing a
nonproteinogenic amino acid 3, 4-dimethylcyclopropylproline
as a P2 residue because it had previously been established to
mimic constrained leucine.12 Incorporation of the 3,4-dimeth-
compound 4 with a Ki* of 0.010 µM and EC90(rep) of 0.2 µM.
Despite their good potencies, compounds of type 4 did not have
appreciable oral bioavailability in rats, dogs, and monkeys.11
To obtain potent compounds with improved PK, we decided
to modify 4 by varying side chain residues at P1′, P3, P1, and
P3-capping sites. We now report herein our efforts toward
modification of compound 4 through extensive truncations and
the discovery of 70 as a selective, potent HCV NS3 protease
inhibitor with oral bioavailability, a potential therapeutic agent
for the treatment of HCV infections.
Chemistry
Syntheses of P1 fragments were accomplished following the
general methods shown in Schemes 1-3. As outlined in Scheme
1, method A using cyanohydrin chemistry was employed for
the syntheses of P1 segments where the corresponding amino
acids were readily available or easily synthesized. Thus,
alkylation of diphenylimine protected glycine derivative 5 with
appropriate alkyl halide followed by hydrolysis and Boc
protection yielded amino esters of type 7. The corresponding
N-Boc protected amino acids were converted to the aldehyde 8
by reduction of the intermediate Wienreb amide using LiAlH4.13
These aldehydes were converted to cyanohydrins of type 9 by
treatment with acetone cyanohydrin and triethylamine.14 Inter-
mediates 9 were hydrolyzed using aqueous or methanolic HCl
to the corresponding hydroxy acids or hydroxy esters, which

6076 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Scheme 1. Cyanohydrin Route: Method Aa
Venkatraman et al.
Scheme 3. Aminohydroxylation Reaction: Method Ca
a Reagents and conditions: (a) (EtO)2P(O)CH2COOtBu, Et3N; (b)
t
(DHQ)2Phal, Cbz-NH2, BuOCl, aqueous NaOH; (c) (i) TFA/CH2Cl2; (ii)
NH4Cl, EDCI, NMM; (iii) H2/Pd/C, MeOH or Me2S, TFA.
Scheme 4a
a Reagents and conditions: (a) KOtBu, THF, -78 °C f room temp,
R3X, room temp, 12 h; (b) (i) 1 M aqueous HCl, THF; (ii) Boc2O; (c) (i)
aqueous LiOH, THF/H2O/MeOH; (ii) BOP or EDCI, CH3NHOCH3‚HCl,
CH2Cl2, DMF; (iii) LiAlH4, THF, 0 °C f room temp, 0.5 h; (d)
(CH3)2C(OH)CN, Et3N, CH2Cl2, room temp; (e) (i) K2CO3, H2O2, DMSO;
(ii) 4 M HCl/dioxane; (f) (i) 6 M methanolic HCl, reflux, 12 h; (ii) Boc2O,
(iPr)2EtN; (iii) aqueous LiOH, THF/H2O/MeOH; (iv) NH4Cl, EDCI, NMM,
DMF; (iv) 4 M HCl/dioxane.
Scheme 2. Henry Reaction: Method Ba
a Reagents and conditions: (a) (i) KHMDS, PhSeCl, -78 °C f room
temp; (ii) H2O2, pyridine; (b) (i) BuLi, [(CH3)2CHPPh3]Br, -78 °C f room
temp, THF; (ii) LiAlH4, THF, reflux; (iii) H2/Pd/C, Boc2O; (c) (i) Jones
oxidation, acetone, -5 °C, 3 h; (ii) TMS-diazomethane, toluene/MeOH;
(iii) 4 M HCl/dioxane; (d) (i) EDCI, HOOBt, NMM, CH2Cl2/DMF, 12 h
or HATU, NMM, CH2Cl2/DMF; (e) aqueous LiOH, THF/H2O/MeOH; (f)
(i) 10, 14, or 15, EDCI, HOOBt, NMM, CH2Cl2/DMF, room temp, 12 h;
(ii) Cl2COOH, EDCI‚HCl, DMSO.
a Reagents and conditions: (a) OHC-COOH‚H2O, MeOH, Et3N; (b)
H2/Pd/C, CH3COOH; (ii) Boc2O, NaHCO3, aqueous dioxane; (c) (i) NH4Cl
or R2H, EDCI, NMM, DMF/CH2Cl2; (ii) 4 M HCl/dioxane; (d) (i) EDCI,
NMM, Gly-OBn‚HCl; (ii) 4 M HCl/dioxane.
14. The benzyl ester was subsequently deprotected and coupled
with various amines.
were protected with the Boc group and subsequently coupled
with ammonium chloride. Deprotection of the Boc group using
HCl/dioxane resulted in a P1 fragment of type 10. In some cases,
intermediates 9 were directly hydrolyzed to the corresponding
primary amides using aqueous basic hydrogen peroxide, which
were then followed by deprotection using 4 M HCl to yield
R-hydroxyamide of type 10.15
The Henry reaction (method B) was employed in the synthesis
of the P1 fragment where the corresponding nitroalkanes were
commercially available (Scheme 2).16 Nitropropane or nitrobu-
tane was condensed with glyoxalic acid to form 3-substituted
2-hydroxy-3-nitropropionic acid derivatives 12. The nitro group
of 12 was catalytically hydrogenated using Pd/C, and the
resulting amines were protected as the tert-butylcarbamate to
form hydroxyacid of type 13. Coupling of 13 with ammonium
chloride followed by Boc deprotection resulted in P1 segments
used for syntheses of inhibitors outlined in Table 3. Compounds
summarized in Table 2 were obtained via coupling acid 13 with
various aliphatic and aromatic amines. P1 segments required
for the preparation of compounds in Table 1 were synthesized
by coupling acid 13 with glycine benzyl ester to form dipeptide
As shown in Scheme 3, Sharpless amino hydroxylation
(method C) was the third method employed for syntheses of P1
fragments of other compounds shown in Table 3.17 Thus,
aldehydes of type 16 were converted to the R,â-unsaturated tert-
butyl esters 17 via the Horner Emmons reaction,18 and the
resultant olefins were subjected to aminohydroxylation to yield
hydroxy esters of type 18. Deprotection of tert-butyl esters of
intermediates 18 generated hydroxy acids, which were used as
described previously to prepare 10.
Scheme 4 outlines representative syntheses of inhibitors 26-
72. The synthesis of the P2 piece was carried out via phenylse-
lenation of pyroglutamic acid derivative 19, followed by
oxidation of intermediate selenide with H2O2 and subsequent
elimination to provide R,â-unsaturated lactam 20.19 Treatment
of lactam 20 with isopropylphosphonium ylide followed by
reduction of the lactam with LiAlH4 resulted in the 3,4-
dimethylcyclopropaned N-benzylprolinol.20 The N-benzyl group
deprotection using catalytic hydrogenation followed by Boc
protection yielded prolinol derivative 21. Jones oxidation of 21,
followed by esterification of the resultant acid with TMS-

Carboxamide for Treatment of Hepatitus C
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6077
Table 1. a
diazomethane and Boc deprotection, yielded P2 amine salt 22.
Coupling of 22 with the appropriately capped P3 amino acid
using the EDCI protocol gave dipeptide 23 that was hydrolyzed
to acid 24 using aqueous LiOH. Subsequent couplings of acids
24 with appropriate P1 segments resulted in intermediate
hydroxyamides, which were oxidized to the corresponding
ketoamides of type 25 using modified Moffat21 conditions with
EDCI, DMSO, and Cl2CHCOOH to yield compounds sum-
marized in Tables 3-7. Alternatively, compounds in Tables 1
and 2 were readily obtained by coupling 24 with either
intermediate 15 or 14 followed by oxidation with Dess-Martin
periodinane reagent.22
Discussion
Synthesized inhibitors shown in Tables 1-7 were evaluated
for their ability to inhibit the hydrolysis of chromogenic
4-phenylazophenyl (PAP) ester from the peptide fragment Ac-
DTEDVVP(Nva)-O-4-PAP in a HCV protease continuous
assay.23 Inhibitors exhibited reversible slow tight binding
kinetics, and Ki* data generated reflected equilibrium binding
efficiency.24 Potent compounds were then evaluated in a
replicon-based cellular assay.25 EC90, the concentration required
for inhibition of 90% of virus replication in hepatocytes, was
recorded as a measure of replicon cellular potency. A multiplex
Taqman RT reaction was done measuring both the HCV and
endogenous GAPDH RNA levels. GAPDH RNA levels did not
change when measured up to a maximum concentration of 5
µM. MTS assay as a mesure of toxicity was conducted on
compounds of interest. Compounds of interest were evaluated
for binding to human neutrophil elastase and the ratio of activity,
HNE/HCV, reflected a measure of selectivity. Neutrophil
elastase was chosen as a measure of selectivity because of its
close structural similarity to HCV NS3 protease. Selected
compounds were evaluated in a rapid rat assay26 to obtain
plasma levels. Compounds with good enzyme binding, selectiv-
ity, cellular potency, and rapid rat plasma levels were evaluated
for full PK in rats, dogs, and monkeys.
a Table notes are as follows. *P1 fragment synthesized using method B.
§Ki* value represents binding for a mixture of diasteromers at P1 and within
2-fold for 95% confidence limit.
was poor with AUC ) 0.09 µM‚h. The role of phenylglycine
NH at P2′ was probed by replacing it with oxygen to give
compound 33 (Ki* ) 0.050 µM). This result was similar to the
activity of NH compound 26, demonstrating that the phenyl-
glycine NH was not critical for maintaining activity. This
suggested that suitably modified P1′ analogues could also
provide potent inhibitors.
P1′ Modified Analogues. Further exploration of P1′ truncated
analogues were carried out retaining norvaline as P1 and
cyclohexylglycine as P3 residues. Initially, we decided to explore
the P1′ site using smaller aliphatic amines. Results of these
modifications are summarized in Table 2.
Replacement of the glycine-phenylglycine-dimethylamide
segment of inhibitor 4 with butylamide resulted in compound
34 with Ki* ) 1.9 µM, 50-fold less potent than 33. However,
introduction of allylamide at the P1′ site resulted in compound
35 with Ki* ) 0.28 µM. Although the potency of 35 was 7-fold
less than that of 33, it was encouraging to see this kind of
activity with a P1′ truncated compound. More interestingly, 35
had an excellent oral PK in rats with AUC ) 26 µM‚h.
Incorporation of a smaller group such as methylamide at P1′
provided compound 36 with Ki* ) 1.5 µM and excellent PK in
rats with AUC ) 13.4 µM‚h. Further truncation to a primary
amide functionality resulted in 37 (Ki* ) 0.100 µM), a 3-fold
improvement in potency over allylamide derivative 35. Com-
pound 37 also had a good PK in rats with AUC ) 2.52 µM‚h.
Enhanced potency of primary amide 37 was hypothesized to
result from multiple hydrogen bondings to the enzyme backbone.
This was confirmed by replacement of the primary amide with
P2′ Modified Analogues. Initially, phenylglycine dimethyl-
amide at P2′ was replaced with benzyl and alkyl derivatives
that mimicked the aryl portion of 4. These modifications were
intended to remove the terminal amide bond from 4, thus making
the resulting inhibitors less peptidic. Results of these modifica-
tions are summarized in Table 1.
Thus, replacement of phenylglycine of 4 with benzylamide
resulted in 26 with Ki* ) 0.056 µM, 5-fold less active than 4.
Introduction of thiophenemethylamide at P2′ gave compound
27 with activity similar to that of inhibitor 26 (Ki* ) 0.060
µM). Both these compounds were evaluated for oral PK in rats
and found to have AUC values of 1.27 and 0.73 µM‚h,
respectively. It was encouraging to observe that truncation of a
single amide resulted in compounds with appreciable plasma
levels in rats. Incorporation of a methyl group at the benzylic
position of 26 resulted in compound 28 with similar potency,
Ki* ) 0.055 µM, but improved rat PK (AUC ) 2.66 µM‚h).
Replacement of phenylglycine dimethylamide at P2′ with
cyclopropylmethylamide resulted in compound 29 with Ki* )
0.130 µM, a 2-fold loss in activity compared to 26. This could
have been due to the fact that the cyclopropylmethyl group was
too small to effect the desired interaction at P2′. Compound 30
lacking the benzyl group or aliphatic group lost potency,
indicating the preference of an aryl or alkyl moiety at P2′.
Introduction of (R)-methylcyclohexylamide at P2′ gave com-
pound 31 (a saturated analogue of 28) that resulted in a 2-fold
loss in activity. Moreover, the rapid rat AUC of inhibitor 31

6078 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Venkatraman et al.
Table 2. a
Table 3. a
a Table notes are as follows. † tert-Butylglycine was used as P3 residue.
] Method B was used for syntheses of P1 fragment. § Ki* value represents
binding for mixture of diasteromers at P1 and within 2-fold for 95%
confidence limit. NA ) data not available.
dimethylamide, resulting in compound 38, which was essentially
inactive (Ki* > 13.0 µM). Isosteric replacement of NH2 with
OH resulted in ketoacid 39 with Ki* ) 0.110 µM, which once
again demonstrated the importance of the P1′ hydrogen bond
in maintaining activity. X-ray crystal structures of compounds
of type 37 bound to protease established the participation of
the primary amide functionality in hydrogen-bonding to
glutamine-41 of the enzyme (Figure 3).27
Modifications at P1. Analysis of the X-ray structure of NS3
enzyme revealed the S1 pocket to be uniquely shallow and
hydrophobic in nature. We sought to explore this pocket by
introducing a variety of motifs. Enzyme binding and cellular
data of P1 modified compounds are tabulated in Table 3.
The carbon bearing P1 residue is adjacent to the electrophilic
ketoamide moiety where serine-139 from the enzyme attacks
to form a covalent bond. The R-proton is acidic, making this
center readily epimerizable. Thus, it was our initial intention to
quarternize this center to alleviate potential epimerization.
Unfortunately, incorporation of 1-aminocyclopropyl P1 resulted
in compound 40 that did not have appreciable activity. This
loss in potency was ascribed to the inability of serine-139 to
attack ketoamide functionality because of steric hindrance.
Consequently, follow-up analogues were mostly prepared with
monosubstituted amino acids at P1.
a Table notes are as follows. ]Methods used for syntheses of P1 fragments
(Scheme 1): a ) cyanohydrin route; b ) Henry reaction; c ) Sharpless
§
method. Ki* value represents binding for a mixture of diasteromers at P1
and within 2-fold for 95% confidence limit. NA ) data not available.
1Highest tested concentration is 5 µM.
Since a propyl group was readily accommodated at the P1
site, we decided to further study the effect of changing the length
of the carbon chain by incorporating a 2-aminobutyric acid
moiety at this position. This resulted in the formation of
compound 41 with Ki* ) 0.74 µM, a 7-fold loss in activity in
comparison to norvaline derivative 37. Incorporation of the
2-aminohex-5-enoic acid derivative resulted in a less potent
inhibitor 42 with Ki* ) 0.150 µM. This demonstrated that
extension of the carbon chain at P1 was undesirable. We
therefore explored the effect of branching substitution at the S1
site. Replacement of norvaline with 2-amino-4-methylpent-4-
enoic acid and isoleucine derivatives resulted in compounds 43
and 44 with Ki* ) 0.3 µM and Ki* ) 0.4 µM, respectively.
The loss in activity indicated that branching at P1 was
detrimental. Incorporation of cyclopropylalanine derived P1
resulted in compound 45 with Ki* ) 0.025 µM, a 4-fold
improvement in activity over 37. This result was encouraging
because, for the first time, we achieved an activity of Ki* less
than 0.1 µM in a truncated compound. The profound effect of
constraining the two methyl groups of leucine into a ring to
form cyclopropylalanine (compare compounds 45 and 44)
resulted in a 16-fold improvement in potency. Introduction of
cyclobutylalanine at the P1 position resulted in compound 46
with further enhancement in activity (Ki* ) 0.008 µM). Further
expansion of the ring size to the cylcopentylalanine derivative

Carboxamide for Treatment of Hepatitus C
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6079
Table 4. a
yielded compound 47 that resulted in a loss in activity with Ki*
) 0.150 µM. Even worse, introduction of cyclohexylalanine at
P1 yielded compound 48 that was not active even at 12 µM.
These studies clearly established that small cyclic groups were
well tolerated at the S1 pocket with the optimum ring being
cyclobutylalanine. Potent inhibitors 45 and 46 were further
profiled for their selectivity against human neutrophil elastase,
HCV replicon cellular activity, and PK in rats and higher
species. As summarized in Table 3, the HNE/HCV selectivities
of these compounds were dependent on the size of the P1
substituents. The greater the size of the P1 residue, the better
was the HNE/HCV selectivity. Comparison of compounds 45-
47 demonstrated that the cyclopentylalanine, analogue 47, was
more selective (HNE/HCV ) 370) than cyclobutylalanine
compound 46 (HNE/HCV ) 138), which was in turn more
selective than cyclopropylalanine derivative 45 (HNE/HCV )
23). The replicon cellular activity of compounds 45 and 46 were
0.4 and 0.7 µM, respectively. In full rat PK studies compound
45 had an AUC of 1.3 µM‚h and bioavailability of 4% whereas
compound 46 had an AUC of 1.5 µM‚h and bioavailability of
28%. However, compound 46 had poor PK in monkeys (Table
7). Introduction of small polar groups, such as oxygen, in the
ring resulted in compounds 49 and 50 with Ki* ) 0.40 and
0.090 µM, respectively, a 50-fold loss in activity compared to
46. This result clearly reinforced the fact that the S1 position
preferred lypophilic groups and introduction of polar functional-
ity at P1 resulted in loss of potency.
Modifications at the P3 Site. The S3 site of HCV NS3
protease is a shallow hydrophobic pocket occupied by isoleucine
in the natural substrate. We explored the tolerance of this site
by introducing a wide variety of substituents. Data for the
resulting compounds are summarized in Table 4. Compounds
described in this table were synthesized using procedures
outlined in Schemes 1-4. In this series, cyclopropylalanine and
cyclobutylalanine were retained as P1 residues.
Truncation of cyclohexylglycine to valine at the S3 site
resulted in inhibitor 52 with Ki* ) 0.210 µM, an 8-fold loss in
potency compared to analogous cyclohexylglycine compound
45. Similarly, introduction of cyclopentylglycine at P3 gave 53,
a slightly less active compound with Ki* ) 0.100 µM. The
decrease in potencies for compounds 52 and 53 in comparison
to P3 cyclohexylglycine was attributed to insufficient van der
Waals interaction of side chains at the S3 site. To achieve better
overlap, indanylglycine was introduced resulting in compound
54 with Ki* ) 0.082 µM. Introduction of indanylglycine at P3
with P1 cyclobutylalanine resulted in compound 55 (Ki* ) 0.27
µM), a 30-fold loss in activity. Further exploration of the P3
site with lypophilic tert-butylglycine resulted in compound 56
(Ki* ) 0.057 µM) containing P1 cyclopropylalanine and
compound 57 (Ki* ) 0.076 µM) containing P1 cyclobutylala-
nine. It is worth mentioning that these compounds had a 3- to
4-fold improvement in binding activity relative to P3 valine
compound 52. P3 tert-butylglycine derived inhibitors 56 and
57 demonstrated improved HNE/HCV selectivity compared to
P3 cyclohexylglycine containing compounds. Once again the
P1 cyclobutylalanine analogue 57 had a better HNE/HCV
selectivity than the P1 cyclopropylalanine analogue 56. Cellular
activity of 56 and 57 in the replicon-based cellular assay was
similar to that of cyclohexylglycine derived compounds 45 and
46 with EC90 ) 0.6 and 0.8 µM, respectively. Introduction of
amino(1-methylcyclopropyl)acetic acid, a constrained tert-
butylglycine analogue at P3, resulted in inhibitors 58 and 59
with diminished activity: Ki* ) 0.300 and 0.700 µM, respec-
tively. Incorporation of 2-amino-3-hydroxy-3-methylbutyric acid
a Table notes are as follows. §Ki* value represents binding for a mixture
of diasteromers at P1 and within 2-fold for 95% confidence limit. NA )
data not available. 1Highest tested concentration is 5 µM.
at P3 gave 60 with Ki* ) 0.220 µM. This demonstrated that the
S3 site was less tolerant to polar substituents.
Modifications at the P3-Capping Site. From X-ray crystal
structure studies of inhibitors bound to NS3 protease, it was
clear that the tert-butyl group of Boc carbamate occupied the
S4 pocket. To explore this site further, a wide variety of
carbamates and ureas were synthesized (Table 5). tert-Butylg-
lycine was retained as the P3 moiety because it demonstrated
improved elastase selectivity in comparison to other P3 residues.
Cyclobutylalanine and cyclopropylalanine were similarly re-
tained as the P1 residues.
Incorporation of isopropyl carbamate capping with the P1
cyclopropylalanine residue resulted in compound 61 with Ki*
) 0.150 µM, 3-fold less active than the base compound 56
containing P3 Boc capping. On the other hand, the cyclopropyl
carbamate P3-capped compound 62 had a slightly lower activity
with Ki* ) 0.300 µM.
Replacement of the Boc group in 56 with isobutyl carbamate
resulted in compound 63 with similar potency (Ki* ) 0.07 µM)

6080 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Venkatraman et al.
Table 5. a
Table 6. a
a Table notes are as follows. §Ki* value represents binding for a mixture
of diasteromers at P1 and within 2-fold for 95% confidence limit.
respectively), elastase selectivities, and cellular potencies.
Inhibitor 70 with the tert-butylurea cap demonstrated HNE/HCV
selectivity of 2200, a 15-fold improvement relative to the
corresponding carbamate derivative 57. tert-Butylurea capped
compound 70 also demonstrated an improved cellular potency
in the replicon assay with EC90 ) 0.350 µM. Introduction of
N-methyl-tert-butylurea capping resulted in compounds 71 and
72 with Ki* ) 0.060 and 0.100 µM, respectively, a 4- to 7-fold
loss in potency suggesting the involvement of urea NH in
hydrogen-bonding to the peptide backbone.
Synergistic Effects of Residues on Binding and Selectivity.
As the SAR of various substituents at P1, P3, and P3-cappings
were investigated, synergistic effects that influenced potency
and selectivity were observed. These synergistic effects are
discussed below.
(a) Effect of P3 and P3-Capping on Binding Activity. As
shown in Table 6, the potency of inhibitors discussed greatly
depended on the substituents at P3 and P3-capping. Thus, in a
given series with P1 cyclopropylalanine, Boc capped compound
45 (Ki* ) 0.025 µM) with P3 cyclohexylglycine had a better
enzyme activity than compound 56 (Ki* ) 0.057 µM) containing
P3 tert-butylglycine. However, tert-butylurea capped compound
68 (Ki* ) 0.013 µM) with P3 tert-butylglycine was found to be
more potent than compound 73 (Ki* ) 0.050 µM) that contained
P3 cyclohexylglycine. A similar observation was also seen in
the P1 cyclobutylalanine containing series of compounds; the
Boc capped P3 cyclohexylglycine containing compound 46 (Ki*
) 0.008 µM) was more potent than Boc capped P3 tert-
butylglycine compound 57 (Ki* ) 0.076 µM) and vice versa,
a Table notes are as follows. §Ki* value represents binding for a mixture
of diasteromers at P1 and within 2-fold for 95% confidence limit. NA )
data not available. 1Highest tested concentration is 5 µM.
and HNE/HCV selectivity (HNE/HCV ) 160). Introduction of
carbamate derived from (R)-3-methylbutan-2-ol resulted in
compound 64 with Ki* ) 0.036 µM, a 2-fold improvement in
activity over the base compound. It was encouraging to see that
the elastase selectivity of 64 (HNE/HCV ) 3800) was greatly
improved while retaining replicon cellular potency (EC90 ) 0.7
µM). Overall, introduction of different carbamate P3 caps did
not appreciably improve enzyme binding or cellular activity;
therefore, urea surrogates were pursued.
The isopropylurea capped compound 65 (Ki* ) 0.140 µM)
did not enhance activity in the P1 cyclopropylalanine series.
However, introduction of isopropylurea in the P1 cyclobutyla-
lanine series resulted in compound 66 with a 3-fold improvement
in potency (Ki* ) 0.016 µM) and 420-fold improvement in
selectivity against elastase. Incorporation of tert-butylurea as a
replacement of the Boc group resulted in compounds 68 and
70 with improved binding (Ki* ) 0.013 and 0.014 µM,

Carboxamide for Treatment of Hepatitus C
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6081
Table 7. PK Data of Selected Potent Compounds
po
compd
AUC (µM‚h)
% bioavailability
Rat
1.3
1.5
0.83
1.00
1.52
45a
46a
66a
68a
70a
3.4
28
13
16
26
Monkey
46c
68b
70c
0.11
0.05
0.12
1
2
4-11
Dog
68b
70c
1.6
3.08
36
30
a 10 mg/kg. b 2 mg/kg. c 3 mg/kg.
and the urea capped P3 tert-butylglycine derivative 70 (Ki* )
0.014 µM) was more potent than P3 cyclohexylglycine derivative
74 (Ki* ) 0.050 µM). This synergy could not be explained by
assessment of the X-ray structures of these compounds bound
to the protease.
Figure 2. Compound 70.
(b) Effect of P3 and P1 Substituents on Selectivity. Another
interesting synergy was observed on selectivity against elastase
by the interaction of P1 and P3 residues. Thus, a close
examination of inhibitors demonstrated that P1 cyclobutylalanine
derived inhibitors had better selectivity against HNE than P1
cyclopropylalanine compounds. Moreover, in the series contain-
ing P1 cyclobutylalanine, inhibitors containing P3 tert-butylg-
lycine (compounds 57 and 70) were more selective than P3
cyclohexylglycine counterparts (compounds 46 and 74).
Pharmacokinetic Studies. On the basis of potency and
selectivity, key compounds were selected and evaluated for their
PK profiles in rats, monkeys, and dogs.
As shown in Table 7, the Boc capped compound 46
containing P1 cyclobutylalanine had a good bioavailability of
28% in rats with AUC ) 1.5 µM‚h, whereas the corresponding
P1 cyclopropylalanine containing compound 45 had a bioavail-
ability of 4% with albeit similar AUC ) 1.3 µM‚h. However,
PK in monkeys for compound 46 was poor with AUC ) 0.11
µM‚h and a short half-life of 1 h. The isopropylurea capped
compound 66 demonstrated moderate PK in rats with AUC )
0.83 µM‚h and bioavailability of 13%. Thus, it was not further
evaluated in monkeys and dogs. Evaluation of tert-butylurea
capped compound 68 and 70 in rats gave AUC ) 1.0 and 1.5
µM‚h and bioavailabilities of 16% and 26%, respectively. The
PK data of these inhibitors in dogs were similar to those in
rats; P1 cyclopropylalanine derived compound 68 had bioavail-
ability of 36% with AUC ) 1.6 µM‚h at 2 mg/kg, and P1
cyclobutylalanine analogue compound 70 had bioavailability of
30% with AUC ) 3.1 µM‚h at 3 mg/kg. Both compounds
showed variable monkey PK with bioavailability ranging
between 2% and 11%. Inhibitor 70 was moderately absorbed
with an absorption of 36% in rats and absorption of 23% in
dogs. It was well distributed in the body especially in the target
organ liver, which demonstrated a liver/plasma ratio of ∼30.
Since compound 70 had good enzyme binding, selectivity
against HNE, desirable PK properties, excellent target organ
exposure, and no hERG or CYP inhibition, it was progressed
into clinical studies in humans (Figure 2).
Figure 3. X-ray structure of inhibitor 70 bound to NS3 protease.
dimethylcyclopropylproline residue adopted a bent conformation
that allowed maximum overlap of the methylenes of proline
and cyclopropyl ring to Ala-156. The conformation adopted by
cyclopropanated proline allowed the methyl group proximal to
the carbonyl to interact with His-57 and the methyl group distal
to carbonyl to interact with Ala-156 and Arg-155. The side chain
of P3 tert-butylglycine occupied the S3 pocket with the two
methyl groups of the tert-butyl group interacting with the
protease effectively and the third methyl group being solvent-
exposed. The tertiary butyl group of P3-urea capping occupied
the S4 pocket, enhancing binding of the inhibitor to protease.
The electrophilic ketoamide reversibly trapped serine-139 to
form a covalent bond with the enzyme and the hydrogen of the
primary amide donated a hydrogen bond to the peptidic
backbone of the protein, locking the inhibitor to the surface.
In addition to van der Waals contacts, inhibitor 70 formed a
series of specific hydrogen bonds with the protein surface. A
schematic representation of these interactions to the protease is
shown in Figure 4. When the various hydrogen-bonding
interactions that existed between inhibitor 70 and NS3 protease
were mapped out, it was evident that the primary ketoamide
donated a hydrogen bond to glutamine-41, and the carbonyl
oxygen in turn made two hydrogen bonds with the nitrogens of
serine-139 and glycine-137. The urea nitrogens donated two
hydrogen bonds to alanine-157, thus improving potency and
HCV specificity. Additionally, the nitrogen of the P1 cyclobu-
The X-ray structure of inhibitor 70 bound to HCV NS3
protease was solved and is shown in Figure 3.
From the structure, it was clear that cyclobutylalanine moiety
occupied most of the space available at the S1 pocket. The P2

6082 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Venkatraman et al.
thin-layer chromatography (TLC) were performed on precoated
silica gel plates available from Analtech. Column chromatography
was performed using Merck silica gel 60 (particle size 0.040-0.055
µm, 230-400 mesh) or using Biotage or Isco chromatographic
systems. Many compounds were further purified using Varian
normal-phase HPLC with YMC-diol column with solvent A
(hexanes) and solvent B (2-propanol, CH2Cl2, and acetonitrile).
Visualization was accomplished with UV light or by staining with
basic KMnO4 solution, methanolic H2SO4, or Vaughn’s reagent.
NMR spectra were recorded in CDCl3 or DMSO-d6 unless otherwise
noted at 300, 400, or 500 MHz (1H NMR) or at 75, 100, or 125
MHz (13C NMR). Mass spectra were obtained using electron spray
FAB ionization methods. Where applicable 1H NMR and 13C NMR
of compounds are reported as two sets of signals (integration adding
up to two molecules) representing two diasteromers.
Figure 4. Hydrogen bond interactions of inhibitor 70 with HCV
protease.
Representative Procedure for the Synthesis of 3-Amino-4-
cyclobutyl-2-hydroxybutyramide Hydrochloride Salt (R3
)
tylalanine donated a hydrogen bond to Arg-155, and the oxygen
of the P3 carbonyl group accepted a hydrogen bond from the
Ala-157. In combination with hydrophobic interaction, the array
of hydrogen bonds also contributed greatly to the binding
potency and selectivity of compound 70.
Cyclobutylmethyl, 10) Using Cyanohydrin Route. Similar meth-
ods were employed for the syntheses of intermediates 10 where R3
was cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, isobu-
tyl, isobutylene, or n-butenyl.
Ethyl 2-tert-Butoxycarbonylamino-3-cyclobutylpropionate (7).
A stirred solution of ketimime 5 (50 g, Aldrich, 187.1 mmol) in
dry THF (400 mL) under N2 was cooled to -78 °C and treated
with a solution of KtBuO (220 mL, 1.15 equiv, 1 M in THF). The
reaction mixture was stirred at 0 °C for 1 h and treated with
bromomethylcyclobutane (28 mL, 249 mmol) and further stirred
at room temperature for 48 h. The reaction mixture was concentrated
in vacuo, and the residue was dissolved in Et2O (300 mL) and
treated with aqueous HCl (2 M, 300 mL). The resulting solution
was stirred at room temperature for 5 h and extracted with Et2O (1
L). The aqueous layer was basified (pH ∼12-14) with aqueous
NaOH (50%) and extracted with CH2Cl2 (3 × 300 mL). The
combined organic layers were dried (MgSO4), filtered, and con-
centrated to give pure cyclobutylalanine ethyl ester (18 g) as a
colorless oil. 1H NMR (CDCl3, 400 MHz) δ 4.13 (q, 2 H, J ) 7.2
Hz), 3.33 (dd, 1 H, J ) 5.5 and 2.2 Hz), 2.42 (h, 1 H, J ) 7.7 Hz),
2.08-1.98 (m, 2 H), 1.89-1.55 (m, 6 H), 1.25 (t, 3 H, J ) 7.2
Hz).
A solution of cyclobutylalanine ethyl ester (18 g, 105.2 mmol)
in CH2Cl2 (350 mL) was treated with di-tert-butyl dicarbonate (23
g, 105.4 mmol) at 0 °C and stirred at room temperature for 12 h.
After the completion consumption of starting material (TLC), the
reaction mixture was concentrated in vacuo and purified by
chromatography (SiO2, EtOAc/hexanes, 0-20% gradient). 1H NMR
(CDCl3, 500 MHz) δ 5.00 (d, 1 H, J ) 7.3 Hz), 4.22 (dq, 3 H, J
) 6.9 and 1.0 Hz), 2.42 (p, 1 H, J ) 8.2 Hz), 2.15-2.10 (m, 2 H),
1.94-1.68 (m, 6 H), 1.49 (s, 9 H), 1.33 (t, 3 H, J ) 7.3 Hz); 13C
NMR (CDCl3, 125 MHz) δ 173.5, 155.7, 80.1, 61.6, 52.9, 40.2,
33.0, 28.9, 28.8, 19.1, 14.6; MS (ESI), 294 [(M + Na)+, 80], 216
(50), 172 (100).
(2-Cyclobutyl-1-formylethyl)carbamic Acid tert-Butyl Ester
(8). Ester 7 was dissolved in THF/H2O (200 mL, 1:1) and treated
with LiOH‚H2O (6.5 g, 158.5 mmol) and stirred at room temperature
for 3 h. The reaction mixture was acidified with concentrated HCl
(pH ∼1-0) and extracted with CH2Cl2. The combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo to
yield Boc protected cyclobutylalanine as a colorless viscous oil,
which was used for next step without further purification.
A solution of acid (15.0 g, 61.7 mmol) in CH2Cl2 (250 mL) was
treated with BOP reagent (41.1 g, 93 mmol), N-methylmorpholine
(27 mL), and N,O-dimethylhydroxylamine hydrochloride (9.07 g,
93 mmol) and stirred at room temperature for 16 h. The reaction
mixture was diluted with 1 N aqueous HCl (250 mL), and the
organic layers were separated. The aqueous layer was extracted
with CH2Cl2 (3 × 300 mL). The combined organic layers were
dried (MgSO4), filtered, concentrated in vacuo, and purified by
chromatography (SiO2, EtOAc/hexanes, 2:3) to yield the Wienreb
amide (15.0 g) as a colorless solid. 1H NMR (CDCl3, 400 MHz) δ
5.11 (d, 1 H, J ) 9.3 Hz), 4.62-4.55 (m, 1 H), 3.77 (s, 3 H), 3.20
Conclusions
A series of systematic truncations and modifications of amino
acid residues of the lead undecapeptide 1 led to the discovery
of 70, a potent, low molecular weight HCV NS3 protease
inhibitor. Initial truncation efforts of inhibitor 1 led to the
discovery of compounds that spanned from P3 to P2′, which
had good enzyme binding as exemplified by compound 4. Lack
of PK and cellular potency in earlier series necessitated further
truncation and depeptidization of initial molecules. The relation-
ship between lower molecular weight inhibitors and rat PK was
established early with the identification of P2′ truncated
analogues. P1′ SAR studies allowed identification of primary
ketoamide moiety as an excellent residue that improved binding
potency of the P3-P1 truncated compounds. The identification
of primary ketoamides series with improved enzyme activity
and PK allowed further optimization to identify 37. Systematic
SAR studies in the P1 site identified cyclopropylalanine and
cyclobutylalanine as excellent P1 groups that provided potent
inhibitors. Cyclopropylalanine and cyclobutylalanine at P1 in
combination with cyclohexylglycine at P3 yielded inhibitors 45
and 46 that were active in enzyme and replicon cellular assays.
Even though cyclohexyglycine was an acceptable P3 residue,
further SAR at this site showed tert-butylglycine as an alterna-
tive group that provided inhibitors with improved HNE selectiv-
ity. In the course of these investigations, we discovered a
synergy between P1, P3, and P3-capping residues that governed
enzyme binding and HNE selectivity. Finally, P3-capping SAR
led to the identification of tert-butylurea as an excellent cap,
which in turn improved potency of the P3 tert-butylglycine series
leading to the identification of compound 70 as a candidate with
excellent binding, selectivity, and cellular potency.
The PK profile of inhibitor 70 demonstrated good oral
bioavailabilties of 26% and 30% in rats and dogs with excellent
distribution in animals. Target organ analysis in rats revealed a
desirable 30-fold liver/plasma concentration of 70 with a
concentration of ∼14 times the replicon EC90.
Finally, the X-ray of 70 was solved and illustrated the major
interactions between the inhibitor and the enzyme. Compound
70 has been advanced to phase 1 clinical trials for the treatment
of hepatitis C viral disease.
Experimental Section
General. Dry solvents were purchased from Aldrich or Acros
and used without further purification. Other solvents or reagents
were used as obtained except when otherwise noted. Analytical

Carboxamide for Treatment of Hepatitus C
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6083
(s, 3 H), 2.40 (p, 1 H, J ) 7.7 Hz), 2.14-2.00 (m, 2 H), 1.91-
1.59 (m, 6 H), 1.43 (s, 9 H).
The reaction mixture was stirred at room temperature for 3 days.
The solvents were removed in vacuo, and the residue was diluted
with aqueous HCl (250 mL) and extracted with CH2Cl2. The
combined organic layers were washed with aqueous saturated
NaHCO3, dried (MgSO4), filtered, and concentrated in vacuo to
obtain (2-carbamoyl-1-cyclobutylmethyl-2-hydroxyethyl)carbamic
acid tert-butyl ester. 1H NMR (CH3OH-d4, 400 MHz) δ 4.13 (d, 1
H, J ) 3.9 Hz), 4.06 (d, 1 H, J ) 3.0 Hz), 3.89-3.70 (m, 2 H),
2.42-2.30 (m, 2 H), 2.26-1.98 (m, 4 H), 1.98-1.55 (m, 1 2 H),
1.42 (s, 9 H), 1.40 (s, 9 H). It was deprotected as described before
to yield 10 as beige solid.
Benzyl (3-tert-Butoxycarbonylamino-2-hydroxyhexanoylami-
no)acetate Hydrochloride Salt (14). To a stirred solution of 1328
(3.00 g, 12.0 mmol) in DMF (15 mL) and CH2Cl2 (15 mL) at -20
°C was added HOOBt (1.97 g, 12.0 mmol), N-methylmorpholine
(4.0 mL, 36.0 mmol), and EDCI (2.79 g, 14.5 mmol). The mixture
was stirred for 10 min, followed by addition of HCl‚H2N-Gly-OBn
(2.56 g, 13.0 mmol). The resulting solution was stirred at -20 °C
for 12 h, then kept in the refrigerator overnight and concentrated
to dryness, followed by dilution with EtOAc (150 mL). The EtOAc
solution was then washed twice with saturated NaHCO3, H2O, 5%
H3PO4, and brine, dried over Na2SO4, filtered, concentrated to
dryness, and purified by chromatography (SiO2, acetone/hexanes,
1:4 f 1:1) (4.5 g, 94%) to yield Boc-protected 14. 1H NMR
(DMSO-d6, 500 MHz, mixture of diastereomers) δ 8.21 and 8.18
(t, 2 H, J ) 6.5 Hz), 7.40-7.33 (m, 10 H), 6.39 and 5.94 (d, 2 H,
J ) 9.0 and 9.5 Hz), 5.89 and 5.76 (d, 2 H, J ) 6 and 5.5 Hz),
5.14 (bs, 4 H), 3.99-3.70 (m, 8 H), 1.39 and1.36 (s, 18 H), 1.48-
1.07 (m, 8 H), 0.85 and 0.77 (t, 6 H, J ) 7.0 and 6.5 Hz); 13C
NMR (DMSO-d6, 125 MHz) δ 173.9, 173.2, 170.5, 170.4, 156.1,
136.8, 129.3, 128.9, 128.8, 128.7, 78.5, 78.4, 74.5, 73.1, 66.6, 53.5,
41.4, 30.5, 29.1, 29.0, 19.8, 19.7, 14.7, 14.6. MS (ESI) 395 [(M +
1)+, 100].
A solution of the amide (15 g, 52.1 mmol) in dry THF (200
mL) was treated with a solution of LiAlH4 (1 M, 93 mL, 93.0 mmol)
at 0 °C. The reaction mixture was stirred at room temperature for
1 h and carefully quenched at 0 °C with a solution of KHSO4 (10%
aqueous) The resultant slurry was filtered, and the organic layer
was diluted with aqueous HCl (1 M, 150 mL) and extracted with
CH2Cl2 (3 × 200 mL). The combined organic layers were washed
with aqueous HCl (1 M), saturated NaHCO3, and brine and dried
(MgSO4). The mixture was filtered and concentrated in vacuo to
yield aldehyde 8 as viscous colorless oil (14 g). 1H NMR (CDCl3,
500 MHz) δ 9.62 (s, 1 H), 5.06 (bs, 1 H), 4.22 (q, 2 H, J ) 6.3
Hz), 2.46 (s, 1 H, J ) 7.9 Hz), 2.17-2.08 (m, 2 H), 2.03-1.83
(m, 3 H), 1.72 (s, 3 H, J ) 10.4 Hz), 1.50 (s, 9 H); 13C NMR
(CDCl3, 125 MHz) δ 200.3, 155.8, 80.4, 59.5, 36.8, 32.9, 29.2,
28.7, 19.0; MS (ESI) 250 [(M + Na)+, 5], 172 (20), 128 (100).
(2-Cyano-1-cyclobutylmethyl-2-hydroxyethyl)carbamic Acid
tert-Butyl Ester (9). A solution of the aldehyde 8 (14 g, 61.6 mmol)
in CH2Cl2 (50 mL) was treated with Et3N (10.73 mL, 74.4 mmol)
and acetone cyanohydrin (10.86 g, 127.57 mmol) and stirred at
room temperature for 12 h. The reaction mixture was concentrated
in vacuo and diluted with aqueous HCl (1 M, 200 mL) and extracted
into CH2Cl2 (3 × 200 mL). The combined organic layers were
washed with H2O and brine, dried (MgSO4), filtered, concentrated
in vacuo, and purified by chromatography (SiO2, EtOAc/hexanes,
1:4) to yield 9 (10.3 g) as a colorless oil as a mixture of
diastereomers.
3-Amino-4-cyclobutyl-2-hydroxybutyramide Hydrochloride
Salt (10). (a) Basic Hydrogen Peroxide Hydrolysis. A solution
of cyanohydrin 9 (4.5 g, 17.7 mmol) in CH3OH (50 mL) was treated
with H2O2 (10 mL) and LiOH (820 mg, 20.8 mmol) and stirred at
0 °C for 3 h. The reaction mixture was diluted with aqueous
Na2S2O3 (5%, 50 mL) and extracted with CH2Cl2 (3 × 150 mL).
The combined organic layers were dried (MgSO4), filtered, and
concentrated in vacuo to yield Boc proteced hydroxyamide (3.25
g, 67%). The residue was dissolved in 4 N HCl in dioxane and
stirred at room temperature for 2 h. The reaction mixture was
concentrated in vacuo to give 10, which was used without further
A solution of Boc protected compound was dissolved in 4 M
HCl in dioxane and stirred at room temperature for 2 h. The reaction
mixture was concentrated in vacuo and dried to yield 14 as a pale-
brown solid.
Representative Procedure for the Synthesis of 3-Amino-2-
hydroxyhexanoic Acid Butylamide Hydrochloride Salt (R3 )
Propyl, R2 ) C4H9NH; 15). Similar methods were employed for
the syntheses of intermediates of type 15 where R3 was propyl and
R2 was NH2, C4H9NH, allylNH, CH3NH, and (CH3)2N.
1
purification. HNMR (500 MHz, DMSO-d6, mixture of diaster-
omers) δ 8.10 (s, 3 H), 7.85 (s, 3 H), 7.56-7.49 (m, 4 H), 6.45 (d,
1 H, J ) 6.0 Hz), 6.28 (d, 1 H, J ) 5.0 Hz), 4.19 (q, 1 H, J ) 2.5
Hz), 3.98 (dd, 1 H, J ) 2.0 and 4.0 Hz), 3.26 (b, 1H), 3.13 (b,
1H), 2.46-2.37 (m, 2 H), 2.1-2.00 (m, 4 H), 1.85-1.45 (m, 8 H);
13C NMR δ 174.4, 174.0, 71.7, 70.2, 52.1, 51.9, 36.5, 35.1, 32.0,
31.9, 28.4, 28.3, 28.1, 18.7, 18.5; MS (ESI) 173 [(M + Na)+, 100].
(b) Acidic Hydrolysis of Cyanohydrin (Alternative Proce-
dure). Saturated methanolic HCl, prepared by bubbling HCl gas
to CH3OH (700 mL) at 0 °C, was treated with cyanohydrin 9 and
heated at reflux for 24 h. The mixture was concentrated in vacuo
to yield methyl[3-amino-4-cyclobutyl-2-hydroxybutyrate]‚HCl, which
was used in the next step without purification. 1H NMR (CH3OH-
d4, 400 MHz) δ 4.48 (d, 1 H, J ) 3.3 Hz), 4.26 (d, 1 H, J ) 3.3
Hz), 3.79 (s, 3 H), 3.51-3.38 (m, 2 H), 3.33 (s, 3 H), 2.5-2.35
(m, 2 H), 2.34-2.00 (m, 4 H), 1.93-1.58 (m, 12 H).
A suspension of the amine salt in CH2Cl2 (200 mL) was treated
with Et3N (45.0 mL, 315 mmol) and Boc2O (45.7 g, 209 mmol) at
-78 °C. The reaction mixture was then stirred at room temperature
overnight and diluted with aqueous HCl (2 M, 200 mL) and
extracted into CH2Cl2. The organic layer was dried (MgSO4),
filtered, concentrated in vacuo, and purified by chromatography
(EtOAc/hexanes, 1:4) to yield methyl 3-tert-butoxycarbonylamino-
4-cyclobutyl-2-hydroxybutyrate.
A solution of the hydroxy acid (300 mg, 1.21 mmol) 13 in CH2-
Cl2 (5 mL) and DMF (5 mL) was treated with n-butylamine (106
mg, 1.46 mmol), EDCI (347 mg, 1.82 mmol), HOOBt (296 mg,
1.81 mmol), and NMM (305 mg, 3.02 mmol). The reaction mixture
was stirred at 0 °C for 16 h. The reaction mixture was concentrated
in vacuo, and the residue was diluted with aqueous HCl (100 mL)
and extracted with EtOAc. The combined organic layers were
washed with aqueous saturated NaHCO3, dried (MgSO4), filtered,
concentrated in vacuo, and purified by chromatography (SiO2,
acetone/hexanes, 2:3) to obtain Boc protected 3-amino-2-hydroxy-
hexanoic acid butylamide (140 mg). 1H NMR (DMSO-d6, 500
MHz) δ 7.72 (t, 1 H, J ) 6.6 Hz), 7.67 (t, 1 H, J ) 7.7 Hz), 6.34
(d, 1 H, J ) 9.5 Hz), 5.95 (d, 1 H, J ) 9.5 Hz), 5.52 (d, 1 H, J )
5.1 Hz), 5.47 (d, 1 H, J ) 6.7 Hz), 3.86-3.83 (m, 2 H), 3.79-
3.60 (m, 2 H), 3.12-2.97 (m, 4 H), 1.36 (s, 18 H), 1.36-1.25 (m,
4 H), 1.28-1.13 (m, 8 H), 1.13-1.06 (m, 4 H), 0.84 (t, 6 H, J )
7.3 Hz), 0.77 (t, 6 H, J ) 7.00 Hz); MS (ESI) 325 [(M + Na)+,
40], 303 [(M + 1)+, 30], 247 (30), 203 (70), 130 (100).
The Boc group was deprotected as described before using 4 M
1
HCl in dioxane to yield 15 as a beige solid. H NMR (DMSO-d6,
400 MHz) δ 8.07 (t, 1 H, J ) 5.9 Hz), 8.04 (t, 1 H, J ) 6.7 Hz),
8.00 (b, 3 H), 7.78 (b, 3 H), 6.40 (d, 1 H, J ) 5.8 Hz), 6.31 (d, 1
H, J ) 5.9 Hz), 4.15 (dd, 1 H, J ) 2.9 and 2.2 Hz), 4.00 (t, 1 H,
J ) 4.4 Hz), 3.68-3.65 (m, 1 H), 3.49-3.44 (m, 1 H), 3.15-3.00
(m, 4 H), 1.59-1.19 (m, 16 H), 0.87-0.79 (2t, 12 H); MS (ESI)
203 [(M + 1)+, 100], 132 (80).
Representative Procedures for the Synthesis of Target
Compounds Described in Tables 2-7. Preparation of {2-[2-(2-
Carbamoyl-1-cyclobutylmethyl-2-oxoethylcarbamoyl)-6,6-di-
A solution of methyl ester obtained in the previous step (3 g,
10.5 mmol) in THF/H2O (1:1) was treated with LiOH‚H2O (645
mg, 15.75 mmol) and stirred at room temperature for 2 h. The
reaction mixture was acidified with aqueous HCl (1 M, 15 mL)
and concentrated in vacuo. The residue was dried in a vacuum.
A solution of the acid in CH2Cl2 (50 mL) and DMF (25 mL)
was treated with NH4Cl (2.94 g, 5.5 mmol), EDCI (3.15 g, 16.5
mmol), HOOBt (2.69 g, 16.5 mmol), and NMM (4.4 g, 43.6 mmol).

6084 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Venkatraman et al.
methyl-3-azabicyclo[3.1.0]hex-3-yl]-1-cyclohexyl-2-oxoethyl}-
carbamic Acid tert-Butyl Ester (46; R4 ) Cyclohexyl, R5 )
OtBu, R3 ) Methylcyclobutyl). Similar methods with minor
modifications were employed for the syntheses of target compounds
described in Tables 2-7. The amino ester 2210 was prepared
following the method of Zhang and Madalengoitia with some
modifications. The introduction of the cyclopropyl ring was
achieved by the reaction of 20 with the corresponding isopropy-
lphosphonium ylide, and the Boc group was cleaved using
methanolic HCl.
A solution of Boc-Chg-OH (Senn Chemicals, 6.64 g, 24.1 mmol)
and amine salt 22 (4.5 g, 22 mmol) in CH2Cl2 (100 mL) at 0 °C
was treated with BOP reagent and stirred at room temperature for
15 h. The reaction mixture was concentrated in vacuo and diluted
with aqueous 1 M HCl. The aqueous layer was extracted with
EtOAc (3 × 200 mL). The combined organic layers were washed
with saturated NaHCO3 (200 mL), dried (MgSO4), filtered,
concentrated in vacuo, and purified by chromatography (SiO2,
EtOAc/hexanes, 3:7) to obtain 23 (R4 ) cyclohexyl, R5 ) OtBu,
6.0 g) as a colorless solid. 1H NMR (500 MHz, DMSO-d6) δ 7.05
(d, 1 H, J ) 8.5 Hz), 4.18 (s, 1 H), 4.01 (d, 1 H, J ) 10 Hz), 3.88
(t, 1 H, J ) 9.0 Hz), 3.76 (dd, 1 H, J ) 5.0 and 5.5 Hz), 3.65 (s,
3 H), 1.79-1.53 (m, 7 H), 1.41 (d, 2 H), 1.35 (s, 9 H), 1.13 (s, 3
H), 0.88 (s, 3 H); 13C NMR (CDCl3, 125 MHz) δ 172.4, 171.6,
156.5, 78.8, 59.7, 57.4, 52.8, 47.4, 39.3, 30.5, 29.6, 29.3, 29.0, 27.9,
26.8, 26.7, 26.4, 26.2; MS (ESI) 431 [(M + Na)+, 60], 409 (90),
353 (80), 309 (100).
28.2, 27.9, 27.5, 27.1, 27.0, 26.84, 26.8, 26.5, 26.4, 26.3, 19.6, 18.7,
18.6. HRMS calcd for C29H47N4O6: 547.3496 (M + H)+. Found:
547.3453
Synthesis of (1R,5S)-N-[3-Amino-1-(cyclobutylmethyl)-2,3-
dioxopropyl]-3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-
3,3-dimethyl-1-oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-
t
t
2(S)-carboxamide (70; R4 ) Bu, R ) BuNH). A solution of
Boc-tert-Lue (5.0 g 21.6 mmol) in dry CH2Cl2/DMF (50 mL, 1:1)
was cooled to 0 °C and treated with the amine salt 22 (5.3 g, 25.7
mmol), NMM (6.5 g, 64.8 mmol), and BOP reagent (11.6 g, 25.7
mmol). The mixture was stirred at room temperature for 24 h,
diluted with aqueous HCl (1 M), and extracted with CH2Cl2. The
combined organic layers were washed with HCl (aqueous, 1 M),
saturated NaHCO3, and brine, dried (MgSO4), filtered, concentrated
in vacuo, and purified by chromatography (SiO2, acetone/hexane,
1:5) to yield 3-[2-(3-tert-butylureido)-3,3-dimethylbutyryl]-6,6-
dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester
as a colorless solid. 1H NMR (CD3OD, 400 MHz) δ 6.48 (d, 1 H,
J ) 9.3 Hz), 4.35 (s, 1 H), 4.21(d, 1 H, J ) 9.9 Hz), 4.04-3.88
(m, 2 H), 3.73 (s, 3 H), 1.58-1.54 (m, 1 H), 1.41 (s, 9 H), 1.49-
1.41 (m, 1 H), 1.07 (s, 3 H), 1.02 (s, 9 H), 0.93 (s, 3 H); 13C NMR
(CDCl3, 125 MHz) δ 172.4, 171.5, 156.4, 80.0, 77.7, 59.6, 59.0,
52.7, 48.1, 35.4, 30.8, 28.7, 27.8, 26.7, 19.9, 12.8; MS (ESI) 405
[(M + Na)+, 100], 383 [(M + 1)+, 40], 327 (70), 283 (100).
A solution of methyl ester (4.0 g, 10.46 mmol) was dissolved in
HCl (4 M solution of dioxane) and stirred at room temperature for
3 h. The reaction mixture was concentrated in vacuo to obtain the
amine hydrochloride salt used in the next step without further
purification. A solution of the amine salt (397 mg, 1.24 mmol) in
CH2Cl2 (10 mL) was cooled to -78 °C and treated with tert-butyl
isocyanate (250 mg, 2.5 mmol) and stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo, and
the residue was diluted with aqueous HCl (1 M) and extracted with
CH2Cl2. The combined organic layers were washed with aqueous
HCl (1 M), saturated NaHCO3, and brine. The organic layers were
dried, filtered, and concentrated in vacuo, and the residue was
purified by chromatography (SiO2, acetone/hexanes, 1:4) to yield
23 as a colorless solid. 1H NMR (CD3OD, 400 MHz) δ 6.05 (s, 1
H), 5.88 (d, 1 H, J ) 9.3 Hz), 4.32 (s, 1 H), 4.27 (d, 1 H, J ) 9.9
Hz), 4.06 (d, 1 H, J ) 10.4 Hz), 3.87 (dd, 1 H, J ) 4.9 and 5.5
Hz), 1.54 (dd, 1 H, J ) 4.9 and 2.2 Hz), 1.44 (d, 1 H, J ) 7.2 Hz),
1.25 (s, 9 H), 1.04 (s, 3 H), 0.99 (s, 9 H), 0.92 (s, 3 H); MS (ESI)
404 [(M + Na)+, 90], 382 [(M + 1)+, 50], 319 (40), 283 (100),
213 (70), 170 (30).
A solution of methyl ester 23 (R4 ) cyclohexyl, R5 ) OtBu, 4.0
g, 9.79 mmol) in THF/H2O (1:1) was treated with LiOH‚H2O (401
mg, 9.79 mmol) and stirred at room temperature for 3 h. The
reaction mixture was acidified with aqueous HCl and concentrated
in vacuo to obtain the free acid that was used in subsequent coupling
1
without further purification. H NMR (CDCl3, 500 MHz) δ 5.23
(d, 2 H, J ) 9.8 Hz), 4.49 (s, 1 H), 4.22 (t, 1 H, J ) 8.8 Hz), 4.06
(d, 1 H, J ) 10.5 Hz), 3.38 (dd, 1 H, J ) 4.7 and 5.8 Hz), 1.88-
1.69 (m, 8 H), 1.56-1.53 (m, 1 H), 1.45 (m, 9 H), 1.33-1.18 (m,
4 H), 1.11 (s, 3 H), 0.98 (s, 3 H); 13C NMR (CDCl3, 125 MHz) δ
173.3, 156.2, 80.1, 77.7, 60.4, 57.3, 48.0, 40.7, 30.1, 29.8, 29.0,
28.7, 27.7, 26.6, 26.3, 26.2, 19.8, 13.0; MS (ESI) 417 [(M + Na)+,
44], 395 [(M + 1)+, 30] 339 (40), 295 (100).
A solution of acid 23 (R4 ) cyclohexyl, R5 ) OtBu, 1.5 g, 3.74
mmol) in DMF/CH2Cl2 (1:1 50 mL) was treated with amine salt
10 (R3 ) cyclobutylmethyl) (772 mg, 3.74 mmol), EDCI (1.07 g,
5.61 mmol), HOOBt (959 mg, 5.61 mmol), and NMM (2.15 mL,
14.96 mmol) at -10 °C. The reaction mixture was stirred at 0 °C
for 48 h and concentrated in vacuo. The residue was diluted with
aqueous 1 M HCl and extracted with CH2Cl2. The combined organic
layers were extracted with aqueous NaHCO3, aqueous HCl, and
brine, dried (MgSO4), filtered, and concentrated in vacuo to obtain
the hydroxyamide (2.08 g) as a tan solid.
A solution of hydroxyamide (2.08 g, 3.79 mmol) in toluene and
DMSO (1:1, 20 mL) at 0 °C was treated with EDCI (7.24 g, 37.9
mmol) and dichloroacetic acid (2.42 g, 19.9 mmol) and stirred at
room temperature. for 4 h. The reaction mixture was diluted with
CH2Cl2 and washed with saturated NaHCO3 and brine. The organic
layer was dried (MgSO4), filtered, concentrated, in vacuo, and
purified by chromatography (SiO2, acetone/hexanes, 3:7) to yield
46 as a colorless solid. 1H NMR (DMSO-d6, 500 MHz) δ 8.30 (d,
1 H, J ) 6.5 Hz), 8.12 (d, 1 H, J ) 7.5 Hz), 8.02 (s, 1 H), 8.00 (d,
1 H, J ) 12.0 Hz), 7.77 (brd, 2 H), 6.96 (d, 1 H, J ) 6.0 Hz), 6.94
(d, 1 H, J ) 6.0 Hz), 4.89-4.96 (m. 2 H), 4.27 (s, 1 H), 4.25 (s,
1 H), 3.94 (d, 2 H, J ) 9.5 Hz), 3.83-3.89 (m, 2 H), 3.76-3.79
(m, 1 H), 3.70-3.73 (m, 1 H), 2.46-2.48 (m, 1 H), 2.31-2.37
(m, 1 H), 1.93-2.01 (m, 4 H), 1.74-1.82 (m, 8 H), 1.56-1.64
(m, 16 H), 1.42-1.47 (m, 2 H), 1.37 (s, 9 H), 1.35 (s, 9 H), 1.37-
1.35 (m, 2 H), 1.10-1.11 (brd, 6 H), 1.02 (s, 3 H), 1.01 (s, 3 H),
0.9 (s, 3 H), 0.86 (s, 3 H), 1.03-0.87 (bm, 4 H); 13C NMR (DMSO-
d6, 125 MHz) δ 198.7, 198.1, 171.8, 171.6, 171.3, 171.2, 163.8,
163.7, 156.5, 78.8, 78.7, 60.7, 60.2, 57.7, 57.6, 52.8, 47.8, 47.7,
39.5, 39.2, 37.7, 37.6, 33.1, 31.6, 29.6, 29.5, 29.0, 28.8, 28.7, 28.3,
A solution of methyl ester (381 mg, 1.0 mmol) in THF/H2O (1:
1, 5 mL) was treated with LiOH‚H2O (62 mg, 1.5 mmol) and stirred
at room temperature for 3 h. The reaction mixture was acidified
with aqueous HCl and concentrated in vacuo to obtain the free acid
1
that was used in a subsequent step without purification. H NMR
(DMSO-d6, 400 MHz) δ 5.94 (s, 1 H), 5.87 (d, 1 H, J ) 10.3 Hz),
4.13 (d, 1 H, J ) 10.3 Hz), 4.08 (s, 1 H), 3.97 (d, 1 H, J ) 10.3
Hz), 3.73 (dd, 1 H, J ) 5.9 and 4.4 Hz), 1.89 (s, 1 H), 1.46 (dd, 1
H, J ) 5.1 and 2.2 Hz), 1.36 (bd, 1 H, J ) 7.3 Hz), 1.15 (s, 9 H),
0.98 (s, 3 H), 0.89 (s, 9 H), 0.80 (s, 3 H); 13C NMR (CDCl3, 125
MHz) δ 174.9, 173.8, 158.1, 77.7, 60.1, 58.3, 51.0, 48.5, 35.4, 30.5,
29.8, 27.9, 26.8, 26.7, 19.9, 12.9; MS (ESI) 390 [(M + Na)+, 20],
368 [(M + 1)+, 20], 269 (100), 312 (20).
A solution of acid (254.9 mg, 0.69 mmol) in DMF/CH2Cl2 (1:1,
5.0 mL) was treated with amine 10 (159 mg, 0.763 mmol), EDCI
(199 mg, 1.04 mmol), HOOBt (169.5 mg, 1.04 mmol), and NMM
(280 mg, 2.77 mmol) at -20 °C. The reaction mixture was stirred
at -20 °C for 48 h and concentrated in vacuo. The residue was
diluted with aqueous 1 M HCl and extracted with EtOAc. The
combined organic layers were extracted with aqueous NaHCO3,
aqueous HCl, and brine, dried (MgSO4), filtered, and concentrated
in vacuo to obtain a tan solid (470 mg) that was used in the next
reaction without further purification.
A solution of hydroxyamide (470 mg, 0.9 mmol) in toluene and
DMSO (1:1 20 mL) at 0 °C was treated with EDCI (1.72 g, 9.0
mmol) and dichloroacetic acid (0.37 mL, 4.5 mmol) and stirred at
room temperature for 4 h. The reaction mixture was diluted with

Carboxamide for Treatment of Hepatitus C
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CH2Cl2 and washed with saturated NaHCO3, and brine. The organic
layer was dried (MgSO4), filtered, concentrated in vacuo, and
purified by chromatography (SiO2, acetone/hexanes, 3:7) to yield
1
70 as a colorless solid as a mixture of diasteromers. H NMR
(DMSO-d6, 500 MHz) δ 8.26 (d, 1 H, J ) 7.0 Hz), 8.00 (s, 1 H),
7.75 (s, 1 H), 5.96 (s, 1 H), 5.84 (d, 1 H, J ) 10 Hz), 4.96 (m, 1
H), 4.28 (s, 1H), 4.11 (d, 1 H, J ) 11 Hz), 3.94 (d, 1H, J ) 10
Hz), 3.73 (dd, 1 H, J ) 10 and 5 Hz), 2.48 (m, 1 H), 1.95 (m, 2
H), 1.61 (m, 1 H), 1.59 (m, 1 H), 1.77 (m, 1 H), 1.57 (m, 1 H),
1.74 (m, 2 H), 1.42 (dd, 1 H, J ) 7.5 and 5 Hz), 1.28 (d, 1 H, J )
7.5 Hz), 1.17 (s, 9 H), 1.01 (s, 3 H), 0.90 (s, 9 H), 0.85 (s, 3 H),
8.15 (d, 1 H, J ) 7.0 Hz), 7.96 (s, 1 H), 7.74 (s, 1 H), 5.96 (s, 1
H), 5.86 (d, 1 H, J ) 10 Hz), 4.85 (m, 1 H), 4.27 (s, 1H), 4.13 (d,
1 H, J ) 11.0 Hz), 3.97 (d, 1H, J ) 10 Hz), 3.76 (dd, 1 H, J ) 10
and 5 Hz), 2.36 (m, 1 H), 1.97 (m, 2 H), 1.60 (m, 2 H), 1.78 (m,
1 H), 1.64 (m, 1 H), 1.75 (m, 2 H), 1.44 (dd, 1 H, J ) 7.5 and 5
Hz), 1.27 (d, 1 H, J ) 7.5 Hz), 1.17 (s, 9 H), 1.00 (s, 3 H), 0.89
(s, 9 H), 0.82 (s, 3 H); 13C NMR (DMSO-d6, 125 MHz) δ 197.8,
170.9, 170.8, 162.8, 157.4, 59.1, 56.8, 51.8, 48.9, 47.4, 36.7, 34.0,
32.0, 30.6, 29.1, 27.8, 27.3, 27.1, 26.4, 26.1, 18.5, 17.7, 12.5. 197.1,
171.1, 170.7, 163.0, 157.3, 59.4, 56.9, 52.1, 48.9, 47.4, 36.6, 34.0,
32.1, 30.5, 29.1, 27.9, 27.4, 26.8, 26.4, 26.1, 18.5, 17.8, 12.4. HRMS
calcd for C27H46O5N5 [M + 1]+ 520.3499. Observed: 520.3514.
Anal Calcd for C27H45N5O5: C, 62.40%; H, 8.73%; N, 13.48%.
Found: C, 62.48%; H, 8.94%; N, 13.19%.
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Note Added after ASAP Publication. This manuscript was
released ASAP on September 7, 2006 with an error in an
author’s name. The correct version was posted on September
20, 2006.
Supporting Information Available: NMR and MS data for
compounds 26-74. This material is available free of charge via
the Internet at http://pubs.acs.org.
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(29) Rats were orally administered with 10 mg of compound in 20%
HPBCD. Blood was withdrawn periodically and pooled. AUC was
calculated over a 0-6 h period.
(30) Synthesis of hydroxy acid 39: Acid 13 (R3 ) C3H7) was converted
to the methyl ester and oxidized using Dess-Martin reagent to yield
intermediate methyl 3-tert-butoxycarbonylamino-2-oxohexanoate.
Deprotection of the Boc group of methyl 3-tert-butoxycarbonylamino-
2-oxohexanoate and coupling with 24 yielded the ketoester, precursor
of 39. Hydrolysis of methyl ester using aqueous NaHCO3 in
2-propanol yielded ketoacid 39.
JM060325B