Potent inhibitors of the hepatitis C virus NS3 protease: Use of a novel P2 cyclopentane-derived template

Article abstract of DOI:10.1016/j.bmc.2006.04.008

The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low nanomolar acti

Full text of DOI:10.1016/j.bmc.2006.04.008

Bioorganic & Medicinal Chemistry 14 (2006) 5136–5151
Potent inhibitors of the hepatitis C virus NS3 protease:
Use of a novel P2 cyclopentane-derived template
Per-Ola Johansson,a Marcus Ba¨ck,a Ingemar Kvarnstro¨m,a Katarina Jansson,b
Lotta Vrang,b Elizabeth Hamelink,b Anders Hallberg,c
a,b,
b,d,
*
˚
*
Asa Rosenquist
and Bertil Samuelsson
aDepartment of Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden
bMedivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
cDepartment of Medicinal Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden
dDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 1 February 2006; revised 30 March 2006; accepted 4 April 2006
Available online 3 May 2006
Abstract—The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising
new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low
nanomolar activities, were identified from a series of novel inhibitors incorporating a trisubstituted cyclopentane dicarboxylic acid
moiety as a surrogate for the widely used N-acyl-(4R)-hydroxyproline in the P2 position.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
chymotrypsin-like serine protease,2b responsible for the
cleavage at four out of five sites of the non-structural
portion of the polyprotein.8 The NS3 protease has been
shown to be essential for viral replication9 and valida-
tion of this drug target was first demonstrated in a
two-day proof-of-concept study in man with the very
potent and highly specific product-based macrocyclic
NS3 protease inhibitor BILN 2061 (Fig. 1).10 BILN
2061 contains a trisubstituted N-acyl-(4R)-hydroxypro-
line moiety in the P2 position, a theme that can be seen
in many potent inhibitors reported in the last
years,2b,10,11 for example, the linear compound A2b
(Fig. 1).
Hepatitis C virus (HCV) infection is a serious and pre-
dominantly chronic disease which over time leads to cir-
rhosis and hepatocellular carcinoma and which today is
the leading cause of liver transplantation in the devel-
oped world.1 An estimated 170 million people, 3% of
the world population, are infected with the virus2 and
the existing therapy consisting of PEGylated a-interfer-
on in combination with the nucleoside analog ribavirin
is only effective in ꢀ50% of genotype 1 infected pa-
tients.3 In addition this therapy is associated with severe
adverse effects leading to discontinuation of treatment in
some patient populations indicating the paramount need
for the development of new, effective, and well-tolerated
treatment paradigms.2b In recent years a vast number of
reports have appeared describing different classes of
compounds targeting key viral enzymes.4,5 One of the
most promising and well-characterized targets to
emerge is the NS3 protease,6,7 a 180 amino acid long
A number of product-based NS3 protease inhibitors
based on other motifs and strategies have also been
described such as those incorporating the cysteine mimic
(S)-2-amino-4,4-difluorobutyric acid in the P1 posi-
tion.12 VX-950 (Fig. 1) is the most advanced NS3 prote-
ase inhibitor belonging to another class of NS3
inhibitors, namely the electrophilic or serine-trap inhib-
itors. The most prominent feature of this class of inhib-
itors is that they contain a reactive electrophilic center at
the cleavage site. Whilst this results in covalent interac-
tion with the catalytic Ser139 of the active site of the
NS3 enzyme, the kinetics observed is generally fully
reversible. VX-950, an a-ketoamide inhibitor of this
Keywords: HCV; NS3; Protease inhibitor; Cyclopentane-derived P2
scaffold.
*
Corresponding authors. Tel.: +46 8 54683100; fax: +46 8 54683199;
e-mail addresses: asa.rosenquist@medivir.se; bertil.samuelsson@
medivir.se
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.008

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5137
NH
N
OMe
OMe
N
O
N
O
S
H
N
O
H
N
O
O
N
N
O
H
N
O
N
H
OH
OH
O
O
O
N
H
O
O
BILN 206110
A2b
Ki = 0.30 nM (HCV 1a)
Ki = 0.66 nM (HCV 1b)
IC50 = 1 nM
OMe
N
O
H
N
H
N
O
O
N
O
O
NH
H
N
OH
NH
N
H
O
O
N
O
O
HN
O
NH
O
O
N
13
Ki = 22 nM (HCV 1a)
VX-95013
Ki = 47 nM (HCV 1a)
Ki = 100 nM (HCV 1b)
EC50 = 400 nM
Figure 1. Two potent HCV NS3 protease inhibitors incorporating a 4-hydroxyproline moiety (BILN 2061 and A), the electrophilic inhibitor VX-950,
and compound 13, one of the most potent inhibitors in our novel series containing a trisubstituted cyclopentane moiety as a hydroxyproline mimic.
class displaying Ki values of 47 and 100 nM for HCV
NS3 1a and 1b, respectively, was recently reported to
have completed phase Ib clinical trials with promising
results.13
N
O
O
O
O
O
O
I
II
III
O
O
The amino acid L-proline has frequently been used as a
building block in peptidomimetic inhibitors targeting a
number of proteases. L-Proline has been incorporated
in inhibitors targeting, for example, HIV,14 angiotensin
converting enzyme (ACE),15 thrombin,16 and also
HCV.17 Previous work on thrombin inhibitors from
our laboratory has shown that the N-acylproline moiety
I (Fig. 2) can be replaced with isosteric five-membered
ring templates, exemplified by structures II and III18
(Fig. 2), delivering inhibitors with low to modest inhib-
itory activities toward thrombin.
N
O
O
O
O
IV
V
Figure 2. N-Acylproline (I), previously reported N-acylproline isos-
teres (II and III), N-acyl-(4R)-hydroxyproline (IV), and our cyclopen-
tane-based N-acyl-(4R)-hydroxyproline isostere (V) used in the
synthesis of novel HCV NS3 protease inhibitors.
2. Results and discussion
2.1. Chemistry
We now report on the synthesis of HCV NS3 protease
inhibitors incorporating a novel trisubstituted cyclopen-
tane moiety (V, Fig. 2) in the P2 position, exemplified by
inhibitor 13 (Fig. 1, Scheme 3, Table 1), displaying a
highly promising Ki value of 22 nM. The synthesis of
these novel inhibitors starting from the pivotal interme-
diate trans-(3R,4R)-bis(methoxycarbonyl)cyclopenta-
none ((ꢁ)-1)19 delivering several target compounds
with nanomolar activity is discussed. In addition, an
extensive SAR analysis of these new inhibitors has been
performed.
Figure 3 depicts the structures of the target molecules
synthesized all encompassing the trisubstituted cyclo-
pentane scaffold V.
The R1 amino acid derivatives B, C, and E were all
commercially available or readily synthesized from suit-
ably protected and commercially available precursors.
The vinylcyclopropane amino acid D was synthesized
according to literature procedure20 (Fig. 3). The R2

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P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
Table 1. Target molecules, synthesis methods, total yields, and inhibition constants
OMe
N
R-OH :
OH
Compound
Structure
Method
Yielda (%) (5 or 6 steps)
Kib (lM) HCV NS3 1a
% Inhc at 10 lM
OR
H
N
H
N
O
O
14
I
30
ND
21
OH
N
H
O
O
O
O
OR
OR
H
N
H
N
O
O
O
15
16
17
18
19
9
I
21
66
42
32
40
61
61
30
37
23
ND
2.3
65
100
35
OH
OH
N
H
O
O
O
O
O
O
O
O
H
N
H
N
O
I
N
H
OR
OR
OR
OR
OR
OR
OR
OR
H
N
H
N
O
O
O
O
O
O
O
O
O
I
ND
ND
6.6
OH
OH
OH
OH
OH
OH
OH
OH
N
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
I
37
N
H
H
N
H
N
I
H
N
N
H
H
N
H
N
I
1.7
H
N
N
H
H
N
H
N
O
20
13
21
22
I
1.2
N
H
O
H
N
H
N
O
O
O
O
II
II
II
0.022
0.016
>10
H
N
N
H
H
N
H
N
O
O
N
H
N
H
N
H
N
H
N
N
(continued on next page)

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5139
Table 1 (continued)
Compound
Structure
Method
Yielda (%) (5 or 6 steps)
Kib (lM) HCV NS3 1a
% Inhc at 10 lM
OR
H
N
H
N
O
O
23
24
25
II
23
2.7
OH
N
H
O
O
OR
H
N
H
N
O
O
II
II
40
16
6.9
OH
N
H
O
O
OR
H
N
H
N
O
O
0.56
H
N
OH
CHF2
N
H
O
O
O
a Total yield over five steps for Method I and over six steps for Method II.
b ND, not determined.
c % Inh at 10 lM for compounds where the Ki values have not been measured or where it is important for the SAR discussion.
2-phenyl-7-methoxy-4-quinolinol moiety (F) was also
synthesized according to a published procedure.2b,21
The R3 dipeptides or capped amino acids, G–Q, were all
synthesized employing standard peptide coupling condi-
tions and standard amino acid protection/deprotection
protocols using commercially available and suitably pro-
tected amino acids with the exception ofthe N-methylated
compound O, where Fmoc-Chg-OH initially was
N-methylated using paraformaldehyde and p-toluene-
sulfonic acid in refluxing toluene followed by the treat-
ment with triethylsilane (Et3SiH) and trifluoroacetic
acid (TFA) in chloroform,22 before using the standard
peptide synthesis protocol. The corresponding dipeptide
where the nitrogen on tert-butyl glycine had been methyl-
ated was also prepared using the same procedure as in the
synthesis of O. However, the properties of an inhibitor
incorporating this R3-group could not be evaluated, since
coupling of the corresponding dipeptide to the template
was unsuccessful in spite of evaluating a large number
of coupling reagents and conditions. The low coupling
reactivity is likely to be due to steric hindrance.
Scheme 2 depicts the synthesis of target molecule 9
according to Method I, also employed in the synthesis
of target compounds 14–20 (Table 1). The lactone (4)
was opened by allowing it to react with H-Nva-OtBu,
diisopropylethylamine (DIEA), and 2-hydroxypyridine25
in refluxing THF togive amide 6 in 96% yield. Mitsunobu-
like conditions using 2-phenyl-7-methoxy-4-quinolinol,
triphenylphosphine, and diisopropyl azodicarboxylate
(DIAD) in THF then provided compound 7 in 78%
yield.11c
The methyl ester in compound 7 was then hydrolyzed
using lithium hydroxide in dioxane/water 1:1, and the
corresponding acid was then coupled with amine L
(Fig. 3) using O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate (HATU) and
DIEA in DMF to give compound 8 in 81% yield. Final-
ly, the tert-butyl ester in 8 was hydrolyzed by reaction
with TFA and Et3SiH in DCM26 yielding 9 in quantita-
tive yield (Scheme 2).
Method II, used for the synthesis of target molecules 13
and 21–25 (Table 1), is described in Scheme 3. Initial
attempts to open the tert-butyl scaffold (5) directly with
the vinylcyclopropyl amino acid D using the same con-
ditions as in Method I (DIEA, 2-hydroxypyridine in
refluxing THF) were not successful. Instead, scaffold 5
was opened by careful treatment with lithium hydroxide
in dioxane/water 1:1 at 0 ꢁC to afford the corresponding
carboxylic acid, which was then coupled with D using
HATU and DIEA in DMF to give the amide 10 in
89% total yield (Scheme 3).
The synthesis of the two bicyclic lactone scaffolds
employed in the synthesis of the target molecules is out-
lined in Scheme 1.
trans-(3R,4R)-bis(Methoxycarbonyl)cyclopentanone ((ꢁ)-
1), used as starting material, was prepared according
to literature procedure.19a Treatment of (ꢁ)-1 with sodi-
um borohydride in methanol furnished the alcohol 223 in
76% yield. Compound 2 was then allowed to react with
sodium hydroxide in methanol to hydrolyze the methyl
esters, followed by reaction with acetic anhydride in pyr-
idine24 to provide the bicyclic lactone 323 in 88% total
yield. The methyl ester protected scaffold (4) was affor-
ded in 81% yield by reacting 3 with methyl iodide and
silver (I) oxide in acetone. Treatment of 3 with di-tert-
butyl dicarbonate (Boc2O) and 4-dimethylaminopyri-
dine (DMAP) in dichloromethane (DCM) yielded the
tert-butyl ester protected scaffold (5) in 52% (Scheme 1).
Applying similar Mitsunobu conditions with 2-
phenyl-7-methoxy-4-quinolinol as used in the synthesis
of compound 7 furnished compound 11 in 68% yield.11c
Hydrolysis of the tert-butyl ester in 11 was then per-
formed with TFA and Et3SiH in DCM,26 yielding the
corresponding acid, which was coupled to dipeptide L
(Fig. 3) using HATU and DIEA in DMF to afford

5140
P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
R2
O
H
N
H
N
R1
R3
O
O
R3-NH2
H2N-R1
O
O
O
O
O
O
O
O
O
O
H2N
H2N
NH2
NH2
N
H
N
H
O
O
O
O
O
O
G
H
B
C
O
O
H2N
H2N
NH2
NH2
N
H
N
H
O
O
CHF2
J
I
E
D
O
O
O
O
O
H
N
H
N
NH2
NH2
NH2
NH2
R2-OH
N
H
N
H
O
O
O
O
O
L
K
OMe
N
N
O
NH2
OH
N
H
N
H
F
M
N
O
H
N
NH2
N
N
H
O
P
O
NH2
N
H
Q
Figure 3. A general picture of the central cyclopentane scaffold and the different R1, R2, and R3 substituents that were used in this report.
O
O
compound 12 in 74% total yield. Finally, the ethyl ester
was hydrolyzed using lithium hydroxide in THF/MeOH/
water 2:1:1 to yield target compound 13 in 67%
(Scheme 3).
O
O
i
O
HO
O
O
O
O
(-)-1
2 (76%)
ii, iii
3. Biological data and structure–activity relationships
O
iv
v
O
O
All products were screened against the HCV NS3 1a
protease and the percent inhibition was determined at
three concentrations: 10, 1, and 0.1 lM. The Ki values
were determined for the most potent inhibitors after
the initial screenings. Table 1 summarizes all inhibitors
synthesized and the methods used for the synthesis of
each individual inhibitor along with the total yields.
The Ki values, if measured, and the percent inhibition
at a concentration of 10 lM for selected compounds
are also included in Table 1.
O
O
O
4 (81 %)
O
OH
O
O
3 (88%)
O
O
5 (52 %)
Scheme 1. Reagents and conditions: (i) NaBH4, MeOH, 0 ꢁC; (ii)
NaOH (1 M), MeOH; (iii) Ac2O, pyridine; (iv) MeI, Ag2O, acetone;
(v) Boc2O, DMAP, CH2Cl2.

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5141
Method I
N
OMe
O
OH
O
i
ii
O
H
N
H
N
O
O
O
O
O
O
O
O
O
O
O
O
4
6 (96%)
7 (78%)
iii, iv
N
OMe
N
O
OMe
O
H
N
H
N
H
N
H
N
O
O
O
O
v
O
H
N
H
N
OH
N
H
N
H
O
O
O
O
O
O
9 (100%)
8 (81%)
Scheme 2. Reagents and conditions: (i) H-Nva-OtBu, DIEA, 2-hydroxypyridine, THF, reflux; (ii) 2-phenyl-7-methoxy-4-quinolinol, PPh3, DIAD,
THF; (iii) LiOH, dioxane/H2O 1:1; (iv) L, HATU, DIEA, DMF; (v) TFA, Et3SiH, CH2Cl2.
Method II
OMe
N
O
OH
O
i, ii
iii
O
H
N
H
N
O
O
O
O
O
O
O
O
O
O
O
O
5
10 (89%)
11 (68%)
iv, v
OMe
OMe
N
N
O
O
vi
H
N
H
N
H
N
O
H
N
O
O
O
O
H
N
H
N
OH
N
H
N
H
O
O
O
O
13 (67%)
12 (74%)
O
O
Scheme 3. Reagents and conditions: (i) LiOH, dioxane/H2O 1:1, 0 ꢁC; (ii) D, HATU, DIEA, DMF; (iii) 2-phenyl-7-methoxy-4-quinolinol, PPh3,
DIAD, THF; (iv) TFA, Et3SiH, CH2Cl2; (v) L, HATU, DIEA, DMF; (vi) LiOH, THF/MeOH/H2O 2:1:1.
All the inhibitors discussed below contain a P2 2-phenyl-
7-methoxy-4-quinoline substituent, which has been
reported to play an important role in stabilizing the cat-
alytic machinery in the correct geometry by shielding
that part of the protease from exposure to solvent,27
and is a substituent found to furnish potent inhibi-
tors,2b,10b,11b,11c for example, compound A2b (Fig. 1).
In addition, the P2 aryl substituent likely interacts
favorably with the helicase domain of the NS3 protein.28
P3–P4 substituents of the compounds incorporating this
new template have a reversed direction, compared with
the general N-C direction seen in, for example, inhibitor
A. Another feature to be considered is that this P2 cyclo-
pentane template is not planar at the 1-position of the
ring, since it is sp3 hybridized, whereas proline has a
nitrogen atom in the corresponding ring position and
is planar.
The X-ray crystal structure, PDB entry 1CU1, of a
bound product of the NS3-mediated cleavage28a (the
C-terminal of the full length single strand NS3 con-
First, it was important to establish the preferred stereo-
chemistry at the P3–P4 positions of these inhibitors. The

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P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
struct) was used as starting point for the modeling to
predict the stereochemical requirements of the substitu-
ents at the P3–P4 positions (Fig. 4).
chain of the product. The same pattern of binding is val-
id for the side chain and the amide of the P4 substituent
(Fig. 6). In view of the molecular modeling performed it
was thus anticipated that the L-configuration would be
generally preferred for both the P3 and P4 amino acids.
Compound 14 (Table 1), having the D-configurations at
the P3 and P4 positions, was thus aligned with the NS3
product using GASP (Genetic Algorithm Similarity Pro-
gram) included in Sybyl 7.1 (Tripos Inc.).29 As depicted
in Figure 5, the side chains having D-configurations in
the P3–P4 positions do not overlay at all well with the
side chains of the product and no good alignment could
be obtained.
To verify the modeling results experimentally, inhibitor
14, with D stereochemistry on both the valine and the
cyclohexyl glycine moieties, and inhibitor 15 with the
L-configuration on the corresponding residues were syn-
thesized. Compounds 14 and 15 are both weakly active
compounds but 15 is clearly the most active of the two
displaying 65% inhibition at a concentration of 10 lM
compared to 21% inhibition exhibited by 14, which is
in agreement with the modeling predictions. The
relatively low potency of these two compounds, and
compound 15 in particular, can obviously be attributed
to the use of the non-optimized Abu P1 substituent as a
cysteine mimic. Compound 16 encompasses the report-
edly better norvaline as the P1 substituent, which yielded
improved activity properties, with a measurable Ki value
In contrast, alignment of compound 15 (Table 1), hav-
ing the L-amino acids at P3 and P4, gives a very good
overlay with the side chains of the bound cleavage prod-
uct (Fig. 6). The carbonyl of the acyl cyclopentyl moiety
adopts the same position as the P3-carbonyl of the prod-
uct and the P3-NH of compound 15 shows the same
interaction as the P2-NH of the product (PDB entry
1CU1) and the P3-side chain is positioned as the P3 side
P4
O
O
OH
P2
O
O
H
N
H
N
H
N
N
H
N
H
OH
OH
O
O
P1
P3
Figure 4. The C-terminus of the helicase domain (a product of the NS3-mediated cleavage of the NS3–NS4A junction of the HCV polyprotein) in its
bound conformation to the NS3 active site obtained from the published X-ray crystal structure (PDB entry 1CU1), and a simple chemical structure
showing the amino acid sequence in question.
Figure 5. A modeled structure of compound 14 containing D-amino acids at the P3 and P4 positions (green) superimposed on the bound C-terminus
helicase residue (magenta). The P3 and P4 side chains do not overlay with the side chains of the product and will not fit in the S3 and S4 pockets of
the NS3 protease.

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5143
Figure 6. Compound 15 (gray) containing the natural L-amino acids in P3 and P4 superimposed on the bound C-terminus helicase residue (magenta).
The picture clearly shows that L-configurations of P3 and P4 are required to get an overlay that fits well with the conformation of the bound product.
of 2.3 lM and 100% inhibition at a concentration of
10 lM. In order to rule out the possibility that combina-
tions of D-L or L-D configurations of the P3–P4 substit-
uents could provide more potent inhibitors, compounds
17 and 18 were prepared with 17 having D-valine and L-
cyclohexyl glycine and 18 having the opposite (L-D) con-
figurations on the two moieties. The two compounds, 17
and 18, were both found to display low activity, 35%
and 37% inhibition, respectively, at 10 lM concentra-
tions, which further confirms the importance of L-con-
figurations of the P3–P4 residues. All compounds
discussed up to this point incorporate a metabolically
labile methyl ester capping group on the P4 amino acid.
Replacing the methyl ester with a methyl amide deliv-
ered inhibitors with slightly lower potency as can be seen
by comparing the methyl amide compound 19 with the
corresponding methyl ester compound 16 having Ki
values of 6.6 and 2.3 lM, respectively. It has previously
been shown that substitution of valine for tert-butyl gly-
cine can yield more potent NS3 inhibitors.2b This mod-
ification furnished compound 9, which is almost four
times more potent than the corresponding valine-con-
taining compound 19, exhibiting a promising Ki value
of 1.7 lM. The corresponding methyl ester capped com-
pound 20 is in this case essentially equipotent with a Ki
value of 1.2 lM. This indicates that the metabolically
more stable methyl amide functions well as a methyl
ester isostere in more optimized compounds.
potent than the corresponding norvaline substituted
compound 9, exhibiting the impressive Ki value of
22 nM. To further investigate the binding modes of
these inhibitors an N-methylation study of the amide
nitrogens in the P3–P4 portion was conducted for inhib-
itors 21 and 22, to examine the impact of hydrogen bond
interactions with the NS3 protein. The methylation
product of the amide nitrogen closest to the cyclopen-
tane ring could however not be obtained, probably
due to steric hindrance impeding the subsequent
coupling reaction.
Compound 21, with an added methyl substituent on the
nitrogen of the capping group, is an almost equipotent
inhibitor compared with compound 9 displaying a Ki
value of 16 nM versus 22 nM, suggesting that the hydro-
gen on the capping methyl amide is not contributing in
hydrogen bond interactions. Whilst this is true for the
terminal amide nitrogen, it is noteworthy that an almost
total loss of activity is encountered when the amide
nitrogen of the cyclohexyl glycine moiety is methylated,
that is, in compound 22, displaying a Ki of >10 lM,
highlighting the importance of this position for hydro-
gen bond interaction.
Compounds 23 and 24 are inhibitors of lower molecular
weights where the P3 residue has been capped with
cyclopentylamine or tert-butylamine. Neither is very
potent with 23 displaying a Ki of 2.7 lM and 24 a Ki
of 6.9 lM.
`
Llinas-Brunet et al. have shown that (1R,2S)-1-amino-2-
vinylcyclopropane carboxylic acid is an outstanding P1
substituent and a cysteine replacement with an optimal
fit in the hydrophobic S1 pocket of the NS3 prote-
ase,11c,d and it has been incorporated in an array of very
potent inhibitors, for example, BILN 2061 and com-
pound A (Fig. 1).
Compound 25 has the (S)-2-amino-4,4-difluorobutyric
acid cysteine mimic reported by Narjes et al.12a in the
P1 position. This P1 substituent provides an increase
in potency compared to the molecules containing Abu
or norvaline, with a Ki of 0.56 lM, but it is still substan-
tially less potent, about 25 times, than the corresponding
inhibitor (13) containing the (1R,2S)-1-amino-2-vinylcy-
clopropane carboxylic acid residue.
This P1 substituent in the current series rendered the
very potent inhibitor 13, which is almost 80 times more

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P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
A general observation regarding this series of inhibi-
tors is that the P1 substituent is crucial to provide po-
tent compounds. Furthermore, we can conclude that
these inhibitors, even though incorporating an opti-
mized P1 residue, still are extremely sensitive to mod-
ifications in the P3–P4 region, for example,
methylation at certain positions (compound 22), and
truncations as in compounds 23 and 24, leading to
serious losses in activity.
6. General methods
NMR-spectra were recorded on a Varian 300 MHz
instrument using CDCl3 and CD3OD as solvents.
TMS was used as reference. Optical rotations were mea-
sured using a Perkin-Elmer 141 polarimeter. TLC was
carried out on Merck precoated 60 F254 plates using
UV-light and charring with ethanol/sulfuric acid/p-anis-
aldehyde/acetic acid 90:3:2:1, and a solution of 0.5%
ninhydrin in ethanol for visualization. Flash column
chromatography was performed using silica gel 60
(0.040–0.063 mm, Merck). Organic phases were dried
over anhydrous magnesium sulfate. Concentrations
were performed under diminished pressure (1–2 kPa)
at a bath temperature of 40 ꢁC. MALDI-TOF-spectra
were recorded on a Voyager-DE STR Biospectrometry
Workstation using a-cyano-4-hydroxycinnamic acid as
a matrix and reference. HPLC was performed on a pre-
parative C-18 column.
4. Conclusion
We have developed efficient synthetic routes to an
interesting and novel series of HCV NS3 protease
inhibitors comprising a trisubstituted cyclopentane N-
acyl-(4R)-hydroxyproline mimic in the P2 position.
Different P1 and P3–P4 substituents were evaluated
in order to determine preferred substituent patterns
for these new cyclopentane-based inhibitors. With
proper choice of substituents, very potent and promis-
ing inhibitors in the low nanomolar range could be
obtained from this cyclopentane-derived scaffold, that
is, compounds 13, 21, and 25, which exhibit Ki values
of 22, 16, and 560 nM, respectively. These results sug-
gest that the frequently used P2 hydroxyproline scaf-
fold (IV, Fig. 2) can successfully be displaced by a
properly substituted cyclopentane scaffold (V,
Fig. 2), and that further refinements of the com-
pounds presented herein could provide even more
effective HCV NS3 protease inhibitors.
7. LC–MS purity measurements
7.1. Chromatography system A
Column: Phenomenex C18 150 · 4.6 mm; Pump: Gilson
gradient pump 322; UV/vis-detector: Gilson 155; MS
detector: Thermo Finnigan Surveyor MSQ; Software:
Gilson UniPoint 4.0 and Xcalibur 1.3. Gradient: metha-
nol 40–100% over 10 min at 1 mL/min followed by 100%
for 5 min at 1 mL/min. To all solvents formic acid (0.1%
v/v) was added. Peaks were detected at 254 nm.
5. Experimental section
7.2. Chromatography system B
5.1. HCV NS3 protease enzyme assay30
As system A except: Gradient: acetonitrile 0–100% over
10 min at 1 mL/min followed by 100% for 5 min at
1 mL/min.
The inhibition assay with HCV protease was performed
using recombinant full length NS3 enzyme (Poliakov
et al.)31 and NS4A (KKGSVVIVGRIVLSGK, Gunnar
Lindeberg, Department of Medicinal Chemistry, Upp-
sala University, Sweden) in final concentrations of
3.5 nM and 14 lM, respectively. The test compounds
were dissolved and diluted in DMSO and were added
to the assay buffer containing 50 mM HEPES, pH 7.5,
40% glycerol, 0.1% CHAPS, and 10 mM DTT. The
maximum final DMSO concentration in the assay was
1%. After a pre-incubation for 30 min at room temper-
ature, the enzyme reaction was started by adding the
FRET substrate Ac-Asp-Glu-Asp(EDANS)-Glu-Glu-
Abu-w-(COO)Ala-Ser-Lys(DABCYL)-NH2 (RET S1,
AnaSpec, San Jose, CA, USA) to a final concentration
of 2 lM. The enzyme activity was continously measured
over time (20 min) in a fluorescence reader (Fluorocan
Ascent, ThermoLab systems, Stockholm, Sweden) with
355 nm as excitation and 500 nm as emission wave-
lengths, respectively.
8. General synthetic procedures (used in the synthesis,
protections, and deprotections of compounds
B, C, E, and G–Q)
8.1. Peptide coupling
To the acid (0.264 mmol) dissolved in DMF (5 mL) were
added the amine (0.313 mmol) and diisopropylethyl-
amine (DIEA) (0.792 mmol). The solution was cooled
to 0 ꢁC. HATU (0.316 mmol) was added and the mix-
ture was stirred for 0.5 h at 0 ꢁC and for an additional
2 h at room temperature. The solvent was then evapo-
rated and the residue extracted with EtOAc, washed
with brine, dried, filtered, and concentrated. The prod-
uct was purified by flash column chromatography or
HPLC.
8.2. Boc protection of amino acids
IC50 values were calculated by non-linear fitting into the
equation (1 ꢁ vi/vo) = (I)/((I) + IC50) and the Ki value
was calculated from the IC50 value using the equation
Ki = IC50/(1 + S/Km) assuming a competitive enzyme
inhibition.
Triethylamine (6.40 mmol) was added dropwise to a stir-
red solution of the amino acid (2.29 mmol) and di-tert-
butyl dicarbonate (2.74 mmol) in dioxane/water 1:1
(8 mL) and the solution was stirred overnight. The mix-

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5145
ture was extracted with petroleum ether (2·) and the
aqueous phase was cooled to 0 ꢁC and carefully acidified
to pH 3 by slow addition of 4 M NaHSO4ÆH2O solution.
The acidified water phase was extracted with EtOAc
(3·) and the combined organic phases were washed with
brine (2·) and was then dried, filtered, and concentrat-
ed. No further purification was needed.
CHCl3 and 30 mL TFA was added Et3SiH
(15.81 mmol). After stirring for 22 h, the solvents were
removed in vacuo. Purification was performed by crys-
tallization from EtOAc/hexane.
8.9. Fmoc deprotection
The Fmoc protected amino acid (3.235 mmol) was dis-
solved in 25 mL DMF containing 20% piperidine, and
the mixture was stirred for 2 h. The solvents were evap-
orated and the remainder was extracted with EtOAc and
washed with brine. The organic phase was dried, filtered,
and concentrated. Purification was performed by flash
column chromatography.
8.3. Boc/tert-butyl ester deprotection
To a solution of the protected compound (0.362 mmol)
in methylene chloride (3 mL) were added triethylsilane
(0.742 mmol) and TFA (3 mL). The mixture was stirred
for 2 h at room temperature and was then evaporated
and coevaporated with toluene.
8.4. tert-Butyl ester protection
9. Synthetic procedures
The acid (9.16 mmol), tert-butanol (9.16 mmol), and
DMAP (4.76 mmol) were dissolved in DCM (40 mL)
at 0 ꢁC. EDC (10.7 mmol) was added and the mixture
was stirred at 0 ꢁC for 2 h, thereafter at room tempera-
ture overnight. The mixture was evaporated, extracted
with EtOAc, and washed with saturated NaHCO3 solu-
tion. After drying, filtration, and evaporation the crude
product was purified by flash column chromatography.
9.1. (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester hydrochloride (D)
Synthesized according to Ref. 20.
9.2. 2-Phenyl-7-methoxy-4-quinolinol (F)
Synthesized according to Refs. 2b and 21.
8.5. Methyl ester protection
9.3. trans-(3R,4R)-bis(Methoxycarbonyl)cyclopentanone
((ꢁ)-1)
22
The acid (5.19 mmol) was dissolved in acetone (28 mL)
and MeI (78.7 mmol) and Ag2O (5.9 mmol) were added.
The mixture was allowed to stir overnight and was
thereafter filtered through Celite, evaporated, and puri-
fied by flash column chromatography.
Synthesized according to Ref. 19 ½aꢂ ꢁ132 (c 1.6,
D
22
CHCl3) (lit. ½aꢂ ꢁ133).
D
9.4. trans-(3R,4R)-bis(Methoxycarbonyl)cyclopentanol
(2)
8.6. Methyl ester deprotection
Sodium borohydride (1.11 g, 0.029 mol) was added to a
stirred solution of (ꢁ)-1 (4.88 g, 0.0244 mol) in metha-
nol (300 mL) at 0 ꢁC. After 1 h, the reaction was
quenched with 90 mL brine, concentrated, and extracted
with ethyl acetate. The organic phases were pooled,
dried, filtered, and concentrated. The crude product
was purified by flash column chromatography (tolu-
ene/ethyl acetate 1:1) to give 2 (3.73 g, 76%) as a yellow
oil. Compound 2: 1H NMR (CDCl3, 300 MHz): d 1.90–
2.30 (m, 4H), 3.20–3.30 (m, 1H), 3.38–3.50 (m, 1H), 3.72
(s, 6H), 4.37–4.45 (m, 1H).
The methyl ester (0.597 mmol) was dissolved in diox-
ane/water (1:1) (11 mL) and the mixture was cooled
to 0 ꢁC. LiOH (1.0 M, 0.815 mmol) was added drop-
wise to the solution and the mixture was stirred at
0 ꢁC for 1 h. After neutralization using 1 M HCl, the
solvents were evaporated and coevaporated with
toluene.
8.7. Z-group deprotection
The protected compound (0.299 mmol) was dissolved in
ethanol (95%) (8 mL). 10% palladium on active carbon
(40 mg) and hydrogen (atmospheric pressure, with flush-
ing) were added and the mixture was stirred for 90 min.
The suspension was filtered through Celite followed by
evaporation of the ethanol.
9.5. 3-Oxo-2-oxa-bicyclo[2.2.1]heptane-5-carboxylic acid
(3)
Sodium hydroxide (1 M, 74 mL, 0.074 mol) was added
to a stirred solution of 2 (3.73 g, 0.018 mol) in methanol
(105 mL) at room temperature. After 4 h, the reaction
mixture was neutralized with 3 M HCl, evaporated
and co-evaporated with toluene several times. Pyridine
(75 mL) and Ac2O (53 mL) were added and the reaction
mixture was allowed to shake overnight at room temper-
ature. The mixture was then co-evaporated with toluene
and purified by flash column chromatography (ethyl
acetate + 1% acetic acid) to give 3 (2.51 g, 88%) as a
slightly yellow oil. All analytical data are in accordance
with Ref. 23.
8.8. N-methylation of Fmoc protected amino acids
The Fmoc protected amino acid (5.271 mmol) was sus-
pended in 80 mL toluene, and paraformaldehyde
(840 mg) followed by p-TsOH (0.242 mmol) were added.
The reaction mixture was refluxed for 1 h with azeotro-
pic removal of water before it was cooled to room tem-
perature, washed with aqueous 1 M NaHCO3, dried,
filtered, and concentrated. To the residue in 30 mL

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P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
9.6. 3-Oxo-2-oxa-bicyclo[2.2.1]heptane-5-carboxylic acid
methyl ester (4)
(580 mg, 2.21 mmol) were dissolved in dry THF, and
the temperature was lowered to 0 ꢁC. DIAD (435 lL,
2.21 mmol) dissolved in 2 mL dry THF, was added to
the mixture during 5 min. After 2 h, the temperature
was raised to room temperature and the solution was
stirred overnight. Evaporation and purification by flash
column chromatography (toluene/EtOAc gradient 6:1 to
4:1) gave 7 (324 mg, 78%). Compound 7: 1H NMR
(CDCl3, 300 MHz) d 0.93 (t, J = 7.1 Hz, 3H), 1.26–
1.40 (m, 2H), 1.46 (s, 9H), 1.57–1.71(m, 1H), 1.76–1.88
(m, 1H), 2.34–2.70 (m, 4H), 3.22–3.32 (m, 1H), 3.45
(q, J = 8.2 Hz, 1H), 3.63 (s, 3H), 3.94 (s, 3H), 4.44–
4.56 (m, 1H), 5.16–5.24 (m, 1H), 6.53 (b, 1H), 6.92–
7.02 (m, 2H) 7.11 (dd, J = 2.5, 9.1 Hz, 1H), 7.40–7.56
(m, 3H), 7.96 (d, J = 9.1 Hz, 1H), 8.03 (d, J = 8.2 Hz,
2H); 13C NMR (CDCl3, 75.5 MHz) d 13.6, 18.3, 27.9,
34.5, 35.6, 46.2, 46.4, 52.1, 52.6, 55.3, 78.2, 81.8, 98.1,
107.4, 115.2, 118.0, 122.8, 127.3, 127.4, 128.5, 128.6,
128.9, 129.0, 140.2, 151.2, 159.0, 160.3, 161.1, 171.5,
172.6, 174.4.
To a solution of 3 (44 mg, 0.282 mmol) in acetone
(1.5 mL) were added methyl iodide (593 mg, 4.17 mmol)
and silver (I) oxide (68 mg, 0.293 mmol). The suspension
was stirred at room temperature overnight. The mixture
was filtered through Celite and the solvent was evapo-
rated. Flash column chromatography (toluene/ethyl ace-
tate 4:1) provided 4 (39 mg, 81%) as colorless crystals.
1
Compound 4: H NMR (CDCl3, 300 MHz) d 1.96 (d,
J = 10.7 Hz, 1H), 2.21–2.25 (m, 3H), 2.91–2.95 (m,
1H), 3.16 (s, 1H), 3.75 (s, 3H), 4.98 (app. s, 1H); 13C
NMR (CDCl3, 75.5 MHz) d 33.3, 38.0, 39.6, 45.8,
52.6, 80.4, 173.1, 176.3.
9.7. 3-Oxo-2-oxa-bicyclo[2.2.1]heptane-5-carboxylic acid
tert-butyl ester (5)
DMAP (14 mg, 0.115 mmol) and di-tert-butyl dicarbon-
ate (Boc2O) (252 mg, 1.44 mmol) were added to a stirred
solution of 3 (180 mg, 1.15 mmol) in 2 mL CH2Cl2 un-
der argon atmosphere at 0 ꢁC. The reaction mixture
was allowed to attain room temperature and was stirred
overnight. The reaction mixture was concentrated and
the crude product was purified by flash column chroma-
tography (toluene/ethyl acetate gradient 15:1, 9:1, 6:1,
4:1, 2:1) to give 5 (126 mg, 52%) as colorless crystals.
10.3. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
ylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
cyclopentanecarbonyl]-amino}-pentanoic acid tert-butyl
ester (8)
Compound 7 (38 mg, 0.066 mmol) was dissolved in
dioxane/water 1:1 (4 mL) and the solution was cooled
to 0 ꢁC and 1 M LiOH (132 lL, 0.132 mmol) was added.
The temperature was raised to room temperature and
the solution was stirred for 2 h after which it was neu-
tralized by addition of 1 M HCl and evaporated and
coevaporated with toluene. The residue and amine L
were dissolved in DMF and coupled using General Syn-
thetic Procedure A (Peptide coupling). Purification with
HPLC (MeOH/water 9:1 + 0.2% TEA) provided com-
pound 8 (44 mg, 81%) as a colorless solid. Compound
1
Compound 5: H NMR (CD3OD, 300 MHz) d 1.45 (s,
9H), 1.90 (d, J = 11.0 Hz, 1H), 2.10–2.19 (m, 3H),
2.76–2.83 (m, 1H), 3.10 (s, 1H), 4.99 (s, 1H); 13C
NMR (CD3OD, 75.5 MHz) d 27.1, 33.0, 37.7, 40.8,
46.1, 81.1, 81.6, 172.0, 177.7.
10. Method I
10.1. (1R,2R,4S)-2-((S)-1-tert-Butoxycarbonyl-butylcar-
bamoyl)-4-hydroxy-cyclopentanecarboxylic acid methyl
ester (6)
1
8: H NMR (CDCl3, 300 MHz) rotamers (5:1) d 0.79
(t, J = 7.3 Hz, 3H), 0.85–1.19 (m, 3H), 0.93 (s, over-
lapped, 9H), 1.20–1.35 (m, 2H), 1.39 (s, 1.5 H), 1.43
(s, 7.5 H), 1.54–1.79 (m, 6H), 2.06–2.28 (m, 3H), 2.39–
2.51 (m, 2H), 2.66–2.78 (m, 1H), 2.74 (d, overlapped,
J = 4.7 Hz, 3H), 3.42–3.68 (m, 2H), 3.84 (s, 2.5 H),
3.88 (s, 0.5 H), 4.19 (t, J = 8.9 Hz, 1H), 4.39–4.59 (m,
1H), 4.68 (d, J = 9.6 Hz, 1H), 5.04–5.14 (m, 1H), 6.77
(s, 1H), 6.88–7.06 (m, 2H), 7.26–7.47 (m, 6H), 7.53 (b,
1H), 7.85–7.97 (m, 3H); 13C NMR (CDCl3, 75.5 MHz)
d 13.7, 18.7, 25.6, 25.7, 26.0, 26.7, 28.0, 28.9, 29,7,
34.5, 34.7, 37.7, 38.0, 39.2, 46.6, 47.7, 52.7, 55.3, 58.5,
60.3, 77.9, 81.7, 98.0, 107.4, 115.0, 117.9, 122.8, 127.4,
128.6, 129.0, 140.2, 151.2, 158.9, 160.6, 161.1, 170.9,
171.6, 171.8, 172.7, 173.3. MALDI-TOF: (M+H)+
calcd: 828.5, found: 828.6; (M+Na)+ calcd: 850.5,
found: 850.6; (M+K)+ calcd: 866.6, found: 866.6.
Compound 4 (263 mg, 1.55 mmol) and H-Nva-OtBu
(420 mg, 2.42 mmol) were dissolved in dry THF
(20 mL). DIEA (530 lL, 3.04 mmol) and 2-hydroxypyri-
dine (260 mg, 2.73 mmol) were added and the mixture
was refluxed for five days. The solvent was evaporated
and the crude product was purified by flash column
chromatography (toluene/EtOAc 1:2) to give
6
(510 mg, 96%). Compound 6: 1H NMR (CDCl3,
300 MHz): d 0.93 (t, J = 7.3 Hz, 3H), 1.29–1.40 (m,
2H), 1.47 (s, 9H), 1.57–1.70 (m, 1H), 1.70–1.83 (m,
1H), 1.83–2.05 (m, 2H), 2.05–2.24 (m, 2H), 3.08–3.18
(m, 1H), 3.23–3.33 (m, 1H), 3.71 (s, 3H), 4.34 (br s,
1H), 4.38–4.49 (m, 2H), 7.03 (b, 1H); 13C NMR (CDCl3,
75.5 MHz) d 13.4, 18.1, 27.6, 34.0, 37.9, 40.0, 46.3, 46.5,
51.7, 52.6, 72.7, 81.5, 171.2, 175.1, 175.5.
10.4. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
ylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
cyclopentanecarbonyl]-amino}-pentanoic acid (9)
10.2. (1R,2R,4R)-2-((S)-1-tert-Butoxycarbonyl-butylcar-
bamoyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclo-
pentanecarboxylic acid methyl ester (7)
Compound 6 (249 mg, 0.725 mmol), 2-phenyl-7-meth-
oxy-4-quinolinol (310 mg, 1.23 mmol), and PPh3
Compound 8 (21 mg, 0.025 mmol) was dissolved in
CH2Cl2 (1.5 mL) and triethylsilane (10 lL, 0.063 mmol)

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5147
1
and TFA (1.5 mL) were added. The solution was stirred
for 2 h at room temperature after which the solvents were
evaporated and coevaporated with toluene to provide
compound 9 (20 mg, 100%) as a colorless solid. Com-
68%). Compound 11: H NMR (CDCl3, 300 MHz) d
1.22 (t, J = 7.1 Hz, 3H), 1.37 (s, 9H), 1.50 (dd, J = 5.5,
9.6 Hz, 1H), 1.89 (dd, J = 5.5, 8.0 Hz, 1H), 2.12 (app
q, J = 8.7 Hz, 1H), 2.24–2.34 (m, 1H), 2.41–2.54 (m,
3H), 3.09–3.19 (m, 1H), 3.30–3.42 (m, 1H), 3.94 (s,
3H), 4.09–4.21 (m, 2H), 5.12 (dd, J = 1.8, 10.3 Hz,
1H), 5.15–5.21 (m, 1H), 5.28 (dd, J = 1.8, 17.2 Hz,
1H), 5.76 (ddd, J = 8.5, 10.3, 17.2 Hz), 7.02 (s, 2H),
7.08 (dd, J = 2.6, 9.2 Hz, 1H), 7.41–7.55 (m, 4H), 7.96
(d, J = 9.1 Hz, 1H), 8.05 (d, J = 6.6 Hz, 2H); 13C
NMR (CDCl3, 75.5 MHz) d 14.3, 23.1, 27.9, 33.5,
35.1, 35.6, 40.2, 45.4, 45.7, 47.2, 55.5, 61.3, 78.6, 81.9,
98.3, 107.5, 115.4, 117.8, 118.0, 123.0, 127.5, 128.8,
129.2, 133.6, 140.4, 151.3, 159.2, 160.5, 161.3, 170.0,
173.9, 174.4.
1
pound 9: H NMR (CD3OD, 300 MHz) d 0.93 (t, over-
lapped, 3H), 0.98 (s, 9H), 0.99–1.25 (m, 4H), 1.30–1.49
(m, 3H), 1.50–1.90 (m, 8H), 2.25–2.39 (m, 2H), 2.54–
2.62 (m, 1H), 2.64 (s, 3H), 2.72–2.87 (m, 1H), 3.34–3.57
(m, 3H), 4.02–4.13 (m, 1H), 4.06 (s, overlapped, 3H),
4.27–4.36 (m, 1H), 4.37–4.47 (m, 1H), 5.57–5.66 (m,
1H), 7.45 (dd, J = 2.3, 9.2 Hz, 1H), 7.48 (s, 1H), 7.54 (d,
J = 2.2 Hz, 1H), 7.69–7.79 (m, 3H), 8.01–8.07 (m, 2H),
8.42 (d, J = 9.3 Hz, 1H); 13C NMR (CD3OD,
75.5 MHz) d 14.0, 20.2, 26.0, 26.9, 27.2, 30.1, 30.7, 34.6,
35.3, 37.2, 39.1, 41.2, 47.7, 53.7, 56.9, 59.4, 59.5, 62.5,
83.7, 100.4, 101.3, 102.2, 116.2, 121.7, 126.7, 129.8,
130.8, 133.3, 133.9, 143.5, 157.9, 166.6, 168.5, 172.5,
173.6, 175.3, 175.4, 175.5. HRMS calcd (M+H)+:
772.4285; found: 772.4311. LC–MS Purity System A:
tR = 7.25 min, 95%; System B: tR = 7.43 min, 95%.
11.3. (1R,2S)-1-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-
methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-pro-
pylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
cyclopentanecarbonyl]-amino}-2-vinyl-cyclopropanecarb-
oxylic acid ethyl ester (12)
11. Method II
To a solution of compound 11 (30 mg, 0.050 mmol) in
methylene chloride (1.5 mL) were added triethylsilane
(21 lL, 0.132 mmol) and TFA (1.5 mL). The mixture
was stirred for 2 h at room temperature and was then
evaporated and coevaporated with toluene. The residue
and amine L were dissolved in DMF and coupled using
General Synthetic Procedure A (Peptide coupling). Puri-
fication using HPLC (MeOH/water 9:1 + 0.2% triethyl-
amine) provided compound 12 (30 mg, 74%) as a
colorless solid. Compound 12: 1H NMR (CD3OD,
300 MHz) d 0.81–1.14 (m, 4H), 0.99 (s, overlapped,
9H), 1.21 (t, J = 7.1 Hz, 3H), 1.35–1.51 (m, 4H), 1.52–
1.65 (m, 3H), 1.66–1.72 (m, 2H), 2.03–2.20 (m, 2H),
2.24–2.39 (m, 1H), 2.46–2.56 (m, 1H), 2.66 (s, 3H),
2.72–2.85 (m, 1H), 3.39–3.48 (m, 2H), 3.90 (s, 3H),
4.03–4.15 (m, 3H), 4.44 (s, 1H), 5.09 (dd, J = 1.9,
10.3 Hz, 1H), 5.19–5.27 (m, 1H), 5.25 (dd, overlapped,
1H), 5.79 (ddd, J = 8.8, 10.3, 17.2 Hz, 1H), 6.99 (s,
1H), 7.07 (dd, J = 2.5, 9.1 Hz, 1H), 7.29 (d, J = 2.5 Hz,
1H), 7.43–7.52 (m, 3H), 7.86–7.98 (m, 2H), 8.05 (d,
J = 9.3 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) d
14.7, 23.4, 26.0, 26.9, 27.1, 27.3, 30.1, 30.7, 35.0, 35.4,
38.3, 38.8, 40.9, 41.0, 47.9, 55.9, 59.6, 62.0, 62.4, 79.8,
99.9, 107.3, 116.4, 118.0, 119.1, 124.4, 128.9, 129.8,
130.5, 135.3, 141.3, 152.1, 161.1, 162.4, 163.0, 171.6,
172.5, 173.7, 175.2, 176.8. MALDI-TOF: (M+H)+
calcd: 810.4, found: 810.5; (M+Na)+ calcd: 832.4,
found: 832.4; (M+K)+ calcd: 848.5, found: 848.4.
11.1. (1R,2R,4S)-2-((1R,2S)-1-Ethoxycarbonyl-2-vinyl-
cyclopropylcarbamoyl)-4-hydroxy-cyclopentanecarboxy-
lic acid tert-butyl ester (10)
Compound 5 (56 mg, 0.264 mmol) was dissolved in diox-
ane/water 1:1 (5 mL) and the mixture was cooled to 0 ꢁC.
1M lithium hydroxide (520 lL, 0.520 mmol) was added
and the mixture was stirred at 0 ꢁC for 45 min, after which
the mixture was neutralized with 1 M hydrochloric acid
and evaporated and coevaporated with toluene. The resi-
due was dissolved in DMF (5 mL) and coupled to
(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester hydrochloride using General Synthetic Proce-
dure A (Peptide coupling). Purification by flash column
chromatography (toluene/EtOAc 1:1) provided com-
pound 10 (86 mg, 89%) as a colorless oil. Compound 10:
1H NMR (CDCl3, 300 MHz) d 1.16 (t, J = 7.3 Hz, 3H),
1.40 (s, 9H), 1.70–1.83 (m, 2H), 1.92–2.00 (m, 2H),
2.03–2.14 (m, 2H), 2.93–3.04 (m, 1H), 3.09–3.19 (m,
1H), 3.93–4.17 (m, 1H), 4.26 (br s, 1H), 5.06 (dd,
J = 1.7, 10.2 Hz, 1H), 5.23 (dd, J = 1.7, 17.0 Hz, 1H),
5.66 (ddd, J = 8.8, 10.2, 17.0 Hz, 1H); 13C NMR (CDCl3,
75.5 MHz) d 14.0, 22.8, 27.9, 33.1, 37.1, 40.1, 40.2, 45.6,
47.8, 61.3, 73.0, 81.2, 117.8, 133.2, 169.8, 174.2, 177.7.
11.2. (1R,2R,4R)-2-((1R,2S)-1-Ethoxycarbonyl-2-vinyl-
cyclopropylcarbamoyl)-4-(7-methoxy-2-phenyl-quinolin-
4-yloxy)-cyclopentanecarboxylic acid tert-butyl ester (11)
11.4. (1R,2S)-1-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-
methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-pro-
pylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
cyclopentanecarbonyl]-amino}-2-vinyl-cyclopropanecarb-
oxylic acid (13)
Compound 10 (73 mg, 0.199 mmol) was dissolved in dry
THF (4 mL) and 2-phenyl-7-methoxy-4-quinolinol
(86 mg, 0.342 mmol) and triphenylphosphine (141 mg,
0.538 mmol) were added. The mixture was cooled to
0 ꢁC and DIAD (111 lL, 0.567 mmol) dissolved in
1 mL THF was added dropwise and the mixture was
stirred for 48 h at room temperature. The solvent was
evaporated and the crude product was purified by flash
column chromatography gradient elution (toluene/
EtOAc 9:1, 6:1, 4:1) to give compound 11 (81 mg,
To a solution of compound 12 (20 mg, 0.025 mmol) in
THF/MeOH/water 2:1:1 (2 mL) at 0 ꢁC was added
1 M LiOH (175 lL, 0.175 mmol) and the solution was
allowed to attain room temperature and was stirred
for 48 h. The solution was acidified to pH 3 with 1 M
HCl and was then evaporated and coevaporated with

5148
P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
toluene. The crude product was purified by HPLC
(MeOH/water 3:2 + 0.2% TFA followed by MeOH/
water 4:1 + 0.2% TFA) to give compound 13 (13 mg,
67%) as a colorless solid. Compound 13: 1H NMR
(CD3OD, 300 MHz) d 0.82–0.98 (m, 1H), 1.01 (s, 9H),
1.05–1.26 (m, 3H), 1.34–1.43 (m, 1H), 1.49–1.77 (m,
8H), 2.10–2.21 (m, 1H), 2.28–2.42 (m, 2H), 2.50–2.61
(m, 1H), 2.64 (s, 3H), 2.68–2.81 (m, 1H), 3.36–3.45 (m,
2H), 4.04–4.11 (m, 1H), 4.06 (s, overlapped, 3H), 4.27
(d, J = 8.8 Hz, 1H), 5.10 (dd, J = 1.8, 10.3 Hz, 1H),
5.28 (dd, J = 1.8, 17.2 Hz, 1H), 5.59–5.68 (m, 1H),
5.82 (ddd, J = 9.1, 10.3, 17.2 Hz, 1H), 7.44 (dd,
J = 2.5, 11.8 Hz, 1H), 7.50 (s, 1H), 7.53 (d, J = 2.5 Hz,
1H), 7.69–7.78 (m, 3H), 8.02–8.07 (m, 2H), 8.39 (d,
J = 9.3 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) d
23.5, 26.0, 26.9, 27.2, 27.3, 30.0, 30.7, 34.7, 35.3, 37.0,
38.7, 41.0, 41.3, 47.4, 56.9, 59.4, 62.7, 83.9, 100.4,
102.2, 116.2, 117.7, 121.7, 126.7, 129.8, 130.8, 133.4,
133.9, 135.6, 143.5, 158.0, 166.6, 168.6, 172.5, 173.4,
173.6, 175.4, 176.4. HRMS calcd (M+H)+: 782.4129;
found: 782.4158. LC–MS Purity System A:
tR = 7.09 min, 98%; System B: tR = 7.45 min, 98%.
3.95 (s, 3H), 4.12–4.23 (m, 2H), 4.28–4.38 (m, 1H),
5.31 (b, 1H), 7.43 (dd, J = 2.2, 9.3 Hz, 1H), 7.47 (s,
1H), 7.51 (s, 1H), 7.66–7.89 (m, 3H), 7.99–8.07 (m,
2H), 8.42 (d, J = 9.1 Hz, 1H); 13C NMR (CD3OD,
75.5 MHz) d 10.7, 18.8, 19.7, 25.8, 27.0, 27.0, 29.7,
30.5, 31.8, 37.7, 38.9, 41.2, 47.9, 52.3, 55.3, 56.9, 58.8,
60.6, 83.6, 100.7, 102.2, 116.3, 121.5, 126.7, 129.8,
130.8, 133.7, 133.8, 143.9, 158.2, 166.4, 168.3, 173.3,
173.8, 175.2, 175.5, 175.6. HRMS calcd (M+H)+:
745.3813; found: 745.3787. LC–MS Purity System A:
tR = 7.86 min, 99%; System B: tR = 7.66 min, 98%.
12.3. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2-methyl-propylcarba-
moyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopen-
tanecarbonyl]-amino}-pentanoic acid (16)
Compound 16 was obtained in 66% total yield as a
colorless solid using Method I. Compound 16: 1H
NMR (CD3OD, 300 MHz) d 0.86–1.00 (m, 9H),
1.01–1.24 (m, 4H), 1.36–1.46 (m, 2H), 1.48–1.75 (m,
8H), 1.70–1.89 (m, overlapped, 1H), 1.96–2.12 (m,
1H), 2.22–2.40 (m, overlapped, 2H), 2.49–2.64 (m,
1H), 2.72–2.91 (m, 1H), 3.26–3.40 (m, overlapped,
1H), 3.50–3.68 (m, overlapped, 1H), 3.62 (s, 3H),
4.05 (s, 3H), 4.09–4.17 (m, 1H), 4.17–4.25 (m, 1H),
4.35–4.45 (m, 1H), 5.62 (b, 1H), 7.44 (dd, J = 2.2,
9.3 Hz, 1H), 7.49 (s, 1H), 7.53 (d, J = 2.2 Hz, 1H),
7.65–7.78 (m, 3H), 7.98–8.06 (m, 2H), 8.41 (dd,
J = 2.8, 9.3 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz)
d 13.9, 18.8, 19.7, 20.2, 27.0, 29.7, 30.5, 31.8, 34.6,
37.7, 38.9, 41.1, 47.8, 52.3, 53.6, 56.9, 58.8, 58.9,
60.3, 83.8, 100.4, 102.2, 116.2, 121.6, 126.7, 129.8,
130.8, 133.3, 133.8, 143.5, 157.9, 166.5, 168.5, 173.3,
173.9, 175.5, 175.5, 175.6. HRMS calcd (M+H)+:
759.3969; found: 759.4001. LC–MS Purity System A:
tR = 8.05 min, 98%; System B: tR = 7.86 min, 100%.
12. Target compounds
12.1. (S)-2-{[(1R,2R,4S)-2-{(R)-1-[((R)-Cyclohexyl-
methoxycarbonyl-methyl)-carbamoyl]-2-methyl-propyl-
carbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
cyclopentanecarbonyl]-amino}-butyric acid (14)
Compound 14 was obtained in 30% total yield as a
colorless solid using Method I. Compound 14: 1H
NMR (CD3OD, 300 MHz) d 0.82–1.02 (m, 9H),
1.04–1.42 (m, 6H), 1.52–1.80 (m, 6H), 1.80–1.96 (m,
overlapped, 1H), 2.00–2.14 (m, 1H), 2.29–2.46 (m,
2H), 2.51–2.65 (m, 1H), 2.68–2.84 (m, 1H), 3.24–3.39
(m, overlapped, 1H), 3.47–3.60 (m, 1H), 3.67 (s,
3H), 4.07 (s, 3H), 4.18–4.27 (m, 2H), 4.28–4.38 (m,
1H), 5.64 (app. br s, 1H), 7.44 (dd, J = 2.3, 6.9 Hz,
1H), 7.42 (s, 2H), 7.67–7.81 (m, 3H), 8.04 (d,
J = 7.8 Hz, 2H), 8.41 (d, J = 9.1 Hz, 1H); 13C NMR
(CD3OD, 75.5 MHz) d 10.8, 18.5, 19.6, 25.7, 27.1,
27.1, 30.1, 30.6, 31.9, 37.3, 38.2, 41.1, 47.8, 52.3,
55.4, 56.9, 59.0, 59.1, 60.2, 83.8, 100.5, 102.2, 116.3,
121.6, 126.8, 129.8, 130.8, 133.6, 133.8, 143.7, 158.1,
166.5, 168.5, 173.4, 173.8, 175.4, 175.7, 175.7. HRMS
calcd (M+H)+: 745.3813; found: 745.3849. LC–MS
Purity System A: tR = 7.58 min, 100%; System B:
tR = 7.78 min, 100%.
12.4. (S)-2-{[(1R,2R,4S)-2-{(R)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2-methyl-propylcarba-
moyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopen-
tanecarbonyl]-amino}-pentanoic acid (17)
Compound 17 was obtained in 42% total yield as a
colorless solid using Method I. Compound 17: 1H
NMR (CD3OD, 300 MHz) d 0.86–0.97 (m, 9H),
1.04–1.19 (m, 2H), 1.20–1.33 (m, 3H), 1.34–1.49 (m,
2H), 1.58–1.69 (m, 3H), 1.70–1.89 (m, 4H), 2.04–2.16
(m, 1H), 2.32–2.45 (m, 2H), 2.46–2.67 (m, 2H),
2.70–2.84 (m, 1H), 3.36–3.46 (m, 1H), 3.47–3.60 (m,
1H), 3.69 (s, 3H), 4.06 (s, 3H), 4.26–4.43 (m, 3H),
5.61–5.70 (m, 1H), 7.45 (dd, J = 1.8, 9.2 Hz, 1H),
7.53 (s, 1H), 7.54 (d, overlapped, 1H), 7.68–7.78 (m,
3H), 8.02–8.09 (m, 2H), 8.40 (d, J = 9.3 Hz, 1H);
13C NMR (CDCl3,75.5 MHz) d 13.8, 18.1, 19.3,
19.7, 26.1, 28.7, 29.7, 30.3, 33.4, 36.0, 36.7, 40.7,
41.2, 43.8, 47.0, 47.5, 52.4, 52.7, 53.5, 56.4, 57.4,
59.4, 82.1, 100.1, 100.2, 114.8, 119.9, 121.1, 124.9,
128.6, 129.4, 131.0, 132.4, 142.7, 155.8, 164.8, 166.6,
171.9, 172.7, 173.4, 173.9, 174.8. HRMS calcd
(M+H)+: 759.3969; found: 759.3998. LC–MS Purity
12.2. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2-methyl-propylcarba-
moyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopen-
tanecarbonyl]-amino}-butyric acid (15)
Compound 15 was obtained in 21% total yield as a col-
orless solid using Method I. Compound 15: H NMR
1
(CD3OD, 300 MHz) d 0.82–0.99 (m, 9H), 0.82–1.40
(m, overlapped, 6H), 1.48–1.78 (m, 6H), 1.80–1.95 (m,
1H), 1.97–2.12 (m, 1H), 2.22–2.40 (m, overlapped,
2H), 2.51–2.64 (m, 1H), 2.71–2.90 (m, 1H), 3.16–3.39
(m, overlapped, 1H), 3.49–3.59 (m, 1H), 3.63 (s, 3H),
System
tR = 8.12 min, 100%.
A:
tR = 8.92 min,
97%;
System
B:

P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
5149
12.5. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((R)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2-methyl-propylcarba-
moyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopen-
tanecarbonyl]-amino}-pentanoic acid (18)
59.0, 62.2, 83.8, 100.4, 102.2, 116.2, 121.7, 126.7,
129.2, 129.8, 130.8, 133.4, 133.8, 143.5, 158.0, 166.5,
168.5, 172.9, 173.2, 175.3, 175.5, 179.8. HRMS calcd
(M+H)+: 773.4126; found: 773.4119. LC–MS Purity
System A: tR = 8.56 min, 100%; System B:
tR = 8.07 min, 100%.
Compound 18 was obtained in 32% total yield as a col-
orless solid using Method I. Compound 18: H NMR
1
(CD3OD, 300 MHz, rotamers) d 0.90–1.04 (m, 9H),
1.13–1.21 (m, 2H), 1.22–1.32 (m, 3H), 1.33–1.49 (m,
2H), 1.52–1.64 (m, 3H), 1.65–1.89 (m, 5H), 2.01–2.14
(m, 1H), 2.26–2.42 (m, 2H), 2.52–2.67 (m, 1H), 2.74–
2.88 (m, 1H), 3.32–3.41 (m, overlapped, 1H), 3.47–3.58
(m, 1H), 3.59 (s, 3H), 4.06 (s, 3H), 4.20–4.34 (m, 2H),
4.36–4.45 (m, 1H), 5.59–5.69 (m, 1H), 7.45 (dd,
J = 2.3, 9.2 Hz, 1H), 7.49 (s, 1H), 7.52 (d, J = 2.3 Hz,
1H), 7.68–7.80 (m, 3H), 8.01–8.08 (m, 2H), 8.44 (d,
J = 9.1 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) d
12.8, 17.2, 18.8, 19.0, 21.1, 25.8, 28.6, 29.5, 30.8, 33.5,
36.4, 37.8, 40.0, 46.6, 51.1, 52.5, 55.7, 57.5, 58.9, 82.5,
99.3, 101.0, 115.1, 120.4, 125.5, 128.6, 129.6, 132.5,
132.6, 142.6, 156.9, 165.3, 167.2, 172.1, 172.5, 174.3.
HRMS calcd (M+H)+: 759.3969; found: 759.3976.
LC–MS Purity System A: tR = 8.39 min, 98%; System
B: tR = 7.93 min, 96%.
12.8. (1R,2S)-1-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-
dimethylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-
propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-
yloxy)- cyclopentanecarbonyl]-amino}-2-vinyl-cyclopro-
panecarboxylic acid (21)
Compound 21 was obtained in 37% total yield as a col-
orless solid using Method II. Compound 21: H NMR
1
(CDCl3, 300 MHz) d 0.75–0.87 (m, 2H), 0.90 (s, 9H),
0.98–1.17 (m, 3H), 1.32–1.63 (m, 8H), 1.81–1.98 (m,
2H), 2.10–2.21 (m, 1H), 2.28–2.40 (m, 1H), 2.74–2.89
(m, 1H), 2.90–3.03 (m, 1H), 2.95 (s, overlapped, 3H),
3.05–3.19 (m, 1H), 3.10 (s, 3H), 3.63–3.78 (m, 1H),
3.99 (s, 3H), 4.47 (d, J = 9.9 Hz, 1H), 4.70–4.80 (m,
1H), 5.11 (d, J = 11.3 Hz, 1H), 5.27 (d, J = 16.7 Hz,
1H), 5.67–5.87 (m, 2H), 6.70 (br s, 1H), 7.21 (b, 1H),
7.29 (dd, J = 1.9, 9.3 Hz, 1H), 7.39 (s, 1H), 7.48–7.60
(m, 3H), 7.74 (s, 1H), 7.92 (d, J = 6.6 Hz, 2H), 8.16 (d,
J = 9.3 Hz, 1H), 8.86 (b, 1H); 13C NMR (CDCl3,
75.5 MHz) d 25.9, 26.1, 27.0, 28.2, 30.0, 32.8, 33.9,
34.8, 36.5, 38.2, 38.8, 40.7, 41.3, 43.7, 44.7, 49.4, 54.1,
56.8, 62.6, 81.7, 100.2, 100.3, 114.9, 118.8, 121.9,
124.7, 128.9, 130.0, 132.0, 133.1, 133.5, 143.1, 156.9,
165.5, 167.1, 171.7, 172.3, 174.1, 174.6, 175.0. HRMS
calcd (M+H)+: 796.4285; found: 796.4312. LC–MS
Purity System A: tR = 7.62 min, 100%; System B:
tR = 7.64 min, 99%.
12.6. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
ylcarbamoyl-methyl)-carbamoyl]-2-methyl-propylcarba-
moyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopen-
tanecarbonyl]-amino}-pentanoic acid (19)
Compound 19 was obtained in 40% total yield as a
colorless solid using Method I. Compound 19: 1H
NMR (CD3OD, 300 MHz) d 0.84–0.99 (m, 9H),
1.00–1.24 (m, 4H), 1.27–1.46 (m, 3H), 1.50–1.74 (m,
7H), 1.75–1.86 (m, 1H), 1.99–2.14 (m, 1H), 2.23–2.40
(m, 2H), 2.52–2.59 (m, 1H), 2.63 (s, 3H), 2.75–2.90
(m, 1H), 3.16–3.26 (m, 1H), 3.46–3.59 (m, 1H),
4.01–4.11 (m, 1H), 4.06 (s, overlapped, 3H), 4.13–
4.21 (m, 1H), 4.35–4.43 (m, 1H), 5.59–5.67 (m, 1H),
7.41–7.54 (m, 3H), 7.68–7.79 (m, 3H), 8.04 (d,
J = 6.6 Hz, 2H), 8.44 (d, J = 9.1 Hz, 1H). HRMS
calcd (M+H)+: 758.4129; found: 758.4145. LC–MS
Purity System A: tR = 6.61 min, 98%; System B:
tR = 7.41 min, 100%.
12.9. (1R,2S)-1-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-
methylcarbamoyl-methyl)-methyl-carbamoyl]-2,2-dimeth-
yl- propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-
yloxy)- cyclopentanecarbonyl]-amino}-2-vinyl-cyclopro-
panecarboxylic acid (22)
Compound 22 was obtained in 23% total yield as a
colorless solid using Method II. Compound 22: 1H
NMR (CD3OD, 300 MHz) d 0.84–1.06 (m, 2H), 1.00
(s, overlapped, 9H), 1.12–1.26 (m, 2H), 1.30–1.41 (m,
4H), 1.42–1.68 (m, 3H), 1.70–1.78 (m, 2H), 1.80–1.92
(m, 1H), 2.11–2.27 (m, 2H), 2.28–2.40 (m, 1H),
2.50–2.63 (m, 1H), 2.67 (s, 3H), 2.70–2.80 (m, 1H),
3.12 (s, 3H), 3.34–3.46 (m, 1H), 3.99 (s, 1H), 4.08
(s, 3H), 4.73 (d, J = 11.3 Hz, 1H), 5.10 (dd, J = 1.9,
10.2 Hz, 1H), 5.28 (dd, J = 1.4, 17.3 Hz, 1H), 5.59–
5.67 (m, 1H), 5.83 (ddd, J = 8.8, 10.2, 17.3 Hz, 1H),
7.45 (dd, J = 2.2, 9.2 Hz, 1H), 7.50 (s, 1H), 7.54 (d,
J = 2.2 Hz, 1H), 7.70–7.80 (m, 3H), 8.00–8.07 (m,
2H), 8.42 (d, J = 9.3 Hz, 1H); 13C NMR (CD3OD,
75.5 MHz) d 23.5, 26.0, 26.5, 26.9, 27.3, 29.5, 30.7,
32.1, 34.7, 36.1, 36.5, 37.1, 38.6, 41.0, 47.3, 56.9,
57.0, 61.7, 83.9, 100.4, 102.2, 116.2, 117.7, 121.7,
126.7, 129.8, 130.8, 133.3, 133.9, 135.5, 143.5, 158.0,
166.6, 168.5, 172.3, 173.2, 174.2, 175.3, 176.3. HRMS
calcd (M+H)+: 796.4285; found: 796.4307. LC–MS
Purity System A: tR = 7.46 min, 99%; System B:
tR = 7.58 min, 100%.
12.7. (S)-2-{[(1R,2R,4S)-2-{(S)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopentane-
carbonyl]-amino}-pentanoic acid (20)
Compound 20 was obtained in 61% total yield as a col-
orless solid using Method I. Compound 20 H NMR
1
(CD3OD, 300 MHz) d 0.94 (t, J = 7.4 Hz, 3H), 0.98–
1.09 (m, 2H), 1.00 (s, 9H), 1.10–1.28 (m, 4H), 1.35–
1.48 (m, 2H), 1.52–1.74 (m, 7H), 1.75–1.89 (m, 1H),
2.26–2.39 (m, 2H), 2.54–2.64 (m, 1H), 2.73–2.87 (m,
1H), 3.34–3.43 (m, 1H), 3.44–3.59 (m, 1H), 3.66 (s,
3H), 4.07 (s, 3H), 4.14–4.22 (m, 1H), 4.34 (d,
J = 9.1 Hz, 1H), 4.36–4.46 (m, 1H), 5.57–5.66 (m, 1H),
7.41–7.58 (m, 3H), 7.66–7.78 (m, 4H), 8.01–8.08 (m,
1H), 8.43 (d, J = 9.1 Hz, 1H); 13C NMR (CD3OD,
75.5 MHz) d 14.0, 20.2, 24.9, 27.0, 27.1, 29.8, 30.5,
34.6, 35.3, 37.2, 39.1, 41.2, 46.9, 47.7, 52.2, 53.7, 56.9,

5150
P.-O. Johansson et al. / Bioorg. Med. Chem. 14 (2006) 5136–5151
12.10. (1R,2S)-1-{[(1R,2R,4S)-2-((S)-1-Cyclopentylcar-
bamoyl-2,2-dimethyl-propylcarbamoyl)-4-(7-methoxy-2-
phenyl-quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-
2-vinyl-cyclopropanecarboxylic acid (23)
J = 2.5, 9.3 Hz, 1H), 7.49 (s, 1H), 7.53 (d, J = 2.2 Hz,
1H), 7.69–7.78 (m, 3H), 7.98–8.06 (m, 2H), 8.41 (d,
J = 9.3 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) d
26.0, 26.9, 27.2, 30.0, 30.7, 35.2, 36.9, 37.2, 38.9, 41.3,
47.6, 56.9, 59.4, 62.6, 83.7, 100.5, 102.2, 116.2, 116.9,
121.7, 126.7, 129.8, 129.9, 130.8, 133.4, 133.9, 143.6,
158.0, 166.6, 168.5, 172.5, 173.5, 175.3, 175.4. HRMS
calcd (M+H)+: 794.3940; found: 794.3926. LC–MS
Purity System A: tR = 7.05 min, 97%; System B:
tR = 7.48 min, 97%.
Compound 23 was obtained in 23% total yield as a col-
orless solid using Method II. Compound 23: H NMR
1
(CD3OD, 300 MHz) d 0.98 (s, 9H), 1.28–1.48 (m, 3H),
1.49–1.76 (m, 5H), 1.78–1.94 (m, 2H), 2.10–2.24 (m,
1H), 2.26–2.45 (m, 2H), 2.50–2.62 (m, 1H), 2.66–2.79
(m, 1H), 3.35–3.48 (m, 2H), 3.94–4.03 (m, 1H), 4.06 (s,
3H), 4.16–4.24 (m, 1H), 5.10 (dd, J = 1.8, 10.3 Hz,
1H), 5.29 (dd, J = 1.8, 17.2 Hz, 1H), 5.62 (b, 1H), 5.82
(ddd, J = 9.1, 10.3, 17.2 Hz, 1H), 7.43 (dd, J = 2.5,
9.3 Hz, 1H), 7.50 (s, 1H), 7.50–7.69 (dd, overlapped,
1H), 7.67–7.80 (m, 3H), 8.01–8.11 (m, 2H), 8.39 (d,
J = 9.3 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) d
24.7, 24.7, 27.3, 33.1, 33.6, 34.7, 35.4, 36.9, 38.7, 41.0,
47.4, 52.3, 56.9, 62.3, 83.9, 100.4, 102.3, 116.2, 117.7,
121.6, 126.7, 129.8, 130.8, 133.4, 133.8, 135.6, 143.5,
158.0, 166.5, 168.6, 171.9, 173.4, 175.2, 176.4. HRMS
calcd (M+H)+: 697.3601; found: 697.3589. LC–MS
Purity System A: tR = 7.34 min, 98%; System B:
tR = 7.63 min, 100%.
Acknowledgments
We gratefully thank Medivir AB, the Swedish Founda-
tion for Strategic Research (SSF), and Knut and Alice
Wallenberg’s Foundation for financial support. We also
thank Jussi Kangasmetsa¨, Medivir UK, for help regard-
ing the HRMS analyses.
References and notes
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12.11. (1R,2S)-1-{[(1R,2R,4S)-2-((S)-1-tert-Butylcarba-
moyl-2,2-dimethyl-propylcarbamoyl)-4-(7-methoxy-2-
phenyl-quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-
2-vinyl-cyclopropanecarboxylic acid (24)
Compound 24 was obtained in 40% total yield as a col-
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1
´
(CD3OD, 300 MHz) d 0.99 (s, 9H), 1.26 (s, 9H), 1.34–
1.42 (m, 1H), 1.69–1.76 (m, 1H), 2.12–2.23 (m, 1H),
2.28–2.44 (m, 2H), 2.51–2.62 (m, 1H), 2.65–2.79 (m,
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1H), 5.10 (dd, J = 1.7, 10.4 Hz, 1H), 5.29 (dd, J = 1.7,
17.3 Hz, 1H), 5.59–5.67 (m, 1H), 5.83 (ddd, J = 9.1,
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7.47–7.55 (m, 2H), 7.68–7.78 (m, 3H), 8.01–8.07 (m,
2H), 8.40 (d, J = 9.3 Hz, 1H); 13C NMR (CD3OD,
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40.9, 46.7, 47.3, 52.2, 56.9, 62.5, 77.3, 83.9, 100.4,
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