Synthesis of novel potent hepatitis C virus NS3 protease inhibitors: Discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a N-acyl-l-hydroxyproline bioisostere

Article abstract of DOI:10.1016/j.bmc.2006.10.044

Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-l-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol. The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a K_i value of 1.1 nM.

Full text of DOI:10.1016/j.bmc.2006.10.044

Bioorganic & Medicinal Chemistry 15 (2007) 827–838
Synthesis of novel potent hepatitis C virus NS3 protease inhibitors:
Discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid
as a N-acyl-L-hydroxyproline bioisostere
a
a
a
b
˚
Fredrik Thorstensson, Fredrik Wangsell, Ingemar Kvarnstrom, Lotta Vrang,
¨
Elizabeth Hamelink,b Katarina Jansson,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, BMC, Uppsala University, Box 574, S-751 23 Uppsala, Sweden
dDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 3 May 2006; revised 17 October 2006; accepted 23 October 2006
Available online 25 October 2006
Abstract—Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-
1,2-dicarboxylic acid as a new N-acyl-L-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps
from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position
and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol.
The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay
using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a Ki value of 1.1 nM.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
virological response in genotype 1 infected patients.5
The limited efficacy of current therapies and the consid-
erable side effects leading to discontinuation of therapy
in some patient populations clearly highlight the need
for new and more efficacious therapeutic options.
HCV-virus infection is a major health issue where infect-
ed are at risk of developing chronic hepatitis, cirrhosis,
and hepatocellular carcinoma.1 Approximately 3% of
the world population (ꢀ200 million people) are infected
by the HCV virus, which spreads through blood contact
and through exchange of bodily fluids.2 After 20 years of
chronic HCV infection ꢀ20% of those infected develop
cirrhosis which may (65%) progress to liver cancer.3
In the western world end-stage liver disease caused by
chronic hepatitis C viral infection is one of the primary
reasons for liver transplantation.4 At present, there are
no vaccines available and the currently most effective
therapy consists of PEGylated a-interferons in combina-
tion with ribavirin, resulting in only ꢀ50% sustained
HCV is a single-stranded positive RNA-virus that en-
codes a polyprotein of approximately 3000 amino acids,
which is proteolytically processed into at least four
structural and six non-structural viral proteins by host
enzymes and viral enzymes (NS2- and NS3-protease).6
The NS3 protein is a bifunctional enzyme, which has
protease activity at its N-terminal domain and RNA
helicase/ATPase activity at its C-terminal domain. The
NS3 protease is responsible for the autocatalytic cis
cleavage of the NS3–NS4A junction and the trans cleav-
age at the NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B
junctions.7 The NS3 protease and NS5B RNA polymer-
ase of HCV are currently the two most promising targets
in the search for antiviral agents against HCV.8
Keywords: HCV; NS3; Protease inhibitor; N-Acyl-L-hydroxyproline
mimic; 4-Hydroxy-cyclopent-2-ene-1,2- dicarboxylic acid;
Cyclopentene.
*
Corresponding authors. Tel.: +46 8 54683120; fax: +46 8 6083199
˚
(A.R.); tel.: +46 8 6083100; fax: +46 8 6083199 (B.S.); e-mail
addresses: asa.rosenquist@medivir.se; bertil.samuelsson@medivir.se
Inhibitors of the NS3 protease generally fall into struc-
tural classes characterized as either non-covalent or
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.044

828
F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
covalent inhibitors, the latter composed of peptides
bearing a reactive center at the cleavage site trapping
the catalytic Ser139 of the NS3 protease active site.
Most advanced in this inhibitor class are SCH5030349
and VX-95010,11, both a-ketoamides (Fig. 1) which are
both being evaluated in phase IIa clinical trials. A sec-
ond structural class of NS3 protease inhibitors are the
product based inhibitors exhibiting a P112 carboxylic
acid,13 for example, tetrapeptide 113h (Fig. 1). The most
promising inhibitor from this class is the highly potent
macrocyclic tripeptide inhibitor, BILN 2061, developed
by researchers at Boehringer Ingelheim (Fig. 1). In a
proof-of-concept study, a two-day, twice-daily treat-
ment of BILN 2061 (2· 200 mg) in patients infected with
HCV genotype 1 decreased the viral load to nearly unde-
tectable levels within 24–28 h after administration.13h,14
Figure 2. The previously reported II and III are examples of N-acyl-
proline (I) mimetics used in inhibitors toward thrombin. Recently, we
reported the successful use of V in HCV NS3 inhibitors. In the present
work we use VI as a N-acyl-hydroxyproline isosteres (IV).
Several peptidomimetic inhibitors feature L-proline as a
key building block, for example,13 inhibitors of HIV-1,15
angiotensin converting enzyme (ACE),16 and throm-
bin.17 In previous work from our laboratory, novel
five-membered carbocyclic ring isosteres of proline have
been disclosed furnishing thrombin inhibitors of low to
modest activity (Fig. 2).18 Recently, we reported the suc-
cessful use of a novel trisubstituted cyclopentane moiety
(V) in HCV NS3 protease inhibitors.19 We now report
on the synthesis of 4-hydroxy-cyclopent-2-ene-1,2-dicar-
boxylic acid and its evaluation as a P2 bioisostere of N-
acyl-L-hydroxy-proline in HCV NS3 protease inhibitors
(Fig. 2). Several highly promising inhibitors have been
developed from this work where one of the most potent
inhibitors 39a exhibits a Ki value of 1.3 nM (Fig. 10).
2. Results and discussion
The ketone 5 was synthesized as previously reported
from commercially available and inexpensive (syn)-tet-
rahydrophthalic anhydride 2 (Scheme 1).20,21 The anhy-
dride 2 was ring-opened to the corresponding diester
and oxidatively cleaved to the dicarboxylic acid 3, which
was cyclized and decarboxylated to the syn-cyclopenta-
none 4. The ketone 4 was a-brominated with cupric bro-
mide and b-eliminated to the olefin 5 in 53% yield, using
calcium carbonate.22 The a,b-unsaturated ketone 5 was
subsequently reduced to give the alcohol 6 in 92% yield
Figure 1. VX-950, SCH 503034, compound 1, and BILN 2061 are potent HCV inhibitors containing the P2-L-hydroxyproline moiety. Compound
38a is the most potent inhibitor in our novel series and is containing the P2-hydroxycyclopentene moiety.

F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
829
Figure 4. The relevant HMBC correlations observed for 7.
Scheme 1. Synthesis of the P2-template. Reagents and conditions: (a)
p-toluene sulfonic acid monohydrate, methanol, reflux; (b) KMnO4,
water, 0 ꢁC; (c) acetic anhydride, 130 ꢁC; (d) NaOAc, 130 ꢁC; (e)
CuBr2, THF, reflux; (f) CaCO3, DMF, 100 ꢁC; (g) NaBH4, methanol,
ꢁ30 ꢁC; (h) LiOH, water/dioxane (1:1), 0 ꢁC.
Figure 5. The P1-building blocks (A–C) (H2NCHR1COOtBu).
in a 6:1 diastereomeric ratio (syn/anti) using sodium
borohydride at ꢁ30 ꢁC.
C was coupled directly with the carboxylic acid 7 with-
out prior purification due to rapid decomposition of the
free amine at room temperature. The alcohols 8–10
were subsequently coupled with 7-methoxy-2-phenyl-
quinolin-4-ol (R2OH) to give the ethers 11–13 in 61–
78% yield using triphenylphosphine (PPh3) and diiso-
propyl azodicarboxylate (DIAD) in THF.26,28 The
methyl esters 11–13 were hydrolyzed with lithium
hydroxide in dioxane/water 1:1 at 0 ꢁC to furnish the
carboxylic acids 14–16 in 72–97% yield. The P3/P4
building blocks D–I were coupled with the acids 14–
16 using HATU and DIPEA in DMF, which resulted
in the tert-butyl esters 17–28 in 30–50% yield (Fig. 6).
The P3/P4 building blocks D–H were synthesized from
commercially available amino acids using standard pro-
tecting group chemistry.24,29 The tert-butylesters 17–28
were hydrolyzed, using TFA and Et3SiH in DCM, to
yield the final products 29–40 in 34–100% yield.30 Sev-
eral of the diasteromeric compounds 17–40 could be
separated31 by chromatography and all isolated prod-
ucts were tested individually to determine HCV NS3/
4A inhibitory properties (Table 1).
The stereochemistry of the syn/anti diasteromers23 of 6
was determined by NOESY NMR.24 The NOESY-spec-
tra of syn-6 show that H1 and H4 interact with the same
methylene hydrogen (H5b), which is consistent with the
syn configuration (Fig. 3). In the NOESY-spectra of
anti-6, the H4 interacts with H5a and the H1 with H5b,
which again is consistent with the anti-6 isomer.
The dimethylester 6 (syn/anti 6:1) was regioselectively25
mono-hydrolyzed using 1.0 equiv of LiOH in dioxane/
water, yielding the diasteromeric acid 7 (syn/anti 6:1),
which was recrystallized (hexane/diethyl ether) to the
pure racemic acid 7 (syn) in 31% yield. The regiochem-
istry of the a,b-unsaturated acid 7 was determined with
HMBC-spectra and the relevant correlations shown in
Figure 4.24
The acid 7 (syn) was coupled with the three P1-building
blocks (A–C) (Fig. 5), using O-(7-azabenzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium
hexafluorophosphate
(HATU) and diisopropylethylamine (DIPEA) in DMF,
to give the amides 8–10 in 52–69% yield (Scheme 2).
The P1-building block A was commercially available, B
was readily synthesized from commercially available ami-
no acids, and C was synthesized from (1R,2S)-1-amino-2-
vinyl-cyclopropanecarboxylic acid ethyl26 using Fmoc-
succinate and TBTA.24,27,37 The vinylcyclopropyl amine
The final products 35a and 35 b are either of the 1R,4R
or the 1S,4S stereochemistry in the cyclopentene ring
(Fig. 7A) and the assignment of the diastereomers was
based on 1H-, 13C-, COSY-, NOESY-, TOCSY-,
HMBC-, HSQC-, and ROESY-spectra.24 According to
computer simulations the main significant NOE-differ-
ences between the diastereomers are the distance be-
tween P2Ha and P3NH (Fig. 7B).24,32 In the ROESY-
spectra33 of 35a and 35b the two different diastereomers
exhibit similar NOE cross-peak patterns and intensities
except at the cross-peak P2Ha M P3NH (Fig. 7C). The
fact that 35a has a weak NOE-interaction and 35b has
a medium one indicates that 35a has the 1R,4R- and that
35b has the 1S,4S-configuration, respectively. This
annotation is also supported by the fact that diastereo-
mer 35a (Ki value of 30 nM), which closely resembles
Figure 3. The relevant NOE-effects observed in 6 (syn) and 6 (anti).

830
F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
Scheme 2. Synthesis of the final products. Reagents and conditions: (a) DIPEA, HATU, the P1-building blocks, (A–C, H2NCHR1COOtBu), 0 ꢁC,
DMF; (b) the P2-substituent (7-methoxy-2-phenyl-quinolin-4-ol, R2OH), PPh3, DIAD, THF; (c) LiOH, dioxane/water (1:1); (d) DIPEA, HATU, the
P3/P4-building blocks, (D–I, NH2R3), 0 ꢁC, DMF; (e) triethyl silane, TFA, DCM.
L-4-hydroxyproline residue exhibited in known HCV
protease inhibitors.8,13,26 Thus inhibitors 34a, 35a, 38a,
and 39a show highly promising potencies, whereas
inhibitors 34b, 35b, 38b, and 39b (Table 1) incorporat-
ing the enantiomeric hydroxycyclopentene are consid-
erably less active. However, 39b (Ki value of 23 nM)
is still having an unexpectedly high potency for having
the non-optimal P2 stereochemistry. Therefore, our
inhibitor 39b was aligned with compound 113f de-
scribed by Boehringer Ingelheim using a molecular
modeling alignment tool (GASPTM) to elucidate
whether it would be possible to find a conformation
that would overlay well with 1 (Fig. 8). The overall
alignment of 39b and 1 is similar, except for the cyclo-
pentene group in 39b that adopts a different angle
compared to the proline of 1, forcing the P1–P2 amide
into a different conformation and thereby losing the
possibility to form a H-bond to the protein backbone
Figure 6. The P3/P4 building blocks (D–I) (R3-NH2).
of Arg 155. Apart from that, all side chains and H-
bonding groups seem to have the same interaction
the L-proline configuration, is a superior HCV NS3
protease inhibitor compared to 35b (Ki > 2000 nM)
(Table 1).
possibilities as 1. Similar SAR in P1 and P3 also indi-
cates that the binding mode of the b-series is similar
to the a-series.
At the P1-position the ethyl Gly amino acid A (Fig. 5) is
roughly 4 times less potent than the corresponding pro-
pyl Gly amino acid B, cf. 32 and 36 (exhibiting Ki values
of 280 and 67 nM, respectively). The most potent inhib-
itors were obtained using the (1R,2S)-1-amino-2-vinyl-
cyclopropylcarboxylic acid C as P1-residue, cf. 35a
and 38a (exhibiting Ki values of 30 and 1.1 nM,
respectively).37
3. Biological data and structure–activity relationships
Inhibitors were evaluated in an in vitro assay compris-
ing the full-length NS3 protein (Table 1). All of the
HCV protease inhibitors synthesized contain the
7-methoxy-2-phenyl-quinolin-4-ol (R2OH) group as
P2-substituent,13a,34 which is reported to stabilize the
catalytic machinery in the correct geometry by shield-
ing the catalytic triad of the protease from exposure
to the solvent35and moreover which likely interacts
with the helicase domain of the NS3 protein enhancing
the overall activity.36 The preferred stereochemistry
of the P2 hydroxycyclopentene is analogous to the
It has previously been reported that the P1-carboxylic
acid forms a hydrogen bond with Ser139 which results
in increased affinity to the HCV NS3 protease.37 The
present inhibitors fall into this general SAR, as can be
seen by comparing, for example, the acids 38a and 39a

F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
831
Table 1. Activity data
Compound
Structure
Ki (nM)
29a
>2000
30 (anti)
523
503
31 (anti)
32 (anti)
280
33 (anti)
>2000
34a
102
35a
30
36 (anti)
67
37 (anti)
24
(continued on next page)

832
F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
Table 1 (continued)
Compound
Structure
Ki (nM)
38a
1.1
39a
1.3
40 (anti)
>5000
34b
2560
35b
>2000
38b
108
39b
23
26 (anti)
>2000
>2000
27 (anti)

F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
833
Figure 7. (A) Diastereomers 35; (B) overlay of energy minimized 35a (red) and 35b (blue). Theoretical NOE interactions of P3NH M P2aH is in 35a
medium (M) and in 35b strong (S); (C) data extracted from ROESY-spectra24 of 35a and 35b displaying the NOE-interactions between
P3NH M P2aH and P1NH M P2aH.
comparing the S,S-inhibitor 30 (Ki value of 523 nM)
corresponding
with the
(Ki > 2000 nM).
R,R-inhibitor
29a
The S3 pocket has a major role to rigidify and prede-
fine the inhibitor in the extended conformation.13f It
has also been shown that the P3 Val can be replaced
by other hydrophobic P3 residues.38 In the present
series enhanced activity was observed when the P3
Val was replaced with tert-butyl glycine, cf. 34 and
35 (displaying Ki values of 102 and 30 nM, respectively).
Attempts to shorten the inhibitors cf. the P1–P3
spanning derivative 33 (Ki > 2000 nM) were not
successful.39
The P4 methyl ester derivative 38a (Ki value of 1.1 nM)
and the P4 methyl amide derivative 39a (Ki value of
1.3 nM) are equipotent, which suggests that no hydro-
Figure 8. Compound 39b (magenta) with the less active enantiomeric
stereochemistry of the cylopentene has been superimpositioned with 1
(gray). The figure shows that it is possible to align 39b with 1, so that
all side chains and all H-bonding groups except the P1–P2 amide
overlay well with 1, indicating a similar binding mode.
(Ki values of 1.1 and 1.3 nM, respectively) with the cor-
responding tert-butylesters 26 and 27, both essentially
devoid of inhibitory activity.
At the P3/P4 positions the S stereochemistry has been
the preferred in all inhibitors reported in the literature.13
In the present work the P2 cyclopentene scaffold intro-
duces a reversal of direction in the peptide chain at the
P2 position, resulting in inhibitors having two carboxy
terminus. Moreover, a one carbon extension is intro-
duced along the P3/P4 amino acid chain. Whilst this
novel P2 hydroxycyclopentene template introduces sub-
stantial structural changes affecting the P3/P4 residues
of the inhibitors it became clear from analyzing the
3D computer overlays of structures 1 and 39a that the
S stereochemistry would indeed be most favored at both
the P3 and P4 positions (Fig. 9). The importance of the
stereochemistry at the P3/P4 residues can be seen by
Figure 9. Compound 39a (orange) with reversed direction of the
peptide in P3 and P4, has been superimpositioned with 1 (gray), for
comparison of the directionality of the side chains. The P3/P4 side
chains of 39a with S stereochemistry overlay well with the P3/P4 side
chains of 1.

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F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
5. Experimental
5.1. HCV NS3 protease enzyme assay41
The inhibition assay with HCV protease was performed
using recombinant full-length NS3 enzyme (Poliakov
et al.) and NS4A (KKGSVVIVGRIVLSGK, Gunnar
Lindeberg, Department of Medicinal Chemistry, Upp-
sala University, Sweden) in final concentrations of
3.5 nM and 14 lM, respectively.42 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 max-
imum final DMSO concentration in the assay was 1%.
After a pre-incubation for 30 min at room temperature,
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, Ana-
Spec, San Jose, CA, USA) to a final concentration of
2 lM. The enzyme activity was continuously 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. 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.
5.2. General methods
1
Standard H NMR and 13C NMR spectra were record-
ed on a Varian 300 instrument using CDCl3 or metha-
nol-d4 with TMS as an internal standard. NOESY
spectra that were used to determine the structure of
syn-6 and anti-6 were recorded on Varian 300 instru-
ment using CDCl3 with TMS as an internal standard.
1H-, 13C-, HMBC-, HSQC-, TOCSY-, NOESY-,
ROESY-, and COSY-NMR that were used to determine
the structure of 35a and 35b, and determining the regio-
chemistry of 7 were recorded on a Varian 500 instru-
ment using methanol-d4 or DMSO-d6. In recorded
NMR spectra with diasteromeric mixtures the presumed
diasteromeric peaks were put into brackets with the gen-
eral formula [nn.n & nn.n] for 13C NMR and [n.nn &
n.nn, (x, yH)] for 1H NMR. Preparative HPLC was per-
formed on a Gynkotek (pump: P580, detector: UVD
170S, software: Chromeleon) using a Kromasil 100-10-
C18 (250· 20 mm) column. Mixtures of deionized water
and methanol were used as mobile phase. TLC was car-
ried out on Merck precoated 60 F254 plates using UV-
light and charring with ethanol/sulfuric acid/acetic
acid/p-anisaldehyde (90:3:1:2) for visualization. MAL-
DI-TOF analysis was performed using a Voyager-DE
STR Biochemistry Workstation, in positive mode.
Using the matrix recipe: 10 mg a-cyano-4-hydroxy-
trans-cinnamic acid dissolved in 0.5 mL of 0.1% TFA
solution and 0.5 mL acetonitrile. LC/MS was performed
Figure 10. NS3/4A inhibition values for the P2-cyclopentene inhibitor
39a and the corresponding P2-cyclopentane inhibitor 41.
gen donating bonds are required at this position. In con-
trast N-methylation of the P4-NH derivative 39a to give
P4-NMe derivative 40 (Ki > 5000 nM) results in loss of
inhibitory activity, suggesting that the P4-NH is in-
volved in a hydrogen bond donation network with the
NS3-enzyme.
As can be seen from Figure 10, it is evident that the
most potent inhibitor in the previously synthesized
P2-cyclopentane series19 is considerably less active
than the corresponding inhibitor of the P2-cyclopen-
tene series.40
4. Conclusion
Eighteen HCV NS3 protease inhibitors have been syn-
thesized all encompassing the novel 4-hydroxy-cyclo-
pent-2-ene-1,2-dicarboxylic
acid,
a
N-acyl-L-
hydroxyproline mimic, in the P2-position. These inhibi-
tors display two carboxy terminus where the P2 tem-
plate induces a reversal of direction of the P3/P4
amino acid chain. In spite of these different features as
compared with inhibitors previously reported in the lit-
erature very potent inhibitors have been identified. The
most potent inhibitor in this series displays an impres-
sive Ki value of 1.1 nM.
on
a Gilson system (Column: Phenomenex C18
250 · 15 mm and Phenomenex C18 150 · 4.6 mm for
preparative and analytical runs, respectively; Pump: Gil-
son gradient pump 322; UV/Vis-detector: Gilson 155;
MS detector: Thermo Finnigan Surveyor MSQ; Gilson

F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
835
Fraction Collector FC204, software: Gilson UniPoint
ver. 4.0 and Xcalibur ver. 1.3) using methanol with
0.1% formic acid and deionized water with 0.1% formic
acid as mobile phase. Concentrations were performed
under diminished pressure (1–2 kPa) at a bath tempera-
ture of 40 ꢁC. All low-temperature reactions were
accomplished by submerging the reaction vessels into
an ethanol bath that had been cooled by additions of li-
quid nitrogen. Organic phases were dried over magne-
sium sulfate monohydrate. All degassing of solvents
were achieved using ultrasonification (Ultrasonik 104·)
for at least 2 h. Optical rotation was performed on a
Perkin-Elmer 141. Drying of solvents and reagents:
THF was refluxed over sodium/benzophenone and dis-
12.3 mmol) dissolved in dioxane and water (1:1,
50 mL), LiOH (0.3 g, 12.3 mmol) was added. After
two and a half hours the mixture was co-evaporated
with toluene and methanol. Purification by flash chro-
matography (toluene/ethyl acetate 3:1 + 1% AcOH)
gave a syn/anti (6:1) mixture (1.6 g, 68%), which was
recrystallized twice from hexane/diethyl ether yielding
pure syn-7 (0.7 g, 31%) as white crystals (mp 94–
1
98 ꢁC). H NMR (300 MHz, CD3OD): d 1.78–1.89 (m,
1H), 2.70–2.84 (m, 1H), 3.56–3.71 (m, 1H), 3.76 (s,
3H), 4.81–4.90 (m, 1H), 6.76–6.81 (m, 1H); 13C NMR
(75.5 MHz, CDCl3): d 38.0, 48.0, 52.4, 75.7, 137.0,
146.2, 165.0 178.4.
˚
tilled onto 4 A MS; filtration was achieved using filter
paper Munktell, OOH quality.
5.4. Method used for synthesis of the amides 8–10
5.4.1. ((3S,5R) and (3R,5S))-5-((S)-1-tert-Butoxycarbon-
yl-butylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylic
acid methyl (9). To an ice cooled solution of 7 (0.20 g,
1.1 mmol) and B (2-amino-pentanoic acid tert-butyl es-
ter) (0.24 g, 1.4 mmol) in DMF (7 mL), DIPEA
(0.18 g, 1.4 mmol) and HATU (0.53 g, 1.4 mmol) were
added. After two hours the solution was concentrated
and purified by flash chromatography (toluene/ethyl
acetate 3:1). This gave 9 as a yellow oil (0.22 g, 63%).
1H NMR (300 MHz, CDCl3): d 0.84–0.96 (m, 3H),
1.14–1.39 (m, 2H), [(1.44 and 1.49) (s, 9H)], 1.50–1.60
(m, 1H), 1.61–1.85 (m, 1H), 1.97–2.10 (m, 1H), 2.11–
2.28 (m, 1H), 3.57–3.68 (m, 1H), [(3.73 and 3.76) (s,
3H)], 4.30–4.50 (m, 1H), 4.63–4.73 (m, 1H), 6.80–6.95
(m, 1H), 6.95–7.00 (m, 1H); 13C NMR (75.5 MHz,
CDCl3): d [13.9 and 14.0], [18.5 and 18.7] [28.1 and
28.2], [34.7 and 34.8], 37.6, [50.3 and 50.5], [52.0 and
52.2], [53.4 and 53.6], [75.0 and 75.1], [82.2 and 82.4],
[137.1 and 137.2], [146.1 and 146.3], 165.1, [171.2 and
171.5], [173.3 and 173.5].
5.3. Synthesis of the P2-template
5.3.1. ((1S,4R) and (1R,4S))-4-Hydroxy-cyclopent-2-ene-
1,2-dicarboxylic acid dimethyl ester (syn-6). To a cold
solution (ꢁ30 ꢁC) of 5 (3.18 g, 16.1 mmol) dissolved in
MeOH (23 mL), NaBH4 (0.66 g, 17.5 mmol) was added.
After seven minutes the excess NaBH4 was destroyed by
adding brine (80 mL). The mixture was concentrated
and extracted with ethyl acetate (3· 80 mL). The com-
bined organic phases were dried, filtered, and concen-
trated which gave the diasteromeric mixture (syn/anti
6:1) of 6 (3.0 g, 92%) as a yellow oil. The major com-
pound syn-6 could further be purified by flash chroma-
tography (toluene/ethyl acetate 3:1) but the anti-6 was
always polluted with the syn-6. 1H NMR (300 MHz,
CDCl3): d 1.69–1.77 (m, 1H), 2.43–2.56 (m, 1H), 3.55
(s, 3H), 3.57 (s, 3H), 3.68–3.70 (m, 1H), 4.66–4.4.69
(m, 1H) 6.70–7.72 (m, 1H); 13C NMR (75.5 MHz,
CDCl3): d 38.3, 48.0, 52.2, 52.9, 75.7, 137.0, 146.3,
164.6, 175.9.
5.5. Synthesis of the ethers 11–13
5.3.2. (1R,4R) and (1S,4R)-Dimethyl-4-hydroxycyclo-
pent-2-ene-1,2-dicarboxylate (anti-6). The alcohol syn-6
(0.23 g, 1.15 mmol), benzoic acid (0.16 mL, 1.4 mmol),
triphenylphosphine (0.37 g, 1.4 mmol) and DIAD
(0.32 mL, 1.6 mmol) were dissolved in dry THF
(5.7 mL) and stirred at room temperature. After two
hours the solvent was removed by evaporation to give
a yellow oil. The residue was purified by flash chroma-
tography (toluene/ethyl acetate 18:1) to give a colorless
oil. The oil was diluted in MeOH (6 mL) and cooled
in an ice bath. To the solution sodium methoxide
(0.57 mmol, 1 M) was slowly added while stirring. After
one hour the reaction was terminated and the solvent
was removed by evaporation. The residue was purified
by flash chromatography (toluene/ethyl acetate 1:1) to
give the alcohol anti-6 as a yellow oil. 1H NMR
(300 MHz CDCl3): d 2.04–2.13 (m, 1H) 2.37–2.46 (m,
1H), 3.65 (s, 3H), 3.71 (s, 3H) 3.87–3.92 (m. 1H),
5.05–5.15 (m, 1H), 6.79–6.81 (m, 1H); 13C NMR
(75.5 MHz CDCl3): d 38.9, 48.6, 52.2, 52.4, 75.7,
136.4, 146.6, 164.7, 174.5.
5.5.1. ((3R,5R) and (3S,5S))-5-((S)-1-tert-Butoxycarbon-
yl-butylcarbamoyl)-3-(7-methoxy-2-phenyl-quinolin-4-
yloxy)-cyclopent-1-enecarboxylic acid methyl ester (12).
To an ice cooled solution of 9 (0.23 g, 0.67 mmol) in
dry THF (5 mL), 7-methoxy-2-phenyl-quinolin-4-ol
(0.22 g, 0.88 mmol) and triphenylphosphine (0.23 g,
0.88 mmol) were added. Then DIAD (0.18 g,
0.88 mmol) was dissolved in THF (2 mL) and added
dropwise to the solution. After one hour the mixture
was concentrated and purified by flash chromatogra-
phy (toluene/ethyl acetate 3:1). This gave 12 as a white
1
powder (0.30 g, 77%). H NMR (300 MHz, CDCl3): d
0.88–1.00 (m, 3H), 1.18–1.43 (m, 2H), [(1.45 and
1.50) (s, 9H)], 1.53–1.65 (m, 1H), 1.66–1.85 (m, 1H),
2.29–2.43 (m, 1H), 3.10–3.25 (m, 1H), [(3.79 and
3.83) (s, 3H)], 3.97 (s, 3H), 4.05–4.20 (m, 1H), 4.38–
4.50 (m, 1H), 6.03–6.13 (m, 1H), 6.65–6.90 (m, 1H),
7.04–7.18 (m, 3H), 7.40–7.56 (m, 4H), 8.00–8.12 (m,
3H); 13C NMR (75.5 MHz, CDCl3): d 14.0, [18.6 and
18.8], [28.2 and 28.3], [34.9 and 35.0], 36.2, [50.2 and
50.5], 52.4, 53.2, 55.7, 82.0, 82.3, 98.4, 107.8, 118.4,
123.1, 127.8, 129.0, 129.1, 129.2, 129.5, 132.2, 138.7,
140.5, 142.4, 152.0, 159.2, [161.0 and 161.4], [164.0
and 164.2], [171.2 and 171.3].
5.3.3. (1S,4R) or (1R,4S)-4-Hydroxy-cyclopent-2-ene-
1,2-dicarboxylic acid 2-methyl ester (7). To an ice-cold
solution of a diasteromeric mixture of 6 (2.5 g,

836
F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
5.6. Method used for synthesis of the carboxylic acids
14–16
5.8. Method used for synthesis of the final compounds 29–40
5.8.1. (S)-2-{[(1R,4R)-2-{(R)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclo-
pent-2-enecarbonyl]-amino}-pentanoic acid (35a) and (S)-
2-{[(1S,4S)-2-{(R)-1-[((S)-Cyclohexyl-methoxycarbonyl-
methyl)-carbamoyl]-2,2-dimethyl-propylcarbamoyl}-4-(7-
methoxy-2-phenyl-quinolin-4-yloxy)-cyclopent-2-enecar-
bonyl]-amino}-pentanoic acid (35b). A mixture of the tert-
butyl ester 23a and 23b (28 mg, 0.034 mmol), triethyl si-
lane (8.7 mg, 0.075 mmol) was dissolved in DCM
(1 mL) and cooled to 0 ꢁC using an ice-bath. TFA
(1 mL) was added and two hours later the mixture was
concentrated. The diastereomers were separated by the
use of preparatory HPLC (65% MeOH + 0.2% TEA) to
the chiral compounds 35a (15 mg, 55%) and 35b (12 mg,
45%). After lyophilization 35a and 35b were collected as
5.6.1. ((3R,5R) and (3S,5S))-5-((S)-1-tert-Butoxycarbon-
yl-butylcarbamoyl)-3-(7-methoxy-2-phenyl-quinolin-4-
yloxy)-cyclopent-1-enecarboxylic acid (15). The methyl
ester 12 (0.35 g, 0.61 mmol) was dissolved in dioxane/
water (1:1, 7 mL) and LiOH (0.031 g, 1.3 mmol) was
added. The reaction mixture was stirred overnight and
then diluted with toluene/methanol (1:1, 10 mL) and
concentrated. This gave the lithium salt of 15 (0.32 g,
90%) as a brown powder. 1H NMR (300 MHz,
CD3OD): d 0.90–1.00 (m, 3H), 1.18–1.30 (m, 1H),
1.38–1.42 (m, 1H), [(1.44 and 1.53) (s, 9H)], 1.60–1.82
(m, 2H), 2.30–2.40 (m, 1H), 3.00–3.17 (m, 1H), 3.98 (s,
3H), 4.01–4.12 (m, 1H), 4.20–4.30 (m, 1H), 5.95–6.05
(m, 1H), 6.78–6.82 (m, 1H), 7.10–7.30 (m, 3H), 7.45–
7.60 (m, 3H), 8.00–8.15 (m, 3H); 13C NMR
(75.5 MHz, CD3OD): d 12.9, (18.7 and 18.80, 23.1,
27.1, 35.7, (50.1 and 50.3), (53.5 and 53.7), 54.8, 81.4,
(82.9 and 83.0), 98.9, 106.3, 117.9, 123.1, 125.2, 127.8,
128.0, 128.6, 128.7, 129.3, 135.3, 135.6, 140.3, 151.0,
160.3, 161.9, 170.7, 174.1.
22
1
white powders. Compound 35a: ½aꢂ +155.8; H NMR
D
(300 MHz, CD3OD): d 0.90–0.97 (m, 3H), 1.03 (s, 9H),
1.05–1.50 (m, 7H), 1.50–1.80 (m, 8H), 2.43–2.55 (m,
1H), 2.77–2.90 (m, 1H), 3.68 (s, 3H), 3.96 (s, 3H), 4.20–
4.30 (m, 2H), 4.31–4.40 (m, 1H), 4.45–4.50 (m, 1H),
6.03–6.11 (m, 1H), 6.98 (s, 1H), 7.12–7.19 (m, 1H), 7.36
(s, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.50–7.60 (m, 3H),
8.03–8.10 (m, 3H); 13C NMR (75.5 MHz, CD3OD): d
13.1, 19.1, 26.1, 28.7, 28.9, 29.5, 34.3, 34.8, 35.9, 40.1,
50.8, 51.2, 54.8, 55.0, 57.9, 60.7, 83.5, 99.1, 106.0, 115.2,
118.2, 123.3, 127.8, 128.0, 128.7, 128.8, 129.7, 135.2,
139.8, 143.7, 150.6, 160.1, 162.2, 165.2, 171.7, 172.2,
173.4. HRMS m/z 771.3988 [(M+H)+ calcd for
5.7. Method used for synthesis of the tert-butylesters
17–28
5.7.1. (S)-2-{[(1R,4R)-2-{(R)-1-[((S)-Cyclohexyl-meth-
oxycarbonyl-methyl)-carbamoyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclo-
pent-2-enecarbonyl]-amino}-pentanoic acid tert-butyl
ester (23a) and (S)-2-{[(1S,4S)-2-{(R)-1-[((S)-Cyclohex-
yl-methoxycarbonyl-methyl)-carbamoyl]-2,2-dimethyl-
propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-
yloxy)-cyclopent-2-enecarbonyl]-amino}-pentanoic acid
tert-butyl ester (23b). To an ice-cold DMF (1 mL) solu-
tion of 15 (60 mg, 0.10 mmol) the amine F (42 mg,
0.15 mmol), DIPEA (19 mg, 0.15 mmol) and HATU
(62 mg, 0.16 mmol) were added. After two and a half
hours the mixture was concentrated and purified by
flash chromatography (toluene/ethyl acetate 3:1)
(25 mg, 50%). The diastereomers 23 were separated
using HPLC (90% MeOH + 0.2% TEA) to 23a (6 mg,
22
D
þ
C43H55N4O9 771.3969]. Compound 35b: ½aꢂ ꢁ72.3;
1H NMR (300 MHz, CD3OD): d 0.90–0.97 (m, 3H),
1.02 (s, 9H), 1.07–1.35 (m, 7H), 1.53–1.90 (m, 8H),
2.46–2.61 (m, 1H), 2.76–2.88 (m, 1H), 3.69 (s, 3H), 3.96
(s, 3H), 4.15–4.35 (m, 2H), 4.37–4.41 (m, 1H), 4.42–4.47
(m, 1H), 6.02–6.12 (m, 1H), 7.02 (s, 1H), 7.16 (dd,
J = 2.47, 9.34 Hz, 1H), 7.32 (s, 1H), 7.40 (d,
J = 2.47 Hz, 1H), 7.48–7.58 (m, 3H), 8.03–8.12 (m, 3H);
13C NMR (75.5 MHz, CD3OD): d 13.0, 18.8, 25.9, 26.0,
28.8, 29.4, 34.2, 34.8, 36.3, 39.9, 48.8, 50.5, 51.1, 54.8,
57.9, 60.5, 82.8, 99.0, 106.0, 115.1, 118.2, 123.1, 127.8,
127.9, 128.7, 129.0, 129.5, 136.7, 139.8, 142.8, 150.6,
160.1, 162.0, 162.2, 164.7, 172.1, 173.5. HRMS m/z
1
6%) and 23 b (9 mg, 10%). Compound 23a: H NMR
þ
(300 MHz, CDCl3): d 0.82–0.90 (m, 3H), 1.01 (s, 9H),
1.05–1.40 (m, 7H), 1.46 (s, 9H), 1.50–1.80 (m, 8H),
2.20–2.35 (m, 1H), 3.07–3.25 (m, 1H), 3.73 (s, 3H),
3.97 (s, 3H), 4.11 (d, J = 7.96 Hz, 1H), 4.38–4.52 (m,
3H), 6.03–6.12 (m, 1H), 6.24 (d, J = 8.79 Hz, 1H), 6.63
(s, 1H), 6.82 (d, J = 9.06 Hz, 1H), 7.07–7.27 (m, 2H),
7.36 (d, J = 7.96 Hz, 1H), 7.41–7.55 (m, 4H), 8.01–8.10
(m, 3H); 13C NMR (75.5 MHz, CD3OD): d 14.0, 18.8,
26.1, 26.8, 28.2, 28.6, 29.6, 34.9, 35.6, 36.2, 40.9, 50.7,
52.4, 53.3, 55.7, 57.3, 60.8, 82.0, 82.7, 98.4, 105.2,
107.7, 115.2, 118.4, 123.2, 127.9, 129.0, 129.4, 131.1,
135.1, 138.4, 142.4, 153.3, 159.6, 161.6, 164.2, 170.1,
171.3, 172.2. Compound 23b: 1H NMR (300 MHz,
CDCl3): d 0.90–0.98 (m, 3H), 1.04 (s, 9H), 1.08–1.40
(m, 7H), 1.44 (s, 9H), 1.55–1.90 (m, 8H), 2.20–2.38 (m,
1H), 3.10–3.22 (m, 1H), 3.73 (s, 3H), 3.97 (s, 3H),
4.02–4.15 (m, 1H), 4.35–4.48 (m, 3H), 6.00–6.08 (m,
1H), 6.72 (s, 1H), 6.90 (d, J = 9.06 Hz, 1H), 7.09–7.20
(m, 3H), 7.44–7.55 (m, 5H), 8.03–8.11 (m, 3H).
771.4006 [(M+H)+ calcd for C43H55N4O9 771.3969].
5.9. Modeling
The alignments of 39a and 39b with compound 1 were
obtained from energy minimizations and GASPTM
(Genetic Algorithm Similarity Program) included in
Sybyl 7.1 (Tripos Inc. 1699 South Hanley Rd., St. Louis,
Missouri, 63144, USA). The compounds (1, 39a, and 39b)
were built in an extended conformation and minimized.
The alignment of 39a and 39b with 1, respectively, was
run in 10 cycles for each compound. The final results from
the alignments are shown in Figures 8 and 9.
Acknowledgments
We gratefully acknowledge financial support from
VINNOVA and the Swedish Foundation for Strategic

F. Thorstensson et al. / Bioorg. Med. Chem. 15 (2007) 827–838
837
10. http://www.vrtx.com/. News may 17 2005: Vertex reports
that oral hepatitis C protease inhibitor, VX-950, dramat-
ically reduces viral levels in phase Ib clinical study.
11. (a) Shu-Hui, C.; Lamar, J.; Yip, Y.; Frantz, V.; Johnson,
R. B.; Wang, Q. M.; Glass, J. I.; Heinz, B.; Colacino, J.
Lett. Drug Des. Discovery 2005, 2, 118–124; (b) Lin, C.;
Lin, K.; Luong, Y.-P.; Rao, B. G.; Wei, Y.-Y.; Brennan,
D. L.; Fulghum, J. R.; Hsiao, H.-M.; Ma, S.; Maxwell, J.
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Research (SSF) from the VINST program, Medivir AB,
and Knut and Alice Wallenberg’s Foundation. We also
thank Professor Pei Zhen Tao, Department of Virology,
Institute of Medicinal Technology, Beijing, China, for
enzymatic testing, Dr Tatiana Maltseva, Medivir AB
Sweden for 2D NMR experiments, and Jussi Kangas-
metsa¨, Medivir UK, for HRMS analysis.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.10.044.
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