Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents

Article abstract of DOI:10.1016/j.bmc.2010.05.027

Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel π-π-interaction between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized that the introduction of a vinyl on the phenylglycine might strengthen this π-π-interaction. Thus, herein is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both d- and l-phenylglycine were found to be effective inhibitors, with a slight preference for the d-epimers. Furthermore, prime-side alkenylic extension of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the nanomolar range (～35 nM), potencies which were retained on mutant variants of the protease.

Full text of DOI:10.1016/j.bmc.2010.05.027

Bioorganic & Medicinal Chemistry 18 (2010) 5413–5424
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors
with alkenylic prime-side substituents
Anna Lampa a, Angelica E. Ehrenberg b, Sofia S. Gustafsson b, Aparna Vema a, Eva Åkerblom a,
Gunnar Lindeberg a, Anders Karlén a, U. Helena Danielson b, Anja Sandström a,
*
a Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
b Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 January 2010
Revised 6 May 2010
Accepted 11 May 2010
Available online 20 May 2010
Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel p–p-interaction
between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized
that the introduction of a vinyl on the phenylglycine might strengthen this -interaction. Thus, herein
is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based
HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both - and -phenylglycine were found to
be effective inhibitors, with a slight preference for the -epimers. Furthermore, prime-side alkenylic exten-
p–p
D
L
D
Keywords:
HCV
Protease inhibitors
Peptidomimetics
Phenylglycine
Resistance
sion of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the
nanomolar range (ꢀ35 nM), potencies which were retained on mutant variants of the protease.
Ó 2010 Elsevier Ltd. All rights reserved.
Alkenylic acylsulfonamides
1. Introduction
in vivo.6 Supplementary to the proteolytical properties of the
NS3 protease, it has also been shown to be involved in processes
Hepatitis C virus (HCV) is considered as a major and growing
global health problem. HCV causes inflammation of the liver and
is carried by an estimated 170 million people worldwide.1 An
HCV infection can lead to serious conditions such as acute liver dis-
ease, cirrhosis, or liver cancer.2 As a consequence, HCV is the lead-
ing cause of liver transplantation. Reinfection of the new liver is
often a problem, and liver transplantation does therefore not lead
to termination of an HCV infection.3 The current treatment
regimen consists of a combination of ribavirin and pegylated
to attenuate and evade the host cell’s natural immune defense.7
Of several potential anti-HCV drug targets, the NS5B polymer-
ase and the NS3 protease have so far been the most extensively
examined points of attack. An advantage with NS3 protease inhib-
itors is the potential two mechanisms of action, as mentioned,
leading to a restored host immune response as well as inhibition
of HCV replication. Thus, a number of NS3 protease inhibitors have
been developed, for example, BILN-2061,8 ciluprevir (Fig. 1). It was
the first HCV NS3 protease inhibitor to enter clinical trials, but it
was later withdrawn due to heart toxicity in animals. At present,
several inhibitors are being evaluated in clinical trials9 (Fig. 1).
HCV NS3 protease inhibitors can be divided into two major clas-
ses, according to their mechanism of action. The product-based
HCV NS3 protease inhibitors, exemplified by BILN-2061, TMC435,
RG7227, and MK-7009 (Fig. 1), are based on the findings that
NS3 protease is inhibited by its own cleavage product.15,16 These
inhibitors are characterized by an acidic functionality such as a car-
boxylic acid or an acyl sulfonamide at the C-terminal. The other
class, the electrophilic HCV NS3 protease inhibitors, exemplified
by VX-950 and SCH 503034 (Fig. 1), are reversibly, but covalently,
modifying the catalytic serine residue of the protease.17 Although
the two classes of inhibitors are very different, both from a struc-
tural and mechanistical point of view, a common feature in the
inhibitors is a P2 proline or a proline mimic.
interferon-a. Unfortunately, the therapy is ineffective for about
40–50% of patients with chronic HCV infection, and it has several
adverse effects.4 Hence, development of new therapies is of great
importance.
HCV contains single stranded (+)-RNA that is released inside the
infected hepatocyte and translated into a polyprotein. The polypro-
tein contains about 3000 amino acids and is proteolytically cleaved
into 10 viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A,
and NS5B. NS3 is an enzyme with dual functionalities. It has serine
protease as well as helicase/NTPase activities. This viral protease is
responsible for the cleavage between NS3/4A, NS4A/4B, NS4B/5A,
and NS5A/5B5 and has proven to be essential for survival of HCV
* Corresponding author. Tel.: +46 18 471 49 57; fax: +46 18 471 4474.
E-mail address: Anja.Sandstrom@orgfarm.uu.se (A. Sandström).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.027

5414
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
O
F
N
O
H
N
N
N
O
O
S
O
N
O
O
NH
O
O
O
H
N
O
N
N
O
NH
O
O
O
N
S
NH
HN
O
NH
O
N
OH
O
H
O
O
O
N
H
N
N
H
O
VX-950, Telaprevir
Ki = 40 nM, EC50 = 350 nM
BILN 2061, Ciluprevir
Ki = 0.30 nM, EC50 = 4 nM
RG7227 (ITMN-191)
IC50 < 0.25 nM, EC50 = 2.1 nM
O
N
N
N
O
O
O
S
O
N
O
O
NH
NH
O
N
H
N
O
O O
O
O
NH
NH
O
O
O
S
S
NH2
O
NH
N
O
O
O
N
H
N
O
H
TMC435
Ki = 0.36 nM, EC50 = 7.8 nM
MK-7009
Ki = 0.06 nM, EC50 = 5 nM
SCH 503034, Boceprevir
Ki* = 14 nM, EC50 = 200 nM
Figure 1. Selection of HCV NS3 protease inhibitors that have entered clinical trials. VX-950,10 SCH 503034,11 BILN-2061,8 TMC435,12 RG7227, formerly referred to as
ITMN-191,13 and MK-7009.14
The large population size and high mutability of HCV, have led
to the emergence of several genotypes and quasispecies of the
virus.18 Moreover, HCV has been shown to evolve into resistant
forms to one or more types of protease inhibitors both in vitro
and in vivo. The proline residue in the P2 position has been shown
to be the basis for the cross-resistance shown by the two classes of
inhibitors for A156 mutated NS3 variants.19,20 Consequently, there
is a big need for new HCV NS3 protease inhibitors based on non-
proline P2 moieties which can possess a different binding mode.
In the search for novel HCV NS3 protease inhibitors, we previ-
ously found phenylglycine (Phg) as a useful P2 residue, with a pos-
different fashion than the proline-based ones — a fact which we
believe can be utilized in the development of inhibitors with a un-
ique resistance profile. However, this necessitates the search for
new optimized phenylglycine-based inhibitors.
The aromatic nature of phenylglycine not only convey possible
p
-interactions, but also allows for chemical decorations. We
hypothesized that introduction of a vinyl substituent on the phen-
ylglycine could increase the -stacking that has been proposed as
p
an important interaction between the phenylglycine and the H57
residue.21 A vinyl can also serve as a handle in forthcoming
metathesis reactions.
sible
p
-stacking to the catalytic H57 residue, exemplified by
Thus, we herein present the synthesis, structure–activity rela-
tionships and biochemical evaluation on both wild-type and mu-
tated enzyme of a series of vinyl substituted phenylglycine-based
HCV NS3 protease inhibitors with different acylsulfonamides at
the C-terminal. The inhibitor modifications explored herein led to
optimized phenylglycine-based inhibitors. The same modifications
were less effective in a proline-based inhibitor. These results fur-
ther strengthen the hypothesis of a different binding mode of
phenylglycine-based inhibitors which can be advantageous in the
development of unique HCV NS3 protease inhibitors.
compound A in Figure 2.21 Moreover, a phenylglycine-based inhib-
itor has shown to retain its potency to the R155Q, A156T, and
D168V mutants of the NS3 protease.22 Phenylglycine-based inhib-
itors were previously found to differ in structure–activity relation-
ship to the well investigated proline-based NS3 protease inhibitors.
Fragments that are optimized for the proline-based inhibitors, for
example, the vinyl-ACCA in the P1 position did not improve po-
tency for phenylglycine-based inhibitors.21 The overall results indi-
cated that phenylglycine-based inhibitors bind to the protease in a
2. Results
O
2.1. Chemistry
N
Compound 123 (Scheme 1) was synthesized according to de-
scribed procedures,23–25 starting by brominating 4-hydroxy-
L-
O
phenylglycine with bromine and hydrobromic acid in acetic acid.
Only monobrominated 3-bromo-4-hydroxy- -phenylglycine was
L
obtained, in our case as the hydrobromide salt. The amine was
Boc-protected,24 and then separated from non-brominated
compound on silica.25 A nucleophilic aromatic substitution reac-
tion using 1, 4-chloro-7-methoxy-2-phenylquinoline26 and potas-
sium-tert-butoxide in DMSO resulted in the heteroaryl ether 2
(Scheme 1) after 6½ weeks of stirring at 64 °C. The extremely long
O
O
O
O
S
H
N
BocHN
N
H
N
H
O
A
Figure 2. Compound A,21 Ki = 230 nM.

A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
5415
O
O
complex resulting in a partly racemized product (Scheme 1, com-
pound 3). An enantiomeric mixture of
L and D in about a 2:1 ratio
was obtained, as proved by chiral HPLC analysis of 3 and also dia-
stereomeric analysis by HPLC after a forthcoming coupling (vide
infra). The tendency of phenylglycine racemization during Suzuki
reactions has been described previously.28
N
N
OH
O
O
Br
Br
The P1–P10 building blocks (4–11) were all synthesized
(Scheme 2) from commercially available Boc protected norvaline
a
b
OH
OH
OH
(Boc-L-Nva) (compounds 4–10), or Boc protected L-cyclopropylala-
BocHN
BocHN
BocHN
O
O
O
nine (Cpa) (compound 11). The amino acids were coupled with sul-
fonamides that were either obtained commercially or synthesized
following literature procedures26,29–31 using carbonyldiimidazole
(CDI) and 1,8-diazabicycloundecene (DBU) as base in dry THF
resulting in compounds 4–8 and 10, 11. The amino acids were acti-
vated by CDI at 60 °C or room temperature for 1 h before addition of
sulfonamide and DBU at room temperature. The reactions where the
amino acids were activated under heat were completed within 2 h.
3
1
2
Scheme 1. Reagents and conditions: (a) 4-chloro-7-methoxy-2-phenylquinoline,
KOtBu, DMSO, 64 °C, 43%; (b) 2,4,6-trivinylcycloboroxane pyridine complex,
Pd(OAc)2, [(tBu)3PH]BF4, K2CO3, DME, H2O, microwave heating at 100 °C for
15 min, 34%.
reaction time was preferred over a shorter reaction with higher
temperature due to unwanted removal of the Boc-protecting group
and decarboxylation. The vinyl was introduced by a microwave as-
sisted Suzuki reaction27 using 2,4,6-trivinylcycloboroxane pyridine
This also resulted in a small amount of racemization at the P1 a-car-
bon as proved by the formation of epimers detected in compounds
19–25. The ortho vinylated P1–P10 building block (compound 9)
O
O
N
N
O
O
O
BocHN
OH
O
O
O
O
O
O
S
O
O
O
O
O
H2N
R
3
H
N
H
N
BocHN
BocHN
S
OH
N
R
S
BocHN
a
c, d BocHN
N
R
c, d
N
H
H
P1
P1
H
O
P1
O
P1
P1
R
4
12
19aa, 19bb
5
6
13
14
20aa, 20bb
21aa, 21bb
7
8
9
15
22aa, 22bb
Br
b
23aa, 23bb
24aa, 24bb
16
17
10
11
25a
18
Scheme 2. Reagents and conditions: (a) CDI, THF, 60 °C/rt, DBU, rt, 15–84% ; (b) 2,4,6-trivinylcycloboroxane pyridine complex, Pd(OAc)2, [(tBu)3PH]BF4, K2CO3, DME, H2O,
a
b
microwave heating at 100 °C for 15 min, 66%; (c) 4.0 M HCl in dioxane; (d) HATU, DIEA, DMF, rt
L-Phg, D-Phg, 20–56%.

5416
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
was made from the corresponding ortho brominated compound
(compound 8) through a microwave assisted Suzuki reaction.27
Deprotection of 4–7 and 9–11 with 4.0 M HCl in dioxane
and coupling with the P2-building block (compound 3) using
N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-meth-
ylene]-N-methylmethanaminium hexafluorophosphate N-oxide
(HATU) and N,N-diisopropylethylamine (DIEA) in DMF resulted in
diastereomeric mixtures of compounds 12–18 (Scheme 2), due to
the previously mentioned racemization on the phenylglycine resi-
due during the Suzuki reaction.
phenylglycine in L-form (compounds 19a–24a, 25), and D-form
(compounds 19b–24b), respectively. The two isomers could be sep-
arated by preparative HPLC on a reversed phase column. The major
isomer eluted earlier than the minor isomer. The ratio between the
major and minor isomers was approximately 2:1, in analogy with
the results shown by chiral HPLC analysis of compound 3. This indi-
cates that the major isomer corresponds to the
the synthesis started with -Phg (compound 1). Throughout the ser-
ies we have also observed in the 1H NMR spectra that the phenylgly-
cine -proton is shifted downfield in the -Phg isomers (compounds
19a–24a, 25) to the corresponding shift for the -Phg isomers (com-
-proton in epi-
L-Phg isomer since
L
a
L
After N-deprotection, compounds 12–18 were coupled with Boc-
t-Leu, using HATU and DIEA in DMF. This resulted in compounds 19–
25 (Scheme 2) in a diastereomeric mixture of two isomers with the
D
pounds 19b–24b). The difference in shift of the Phg
a
meric pairs has been described before by Salituro and Townsend,24
and is in analogy with our findings. The degree of RP-HPLC separa-
tion of the L- and D-isomers of the final compounds varied a lot.
O
O
The yields of the final compounds depended on how well the iso-
mers separated and are therefore indefinitely calculated, and some-
times deceptively low. Some inhibitors were better separated in TFA
buffer than formic acid buffer. With TFA present in the eluent the
inhibitors were obtained as TFA-salt.
Compound 26 (Scheme 3), synthesized according to a previously
published procedure26 was coupled with the N-deprotected HCl
salt of 4. This yielded compound 27 (Scheme 3) which was used
as a reference compound comprising proline in the P2 position.
N
N
O
O
4
N
N
a, b
O
OH
NH
O
S
BocHN
BocHN
O
O
O
O
O
HN
26
27
2.2. Biochemical evaluation
Ki SD = 300 46 nM
Compounds 19–25 were biochemically evaluated and Ki-values
Scheme 3. Reagents and condition: (a) 4.0 M HCl/dioxane; (b) HATU, DIEA, DMF, rt,
determined with an activity-based inhibition assay containing the
21%.
Table 1
Enzyme inhibition constants (Ki-values) and inhibition of replication (EC50) measured on wild-type enzyme and in a subgenomic HCV replicon assay, respectively
O
O
L-Phg
D-Phg
N
N
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
O
S
O
S
R
R
N
H
N
N
H
N
H
H
O
O
O
O
R
Compound
Ki SD (nM)
63
EC50
(
lM)
R
Compound
Ki SD (nM)
48
EC50 (lM)
19a
20a
9
>10
>10
19b
20b
2
8.9
4.7
120 26
100 14
21a
22a
23a
330 60
nd
21b
22b
23b
190 40
nd
nd
5.4
290 40
120 20
nd
130 20
>10
47
35
6
2
24a
25a
140 40
>10
nd
24b
3
500 130
SD, standard deviation.
nd, not determined.
a
P1 Cpa.

A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
5417
full-length NS3 protein and a peptide corresponding to the activat-
ing region of NS4A32 (Table 1). Additionally, compounds 19a, 19b,
20a, 20b, 23a, and 23b were evaluated on A156T and D168V mu-
tant forms of the protease22 (Table 2). Inhibition constants were
measured and vitality values calculated. Vitality values describe
the influence the mutant substitution has on the enzyme when
the inhibitor is present. A vitality value >1 means that the mutation
is favorable for the enzyme in the presence of inhibitor, while a
vitality value <1 means that the mutation is disadvantageous.
Compounds 19a, 19b, 20a, 20b, 23a, 23b, 24a, 24b, and 26 were
also tested for EC50-values in a replicon system using the Huh-7
cell line containing subgenomic HCV RNA genotype 1b replicon
with firefly luciferase (Table 1).33
elongated phenyl substituents were 2–4 times more potent than
the non-elongated phenyl substituents (compounds 22a
Ki = 290 nM and 22b Ki = 130 nM). The same increase in potency
was seen when the cyclopropyl was extended with an allyl (com-
pound 21a Ki = 330 nM), which gave a 1.5-fold more potent com-
pound than the non-elongated cyclopropyl (compound 25
Ki = 500 nM), even though these compounds have different P1 sub-
stituents and cannot be fairly compared. Cyclopropyl is commonly
used as a P10 substituent in proline-based inhibitors (see Fig. 1).
However, in this series the compounds containing an elongated
cyclopropyl (compounds 21a and 21b) or a cyclopropyl (compound
25) in P10 were among the least potent.
Generally, there was not very much difference in inhibitory po-
tency between the epimeric pairs (a and b). Surprisingly, the
isomers (b-compounds) were slightly more potent than the
D
-Phg
-Phg
L
3. Discussion
isomers (a-compounds) throughout the whole series. According
to modeling, both the -form and the -form can fit into the binding
pocket with a slight adjustment in the backbone position (Fig. 3).
Modeling of compounds 19a and 19b also shows that both the
and -form might have a good -overlap with H57. Although, the
phenylglycine seems to contribute more to the -overlap for the
-form, while the vinyl is the major -contributor in the -form.
Comparison of the equipotent compounds 22a and A (Fig. 2)
shows that the vinyl substitution on the phenylglycine does not
have a large impact on the inhibitory potency, at least not for
Phg compounds. Since the vinyl is more involved in the -overlap
with H57 according to modeling, the impact may be larger for the
-form as indicated by the twofold potency improvement from
230 nM (compound A) and 290 nM (compound 22a) to 130 nM
for 22b. In the model (Fig. 3), the sulfonamide pentenylic substitu-
ent is positioned in between the residues K136 and Q41 and there-
by maximizing hydrophobic contacts. Modeling also suggests that
the elongated inhibitors can adopt a c-clamp shape in analogy with
an X-ray crystal structure of a P2–P4 macrocyclic HCV NS3 prote-
ase inhibitor presented by Venkatraman et al.34 This shape immo-
bilizes K136 and enhances binding efficiency.
D
L
Aiming at developing novel HCV NS3 protease inhibitors, dis-
tinct from the already well explored P2 proline-based inhibitors,
we decided to further optimize P2 phenylglycine-based inhibitors
that had shown a potential from previous studies.21 Phenylgly-
cine-based inhibitors seem to bind with a slightly different binding
mode as compared with proline-based inhibitors and is thus not
suitable with building blocks that are optimized for the proline
L
-
D
p
p
L
p
D
inhibitors. Hence, new inhibitors based on vinyl decorated
D- and
L-
L-phenylglycine with C-terminal acylsulfonamide substituents of
varying sizes and lengths were synthesized. The P10 sulfonamide
substituents ranged from straight alkenylic chains of five (com-
pounds 19a and 19b) or six carbons (compounds 20a and 20b) to
cyclopropyl substituents, either allyl substituted (compounds 21a
and 21b) or non-substituted (compound 25). A variety of phenyl
substituents were also made, ranging from the unsubstituted phe-
nyl (compounds 22a and 22b) to phenyl that were ortho
substituted with a vinyl (compounds 23a and 23b) or an allyl
(compounds 24a and 24b).
p
D
The biochemical evaluation revealed that the new series of
inhibitors were equipotent or more potent to the previous series
with the best inhibitor possessing a Ki value of 35 nM (compared
to compound A, with a Ki of 230 nM). Within both epimeric series
(a and b), all inhibitors encompassing substituents with a vinylic-
or allylic elongation were slightly more potent than their non-elon-
gated counterparts. The inhibitors with linear alkenylic substitu-
ents (compounds 19a, 19b, 20a, and 20b) in P10 and elongated
phenyl substituents (compounds 23a, 23b, 24a, and 24b) proved
to be the most successful inhibitors in the series. Among the linear
alkenylic substituents, the substituents with five carbons (com-
pounds 19a Ki = 63 nM and 19b Ki = 48 nM) were preferred over
the substituents with six carbons (compounds 20a Ki = 120 nM
and 20b Ki = 100 nM). For the elongated phenyl substituents, the
differences in activities were not large going from ortho-vinylic
(compounds 23a Ki = 120 nM and 23b Ki = 47 nM) to ortho-allylic
(compounds 24a Ki = 140 nM and 24b Ki = 35 nM). Although, the
The
D-Phg inhibitors 19b, 20b, 23b, and 24b were the only
inhibitors that showed measurable inhibitory effects in the cell-
based assay. Considering the fact that the inhibitors in this series
are acyclic and flexible, it must be noted that these inhibitors still
shows micromolar cell activity. By comparing the activities of com-
pounds 19a (Ki = 63 nM), 19b (Ki = 48 nM), and 27 (300 nM) it is
Table 2
Inhibition constants and vitality values evaluated with A156T and D168V substituted
NS3
Compound
A156T
Ki SD (nM)
D168V
Ki SD (nM)
V
V
19a
19b
20a
20b
23a
23b
180 20
86 20
380 80
270 60
250 70
180 10
0.9
0.9
1.6
1.4
1.1
0.9
140 10
380 30
390 70
530 40
450 60
230 30
1.0
6.2
2.5
3.0
2.8
2.0
Figure 3. Overlay of compound 19a (green) and 19b (orange) docked in the full-
length NS3 protease binding site (PDB code 1CU1). The protease residues are
displayed as sticks.
SD, standard deviation.
V, vitality values calculated as described in Ref. 21.

5418
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
shown that the phenylglycine-based inhibitors are fivefold as po-
tent as the corresponding proline-based analog. Thus, elongated
P10 alkenylic- and phenyl acylsulfonamides seems to potentiate
the inhibition potency of phenylglycine-based inhibitors, as did vi-
(Zorbax SB-C8 (4.8 ꢁ 50 mm)) column using UV (220 or 230 nm)
detection. Preparative HPLC-MS was performed on a Gilson-Finni-
gan ThermoQuest AQA system equipped with a C8 (Zorbax SB-C8
(5
l
m, 150 ꢁ 21.2 mm)) column using MeCN/H2O (0.05% HCOOH)
nyl substitution for the
D
-Phg epimer.
as the mobile phase with UV (254 nm) and MS (ESI) detection or
a system equipped with a C8 (Zorbax SB-C8 (21.2 ꢁ 150 mm)) col-
umn using UV (220 or 230 nm) detection and MeCN/H2O (0.05%
HCOOH) or MeCN/H2O (0.09% TFA) as the mobile phase. Chiral
analysis was performed on a HPLC system equipped with a Repro-
Contrary to some P2 proline-based inhibitors,22 biochemical
evaluation on mutated forms of NS3 show that the inhibitory ef-
fects of the tested inhibitors herein, 19a, 19b 20a, 20b, 23a, and
23b are more or less unaffected by the A156T mutation as shown
by vitality values around 1, and slightly less efficient on the
D168V mutations compared to wild-type (V = 1–6). As a compari-
son it could be mentioned that the vitality values for BILN-2061
(Fig. 1) is 1600 for A156T and 3200 for D168V. The pentenylic
inhibitor 19b retains potency against A156T (Ki = 180 nM, V =
0.90) while it loses potency against D168V (Ki = 380 nM, V = 6.2).
Interestingly, compound 19a retains potency to both A156T and
D168V. The larger drop in potency on D168V for 19b compared
to 19a must be attributed to loss of important interactions or in-
sil CHIRAL-NR (250 ꢁ 4.6 mm, 8
lm) column. The purities of the
inhibitors were determined by RP-HPLC on the following systems
(UV detection at 220 nm): System 1 (Zorbax SB-C8, 4.8 ꢁ 5 mm,
MeCN/H2O with 0.05% HCOOH) and system 2 (Zorbax SB-C8,
4.8 ꢁ 5 mm, MeCN/H2O with 25 mM NH4OAc, pH 6.3). Microwave
reactions were performed in a SmithSyntheSizer™ or in an Initia-
tor™ single-mode microwave cavity producing controlled irradia-
tion at 2450 MHz. NMR spectra were recorded on a Varian
Mercury plus spectrometer (1H at 399.8 MHz, 13C at 100.5 MHz)
at ambient temperature. Chemical shifts (d) are reported in ppm,
indirectly referenced to tetrametylsilane (TMS) via the solvent sig-
nal (1H: CHCl3 d 7.26, CD2HOD d 3.31; 13C: CDCl3 d 77.16, CD3OD d
49.00). Exact molecular masses were determined on Micromass Q-
Tof2 mass spectrometer equipped with an electrospray ion source.
creased steric conflict on V168 from the D-Phg 19b. Altogether,
these results further strengthen the hypothesis that phenylgly-
cine-based inhibitors have a resistance profile differing from
proline-based inhibitors.
4. Conclusion
5.2. Compound 2
In summary, an attempt to optimize P2 phenylglycine-based
HCV NS3 protease inhibitors by incorporating a vinyl fragment re-
sulted in a series of acyclic tripeptidic inhibitors. Due to the phen-
ylglycine’s tendency to racemize, the inhibitors were obtained both
Compound
1 (2.08 g, 6.00 mmol), 4-chloro-7-methoxy-2-
phenylquinoline (0.810 g, 3.00 mmol) and KOtBu (1.34 g, 12.0
mmol) were dried over night in vacuo over P2O5. Dry DMSO
(H2O < 0.005%) (10.5 mL) was added to a vial containing 1 and
KOtBu. The vial was sealed under nitrogen and stored for 40 min
before 4-chloro-7-methoxy-2-phenylquinoline dissolved in DMSO
(7.5 mL) was added. The reaction was heated at 64 °C and stirred
for 6½ weeks until no more product was formed. The reaction mix-
ture was filtered, diluted with water (230 mL), and extracted with
diethyl ether to remove unreacted 4-chloro-7-methoxy-2-phenyl-
quinoline. The aqueous phase was acidified with 1 M HCl
(6.0 mL) to pH 5.3. Centrifugal filtration of the solid, yielded 2
(753 mg, 43%) as a beige powder. To obtain a concentration of 2,
high enough to record a 13C NMR spectrum, TFA had to be added
to the NMR sample. Therefore, the 13C NMR shifts of 2 is reported
as a TFA-salt. 1H NMR (CD3OD): d 8.30 (d, J = 9.2 Hz, 1H), 7.86 (d,
J = 2.1 Hz, 1H), 7.83–7.76 (m, 2H), 7.57 (dd, J = 2.1, 8.4 Hz, 1H),
7.50 (d, J = 2.5 Hz, 1H), 7.48–7.43 (m, 3H), 7.38 (d, J = 8.4 Hz, 1H),
7.30 (dd, J = 9.2, 2.5 Hz, 1H), 6.72 (s, 1H), 5.24 (s, 1H), 4.00 (s,
3H), 1.46 (s, 9H). 13C NMR (CD3OD, as TFA-salt): d 173.0, 167.7,
167.0, 158.6, 157.5, 150.5, 144.9, 140.8, 134.8, 133.8, 133.5,
130.8, 130.4, 129.7, 126.0, 124.5, 122.7, 116.6, 115.5, 103.0,
101.2, 81.2, 58.2, 57.1, 28.7. MS calcd for C29H27BrN2O6 [M+H]+
580.1, found: 579.2, 581.2.
as the
tency, with a slight preference for the
of the inhibitors possessed cell-activities below 10
L
- and
D
-Phg epimer. The epimers proved to be of equal po-
-Phg epimer. Notably, some
M. Some
D
l
inhibitors were evaluated on A156T and D168V substituted en-
zyme and showed the important ability to retain their potency
against these mutant variants. C-terminal acylsulfonamides with
alkenylic substituents proved to be beneficial in combination with
the vinylated P2 phenylglycine, yielding inhibitors with inhibition
constants of 35 nM. The same elongated alkenylic acylsulfonamide
was not advantageous on a P2 proline-based inhibitor which was
five times less potent. This indicates that the P2 phenylglycine-
based inhibitors bind in a different fashion compared to the P2 pro-
line-based inhibitors, and has different optimization points. This
will be explored in further optimization of inhibitors with an antic-
ipated unique resistance profile.
5. Experimental
5.1. Chemistry
Reagents and solvents were obtained commercially and used
without further purification. Thin layer chromatography (TLC)
was performed on aluminum sheets precoated either with silica
gel 60 F254 (0.2 mm, Merck) or aluminum oxide 60 F254 neutral
(0.2 mm, Merck). Chromatographic spots were visualized using
UV detection and/or 2% ninhydrin in ethanol solution followed
by heating. Column chromatography was performed using silica
5.3. Compound 3
In a microwave process vial, 2 (381 mg, 0.659 mmol), 2,4,6-trivi-
nylcycloboroxane pyridine complex (294 mg, 1.32 mmol), Pd(OAc)2
(15.8 mg, 0.0712 mmol), [(tBu)3PH]BF4 (38.2 mg, 0.132 mmol),
K2CO3 (547 mg, 3.96 mmol), DME (13.2 mL), and water (3.96 mL)
were mixed. The vial was sealed under N2 and exposed to microwave
heating at 100 °C for 15 min. After centrifugal filtration, the DME
was evaporated. Water (16 mL) was added and the mixture was
acidified with 1 M HCl to pH 1. The mixture was extracted with ethyl
acetate and the organic phase was collected and evaporated. Purifi-
cation by preparative HPLC-MS gave 3 (118 mg, 34%) as a yellow so-
lid. 1HNMR (CD3OD): d 8.29 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 2.4 Hz, 1H),
7.76–7.73 (m, 2H), 7.49–7.46 (m, 2H), 7.43–7.40 (m, 3H), 7.28 (dd,
J = 9.1, 2.5 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.77 (dd, J = 17.5,
gel 60 (40–63
mal (63–200
l
m, Merck) or aluminum oxide 90 standardized nor-
l
m, Merck) or silica gel 60 RP-18 (40–63 m, Merck).
l
Analytical HPLC-MS was performed on a Gilson-Finnigan Thermo-
Quest AQA system equipped with a C18 (Onyx Monolithic C18
(50 ꢁ 4.6 mm) or a C4 (Hichrom ACE C4 (5 m, 50 ꢁ 4.6 mm)) col-
l
umn using MeCN/H2O (0.05% HCOOH) as the mobile phase with UV
(254 nm) MS (ESI) detection or on a Gilson Thermo-Finnigan
Surveyor MSQ system using MeCN/H2O (0.05% HCOOH) as the mo-
bile phase with ELSD detection or on a system equipped with a C8

A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
5419
11.3 Hz, 1H), 6.67 (s, 1H), 5.89 (dd, J = 17.5, 1.2 Hz, 1H), 5.26–5.22
(m, 2H), 3.99 (s, 3H), 1.46 (s, 9H). 13C NMR (CD3OD) d 174.3, 164.5,
164.0, 160.6, 157.4, 151.6, 151.4, 139.6, 138.2, 131.9, 131.1, 130.9,
129.9, 129.9, 128.7, 127.5, 124.1, 123.2, 120.4, 117.9, 115.8, 106.7,
102.0, 80.9, 59.2, 56.2, 28.7. MS calcd for C31H30N2O6 [M+H]+
527.2, found: 527.3.
1.47 mmol), CDI (715 mg, 4.41 mmol), DBU (662
l
L, 4.41 mmol),
and dry THF (21 mL). Purification on silica gel (CH2Cl2/MeOH
97:3) and then aluminum oxide (CH2Cl2/MeOH 95:5, then
CH2Cl2/MeOH/HCOOH 95:5:1) gave 6 (59 mg, 15%). 1H NMR
(CDCl3): d 9.29 (s, 1H), 5.69 (m, 1H), 5.14–5.10 (m, 2H), 5.09 (s,
1H), 4.15 (m, 1H), 2.64 (dd, J = 3.2, 7.4 Hz, 2H), 1.81 (m, 1H),
1.70–1.54 (m, 3H), 1.45 (s, 9H), 1.44–1.37 (m, 2H), 0.97–0.93 (m,
2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3): d 170.8, 156.3,
132.4, 119.4, 81.1, 55.1, 40.1, 34.7, 33.6, 28.4, 18.9, 13.8, 11.2,
11.1. MS calcd for C16H28N2O5S [M+H]+ 361.2, found: 361.0.
5.4. General procedure for synthesis of compounds 4–8, 10–11
Boc-L-Nva, CDI and sulfonamides were dried in vacuo over P2O5
over night. Boc-
dissolved in dry THF under N2 atmosphere.
L
-Nva or Boc- -cyclopropylalanine and CDI were
L
5.8. Compound 7
Method A: The solution was stirred at room temperature for 1 h
before the sulfonamide, dissolved in dry THF was added together
with DBU. The reaction was left over night.
Method B: The solution was stirred at 60 °C for 1 h before the
sulfonamide, dissolved in dry THF was added together with DBU.
The reaction was stirred in room temperature for 2 h.
Method A and B: The solvent was evaporated and CH2Cl2 was
added. The solution was washed with 5% citric acid solution before
the organic phase was evaporated.
Purification: Most of the compounds were firstly purified on sil-
ica gel to remove rests of non-reacted amino acid. During a second
purification on aluminum oxide the sulfonamide was washed out
with the eluent (CH2Cl2/MeOH) while the product was adsorbed
to the aluminum oxide. By adding 1% formic acid to the eluent,
the pure product could be eluted from the aluminum oxide.
Prepared according to method B described in the general proce-
dure. Boc-
L
-Nva (65.2 mg, 0.300 mmol), benzenesulfonamide
L,
(142 mg, 0.900 mmol), CDI (97.3 mg, 0.600 mmol), DBU (135
l
0.903 mmol), and dry THF (9 mL). After purification on silica gel
(CH2Cl2/MeOH 95:5) followed by aluminum oxide (CH2Cl2/MeOH
95:5, then CH2Cl2/MeOH/HCOOH 95:5:1) and then silica gel RP-
C18 (MeCN/H2O 30:70, then MeCN/H2O 50:50), 7 was obtained
as a white solid (47 mg, 44%). 1H NMR (CDCl3): d 7.99 (d,
J = 7.6 Hz, 2H), 7.57 (m, 1H), 7.50–7.45 (m, 2H), 3.96 (br s, 1H),
1.62 (m, 1H), 1.35 (s, 9H), 1.24–1.17 (m, 2H), 0.82 (t, J = 7.4 Hz,
3H). 13C NMR (CDCl3): d 171.6, 156.1, 139.1, 133.7, 128.9, 128.2,
80.7, 54.4, 34.3, 28.2, 18.4, 13.6. MS calcd for C16H25N2O5S
[M+H]+ 357.1, found: 357.1.
5.9. Compound 8
5.5. Compound 4
Prepared following method B in the general procedure de-
scribed previously. Boc-
zenesulfonamide (2.0 g, 8.48 mmol), CDI (918 mg, 5.66 mmol),
DBU (1260 L, 8.48 mmol), and dry THF (70 mL). Purification on
L-Nva (615 mg, 2.83 mmol), 1-bromoben-
Prepared according to the general procedure described above,
using method B. Boc-L-Nva (435 mg, 2.00 mmol), pent-4-ene-1-
l
sulfonamide, prepared according to a previously described meth-
silica gel (CH2Cl2/MeOH 95:5) then aluminum oxide (CH2Cl2/
MeOH 95:5, then CH2Cl2/MeOH/HCOOH 95:5:1) yielded the prod-
uct as a white solid (1.0 g, 84%). 1H NMR (CD3OD): d 8.23 (dd,
J = 2.3, 7.4 Hz, 1H), 7.80 (dd, J = 1.7, 7.4 Hz, 1H), 7.59–7.50 (m,
2H), 4.07 (dd, J = 5.0, 9.1 Hz, 1H), 1.69 (m, 1H), 1.51 (m, 1H), 1.41
(s, 9H), 1.34–1.24 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR
(CD3OD): d 173.5, 157.8, 139.7, 136.4, 135.9, 134.2, 128.8, 121.0,
80.7, 56.1, 34.7, 28.7, 19.9, 13.9. MS calcd for C16H23BrN2O5S
[M+H]+ 436.1, found: 435.1, 437.1.
od29 (895 mg, 6.00 mmol), CDI (649 mg, 4.00 mmol), DBU
(900 lL, 6.01 mmol), and dry THF (48 mL). The product was puri-
fied on silica gel (CH2Cl2/MeOH 95:5), then on an aluminum oxide
column (CH2Cl2/MeOH 97:3, then CH2Cl2/MeOH/HCOOH 95:5:1)
and was yielded as a white solid (545 mg, 78%). 1H NMR (CDCl3):
d 9.50 (s, 1H), 5.73 (m, 1H), 5.13 (s, 1H), 5.08–5.01 (m, 2H), 4.20–
4.09 (m, 1H), 3.45–3.38 (m, 2H), 2.22–2.15 (m, 2H), 1.96–1.86
(m, 2H), 1.85–1.73 (m, 1H), 1.66–1.54 (m, 1H), 1.44 (s, 9H), 1.43–
1.32 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) d 172.1,
156.3, 136.3, 116.7, 81.3, 55.0, 52.7, 33.5, 31.9, 28.4, 22.4, 18.9,
13.7. MS calcd for C15H28N2O5S [M+H]+ 349.2, found: 349.1.
5.10. Compound 9
In a microwave process vial, 8 (330 mg, 0.758 mmol), 2,4,6-trivi-
nylcycloboroxane pyridine complex (338 mg, 1.52 mmol), Pd(OAc)2
(18.0 mg, 0.0748 mmol), [(tBu)3PH]BF4 (44 mg, 0.152 mmol), K2CO3
(944 mg, 6.83 mmol), DME (12 mL), and water (3.6 mL) were mixed.
The vial was sealed under N2 and the vessel was heated by micro-
waves to 100 °C for 15 min. DME was evaporated, water (16 mL)
was added and the mixture was acidified with 1 M HCl to pH 1. After
extraction with ethyl acetate and evaporation of the organic phase,
the product was purified on preparative RP-HPLC-MS giving 9
(192 mg, 66%) as a white solid. 1H NMR (CD3OD): d 8.09 (m, 1H),
7.72 (m, 1H), 7.63 (m, 1H), 7.54 (dd, J = 10.9, 17.3 Hz, 1H), 7.45 (m,
1H), 5.79 (d, J = 17.3 Hz, 1H), 5.48 (dd, J = 1.3, 10.9 Hz, 1H), 3.98
(dd, J = 5.2, 8.8 Hz, 1H), 1.57 (m, 1H), 1.43 (m, 1H), 1.39 (s, 9H),
1.25–1.19 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (CD3OD) d
173.4, 157.7, 138.6, 137.3, 135.0, 134.5, 131.9, 128.8, 128.7, 119.5,
80.7, 56.0, 34.8, 28.6, 19.8, 13.9. MS calcd for C18H26N2O5S [M+H]+
383.1, found: 383.1.
5.6. Compound 5
Prepared according to the general procedure previously de-
scribed, using method B. Boc-
ene-1-sulfonamide, prepared as described previously29 (871 mg,
5.34 mmol), CDI (577 mg, 3.56 mmol), DBU (798 L, 5.33 mmol),
L-Nva (387 mg, 1.78 mmol), hex-5-
l
and dry THF (54 mL). Purification on silica gel (CH2Cl2/MeOH 95:5)
followed by aluminum oxide (CH2Cl2/MeOH 95:5, then CH2Cl2/
MeOH/HCOOH 95:5:1) gave 5 as a white sticky solid (515 mg
80%). 1H NMR (CDCl3): d 5.75 (m, 1H), 5.03 (m, 1H), 4.99 (m, 1H),
4.96 (m, 1H), 4.11 (br s, 1H), 3.45–3.40 (m, 2H), 2.11–2.05 (m, 2H),
1.87–1.79 (m, 2H), 1.66–1.49 (m, 4H), 1.46 (s, 9H), 1.44–1.34 (m,
2H), 0.94 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3) d 171.8, 156.3, 137.6,
115.6, 81.5, 55.1, 53.3, 33.2, 33.1, 28.4, 27.4, 22.6, 18.9, 13.8. MS calcd
for C16H30N2O5S [M+H]+ 363.2, found: 363.8.
5.7. Compound 6
5.11. Compound 10
Prepared following method A, described in the general proce-
Prepared according to method A described in the general proce-
dure earlier. Boc-L-Nva (163 mg, 0.750 mmol), 2-allylbenzenesulf-
dure. Boc-L-Nva (480 mg, 2.21 mmol), 1-allylcycloproane-1-sulfon-
amide, prepared the same way as described previously,30 (237 mg,

5420
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
onamide (99 mg, 0.502 mmol), prepared according to a previously
described procedure,31 CDI (243 mg, 1.50 mmol), DBU (225
L,
5.15. Compound 13
l
1.50 mmol), and dry THF (10.5 mL). Purification on silica gel
(EtOAc/i-hexane 40:60) yielded 10 (113 mg, 38%). 1H NMR
(CD3OD): d 8.09 (dd, J = 1.5, 8.0 Hz, 1H), 7.57 (dt, J = 1.4, 7.5 Hz,
1H), 7.41–7.36 (m, 2H), 6.04 (m, 1H), 5.11 (m, 1H), 5.09 (m, 1H),
3.98 (dd, J = 5.4, 8.7 Hz, 1H), 3.89 (dd, J = 6.8, 6.1 Hz, 1H), 3.81
(dd, J = 6.5, 16.2 Hz, 1H), 1.65–1.44 (m, 2H), 1.38 (s, 9H), 1.34–
1.26 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (CD3OD) d 172.8,
156.6, 139.8, 137.5, 136.4, 133.5, 131.3, 130.9, 126.2, 116.1, 79.4,
55.0, 36.4, 33.7, 27.5, 18.7, 12.8. MS calcd for C19H28N2O5S
[M+H]+ 397.2, found: 397.1.
Prepared according to the general procedure using 3 (30.0 mg,
0.0570 mmol), the N-deprotected HCl salt of 5 (26.9 mg, 0.09
mmol), HATU (25.8 mg, 0.0680 mmol), DIEA (64.4 lL, 0.370 mmol),
and DMF (1 mL). Purification on silica gel (CH2Cl2/MeOH 93:7)
gave a 4:3 diastereomeric mixture of 13 as a white solid (34 mg,
49%). 1H NMR (CD3OD, [both isomers reported]): d [8.25 (d,
J = 9.2 Hz), 8.24 (d, J = 9.2 Hz), 1H], [7.88 (d, J = 2.4 Hz), 7.86 (d,
J = 2.4 Hz), 1H], [7.79–7.69 (m, 2H)], [7.52–7.46 (m, 2H)], [7.43–
7.39 (m, 3H)], [7.26–7.16 (m,2H)], [6.77 (dd, J = 11.3, 17.5 Hz,
1H)], [6.65 (s), 6.61 (s), 1H], [5.94 (dd, J = 1.2, 17.5 Hz), 5.93 (dd,
J = 1.2, 17.5 Hz), 1H], [5.68 (m, 1H)], 5.35 (s, 1H), [5.27 (dd,
J = 1.4, 11.2 Hz), 5.26 (dd, J = 1.4, 11.2 Hz), 1H], [4.92–4.82 (m,
2H)], [4.33 (m, 1H)], 3.97 (s, 3H), [3.36–3.16 (m, 2H)], [2.11–1.92
(m, 2H)], [1.84–1.56 (m, 6H)], 1.45 (s, 9H), [1.41–1.34 (m, 2H)],
[0.94 (t, J = 7.4 Hz), 0.78 (t, J = 7.4 Hz), 3H]. 13C NMR (CD3OD, major
isomer reported): d 174.6, 172.8, 169.9, 163.9, 157.4, 152.4, 151.9,
140.6, 139.0, 137.6, 132.1, 131.0, 130.7, 130.0, 129.9, 128.7, 128.6,
127.6, 123.9, 123.3, 120.1, 117.9, 115.8, 115.5, 107.6, 101.8, 81.1,
59.0, 56.1, 55.5, 53.5, 34.8, 34.1, 28.7, 28.4, 23.8, 20.0, 14.0. MS
calcd for C42H50N4O8S [M+H]+ 771.3, found: 771.4.
5.12. Compound 11
Prepared according to method A described in the general proce-
dure.
earlier described.24 Boc-
cyclopropylsulfonamide (878 mg, 7.25 mmol) prepared as de-
scribed previously,26 CDI (1.18 mg, 7.27 mmol), DBU (1100
L,
L
-cyclopropylalanine was Boc protected in the same way as
L
-cyclopropylalanine (830 mg, 3.62 mmol),
l
7.35 mmol), and dry THF (30 mL). Purification on RP-C18 silica
(MeCN/H2O 1:2 to MeCN/H2O 1:1) followed by silica gel (CH2Cl2/
MeOH 95:5) yielded 11 as a white solid (549 mg, 46%). 1H NMR
(CD3OD): d 4.12 (m, 1H), 2.96 (m, 1H), 1.66 (m, 1H), 1.52 (m,
1H), 1.45 (s, 9H), 1.30–1.18 (m, 2H), 1.14–1.06 (m, 2H), 0.80 (m,
1H), 0.56–0.44 (m, 2H), 0.21–0.10 (m, 2H). 13C NMR (CD3OD): d
174.4, 157.8, 80.7, 56.9, 37.9, 31.8, 28.7, 8.4, 6.4, 6.2, 5.2, 4.8. MS
calcd for C14H24N2O5S [M+H]+ 333.1, found: 333.0. Anal. Calcd for
5.16. Compound 14
Prepared according to the general procedure using 3 (80.0 mg,
0.151 mmol), the N-deprotected HCl salt of 6 (38.5 mg, 0.130
mmol), HATU (69.1 mg, 0.182 mmol), DIEA (140 lL, 0.820 mmol),
C14H24N2O5S: C, 50.58; H, 7.28; N, 8.43. Found: C, 50.77; H, 7.24;
and DMF (1.5 mL). Purification on silica gel (CH2Cl2/MeOH 95:5)
yielded 14 in a 5:3 diastereomeric mixture (84 mg, 84%). 1H NMR
(CD3OD, major isomer reported): d 8.28 (d, J = 9.3 Hz, 1H), 7.89 (d,
J = 2.3 Hz, 1H), 7.79–7.73 (m, 2H), 7.51 (m, 1H), 7.48 (d, J = 2.5 Hz,
1H), 7.45–7.41 (m, 3H), 7.27 (dd, J = 2.5, 9.2 Hz, 1H), 7.21 (d,
J = 8.4 Hz, 1H), 6.79 (dd, J = 11.3, 17.7 Hz, 1H), 6.67 (s, 1H), 6.29
(dd, J = 1.1, 11.2 Hz, 1H), 5.97 (dd, J = 1.1, 17.7 Hz, 1H), 5.67 (m,
1H), 5.36 (s, 1H), 5.11–4.94 (m, 2H), 4.37 (dd, J = 5.4, 8.8 Hz, 1H),
4.00 (s, 3H), 2.63–2.49 (m, 2H), 1.85–1.61 (m, 2H), 1.60–1.39 (m,
2H), 1.47 (s, 9H), 1.39–1.22 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H), 0.84–
0.77 (m, 2H). 13C NMR (CD3OD, major isomer reported) d 173.2,
172.8, 163.9, 163.6, 161.1, 157.6, 152.5, 152.0, 140.7, 137.6, 134.3,
132.0, 130.9, 130.7, 129.9, 129.9, 128.7, 127.6, 123.9, 123.3, 120.2,
119.2, 118.0, 115.8, 107.6, 101.9, 81.1, 59.0, 56.1, 55.3, 41.0, 35.8,
34.8, 28.7, 20.0, 14.0, 12.3, 11.0. MS calcd for C42H48N4O8S [M+H]+
769.3, found: 769.1.
N, 8.45.
5.13. General procedure for synthesis of compounds 12–18
Removal of the Boc-protecting group of compounds 4–7 and 9–
11 were performed by dissolving them in 4.0 M HCl in 1,4-dioxane
(10 mL/mmol) and stirring until no starting material could be de-
tected by LC–MS. The solvent was evaporated and the HCl salt of
the product was used without further purification in the forthcom-
ing couplings. Compound 3, HATU, and the HCl salt of the N-depro-
tected 4–7 and 9–11 were dissolved in DMF. DIEA was added until
the pH exceeded 10. The reactions were stirred until no starting
material could be detected by LC–MS. After addition of EtOAc
and washing with 0.1 M NaHSO4 followed by 0.035 M NaHSO4,
the organic phase was evaporated.
5.14. Compound 12
5.17. Compound 15
Prepared according to the general procedure using 3 (100 mg,
0.190 mmol) the N-deprotected HCl salt of 4 (65.5 mg, 0.230
mmol), HATU (87.4 mg, 0.230 mmol), DIEA (190 lL, 1.11 mmol),
Prepared according to the general procedure using 3 (29.2 mg,
0.0555 mmol), the N-deprotected HCl salt of
0.0886 mmol), HATU (25.3 mg, 0.0665 mmol), DIEA (63.9
7
(24.2 mg,
L,
l
and DMF (2 mL). Purification on silica gel (CH2Cl2/MeOH 95:5)
yielded 12 as a 2:1 diastereomeric mixture (124 mg, 86%). 1H
NMR (CD3OD, major isomer reported): d 8.28 (d, J = 9.2 Hz, 1H),
7.87 (d, J = 2.3 Hz, 1H), 7.79–7.75 (m, 2H), 7.51 (m, 1H), 7.48 (d,
J = 2.5 Hz, 1H), 7.46–7.41 (m, 3H), 7.27 (dd, J = 2.5, 9.2 Hz, 1H),
7.21 (d, J = 8.4 Hz, 1H), 6.79 (dd, J = 11.2, 17.7 Hz, 1H), 6.66 (s,
1H), 5.96 (dd, J = 1.0, 17.6 Hz, 1H), 5.67 (m, 1H), 5.35 (s, 1H), 5.28
(dd, J = 1.1, 11.2 Hz, 1H), 4.99–4.89 (m, 2H), 4.35 (dd, J = 5.5,
8.5 Hz, 1H), 3.99 (s, 3H), 3.43–3.25 (m, 2H), 2.10–2.03 (m, 2H),
1.91–1.62 (m, 4H), 1.48–1.38 (m, 2H), 1.46 (s, 9H), 0.95 (t,
J = 7.3 Hz, 3H). 13C NMR (CD3OD, major isomer reported) d 174.0,
172.9, 164.0, 163.7, 160.9, 157.6, 152.3, 151.9, 140.4, 138.0,
132.0, 130.9, 130.9, 130.8, 130.0, 129.9, 128.7, 127.6, 123.9,
123.3, 120.2, 118.0, 116.6, 115.8, 107.5, 101.8, 81.1, 59.0, 56.1,
55.3, 53.0, 34.6, 32.9, 28.7, 23.6, 20.1, 14.0. MS calcd for
0.366 mmol), and DMF (0.8 mL). Purification on preparative
HPLC-MS (MeCN/H2O (0.05% HCOOH)) yielded 15 as a diastereo-
meric mixture (26 mg, 61%). 1H NMR (CD3OD, major isomer re-
ported): d 8.31 (d, J = 9.15 Hz, 1H), 7.92 (m, 1H), 7.80–7.75 (m,
3H), 7.53–7.47 (m, 3H), 7.46–7.39 (m, 5H), 7.35 (dd, J = 2.4,
8.6 Hz, 1H), 7.30 (dd, J = 2.6, 9.4 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H),
6.76 (dd, J = 11.1, 17.4 Hz, 1H), 6.64 (s, 1H), 5.89 (d, J = 17.4 Hz,
1H), 5.27 (dd, J = 1.3, 11.4 Hz, 2H), 4.31 (dd, J = 5.5, 8.6 Hz, 1H),
4.01 (s, 3H), 1.78–1.55 (m, 2H), 1.45 (s, 9H), 1.37–1.26 (m, 2H),
0.88 (t, J = 7.5 Hz, 3H). 13C NMR (CD3OD, major isomer reported)
173.7, 172.6, 164.0, 163.7, 161.0, 156.3, 152.4, 151.9, 141.6,
140.6, 137.2, 134.2, 132.0, 130.9, 130.8, 129.9, 129.8, 129.7,
128.9, 128.7, 127.7, 123.9, 123.3, 120.2, 118.0, 115.8, 107.5,
101.9, 82.1, 59.0, 56.2, 55.4, 34.8, 28.7, 19.8, 14.0. MS calcd for
41H48N4O8S [M+H]+ 757.3, found: 757.3.
C
42H44N4O8S [M+H]+ 765.3, found: 765.5.
C

A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
5421
5.18. Compound 16
(CD3OD, major isomer reported): d 173.3, 172.7, 164.9, 164.0,
163.6, 161.1, 152.5, 152.0, 140.7, 137.3, 132.1, 131.0, 130.7, 130.0,
129.9, 128.7, 127.6, 124.0, 123.4, 120.2, 117.9, 115.8, 107.6, 101.8,
81.1, 59.1, 56.1, 55.9, 38.8, 31.8, 28.7, 8.4, 6.4, 6.1, 5.3, 5.0. MS calcd
for C40H44N4O8S [M+H]+ 741.3, found: 741.1.
Prepared according to the general procedure using 3 (100 mg,
0.190 mmol), the N-deprotected HCl salt of 9 (95.5 mg, 0.300
mmol), HATU (87.4 mg, 0.230 mmol), DIEA (218 lL, 1.25 mmol),
and DMF (3.5 mL). Purification on preparative RP-HPLC-MS
(MeCN/H2O (0.05% HCOOH)) yielded 16 as a white solid in a 3:2 dia-
stereomeric mixture (108 mg, 72%). 1H NMR (CD3OD, [both diaste-
reomers reported]) d [8.27 (d, J = 9.1 Hz), 8.26 (d, J = 9.1 Hz), 1H],
[8.07 (dd, J = 1.4, 8.0 Hz), 7.97 (dd, J = 1.4, 8.0 Hz), 1H], [7.84–7.71
(m), 3H], [7.65–7.45 (m), 5H], [7.43–7.33 (m), 4H], [7.29–7.23
(m), 1H], [7.16 (d, J = 8.5 Hz), 7.09 (d, J = 8.5 Hz), 1H], [6.75 (t,
J = 10.4 Hz), 6.71 (t, J = 10.4 Hz), 1H], [6.62 (s), 6.60 (s), 1H], [5.87
(dd, J = 6.7, 17.6 Hz), 1H], [5.71 (m), 1H], [5.44–5.38 (m), 1H],
[5.31–5.20 (m), 2H], [4.38 (m), 1H], [3.99 (s), 3.98 (s), 3H], [1.75–
1.66 (m), 1H], [1.63–1.53 (m), 1H], [1.44 (s), 9H], [1.36–1.25 (m),
2H], [0.87, (t, J = 7.3 Hz), 0.70 (t, J = 7.3 Hz), 3H]. 13C NMR (CD3OD)
d [173.7], [173.1], [172.5], [164.3, 164.2], [163.8], [160.7, 160.6],
[157.3], [151.8], [151.6,], [139.7], [138.3], [138.1], [137.4], [134.8,
134.7], [134.4, 134.3], [132.0, 131.9], [131.5, 131.4], [131.0,
130.9], [130.7], [129.9], [128.8, 128.7], [128.5], [127.6, 127.5],
[124.0], [123.3, 123.2], [120.3], [119.0, 118.8], [118.2, 118.1],
[115.7], [107.0, 106.9], [101.9, 101.8], [81.1], [59.6, 58.9], [56.2],
[55.1, 54.9], [34.9], [28.7], [19.8, 19.6], [14.0, 13.8]. MS calcd for
5.21. General procedure for preparation of compounds 19–25
For removal of the Boc-protecting group, compounds 12–18
were dissolved in a 4.0 M HCl in dioxane solution (10 mL/mmol).
The solutions were stirred until starting material no longer could
be visualized by LC–MS. The solvent was evaporated and the N-
deprotected HCl salts of 12–17 were used without further purifica-
tion. They were mixed with Boc-t-Leu, HATU, DIEA and DMF and
stirred until no starting material could be visualized by LC–MS.
After addition of ethyl acetate, washing with 0.1 M sodium acetate
buffer, pH 4, the organic phase was evaporated. Purification and
partial separation of the diastereomers (compounds 19–24) was
made by preparative HPLC.
5.22. Compounds 19a and 19b
Prepared according to the general procedure using the N-depro-
tected HCl salt of 12 (16.8 mg, 0.0243 mmol), Boc-t-Leu (11.1 mg,
0.0479 mmol), HATU (22.0 mg, 0.0578 mmol), DIEA (32.0 lL,
C
44H46N4O8S [M+H]+ 791.3, found: 791.7.
0.187 mmol), and DMF (0.7 mL). Purification on preparative HPLC
(MeCN/H2O (0.05% HCOOH)) yielded 19a (8.2 mg, 39%) and 19b
(3.5 mg, 17%) as white solids. 19a: 1H NMR (CD3OD): d 8.29 (d,
J = 9.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.78–7.75 (m, 2H), 7.53
(dd, J = 2.4, 8.5 Hz, 1H), 7.49 (d, J = 2.5 Hz, 1H), 7.47–7.42 (m, 3H),
7.28 (dd, J = 2.5, 9.2 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.79 (dd,
J = 11.2, 17.6 Hz, 1H), 6.65 (s, 1H), 6.00 (d, J = 18.0 Hz, 1H), 5.71–
5.60 (m, 1H), 5.69 (s, 1H), 5.28 (dd, J = 1.2, 11.2 Hz, 1H), 4.97–
4.88 (m, 2H), 4.35 (dd, J = 5.4, 8.8 Hz, 1H), 4.03 (s, 1H), 4.00 (s,
3H), 3.38–3.16 (m, 2H), 2.10–2.02 (m, 2H), 1.89–1.65 (m, 4H),
1.55–1.41 (m, 2H), 1.42 (s, 9H), 1.02 (s, 9H), 0.96 (t, J = 7.3 Hz,
3H). 13C NMR (CD3OD): 173.9, 173.2, 171.9, 164.0, 163.7, 161.0,
157.9, 152.4, 151.9, 140.6, 138.0, 136.9, 132.0, 130.9, 130.7,
130.2, 139.9, 128.7, 127.8, 123.9, 123.3, 120.2, 118.1, 116.6,
115.8, 107.5, 101.9, 80.7, 63.8, 57.3, 56.1, 55.3, 53.0, 35.3, 34.6,
32.9, 28.7, 27.2, 23.6, 20.1, 14.0. HRMS calcd for C47H59N5O9S
[M+H]+ 870.4112, found: 870.4097. HPLC purity (system 1: 93%,
system 2: 98%). 19b: 1H NMR (CD3OD): d 8.31 (d, J = 9.1 Hz, 1H),
7.90 (d, J = 2.3 Hz, 1H), 7.76–7.72 (m, 2H), 7.51 (dd, J = 2.3, 8.3 Hz,
1H), 7.49 (d, J = 2.5 Hz, 1H), 7.46–7.42 (m, 3H), 7.30 (dd, J = 2.5,
9.2 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 6.80 (dd, J = 11.2, 17.7 Hz,
1H), 6.60 (s, 1H), 5.96 (d, 17.7 Hz, 1H), 5.73 (m, 1H), 5.61 (s, 1H),
5.31 (dd, J = 1.2, 11.1 Hz, 1H), 5.05–4.90 (m, 2H), 4.32 (dd, J = 5.4,
8.9 Hz, 1H), 4.01 (s, 3H), 3.97 (s, 1H), 3.44–3.33 (m, 2H), 2.18–
2.11 (m, 2H), 1.95–1.65 (m, 4H), 1.44 (s, 9H), 1.38–1.24 (m, 2H),
0.96 (s, 9H), 0.84 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD): 173.9,
173.2, 172.4, 164.0, 163.7, 161.1, 157.9, 152.5, 152.1, 140.8,
138.0, 137.3, 132.2, 130.8, 130.7, 130.7, 129.8, 128.6, 128.0,
123.9, 123.5, 120.3, 118.0, 116.6, 115.8, 107.5, 101.8, 80.7, 63.9,
58.1, 56.1, 55.4, 53.1, 35.4, 34.4, 33.0, 28.8, 27.2, 23.6, 19.9, 13.8.
HRMS calcd for C47H59N5O9S [M+H]+ 870.4112, found: 870.4095.
HPLC purity (system 1: 95%, system 2: 99%).
5.19. Compound 17
Prepared according to the general procedure using 3 (68.0 mg,
0.129 mmol), the N-deprotected HCl salt of 10 (43.2 mg, 0.130
mmol), HATU (59.0 mg, 0.155 mmol), DIEA (120 lL, 0.702 mmol),
and DMF (1.3 mL). Purification on silica gel (CH2Cl2/MeOH 95:5)
yielded 17 as a white solid in a 3:1 diastereomeric mixture
(86 mg, 82%). 1H NMR (CD3OD, major isomer reported) d 8.29 (d,
J = 9.1 Hz, 1H), 7.98 (dd, J = 1.4, 7.9 Hz, 1H), 7.79–7.74 (m, 3H),
7.49 (d, J = 2.5 Hz, 1H), 7.45–7.41 (m, 4H), 7.36 (ddd, J = 1.4, 7.5,
7.5 Hz, 1H), 7.32 (m, 1H), 7.28 (dd, J = 2.5, 9.1 Hz, 1H), 7.20 (m,
1H), 7.05 (d, J = 8.4 Hz, 1H), 6.71 (dd, J = 11.2, 17.7 Hz, 1H), 6.61
(s, 1H), 6.00 (m, 1H), 5.83 (dd, J = 1.2, 17.6 Hz, 1H), 5.29 (s, 1H),
5.21 (dd, J = 1.1, 11.2 Hz, 1H), 4.98–4.91 (m, 2H), 4.34 (dd, J = 5.1,
8.6, 1H), 4.00 (s, 3H), 3.84 (dd, J = 6.5, 16.1 Hz, 1H), 3.71 (dd,
J = 6.3, 16.1 Hz, 1H), 1.80–1.53 (m, 2H), 1.44 (s, 9H), 1.44–1.27 (m,
2H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (CD3OD, major isomer re-
ported) d 172.5, 171.3, 162.9, 162.5, 159.8, 156.3, 151.1, 150.6,
139.5, 139.2, 138.2, 136.6, 136.1, 133.0, 131.3, 130.7, 130.4, 129.6,
128.7, 128.5, 127.6, 127.5, 126.4, 126.0, 122.8, 122.0, 119.1, 116.9,
115.9, 114.6, 106.2, 100.7, 79.9, 57.7, 55.0, 54.1, 36.4, 33.9, 27.5,
18.7, 12.9. MS calcd for C45H49N4O8S [M+H]+ 805.3, found: 805.5.
5.20. Compound 18
Prepared according to the general procedure described using 3
(15.3 mg, 0.0290 mmol), the N-deprotected HCl salt of 11
(7.79 mg, 0.0290 mmol), HATU (13.2 mg, 0.0348 mmol), DIEA
(27.4
(CH2Cl2/MeOH 97:3) yielded the pure
(6 mg, 28%) as well as a diastereomeric mixture of the
and the
-Phg form (8.7 mg, 41%). 1H NMR (CD3OD, major isomer
l
L, 0.157 mmol), and DMF (0.7 mL). Purification on silica gel
-Phg diastereomer of 17
-Phg form
L
L
D
reported): d 8.30 (d, J = 9.2 Hz, 1H), 7.88 (d, J = 2.3 Hz, 1H), 7.78
(m, 1H), 7.52–7.48 (m, 2H), 7.46–7.42 (m, 2H), 7.29 (dd, J = 2.4,
9.1 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 6.79 (dd, J = 11.2, 17.7 Hz, 1H),
6.66 (s, 1H), 5.97 (dd, J = 1.1, 17.7 Hz, 1H), 5.36 (s, 1H), 5.29 (dd,
J = 1.1, 11.1 Hz, 1H), 4.45 (dd, J = 6.3, 7.6 Hz, 1H), 4.00 (s, 3H), 2.99
(s, 1H), 2.86 (d, J = 0.8 Hz, 1H), 1.79 (m, 1H), 1.61 (m, 1H), 1.47 (s,
9H), 1.30 (m, 1H), 1.22 (m, 1H), 1.11 (m, 1H), 1.01–0.93 (m, 2H),
0.85 (m, 1H), 0.51–0.43 (m, 2H), 0.20–0.09 (m, 2H). 13C NMR
5.23. Compounds 20a and 20b
Prepared according to the general procedure using the N-depro-
tected HCl salt of 13 (29.2 mg, 0.0422 mmol), Boc-t-Leu (27.7 mg,
0.119 mmol), HATU (53.2 mg, 0.140 mmol), DIEA (71.1 lL,
0.408 mmol), and DMF (0.9 mL). Purification on preparative HPLC
(MeCN/H2O (0.05% HCOOH)) yielded 20a (6 mg, 17%) and 20b
(3.5 mg, 10%) as white solids. 20a: 1H NMR (CD3OD): d 8.29 (d,

5422
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
J = 9.2 Hz, 1H), 7.92 (d, 2.4 Hz, 1H), 7.79–7.75 (m, 2H), 7.54 (dd,
J = 2.4, 8.5 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.47–7.42 (m, 3H),
7.28 (dd, J = 2.4, 9.1 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 6.79 (dd,
J = 11.2, 17.7 Hz, 1H), 6.65 (s, 1H), 5.99 (d, J = 17.7 Hz, 1H), 5.73–
5.62 (m, 2H), 5.28 (dd, J = 1.0, 11.2 Hz, 1H), 4.93–4.87 (m, 2H),
4.35 (dd, J = 5.0, 8.6 Hz, 1H), 4.06–3.98 (m, 4H), 3.29–3.12 (m,
2H), 2.00–1.92 (m, 2H), 1.85–1.64 (m, 4H), 1.50–1.43 (m, 2H),
1.42 (s, 9H), 1.41–1.28 (m, 2H), 1.02 (s, 9H), 0.96 (t, J = 7.3 Hz,
3H). 13C NMR (CD3OD): 175.2, 173.2, 171.7, 163.9, 163.6, 161.1,
157.9, 152.6, 152.0, 140.8, 139.1, 136.9, 132.0, 130.9, 130.7,
130.1, 129.9, 128.7, 127.7, 123.9, 123.3, 120.1, 118.0, 115.8,
115.5, 107.7, 101.8, 80.7, 63.8, 57.4, 56.1, 55.7, 53.4, 35.3, 35.0,
34.2, 28.7, 28.5, 27.2, 23.9, 20.1, 14.0. HRMS calcd for C48H61N5O9S
[M+]H+ 884.4268, found: 884.4260. HPLC purity (system 1: 96%,
system 2: 94%) 20b: 1H NMR (CD3OD): d 8.31 (d, J = 9.2 Hz, 1H),
7.90 (d, J = 2.4 Hz, 1H), 7.76–7.73 (m, 2H), 7.53–7.48 (m, 2H),
7.46–7.43 (m, 3H), 7.29 (dd, J = 2.4, 9.1 Hz, 1H), 7.24 (d, 8.3 Hz,
1H), 6.80 (dd, J = 11.2, 17.7 Hz, 1H), 6.60 (s, 1H), 5.96 (d,
J = 17.7 Hz, 1H), 5.74 (m, 1H), 5.62 (s, 1H), 5.31 (dd, J = 1.0,
11.2 Hz, 1H), 5.01–4.48 (m, 2H), 4.33 (dd, J = 5.1, 8.7 Hz, 1H), 4.01
(s, 3H), 3.96 (s, 1H), 3.44–3.33 (m, 2H), 2.10–1.97 (m, 2H), 1.85–
1.65 (m, 4H), 1.51–1.39 (m, 11H), 1.35–1.24 (m, 2H), 0.95 (s, 9H),
0.83 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD) d 173.8, 173.2, 172.3,
163.9, 163.6, 161.1, 157.9, 152.6, 152.1, 140.9, 139.1, 137.4,
132.2, 130.9, 130.7, 130.7, 129.8, 128.6, 128.0, 123.9, 123.5,
120.2, 118.0, 115.8, 115.6, 107.7, 101.8, 80.7, 64.0, 58.1, 56.1,
55.5, 53.5, 35.4, 34.7, 34.2, 28.8, 28.5, 27.2, 23.9, 19.9, 13.9. HRMS
calcd for C48H61N5O9S [M+H]+ 884.4268, found: 884.4262. HPLC
purity (system 1: 97%, system 2: 99%).
5.25. Compounds 22a and 22b
Prepared according to the general procedure using the N-depro-
tected HCl salt of 15 (11.9 mg, 0.0179 mmol), Boc-t-Leu (6.60 mg,
0.0285 mmol), HATU (8.30 mg, 0.0218 mmol), DIEA (20.8 lL,
0.119 mmol), and DMF (0.5 mL). Purification on preparative HPLC
(MeCN/H2O (0.05% HCOOH)) yielded 22a (1.6 mg, 19%) and 22b
(2.2 mg, 29%) as white solids. 22a: 1H NMR (CD3OD): d 8.32 (d,
J = 9.0 Hz, 1H), 7.92–7.87 (m, 2H), 7.83 (d, J = 2.4 Hz, 1H), 7.79–
7.75 (m, 2H), 7.50 (d, J = 2.4 Hz, 1H), 7.48–7.37 (m, 5H), 7.30 (dd,
J = 2.3, 9.0 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 6.77 (dd, J = 11.7,
18.2 Hz, 1H), 6.63 (s, 1H), 6.51–6.46 (m, 1H), 5.97–5.89 (m, 1H),
5.64 (s, 1H), 5.27 (dd, J = 1.2, 11.3 Hz, 1H), 4.31 (dd, J = 5.8,
8.8 Hz, 1H), 4.02 (s, 4H), 1.77–1.54 (m, 2H), 1.42 (s, 9H), 1.39–
1.28, (m, 2H), 1.00 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3):
d 173.1, 171.1, 163.9, 163.6, 161.2, 157.4, 152.6, 151.8, 140.9,
137.1, 132.9, 131.9, 130.9, 130.6, 130.0, 129.8, 129.3, 128.7,
128.4, 127.5, 123.9, 123.2, 120.1, 118.2, 118.0, 115.9, 111.2,
107.7, 101.9, 80.6, 63.9, 57.1, 56.5, 56.1, 35.8, 35.5, 28.7, 27.3,
19.8, 14.1. HRMS calcd for C48H55N5O9S [M+H]+ 878.3799, found:
878.3807. HPLC purity (system 1: 94%, system 2: 92%) 22b: 1H
NMR (CD3OD): d 8.31 (d J = 9.3 Hz, 1H), 7.95–7.91 (m, 2H), 7.84
(m, 1H), 7.76–7.72 (m, 2H), 7.50–7.41 (m, 7H), 7.29 (dd, J = 2.6,
9.2 Hz, 1H), 7.19 (d, J = 9.0 Hz), 6.77 (dd, J = 11.3, 17.7 Hz, 1H),
6.59 (s, 1H), 5.89 (d, J = 17.3 Hz, 1H), 5.61 (s, 1H), 5.27 (d,
J = 11.2 Hz, 1H), 4.30 (dd, J = 5.1, 8.6 Hz, 1H), 4.01 (s, 3H), 3.98–
3.94 (m, 1H), 1.80–1.51 (m, 2H), 1.42 (s, 9H), 1.32–1.26 (m, 2H),
0.95 (s, 9H), 0.73 (t, J = 7.7 Hz, 3H). HRMS calcd for C48H55N5O9S
[M+H]+ 878.3799, found: 878.3804. HPLC purity (system 1: 95%,
system 2: 98%)
5.24. Compounds 21a and 21b
5.26. Compounds 23a and 23b
Prepared according to the general procedure using the N-
deprotected HCl salt of 14 (14.0 mg, 0.0200 mmol), Boc-t-Leu
(9.25 mg, 0.0400 mmol), HATU (18.2 mg, 0.048 mmol), DIEA
Prepared according to the general procedure using the N-depro-
tected HCl salt of 16 (11.6 mg, 0.0160 mmol), Boc-t-Leu (11.1 mg,
0.0479 mmol), HATU (21.8 mg, 0.0573 mmol), DIEA (28.4 lL,
(26.6 lL, 0.156 mmol), and DMF (0.7 mL). Three purification on
0.163 mmol), and DMF (0.9 mL). Purification on preparative HPLC
(MeCN/H2O (0.05% HCOOH)) yielded 23a (2.1 mg, 14%) and 23b
(1.4 mg, 9.6%) as white solids. 23a: 1H NMR (CD3OD): d 8.32 (dd,
J = 1.4, 9.2 Hz, 1H), 7.98 (dd, J = 1.4, 8.0 Hz, 1H), 7.81 (d,
J = 2.4 Hz, 1H), 7.77–7.72 (m, 2H), 7.59 (m, 1H), 7.56–7.50 (m,
2H), 7.49–7.41 (m, 4H), 7.36 (dd, J = 2.3, 8.5 Hz, 1H), 7.32 (ddd,
J = 1.2, 2.5, 9.0 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 8.3 Hz,
1H), 6.72 (dd, J = 11.2, 17.5 Hz, 1H), 6.61 (s, 1H), 5.89 (d,
J = 17.6 Hz, 1H), 5.69 (dd, J = 1.2, 17.4 Hz, 1H), 5.65 (s, 1H), 5.41
(dd, J = 1.2, 10.9 Hz, 1H), 5.24 (d, J = 11.2 Hz, 1H), 4.36 (dd, J = 4.8,
8.8 Hz, 1H), 4.02 (s, 4H), 1.69 (m, 1H), 1.55 (m, 1H), 1.41 (s, 9H),
1.37–1.28 (m, 2H), 0.99 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR
(CD3OD): d 173.1, 172.6, 171.6, 164.5, 164.0, 160.7, 157.9, 151.7,
151.6, 139.7, 138.5, 137.7, 136.9, 134.7, 134.6, 131.9, 131.7,
131.0, 130.6, 130.0, 128.8, 128.7, 128.6, 127.8, 124.1, 123.2,
120.5, 119.3, 118.3, 115.8, 106.8, 106.2, 102.0, 80.6, 63.8, 57.2,
56.3, 54.9, 35.3, 34.8, 28.7, 27.2, 19.9, 13.9. HRMS calcd for
preparative HPLC (MeCN/H2O (0.05% HCOOH)) had to be per-
formed to yield 21a (2.7 mg, 15.3%) and 21b (0.8 mg, 4.5%) as
white solids. 21a: 1H NMR (CD3OD): d 8.30 (d, J = 9.1 Hz, 1H),
7.94 (d, J = 2.3 Hz, 1H), 7.78–7.75 (m, 2H), 7.54 (dd, J = 2.3,
8.5 Hz, 1H), 7.49 (d, J = 2.5 Hz, 1H), 7.46–7.41 (m, 3H), 7.29 (dd,
J = 2.5, 9.1 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.79 (dd, J = 11.3,
17.7 Hz, 1H), 6.65 (s, 1H), 6.01 (d, J = 17.7 Hz, 1H), 5.72–5.61
(m, 1H), 5.69 (s, 1H), 5.29 (dd, J = 1.2, 11.2, 1H), 5.05–4.93 (m,
2H), 4.37 (dd, J = 5.2, 9.0 Hz, 1H), 4.03 (s, 1H), 4.01 (s, 3H),
2.64–2.50 (m, 2H), 1.84–1.63 (m, 2H), 1.54–1.28 (m, 4H), 1.49
(s, 9H), 1.02 (s, 9H), 0.97 (t, J = 7.3 Hz, 3H), 0.84–0.79 (m, 2H).
13C NMR (CD3OD): d 173.2, 172.7, 171.8, 163.9, 163.6, 161.2,
156.2, 152.6, 152.0, 140.8, 136.9, 134.3, 132.1, 130.9, 130.7,
129.9, 128.7, 127.5, 123.9, 123.2, 120.2, 119.3, 118.1, 115.8,
107.6, 101.9, 80.7, 63.8, 57.3, 56.1, 55.2, 41.0, 35.8, 35.3, 35.3,
34.7, 28.7, 27.2, 20.1, 14.0, 12.3, 10.9. HRMS calcd for
48H59N5O9S [M+H]+ 882.4112, found: 882.4131. HPLC purity
C
50H57N5O9S [M+H]+ 904.3955, found: 904.3973. HPLC purity (sys-
C
(system 1: 96%, system 2: 96%). 21b: 1H NMR (CD3OD): 8.31
(d, J = 9.1 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.77–7.72 (m, 2H),
7.52 (dd, J = 2.1, 8.4 Hz, 1H), 7.49 (d, J = 2.5 Hz, 1H), 7.45–7.42
(m, 3H), 7.29 (dd, J = 2.5, 9.2 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H),
6.80 (dd, J = 11.1, 17.7 Hz, 1H), 6.59 (s, 1H), 5.96 (d, J = 17.7 Hz,
1H), 5.75 (m, 1H), 5.62 (s, 1H), 5.31 (dd, J = 1.2, 11.1 Hz, 1H),
5.09–5.00 (m, 2H), 4.36 (dd, J = 5.2, 9.0 Hz, 1H), 4.01 (s, 3H),
3.97 (s, 1H), 2.65 (dt, J = 1.3, 7.3 Hz, 2H), 1.85–1.62 (m, 2H),
1.56 (m, 1H), 1.47–1.31 (m, 2H), 1.43 (s, 9H), 1.33–1.23 (m,
2H), 0.96 (s, 9H), 0.88 (m, 1H), 0.83 (t, J = 7.4 Hz, 3H). HRMS calcd
for C48H59N5O9S [M+H]+ 882.4112, found: 882.4094. HPLC purity
(system 1: 91%, system 2: 96%).
tem 1: 96%, system 2: 97%). 23b: 1H NMR (CD3OD): d 8.32 (d,
J = 9.1 Hz, 1H), 8.08 (dd, J = 1.5, 8.1 Hz, 1H), 7.84 (d, J = 2.5 Hz,
1H), 7.77–7.72 (m, 2H), 7.66 (m, 1H), 7.56 (dd, J = 11.1, 17.4 Hz,
2H), 7.50 (d, J = 2.4 Hz, 1H), 7.48–7.39 (m, 5H), 7.31 (dd, J = 2.6,
9.2 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 11.3, 17.6 Hz,
1H), 6.59 (s, 1H), 5.88 (dd, J = 1.1, 17.6 Hz, 1H), 5.76 (dd, J = 1.2,
17.2 Hz, 1H), 5.56 (s, 1H), 5.46 (dd, J = 1.2, 11.0 Hz, 1H), 5.28 (dd,
J = 1.1, 11.1 Hz, 1H), 4.34 (dd, J = 4.8, 9.0 Hz, 1H), 4.01 (s, 3H),
3.95 (s, 1H), 1.67 (m, 1H), 1.54 (m, 1H), 1.41 (s, 9H), 1.18–1.06
(m, 2H), 0.94 (s, 9H), 0.73 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD): d
173.2, 172.8, 172.0, 164.4, 164.0, 160.8, 157.8, 151.9, 151.7,
140.0, 138.6, 137.8, 137.5, 134.8, 134.6, 132.2, 131.8, 131.0,

A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
5423
130.7, 130.6, 130.0, 128.7, 128.7, 128.6, 127.9, 124.1, 123.4, 120.5,
119.4, 118.2, 115.7, 106.9, 101.9, 80.7, 63.9, 58.0, 56.2, 54.8, 35.4,
34.5, 28.8, 27.1, 19.7, 13.8. HRMS calcd for C50H57N5O9S [M+H]+
904.3955, found: 904.3940. HPLC purity (system 1: 96%, system
2: 97%).
173.0, 171.6, 169.0, 167.1, 158.4, 155.5, 150.5, 144.4, 138.6,
133.9, 133.1, 132.2, 131.8, 130.9, 130.2, 129.6, 126.0, 123.1,
122.7, 199.5, 115.5, 103.1, 100.8, 80.7, 63.8, 57.2, 57.1, 55.8, 37.5,
35.3, 31.9, 28.7, 27.2, 8.4, 6.4, 6.2, 5.3, 5.0. HRMS calcd for
C
46H55N5O9S [M+H]+ 854.3799, found: 854.3787. HPLC purity (sys-
tem 1: 95%, system 2: 99%)
5.27. Compounds 24a and 24b
5.29. Compound 27
Prepared according to the general procedure using the N-depro-
tected HCl salt of 17 (18.5 mg, 0.0250 mmol), Boc-t-Leu (11.6 mg,
0.0501 mmol), HATU (22.8 mg, 0.0600 mmol), DIEA (33.3 lL,
Compound 4 (14 mg, 0.0401 mmol) was dissolved in 4.0 M HCl/
dioxane and stirred for 4 h. The solvent was then evaporated. The
HCl salt of 4 was stirred with 2626 (15.4 mg, 0.0267 mmol), HATU
0.195 mmol), and DMF (0.7 mL). Purification by preparative HPLC
(MeCN/H2O (0.1% TFA)) yielded 24a (8.6 mg, 37%) as TFA-salt. Rep-
urification of the remaining diastereomeric mixture by preparative
HPLC (MeCN/H2O (0.05% HCOOH)) yielded 24b (1.84 mg, 8%) as a
white solid. 24a: 1H NMR (CD3OD (TFA-salt)): d 8.57 (d, J = 9.3 Hz,
1H), 7.96 (dd, J = 1.4, 8.2 Hz, 1H), 7.81 (dd, J = 2.4, 9.5 Hz, 1H),
7.78 (d, J = 1.6 Hz, 1H), 7.68 (m, 1H), 7.62–7.60 (m, 4H), 7.58 (dd,
J = 2.3, 9.3 Hz, 1H), 7.45 (ddd, J = 1.4, 6.9, 8.3 Hz, 1H), 7.37 (dd,
J = 2.2, 8.4 Hz, 1H), 7.28 (dd, J = 1.0, 6.9 Hz, 1H), 7.22 (dt, J = 2.0,
8.3 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 6.68 (dd, J = 11.2,
17.7 Hz, 1H), 5.99 (m, 1H), 5.89 (dd, 0.9, 17.7 Hz, 1H), 5.60 (s, 1H),
5.28 (dd, J = 0.9, 11.2 Hz, 1H), 5.02–4.97 (m, 2H), 4.33 (dd, J = 5.2,
8.9 Hz, 1H), 4.11 (s, 3H), 3.98 (s, 1H), 3.86 (dd, J = 6.6, 16.3 Hz,
1H), 3.74 (dd, J = 6.6, 16.2 Hz, 1H), 1.74–1.55 (m, 2H), 1.42 (s, 9H),
1.41–1.40 (m, 2H), 1.00 (s, 9H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR
(CD3OD): d 173.1, 172.4, 171.5, 168.5, 166.8, 158.5, 157.9, 150.5,
145.1, 141.0, 138.6, 138.2, 137.6, 134.6, 133.7, 133.6, 132.6, 132.0,
131.7, 130.8, 130.4, 130.0, 129.6, 128.4, 127.3, 125.8, 123.0, 122.5,
119.5, 117.3, 115.5, 102.9, 101.5, 80.6, 63.8, 57.2, 57.0, 54.9, 37.5,
35.3, 34.5, 28.7, 27.2, 20.0, 13.9. HRMS calcd for C51H59N5O9S
[M+H]+ 918.4112, found: 918.4131. HPLC purity (system 1: 97%,
system 2: 98%). 24b: 1H NMR (CD3OD): d 8.31 (d, J = 9.2 Hz, 1H),
8.08 (dd, J = 1.2, 8.0 Hz, 1H), 7.82 (s, 1H), 7.75 (m, 2H), 7.50 (d,
J = 2.5 Hz, 1H), 7.45–7.40 (m, 5H), 7.36–7.28 (m, 3H), 7.17 (d,
J = 8.2 Hz, 1H), 6.75 (dd, J = 11.3, 17.8 Hz, 1H), 6.58 (s, 1H), 6.01
(m, 1H), 5.87 (dd, J = 1.0, 17.7 Hz, 1H), 5.56 (s, 1H), 5.26 (dd,
J = 0.9, 11.3 Hz, 1H), 5.10–5.00 (m, 2H), 4.34 (dd, J = 4.9, 8.9, 1H),
4.01 (s, 3H), 3.94 (s, 1H), 3.88 (dd, J = 6.5, 15.7 Hz, 1H), 3.81 (dd,
J = 6.5, 15.9 Hz, 1H), 1.72 (m, 1H), 1.61 (m, 1H), 1.42 (s, 9H), 1.25–
1.11 (m, 2H), 0.94 (s, 9H), 0.77 (t, J = 7.0 Hz, 3H). 13C NMR (CD3OD):
d 173.1, 171.9, 164.0, 163.7, 161.1, 157.8, 152.5, 152.0, 140.8, 140.7,
140.0, 137.9, 137.4, 134.1, 132.5, 132.2, 131.7, 130.8, 130.7, 130.6,
129.9, 128.7, 128.0, 127.2, 123.9, 123.5, 120.3, 118.1, 117.1, 115.8,
107.5, 101.8, 80.7, 64.0, 58.1, 56.1, 54.6, 37.7, 35.4, 34.9, 28.8,
27.2, 19.8, 13.9. The Nva carbonyl carbon could not be found due
to low concentration of NMR sample. HRMS calcd for C49H57N5O9S
[M+H]+ 918.4112, found: 918.4124. HPLC purity (system 1: 98%,
system 2: 98%).
(12.3 mg, 0.0324 mmol), DIEA (22.9 lL, 0.132 mmol), and DMF
(0.7 mL) for 2 h. EtOAc was added and the solution was washed
with sodium acetate buffer, pH 4. Purification on silica gel
(EtOAc/i-hexane 2:1), then preparative HPLC (MeCN/H2O (0.05%
HCOOH)) gave 27 (4.6 mg, 21%) as a white solid. 1H NMR (CD3OD):
d 8.15 (d, J = 9.0 Hz, 1H), 8.08–8.05 (m, 2H), 7.63–7.56 (m, 3H), 7.43
(d, J = 2.5 Hz, 1H), 7.36 (s, 1H), 7.15 (dd, J = 2.5, 9.0 Hz, 1H), 5.78 (m,
1H), 5.60 (s, 1H), 5.09–4.98 (m, 2H), 4.75 (m, 1H), 4.60 (m, 1H), 4.30
(dd, J = 5.1, 8.9 Hz, 1H), 4.23 (s, 1H), 4.06 (m, 1H), 3.98 (s, 3H), 3.36
(m, 2H), 2.77 (m, 1H), 2.42 (m, 1H), 2.21–2.15 (m, 2H), 1.98–1.61
(m, 3H), 1.59–1.42 (m, 2H), 1.28 (s, 9H), 1.04 (s, 9H), 0.99 (s, 1H),
0.95 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD): d 174.2, 173.6, 173.4,
164.0, 160.7, 158.0, 151.7, 139.4, 138.1, 138.1, 131.4, 130.1,
129.3, 124.9, 119.9, 116.5, 116.5, 105.6, 100.7, 80.5, 78.9, 60.7,
60.0, 56.2, 55.3, 55.2, 53.1, 36.0, 35.9, 34.8, 33.0, 28.6, 26.9, 23.7,
20.0, 14.0. HRMS calcd for C42H57N5O9S [M+H]+ 808.3955, found:
808.3969. HPLC purity (system 1: 95%, system 2: 98%).
5.30. Enzyme inhibition
The protease activity of the full-length HCV NS3 protein (prote-
ase-helicase/NTPase) was measured using
a FRET-assay as
previously described.32,35 In short, 1 nM enzyme was incubated
for 10 min at 30 °C in 50 mM HEPES, pH 7.5, 10 mM DTT, 40% glyc-
erol, 0.1% n-octyl-b-
peptide cofactor 2K-NS4A (KKGVVIVGRIVLSGK), and inhibitor.
The reaction was started by the addition of 0.5 M substrate (Ac-
DED(Edans)EEAbu [COO]ASK(Dabcyl)-NH2) obtained from Ana-
D-glucoside, 3.3% DMSO with 25 lM of the
l
w
Spec Inc. (San Jose, USA). Non-linear regression analysis of the data
was made using Grafit 5.0.13 (Erithacus software limited). Evalua-
tion of anti replicative effect in cells was performed as described by
Lohmann et al.33
5.31. Computational methodology
All the ligands were built in maestro and geometry optimization
is carried out using OPLS-2005 force field. The crystal structure of
HCV bifunctional protease helicase (1CU1) is used in the study.
This NS3 protein complex consists of the C-terminal helicase do-
main and the N-terminal protease domain with a covalently linked
NS4A cofactor. The protease active site is occupied by the substrate
(NS3 C-terminus which is part of the helicase domain). The active
site was made available for the docking studies by deleting these
terminal residues (624–631) occupying the active site.
5.28. Compound 25
Prepared according to the general procedure using the N-depro-
tected HCl salt of 18 (5.14 mg, 0.00760 mmol), Boc-t-Leu (8.00 mg,
0.0152 mmol), HATU (6.92 mg, 0.0182 mmol), DIEA (11.7 lL,
0.0669 mmol), and DMF (0.5 mL). After purification on preparative
HPLC (MeCN/H2O (0.05% HCOOH)), 25 was yielded as a white solid
(2.48 mg, 38%). 1H NMR (CD3OD) d 8.31 (d, J = 9.2 Hz, 1H), 7.93 (d,
J = 2.3 Hz, 1H), 7.79–7.75 (m, 2H), 7.55 (dd, J = 2.2, 8.4 Hz, 1H), 7.49
(d, J = 2.5 Hz, 1H), 7.48–7.42 (m, 3H), 7.29 (dd, J = 2.5, 9.2 Hz, 1H),
7.23 (d, J = 8.3 Hz, 1H), 6.79 (dd, J = 11.2, 17.6 Hz, 1H), 6.64 (s,
1H), 6.00 (d, J = 17.6 Hz, 1H), 5.70 (s, 1H), 5.29 (d, J = 11.4 Hz,
1H), 4.86 (s, 1H), 4.45 (dd, J = 7.8, 6.3 Hz, 1H), 4.01 (s, 3H), 2.85
(m, 1H), 1.79 (m, 1H), 1.60 (m, 1H), 1.42 (s, 9H), 1.23–1.16 (m,
1H), 1.14–1.07 (m, 1H), 1.01 (s, 9H), 0.97–0.80 (m, 3H), 0.54–0.42
(m, 2H), 0.22–0.10 (m, 2H). 13C NMR (CD3OD (TFA-salt)): d 173.3,
All the crystallographic waters were removed and the protein
was prepared for docking using the protein preparation tool imple-
mented in the Schrodinger Suite 2008.36 The generated structures
were subjected to restrained minimization using the OPLS-2005
force field with ‘normal’ BatchMin cutoffs (7.0 Å VDW; 12.0 Å
ELE). To account for the conformational changes in the protein, In-
duced fit docking is carried out using FLO (also called QXP).37
A
truncated protein structure that included only amino acids within
9 Å from the residues 624–631 (C-terminus) was used in the dock-
ing studies using FLO. Protein flexibility is accounted by allowing

5424
A. Lampa et al. / Bioorg. Med. Chem. 18 (2010) 5413–5424
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M.; Vrang, L.; Vendeville, S.; Van de Vreken, W.; McGowan, D.; Tahri, A.; Hu, L.;
Boutton, C.; Lenz, O.; Delouvroy, F.; Pille, G.; Surleraux, D.; Wigerinck, P.;
Samuelsson, B.; Simmen, K. Bioorg. Med. Chem. Lett. 2008, 18, 4853.
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14. Liverton, N. J.; Carroll, S. S.; DiMuzio, J.; Fandozzi, C.; Graham, D. J.; Hazuda, D.;
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crucial amino acid residues in the binding pocket to move freely up
to 0.2 Å. Movement larger than 0.2 Å was penalized by 20.0 kJ/mol/
Å2. Flexible residues occupying the active site viz., R155, Q526 and
K136 were given full conformational freedom. Constraints were
applied to the inhibitor to restrict its translation away from the ac-
tive site. For each inhibitor, 10 unique binding poses were gener-
ated using 2000 Monte Carlo perturbation cycles. These poses
were further subjected to 20 steps of simulated annealing followed
by energy minimization. Each cycle involves 400 rapid Monte Carlo
steps generating unique conformations within an energy window
of 50 kJ/mol/Å and an RMSD > 0.5 Å. Each of the complexes was
submitted to a 3 fs dynamics at 600 K after 3000 fs of equilibration
steps. The maximum movement of an atom in any single step was
limited to 0.1 Å. Hydrogen vibrations were damped by assigning an
atomic weight of 10. Among the 10 best poses generated, the most
plausible binding mode conformation for each compound was se-
lected based on visual inspection.
16. Steinkuhler, C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch, U.; Cortese, R.; Pessi,
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The authors gratefully acknowledge support from Knut and
Alice Wallenberg’s Foundation. The authors also thank Dr. Aleh
Yahorau, Department of Pharmaceutical Biosciences, Uppsala Uni-
versity, for help with HRMS analyses and Medivir AB, Huddinge,
Sweden for performing EC50-determinations. Also Johanna Grandin
for assistance in the synthetic work.
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