Vinylated linear P2 pyrimidinyloxyphenylglycine based inhibitors of the HCV NS3/4A protease and corresponding macrocycles

Article abstract of DOI:10.1016/j.bmc.2014.10.010

With three recent market approvals and several inhibitors in advanced stages of development, the hepatitis C virus (HCV) NS3/4A protease represents a successful target for antiviral therapy against hepatitis C. As a consequence of dealing with viral diseases in general, there are concerns related to the emergence of drug resistant strains which calls for development of inhibitors with an alternative binding-mode than the existing highly optimized ones. We have previously reported on the use of phenylglycine as an alternative P2 residue in HCV NS3/4A protease inhibitors. Herein, we present the synthesis, structure-activity relationships and in vitro pharmacokinetic characterization of a diverse series of linear and macrocyclic P2 pyrimidinyloxyphenylglycine based inhibitors. With access to vinyl substituents in P3, P2 and P1′ positions an initial probing of macrocyclization between different positions, using ring-closing metathesis (RCM) could be performed, after addressing some synthetic challenges. Biochemical results from the wild type enzyme and drug resistant variants (e.g., R155 K) indicate that P3-P1′ macrocyclization, leaving the P2 substituent in a flexible mode, is a promising approach. Additionally, the study demonstrates that phenylglycine based inhibitors benefit from p-phenylpyrimidinyloxy and m-vinyl groups as well as from the combination with an aromatic P1 motif with alkenylic P1′ elongations. In fact, linear P2-P1′ spanning intermediate compounds based on these fragments were found to display promising inhibitory potencies and drug like properties.

Full text of DOI:10.1016/j.bmc.2014.10.010

Bioorganic & Medicinal Chemistry 22 (2014) 6595–6615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Vinylated linear P2 pyrimidinyloxyphenylglycine based inhibitors
of the HCV NS3/4A protease and corresponding macrocycles
Anna Lampa a, Hiba Alogheli a, Angelica E. Ehrenberg b, Eva Åkerblom a, Richard Svensson c,d
,
Per Artursson c,d, U. Helena Danielson b, Anders Karlén a, Anja Sandström a,
⇑
a Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
b Department of Chemistry-BMC, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
c Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden
d The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Uppsala University, A Node of the Chemical Biology Consortium Sweden (CBCS), Box 580, 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:
With three recent market approvals and several inhibitors in advanced stages of development, the hep-
atitis C virus (HCV) NS3/4A protease represents a successful target for antiviral therapy against hepatitis
C. As a consequence of dealing with viral diseases in general, there are concerns related to the emergence
of drug resistant strains which calls for development of inhibitors with an alternative binding-mode than
the existing highly optimized ones. We have previously reported on the use of phenylglycine as an alter-
native P2 residue in HCV NS3/4A protease inhibitors. Herein, we present the synthesis, structure–activity
relationships and in vitro pharmacokinetic characterization of a diverse series of linear and macrocyclic
P2 pyrimidinyloxyphenylglycine based inhibitors. With access to vinyl substituents in P3, P2 and P10
positions an initial probing of macrocyclization between different positions, using ring-closing metathe-
sis (RCM) could be performed, after addressing some synthetic challenges. Biochemical results from the
wild type enzyme and drug resistant variants (e.g., R155 K) indicate that P3–P10 macrocyclization, leaving
the P2 substituent in a flexible mode, is a promising approach. Additionally, the study demonstrates that
phenylglycine based inhibitors benefit from p-phenylpyrimidinyloxy and m-vinyl groups as well as from
the combination with an aromatic P1 motif with alkenylic P10 elongations. In fact, linear P2–P10 spanning
intermediate compounds based on these fragments were found to display promising inhibitory potencies
and drug like properties.
Received 15 July 2014
Revised 4 October 2014
Accepted 9 October 2014
Available online 18 October 2014
Keywords:
HCV
NS3
Protease inhibitors
Macrocyclization
Phenylglycine
Metathesis
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
for the prevalent genotype 1.4 The significance of the disease and
poor treatment options have made HCV a prioritized research area
Hepatitis C is a major global health issue with an estimated glo-
bal prevalence of ca. 2.2% and 350 000 related deaths every year.1
The causative agent of the disease is the hepatitis C virus (HCV).
The virus infects the liver, and can cause acute and chronic liver
disease. HCV constitutes a major reason for liver transplantations
in the western world.2 The mainstay treatment has for long been
within the pharmaceutical industries the last decades, and from
which results now are starting to be seen. In 2012 two direct acting
antiviral drugs (DAAs), the protease inhibitors boceprevir5 (Victrel-
is™, Merck & Co.) and telaprevir6 (Incivek™,Vertex) were approved
for the treatment of genotype 1, thus resulting in a significant bet-
ter cure rate in combination with the standard therapy (up to 80%)
and shortened treatment periods.7 In 2013 two more DAAs, sime-
previr,8 a protease inhibitor, and sofosbuvir,9 a polymerase inhibi-
tor, were launched. In addition, several DAAs are currently in
advanced clinical stages and these are evaluated in various combi-
nations, including interferon-free alternatives. The treatment
alternatives for HCV patients are definitely improving. However,
the choice of treatment for a particular patient is complex, depend-
ing on several individual and viral factors such as genotype,
subtype, disease progress, treatment history, tolerability etc., as
based on the immunomodulating pegylated interferon-a alongside
the broad-spectrum antiviral agent ribavirin. The treatment could
progress up to 72 weeks and is associated with severe side-effects
such as pancytomia, depression and flu-like symptoms.3 Moreover,
the success rate of the treatment is highly dependent on the viral
genotype (i.e., genotype 1–6) with a success rate of ca. 45–50%
⇑
Corresponding author. Tel.: +46 18 471 42 85; fax: +46 18 471 4474.
E-mail address: anja.sandstrom@orgfarm.uu.se (A. Sandström).
http://dx.doi.org/10.1016/j.bmc.2014.10.010
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6596
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
well as cost. Besides, the efficacy of these medications can be ham-
pered by emergence of drug resistance and more seriously cross
resistance, as indicated in cell-based as well as clinical studies.10,11
Thus, efforts to design and develop coming generation of DAAs that
retain activity against resistant variants must be taken into
consideration.
humans after oral administration.22 Unfortunately, the develop-
ment of ciluprevir was halted due to cardiotoxicity.23,24 After cilu-
previr, several NS3/4A protease inhibitors have entered clinical
trials, all showing excellent inhibitory potencies (Fig. 1).
The main structural variations of the clinical candidates are the
product-based or electrophilic functionality and if they are linear
or macrocyclized between different positions. A consequence of
the development of direct-acting antiviral HCV NS3/4A protease
inhibitors is the possible emergence of resistant mutants which
is a common setback among viral diseases in general. In vitro
and in vivo studies have revealed mutations leading to protease
variants with amino acid substitutions particularly in areas which
interact with the P2 region of the protease inhibitor, both in the
product-based and electrophilic class of inhibitors.10,11,28,29 A com-
mon attribute among the inhibitors that have entered clinical trials
so far, is that they carry a proline or proline mimic in the P2 posi-
tion, the latter in the case of simeprevir (Fig. 1). These proline-
based inhibitors are highly optimized and outstanding regarding
antiviral efficacy. As an alternative to proline, we have previously
presented inhibitors holding a phenylglycine (Phg) in the P2 posi-
tion.30–32 This inhibitor class has a different binding-conformation
than proline-based inhibitors30 and is therefore less affected by
mutations that emerge in the presence of proline-based inhibi-
tors.33,34 In an attempt to optimize phenylglycine-based inhibitors,
we have previously found that alkenylic elongations at the acyl-
sulfonamide were more beneficial in phenylglycine-based inhibi-
tors than proline-based inhibitors (A and B, Fig. 2). It was also
found that the commonly utilized P1-residues norvaline, or vinyl
cyclopropyl carboxylic acid (vinyl ACCA)35 were not beneficial in
combination with P2 phenylglycine.30,31 However, incorporation
of an aromatic P1 residue was more suitable (D, Fig. 2), and seems
to be an interesting optimization point for phenylglycine-based
inhibitors.34 This leads us to believe that there are still many
unraveled points for potentiation of phenylglycine-based
inhibitors.
The genome of HCV contains single-stranded (+)-RNA that is
translated into a polyprotein of approximately 3000 amino acids.
The polyprotein consists of 10 viral proteins. The first third of the
N-terminal part of the polyprotein encodes the structural region
consisting of proteins C, E1, E2, and p7 which are involved in the
structural build-up of the virion. The rest of the polyprotein
encodes for the non-structural proteins NS2, NS3, NS4A, NS4B,
NS5A and NS5B, which are essential in the viral replication machin-
ery.12,13 NS3 is a bifunctional enzyme containing a serine protease
domain and a RNA helicase domain.14 The NS3 protease is, when
combined with its cofactor NS4A, responsible for deliberating the
proteins NS4A, NS4B, NS5A and NS5B from the polyprotein. Several
targets are explored for anti-HCV therapy, and the NS3/4A protease,
along with NS5B polymerase, are among the more well-studied and
promising considering its central role in the viral replication
machinery. Moreover, the NS3 protease is involved in interference
with the infected cell’s natural antiviral response, thereby facilitat-
ing an evasion of the immune system for the virus.15
A specific characteristic of the NS3 protease is that it is inhibited
by its own cleavage products.16,17 Since this discovery, several so-
called product-based HCV NS3/4A protease inhibitors have been
developed.18 The product-based inhibitors are characterized by
an acidic functionality,19 most often a carboxylic acid or an acyl-
sulfonamide20 in the C-terminal (Fig. 1). Another class of HCV
NS3/4A protease inhibitors, the so-called electrophilic inhibitors
utilizes an electrophilic functionality to interact covalently, and
reversibly with the serine residue of the protease.21 Proof-of-con-
cept was established with ciluprevir, BILN-2061. This was the first
NS3/4A protease inhibitor to show reduced HCV RNA levels in
H
S
N
O
N
O
Br
N
N
O
H
N
N
N
O
O
O
S
O
O
O
H
N
N
O
NH
N
N
H
N
O
O
O
O
O
NH
O
S
O
NH
OH
O
O
O
O
N
H
O
O
O
N
OH
H
Ciluprevir, BILN 2061
Faldaprevir, BI 201335
Ki (1a)= 1.2 nM
Vaniprevir, MK-7009
Ki = 0.05 nM, EC50 = 5 nM
Ki = 0.30 nM, EC50 = 4 nM
Ki (1b) = 2.8 nM
O
EC50 (1a)=6.5 nM
EC50 (1b) = 3.1 nM
N
N
S
O
N
O
O
NH
N
O
H
N
O
O
O
O
S
NH
NH
O
O
O
N
HN
O
NH
NH
N
O
O
O
N
NH2
NH
O
H
N
O
Telaprevir, IncivekTM
Ki = 40 nM, EC50 = 350 nM
Boceprevir, VictrelisTM
Ki* = 14 nM, EC50 = 200 nM
Simeprevir, Olysio TM
Ki = 0.36 nM, EC50 = 7.8 nM
Figure 1. A selection of macrocyclic and linear product-based, and electrophilic HCV NS3/4A protease inhibitors that have entered clinical trials. Ciluprevir,22 vaniprevir,25,26
faldaprevir,27 simeprevir,8 telaprevir,6 and boceprevir.5

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6597
O
O
N
N
O
O
N
O
O
O
O
O
NH
H
O
BocHN
N
S
BocHN
O
O
O
S
N
H
N
HN
H
O
A
B
Ki = 300 nM
Ki = 63 nM
O
O
N
N
O
N
O
H
N
O
O
NH
S
O
O
S
NH
H
O
O
BocHN
BocHN
N
N
O
O
O
H
O
C
D
Ki = 830 nM
Ki = 310 nM
Figure 2. Two examples of fragments better suited with phenylglycine than proline. (1) Alkenylic P10 elongations (B vs A); (2) aromatic P1 moiety (D vs C).
In a previous attempt to pin down the binding mode of phenyl-
glycine-based inhibitors, we introduced conformational strain by
macrocyclization between the P10 and P2 positions. Disappoint-
ingly, potentiation of the inhibitory activity was only found in
one case compared to the acyclic analogues possibly explained
provide us with a better understanding of the binding mode of
phenylglycine-based inhibitors. Herein, we present the synthesis,
structure–activity relationships and in vitro pharmacokinetic char-
acterization for a diverse series of linear and macrocyclic HCV NS3/
4A protease inhibitors.
by a disruption of a beneficial p–p interaction between phenylgly-
cine and H57 of the protease, caused by macrocyclization.32 Also,
the synthesis of the P2 building block containing a quinolinyl-
oxy-type of substitution36 on the phenylglycine was difficult and
very slow. Thus, we were encouraged to find alternatives to the
quinolinyloxy P2 substituent and combine this new alternative
with the advantageous alkenylic acylsulfonamides and the aro-
matic P1 moieties. The 2-phenyl-4-methoxy-pyrimidinyloxy sub-
2. Chemistry
The different P1–P10 building blocks, shown in Scheme 1,
were synthesized by coupling commercially available Boc-L-Nva,
2-(t-butoxycarbonylamino)benzoic acid (Boc-Abz-OH) with but-
3-ene-1-sulfonamide39 or pent-4-ene-1-sulfonamide,39 using car-
bonyldiimidazole (CDI) and 1,8-diazabicycloundecene (DBU) in
THF. CDI activation of the acid was performed under 60 °C for 1 h
before addition of sulfonamide and DBU. Purification on silica gel
removed traces of unreacted acid. This was followed by purifica-
tion on aluminium oxide to remove any of the remaining sulfon-
amide. Building blocks 1–3 were obtained in a yield of 59–80%
(Scheme 1).
The new P2 building block with a pyrimidine substituent was
synthesized according to Scheme 2. To attain the P2-substituent
containing a methoxy group in the 6-position of the pyrimidine,
commercially available fenclorim needed reflux with sodium
methoxide in methanol, contrary to a previously published synthe-
sis in room temperature40 After 7 h of reflux, the disubstituted
byproduct (4,6-dimethoxy-2-phenylpyrimidin) started to be
stituent was chosen as an interesting candidate for
a P2
substituent.37 The synthesis of the pyrimidine based P2 substituent
opened up for diversification of the P2 substituent such as vinyl
functionalities, inviting to macrocyclization to the P2 substituent
from P3, which is an interesting option for macrocyclization.38 Fur-
thermore, by the use of alkenylic P10 substituents, macrocycliza-
tions connecting P10 with the P3, as well as with the P2
substituent were interesting options. Indeed, modeling suggested
a proximity between P3 and the alkenylic P10 which motivated
us to explore such a macrocyclization. We hoped that access to
these different alternatives, as summarized in Figure 3, would
Ph
P2-P1'
N
H
O
OH
O
N
N
S
n
O
O
BocHN
BocHN
BocHN
BocHN
P4-P2
O
P2-P1'
O
O
n=0 1 (59%)
S
2
n=1
(80%)
H
N
H2N
n
O
n
O
S
or
O
H
N
H
N
O
O
a
n = 0,1
O
P3
N
H
O
O
O
S
O
O
OH
3 (78%)
N
P3-P1'
H
Figure 3. Summarized view of the different macrocyclization alternatives covered
in this study.
Scheme 1. Reagents: (a) CDI, DBU, THF, 60 °C ? rt.

6598
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
an intermediate useful in both the synthesis of linear and cyclized
variants and also since we were interested in both epimers. Thus,
R2 = Cl, MeO
N
compound 7 was coupled directly with the P3 building block,
R2
N
N-[(but-3-en-1-yloxy)carbonyl]-3-methyl-L
-valine,44 for further
R2
N
O
coupling with the P1–P10 building block (Scheme 4). To enable this,
7 was first transformed into the methyl ester 20 (56%), under mild
conditions using methyl iodide and cesium carbonate in DMF.46
This was followed by Boc-deprotection using TFA in CH2Cl2 to
avoid addition of HCl to the pyrimidine vinyl and HATU mediated
coupling in DMF to give P2–P4 fragment 21 in a 62% yield. Micro-
wave assisted ester hydrolysis of 21 using potassium carbonate in
acetonitrile and coupling with the N-deprotected HCl salt of 3 gave
both diastereomers 22L and 22D in a yield of 6% and 3.5%, respec-
tively. Unfortunately, it was observed during this last step of the
synthesis that byproducts were formed, with masses indicating
that the ester deprotected P4–P2 starting material is decomposed
during activation.30,45
Access of a vinylated pyrimidine-based P2 substituent, and alk-
enylic P1–P10 building blocks invited to a new P2–P10 macrocycli-
zation connecting the pyrimidine-based substituent with P10. A
similar macrocyclization connecting the P2 substituent with P10
has been investigated in a recent patent application.47 The ring-
closing metathesis reactions forming macrocycles connecting the
P2 pyrimidine substituent and P10 side chains are outlined in
Scheme 5. Diolefinic compounds 11 and 12 were treated with Hov-
eyda–Grubbs 2nd generation catalyst in trifluorotoluene under
microwave irradiation to obtain macrocycles 23 and 24 (Scheme 5).
During the ring closure of 11 and 12, a precipitate, most probably
dimeric and polymeric starting material was formed apart from the
desired product. The precipitation was more pronounced in the
ring-closure of 12, leading us to believe that the ring-closure of a
compound containing an aromatic P1 moiety is more difficult than
with a non-aromatic P1. After silica purification, compounds 23
and 24 were obtained in a 43% and 11% yield, respectively, in trans
N
OH
R1
R1
Cl
a
OH
OH
BocHN
BocHN
O
O
4 R1 = H
5 R1 = Br
6 R1 = H, R2 = Cl (91%)
b
b
7 R1 = H, R2 = vinyl (83%)
8 R1 = H, R2 = MeO (70%)
9 R1 = Br, R2 = MeO (87%)
10 R1 = vinyl, R2 = MeO (82%)
Scheme 2. Reagents: (a) KOtBu, DMSO, rt. (R2 = Cl)/64 °C (R2 = OMe); (b) 2,4,6-
trivinylcycloboroxane pyridine complex, Pd(OAc)2, [(tBu)3PH]BF4, K2CO3, DME, H2O,
MW, 100 °C, 15 min.
formed and the reaction was interrupted. After extraction, 4-
chloro-6-methoxy-2-phenylpyrimidine
characterized.
was
obtained
and
Starting with the Boc-protected 4-hydroxy-
L
-phenylglycine
-phenylglycine
(4),41 or the Boc-protected 3-bromo-4-hydroxy-
L
(5),42 the P2 building blocks 6, 8 and 9 were synthesized through
a nucleophilic aromatic substitution reaction using commercially
available fenclorim or 4-chloro-6-methoxy-2-phenylpyrimidine
together with potassium-tert-butoxide in DMSO at either 64 °C (8
and 9), or room temperature (6). Completion of the reactions was
established by LC–MS and P2 building blocks 6, 8 and 9 were
obtained in 91%, 70%, and 87% yield, respectively. A microwave
assisted Suzuki reaction using 6, 2,4,6-trivinylcyclotriboroxane
pyridine complex, and [(tBu)3PH]BF4 (Fu salt),43 Pd(OAc)2 in DME
produced 7 in only 15 min at 100 °C and in a yield of 83% after pre-
cipitation. Compound 10 was generated through the same micro-
wave assisted Suzuki reaction as 7 in a yield of 82%.
configuration only. Further coupling of 23 with Boc-L-tLeu yielded
the P2–P10 macrocyclic inhibitor 26 in a yield of 61%. The same
coupling of 24 produced the P2–P10 macrocyclic inhibitor 27 in a
20% yield.
Synthesis of the P2–P1–P10 compounds 11–15 (Scheme 3) were
performed by peptide coupling of P2 building blocks 7, 8 or 10 with
the N-deprotected HCl-salts of 1, 2 or 3 using N-[(dimethylamino)-
1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylme-
thanaminium hexafluorophosphate N-oxide (HATU) and diisopro-
pylethylamine (DIEA). The P1–P10 building blocks comprising an
aromatic moiety (1 and 2) were coupled in dry DCM at 45 °C, while
the coupling of the norvaline containing building block (3) was per-
formed in dry DMF at room temperature After silica gel purification,
compounds 11–15 were obtained in a yield of 18–96%. For the fur-
ther coupling of 12, the N-deprotection had to be performed using
TFA in DCM, since the alternative deprotection using HCl/dioxane
resulted in addition of HCl over the pyrimidine vinyl. The TFA-salt
of 12 was coupled with N-[(But-3-en-1-yloxy)carbonyl]-3-methyl-
Another type of P2–P10 macrocyclization, is where the phenyl-
glycine part of the P2 is connected with the alkenylic P10 side-
chain. A P2–P10 type of cyclization has been reported before by
us32 and others.48 The ring closure of 14 (Scheme 5) required a high
(30 mol %) catalyst load before getting a satisfying amount con-
verted starting material. Again, we experienced that ring-closure
of 14, with an aromatic P1 was complicated, leaving more unre-
acted linear starting material than ring-closure of compounds with
norvaline P1.32 Some dimeric and ring-contracted products were
formed besides compound 25, which was obtained in a 28% yield,
in cis configuration. The P2–P10 macrocyclic tripeptidic inhibitor 28
was obtained in a 33% yield by coupling 25 with Boc-L-tLeu.
The use of Boc-2-amino-8-nonenoic acid in P3 enabled ring-
closure between P3 and P10. P3–P10 macrocycles have previously
been investigated in P2 proline based inhibitors in a patent appli-
cation.49,50 The ring closure of 17 (Scheme 6) proved to be compli-
cated. Full conversion of starting material was not obtained and
large amounts of dimer, trimer, and ring-closed variants of the
dimer and trimer were formed according to LC–MS. An interesting
observation was made when a small fraction of 17 was purified on
preparative HPLC with acetonitrile/water containing 0.05% formic
acid as eluent, for biochemical evaluation. When comparing the
1H NMR of 17, purified on silica gel with 17, purified on preparative
HPLC, it was clear that the 1H NMR were different; in particular the
protons around the aromatic P1 moiety (Table 1). Thus, this indi-
cates that 17 exists in two different conformations in where one
of the conformations contains an internal hydrogen bond between
the P1 amine hydrogen and the P1 carbonyl (Fig. 4), which would
L
-valine, prepared according to a published patent,44 using HATU
in DMF resulting in 16 that was further purified by HPLC. Compound
13 was coupled both with Boc-2-amino-8 nonenoic acid and Boc-
tLeu to generate 17 and 18 in 79% and 35% yield, respectively. The
coupling of 15 with Boc- -tLeu resulted in both diastereomers 19L
L
-
L
and 19D in a yield of 15% and 10%, respectively.
Peptide coupling in the synthesis of phenylglycine-based inhib-
itors is normally performed in a fashion to retain the Boc-protect-
ing group on the phenylglycine as long as possible throughout the
synthesis; which is demonstrated and exemplified in Scheme 3.
This is to avoid racemization on the phenylglycine
a
-carbon.45 In
the case of linear compounds 22 encompassing three vinyl groups
(of which the corresponding P4–P2 cyclized variants will be
prepared, vide infra), another approach was attempted to generate

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6599
P1-P1'
P2-P1-P1'
P3-P2-P1-P1'
P2
N
N
N
R2
N
O
O
H
N
R1
O
O
S
O
O
H
N
H
N
H
O
O
N
S
O
N
H
O
n
H
N
O
O
N
BocHN
1 or 2
16
(4%)
R2
N
O
O
d, e
a, f
a, c
12
13
14
15
R1 = H, R2 = vinyl, n = 1 (96%)
R1 = H, R2 = MeO, n = 1 (86%)
N
R1
O
N
O
OH
R
1 = vinyl, R2 = MeO, n = 0 (45%)
BocHN
H
N
3
O
7
O
O
S
R1 = vinyl, R2 = MeO, n = 1 (18%)
H
N
a, b
O
O
BocHN
8
a, g
a, g
N
H
10
O
17
(79%)
N
N
O
N
O
N
O
O
O
S
H
N
O
BocHN
N
R1
H
O
H
O
N
O
S
O O
H
N
H
N
11
(77%)
O
N
H
O
O
18
R1 = H (35%)
R1 = vinyl (15%)
R1 = vinyl (10%)
19L
19D
Scheme 3. Reagents: (a) 4.0 M HCl in dioxane; (b) HATU, DIEA, DMF, rt; (c) HATU, DIEA, DCM 45 °C; (d) TFA, DCM; (e) HATU, N-[(But-3-en-1-yloxy)carbonyl]-3-methyl-
L-
valine, DIEA, DMF, rt; (f) HATU, Boc-2-amino-8-nonenoic acid, 4-methylmorpholine, DMF; (g) HATU, Boc- -tLeu, DIEA, DMF, rt.
L
N
N
N
N
N
O
N
O
N
O
N
O
O
O
O
H
N
OH
3
b, c
O
O
O
a
d, e, f
O
O
O
O
O O
H
N
H
N
H
N
O
O
S
OH
BocHN
N
H
N
H
N
BocHN
H
O
O
O
O
7
21
20
(62%)
22L
(6%)
(56%)
22D (3.5%)
Scheme 4. Reagents: (a) CsCO3, MeI, DMF; (b) TFA, DCM; (c) HATU, DIEA, DMF, rt; (d) K2CO3, MeCN, H2O; (e) 4.0 M HCl in dioxane; (f) HATU, DIEA, DMF, rt.
create an unfavourable pose for ring-closure between P10 and P3. In
a previous publication it is shown that the proton next to the acet-
anilide (H1 in Fig. 4) exhibit signals at unusually low field as an
effect of the internal hydrogen bond.51 The same observation was
seen in the 1H NMR of the two conformations of 17; in the 1H
NMR from silica purified 17 H1 resulted in a signal at a higher shift
than in the corresponding HPLC (acidic eluent) purified 17. This
can be a result of that the internal hydrogen bond is weakened,
or disrupted in the presence of acid. By adding formic acid to the
reaction mixture during ring-closure of 17 it was evident from
LC–MS that less dimerization and polymerization occurred and
that the desired product 29 was the major product beside a small
amount of ring-contracted product (30) (Fig. 5). After repeated
purification on preparative HPLC the two diastereomers 29L and
29D could be isolated in 11% and 6% yield, respectively. Addition-
ally, the ring contracted product 30 was isolated as a diastereo-
meric mixture in
a yield of 3%. The mixed fractions from
purification of 29 were pooled and subjected to reduction using

6600
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
O
BocHN
OH
RCM
Tripeptidic macrocycle
Diolefin
Macrocycle
N
N
N
N
N
N
O
O
O
b, c
a
O
O
O
O
S
NH
O
H
O
S
NH
O
O
O
O
S
H
N
H
N
N
BocHN
BocHN
N
H
BocHN
N
H
O
26 (61%)
23 (43%)
O
O
11
N
N
N
N
O
N
O
N
O
H
b, c
a
H
N
H
N
O
N
S
O
O
O
S
O
O
S O
O
H
N
O
O
H
H
N
BocHN
N
BocHN
BocHN
N
N
H
O
O
O
27 (20%)
12
24 (11%)
N
N
O
N
O
O
N
O
N
O
O
O
O
O
S
S
H
N
a
d, c
O
O
O
O
NH
NH
S
O
O
H
N
H
H
O
N
BocHN
N
BocHN
BocHN
N
H
O
O
O
25 (28%)
28 (33%)
14
Scheme 5. Reagents: (a) Hoveyda–Grubbs 2nd generation catalyst, trifluorotoluene; (b) TFA, DCM; (c) HATU, Boc-
L
-tLeu, DIEA, DMF, rt; (d) 4.0 M HCl in dioxane.
N
N
O
N
O
O
N
O
17
b
O
a
O
HN
O
O
O
HN
O
BocHN
BocHN
N
H
N
H
31L n=1 (14%)
31D n=1 (12%)
32 n=0 (15%)
29L n=1 (11%)
29D n=1 (6%)
30 n=0 (3%)
NH
O
NH
O
n
n
S
O
S
O
Scheme 6. Reagents: (a) Hoveyda–Grubbs 2nd generation catalyst, trifluorotoluene, formic acid; (b) Mixed fractions from purification of 29, H2, Pd/C, THF.
H2 and palladium on charcoal in methanol. After reduction and
purification saturated inhibitors 31L, 31D and 32 were obtained
in a yield of 14%, 12% and 15% (over the last step), respectively.
formed beside the main product 33 which was obtained in a yield
of 68%. Ester hydrolysis of 33 followed by coupling with Boc depro-
tected 2 and 3 in the same inverse type of peptide synthesis as per-
formed with compound 21, resulted in P4–P2 macrocyclic
inhibitors 34 and 35 in a respective yield of 45% and 32%. The
decomposition of the P4–P2 building block that occurred for com-
pound 21 was not observed in this case. We therefore believe that
to avoid not only racemization, but also degradation, of the
The use of N-[(but-3-en-1-yloxy)carbonyl]-3-methyl-L-valine in
P3 also enabled P4–P2 macrocyclization. This cyclization is of the
same type as in vaniprevir.26 The ring-closure of 21 (Scheme 7)
went rather smooth and without precipitation, although trace
amounts of ring-contracted product and polymeric material were

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6601
Table 1
or to involve the N-terminal in a macrocycle which evidently pro-
tects the P4–P2 building-block from decomposition, but also from
racemization since compounds 34 and 35 were only obtained as
one diastereomer in trans conformation.
To evaluate the effectiveness of the new pyrimidine-based P2
substituent, the two reference analogues to compounds 19L and
Variations in 1H NMR shifts of H1–H4 of compound 17, as
a consequence of
purification method used
17 after silica purification
1H NMR d
17 after acidic HPLC purification
1H NMR d
Proton
H1
H2
H3
H4
8.45
7.40
7.07
8.14
8.17
7.58
7.25
7.73
19D containing a quinoline-based P2 substituent were synthesized
31
(Scheme 8). Coupling of compound 36
with the N-deprotected
HCl salt of 2, using HATU and DIEA in DCM and subsequent cou-
pling with P3 Boc- -tLeu and HPLC purification yielded compounds
L
38L and 38D in a yield of 10%, respectively.
3. Biochemical evaluation
N
O
N
O
The inhibitory effects (Ki-values) for compounds 12–19, 22, 25–
32, 34–35 and 38 were evaluated in a biochemical inhibition assay
using full length NS3 protein from genotype 1a and a sixteen
amino acid fragment, which corresponds to the activating region
of NS4A (Scheme 8, and Tables 2–6).52 Additionally, compound
29D was evaluated with the drug resistant A156T, D168V and
R155 K variants of the protease.33 Inhibition constants were mea-
sured and vitality values (V) calculated (Table 7). Vitality values
represent normalized inhibitory effects of the inhibitors with
respect to the effects from amino acid substitutions on catalytic
efficiency (kcat/Km) of the enzyme variants. A vitality value less
than 1 demonstrates a more efficient inhibitor against the mutated
variant compared to the wild type enzyme, while if V > 1 the inhib-
itor is less efficient against the mutated virus.33 A mixture of com-
pounds 19L and 19D, 38L and 38D and compound 29D were also
tested for EC50-values using the Huh-7 cell line containing subge-
nomic HCV RNA genotype 1b replicon with firefly luciferase result-
O
O
NH
S
O
O
H
N
BocHN
H4
N
H
17
O
H1
H3
H2
Figure 4. An internal hydrogen bond in compound 17 influencing the 1H NMR
shifts of H1–H4.
ing EC50 > 10
lM (19L and 19D), EC50 = 5.4 lM (38L and 38D) and
EC50 = 15
l
M (29D).53
4. In silico and in vitro physicochemical and pharmacokinetic
profiling
A few representative compounds (i.e., 13, 15, 17, 18, 19, 29L and
29D) with good inhibition potencies were selected for in silico and
in vitro physicochemical and pharmacokinetic profiling. The pre-
dicted pKa and logD7.4 values for compounds 13, 15, 17, 18, 19,
29L and 29D were calculated using ADMET predictor v.5.5 and
are presented in Table 8. The calculated pKa-values span between
5.8 and 6.2 for the acidic acylsulfonamide group in the inhibitors
and the corresponding logD7.4-values between 3.5 and 4.8. From
these logD7.4 values low solubility, high permeability and a moder-
ate to high metabolism would be expected.54
All compounds (13, 15, 17, 18, 19, 29L and 29D) showed a mod-
erate (>20
to more than 100
pounds 13 and 15 having solubilities close to or above 100
l
M) to good (>100
M; the latter exemplified by the truncated com-
M as
lM) solubility ranging from 33 lM
l
l
this concentration is the limit of the assay. Metabolic stability
was determined by incubating the compounds with pooled human
liver microsomes. The in vitro half-life (t1/2) and the in vitro intrin-
sic clearance (Clint) were calculated utilizing previously published
models55,56 and the results are presented in Table 8. The Clint pri-
marily describes the compound sensitivity towards oxidative
metabolism and can be used as a prediction of the in vivo Clint if
the total clearance mechanism is mainly hepatic and when oxida-
tive metabolism dominates. The cut-off values that were used to
classify the compounds regarding metabolic stability are as fol-
Figure 5. LC-UV chromatograms (254 nm) on crude products after ring-closure of
17 showing product (pro), dimers (di) and trimers (tri). Above: 17, Hoveyda–Grubbs
2nd generation catalyst, trifluorotoluene, 100 °C, 5 min. Below: 17, Hoveyda–
Grubbs 2nd generation catalyst, formic acid, trifluorotoluene, 100 °C, 5 min.
lows: Clint < 47 (ll/min/mg) indicates a low risk for high first
metabolism in vivo, 47 < Clint < 92 a moderate risk, and Clint > 92
a high risk. All the linear inhibitors 13, 15, 17 and 18 showed a
moderate risk of oxidative metabolism (Clin = 60–86 lL/min/mg),
phenylglycine-containing fragment it is necessary to keep the
Boc-protecting group as long as possible throughout the synthesis,

6602
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
N
N
O
H
N
O
O
O
S
H
N
N
O
O
NH
N
O
N
H
N
O
N
O
34 (45%)
2
O
b, c, d
a
O
O
O
O
O
3
H
N
N
O
NH
O
N
H
N
O
N
H
b, c, e
O
O
O
33 (68%)
21
O
O
O
O
O
H
NH
N
S
O
N
N
H
H
O
35 (32%)
Scheme 7. Reagents: (a) Hoveyda–Grubbs 2nd generation catalyst, trifluorotoluene; (b) K2CO3, MeCN, H2O (c) 4.0 M HCl in dioxane; (d) HATU, DIEA, DCM, 45 °C; (e) HATU,
DIEA, DMF, rt.
O
O
O
N
N
N
O
BocHN
O
O
O
OH
2
H
N
H
N
O
O
S
O
S
a, b
a, c
H
H
O
O
O
O
OH
N
BocHN
N
BocHN
BocHN
N
H
O
O
O
36
37 (50%)
38L L-Phg Ki = 290 nM (10%)
38D D-Phg Ki = 120 nM (10%)
Scheme 8. Reagents: (a) 4.0 M HCl in dioxane; (b) HATU, DIEA, DCM, 45 °C; (c) HATU, DIEA, DMF, rt.
except for compound 19 (Clin = 17
clic compounds 29L and 29D proved to be moderate to highly sus-
ceptible for oxidative metabolism (Clin = 87 and 137 L/min/mg,
lL/min/mg). The two macrocy-
5. Discussion
l
In an attempt to further depeptidize, optimize, and attain infor-
mation about the binding-mode of P2 phenylglycine-based HCV
NS3/4A protease inhibitors, we decided to combine a few previ-
ously discovered promising fragments for this type of inhibitors
such as alkenylic P10 elongations and aromatic P1 moieties.31,61
Furthermore, the synthesis of the P2 building block was simplified
and the reaction time was considerably shortened by introduction
of a pyrimidine-based P2 substituent as compared to the previ-
ously used quinoline (see Fig. 2). Through the possibility of intro-
ducing vinyls on both the P2 phenylglycine part itself and on its
pyrimidine substituent, and utilizing alkenylic substituents at the
P3 and P10 positions, macrocyclization could be performed
between several positions in the molecules, which eventually
opened up for an initial probing of the impact of different types
of macrocycles in these inhibitors. Molecular modeling especially
invited to some of the attempted macrocylizations. For example,
modeling of compound 34 overlaid with vaniprevir showed that
34 occupies the same space, except for the phenyl substituent on
the pyrimidine which sticks out into a pocket formed by the heli-
case. Though, both macrocycles show a good overlap and form
the same backbone interactions with the protein (Fig. 6). Thus, this
model motivated us to explore the P4–P2 cyclization. Modeling of
compound 17 furthermore indicated a proximity between the
respectively). Prediction of the intestinal epithelial permeability,
presented in Table 8, expressed as apparent permeability coeffi-
cients (Papp), for compounds 13, 15, 17, 18, 19 , 29L and 29D, were
determined from transport rates across Caco-2 cell monolayers, as
described previously.57 However, a modified assay based on more
physiological media had to be devised because all tested com-
pounds displayed very strong adsorption to the plastic ware.
Briefly, to minimize the nonspecific binding, fasted state simulated
intestinal fluid (FASSIF)58 was used in the apical (donor) compart-
ment and a 1% (w/v) bovine serum albumin (BSA) solution of
Hank’s Buffered Salt Solution (HBSS)59 was used in the basolateral
(receiver) compartment. Using this format, the experiments could
be run with reasonable mass balances above 64%. In our calibrated
assay setup, a Papp value below 0.2 ꢀ 10ꢁ6 cm/s indicates low per-
meability, a Papp value between 0.2 ꢀ 10ꢁ6 cm/s to 1.6 ꢀ 10ꢁ6 cm/s
indicates moderate permeability, and a Papp value above 1.6 ꢀ 10ꢁ6
cm/s indicates high permeability.60 The truncated linear P2–P10
spanning inhibitor 13 penetrated the intestinal epithelial cells very
well (Papp = 4.0 ꢀ 10ꢁ6 cm/s). The remaining compounds had mod-
erate to low permeability, for example, the macrocycles 29L and
29D displayed Papp-values of 0.22 ꢀ 10ꢁ6 and 0.15 ꢀ 10ꢁ6 cm/s,
respectively.

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6603
Table 2
Inhibitory potencies (Ki) of linear inhibitors measured with full-length NS3/4A protein from genotype 1a
N
N
O
R3
R2
O
O
O
S
H
N
R1
N
H
P1
N
n
H
O
Compound
R1
R2
R3
P1
n
Ki SD (lM)
12
Boc
H
H
1
0.22 0.022
0.60 0.14
O
O
O
13
Boc
1
0.37 0.06
0.19 0.02
14
15
Boc
Boc
0
1
O
16
17
O
O
H
1
1
0.11 0.011
H
N
O
BocHN
O
H
H
0.23 0.03
O
O
O
BocHN
BocHN
BocHN
18a
19L
19D
1
1
1
0.095 0.013
0.026 0.0025
0.033 0.0033
O
O
O
O
22L
O
H
H
H
1
1
0.53 0.09
N
O
O
22D
O
H
0.42 0.06
N
O
SD, standard deviation. aDiasteromeric mixture L:D 5:2.
alkenylic P3 and P10 substituents, with a distance that invites to
macrocyclization. Modeling of 29L shows that a macrocycle con-
necting P3 and P10 can be accommodated by the protease and that
it forms a distinct hydrophobic interaction with the side chain of
Lys136 (Fig. 7).
The synthetic routes were designed to enable development of
linear inhibitors as well as macrocyclic inhibitors connecting the
P4–P2, P3–P10 and P2–P10 positions (Fig. 3). Our previous attempts
of macrocyclizations using ring closing metathesis reactions (RCM)
have proven to be highly substrate dependent. Thus, difficult

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A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
Table 3
Table 5
Inhibitory potencies (Ki) of P2–P10 macrocycles measured with full-length NS3/4A
protein from genotype 1a
Inhibitory potencies (Ki) of P3–P10 macrocyclic inhibitors measured with full-length
NS3/4A protein from genotype 1a
N
N
N
O
N
O
O
S
O
O
O
O
HN
P1
H
N
BocHN
R
N
H
N
H
O
HN
O
O
NH
O
n=0,1
S
O
Compound
R
P1
Ring size
Ki SD (lM)
n
O
BocHN
26
20
21
2.4 0.3
Compd
Ring size
Ki SD (lM)
29L
29D
30
31L
31D
32
21
21
20
0.11 0.02
0.08 0.009
0.22 0.048
0.11 0.01
0.11 0.01
0.13 0.02
O
BocHN
27
0.66 0.22
21 saturated
21 saturated
20 saturated
SD, standard deviation.
SD, standard deviation.
Table 4
Table 6
Inhibitory potencies (Ki) of P2–P10 macrocyclic inhibitors measured with full-length
NS3/4A protein from genotype 1a
Inhibitory potencies (Ki) of P4–P2 macrocyclic inhibitors measured with full-length
NS3/4A protein from genotype 1a
N
N
N
O
N
O
O
19-ring
O
O
O
S
NH
O
H
N
O
R
H
N
O
N
H
R1
O
N
H
O
Compound
R
Ki SD (lM)
Compound
R1
Ring size
15
Ki SD (lM)
H
25
Boc
1.3 0.27
O
N
S
S
O
H
N
34
0.39 0.08
1.57 0.29
O
O
BocHN
28
15
0.63 0.14
O
O
O
H
N
SD, standard deviation.
N
35
H
substrates without a preorganized conformation that facilitates
contact between the two vinyls, can be associated with the forma-
tion of side products such as the ones resulting from ring contrac-
tion and isomerization.32 In this study another complicated factor
arose through the introduction of a rigid ortho acylanilide type of
P1–P10 buildning block that retains an internal hydrogen bond.
RCM between the P1–P10 building block and the vinyl in P3 posi-
tion was accompanied with side reactions and polymerization
issues in particular. However, the outcome was found to be dra-
matically improved by the addition of acid that interrupted the
internal hydrogen bond and thus improved flexibility of the mole-
cule. In this context it could be worth mentioning that Grubbs and
coworkers have observed that presence of acetic acid also could
prevent undesirable isomerization during olefin metathesis.62
The inhibitory potencies of the linear and cyclic inhibitors pro-
duced in this study were evaluated and found to be in the range of
SD, standard deviation.
26 nM to 1.6 lM. The inhibitory potencies of compounds 19L and
19D (Ki = 26 nM, and 33 nM), comprising the pyrimidine-based
P2-substituent and aromatic P1 moiety, revealed that the new
2-phenyl-4-methoxy-pyrimidinyloxy P2 substituent was tolerated
and led to more potent inhibitors compared to compounds 38L and
38D (Ki = 290 nM and 120 nM, Scheme 8), containing the quino-
line-based P2 substituent. This improvement in inhibitory activity
and the considerably simplified synthesis merit further use of the
pyrimidine-based P2 substituent.
The combination of a P2 substituent based on pyrimidine with an
aromatic P1 moiety was also revealed to be well-tolerated by the
comparison of quinoline-substituted compound B with a norvaline

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6605
Table 7
Inhibition constants (Ki) and vitality values (V) evaluated with A156T, D168V, and
R155K variants of full-length NS3/4A
Compound
29D
A156T
Ki SD (nM)
160
D168V
R155K
V
Ki SD (nM)
V
Ki SD (nM)
V
2.2 600
3.0 220
0.50
SD, standard deviation.
V, vitality values.33
in the P1 position (Ki = 63 nM), to compound 19L (Ki = 26 nM). Thus,
the choice of an aromatic P1 fragment seems as a promising alterna-
tive to norvaline. This was further strengthened when comparing
the linear compounds, with a 2-phenyl-4-vinyl-pyrimidinyloxy P2
substituent and a P3 N-[(but-3-en-1-yloxy)carbonyl]-3-methyl-L-
valine. Compound 16 (Ki = 110 nM), containing the aromatic P1
moiety is almost five times as potent as the norvaline containing
compound 22L (Ki = 530 nM). The differences between an aromatic
P1 group and norvaline in P1 are also shown in the macrocyclic com-
pounds. When comparing the P2–P10 macrocyclic compound 27
(Ki = 660 nM), with an aromatic P1 residue to the norvaline
containing analogue 26 (Ki = 2400 nM), a fourfold improvement in
inhibitory potency is shown for the compound with an aromatic
P1. The same fourfold difference is seen when comparing the
P4–P2 macrocyclic compound 34 (Ki = 390 nM) containing an
aromatic P1 with the norvaline analogue 35 (Ki = 1570 nM).
Figure 6. Overlay of compound 34 (green) and Vaniprevir (yellow) docked in the
full-length NS3 protease binding site of the 1CU1 crystal structure.14 The protease
domain is shown in grey, the NS3 helicase domain is shown in beige.
A beneficial
p–p interaction between the vinyl substituent on
the phenylglycine and the catalytic H57 has previously been pro-
posed,31,32 and this hypothesis is further strengthened herein.
Thus, when comparing the P2 2-phenyl-4-methoxy-pyrimidinyl-
oxy substituted P2–P10spanning compounds 13 (Ki = 600 nM) and
15 (Ki = 190 nM), a threefold preference is seen for compound 15
that has a vinyl substituent on the phenylglycine ring. The same
trend is seen when comparing the longer P3-P10 spanning equiva-
lents 18 (Ki = 95 nM) and 19L (Ki = 26 nM), that yet again shows a
threefold advantage for vinylated compound 19L. An obvious drop
in potency comes with the P2–P10 ring-closure of the P2–P10span-
ning compound 14 (Ki = 370 nM) into macrocyclic 25 (Ki = 1300
nM) as well as for the longer P3-P10spanning compound 19L
(Ki = 26 nM) and macrocyclic 28 (Ki = 630 nM). Thus, constraining
the P2 vinyl and the alkenylic P10 results in a less efficient inhibi-
tors, which is in line with our previous observations.32
Figure 7. Overlay of acyclic compounds 17 (pink) and 29L (yellow) docked in the
full-length NS3 protease binding site of the 1CU1 crystal structure.14 The protease
domain is shown in grey, the NS3 helicase domain is shown in beige.
Another example of reduction in inhibitory activity after the
alkenylic P10 is constrained is seen for the linear P2–P10 spanning
compound 12 (Ki = 220 nM) with a 2-phenyl-4-vinyl-pyrimidinyl-
oxy P2 substituent. After P2–P10 macrocyclization, and now with
the vinyl on the pyrimidine ring, and further coupling with P3
From modeling (Fig. 6), the macrocyclization connecting the P3
with the P2 substituent seemed very promising in line with vani-
previr. The biochemical evaluation was thus disappointing. Never-
theless, the P4–P2 macrocyclic inhibitors 34 (Ki = 390 nM), with an
aromatic P1 residue, and 35 (Ki = 1570 nM), with norvaline in P1
position, showed a slightly better inhibitory potency than the
corresponding P2-P10 macrocyclic inhibitors 27 (Ki = 660 nM),
Boc-L
-tLeu, the P3–P10 spanning P2–P10 macrocyclic compound
27 (Ki = 660 nM), shows a threefold drop in potency, even though
it holds a P3 substituent.
Table 8
Measured solubility, metabolic stability in human microsomes, and Caco-2 permeability of compounds 13, 15, 17, 18, 19, 29L and 29D, and calculated acidic pKa values (acyl
sulfonamides), and logD7.4-values
In vitro
In silico
Metabolic stability
L/min/mg)
td1/2 (min)
Compd
Solubility (
l
M) pH 7.4
Caco-2 Permeability Papp (10ꢁ6 cm/s)a a-bb
Clcint
(l
pKa
LogD7.4
13
15
17
18
93
101
91
33
66
51
51
4.0
86
66
60
64
17
87
137
16
21
23
22
83
16
10
5.8
5.8
6.0
6.0
6.0
6.2
6.2
3.5
4.0
4.8
4.3
4.7
4.0
4.0
1.0 0.7
0.22 0.2
0.22 0.04
1.7
0.35 0.12
0.15 0.11
19e
29L
29D
d
aPapp = apparent permeability coefficient. ba–b = apical to basolateral. cClint = in vitro clearance. t1/2 = in vitro half-life. eA mixture of 19L and 19D.

6606
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
and 26 (Ki = 2400 nM). Again, the impairment caused by straining
the alkenylic P10 is shown.
inhibitory activities, which stresses the importance of having a flex-
ible P2 moiety in this type of inhibitors. On the other hand, P10 to P3
macrocyclization were allowed in terms of inhibitory potency on
the wildtype and drug resistant strains of the enzyme, although fur-
ther optimizations of this class of inhibitors are necessary to
improve apparent intestinal permeability and metabolic stability.
It is further shown herein that the RCM reactions which are ham-
pered by internal hydrogen bonds could be made feasible by the
addition of formic acid.
As discussed above the P10–P3 cyclization, which is overall poorly
explored in HCV NS3/4A protease inhibitors, looked advantageous
for the phenylglycine-based inhibitor 17when it comes to molecular
modelling (Fig. 7). Actually, biochemical evaluations of the formed
macrocyclic products, that is, 29L (Ki = 110 nM), 29D (Ki = 80 nM),
and ring contracted product 30 (Ki = 220 nM), reveal the first exam-
ples in this study of where ring-closure is slightly favored over the
linear analogue 17 (Ki = 230 nM). 29D was also found to retain
decent potency on the A156T (Ki = 160 nM, V = 2.2) and D168V
(Ki = 600 nM, V = 3.0) and R155 K (Ki = 220 nM, V = 0.50) mutants.
The vitality value of 0.5 for the R155K variant; that is considered
as one of the most serious drug resistant enzyme variants arising
after treatment with DAAs,10,63 is particularly worthy to note.
A driving force for the development of macrocycles in drug devel-
opment are, besides a possible preorganization of bioactive confor-
mation with positive entropic effect for the binding efficiency as a
result, the pharmacokinetic advantages such as increased cell per-
meability and metabolic stability often associated with such com-
pounds. Thus, to support further exploration of compounds
derived from the promising macrocycles 29L/D we were eager to
see its in vitro pharmacokinetic characteristics. Unfortunately, both
macrocycles proved to have poor pharmacokinetic properties as
shown by high metabolic susceptibility and poor intestinal perme-
ability (Table 8), which partly can explain the inhibitory efficiency
7. Experimental section
7.1. Chemistry
Reagents and solvents were obtained commercially and used
without further purification. Thin layer chromatography (TLC)
was performed on aluminium sheets precoated with silica gel 60
F254 (0.2 mm). Chromatographic spots were visualized using
UV-detection and/or 2% ninhydrin in ethanol solution followed
by heating. Column chromatography was performed using silica
gel 60 (40–63 lM). Analytical HPLC–MS was performed on a
Gilson-Finnigan ThermoQuest AQA system equipped with a C18
(Onyx Monolithic C18 (50 ꢀ 4.6 mm)) or a C4 (Hichrom ACE C4
(5
lm, 50 ꢀ 4.6 mm)) column using MeCN/H2O (0.05% HCOOH)
or MeOH/H2O (0.05% HCOOH) with UV (254 nM) and MS (ESI)
detection or on a Dionex UltiMate 3000 equipped with a C18 (Phe-
nomenex Kinetex C18 (50 ꢀ 3.00 mm) column using MeCN/H2O
(0.05% HCOOH) UV (254 ) and MS (ESI IonTrap) detection or on a
manual system equipped with a C8 (Zorbax SB-C8 (4.8 ꢀ 50
mm)) column using UV (220 nm) detection. Preparative HPLC
was performed on a Gilson-Finigan ThermoQuest AQA system
drop to 15 lM (EC50-value) for 29D in the replicon assay.
In regard to the overall macrocyclic scan performed in this
study it appears that macrocyclizations involving either the P2
substituent or the P2 phenylglycine part are damaging for inhibi-
tory activity, and that P10 to P3 macrocyclizations seem most
promising to pursue in terms of inhibitory potency on wildtype
and drug resistant strains of the enzyme. Thus, the importance of
the retaining a flexible P2 side-chain in this type of inhibitors is
established. The importance of flexible P2 substituents in retaining
inhibitory potency for drug resistance enzyme variants has
recently been proposed.11
equipped with a C8 (Zorbax SB-C8 (5
using MeCN/H2O (0.05% HCOOH) as the mobile phase with UV
l
m, 150 ꢀ 21.2 mm)) column
(254 nm) detection or on a manual system equipped with a C8
(Zorbax SB-C8 (5
lm, 150 ꢀ 21.2 mm)) column using UV detection
and MeCN/H2O (0.05% HCOOH), MeCN/H2O (0.1% TFA), or MeCN/
H2O with 25 mM NH4OAc, pH 6,3 as the mobile phase. Purity
determinations were done by RP-HPLC using the following condi-
tions (UV detection at 254 nm): System 1 (ACE 5 C8 A3071,
50 ꢀ 4.6 mm, MeCN/H2O with 0.1% TFA) and system 2 (Restec
Allure Biphenyl, 50 ꢀ 4.6 mm, MeCN/H2O with 0.1% TFA). Micro-
wave reactions were carried out in a SmithSynthesizer™ or in an
Initiator™ single-mode microwave cavity producing controlled
irradiation 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 tetramethylsilane (TMS) via the solvent
signal (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.
What is particularly noteworthy in this study was that the
intermediate compounds in synthesis, that is, the linear P2–P10
spanning compounds with aromatic P1 residues, that is, 12
(Ki = 220 nM), 13 (Ki = 600 nM), 14 (Ki = 370 nM), and 15 (Ki = 190 -
nM) retained impressive inhibitory potency without a P3 building
block. Considering the smaller size combined with the good solu-
bility and somewhat better intestinal permeability of compound
13 (solubility = 93
l
M, Papp = 4.0 ꢀ 10ꢁ6 cm/s) and 15 (solubil-
ity = 101
l
M, Papp = 1.0 ꢀ 10ꢁ6 cm/s) it seemed worthwhile to
further explore the P2–P10 spanning inhibitors with the 2-phe-
nyl-4-methoxy-pyrimidinyloxy P2 substituent, a vinylated phenyl-
glycine in P2, an aromatic P1 and alkenylic elongation in P10.64
6. Conclusion
7.1.1. Compound 1 (tert-butyl (2-((but-3-en-1-
In summary, access to vinylated P3, P2 and P1 building blocks
combined with a careful synthetic planning allowed preparation
of a diverse set of RCM produced macrocycle-based HCV NS3/4A
protease inhibitors and their corresponding linear analogues. Bio-
chemical results proved that it is beneficial to substitute the P2
phenylglycine with p-phenylpyrimidinyloxy and m-vinyl groups.
The inhibitors were further successfully combined with an aromatic
P1 residue with alkenylic P10 elongations, which eventually led to
the discovery of truncated P2–P10 based inhibitors with promising
inhibitory potencies and in vitro pharmacokinetic properties.
Probing of various macrocycles in P2 phenylglycine based inhibitors
revealed that macrocyclizations involving either the P2 phenylgly-
cine part or its p-pyrimidine substituent are detrimental for the
ylsulfonyl)carbamoyl)phenyl)carbamate)
Boc-Abz-OH (522 mg, 2.2 mmol) was dried in vacuo overnight
over P2O5. Boc-Abz-OH and CDI (714 mg, 4.4 mmol) were dissolved
in dry THF (40 mL) under N2 atmosphere. The solution was stirred at
66 °C for 2 h before but-3-ene-1-sulfonamide (595 mg, 4.4 mmol),
prepared as described earlier,39 dissolved in dry THF (4 mL) was
added together with DBU (991 lL, 6.6 mmol) under N2 atmosphere.
The reaction was then stirred for 2 h in r.t. THF was evaporated and
DCM (40 mL) was added. The solution was washed with 5% citric
acid and brine before the organic phase was evaporated. The product
was purified on silica gel (CH2Cl2/MeOH 93:7) yielding 1 (464 mg,
59%) as a white solid. 1H NMR (CDCl3) d: 8.44 (dd, J = 1.2, 8.6 Hz,
1H), 7.58–7.51 (m, 2H), 7.05 (ddd, J = 1.2, 7.2, 8.1 Hz, 1H), 5.82 (m,

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6607
1H), 5.21–5.10 (m, 2H), 3.70–3.65 (m, 2H), 2.69–2.62 (m, 2H), 1.52 (s,
9H). 13C NMR (CDCl3/CD3OD 50:50) d: 169.0, 153.2, 141.4, 134.7,
133.7, 129.1, 121.8, 120.1, 117.6, 117.1, 81.2, 52.7, 28.3, 27.5. MS
calcd for C16H22N2O5S [M+H]+ 355.1, found: 355.1.
129.6, 129.5, 122.8, 106.4, 80.9, 58.9, 28.7. MS calcd for C23H22ClN3-
O5 [M+H]+ 455.1, 457.1, found: 455.8, 457.8.
7.1.8. Compound 7 ((S)-2-((tert-butoxycarbonyl)amino)-2-(4-
((2-phenyl-6-vinylpyrimidin-4-yl)oxy)phenyl)acetic acid)
Compound 6 (402 mg, 0.9 mmol), 2,4,6-trivinylcyclotriborox-
ane pyridine complex (432 mg, 1.8 mmol), Pd(OAc)2 (21.6 mg,
0.09 mmol), [(tBu)3PH]BF4 (52.2 mg, 0.18 mmol), K2CO3 (744 mg,
5.4 mmol), H2O (5.4 mL), and DME (18 mL) were mixed and
divided into six 5 mL microwave process vials. The vials were
sealed and exposed to microwave heating at 100 °C for 15 min.
After filtration, the reaction mixtures were pooled and H2O
(55 mL) was added. 1 M NaOH was added until the pH was 10.
The aqueous phase was washed with Et2O and acidified with 2 M
HCl to pH 2.85. Filtration of the formed precipitate yielded 7
(333 mg, 83%) as a white solid. 1H NMR (CD3OD): d 8.27–8.24 (m,
2H), 7.53 (d, J = 8.5 Hz, 2H), 7.46–7.36 (m, 3H), 7.25 (d, J = 8.5 Hz,
2H), 6.87–6.79 (m, 2H), 6.61 (dd, J = 1.5, 17.3 Hz, 1H), 5.70 (dd,
J = 1.5, 10.5 Hz, 1H), 5.27 (s, 1H), 1.47 (s, 9H). 13C NMR (CD3OD):
d 173.0, 170.8, 165.1, 164.2, 156.3, 152.8, 137.2, 135.1, 135.0,
130.8, 128.8, 128.2 (2 carbons), 122.0, 121.7, 102.6, 79.7, 57.6,
27.5. MS calcd for C25H25N3O5 [M+H]+ 448.2, found: 447.9.
7.1.2. Compound 2 (tert-butyl(2-((pent-4-en-1-
ylsulfonyl)carbamoyl)phenyl)carbamate)
Boc-Abz-OH (522 mg, 2.2 mmol) was dried in vacuo overnight
over P2O5. Boc-Abz-OH and CDI (714 mg, 4.4 mmol) were dissolved
in dry THF (40 mL) under N2 atmosphere. The solution was stirred
at 66 °C for 2.5 h before pent-4-ene-1-sulfonamide (657 mg,
4.4 mmol), prepared as described earlier,39 dissolved in dry THF
(4 mL) was added together with DBU (991 lL, 6.6 mmol) under
N2 atmosphere. The reaction was then stirred for 4 h. THF was
evaporated and DCM (40 mL) was added. The solution was washed
with 5% citric acid and brine before the organic phase was evapo-
rated. The product was purified on silica gel (CH2Cl2/MeOH 93:7)
followed by aluminium oxide (CH2Cl2/MeOH 93:7 then CH2Cl2/
MeOH/HCOOH 95:5:1) yielding 2 (648 mg, 80%) as a white solid.
1H NMR (CDCl3) d: 9.82 (s, 1H), 8.41 (dd, J = 1.2, 9.2 Hz, 1H),
7.56–7.51 (m, 2H), 5.80–5.69 (m, 2H), 5.11–5.04 (m, 2H), 3.60–
3.55 (m, 2H), 2.27–2.20 (m, 2H), 2.04–1.95 (m, 2H), 1.52 (s, 9H).
13C NMR (CDCl3) d: 167.7, 153.0, 142.1, 136.1, 135.2, 128.0,
121.8, 120.6, 117.0, 116.0, 81.2, 53.2, 31.9, 28.4, 22.5. MS calcd
for C17H24N2O5S [M+H]+, 369.1, found: 369.2.
7.1.9. Compound 8 ((S)-2-((tert-butoxycarbonyl)amino)-2-(4-
((6-methoxy-2-phenylpyrimidin-4-yl)oxy)phenyl)acetic acid)
Compound 4 (962 mg, 3.6 mmol), 4-chloro-6-methoxy-2-phe-
nylpyrimidine (396 mg, 1.8 mmol) and KOtBu (806 mg, 7.2 mmol)
were dried overnight in vacuo over P2O5. The amounts of 4 and
KOtBu were divided into two reaction vials. Dry DMSO (H2O
<0.005%) (4 mL) was added to each reaction vial containing 4 and
KOtBu. The vials were sealed under nitrogen and the reaction mix-
ture stirred for 40 min before 4-chloro-6-methoxy-2-phenylpyrim-
idine dissolved in DMSO (5 mL) was added to each vial. The
reactions were heated at 64 °C overnight. The reactions were
pooled, filtered and diluted with water (150 mL) before extraction
with ethyl acetate. After evaporation of the organic phase and puri-
fication on silica gel (CH2Cl2/MeOH 90:10, then CH2Cl2/MeOH/
AcOH 95:5:3) 8 (596 mg, 70%) was obtained as a white solid. 1H
NMR (CD3OD): d 8.26–8.24 (m, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.45
(m, 1H), 7.42–7.37 (m, 2H), 7.21–7.18 (m, 2H), 6.06 (s, 1H), 4.86
(s, 1H), 4.06 (s, 3H), 1.46 (s, 9H). 13C NMR (CD3OD): d 174.1,
173.7, 172.6, 165.2, 157.5, 154.3, 138.1, 136.0, 132.1, 130.0, 129.3
(2 carbons), 122.8, 90.0, 81.0, 58.7, 54.7, 28.7. MS calcd for
7.1.3. Compound 3 ((S)-tert-butyl (1-oxo-1-(pent-4-en-1-
ylsulfonamido)pentan-2-yl)carbamate)
Prepared as described by us previously.31
7.1.4. 4-Chloro-6-methoxy-2-phenylpyrimidine
4,6-Dichloro-2-phenylpyrimidine (5.0 g, 22.2 mmol) and
NaOCH3 (3.6 g, 66.6 mmol) was stirred in methanol (500 mL) on
an ice bath for 1 h, then brought to reflux for 7 h. After evaporation,
the white solid was dissolved in DCM (100 mL) and washed with
water. Evaporation of the organic phase yielded 8 (3.98 g, 81%) as
a white solid. 1H NMR (CD3OD): d 8.39–8.36 (m, 2H), 7.53–7.44
(m, 3H), 6.76 (s, 1H), 4.08 (s, 3H). 13C NMR (CD3OD): d 172.3,
165.9, 162.2, 137.4, 132.6, 129.5, 129.5, 106.0, 55.0. MS calcd for
C
11H9ClN2O [M+H]+ 221.0, found: 221.0.
7.1.5. Compound 4 ((S)-2-((tert-butoxycarbonyl)amino)-2-(4-
hydroxyphenyl)acetic acid)
Prepared as described by us previously.30
C
24H25N3O6 [M+H]+ 452.1, found: 452.1.
7.1.6. Compound 5 ((S)-2-(3-bromo-4-hydroxyphenyl)-2-((tert-
butoxycarbonyl)amino)acetic acid)
7.1.10. Compound 9 ((S)-2-(3-bromo-4-((6-methoxy-2-
phenylpyrimidin-4-yl)oxy)phenyl)-2-((tert-
Prepared as described by us previously.31
butoxycarbonyl)amino)acetic acid)
7.1.7. Compound 6 ((S)-2-((tert-butoxycarbonyl)amino)-2-(4-
((6-chloro-2-phenylpyrimidin-4-yl)oxy)phenyl)acetic acid)
Compound 4 (535 mg, 2.0 mmol), commercially available fenclo-
rim (900 mg, 4.0 mmol) and KOtBu (449 mg, 4.0 mmol) were dried
overnight in vacuo over P2O5. Dry DMSO (8 mL) was added to the
sealed reaction vial containing KOtBu under N2 atmosphere. Com-
pound 4, dissolved in DMSO (6 mL) was added to the reaction vial,
and the reaction mixture was stirred for 30 min. Fenclorim, dis-
solved in DMSO (12 mL) was added to the reaction mixture and stir-
red at rt for 4 h. Water (150 mL) was added and pH adjusted to 11
with 2 M KOH. The mixture was washed with Et2O before the aque-
ous phase was acidified using 1 M HCl to pH 2.75 so a precipitate was
formed. Filtration of the precipitate yielded 6 (833 mg, 91%) as a
white solid. 1H NMR (CD3OD): d 8.20–8.16 (m, 2H), 7.55 (d,
J = 8.7 Hz, 2H), 7.47 (m, 1H), 7.41–7.36 (m, 2H), 7.25 (d, J = 8.7 Hz,
2H), 6.94 (s, 1H), 5.27 (s, 1H), 1.47 (s, 9H). 13C NMR (CD3OD): d
174.2, 171.9, 166.1, 163.4, 157.5, 153.6, 137.0, 136.8, 132.8, 130.0,
Compound 5 (2.0 g, 5.7 mmol), 4-chloro-6-methoxy-2-phenyl-
pyrimidine (1.9 g, 8.6 mmol) and KOtBu (1.5 g, 2.2 mmol) were
dried overnight in vacuo over P2O5. Dry DMSO (H2O <0.005%)
(40 mL) was added to a vial containing 5 and KOtBu. The vial was
sealed under nitrogen and stirred for 40 min before 4-chloro-6-
methoxy-2-phenylpyrimidine dissolved in DMSO (40 mL) was
added. The reaction was heated at 64 °C for 2 days. The reaction mix-
ture was filtered, diluted with water (500 mL), and extracted with
petroleum ether. The aqueous phase was acidified with 1 M HCl to
pH 3. Filtration of the precipitate yielded 9 (2.6 g, 87%) as a white
solid. 1H NMR (CD3OD) d 8.17 (m, 2H), 7.78 (d, J = 2.2 Hz, 1H), 7.51
(dd, J = 2.1, 8.3 Hz, 1H), 7.42 (m, 1H), 7.38–7.33 (m, 2H), 7.29 (d,
J = 8.4 Hz, 1H), 6.14 (s, 1H), 5.28 (s, 1H), 4.07 (s, 3H), 1.47 (s, 9H).
13C NMR (CDCl3) d: 172.8, 172.3, 170.4, 163.9, 156.9, 150.1, 137.3,
136.9, 132.4, 131.1, 128.5, 128.4, 127.3, 124.1, 116.7, 89.1, 82.4,
58.1, 54.3, 28.3. MS calcd for C24H24BrN3O6 [M+H]+: 530.1, 532.1,
found: 530.1, 532.1.

6608
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
7.1.11. Compound 10 ((S)-2-((tert-butoxycarbonyl)amino)-2-(4-
((6-methoxy-2-phenylpyrimidin-4-yl)oxy)-3-vinylphenyl)acetic
acid)
45 °C for 2 h. Addition of DCM before workup and purification on sil-
ica gel (CH2Cl2/MeOH 93:7), gave 12 (67 mg, 96%) as a whitesolid. 1H
NMR (CD3OD):d 8.48 (m, 1H), 8.22–8.18 (m, 2H), 8.11 (m, 1H), 7.70
(d, J = 8.6 Hz, 2H), 7.44–7.35 (m, 2H), 7.31–7.21 (m, 4H), 7.08 (ddd,
J = 1.2, 7.3, 8.0 Hz, 1H), 6.82 (s, 1H), 6.81 (dd, J = 10.5, 17.3 Hz, 1H),
5.75–5.63 (m, 2H), 5.34 (s, 1H), 4.98–4.85 (m, 2H), 3.31–3.24 (m,
2H), 2.12–2.04 (m, 2H), 1.91–1.82 (m, 2H), 1.48 (s, 9H). 13C NMR
(CD3OD): d 173.8, 172.0, 171.3, 166.3, 165.3, 157.5, 154.1, 140.7,
138.5, 138.3, 136.3, 136.1, 133.2, 133.9, (2 carbons), 130.3, 129.4,
129.3, 125.0, 124.0, 123.2, 123.1, 121.2, 116.1, 103.7, 81.1, 61.8,
53.0, 33.5, 28.8, 24.3. MS calcd for C37H39N5O7S [M+H]+ 698.3, found:
698.3. HPLC purity (system 1: 96.2%, system 2: 97.9%).
Compound 9 (705.3 mg, 1.33 mmol), 2,4,6-trivinylcycloborox-
ane pyridine complex (591.5 mg, 2.66 mmol), Pd(OAc)2 (32.2 mg,
0.133 mmol), [(tBu)3PH]BF4 (77 mg, 0.266 mmol), K2CO3 (1.11 g,
8.1 mmol), DME (26.6 mL) and water (8 mL) were mixed in seven
5 mL microwave vials. The vials were sealed and exposed to micro-
wave heating at 100 °C for 15 min. After centrifugal filtration, and
pooling of the reaction mixtures, the DME was evaporated. Water
(35 mL) was added and the reaction mixture was acidified with
5% HCl to pH 1. Filtration of the precipitate yielded 10 (518 mg,
82%) as a white solid. 1H NMR (CD3OD) d: 8.26–8.22 (m, 2H),
7.78 (d, J = 2.3 Hz, 1H), 7.47–7.36 (m, 4H), 7.15 (d, J = 8.3 Hz, 1H),
6.79 (dd, J = 11.4, 17.4 Hz, 1H), 6.00 (s, 1H), 5.86 (dd, J = 1.3,
17.8 Hz, 1H), 5.28 (dd, J = 1.3, 11.4 Hz, 1H), 5.20 (s, 1H), 4.06 (s,
3H), 1.46 (s, 9H). 13C NMR (CD3OD) d: 174.6, 173.8, 172.7, 165.3,
157.5, 151.0, 138.1, 137.5, 132.1, 132.0, 131.5, 129.3 (2 carbons),
129.1, 126.9, 123.8, 117.1, 89.5, 80.8, 59.3, 54.7, 28.7. MS calcd
for C26H27N3O6 [M+H]+ 478.2, found: 478.2.
7.1.16. Compound 13 ((S)-tert-butyl(1-(4-((6-methoxy-2-
phenylpyrimidin-4-yl)oxy)phenyl)-2-oxo-2-((2-((pent-4-en-1-
ylsulfonyl)carbamoyl)phenyl)amino)ethyl)carbamate)
Prepared according to the general procedure B, using the N-
deprotected HCl-salt of
0.27 mmol), HATU (124 mg, 0.33 mmol), and DIEA (269
2
(99.3 mg, 0.33 mmol),
8
(144 mg,
L,
l
1.57 mmol). The reaction was stirred in DCM for 3 h. Purification
on silica gel (CH2Cl2/MeOH 90:10) yielded 13 (162 mg, 86%) as a
white solid. 1H NMR (CD3OD): d 8.49 (m, 1H), 8.20–8.17 (m, 2H),
8.15 (m, 1H), 7.69–7.66 (m, 2H), 7.43–7.35 (m, 2H), 7.31–7.26
(m, 2H), 7.17 (d, J = 8.6 Hz, 2H), 7.04 (m, 1H), 6.06 (s, 1H), 5.67
(m, 1H), 5.33 (s, 1H), 4.96–4.84 (m, 2H), 4.03 (s, 3H), 3.26–3.18
(m, 2H), 2.11–2.02 (m, 2H), 1.92–1.83 (m, 2H), 1.46 (s, 9H). 13C
NMR (CD3OD): d 174.2, 173.7, 172.5, 171.2, 165.1, 157.4, 154.4,
140.8, 138.5, 138.0, 136.0, 133.3, 132.2, 132.0, 130.3, 129.3 (2 car-
bons), 124.8, 123.9, 123.0, 121.1, 116.1, 90.0, 81.1, 61.8, 54.7, 53.2,
33.4, 28.7, 24.2. HRMS calcd for C36H39N5O8S [M+H]+ 702.2598,
found: 702.2601. HPLC purity (system 1: 98.6%, system 2: 99.6%).
7.1.12. General procedure A, for N-deprotection (compounds 1–
3, 13–15, 23–25, and 37)
The respective carbamate was dissolved in 4.0 M HCl in 1,4-
dioxane (10 mL/mmol starting material) and stirred at room tem-
perature until starting material could no longer be visualized by
LC–MS. The solvent was thereafter evaporated and the hydrochlo-
ride salt of the N-deprotected product was used in forthcoming
couplings without further purification.
7.1.13. Compound 11 (tert-butyl ((S)-2-oxo-2-(((S)-1-oxo-1-
(pent-4-en-1-ylsulfonamido)pentan-2-yl)amino)-1-(4-((2-
phenyl-6-vinylpyrimidin-4-yl)oxy)phenyl)ethyl)carbamate)
Compound 7 (64 mg, 0.143 mmol) was mixed with the N-depro-
7.1.17. Compound 14 ((S)-tert-butyl(1-(4-((6-methoxy-2-
phenylpyrimidin-4-yl)oxy)-3-vinylphenyl)-2-oxo-2-((2-((pent-
4-en-1-ylsulfonyl)carbamoyl)phenyl)amino)ethyl)carbamate)
Prepared according to the general procedure B, using 10 (119 mg,
0.25 mmol), the N-deprotected HCl-salt of 1 (110 mg, 0.38 mmol),
tected HCl-salt of
0.172 mmol) and DIEA (142
3
(26.4 mg, 0.172 mmol), HATU (65 mg,
L, 0.829) and stirred in DMF for 2 h.
l
Addition of ethyl acetate (20 mL) before work up and purification
on silica gel (CH2Cl2/MeOH 95:5) yielded 11 (74.2 mg, 77%) as a
white solid. 1H NMR (CDCl3): d 10.12 (s, 1H), 8.35–8.30 (m, 2H),
7.47–7.38 (m, 5H), 7.24 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 7.7 Hz, 1H),
6.75 (dd, J = 10.4, 17.2 Hz, 1H), 6.62 (s, 1H), 6.60 (dd, J = 1.7,
17.2 Hz, 1H), 5.78–5.62 (m, 3H), 5.26 (s, 1H), 5.04–4.96 (m, 2H),
4.58 (m, 1H), 3.41–3.26 (m, 2H), 2.15–2.08 (m, 2H), 1.92–1.81 (m,
3H), 1.71 (m, 1H), 1.46 (s, 9H), 1.42–1.33 (m, 2H), 0.91 (t, J = 7.3 Hz,
3H). 13C NMR (CDCl3): d 171.2, 170.2, 164.9, 164.4, 162.9, 155.9,
153.1, 137.2, 136.4, 135.2, 133.7, 131.0, 128.7, 128.5 (2 carbons),
122.9, 122.4, 116.6, 102.7, 81.4, 59.0, 54.1, 52.7, 33.7, 31.9, 28.4,
22.2, 18.8, 13.7. MS calcd for C37H45N5O7S [M+H]+ 678.3, found: 677.9.
HATU (114 mg, 0.3 mmol), and DIEA (273 lL, 1.6 mmol). The reac-
tion was stirred in DCM for 2 h. Purification on silica gel (CH2Cl2/
MeOH 94:6) followed by purification on preparative HPLC (MeCN/
H2O (0.05% HCOOH)) yielded 14 (13.1 mg, 7%) as a white solid. 1H
NMR (CD3OD): d 8.24 (dd, J = 1.2, 8.4 Hz, 1H), 8.20 (dd, J = 1.4,
8.5 Hz, 2H), 7.86 (d, J = 2.4 Hz, 1H), 7.75 (dd, J = 1.6, 7.9 Hz, 1H),
7.59 (dt, J = 1.6, 7.5 Hz, 1H), 7.48 (dd, J = 2.4, 8.5 Hz, 1H), 7.41 (m,
1H), 7.34–7.30 (m, 2H), 7.25 (ddd, J = 1.1, 7.4, 7.9 Hz, 1H), 7.20 (d,
J = 8.3 Hz, 1H), 6.81 (dd, J = 11.3, 17.8 Hz, 1H), 6.05 (s, 1H), 5.94 (d,
J = 17.8 Hz, 1H), 5.76 (m, 1H), 5.40 (s, 1H), 5.31 (d, J = 11.2 Hz, 1H),
5.09 (d, J = 17.2 Hz, 1H), 4.98 (d, J = 10.2 Hz, 1H), 4.05 (s, 3H), 3.55–
3.50 (m, 2H), 2.58–2.51 (m, 2H), 1.49 (s, 9H). 13C NMR (CD3OD): d
173.7, 173.5, 172.6, 171.3, 165.2, 157.5, 151.3, 140.7, 138.0, 136.7,
136.6, 133.2, 132.3, 132.0, 131.8, 131.3, 129.4, 129.3, 127.3, 124.9,
124.2, 124.0, 121.2, 117.7, 116.5, 89.0, 81.1, 61.9, 54.7, 52.6, 29.1,
7.1.14. General procedure B, for synthesis of compounds 12–15
and 37
The N-deprotected HCl-salts of P1–P10 building blocks 1 or 2
were mixed with P2 building blocks 7, 8, 10 or 36 and HATU in
dry DCM. DIEA was added until the pH exceeded 10. The reactions
were stirred at 45 °C. When starting material no longer could be
visualized by LC–MS, DCM was added and the reaction mixture
was washed with 0.1 M NaHSO4 followed by 0.035 M NaHSO4.
The organic phase was thereafter evaporated before purification.
28.7. HRMS calcd for
C
37H39N5O8S [M+H]+ 714.2598, found:
714.2606. HPLC purity (system 1: 100%, system 2: 100%).
7.1.18. Compound 15 ((S)-tert-butyl(1-(4-((6-methoxy-2-
phenylpyrimidin-4-yl)oxy)-3-vinylphenyl)-2-oxo-2-((2-((pent-
4-en-1-ylsulfonyl)carbamoyl)phenyl)amino)ethyl)carbamate)
Prepared according to the general procedure B using the N-
7.1.15. Compound 12 ((S)-tert-butyl(2-oxo-2-((2-((pent-4-en-1-
ylsulfonyl)carbamoyl)phenyl)amino)-1-(4-((2-phenyl-6-
vinylpyrimidin-4-yl)oxy)phenyl)ethyl)carbamate)
Prepared according to the general procedure B described above,
using 7 (44.7 mg, 0.10 mmol), the N-deprotected HCl-salt of 2
(36.6 mg, 0.12 mmol), HATU (45.6 mg, 0.12 mmol), and DIEA
deprotected HCl-salt of
0.083 mmol), HATU (38 mg, 0.10 mmol) and DIEA (92
2
(40.5 mg, 0.13 mmol), 10 (40 mg,
L,
l
0.53 mmol). The reaction was stirred in DCM overnight. Purifica-
tion on preparative HPLC (MeCN/H2O (0.05% HCOOH)) yielded 15
(10.8 mg, 18%) as a white solid. 1H NMR (CD3OD): d 8.24 (d,
J = 8.4 Hz, 1H), 8.20 (dd, J = 1.5, 8.3 Hz, 2H), 7.86 (d, J = 2.4 Hz,
1H), 7.75 (dd, J = 1.5, 8.0 Hz, 1H), 7.60 (m, 1H), 7.48 (dd, J = 2.4,
(99 lL, 0.58 mmol). The reaction was stirred in dry DCM (5 mL) at

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6609
8.4 Hz, 1H), 7.41 (dt, J = 1.2, 7.5 Hz, 1H), 7.33–7.29 (m, 2H), 7.25
(ddd, J = 1.2, 8.0 Hz, 1H), 7.20 (d, J = 8.4, 1H), 6.81 (dd, J = 11.1,
17.6 Hz, 1H), 6.06 (s, 1H), 5.94 (dd, J = 1.2, 17.6 Hz, 1H), 5.75 (m,
1H), 5.40 (s, 1H), 5.31 (dd, J = 1.2, 11.1 Hz, 1H), 5.05–4.95 (m,
2H), 4.06 (s, 3H), 3.44 (m, 2H), 2.15 (m, 2H), 1.88 (m, 2H), 1.49
(s, 9H). 13C NMR (CD3OD): d 173.8, 172.6, 171.2, 169.9, 165.3,
157.7, 151.4, 139.7, 138.1, 136.6, 134.9, 132.4, 132.1, 131.4,
130.3, 129.4, 129.3 (2 carbons), 127.6, 125.2, 124.3, 123.2, 122.4,
117.6, 116.5, 89.7, 81.3, 61.1, 54.8, 53.5, 33.0, 28.7, 23.6. HRMS
calcd for C38H41N5O8S [M+H]+ 728.2676, found 728.2150. HPLC
purity (system 1: 98.8%, system 2: 98.7%).
7.73 (dd, J = 1.8, 8.2 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.58 (m, 1H),
7.43 (m, 1H), 7.39–7.33 (m, 2H), 7.29–7.24 (m, 2H), 7.23 (d,
J = 1.2 Hz, 1H), 6.11 (s, 1H), 5.83–5.69 (m, 2H), 5.64 (s, 1H), 5.04–
4.87 (m, 4H), 4.17 (m, 1H), 4.07 (s, 3H), 3.49–3.40 (m, 2H), 2.19–
2.11 (m, 3H), 2.04–1.97 (m, 2H), 1.92–1.83 (m, 3H), 1.71–1.61 (m,
2H), 1.42 (s, 9H), 1.38–1.30 (m, 2H). 13C NMR (CD3OD, purified on
HPLC): d 175.3, 173.8, 172.6, 170.4, 170.0, 165.3, 158.1, 154.6,
140.0, 139.4, 138.1(2 carbon), 135.5, 134.7, 132.1, 130.8, 130.6,
130.3, 129.4 (2 carbons), 125.3, 123.5, 123.1, 116.5, 114.8, 90.1,
80.7, 59.6, 56.1, 54.7, 53.5, 34.8, 33.0, 30.0 (2 carbons), 28.7, 26.7,
23.6. HRMS calcd for
C
45H54N6O9S [M+H]+ 855.3751, found:
855.3748. HPLC purity (system 1: 96.9%, system 2: 97.8%).
7.1.19. Compound 16 (but-3-en-1-yl((S)-3,3-dimethyl-1-oxo-1-
(((S)-2-oxo-2-((2-((pent-4-en-1-ylsulfonyl)carbamoyl)phenyl)
amino)-1-(4-((2-phenyl-6-vinylpyrimidin-4-yl)oxy)phenyl)ethyl)
amino)butan-2-yl)carbamate)
7.1.21. General procedure C, for synthesis of compounds 18–19,
26–28, and 38
The N-deprotected HCl-salts of the P2–P1–P10 building blocks
Compound 12 (56.7 mg, 0.081 mmol) was stirred in CH2Cl2/TFA
1:1 (2 mL) for 1 h. After evaporation of the solvent the N-deprotec-
ted TFA-salt of 12 was mixed with N-[(But-3-en-1-yloxy)car-
were mixed with HATU and Boc-L-tLeu, or Boc-2-amino-8 nonenoic
acid in dry DMF. DIEA was added until the pH exceeded 10. The reac-
tions were stirred in room temperature until starting material could
no longer be detected by LC–MS. Ethyl acetate was added and the
reaction mixture washed with sodium acetate buffer pH 4. The
organic phase was evaporated before purification on preparative
HPLC.
bonyl]-3-methyl-
0.14 mmol), and DIEA (96
L
-valine44 (26 mg, 0.11 mmol), HATU (52 mg,
l
L). The reaction was stirred in dry
DMF at room temperature for 1 h. Ethyl acetate (15 mL) was added
and the reaction mixture was washed with 0.1 M NaHSO4 followed
by 0.035 M NaHSO4 before evaporation of the organic phase. Puri-
fication by preparative HPLC (MeCN/H2O (0.1% TFA)) yielded 16
(2.9 mg, 4%) as a white solid. 1H NMR (CD3OD): d 8.25–8.22 (m,
2H), 8.15 (m, 1H), 7.71 (dd, J = 1.4, 7.8 Hz, 1H), 7.67–7.65 (m,
2H), 7.59 (ddd, J = 1.5, 7.4, 8.3 Hz, 1H), 7.41 (m, 1H), 7.36–7.30
(m, 4H), 7.25 (ddd, J = 1.2, 7.5, 7.9 Hz, 1H), 6.88 (s, 1H), 6.84 (dd,
J = 10.7, 17.3 Hz, 1H), 5.86–5.69 (m, 3H), 5.66 (s, 1H), 5.12–4.93
(m, 4H), 4.13 (s, 1H), 4.11–4.04 (m, 2H), 3.46–3.40 (m, 2H), 2.40–
2.33 (m, 2H), 2.17–2.10 (m, 2H), 1.91–1.82 (m, 2H), 1.06 (s, 9H).
13C NMR (CD3OD): d173.1, 171.9, 170.2, 169.9, 166.3, 165.7,
165.4, 154.4, 139.5, 138.4, 138.1, 136.3, 135.5, 135.2, 134.7,
132.0, 130.7, 130.3, 129.4 (2 carbons), 125.3, 123.6, 123.3, 117.5,
116.5, 103.9, 65.5, 64.2, 59.7, 53.5, 35.5, 34.7, 33.0, 27.3, 23.6.
HRMS calcd for C43H48N6O8S [M+H]+ 809.3333, found: 809.3340.
HPLC purity (system 1: 98.8%, system 2: 98.2%).
7.1.22. Compound 18 (tert-butyl ((S)-1-(((S)-1-(4-((6-methoxy-
2-phenylpyrimidin-4-yl)oxy)phenyl)-2-oxo-2-((2-((pent-4-en-
1-ylsulfonyl)carbamoyl)phenyl)amino)ethyl)amino)-3,3-
dimethyl-1-oxobutan-2-yl)carbamate)
Prepared according to the general procedure C, using the N-
deprotected HCl-salt of 13 (17.0 mg, 0.027 mmol), Boc-
(18.7 mg, 0.081 mmol), HATU (36.9 mg, 0.097 mmol), and DIEA
(38.5 L, 0.22 mmol). The reaction was stirred in DMF for 1.5 h.
Purification on preparative HPLC (MeCN/H2O with 25 mM NH4OAc,
pH 6.3) yielded 18 (7.9 mg, 35%), as the NH+4-salt, in a 5:2
diaste-
L-tLeu
l
L:D
reomeric mixture. 1H NMR (CDCl3, major isomer reported): d 8.55
(m, 1H), 8.32–8.25 (m, 3H), 7.61–7.52 (m, 3H), 7.42 (m, 1H), 7.37–
7.31 (m, 2H), 7.23–7.20 (m, 2H), 7.13 (m, 1H), 5.98 (s, 1H), 5.72 (m,
1H), 5.24 (s, 1H), 5.08–5.00 (m, 2H), 4.06 (s, 3H), 3.99 (m, 1H),
3.56–3.40 (m, 2H), 2.22–2.15 (m, 2H), 1.99–1.90 (m, 2H), 1.40 (s,
9H), 1.05 (s, 9H). 13C NMR (CDCl3): d 172.2, 171.2, 170.9, 168.6,
168.3, 164.2, 156.0, 153.4, 150.9, 140.3, 137.0, 136.4, 134.8,
134.1, 131.2, 129.1, 128.5 (2 carbons), 123.9, 122.3, 121.9, 118.5,
116.8, 89.2, 80.2, 62.7, 58.4, 54.2, 53.2, 34.7, 32.0, 28.5, 26.9,
7.1.20. Compound 17 (tert-butyl((S)-1-(((S)-1-(4-((6-methoxy-2-
phenylpyrimidin-4-yl)oxy)phenyl)-2-oxo-2-((2-((pent-4-en-1-
ylsulfonyl)carbamoyl)phenyl)amino)ethyl)amino)-1-oxonon-8-
en-2-yl)carbamate)
The N-deprotected HCl-salt of 13 (73 mg, 0.11 mmol) was mixed
with Boc-2-amino-8-nonenoic acid (46 mg, 0.17 mmol), HATU
22.5. HRMS calcd for C
42H50N6O9S [M+H]+ 815.3438, found:
815.3436. HPLC purity (system 1: 98.2%, system 2: 99.7%).
(78 mg, 0.21 mmol), and 4-methylmorpholine (116 lL, 0.88 mmol).
The reaction was stirred in DMF (2 mL) at room temperature for 2 h.
Ethyl acetate (15 mL) was added and the reaction mixture was
washed with 0.1 M NaHSO4 followed by 0.035 M NaHSO4, whereaf-
ter the organic phase was evaporated. Purification on silica gel (CH2-
Cl2/MeOH 93:7) yielded 17 (76.4 mg, 79%. A small fraction was
purified on HPLC (MeCN/H2O (0.05% HCOOH)). 1H NMR (CD3OD,
purified on silica gel): d 8.45(dd, J = 1.3, 8.1 Hz, 1H), 8.23–8.19
(m, 2H), 8.15 (dd, J = 1.7, 8.0 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.42–
7.37 (m, 2H), 7.35–7.29 (m, 2H), 7.22–7.19 (m, 2H), 7.07 (m, 1H),
6.08 (s, 1H), 5.85–5.61 (m, 3H), 4.97–4.86 (m, 4H), 4.21 (m, 1H),
4.05 (s, 3H), 3.78 (s, 1H), 3.28–3.16 (m, 2H), 2.11–2.01 (m, 3H),
1.99–1.92 (m, 2H), 1.88–1.79 (m, 3H), 1.74–1.64 (m, 2H), 1.42 (s,
9H), 1.33–1.25 (m, 2H). 13C NMR (CD3OD, purified on silica gel): d
175.3, 173.8, 173.7, 172.5, 170.3, 165.1, 157.9, 154.5, 140.6, 140.0,
138.6, 138.0, 135.6, 133.0, 132.0, 131.8, 130.3, 129.4, 129.3, 125.2,
124.1, 123.1, 121.3, 116.0, 114.7, 90.0, 80.7, 60.4, 56.5, 54.7, 52.9,
34.7, 33.5, 33.2, 29.9, 28.7, 26.8, 24.3. 1H NMR (CD3OD, purified
on HPLC): d 8.26–8.22 (m, 2H), 8.17 (m, 1H), 7.73 (dd, J = 1.8,
8.2 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.58 (m, 1H), 7.43 (m, 1H),
7.1.23. Compound 19L (tert-butyl((S)-1-(((S)-1-(4-((6-methoxy-
2-phenylpyrimidin-4-yl)oxy)-3-vinylphenyl)-2-oxo-2-((2-((pent-
4-en-1-ylsulfonyl)carbamoyl)phenyl)am ino)ethyl) amino)-3,3-
dimethyl-1-oxobutan-2-yl)carbamate) and 19D (tert-butyl((S)-
1-(((R)-1-(4-((6-methoxy-2-phenylpyrimidin-4-yl)oxy)-3-vinylp
henyl)-2-oxo-2-((2-((pent-4-en-1-ylsulfonyl)carbamoyl)phenyl)
amino)ethyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)carbamate)
Prepared following the general procedure C, using the N-depro-
tected HCl-salt of 15 (41.8 mg, 0.063 mmol), Boc-
0.10 mmol), HATU (28.7 mg, 0.075 mmol) and DIEA (94
L
-tLeu (23.3 mg,
L,
l
0.54 mmol). The reaction was stirred in DMF for 5 h. Purification
and separation of the diastereomers on preparative HPLC (MeCN/
H2O (0.05% HCOOH)) gave 19L (8.1 mg, 15%) and 19D (5.5 mg,
10%) as white solids. 19L 1H NMR (CD3OD): d 8.77 (d, J = 6.6 Hz,
1H), 8.20 (dd, J = 1.2, 8.3 Hz, 2H), 8.14 (dd, J = 1.2, 8.5, 1H), 7.92
(d, J = 2.4 Hz, 1H), 7.72 (dd, J = 1.6, 8.0 Hz, 1H), 7.59 (ddd, J = 1.5,
7.3, 8.2 Hz, 1H), 7.37 (dd, = 2.7, 8.4 Hz, 1H), 7.41 (m, 1H), 7.31 (t,
J = 7.7 Hz, 2H), 7.25 (dt, J = 1.5, 7.8 Hz, 1H), 7.21 (d, J = 8.4 Hz,
1H), 6.82 (dd, J = 11.2, 17.9 Hz, 1H), 6.07 (s, 1H), 5.99 (d,

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A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
J = 17.6 Hz, 1H), 5.77–5.66 (m, 2H), 5.32 (dd, J = 1.2, 11.3 Hz, 1H),
5.02–4.93 (m, 2H), 4.06 (s, 3H), 3.44–3.32 (m, 2H), 2.12–2.05 (m,
2H), 1.88–1.79 (m, 2H), 1.42 (s, 9H), 1.05 (s, 9H). 13C NMR (CD3OD):
d 173.8, 173.4, 173.3, 172.6, 170.0, 169.8, 165.3, 158.0, 155.1,
151.5, 139.4, 138.1, 138.0, 135.9, 134.7, 132.5, 132.4, 132.1,
131.4, 130.2, 129.4, 129.3, 127.8, 125.3, 124.4, 123.6, 117.8,
116.5, 89.7, 80.8, 63.9, 59.6, 54.8, 35.3, 32.9, 28.7, 27.3, 23.5. HPLC
purity (system 1: 100%, system 2 100%) 19D 1H NMR (CD3OD): d
8.84 (d, J = 6.9 Hz, 1H), 8.19 (dd, J = 2.0, 8.2 Hz, 2H), 8.05 (m, 1H),
7.91 (d, J = 2.3 Hz, 1H), 7.71 (dd, J = 1.5, 7.8 Hz, 1H), 7.59 (m, 1H),
7.52 (dd, J = 2.4, 8.2 Hz, 1H), 7.42 (tt, J = 1.5, 7.3 Hz, 1H), 7.32 (t,
J = 7.6 Hz, 2H), 7.27 (dt, J = 1.2, 7.6 Hz, 1H), 7.22 (d, J = 8.2 Hz,
1H), 6.82 (dd, J = 11.3, 17.9 Hz, 1H), 6.08 (s, 1H), 5.98 (d,
J = 17.8 Hz, 1H), 5.70 (dt, J = 6.7, 17.0 Hz, 2H), 5.32 (dd, J = 1.2,
11,3 Hz, 1H), 5.02–4.92 (m, 2H), 4.07(s, 3H), 3.40–3.30 (m, 2H)
2.06 (q, J = 7.1 Hz, 2H), 1.88–1.79 (m, 2H), 1.43 (s, 9H), 1.00 (s,
9H). 13C NMR (CD3OD): d 179.1, 177.4, 173.8, 172.6, 169.7, 168.7,
165.3, 157.9, 151.5, 138.2, 138.1, 136.1, 134.4, 132.5, 132.1,
131.3, 130.1, 130.0, 129.4, 129.3, 127.9, 125.6, 124.4, 117.8,
116.4, 96.5, 89.7, 80.7, 64.1, 59.4, 54.8, 53.4, 35.4, 32.9, 28.8,
27.2, 23.6. HRMS calcd for C44H52N6O9S [M+H]+ 841.3594, found:
841.3596. HPLC purity (system 1: 100%, system 2 100%).
7.1.26. Compound 22L and 22D
In a microwave process vial, compound 21 (50 mg, 0.087 mmol)
was mixed with K2CO3 (18 mg, 0.131 mmol) in MeCN (3.2 mL) and
H2O (1.6 mL). The vial was sealed and irradiated by microwaves to
100 °C for 25 min. H2O (5 mL) and TFA (20 lL) was added to the
reaction mixture to pH 2. The reaction mixture was thereafter
extracted with EtOAc before the organic phase was evaporated.
This was mixed with the N-deprotected HCl-salt of 3 (28.7 mg,
0.101 mmol), HATU (38.4 mg, 0.101 mmol), and DIEA (83 lL,
0.487 mmol) in dry DMF. The reaction was stirred for 4 h before
EtOAc (20 mL) was added and the reaction mixture was washed
with 0.1 M NaHSO4 followed by 0.035 M NaHSO4. Thereafter, the
organic phase was evaporated. Purification on silica gel (CH2Cl2/
MeOH 95:5) followed by purification and separation on prepara-
tive HPLC (MeCN/H2O (0.1% TFA)) yielded the two diastereomers
22L (3.96 mg, 6%) and 22D (2.32 mg, 3.5%) as white solids. 22L
1H NMR (CD3OD): d 8.28–8.23 (m, 2H), 7.59–7.55 (m, 2H), 7.46–
7.38 (m, 3H), 7.26–7.22 (m, 2H), 6.82 (dd, J = 10.6, 17.3 Hz, 1H),
6.61 (dd, J = 1.6, 17.3 Hz, 1H), 5.80 (m, 1H), 5.71 (dd, J = 1.6,
10.6 Hz, 1H), 5.67 (dd, J = 10.3, 17.1 Hz, 1H), 5.62 (s, 1H), 5.12–
4.87 (m, 4H), 4.35 (m, 1H), 4.12–4.04 (m, 3H), 3.38–3.19 (m, 2H),
2.40–2.32 (m, 2H), 2.11–2.04 (m, 2H), 1.85–1.68 (m, 4H), 1.55–
1.38 (m, 2H), 1.02 (s, 9H), 0.96 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD):
d 173.3, 172.7, 172.0, 171.8, 166.1, 165.3, 158.5, 153.9, 138.2,
137.7, 136.1, 135.3, 131.9, 130.2, 129.4, 129.3, 123.3, 122.8,
117.5, 116.6, 103.6, 65.3, 63.9, 57.4, 55.0, 53.0, 35.4, 34.5, 34.4,
32.8, 27.1, 23.4, 19.9, 13.9. HRMS calcd for C41H52N6O8S [M+H]+
789.3646, found: 789.3649. HPLC purity (system 1: 100%, system
2: 100%). 22D 1H NMR (CD3OD): d 8.26–8.22 (m, 2H), 7.58–7.55
(m, 2H), 7.47–7.36 (m, 3H), 7.29–7.25 (m, 2H), 6.87 (s, 1H), 6.84
(dd, J = 10.5, 17.3 Hz, 1H), 6.63 (dd, J = 1.6, 17.3 Hz, 1H), 5.83 (m,
1H), 5.72 (dd, J = 1.6, 10.5 Hz, 1H), 5.56 (s, 1H), 5.14–4.94 (m,
4H), 4.32 (m, 1H), 4.17–4.01 (m, 3H), 3.45–3.29 (m, 2H), 2.42–
2.34 (m, 2H), 2.19–2.11 (m, 2H), 1.94–1.68 (m, 4H), 1.41–1.26
(m, 2H), 0.99 (s, 9H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD): d
173.2, 172.7, 171.9 (2 carbons), 166.3, 165.3, 158.7, 154.2, 138.5,
138.0, 136.3, 135.8, 135.5, 131.9, 130.7, 129.4, 129.3, 123.2,
123.1, 117.6, 116.6, 103.9, 65.5, 64.4, 58.3, 55.4, 53.1, 35.3, 34.6,
34.3, 32.9, 27.1, 23.6, 19.9, 13.9. HRMS calcd for C41H52N6O8S
[M+H]+ 789.3646, found: 789.3638. HPLC purity (system 1: 100%,
system 2: 100%).
7.1.24. Compound 20 ((S)-methyl 2-((tert-butoxycarbonyl)
amino)-2-(4-((2-phenyl-6-vinylpyrimidin-4-yl)oxy)phenyl)
acetate)
7
(191 mg, 0.43 mmol) was mixed with Cs2CO3 (278 mg,
0.85 mmol) in a round bottomed flask. The flask was sealed under
nitrogen. DMF (12 mL) was added and the reaction mixture was
stirred for 30 min before MeI (53.4 lL, 0.85 mmol) was added.
The reaction was then stirred at room temperature overnight.
The reaction mixture was filtered before ethyl acetate (25 mL)
was added. The reaction mixture was washed with saturated
NaHCO3 before the organic phase was evaporated. Purification
on silica gel (EtOAc/i-hexane 25:75) yielded 20 (110.5 mg, 56%)
as a white solid. 1H NMR (CDCl3): d 8.36–8.32 (m, 2H), 7.47–
7.38 (m, 5H), 7.26–7.23 (m, 2H), 6.77 (dd, J = 10.4, 17.1 Hz, 1H),
6.65 (s, 1H), 6.61 (dd, J = 1.5, 17.1 Hz, 1H), 5.70 (dd, J = 1.5,
10.5 Hz, 1H), 5.62 (s, 1H), 5.39 (s, 1H), 3.77 (s, 3H), 1.46 (s, 9H).
13C NMR (CDCl3): d 171.8, 170.5, 164.9, 164.5, 155.1, 152.9,
137.4, 135.3, 134.2, 131.1, 128.6 (3 carbons), 122.9, 122.2,
102.8, 80.6, 57.3, 53.0, 28.5. MS calcd for C26H27N3O5 [M+H]+
462.2, found: 461.9.
7.1.27. Compound 23
In
a
microwave process vial, compound 11 (60 mg,
7.1.25. Compound 21 (S)-methyl 2-((S)-2-(((but-3-en-1-yloxy)
carbonyl)amino)-3,3-dimethylbutanamido)-2-(4-((2-phenyl-6-
vinylpyrimidin-4-yl)oxy)phenyl)acetate
0.0885 mmol), was mixed with Hoveyda–Grubbs 2nd generation
catalyst (14 mg, 0.0223 mmol) in trifluorotoluene (20 mL). The vial
was sealed under N2 and irradiated by microwaves to 110 °C for
5 min. After filtration, the solvent was evaporated. Purification on
silica gel (CH2Cl2/MeOH 88:12) yielded 23 (24.4 mg, 43%) as a
white solid. 1H NMR (CD3OD): d 8.40–8.36 (m, 2H), 7.71–7.68 (m,
2H), 7.52–7.46 (m, 3H), 7.26–7.22 (m, 2H), 6.52 (d, J = 16.1 Hz,
1H), 6.34 (m, 1H), 6.29 (s, 1H), 5.38 (s, 1H), 4.38 (m, 1H), 3.27–
3.22 (m, 2H), 2.47–2.40 (m, 2H), 1.96 (m, 1H), 1.90–1.79 (m, 2H),
1.72 (m, 1H), 1.46 (s, 9H), 0.98 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD):
d 174.7, 173.4, 172.2, 167.9, 166.7, 157.3, 153.9, 140.0, 138.3,
138.1, 132.1, 132.0, 130.2, 129.5 (2 carbons), 123.0, 98.6, 81.0,
58.8, 54.9, 52.4, 35.4, 31.0, 28.7, 23.2, 20.0, 14.0. MS calcd for C33-
H39N5O7S [M+H]+ 650.3, found: 650.0.
Compound 20 (65 mg, 0.14 mmol) was stirred in CH2Cl2/TFA
1:1 (4 mL) for 1 h. After evaporation of the solvent the N-deprotec-
ted TFA-salt of 20 was mixed with N-[(But-3-en-1-yloxy)car-
bonyl]-3-methyl-
0.19 mmol), and DIEA (113
L
-valine44 (36 mg, 0.16 mmol), HATU (71 mg,
l
L, 1.25 mmol). The reaction was stir-
red in dry DMF (1 mL) at room temperature for 1 h. Ethyl acetate
(20 mL) was added and the reaction mixture was washed with
0.1 M NaHSO4 followed by 0.035 M NaHSO4 before evaporation
of the organic phase. Purification on silica gel (CH2Cl2/MeOH
98:2) yielded 21 (50 mg, 62%). 1H NMR (CDCl3): d 8.35–8.31 (m,
2H), 7.44–7.40 (m, 5H), 7.26–7.23 (m, 2H), 6.76 (dd, J = 10.4,
17.2 Hz, 1H), 6.64 (s, 1H), 6.61 (dd, J = 1.7, 17.2 Hz, 1H), 5.74 (m,
1H), 5.69 (dd, J = 1.7, 10.4 Hz, 1H), 5.58 (d, J = 6.7 Hz, 1H), 5.41 (d,
J = 9.4 Hz), 5.10–5.01 (m, 2H), 4.13–4.00 (m, 3H), 3.77 (s, 3H),
2.36–2.30 (m, 2H), 1.05 (s, 9H). 13C NMR (CDCl3): d 171.0, 170.4,
170.3, 164.8, 164.4, 156.7, 153.0, 137.2, 135.2, 134.2, 133.0,
131.1, 128.8, 128.5 (2 carbons), 123.0, 122.3, 117.3, 102.7, 64.4,
62.7, 56.2, 53.1, 35.0, 33.5, 26.7. MS calcd for C32H36N4O6 [M+H]+
573.3, found 573.0.
7.1.28. Compound 24
In two microwave process vials, compound 12 (70 mg,
0.10 mmol) was mixed with Hoveyda–Grubbs 2nd generation cat-
alyst (4.4 mg, 0.0070 mmol) in trifluorotoluene (37.6 mL). The vials
were sealed under N2 and irradiated by microwaves to 100 °C for
7.5 min. After filtration, the reaction mixtures were pooled and
evaporated. Purification on silica gel (CH2Cl2/MeOH 90:10) yielded

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6611
24 (7.3 mg, 11%) as a white solid. 1H NMR (CD3OD): d 8.68 (dd,
J = 1.2, 8.4 Hz, 1H), 8.38–8.35 (m, 2H), 8.22 (dd, J = 1.7, 8.0 Hz,
1H), 7.64 (m, 1H), 7.65–7.63 (m, 2H), 7.49–7.45 (m, 3H), 7.41
(ddd, J = 1.8, 7.3, 8.5 Hz, 1H), 7.33–7.26 (m, 2H), 7.05 (ddd, J = 1.3,
7.3, 8.1 Hz, 1H), 6.52 (d, J = 16.1 Hz, 1H), 6.48 (s, 1H), 5.48 (s, 1H),
3.06–2.94 (m, 2H), 2.52 (m, 1H), 2.37 (m, 1H), 2.01 (m, 1H), 1.88
(m, 1H), 1.47 (s, 9H). 13C NMR (CD3OD): d 176.7, 173.0, 170.0,
168.5, 166.6, .4, 154.3, 141.6, 141.0, 138.4, 137.3, 132.9, 132.5,
132.0, 131.6, 129.5 (2 carbons), 123.6, 123.0, 122.9, 120.5, 99.2,
80.7, 60.7, 51.3, 31.4, 28.7, 24.2. MS calcd for C35H35N5O7S
[M+H]+ 670.2, found: 670.4.
1H), 3.54 (m, 1H), 3.20 (m, 1H), 2.55 (m, 1H), 2.44 (m, 1H), 2.09
(m, 1H), 1.91 (m, 1H), 1.44 (s, 9H), 1.04 (s, 9H). 13C NMR (CD3OD):
d 173.0, 172.9, 169.8, 168.4, 166.7, 157.9, 154.5, 141.4, 140.2,
138.4, 136.3, 134.4, 132.1, 131.9, 131.3, 129.5 (2 carbons), 124.1,
123.2, 123.1, 121.5, 99.3, 80.6, 63.7, 59.3, 52.1, 35.5, 31.1, 28.7,
27.2, 23.6. HRMS calcd for C41H46N6O8S [M+H]+ 783.3176, found:
783.3174. HPLC purity (system 1: 100%, system 2: 100%).
7.1.32. Compound 28
Prepared according to the general procedure C, using the N-
deprotected TFA-salt of 25 (29.9 mg, 0.043 mmol), Boc-
(19.8 mg, 0.085 mmol), HATU (39.0 mg, 0.102 mmol), DIEA
(57 L, 0.24 mmol), and DMF (2 mL). The reaction was stirred in
L-tLeu
7.1.29. Compound 25
l
In eight microwave process vials, compound 14 (133.6 mg,
0.19 mmol) was mixed with Hoveyda–Grubbs 2nd generation cat-
alyst (36.3 mg, 0.058 mmol) in trifluorotoluene (92 mL). The vials
were sealed under N2 and irradiated by microwaves to 110 °C.
The reaction mixtures were pooled and evaporated. Purification
on silica gel (CH2Cl2/MeOH 95:5 to 90:10) yielded 25 (31.4 mg,
28%). A small fraction was purified on HPLC (MeCN/H2O with
25 mM NH4OAc, pH 6.3). 1H NMR (CD3OD, NH+4-salt): d 8.31 (m,
1H), 8.15–8.11 (m, 2H), 7.79 (m, 1H), 7.67 (s, 1H), 7.56–7.51 (m,
2H), 7.36 (m, 1H), 7.25–7.17 (m, 4H), 6.45 (d, J = 11.5 Hz, 1H),
6.02 (s, 1H), 5.70 (m, 1H), 5.24 (s, 1H), 4.04 (s, 3H), 3.69–3.63 (m,
2H), 2.54 (m,1H), 2.43 (m, 1H), 1.47 (s, 9H). 13C NMR (CD3OD): d
174.5, 173.6, 172.5, 170.7, 164.9, 157.5, 152.2, 140.2, 137.8,
136.8, 132.9, 132.8, 132.2, 132.0, 131.5, 131.1, 129.2 (2 carbons),
129.1, 127.7, 126.7, 125.5, 124.1, 124.0, 121.3, 89.4, 81.1, 62.1,
room temperature for 2 h. Purification on preparative HPLC yielded
28 (11.7 mg, 33%) as the NH+4-salt. 1H NMR (CD3OD): d 8.29 (d,
J = 8.1 Hz, 1H), 8.12 (dd, J = 1.3, 8.1 Hz, 2H), 7.81 (m, 1H), 7.73 (s,
1H), 7.56–7.48 (m, 3H), 7.35 (m, 1H), 7.24–7.15 (m, 4H), 6.44 (d,
J = 11.7 Hz, 1H), 6.02 (s, 1H), 5.71 (m, 1H), 5.52 (s, 1H), 4.12 (s,
1H), 4.03 (s, 3H), 3.74 (m, 1H), 3.48 (m, 1H), 2.59 (m, 1H), 2.36
(m, 1H), 1.43 (s, 9H), 1.08 (s, 9H). 13C NMR (CD3OD): d 173.7,
173.4, 172.4, 170.0, 165.1, 157.9, 152.3, 139.6, 137.9, 136.0,
133.7, 132.1, 131.7, 131.2, 130.9, 129.3, 129.2, 128.5, 127.4, 124.7
(2 carbons), 124.1, 122.4, 89.5, 80.7, 63.5, 60.8, 54.7, 53.1, 35.5,
28.7, 27.3, 25.0. HRMS calcd for C41H46N6O9S [M+H]+ 799.3047,
found; 799.3121. HPLC purity (system 1: 100%, system 2: 99.6%).
7.1.33. Compounds 29L, 29D and 30
Compound 17 (84.1 mg, 0.098 mmol) was lyophilized in CH3CN
54.7, 52.4, 28.7, 25.3. HRMS calcd for
C
35H35N5O8S [M+H]+
and HCOOH (3.7
lL, 0.098 mmol). This was mixed with HCOOH
686.2285, found: 686.2288. HPLC purity (system 1: 100%, system
2: 95.4%).
(3.7 L, 0.098 mmol), Hoveyda–Grubbs 2nd generation catalyst
l
(6.1 mg, 0.0098 mmol) and trifluorotoluene (64 mL) in four micro-
wave process vials. The vials were heated by microwaves to 100 °C
for 5 min. The reactions were thereafter pooled and evaporated.
Purification on silica gel (CH2Cl2/MeOH 90:10) followed by purifi-
cation on preparative HPLC (MeCN/H2O with 25 mM NH4OAc, pH
6.3) yielded 29L (9.15 mg, 11%), 29D (4.93 mg, 6%), and 30
(2.24 mg, 3%) as the NH+4-salts. 29L 1H NMR (CD3OD): d 8.27 (m,
3H), 7.94 (m, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.46–7.36 (m, 4H), 7.26
(d, J = 8.6 Hz, 2H), 7.16 (m, 1H), 6.09 (s, 1H), 5.53 (s, 1H), 5.41–
5.29 (m, 2H), 4.28 (m, 1H), 4.07 (s, 3H), 3.57 (m, 1H), 3.23 (m,
1H), 2.31–2.06 (m, 6H), 2.05 (m, 2H), 1.62 (m, 1H), 1.44 (s, 9H),
1.42–1.35 (m, 3H). 13C NMR (CD3OD): d 176.3, 173.8, 172.6,
171.4, 165.3, 158.1, 154.6, 139.4, 138.1, 134.6, 133.5, 133.2,
132.1, 130.8, 130.6, 129.4 (2 carbons), 124.6, 123.1, 122.5, 121.2,
119.9, 90.0, 80.4, 61.4, 55.6, 54.7, 52.7, 33.5, 32.4, 32.0, 29.0, 28.8,
28.5, 26.3, 23.8. HRMS calcd for C43H50N6O9S [M+H]+ 827.3438,
found: 827.3436. HPLC purity (system 1: 99.1%, system 2: 98.4%).
29D 1H NMR (CD3OD): d 8.43 (m, 1H), 8.27–8.22 (m, 2H), 7.94
(m, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.52 (ddd, J = 1.5, 7.4, 8.4 Hz, 1H),
7.46–7.36 (m, 3H), 7.27–7.23 (m, 2H), 7.19 (m, 1H), 6.09 (s, 1H),
5.57 (s, 1H), 5.42–5.29 (m, 2H), 4.07 (s, 3H), 4.04 (m, 1H), 3.59
(m, 1H), 3.23 (m, 1H), 2.27–2.22 (m, 2H), 2.07–1.97 (m, 4HV),
1.92–1.83 (m, 2H), 1.51 (m, 1H), 1.38 (s, 9H), 1.33–1.24 (m, 3H).
13C NMR (CD3OD): J = 176.5, 173.8, 172.6, 171.0, 165.3, 157.8,
154.6, 139.7, 138.1, 134.9, 133.8, 133.5, 132.1, 131.0, 130.8,
130.6, 129.4 (2 carbons), 124.7, 124.1, 123.0, 122.3, 90.1, 80.8,
60.7, 58.0, 54.7, 52.9, 33.8, 32.3, 31.9, 28.8, 28.7, 28.6, 26.4, 24.0.
HRMS calcd for C43H50N6O9S [M+H]+ 827.3438, found: 827.3441.
HPLC purity (system 1: 100%, system 2: 96.6%). Compound 30 1H
NMR (CD3OD, major isomer reported): d 8.38 (m, 1H), 8.27–8.23
(m, 2H), 7.98 (m, 1H), 7.64–7.58 (m, 2H), 7.52–7.36 (m, 4H),
7.28–7.22 (m, 2H), 7.18 (m, 1H), 6.10 (s, 1H), 5.58 (s, 1H), 5.41–
5.33 (m, 2H), 4.25 (m, 1H), 4.07 (s, 3H), 3.60 (m, 1H), 3.26 (m,
1H), 2.59 (m, 1H), 2.23–2.15 (m, 2H), 2.07–1.89 (m, 6H), 1.70 (m,
1H), 1.40 (s, 9H). HRMS calcd for C42H48N6O9S [M+H]+ 813.3282,
found: 813.3284. HPLC purity (system 1: 100%, system 2: 98.1%).
7.1.30. Compound 26
Prepared according to the general procedure C, using the N-
deprotected TFA-salt of 23 (20.4 mg, 0.031 mmol), Boc-
(14.2 mg, 0.062 mmol), HATU (28 mg, 0.074 mmol), DIEA (61
L
-tLeu
L,
l
0.36 mmol) and DMF (1.5 mL). The reaction was stirred 2 h in room
temperature. EtOAc (20 mL) was added, and the reaction mixture
washed with 0.1 M NaHSO4 follwed by 0.035 M NaHSO4 before
the organic phase was evaporated. Purification on preparative
HPLC (MeCN/H2O (0.05% HCOOH)) yielded 26 (14.3 mg, 61%) as a
white solid. 1H NMR (CD3OD): d 8.41–8.38 (m, 2H), 7.75–7.71 (m,
2H), 7.53–7.47 (m, 3H), 7.27–7.22 (m, 2H), 6.56 (d, J = 16.1 Hz,
1H), 6.36 (m, 1H), 6.32 (s, 1H), 5.63 (s, 1H), 4.40 (dd, J = 3.9,
10.0 Hz, 1H), 4.08 (s, 1H), 3.27–3.24 (m, 2H), 2.50–2.43 (m, 2H),
1.99 (m, 1H), 1.92–1.78 (m, 2H), 1.71 (m, 1H), 1.57–1.47 (m, 2H),
1.45 (s, 9H), 1.04 (s, 9H), 0.98 (t, J = 7.3 Hz, 3H). 13C NMR (CD3OD):
d 173.7, 173.4, 173.2, 171.9, 168.0, 166.7, 158.0, 154.0, 139.9,
138.4, 137.3, 132.2, 132.1, 130.5, 129.5 (2 carbons), 123.0, 98.6,
80.7, 63.7, 57.9, 54.5, 52.6, 35.4, 35.1, 31.0, 28.7, 27.2, 23.1, 20.1,
13.9. HRMS calcd for
C
39H50N6O8S [M+H]+ 763.3489, found:
763.3497. HPLC purity (system 1: 100%, system 2: 99.0%).
7.1.31. Compound 27
Prepared according to the general procedure C, using the N-
deprotected TFA-salt of 24 (7.5 mg, 0.011 mmol), Boc-
(5.0 mg, 0.022 mmol), HATU (9.9 mg, 0.026 mmol), DIEA (14.5
L
-tLeu
L,
l
0.085 mmol), and DMF (1 mL). The reaction was stirred in room
temperature for 3 h. EtOAc (10 mL) was added and the reaction
mixture was washed with 0.1 M NaHSO4 follwed by 0.035 M
NaHSO4 before the organic phase was evaporated. Purification on
preparative HPLC (MeCN/H2O (0.05% HCOOH)) yielded 27
(1.73 mg, 20%) as a white solid. 1H NMR (CD3OD): d 8.68 (m, 1H),
8.41–8.37 (m, 2H), 7.98 (dd, J = 1.7, 7.9 Hz, 1H), 7.70–7.66 (m,
2H), 7.56–7.45 (m, 4H), 7.33–7.30 (m, 2H), 7.14 (m, 1H), 6.56 (d,
J = 16.2 Hz, 1H), 6.50 (s, 1H), 6.42 (m, 1H), 5.76 (s, 1H), 4.03 (s,

6612
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
7.1.34. Compounds 31L, 31D and 32
(CDCl3): d 172.0, 170.1, 167.8, 167.7, 165.8, 165.3, 156.5, 152.8,
140.3, 137.0, 136.5, 136.1, 135.6, 133.2, 131.6, 130.3, 128.8,
128.7, 128.2, 127.8, 124.1, 123.4, 122.1, 117.0, 116.8, 96.9, 63.6,
63.2, 58.1, 53.3, 34.5, 32.1, 31.9, 26.5, 22.4. HRMS calcd for C41H44-
N6O8S [M+H]+ 781.3020, found: 781.3017.
Mixed fractions from the purification of 29 (7.4 mg,
0.0089 mmol), and Pd/C (10%) (2 mg, 0.0009 mmol) was mixed in
THF (4 mL). The reaction was stirred under H2-atmosphere for
27 h. Filtration followed by purification on preparative HPLC
(MeCN/H2O with 25 mM NH4OAc, pH 6.3) yielded 31L (1.02 mg,
14%), 31D (0.86 mg, 12%), and 32 (1.13 mg, 15%) as the NH+4-salts.
31L 1H NMR (CD3OD): d 8.26–8.23 (m, 2H), 8.15 (d, J = 8.4 Hz,
1H), 7.72 (d, J = 8.5 Hz, 1H), 7.61–7.56 (m, 3H), 7.46–7.35 (m,
3H), 6.04 (s, 1H), 5.52 (s, 1H), 4.25 (m, 1H), 4.06 (s, 3H), 6.04 (s,
1H), 5.52 (s, 1H), 4.25 (m, 1H), 4.06 (s, 3H), 3.63 (m, 1H), 3.50
(m, 1H), 1.99–1.88 (m, 3H), 1.66 (m, 1H), 1.61–1.50 (m, 2H), 1.41
(s, 9H), 1.35–1.24 (m, 12H). HRMS calcd for C43H52N6O9S [M+H]+
829.3595, found: 829.3593. HPLC purity (system 1: 100%, system
2: 100%). Compound 31D (CD3OD): d 8.27–8.23 (m, 2H), 8.16 (m,
1H), 7.85 (m, 1H), 7.62–7.58 (m, 2H), 7.54 (m, 1H), 7.46–7.36 (m,
4H), 7.28–7.20 (m, 3H), 6.11 (s, 1H), 5.59 (s, 1H), 4.13 (m, 1H),
4.07 (s, 3H), 3.56 (m, 1H), 3.26 (m, 1H), 1.97–1.90 (m, 2H), 1.86–
1.71 (m, 3H), 1.59–1.51 (m, 2H), 1.39 (s, 9H), 1.35–1.28 (m, 5H).
HRMS calcd for C43H52N6O9S [M+H]+ 829.3595, found: 829.3593.
HPLC purity (system 1: 100%, system 2: 100%). Compound 32 1H
NMR (CD3OD): d 8.27–8.23 (m, 2H), 8.08 (m, 1H), 7.87 (m, 1H),
7.67–7.62 (m, 2H), 7.50 (m, 1H), 7.45–7.35 (m, 4H), 7.27–7.23
(m, 2H), 7.20 (m, 1H), 6.10 (s, 1H), 5.60 (s, 1H), 4.16 (m, 1H),
4.07 (s, 3H), 3.48 (m, 1H), 3.26 (m, 1H), 1.97–1.90 (m, 2H), 1.80–
1.72 (m, 2H), 1.58–1.49 (m, 2H), 1.39 (s, 9H), 1.37–1.34 (m, 4H).
HRMS calcd for C42H50N6O9S [M+H]+ 815.3438, found: 815.3447.
HPLC purity (system 1: 100%, system 2: 100%).
7.1.37. Compound 35
In
a
microwave process vial, compound 33 (5.0 mg,
0.009 mmol) was mixed with K2CO3 (1.9 mg, 0.014 mmol) in MeCN
(0.5 mL) and H2O (0.25 mL). The vial was sealed and irradiated by
microwaves to 100 °C for 15 min. H2O (1 mL) and TFA (10 lL) was
added to the reaction mixture to pH 2. The reaction mixture was
thereafter extracted with EtOAc before evaporation of the organic
phase. This was mixed with the N-deprotected HCl-salt of 3
(1.54 mg, 0.010 mmol), HATU (3.8 mg, 0.010 mmol), DIEA (8.4 lL,
0.05 mmol) in dry DMF (0.25 mL). The reaction was stirred in room
temperature for 1 h. EtOAc was added and the reaction mixture
was washed with sodium acetate buffer pH 4 before evaporation
of the organic phase. Purification on preparative HPLC (MeCN/
H2Ob (0.1% TFA)) yielded 35 (2.1 mg, 32%) as a white solid. 1H
NMR (CD3OD): d 8.40–8.37 (m, 2H), 7.70–7.64 (m, 2H), 7.53–7.47
(m, 3H), 7.22–7.19 (m, 2H), 6.57 (d, J = 16.4 Hz, 1H), 6.33 (m, 1H),
6.19 (s, 1H), 5.67 (m, 1H), 5.63 (s, 1H), 5.09–4.09 (m, 2H), 4.48
(m, 1H), 4.37 (m, 1H), 4.08 (s, 1H), 3.87 (m, 1H), 3.28–3.25 (m,
2H), 2.66 (m, 1H), 2.52 (m, 1H), 2.20–2.14 (m, 2H), 1.89–1.81 (m,
2H), 1.79–1.64 (m, 2H), 1.51–1.37 (m, 2H), 1.01 (s, 9H), 0.97 (t,
J = 7.4 Hz, 3H). 13C NMR (CD3OD): d 173.6, 173.2, 172.1, 171.3,
171.0, 167.7, 166.7, 158.4, 153.8, 139.0, 138.3, 138.1, 138.0,
132.2, 131.5, 129.6, 129.5, 116.7, 97.9, 88.3, 64.5, 63.7, 57.0, 55.2,
53.1, 35.1, 34.4, 33.5, 32.9, 26.9, 23.6, 20.1, 14.0. HRMS calcd for
7.1.35. Compound 33
In a microwave process vial, 21 (21.5 mg, 0.0375 mmol) and
Hoveyda–Grubbs 2nd generation catalyst (1.6 mg, 0.0026 mmol)
were mixed in trifluorotoluene (18.5 mL). The vial was sealed
under N2 and irradiated by microwaves to 110 °C for 5 min. The
reaction mixtures were pooled and filtrated on a small plug of RP
silica gel before evaporation. Purification on silica gel (EtOAc/
i-hexane 2:3) yielded 33 (13.9 mg, 68%) as a white solid. 1H NMR
(CDCl3): d 8.49–8.46 (m, 2H), 7.50–7.45 (m, 5H), 7.22–7.15 (m,
2H), 7.06 (d, J = 6.8 Hz, 1H), 6.56 (d, J = 16.3 Hz, 1H), 6.16 (m, 1H),
6.04 (s, 1H), 5.56 (d, J = 7.3 Hz, 1H), 5.35 (d, J = 9.7 Hz, 1H), 4.50
(m, 1H), 3.91–3.85 (m, 2H), 3.81 (s, 3H), 2.63 (m, 1H), 2.47 (m,
1H), 1.02 (s, 9H). 13C NMR (CDCl3): d 171.9, 170.8, 169.9, 166.4,
165.6, 156.4, 152.8, 137.0, 135.9, 135.8, 131.4, 131.1, 128.6 (2
carbons), 96.8, 63.6, 63.1, 56.2, 53.5, 34.6, 32.0, 26.4. MS calcd for
C
39H48N6O8S [M+H]+ 761.3333, found: 761.3336. HPLC purity (sys-
tem 1: 100%, system 2: 98.7%).
7.1.38. Compound 36 (2-((tert-butoxycarbonyl)amino)-2-(4-((7-
methoxy-2-phenylquinolin-4-yl)oxy)-3-vinylphenyl)acetic
acid)
Prepared as described previously by us.31
7.1.39. Compound 37
Prepared according to the general procedure, using 36 (54.5 mg,
0.103 mmol), the N-deprotected HCl-salt of
0.103 mmol), HATU (62.7 mg, 0.165 mmol) and DIEA (154
2
(31.4 mg,
L,
l
0.886 mmol). The reaction was strirred for 3.5 h. Purification on sil-
ica gel (CH2Cl2/MeOH 93:7) gave 37 (39.5 mg, 50%) as a white
solid. 1H NMR (CD3OD): d 8.45 (m, 1H), 8.21 (d, J = 9.2 Hz, 1H),
8.11 (m, 1H), 7.99 (d, J = 2.3 Hz, 1H), 7.72–7.76 (m, 2H), 7.63 (dd,
J = 2.3, 8.5 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.41–7.28 (m, 4H),
7.20 (dd, J = 2.5, 9.1 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.99 (m, 1H),
6.72 (dd, J = 11.6, 18.0 Hz, 1H), 6.62 (s, 1H), 5.96 (d, J = 18.0 Hz,
1H), 5.66 (m, 1H), 5.38 (s, 1H), 5.22 (d, J = 11.8 Hz, 1H), 4.94–4.80
(m, 2H), 3.95 (s, 3H), 3.30–3.16 (m, 2H), 2.08–1.99 (m, 2H), 1.91–
1.79 (m, 2H), 1.45 (s, 9H). 13C NMR (CD3OD): d 173.9, 170.9,
164.0, 163.6, 160.7, 152.1, 143.9, 141.4, 140.7, 140.1, 138.6,
138.5, 137.2, 133.2, 132.2, 130.7, 130.1, 129.8, 128.6, 127.9,
124.8, 123.9, 123.5, 121.1, 120.2, 118.4, 116.1, 115.8, 107.3,
106.2, 101.9, 81.1, 61.7, 56.1, 40.6, 38.9, 33.4, 28.7, 24.1. MS calcd
for C43H44N4O8S [M+H]+ 777.3, found: 777.4.
C
30H32N4O6 [M+H]+ 545.2, found: 545.0.
7.1.36. Compound 34
In a microwave process vial, 33 (14.8 mg, 0.027 mmol) was
mixed with K2CO3 (5.5 mg, 0.040 mmol) in MeCN (1.5 mL) and
H2O (5 mL). The vial was sealed and irradiated by microwaves to
100 °C for 15 min. H2O (3 mL) and TFA (10 lL) was added to the
reaction mixture to pH 2. The reaction mixture was thereafter
extracted with EtOAc before the organic phase was evaporated.
This was mixed with the N-deprotected HCl-salt of 2 (9.4 mg,
0.031 mmol), HATU (11.7 mg, 0.031 mmol), DIEA (25.4 lL,
0.15 mmol) in dry DCM. The reaction was stirred at 45 °C for 2 h.
CH2Cl2 (6 mL) was added and the reaction mixture was washed
with 0.1 M NaHSO4 followed by 0.035 M NaHSO4 before the
organic phase was evaporated. Purification by preparative HPLC
(MeCN/H2O (0.1% TFA)) yielded 34 (9.0 mg, 45%). 1H NMR (CDCl3):
d 11.06 (s, 1H), 8.61 (m, 1H), 8.44–8.40 (m, 2H), 7.65–7.60 (m, 3H),
7.52–7.43 (m, 6H), 7.17 (m, 1H), 6.62 (d, J = 16.9 Hz, 1H), 6.22 (m,
1H), 6.11 (s, 1H), 5.77 (m, 1H), 5.61 (d, J = 6.2 Hz, 1H), 5.45 (d,
J = 9.6 Hz, 1H), 5.15–5.06 (m, 2H), 4.50 (m, 1H), 3.97 (m, 1H),
3.88 (m, 1H), 3.55–3.41 (m, 2H), 2.65 (m, 1H), 2.48 (m, 1H),
2.29–2.22 (m, 2H), 2.01–1.88 (m, 2H), 1.04 (s, 9H). 13C NMR
7.1.40. Compound 38L and 38D
Prepared according to the general procedure C, using the N-
deprotected HCl-salt of 37 (42 mg, 0.059 mmol), Boc-
(21.9 mg, 0.095 mmol), HATU (27.0 mg, 0.071 mmol) and DIEA
(89 L, 0.51 mmol) the reaction was stirred in DMF for 1 h. Purifi-
L-tLeu
l
cation and separation of the diastereomers was performed on RP-
HPLC (MeCN/H2O (0.05% HCOOH)) giving compounds 38L
(5.1 mg, 10%) and 38D (5.3 mg, 10%) as white solids. Compound

A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
6613
38L 1H NMR (CD3OD): d 8.77 (m, 1H), 8.41 (d, J = 9.3 Hz, 1H), 8.17
(m, 1H), 8.02 (d, J = 2.8 Hz, 1H), 7.82 (m, 1H9, 7.72 (dd, J = 1.5,
6.9 Hz, 2H), 7.67 (m, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.53–7.48 (m,
2H), 7.46–7.39 (m, 2H), 7.32 (d, J = 8.6 Hz, 1H), 7.21 (dt, 1.4,
7.5 Hz, 1H), 6.78 (s, 1H), 6.77 (dd, J = 11.1, 17.8 Hz, 1H) 6.53 (m,
1H), 6.04 (m, 1H), 5.77–5.63 (m, 2H), 5.34 (dd, J = 1.2, 11.1 Hz,
1H), 5.01–4.70 (m, 2H), 4.04 (s, 3H), 3.36–3.17 (m, 2H), 2.12–2.05
(m, 2H), 1.87–1.77 (m, 2H), 1.40 (s, 9H), 1.04 (s, 9H). HPLC purity
(system 1: 100%, system 2: 99.5%). Compound 38D 1H NMR (CD3-
OD): d 8.76 (m, 1H), 8.38 (d, J = 9.0 Hz, 1H), 8.12 (d, J = 8.1 Hz,
1H), 8.00 (d, J = 2.5 Hz, 1H), 7.80 (dd, J = 8.0, 1.7 Hz, 1H), 7.76–
7.72 (m, 2H), 7.63 (dd, J = 2.2, 8.4 Hz, 1H), 7.55–7.40 (m, 5H),
7.37 (dd, J = 2.4, 9.1 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.22 (dt, 1.1,
7.6 Hz, 1H), 6.80 (dd, 11.3, 17.7 Hz, 1H), 6.72 (s, 1H), 6.03 (d,
J = 18.1, 1H), 5.78–5.65 (m, 2H), 5.33 (dd, J = 1.2, 11.1 Hz, 1H),
4.95(m, 1H), 4.06 (s, 1H), 4.03 (s, 3H), 3.39–3.32 (m, 2H), 2.13–
2.04 (m, 2H), 1.89–1.79 (m, 2H), 1.41 (s, 9H), 0.98 (s, 9H). HPLC
purity (system 1: 100%, system 2: 99.7%). HRMS calcd for
used to classify the compounds regarding metabolic stability fol-
lows: Clint <47 ( l/min/mg) indicates a low risk for high first
metabolism in vivo, 47 < Clint < 92 a moderate risk, and Clint >92
l
a high risk.
7.4.2. Solubility determinations
Determination of solubility was performed by addition of an ali-
quot of DMSO stock (10 mM) to phosphate buffered saline (PBS)
(final DMSO concentration 1%). Incubation with rotation in HPLC
glass vials for for 2 h at 37 °C. The samples were then centrifuged
at 10,000ꢀg for 30 min and the supernatant analyzed with
LC–MS/MS. This medium throughput method can thus only detect
solubilities below 100 lM.
7.4.3. Cell culture
Caco-2 cells, obtained from American Tissue Collection, Rock-
ville, MD, were maintained in an atmosphere of 90% air and 10%
CO2, as described previously.57 For transport experiments,
3.0ꢀ105 cells were seeded on polycarbonate filter inserts (12 mm
C
49H55N5O9S [M+H]+ 890.3799, found: 890.3801 (on a mixture of
38L and 38D).
diameter; pore size 0.4 lm; Costar, Cambridge, MA) and allowed
to grow and differentiate for 21–24 days. The monolayers integrity
7.2. Enzyme Inhibition
was assessed by measuring the paracellular marker [14C]-Mannitol
(1.0 lCi/mmol; Perkin–Elmer Life Sciences, Boston, MA) transport
The protease activity of the full-length HCV NS3/4A protein
(protease–helicase/NTPase from genotype 1a was measured using
a FRET-assay as described previously.52,65 In short, 1nM enzyme
was incubated for 10 min at 30 °C in 50 mM HEPES, pH 7.5,
and the transepithelial electrical resistance (TEER) before and after
the experiments.
7.4.4. Permeability experiments
10 mM DDT, 40% glycerol, 0.1% n-octyl-b-
with 25 of the peptide cofactor 2K-NS4A (KKGSVVIV-
GRIVLSGK), and inhibitor. The reaction was started by the addition
of 0.5 substrate (Ac-DED(Edans)EEAbu [COO]ASK(Dabcyl)-
NH2) obtained from AnaSpec Inc. (San Jose, USA). Non-linear
regression analysis of the data was made using Grafit 5.0.13 (Erith-
acus software limited).
D
-glucoside, 3.3% DMSO
Stock solutions (10 mM) of the compounds were prepared in
DMSO and diluted to 10 lM (final DMSO concentration of 0.1%)
l
M
in fasted state simulated intestinal fluid (FASSIF) at pH 7.4. HBSS
pH 7.4 supplemented with 1% (w/v) BSA was prepared to be used
in the receiving, basolateral, compartment.
The Caco-2 study was performed in accordance with published
protocols.57 Caco-2 cell monolayers (passage 94–105) were grown
on permeable filter support and used for transport study on day 21
l
M
w
7.3. In silico predictions
after seeding. Prior to the experiment a drug solution of 10 lM was
prepared and warmed to 37 °C. The Caco-2 filters were washed
with pre-warmed Hank’s balanced salt solution (HBSS) prior to
the experiment, and thereafter the experiment was started by
applying the donor solution on the apical side. The experiments
were performed at 37 °C and with a stirring rate of 500 rpm. The
receiver compartment was sampled at 15, 30 and 60 min, and at
60 min also a final sample from the donor chamber was taken in
order to calculate the mass balance of the compound. Directly after
the termination of the experiment the filter inserts were washed
with pre-warmed HBSS and the membrane integrity was checked.
This was performed by transepithelial electrical resistance (TEER)
measurement and by measurement of Mannitol permeability,
which is a paracellular marker used for integrity measurements.
For transport studies performed under sink conditions, where
less than 10% of the compound was transported across the Caco-
The predicted pKa and logD7.4 values for compounds 13, 15, 17,
18, 19, 29L and 29D were calculated using ADMET predictor v.5.5
and are presented in Table 8 in the manuscript.
7.4. In vitro preclinical profiling
7.4.1. Metabolic stability
The Clint can be used predictively as measurement of the in vivo
metabolic stability when the total clearance mechanism can be
described as hepatic and metabolic, and when oxidative metabo-
lism dominates. Compounds 13, 15, 17, 18, 19, 29L and 29D
(1 lM) were pre-incubated for 5 min at 37 °C with pooled human
liver microsomes (0.5 mg/mL; Xenotech, Kansas, KS) in 0.1 M
potassium phosphate buffer pH 7.4 prior to the addition of NADPH
(1 mM final concentration) to initiate the reaction. The reaction
was then incubated for 0, 5, 15 and 40 min and at each time point
the reaction was quenched by addition of ice-cold acetonitrile (35%
final concentration) containing 0.1 lM Warfarin as an internal
standard. Plates were centrifuged at 3500 rpm for 20 min at 4 °C,
2 cell monolayers, the apparent permeability coefficients (Papp
)
were calculated from the equation
D
D
Q
t
1
AC0
Papp
¼
ꢀ
D
Q
t
Where
is the steady-state flux (mol/s), C0 is the initial con-
and the supernatants were subjected to LC/MS/MS.
centration Din the donor chamber at each time interval (mol/mL),
and A is the surface area of the filter (cm2). Papp was obtained from
nonlinear regression of the accumulated dose in the receiver com-
partment over time, minimizing the sum of squared residuals in
the equation.
The natural logarithm of the analytical peak area ratio (relative
to 0 min sample which was considered as 100%) was plotted
against time and analyzed by linear regression. In vitro half-life
(t1/2) and in vitro intrinsic clearance (Clint) were calculated on the
basis of first-order reaction kinetics of the percentage of remaining
compound according to previously published models.55,56
Dextromethorphan (3 lM) and Midazolam (5 lM) were used as
positive controls for cytochrome P450 enzymes (CYP) isoforms
CYP2D6 and CYP3A4, respectively. The cut-off values that were
A Papp value below 0.2 ꢀ 10ꢁ6 cm/s indicates low permeability,
a Papp value between 0.2 ꢀ 10ꢁ6 cm/s to 1.6 ꢀ 10ꢁ6 cm/s indicates
moderate permeability, and a Papp value above 1.6 ꢀ 10ꢁ6 cm/s
indicates high permeability.60

6614
A. Lampa et al. / Bioorg. Med. Chem. 22 (2014) 6595–6615
Table 9
400 rapid Monte Carlo steps generating unique conformations
Mass spectrometric specific settings used for detection
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 move-
ment of an atom in any single step was limited to 0.1 Å. Hydro-
gen vibrations were damped by assigning an atomic weight of 10
to the hydrogens. Among the 10 best poses generated, the most
plausible binding mode conformation for each compound was
selected based on visual inspection.
Compound ESI
( )
m/z
(parent)
m/z
(product)
Cone
voltage (V)
Collision
energy (V)
13
15
17
18
19
+
+
+
+
+
+
+
702.4
728.4
855.5
815.4
841.5
827.5
827.5
646.3
672.3
755.4
715.4
741.4
727.4
727.4
22
24
20
20
22
20
20
16
16
18
16
18
18
18
29L
29D
Acknowledgements
Radioactive samples ([14C]-Mannitol) were analyzed with a liquid scintillation
counter (TopCount NXT, Perkin–Elmer Life Sciences, Boston).
We gratefully acknowledge support from Swedish Research
Council (grant nos. 9478, 21386 and D0571301), Knut and Alice
Wallenberg’s foundation, Science for Life Laboratory and Chemical
Biology Consortium of Sweden (CBCS). We also thank Simulations
Plus for providing access to the ADMET Predictor software, Johanna
Grandin and Gunnar Lindeberg for technical support and Dr Aleh
Yahorau, Department of Pharmaceutical Biosciences, Uppsala Uni-
versity, for help with HRMS analyzes and Medivir AB, Huddinge,
Sweden for EC50-determinations and financial support.
7.4.5. Data analysis
All experiments were performed in, at least, triplicates, and
samples were subjected to liquid chromatography/mass spectrom-
etry analysis with a Waters XEVO TQ triple-quadrupole mass spec-
trometer (electrospray ionization, ESI) coupled to a Waters Acquity
UPLC (Waters Corp.). For chromatographic separation a general
gradient was used (1% mobile phase B to 90% over 2.5 min total
References
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in partial injection mode using the MS settings in Table 9. In gen-
eral, a standard curve between 1–1000 nM was prepared for quan-
tization. Warfarin was used as an internal standard in all samples.
l
m column 2 ꢀ 50 mm (Waters Corp.).
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